synthesis, characterization and …shodhganga.inflibnet.ac.in/bitstream/10603/3446/11/11_chapter...

119

SYNTHESIS, CHARACTERIZATION AND

BIOLOGICAL EVALUATION OF SOME

NEW 2 - AMINO - 4, 6 - DIARYL

PYRIMIDINES

CHAPTER - 2

120

2.1 LITERATURE REVIEW

Pyrimidine was first isolated by Gabriel and Colman in 1899.

The chemistry of pyrimidine1 and its derivatives have been studied

since the past century due to their diverse pharmacological properties.

Pyrimidine (50) and purine (51), the two nitrogen containing

heterocyclic aromatic compounds are the parents of the “bases” that

constitute a key structural unit of nucleic acids, even though

pyrimidine itself does not exist in nature. Both pyrimidine and purine

are planar and this flat shape is very important when we consider the

structure of nucleic acids.

N

N

1

2

3

4

5

6

N

N

1

2

3

N

N

H

4

5

9

8

7

6

(50) (51)

In terms of their chemistry, pyrimidine and purine resemble

pyridine. They are weak bases and relatively unreactive towards

electrophilic aromatic substitution. There is an important structural

difference between pyrimidine derivatives that bear –OH groups and

those with –NH2 groups. The structure of a pyrimidine that bears an

amino group follows directly from the structure of the parent ring

system as seen in the case of cytosine. An equilibrium exists in the

aminopyrimidines between the amino (52) and imino forms (53).

121

N

N

NH

H

N

N

NH2

(52) (53)

However, the corresponding pyrimidine having a hydroxyl group

(54) resemble an enol, but exist instead in its keto form (55), contrary

to the stable isomers with the hydroxyl groups on benzene- like rings.

This is because the keto form of the pyrimidine is also aromatic and

stable owing to amide resonance2 as shown in Scheme 12.

N

N

HO N

N

O

H

(54) (55)

N

N

H

O N

N

O

H

Scheme 12. Resonance in keto form of 4-hydroxypyrimidine

122

GENERAL METHODS OF SYNTHESIS OF PYRIMIDINES

The general methods employed in the synthesis of pyrimidines

are briefly reviewed below:

1. 1, 4-dihydro-4-phenyl-2, 6-dimethyl-3, 5-diacetylpyridines were

converted into chalcones by Claisen-Schmidt condensation with

aldehydes. The resulted chalcones were cyclized with guanidine to

give aminopyrimidines3 (Scheme 13).

NH3C CH3

CH3H3C

C6H5O O

H

2ArCHO

NH3C CH3

C6H5O O

H

ArAr

NH2H2N

NH

NH3C CH3

C6H5

H

NN NN

NH2 NH2

ArAr

Scheme 13

2. 1, 3-diaryl-propenones react with guanidine by refluxing them

together in a basic alcoholic medium to give dihydropyrimidines,

which on oxidation with H2O2 yield 4, 6-diaryl-2-

aminopyrimidines4 (Scheme 14).

123

Ar1 Ar2

O

+

NH2H2N

NH

N NH

Ar1

NH2

Ar2

N N

Ar1

NH2

Ar2H2O2

Scheme 14

3. Guanidine reacts with -ketoesters, -diketones, cyanoacetic esters

and , -unsaturated carbonyl compounds to give 2–amino

pyrimidines usually in good yields5 (Scheme 15).

H2N CNH

NH2

CH2

COCH3

COOC2H5

N

N

OH

H2N CH3

Scheme 15

4. Urea reacts with , -unsaturated esters to form dihydrouracil or

uracil6 (Scheme 16).

CO

NH2

NH2+ CH

CHCH3

COOC2H5

C

HN CH2

CHCH3

N

H

O

O

N

NHO

OH

CH3

Scheme 16

5. Ureidoethylenes undergo ready cyclization to pyrimidines in the

presence of a basic catalyst7 (Scheme 17). The required

ureidoethylenes can be prepared from N, N′-dicarbamylformamidines

by reaction with cyanoethylacetate. N, N′-dicarbamylformamidines

can be obtained by the reaction of urea with ethyl orthoformate.

124

CONH2

NH2

C2H5OCHO H2NCNCHNHCNH2

O O

H2CCOOC2H5

CN

N

CNH2

CC

H

H

O

COOC2H5

CN

NaOC2H5N

NO

H

H

NH

COOC2H5

2 H

Scheme 17

125

SPECTRAL FEATURES OF PYRIMIDINES:

UV SPECTRA OF PYRIMIDINES:

Two bands, one at 243 nm and the other at 298 nm are

generally observed in the UV spectrum of pyrimidines. When an

electron releasing substituent is present, a bathochromic shift of the

second band (n-*) usually occur while the electron withdrawing

substituent produces an opposite effect.

The more intense band at 243 nm is due to -* transition

which undergoes a bathochromic shift by both type of substituents

with an increase in intensity. Two bands, one in between 330-350 nm

and the other in between 250-260 nm are usually observed in the

case of 3, 5-diaryl pyrimidines8-10 , whereas 4,6-diarylpyrimidines

exhibit characteristic UV maxima at 254 nm for -* transition and at

350 nm for n-* transition.

IR SPECTRA OF PYRIMIDINES:

The 2-aminopyrimidine system can readily be characterized by

the appearance of absorption bands11 in between 1650 - 1630 cm-1

(C=N) , 1584-1570 cm1 (C=C) and three sharp bands in the region

3490-3460 cm-1, 3352-3350 cm-1and 3200-3100 cm-1 (NH2 free and

hydrogen bonded).The C-H stretching modes occur in between 3100-

3050 cm-1. The characteristic ring-breathing mode occurs in between

1020-990 cm-1 and another characteristic band near 800 cm-1.

NMR SPECTRA OF PYRIMIDINES:

The spectrum of 2-aminopyrimidine consists of a doublet

centered at 5.7 ppm (equivalent 4 & 6 hydrogens) and a triplet (5-

126

hydrogen) centered at 4.1 ppm superimposed on a broad peak (NH2).

The doublet and triplet splitting are both 48 Hz, and the relative areas

are estimated to be 2:1. The spectrum may be interpreted as

indicating the predominance of the amino form of the molecule in

DMSO solution. But the unsymmetrical imino form cannot be

completely ruled out from the NMR spectrum; however, the amino

structure is considered to be the prevalent species.

In the 1H NMR spectra, the relative deshielding of 4 protons is in

the order C2 - H > C4 - H = C6-H > C5-H as expected. Thus the C2-H

resonates at 8.78 and C5-H at 7.46 12, 13. The 2-amino substituted

pyrimidines show a doublet and triplet typical of A2X system with the

C4-H / C6-H signal broadened by coupling to the adjacent nitrogen14.

The 2-amino 4, 6-diarylpyrimidines show the C5-H proton as a singlet

around 7.2 – 7.47 and a broad signal in between 5.47 –5.80 due

to the amino protons, which disappears when the CDCl3 solution is

shaken with D2O15.

MASS SPECTRA OF PYRIMIDINES:

The mass spectra of pyrimidines are generally simple. The

dominant fragmentation mode of pyrimidines involves sequential loss

of two HCN molecules to give ionized acetylene at m/z 26, as the base

peak16. A similar sequential loss of two molecules of HCN 17, 18 was

observed in the fragmentation of 2-aminopyrimidines.

127

THERAPEUTIC POTENTIAL OF PYRIMIDINES:

A literature survey revealed that various substituted

pyrimidines are known to possess antimicrobial, anti-inflammatory,

anticancer, antiviral, antitubercular, antimalarial and other

miscellaneous activities. Given below is a brief account of various

modifications reported on pyrimidine nucleus, which showed a variety

of biological and pharmacological activities.

Antimicrobial activity:

The finding that 2,4-diaminopyrimidines inhibit the growth of

microorganisms by interfering with their utilization of folic acid led to

an intensive search for antiinfective agents in this class of heterocyclic

compounds. Trimethoprim developed as an antimalarial drug had

unique broad spectrum antimicrobial action. The pioneering work of

Hitchings19 led to the combination of trimethoprim with sulfa drug,

sulfamethoxazole constituting an important advance in the

development of clinically effective antimicrobial agents. Chemical

modification of trimethoprim led to potent antibacterial compound

tetroxoprim (56)

H3CO

H3COH2CH2CO

OCH3

CH2

N

NH2N NH2

(56)

128

Patel et al.20 synthesized some new 2-amino-4-

substitutedphenyl-6-(8-quinolinol-5-yl) pyrimidines (57) which

showed moderate to potent antimicrobial activity.

N N

NH2

R

NOH

(57)

Mishra et al.21 reported the synthesis of a series of pyrimidine-2-one

derivatives (58) which showed antimicrobial activity relative to

norfloxacin against Gram-positive and Gram-negative bacteria using

serial dilution technique.

N

N

R

O

H

R2

N

NN

R1

CH3

H

(58)

Bodke et al.22 reported the synthesis of some new benzofuro [3, 2-d]

pyrimidines (59). These compounds were screened for antibacterial

and antifungal activity.

129

O

NNH

OH

O

N

(59)

Murthy et al.23 synthesized some new chromanopyrimidines

(60). They were tested against Gram -positive bacteria B. subtillis and

B. pumilus and Gram -negative bacteria E. coli and P. vulgaris.

O

N N

OHMe

Me

NH2

R

(60)

Kudari et al. 24 reported the synthesis and antimicrobial

properties of bis- 2-thioxo-1,2,3,4,5,6-hexahydropyrimidine 4,6-diones

(61).

NN

OO

S

C CH2O O CH2C

OO

N N

OO

S

RR

(61)

130

Anticancer activity:

Some novel substituted pyrimidines (62) bearing a benzofuran

substituent were synthesized and evaluated for antitumor activity by

Babu et al 25, of which compounds with R2=SH and R1=4-MeOC6H4

showed significant antitumor activity.

O N

N

R1

R2

(62)

Fathalla et al. 26 synthesized a series of some new pyrimidine

derivatives ( 63 & 64) and evaluated them for antitumor activity. The

results indicated that some of these compounds showed antitumor

activity against liver cancer (HEPG 2) tumor cell line tested, but with

varying intensities in comparison to the known anticancer drugs, 5-

fluoro uracil and doxorubicin.

N

NS

H

R

CN

NH NH2

N

N

CN

H

S

OCH3

N

O

(63) (64)

131

Wang et al. 27 synthesized some 2-anilino-4-(1H-pyrrol-3-yl)

pyrimidines (65) inhibiting CDK2 & CDK4.

N

N

N

H

Me

NO2

NH

MeMe

Me

(65)

Fahmy et al.28 synthesized new fluorinatedthiazolo [4,5-

d]pyrimidines (66-68) which showed anticancer activity.

N

N

N

SS

Me

F

ON

F

F

N

N

N

SS

Me

F

NHF

N

N

N

SS

Me

F

Cl

(66) (67) (68)

Kaldirkyan et al.29 synthesized disubstituted 5-(3-methyl-4-

alkoxy- benzyl) pyrimidines (69), which were screened for their

anticancer activity.

R= Me, Et, Pr, Me2CH

(69)

N

N

CH2

Me

OR

OHHS

OH

132

Grigoryan et al.30 synthesized some novel 2, 5-substituted

pyrimidines (70). These compounds were tested for their antitumor

activity.

N

N

OH

OHS R

(70)

Chan et al.31 synthesized new diaminopyrimidine (71) with ω-

carboxyalkoxy or ω-carboxy-1-alkynyl substitution and these

compounds possessed significant and selective inhibition of DHFR.

N

N N

NH2

H2N

Me OMe

C

C

COOH

(71)

Antimalarial acitivity:

Vishwanadhan et al.32 reported the synthesis of some novel 5-

substituted amino-2, 4-diamino-8-chloropyrimido-[4, 5-b] quinolines

(72). These molecules were evaluated for blood schizonticidal activity

in mice infected by Plasmodium berghei. Some of these compounds

had significant curative potential when compared with chloroquine.

133

N N

N

NH

O2S

NH

R

NH2

NH2Cl

(72)

Chauhan et al.33 synthesized a series of 2, 4, 6-trisubstituted

pyrimidines ( 73) and evaluated them for their in vitro antimalarial

activity against Plasmodium falciparum. Of the 18 compounds

synthesized, 14 compounds showed MIC in the range of 0.25- 2

μg/mL and were several fold more active than pyrimethamine.

N

N

N

NH

R1

R

(73)

134

Antiviral activity:

Chern et al.34 synthesized some fused pyrimidines (74)

possessing specificity against human enteroviruses.

N

N

NN

NNPh

Ph

Ph

(74)

Sheriff et al.35 synthesized 2-(benzoxazol-2-yl-amino)-3H-4-

oxopyrimidines (75) and screened them for in vitro anti-HIV activity.

N

O

N

HN

N

H

O

H

HO

(75)

Novikov et al.36 synthesized new 1-[{2-(phenoxy) ethoxy}methyl]-

uracil derivatives (76). These compounds showed viral inhibition

properties against HIV-1.

N

N

Br

O

O Me

OO

R

R = Cl or Me

(76)

135

Zhou et al.37 synthesized some novel (Z) - and (E)-[2-fluoro-2-

(hydroxy methyl) cyclopropylidienemethyl] pyrimidines (77) which are

methylenecyclopropane analogues of nucleosides.

N

N

NN

NH2

F

HO

(77)

Anti-inflammatory activity:

Pirisino et al.38 have synthesized a new 2- phenylpyrazolo-4-

ethyl-4, 7-dihydro [1,5-a]-pyrimidine-7-one (78) and was evaluated

for anti-inflammatory activity by carrageenan-induced paw oedema

and cotton pellet-induced granuloma methods. This compound was

found to possess the activity similar to indomethacin, phenylbutazone

and isoxicam.

N

N

N

C2H5

O

(78)

Cenieola et al.39 evaluated some imidazolo [1, 2-c] pyrimidines

(79) for anti-inflammatory activity by carrageenan-induced paw

oedema method and found to show activity comparable to

indomethacin. These compounds were devoid of ulcerogenic property.

136

N

N

H

R1

CH3

R2

N

R1= Cl, OCH3, CH3

(79) R2=COOH, CH2COOH

Nargund et al.40 reported the synthesis of few substituted 2-

mercapto-3-(N-alkyl) pyrimido [5, 4-c] cinnolin-4- ones (80) and

screened for anti-inflammatory activity by carrageenan-induced rat

paw oedema method. Some of these compounds showed significant

reduction in paw oedema when compared to phenylbutazone.

NN

NN

S

R1

O

R

H

R= H, CH3

(80) R1= H, OCH3, CH3, Cl

Several pyrazolo [3, 4-d] pyrimidine derivatives were synthesized

as potential inhibitors of adenosine kinase by Cottam et al.41. One of

the compounds (81), was found to display good anti-inflammatory

activity at a dose of 30 mg/kg when evaluated in vivo in rat pleurisy

inflammation model.

137

N

N N

N

I

O

HO

OH

CH2OH

NH2

(81)

Bruni et al.42 synthesized a series of pyrazolo [1,5-a] pyrimidin-

7-one (82), which when evaluated for anti-inflammatory activity by

carrageenan-induced rat paw oedema method, significant activity was

observed, when phenyl group at 2- position was replaced by 2-theinyl

or 2-pyridinyl group. Compounds with alkyl and saturated ring

systems in the place of R at 2-position showed reduction in activity.

N

NN

R

CH3

O

(82)

Vidal et al.43 have studied the effects of some hexahydroimidazo

[1, 2-c] pyrimidine derivatives (83) on leukocyte function in vitro and

screened for anti-inflammatory activity in two models of inflammation.

The compound having the fluorine in the place of R showed significant

inhibition of paw swelling with reduced PGE2 levels in paw

homogenates.

138

O

O N

N

N

N SO2 CH3

OH

H3C

H3C

R

(83) R= H, p-Br, p- Cl, o- Cl

Bruni et al.44 reported the synthesis of some new 2, 5-

cycloamino-5H-benzopyrano[4,3-d] pyrimidines (84), which showed

anti-inflammatory activity at 100 mg/kg dose level.

O

NR2

N N

NR1

(84)

NR1= NR2= pyrrolo, piperidino, morpholino

Ferri et al.45 synthesized some 2-tosylamino and 2-

tosyliminopyrimidine derivatives (85 & 86) and studied their

interference with some leukocyte functions and 5-lipooxygenase ( 5-

LO) activity. The study demonstrated that all the compounds

inhibited cell free 5-LO activity and reduced activation of neutrophils,

which may have relevance for the modulation of the inflammatory

response.

139

N

N NTs

CH2 CONHR

(85)

N

N N Ts

CH2

H

CONHR

(86)

Ihsan et al.46 synthesized some new 1,2,4-triazolo[1,5-c]-

pyrimidines (87-89), having anti-inflammatory activity against

carrageen-induced rat paw oedema.

N N

R1

R2

N

N

Ph N N

N

N

CN

Ph

OMe

O

O

N

N

N

NN

CN

Ph

Br

(87) (88) (89)

Kandeel et al.47 synthesized some thienopyrimidines (90)

exhibiting anti- inflammatory activity.

N

N

N

NS

R2

Me

Me

R2= H, Me, SH, Ph, 2-Br C6H4,

4- Br C6H4, 4-Me C6H4

(90)

140

Carmen et al.48 synthesized pyrazolo [1, 5-a] pyrimidines (91)

possessing significant and selective COX-2 inhibitory activity.

N N

NR1

R2

R3

SO2Me

F

(91)

Miscellaneous activities:

Pandey et al.49 synthesized some novel terpenylpyrimidines (92

& 93) having antileishmanial activity.

Me Me N N

R1

NH2

Me

Me Me N N

R2

NH2

Me

(92) (93)

Joubran et al.50 synthesized new arylpropanolamines contaning

dipyrrolidinyl pyrimidines (94 & 95). These compounds showed

antioxidant activity and proved as neuroprotective agents.

NH

N

N

N

N

OH

R

NH N

N

N

N

OH

R

(94) (95)

141

Therapeutically important drugs51,52 containing pyrimidine

moiety along with their structures are given below:

DRUG ACTIVITY

N

O

I

O

HO

HO

O

NH

Idoxuridine (96)

Antiviral

N

O

CF3

O

HO

HO

O

NH

Triflouridine (97)

Antiviral

N

O

CH3

O

N3

HO

O

N

H

Zidovudine (98)

Antiviral(AIDS)

142

OHO

N

N NH2O

Zalcitabine (99)

Antiviral(AIDS)

N

N

CH3

O

O

H

OHO

Stavudine (100)

Antiviral(AIDS)

N

NO

S

OHO

NH2

Lamivudine (101)

Antiviral(AIDS)

N

N

F

H

H

O

O

5-fluorouracil (102)

Antiviral(AIDS) and

Anticancer

143

N

N

NH2

OO

HO

HOHO

Cytarabine (103)

Antiviral(AIDS)

N

NO

NH

O

HO

HO

Acitabine (104)

Antiviral(AIDS)

HCl

N

N

NN

O

N

O

Busiprone (105)

Antidepressant

CH3CH2

N

N

H

H

O

O

Primidone (106)

Anticonvulsant

144

N

NN

N N

N

CH2CH2OH

CH2CH2OH

N

N

HOH2CH2C

HOH2CH2C

Dipyridamole (107)

Vasodilator

N

N

N

NH2

H2N

O

Minoxidil (108)

Antihypertensive

CH2NCH2CH2N(CH3)2

N NCH3O

Thonzylamine (109)

Antihistaminic

145

O

NH

N

N

N

H

H

Alniditan (110)

Antihistaminic

N

N

H2N CH2

NH2OCH3

OCH3

OCH3

Trimethoprim (111)

Antiinfective

N

N

H2N

NH2

CH2CH3

Cl

Pyrimethamine (112)

Antimalarial

H2N S

O

O

NH N

N

H3CO OCH3

Sulfadoxine (113)

Antimalarial

146

N

NO

H

F

NH2

Flucytosine (114)

Antifungal

H2N S

O

O

N

N

N

CH3

CH3

H

Sulfamethazine (115)

Antibacterial

H2N S

O

O

N

N

N

H

Sulfadiazine (116)

Antibacterial

147

2.2 EXPERIMENTAL WORK

Aims and Objectives:

It could be seen from the literature that pyrimidines and their

derivatives were found to possess different biological activities. The

pyrimidines synthesized earlier in our laboratories also possessed

significant anti-inflammatory, anticancer and antimicrobial activities.

In continuation of our earlier work on pyrimidines, it is thought of

synthesizing some more new pyrimidines from the chalcones

synthesized in chapter-1, in order to consolidate the results in the

substituted pyrimidine series.

1. To synthesize and purify the 2, 4, 6-trisubstituted pyrimidines

from the chalcones obtained from 3'-methyl-4'-

hydroxyacetophenone.

2. To characterize the compounds using spectral (IR, 1H NMR and

Mass) methods and Elemental analysis. The data related to

structural characterization are given individually.

3. To screen the synthesized pyrimidines for their toxicity and

possible biological activities like anti-inflammatory,

antibacterial, antifungal and anticancer.

4. To identify the active compounds for further exploitation.

148

Materials and methods:

The same chemicals, solvents, procedures and instruments that

were mentioned in chapter-1 also used here. Guanidine hydrochloride

was obtained from the local supplier and the chalcones used were

obtained from 3'-methyl-4'-hydroxyacetophenone as described in

chapter-1.

General procedure for the synthesis of 2, 4, 6-trisubstituted

pyrimidines 53-57

The condensation of the chalcones with Guanidine

hydrochloride in an alkaline medium viz., in potassium hydroxide in

the presence of ethanol, at reflux temperatures (2 to 6 hours) resulted

in the formation of corresponding pyrimidines (Scheme 18).

Completion of the reaction was established by TLC using silica gel-G.

After completion of the reaction, the reaction mixture was poured onto

crushed ice with constant stirring. The solid that separated was

filtered, dried and purified by column chromatography on silica gel,

using a mixture of ethyl acetate and hexane as the mobile phase. The

purified pyrimidine derivatives were obtained as light to bright yellow

fine powders.

The chalcones that were used in the synthesis of pyrimidines:

1. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-pyridinyl)-2-propen-1-one (B1)



4. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-furyl)-2-propen-1-one (B4)

149

5. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-pyrrolyl)-2-propen-1-one (B5)

6. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-thienyl)-2-propen-1-one (B6)

7. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-indolyl)-2-propen-1-one (B7)

8. 1-(3'–methyl -4'-hydroxyphenyl)-3-(2''-quinolinyl)-2-propen-1-one (B8)

9. 1-(3–methyl -4'-hydroxyphenyl)-3-(9''-anthracenyl)-2-propen-1-one

(B9)

10. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-fluorophenyl)-2-propen-1-one

(B10)

11. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-chlorophenyl)-2-propen-1-one

(B11)

12. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-bromophenyl)-2-propen-1-one

(B12)

13. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-methylphenyl)-2-propen-1-

one (B13)

14. 1-(3'–methyl -4'-hydroxyphenyl)-3-(4''-methoxyphenyl)-2-propen-1-

one (B14)

15. 1-(3'–methyl -4'-hydroxyphenyl)-3-(3'', 4'', 5''- trimethoxyphenyl)-2-

propen-1- one (B15)

Chalcone Guanidine 2, 4, 6 – trisubstituted

(as hydrochloride salt) pyrimidine

Scheme 18

H3C

HO

C

O

C C Ar

HH

+ H2N C NH2

NH

KOH

NN

NH2

H3C

HO

Ar

12

3

4

56

1' 2'

3'

4'5'

6'

Ethanol

2 - 4 hours

Reflux

150

Where Ar =

N

N

N

B1 B2 B3

O

N

H

S

B4 B5 B6

N

H

N

B7 B8 B9

F

Cl

Br

B10 B11 B12

CH3

OCH3

OCH3

OCH3

OCH3

B13 B14 B15

151

Procedure:

Synthesis of 2-amino-4-(3'–methyl-4'-hydroxyphenyl)-6-(2''-

pyridinyl) pyrimidine (B1P1):

1-(3'–methyl-4'-hydroxyphenyl)-3-(2''-pyridinyl)-2-propen-1-one

(B1)(0.001 mol ) was condensed with guanidine hydrochloride (0.001

mol ) in the presence of potassium hydroxide (0.002 mol ) in absolute

ethanol (5 ml) at reflux temperature on a water bath for 6 hours. The

solvent was evaporated in vacuo and crushed ice was added to the

residue while mixing thoroughly, whereupon a bright yellow solid

separated out. This solid was filtered under vacuum, dried and

purified by column chromatography to give pure pale yellow solid.

The compound B1P1 was analyzed for molecular formula as

C16H14N4O, m.p. 1570C, well supported by a [M+H]+ ion at m/z 279 in

its positive mode electrospray ionization mass spectrum (Fig. 21). IR

(KBr disc, cm-1) spectrum (Fig. 19) showed the characteristic bands at

3361 (NH2), 3499 (O-H), 1602 (C=N) and 1573 (C=C) and 1508

(CH=CH).

The 1H NMR spectrum (400 MHz, CDCl3, Fig. 20) of compound

B1P1 showed the characteristic C-5-H of the pyrimidine around δ 8.28

as singlet and C-2-NH2 at δ 5.40 as singlet. The spectrum also

accounted for the other aromatic protons of the hetero aromatic and

phenyl rings in between δ 7.20 - 8.68. The spectrum also showed an

aromatic methyl group as a singlet at δ 2.32 integrating for three

protons.

152

The results of Elemental analysis were also in agreement

with those of the calculated values.

Based on the above spectral data and elemental analysis, the

structure of the compound B1P1 was confirmed as 2-amino-4-(3' –

methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl) pyrimidine.

By adopting the above synthetic procedure, pyrimidines of 3'-

methyl-4'-hydroxyacetophenone chalcones (chapter-1) were

synthesized, the physical and spectral characteristics of these

pyrimidines (B1P1-B15P15) were presented separately in detail.

153

The new 2, 4, 6 - trisubstituted pyrimidines thus synthesized:

1. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl)

pyrimidine (B1P1)


pyrimidine (B2P2)


pyrimidine (B3P3)

4. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-furyl)

pyrimidine (B4P4)

5. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyrrolyl)

pyrimidine (B5P5)

6. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-thienyl)

pyrimidine (B6P6)

7. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-indolyl)

pyrimidine (B7P7)

8. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-quinolinyl)

pyrimidine (B8P8)

9. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(9''-anthracenyl)

pyrimidine (B9P9)

10. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-fluorophenyl)

pyrimidine (B10P10)

154

11. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-chlorophenyl)

pyrimidine (B11P11)

12. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-bromophenyl)

pyrimidine (B12P12)

13. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-methylphenyl)

pyrimidine (B13P13)

14. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-methoxyphenyl)

pyrimidine (B14P14)

15. 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(3'',4'',5''-

trimethoxyphenyl) pyrimidine (B15P15)

155

Characterization of the new 2, 4, 6-trisubstituted pyrimidines

Table 8. Physical characterization data of 2, 4, 6-trisubstituted

pyrimidines

N N

NH2

Ar

HO

H3C

Compound

Ar

Molecular

Formula

Relative

Molecular

Mass

( RMM)

Melting

Point

( oC)

Yield

(%)

B1P1

N

C16H14 N4O

278

157

76

B2P2

N

C16H14 N4O

278

160

74

B3P3

N

C16H14 N4O

278

149

75

B4P4 O

C15H13 N3O2

267

173

70

B5P5 N

H

C15H14 N4O

266

159

72

156

B6P6 S

C15H13 N3SO

283

162

78

B7P7 N

H

C19H16 N4O

316

195

68

B8P8

N

C20H16 N4O

328

183

70

B9P9

C25H19 N3O

377

170

66

B10P10

F

C17H14 N3FO

256

260

71

B11P11

Cl

C17H14 N3ClO

311.5

145

73

B12P12

Br

C17H14 N3BrO

356

252

70

B13P13

CH3

C18H17 N3O

291

110

66

157

B14P14 OCH3

C18H17 N3O2

307

155

68

B15P15

OCH3

OCH3

OCH3

C20H21 N3O4

367

165

72

158

Table 9. Elemental analysis data of 2, 4, 6-trisubstituted

pyrimidines

Compound ( % Calculated)

C H N

( % Found)

C H N

B1P1 69.05 5.07 20.13 69.02 5.04 20.12

B2P2 69.05 5.07 20.13 69.01 5.02 20.10

B3P3 69.05 5.07 20.13 69.01 5.00 20.07

B4P4 67.40 4.90 15.70 67.00 4.87 15.20

B5P5 67.16 5.97 20.89 67.11 5.92 20.84

B6P6 63.58 4.62 14.83 63.55 4.58 14.80

B7P7 72.13 5.10 17.17 72.10 5.06 17.13

B8P8 73.15 4.91 17.06 73.14 4.86 17.02

B9P9 79.55 5.07 11.13 79.50 5.00 11.15

B10P10 69.15 5.09 14.23 69.13 5.05 14.25

B11P11 65.49 4.53 13.48 65.44 4.50 13.45

B12P12 57.32 3.96 11.80 57.30 3.92 11.75

B13P13 74.20 5.88 14.42 74.17 5.84 14.47

B14P14 70.35 5.54 13.68 70.30 5.51 13.63

B15P15 65.38 5.76 11.44 65.36 5.72 11.40

159

Table 10. IR spectral data of 2, 4, 6-trisubstituted

pyrimidines

Compound Position of absorption band ( cm-1)

B1P1 3361 (NH2), 3499 (O-H), 1602 (C=N),

1573 (C=C Quadrant of Ar), 1508 (CH=CH)

B2P2 3362 (NH2), 3497 (O-H), 1600 (C=N),

1575 (C=C Quadrant of Ar),1505 (CH=CH)

B3P3 3364 (NH2), 3496 (O-H), 1602 (C=N),


B4P4 3360 (NH2), 3498 (O-H), 1602 (C=N),

1573 (C=C Quadrant of Ar), 1504 (CH=CH), 1200 (-C-O-)

B5P5 3365 (NH2), 3499 (O-H), 1605 (C=N),


B6P6 3361 (NH2), 3497 (O-H), 1602 (C=N),

1572 (C=C Quadrant of Ar), 1504 (CH=CH), 704 (C-S)

B7P7

3365 (NH2), 3495 (O-H), 1604 (C=N),


B8P8 3361 (NH2), 3496 (O-H), 1603 (C=N),

1570 (C=C Quadrant of Ar),1503 (CH=CH)

B9P9 3363 (NH2), 3495 (O-H), 1601 (C=N),


B10P10 3361 (NH2), 3498 (O-H), 1605 (C=N),

1574 (C=C Quadrant of Ar), 1505 (CH=CH), 1120 (C-F)

160

B11P11 3360 (NH2), 3496 (O-H), 1600 (C=N),

1572 (C=C Quadrant of Ar), 1505 (CH=CH), 853 (C-Cl)

B12P12 3362 (NH2), 3499 (O-H), 1604 (C=N),

1575 (C=C Quadrant of Ar), 1506 (CH=CH), 1020 (C-Br)

B13P13 3360 (NH2), 3497 (O-H), 1603 (C=N),


B14P14 3361 (NH2), 3495 (O-H), 1602 (C=N),

1571 (C=C Quadrant of Ar), 1507 (CH=CH), 1070 (-O-CH3)

B15P15 3365 (NH2), 3498 (O-H), 1604 (C=N),

1570 (C=C Quadrant of Ar), 1504 (CH=CH), 1072 (-O-CH3)

161

Table 11. 1 H NMR spectral data of 2, 4, 6-trisubstituted pyrimidines

Compound Chemical shift ( δ ) in ppm

B1P1 8.28 (1H,s, C-5-H)

5.40 (2H,s, C-2-NH2)

7.87 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.20 (1H,d,J=8.8Hz,C-5'-H)

7.90 (1H,d,J=8.8Hz,C-6'-H)

8.57 (1H,d,J=8Hz,C-3''-H)

7.67 (1H,t,J=8Hz,C-4''-H)

7.47 (1H,m ,J=7.5Hz,C-5''-H)

8.68 (1H,d,J=7.8Hz, C-6''-H)

B2P2 7.71 (1H,s, C-5-H)

5.30 (2H,s, C-2-NH2)

7.84 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.20 (1H,d,J=8.8Hz,C-5'-H)

7.94 (1H,d,J=8.8Hz,C-6'-H)

9.19 (1H,s,C-2''-H)

8.31 (1H,d,J=8.2Hz,C-4''-H)

7.42 (1H,m ,J=8.2Hz,C-5''-H)

8.69 (1H,d,J=7.8Hz, C-6''-H)

B3P3 7.86 (1H,s, C-5-H)

5.30 (2H,s, C-2-NH2)

7.83 (1H,s,C-2'-H)

2.33 (3H,s,C-3'-CH3)

7.20 (1H,d,J=8.8Hz,C-5'-H)

7.94 (1H,d,J=8.8Hz,C-6'-H)

8.75 (1H,d, J=8.1Hz ,C-2''-H)

7.93 (1H,d,J=8.1Hz,C-3''-H)

8.09 (1H,d ,J=8.1Hz,C-5''-H)

8.61 (1H,d,J=8.2Hz, C-6''-H)

162

B4P4 8.00 (1H,s, C-5-H)

5.25 (2H,s, C-2-NH2)

7.83 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.20 (1H,d,J=8.8Hz,C-5'-H)

7.87 (1H,d,J=8.8Hz,C-6'-H)

7.00 (1H,d, J=6.8Hz ,C-3''-H)

7.41 (1H,d,J=6.7Hz,C-4''-H)

7.70 (1H,d,J=6.7Hz,C-5''-H)

B5P5 7.80 (1H,s, C-5-H)

5.30 (2H,s, C-2-NH2)

7.77 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.20 (1H,d,J=8.8Hz,C-5'-H)

8.00 (1H,d,J=8.8Hz,C-6'-H)

6.87 (1H,d, J=6.7Hz ,C-3''-H)

6.18 (1H,m,J=6.8Hz,C-4''-H)

6.69 (1H, d, J=6.7Hz,C-5''-H)

B6P6 7.40 (1H,s, C-5-H)

5.18 (2H,s, C-2-NH2)

7.82 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.20 (1H,d,J=8.8Hz,C-5'-H)

7.90 (1H,d,J=8.8Hz,C-6'-H)

7.89 (1H,d, J=6.6Hz ,C-3''-H)

7.21 (1H,m,J=6.7Hz,C-4''-H)

7.57 (1H, d, J=6.6Hz,C-5''-H)

163

B7P7

7.38 (1H,s, C-5-H)

5.44 (2H,s, C-2-NH2)

7.86 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.15 (1H,d,J=8.8Hz,C-5'-H)

7.91 (1H,d,J=8.8Hz,C-6'-H)

8.21 (1H,m,C-2''-H)

8.60 (1H,d,J=7.7Hz,C-4''-H)

7.05 (1H,t, J=7.7Hz,C-5''-H)

7.22 (1H,t, J=7.9Hz,,C-6''-H)

7.44 (1H,d, J=7.9Hz,C-7''-H)

B8P8 8.31 (1H,s, C-5-H)

5.35 (2H,s, C-2-NH2)

7.87 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.20 (1H,d,J=8.8Hz,C-5'-H)

7.90 (1H,d,J=8.8Hz,C-6'-H)

8.50 (1H,d, J=8.1Hz, C-3''-H)

8.30 (1H,d,J=8.1Hz,C-4''-H)

7.85(1H,d, J=7.9Hz,C-5''-H)

7.55 (1H,t, J=7.9Hz,C-6''-H)

7.73 (1H,t, J=7.9Hz,C-7''-H)

8.26 (1H,d,J=7.9Hz, C-8''-H)

B9P9 7.82 (1H,s, C-5-H)

5.02 (2H,s, C-2-NH2)

7.90 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.10 (1H,d,J=8.8Hz,C-5'-H)

7.91 (1H,d,J=8.8Hz,C-6'-H)

7.81 (1H,d, J=7.8Hz ,C-1''-H)

7.18 (1H,m, J=8.1Hz ,C-2''-H)

164

7.48 (1H,m, J=8.1Hz ,C-3''-H)

8.23 (1H,d,J=8.1Hz,C-4''-H)

8.23 (1H,d, J=8.1Hz,C-5''-H)

7.48 (1H,m, J=8.1Hz,C-6''-H)

7.18 (1H,m, J=7.8Hz,C-7''-H)

7.82 (1H,d,J=7.8Hz, C-8''-H)

8.70 (1H,s, C-10''-H)

B10P10 7.84 (1H,s, C-5-H)

5.45 (2H,s, C-2-NH2)

7.84 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

6.89 (1H,d,J=8.8Hz,C-5'-H)

7.94 (1H,d,J=8.8Hz,C-6'-H)

8.10 (1H,d,J=8.6Hz,C-2''-H)

7.29 (1H,d,J=8.6Hz,C-3''-H)

7.29 (1H,d, J=8.6Hz,C-5''-H)

7.99 (1H,d, J=8.6Hz,C-6''-H)

B11P11

7.81 (1H,s, C-5-H)

5.42 (2H,s, C-2-NH2)

7.84 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.15 (1H,d,J=8.8Hz,C-5'-H)

7.94 (1H,d,J=8.8Hz,C-6'-H)

7.85 (1H,d,J=8.4Hz,C-2''-H)

7.60 (1H,d,J=8.4Hz,C-3''-H)

7.60 (1H,d, J=8.4Hz,C-5''-H)

7.99 (1H,d, J=8.4Hz,C-6''-H)

165

B12P12 7.79 (1H,s, C-5-H)

5.30 (2H,s, C-2-NH2)

7.84 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.30 (1H,d,J=8.8Hz,C-5'-H)

7.94 (1H,d,J=8.8Hz,C-6'-H)

8.12 (1H,d,J=8.2Hz,C-2''-H)

7.53 (1H,d,J=8.2Hz,C-3''-H)

7.53 (1H,d, J=8.2Hz,C-5''-H)

8.12 (1H,d, J=8.2Hz,C-6''-H)

B13P13

7.72 (1H,s, C-5-H)

5.43 (2H,s, C-2-NH2)

7.84 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

6.89 (1H,d,J=8.8Hz,C-5'-H)

7.94 (1H,d,J=8.8Hz,C-6'-H)

8.05 (1H,d,J=8.1Hz,C-2''-H)

7.36 (1H,d,J=8.1Hz,C-3''-H)

2.41 (3H,s, C-4''-CH3)

7.36 (1H,d, J=8.1Hz,C-5''-H)

8.05 (1H,d, J=8.1Hz,C-6''-H)

166

B14P14

7.75 (1H,s, C-5-H)

5.45 (2H,s, C-2-NH2)

7.84 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.10 (1H,d,J=8.8Hz,C-5'-H)

7.94 (1H,d,J=8.8Hz,C-6'-H)

8.10 (1H,d,J=7.9Hz,C-2''-H)

7.05 (1H,d,J=7.9Hz,C-3''-H)

3.80 (3H,s, C-4''-OCH3)

7.05 (1H,d, J=7.9Hz,C-5''-H)

7.99 (1H,d, J=7.9Hz,C-6''-H)

B15P15

7.57 (1H,s, C-5-H)

5.25 (2H,s, C-2-NH2)

7.84 (1H,s,C-2'-H)

2.32 (3H,s,C-3'-CH3)

7.15 (1H,d,J=8.8Hz,C-5'-H)

7.94 (1H,d,J=8.8Hz,C-6'-H)

7.61 (1H,s,C-2''-H)

3.93 (3H,s,C-3''-OCH3)

3.94 (3H,s, C-4''-OCH3)

3.93 (3H,s, C-5''-OCH3)

7.61 (1H,s, C-6''-H)

167

Fig.19. IR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl) pyrimidine (B1P1)

168

Fig.20. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl) pyrimidine (B1P1)

169

Fig.21. Mass spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyridinyl) pyrimidine (B1P1)

170


171


172

Fig.24. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-furyl) pyrimidine (B4P4)

173

Fig.25. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-pyrrolyl) pyrimidine (B5P5)

174

Fig.26. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-thienyl) pyrimidine (B6P6)

175

Fig.27. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-indolyl) pyrimidine (B7P7)

176

Fig.28. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(2''-quinolinyl) pyrimidine (B8P8)

177

Fig.29. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(9''-anthracenyl) pyrimidine (B9P9)

178

Fig.30. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-fluorophenyl) pyrimidine (B10P10)

179

Fig.31. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-chlorophenyl) pyrimidine (B11P11)

180

Fig.32. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-bromophenyl) pyrimidine (B12P12)

181

Fig.33. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-methylphenyl) pyrimidine (B13P13)

182

Fig.34. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(4''-methoxyphenyl) pyrimidine (B14P14)

183

Fig.35. 1H NMR spectrum of 2-amino-4-(3' –methyl -4'-hydroxyphenyl)-6-(3'', 4’, 5’’-trimethoxyphenyl) pyrimidine (B15P15)

175

2.3 BIOLOGICAL EVALUATION

PRESENT WORK

A number of 2, 4, 6-trisubstituted pyrimidines were reported to

possess diverse biological activities like antimicrobial, antidepressant,

analgesic, anti-inflammatory, anticancer, antiviral, antileishmanial,

antitubercular, anti-HIV and antimalarial. In view of the varied

biological and pharmacological importance of pyrimidine derivatives, it

is felt worthwhile to evaluate them for possible activities. These

compounds therefore were screened for anti-inflammatory,

antibacterial, antifungal and anticancer activities.

EXPERIMENTAL METHODS

ACUTE TOXICITY

The same protocols and procedures that have been followed in

Chapter-1 are used to study acute toxicity of 2, 4, 6-trisubstituted

pyrimidines.

All the pyrimidines employed in the pharmacological screening

have been found to be free form toxicity as well as toxic symptoms

even at a high dose of 1000 mg/kg body weight and hence they are

considered safe.

2.3.1 ANTI-INFLAMMATORY ACTIVITY


Chapter-1 are used to study anti-inflammatory activity of pyrimidines

(B1P1-B15P15). The results are presented in Table 12.

176

Table12. Anti-inflammatory activity of 2, 4, 6-trisubstituted

pyrimidines

Compound

Ar

% inhibition ± SEM at

various time intervals

0.5 h 1.0h 2.0h 3.0h 4.0h 6.0h

B1P1 2''-pyridinyl 7±1 8±1 38±1 45±1 77±1 80±1

B2P2 3''-pyridinyl 7±1 8±1 37±1 47±1 76±1 79±1

B3P3 4''-pyridinyl 6±1 7±1 36±1 47±1 75±1 79±1

B4P4 2''-furyl 6±1 7±1 35±1 46±1 74±1 77±1

B5P5 2''-pyrrolyl 15±1 16±1 54±1 65±1 95±1 97±1

B6P6 2''-thienyl 8±1 10±1 40±1 51±1 81±1 82±1

B7P7 2''-indolyl 15±1 17±1 55±1 67±1 96±1 98±1

B8P8 2''-quinolinyl 8±1 9±1 40±1 49±1 78±1 81±1

B9P9 9''-anthracenyl 11±1 14±1 46±1 55±1 87±1 91±1

B10P10 4''-fluorophenyl 10±1 13±1 45±1 56±1 84±1 89±1

B11P11 4''-chlorophenyl 9±1 12±1 44±1 55±1 82±1 86±1

B12P12 4''-bromophenyl 8±1 10±1 42±1 53±1 80±1 83±1

B13P13 4''-methylphenyl 11±1 14±1 50±1 60±1 91±1 95±1

B14P14 4''-methoxyphenyl 12±1 14±1 48±1 59±1 90±1 93±1

B15P15 3'',4'',5''-

trimethoxyphenyl

14±1 15±1 52±1 63±1 94±1 96±1

Aceclofenac (standard) 21±1 23±1 56±1 67±1 96±1 99±1

All values are represented as mean±SEM (n=6). *P<0.01 compared to

reference standard Aceclofenac. Student’s t-test. Dosage: Aceclofenac-

2 mg/kg and test compounds-10 mg/kg body weight of rat.

177

DISCUSSION ON THE RESULTS:

The anti-inflammatory activity of all the 2, 4, 6-trisubstituted

pyrimidines have been evaluated by using carrageenan-induced rat

paw oedema method. The results of this activity shown in Table 12.

From the results, it is evident that a number of these

pyrimidines possessed some degree of anti-inflammatory activity,

when compared to the standard drug, aceclofenac, but not at an

identical dose level, since the compounds were tested at 10 mg/kg,

whereas the drug tested at 2 mg/kg body weight dose levels. In

particular, compound B7P7 and B5P5 possessed maximum activity

Compounds B15P15, B13P13 and B14P14 carrying the electron releasing

substituents on the aromatic ring also enhanced the activity.

This suggest that pyrimidines having a number of these

substituents at different positions of the aromatic ring can be

synthesized with a hope to get promising leads in this series of

compounds. Another interesting observation emerged out of this study

is that the anti-inflammatory activity of the pyrimidines was more

than that of chalcones from which they were obtained. All these

observations were in agreement with the reports cited in the literature.

178

2.3.2 ANTIBACTERIAL ACTIVITY

Since pyrimidines have been reported to possess antibacterial

activity and hence the new 2, 4, 6-trisubstituted pyrimidines prepared

in the present work were tested for their antibacterial activity. The

same protocols and procedures that have been followed in Chapter-1

are used to study antibacterial activity of newly synthesized

pyrimidines (B1P1-B15P15). The results are presented in Table 13.

179

Table 13. Antibacterial activity of 2, 4, 6 – trisubstituted

pyrimidines

Compound

Ar

Zone of inhibition in mm

Quantity in µg/ml

B.pumilis B.subtilis E. coli P.vulgaris

50 100 50 100 50 100 50 100

B1P1 2''-pyridinyl 04 06 05 07 05 09 09 10

B2P2 3''-pyridinyl 04 05 05 06 05 07 08 09

B3P3 4''-pyridinyl 05 06 05 07 06 08 08 10

B4P4 2''-furyl 12 14 13 17 15 17 13 15

B5P5 2''-pyrrolyl 05 08 06 09 07 11 09 12

B6P6 2''-thienyl 05 07 05 08 06 10 10 11

B7P7 2''-indolyl 09 12 10 13 13 16 10 12

B8P8 2''-quinolinyl 13 15 14 17 16 18 14 16

B9P9 9''-anthracenyl 04 05 04 05 05 06 07 08

B10P10 4''-fluorophenyl 12 15 13 16 16 17 12 15

B11P11 4''-chlorophenyl 11 14 12 16 15 16 12 14

B12P12 4''-bromophenyl 10 14 12 15 14 16 11 13

B13P13 4''-methylphenyl 06 09 08 11 09 14 09 11

B14P14 4''-methoxyphenyl 05 09 07 10 08 13 09 10

B15P15 3'',4'',5''-trimethoxyphenyl

07 10 08 12 10 15 08 12

Benzyl penicillin(standard) 15 18 16 19 18 22 17 20

Control (DMSO) - - - - - - - -

180


All the pyrimidine derivatives (B1P1-B15P15), have been evaluated

for their antibacterial activity against, Bacillus pumilis, Bacillus

subtilis (Gram-positive) and Escherichia coli, Proteus vulgaris (Gram-

negative) using cup-plate method. The results of this evaluation have

been compared by taking benzyl penicillin as reference standard.

The antibacterial activity data of pyrimidine derivatives (B1P1-

B15P15, Table 13) indicated that the compounds have some degree of

inhibitory activity on all the bacteria at both 50 µg (0.05 ml) and 100

µg (0.1ml) dose levels, when compared with the reference standard.

From the results, it is evident that the compounds B8P8, B4P4

and B10P10 exhibited significant antibacterial activity, at a

concentration of 0.1 ml dose level and is comparable to that of

standard drug, benzyl penicillin at a concentration of 100 µg/ml.

Compounds B11P11, B12P12 and B7P7 showed moderate antibacterial

activity. The results are consistent with the biological activity of

existing drugs. Further studies have to be conducted to explore the

mechanism of action of these compounds.

181

2.3.3 ANTIFUNGAL ACTIVITY


Chapter-1 are used for antifungal activity of pyrimidines (B1P1-B15P15)

and the results are given in Table 14.

182

Table 14. Antifungal activity of 2, 4, 6 – trisubstituted

pyrimidines

Compound

Ar

Zone of inhibition in mm

Quantity in µg/ml

A.niger P.crysogenium

50 100 50 100

B1P1 2''-pyridinyl 04 05 05 07

B2P2 3''-pyridinyl 04 06 05 06

B3P3 4''-pyridinyl 04 05 06 07

B4P4 2''-furyl 09 12 08 10

B5P5 2''-pyrrolyl 05 07 05 06

B6P6 2''-thienyl 05 06 05 07

B7P7 2''-indolyl 08 11 07 09

B8P8 2''-quinolinyl 10 12 09 11

B9P9 9''-anthracenyl 04 05 05 06

B10P10 4''-fluorophenyl 13 16 12 14

B11P11 4''-chlorophenyl 12 15 12 13

B12P12 4''-bromophenyl 11 14 11 12

B13P13 4''-methylphenyl 06 09 06 07

B14P14 4''-methoxyphenyl 05 08 06 07

B15P15 3'',4'',5''-trimethoxyphenyl

07 10 07 08

Fluconazole (standard) 17 21 15 18

Control (DMSO) - - - -

183


The antifungal activity of substituted pyrimidines obtained in

the present study was evaluated against A. niger and P.crysogenium

employing fluconazole as the standard drug and using cup-plate

method.

A close examination of Table 14, pertaining to the antifungal

data of pyrimidine derivatives (B1P1-B15P15) revealed that all the

compounds in this series have been found to be effective against all

fungi at both 50 µg (0.05 ml and 100 µg (0.1 ml dose levels, when

compared with reference standard fluconazole.

Among the compounds tested, compounds B10P10, B11P11 and

B12P12 were found to be more potent than other compounds. This

clearly revealed the contribution of electron withdrawing groups (like

halogens) on the aromatic ring in enhancing the antifungal activity.

Literature reports were also in agreement with these observations.

184

2.3.4 ANTICANCER ACTIVITY

A number of pyrimidines synthesized earlier in our laboratory,

when screened for anticancer activity, found to be active on only

prostate cancer cell lines and infact some of them were found to

possess significant activity. Moreover, several reports on the

usefulness of pyrimidines as anticancer agents were available in

literature.

The synthesized pyrimidines have been screened for anticancer

activity on prostate cancer cell lines (DU-145) using MTT based

cytotoxicity assay described by Mosmann58 in 1983.

Chemicals/Biochemicals used in the present study:

(a) Media: DMEM (Dulbecco’s Modified Eagles Medium)

(b) 10% Fetal bovine serum (FBS)

(c) MTTreagent:

[3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide]

(d) Cell lines: DU-145 cell lines, were obtained from the National

Centre for Cell Science ( NCCS), Pune ( India)

Procedure:

This method is based on a colorimetric assay which takes

into account the ability of a mitochondrial dehydrogenase

enzyme from viable cells to cleave the tetrazolium rings of the

pale yellow MTT and form dark blue formazan crystals which is

largely impermeable to cell membranes, thus resulting in its

accumulation within healthy cells. The level of formazan created

185

is a reflection of the number of surviving cells and shows a

proportionality relationship between them.

The required cell proliferation assay kit was

obtained from Roche Applied Sciences, Germany. The procedure

consists of seeding an equal number of DU-145 cells in each

well of a 96- well microplate and incubating at 37○C in the

presence of 5% CO2. Various concentrations of the test

substances were added to the cells. For every 24 hours the

culture medium was renewed with the test substances. 0.5%

DMSO was added into the vehicle control culture wells. After 72

hours treatment, 5 µl of MTT reagent (R&D Systems, USA) along

with 45 µl of phenol red and FBS free DMEM (Sigma Life

Science, USA) was added to each well and incubated for 4 hours

at 37○C in presence of 5% CO2. Then 50 µl of solublization

buffer (R&D Systems, USA) was added to each well to solubilize

the coloured formazan crystals produced by the reduction of

MTT. After 24 hours, the optical density was measured at 550

nm using a microplate reader (BioRad, USA). The results (mean

O.D.± SD) obtained from quadruplicate wells were used in

calculation to determine the IC50 of the test compounds.

186

The percent inhibition is then calculated from the

formula:

% inhibition = Control O.D. – Sample O.D. × 100

Control O.D.

The IC50 values of the newly synthesized pyrimidine derivatives

were shown in Table 15.

Table 15. Anticancer activity of selective 2, 4, 6 – trisubstituted

Pyrimidines on DU-145 cell lines

S.No.

Compound

IC50 for cell proliferation (50µg/ml)

1 B5P5 10.56

2 B7P7 24.79

3 B8P8 32.18

4 B10P10 185.23

5 B11P11 55.46

6 B12P12 42.17

187


The above IC50 values for pyrimidines revealed that they did not

have any significant anticancer activity against the cell lines (DU-145)

tested. Of all the compounds B10P10 showed maximum activity. The

fluorine substituent present on the phenyl ring in B10P10 contributed

favorably to the observed anticancer activity, which is consistent with

the literature reports. In fact, a number of anticancer drugs currently

used in therapy possessed one or more fluorine substituents in their

structures. Pyrimidines having number of substituents at different

positions of the phenyl ring can be synthesized as the resulting

compounds are likely to possess significant activity. A QSAR study on

a large set of compounds need to be carried out in order to arrive at

the structural requirements and contributing physico-chemical

properties for the anticancer activity of 2,4,6-trisubstituted

pyrimidines. These compounds also need to be tested on other cancer

cell lines in order to predict their activity and therapeutic usefulness.

188

2.4 REFERENCES

1. Kidwai, M., Saxena, S., Rastogi, S. and Venkataramanan, R.,

Current Med. Chem. Anti-infective agents, 2, 269 (2003).

2. Carey, F.A., in: Organic Chemistry, The McGraw- Hill companies

(7th Indian Edition), New delhi, 1164 ( 2008).

3. Thore, S.N. et al., Asian J. Chem., 19, 4429 (2007).

4. Varga, L., Nagy, T., Kovesdi, I., Benet, B. J., Dorman, G., Urge, L.

and Darvis, F., Tetrahedron, 59, 655 (2003).

5. Kenner, G.W. and Todd, A., in: Hetrocyclic compounds, Chapman

and Hall, New York, 242 (1957).

6. Phillippi, E., Hendgen, F. and Hernler, F., Monatsch. Chemie., 69,

270 (1936).

7. Whitehead, C. and Traverso, J., J.Am.Chem. Soc., 75, 671 (1953).

8. Armarego, W.L.F., in: Physical Methods in Hetrocyclic Chemistry,

Academic Press, New York, 90 (1971).

9. Vanderhaeghe, H. and Claesen, M., Bull. Sci. Chim. Belg., 66, 276

(1957).

10. Simson, M.M., J. Am. Chem. Soc., 71, 1470 (1949)

11. Baddar, F.G., Al-Hajjar, F.H and El-Rayyes, N.R., J. Het. Chem.,

13, 257 (1971).

189

12. Brown, D.J., in: Comprehensive Heterocyclic Chemistry, Pergman,

3, 57 (1984).

13. Al-Hajjar, F.H. and Sabir, S.S., J. Het. Chem., 19, 1087 (1982).

14. Reddy, G.S.I., Hobgood, R.T. and Gold Stein, J.H., J. Am. Chem.

Soc., 84, 336 (1962).

15. El-Rayyes, N. R. and Al-Hajjar, F.H., J. Het. Chem., 14, 367

(1977).

16. Rice, J.M., Dudek, G.O. and Barber, M., J. Am. Chem. Soc., 87,

4569 (1965).

17. Nishiwaki, T., Tetrahedron, 22, 3117 (1966).

18. Spitller, G., in: Physical Methods in Hetrocyclic Chemistry,

Academic Press, New York, 3, 286 (1971).

19. Hitchings, G.H., Ann. N.Y. Acad. Sci., 23, 700 (1961).

20. Patel, V.G. and Patel, J.C., Ultra Scientist of Physical Sci., 16, 111

(2004).

21. Mishra, B.K., Mishra, R. and Harinarayana Moorthy, N.S., Trends

in Applied Sciences Research, 3, 203 (2008).

22. Bodke, Y. and Sangapure, S.S., J. Indian Chem. Soc., 80, 187

(2003).

23. Murthy, Y.L.N., Rani, N., Ellaiah, P. and Bhavani Devi, R., Het.

Comm., 11, 189 (2005).

190

24. Kudari, S.M., Munera, W. and Beede, S.M., Oriental J. Chem., 12,

167 (1996).

25. Babu, V., Harinadha Kumar, P., Senthil Srinivasan, K.K. and

Bhat, G. V., Indian J. Pharm. Sci., 66, 647 (2004).

26. Fathalla, O.A., Zeid, I.F., Haiba, M.E., Soliman, A.M., Abid-

Elmoez, S.I.A. and EI-Serwy, W.S., World J. Chemistry., 4, 127

(2009).

27. Wang, S. et al., Bioorg. Med. Chem. Lett., 14, 4237 (2004).

28. Fahmy, H.T.Y., Rostom Sherif, A.F., Saudi Manal, N., Zjawiony

Jordan, K. and Robins David, J., Archiv der Pharmazie., 336,

216 (2003).

29. Kaldirkyan, M.A., Grigoryan, L.A., Geboyan, V.A., Arsenyan, F.G.,

Stepayan, G.M. and Garibdzhanyan, B.T., Pharm. Chem. J., 34,

521 (2006).

30. Grigoryan, L.A., Kaldrikyan, M.A., Melik-Ogandzhanyan, R.G.,

Arsenyan, F.G., Stepayan, G.H. and Garibdzhanyan, B.G., Pharm.

Chem. J., 33, 468 (2005).

31. Chan, D.C.M., Fu, H. F., Ronalf, A., Queener, S. F. and

Rosowsky, A., J. Med. Chem., 48, 4426 (2005).

32. Vishwanadhan, C.L., Joshi, A.A. and Sachin, S.N., Bioorg. Med.

Chem. Lett., 15, 73 (2005).

191

33. Chauhan, P.M.S., Anu, A., Srivastava, K. and Puri, S.K., Bioorg.

Med. Chem., 13, 6226 (2005).

34. Chern, J.H. et al., Bioorg. Med. Chem. Lett., 14, 2519 (2004).

35. Sheriff, A.F.R., Fahmy Hesham, T.Y. and Saudi Manal, N.S.,

Scientia Pharmaceutica, 71, 57 (2003).

36. Novikov, M.S., Ozerov, A.A., Orlova, Y.A. and Buckheit, R.W.,

Chem. Het. Compounds, 41, 625 (2005).

37. Zhou, S., Kern, E. R., Gullen, E., Yung-Chi, C., Drach, J.,

Matsumi, S., Mitsuya, H. and Zem Licka J., J. Med. Chem., 47,

6964 (2004).

38. Pirisino, R., Bainchini, F., Banchelli, J., Ignesti, G. and Ramondi,

L., Pharmacol. Res. Comm., 18, 241 (1996).

39. Cenieola, M. L., Donnoli, D., Stella, L., Paola, C.D., Constantino,

M., Anignente, E., Arena, F., Luraschi, E. and Saturnino, C.,

Pharmacol. Res., 22, 80 (1990).

40. Nargund, L.V.G., Badiger, V.V. and Yarnal, S.M., J. Pharm. Sci.,

81, 365 (1992).

41. Cottam, H.B., Wasson, D.B., Shih, H.C., Raychaudhary, A.,

Pasquale, G.D. and Carson, D.A., J. Med. Chem., 36, 3424 (1993).

42. Bruni, F., Costazo, A., Selleri, S., Guerrini, G., Fantozzi, R.,

Pirisino, R. and Brunelleschi, S., J. Pharm. Sci., 82, 480 (1993).

192

43. Vidal, A., Ferrandiz, M.L., Ubeda, A., Alarcon, A.A., Arques, J.S.

and Alcarz, M.J., J. Pharm. Pharmacol., 53, 1379 (2001).

44. Bruni, O., Brullo, C., Ranise, A., Schenone, S., Bondavalls, S.,

Barvocelli, E., Ballabeni, V., Chivarani, M., Tognolini, M. and

Jmpicciatore, M., Bioorg. Med. Chem. Lett., 11, 1397 (2001).

45. Ferri, P.F., Ubeda, A., Guillen, J., Lasri, J., Rosende, M.E.G.,

Akssir, M. and Arques, J.S., Eur. J. Med. Chem., 38, 289 (2003).

46. Ihsan, A.S. et al., J. Saudi Chem. Society, 7, 207 (2003).

47. Kandeel, M.M. and Omar, A.H., Bull. Faculty Pharmacy, 41, 43

(2003).

48. Carmen, A. et al., J. Med. Chem., 44, 350 (2001).

49. Pandey, S., Suryawanshi, S.N., Suman, G. and Srivastavam,

V.M.L., Eur. J. Med. Chem., 39, 969 (2004).

50. Joubran, L., Jackson, W. R., Compi, E. M., Robinson, A. J.,

Wells, B. A., Godfreay, P. D., Callaway, J. K. and Jaraott, B.,

Austrian J. Chem., 56, 597 (2003).

51. Burger, A., in: Burger’s Medicinal Chemistry and Drug Discovery,

John- Wiley publications Inc. 5th Edition (1995).

52. Foye, W.O., Lemke, T.L. and David, W., in: Principles of Medicinal

Chemistry, B. I. Waverly pvt. Ltd, 4th edition (1995).

193

53. Reddy, C.S. and Nagaraj, A., J. Heterocycl. Chem., 44, 1181

(2007).

54. Suryawanshi, S.N., Bhat, B.A., Susmita, P., Naveen, C. and

Suman, G., Eur. J. Med. Chem., 42, 1211 (2007).

55. Chauhan, P.M.S., Naresh, S., Agarwal, A., Sanjay babu, K.,

Nishi, N. G. and Suman, G., Bioorg. Med. Chem., 14, 7706 (2006).

56. Akbar, M., Naser, F., Golnar, K. and Neda, F., Synthesis and

Reactivity in Inorganic, Metal-Organic and Nano-Metal Chemistry,

37, 279 (2007).

57. Shujang, Tu, Fang, F., Chunbao, M., Hong, J., Youjian, F.,

Daqing, S. and Xiangshan, W., Tetrahedron Lett.,44, 6153 (2003).

58. Mosmann, T., J.Immunol Methods, 65, 55 (1983).

synthesis, characterization and …shodhganga.inflibnet.ac.in/bitstream/10603/3446/11/11_chapter...

Documents