section 4section 4 results and ...shodhganga.inflibnet.ac.in/bitstream/10603/13585/8/08...inhibitory...

92

SECTION 4SECTION 4SECTION 4SECTION 4

RESULTS AND RESULTS AND RESULTS AND RESULTS AND

DISCUSSIONDISCUSSIONDISCUSSIONDISCUSSION

93

4.1 Pyrrolo-isooxazole benzoic acid derivatives

4.1.1 Chemistry

Ring opening cyclization of p-aminobenzoic acid with maleic anhydride in presence of

tetrahydrofurane (THF) provided maleanilic acid (P1). Subsequently, refluxing of maleanilic

acid (P1) in acetic anhydride with an equimolecular amount of sodium acetate via ring closed

cyclization afforded N-arylmaleimide (P2) (Figure 42) [9, 10, 11]. Reduction of substituted

nitrobenzene with zinc in presence of water and ammonium chloride yielded N–

arylhydroxylamine (P3). Condensation of N- arylhydroxylamine (P3) with substituted

benzaldehyde (P4a-k) in presence of chloroform gave respective azomethine-N- oxides (P5a-

k, P6a-k) (Figure 43). Refluxing of substituted azomethine-N-oxides (P5a-k, P6a-k) with

N-arylmaleimide (P2) in presence of toluene and ethanol afforded products (P7a-k, P8a-k)

which on fractional crystallization from toluene provided two stereoisomers (Figure 44).

These stereoisomers were characterized as cis- and trans- isomers (a and a’), respectively.

4.1.2 Biological activity

All synthesised pyrrolo-isooxazole benzoic acid derivatives demonstrated higher

inhibitory activity against AChE than p-aminobenzoic acid (PABA) in in-vitro tests. Most of

compounds exhibited similar activity to donepezil and four of them (P7h, P7i, P8i, P8h, IC50

= 19.1 ± 1.9 -17.5 ± 1.5 nM) displayed higher inhibitory activity as compared to donepezil

(21.5 ± 3.2 nM) with test compound P8ia (IC50 = 17.5 ± 1.5 nM) being the most active one.

Furthermore, the cis-isomers displayed equipotency or slightly more potency than

corresponding trans-isomers with respect to AChE inhibition. From IC50 values of tested

compounds, it appears that in this series the electronic effects of the susbstituents in the

aromatic rings is almost negligible, and have almost no effect on the biological activities. The

compounds with methoxy substitution group (P7i and P8i) were highly potent than

94

compounds with others substituted groups like hydroxy, halogen, nitro group. Besides, the

shifting of substituted group from para- to meta- and ortho- position resulted in drop in AChE

inhibitory potency (Table 1). The compound P8ia was also evaluated for memory restoration

in scopolamine-induced amnesia in mice. Administration of scopolamine significantly

decreased day 4 ELT and TSTQ on day 5 indicating an impairment of memory as assessed on

Morris water maze as compared to normal mice. However, treatment with test compound

P8ia (5 and 10 mg/kg) along with donepezil (25 mg/kg) attenuated scopolamine-induced

decrease in day 4 ELT and TSTQ on day 5 in a significant manner (Table 2 and Figure 45).

4.1.3 Molecular docking

To disclose a possible binding mode of compound P8ia with human AChE enzyme’s

binding pockets, docking simulations were performed using the available crystallographic

structure of enzyme (PDB code 1B41) using Molegro Virtual Docker. The docking

simulation revealed that the enzyme and compound 8ia interacted through π-π aromatic

interactions and hydrogen bonding (Figure 46). One of the oxygen of terminal carboxyl

group attached to phenyl ring may form a hydrogen bond with –NH- group of Arg 296 (3.58

A°) which is in-turn is a part of Leu289, Pro290, Arg296 loop that helps in completing the

binding by creating a hydrophobic environment [234]. The nitrogen of pyrrolo ring may

interact with terminal hydroxyl group of Tyr 337 through hydrogen bond (3.53 A°) which is a

constituent of “anionic” sub-site in the gorge and is involved in optimally positioning the

ester at the acylation site along with binding to trimethylammonium choline through p-cation

interactions. The nitrogen of pyrrolo ring may also form hydrogen bond with terminal

hydroxyl group of Tyr 124 (2.71 A°), one of the five residues of peripheral anionic site which

in turn is clustered around the entrance to the active site gorge. One of the oxo moiety

attached to the pyrrolo ring may interact with terminal hydroxyl of Tyr 341 through hydrogen

bond (3.35 A°), which is again an important residue of peripheral anionic site [235]. An

95

oxygen of methoxy substituent attached to one of the phenyl ring may form a favourable

hydrogen bond with amino terminal of Asn 87 (3.41 A°) which is a part of a disulphide-

linked loop (Cys69–Cys96) (omega loop) covering an active site of AChE buried at the

bottom of a 20 A° deep gorge [236]. This loop is associated with peripheral anionic site and

forms the part of the outer wall of the gorge and it also includes Trp 86 which is a principal

component of “anionic” sub-site. The docking results also revealed the potential π-π aromatic

interactions between compound 8ia and amino acid residues of human AChE. The phenyl

ring of terminal benzoic acid may show π-π interactions with Phe 338, a part of “anionic sub-

site” along with constituents of peripheral anionic site i.e, Tyr 341 and Tyr 72. One of the

phenyl ring attached to isooxazole moiety may also show π-π interactions with ring structure

of Trp 86, the principal component of “anionic sub-site”.

4.2 Carbamate Substituted Coumarin derivatives

4.2.1 Chemistry

The synthesis of coumarin derivatives with carbamate moiety is illustrated as a

representative case in Figure 47. Benzoylation of 7-hydroxy-4-methyl coumarin (C1) with

substituted benzoyl chloride (C2a-C2h) in the presence of cold 5% NaOH solution provided

substituted 4-methyl-2-oxo-2H-chromen-7-ylbenzoate (C3a-C3h) that in turn was treated

with H2SO4 and NaN3 to afford substituted 4-methyl-2-oxo-2H-chromen-7-

ylphenylcarbamates (C4a-C4h) in a overall good yield.


All synthesized coumarin derivatives with substituted benzoate moiety (C3a-C3h)

and coumarin derivatives with substituted phenylcarbamate moiety (C4a-C4h) demonstrated

higher inhibitory activity against AChE than 7-hydroxy-4-methylcoumarin (parent

96

compound) in in-vitro tests. Three compounds (C4f, C4g, C4h with IC50 in the range of 23.1

± 1.1–18.4 ± 3.3 nM ) exhibited similar activity to donepezil and two of them (C4d, C4e with

IC50 = 13.5 ± 1.7, 14.9 ± 1.5 nM, respectively) displayed higher inhibitory activity as

compared to donepezil (21.5 ± 3.2 nM) (Table 3). Furthermore, it was demonstrated that

coumarin with substituted phenylcarbamate group (C4a-C4h) displayed more potency than

corresponding benzoate derivatives (C3a-C3h) with respect to AChE inhibition. From IC50

values of tested compounds, the potency of AChE inhibition was mainly influenced by

change of substituents in the phenylcarbamate moiety as well as by the position of substituted

groups. However, the effect of electron-donating (-OCH3) and electron-withdrawing (-NO2

and -Cl) substituents at the phenyl ring did not show regularity to the inhibition of AChE.

The compounds with nitro substitution group (C4d, C4e) were highly potent than compounds

with others substituted groups like halogen and methoxy group. Besides, the shifting of

substituted group from ortho- to meta- and para- position led to drop in AChE inhibitory

potency (Table 3). The compound C4d was also evaluated for memory restoration activity in

scopolamine-induced amnesia in mice. Administration of scopolamine significantly

decreased day 4 ELT and TSTQ on day 5 indicating an impairment of memory as assessed on

Morris water maze as compared to normal mice. However, treatment with test compound

C4d (5 and 10 mg/kg) along with donepezil (25 mg/kg) attenuated scopolamine-induced

decrease in day 4 ELT and TSTQ on day 5 in a significant manner (Table 4 and Figure 48).


To disclose a possible binding mode of compound C4d with human AChE enzyme’s

binding pockets, the docking simulation were performed using the available crystallographic


simulation revealed that the enzyme and compound C4d interacted through π-π aromatic

97

interactions and hydrogen bonding (Figure 49). Ser 203 and His 447 constitute an important

part of an active centre (catalytic/acylation site) of AChE, which is located centrosymmetric

to the sub-unit and at the bottom of 20 A° deep, narrow gorge [237]. The docking studies

revealed that oxygen of nitro group attached to phenyl ring may interact with hydroxyl group

of Ser 203 with distance of 2.47 A°. Furthermore, the phenyl ring of carbamate moiety may

interact with His 441 through π-π interactions. The oxygen of nitro group may also interact

with hydroxyl groups of Gly 121 and Gly 122 through hydrogen bonding with distance of

3.03 A° and 2.91 A°, respectively. Gly 121 and Gly 122 are the important groups of “oxy-

anionic hole” which in turn provide hydrogen bond donors to stabilize tetrahedral transition

state of substrate [238]. The oxygen of heterocyclic coumarin ring may also show hydrogen

bonding with hydroxyl group of Ser 125 of enzyme with the distance of 3.03 A°. Trp 86, Tyr

133 and Phe 338 constitute an important part of “anionic sub-site” which binds to quaternary

trimethylammonium choline moiety of substrate/inhibitor through π-cation interactions [239],

thus, its major role is in optimal positioning of ester at the acylation/catalytic site. The

compound C4d may show different interactions with these amino acid units of “anionic sub-

site”. The oxo group of carbamate moiety of compound C4d may interact with hydroxyl

group of Tyr 133 with the distance of 3.12 A°; its phenyl ring may also show π- π interaction

with Phe 338 and Tyr 133 along with its interaction with Trp 86. It also appears from the

docking results that one of the coumarin ring may also show π- π interaction with Phe 297, a

part of acyl pocket, which is responsible for substrate selectivity by preventing access of

other larger members of choline ester series.

4.3 Flavonoid Derivatives

4.3.1 Chemistry

98

The synthesis of carbamate substituted flavanone derivatives is illustrated as a

representative case in Figures 50 and 51. The base-catalysed Claisen-Schmidt condensation

reaction of 2-hydroxy acetophenone (F1) or 2-hydroxy-4,6-dimethoxyacetophenone (F1’)

with differently substituted benzaldehydes (F2a-F2g) in the presence of ethyl alcohol and

60% KOH followed by neutralisation in presence of cold acetic acid yielded differently

substituted chalcones (F3a-F3g) and (F3a’-F3g’), respectively [240]. Subsequently, the

differently substituted chalcones (F3a-F3g) and (F3a’-F3g’) underwent intra-molecular

oxidative cyclization on refluxing with glacial acetic acid to yield flavanone compounds

(F4a-F4g) and (F4a’-F4g’), respectively [241]. Thereafter refluxing of flavanone compounds

(F4a-F4g) and (F4a’-F4g’) with phenyl isocyanate in the presence of petroleum-ether and

triethylamine (2 or 3 drops) provided phenyl carbamate substituted flavanone derivatives

(F5a-F5g) and (F5a’-F5g’), respectively.


All the synthesized flavanone derivatives were screened for AChE inhibitory activity

in the rat cortex homogenate using modified Ellman method with donepezil as the standard

AChE inhibitor [242, 243]. All the compounds exhibited AChE inhibitory activity with

carbamate substituted 5,7-dimethoxy flavanone derivatives (F5b’-F5g’) being the most

potent compounds with IC50 ranging from 19.6 ± 1.8 to 9.9 ± 1.6 nM (Table 5 and Table 6).

Furthermore, the compound F5f’ was found to be the most potent AChE inhibitor with IC50

9.9 ± 1.6 nM. The replacement of –OH group of ring B of flavanone scaffold (F4a-F4g ,

F4a’-F4g’) with the phenyl carbamate moiety (F5a-F5g, F5a’-F5g’) led to dramatic increase

in AChE inhibitory activity suggesting phenyl carbamate as an important moiety that may

influence the AChE activity. The presence of two electron releasing methoxy groups at 5th

and 7th position of phenyl ring A of carbamate substituted flavanones conferred greater AChE

inhibitory activity in compound F5a’-F5g’ as compared to compound F5a-F5g without

99

dimethoxy groups at 5th and 7th positions of carbamate substituted flavanones. Furthermore,

the position of carbamate moiety linked to ring B of flavanone scaffold also influenced the

AChE inhibitory activity with higher potency for compounds with carbamate attached to

flavanone at para- position (F5e’) as compared to corresponding compounds with carbamate

moiety at meta positions (F5d’). The nature of substituents i.e., electron

releasing/withdrawing groups attached to ring B of flavanone moiety also influenced the

AChE inhibitory activity. The compound with two –OCH3 groups (electron releasing) at ring

B (F5f’, IC50 9.9 ± 1.6 nM) demonstrated higher AChE inhibition as compared to

corresponding compounds with one –OCH3 group (F5d’, IC50 16.3 ± 2.1 nM; F5e’, IC50 12.8

± 1.8 nM) and -NO2 group (electron withdrawing) (F5g’ IC50 18.2 ± 1.9 nM). The compound

(F5f’) was also evaluated for memory restoration in scopolamine-induced amnesia in mice.

Administration of scopolamine significantly decreased day 4 ELT and TSTQ on day 5

indicating an impairment of memory as assessed on Morris water maze as compared to

normal mice. However, treatment with test compound F5f’ (5 and 10 mg/kg) along with

donepezil (25 mg/kg) attenuated scopolamine-induced decrease in day 4 ELT and TSTQ on

day 5 in a significant manner (Table 7 and Figure 52).


To disclose a possible binding mode of compound 5f’ with human AChE enzyme’s

binding pockets, the docking simulation were performed using the available crystallographic


simulation revealed that the enzyme and compound 5f’ interacted through π-π aromatic

interactions and hydrogen bonding (Figure 53). The oxygen atom of methoxy group attached

to the ring B of flavonoid may interact with –NH- group of Gly 121; -NH- of Gly 122 and –

OH group of Ser 203 through hydrogen bonding with a distance of 3.46 A°, 3.15 A° and 2.77

100

A°, respectively. Gly 121 and Gly 122 are important groups of “oxy-anionic hole” which in

turn provide hydrogen bond donors to stabilize tetrahedral transition state of substrate. Ser

203 is an important constituent of catalytic site lying deep with in the molecule at the base of

an narrow 20 A° deep gorge. The oxygen of other methoxy group attached to ring B of

flavonoid scaffold may show hydrogen bonding with hydroxyl group of Tyr 337 (a

constituent of “anionic” sub-site in the gorge and involved in optimally positioning the ester

at the acylation site along with binding to trimethylammonium choline through π-cation

interactions) at the distance of 3.20 A°. The heterocyclic oxygen of ring C of flavonoid

scaffold may interact with hydroxyl group of Tyr 124 (one of the five residues of peripheral

anionic site clustered around the entrance to the active site gorge) at a distance of 2.89 A°.

The docking results also revealed the potential π-π aromatic interactions between compound

5f’ and amino acid residues of human AChE. The heteocyclic phenyl C- ring of the

compound 5f’ may show π- π interactions with Phe 338 (constitute “anionic sub-site” along

with constituents of peripheral anionic site Tyr 341 and Tyr 72). The phenyl A- ring of

flavonoid scaffold may also show π- π interactions with Phe 295 (a part of acyl pocket,

which is responsible for substrate selectivity by preventing access of other larger members of

choline ester series) and Trp 286 (one of the five residues of peripheral anionic site clustered

around the entrance to the active site gorge).

101

NH2

O OH

+

OOO

OH

O

NH

OOOH

p-amino benzoic acid Maleic anhydride 4{[3-carboxyprop-2-enoyl]amino}benzoic acid

(Maleanilic acid) (P1)

OH

O

N

O

O

4-(2,5-dioxo-2,5-dihydo-1H-pyrrol-1-yl)bezoic acid

(Maleimide) (P2)

Toluene

Stirring at room temp, 1 hour

Sodium acetate, acetic anhydride

Refluxing for one and half hour

Figure 42: Schematic diagram describing the steps in the synthesis of maleimide from p-

aminobenzoic acid.

102

NHOH

+

CHO

Y Y

N-phenylhydroxylamine substituted benzaldehyde (P4a-P4k) Diarylnitrone (azomethine N-oxide)

(P3) For X=H; P5a-P5k; X=CH3;P6a-P6k

N

X

X

Chloroform,stirring, 2-8 h

room temperature

O

Figure 43: Synthesis of diarylnitrone from nitrobenzene and substituted benzaldehydes.

NO2

X

X = H , CH3

NHOH

Zn / NH4Cl /H2O

reduction

X

N- phenylhydroxylamine(P3)Nitrobenzene

103

N

O

+

OH

O

N

O

OReflux, 7-8 h

Toulene

NO

NOO

OH O

H

H H

Y

azomethine N-oxide (P5a-P5k , P6a-P6k) Maleimide (P2) Cis isomers of substituted

pyrrolo-isoxazole benzoic

acid derivatives (P7aa-P8ka)

X

Y

X

+

NO

NOO

OH O

H

H H

Trans-isomers of substituted

pyrrolo-isoxazole benzoic acid derivatives

(P7aa'-P8ka')

Y

X

Figure 44: Schematic diagram describing the steps in the synthesis of substituted cis- and

trans- isomers from differently substituted diaryl nitrones and maleimide.

104

Table 1: Acetylcholinesterase inhibitory activity of pyrrolo-isooxazole benzoic acid

derivatives

N

O

N

O

O

OH

O

X

Y

Sr. No. Compound X Y Cis-

isomer

IC50

(nM )

Trans-

isomer

IC50 (nM )

1 PABA 35.2 ± 2.1

2 Donepezil 21.5 ± 3.2

3 P7a H H P7aa 23.9± 1.8 P7aa’ 24.4 ± 2.2

4 P7b H 2-OH P7ba 23.9± 1.7 P7ba’ 24.8 ± 2.2

5 P7c H 4-OH P7ca 22.5 ± 2.1 P7ca’ 22.7 ± 1.6

6 P7d H 2-Cl P7da 21.8 ± 1.2 P7da’ 22.4 ± 1.5

7 P7e H 3-Cl P7ea 20.1 ± 2.2 P7ea’ 21.8 ± 1.9

8 P7f H 4-Cl P7fa 19.7 ± 1.8 P7fa’ 20.9 ± 1.8

9 P7g H 2-OMe P7ga 19.2 ± 1.9 P7ga’ 20.6 ± 1.8

10 P7h H 3-OMe P7ha 19.1 ± 2.1 P7ha’ 20.5 ± 2.2

11 P7i H 4-OMe P7ia 18.8 ± 2.1 P7ia’ 19.1 ± 1.9

105

12 P7j H 2-NO2 P7ja 26.1 ± 2.4 P7ja’ 26.9 ± 2.8

13 P7k H 4-NO2 P7ka 25.8 ± 2.6 P7ka’ 26.4 ± 2.2

14 P8a CH3 H P8aa 22.8 ± 2.9 P8aa’ 22.4 ± 2.8

15 P8b CH3 2-OH P8ba 23.2 ± 2.8 P8ba’ 24.1± 2.1

16 P8c CH3 4-OH P8ca 21.9 ± 2.4 P8ca’ 22.5 ± 2.5

17 P8d CH3 2-Cl P8da 21.3 ± 2.8 P8da’ 22.1 ± 2.9

18 P8e CH3 3-Cl P8ea 22.2± 2.8 P8ea’ 23.8 ± 2.1

19 P8f CH3 4-Cl P8fa 19.2 ± 2.4 P8fa’ 20.4 ± 2.2

20 P8g CH3 2-OMe P8ga 19.1 ± 2.4 P8ga’ 20.2 ± 2.5

21 P8h CH3 3-OMe P8ha 18.8 ± 1.8 P8ha’ 19.9 ± 1.9

22 P8i CH3 4-OMe P8ia 17.5 ± 1.5 P8ia’ 18.0 ± 1.8

23 P8j CH3 2-NO2 P8ja 24.3 ± 1.9 P8ja’ 25.2 ± 2.1

24 P8k CH3 4-NO2 P8ka 23.4 ± 1.8 P8ka’ 24.1 ± 1.9

106

Table 2: Effect of different interventions on escape latency time (ELT) using Morris water

maze for memory evaluation. Values are expressed as mean ± S.E.M. for six animals. a=

p<0.05 vs day 1 ELT in normal; b= p<0.05 vs day 4 ELT in normal; c= p<0.05 vs day 4

ELT in scopolamine.

S.

No

Group Dose Day 1 ELT Day 4 ELT

1. Normal ---- 86.2 ± 5.5 37.2 ±5.2a

2. Scopolamine 0.5 mg/kg (i.p) 89.8 ± 4.8 79.3 ± 6.3b

3. Compound P8ia in

scopolamine

2 mg/kg (i.p) 83.7 ± 3.8 74.8 ± 5.6

4. Compound P8ia in

scopolamine

5 mg/kg (i.p) 85.2 ± 6.1 69.5 ± 6.9

5. Compound P8ia in

scopolamine

10 mg/kg (i.p) 81.2 ± 4.9 49.1 ± 5.7c

6. Vehicle in scopolamine 5 ml/kg (i.p.) 89.7 ± 3.8 78.7 ± 5.7

7. Donepezil 25 mg/kg (i.p.) 84.8 ± 4.1 52.6 ± 4.0c

107

0

10

20

30

40

50

60

70

Normal

Sco

Comp P8ia (2 mg/kg) in sco



Vehicle in sco

Donel in sco

Tim

e spent in target quadrant(s)

Q1 Q2 Q3 Q4

a

b

c

Figure 45: Effect of different interventions on time spent in target quadrant (TSTQ) i.e., Q4

in Morris water maze test for memory evaluation. Values are expressed as mean ± S.E.M for

six animals. Sco= scopolamine; Donel=donepezil. a= p<0.05 vs time spent in other quadrants

(Q1, Q2, Q3) in normal; b= p<0.05 vs TSTQ in normal; c= p<0.05 vs TSTQ in scopolamine

treated. The data were analysed using One way ANOVA followed by Tukey’s multiple range

test.

c

108

Figure 46: The docking view of compound P8ia with AChE (PDB code 1B41) showing five

hydrogen bond interactions (shown by broken lines) among the different amino acid residues

and structural parts of compound. The different atoms are shown in different colours i.e.,

nitrogen with blue, oxygen with red and carbon with white.

109

O O

CH3

OH

+

COCL

R

x

O

O

CH3

O O

R

7-hydroxy-4-methylcoumarin(C1)

substituted benzoylchloride(C2a-C2h) 4-methyl-2-oxo-2H-chromen-7

-yl benzoate (C3a-C3h)

a: R = H , b: 2-Cl

O

O

CH3

O O

NH

R

4-methyl-2-oxo-2H-chromen-7-yl phenylcarbamate(C4a-C4h)

c: R = 4-Cl , d : R= 2-NO2

e: R= 4-NO2 , f : R = 4-OMe

y

g: R= 3-OMe ,h: R= 2-OMe

Figure 47: Schematic diagram describing the steps in the synthesis of substituted

carbamates derivatives of coumarin from 7-hydroxy-4-methylcoumarin.

Reagents and Conditions: (x) 40 ml cold 5% NaOH solution with stirring at room temperature

for 8 hours; (y) NaN3 and H2SO4 with stirring at room temperature for 6 hours.

110

Table 3: Acetylcholinestease inhibitory activity of coumarin derivatives with carbamate

moiety

O

O

CH3

O O

R

O

O

CH3

O O

NH

R

(C3a-C3h) (C4a- C4h)

Compound R IC50 (nM)

7-hydroxy-4 methyl

coumarin

- 375 ± 7.8

Donepezil - 21.5 ± 3.2

C3a H 238 ± 4.5

C3b 2-Cl 210 ± 4.9

C3c 4-Cl 230 ± 6.8

C3d 2-NO2 102 ± 8.6

C3e 4-NO2 115 ± 5.4

C3f 4-Ome 195 ± 3.5

C3g 3-OMe 178 ± 5.9

111

C3h 2-OMe 157 ± 7.3

C4a H 35.1 ± 3.4

C4b 2-Cl 26.5 ± 2.1

C4c 4-Cl 30.3 ± 2.3

C4d 2-NO2 13.5 ± 1.7

C4e 4-NO2 14.9 ± 1.5

C4f 4-OMe 23.1 ± 1.1

C4g 3-OMe 19.3 ± 2.5

C4h 2-OMe 18.4 ± 3.3

112




ELT in scopolamine.

S.

No

Group Dose Day 1

ELT

Day 4 ELT

1. Normal ---- 86.2 ± 5.5 37.2 ±5.2a


3. Compound C4d in

scopolamine

2 mg/kg (i.p) 82.1 ± 3.1 75.2 ± 4.5

4. Compound C4d in

scopolamine

5 mg/kg (i.p) 86.1 ± 5.1 65.3 ± 5.9c

5. Compound C4d in

scopolamine

10 mg/kg (i.p) 85.1 ± 4.2 48.4 ± 4.7c



113

0

10

20

30

40

50

60

70

Normal

Sco

Comp C4f (2 mg/kg) in sco



Vehicle in sco

Donel in sco Tim

e spent in target quadrant(s) Q1 Q2 Q3 Q4

a

b

cc






test.

c

114

Figure 49: The docking view of compound C4d with AChE (PDB code 1B41) showing five

hydrogen bond interactions (shown by broken green lines) among the different amino acid

residues and structural parts of compound. The different atoms are shown in different colours

i.e., nitrogen with blue, oxygen with red and carbon with white.

115

OH

O

CH3

+

O

H

R

R

OH

O

R

R '

2-hydroxyacetophenone

(F1)

substituted benzaldehyde

(F2a- F2g)

glacial acetic acid

substituted chalcones(F3a-F3g)

O

O

R

R 'O

O

R '

COONHC6H5

compound 2a 2b 2c 2d 2e 2f 2g

R 2-OH 3-OH 4-OH 3-OH 4-OH 4-OH 4-OH

substituted flavanones (F4a-F4g )substituted flavanones with carbamate moiety (F5a-F5g)

refluxing, 72hr

EtOH, KOH

stirring at 00C, 1 day

phenylisocyanate

refluxing,15-20 min.

,

R' H H H 4-OMe 3-OMe 3 and 5-OMe 3-OMe and 5- NO2

Figure 50: Schematic diagram describing the steps in the synthesis of substituted flavanone

derivatives (F4a-F4g) and substituted flavanone derivatives with carbamate moiety (F5a-

F5g) from 2-hydroxyacetophenone (F1) and substituted benzaldehydes (F2a-F2g)

116

OH

O

CH3

O

CH3

OCH3

+

O

H

R

R '

R

R

2-hydroxy-4,6-dimethoxy acetophenone (F1')

substituted benzaldehyde

(F2a- F2g)

glacial acetic acid

O

O

O

CH3

OCH3

R

O

O

O

OCH3

CH3

R '

COONHC6H5

substituted flavanones (F4a'-F4g' )substituted flavanones with carbamate moiety (F5a'-F5g')

OH

O

O

CH3

OCH3

compound 2a 2b 2c 2d 2e 2f 2g

R 2-OH 3-OH 4-OH 3-OH 4-OH 4-OH 4-OH

EtOH , KOH

stirring at 00 C, 1 day

substituted chalcones (F3a'-F3g' )

refluxing, 72 hr

phenylisocyanate

refluxing, 15_20 min

'

R '

R' H H H 4-OMe 3-OMe 3 and 5-OMe 3-OMe and 5 - NO2

Figure 51: Schematic diagram describing the steps in the synthesis of substituted 5,7-

dimethoxyflavanone derivatives (F4a’-F4g’) and substituted 5,7-dimethoxyflavanone

derivatives with carbamate moiety (F5a’-F5g’) from 2-hydroxy-4,6-dimethoxyacetophenone

(F1’) and substituted benzaldehydes (F2a-F2g)

117

Table 5: AChE inhibitory activity of flavanone derivatives (F4a-F4g) and flavanone

derivatives with carbamate moiety (F5a-F5g).

O

O

R

R

substituted flavanones (4a-4g )

'

O

O

R '

COONHC6H5

substituted flavanones with carbamate moiety (5a-5g)

Sr.

No.

compound R R’ -COONHC6H5

( carbamate moiety)

IC50 (nM)

1 F4a 2’-OH H - 149 ± 1.2

2 F4b 3’-OH H - 147 ± 1.1

3 F4c 4’-OH H - 145 ± 1.6

4 F4d 3’-OH 4’-OMe - 140 ± 1.5

5 F4e 4’-OH 3’-OMe - 138 ± 1.6

6 F4f 4’-OH 3’,5’-di–OMe - 131 ± 1.1

7 F4g 4’-OH 3’-OMe and 5’ -NO2 - 144 ± 2.1

8 F5a - H ortho 39.9 ± 2.5

9 F5b - H meta 38.8 ± 1.8

10 F5c - H para 36.7 ± 1.9

11 F5d - 4’-OMe meta 27.6 ± 1.2

12 F5e - 3’-OMe para 21.9 ± 1.3

13 F5f - 3’,5’-di-OMe para 19.3 ± 1.8

14 F5g - 3’-OMe and 5’-NO2 para 30.5 ± 1.2

118

15 donepezil - - - 21.5 ± 3.2

119

Table 6: AChE inhibitory activity of 5,7-dimethoxyflavanone derivatives (F4a’-F4g’) and

5,7-dimethoxyflavanone derivatives with carbamate moiety (F5a’-F5g’)

O

O

O

CH3

OCH3

R

R

substituted flavanones (4a'-4g' )

'

O

O

O

OCH3

CH3

R '

COONHC6H5

substituted flavanones with carbamate moiety (5a'-5g')

Sr.

No.

Compound R R’ -COONHC6H5 IC50 (nM)

1 F4a’ 2’-OH H - 120 ± 1.7

2 F4b’ 3’-OH H - 119 ± 2.1

3 F4c’ 4’-OH H - 118 ±1.2

4 F4d’ 3’-OH 4’-OMe - 114 ± 1.1

5 F4e’ 4’-OH 3’-OMe - 112 ± 1.3

6 F4f’ 4’-OH 3,’5’-di -OMe - 107 ± 1.8

7 F4g’ 4’-OH 3’-OMe and 5’ -NO2 - 116 ± 1.2

8 F5a’ - H Ortho 21.5 ± 1.3

9 F5b’ - H Meta 19.6 ± 1.8

10 F5c’ - H Para 18.8 ± 1.6

11 F5d’ - 4’-OMe Meta 16.3 ± 2.1

12 F5e’ - 3’-OMe Para 12.8 ± 1.8

13 F5f’ - 3’,5’-di-OMe Para 9.9 ± 1.6

14 F5g’ - 3’-OMe and 5’-NO2 Para 18.2 ± 1.9

120




ELT in scopolamine.

S.

No

Group Dose Day 1 ELT Day 4 ELT

1. Normal ---- 86.2 ± 5.5 37.2 ±5.2a


3. Compound F5f' in

scopolamine

2 mg/kg (i.p) 84.3 ± 2.8 72.1 ± 3.6

4. Compound F5f' in

scopolamine

5 mg/kg (i.p) 87.5 ± 4.1 66.4 ± 5.9

5. Compound F5f' in

scopolamine

10 mg/kg (i.p) 83.1 ± 3.9 45.4 ± 3.7c



121

0

10

20

30

40

50

60

70

Normal

Sco

Comp CF5f' (2 mg/kg) in sco



Vehicle in sco

Donel in sco Tim

e spent in target quadrant(s) Q1 Q2 Q3 Q4

a

b

c






test.

c

122

Figure 53: The docking view of compound F5f’ with AChE (PDB code 1B41) showing

eleven hydrogen bond interactions (shown by broken lines) among the different amino acid

residues and structural parts of compound. The different atoms are shown in different colours

i.e., nitrogen with blue, oxygen with red and carbon with white.

section 4section 4 results and ...shodhganga.inflibnet.ac.in/bitstream/10603/13585/8/08...inhibitory...

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