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.
4.2.2 Biological activity
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).
4.2.3 Molecular docking
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
structure of enzyme (PDB code 1B41) using Molegro Virtual Docker. The docking
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.
4.3.2 Biological activity
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).
4.3.3 Molecular docking
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
structure of enzyme (PDB code 1B41) using Molegro Virtual Docker. The docking
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
Comp P8ia (5 mg/kg) in sco
Comp P8ia (10 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
Table 4: 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 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
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
113
0
10
20
30
40
50
60
70
Normal
Sco
Comp C4f (2 mg/kg) in sco
Comp C4f (5 mg/kg) in sco
Comp C4f (10 mg/kg) in sco
Vehicle in sco
Donel in sco Tim
e spent in target quadrant(s) Q1 Q2 Q3 Q4
a
b
cc
Figure 48: 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
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
Table 7: 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 0.4 mg/kg (i.p) 89.8 ± 4.8 79.3 ± 6.3b
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
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
121
0
10
20
30
40
50
60
70
Normal
Sco
Comp CF5f' (2 mg/kg) in sco
Comp CF5f' (5 mg/kg) in sco
Comp CF5f' (10 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 52: 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
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.
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