Neurobehavioral investigation and acetylcholinesterase inhibitory activity study for some new coumarin derivatives

Article abstract of DOI:10.1016/j.ejmech.2019.111651

Twenty four 6-aminocoumarin based derivatives were synthesized according to two schemes. All the compounds were screened for their acetylcholinesterase inhibitory activity where compound 5b proved to be the most potent AChE inhibitor with (IC₅₀

Full text of DOI:10.1016/j.ejmech.2019.111651

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Neurobehavioral investigation and acetylcholinesterase inhibitory activity study for
some new coumarin derivatives
Walaa R. Mahmoud, Yassin M. Nissan, Menna M. Elsawah, Rana H. Refaey, Mai F.
Ragab, Kamilia M. Amin
PII:
S0223-5234(19)30791-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111651
EJMECH 111651
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 July 2019
Revised Date: 19 August 2019
Accepted Date: 27 August 2019
Please cite this article as: W.R. Mahmoud, Y.M. Nissan, M.M. Elsawah, R.H. Refaey, M.F. Ragab, K.M.
Amin, Neurobehavioral investigation and acetylcholinesterase inhibitory activity study for some new
coumarin derivatives, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111651.
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AChE
X
AChE
AChE
AChE
X
AChE
X
AChE
5b
A42
10
8
***
IC50
37 nM
6
+++
X
+++
+++
+++
4
2
0
l
i
Z
T
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2f
4b
5b
z
e
β Amyloid
S
ma
AChE
p
r
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o
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Molecular docking of 5b in the active site of AChE

Neurobehavioral investigation and acetylcholinesterase inhibitory activity
study for some new coumarin derivatives
Walaa R. Mahmouda*, Yassin M. Nissana,b, Menna M. Elsawahc Rana H. Refaeyb, Mai F. Ragabd
,
and and Kamilia M. Amina
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Elini St.,
Cairo 11562, Egypt.
bPharmaceutical Chemistry Department, Faculty of Pharmacy, October University for Modern
Sciences and Arts (MSA), Giza, Egypt.
cDrug Research Center, Cairo, Egypt.
dPharmacology and Toxicology Department, Faculty of Pharmacy, October University for
Modern Sciences and Arts (MSA), Giza, Egypt.
*Corresponding author: Walaa R. Mahmoud, Pharmaceutical Chemistry Department, Faculty of
Pharmacy, Cairo University, Kasr Elini St., Cairo 11562, Egypt.
E-mail address: walaa.abozaid@pharma.cu.edu.eg
ABSTRACT.
Twenty four 6-aminocoumarin based derivatives were synthesized according to two schemes. All
the compounds were screened for their acetylcholinesterase inhibitory activity where compound
5b proved to be the most potent AChE inhibitor with (IC50= 37 nM) compared to tacrine and
donepezil (IC50= 55.0 and 59.0 nM, respectively). Six compounds 2f, 2g, 4b, 5b, 8b and 9b
revealed superior activity over donepezil and a conclusive structure activity relationship study
was conducted explaining the obtained results. Furthermore, compounds 2f, 4b and 5b were
investigated for their neurobehavioral effect in vivo. All the tested compounds showed
improvement of neurobehavioral experiments using donepezil as reference drug. In addition,
compounds 2f, 4b and 5b were able to reduce extracellular deposition of amyloid beta 42 in a
comparable manner to donepezil. The binding modes of the synthesized compounds were
evaluated in silico via molecular docking in the active site of AChE, as well as molecular
dynamics simulation study. A pharmacophore model was generated for the newly synthesized
compounds.
1

Keywords: acetylcholinesterase inhibitors; coumarin derivatives; Alzheimer’s disease
1.
Introduction
Alzheimer’s disease (AD) is a brain disease accompanied with severe neuronal damage in
different parts of the brain where symptoms vary according to the degree of damage to the
[1]
neurons . The disease is the leading cause of dementia[2] with estimated threefold increase in
elderly patients by 2050[3] therefore, the discovery of disease modifying therapies preventing or
5]
even slowing the disease progression is a pertinacious goal to all researchers.[4,
The
neurotransmitter acetylcholine (ACh) has a crucial role in modulating behavioral and physiologic
functions of the cholinergic neurons in the nervous system and decreased levels of ACh has a
significant role in the disease development.[6, 7] One major strategy in AD treatment is developing
acetylcholinesterase inhibitors (AChEIs) and determining their inhibitory activity against
AChE[8] accompanied with different behavioral tests on animal models to assess potential in vivo
efficacy.[9-11] Nowadays, donepezil is the gold standard for managing AD. It is a selective and
reversible AChEI that was proven to be effective in improving memory and cognition with a
lower incidence of side effects compared to other AChEIs.[12] It is capable of attenuating and
delaying the progression of the cognitive deficits observed in AD.[13] However, donepezil is not a
cost-effective treatment option, so there in an increasing demand to find other AD treatments that
could be beneficial on both treatment and cost levels.[12]
On the other hand, drugs derived from natural sources are superior to totally synthetic drugs in
many aspects of safety and efficacy[14] among which coumarins emerge as natural compounds
with variable biological activities and fortunately low toxicity.[14, 15] The natural coumarins
scopoletin I and esculetin II as well as many synthetic coumarin derivatives comprise a well-
pursued class of CNS active agents with memory ameliorating properties.[16-31] The synthetically
modified coumarin ensaculin III (KA-672 HCl) was nominated as potent AChEI with anti-
dementia activity that led to extensive focusing on coumarin compounds as possible route for
AD management[32, 33] and several attempts were made afterwards to develop other AChEIs as
[34, 35]
compounds IV and V.
Interestingly, potent AChE inhibitors were achieved via
hybridization of the 6-amino coumarin scaffold with different moieties such aryl piperazine with
different spacers from the coumarin nucleus as ensaculin III[33] and VI[34], aryl acetamide VII[36]
,
2

propyl pyrrolidine VIII[37] and halophenyl alkyl amide IX[38] all linked to position 6 via an ether
linkage or its carbon isostere (Figure 1) that encouraged the authors to mimic such substitutions
aiming to obtain potential activity. Additionally, searching the literature for other bioactive
moieties that would augment the potential activity of 6-coumarin derivatives pointed toward
imines, thiazoles and thiazolidinones either directly attached or separated via variable spacers
from the coumarin nucleus.[39-44]
Figure 1. The structure of different natural and synthetic coumarin compounds with potential
AChE inhibitory activity
Based on the previous facts and in a trial to develop new AChEIs based on coumarin scaffold, 6-
aminocoumarin was manipulated to different nitrogenous spacers linked to aromatic/aliphatic
moieties. These manipulations included: Schiff’s bases 2a-g, thiazolidinone derivatives 3b-d, 8a-
g and acetamide derivatives 4a&b, 5a&b and 9a-c (Figure 2).
3

Figure 2. Representation of the newly synthesized compounds diversity of nitrogenous spacers
The newly synthesized derivatives were evaluated for their AChE inhibitory activity and the
most active compounds were subjected to in vivo neurobehavioral assessment and tested for their
capability to decrease the deposition of extracellular beta amyloid 42. The binding mode of the
newly synthesized compounds was explored through in silico molecular modeling study.
2.
2.1. Chemistry
The target coumarin compounds 2a-g, 3b-d, 4a&b, 5a&b, 8a-g, 9a-c were synthesized
Results and Discussion
according to two schemes (1 and 2) using 6-amino coumarin 1 as a key starting compound. In
scheme 1, various Schiff’s bases were obtained via the condensation between the amino group in
compound 1 with different aromatic aldehydes to furnish target compounds 2a-g with a
1
characteristic azomethine singlet signal at 8.45-8.71 ppm in their H NMR spectra. Afterwards,
heterocyclization of the Schiff’s bases with thioglycolic acid under dry conditions afforded the
1
aryl thiazolidinones 3b-d that were featured by the CH2, CH pattern in their H NMR in the
range of 3.98-4.64 and 7.04-7.98 ppm, respectively. In the same context, a 2-acetyl moiety was
inserted as a spacer between the amino group and a terminal aryl/heterocyclic ring via the
4

reaction of 6-aminocoumarin 1 with either phenacyl bromides or the chloroacetamide derivative
of secondary amines to yield target compounds 4a&b and 5a&b with prominent aliphatic CH2
signal at 4.74, 4.65 and 3.98, 3.92 ppm, respectively (Scheme 1). In scheme 2, the 6-
chloroacetamido derivative 6 obtained by chloroacetylation of 6-aminocoumarin I was cyclized
to an active methylene containing compound 7 that was subsequently condensed with various
aromatic aldehydes to yield the corresponding arylidene compounds 8a-g with characteristic
methine protons at 7.23-7.87 ppm. Further elongation of the 6-attached side chain was achieved
via alkylation of different primary amines with the chloroacetamido derivative 6 to give
compounds 9a-c where the methine protons appeared as a singlet signal at 4.00, 4.26 and 4.26
ppm, respectively (Scheme 2).
Scheme 1: Reagents and reaction conditions; (a) aromatic aldehydes, piperidine, ethanol (b)
mercaptoacetic acid, 1,4-dioxane, ZnCl2 (c) phenacyl bromides, absolute ethanol (d)
chloroacetamides, dry acetone, anhydrous potassium carbonate and potassium iodide
5

Scheme 2: Reagents and reaction conditions; (a) chloroacetyl chloride, glacial acetic acid,
anhydrous sodium acetate (b) ammonium thiocyanate, methanol (c) aromatic aldehydes, glacial
acetic acid, anhydrous sodium acetate (d) anilines, dimethyl formamide, potassium carbonate
2.2. Biological Evaluation
2.2.1. Acetylcholine esterase (AChE) inhibition
A modified Ellman’s method[45] was used for the in vitro biological evaluation of the
AChE inhibitory activity of the twenty four synthesized compounds and the results are presented
in (Table 1). All the synthesized compounds showed activity as inhibitors for the enzyme in the
range of IC50 values (37-276.4 nM) compared to tacrine and donepezil as reference standards
with IC50 values 55 and 59 nM, respectively.
6

Table 1. AChE inhibitory activity for the twenty four synthesized compounds as IC50 values
(nM)
Compound no.
AChE IC50
(nM±SE)
Compound no.
AChE IC50
(nM±SE)
114.4 ± 8.6
135.1 ± 11.1
226.0 ± 15
172.1 ± 14.2
78.9 ± 6.7
41.0 ± 3
37.0 ± 2
2a
2b
2c
2d
2e
2f
5b
94.1 ± 7.3
8a
49.8 ± 4.8
8b
216.0 ± 14.43
150.7 ± 12.6
173.5 ± 14.6
8c
8d
8e
8f
56.9 ± 5.6
71.1± 7.2
84.0 ± 4
2g
3b
3c
3d
4a
4b
5a
276.4 ± 19.5
55.2 ± 4.4
62.0 ± 5
8g
102.0 ±
6
9b
311.0 ± 24
148.0 ± 11
55.0 ± 3.18
80.0 ± 5
9c
55.0 ± 3.18
59.0 ± 3.68
Tacrine
Donepezil
All results were found to be statistically significant at P < 0.05
The results revealed that in the aromatic imine derivatives 2a-g, the bulkier the para
substituent, the more the inhibitory activity exhibited. Consequently, the most active compounds
of this series were the dimethyl amino 2f derivative with IC50 = 41 nM while the fluoro
derivative 2c showed the least activity with IC50 = 226 nM. The rest of the compounds showed
moderate to weak activity. On the other hand, the activity of the arylthiazolidinone derivatives
3b-d was less than the imine derivatives where the methoxy derivative 3b was the most active
compound with IC50 value = 71.1 nM while the SCH3 derivatives showed three folds less activity
with IC50 value = 311 nM. A closer look on the acetamido and amino derivatives 4a&b, 5a&b
and 9a-c indicated that the unsubstituted aniline derivative 9a was the least active compound
with IC50 = 186.9 nM. Moreover, the secondary amine derivatives showed promising activity
particularly the morpholine derivative 5b that was the most active compounds among all the
synthesized compounds with IC50 = 37 nM. The replacement of anilines with a direct attached
7

phenyl group resulted in enhancement of the activity where the methoxy phenyl derivative 4b
exhibited remarkable activity with IC50 = 55 nM. Concerning the arylidenethiazolidinone
derivatives 8a-g, the methoxy derivative 8b proved to be the most active compound with IC50 =
49.8 nM. On the other hand, the replacement of the methoxy group with halide resulted in
dramatic drop in the activity as the bromo derivative 8g with IC50 = 276.4 nM. Interestingly, the
N,N-dimethyl derivative 8f showed moderate activity with IC50 = 84 nM superior to the
unsubstituted derivative 8a with IC50 = 94.1 nM. It is worth mentioning that the N,N-dimethyl
derivative 8f was the most active compound among the imine derivatives series.
2.2.2. Neurobehavioral tests
The most active compounds 2f, 4b and 5b in the previous AChE inhibition screening were
further subjected to in vivo neurobehavioral tests to explore their capability of improving
neurobehavioral response in the test animals.
2.2.2.1. Morris Water Maze (MWM):
The MWM is a water-based model employed to asses learning capacity and spatial
memory. The animals learn to escape from the water using distal clues that help them navigate
from the start position to reach a hidden submerged platform.[46, 47] The procedure was carried
out for four consecutive days, and the mean escape latency (MEL, the time taken by each mouse
to reach the hidden platform) was recorded daily for each mouse. The probe test was conducted
on day 5 where the hidden platform was removed and the animal was allowed to swim freely for
60 seconds. The time spent swimming in the target quadrant where the platform was previously
placed was recorded.[48, 49]
-Probe test: Compound 2f showed the best results indicated by the significantly long time spent
by the mice in the target quadrant compared to all the other tested compounds. In addition, it
showed a significant increase in the time spent in the target quadrant compared to the standard
compound donepezil (Figure 3).
-Day 4 MEL: Compound 2f showed the best results compared to the other tested compounds as
indicated by the least time required by the mice to reach the target quadrant in day 4, hence
providing the lowest day 4 MEL (Figure 4).
8

Probe Test
40
30
20
10
0
+++
+++
+++
+++
***
Figure 3. Effect of compounds 2f, 4b and 5b on learning and memory in mice following ICV
administration of STZ to induce cognitive impairment.
Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way
ANOVA followed by Tukey-Kramer multiple comparison test.
+++ statistically significant from the STZ injured group at p ≤ 0.001
*** statistically significant (p ≤ 0.001) versus normal group
9

Day 4 MEL
20
15
10
5
***
MEL
60
40
20
0
+++
Normal
+++
+++
STZ
Donepezil
+++
2f
4b
5b
0
Days
MEL
60
40
20
0
Normal
STZ
Donepezil
2f
4b
5b
Days
Figure 4. Effect of compounds 2f, 4b, and 5b on learning and memory in mice following ICV
administration of STZ to induce cognitive impairment by measuring MEL escape latency of the tested
compounds in days 1, 2, 3 and 4
Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way
ANOVA followed by Tukey-Kramer multiple comparison test.
+++ statistically significant from the STZ injured group at p ≤ 0.001
*** statistically significant (p ≤ 0.001) versus normal group
2.2.2.2. Novel Object Recognition Test (NOR):
Novel Object Recognition Test (NOR) relies on the fact that rodents are curious and by instinct
tend to explore novel objects thus this test is useful in evaluating recognition memory.[50-52] The
time spent by the animal exploring the novel object was recorded and used to calculate
10

exploratory preference. Significance of the test depends on calculating % exploratory preference
where greater exploratory preference indicates better memory retention.[53]
NOR test results:
Animals in the ICV STZ group showed poor memory retention indicated by a significant
decrease in the % exploratory preference compared to the control groups. The standard
compound, donepezil was able to significantly improve recognition memory shown by a
significant increase in the % exploratory preference of the animals compared to the injured ICV
STZ group. The 3 tested compounds showed great potential as they led to a significant (p ≤
0.001) increase in the % exploratory preference compared to the ICV STZ injured group which
indicates their ability to improve memory and recognition (Figure 5). Compound 4b provided
the best results indicated by the highest % exploratory preference.
Novel Object Recognition
+++
100
+++
+++
+++
80
60
*
40
20
0
Figure 5. Effect of compounds 2f, 4b and 5b on recognition and novelty in mice following ICV
administration of STZ to induce cognitive impairment.
Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way
ANOVA followed by Tukey-Kramer multiple comparison test.
+++ statistically significant from the STZ injured group at p ≤ 0.001
* statistically significant versus Normal group at p ≤ 0.05
11

2.2.2.3. Y-maze test:
The Y-maze spontaneous alteration test is used to evaluate spatial memory and learning
as it relies on the intuitive nature of rodents to explore new routes rather than familiar ones.[51, 54-
56] The spontaneous alternation was described as the successive entry into the maze arms without
repetitions on overlapping triplet sets therefore, the index for spatial memory was the percentage
spontaneous alternation.[51, 57]
Y-maze test results:
The effect of the tested compounds on spatial memory was assessed by the Y-maze test. The
ICV STZ group showed a significant reduction in the % Spontaneous alternation (p ≤ 0.001)
when compared to the control groups. On the other hand, donepezil treatment caused as
significant increase in the % spontaneous alteration compared to the injured group. Our test
compounds were able to attenuate the injury inflicted by STZ as shown by a significant
improvement in the % spontaneous alteration compared to the injured group. Compound 5b gave
the best results compared to the other test compounds indicated by a high % spontaneous
alteration.
Y maze
100
80
60
40
20
0
+++
+++
+++
+++
***
Figure 6. Effect of compounds 2f, 4b and 5b on spatial working memory and learning in mice
following ICV administration of STZ to induce cognitive impairment.
Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way
ANOVA followed by Tukey-Kramer multiple comparison test.
12

+++ statistically significant from the STZ injured group at p ≤ 0.001
*** statistically significant (p ≤ 0.001) versus normal group
2.2.3. Beta amyloid 42 deposition
Extracellular deposition of the neurotoxic insoluble Aβ42 is one of the characteristic markers of
AD.[58, 59] These amyloid plaques result from the sequential cleavage of the amyloid precursor
protein (APP) by β and γ secretases[58], which release several Aβ peptides including Aβ4.[60]
These accumulated plaques lead to synaptic damage and neuronal cell death.[61, 62] In the present
study, ICV administration of STZ caused a significant increase in the levels of the neurotoxic
Aβ42 compared to the control groups, while the cholinesterase inhibitor donepezil produced a
significant decrease in Aβ42 levels in the brain homogenates compared to the ICV STZ injured
group. The test compounds were able to significantly (p ≤ 0.001) reduce Aβ42 levels compared
to the injured group (Figure 7) and compound 5b gave the best results as it exhibited the least
concentrations of the detrimental Aβ-42.
Aβ42
10
***
8
6
+++
+++
+++
+++
4
2
0
Figure 7. Effect of compounds 2f, 4b and 5b on Aβ levels in mouse brain homogenates
following ICV administration of STZ to induce cognitive impairment.
Each result represents the mean value for 10 mice ± SE of the mean. Statistical analysis was carried out by one-way
ANOVA followed by Tukey-Kramer multiple comparison test.
13

+++ statistically significant from the STZ injured group at p ≤ 0.001
*** statistically significant (p ≤ 0.001) versus normal group
In a closer look to figures (3-7) we can understand the test results based on the effect of ICV
STZ administration that produced a significant deterioration in memory and learning indicated
by the poor performance of the mice in all the neurobehavioral tests conducted. It also led to a
significant increase in the levels of Aβ42 in the brain. These results are in agreement with other
studies that reported cognitive deficits in this SAD animal model such as memory and learning
difficulties. These deficits are mainly due to the injury STZ produces upon ICV administration
such as oxidative stress, production of inflammatory mediators[23, 63, 64], accumulation of Aβ and
hyperphosphorylated tau[61, 65] which eventually lead to neuronal cell death [66-68]. The results of
the behavioral tests conducted revealed that the test compounds 2f, 4b and 5b possess potential
symptomatic treatment of dementia of AD as they improved the memory and learning deficits
induced by the ICV injection of STZ. They improved the performance of the animals in the
MWM, NOR and Y-maze test and reduced the levels of accumulated Aβ42. In Morris Water
Maze, compound 2f showed best time for experimental mice followed by compound 5b among
the tested compounds. Compound 5b was also the best compound in Y maze test while it comes
second in novel object recognition. The fact that compound 5b was the best compound in
reducing accumulation of Aβ42 and the most active compound in AChE inhibition is
encouraging for further biological investigation bearing in mind its natural coumarin source and
efficacy over donepezil.
2.3. In silico studies:
2.3.1. Molecular docking
A molecular docking study was carried out to gain structural insight in the mechanism of action
of the inhibitors and their binding modes. The coumarin ring in all the synthesized compounds,
with the exception of compounds 2a, 2b and 5b formed π-π stacking with the indole ring of the
Trp 279 residue, similar to those reported to be formed by dual binding site inhibitors such as
donepezil and decemathonium[69], and the phenyl ring of Tyr 334 in the peripheral site of the
enzyme. Compound 5b formed the same interactions mentioned with Trp 279 and Tyr 334, but
adopted a slightly different conformation, where the phenyl group formed the π-π stacking rather
than the coumarin moiety as observed with the remaining compounds formed these interactions.
14

The conformation of the docked compounds was compared to that of the native crystal ligand,
donepezil. It was found that all the synthesized compounds adopted similar conformations to that
of donepezil, although not all of them were able to occupy both the peripheral and the catalytic
sites. Of the compounds showing the highest biological activity, only compounds 4b, 5b, 8b and
9b were able to occupy both binding sites of the enzyme (Figure 8).
Figure 8. Orientation of compounds 4b (pink), 5b (blue), 8b (orange), 9b (green) and donepezil
(dark grey) in the active site of AChE enzyme while hydrogen atoms are not shown for clarity
Furthermore, compounds 4b, 5b, 8b and 9b formed an additional π-π stacking interaction with
Trp 84 in the catalytic site of the enzyme, which is also similar to that reported to be formed by
donepezil.[69] In addition, compounds 4b and 9b were also able to form an ionic bond with Asp
72, which is a key residue in the mechanism of action of AChEIs. Asp 72 is thought to determine
the correct orientation of the ligands in the catalytic site of the enzyme.[70] Compound 4b showed
an additional hydrogen bond between the carbonyl group in the connecting chain and Tyr 121
residue. This interaction has been previously reported to be formed by novel dual acting
coumarin derivatives[71] , as well as by huprines[72] , and is thought to positively affect the
biological activity of acetylcholine esterase inhibitors (Figure 9), (Table 2).
15

(a)
(b)
(c)
(d)
Figure 9. (a-d) Docking of compounds 4b, 5b, 8b and 9b respectively, into the binding pocket
of AChE enzyme, synthesized compounds shown in purple and key interacting residues are
shown in yellow while hydrogen atoms are not shown for clarity
Table 2. The docking scores and interactions of the most active compounds
Compound Energy score
(Kcal/mol)
Estimated
Ki value
(nM)
Amino acid
Interacting
group
Length
(Å)
-9.03
408.41
Asp 72
(ionic bond)
Tyr 334
(π-π stacking)
Trp 279
Piperidine
nitrogen
Coumarin ring
3.48
4.16
3.94
3.90
4b
Coumarin ring
Benzene ring
(π-π stacking)
Trp 84
16

(π-π stacking)
Tyr 121
Carbonyl group
2.90
(hydrogen
bond)
-9.01
-9.70
425.5
Tyr 334
(π-π stacking)
Trp 279
(π-π stacking)
Trp 84
(π-π stacking)
Tyr 334
(π-π stacking)
Trp 279
(π-π stacking)
Trp 84
(π-π stacking)
Asp 72
(ionic bond)
Tyr 334
Phenyl ring
Phenyl ring
3.91
3.85
3.86
3.96
3.45
3.84
3.50
3.50
3.78
3.75
5b
8b
Coumarin ring
Coumarin ring
Coumarin ring
Benzene ring
Amide nitrogen
Coumarin ring
Coumarin ring
Benzene ring
137.73
9b
-8.42
1107.51
(π-π stacking)
Trp 279
(π-π stacking)
Trp 84
(π-π stacking)
2.3.2. Pharmacophore feature generation
Pharmagist generated 11 pharmacophore models using the compounds showing a similar
orientation to donepezil, 2a-g, 3b, 3c 4a, 5a and 5b. The highest scoring model, with a score of
44.9, had 5 spatial feature; 2 aromatic features, 2 hydrogen bond acceptors and one positive
charged feature. The aromatic features referred to the coumarin and phenyl ring, both of which
were found to be necessary for interaction and correct orientation of the molecule within the
binding pocket. The 2 hydrogen bond donors referred to the oxygen atoms of the coumarin ring,
although they were not involved in any interactions with the binding pocket, but are thought to
play a role in the correct positioning of the molecule in the hydrophilic peripheral binding site.[72]
The positively charged feature referred to the positively charged nitrogen in the chain connecting
the coumarin and phenyl moieties, which should interact through an ionic bond with Asp72
17

residue. This feature was slightly misaligned in the pharmacophore model, this is in agreement
with the docking results in which showed that only compounds 4b and 9b were able to form an
ionic bond with Asp 72 (Figure 10).
Figure 10. Pharmacophore model generated by alignment of the 11 compounds, showing 5
feature, 2 aromatic features shown in blue, 2 hydrogen bond donor features shown in pink and on
ionic feature shown in green. Pharmacohore model created using PharmaGist webserver and
visualized using Accelrys.
2.3.3. Molecular dynamics simulation
Molecular dynamics simulations were carried out using the binding pose of compound 5b, to
confirm the molecular docking results. The root-mean square deviation (RMSD) of both the
ligand and the choline esterase are shown in figure 11, the fluctuations for both the ligand and
the protein deviated from the average by 1 . This confirms the stability of the ligand in the
binding pocket of the enzyme. Additionally, visual inspection of the trajectory of the binding
pose confirmed the stability of the ligand in the binding pocket.
18

Figure 11. RMSD of the acetylcholine esterase enzyme (shown in black) and the ligand (shown
in red)
3. Conclusion
Twenty four 6-aminocoumarin based derivatives were synthesized and screened for their in vitro
AChE inhibitory activity. Among the tested compounds, the best IC50 value was for compound
5b with (IC50= 37 nM) compared to donepezil and tacrine as reference drugs (IC50= 55.0 and
59.0 µM, respectively). The study led to conclusions regarding the structure activity relationship
for the synthesized compounds where the substitution on the amino group of 6-aminocoumarin
with acetophenones or acetamido group in aliphatic heterocycle or arylacetamide was in general
better for the activity for the para substituted derivatives. In the same vein, arylimine
substitutions gave less activity than all other substitutions with the exception of the N,N dimethyl
19

amino and the bromo para substitutions. Finally, Arylidine thiazolidinone derivatives were better
than the 4-phenyl-thiazolidinone analogues with superior activity to the methoxy substitution.
Furthermore, in vivo neurobehavioral tests were conducted for the most active compounds 2f, 4b
and 5b where they caused better neurobehavioral response in the tested animals especially
compounds 2f and 5b. Compound 5b also decreased accumulation of beta amyloid Aβ42 in a
comparable manner to donepezil. In silico molecular docking revealed the binding mode of the
most active compounds with good interactions with the active site of AChE enzyme. This was
further confirmed through the use of molecular dynamics simulations, which confirmed the
stability of the docked pose within the active site of the enzyme. Interestingly, compound 5b
may be also given at lower doses than the standard compound donepezil which might lead to
fewer side effects and better tolerability taking in consideration its natural coumarin base.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were measured on a Stuart SMP10 digital apparatus and the values were
uncorrected. IR spectra were recorded on Shimadzu FT-IR 8400S infrared spectrophotometer at
the Microanalytical Unit-Faculty of Pharmacy-Cairo University. Mass spectra are expressed as
m/z (Intensity %). Both 1H and 13C NMR spectra were recorded in deuterated
dimethylsulfoxide (DMSO-d6) on a Bruker Ascend 400/ R spectrometer at 400 and 100 MHz,
respectively at the Microanalytical Unit, Faculty of Pharmacy, Cairo University or at the
chemical warfare department, Ministry of Defense, Egypt. Chemical shifts are expressed in
δ
values (ppm) and coupling constant (J) values are given in hertz and the abbreviations used for
the presented signals are: s, singlet; d, doublet; m, multiplet. Elemental analyses were computed
at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt. Reaction
were routinely monitored by Thin Layer Chromatography (TLC) on silica gel precoated F254
Merck plates. Compounds 1[73], 2a[73], 2d[74], 2e[74], 2f[75] and 6[76] were prepared as reported in
the literature.
4.1.2. General procedure for the preparation of compounds 2a-g
20

An equimolar mixture of 6-amino-2H-chromen-2-one 1 (1.61 gm, 10 mmol.) and
different aromatic aldehydes (10 mmol.) dissolved in absolute ethanol (50 mL) containing few
drops of piperidine was refluxed for 4-8 h. After cooling to room temperature, the solution was
left to stand for 12 h and the precipitated solid was filtered off and recrystallized from ethanol to
give the target compounds 2a-g.
4.1.2.1. 6-(Benzylideneamino)-2H-chromen-2-one (2a) [73]
Yellowish white powder, yield: 63 %,
.
m.p. 150-152 °C, IR (cm-1, KBr): 3082 (CH aromatic), 2985 (CH aliphatic), 1701 (C=O), 1624
1
(C=N). H NMR (DMSO-d6, 400 MHz) δ, ppm: 6.52 (d, 1H, coumarin H-3,
J
= 9.6), 7.43 (d,
= 8.8), 7.50-7.63 (m, 4H, aromatic H + coumarin H-5), 7.93 (dd, 1H,
coumarin H-7, Ja = 8.0, Jb = 2.0), 8.06 (d, 3H, aromatic H + coumarin H-4, = 9.6), 8.68 (s, 1H,
1H, coumarin H-8,
J
J
CH=N). Anal. Calcd. for C16H11NO2 (249.26): C, 77.10; H, 4.45; N, 5.62 %. Found: C, 76.87; H,
4.61; N, 5.90 %.
4.1.2.2. 6-[(4-Methoxybenzylidene)amino]-2H-chromen-2-one (2b). Reddish brown powder,
yield: 65 %, m.p. 157-159 °C, IR (cm-1, KBr): 3047 (CH aromatic), 2962 (CH aliphatic), 1701
1
(C=O), 1624 (C=N). H NMR (DMSO-d6, 400 MHz) δ, ppm: 3.86 (s, 3H, OCH3), 6.53 (d, 1H,
coumarin H-3,
(dd, 1H, coumarin H-7, Ja = 8.7, Jb = 2.4), 7.61 (d, 1H, coumarin H-5,
H, = 8.6), 8.08 (d, 1H, coumarin H-4, = 9.6), 8.61 (s, 1H, CH=N). Anal. Calcd. for
J
= 9.5), 7.09 (d, 2H, Ar-H,
J
= 8.7), 7.43 (d, 1H, coumarin H-8,
J = 8.7), 7.53
J
= 2.3), 7.90 (d, 2H, Ar-
J
J
C17H13NO3 (279.29): C, 73.11; H, 4.69; N, 5.02 %. Found: C, 73.29; H, 4.88; N, 5.18 %.
4.1.2.3. 6-[(4-Flurobenzylidene)amino]-2H-chromen-2-one (2c). Yellowish brown powder,
yield: 67 %, m.p. 180-182 °C, IR (cm-1, KBr): 3039 (CH aromatic), 2978 (CH aliphatic), 1701
1
(C=O), 1627 (C=N). H NMR (DMSO-d6, 400 MHz) δ, ppm: 6.52 (d, 1H, coumarin H-3,
J =
9.6), 7.43 (d, 1H, coumarin H-8,
(d, 1H, H-5 coumarin, = 2.4 Hz), 7.93 (dd, 1H, coumarin H-7, Ja = 7.6, Jb = 2.4), 8.06 (d, 2H,
aromatic H, = 10 Hz), 8.68 (s, 1H, CH=N). Anal. Calcd. for C16H10FNO2 (267.25): C, 71.91;
J = 8.8), 7.50-7.56 (m, 3H, aromatic H + coumarin H-4), 7.63
J
J
H, 3.77; N, 5.24 %. Found: C, 72.03; H, 3.84; N, 5.45 %.
4.1.2.4. 6-[(4-(Methylthio)benzylidene)amino]-2H-chromen-2-one (2d).[74] Brownish orange
crystals, yield: 61 %, m.p. 155-157 °C, IR (cm-1, KBr): 3039 (CH aromatic), 2916 (CH aliphatic)
1
1708 (C=O), 1624 (C=N). H NMR (DMSO-d6, 400 MHz) δ, ppm: 2.52 (s, 3H, SCH3), 6.52 (d,
21

1H, coumarin H-3,
8.8), 7.54 (dd, 1H, coumarin H-7, Ja = 8.8, Jb = 2.8), 7.61 (d, 1H, coumarin H-5,
(d, 2H, aromatic H, = 8.4), 8.05 (d, 1H, coumarin H-4,
= 9.6), 8.63 (s, 1H, CH=N). 13C NMR
J
= 9.6), 7.37 (d, 2H, aromatic H,
J
= 8.4), 7.42 (d, 1H, coumarin H-8,
J =
J
= 2.4), 7.85
J
J
(DMSO-d6) δ: 14.5, 110.6, 117.5, 119.6, 120.4, 125.8, 129.5, 132.7, 143.7, 144.8, 148.1, 152.1,
160.4, 161.1. Anal. Calcd. for C17H13NO2S (295.36): C, 69.13; H, 4.44; N, 4.74 %. Found: C,
69.40; H, 4.61; N, 4.98 %.
4.1.2.5. 6-[(4-Chlorobenzylidene)amino]-2H-chromen-2-one (2e).[74] Buff powder, yield: 69 %,
m.p. 190-192 °C, IR (cm-1, KBr): 3078 (CH aromatic), 2951 (CH aliphatic), 1716 (C=O), 1627
(C=N). 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 6.52 (d, 1H, coumarin H-3,
aromatic H, = 8.8), 7.56 (dd, 1H, coumarin H-7, Ja = 8.8, Jb = 2.4), 7.58-7.64 (m, 2H, coumarin
H-5, H-8), 7.94 (d, 2H, aromatic H, = 9.2), 8.06 (d, 1H, coumarin H-4, = 9.2), 8.70 (s, 1H,
J = 10), 7.44 (d, 2H,
J
J
J
CH=N). Anal. Calcd. for C16H10ClNO2 (283.71): C, 67.74; H, 3.55; N, 4.94 %. Found: C, 67.96;
H, 3.49; N, 5.17 %.
4.1.2.6. 6-[(4-(Dimethylamino)benzylidene)amino]-2H-chromen-2-one (2f).[75] Brown powder,
yield: 68 %, m.p. 196-198 °C, IR (cm-1, KBr): 3043 (CH aromatic), 2869 (CH aliphatic), 1701
1
(C=O), 1624 (C=N). H NMR (DMSO-d6, 400 MHz) δ, ppm: 3.30 (s, 6H, 2CH3), 6.49 (d, 1H,
coumarin H-3,
7.46 (dd, 1H, coumarin H-7, Ja = 8.8, Jb = 2.8), 7.53 (d, 1H, coumarin H-5,
aromatic H, = 8.8), 8.04 (d, 1H, coumarin H-4,
= 9.2), 8.45 (s, 1H, CH=N). 13C NMR
J
= 9.6), 6.78 (d, 2H, aromatic H,
J
= 9.2), 7.38 (d, 1H, coumarin H-8,
J = 8.8),
J
= 2), 7.74 (d, 2H,
J
J
(DMSO-d6) δ: 42.0, 111.9, 119.9, 125.6, 129.3, 130.8, 132.1, 136.2, 144.7, 149.1, 151.5, 152.9,
160.5, 161.9. Anal. Calcd. for C18H16N2O2 (292.33): C, 73.95; H, 5.52; N, 9.58 %. Found: C,
74.09; H, 5.48; N, 9.82 %.
4.1.2.7. 6-[(4-Bromobenzylidene)amino]-2H-chromen-2-one (2g). Light yellow powder, yield:
64 %, m.p. 190-192 °C, IR (cm-1, KBr): 3078 (CH aromatic), 2889 (CH aliphatic), 1716 (C=O),
1627 (C=N). 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 6.55 (d, 1H, coumarin H-3,
(d, 1H, coumarin H-8, = 8.7), 7.59 (dd, 1H, coumarin H-7, Ja = 8.7, Jb = 2.5), 7.67 (d, 1H,
coumarin H-5, = 2.4), 7.76 (d, 2H, aromatic H, = 8.4), 7.90 (d, 2H, aromatic H, = 8.4), 8.08
(d, 1H, coumarin H-4, = 9.6), 8.71 (s, 1H, CH=N). Anal. Calcd. for C16H10BrNO2 (328.16): C,
J = 9.5), 7.47
J
J
J
J
J
58.56; H, 3.07; N, 4.27 %. Found: C, 58.75; H, 3.23; N, 4.05 %.
4.1.3. General procedure for the preparation of compounds 3b-d
22

A mixture of compound 2b-d (10 mmol), thioglycolic acid (1.05 mL, 15 mmol) and a few
crystals of anhydrous zinc chloride in dry 1,4-dioxane (50 mL) was refluxed for 12–14 h and
monitored by TLC. The reaction mixture was cooled and neutralized with 10 % sodium
bicarbonate solution till complete precipitation of the product. The separated solid was filtered,
washed with water and recrystallized from ethanol.
4.1.3.1. 2-(4-Methoxyphenyl)-3-(2-oxo-2H-chromen-6-yl)thiazolidin-4-one (3b).White powder,
yield: 30 %, m.p. 250-252 °C, IR (cm-1, KBr): 3074 (CH aromatic), 2924 (CH aliphatic), 1759,
1681 (2 C=O). 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 3.85 (s, 3H, OCH3), 4.15 (d, 2H, CH2),
6.58 (d, 1H, coumarin H-3,
8, = 8.8), 7.67 (d, 2H, aromatic H,
7.85 (d, 1H, coumarin H-5, = 2.3), 7.98 (s, 1H, CH), 8.11 (d, 1H, coumarin H-4,
J
= 9.6), 7.15 (d, 2H, aromatic H,
= 8.8), 7.71 (dd, 1H, coumarin H-7, Ja = 8.8, Jb = 2.4),
= 9.6). Anal.
J = 8.8), 7.58 (d, 1H, coumarin H-
J
J
J
J
Calcd. for C19H15NO4S (353.39): C, 64.58; H, 4.28; N, 3.96 %. Found: C, 64.80; H, 4.49; N, 4.28
%.
4.1.3.2. 2-(4-Fluorophenyl)-3-(2-oxo-2H-chromen-6-yl)thiazolidin-4-one (3c)
. Light brown
powder, yield: 34 %, m.p. 266-268 °C, IR (cm-1, KBr): 3040 (CH aromatic), 2960 (CH
aliphatic), 1724, 1685 (2 C=O). 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 3.98 (d, 2H, CH2), 6.36
(d, 1H, coumarin H-3,
8.4), 7.04 (dd, 1H, coumarin H-7, Ja = 8.8, Jb = 2.4), 7.14-7.24 (m, 4H, aromatic H+ coumarin
H-5+ CH), 7.87 (d, 1H, coumarin H-4, = 10). Anal. Calcd. for C18H12FNO3S (341.36): C,
J = 9.6), 6.80 (d, 1H, coumarin H-8, J = 8.8), 6.95 (d, 2H, aromatic H, J =
J
63.33; H, 3.54; N, 4.10 %. Found: C, 63.57; H, 3.72; N, 4.39 %.
4.1.3.3. 2-[4-(Methylthio)phenyl]-3-(2-oxo-2H-chromen-6-yl)thiazolidin-4-one (3d). Brown
powder, yield: 31 %, m.p. 245-247 °C, IR (cm-1, KBr): 3066 (CH aromatic), 2951 (CH
1
aliphatic), 1724, 1708 (2 C=O). H NMR (DMSO-d6, 400 MHz) δ, ppm: 2.48 (s, 3H, SCH3),
4.64 (s, 2H, CH2), 6.36 (d, 1H, coumarin H-3,
7.08 (m, 4H, aromatic H + coumarin H-7 + CH), 7.15 (d, 1H, coumarin H-8,
2H, aromatic H, = 9.6), 8.04 (d, 1H, coumarin H-4, = 8.8). Anal. Calcd. for C19H15NO3S2
J
= 10), 6.83 (d, 1H, H-5 coumarin,
J = 2.4), 7.04-
J
= 8.8), 7.86 (d,
J
J
(369.46): C, 61.77; H, 4.09; N, 3.79 %. Found: C, 62.01; H, 4.21; N, 3.94 %.
4.1.4. General procedure for the preparation of compounds 4a&b
23

A mixture of the key starting material 1, and (10 mmol.) of either phenacyl bromide or p-
methoxy phenacyl bromide in (50 mL) of absolute ethanol was heated for 4 h under reflux. The
formed precipitate was filtered, and recrystallizes from methanol.
4.1.4.1. 6-[(2-Oxo-2-phenylethyl)amino]-2H-chromen-2-one (4a). Yellow powder, yield: 90 %,
m.p. 210-212 °C, IR (cm-1, KBr): 3380 (NH), 3064 (CH aromatic), 2826 (CH aliphatic), 1716,
1
1680 (2 C=O). H NMR (DMSO-d6, 400 MHz) δ, ppm: 4.74 (s, 2H, CH2), 6.13 (t, 1H, NH
exchanged with D2O), 6.38 (d, 1H, coumarin H-5,
J
= 9.5), 6.86 (d, 1H, coumarin H-5,
= 8.9), 7.58 (t, 2H,
= 6.2), 7.89 (d, 1H, coumarin H-4, = 9.4), 8.08
J = 2.7),
7.08 (dd, 1H, coumarin H-7, Ja = 8.9, Jb = 2.7), 7.19 (d, 1H, coumarin H-8,
J
aromatic H,
J
= 7.6), 7.69 (t, 1H, aromatic H,
J
J
13
(t, 2H, aromatic H,
J
= 7.1). C NMR (DMSO-d6) δ: 50.6, 108.6, 116.4, 117.0, 118.7, 119.5,
128.3, 129.2, 134.0, 135.5, 144.8, 145.6, 146.0, 160.9, 196.8. Anal. Calcd. for C17H13NO3
(279.29): C, 73.11; H, 4.69; N, 5.02 %. Found: C, 73.47; H, 4.58; N, 5.29 %.
4.1.4.2. 6-[(2-(4-Methoxyphenyl)-2-oxo-ethyl]amino)-2H-chromen-2-one (4b). Yellow powder,
yield: 88 %, m.p. 226-228°C, IR (cm-1, KBr): 3367 (NH), 3078 (CH aromatic), 2978 (CH
1
aliphatic), 1718, 1670 (2 C=O). H NMR (DMSO-d6, 400 MHz) δ, ppm: 3.82 (s, 3H, OCH3),
4.64 (s, 2H, CH2), 6.36 (d, 1H, H-3 coumarin, J = 10), 6.83 (d, 1H, coumarin H-5, J = 2.4), 7.04-
7.08 (m, 3H, coumarin H-7 + coumarin H-8 + NH exchanged with D2O), 7.15 (d, 2H, aromatic
13
H,
J
= 8.8), 7.86 (d, 1H, coumarin H-4,
J
= 9.6), 8.04 (d, 2H, aromatic H,
J
= 8.8). C NMR
(DMSO-d6) δ: 50.2, 56.0, 108.6, 114.4, 116.4, 117.0, 118.7, 119.5, 128.4, 130.6, 144.8, 145.6,
146.0, 160.9, 163.9, 195.0. Anal. Calcd. for C18H15NO4 (309.32): C, 69.89; H, 4.89; N, 4.53 %.
Found: C, 70.12; H, 5.01; N, 4.78 %.
4.1.5. General procedure for the preparation of compounds 5a&b
6-Amino-2H-chromen-2-one 1 (1.61 gm, 10 mmol.) and the corresponding
chloroacetamide (10 mmol.) were dissolved in dry acetone (30 mL) containing (2 gm, 15 mmol.)
of potassium carbonate and traces of potassium iodide and heated under reflux for 12-20 h. The
precipitated product was filtered, washed with water and recrystallized with ethanol.
4.1.5.1. 6-[(2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl)amino]-2H-chromen-2-one (5a). Florescent
yellow powder, yield: 57 %, m.p. 284-286 °C, IR (cm-1, KBr): 3356 (NH), 3059 (CH aromatic),
1
2974 (CH aliphatic), 1708, 1654 (2 C=O). H NMR (DMSO-d6, 400 MHz) δ, ppm: 3.63 (s, 8H,
24

piprazine H), 3.98 (d, 2H, CH2), 5.85 (t, 1H, NH exchanged with D2O), 6.36 (d, 1H, H-3
coumarin, = 9.6), 6.78-6.81 (m, 2H, coumarin H-8 + aromatic H), 6.95 (d, 2H, aromatic H,
8.4), 7.04 (dd, 1H, coumarin H-7, Ja = 8.8, Jb = 2.4), 7.15 (d, 1H, coumarin H-5, = 9.2), 7.22
(t, 2H, aromatic H, = 8), 7.87 (d, 1H, coumarin H-4, = 10). Anal. Calcd. for C21H21N3O3
(363.41): C, 69.41; H, 5.82; N, 11.56 %. Found: C, 69.70; H, 5.97; N, 11.82 %.
J
J =
J
J
J
4.1.5.2. 6-[(2-Morpholino-2-oxoethyl) amino]-2H-chromen-2-one (5b). Yellow powder, yield:
51 %, m.p. 230-232 °C, IR (cm-1, KBr): 3390 (NH), 3050 (CH aromatic), 2989 (CH aliphatic),
1728, 1651 (2 C=O). 1H NMR (DMSO-d6, 400 MHz) δ, ppm: 3.46-3.60 (m, 8H, morpholine H),
3.92 (d, 2H, CH2,
H-3, = 9.6), 6.78 (d, 1H, coumarin H-5,
2.8), 7.14 (d, 1H, coumarin H-8, = 8.8), 7.86 (d, 1H, coumarin H-4,
J
= 5.2), 5.83 (t, 1H, NH exchanged with D2O,
= 2.8), 7.02 (dd, 1H, coumarin H-7, Ja = 8.8, Jb
= 10). 13C NMR (DMSO-
J = 5), 6.36 (d, 1H, coumarin
J
J
=
J
J
d6) δ: 42.2, 45.2, 66.5, 108.5, 116.4, 117.0, 118.6, 119.5, 144.8, 145.6, 145.9, 160.9, 168.2. Anal.
Calcd. for C15H16N2O4 (288.30): C, 62.49; H, 5.59; N, 9.72 %. Found: C, 62.75; H, 5.73; N, 9.98
%.
4.1.6. General procedure for the preparation of compound 7
A mixture of compound 6 (1 gm, 4.21 mmol.) and ammonium thiocyanate (3 gm, 39.4
mmol.) in methanol (20 mL) was refluxed for 4 h then filtered, washed with water and
crystallized from ethanol.
4.1.6.1. Buff powder, yield: 65%, m.p. 250-252 °C IR (cm-1, KBr): 3298 (NH), 3049 (CH
1
aromatic), 2935 (CH aliphatic), 1734, 1708 (2 C=O), 1633 (C=N). H NMR (DMSO-d6, 300
MHz) δ, ppm: 3.95 (s, 2H, CH2), 6.47 (d, 1H, coumarin H-3,
J
= 9.3), 7.29-7.41 (m, 2H,
= 9.3),
coumarin H-7+H-8), 7.74 (d, 1H, coumarin H-5, = 2.4), 7.95 (d, 1H, coumarin H-4,
J
J
11.84 (s, 1H, NH exchanged with D2O). Anal. Calcd. for C12H8N2O3S (260.27): C, 55.38; H,
3.10; N, 10.76 %. Found: C, 55.45; H, 3.06; N, 10.81 %.
4.1.7. General procedure for the preparation of compounds 8a-g
To a well stirred solution of the coumarin thiazolidione derivative 7 (0.99 gm, 3.8 mmol.)
in glacial acetic acid (35 mL) buffered with sodium acetate, the appropriate aryl aldehyde (40
mmol.) was added and the solution was refluxed for 3 h then cooled to room temp. The solid was
25

filtered, washed with water, dried and crystallized from ethanol to give compounds 8a-g in good
yields.
4.1.7.1. 5-Benzylidene-2-[(2-oxo-2H-chromen-6-yl)imino]thiazolidin-4-one (8a). Greenish
brown powder, yield: 70 %, m.p. 272-274 °C, IR (cm-1, KBr): 3232 (NH), 3037 (CH aromatic),
2987 (CH aliphatic), 1743, 1672 (2 C=O), 1635 (C=N). 1H NMR (DMSO-d6, 300 MHz) δ, ppm:
6.52 (d, 1H, coumarin H-3,
J
= 10.2), 7.29 (d, 2H, aromatic H,
J
= 9), 7.40-7.73 (m, 7H,
= 9), 8.13 (s, 1H, NH
aromatic H + coumarin H + CH=C), 8.06 (d, 1H, H-4 coumarin,
J
exchanged with D2O). MS m/z: 348.87 [M] +. Anal. Calcd. for C19H12N2O3S (348.38): C, 65.51;
H, 3.47; N, 8.04 %. Found: C, 65.72; H, 3.44; N, 8.12 %.
4.1.7.2. 5-(4-Methoxybenzylidene)-2-[(2-oxo-2H-chromen-6-yl)imino]thiazolidin-4-one (8b).
Yellow powder, yield: 62 %, m.p. 280-282 °C, IR (cm-1, KBr): 3427 (NH), 3049 (CH aromatic),
2956 (CH aliphatic), 1759, 1708 (2 C=O), 1651 (C=N). 1H NMR (DMSO-d6, 300 MHz) δ, ppm:
3.85 (s, 3H, OCH3), 6.49 (d, 1H, coumarin H-3,
J
= 10.2), 6.99 (d, 2H, aromatic H,
= 8.1), 7.23-7.51 (m, 3H, coumarin H-5 + coumarin H-7 + CH=C), 7.86
= 9.3), 8.04 (d, 2H, aromatic H, = 9.6), 12.00 (s, 1H, NH exchanged
J = 8.7), 7.05
(d, 1H, coumarin H-8,
(d, 1H, coumarin H-4,
J
J
J
with D2O). Anal. Calcd. for C20H14N2O4S (378.40): C, 63.48; H, 3.73; N, 7.40 %. Found: C,
63.65; H, 3.79; N, 7.62 %.
4.1.7.3. 5-(4-Fluorobenzylidene)-2-[(2-oxo-2H-chromen-6-yl)imino]thiazolidin-4-one (8c).
Yellowish white powder, yield: 81 %, m.p. 293-295 °C, IR (cm-1, KBr): 3398 (NH), 3051 (CH
1
aromatic), 2924 (CH aliphatic), 1759, 1718 (2 C=O), 1668 (C=N). H NMR (DMSO-d6, 300
MHz) δ, ppm: 6.52 (d, 1H, coumarin H-3,
J
= 9.3), 7.27 (d, 2H, aromatic H,
J = 9), 7.31-7.87 (m,
5H, aromatic H + CH=C), 8.05 (d, 2H, aromatic H,
J
= 9.9), 8.12 (s, 1H, NH exchanged with
D2O). Anal. Calcd. for C19H11FN2O3S (366.37): C, 62.29; H, 3.03; N, 7.65 %. Found: C, 62.44;
H, 3.07; N, 7.82 %.
4.1.7.4. 5-[4-(Methylthio)benzylidene]-2-[(2-oxo-2H-chromen-6-yl)imino]thiazolidin-4-one (8d).
Brown powder, yield: 68 %, m.p. 281-283 °C, IR (cm-1, KBr): 3260 (NH), 3080 (CH aromatic),
2924 (CH aliphatic), 1739, 1697 (2 C=O), 1651 (C=N). 1H NMR (DMSO-d6, 300 MHz) δ, ppm:
2.53 (s, 3H, SCH3), 6.53 (d, 1H, coumarin H-3,
J = 9.6), 7.31 (d, 2H, aromatic H, J = 6.9), 7.42
(d, 1H, coumarin H-8, = 6.3), 7.54-7.65 (m, 4H, aromatic H + coumarin H-5 + CH=C), 7.69 (s,
J
1H, NH exchanged with D2O), 7.80 (dd, 1H, coumarin H-7, Ja = 6.6, Jb = 2.1), 8.05 (d, 1H,
26

coumarin H-4,
J = 9.3). Anal. Calcd. for C20H14N2O3S2 (394.47): C, 60.90; H, 3.58; N, 7.10 %.
Found: C, 61.17; H, 3.62; N, 7.23 %.
4.1.7.5. 5-(4-Chlorobenzylidene)-2-[(2-oxo-2H-chromen-6-yl)imino]thiazolidin-4-one (8e).
Greenish grey powder, yield: 76 %, m.p. 288-290 °C, IR (cm-1, KBr): 3398 (NH), 3049 (CH
1
aromatic), 2960 (CH aliphatic), 1759, 1720 (2 C=O), 1653 (C=N). H NMR (DMSO-d6, 300
MHz) δ, ppm: 6.52 (d, 1H, coumarin H-3,
J
= 9.3), 7.28 (d, 2H, aromatic H,
J
= 9.9), 7.39-7.63
= 9.9),
(m, 6H, aromatic H + coumarin H-5, H-7, H-8 + CH=C), 8.05 (d, 1H, coumarin H-4,
J
12.15 (s, 1H, NH exchanged with D2O). Anal. Calcd. for C19H11ClN2O3S (382.82): C, 59.61; H,
2.90; N, 7.32 %. Found: C, 59.84; H, 2.87; N, 7.49 %.
4.1.7.6. 5-[4-(Dimethylamino)benzylidene]-2-[(2-oxo-2H-chromen-6-yl)imino]thiazolidin-4-one
(8f). Reddish brown powder, yield: 53 %, m.p. 280-282 °C, IR (cm-1, KBr): 3236 (NH), 3066
1
(CH aromatic), 2954 (CH aliphatic), 1730, 1670 (2 C=O), 1641 (C=N). H NMR (DMSO-d6
,
300 MHz) δ, ppm: 3.02 (s, 6H, 2CH3), 6.51 (d, 1H, H-3 coumarin,
H, = 8.4), 7.32-7.62 (m, 5H, aromatic H + coumarin H-5, H-7, H-8), 7.87 (s, 1H, CH=C), 8.08
(d, 1H, coumarin H-4,
= 9.6), 12.19 (s, 1H, NH exchanged with D2O). 13C NMR (DMSO-d6) δ:
J = 9.6), 6.76 (d, 2H, aromatic
J
J
42.1, 111.5, 112.7, 114.5, 117.5, 118.86, 119.5, 126.4, 132.2, 144.7, 146.5, 149.2, 155.7, 159.2,
163.9, 173.7. Anal. Calcd. for C21H17N3O3S (391.44): C, 64.43; H, 4.38; N, 10.73 %. Found: C,
64.62; H, 4.49; N, 10.92 %.
4.1.7.7. 5-(4-Bromrobenzylidene)-2-[(2-oxo-2H-chromen-6-yl)imino]thiazolidin-4-one (8g).
Yellowish brown powder, yield: 84 %, m.p. >300 °C, IR (cm-1, KBr): 3412 (NH), 3064 (CH
1
aromatic), 2958 (CH aliphatic), 1759, 1718 (2 C=O), 1654 (C=N). H NMR (DMSO-d6, 300
MHz) δ, ppm: 6.53 (d, 1H, coumarin H-3,
1H, coumarin H-8, = 8.7), 7.56-7.69 (m, 3H, coumarin H-7 + aromatic H), 7.74 (d, 1H,
coumarin H-5, = 7.8), 7.83 (s, 1H, CH=C), 8.05 (d, 1H, coumarin H-4, = 9.3), 12.23 (s, 1H,
J = 9.6), 7.29 (d, 2H, aromatic H, J = 8.4), 7.45 (d,
J
J
J
NH exchanged with D2O). Anal. Calcd. for C19H11BrN2O3S (427.27): C, 53.41; H, 2.59; N, 6.56
%. Found: C, 53.58; H, 2.58; N, 6.74 %.
4.1.8. General procedure for the preparation of compounds 9a-c
An equivalent mixture of compound 6 (2.37 gm, 10 mmol.) and the corresponding
primary amine (10 mmol.) with (2 gm, 15 mmol.) of potassium carbonate in the presence of
27

traces of potassium iodide in dry DMF (30 mL) was heated for 24-48 h under reflux as
monitored by TLC. The reaction was poured on acidified cold water, filtered, washed with water
and recrystallized from ethanol to give the target compounds 9a-c.
4.1.8.1. 2-[(2-Oxo-2H-chromen-6-yl)amino]-N-phenylacetamide (9a). Reddish brown powder,
yield: 64 %, m.p. 224-226 °C, IR (cm-1, KBr): 3394, 3273 (NHs), 3068 (CH aromatic), 2983
(CH aliphatic), 1747, 1718 (2 C=O). 1H NMR (DMSO-d6, 300 MHz) δ, ppm: 4.00 (s, 2H, CH2),
6.53 (d, 1H, coumarin H-3,
aromatic H + coumarin H-7), 7.63 (d, 1H, coumarin H-5,
with D2O), 8.05 (d, 2H, aromatic H, = 9.6), 8.15 (d, 1H, coumarin H-4,
J
= 9), 7.28 (d, 1H, coumarin H-8,
= 10.8), 7.74 (s, 1H, NH exchanged
= 8.7), 12.25 (s, 1H,
J = 8.4), 7.40-7.57 (m, 4H,
J
J
J
NH exchanged with D2O). Anal. Calcd. for C17H14N2O3 (294.30): C, 69.38; H, 4.79; N, 9.52 %.
Found: C, 69.45; H, 4.69; N, 9.49 %.
4.1.8.2. N-(4-Methoxyphenyl)-2-[(2-oxo-2H-chromen-6-yl)amino]acetamide (9b). Orange
crystalline powder, yield: 58 %, m.p. 228-230 °C, IR (cm-1, KBr): 3271 (br.) (NHs), 3097 (CH
aromatic), 2940 (CH aliphatic), 1724, 1685 (2 C=O). 1H NMR (DMSO-d6, 300 MHz) δ, ppm:
3.31 (s, 3H, OCH3), 4.26 (s, 2H, CH2), 5.68 (d, 1H, coumarin H-3,
aromatic H + coumarin H-4, H-7, H-8), 8.16 (d, 2H, aromatic H, = 9), 8.51 (d, 1H, coumarin
H-5, = 2.4), 12.05 (s, 1H, NH exchanged with D2O), 12.49 (s, 1H, NH, exchanged with D2O).
J = 9), 6.85-7.40 (m, 5H,
J
J
Anal. Calcd. for C18H16N2O4 (324.33): C, 66.66; H, 4.97; N, 8.64 %. Found: C, 66.89; H, 4.89;
N, 8.53 %.
4.1.8.3. N-(4-Fluorophenyl)-2-[(2-oxo-2H-chromen-6-yl)amino]acetamide (9c). Light yellow
powder, yield: 61 %, m.p. 254-256 °C, IR (cm-1, KBr): 3377 (br.) (NHs), 3050 (CH aromatic),
1
2993 (CH aliphatic), 1728, 1718 (2 C=O). H NMR (DMSO-d6, 300 MHz) δ, ppm: 4.26 (s, 2H,
CH2), 6.56 (d, 1H, coumarin H-3,
exchanged with D2O), 7.83 (d, 1H, coumarin H-5,
coumarin H-7), 8.13 (d, 1H, coumarin H-4, = 9.9) 13.01 (s, 1H, NH exchanged with D2O).
J
= 9.6), 7.55-7.72 (m, 4H, aromatic H + coumarin H-8 + NH
J
= 2.4), 7.90-8.00 (m, 3H, aromatic H +
J
Anal. Calcd. for C17H13FN2O3 (312.30): C, 65.38; H, 4.20; N, 8.97 %. Found: C, 65.26; H, 4.27;
N, 8.84 %.
4.2. Biological Evaluation
4.2.1. Acetylcholine esterase inhibitory activity
28