Synthesis, pharmacological assessment, molecular modeling and in silico studies of fused tricyclic coumarin derivatives as a new family of multifunctional anti-Alzheimer agents

Article abstract of DOI:10.1016/j.ejmech.2015.10.046

A series of fused tricyclic coumarin derivatives bearing iminopyran ring connected to various amido moieties were developed as potential multifunctional anti-Alzheimer agents for their cholinesterase inhibitory and radical scavenging activities. In vitro studies revealed that most of these compounds exhibited high inhibitory activity on acetylcholinesterase (AChE), with IC₅₀ values ranging from 0.003 to 0.357 μM which is 2-220 folds more potent than the positive control, galantamine. Their inhibition selectivity against AChE over butyrylcholinesterase (BuChE) has increased about 194 fold compared with galantamine. The developed compounds also showed potent ABTS radical scavenging activity (IC₅₀ 7.98-15.99 μM). Specifically, the most potent AChE inhibitor 6n (IC₅₀ 0.003 ± 0.0007 μM) has an excellent antioxidant profile as determined by the ABTS method (IC₅₀ 7.98 ± 0.77 μM). Moreover, cell viability studies in SK N SH cells showed that the compounds 6m-q have significant neuroprotective effects against H₂O₂-induced cell death, and are not neurotoxic at all concentrations except 6n and 6q. The kinetic analysis of compound 6n proved that it is a mixed-type inhibitor for EeAChE (K_i1 0.0103 μM and K_i2 0.0193 μM). Accordingly, the molecular modeling study demonstrated that 6m-q with substituted benzyl amido moiety possessed an optimal docking pose with interactions at catalytic active site (CAS) and peripheral anionic site (PAS) of AChE simultaneously and thereby they might prevent aggregation of Aβ induced by AChE. Furthermore, in silico ADMET prediction studies indicated that these compounds satisfied all the characteristics of CNS acting drugs. Most active inhibitor 6n is permeable to BBB as determined in the in vivo brain AChE activity. To sum up, the multipotent therapuetic profile of these novel tricyclic coumarins makes them promising leads for developing anti-Alzheimer agents.

Full text of DOI:10.1016/j.ejmech.2015.10.046

Accepted Manuscript
Synthesis, pharmacological assessment, molecular modeling and in silico studies of
fused tricyclic coumarin derivatives as a new family of multifunctional anti-Alzheimer
agents
Shaik Jeelan Basha, Palaka Bhagath Kumar, Penumala Mohan, Kotapati Kasi
Viswanath, Devineni Subba Rao, Eadlapalli Siddhartha, Darla M. Manidhar,
Ampasala Dinakara Rao, Vadde Ramakrishna, Amooru G. Damu
PII:
S0223-5234(15)30328-7
10.1016/j.ejmech.2015.10.046
EJMECH 8181
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 July 2015
Revised Date: 23 October 2015
Accepted Date: 28 October 2015
Please cite this article as: S.J. Basha, P.B. Kumar, P. Mohan, K.K. Viswanath, D.S. Rao, E. Siddhartha,
D.M. Manidhar, A.D. Rao, V. Ramakrishna, A.G. Damu, Synthesis, pharmacological assessment,
molecular modeling and in silico studies of fused tricyclic coumarin derivatives as a new family of
multifunctional anti-Alzheimer agents, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.10.046.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis, pharmacological assessment, molecular modeling and in silico studies of fused tricyclic
coumarin derivatives as a new family of multifunctional anti-Alzheimer agents
Shaik Jeelan Basha, Palaka Bhagath Kumar, Penumala Mohan, Kotapati Kasi Viswanath, Devineni Subba
Rao, Eadlapalli Siddhartha, Darla M. Manidhar, Ampasala Dinakara Rao, Vadde Ramakrishna, Amooru G.
Damu
O
O
O
O
HN
Br
N
H
6n
AChE: IC50 0.003 ± 0.0007 µm
ABTS: IC50 7.98 ± 0.77 µm
Angularly fused tricyclic coumarin derivatives were evaluated as novel multifunctional anti AD agents with
selective AChE and ABTS inhibitory activities. N-(3-bromobenzyl) amide analog 6n was 220 fold superior to
galantamine.

ACCEPTED MANUSCRIPT
Synthesis, pharmacological assessment, molecular modeling and in silico studies of fused
tricyclic coumarin derivatives as a new family of multifunctional anti-Alzheimer agents
Shaik Jeelan Bashaa, Palaka Bhagath Kumarb, Penumala Mohana, Kotapati Kasi Viswanathb,
Devineni Subba Raoc, Eadlapalli Siddharthad, Darla M. Manidharc, Ampasala Dinakara Raob,
Vadde Ramakrishnad, Amooru G. Damua,*
aDepartment of Chemistry, Yogi Vemana University, Kadapa, India.
bCentre for Bioinformatics, School of life Sciences, Pondicherry Central University, Puducherry, India.
cDepartment of Chemistry, University College of Sciences, Sri Venkateswara University,Tirupati, India.
dDepartment of Biotechnology and Bioinformatics, Yogi Vemana University, Kadapa, India.
ABSTRACT
A series of fused tricyclic coumarin derivatives bearing iminopyran ring connected to various
amido moieties were developed as potential multifunctional anti-Alzheimer agents for their
cholinesterase inhibitory and radical scavenging activities. In vitro studies revealed that most of
these compounds exhibited high inhibitory activity on acetylcholinesterase (AChE), with IC50
values ranging from 0.003 to 0.357 µM which is 2–220 folds more potent than the positive
control, galantamine. Their inhibition selectivity against AChE over butyrylcholinesterase
(BuChE) has increased about 194 fold compared with galantamine. The developed compounds
also showed potent ABTS radical scavenging activity (IC50 7.98–15.99 µM). Specifically, the
most potent AChE inhibitor 6n (IC50 0.003 ± 0.0007 µM) has an excellent antioxidant profile as
determined by the ABTS method (IC50 7.98 ± 0.77 µM). Moreover, cell viability studies in SK N
SH cells showed that the compounds 6m–q have significant neuroprotective effects against
H2O2-induced cell death, and are not neurotoxic at all concentrations except 6n and 6q. The
kinetic analysis of compound 6n proved that it is a mixed-type inhibitor for EeAChE (Ki1
0.0103 µM and Ki2 0.0193 µM). Accordingly, the molecular modeling study demonstrated that
6m–q with substituted benzyl amido moiety possessed an optimal docking pose with interactions
at catalytic active site (CAS) and peripheral anionic site (PAS) of AChE simultaneously and
thereby they might prevent aggregation of Aβ induced by AChE. Furthermore, in silico ADMET
prediction studies indicated that these compounds satisfied all the characteristics of CNS acting
drugs. Most active inhibitor 6n is permeable to BBB as determined in the in vivo brain AChE
1

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activity. To sum up, the multipotent therapuetic profile of these novel tricyclic coumarins makes
them promising leads for developing anti-Alzheimer agents.
Keywords: Alzheimer’s disease, Fused tricyclic coumarin, Antioxidant, Acetylcholinesterase,
Butyrylcholinesterase, Neuroprotection, Molecular modeling study, ADMET
*Corresponding author. Tel: +91- 8562-225410; Fax: +91- 8562-225419.
E-mail address: agdamu01@gmail.com (A.G. Damu).
1. Introduction
Alzheimer's disease (AD) is a deadly neurodegenerative disorder that attacks the central
nervous system through progressive degeneration of its neurons [1]. It is the most common form
of dementia affecting over 35 million people worldwide. AD is characterized by the loss of
memory, progressive and irreversible cognitive impairments, language deterioration, severe
behavioral abnormalities, and ultimately causing death [2]. Despite AD has been found for more
than a century, it is still incurable as its etiology has not yet been fully explored. However, low
levels of acetylcholine (ACh), oxidative stress, the inflammation of neurons and β-amyloid (Aβ)
deposits are thought to play definitive roles in AD pathogenesis. Based on these factors several
hypotheses which include cholinergic and noncholinergic interventions have been emerged over
the past decades [3]. However, the cholinergic hypothesis which enhances the central cholinergic
function by maintaining ACh levels via inhibition of acetylcholinesterase (AChE) is the only
approved therapeutic strategy for the treatment of AD.
Recently it has been shown that AChE involves in the extraneous non-cholinergic
function in the early phases of AD by binding to Aβ, thereby accelerating its polymerization into
oligomers and fibrils and increasing the neurotoxicity of Aβ aggregates [4,5]. The peripheral
anionic site (PAS) has been shown to play a crucial role in Aβ pro-aggregating action of AChE.
It is located at the mouth of a 20 Å deep gorge and leads to the catalytic anionic site (CAS) of the
enzyme. Considering these aspects, AChE inhibitors that bind to either PAS or to both the CAS
and PAS may simultaneously alleviate the cognitive deficit and delay the neurodegenerative
process by preventing the assembly of Aβ-peptide [6-10].
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Furthermore, inhibition of butyrylcholinesterase (BuChE), an enzyme closely related to
AChE has been found to be a desirable activity in design of anti-Alzheimer agents [11].
However, serious inhibition of BuChE causes peripheral side effects including gastrointestinal
events, nausea, vomiting, diarrhea and dizziness as it is mainly localized in the peripheral tissues
including plasma and several regions of the brain. For example, the dual AChE and BuChE
inhibitor, tacrine showed severe hepatotoxicity as well as other adverse effects and so withdrawn
from the use. Therefore, the development of selective AChE inhibitors to reduce side effects may
be a more suitable therapeutic strategy for the treatment of AD [12].
Increasing evidence supports the significant impact of oxidative stress in initiating the
aggregation of Aβ and tau protein hyperphosphorylation, involved in the early stage of the
pathologic cascade [13]. Oxidative cell damage marked by lipid peroxidation, nitration, reactive
carbonyls, and nucleic acid oxidation is at higher level in vulnerable neurons in AD [14]. Thus,
neuroprotection against oxidative stress induced cell damage in neuronal cells has become the
important target for AD treatment. Therefore, the combination of selective AChEIs, antioxidants
and neuroprotectives represents an additional rational approach to develop new multifunctional
agents for AD treatment [15,16].
Naturally occurring and chemically synthesized coumarins have received intense
attention in recent years because of their wide ranging biological activities [17]. Researchers
have attempted to explore the coumarin template for developing novel AChE inhibitors with
additional pharmacological activities such as decrease in Aβ deposition and β-secretase
inhibition that are also important for AD management [18-21]. Coumarin derivatives have also
been protecting neurons against Aβ-induced oxidative stress and free radicals [22,23]. Since
coumarins primarily interact with PAS of AChE, several coumarin based dual inhibitors of
AChE were designed by incorporating a catalytic site interacting moiety through an appropriate
spacer [24-31]. As a result, the 3rd or 4th position of coumarin moiety has been described as the
most favorable for linking CAS interacting moiety but not 6th or 7th to obtain potent dual site
AChE inhibitors [18]. However, we focused our attention for the first time on angularly fused
iminopyran at 7 and 8 positions of 7-hydroxy-4-methylcoumarin bearing various N-substituted
amides on 9th position. Thus we report herein synthesis, biological evaluation, molecular
modeling and in silico studies of a series of fused tricyclic coumarin amide derivatives as novel
3

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multifunctional anti-AD agents with potent and selective AChE over BuChE inhibitory and
antioxidant activities.
2. Results and discussion
2.1. Chemistry
The synthetic pathway of fused tricyclic coumarins (6a–s) was outlined in Scheme I. The
target molecules consist of three parts, therefore the synthetic strategy involves the preparation of
cyano acetamide derivative by reacting different amines with ethylcyanoacetate and then fused it
to the formyl coumarin. At first, a series of N-substituted cyano acetamide derivatives (2a–s)
were prepared by simple reaction in readily available screw cap bottle by treating amines with
equivalent amount of ethylcyanoacetate [32,33]. Resorcinol (3) and ethylacetoacetate were
treated under Pechmann conditions to give 7-hydroxy-4-methylcoumarin (4) [34], and compound
4 was treated with hexamethylenetetramine in glacial acetic acid, undergoes Duff formylation, to
provide 8-formyl-7-hydroxy-4-methylcoumarin (5) [35]. Subsequently, compound 5 was
condensed with different N-substituted cyano acetamide derivatives (2a–s) in the presence of
Et3N afforded the final products (6a–s) [36]. An interesting feature of this procedure is the
simple formation of final product without further purification. All the synthesized compounds
1
were characterized by IR, H NMR, 13C NMR spectra and Mass spectrometry. In the IR
spectrum, characteristic N–H absorption band of the imino group observed between 3415 and
3471 cm-1, C=N characteristic absorption band between 1667 and 1689 cm-1 and a singlet of the
imino group proton resonated at 7.67-8.51 ppm in the 1H NMR spectrum further supported that –
CHO, –OH and –CN groups involved in the reaction that leads to an extended iminopyran ring
on coumarin moiety in the final product [36].
2.2 In vitro cholinesterase inhibition activity
To evaluate the potential application of fused tricyclic coumarins in the treatment of AD,
the inhibition of AChE and BuChE activities was measured according to the spectrophotometric
method of Ellman using galantamine, tacrine, donepezil and rivastigmine as positive control
[37,38]. The IC50 values and selectivity index for the inhibition of AChE and BuChE are
4

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summarized in Table 1. The target compounds showed different amounts of inhibitory activity to
AChE with IC50 values ranging from the submicromolar to micromolar range (IC50 0.003 ±
0.0003–2.6 ± 0.249 µM) and good inhibition selectivity (9.54–5793) against AChE over BuChE.
Moreover, most of the synthetic derivatives exhibited AChE inhibitory activity (IC50 0.003 ±
0.0003–0.357 ± 0.027 µM) more potent than the positive control, galantamine, by 2-220 fold and
their inhibition selectivity towards AChE increased about 194 fold compared with galantamine.
Analogs 6c, 6e and 6m-q showed stronger anti-AChE properties than all reference compounds.
As far as BuChE is concerned, all analogs showed lesser inhibitory activity than galantamine,
except compound 6b, whereas all are less potent than donepezil, tacrine and rivastigmine. It was
also demonstrated that 6n displayed the most potent inhibitory activity on AChE with IC50 value
of 0.003 ± 0.0007µM while 6b was the most potent inhibitor of BuChE with IC50 of 11.32 ± 1.38
µM. On the other hand, compounds 6b, 6d and 6r could be considered as dual inhibitors of both
enzymes.
R1
11
OEt
5
4
N
i
NC
4a
1a
6
NC
R2
3
O
1
6a
2 a-s
O
2
iv
7O
O
O
10a
10
1
8
HN
9
iii
ii
12
R2
O
N13
R1
HO
O
O
HO
OH
6a-s
HO
O
O
3
5
4
CHO
N
H
N
O
NH
N
R1R2N =
NHCH3
NHCH2CH3
NH(CH2)11CH3
NH
N
H
a
g
b
c
d
f
e
h
i
Cl
Br
OCH3
Br
N
H
N
H
N
H
N
H
N
H
OCH3
OCH3
n
l
m
o
k
N
H
OCH3
NO2
Cl
O
HN
N
H
N
H
j
HN
p
q
r
s
Scheme 1. Synthetic pathway of target compounds 6a-s. Reagents and conditions: (i) corresponding amine, EtOH; (ii) ethylacetoacetate, H2SO4, 18 h;
(iii) HMTA, glacial acetic acid, 90 oC, reflux, 6h; (iv) Et3N, EtOH
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From the data in Table 1 several conclusions can be drawn concerning the structure-
activity relationships, and the effect of the substituents in phenyl ring of amide moiety. The
change of the cyclopropyl moiety (6d) to cyclohexyl (6e) on amido part increased anti-AChE
activity about 35 fold.
Table 1
An in vitro AChE and BuChE inhibitory activities and ABTS radical scavenging capacity of synthetic fused tricyclic
coumarin based compounds 6a–s and reference compounds
IC50 (µM) ± S.E.M.a
IC50 (µM) ± S.E.M.a
ABTS radical
scavenging activity
Compounds
AChEb
BuChEb
Selectivity
for AChEc
1.4 ± 0.087
>100
11.32 ± 1.38
>100
38.9 ± 2.83
42.05 ± 2.94
73.08 ± 3.80
NA
>100
35.84 ± 3.46
NA
NA
>100
>100
>100
>100
>100
57.93 ± 1.81
24.8 ± 2.05
>100
–
38.5
–
42.74
1557.4
74.26
–
34.16 ± 1.71
>50
>50
>50
>50
>50
11.27 ± 0.71
>50
14.1 ± 1.10
>50
9.04 ± 0.77
>50
11.94 ± 1.21
7.98 ± 0.77
15.99 ± 1.32
10.32 ± 0.84
13.39 ± 0.25
12.98 ± 1.07
>50
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
0.294 ± 0.024
0.022 ± 0.0017
0.91 ± 0.036
0.027 ± 0.004
0.984 ± 0.008
0.115 ± 0.013
2.13 ± 0.0517
0.247 ± 0.019
0.09 ± 0.005
1.9 ± 0.115
0.357 ± 0.027
0.016 ± 0.0021
0.003 ± 0.0007
0.012 ± 0.0018
0.019 ± 0.001
0.01 ± 0.0018
2.6 ± 0.249
–
145.1
–
–
–
–
–
–
–
5793
9.54
–
0.226 ± 0.0136
Galantamine 0.665 ± 0.02
19.78 ± 0.83
7.69 ± 0.42
1.77 ± 0.13
5.5 ± 1.3
–
29.74
202.36
4.75
1.61
–
–
–
–
–
Donepezil
Tacrine
0.038± 0.002
0.372 ± 0.14
Rivastigmine 3.4 ± 0.83
Trolox
–
27.35 ± 1.34
a Concentration required to produce 50% inhibition of enzyme activity. IC50 values are given as the mean of three
independent determinations.
bAChE from Electric Eel and BuChE from equine serum were used.
cSelectivity ratio = (IC50 of BuChE)/(IC50 of AChE).
Meanwhile, elongation of aliphatic side chain in case of compounds 6a, 6b and 6c
increased the AChE inhibitory activity due to the more lipophilic property of these substituents.
However, improvement in BuChE inhibitory activity was limited to N-ethyl group from N-
6

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methyl group. Replacement of the aliphatic methyl group (6i) at benzylic position by aromatic
substituent (6j) led to an increase in the activity toward AChE but the BuChE inhibition activity
was diminished. Interestingly, monosubstituted benzyl amide inhibitors 6m-6p bearing electron
withdrawing groups such as –Cl or –Br at ortho or meta positions were 110-700 fold more potent
than inhibitor 6h with simple benzyl moiety. Further, compound 6q with para-(ter-butyl) benzyl
moiety was also 210 folds more potent than compound 6h. 2,4-Dimethoxy substitution at
benzylamido moiety as in 6l enhanced inhibition potency while 4-methoxy group on methyl
benzylamido moiety (6k) diminished inhibition potency compared to the unsubstituted
counterparts 6h and 6i, respectively.
2.3 In vitro antioxidant activity
The reduction of the oxidative stress is another crucial aspect in designing agents for AD
treatment. We examined the antioxidant activities of our synthetic derivatives by using the ABTS
(2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) radical scavenging method [39]. Trolox, a
water-soluble vitamin E analog, was used as a reference standard. The ability of compounds to
scavenge radicals was shown as IC50, the test compound’s concentration resulting in 50%
inhibition of free radical (Table 1). According to the data, ten synthetic derivatives were found to
possess potent ABTS radical scavenging capacities with IC50 values in the range of 7.98 to 34.16
µM. Among all nineteen synthetic analogs, nine compounds showed higher radical scavenging
activities (IC50 range of 7.98 –15.99 µM) than trolox (IC50 of 27.35 µM) which is 1.7 to 3.4 fold
of trolox. The best results were obtained with derivatives bearing 3-bromo or 4-methoxy group
on phenyl ring of benzyl amido moiety. Compound 6g with heterocyclic amide moiety displayed
higher radical scavenging property than their alicyclic counterparts. Converting methyl to benzyl
as in 6a to 6n diminishes the antioxidant activity. Introduction of methoxy group on 4-position of
phenyl ethyl amido moiety as in 6k enhances the radical scavenging activity by 1.5 fold. The
radical scavenging capacity of compounds increased from ortho to meta halo substitution as can
be seen in 6m-6p.
2.4 Neurotoxicity of compounds in SK N SH cells
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To investigate the effect of the selected compounds (6m, 6n, 6o, 6p and 6q) and
galantamine as reference on cell viability, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was conducted on SK N SH cells (human neuroblastoma cell
line) [40,41]. The SK N SH cells were incubated with varying concentrations (40, 80, 120, 160
and 200 µM) of the test compounds and galantamine for 24 h and measured cell viability. The
results depicted in figure x revealed that under these conditions all of the tested compounds were
nontoxic to SK N SH cells at any of the concentrations tested except 6n and 6q.
2.5 Neuroprotective effect against H2O2-induced cell death in SK N SH cells
The neuroprotective capacities of selected compounds against oxidative stress were
evaluated by assessing the ability of the tested compounds to protect against H2O2-induced
induced SK N SH cell injury and using the MTT assay to assess cell viability [40,41]. As
estimated by MTT assay, cell viability is markedly decreased to 60% after 1.0 mM H2O2
exposure for 24 h. However, when cells were preincubated with compounds (40, 80, 120 µM) for
3 h, and then exposed to H2O2, cell toxicity was significantly attenuated in a dose-dependent
manner and also brought to control levels at higher concentrations. Addition of compound to the
medium prior to H2O2 addition increases survival cells by preventing H2O2 induced oxidative
stress caused cell death indicating that these compounds act as a potential oxidative suppressors.
Catechin was included as a reference antioxidant compound.
140
120
100
*
*
*
*
*
80
60
40
20
0
*
*
*
*
*
*
*
*
*
*
*
*
*
*
6M
6N
6O
6P
6Q
Galantamine
Fig. 1. Neurotoxic effects of 6m, 6n, 6o, 6p and 6q compounds on SK N SH cells (human neuroblastoma cell line).
Bar chart show the percentage of cell viability in the presence or absence (control) of indicated concentrations of
6m, 6n, 6o, 6p, 6q, and Galantamine. MTT assay was done to assess cell viability. Cell viability corresponding to
control cells was considered as 100%. The values represent the mean SEM of three independent experiments, each
8

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one performed by triplicate in different cell batches. Statistics show neurotoxic effects of these compounds versus
controls. *P < 0.05 versus 100% cell viability (one-way ANOVA test).
120
100
*
*
*
80
60
40
20
0
*
*
*
6M
6N
6O
6P
6Q
Catechin
Fig. 2. Neuroprotective activity of compounds 6m, 6n, 6o, 6p, 6q and catechin against H2O2 induced cell death in SK
N SH cells. Cells were treated with different concentrations 40 µM to 120 µM of the compound for 3 h. After 24 h,
cell viability was determined by MTT assay in the presence 1.0 mM H2O2. The values represent the mean SEM of
three independent experiments, each one performed by triplicate in different cell batches. Statistics show neurotoxic
effects of these compounds versus controls. *P < 0.05 versus H2O2 treatment.
2.6 Kinetic study of the AChE inhibition
To gain further insight into the mechanism of action on AChE of this family of
compounds, a kinetic study was carried out on compound 6n, the most potent AChE inhibitor.
For this purpose, the rate of the enzyme activity was measured at three different concentrations
of compound 6n using different concentrations of the substrate acetylthiocholine iodide (ATCh).
In each case, the initial velocity was measured at different concentrations of the substrate (S), and
the reciprocal of the initial velocity (1/v) was plotted against the reciprocal of [ATCh].
10
9
8
7
6
y = 302.0x + 3.127
R² = 0.919
5
4
3
2
1
0
-0.02
0
0.02
Concentration (µM)
Fig. 3. Steady-state inhibition of AChE by compound 6n. (Left) Lineweaver Burk plot of reciprocal of initial
velocities versus reciprocal of acetylthiocholine iodide concentrations (0.1-0.5 mM) in the absence and presence of
9

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6n at 0.002µM, 0.005µM and 0.02µM; (right) secondary plots of the Lineweaver Burk plot, slope versus various
concentrations of 6n.
Graphical analysis of the reciprocal Lineweaver Burk plots (Fig. 3) showed both
increased slopes and intercepts at increasing concentration of the inhibitor and intersection of
double reciprocal lines in the upper left quadrant. This pattern indicates a mixed-type inhibition
[42]. Generally, this type of inhibition suggests binding to free enzyme and enzyme-substrate
complex. Thus, this result indicated that compound 6n was able to bind at both the catalytic and
peripheral site of AChE. The inhibitory constants (Ki1 and Ki2) for the compound 6n were
calculated as 0.0103 µM (binding to free enzyme) and 0.0193 µM (binding to enzyme–substrate
complex) using secondary plots.
2.7 Molecular modeling study
A molecular modeling study was performed to investigate possible binding mode of fused
tricyclic coumarin derivatives to the cholinesterase enzymes utilizing a docking program,
Schrödinger maestro software (version 9.2; Schrödinger LLC, NewYork). For this purpose, five
most potent compounds (6m–q) with IC50 values below 0.02 µM were docked into the active site
of the EeAChE. The 3D structure of EeAChE (from Electric eel) was retrieved from PDB (PDB
ID: 1C2O) [43]. The results were characterized by the G Scores obtained from GLIDE (Grid-
based Ligand Docking with Energetics) docking [44]. The best docked poses in terms of the
Glide energy of binding were further analyzed to clarify interactions between target compounds
and AChE. Exhaustive analysis of the docking poses showed that compounds 6m, 6o and 6q
were similarly oriented in the active site. However, the binding modes of 6n and 6p were
significantly different from the binding modes of 6m, 6o and 6q at the AChE active site gorge.
The binding poses within AChE along with the residues surrounding derivatives 6m, 6o and 6q
are depicted in Fig. 4.
As can be seen in Fig. 4, the docking orientation of 6m, 6o and 6q places the ligand in the
PAS with the fused tricyclic coumarin moiety sandwiched between Trp 286 (4.7 Å) and Tyr 341
(4.5 Å) residues by π−π stacking. The 2-bromo benzylamido moiety extended to the opening of
the PAS and forms CH...π interaction with side chain of Glu 292 (4.0 Å). In this orientation, the
coumarin carbonyl group establishes hydrogen bond interaction with the hydroxyl group of Phe
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295. Also compounds 6m, 6o and 6q were all able to protrude out of the AChE gorge, a feature
which is considered important for the AChE-induced Aβ aggregation inhibition.
The orientation of ligands 6n and 6p is reversed comparing to the pose of 6m, 6o and 6q.
As shown in Fig. 4, the ligand was well accommodated in the gorge of AChE active site so that
the benzylamido moiety was leaning toward the CAS. Specifically, 3-halo phenyl ring of
benzylamido moiety stacks against the Phe 338 via T-shape (edge-to-face) π−π interaction and
potentially induce hydrophobic interactions with residues Tyr 337, Trp 86, Phe 295, Gly 121 and
Ser 203. However, no interactions with the catalytic triad residues have been found. In this
binding mode, the tricyclic coumarin scaffold formed π-stacking with aromatic ring of indole
moiety of Trp 286 in the PAS. The coumarin carbonyl group in 6n was hydrogen-bonded to Tyr
72. In addition, amide –NH group establishes hydrogen bond interaction with –OH group of Tyr
124 in the mid gorge site. This binding mode is in agreement with mixed-mode inhibition pattern
of 6n, in which both CAS and PAS are occupied by the ligand. However, instead 6p disclosed
only one hydrogen bonding interactions between imino –NH and Phe 295.
With respect to BuChE, in the absence of X-ray structure of EqBuChE (from Horse serum), a 3D
structure was predicted by homology modeling approach [43]. The template considered for
homology modeling of BuChE yielded 2PM8 as the suitable structural template with 94% query
coverage and 90% sequence similarity. To explore possible binding modes for the most active
compounds 6b and 6r toward BuChE, they were docked into BuChE active site. The best ranked
docking results revealed that BuChE can effectively accommodate the compounds 6b and 6r
inside the active site gorge (Fig. 5). The aromatic amino acid residues like Tyr 72, Tyr 124, Trp
286, Phe 295, Phe 297 and Tyr 337 in AChE are mostly replaced by aliphatic ones: Asn 68, Gln
119, Ala 277, Leu 286, Val 288, and Ala 328 in BuChE. This variation might have faded the
ability of BuChE to form π−π stacking interactions compared to AChE. The 6r–BuChE complex
was found to be stabilized mainly by the hydrophobic interactions with all the active site residues
existing along the wall of the active site gorge which includes PAS, Anionic subsite, Oxyaninon
hole, catalytic triad, and choline binding region of the enzyme. The 2-(2-methoxyphenoxy)ethyl
amide moiety of 6r found to be protruding into the space between the residues Trp 110 and Ala
356 of anionic subsite and forming hydrophobic interactions with residues Ala 356, Phe 357, Trp
458, and Met 465. In the complex, Trp 110 was found to allow for further π–π stacking
11

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interactions with the iminopyran ring of the ligand. The oxygen atom of the phenoxy is also
forming a hydrogen bond with His 466 of the catalytic triad.
6m
6n
6p
6o
6q
Fig. 4. Docking pose of the test compounds 6m
–
q at the binding site of AChE predicted by molecular modeling
12

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6b
6r
Fig. 5. Docking pose of the test compounds 6b and 6r at the binding site of BuChE predicted by molecular modeling
The binding mode of compound 6b, shown in Fig. 5, places the ligand within the central region
of the active site gorge where substrate or inhibitors reported to bind effectively. In this
orientation, Phe 357 allowed a π–π stacking interaction with the tricyclic coumarin moiety at
choline binding site of the BuChE. Ligand 6b exhibited two strong hydrogen bond interactions
with amino acids of the catalytic triad, they are between oxygen of coumarin moiety and –OH
group of Ser 226 and between amide carbonyl and –NH group of His 466. The N-ethyl group on
the amide part forms CH….π interaction with Trp 110. In addition 6b interact with Trp110, Leu
313, Leu 314, Val 316, Ala 356, Ile 426, Trp 458, Met 465, and Tyr 468 residues via
hydrophobic interactions. Conversely to the 6r-BuChE complex, the favorable position of 6b
within the gorge accounts for a higher affinity of 6b towards BuChE and makes it a more potent
BuChE inhibitor than compound 6r.
2.8 Theoretical ADMET analysis of fused coumarins
Finally, a series of theoretical calculations using QikProp (Version 3.5, Schrödinger,
LLC, New York, NY, 2012) and LC50 values (Lethal concentration 50%) using http://lazar.in-
silico.de/predict allowed us to predict the ADMET (Absorption, Distribution, Metabolism,
Excretion and Toxicity) properties of target compounds (6a–s) within the organism [31,45]. The
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four criterion ADME influence the drug levels, its kinetics and exposure to the tissues, and hence
the performance and pharmacological activity of a drug. About 45 physically significant
descriptors and pharmacologically relevant properties of these compounds were predicted and
some of the important properties were analyzed (Table S1, Supplementary Material). Based on
the predicted values for BBB penetration, all compounds might be able to penetrate into the CNS
and therefore, are considered as active compounds on the CNS. CNS drugs show values of MW
<450, HB donor <3, HB acceptors <10, QPlogPo/w <6, TPSA <140, number of rotatable bonds
<8 and hydrogen bonds <8 CNS, in a smaller range than general therapeutics [46,47]. All the
active compounds satisfied the characteristics of CNS acting drugs as all predicted values are in
the limits. The solubility (QPlogS) of organic molecules in water has a significant impact on
many ADME-related properties like uptake, distribution, transport, and eventually
bioavailability. All compounds showed solubility values within the limits [48]. Moreover, all the
compounds may not show either acute toxicity according to the calculated LC50 values nor
mutagenic effect with respect to the AMES test data. In addition, the Lipinski criteria for the
target compounds were predicted. According to Lipinski criteria of drug likeness, all the
compounds (6a-6s) within the range set by Lipinski’s rule of five and could be good candidate
for drug development.
2.9 Determination of brain AChE activity
Anti-Alzheimer drug candidate, like any other CNS drug, must be able to efficiently enter
into the brain, for which a major requirement is to cross the blood brain barrier (BBB). Thus, to
determine whether the compound 6n could penetrate the BBB, the brain AChE activity was
measured as an indirect method [49]. Different concentrations of most active compound 6n (16,
50, 100 and 200 mg/kg) was administered via intraperitoneal injection to male mice (30 ± 2g).
After 48 h of administration, mice were sacrificed and brain homogenates were prepared.
Supernatant of brain homogenate was used to determine brain AChE activity. The rate of
hydrolysis of ATCh (15 µL of 1mM) by brain AChE (10, 20, 30, 40 and 50 µL) at was measured
using Ellman method. As presented in fig.6, the rate of hydrolysis of ATCh by brain AChE was
decrease with increase in administratered dose of 6n. Therefore, after treatment with 6n, the
brain AChE activity was found to be down regulated in dose dependent manner which is possible
only if 6n had penetrated the blood-brain barrier and inhibit brain AChE activity.
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0.6
0.5
0.4
0.3
0.2
0.1
0
100
90
80
70
0 mg/kg
60
50
40
30
20
10
0
16 mg/kg
50 mg/kg
100 mg/kg
200 mg/kg
16
50
100
200
10 20 30 40 50
supernant in µL
mg of 6n/kg
Fig. 6. Determination of brain AChE activity to assess BBB penetration of 6n. (left) Rate of hydrolysis of ATCh by
AChE in brain homogenates from mice injected with 6n; (right) AChE activity of brain homogenates from mice
injected with 6n, expressed as a percentage of the AChE activity of corresponding preparations from control mice.
3. Conclusion
In summary, nineteen new iminopyran fused tricyclic coumarin derivatives were
synthesized and evaluated as novel multifunctional agents against AD with selective, dual
binding site AChE inhibitory and antioxidant activities. The biological evaluation showed that
most of these molecules were potent and selective AChE inhibitors, which is 2–220 folds more
potent than the positive control, galantamine. Especially 6n bearing 2-bromobenzyl amide
moiety was found to be most potent AChE inhibitor (IC50 0.003 ± 0.0007 µM) with strong ABTS
radical scavenging activity (7.98 ± 0.77 µM). Compound 6b exhibited the highest BuChE
inhibition potency. Besides, compound 6q with ter-butyl group on para position of benzyl amido
moiety was the highest selective AChE inhibitor (SI 5793). It is worth highlighting that all
assayed compounds exhibiting not only no neurotoxicity but even a strong neuroprotective effect
than catechin, reference compound. Both the inhibition kinetic and docking studies demonstrated
the capability of prototype compounds to bind with the CAS and PAS of AChE simultaneously,
and inducing a strong inhibitory effect. In silico ADMET prediction indicated that compounds
are CNS active and comply with the Lipinski's rule of five. An in vivo brain AChE activity study
indicated that 6n would probably cross the Blood Brain Barrier. Altogether, the results suggest
that the tricyclic fused coumarin derivatives, in particular compound 6n, can be considered as
potential therapeutic agents for AD. The work also presents a new family of multifunctional
leading structures for developing novel anti-AD drugs.
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4. Experimental section
4.1 General information
All commercially available starting materials and reagents were purchased from Merck and were
used without further purification. Reaction progress was monitored using analytical thin layer
chromatography (TLC) on precoated silica gel 60 F254 aluminum plates and the spots were
detected under UV light (254 nm). Melting points were measured on GUNA melting point
apparatus and were uncorrected. The IR spectra were taken as KBr discs using Perkin Elmer
Spectrum 2 spectrophotometer (Singapore). The NMR spectra were recorded on a Bruker
spectrometer operating at 400MHz (1H) and at 100 MHz (13C). The chemical shifts (δ) and
coupling constants (J) are expressed in parts per million and hertz, respectively. Splitting patterns
are designated as s, singlet; d, doublet; t, triplet; m, multiplet. The atoms numbering of the
intermediate and target compounds used for NMR data are depicted in scheme 1. Mass spectra
were determined on an Agilent LC/MSD trap SL 1100 series spectrometer with a 70 eV (ESI
probe) and high-resolution mass spectra were obtained by using ESI-QTOF mass spectrometry.
4.2 General procedure for the synthesis of N-substituted cyano acetamide derivatives (2a–s)
To a solution of different amines (1 mmol) in ethanol (10mL), ethylcyanoacetate (1.2 mmol) was
added. The reaction mixture was stirred for 5-8 h under reflux. Progress of the reaction was
0
monitored by TLC. After completion of the reaction, the resulting mixture was cooled to 0–5 C
in an ice bath and the solid separated was filtered off [32,33].
4.3 Representative procedure for the preparation of 7-hydroxy-4-methylcoumarin (4)
0
In a round bottom flask (100 mL), charged conc. sulfuric acid (20.00 ml) and cooled to 0–5 C
in an ice bath. A solution of resorcinol (1 mmol) in ethylacetoacetate (1.5 mmol) was added to
0
sulfuric acid under constant stirring at 0–5 C. The reaction mixture was stirred overnight at
room temperature, then it was slowly poured into crushed ice and the resulting solid was
separated by filtration and washed successively with water. The product was dried and
recrystallized with ethanol to get compound 4 [34].
4.4 Synthetic procedure for 8-formyl-7-hydroxy-4-methylcoumarin (5)
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A reaction mixture of 4 (20 g, 114 mmol) and hexamethylenetetramine (40 g, 285 mmol) in
glacial acetic acid (150 mL) was stirred for 5.5 h at 950C. Thereafter, aqueous solution of HCl
(300 mL, conc. HCl/H2O = 84:100, v/v) was added, stirred further for 0.5 h at 700C and then the
final reaction mixture after cooling was poured into ice water. The resulting solution was
exhaustively extracted with ethyl acetate. The combined organic extracts were dried over Na2SO4
and the solvent was removed under reduced pressure. The crude solid was purified by
crystallization from ethylacetate [35].
4.5 General synthetic procedure for Synthesis of final product: (6a–s)
To a solution of 5 (1.5 mmol) and 2a–s (3.1 mmol) in ethanol (10 mL) was added Et3N (0.1
mmol) drop wise at room temperature. The resulting mixture was stirred at 50 °C for 2-3 h and
then allowed to cool to room temperature. The product was precipitated from the reaction
mixture, collected by filtration and washed with methanol (2-3 mL) to yield corresponding final
products (6a–s) [36].
4.5.1. N9,4-dimethyl-8-imino-2-oxo-2H,8H-pyrano[2,3-f]chromene-9-carboxamide (6a)
Off white solid; yield 69%; mp: 150–152 °C; IR (KBr) vmax: 3415, 3309 (N–H), 2986, 2927,
1
1738 (C=O), 1685 (C=N), 1631 (C=O), 1528, 1386, 1284, 1068, 862, 817, 579 cm-1; H NMR
(400 MHz, DMSO-d6) δ: 9.99 (br s, 1H, 13-NH), 9.29 (s, 1H, 10-H), 8.51(s, 1H, 8-NH), 7.88 (d,
1H, J = 8.4 Hz, 5-H), 7.16 (d, 1H, J = 8.4 Hz, 6-H), 6.40 (s, 1H, 3-H), 2.85 (br s, 3H, 1'-CH3),
2.43 (s, 3H, 11-CH3); 13C NMR (100 MHz, DMSO-d6) δ: 167.9 (C-2), 164.9 (C-8), 161.8 (C-12),
159.7 (C-1a), 153.3 (C-4), 152.5 (C-6a), 151.3 (C-10), 125.4 (C-5), 116.6 (C-4a), 115.6 (C-9),
113.7 (C-3), 112.9 (C-10a), 109.3 (C-6), 32.4 (C-1'), 19.0 (C-11); ESI-MS m/z: 286.1 [M+H] +;
HRMS calcd. for C15H13O4N2: 285.0876, found: 285.0885.
4.5.2. N9-ethyl-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9-carboxamide (6b)
Reddish brown solid; yield 70%; mp: 151–154 °C; IR (KBr) vmax: 3443, 3250 (N–H), 3079,
2853, 2854, 2926, 1733 (C=O), 1671 (C=N), 1627 (C=O), 1552, 1445, 1387, 1191, 1072, 847,
1
772, 585 cm-1; H NMR (400 MHz, CDCl3) δ: 9.97 (br s, 1H, 13-NH), 8.93 (s, 1H, 10-H), 7.71
(s, 1H, 8-NH), 7.63 (d, 1H, J = 8.8 Hz, 5-H), 7.03 (d, 1H, J = 8.8 Hz, 6-H), 6.24 (s, 1H, 3-H),
13
3.46 (m, 2H, 1'-CH2), 2.42 (s, 3H, 11-CH3), 1.23 (t, 3H, J = 13.2 Hz, 2'-CH3); C NMR (100
17

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MHz, CDCl3) δ: 161.3 (C-2), 159.2 (C-8), 156.4 (C-12), 155.4 (C-1a), 152.0 (C-4), 150.9 (C-6a),
134.6 (C-10), 127.9 (C-5), 121.3 (C-4a), 115.8 (C-9), 114.0 (C-3), 111.5 (C-10a), 108.6 (C-6),
34.8 (C-1'), 18.9 (C-11), 14.7 (C-2'); ESI-MS m/z: 299.1 [M+H] +; HRMS calcd. for C16H15O4N2:
299.10263, found: 299.10267.
4.5.3. N-dodecyl-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9-carboxamide (6c)
Orange solid; yield 50%; mp: 164–167 °C; IR (KBr) vmax: 3440, 3321 (N–H), 3204, 3067, 2956,
1
2921, 2852, 1726 (C=O), 1679 (C=N), 1642, 1625 (C=O), 1584, 1201, 1080, 858, 771 cm-1; H
NMR (400 MHz, CDCl3) δ: 10.00 (br s, 1H, 13-NH), 8.89 (s, 1H, 10-H), 7.71 (s, 1H, 8-NH),
7.63 (d, 1H, J = 8.8 Hz, 5-H), 7.02 (d, 1H, J = 8.8 Hz, 6-H), 6.22 (s, 1H, 3-H), 3.41 (q, 2H, J = 6
Hz, 1'-CH2), 2.41 (s, 3H, 11-CH3), 1.58 (m, 2H, 2'- CH2), 1.24 (br s, 18H, 3'–11'- CH2) 0.86 (t,
13
3H, J = 6.8 Hz, 12'-CH3); C NMR (100 MHz, CDCl3) δ: 161.3 (C-2), 159.2 (C-8), 156.4 (C-
12), 155.4 (C-1a), 152.0 (C-4), 150.8 (C-6a), 134.4 (C-10), 127.9 (C-5), 121.3 (C-4a), 115.8 (C-
9), 114.0 (C-3), 111.5 (C-10a), 108.5 (C-6), 40.0 (C-1'), 32.0 (C-2'), 29.7 (C-3'), 29.7 (C-4'), 29.7
(C-5'), 29.6 (C-6'), 29.4 (C-7'), 29.4 (C-8',9'), 27.2 (C-10'), 22.8 (C-11'), 18.9 (C-11), 14.2 (C-
12'); ESI-MS m/z: 439.4 [M + H] +; HRMS calcd. for C26H35O4N2: 439.2598, found: 439.2604.
4.5.4. N9-cyclopropyl-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9-carboxamide
(6d)
Yellow solid; yield 70%; mp: 115–117 °C; IR (KBr) vmax: 3447, 3289 (N–H), 3063, 2950, 2876,
1739 (C=O), 1675 (C=N), 1625 (C=O), 1574, 1452, 1386, 1346, 1203, 1195, 820, 767, 598 cm-1;
1H NMR (400 MHz, CDCl3) δ: 10.05 (d, 1H, J = 2.4 Hz, 13-NH), 8.92 (s, 1H, 10-H), 7.67 (s,
1H, 8-NH), 7.64 (d, 1H, J = 8.8 Hz, 5-H), 7.02 (d, 1H, J = 8.8 Hz, 6-H), 6.24 (s, 1H, 3-H), 2.97-
13
2.94 (m, 1H, 1'-H), 2.42 (s, 3H, 11-CH3), 0.86-0.59 (m, 4H, 2'- CH2, 3'- CH2); C NMR (100
MHz, CDCl3) δ: 162.8 (C-2), 159.2 (C-8), 156.4 (C-12), 155.4 (C-1a), 152.1 (C-4), 150.9 (C-6a),
134.5 (C-10), 128.0 (C-5), 121.0 (C-4a), 115.8 (C-9), 114.0 (C-3), 111.5 (C-10a), 108.5 (C-6),
+
23.0 (C-1'), 18.9 (C-11), 6.65 (C-2', C-3'); ESI-MS m/z: 311.1 [M+H] ; HRMS calcd. for
C17H15O4N2: 311.1032, found: 311.1036.
4.5.5.N9-cyclohexyl-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9-carboxamide (6e)
18

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Orange solid; yield 71%; mp: 148–151 °C; IR (KBr) vmax: 3461, 3285 (N–H), 3190, 2924, 1737
(C=O), 1675 (C=N), 1626 (C=O), 1578, 1447, 1391, 1347, 1279, 1172, 821, 770 cm-1; 1H NMR
(400 MHz, CDCl3) δ: 10.02 (d, 1H, J = 7.2 Hz, 13-NH), 8.90 (s, 1H, 10-H), 7.71 (s, 1H, 8-NH),
7.63 (d, 1H, J = 8.8 Hz, 5-H), 7.02 (d, 1H, J = 8.8 Hz, 6-H), 6.23 (s, 1H, 3-H), 3.97 (m, 1H, 1'-
H), 2.42 (s, 3H, 11-CH3), 1.96 (m, 4H, 2'- CH2, 6'-CH2), 1.24 (m, 6H, 3'–5'-CH2); 13C NMR (100
MHz, CDCl3) δ: 160.3 (C-2), 159.2 (C-8), 156.4 (C-12), 155.4 (C-1a), 152.1 (C-4), 150.8 (C-6a),
134.5 (C-10), 127.8 (C-5), 121.5 (C-4a), 115.7 (C-9), 114.0 (C-3), 111.5 (C-10a), 108.6 (C-6),
48.4 (C-1'), 32.7 (C-2',5'), 25.8 (C-4'), 24.6 (C-3',5'), 18.9 (C-11); ESI-MS m/z: 353.1 [M+H] +;
HRMS calcd. for C20H21O4N2: 353.1502, found: 353.1510.
4.5.6. 8-imino-4-methyl-9-(tetrahydro-1H-1-pyrrolylcarbonyl)-2H,8H-pyrano[2,3-f]chromen-
2-one (6f)
Pale reddish brown solid; yield 63%; mp: 140–144 °C; IR (KBr) vmax: 3439, 3057, 2956, 2924,
2855, 1732 (C=O), 1686 (C=N), 1627 (C=O), 1591, 1575, 1449, 1388, 1169, 1085, 834, 592 cm-
1; 1H NMR (400 MHz, CDCl3) δ: 8.49 (s, 1H, 10-H), 7.82 (s, 1H, 8-NH), 7.77 (d, 1H, J = 8.8 Hz,
5-H), 7.10 (d, 1H, J = 8.8 Hz, 6-H), 6.31 (s, 1H, 3-H), 3.63 (m, 2H, 2'-CH2), 3.44 (m, 2H, 5'-
CH2), 2.47 (s, 3H, 11-CH3), 1.95 (m, 4H, 3'- CH2, 4'- CH2); 13C NMR (100 MHz, CDCl3) δ:
162.6 (C-2), 158.8 (C-8) 156.9 (C-12), 155.8 (C-1a), 152.3 (C-4), 150.4 (C-6a), 136.1 (C-10),
128.2 (C-5), 126.4 (C-4a), 116.1 (C-9), 114.4 (C-3), 112.8 (C-10a), 108.1 (C-6), 47.8 (C-2'), 46.3
+
(C-5'), 26.0 (C-3'), 24.4 (C-4'), 19.0 (C-11); ESI-MS m/z: 326.1 [M+H] ; HRMS calcd. for
C18H17O4N2: 326.10278, found: 326.10268.
4.5.7. 8-imino-4-methyl-9-(morpholinocarbonyl)-2H,8H-pyrano[2,3-f]chromen-2-one (6g)
Pale yellow solid; yield 58%; mp: 137–140 °C; IR (KBr) vmax: 3436, 3073, 2924, 2853, 1727
(C=O), 1667 (C=N), 1637 (C=O), 1593, 1573, 1466, 1373, 1275, 1201, 1108, 1067, 847, 770,
593 cm-1; 1H NMR (400 MHz, CDCl3) δ: 8.50 (s, 1H, 10-H), 7.82 (s, 1H, 8-NH), 7.60 (d, 1H, J =
8.8 Hz, 5-H), 7.03 (d, 1H, J = 8.8 Hz, 6-H), 6.25 (s, 1H, 3-H), 3.77 (t, 4H, 3'-CH2, 5'-CH2), 3.58
(t, 4H, 2'-CH2, 6'-CH2), 2.43 (s, 3H, 11-CH3); 13C NMR (100 MHz, CDCl3) δ: 164.3 (C-2,8),
159.5 (C-12), 155.4 (C-1a), 152.4 (C-4), 150.1 (C-6a), 137.0 (C-10), 127.5 (C-5), 121.5 (C-4a),
115.7 (C-9), 113.8 (C-3), 111.9 (C-10a), 107.6 (C-6), 66.8 (C-3',5'), 47.4 (C-2',6'), 18.9 (C-11);
ESI-MS m/z: 341.1 [M + H] +; HRMS calcd. for C18H17O5N2: 341.11320, found: 341.11347.
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4.5.8. N9-benzyl-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9-carboxamide (6h)
Pale yellow solid; yield 80%; mp: 157–161 °C; IR (KBr) vmax: 3440, 3297 (N–H), 3076, 2926,
1722 (C=O), 1677 (C=N), 1624 (C=O), 1577, 1540, 1392, 1243, 1204, 1177, 1124, 1072, 850,
770, 593 cm-1; 1H NMR (400 MHz, CDCl3) δ: 10.43 (br s, 1H, 13-NH), 8.96 (s, 1H, 10-H), 7.68
(s, 1H, 8-NH), 7.63 (d, 1H, J = 8.4 Hz, 5-H), 7.35-7.25 (m, 5H, 2'-H, 3'-H, 4'-H, 5'-H, 6'-H), 7.02
(d, 1H, J = 8.4 Hz, 6-H), 6.23 (s, 1H, 3-H), 4.64 (d, 2H, J = 4 Hz, 14-CH2), 2.41 (s, 3H, 11-CH3);
13C NMR (100 MHz, CDCl3) δ: 161.5 (C-2), 159.2 (C-8), 156.3 (C-12), 155.4 (C-1a), 152.0 (C-
4), 150.9 (C-6a), 138.4 (C-1'), 135.0 (C-10), 128.7 (C-3',5'), 128.0 (C-5), 127.8 (C-2',6'), 127.4
(C-4'), 121.1 (C-4a), 115.8 (C-9), 114.0 (C-3), 111.5 (C-10a), 108.5 (C-6), 44.0 (C-14), 18.9 (C-
11); ESI-MS m/z: 361.1 [M + H] +; HRMS calcd. for C21H17O4N2: 361.11828, found: 361.11866.
4.5.9. 8-imino-4-methyl-2-oxo-N-(1-phenylethyl)-2H,8H-pyrano[2,3-f]chromene-9-
carboxamide (6i)
Pale yellow solid; yield 66%; mp: 158–160 °C; IR (KBr) vmax: 3444, 3273 (N–H), 3061, 3028,
2969, 2927, 1727 (C=O), 1677 (C=N), 1626 (C=O), 1579, 1550, 1494, 1448, 1389, 1251, 1201,
1062, 823, 770, 701, 598 cm-1; 1H NMR (400 MHz, CDCl3) δ: 10.53 (d, 1H, J = 7.2 Hz, 13-NH),
8.96 (s, 1H, 10-H), 7.73 (s, 1H, 8-NH), 7.63 (d, 1H, J = 8.8 Hz, 5-H), 7.41-7.25 (m, 5H, 2'-H, 3'-
H, 4'-H, 5'-H, 6'-H ), 7.03 (d, 1H, J = 8.8 Hz, 6-H), 6.24 (s, 1H, 3-H), 5.31-5.27 (m, 1H, 14-H),
2.41 (s, 3H, 11-CH3) 1.57 (d, 3H, J = 7.2 Hz, 15-CH3); 13C NMR (100 MHz, CDCl3) δ: 160.5 (C-
2), 159.1 (C-8), 156.4 (C-12), 155.3 (C-1a), 152.0 (C-4), 150.8 (C-6a), 143.7 (C-1'), 134.8 (C-
10), 128.7 (C-3',5'), 128.0 (C-5), 127.2 (C-4'), 126.3 (C-2',6'), 121.1 (C-4a), 115.8 (C-9), 114.0
(C-3), 111.5 (C-10a), 108.4 (C-6), 49.6 (C-14), 22.7 (C-15), 18.9 (C-11); ESI-MS m/z: 375.2
[M + H] +; HRMS calcd. for C22H19O4N2: 375.1345, found: 375.1353.
4.5.10. N9-benzhydryl-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9-
carboxamide (6j)
Pale yellow solid; yield 74%; mp: 174–176 °C; IR (KBr) vmax: 3454, 3317 (N–H), 3201, 3209,
3060, 2925, 1747 (C=O), 1675 (C=N), 1624 (C=O), 1574, 1453, 1389, 1225, 1197, 1067, 832,
760, 596 cm-1; 1H NMR (400 MHz, CDCl3) δ: 11.07 (d, 1H, J = 8.4 Hz, 13-NH), 8.91 (s, 1H, 10-
H), 7.74 (s, 1H, 8-NH), 7.60 (d, 1H, J = 8.8 Hz, 5-H), 7.35-7.27 (m, 10H, 2'-H, 3'-H, 4'-H, 5'-H,
20

ACCEPTED MANUSCRIPT
6'-H and 2''-H, 3''-H, 4''-H, 5''-H, 6''-H), 7.01 (d, 1H, J = 8.8 Hz, 6-H), 6.46 (d, 1H, J = 8.4 Hz,
13
14-H), 6.20 (s, 1H, 3-H), 2.37 (s, 3H, 11-CH3); C NMR (100 MHz, CDCl3) δ: 160.7 (C-2),
159.1 (C-8), 156.4 (C-12), 155.4 (C-1a), 152.0 (C-4), 150.8 (C-6a), 142.1 (C-1',1''), 135.1(C-10),
128.7 (C-3',5' and C-3'',5''), 128.1(C-5), 127.5 (C-2',6' and C-2'', 6''), 127.0 (C-4',4''), 121.0 (C-
4a), 115.8 (C-9), 114.0 (C-3), 111.5 (C-10a), 108.4 (C-6), 57.5 (C-14), 18.91 (C-11); ESI-MS
m/z: 437.1 [M + H] +; HRMS calcd. for C27H21O4N2: 437.14958, found: 437.14966.
4.5.11. N9-[1-(4-methoxyphenyl)ethyl]-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9
carboxamide (6k)
Pale yellow solid; yield 80%; mp: 139–142 °C; IR (KBr) vmax: 3447, 3280 (N–H), 3272, 3182,
3094, 3034, 2972, 2930, 2835, 1728 (C=O), 1675 (C=N), 1626 (C=O), 1578, 1446, 1390, 1355,
1
1201, 1175, 1062, 831, 770, 596 cm-1; H NMR (400 MHz, CDCl3) δ: 10.44 (d, 1H, J = 7.6 Hz,
13-NH), 8.91 (s, 1H, 10-H), 7.70 (s, 1H, 8-NH), 7.61 (d, 1H, J = 8.8 Hz, 5-H), 7.32 (d, 2H, J =
8.8 Hz, 2'-H, 6'-H), 7.01 (d, 1H, J = 8.8 Hz, 6-H), 6.87 (d, 2H, J = 8.8 Hz, 3'-H, 5'-H), 6.22 (s,
1H, 3-H), 5.25-5.21 (m, 1H, 14-H), 3.78 (s, 3H, 4'-OCH3), 2.40 (s, 3H, 11-CH3), 1.55 (d, 3H, J =
6.8 Hz, 15-CH3); 13C NMR (100 MHz, CDCl3) δ: 160.5 (C-2), 159.2 (C-8), 158.7 (C-12), 156.4
(C-4'), 155.4 (C-1a), 152.0 (C-4), 150.8 (C-6a), 135.9 (C-1'), 134.8 (C-10), 128.0 (C-5), 127.4
(C-2', C-6'), 121.2 (C-4a), 115.8 (C-9), 114.0 (C-3',5'), 114.0 (C-3), 111.5 (C-10a), 108.5 (C-6),
55.4 (4'-OCH3), 48.9 (C-14), 22.6 (C-15), 18.9 (C-11); ESI-MS m/z: 405.2 [M + H] +; HRMS
calcd. for C23H21O5N2: 405.14450, found: 405.14473.
4.5.12. N-(2,4-dimethoxybenzyl)-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9-
carboxamide (6l )
Brick red solid; yield 81%; mp: 152–155 °C; IR (KBr) vmax: 3447, 3297 (N–H), 3086, 2935,
1740 (C=O), 1675 (C=N), 1626 (C=O), 1578, 1389, 1347, 1225, 1198, 1069, 834, 768, 596 cm-1;
1H NMR (400 MHz, CDCl3) δ: 10.34 (t, 1H, J = 5.6 Hz, 13-NH), 8.89 (s, 1H, 10-H), 7.67 (s, 1H,
8-NH), 7.59 (d, 1H, J = 8.8 Hz, 5-H), 7.24 (s, 1H, 3'-H), 6.98 (d, 1H, J = 8.8 Hz, 6-H), 6.46-6.42
(m, 2H, 5'-H, 6'-H), 6.21 (s, 1H, 3-H), 4.55 (d, 2H, J = 5.6 Hz, 14-CH2), 3.84 (s, 3H, 4'-OCH3),
3.78 (s, 3H, 2'-OCH3), 2.40 (s, 3H, 11-CH3); 13C NMR (100 MHz, CDCl3) δ: 161.1 (C-2), 160.4
(C-8), 159.2 (C-12), 158.7 (C-4'), 156.2 (C-2'), 155.4 (C-6a), 152.1 (C-4), 150.8 (C-1a), 134.5
(C-6'), 130.2 (C-10), 127.9 (C-5), 121.4 (C-1'), 119.1 (C-10a), 115.7 (C-9), 113.9 (C-4a), 111.5
21

ACCEPTED MANUSCRIPT
(C-6), 108.5 (C-5'), 103.9 (C-3'), 98.6 (C-6), 55.5 (2', 4' -OCH3), 39.1 (C-14), 18.9 (C-11); ESI-
MS m/z: 421.2 [M + H] +; HRMS calcd. for C23H21O6N2: 421.13941, found: 421.13980.
4.5.13. N-(2-bromobenzyl)-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9
carboxamide (6m)
Orange solid; yield 73%; mp: 207–209°C; IR (KBr) vmax: 3439, 3309 (N–H), 3209, 3076, 2924,
2853, 1721 (C=O), 1689 (C=N), 1623 (C=O), 1575, 1556, 1441, 1393, 1204, 1025, 771 cm-1; 1H
NMR (400 MHz, CDCl3) δ: 10.56 (br s, 1H, 13-NH), 9.02 (s, 1H, 10-H), 7.75 (s, 1H, 8-NH),
7.65 (d, 1H, J = 8.8 Hz, 5-H), 7.56 (d, 1H, J = 9.2 Hz, 3'-H), 7.47 (d, 1H, J = 9.2 Hz, 6'-H), 7.29
(t, 1H, J = 7.6 Hz, 4'-H), 7.14 (t, 1H, J = 7.6 Hz, 5'-H), 7.04 (d, 1H, J = 8.8 Hz, 6-H), 6.26 (s,
1H, 3-H), 4.71 (d, 2H, J = 6 Hz, 14-CH2), 2.43 (s, 3H, 11-CH3); 13C NMR (100 MHz, DMSO-d6)
δ: 161.5 (C-2), 159.2 (C-8), 155.6 (C-12), 154.5 (C-1a), 153.8 (C-4), 150.5 (C-6a), 137.7 (C-1'),
133.0 (C-10), 132.9 (C-3'), 130.5 (C-6'), 129.9 (C-4'), 128.7 (C-5), 128.3 (C-5'), 123.2 (C-2'),
120.8 (C-4a), 115.7 (C-9), 113.3 (C-3), 111.7 (C-10a), 109.2 (C-6) 43.8 (C-14), 18.7 (C-11);
ESI-MS m/z: 439.1 [M + H] +; HRMS calcd. for C21H16BrO4N2: 439.0294, found: 439.0298.
4.5.14. N-(3-bromobenzyl)-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-
9- carboxamide (6n)
Orange solid; yield 68%; mp: 188–190°C; IR (KBr) vmax: 3448, 3300 (N–H), 3206, 3062, 2923,
1726 (C=O), 1678 (C=N), 1625 (C=O), 1574, 1589, 1428, 1389, 1199, 1071, 771 cm-1; 1H NMR
(400 MHz, CDCl3) δ: 10.49 (t, 1H, J = 11.2 Hz, 13-NH), 8.89 (s, 1H, 10-H), 7.71 (s, 1H, 8-NH),
7.63 (d, 1H, J = 8.8 Hz, 5-H), 7.48 (s, 1H, 2'-H), 7.37 (d, 1H, J = 7.6 Hz, 4'-H), 7.27 (d, 1H, J =
7.6 Hz, 6'-H), 7.19 (t, 1H, J = 7.6 Hz, 5'-H), 7.01 (d, 1H, J = 8.8 Hz, 6-H), 6.22 (s, 1H, 3-H), 4.59
(d, 2H, J = 5.6 Hz, 14- CH2), 2.40 (s, 3H, 11-CH3); 13C NMR (100 MHz, CDCl3) δ: 161.7 (C-2),
159.1 (C-8), 156.2 (C-12), 155.4 (C-1a), 152.1 (C-4), 150.8 (C-6a), 140.8 (C-1'), 135.1 (C-10),
130.7 (C-2'), 130.4 (C-4'), 130.2 (C-5'), 128.2 (C-5), 126.4 (C-6'), 122.7 (C-3'), 120.8 (C-4a),
115.8 (C-9), 114.0 (C-3), 111.5 (C-10a), 108.3 (C-6) 43.2 (C-14), 18.9 (C-11); ESI-MS m/z:
439.1 [M + H] +; HRMS calcd. for C21H16BrO4N2: 439.0294, found: 439.0300.
4.5.15. N-(2-chlorobenzyl)-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-
9-carboxamide (6o)
22

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Orange solid; yield 81%; mp: 204–206°C; IR (KBr) vmax: 3443, 3316 (N–H), 3074, 2925, 2854,
1721 (C=O), 1689 (C=N), 1624 (C=O), 1576, 1556, 1443, 1204, 1058, 771 cm-1; 1H NMR (400
MHz, CDCl3) δ: 10.55 (br s, 1H, 13-NH), 9.02 (s, 1H, 10-H), 7.75 (s, 1H, 8-NH), 7.64 (d, 1H, J
= 8.8 Hz, 5-H), 7.46 (d, 1H, J = 9.2 Hz, 3'-H), 7.37 (d, 1H, J = 9.2 Hz, 6'-H), 7.24-7.19 (m, 2H,
4'-H, 5'-H), 7.04 (d, 1H, J = 8.8 Hz, 6-H), 6.26 (s, 1H, 3-H), 4.73 (d, 2H, J = 6.0 Hz, 14- CH2),
2.43 (s, 3H, 11-CH3); 13C NMR (100 MHz, DMSO-d6) δ: 161.6 (C-2), 159.7 (C-8), 159.1(C-12),
155.5 (C-1a), 153.7 (C-4), 150.4 (C-6a), 136.1 (C-1'), 132.8 (C-10), 132.2 (C-2'), 130.9 (C-3'),
129.9 (C-6'), 129.7 (C-5), 129.4 (C-4'), 127.8 (C-5'), 120.7 (C-4a), 115.7 (C-9), 113.3 (C-3),
+
111.6 (C-10a), 109.2 (C-6) 46.2 (C-14), 18.6 (C-11); ESI-MS m/z 395.2; [M + H] ; HRMS
calcd. for C21H16ClO4N2: 395.0799, found: 395.0804.
4.5.16. N-(3-chlorobenzyl)-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-
9-carboxamide (6p)
Orange solid; yield 65%; mp: 186–188°C; IR (KBr) vmax: 3444, 3312 (N–H), 3210, 3075, 2924,
1
2855, 1725 (C=O), 1683 (C=N), 1624 (C=O), 1576, 1556, 1431, 1390, 1201, 1073, 771 cm-1; H
NMR (400 MHz, CDCl3) δ: 10.49 (t, 1H, J = 11.2 Hz, 13-NH), 8.88 (s, 1H, 10-H), 7.71 (s, 1H,
8-NH), 7.63 (d, 1H, J = 8.8 Hz, 5-H), 7.32 (s, 1H, 2'-H), 7.27-7.20 (m, 3H, 4'–6'-H), 7.01 (d, 1H,
J = 8.8 Hz, 6-H), 6.21 (s, 1H, 3-H), 4.60 (d, 2H, J = 6.0 Hz, 14-CH2), 2.40 (s, 3H, 11-CH3); 13C
NMR (100 MHz, CDCl3) δ: 161.7 (C-2), 159.1 (C-8), 156.2 (C-12), 155.3 (C-1a), 152.1(C-4),
150.8 (C-6a), 140.6 (C-1'), 135.0 (C-10), 134.5 (C-3'), 129.9 (C-2'), 128.2 (C-5), 127.7 (C-6'),
127.5 (C-4'), 125.9 (C-5'), 120.7 (C-4a), 115.8 (C-9), 113.9 (C-3), 111.5 (C-10a), 108.3 (C-6),
+
43.3 (C-14), 18.9 (C-11); ESI-MS m/z: 395.2 [M + H] ; HRMS calcd. for C21H16ClO4N2:
395.0799, found: 395.0809.
4.5.17. N-(4-(tert-butyl)benzyl)-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-9-
carboxamide (6q)
Pale yellow solid; yield 65%; mp: 216–218°C; IR (KBr) vmax: 3447, 3302 (N–H), 3075, 3048,
1
2926, 2958, 1743 (C=O), 1677 (C=N), 1624 (C=O), 1575, 1540, 1201, 1069, 770 cm-1; H
NMR (400 MHz, CDCl3) δ: 10.41 (t, 1H, J = 9.6 Hz, 13-NH), 8.99 (s, 1H, 10-H), 7.70 (s, 1H, 8-
NH), 7.64 (d, 1H, J = 8.8 Hz, 5-H), 7.49 (d, 2H, J = 8.4 Hz, 2'-H, 6'-H), 7.29 (d, 2H, J = 8.4 Hz,
3'-H, 5'-H), 7.03 (d, 1H, J = 8.8 Hz, 6-H), 6.25 (s, 1H, 3-H), 4.62 (d, 2H, J = 5.6 Hz, 14-CH2),
23

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2.42 (s, 3H, 11-CH3), 1.31 (s, 9H, 4'-C(CH3)3); 13C-NMR (100 MHz, CDCl3) δ: 161.5 (C-2),
159.2 (C-8), 156.3 (C-12), 155.4 (C-1a), 152.0 (C-4), 150.9 (C-6a), 150.3 (C-4'), 135.3 (C-1')
135.0 (C-10), 128.0 (C-5), 127.5 (C-2') 125.6 (C-3') 121.1 (C-4a), 115.8 (C-9), 114.0 (C-3),
111.5 (C-10a), 108.5 (C-6), 43.7 (C-14), 34.6 (3'-C(CH3)3), 31.4 (3'-C(CH3)3), 18.9 (C-11); ESI-
MS m/z: 417.3 [M + H] +; HRMS calcd. for C25H25O4N2: 417.1815, found: 417.1825.
4.5.18. N9-[2-(2-methoxyphenoxy)ethyl]-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-
f]chromene-9-carboxamide (6r)
Off white solid; yield 79%; mp: 124–128°C; IR (KBr) vmax: 3471, 3293 (N–H), 3065, 2953,
2932, 2877, 2839, 1743 (C=O), 1674 (C=N), 1625 (C=O), 1574, 1454, 1388, 1348, 1222, 1196,
1
819, 769, 596 cm-1; H NMR (400 MHz, CDCl3) δ: 10.42 (t, 1H, 13-NH), 8.90 (s, 1H, 10-H),
7.78 (s, 1H, 8-NH), 7.63 (d, 1H, J = 8.8 Hz, 5-H), 7.02-6.90 (m, 5H, 6-H, 3'–6'-H), 6.23 (s, 1H,
13
3-H), 4.21 (m, 2H, 15-CH2), 3.86 (m, 5H, 14- CH2, 2'-OCH3), 2.41 (s, 3H, 11-CH3); C-NMR
(100 MHz, CDCl3) δ: 161.9 (C-2), 159.2 (C-8), 156.0 (C-12), 155.4 (C-1a), 152.1 (C-4), 150.8
(C-1'), 150.0 (C-2'), 148.2 (C-6a), 134.7 (C-10), 128.0 (C-5), 121.9 (C-4'), 121.1 (C-5'), 121.1
(C-3'), 115.7 (C-4a), 114.8 (C-9), 113.9 (C-3), 112.4 (C-10a), 111.5 (C-6'), 108.4 (C-6), 68.2 (C-
15), 56.1 (2'-OCH3), 39.5 (C-14), 18.9 (C-11); ESI-MS m/z: 421.2 [M + H]+; HRMS calcd. for
C23H21O6N2: 421.13941, found: 421.13923.
4.5.19. N9-(2-nitrophenyl)-8-imino-4-methyl-2-oxo-2H,8H-pyrano[2,3-f]chromene-
9-carboxamide (6s)
Yellow crystal; yield 64%; mp: 81–84 °C; IR (KBr) vmax: 3440, 3295 (N–H), 3061, 2951, 2877,
1743 (C=O), 1673 (C=N), 1630 (C=O), 1573, 1452, 1385, 1346, 1205, 1192, 820, 767, 595 cm-1;
1H NMR (400 MHz, CDCl3) δ: 10.61 (br s, 1H, 13-NH), 8.43 (d, 1H, J = 8.0 Hz, 3'-H), 8.35 (m,
3H, 8-NH, 10-H, 6'-H), 7.72 (d, 1H, J = 8.8 Hz, 5-H), 6.90 (d, 1H, J = 8.8 Hz, 6-H), 6.74 (m,
2H, 4'-H, 5'-H), 6.20 (s, 1H, 3-H), 2.42 (s, 1H, 11-CH3); 13C NMR (100 MHz, CDCl3) δ: 168.1
(C-2), 167.9 (C-8), 164.9 (C-12), 164.5 (C-1a), 161.8 (C-4), 159.6 (C-6a), 153.4 (C-2'), 143.4 (C-
5'), 143.2 (C-1'), 132.8 (C-10), 128.6 (C-6'), 128.4 (C-5), 115.6 (C-4a), 115.5 (C-3'), 115.3 (C-4'),
115.0(C-9), 113.3 (C-3), 113.1 (C-10a), 109.4 (C-6), 18.6 (C-11); ESI-MS m/z: 392.1 [M+H] +;
HRMS calcd. for C20H14O4N2: 392.1026, found: 392.1028.
24

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4.6 Biological Assays
4.6.1. Determination of AChE and BuChE inhibitory activities
Inhibitory potency of target compounds against AChE and BuChE were assayed using
spectrophotometric method developed by Ellman et al. with slight modification [37,38]. EeAChE
(EC. 3.1.1.7, Electric eel) EqBuChE (EC 3.1.1.8, horse serum), DTNB, ATC and BTC iodide
were purchased from Sigma Aldrich, USA. In brief, reaction mixture composed of 10 µL of test
sample of five different concentrations (0.2, 0.4, 0.6, 0.8 and 1.0 µM for AChE assay; 15, 30, 60,
90, 120 µM for BuChE assay), 145 µL phosphate buffer 200mM (pH 7.7), 80µL of
dithiobisnitrobenzoic acid (DTNB) (18.5 mg of DTNB dissolved in 10 mL phosphate buffer pH
7.7), and 10µL of enzyme (0.4 U/mL). The mixture was incubated at 25 ◦C for 5 min.
Subsequently, the enzymatic reaction was initiated by addition of 15µL of 1mM of
acetylthiocholine iodide or butyrylthiocholine iodide (according to the respective enzyme) and
the mixture was again incubated for 5 min at 25 ◦C. The rate of absorbance change was measured
at 412 nm for 6 min using a microplate reader (Bio-Rad 680). Inhibition percentage was
calculated as follows: % inhibition = (E – S) / E × 100, where E is the enzymatic activity without
test compound and S is the enzymatic activity with the test compound. The IC50 values were
determined graphically from inhibition curves (log inhibitor concentration Vs. percent of
inhibition).
4.6.2. In vitro antioxidant activity assay
The radical scavenging activity of the test compounds was measured by the ABTS method [39].
The ABTS was dissolved in water to obtain an 8 mM concentration of ABTS stock solution.
ABTS radical cation (ABTS•+) was generated by adding 3 mM potassium persulfate to the ABTS
stock solution and keeping it in the dark at room temperature for 12-18 h. The ABTS•+ solution
was diluted with ethanol to give an absorbance of 0.48 ± 0.07 at 734 nm. The 10 µL of the test
compounds were allowed to react with 290 µL of ABTS•+ solution. The absorbance was taken 30
min after initial mixing. Trolox was used as a standard.
4.6.3. Cell culture, treatment and cell viability (MTT) assay
25

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SK N SH, human neuroblastoma cells were obtained from National Centre for Cell Sciences.
(Pune, India). The cells were cultured in MEM containing 0.5 mM L-glutamine, 0.1 mM sodium
pyruvate and 1 mM non-essential amino acids with 10% FBS in a highly humidified atmosphere
having 5% CO2 at 37 °C. Cell cultures at approximately 80% confluence were used for all in
vitro experimental procedures.
4.6.3.1. Measurement of Cell viability by MTT assay:
Cytotoxic effects of selected compounds (6m, 6n, 6o, 6p and 6q) on the cell viability was
measured by using MTT assay as described in our previous studies [40,41].SK N SH cells (0.2 X
106 cells per well) in 200 ꢀL of corresponding medium with 10% FBS were seeded into 96-
wellplate. Increasing concentrations of compounds or galantamine dissolved in DMSO were
added to the cells and incubated at 37°C for 24 h in a humidified CO2 incubator with 5% CO2.
The medium was replaced along with 20 ꢀL of 5 mg/mL MTT (3-(4, 5-dimethylthiozol-2-yl)-
2,5-diphenyltetrazoliumbromide). It was further incubated for 4 h in humidified atmosphere, the
medium was removed and 200 ꢀL of 0.1 N acidic isopropyl alcohol was added to the wells to
dissolve the MTT formazan crystals. Absorbance was recorded at 570 nm immediately after the
development of purple color. Relative cell viability was evaluated according to the quantity of
MTT converted into insoluble formazan salt. The optical density of the formazan generated in the
control cells was considered to represent100% viability. The results were expressed as mean
percent of viable cells versus respective control. Experiments were repeated three times and the
results represented as averages with standard error.
4.6.3.2. Protection against H2O2 induced cell death in SK N SH cell
Neuroprotective effect of selected compounds (6m, 6n, 6o, 6p and 6q) against H2O2 induced
oxidative injury in SK N SH cells was determined [40,41]. Thus, SK N SH cells were pretreated
with different concentrations of compounds (40 – 120 µM) and galantamine as reference
compound for 3 h before treatment with H2O2 (1.0mM). H2O2 was added to the medium to
induce oxidative stress and incubated for 24 hours in humidified atmosphere. The cell viability
was measured by MTT colorimetry as described above.
4.7 Kinetic study
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To obtain estimates of the inhibition model and inhibition constant Ki, reciprocal plots of 1/V
versus 1/[S] were constructed at different concentrations of the substrate acetylthiocholine iodide
(0.1–0.5 mM) by using Ellman’s method [42]. The assay solution (250 µL) consists of 145 µL of
200 mM phosphate buffer (pH 7.7), with the addition of 80 µL of DTNB (18.5 mg of DTNB
dissolved in 10 mL phosphate buffer pH 7.7), 10 µL of 0.4 units/mL AChE, and 15 µL of
substrate (ATCh). Three different concentrations of inhibitors (0.002, 0.005 and 0.02 µM) were
added to the assay solution and pre-incubated for 5 min at 25 ◦C with the AChE followed by the
addition of substrate in different concentrations. The parallel control experiments were
performed without inhibitor in the assay. Progress curves were monitored at 412 nm over 6 min.
Then, double reciprocal plots (1/v Vs. 1/[s]) were constructed using Graph pad prism version 5.
The re-plots of the slopes and intercepts of the double reciprocal plots against inhibitor
concentrations gave the inhibitor constants (Ki1 and Ki2, for the binding to free enzyme and
enzyme substrate complex) as the intercepts on the x-axis. Data analysis was performed using
Microsoft Excel.
4.8 Molecular modeling study
To predict the binding modes of target compounds in the active sites of AChE and BuChE
enzymes, molecular docking studies were performed using Schrödinger maestro software
(version 9.2; Schrödinger LLC, NewYork). The proteins used for docking are the crystal
structure of Electric eel acetyl cholinesterase (PDB ID: 1C2O) and the predicted 3D model of
horse butyrylcholinesterase [43]. The results were characterized by the G Scores obtained from
GLIDE (Grid-based Ligand Docking with Energetics) docking [44].
4.8.1. Protein Preparation
Prior to docking, proteins were prepared using protein preparation wizard. Hydrogen atoms were
added to the proteins and charges were assigned. Water molecules and other heteroatoms were
excluded from the crystal structure as they were not significant for the proteins function.
Subsequently, the proteins were optimized and energy minimized using OPLS2005 force field. A
grid was generated around the active site of the enzyme by specifying the key active site residues
reported in the literature using GLIDE module of Schrodinger.
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4.8.2. Ligand Preparation
The ligands used as inputs for docking were designed using ChemBioOffice 2010 software and
prepared using LigPrep (version 2.5, Schrödinger Inc.). Preparation of ligands includes
assigning bond orders, generating various tautomers, ring conformations and stereo chemistries.
All the conformations generated were minimized using OPLS-AA force field before proceeding
for docking studies.
4.8.3. Receptor grid generation
A receptor grid was generated around the protein active site by choosing the residues D74, T83,
W86, G120, G121, G122, Y124, Y133, E202, S203, W286, F295, F297, Y337, and Y341
reported in the literature. Grid box size was set at 20 Å × 20 Å ×20 Å and Vander Waal radii of
receptor atoms were scaled to 1.00 Å with a partial atomic charge of 0.25.
4.8.4. Docking
Glide module of Schrodinger was used for the docking studies. All docking calculations were
performed using Standard Precision (SP) mode. A scaling factor of 0.8 and a partial atomic
charge of less than 0.15 was applied to the atoms of the protein. The poses generated per ligand
were set to 10,000 and the pose chosen for energy minimization was set to 10. Glide docking
score was used to determine the best docked structure from the output. The interactions of these
docked complexes were investigated further.
4.9 In vivo brain AChE activity assay
4.9.1. Animals
Sixteen adult male BALB/c ByJ mice were used in this study. They were kept in the Central
Animal House facility of Sri Venkateswara University, Tirupati, India. The animals were housed
in a propylene cages in a room maintained at 25 ºC, 45-50% relative humidity under a 12 h
light/dark cycle with ad libitum access to standard rodent pellet diet and water. All experimental
procedures involving the animals were performed in accordance with the Animal Ethics
Committee of Sri Venkateswara University (No. 55/2012/(i)/a/CPCSEA/IAEC/SVU/MBJ, dated
08-07-2012), Tirupati, approved by the Government of India.
4.9.2. Drug adminstrattion
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