CHAPTER ndash 5

Inclusion complexation of sulfadimethoxine sulfamerazine and

sulfapyridine with α- and β-cyclodextrinslowast

In this chapter the spectral properties of sulfadimethoxine (4-amino-N-(26-

dimethoxypyrimidin-4-yl) benzenesulfonamide SDMO) sulfamerazine (4-amino-N-(4-

methylpyrimidin-2-yl) benzenesulfonamide SMRZ) and sulfapyridine (4-amino-N-pyridin-

2-ylbenzenesulfonamide SFP) molecules in α-CD β-CD and different solvents have been

investigated using absorption fluorescence and time-resolved fluorescence methods The

formation of solid inclusion complexes of the drugs with α-CD and β-CD were characterized

by SEM TEM FTIR DSC XRD and 1H NMR techniques PM3 method was also applied

to study the inclusion process of the drugs within CD cavity and the minimum energy

structures of drugCD complexes were proposed The chemical structures of SDMO SMRZ

and SFP are given below

Chemical structures of (a) SDMO (b) SMRZ and (c) SFP

51 Effect of CDs with sulfonamides

Table 51 depicts the absorption and fluorescence maxima of SDMO SMRZ and

SFP recorded in different concentrations of α-CD and β-CD at pH ~7 The absorption and

fluorescence spectra of the above drug molecules in the presence and absence of CDs are

lowast Journal of Molecular Structure ndash 1054-1055 (2013) 215 Journal of Carbohydrate Polymers ndash 101 (2014) 828

(a) (b)

(c)

HH22NN SS

OO

OO

NNHHNN

NN

OO

OO

HH22NN SS

OO

OO

NNHHNN

NN

HH22NN SS

OO

OO

NNHHNN

131

shown in Figs 51-54 SDMO gives a structureless broad absorption band at 268 nm

whereas two absorption bands (at 268 and 243 nm) are obtained for SMRZ in aqueous

solution However in the spectrum of SFP in water three absorption bands are found at 310

nm (πrarrπ transition of the double bond) 261 nm (πrarrπ transition of the aniline group)

and 243 nm (πrarrπ transition of the aromatic ring) With increasing concentrations of β-CD

the absorption maxima of the above drugs are red shifted (about ~8 nm) with gradual

decrease in the molar extinction coefficient whereas the absorbance increased at the same

wavelength in the α-CD The decrease or increase in the absorbance is due to the

encapsulation of the above sulfa drugs into the CD cavity and it is attributed to the detergent

action of CDs [87-93] Further when recorded after 24 hours no significant change was

observed in the absorbance indicating that these sulfonamides do not decompose in the CDs

solution The above behaviour is attributed to the enhanced dissolution of the sulfonamide

molecules through the hydrophobic interaction between the guest and non-polar cavity of

the CD These results suggest that sulfonamides are entrapped into the CD cavity to form the

stable inclusion complex Moreover the red shift observed in β-CD reveals that the

pyrimidine nitrogen atom interacts with β-CD hydroxyl groups because it is well known

that CDs are good hydrogen donors [1-3] Additionally in all the cases a clear isosbestic

point was observed in the absorption spectra In general the existence of an isosbestic point in

the absorption spectra is an indication of the formation of well defined 11 complex [87-93]

Figs 53 and 54 display the typical fluorescence spectra of SDMO SMRZ and SFP

in aqueous solution as a function of α-CD and β-CD concentrations Both SDMO and

SMRZ drugs exhibit dual fluorescence with the maximum excitation wavelength of 270 nm

Likewise SFP emits two emission peaks at 348 and 434 nm with a shoulder at 372 nm when

excited at 310 nm It has been shown in our earlier studies [88] that sulfonamide derivatives

undergo normal as well as highly Stokes shifted fluorescence The normal fluorescence

takes place from locally excited (LE) state while twisted intramolecular charge transfer

(TICT) is responsible for highly Stokes shifted fluorescence The PM3 calculations have

suggested that the large stabilization of excited singlet state of the above sulfa drugs with

twisted conformation occurring at the amide SndashN bond between the electron donor group

(aniline ring) and the electron acceptor group (heterocyclic ring) In water the intensity of

LE band is greater than TICT band However on addition of CDs both the LE and TICT

intensities are equally increased With an increase in the β-CD concentration a regular red

shift is observed in the TICT band (434 nm to 445 nm) in all the drugs

132

Table 51

133

Fig 51 Absorption spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of absorbance vs concentration of α-CD

Abs

orba

nce

Wavelength (nm)

080

040

0 200 290 380

[α-CD] times 10-3 M

073

075

077

0 5 10 15

Abs

268 nm

1

7

SMRZ

Abs

orba

nce

Wavelength (nm)

150

075

0 200 275 350

SDMO

1

7 [α-CD] times 10-3 M

066

072

078

0 5 10 15

Abs

268 nm

Abs

orba

nce

Wavelength (nm)

080

040

0 200 300 400

Abs

[α-CD] times 10-3 M

027 028 029

0 4 8 12

040

310 nm

SFP

1

7

134

Fig 52 Absorption spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of absorbance vs concentration of β-CD

Abs

orba

nce

Wavelength (nm)

150

075

0 200 275 350

7

1

SDMO

[β-CD] times 10-3 M

047

061

075

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

100

050

0 200 275 350

SMRZ

7

1 [β-CD] times 10-3 M

068

072

076

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

080

040

0 200 300 400

SFP

Abs

[β-CD] times 10-3 M

027 031 035

0 4 8 12

039 310 nm

7

1

7

1

135

Fig 53 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of α-CD

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520 340 460

SDMO

1

7 [α-CD] times 10-3 M

260

330

400

0 5 10 15

If

341 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

480

240

0 290 405 520

SMRZ If

[α-CD] times 10-3 M

200

260

0 4 8 12

320

341 nm

1

7

Fluo

resc

ence

inte

nsity

(au)

Wavelength (nm)

300

150

0 320 450 580

SFP

1

7

If

[α-CD] times 10-3 M

50

110

0 4 8 12

170

310 nm

136

Fig 54 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of β-CD

340 460

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520

[β-CD] times 10-3 M

250

400

550

0 5 10 15

If

340 nm

1

7

SDMO

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

300

150

0 320 450 580

SFP If

[β-CD] times 10-3 M

0 100 200

0 4 8 12

300

434 nm

1

7

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 290 405 520

1

7 SMRZ If

[β-CD] times 10-3 M 0

300

0 4 8 12

600

452 nm

137

whereas no significant shift was observed in the LE band (341 nm) In SFP the LW and

MW emission bands are regularly red shifted on increasing the β-CD concentrations

accompanied by the increase in the intensity and the SW band became blurred In contrast to

β-CD both LE and TICT bands are not changed in α-CD However the emission intensity is

gradually increased Moreover SDMO SMRZ and SFP molecules show larger enhancement

in β-CD solution when compared to α-CD In CDs the enhancement of the LE and TICT

bands of sulfonamides may be explained as follows The enhancement of the LE band in CD

may be due to lowering of solvent polarity at higher CD concentration [154] Inside the CD

cavity sulfonamides have much less polar environment and the main non-radiative path of

the LE band through ICT or TICT is restricted which also causes an enhancement of the LE

and TICT band Further the geometrical restriction of the CD cavity would restrict the free

rotation of the heterocyclic ring (pyrimidinepyridine) in the CD cavity and thus favours the

formation of TICT state From the earlier studies [88] it is found that in aqueous CD

solutions containing the drugs hydrophobicity is the driving force for encapsulation of the

molecule inside the cavity and naturally the hydrophobic part prefer to go inside the deep

core of the less polar CD cavity and the polar group will be projected in the hydrophilic part

of the CD cavity [154] From the above findings it is clear that in CD the surrounding

polarity of SO2minusNH group does not change very much as there is no hypsochromic shift of

the most polar (TICT) state [170] This may be possible only if the orientation of

sulfonamide is such that the aromatic ring present inside the CD cavity and the amino group

is present outside of the CD nanocavity

The above results indicate that the SDMO SMRZ and SFP molecules are partially

entrapped into the CD cavity Since the size of SDMO SMRZ and SFP molecules are

larger than CD cavity the guests are partially entrapped into the CD It is noteworthy that

the LW emission intensity gradually increased along with red shift on increasing the

concentrations of α-CD and β-CD It can be suggested that the LW emission band is related

to the formation of CD inclusion complex Such a spectral shift may correspond to an

energy stabilization of the emitting state and is characteristic for the fluorescence of TICT

state The LW emission for the above sulfonamides originates from the TICT state with

twisting occurring at the amide SndashN bond between the aniline ring (electron donor) and the

SO2 group or pyrimidine ring (electron acceptor) Further Rajendiran et al [171 172]

reported whenever two aromatic rings are separated by the groups like SO2 CH2 CO NH

etc they form a TICT state Thus it can be speculated that the enhancement of the band at

138

445 nm emission should have originated from the TICT state The LW emission (445 nm) in

both CD solutions suggests that the inclusion process plays a major role in the TICT

emission

In all the three drugs the presence of isosbestic point in the absorption spectra

suggests the formation of 11 inclusion complex between the drugs and CDs The binding

constant for the formation of inclusion complex was determined by analyzing the changes in

the absorbance and fluorescence intensities with the different concentrations of CD The

binding constant (K) values determined using Benesi-Hildebrand relation [120] indicates

that 11 complexes are formed between the sulfonamides and CDs Fig 55 depicts the plot

of 1AndashA0 and 1IndashI0 as a function of 1[CD] for the SDMO SMRZ and SFP molecules The

plot of 1AndashA0 and 1IndashI0 as a function of 1[CD] gives linear line This analysis reflects the

formation of 11 inclusion complex formed in all sulfonamides The calculated binding

constant (K) values are listed in the Table 51 The binding constant values are small when

compared to previously reported hostguest complexes such as dialkyl aminobenzonitrile

and its derivatives [154] This is probably due to (i) sulfonamide molecules are partially

entrapped into the cavity (ii) heterocyclic ring (pyrimidinepyridine) might have entrapped

in the cavity and (iii) these molecules might not be tightly encapsulated in the CDs cavities

The Gibbs free energy changes (∆G) of the inclusion complexes are calculated using

the Eqn 25 As can be seen from Table 51 the calculated ∆G values for SDMOCD

SMRZCD and SFPCD inclusion complexes are negative which suggests that the inclusion

process proceeded spontaneously at 303 K The hydrophobic interaction between the

internal wall of CDs and guest molecules is an important factor for the stability of inclusion

complexes In sulfonamides it is considered that the change in the magnitude of the

hydrophobic interaction is related to that of the contact area of the guest molecule for the

internal wall of CD cavity

52 Effects of solvents

In order to investigate the TICT emission of SDMO SMRZ and SFP the absorption

and fluorescence maxima of the above molecules in the solvents having different polarities

and hydrogen bond forming tendency have been recorded The spectral data are listed in

Table 52 Due to the low solubility of the drug in non-polar cyclohexane solvent its

spectrum was recorded using 1 dioxane solution of cyclohexane The data in Table 52

indicate that the absorption bands are more intensed with less features where the relative

intensity of each is sensitive to solvent polarity This more intensed bands are caused by

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

131

shown in Figs 51-54 SDMO gives a structureless broad absorption band at 268 nm

whereas two absorption bands (at 268 and 243 nm) are obtained for SMRZ in aqueous

solution However in the spectrum of SFP in water three absorption bands are found at 310

nm (πrarrπ transition of the double bond) 261 nm (πrarrπ transition of the aniline group)

and 243 nm (πrarrπ transition of the aromatic ring) With increasing concentrations of β-CD

the absorption maxima of the above drugs are red shifted (about ~8 nm) with gradual

decrease in the molar extinction coefficient whereas the absorbance increased at the same

wavelength in the α-CD The decrease or increase in the absorbance is due to the

encapsulation of the above sulfa drugs into the CD cavity and it is attributed to the detergent

action of CDs [87-93] Further when recorded after 24 hours no significant change was

observed in the absorbance indicating that these sulfonamides do not decompose in the CDs

solution The above behaviour is attributed to the enhanced dissolution of the sulfonamide

molecules through the hydrophobic interaction between the guest and non-polar cavity of

the CD These results suggest that sulfonamides are entrapped into the CD cavity to form the

stable inclusion complex Moreover the red shift observed in β-CD reveals that the

pyrimidine nitrogen atom interacts with β-CD hydroxyl groups because it is well known

that CDs are good hydrogen donors [1-3] Additionally in all the cases a clear isosbestic

point was observed in the absorption spectra In general the existence of an isosbestic point in

the absorption spectra is an indication of the formation of well defined 11 complex [87-93]

Figs 53 and 54 display the typical fluorescence spectra of SDMO SMRZ and SFP

in aqueous solution as a function of α-CD and β-CD concentrations Both SDMO and

SMRZ drugs exhibit dual fluorescence with the maximum excitation wavelength of 270 nm

Likewise SFP emits two emission peaks at 348 and 434 nm with a shoulder at 372 nm when

excited at 310 nm It has been shown in our earlier studies [88] that sulfonamide derivatives

undergo normal as well as highly Stokes shifted fluorescence The normal fluorescence

takes place from locally excited (LE) state while twisted intramolecular charge transfer

(TICT) is responsible for highly Stokes shifted fluorescence The PM3 calculations have

suggested that the large stabilization of excited singlet state of the above sulfa drugs with

twisted conformation occurring at the amide SndashN bond between the electron donor group

(aniline ring) and the electron acceptor group (heterocyclic ring) In water the intensity of

LE band is greater than TICT band However on addition of CDs both the LE and TICT

intensities are equally increased With an increase in the β-CD concentration a regular red

shift is observed in the TICT band (434 nm to 445 nm) in all the drugs

132

Table 51

133

Fig 51 Absorption spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of absorbance vs concentration of α-CD

Abs

orba

nce

Wavelength (nm)

080

040

0 200 290 380

[α-CD] times 10-3 M

073

075

077

0 5 10 15

Abs

268 nm

1

7

SMRZ

Abs

orba

nce

Wavelength (nm)

150

075

0 200 275 350

SDMO

1

7 [α-CD] times 10-3 M

066

072

078

0 5 10 15

Abs

268 nm

Abs

orba

nce

Wavelength (nm)

080

040

0 200 300 400

Abs

[α-CD] times 10-3 M

027 028 029

0 4 8 12

040

310 nm

SFP

1

7

134

Fig 52 Absorption spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of absorbance vs concentration of β-CD

Abs

orba

nce

Wavelength (nm)

150

075

0 200 275 350

7

1

SDMO

[β-CD] times 10-3 M

047

061

075

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

100

050

0 200 275 350

SMRZ

7

1 [β-CD] times 10-3 M

068

072

076

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

080

040

0 200 300 400

SFP

Abs

[β-CD] times 10-3 M

027 031 035

0 4 8 12

039 310 nm

7

1

7

1

135

Fig 53 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of α-CD

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520 340 460

SDMO

1

7 [α-CD] times 10-3 M

260

330

400

0 5 10 15

If

341 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

480

240

0 290 405 520

SMRZ If

[α-CD] times 10-3 M

200

260

0 4 8 12

320

341 nm

1

7

Fluo

resc

ence

inte

nsity

(au)

Wavelength (nm)

300

150

0 320 450 580

SFP

1

7

If

[α-CD] times 10-3 M

50

110

0 4 8 12

170

310 nm

136

Fig 54 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of β-CD

340 460

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520

[β-CD] times 10-3 M

250

400

550

0 5 10 15

If

340 nm

1

7

SDMO

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

300

150

0 320 450 580

SFP If

[β-CD] times 10-3 M

0 100 200

0 4 8 12

300

434 nm

1

7

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 290 405 520

1

7 SMRZ If

[β-CD] times 10-3 M 0

300

0 4 8 12

600

452 nm

137

whereas no significant shift was observed in the LE band (341 nm) In SFP the LW and

MW emission bands are regularly red shifted on increasing the β-CD concentrations

accompanied by the increase in the intensity and the SW band became blurred In contrast to

β-CD both LE and TICT bands are not changed in α-CD However the emission intensity is

gradually increased Moreover SDMO SMRZ and SFP molecules show larger enhancement

in β-CD solution when compared to α-CD In CDs the enhancement of the LE and TICT

bands of sulfonamides may be explained as follows The enhancement of the LE band in CD

may be due to lowering of solvent polarity at higher CD concentration [154] Inside the CD

cavity sulfonamides have much less polar environment and the main non-radiative path of

the LE band through ICT or TICT is restricted which also causes an enhancement of the LE

and TICT band Further the geometrical restriction of the CD cavity would restrict the free

rotation of the heterocyclic ring (pyrimidinepyridine) in the CD cavity and thus favours the

formation of TICT state From the earlier studies [88] it is found that in aqueous CD

solutions containing the drugs hydrophobicity is the driving force for encapsulation of the

molecule inside the cavity and naturally the hydrophobic part prefer to go inside the deep

core of the less polar CD cavity and the polar group will be projected in the hydrophilic part

of the CD cavity [154] From the above findings it is clear that in CD the surrounding

polarity of SO2minusNH group does not change very much as there is no hypsochromic shift of

the most polar (TICT) state [170] This may be possible only if the orientation of

sulfonamide is such that the aromatic ring present inside the CD cavity and the amino group

is present outside of the CD nanocavity

The above results indicate that the SDMO SMRZ and SFP molecules are partially

entrapped into the CD cavity Since the size of SDMO SMRZ and SFP molecules are

larger than CD cavity the guests are partially entrapped into the CD It is noteworthy that

the LW emission intensity gradually increased along with red shift on increasing the

concentrations of α-CD and β-CD It can be suggested that the LW emission band is related

to the formation of CD inclusion complex Such a spectral shift may correspond to an

energy stabilization of the emitting state and is characteristic for the fluorescence of TICT

state The LW emission for the above sulfonamides originates from the TICT state with

twisting occurring at the amide SndashN bond between the aniline ring (electron donor) and the

SO2 group or pyrimidine ring (electron acceptor) Further Rajendiran et al [171 172]

reported whenever two aromatic rings are separated by the groups like SO2 CH2 CO NH

etc they form a TICT state Thus it can be speculated that the enhancement of the band at

138

445 nm emission should have originated from the TICT state The LW emission (445 nm) in

both CD solutions suggests that the inclusion process plays a major role in the TICT

emission

In all the three drugs the presence of isosbestic point in the absorption spectra

suggests the formation of 11 inclusion complex between the drugs and CDs The binding

constant for the formation of inclusion complex was determined by analyzing the changes in

the absorbance and fluorescence intensities with the different concentrations of CD The

binding constant (K) values determined using Benesi-Hildebrand relation [120] indicates

that 11 complexes are formed between the sulfonamides and CDs Fig 55 depicts the plot

of 1AndashA0 and 1IndashI0 as a function of 1[CD] for the SDMO SMRZ and SFP molecules The

plot of 1AndashA0 and 1IndashI0 as a function of 1[CD] gives linear line This analysis reflects the

formation of 11 inclusion complex formed in all sulfonamides The calculated binding

constant (K) values are listed in the Table 51 The binding constant values are small when

compared to previously reported hostguest complexes such as dialkyl aminobenzonitrile

and its derivatives [154] This is probably due to (i) sulfonamide molecules are partially

entrapped into the cavity (ii) heterocyclic ring (pyrimidinepyridine) might have entrapped

in the cavity and (iii) these molecules might not be tightly encapsulated in the CDs cavities

The Gibbs free energy changes (∆G) of the inclusion complexes are calculated using

the Eqn 25 As can be seen from Table 51 the calculated ∆G values for SDMOCD

SMRZCD and SFPCD inclusion complexes are negative which suggests that the inclusion

process proceeded spontaneously at 303 K The hydrophobic interaction between the

internal wall of CDs and guest molecules is an important factor for the stability of inclusion

complexes In sulfonamides it is considered that the change in the magnitude of the

hydrophobic interaction is related to that of the contact area of the guest molecule for the

internal wall of CD cavity

52 Effects of solvents

In order to investigate the TICT emission of SDMO SMRZ and SFP the absorption

and fluorescence maxima of the above molecules in the solvents having different polarities

and hydrogen bond forming tendency have been recorded The spectral data are listed in

Table 52 Due to the low solubility of the drug in non-polar cyclohexane solvent its

spectrum was recorded using 1 dioxane solution of cyclohexane The data in Table 52

indicate that the absorption bands are more intensed with less features where the relative

intensity of each is sensitive to solvent polarity This more intensed bands are caused by

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

132

Table 51

133

Fig 51 Absorption spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of absorbance vs concentration of α-CD

Abs

orba

nce

Wavelength (nm)

080

040

0 200 290 380

[α-CD] times 10-3 M

073

075

077

0 5 10 15

Abs

268 nm

1

7

SMRZ

Abs

orba

nce

Wavelength (nm)

150

075

0 200 275 350

SDMO

1

7 [α-CD] times 10-3 M

066

072

078

0 5 10 15

Abs

268 nm

Abs

orba

nce

Wavelength (nm)

080

040

0 200 300 400

Abs

[α-CD] times 10-3 M

027 028 029

0 4 8 12

040

310 nm

SFP

1

7

134

Fig 52 Absorption spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of absorbance vs concentration of β-CD

Abs

orba

nce

Wavelength (nm)

150

075

0 200 275 350

7

1

SDMO

[β-CD] times 10-3 M

047

061

075

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

100

050

0 200 275 350

SMRZ

7

1 [β-CD] times 10-3 M

068

072

076

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

080

040

0 200 300 400

SFP

Abs

[β-CD] times 10-3 M

027 031 035

0 4 8 12

039 310 nm

7

1

7

1

135

Fig 53 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of α-CD

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520 340 460

SDMO

1

7 [α-CD] times 10-3 M

260

330

400

0 5 10 15

If

341 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

480

240

0 290 405 520

SMRZ If

[α-CD] times 10-3 M

200

260

0 4 8 12

320

341 nm

1

7

Fluo

resc

ence

inte

nsity

(au)

Wavelength (nm)

300

150

0 320 450 580

SFP

1

7

If

[α-CD] times 10-3 M

50

110

0 4 8 12

170

310 nm

136

Fig 54 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of β-CD

340 460

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520

[β-CD] times 10-3 M

250

400

550

0 5 10 15

If

340 nm

1

7

SDMO

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

300

150

0 320 450 580

SFP If

[β-CD] times 10-3 M

0 100 200

0 4 8 12

300

434 nm

1

7

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 290 405 520

1

7 SMRZ If

[β-CD] times 10-3 M 0

300

0 4 8 12

600

452 nm

137

whereas no significant shift was observed in the LE band (341 nm) In SFP the LW and

MW emission bands are regularly red shifted on increasing the β-CD concentrations

accompanied by the increase in the intensity and the SW band became blurred In contrast to

β-CD both LE and TICT bands are not changed in α-CD However the emission intensity is

gradually increased Moreover SDMO SMRZ and SFP molecules show larger enhancement

in β-CD solution when compared to α-CD In CDs the enhancement of the LE and TICT

bands of sulfonamides may be explained as follows The enhancement of the LE band in CD

may be due to lowering of solvent polarity at higher CD concentration [154] Inside the CD

cavity sulfonamides have much less polar environment and the main non-radiative path of

the LE band through ICT or TICT is restricted which also causes an enhancement of the LE

and TICT band Further the geometrical restriction of the CD cavity would restrict the free

rotation of the heterocyclic ring (pyrimidinepyridine) in the CD cavity and thus favours the

formation of TICT state From the earlier studies [88] it is found that in aqueous CD

solutions containing the drugs hydrophobicity is the driving force for encapsulation of the

molecule inside the cavity and naturally the hydrophobic part prefer to go inside the deep

core of the less polar CD cavity and the polar group will be projected in the hydrophilic part

of the CD cavity [154] From the above findings it is clear that in CD the surrounding

polarity of SO2minusNH group does not change very much as there is no hypsochromic shift of

the most polar (TICT) state [170] This may be possible only if the orientation of

sulfonamide is such that the aromatic ring present inside the CD cavity and the amino group

is present outside of the CD nanocavity

The above results indicate that the SDMO SMRZ and SFP molecules are partially

entrapped into the CD cavity Since the size of SDMO SMRZ and SFP molecules are

larger than CD cavity the guests are partially entrapped into the CD It is noteworthy that

the LW emission intensity gradually increased along with red shift on increasing the

concentrations of α-CD and β-CD It can be suggested that the LW emission band is related

to the formation of CD inclusion complex Such a spectral shift may correspond to an

energy stabilization of the emitting state and is characteristic for the fluorescence of TICT

state The LW emission for the above sulfonamides originates from the TICT state with

twisting occurring at the amide SndashN bond between the aniline ring (electron donor) and the

SO2 group or pyrimidine ring (electron acceptor) Further Rajendiran et al [171 172]

reported whenever two aromatic rings are separated by the groups like SO2 CH2 CO NH

etc they form a TICT state Thus it can be speculated that the enhancement of the band at

138

445 nm emission should have originated from the TICT state The LW emission (445 nm) in

both CD solutions suggests that the inclusion process plays a major role in the TICT

emission

In all the three drugs the presence of isosbestic point in the absorption spectra

suggests the formation of 11 inclusion complex between the drugs and CDs The binding

constant for the formation of inclusion complex was determined by analyzing the changes in

the absorbance and fluorescence intensities with the different concentrations of CD The

binding constant (K) values determined using Benesi-Hildebrand relation [120] indicates

that 11 complexes are formed between the sulfonamides and CDs Fig 55 depicts the plot

of 1AndashA0 and 1IndashI0 as a function of 1[CD] for the SDMO SMRZ and SFP molecules The

plot of 1AndashA0 and 1IndashI0 as a function of 1[CD] gives linear line This analysis reflects the

formation of 11 inclusion complex formed in all sulfonamides The calculated binding

constant (K) values are listed in the Table 51 The binding constant values are small when

compared to previously reported hostguest complexes such as dialkyl aminobenzonitrile

and its derivatives [154] This is probably due to (i) sulfonamide molecules are partially

entrapped into the cavity (ii) heterocyclic ring (pyrimidinepyridine) might have entrapped

in the cavity and (iii) these molecules might not be tightly encapsulated in the CDs cavities

The Gibbs free energy changes (∆G) of the inclusion complexes are calculated using

the Eqn 25 As can be seen from Table 51 the calculated ∆G values for SDMOCD

SMRZCD and SFPCD inclusion complexes are negative which suggests that the inclusion

process proceeded spontaneously at 303 K The hydrophobic interaction between the

internal wall of CDs and guest molecules is an important factor for the stability of inclusion

complexes In sulfonamides it is considered that the change in the magnitude of the

hydrophobic interaction is related to that of the contact area of the guest molecule for the

internal wall of CD cavity

52 Effects of solvents

In order to investigate the TICT emission of SDMO SMRZ and SFP the absorption

and fluorescence maxima of the above molecules in the solvents having different polarities

and hydrogen bond forming tendency have been recorded The spectral data are listed in

Table 52 Due to the low solubility of the drug in non-polar cyclohexane solvent its

spectrum was recorded using 1 dioxane solution of cyclohexane The data in Table 52

indicate that the absorption bands are more intensed with less features where the relative

intensity of each is sensitive to solvent polarity This more intensed bands are caused by

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

133

Fig 51 Absorption spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of absorbance vs concentration of α-CD

Abs

orba

nce

Wavelength (nm)

080

040

0 200 290 380

[α-CD] times 10-3 M

073

075

077

0 5 10 15

Abs

268 nm

1

7

SMRZ

Abs

orba

nce

Wavelength (nm)

150

075

0 200 275 350

SDMO

1

7 [α-CD] times 10-3 M

066

072

078

0 5 10 15

Abs

268 nm

Abs

orba

nce

Wavelength (nm)

080

040

0 200 300 400

Abs

[α-CD] times 10-3 M

027 028 029

0 4 8 12

040

310 nm

SFP

1

7

134

Fig 52 Absorption spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of absorbance vs concentration of β-CD

Abs

orba

nce

Wavelength (nm)

150

075

0 200 275 350

7

1

SDMO

[β-CD] times 10-3 M

047

061

075

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

100

050

0 200 275 350

SMRZ

7

1 [β-CD] times 10-3 M

068

072

076

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

080

040

0 200 300 400

SFP

Abs

[β-CD] times 10-3 M

027 031 035

0 4 8 12

039 310 nm

7

1

7

1

135

Fig 53 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of α-CD

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520 340 460

SDMO

1

7 [α-CD] times 10-3 M

260

330

400

0 5 10 15

If

341 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

480

240

0 290 405 520

SMRZ If

[α-CD] times 10-3 M

200

260

0 4 8 12

320

341 nm

1

7

Fluo

resc

ence

inte

nsity

(au)

Wavelength (nm)

300

150

0 320 450 580

SFP

1

7

If

[α-CD] times 10-3 M

50

110

0 4 8 12

170

310 nm

136

Fig 54 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of β-CD

340 460

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520

[β-CD] times 10-3 M

250

400

550

0 5 10 15

If

340 nm

1

7

SDMO

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

300

150

0 320 450 580

SFP If

[β-CD] times 10-3 M

0 100 200

0 4 8 12

300

434 nm

1

7

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 290 405 520

1

7 SMRZ If

[β-CD] times 10-3 M 0

300

0 4 8 12

600

452 nm

137

whereas no significant shift was observed in the LE band (341 nm) In SFP the LW and

MW emission bands are regularly red shifted on increasing the β-CD concentrations

accompanied by the increase in the intensity and the SW band became blurred In contrast to

β-CD both LE and TICT bands are not changed in α-CD However the emission intensity is

gradually increased Moreover SDMO SMRZ and SFP molecules show larger enhancement

in β-CD solution when compared to α-CD In CDs the enhancement of the LE and TICT

bands of sulfonamides may be explained as follows The enhancement of the LE band in CD

may be due to lowering of solvent polarity at higher CD concentration [154] Inside the CD

cavity sulfonamides have much less polar environment and the main non-radiative path of

the LE band through ICT or TICT is restricted which also causes an enhancement of the LE

and TICT band Further the geometrical restriction of the CD cavity would restrict the free

rotation of the heterocyclic ring (pyrimidinepyridine) in the CD cavity and thus favours the

formation of TICT state From the earlier studies [88] it is found that in aqueous CD

solutions containing the drugs hydrophobicity is the driving force for encapsulation of the

molecule inside the cavity and naturally the hydrophobic part prefer to go inside the deep

core of the less polar CD cavity and the polar group will be projected in the hydrophilic part

of the CD cavity [154] From the above findings it is clear that in CD the surrounding

polarity of SO2minusNH group does not change very much as there is no hypsochromic shift of

the most polar (TICT) state [170] This may be possible only if the orientation of

sulfonamide is such that the aromatic ring present inside the CD cavity and the amino group

is present outside of the CD nanocavity

The above results indicate that the SDMO SMRZ and SFP molecules are partially

entrapped into the CD cavity Since the size of SDMO SMRZ and SFP molecules are

larger than CD cavity the guests are partially entrapped into the CD It is noteworthy that

the LW emission intensity gradually increased along with red shift on increasing the

concentrations of α-CD and β-CD It can be suggested that the LW emission band is related

to the formation of CD inclusion complex Such a spectral shift may correspond to an

energy stabilization of the emitting state and is characteristic for the fluorescence of TICT

state The LW emission for the above sulfonamides originates from the TICT state with

twisting occurring at the amide SndashN bond between the aniline ring (electron donor) and the

SO2 group or pyrimidine ring (electron acceptor) Further Rajendiran et al [171 172]

reported whenever two aromatic rings are separated by the groups like SO2 CH2 CO NH

etc they form a TICT state Thus it can be speculated that the enhancement of the band at

138

445 nm emission should have originated from the TICT state The LW emission (445 nm) in

both CD solutions suggests that the inclusion process plays a major role in the TICT

emission

In all the three drugs the presence of isosbestic point in the absorption spectra

suggests the formation of 11 inclusion complex between the drugs and CDs The binding

constant for the formation of inclusion complex was determined by analyzing the changes in

the absorbance and fluorescence intensities with the different concentrations of CD The

binding constant (K) values determined using Benesi-Hildebrand relation [120] indicates

that 11 complexes are formed between the sulfonamides and CDs Fig 55 depicts the plot

of 1AndashA0 and 1IndashI0 as a function of 1[CD] for the SDMO SMRZ and SFP molecules The

plot of 1AndashA0 and 1IndashI0 as a function of 1[CD] gives linear line This analysis reflects the

formation of 11 inclusion complex formed in all sulfonamides The calculated binding

constant (K) values are listed in the Table 51 The binding constant values are small when

compared to previously reported hostguest complexes such as dialkyl aminobenzonitrile

and its derivatives [154] This is probably due to (i) sulfonamide molecules are partially

entrapped into the cavity (ii) heterocyclic ring (pyrimidinepyridine) might have entrapped

in the cavity and (iii) these molecules might not be tightly encapsulated in the CDs cavities

The Gibbs free energy changes (∆G) of the inclusion complexes are calculated using

the Eqn 25 As can be seen from Table 51 the calculated ∆G values for SDMOCD

SMRZCD and SFPCD inclusion complexes are negative which suggests that the inclusion

process proceeded spontaneously at 303 K The hydrophobic interaction between the

internal wall of CDs and guest molecules is an important factor for the stability of inclusion

complexes In sulfonamides it is considered that the change in the magnitude of the

hydrophobic interaction is related to that of the contact area of the guest molecule for the

internal wall of CD cavity

52 Effects of solvents

In order to investigate the TICT emission of SDMO SMRZ and SFP the absorption

and fluorescence maxima of the above molecules in the solvents having different polarities

and hydrogen bond forming tendency have been recorded The spectral data are listed in

Table 52 Due to the low solubility of the drug in non-polar cyclohexane solvent its

spectrum was recorded using 1 dioxane solution of cyclohexane The data in Table 52

indicate that the absorption bands are more intensed with less features where the relative

intensity of each is sensitive to solvent polarity This more intensed bands are caused by

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

134

Fig 52 Absorption spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of absorbance vs concentration of β-CD

Abs

orba

nce

Wavelength (nm)

150

075

0 200 275 350

7

1

SDMO

[β-CD] times 10-3 M

047

061

075

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

100

050

0 200 275 350

SMRZ

7

1 [β-CD] times 10-3 M

068

072

076

0 5 10 15

Abs 268 nm

Abs

orba

nce

Wavelength (nm)

080

040

0 200 300 400

SFP

Abs

[β-CD] times 10-3 M

027 031 035

0 4 8 12

039 310 nm

7

1

7

1

135

Fig 53 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of α-CD

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520 340 460

SDMO

1

7 [α-CD] times 10-3 M

260

330

400

0 5 10 15

If

341 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

480

240

0 290 405 520

SMRZ If

[α-CD] times 10-3 M

200

260

0 4 8 12

320

341 nm

1

7

Fluo

resc

ence

inte

nsity

(au)

Wavelength (nm)

300

150

0 320 450 580

SFP

1

7

If

[α-CD] times 10-3 M

50

110

0 4 8 12

170

310 nm

136

Fig 54 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of β-CD

340 460

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520

[β-CD] times 10-3 M

250

400

550

0 5 10 15

If

340 nm

1

7

SDMO

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

300

150

0 320 450 580

SFP If

[β-CD] times 10-3 M

0 100 200

0 4 8 12

300

434 nm

1

7

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 290 405 520

1

7 SMRZ If

[β-CD] times 10-3 M 0

300

0 4 8 12

600

452 nm

137

whereas no significant shift was observed in the LE band (341 nm) In SFP the LW and

MW emission bands are regularly red shifted on increasing the β-CD concentrations

accompanied by the increase in the intensity and the SW band became blurred In contrast to

β-CD both LE and TICT bands are not changed in α-CD However the emission intensity is

gradually increased Moreover SDMO SMRZ and SFP molecules show larger enhancement

in β-CD solution when compared to α-CD In CDs the enhancement of the LE and TICT

bands of sulfonamides may be explained as follows The enhancement of the LE band in CD

may be due to lowering of solvent polarity at higher CD concentration [154] Inside the CD

cavity sulfonamides have much less polar environment and the main non-radiative path of

the LE band through ICT or TICT is restricted which also causes an enhancement of the LE

and TICT band Further the geometrical restriction of the CD cavity would restrict the free

rotation of the heterocyclic ring (pyrimidinepyridine) in the CD cavity and thus favours the

formation of TICT state From the earlier studies [88] it is found that in aqueous CD

solutions containing the drugs hydrophobicity is the driving force for encapsulation of the

molecule inside the cavity and naturally the hydrophobic part prefer to go inside the deep

core of the less polar CD cavity and the polar group will be projected in the hydrophilic part

of the CD cavity [154] From the above findings it is clear that in CD the surrounding

polarity of SO2minusNH group does not change very much as there is no hypsochromic shift of

the most polar (TICT) state [170] This may be possible only if the orientation of

sulfonamide is such that the aromatic ring present inside the CD cavity and the amino group

is present outside of the CD nanocavity

The above results indicate that the SDMO SMRZ and SFP molecules are partially

entrapped into the CD cavity Since the size of SDMO SMRZ and SFP molecules are

larger than CD cavity the guests are partially entrapped into the CD It is noteworthy that

the LW emission intensity gradually increased along with red shift on increasing the

concentrations of α-CD and β-CD It can be suggested that the LW emission band is related

to the formation of CD inclusion complex Such a spectral shift may correspond to an

energy stabilization of the emitting state and is characteristic for the fluorescence of TICT

state The LW emission for the above sulfonamides originates from the TICT state with

twisting occurring at the amide SndashN bond between the aniline ring (electron donor) and the

SO2 group or pyrimidine ring (electron acceptor) Further Rajendiran et al [171 172]

reported whenever two aromatic rings are separated by the groups like SO2 CH2 CO NH

etc they form a TICT state Thus it can be speculated that the enhancement of the band at

138

445 nm emission should have originated from the TICT state The LW emission (445 nm) in

both CD solutions suggests that the inclusion process plays a major role in the TICT

emission

In all the three drugs the presence of isosbestic point in the absorption spectra

suggests the formation of 11 inclusion complex between the drugs and CDs The binding

constant for the formation of inclusion complex was determined by analyzing the changes in

the absorbance and fluorescence intensities with the different concentrations of CD The

binding constant (K) values determined using Benesi-Hildebrand relation [120] indicates

that 11 complexes are formed between the sulfonamides and CDs Fig 55 depicts the plot

of 1AndashA0 and 1IndashI0 as a function of 1[CD] for the SDMO SMRZ and SFP molecules The

plot of 1AndashA0 and 1IndashI0 as a function of 1[CD] gives linear line This analysis reflects the

formation of 11 inclusion complex formed in all sulfonamides The calculated binding

constant (K) values are listed in the Table 51 The binding constant values are small when

compared to previously reported hostguest complexes such as dialkyl aminobenzonitrile

and its derivatives [154] This is probably due to (i) sulfonamide molecules are partially

entrapped into the cavity (ii) heterocyclic ring (pyrimidinepyridine) might have entrapped

in the cavity and (iii) these molecules might not be tightly encapsulated in the CDs cavities

The Gibbs free energy changes (∆G) of the inclusion complexes are calculated using

the Eqn 25 As can be seen from Table 51 the calculated ∆G values for SDMOCD

SMRZCD and SFPCD inclusion complexes are negative which suggests that the inclusion

process proceeded spontaneously at 303 K The hydrophobic interaction between the

internal wall of CDs and guest molecules is an important factor for the stability of inclusion

complexes In sulfonamides it is considered that the change in the magnitude of the

hydrophobic interaction is related to that of the contact area of the guest molecule for the

internal wall of CD cavity

52 Effects of solvents

In order to investigate the TICT emission of SDMO SMRZ and SFP the absorption

and fluorescence maxima of the above molecules in the solvents having different polarities

and hydrogen bond forming tendency have been recorded The spectral data are listed in

Table 52 Due to the low solubility of the drug in non-polar cyclohexane solvent its

spectrum was recorded using 1 dioxane solution of cyclohexane The data in Table 52

indicate that the absorption bands are more intensed with less features where the relative

intensity of each is sensitive to solvent polarity This more intensed bands are caused by

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

135

Fig 53 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of α-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of α-CD

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520 340 460

SDMO

1

7 [α-CD] times 10-3 M

260

330

400

0 5 10 15

If

341 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

480

240

0 290 405 520

SMRZ If

[α-CD] times 10-3 M

200

260

0 4 8 12

320

341 nm

1

7

Fluo

resc

ence

inte

nsity

(au)

Wavelength (nm)

300

150

0 320 450 580

SFP

1

7

If

[α-CD] times 10-3 M

50

110

0 4 8 12

170

310 nm

136

Fig 54 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of β-CD

340 460

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520

[β-CD] times 10-3 M

250

400

550

0 5 10 15

If

340 nm

1

7

SDMO

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

300

150

0 320 450 580

SFP If

[β-CD] times 10-3 M

0 100 200

0 4 8 12

300

434 nm

1

7

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 290 405 520

1

7 SMRZ If

[β-CD] times 10-3 M 0

300

0 4 8 12

600

452 nm

137

whereas no significant shift was observed in the LE band (341 nm) In SFP the LW and

MW emission bands are regularly red shifted on increasing the β-CD concentrations

accompanied by the increase in the intensity and the SW band became blurred In contrast to

β-CD both LE and TICT bands are not changed in α-CD However the emission intensity is

gradually increased Moreover SDMO SMRZ and SFP molecules show larger enhancement

in β-CD solution when compared to α-CD In CDs the enhancement of the LE and TICT

bands of sulfonamides may be explained as follows The enhancement of the LE band in CD

may be due to lowering of solvent polarity at higher CD concentration [154] Inside the CD

cavity sulfonamides have much less polar environment and the main non-radiative path of

the LE band through ICT or TICT is restricted which also causes an enhancement of the LE

and TICT band Further the geometrical restriction of the CD cavity would restrict the free

rotation of the heterocyclic ring (pyrimidinepyridine) in the CD cavity and thus favours the

formation of TICT state From the earlier studies [88] it is found that in aqueous CD

solutions containing the drugs hydrophobicity is the driving force for encapsulation of the

molecule inside the cavity and naturally the hydrophobic part prefer to go inside the deep

core of the less polar CD cavity and the polar group will be projected in the hydrophilic part

of the CD cavity [154] From the above findings it is clear that in CD the surrounding

polarity of SO2minusNH group does not change very much as there is no hypsochromic shift of

the most polar (TICT) state [170] This may be possible only if the orientation of

sulfonamide is such that the aromatic ring present inside the CD cavity and the amino group

is present outside of the CD nanocavity

The above results indicate that the SDMO SMRZ and SFP molecules are partially

entrapped into the CD cavity Since the size of SDMO SMRZ and SFP molecules are

larger than CD cavity the guests are partially entrapped into the CD It is noteworthy that

the LW emission intensity gradually increased along with red shift on increasing the

concentrations of α-CD and β-CD It can be suggested that the LW emission band is related

to the formation of CD inclusion complex Such a spectral shift may correspond to an

energy stabilization of the emitting state and is characteristic for the fluorescence of TICT

state The LW emission for the above sulfonamides originates from the TICT state with

twisting occurring at the amide SndashN bond between the aniline ring (electron donor) and the

SO2 group or pyrimidine ring (electron acceptor) Further Rajendiran et al [171 172]

reported whenever two aromatic rings are separated by the groups like SO2 CH2 CO NH

etc they form a TICT state Thus it can be speculated that the enhancement of the band at

138

445 nm emission should have originated from the TICT state The LW emission (445 nm) in

both CD solutions suggests that the inclusion process plays a major role in the TICT

emission

In all the three drugs the presence of isosbestic point in the absorption spectra

suggests the formation of 11 inclusion complex between the drugs and CDs The binding

constant for the formation of inclusion complex was determined by analyzing the changes in

the absorbance and fluorescence intensities with the different concentrations of CD The

binding constant (K) values determined using Benesi-Hildebrand relation [120] indicates

that 11 complexes are formed between the sulfonamides and CDs Fig 55 depicts the plot

of 1AndashA0 and 1IndashI0 as a function of 1[CD] for the SDMO SMRZ and SFP molecules The

plot of 1AndashA0 and 1IndashI0 as a function of 1[CD] gives linear line This analysis reflects the

formation of 11 inclusion complex formed in all sulfonamides The calculated binding

constant (K) values are listed in the Table 51 The binding constant values are small when

compared to previously reported hostguest complexes such as dialkyl aminobenzonitrile

and its derivatives [154] This is probably due to (i) sulfonamide molecules are partially

entrapped into the cavity (ii) heterocyclic ring (pyrimidinepyridine) might have entrapped

in the cavity and (iii) these molecules might not be tightly encapsulated in the CDs cavities

The Gibbs free energy changes (∆G) of the inclusion complexes are calculated using

the Eqn 25 As can be seen from Table 51 the calculated ∆G values for SDMOCD

SMRZCD and SFPCD inclusion complexes are negative which suggests that the inclusion

process proceeded spontaneously at 303 K The hydrophobic interaction between the

internal wall of CDs and guest molecules is an important factor for the stability of inclusion

complexes In sulfonamides it is considered that the change in the magnitude of the

hydrophobic interaction is related to that of the contact area of the guest molecule for the

internal wall of CD cavity

52 Effects of solvents

In order to investigate the TICT emission of SDMO SMRZ and SFP the absorption

and fluorescence maxima of the above molecules in the solvents having different polarities

and hydrogen bond forming tendency have been recorded The spectral data are listed in

Table 52 Due to the low solubility of the drug in non-polar cyclohexane solvent its

spectrum was recorded using 1 dioxane solution of cyclohexane The data in Table 52

indicate that the absorption bands are more intensed with less features where the relative

intensity of each is sensitive to solvent polarity This more intensed bands are caused by

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

136

Fig 54 Fluorescence spectra of SDMO SMRZ and SFP in different concentrations of β-CD (M) (1) 0 (2) 0001 (3) 0002 (4) 0004 (5) 0006 (6) 0008 and (7) 001 Inset Fig Plot of fluorescence intensity vs concentration of β-CD

340 460

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 400 520

[β-CD] times 10-3 M

250

400

550

0 5 10 15

If

340 nm

1

7

SDMO

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

300

150

0 320 450 580

SFP If

[β-CD] times 10-3 M

0 100 200

0 4 8 12

300

434 nm

1

7

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 290 405 520

1

7 SMRZ If

[β-CD] times 10-3 M 0

300

0 4 8 12

600

452 nm

137

whereas no significant shift was observed in the LE band (341 nm) In SFP the LW and

MW emission bands are regularly red shifted on increasing the β-CD concentrations

accompanied by the increase in the intensity and the SW band became blurred In contrast to

β-CD both LE and TICT bands are not changed in α-CD However the emission intensity is

gradually increased Moreover SDMO SMRZ and SFP molecules show larger enhancement

in β-CD solution when compared to α-CD In CDs the enhancement of the LE and TICT

bands of sulfonamides may be explained as follows The enhancement of the LE band in CD

may be due to lowering of solvent polarity at higher CD concentration [154] Inside the CD

cavity sulfonamides have much less polar environment and the main non-radiative path of

the LE band through ICT or TICT is restricted which also causes an enhancement of the LE

and TICT band Further the geometrical restriction of the CD cavity would restrict the free

rotation of the heterocyclic ring (pyrimidinepyridine) in the CD cavity and thus favours the

formation of TICT state From the earlier studies [88] it is found that in aqueous CD

solutions containing the drugs hydrophobicity is the driving force for encapsulation of the

molecule inside the cavity and naturally the hydrophobic part prefer to go inside the deep

core of the less polar CD cavity and the polar group will be projected in the hydrophilic part

of the CD cavity [154] From the above findings it is clear that in CD the surrounding

polarity of SO2minusNH group does not change very much as there is no hypsochromic shift of

the most polar (TICT) state [170] This may be possible only if the orientation of

sulfonamide is such that the aromatic ring present inside the CD cavity and the amino group

is present outside of the CD nanocavity

The above results indicate that the SDMO SMRZ and SFP molecules are partially

entrapped into the CD cavity Since the size of SDMO SMRZ and SFP molecules are

larger than CD cavity the guests are partially entrapped into the CD It is noteworthy that

the LW emission intensity gradually increased along with red shift on increasing the

concentrations of α-CD and β-CD It can be suggested that the LW emission band is related

to the formation of CD inclusion complex Such a spectral shift may correspond to an

energy stabilization of the emitting state and is characteristic for the fluorescence of TICT

state The LW emission for the above sulfonamides originates from the TICT state with

twisting occurring at the amide SndashN bond between the aniline ring (electron donor) and the

SO2 group or pyrimidine ring (electron acceptor) Further Rajendiran et al [171 172]

reported whenever two aromatic rings are separated by the groups like SO2 CH2 CO NH

etc they form a TICT state Thus it can be speculated that the enhancement of the band at

138

445 nm emission should have originated from the TICT state The LW emission (445 nm) in

both CD solutions suggests that the inclusion process plays a major role in the TICT

emission

In all the three drugs the presence of isosbestic point in the absorption spectra

suggests the formation of 11 inclusion complex between the drugs and CDs The binding

constant for the formation of inclusion complex was determined by analyzing the changes in

the absorbance and fluorescence intensities with the different concentrations of CD The

binding constant (K) values determined using Benesi-Hildebrand relation [120] indicates

that 11 complexes are formed between the sulfonamides and CDs Fig 55 depicts the plot

of 1AndashA0 and 1IndashI0 as a function of 1[CD] for the SDMO SMRZ and SFP molecules The

plot of 1AndashA0 and 1IndashI0 as a function of 1[CD] gives linear line This analysis reflects the

formation of 11 inclusion complex formed in all sulfonamides The calculated binding

constant (K) values are listed in the Table 51 The binding constant values are small when

compared to previously reported hostguest complexes such as dialkyl aminobenzonitrile

and its derivatives [154] This is probably due to (i) sulfonamide molecules are partially

entrapped into the cavity (ii) heterocyclic ring (pyrimidinepyridine) might have entrapped

in the cavity and (iii) these molecules might not be tightly encapsulated in the CDs cavities

The Gibbs free energy changes (∆G) of the inclusion complexes are calculated using

the Eqn 25 As can be seen from Table 51 the calculated ∆G values for SDMOCD

SMRZCD and SFPCD inclusion complexes are negative which suggests that the inclusion

process proceeded spontaneously at 303 K The hydrophobic interaction between the

internal wall of CDs and guest molecules is an important factor for the stability of inclusion

complexes In sulfonamides it is considered that the change in the magnitude of the

hydrophobic interaction is related to that of the contact area of the guest molecule for the

internal wall of CD cavity

52 Effects of solvents

In order to investigate the TICT emission of SDMO SMRZ and SFP the absorption

and fluorescence maxima of the above molecules in the solvents having different polarities

and hydrogen bond forming tendency have been recorded The spectral data are listed in

Table 52 Due to the low solubility of the drug in non-polar cyclohexane solvent its

spectrum was recorded using 1 dioxane solution of cyclohexane The data in Table 52

indicate that the absorption bands are more intensed with less features where the relative

intensity of each is sensitive to solvent polarity This more intensed bands are caused by

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

137

whereas no significant shift was observed in the LE band (341 nm) In SFP the LW and

MW emission bands are regularly red shifted on increasing the β-CD concentrations

accompanied by the increase in the intensity and the SW band became blurred In contrast to

β-CD both LE and TICT bands are not changed in α-CD However the emission intensity is

gradually increased Moreover SDMO SMRZ and SFP molecules show larger enhancement

in β-CD solution when compared to α-CD In CDs the enhancement of the LE and TICT

bands of sulfonamides may be explained as follows The enhancement of the LE band in CD

may be due to lowering of solvent polarity at higher CD concentration [154] Inside the CD

cavity sulfonamides have much less polar environment and the main non-radiative path of

the LE band through ICT or TICT is restricted which also causes an enhancement of the LE

and TICT band Further the geometrical restriction of the CD cavity would restrict the free

rotation of the heterocyclic ring (pyrimidinepyridine) in the CD cavity and thus favours the

formation of TICT state From the earlier studies [88] it is found that in aqueous CD

solutions containing the drugs hydrophobicity is the driving force for encapsulation of the

molecule inside the cavity and naturally the hydrophobic part prefer to go inside the deep

core of the less polar CD cavity and the polar group will be projected in the hydrophilic part

of the CD cavity [154] From the above findings it is clear that in CD the surrounding

polarity of SO2minusNH group does not change very much as there is no hypsochromic shift of

the most polar (TICT) state [170] This may be possible only if the orientation of

sulfonamide is such that the aromatic ring present inside the CD cavity and the amino group

is present outside of the CD nanocavity

The above results indicate that the SDMO SMRZ and SFP molecules are partially

entrapped into the CD cavity Since the size of SDMO SMRZ and SFP molecules are

larger than CD cavity the guests are partially entrapped into the CD It is noteworthy that

the LW emission intensity gradually increased along with red shift on increasing the

concentrations of α-CD and β-CD It can be suggested that the LW emission band is related

to the formation of CD inclusion complex Such a spectral shift may correspond to an

energy stabilization of the emitting state and is characteristic for the fluorescence of TICT

state The LW emission for the above sulfonamides originates from the TICT state with

twisting occurring at the amide SndashN bond between the aniline ring (electron donor) and the

SO2 group or pyrimidine ring (electron acceptor) Further Rajendiran et al [171 172]

reported whenever two aromatic rings are separated by the groups like SO2 CH2 CO NH

etc they form a TICT state Thus it can be speculated that the enhancement of the band at

138

445 nm emission should have originated from the TICT state The LW emission (445 nm) in

both CD solutions suggests that the inclusion process plays a major role in the TICT

emission

In all the three drugs the presence of isosbestic point in the absorption spectra

suggests the formation of 11 inclusion complex between the drugs and CDs The binding

constant for the formation of inclusion complex was determined by analyzing the changes in

the absorbance and fluorescence intensities with the different concentrations of CD The

binding constant (K) values determined using Benesi-Hildebrand relation [120] indicates

that 11 complexes are formed between the sulfonamides and CDs Fig 55 depicts the plot

of 1AndashA0 and 1IndashI0 as a function of 1[CD] for the SDMO SMRZ and SFP molecules The

plot of 1AndashA0 and 1IndashI0 as a function of 1[CD] gives linear line This analysis reflects the

formation of 11 inclusion complex formed in all sulfonamides The calculated binding

constant (K) values are listed in the Table 51 The binding constant values are small when

compared to previously reported hostguest complexes such as dialkyl aminobenzonitrile

and its derivatives [154] This is probably due to (i) sulfonamide molecules are partially

entrapped into the cavity (ii) heterocyclic ring (pyrimidinepyridine) might have entrapped

in the cavity and (iii) these molecules might not be tightly encapsulated in the CDs cavities

The Gibbs free energy changes (∆G) of the inclusion complexes are calculated using

the Eqn 25 As can be seen from Table 51 the calculated ∆G values for SDMOCD

SMRZCD and SFPCD inclusion complexes are negative which suggests that the inclusion

process proceeded spontaneously at 303 K The hydrophobic interaction between the

internal wall of CDs and guest molecules is an important factor for the stability of inclusion

complexes In sulfonamides it is considered that the change in the magnitude of the

hydrophobic interaction is related to that of the contact area of the guest molecule for the

internal wall of CD cavity

52 Effects of solvents

In order to investigate the TICT emission of SDMO SMRZ and SFP the absorption

and fluorescence maxima of the above molecules in the solvents having different polarities

and hydrogen bond forming tendency have been recorded The spectral data are listed in

Table 52 Due to the low solubility of the drug in non-polar cyclohexane solvent its

spectrum was recorded using 1 dioxane solution of cyclohexane The data in Table 52

indicate that the absorption bands are more intensed with less features where the relative

intensity of each is sensitive to solvent polarity This more intensed bands are caused by

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

138

445 nm emission should have originated from the TICT state The LW emission (445 nm) in

both CD solutions suggests that the inclusion process plays a major role in the TICT

emission

In all the three drugs the presence of isosbestic point in the absorption spectra

suggests the formation of 11 inclusion complex between the drugs and CDs The binding

constant for the formation of inclusion complex was determined by analyzing the changes in

the absorbance and fluorescence intensities with the different concentrations of CD The

binding constant (K) values determined using Benesi-Hildebrand relation [120] indicates

that 11 complexes are formed between the sulfonamides and CDs Fig 55 depicts the plot

of 1AndashA0 and 1IndashI0 as a function of 1[CD] for the SDMO SMRZ and SFP molecules The

plot of 1AndashA0 and 1IndashI0 as a function of 1[CD] gives linear line This analysis reflects the

formation of 11 inclusion complex formed in all sulfonamides The calculated binding

constant (K) values are listed in the Table 51 The binding constant values are small when

compared to previously reported hostguest complexes such as dialkyl aminobenzonitrile

and its derivatives [154] This is probably due to (i) sulfonamide molecules are partially

entrapped into the cavity (ii) heterocyclic ring (pyrimidinepyridine) might have entrapped

in the cavity and (iii) these molecules might not be tightly encapsulated in the CDs cavities

The Gibbs free energy changes (∆G) of the inclusion complexes are calculated using

the Eqn 25 As can be seen from Table 51 the calculated ∆G values for SDMOCD

SMRZCD and SFPCD inclusion complexes are negative which suggests that the inclusion

process proceeded spontaneously at 303 K The hydrophobic interaction between the

internal wall of CDs and guest molecules is an important factor for the stability of inclusion

complexes In sulfonamides it is considered that the change in the magnitude of the

hydrophobic interaction is related to that of the contact area of the guest molecule for the

internal wall of CD cavity

52 Effects of solvents

In order to investigate the TICT emission of SDMO SMRZ and SFP the absorption

and fluorescence maxima of the above molecules in the solvents having different polarities

and hydrogen bond forming tendency have been recorded The spectral data are listed in

Table 52 Due to the low solubility of the drug in non-polar cyclohexane solvent its

spectrum was recorded using 1 dioxane solution of cyclohexane The data in Table 52

indicate that the absorption bands are more intensed with less features where the relative

intensity of each is sensitive to solvent polarity This more intensed bands are caused by

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

139

Fig 55

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

140

Table 52

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

141

the (π π) transition of benzene ring and not by the (n π) transition of the carbonyl group

[155 156] The molar extinction coefficient is very high (~10-4 cm-1) at these maxima It is

observed that the absorption spectra of SDMO are red shifted from non-polar cyclohexane to

methanol due to the most proton accepting nature of solvents with increasing the polarity

Further water can act as proton donor solvent and thus produce a blue shift in the absorption

spectra of the drug molecule The spectral shifts observed in the absorption spectra of

SDMO in protic and aprotic solvents are consistent with the characteristic behaviour of

chromophore containing amino group [152c]

In addition the absorption spectral characteristics of SDMO are red shifted with

respect to that of sulfisomidine (SFM cyclohexane asymp λabs ~262 nm λflu ~327 nm

acetonitrile asymp λabs ~268 nm λflu ~340 nm methanol asymp λabs ~269 nm λflu ~318s 345 nm

water asymp λabs ~263 nm λflu ~342 432 nm) and sulfanilamide (SAM cyclohexane asymp λabs ~260

nm λflu ~320 nm acetonitrile asymp λabs ~261 nm λflu ~336 nm methanol asymp λabs ~261 nm λflu

~340 nm water asymp λabs ~258 nm λflu ~342 nm) [88] Likewise in SMRZ the absorption

maxima are red shifted compared to sulfadiazine (SDA cyclohexane asymp λabs ~262 nm λflu

~326 nm acetonitrile asymp λabs ~268 nm λflu ~330 nm methanol asymp λabs ~269 nm λflu ~318s 345

nm water asymp λabs ~263 nm λflu ~342 432 nm) [88] This shows that the replacement of

dimethoxy or methyl substituted pyrimidine ring on hydrogen atom of SO2ndashNH2 group has a

strong effect on the electronic energy levels of SAM The decrease in red shift of the above

sulfa drugs are in the order SDMO = SMRZ gt SFM = SDA gt SAM indicates the

substitution of pyrimidine ring and methoxy group is the key factor for absorption and

emission characteristics This is because of the different electronic densities of the HOMO

on each atom Further when compared to aniline [88] (cyclohexane asymp λabs ~283 235 nm λflu

~320 nm methanol asymp λabs ~278 230 nm λflu ~335 nm) the absorption spectra are blue

shifted while a red shift is observed in the fluorescence spectra However in polar solvents a

small shift was observed in the absorption and emission spectra for SDMO and SMRZ

suggesting that tautomeric structures present in sulfonamides [173] which is realistic reason

for insoluble nature of the drug in non-polar cyclohexane solvent

The typical fluorescence spectra of SDMO SMRZ and SFP in different solvents at

303 K are shown in Fig 56 In contrast to the weak solvent dependent absorption spectra

the emission properties of SDMO SMRZ and SFP molecules are strongly solvent dependent

indicating the possibility of a change in the character of the electronic state In all non

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

142

Fig 56 Fluorescence spectra of SDMO SMRZ and SFP in different solvents at 303 K (Conc = 4 times 10-5 M) (1) cyclohexane (2) ethyl acetate (3) acetonitrile (4) 2-propanol (5) methanol and (6) water

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 280 390 500

1

2 3 4

5

6

335 445

SDMO λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

750

375

0 320 460 600

4 1

2

3

5

6

SFP λexci = 290 nm

Fluo

resc

ence

inte

nsity

(au

)

Wavelength (nm)

600

300

0 270 345 410

1

2

3 4 5

6

SMRZ λexci = 290 nm

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

143

aqueous solvents sulfonamide molecules give a single emission maximum whereas in water

they give two emission maxima Among the two maxima one occurs in shorter wavelength

region (SW) around ~340 nm and the other in longer wavelength region (LW ~430 nm) The

intensity of SW band is greater than LW band Further the fluorescence intensity is weaker

in water than other non-aqueous polar solvents As the solvent polarity is increased the

emission maxima of the above molecules undergo a considerable red shift Further the

excitation spectra corresponding to SW and LW emission maxima resembles the absorption

spectra of SDMO and SMRZ

The appearance of LW fluorescence in glycerol and CD solutions implies that the

spectral behaviour of the sulfonamides is not due to solute-solvent specific interaction

ie complex formation In non-aqueous solvents the LW emission from SDMO and SMRZ

seems to be very weak or absent as compared with the MW band and therefore the LW

band is assumed to be TICT state This is because the LW is red shifted in water suggesting

that the emitting state is TICT type This is indicated by large increase in dipole moment

upon excitation The increase in dipole moment is caused by a change in the electronic

configuration of the amino group from tetrahedral (Sp3) in S0 state to the trigonal (Sp2) in

the excited state In order to check the TICT the fluorescence spectrum of SDMO was

recorded in solutions consisting of different composition of glycerol-water mixtures As

expected the fluorescence intensity of MW band increased with an increase of glycerol

content This is according to the fact that as the viscosity increases the free rotation of

aniline group decreases In addition the larger Stokes shifts imply that in aprotic solvents

the unspecific interactions are the key factors in shifting the fluorescence maxima to the

longer wavelength for these sulfa drug molecules As evident from the above results these

interactions are large in case of the above drugs and may be attributed to increasing dipole

moment of S1 state and hence a strong charge transfer (CT) character of the emitting state In

the excited state rigid molecules with limited internal degrees of freedom change the

structure of the solvent cage with their dipole moment change This process induces a large

Stokes shifts in these sulfa drugs in the polar solvents

53 Prototropic reactions in aqueous and CD medium

The influence of different acid and base concentrations in the H0pHHndash range of -10

to 160 on the absorption and fluorescence spectra of SDMO SMRZ and SFP were studied

(Table 53) During the study of the pH dependence of absorption and emission the self

aggregation of drug molecule should be avoided Therefore the spectroscopic titrations

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

144

(absorption and emission) were performed in aqueous medium with the concentration of

2 times 10-5 M SDMO is likely to exist in different forms of the protonation and deprotonation

depending on the pH of aqueous solutions neutral monocation dication monoanion The

absorption and fluorescence intensity of SDMO were constant between pH ~75 and 95

indicating that in this range neutral species only present With a decrease on pH from 75

the absorption maxima are regularly red shifted The red shifts in SDMO with an increase of

hydrogen ion concentration suggest the formation of monocation formed by the protonation

of tertiary nitrogen in the pyrimidine ring The monocation can be formed either by

protonation of tertiary nitrogen or ndashNH2 group It is well established that protonation of ndash

NH2 group will lead to the blue shift whereas protonation at =Nndash atom will lead to the red

shift in the absorption and fluorescence spectra of the neutral species Dogra et al [174] have

clearly demonstrated that first protonation takes place on the pyrimidine nitrogen atom and a

regular red shift observed for dication reveals intramolecular proton transfer (IPT) process

between =Nndash atoms and amino group Further increase in the acid concentration from pH

~25 a large red shift was noticed in the absorption maxima suggested that the formation of

dication While increasing the pH above 8 a slight red shifted absorption maximum is

noticed at pH ~11 suggesting the formation of monoanion Further no significant change in

the absorption maxima was observed while increasing the pH of the solutions The same

trend was observed in the pH dependence of absorption and fluorescence spectra of SMRZ

and SFP molecules

The fluorescence spectral behavior of protonated species (monocation and dication)

of SDMO observed is similar to the absorption spectra On increasing the pH above 10 the

emission intensity of the fluorescent band decreased at the same wavelength without the

appearance of any new band This may be due to the formation of monoanion In general

the monoanion of many aromatic amino compounds are reported to be non-fluorescent in the

S1state [174]

To know the effect of CD on the prototropic equilibrium between neutral

monocation and dication the pH dependent changes in the absorption and emission spectra

of the above sulfonamides in aqueous solution containing α-CD and β-CD were recorded

and the spectral data are shown in Table 53 The absorption and emission maxima of these

sulfonamides were studied in 6 times 10-3 M CD solutions in the pH range 01 to 11 In CD

solutions the absorption and emission maxima of these sulfonamides neutral maxima were

red shifted on comparison with aqueous medium (Table 53)

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

145

Table 53

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

146

54 Possible inclusion complex

From the above results the possible inclusion mechanism is proposed as follows

According to the molecular dimension of the guests (long axis SDMO = 134013 Aring SMRZ =

120564 Aring and SFP = 121835 Aring) it is too large to fit entirely in the CD cavity and the entire

guest molecule cannot be fully entrapped within the hydrophobic CD cavity Since the internal

diameter of α-CD and β-CD was found to be approximately ~56 Aring and 65 Aring respectively

and its height ~78 Aring Therefore naturally two different types of inclusion complex formation

between the sulfa drugs and CDs are possible (i) the aniline ring is captured in the CD cavity

and (ii) the heterocyclic (pyrimidinepyridine) ring is captured in the CD cavity Let us consider

the type I arrangement where the aniline part of the drug molecules is encapsulated within the

CD cavity if this type of inclusion complex was formed the above drug molecules should not

show any dual emission in CD medium Further if the amino group is entrapped within the CD

cavity the dication maxima (ie protonation of amino group) should be blue shifted in α-CD

and β-CD than in aqueous medium [174 175] The present results indicate that no significant

difference in the spectral shifts in CDs and aqueous medium ie dication maxima follow the

same trend both in aqueous and CDs medium (Table 53)

In type II encapsulation if the heterocyclic ring (pyrimidinepyridine) is included into

the CD cavity the monocation maxima of the sulfonamide molecule (ie protonation of

tertiary nitrogen atom) should be red shifted in CD solution than aqueous medium The results

in Table 53 indicate that the heterocyclic ring interacts with CDrsquos hydroxyl groups confirming

the fact that CDs are good hydrogen donors The tendencies of these shifts in λabs and λflu for

the above molecules can be attributed to their inclusion into the CD cavity Further similar

results were observed in our earlier reports [88] Moreover in type II complex the CD cavity

impose a restriction on the free rotation of the methyl group or heterocyclic ring in the excited

state In this type of inclusion TICT emission should increase in CD medium Further another

question may arise why SDMO SMRZ and SFP does not exhibit the TICT emission in non-

aqueous and non-polar solvents It may be because of a weak dipole-dipole interaction between

RndashSO2ndashNH group with the solvent and fast back charge transfer These features support the

idea that the TICT state in CD medium was stabilized through complex formation between

SDMO SMRZ and SFP with CDs This confirms that the environments around the aniline

group in both CD medium were same as in the bulk aqueous medium Hence type II complex

formation is favored than type I complex formation These results reveal that the part of the

aniline and heterocyclic ring might be present inside of the CD cavity

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

147

55 Fluorescence life time analysis

The fluorescence lifetime values of SDMO SMRZ and SFP in water α-CD and

β-CD (001 M) have been monitored and the relevant data are compiled in Table 54 The

fluorescence decay curve for the free drugs in water was fitted to biexponential function with

χ2 values of ~11 This indicates that these drugs have two lifetime components One causative

maximum of the total fluorescence (τ1) and the other causative is the rest of fluorescence (τ2)

The first with a longer lifetime is assigned to LE emission and the other to TICT state of guest

molecule It is noteworthy that the decay time of the slow component is different to that of

TICT emission within experimental uncertainty This indicates that an equilibrium between

the locally excited (LE) state and the TICT state is achieved in water rather in a short period

By the addition of CDs biexponential decay curve becomes triexponential Three lifetimes (τ1

τ2 and τ3) were obtained in the presence of both α-CD and β-CD Fig 57 reflects that the

observed lifetime values increase with the addition of CD concentration in aqueous solution

due to the inclusion complex formation between the drugs and CDs The enhancement of

lifetime values indicate that SDMO SMRZ and SFP molecules experience less polar

hydrophobic environments within the CD cavity resulting reduced non-radiative decay

processes Thus the increase in fluorescence lifetimes is a result of the significant interactions

of the sulfa drugs with hydrophobic CD nanocavities The lifetime enhancement is found to be

higher with β-CD than that of α-CD suggests that the stronger hydrophobic interactions

between the sulfonamides and β-CD cavity

Table 54 Fluorescence decay parameters of SDMO SMRZ and SFP in water and 001 M CD solutions (λexcitation = 295 nm and λemission = 340 nm)

Drugs Medium Lifetime (ns) Pre exponential factor

ltτgt τ1 τ2 τ3 a1 a2 a3

SDMO Water 029 328 017 006 268 α-CD 043 114 483 012 007 003 294 β-CD 055 189 558 011 005 002 313

SMRZ Water 093 325 023 005 193 α-CD 052 179 672 012 007 002 360 β-CD 034 254 856 020 009 002 442

SFP Water 027 367 029 004 248 α-CD 036 182 521 027 008 003 279 β-CD 019 272 875 022 007 001 403

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

148

Fig 57 Fluorescence decay curves of (a) SDMO (b) SMRZ and (c) SFP in water and 001 M CD solutions

(a)

(b)

(c)

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

149

56 Molecular modeling studies

The energy features thermodynamic parameters (enthalpy entropy and free energy

change) HOMO-LUMO energy calculations and dipole moment values for the guests

(SDMO SMRZ and SFP) and their corresponding inclusion complexes are summarized in

Table 55 The PM3 level optimized structures of the inclusion complexes are shown in Figs

58 and 59 It can be seen from Figs 58 and 59 that the guest molecules formed stable

inclusion complexes with α-CD and β-CD Interestingly it can also be seen that the

structures of SDMOα-CD SDMOβ-CD SMRZα-CD SMRZβ-CD SFPα-CD and

SFPβ-CD inclusion complexes are all similar to each other

Table 56 depicted the bond distances bond angles and the most interesting dihedral

angles of the drugs (guests) before and after complexation within CDs obtained from the

PM3 optimized most stable structures (Figs 58 and 59) It was evident that in the CD

cavity the geometries of SDMO SMRZ and SFP drugs were slightly altered The

alterations were significant in the dihedral angles which indicate that the drugs adopted

a specific conformation to form a stable complex The internal diameter of α-CD is

approximately 50 Aring and β-CD is approximately 65 Aring considering the shape and

dimensions of both CD the drugs may not be completely embedded into the CD cavity

Since the vertical distance and length of the SDMO SMRZ and SFP drugs are greater than

the dimensions of both CD the guest molecule cannot be fully present inside of the CD

cavity Further the optimized structures of the inclusion complexes were also confirmed that

the guest molecules partially included in the α-CD and β-CD cavity

The optimized inclusion structures in Figs 58 and 59 demonstrated that hydrogen

bond is formed in all the inclusion complexes The intermolecular hydrogen bonds were

formed between hydrogen atom of amino group of the drugs and oxygen atom of primary

hydroxyl group of the CD with a dHO distance less than 30 Aring This justified the importance

of both interaction energy between the drugs and CD necessary to ensure a better inclusion

of the guest into the host molecules The above values were supported by the fact that the

flexibility of the host molecule may be one of the structural requirements for inclusion

complexes formation The present calculations also explained that hydrogen bond brings the

difference between the binding energies of both α-CD and β-CD complexation with the

SDMO SMRZ and SFP drugs

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

150

Table 55

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

151

Fig 58 PM3 optimized structures of 11 inclusion complexes of (a) SDMOα-CD (b) SDMOβ-CD and (c) SMRZα-CD

Side view

2965 Aring

1808 Aring

Upper view

1876 Aring 1840 Aring

2784 Aring

(c)

(b)

1825 Aring

2699 Aring

(a)

1807 Aring

2566 Aring

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

152

Fig 59 PM3 optimized structures of 11 inclusion complexes of (a) SMRZβ-CD (b) SFPα-CD and (c) SFPβ-CD

Side view

1623 Aring

1621 Aring

1821 Aring

2986 Aring

1741 Aring 2441 Aring

Upper view

(a)

(b)

(c)

2986 Aring

1826 Aring

2966 Aring

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

153

Table 56

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

154

In all the six inclusion complex structures in Figs 58 and 59 It is noticed that

drugCD complexes contain three hydrogen bonds one between nitrogen atom of

pyrimidine ring and H atom of the CD with hydrogen bonding dHO distance ranging from

1821 to 2986 Aring The two oxygen atoms of sulphonyl group (ndashSO2) and two hydrogen

atoms of the CD have hydrogen bonding with dHO distance of 162-296 Aring (Figs 58 and

59) Comparatively a careful analysis of the energetic values obtained from PM3 method

suggests that the mutual host-guest hydrogen bonding interactions contribute greatly to

Ecomplexation and are crucial in determining the stability of inclusion complexes [141]

It is well known that the van der Waals forces including the dipole-induced dipole

interactions are proportional to the distances between the guests the wall of the CD cavities

and the polarizabilities of the two components The interaction of the phenyl ring with CD

would play an important role because the phenyl moiety may achieve a maximum contact

area with the internal surface of the cavity of CD The above results imply that the inclusion

of the drug molecules within the CD cavity is affected by hydrophobic and electronic

interactions Since CD has a permanent dipole the primary hydroxyl end is positive and the

secondary hydroxyl end is negative in the glucose units of CD The stability of binding by

hydrophobic interactions is partly the result of van der Waals force but is mainly due to the

effects of entropy produced on the water molecules [176]

The dipole moment of SDMO (695 D) is significantly higher than the other isolated

SMRZ and SFP molecules (Table 55) The dipole moment of the inclusion complexes are

present in the following order SDMOβ-CD gt SDMOα-CD gt SFPβ-CD gt SFPα-CD

gt SMRZβ-CD gt SMRZα-CD All the inclusion complexes have dipole moment values

higher or lower than the corresponding isolated drugs molecules This indicates that the

polarity of the CD cavity changed after the drugs entered into the CD cavities From these

results it is concluded that the dipole moment shows a strong correlation with the

complexation behaviour of the guest and host molecules

The EHOMOndashELUMO gap is an important scale of stability [145] and chemicals with

larger EHOMOndashELUMO gap values tend to have higher stability Therefore we investigated

the electronic structure of the isolated drugs and the inclusion complexes using the PM3

method HOMO and LUMO energies of the sulfonamides and their inclusion complexes

are given in Table 55 and Fig 510 which exposed the energy gap and the chemical

activity of the molecules The LUMO as an electron acceptor represents the ability to

obtain an electron and HOMO represents the ability to donate electron Fig 510 illustrates

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

155

Fig 510

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

156

the LUMO energy orbital pictures of all the isolated drugs are similar to each other whereas

the HOMO is significantly varied The energy gap between HOMO and LUMO of each

complex suggest that there will be a significant change in the electronic structures of guest

molecules The EHOMOndashELUMO gap for SFPβ-CD inclusion complex (-843 eV) was more

negative which advocate that this complex is more stable than other inclusion complexes

Though the results are not readily understandable according to the driving forces

listed in the earlier reports the Morokuma theory of energy decomposition analysis [177]

can offer a reasonable explanation According to the theory when a supramolecule was

formed electrons will lose their identity as belonging to one or other component molecule

Four types of interactions should be considered in the formation of a supramolecule

(a) electrostatic interaction which is favoured by large permanent charges and dipoles

(b) polarization interaction which is favoured by large volume and polarizability of the

molecules (c) exchange energy or Pauli repulsion and (d) charge-transfer interaction which

is contributed from the mixing of the filled orbital of one component molecule with the

vacant orbital of the other The charge transfer is always attractive and the most important

terms in this interaction are contributed from the charge-transfer between the HOMO of one

component and the LUMO of the other These first three interactions constitute the

canonical driving forces in CD chemistry ie dipolendashdipole interaction dipole-induced

dipole interaction and steric effect However they cannot explain the unexpected theoretical

and experimental observations The higher HOMO of the guest molecule the stronger is the

charge transfer in the complexation Herein the PM3 calculations showed that no charge

transfer is formed between the guest and host molecules [176]

To investigate the thermodynamic parameters of the binding process the statistical

thermodynamic calculations were carried out at 1 atm pressure and 29815 K temperature by

PM3 method The thermodynamic quantities the binding energies (ΔE) Gibbs free energy

changes (ΔG) enthalpy changes (ΔH) and entropy changes (ΔS) are depicted in Table 55

The PM3 calculations express that energies of the complexation are lower than those for the

isolated host and guest molecules The complexation energy allowed us to evaluate the

inclusion process and to find the most stable inclusion complex between the complexes

under study The complexation reactions of SDMO SMRZ and SFP with CDs were

exothermic which was judged from the negative ΔE and ΔH values The negative energy

and enthalpy changes suggested that the inclusion processes were energetically and

enthalpically favorable in nature Among the six inclusion complexes SMRZβ-CD

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

157

complex has lowest energy (-1976 kcal mol-1) when compared with other complexes SFP

α-CD (-389 kcal mol-1) SFPβ-CD (-1742 kcal mol-1) SMRZα-CD (-1057 kcal mol-1)

SDMOα-CD (-671 kcal mol-1) and SDMOβ-CD (-1031 kcal mol-1) The above results

indicate that SMRZ and β-CD formed more stable inclusion complex than other two drugs

(because high negative value is more stable) The calculated interaction energies as

discussed above can be promptly understood in terms of the formation of intermolecular

hydrogen bonds only In this respect CD hydrogen atoms (particularly the inner protons)

play a major role in stability of these sulfonamide inclusion complexes For all these

complexes favourable hydrogen bonding interactions have been predicted based on the

distance between hydrogen atoms of hosts (α-CDβ-CD) and nitrogen atoms in heterocyclic

ring or sulphonyl (SO2) groups of the guest molecules For these six inclusion complexes

several hydrogen bonds are possible but only minimum number of hydrogen bonds were

identified (Figs 58 and 59) These findings suggest that dispersion forces must play an

important role in the complex formations [178] The significant ΔE values which may vary

with the length of hydrogen bonds has been predicted From Table 55 the interaction

energies of the six inclusion systems differs from each other this is due to the hydrogen

bond between different groups (Figs 58 and 59) It is often difficult to explain the

difference in interaction energies of these complexes at PM3 level of computing

The experimentally determined free energy change (ΔG) values for all of the

inclusion complexes from the stability constant are negative which indicates that the

encapsulation process was a spontaneous and thermodynamically favoured in the

experimental temperature (303 K) When compared with the experimental ΔG values a

similar negative magnitude was obtained for SMRZβ-CD complex by PM3 calculation On

the contrary positive magnitudes were observed for other inclusion complexes suggesting

that the inclusion reactions were non-spontaneous processes The difference in ΔG values

can be explained by the solvent effect The actual experiments were conducted in aqueous

medium but the computational work was done at vacuum phase Unfortunately because of

limitations in the calculation ability of our computer and the large molecular size of CD the

statistical thermodynamic calculations for these systems could not be performed in aqueous

phase as well as in excited state However it was observed that the solvent effect on the

host-guest interactions easily changes the inclusion process from a non-spontaneous process

in the gas phase to a spontaneous one in the aqueous phase The host-guest interaction

causes an enthalpy-entropy compensating process in the gas phase whereas the same

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

158

interaction causes an enthalpy-entropy co-driven process in aqueous solution because

inclusion complexation releases a number of water molecules from the cavities of CD

To the best of our knowledge the very small negative ΔH values indicate that the

inclusion formation of drugs with CD is exothermic and enthalpy driven It should be noted

that ΔH and ΔS values relyon (i) release of water bound in the cavity (ii) partial destruction

of hydration shells of the reagents (iii) non covalent interactions (van der Waals

hydrophobic and electrostatic interactions as well as hydrogen bonding) and (iv) hydration

of the inclusion complexes All these processes should be taken into account while

discussing thermodynamic parameters of complex formation The ΔH value for SMRZ

β-CD complex is more negative (-1767 kcal mol-1) compared to other inclusion complexes

Probably geometrical factor plays a considerable role in complexation process The small

negative ΔS values are the confirmation of restriction of freedom of the molecule and

formation of less compact structures As it is evident from Table 55 hydroxyl groups

reduce the binding affinity of CD with the drugs making the complexation process more

enthalpy and less entropy favourable [179] It is believed that both the cavity size of CD and

substituents of the drug molecule cause steric hinderence during the inclusion process

The observed small negative ΔS values are due to enhancement of disorder in the

system Moreover hydrophobic interactions which are long range interactions can be

important in the CD complex formation The inclusion complex structure in Figs 58 and

59 also suggest that the benzene ring is partially encapsulated inside the CD cavity and

interacts with it through hydrophobic interactions Further the small ΔH values can be

explained by the prevalence of hydrophobic interactions Based on the ΔS and ΔH values of

the three drugs with CDs it is confirmed that the six inclusion complexes are stable

Comparison of ΔH and ΔS confirmed that enthalpy changes are higher and entropy changes

are lower for the complexation Therefore complexes of the drugs with both α-CD and β-CD

are more enthalpy stabilized

57 Solid inclusion complex studies

571 Scanning electron microscopic morphological observations

In order to study the morphological changes the images of the powdered form of the

drugs α-CD β-CD and powdered form of inclusion complexes observed by SEM

(Fig 511) These images were utilized to evaluate the effect of coprecipitation process on

the surface morphology of the solids used for the formation of solid inclusion systems The

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

159

SEM photographs clearly elucidated the difference between the pure components and their

inclusion complexes It can be seen in the SEM image Fig 511a that pure SDMO crystals

are in the form of sheets with smooth surface whereas the images of pure SMRZ crystals

are well-defined bar shape and a mean size of about ~5-10 micron (Fig 511d) However

the examination of SEM photographs of SFP showed micronized crystals compact

structures can be observed with irregular shapes and different sizes characterized by smooth

surface and obtuse corners (Fig 511g) In contrast the morphologies of the inclusion

complexes are quite different in shapes and sizes from pure drugs and CDs The SDMO

α-CD solid complex (Fig 511b) was appeared in irregular shapes with variable size

whereas a bar like shape was observed for SDMOβ-CD complex (Fig 511c) The

SMRZα-CD complex (Fig 511e) exhibited cylindrical shaped particles and irregular

shaped crystals were observed for the SMRZβ-CD complex (Fig 511f) Likewise the

morphology of SFP drug is different from their inclusion complexes Both the SFPCD solid

crystals show cracks and dimples on the surface which confirms that the coprecipitation

method is able to encapsulate the SFP into the CD cavity Modification of these structures

can be taken as a proof of the formation of new inclusion complexes by molecular

encapsulation

572 Transmission electron microscopic morphological observations

TEM was used to investigate the micro-structural features of the wall of encapsulated

sulfa drugs (Fig 512) In all the inclusion complexes round (presumably spherical) shaped

nanoparticles with the average size of about 30-90 nm were observed In the case of the

SDMOβ-CD inclusion complex the nano particles were partly agglomerated (Fig 512b)

due to this it was somewhat complicated to predict the shape of nano particles A similar

guest encapsulation associated with nanoparticle formation was found for another

hydrophobic drug molecule [180] Preparation of nanoparticles from preformed inclusion

complexes of the sulfa drug and α-CD or β-CD have been proved to be an effective method

to enhance drug loading to nanoparticles These results suggest that the CD serve as a

delivery carrier for hydrophobic drugs It is clearly demonstrated by Bonini et al [181] that

depending upon the concentration the β-CD monomers aggregated in different shapes in

aqueous medium at 298 K According to their investigation β-CD at lower concentration (3

mM) displays a well developed sphere-like shaped nano aggregation with diameter of 100

nm whereas at higher concentration (6 mM) micro-planar aggregates were observed From

the SAED pattern shown in Fig 512b (inset) it is confirmed that the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

160

of SDMO with CDs led the crystalline drug completely transferred to amorphous nature

which is also supported by XRD results

Fig 511 SEM photographs of (a) SDMO (b) SDMOα-CD complex (c) SDMO β-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex

100 microm

20 microm

(b) (e)

(d) (a)

(c) (f)

(h)

(g)

(i)

50 microm

30 microm

20 microm

10 microm 30 microm

10 microm

20 microm

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

161

Fig 512 TEM photographs of (a) SDMOα-CD complex (b) SDMOβ-CD complex (c) SMRZα-CD complex (d) SMRZβ-CD complex (e) SFPα-CD complex and (f) SFPβ-CD complex Inset in figure (b) shows SAED pattern of SDMOβ-CD complex

(a) (b) 90 nm

20 microm 50 microm

02 microm

95 nm

(f) (e)

(c) (d)

200 nm

1 microm 500 nm

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

162

573 FTIR spectral analysis

The complexation between the sulfonamides and CDs was investigated using FTIR

spectroscopy Figs 513 and 514 depict the FTIR spectra of free SDMO SMRZ SFP and

their corresponding inclusion complexes The investigation of the inclusion complexes was

complicated due to the strong stretching frequency of CDs overlapping with the bands of the

drugs The IR spectrum of SDMO drug (Fig 513a) was characterised by principal

absorption peaks at 3450 and 3348 cm-1 (for NH stretching in SO2ndashNH group) 3231 cm-1

(for NH2 stretching) 1651 cm-1 (for NH2 deformation) 1593 cm-1 (for C=C stretching)

1444 and 1352 cm-1 (for CH3 deformation) 1305 cm-1 (for SO2 asymmetric stretching)

1260 cm-1 (for CndashO stretching in ndashCOCH3 group) 1186 cm-1 (for SO2 symmetric

stretching) 1089 and 810 cm-1 (for CndashH deformation in phenyl ring) 1005 cm-1 (for CndashN

stretching) 875 cm-1 (for SndashN stretching) and 545 cm-1 (for SO2 deformation) The FTIR

spectra of CD consisted of the prominent absorption peaks of OndashH stretching (3392 cm-1)

CndashH stretching (2926 cm-1) HndashOndashH bending (1646 cm-1) CndashO stretching (1157 cm-1) and

CndashOndashC bending (1028 cm-1) In contrast the IR spectral features of the pure drug had

completely disappeared in the inclusion complexes The band assigned to the NH stretching

in SO2ndashNH group and NH2 stretching had completely disappeared in the inclusion

complexes (Figs 513b and c) The bands positioned at ~1651 1593 1260-1089 cm-1 were

shifted and their intensities were diminished The NH2 deformation and CH3 deformation

frequencies were shifted in the inclusion complexes to 1654 and 1446 1355 cm-1

respectively However the CndashN stretching was shifted to lower frequency (992 cm-1) in the

inclusion complexes The SndashN stretching and SO2 deformation bands became shorter and

shifted to lower frequencies of 871 and 542 cm-1 respectively The above changes can be

due to the formation of SDMOCD inclusion complexes in solid state According to the

above FTIR analysis of inclusion complexes we might suggest that the aniline ring of the

SDMO was involved in the complexation and the amino group is present outside of the CD

cavity Nevertheless no new chemical bonds were formed which revealed that the non-

covalent interactions between the drug and CDs These results may be due to the presence of

van der Waals interactions between CD and SDMO In this regard van der Waals

interactions are generally considered to contribute to the formation of inclusion complexes

with CDs

Fig 513d shows the characteristic bands of SMRZ at 3483 cm-1 and 3379 cm-1

for amido stretching and amino stretching vibrations respectively 1246 cm-1 for primary

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

163

Fig 513 FTIR spectra of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex and (f) SMRZβ-CD complex

Tra

nsm

ittan

ce

4000 40000 30000 20000 10000

Wavenumber (cm-1)

(a)

3231

06

2953

28

1444

81

1186

33

1143

89

682

86

542

05

1651

22

1089

88

3450

96

3348

73

1593

35

1417

81

1352

22

567

12

875

76

810

18

(d)

(f)

(e)

3254

21

3078

67

1408

16

1302

07

1153

54 68

286

522

05

1630

01

1091

81

3483

76

3379

59

1597

20

1493

04

1327

14

583

12

835

25

2874

19

2779

67

2955

21

891

19

2926

28

3379

59

2773

89

1630

10

1597

28

1570

20

1493

05

1408

16

1329

85

1157

39

1029

15 89

119

83

718

679

47

578

70

545

90

2920

49

3380

23

1641

57

1597

20

1570

20

1493

15

1410

09

1329

55

1157

39

1302

07

889

26

837

18

680

93

576

77

545

90

(b)

(c)

2928

12

3358

24

1653

22

1593

35

1446

78

1355

26

1157

06

1030

31

684

86

569

47

2928

01

3359

21

1654

35

1593

35

1445

85

1355

28

1156

98

1030

68

684

79

569

82

1246

89

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

164

Fig 514 FTIR spectra of (a) SFP (d) SFPα-CD complex and (e) SFPβ-CD complex aromatic amine (CndashN stretching) 1630 cm-1 for secondary amine stretching 1597 cm-1 for

C=C bending and 795-637 cm-1 for CndashH bending vibrations The SO2 symmetric and

asymmetric vibrations are observed at 1153 cm-1 and 1302 cm-1 respectively It can be seen

from Figs 513e and f the characteristic stretching vibrations for C=C 1607 cm-1 and 1502

cm-1 band intensities are reduced and shifted in the solid inclusion complexes (1597 cm-1

and 1493 cm-1) due to the encapsulation of phenyl ring into the CD cavity The symmetric

and asymmetric vibrations of SO2 group are shifted to 1157 cm-1 and 1306 cm-1 in the

inclusion complexes However the band at 1246 cm-1 (for CndashN stretching) diminished

considerably Further the amino and amido stretching vibrations of SMRZ disappeared in

the inclusion complexes The similar changes in the corresponding functional group

frequencies of SFP after inclusion complexation with CDs were also observed (Fig 514)

4000

Tra

nsm

ittan

ce

40000 30000 20000 10000 Wavenumber (cm-1)

3244

56

3310

15

1263

49

679

00

504

05

999

22

3418

17

1583

70

947

13

769

67

1890

41

1772

74

1637

71

1504

61 14

525

3 13

676

5 13

232

9

1222

98

1124

60

1076

38

869

97

825

61

642

35

592

05

(a)

3379

59

2928

56

1643

59

1523

42

1504

51

1244

58

827

48

(c)

3383

45

1585

88

1369

59

1263

21

1157

91

1128

29

1078

12

1028

42

947

35

767

18

680

51

613

87

567

56

2929

35

1643

57

1585

45

1523

39

1504

75

1369

21

1263

78

1157

46

1244

41

1128

23

1078

11

1028

27 94

726

827

92

767

83

680

45

613

57

567

42

(b)

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

165

The bands positioned at 1637 and 1583 cm-1 of SFP shifted to longer frequencies and their

intensities diminished in the inclusion complexes The bands in the region 1124-999 cm-1

completely disappeared in the spectra of SFPCD complexes The CndashS stretching and SO2

stretching frequencies of SFP shifted to 1644 and 1370 cm-1 in the inclusion complexes

respectively and their intensities decreased These IR spectral changes suggest that the

aromatic moiety with SO2ndashNH group of the SMRZ and SFP is probably encapsulated into

the CD cavity and formed stable inclusion complexes in the solid state

574 Differential scanning calorimetry

The DSC thermograms of pure drugs and the solid inclusion complexes are

presented in Fig 515 The thermal curve of SDMO was typical of a crystalline anhydrous

substance with a sharp endotherm at 2034 degC indicating the melting point of the drug On

contrary the complete disappearance of SDMO characteristic endotherm in the solid

inclusion systems is an indicative of a strong interaction between the drug and CD

However the endotherms of α-CD and β-CD shifted to 584 995 1042 degC and 1012 degC

respectively along with significant decrease in the intensity was observed This finding

probably indicates the formation of true inclusion complex of CD with the guest in solid

dispersion andor inclusion complexes with different properties The thermal features of CD

shifted to lower temperature in the complex may be due to the reorientation of CD by the

insertion of the drug molecules

The thermal curve of SMRZ (Fig 515d) was typical of a crystalline anhydrous

substance with a sharp fusion endotherm at 2368 ordmC corresponding to the melting point of

the drug A comparison of the DSC curves of pure CDs with those of encapsulated solid

samples (in 11 mole ratio) clearly showed that the displacement of the endothermic peaks

are due to loss of water into the lower temperature However the complete disappearance of

the characteristic endothermic peak of SMRZ in the DSC curves of inclusion complexes

(Figs 515e and f) indicate a strong interaction of the drug with CD nanocavities and the

stable encapsulated complexes are formed between SMRZ and CDs Similar phenomena

were also obtained for the thermal properties of SFPCD complexes (Figs 515h and i) The

overall DSC results suggest that complexation of the sulfonamides with both CDs was

achieved in the solid state by coprecipitation method

575 X-ray diffraction analysis

Powder XRD has been used to assess the degree of crystallinity of the given samples

When the inclusion complexes of drug and CDs are formed the overall numbers of

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

166

crystalline structure have reduced and more number of amorphous structures have increased

So the solid complexes show less number as well as less intensity of peaks This shows that

overall crystallinity of complexes decreased and due to amorphous halo nature the solubility

increased The XRD patterns of pure SDMO SMRZ and SFP exhibited several sharp and

intense peaks indicating the crystalline nature of the drugs (Fig 516) SDMO exhibits

characteristic peaks at 2θ values of 1028deg 1210deg 1410deg 1902deg 2093deg 2202deg 2494deg

2813deg 3177deg 3295deg 3514deg and 3923deg Most of the characteristic peaks of SDMO were

present in the diffraction patterns of physical mixtures of the drug with CDs without loss of

intensity In contrast the XRD patterns of the SDMOα-CD and SDMOβ-CD complexes

differed considerably from those of the drug and CD alone with a completely diffuse

diffraction pattern which reveals the amorphous nature of the samples The intensity of

peaks at 2θ = 1028deg 1410deg 1902deg 2093deg and 2494deg of SDMO reduced significantly in

SDMOCD inclusion complex diffraction patterns which suggests reduction in crystallinity

of SDMO Some low intensity peaks of SDMO at 2θ = 3295deg 3514deg 3923deg were absent

after complexation with CDs This different pattern might be due to inclusion of SDMO

molecule into the cavity of CD Further appearance of new peaks at 2θ values for the

patterns of SDMOα-CD (1219deg and 1319deg) and SDMOβ-CD (1529deg 2485deg and 2822deg)

inclusion complex indicates the change in SDMO and CD environment after inclusion

complexation

In the XRD pattern of SMRZ (Fig 516d) the intense peaks at 2θ values of 1228deg

1456deg 1702deg 2421deg 3086deg and 3245deg indicate the higher degree of crystallinity of drug

The diffractogram of the SMRZα-CD and SMRZβ-CD complexes (Figs 516e and f)

differed from that of the corresponding SMRZ drug where the characteristic peaks of

SMRZ particularly the major intense diffraction peaks at 2θ = 1228deg 1702deg and 3245deg are

dramatically affected in the inclusion complexes indicating the formation of inclusion

complexes In addition it can be observed the disappearance of characteristic peaks of

SMRZ in the encapsulated complexes suggesting that the crystallinity of the drug further

reduced to a greater degree These phenomena are an indication of the existence of

molecular interactions between the drug and CDs resulting in an amorphous state

Similarly the diffraction pattern of the SFPCD complexes (Figs 516h and i) was more

diffused as compared to pure components (SFP and CD) These observations were indicative

of the transformation of SFP from crystalline to amorphous state which might be because of

inclusion of SFP into CD nanocavities

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

167

Fig 515 DSC thermograms of (a) SDMO (b) SDMOα-CD complex (c) SDMOβ-CD complex (d) SMRZ (e) SMRZα-CD complex (f) SMRZβ-CD complex (g) SFP (h) SFPα-CD complex and (i) SFPβ-CD complex (25-260 oC at 10 oCmin)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200

1922 degC

672 degC 1064 degC 1232 degC

1143 degC

(g)

(h)

(i)

Temperature (oC)

Exo

ther

mic

(mW

mg)

40 60 80 100 120 140 160 180 200 220

(a)

(b)

(c)

2034 degC

1042 degC 995 degC 584 degC

1012 degC

Exo

ther

mic

(mW

mg)

40

(d)

(e)

(f)

2368 degC

60 80 100 120 140 160 180 200 220 240 260

1182 degC

1238 degC 1064 degC

663 degC

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

168

Fig 516

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

169

576 1H NMR analysis

Further to gain more information about the structure of the inclusion complexes

between sulfonamides and CDs 1H NMR spectra for the free drugs and the inclusion

complexes were recorded [152] Figs 517-519 illustrated the chemical shift change of

hydrogen atoms of SDMO SMRZ SFP and their inclusion complexes and the assignations of

protons of the drugs are shown as inset The values of chemical shift (δ) and changes of

chemical shift (Δδ) values are listed in Table 57 The chemical shift of CDs protons reported

by different authors are very close to the values reported in this work When the guest

molecule was inserted into the CD cavity the NMR chemical signals of the guests shifted to

downfield and the CD protons shifted to upfield The H-3 and H-5 protons are located in the

interior of the CDs cavity and it is therefore likely to interact with the protons of drug It can

be seen from Fig 517 and Table 57 that Ha Hc and Hd protons of SDMO showed larger

chemical shifts because of the diminished freedom of rotation caused by the insertion of

SDMO molecule into the CDs cavities This reveals that a part of aniline moiety and

pyrimidine moiety has entered into the CDs cavity However the He proton exhibited

comparably less chemical shift changes in the inclusion complexes The NMR signals of CD

protons placed inside the cavity (H-3 and H-5) are hardly affected by the benzene protons of

the guest suggesting that the SDMO molecule entered through the wider side of the CD cavity

As can be seen from Fig 518 the chemical shift data for the SMRZCD inclusion

complexes were different from those of pure SMRZ drug In particular the resonance of the

aromatic protons (Hb Hc) of SMRZ showed remarkably upfield shift (α-CD asymp -014 -017

ppm and β-CD asymp -015 -019 ppm) in the SMRZCD complexes which suggest that the

resonance of Hb and Hc protons are shielded considerably in the complex and thus the

aromatic part should enter deeply into the cavity However a major shift was observed for

the amino protons of SMRZ (Ha and Hd) in the inclusion complexes which clearly

indicated that the guest molecule has been encapsulated within the CDs cavities Further the

inclusion complexation with SMRZ had significant changes (upfield shift) in the resonance

of H-3 and H-5 protons of CD It is noteworthy that after guest inclusion the H-3 protons

shifted to 003-006 ppm while H-5 protons shifted to 001-003 ppm Both H-3 and H-5

protons are located in the inner part of the CD and H-3 protons are nearer to the wider rim

side of the cavity whereas H-5 protons are nearer to the narrow rim side

The signal assignment for individual proton chemical shift of SFP is shown as inset

in Fig 519 The δ value of Ha is due to the amino group (ndashNH) protons of SFP drug which

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

170

Fig 517 1H NMR spectra of (a) SDMO (b) SDMOα-CD complex and (c) SDMO β-CD complex Inset Fig Assignation of protons of SDMO

(c)

(b)

(a) a

H3CO NH2 a

S O O

N

H N

N

H3CO b

b c

c

d

e

f

g

b c d e

f g

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

171

Fig 518 1H NMR spectra of (a) SMRZ (b) SMRZα-CD complex and (c) SMRZβ-CD complex Inset Fig Assignation of protons of SMRZ

a b

c

e

f d

(c)

(b)

(a)

g

H3C NH2

H

O O

N S

N

N a b

b c

c

d

e

f

g

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

172

Fig 519 1H NMR spectra of (a) SFP (b) SFPα-CD complex and (c) SFPβ-CD complex Inset Fig Assignation of protons of SFP

a b c

d

e f

g j

(c)

(b)

(a)

NH2 H

O O

N S

N a b

g

j

c e

f d

d g

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

173

Table 57

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process

174

shifts to -0046 ppm up field in α-CD complex and -0053 ppm in β-CD complex

(Table 57) The aromatic protons of the SFP molecule (Hd and Hg) shifted to up field as

compared with the corresponding values those for free drug These results indicate that these

above aromatic protons interact with CD cavity protons Further considering the higher

chemical shift of the Ha proton when compared to other protons suggests that the Ha proton

of the SFP molecule considerably has more interactions with the CDs

58 Conclusion

The effects of α-CD and β-CD on the absorption and fluorescence spectra of three

sulfa drugs were investigated Both experimental and theoretical studies have demonstrated

that both CDs form 11 inclusion complexes with sulfonamides In aqueous medium SDMO

SMRZ and SFP showed dual emission one is due to locally excited (LE) and another is due

to TICT Solvent studies revealed that the position of the substituent (dimethoxymethyl

pyrimidine or pyridine group) in the sulfonamide molecule (RminusSO2minusNHminus) is the key factor

to change the absorption and emission behaviour of these molecules The red shift and the

presence of TICT in the CD medium confirmed that the heterocyclic (pyrimidine or

pyridine) ring is encapsulated in the CD cavity and the aniline ring is present outside of the

CD cavity SEM FTIR DSC XRD and 1H NMR results also indicated the formation of

inclusion complexes in the solid state TEM examinations revealed that the nano-sized

particles could be produced from the molecular encapsulation of drugs within the CDs

cavities The various complexation reactions of SDMO SMRZ and SFP with α-CD and β-

CD were studied by semiempirical quantum mechanical calculations (PM3 method) The

optimized host-guest inclusion complex structures proved SDMO SMRZ and SFP

molecules are partially embedded into the CD nanocavities The statistical thermodynamic

calculations advocated that the formations of these inclusion complexes were enthalpy-

driven process The overall theoretical results suggested that hydrogen bonding and

hydrophobic effect play an important role in the complexation process