Novel coumarin-3-carboxamides bearing N-benzylpiperidine moiety as potent acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.10.024

Abstract Some novel coumarin-3-carboxamide derivatives linked to N-benzylpiperidine scaffold were synthesized and evaluated as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. The screening results showed that most of compounds exhibited potent anti-AChE activity in the range of nM concentrations. Among them, compound 10c bearing an N-ethylcarboxamide linker and a 6-nitro substituent showed the most potent activity (IC₅₀ = 0.3 nM) and the highest selectivity (SI = 26,300). Compound 10c was 46-fold more potent than standard drug donepezil against AChE. The kinetic study revealed that compound 10c exhibited mixed-type inhibition against AChE. Protein-ligand docking study demonstrated that the target compounds have dual binding site interaction mode and these results are in agreement with kinetic study.

Full text of DOI:10.1016/j.ejmech.2013.10.024

European Journal of Medicinal Chemistry 70 (2013) 623e630
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel coumarin-3-carboxamides bearing N-benzylpiperidine moiety
as potent acetylcholinesterase inhibitors
a
b
b
b
c
Ali Asadipour , Masoumeh Alipour , Mona Jafari , Mehdi Khoobi , Saeed Emami ,
d
,
e
d,
e
d,
e
a
Hamid Nadri , Amirhossein Sakhteman , Alireza Moradi , Vahid Sheibani ,
e
b
a,b,
*
Farshad Homayouni Moghadam , Abbas Shafiee , Alireza Foroumadi
Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran
a
b
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran,
Iran
c
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari,
Iran
d
Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Neurobiomedical Research Center, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 May 2013
Received in revised form
Some novel coumarin-3-carboxamide derivatives linked to N-benzylpiperidine scaffold were synthesized
and evaluated as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. The
screening results showed that most of compounds exhibited potent anti-AChE activity in the range of nM
concentrations. Among them, compound 10c bearing an N-ethylcarboxamide linker and a 6-nitro sub-
stituent showed the most potent activity (IC50 ¼ 0.3 nM) and the highest selectivity (SI ¼ 26,300).
Compound 10c was 46-fold more potent than standard drug donepezil against AChE. The kinetic study
revealed that compound 10c exhibited mixed-type inhibition against AChE. Protein-ligand docking study
demonstrated that the target compounds have dual binding site interaction mode and these results are
in agreement with kinetic study.
7
October 2013
Accepted 10 October 2013
Available online 23 October 2013
Keywords:
Acetylcholinesterase
Butyrylcholinesterase
Coumarin
Ó 2013 Elsevier Masson SAS. All rights reserved.
N-Benzylpiperidine
Alzheimer’s disease
Docking
Kinetic study
1. Introduction
reported that dual inhibition of AChE and butyrylcholinesterase
(BuChE) might improve the signs of AD and symptoms owing to the
Alzheimer’s disease (AD) is a neurodegenerative disease with
symptoms of memory loss, cognition defect and behavioral
impairment [1]. AD is mainly characterized by the pre-synaptic
decrease of acetylcholine (ACh) due to damage of cholinergic
neurons in some especial parts of the brain such as hippocampus
and cortex (cholinergic hypothesis) [2,3]. Concerning the cholin-
ergic hypothesis one of the rational and effective approaches to
treat the AD’s symptoms, is raising the ACh through inhibition of
acetylcholinesterase (AChE) that is responsible for hydrolysis of
ACh in pre-synaptic areas [4]. Moreover, it has been recently
key role of BuChE in hydrolysis of ACh. Indeed in damaged brain,
preserving the AChE/BuChE activity ratios is essential for successful
treatment of AD [5,6]. Based on these findings, several AChE in-
hibitors comprising different chemical scaffolds such as donepezil
[7], galantamine [8] and ensaculin [9] were synthesized and used
clinically to prevent progression of AD in early stages (Fig. 1).
Previously, due to the straightforward functionalization of
coumarin ring, several coumarin derivatives have been prepared
and evaluated as AChE inhibitors [10]. It has been observed that
coumarins such as AP2238 (Fig. 1) encompassing substitution at
position 3 or 4 have shown increased activity against AChE rather
than 6 or 7 substituted coumarins [10].
*
Corresponding author. Department of Medicinal Chemistry, Faculty of Phar-
Regarding the X-ray crystallographic structure of AChE (PDB ID:
EVE), three main binding sites are determined: the catalytic triad
at the bottom of active site including Ser200, His440, and Glu327,
the catalytic anionic site (CAS) at the vicinity of the catalytic triad
macy and Pharmaceutical Sciences Research Center, Tehran University of Medical
Sciences, Tehran, Iran. Tel.: þ98 21 66954708; fax: þ98 21 66461178.
1
E-mail
addresses:
aforoumadi@yahoo.com,
aforoumadi@tums.ac.ir
(
A. Foroumadi).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.024

624
A. Asadipour et al. / European Journal of Medicinal Chemistry 70 (2013) 623e630
OH
O
O
H CO
3
N
H CO
3
N
H CO
CH
3
3
Donepezil
Galantamine
N
CH3
N
H CO
3
N
O
CH3
O
H CO
3
CH3
H CO
O
3
H CO
O
O
3
Ensaculine
AP2238
O
O
N
H
n
R
N
O
Designed compounds
(
9a-f: n = 0, 10a-g: n = 2)
Fig. 1. Structures of well-known cholinesterase inhibitors and designed compounds.
consisting of Trp84, Tyr130, Gly199, His441, His444 and peripheral
anionic site (PAS) at the gorge rim comprising Tyr70, Asp72, Tyr121,
Trp279 and Tyr334. Furthermore, the key residue Phe330 in the
midgorge is also involved in the recognition of ligands [11,12]. It
was proved that dual binding site (DBS) AChE inhibitors were more
potent inhibitors compared to the compounds that interact with
only one site of the enzyme [13]. Several studies have shown that
coumarin moiety can bind primarily to PAS of AChE [10]. Accord-
ingly, different groups such as benzylamino, phenylpiperazine, and
aniline, which interact with the catalytic site, have been success-
fully connected to the coumarin scaffold using different spacers to
obtain dual binding site inhibitors [14]. In continuation of our
previous work on coumarin AChE inhibitors [15], a novel series of
coumarins have been designed through hybridization approach in
which N-benzylpiperidine moiety of donepezil as a CAS binder and
the coumarin scaffold as PAS binding core were connected together
with amide linker (Fig. 1). Herein, the synthesis, biological evalu-
ation and in silico studies of some substituted coumarins 9aef and
carboxylic acid 3aeg (Supplementary material) [16]. The amine
intermediate 2-(1-benzylpiperidin-4-yl)ethanamine (8b) was pre-
pared from commercially available 1-benzyl-4-piperidone (4) and
malonitrile in 60% yield over four steps [17]. Thus, simple
condensation of malonitrile with compound 4 provided 2-(1-
benzylpiperidin-4-ylidene)malononitrile (5). The latter compound
was then converted to ethyl cyanoacetate 6 through reduction and
simultaneous formation of esteric bond. Then, compound 8b can be
prepared from hydrolysis and decarboxylation of compound 6
4
followed by reduction with LiAlH (Supplementary material).
Finally, coumarin-3-carboxylic acids3aeg, were chlorinated using
thionyl chloride to give the appropriate coumarin-3-carbonyl chlo-
ride and then coupled with 1-benzylpiperidin-4-amine 8a or 2-(1-
benzylpiperidin-4-yl)ethanamine 8b to yield corresponding amides
9 or 10 in moderate yields, respectively (Scheme 1). Activation of the
O
N
Ph
N
H
10aeg as novel dual binding site inhibitors of AChE are reported.
R
a, b
a, c
O
O
O
O
9
a-f
2
. Results and discussion
OH
R
O
2.1. Chemistry
3
a-g
O
O
N
Ph
The synthetic routes from key intermediates 3aeg and 8a,b to
N
R
H
target compounds 9aef and 10aeg have been shown in Scheme 1.
Initially, several ethyl coumarin-3-carboxylate derivatives 2aeg
were synthesized using commercial 2-hydroxybenzaldehydes 1ae
g and diethylmalonate in the presence of catalytic amount of
piperidine which was then hydrolyzed with aqueous solution of
sodium hydroxide to furnish the corresponding coumarin-3-
O
1
0a-g
Scheme 1. Synthesis of target compounds 9 and 10. Reagents and conditions: (a)
thionyl chloride, reflux; (b) 1-benzylpiperidin-4-amine (8a), K CO , toluene, reflux; (c)
2-(1-benzylpiperidin-4-yl) ethanamine (8b), K CO , toluene, reflux.
2
3
2
3

A. Asadipour et al. / European Journal of Medicinal Chemistry 70 (2013) 623e630
625
carboxylic groups of coumarin-3-carboxylic acids 3aeg were
screened by various reagents like N-(3-dimethylaminopropyl)-N -
inhibitory activity of 7-methoxy analog 9d was superior to 8-
methoxy isomer 9e.
0
ethylcarbodiimide hydrochloride (EDC), carbonyldiimidazole (CDI)
and dicyclohexylcarbodiimide (DCC) in various solvents, but the best
result was obtained by using thionyl chloride.
The IC50 values of N-(1-benzylpiperidin-4-yl)-carboxamide se-
ries (compounds 9aef) against BuChE were ꢀ20
mM. Among the N-
ethylcarboxamide series (compounds 10aeg), compounds 10a, 10b
and 10e showed high inhibitory activity against BuChE with IC50
values ꢁ420 nM. The 7-methoxycoumarin 10e was the most active
compound against BuChE (IC50 ¼ 357 nM). Interestingly, the com-
parison of anti-cholinesterase activities of 7- and 8-methoxy iso-
mers 10e and 10g revealed that the position of methoxy group had
high impact on BuChE/AChE selectivity (71 versus 9000).
It is worthwhile to note that the highest selectivity index was
attributed to the most active compound against AChE, 10c with 6-
nitro substituent (SI ¼ 26,300). Compound 10c was 46-fold more
potent than standard drug donepezil against AChE. Besides 6-nitro-
compound 10c, 6-bromo-, 7-methoxy-, and 8-methoxy-derivatives
(compounds 10b, 10e and 10g, respectively) in the N-ethyl-
carboxamide series exhibited superior anti-AChE activity respect to
donepezil.
Structural elucidations of all compounds were done using 1H
NMR and IR spectroscopy and elemental analysis for H, C and N.
2
2
.2. Pharmacology
.2.1. Inhibitory activity against AChE and BuChE
The inhibitory activity of the target compounds against AChE
and BuChE is displayed in Table 1. Based on the IC50 values for AChE,
all compounds with the exception of 9a and 9f exhibited potent
activity in the range of sub-micromolar concentrations (0.3e
6
1
33 nM). However, compound 9a and 9f with IC50 values of 1.2e
.7 M had remarkable anti-AChE activity.
The designed compounds are composed of two fragments; a
CAS binding motif (N-benzylpiperidine) and a PAS binding motif
coumarin) which are joined either via a carboxamide (compounds
aef) or N-ethylcarboxamide (compounds 10aeg) linkers. It was
m
(
9
However, it has been recently reported that dual inhibition of
AChE/BuChE might improve the signs of AD [5,6]. Among the
synthesized compounds, compound 10a was shown to be dual
cholinesterase inhibitor with more favorable balancing between
AChE/BuChE inhibitions compared to the standard drug donepezil.
In this point of view, compound 10a prototype could be considered
as promising lead for future studies.
observed that all compounds with N-ethylcarboxamide linker are
more active than their counterparts with carboxamide linker. The
most active derivative in this series was compound 10c bearing a
nitro group at position 6 of the coumarin ring (IC50 ¼ 0.3 nM).
The IC50 values of substituted coumarins against AChE were
significantly less than those of corresponding unsubstituted cou-
marins 9a and 10a. These data revealed that the bromo, nitro, and
methoxy substituents increase the anti-AChE activity. Exception-
ally, introduction of hydroxyl group on the 6- or 7-position of
coumarin ring diminishes the inhibitory activity against AChE. The
comparison of the IC50 values of 7-methoxy analogs 9d and 10e
with those of 7-hydroxy counterparts 9f and 10f demonstrated that
O-methylation of 7-hydroxycoumarins improved the activity. The
2 2
2.2.2. Protection against H O -induced cell death in PC12 neurons
The neuroprotective activity of the selected compounds (10b,
10c and 10e) against oxidative stress-induced cell death in differ-
entiated PC12 cells was evaluated. Thus, differentiated PC12 cells
were pretreated with different concentrations (1, 10 and 100
the compounds and quercetine as reference compound for 3 h,
before treatment with H (250 M). H was added to the
mM) of
2
O
2
m
2 2
O
8
-methoxycoumarin derivative 10g with IC50 of 2 nM was more
medium for induction of apoptosis and occurrence of apoptosis was
established after staining with DAPI, and cell viability was
measured after 24 h by using the MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assay. It should be
mentioned that the selected compounds did not show any toxicity
active than its 7-methoxy isomer 10e (IC50 ¼ 5 nM). In contrast, in
the N-(1-benzylpiperidin-4-yl)-carboxamide series, the AChE
Table 1
The IC50 values of the target compounds 9aef and 10aeg against AChE and BuChE.
at concentrations of 1e100
of the compounds were evaluated within the range of 1e100
Based on the results (Fig. 2), H significantly reduced the cell
mM. Therefore, neuroprotective activity
mM.
2 2
O
viability to 47% compared with control, whereas pretreatment with
the compounds significantly protected neurons against cell death
in all used concentrations (P value <0.05).
2
.3. Docking studies
a
SId
Compound
R
n
IC50 (nM)
AChEb
BuChEc
Docking studies were performed in order to obtain more insight
into the binding mode of the compounds and also elucidate the
influence of structural modifications on the inhibitory activities of
the compounds against AChE. Several Ligand-bounded crystallo-
graphic structures of AChE are available. For the docking procedure,
the pdb structure of 1EVE was taken from the Brookhaven protein
database (http://www.rcsb.org) as a complex bound with inhibitor
donepezil. To confirm the validity of the used docking protocol, the
crystal structure of donepezil was re-docked on the target enzyme
9
9
9
9
9
9
1
1
1
1
1
1
1
a
b
c
d
e
f
0a
0b
0c
0d
0e
0f
0g
H
6-Br
6-NO
7-OMe
8-OMe
7-OH
H
6-Br
6-NO
6-OH
7-OMe
7-OH
8-OMe
0
0
0
0
0
0
2
2
2
2
2
2
2
1200
30
17,000
42,000
22,000
27,000
20,000
55,000
371
14
1400
51
147
32
32
2
430
183
633
1700
26
2
0.3
70
14
420
7900
210
26,300
222
71
321
9000
384
2
ꢀ
15,610
357
46,000
18,000
5380
and the RMSD value (0.75 A) showed the predictive ability of the
used method.
5
143
2
Subsequently, the two most active compounds of each category
(9b, 10c) were subjected to Autodock vina 1.1.1 using the optimized
Donepezil
14
parameters. As shown in Fig. 3, the orientation of both compounds
in the active site was much similar to that of donepezil.
Since the piperidine moiety of the ligands is protonated at
physiologic pH (7.4), the ligands were recognized by the anionic
a
IC50 values are mean of the three independent experiments.
AChE from Electrophorus electricus.
BuChE from equine serum.
SI ¼ selectivity index (IC50 BuChE/IC50 AChE).
b
c
d

626
A. Asadipour et al. / European Journal of Medicinal Chemistry 70 (2013) 623e630
Fig. 2. Neuroprotective activity of compounds 10b, 10c and 10e against H
2
O
2
-induced cell death in PC12 neurons. Differentiated PC12 cells were treated with different concen-
. Quercetine was used as reference drug.
trations (1e100 M) of the compound for 3 h. After 24 h, cell viability was determined by MTT assay in the presence H O
m
2 2
subsite of the enzyme. It was reported that, this feature of the
molecules takes part in recognition and orientation of the ligands
towards the active site [12]. Furthermore, it was observed that the
benzyl moiety is oriented towards the CAS of the enzyme. It was
therefore concluded that the compounds are anchored in the mid-
gorge of the enzyme through N-benzylpiperidine fragment, while
the coumarin parts of the molecules are accommodated in the
gorge rim. A more detailed view for the interaction of the two
compounds 9b and 10c is also displayed in Fig. 4. As presented, the
phenyl ring of benzyl moiety in both compounds is in parallel
an N-ethylcarboxamide linker and a 6-nitro substituent was the
most active compound (IC50 ¼ 0.3 nM) with high selectivity
(SI ¼ 26,300). Docking studies have also revealed that the higher
flexibility of N-ethylcarboxamide linker in compound 10c proto-
type might lead to the more facile accommodation of the com-
pounds in the active site and dual binding site inhibition of the
enzyme. Moreover, the neuroprotective activity of the target
compounds against oxidative stress-induced cell death as an
additional effect might be important for AD management.
disposition to Trp84 showing aromatic
similar interactions including a ecation between the quaternary
nitrogen of piperidine ring with Phe330 and a stacking of
p
e
p
interaction. Two other
4. Experimental
p
pe
p
4.1. Chemistry
coumarin ring and Trp279 were also observed (Figs. 4 and 5).
Compound 10c displayed a hydrogen bond interaction between
carbonyl group of coumarin moiety and the hydroxyl group of
Tyr121. Consequently, the carbonyl part of amide group is oriented
towards the hydrophilic pocket composed of Ser286, Phe290, and
Arg289. These additional interactions were not observed in 9b. It
seems that the more flexibility of the N-ethylcarboxamide linker in
All commercially available starting materials and reagents were
from Merck and were used without further purification. Coumarin-
3-carboxylic acid derivatives 3aeg were synthesized by using re-
ported method [16]. Compound 8 was prepared from commercially
available 1-benzyl-4-piperidone 4 and malonitrile according to the
literature method [17]. Thin-layer chromatography was performed
using silica gel 250 micron, F254 plates. Melting points were
measured on a Kofler hot stage apparatus and are uncorrected. The
IR spectra were recorded using Nicolet FT-IR Magna 550 spectro-
10c led to its better interactions with the enzyme compared to 9b.
The higher potency of the compounds with N-ethylcarboxamide
linker could be due to the more favorable interactions of com-
pounds with the target enzyme.
1
graph (KBr disks). H NMR spectra were recorded on an NMR in-
strument Bruker 400 or 500 MHz. The chemical shifts (d) and
2.4. Kinetic of the enzyme inhibition
coupling constants (J) are expressed in parts per million and hertz,
The kinetic studies for the most active compound 10c, were
carried out at three fixed inhibitor concentrations (0.357 nM,
.57 nM and 11.9 nM). In each case, the initial velocity measure-
ments were achieved at different substrate (S), acetylthiocholine
ATCh) concentrations and the reciprocal of the initial velocity (1/v)
3
(
was plotted against the reciprocal of [ATCh] [18]. The double-
reciprocal (LineweavereBurk) plot showed a pattern of increasing
slopes and increasing intercepts with higher inhibitor concentra-
tion (Fig. 6). This pattern indicates mixed-type of inhibition for 10c
resulting from a more enhanced inhibitor interaction with both the
free enzyme and the acetylated enzyme.
3
. Conclusion
A series of novel coumarin derivatives bearing N-benzylpiper-
idinyl moiety were synthesized and evaluated for AChE and BuChE
inhibitory activity using a modified Ellman’s method. The results
showed that most of compounds exhibited potent anti-AChE ac-
tivity. Among the synthesized derivatives, compound 10c bearing
Fig. 3. Similar orientation of the best docked poses of 9b (yellow) and 10c (red) along
with native ligand, donepezil (blue), in the active site of AChE. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)

A. Asadipour et al. / European Journal of Medicinal Chemistry 70 (2013) 623e630
627
Fig. 4. Representative model for interactions of compounds 9b (A) and 10c (B) docked into the binding site of AChE. Hydrogen bond is indicated as red dotted line. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
respectively. Mass spectroscopy was conducted with an HP (Agilent
technologies) 5937 Mass Selective Detector. Elemental analyses
were carried out by a CHN-Rapid Heraeus elemental analyzer. The
results of elemental analyses (C, H, N) were within ꢂ0.4% of the
calculated values.
4.1.1.1. N-(1-Benzylpiperidin-4-yl)-2-oxo-2H-chromene-3-
carboxamide (9a). Yield: 65%; pale yellow solid; mp 198e200 C; IR
ꢃ
ꢄ1
1
(KBr, cm
, 400 MHz)
(d, 1H, J ¼ 7.5 Hz, H
.51 (d, 1H, J ¼ 7.5 Hz, H
coumarin), 7.32e7.22 (m, 5H Ph), 3.49 (s, 2H, CH
3.09 (m, 1H, NCH piperidine), 2.79e2.11 (m, 4H, 2CH
1.95e1.47 (m, 4H, 2CH CH
271 (16), 189 (48), 173 (97), 91 (100). Anal. Calcd for C22
72.91; H, 6.12; N, 7.73. Found: C, 72.74; H, 6.33; N, 7.51.
)
n
max: 3312 (NH),1711 and 1648 (C]O). H NMR (DMSO-
: 8.85 (s, 1H, H coumarin), 8.63 (br s, 1H, NH), 7.99
coumarin), 7.75 (t, 1H, J ¼ 7.5 Hz, H coumarin),
coumarin), 7.44 (t, 1H, J ¼ 7.5 Hz, H
benzylic), 3.13e
d
6
d
4
5
7
7
8
6
4.1.1. General procedure for the preparation of compounds 9 and 10
2
Compound 3 (1 mmol) was added to thionyl chloride (5 ml) and
2
N piperidine),
þ
the mixture was refluxed for 3e5 h. After completion of the reac-
tion, thionyl chloride was removed with simple distillation to give
appropriate coumarin-3-carbonyl chloride derivative. The crude
product was used directly without further purification. In the next
step, appropriate amine (1-benzylpiperidin-4-amine or 2-(1-
benzylpiperidin-4-yl)ethanamine) (1 mmol) and potassium car-
bonate (2 mmol) were dissolved in dry toluene (15 ml). The mixture
was refluxed for 6e12 h, and then the solvent was removed under
reduced pressure. The residue was purified by silica gel column
chromatography using petroleum ether-ethyl acetate (8:2) as a
mobile phase to give the pure product.
2
2
N piperidine). MS (m/z, %) 362 (M , 16),
22 2 3
H N O : C,
4.1.1.2. N-(1-Benzylpiperidin-4-yl)-6-bromo-2-oxo-2H-chromene-3-
ꢃ
carboxamide (9b). Yield: 68%; pale yellow solid; mp 200e201 C; IR
ꢄ1
1
(KBr, cm
, 400 MHz)
(d,1H, J ¼ 7.5 Hz, H
7.48 (d, 1H, J ¼ 7.5 Hz, H
2H, CH benzylic), 3.63e3.60 (m, 1H, NCH piperidine), 2.87e2.13
m, 4H, 2CH
)
n
max: 3319 (NH),1716 and 1657 (C]O). H NMR (DMSO-
: 8.78 (s, 1H, H coumarin), 8.66 (br s, 1H, NH), 8.24
coumarin), 7.88 (d,1H, J ¼ 7.5 Hz, H coumarin),
coumarin), 7.41e7.14 (m, 5H Ph), 3.85 (s,
d
6
d
4
5
7
8
2
(
2
N piperidine), 1.98e1.59 (m, 2H, 2CH
2
CH
2
N piperi-
þ
dine). MS (m/z, %) 442 (M þ 2, 2), 440 (M , 2), 253 (22), 251 (21),
189 (55), 172 (77), 91 (100). Anal. Calcd for C22
2 3
H21BrN O : C, 59.87;
H, 4.80; N, 6.35. Found: C, 59.71; H, 4.65; N, 6.21.
4
.1.1.3. N-(1-Benzylpiperidin-4-yl)-6-nitro-2-oxo-2H-chromene-3-
ꢃ
carboxamide (9c). Yield: 66%; pale yellow solid; mp 224e225 C; IR
ꢄ1
(KBr, cm
)
n
max: 3341 (NH), 1724 and 1656 (C]O), 1535, 1344
, 400 MHz) : 8.96 (s, 1H, H coumarin),
coumarin), 8.51 (d, 1H, J ¼ 8.4 Hz, H coumarin), 7.56
coumarin), 7.41e7.15 (m, 5H Ph), 4.05 (s, 2H,
benzylic), 3.64e3.54 (m, 1H, NCH piperidine), 2.88e2.04 (m,
1
(NO
2
). H NMR (DMSO-d
6
d
5
8
.62 (s, 1H, H
4
7
(
CH
d, 1H, J ¼ 8.4 Hz, H
8
2
4
H, 2CH
2
N piperidine), 1.79e1.25 (m, 4H, 2CH
2
CH
2
N piperidine).
þ
MS (m/z, %) 407 (M , 3), 189 (31), 172 (51), 91 (100). Anal. Calcd for
22 21 3 5
C H N O : C, 64.86; H, 5.20; N, 10.31. Found: C, 64.61; H, 5.08; N,
10.52.
4
.1.1.4. N-(1-Benzylpiperidin-4-yl)-7-methoxy-2-oxo-2H-chromene-
ꢃ
3-carboxamide (9d). Yield: 50%; pale yellow solid; mp 193e195 C;
Fig. 5. The relative orientation of carboxamide linker of the best docking poses of 9b
and 10c. The carboxamide linker of 10c oriented to a hydrophillic pocket (red surface)
of the active site. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
ꢄ1
1
IR (KBr, cm
)
n
max: 3311 (NH), 1705 and 1651 (C]O). H NMR
: 8.80 (s, 1H, H coumarin), 8.66 (br s, 1H,
NH), 8.25 (d, 1H, J ¼ 7.5 Hz, H coumarin), 7.87 (d, 1H, J ¼ 7.5 Hz, H
(DMSO-d
6
, 400 MHz)
d
4
5
6

628
A. Asadipour et al. / European Journal of Medicinal Chemistry 70 (2013) 623e630
Fig. 6. Left: LineweavereBurk plot for the inhibition of AChE by 10c at different concentrations with acetylthiocholine (ATCh) as substrate, Right: Steady-state inhibition constant
Ki) of 10c.
(
coumarin), 7.35e7.54 (m, 5H Ph), 3.92 (s, 3H, OCH
3
), 3.63 (s, 2H, CH
2
piperidine), 1.49e1.46 (m, 2H, NHCH
2
2
CH ), 1.23e1.11 (m, 3H,
benzylic), 2.91e2.83 (m, 1H, NCH piperidine), 2.37e2.01 (m, 4H,
CHCH
2
CH
2
N piperidine and 2CHCH
2
N piperidine). MS (m/z, %) 390
þ
2
CH
2
N piperidine), 1.72e1.21 (m, 4H, 2CH
2
CH
2
N piperidine). MS
(M , 15), 299 (50), 217 (98), 173 (44), 91 (100). Anal. Calcd for
C H N O : C, 73.82; H, 6.71; N, 7.17. Found: C, 73.64; H, 6.51; N,
24 26 2 3
þ
(m/z, %) 392 (M , 23), 365 (26), 290 (35), 178 (53), 129 (100). Anal.
Calcd for C23H N
24 2
O
4
: C, 70.39; H, 6.16; N, 7.14. Found: C, 70.51; H,
7.21.
6
.02; N, 7.21.
4
.1.1.8. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-6-bromo-2-oxo-2H-
4
.1.1.5. N-(1-Benzylpiperidin-4-yl)-8-methoxy-2-oxo-2H-chromene-
chromene-3-carboxamide (10b). Yield: 55%; pale yellow solid; mp
ꢃ
ꢃ
ꢄ1
n
3-carboxamide (9e). Yield: 76%; pale yellow solid; mp 198e200 C;
189e190 C; IR (KBr, cm
)
max: 3342 (NH), 1712 and 1651 (C]O).
: 8.80 (s, 1H, H coumarin), 8.66 (s,
coumarin), 8.26 (br s, 1H, NH), 7.88 (d, J ¼ 7.5 Hz, 1H, H
coumarin), 7.58e7.25 (m, 6H, 5H Ph and H coumarin), 3.56 (s, 2H,
CH benzylic), 3.09e3.01 (m, 2H, NHCH ), 2.79e2.62 (m, 2H, 2CHN
piperidine), 2.25e2.11 (m, 2H, 2CHN piperidine), 2.09e2.00 (m, 2H,
2CHCH N piperidine), 1.81e1.62 (m, 2H, NCH CH ), 1.59e1.32 (m,
3H, CHCH N piperidine). MS (m/z, %)
ꢄ1
1
1
IR (KBr, cm
)
n
max: 3312 (NH), 1705 and 1648 (C]O). H NMR
: 8.90 (s, 1H, H coumarin), 8.84 (br s, 1H,
NH), 7.72e7.64 (m, 2H, H5,7 coumarin), 7.43e7.34 (m, 6H, 5H Ph and
coumarin), 4.03 (s, 3H, OCH ), 3.60e3.55 (m, 3H, CH benzylic
and NCH piperidine), 2.89e2.23 (m, 4H, 2CH
.65 (m, 4H, 2CH CH
H NMR (DMSO-d
6
, 400 MHz)
d
4
(
DMSO-d
6
, 400 MHz)
d
4
1H, H
5
7
8
H
6
3
2
2
2
2
N piperidine), 2.04e
N piperidine). MS (m/z, %) 392 (M , 13), 260
þ
1
2
2
2
2
2
(
C
15), 203 (81), 172 (50), 91 (100), 77 (11). Anal. Calcd for
: C, 70.39; H, 6.16; N, 7.14. Found: C, 70.51; H, 6.22; N,
2
CH
2
N piperidine and 2CHCH
2
þ
H N
23 24 2
O
4
470 (M þ 2, 12), 468 (M , 13), 379 (15), 377 (13), 217 (98), 172 (15),
7.31.
2 3
91 (100). Anal. Calcd for C24H25BrN O : C, 61.41; H, 5.37; N, 5.97.
Found: C, 61.31; H, 5.52; N, 5.72.
4
3
.1.1.6. N-(1-Benzylpiperidin-4-yl)-7-hydroxy-2-oxo-2H-chromene-
ꢃ
-carboxamide (9f). Yield: 55%; pale yellow solid; mp 185e186 C;
4.1.1.9. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-6-nitro-2-oxo-2H-chro-
mene-3-carboxamide (10c). Yield: 75%; pale yellow solid; mp 199e
ꢄ1
IR (KBr, cm
)
n
max: 3439 (OH), 3232 (NH), 1714 and 1646 (C]O).
, 400 MHz) : 8.77 (s, 1H, H coumarin), 8.59 (br
s, 1H, NH), 7.81 (d, 1H, J ¼ 7.5 Hz, H coumarin), 7.39e7.11 (m, 5H
Ph), 6.87e6.80 (m, 2H, H6,8 coumarin), 3.83 (s, 2H, CH benzylic),
.53e3.51 (m, 1H, NCH piperidine), 2.78e2.11 (m, 4H, 2CH
1
ꢃ
ꢄ1
n
H NMR (DMSO-d
6
d
4
200 C; IR (KBr, cm
)
max: 3340 (NH), 1724 and 1651 (C]O), 1531
, 400 MHz) : 8.97 (br s, 2H, H4,5
coumarin),
coumarin), 7.58e7.31 (m, 5H Ph), 3.63 (s,
benzylic), 3.11e3.02 (m, 2H, NCH ), 2.81e2.64 (m, 2H,
2CHN piperidine), 2.27e2.13 (m, 2H, 2CHN piperidine), 2.09e2.00
(m, 2H, 2CHCH N piperidine), 1.80e1.61 (m, 2H, NCH CH ), 1.58e
N piperidine). MS
1
5
and 1344 (NO
2
). H NMR (DMSO-d
6
d
2
coumarin), 8.63 (br s, 1H, NH), 8.51 (d, 1H, J ¼ 7.5 Hz, H
7.72 (d, 1H, J ¼ 7.5 Hz, H
2H, CH
7
3
2
N
8
piperidine), 1.89e1.50 (m, 4H, 2CH
2
CH
2
N piperidine). MS (m/z, %)
2
2
þ
378 (M , 11), 287 (42), 189 (51), 172 (70), 91 (100). Anal. Calcd for
C
7.27.
22
H
22
N
2
O
4
: C, 69.83; H, 5.86; N, 7.40. Found: C, 69.67; H, 5.98; N,
2
2
2
1.31 (m, 3H, CHCH
2
CH
2
N piperidine and 2CHCH
2
þ
(
m/z, %) 435 (M , 10), 344 (16), 217 (100), 172 (22), 91 (83). Anal.
4
.1.1.7. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-2-oxo-2H-chromene-3-
Calcd for C24
5.51; N, 9.44.
25 3 5
H N O : C, 66.19; H, 5.79; N, 9.65. Found: C, 66.33; H,
ꢃ
carboxamide (10a). Yield: 53%; pale yellow solid; mp 145e146 C;
ꢄ1
1
IR (KBr, cm
DMSO-d , 500 MHz)
NH), 7.98 (d, 1H, J ¼ 7.5 Hz, H
)
n
max: 3350 (NH), 1712 and 1650 (C]O). H NMR
: 8.84 (s, 1H, H coumarin), 8.66 (br s, 1H,
coumarin), 7.75 (t, 1H, J ¼ 7.5 Hz, H
coumarin), 7.44 (t, 1H,
(
6
d
4
4.1.1.10. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-6-hydroxy-2-oxo-2H-
5
7
chromene-3-carboxamide (10d). Yield: 53%; pale yellow solid; mp
ꢃ
ꢄ1
) n
coumarin), 7.50 (d, 1H, J ¼ 7.5 Hz, H
8
185e186 C; IR (KBr, cm
max: 3413 (OH), 3313 (NH), 1701 and
, 500 MHz) : 8.96 (br s, 1H, NH),
coumarin), 7.31e7.23 (m, 6H, 5H Ph and H
coumarin), 7.14 (d, 1H, J ¼ 7.5 Hz, H coumarin), 7.05 (s, 1H, H
1
J ¼ 7.5 Hz, H
6
coumarin), 7.32e7.22 (m, 5H Ph), 3.45e3.38 (m, 4H,
benzylic), 2.82e2.71 (m, 2H, 2CHN piperidine),1.84e
1642 (C]O). H NMR (DMSO-d
6
d
NCH and CH
2
2
8.76 (s, 1H, H
4
8
5
1.98 (m, 2H, 2CHN piperidine), 1.74e1.63 (m, 2H, 2CHCH
2
N
7

A. Asadipour et al. / European Journal of Medicinal Chemistry 70 (2013) 623e630
629
coumarin), 3.52 (s, 2H, CH
.96e2.92 (m, 2H, 2CHN piperidine), 2.02e2.00 (m, 2H, 2CHN
piperidine), 1.74e1.69 (m, 3H, CHCH CH piperidine and
CHCH N piperidine), 1.60e1.55 (m, 4H, NCH CH and 2CHCH
piperidine). MS (m/z, %) 406 (M , 7), 315 (33), 217 (53),189 (38),106
36), 91 (100). Anal. Calcd for C24 : C, 70.92; H, 6.45; N, 6.89.
Found: C, 70.78; H, 6.33; N, 6.71.
2
benzylic), 3.45e3.38 (m, 2H, NCH
2
),
simulations were finally carried out by means of a batch script
using Autodock vina (ver. 1.1.1) [21] with default parameters and
the exhaustiveness value set as 80. The active site for docking was
established as a box at geometrical center of the native ligand
present in the above mentioned PDB structure with the di-
mensions 40,40,40. The coordinates x, y, z, for the center of grid
box were set as 2.023, 63.29 and 67.062, respectively. The lowest
energy conformation of each ligandeenzyme complex was
selected for analyzing the interactions between AChE and the
inhibitor. The results were visualized using Chimera 1.6 [22]
(Molecular graphics and analyses were performed with the
UCSF Chimera package. Chimera is developed by the Resource for
Biocomputing, Visualization, and Informatics at the University of
California, San Francisco).
2
2
2
N
2
2
2
2
2
N
þ
(
26 2 4
H N O
4
.1.1.11. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-7-methoxy-2-oxo-2H-
chromene-3-carboxamide (10e). Yield: 65%; pale yellow solid; mp
ꢃ
ꢄ1
n
1
68e169 C; IR (KBr, cm
)
max: 3360 (NH), 1706 and 1646 (C]O).
, 400 MHz) : 8.80 (s, 1H, H coumarin), 8.75 (br
s, 1H, NH), 7.59e7.57 (m, 3H, 2H Ph, H coumarin), 7.25e7.23 (m, 3H
Ph), 6.94 (d, 1H, J ¼ 7.5 Hz, H coumarin), 6.86 (s, 1H, H coumarin),
.08 (s, 2H, CH benzylic), 3.91 (s, 3H, OCH ), 3.57e3.28 (m, 4H,
NCH and 2CHN piperidine), 2.60e2.52 (m, 2H, 2CHN piperidine),
.05e1.92 (m, 2H, 2CHCH piperidine), 1.79e1.45 (m, 3H,
1
H NMR (DMSO-d
6
d
4
5
6
8
4
2
3
2
4.3. AChE and BuChE inhibition assay
2
2
N
NCH
2
CH
2
and CHCH
2
CH
2
N piperidine), 1.27e1.22 (m, 2H, 2CHCH
2
N
Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized
þ
piperidine). MS (m/z, %) 420 (M , 9), 404 (5), 388 (23), 172 (26), 91
powder, from electric eel, 1000 unit), butyrylcholinesterase (BuChE,
E.C. 3.1.1.8, from equine serum) and butyrylthiocholine iodide (BTC)
were provided from SigmaeAldrich. 5,5-Dithiobis-(2-nitrobenzoic
acid) (DTNB), potassium dihydrogen phosphate, dipotassium
hydrogen phosphate, potassium hydroxide, sodium hydrogen car-
bonate, and acetylthiocholine iodide were purchased from Fluka.
The solutions of the target compounds were prepared in a mixture
(
100). Anal. Calcd for C25
28 2 4
H N O : C, 71.41; H, 6.71; N, 6.66. Found:
C, 71.33; H, 6.87; N, 6.45.
4
.1.1.12. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-7-hydroxy-2-oxo-2H-
chromene-3-carboxamide (10f). Yield: 62%; pale yellow solid; mp
ꢃ
ꢄ1
) n
185e186 C; IR (KBr, cm
max: 3449 (OH), 3328 (NH), 1720 and
648 (C]O). H NMR (DMSO-d , 500 MHz) : 8.89 (br s, 1H, NH),
.77 (s, 1H, H coumarin),
coumarin), 7.58 (d, 1H, J ¼ 7.5 Hz, H
.29e7.24 (m, 5H, Ph), 6.86e6.81 (m, 2H, H and H coumarin), 3.61
s, 2H, CH benzylic), 3.50e3.41 (m, 2H, NCH ), 2.66e2.61 (m, 2H,
CHN piperidine), 2.04e2.00 (m, 2H, 2CHN piperidine), 1.92e1.87
CH N piperidine and 2CHCH N piperidine), 1.60e
.54 (m, 4H, NCH CH and 2CHCH N piperidine). MS (m/z, %) 406
M , 14), 315 (40), 217 (83), 189 (33), 172 (21), 91 (100). Anal. Calcd
for C24 : C, 70.92; H, 6.45; N, 6.89. Found: C, 70.98; H, 6.63;
N, 7.11.
1
1
8
7
(
6
d
of DMSO (1 ml) and methanol (9 ml) and diluted in 0.1 M KH
2 4
PO /
4
5
K
2
HPO buffer (pH 8.0) to obtain final assay concentrations. The
4
6
8
ꢃ
temperature condition was adjusted as 25 C during all experi-
ments. Five different concentrations were tested for each com-
pound in triplicate to obtain the range of 20%e80% inhibition for
AChE and BuChE. The assay medium was composed of 3 ml of 0.1 M
2
2
2
(
m, 3H, CHCH
2
2
2
1
(
2
2
2
phosphate buffer pH 8.0, 100
.5 unit/mL enzyme solution (AChE, E.C. 3.1.1.7, Type V-S, lyophi-
lized powder, from electric eel). 100 l of each tested compound was
added to the assay tube and incubated at 25 C for 15 min prior to
adding 20 l of substrate (acetylthiocholine iodide). The rate of
ml of 0.01 M DTNB, and 100 ml of
þ
2
26 2 4
H N O
m
ꢃ
m
4
.1.1.13. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-8-methoxy-2-oxo-2H-
absorbance change was measured at 412 nm for 6 min on the
baseline obtained by blank reading of the solutions with non-
enzymatic hydrolysis. The blank was contained 3 ml buffer,
00 ml water, 100 ml DTNB and 20 ml substrate. The IC50 values were
determined graphically from inhibition curves (log inhibitor con-
centration versus percent of inhibition). Spectrophotometric mea-
chromene-3-carboxamide (10g). Yield: 72%; pale yellow solid; mp
ꢃ
ꢄ1
) n
2
12e213 C; IR (KBr, cm
max: 3340 (NH), 1736 and 1660 (C]O).
, 400 MHz) : 8.97 (s, 1H, H coumarin), 8.63e
.57 (m, 2H, NH and H
coumarin), 8.51 (d, 1H, J ¼ 8.4 Hz, H
coumarin), 7.55 (d, 1H, J ¼ 8.4 Hz, H coumarin), 7.39e7.31 (m, 5H
Ph), 3.61 (s, 2H, CH benzylic), 3.51 (s, 3H, OCH ), 3.09e2.95 (m, 2H,
NHCH ), 2.18e2.04 (m, 2H, 2CHN piperidine), 1.78e1.72 (m, 2H,
CHN piperidine), 1.64e1.59 (m, 2H, 2CHCH N piperidine), 1.47e
.40 (m, 2H, NCH CH ), 1.27e1.22 (m, 3H, CHCH CH N piperidine
and 2CHCH N piperidine). MS (m/z, %) 420 (M , 5), 217 (91), 172
39), 106 (36), 91 (100). Anal. Calcd for C25 : C, 71.41; H,
1
H NMR (DMSO-d
8
6
d
4
2
6
5
7
2
3
surements were performed on
a UV Unico Double Beam
2
Spectrophotometer [23]. The described method was also taken for
BuChE inhibition assay.
2
1
2
2
2
2
2
þ
2
(
28
H N
2
O
4
4.4. Cellular biology
6
.71; N, 6.66. Found: C, 71.27; H, 6.61; N, 6.45.
4.4.1. Cell culture, differentiation, and treatment conditions
4.2. Molecular modeling studies
Rat undifferentiated PC12 cells were cultured in RPMI 1640
media with 10% FCS containing 100 units/ml penicillin and 100 mg/
The conformational search studies were performed with
ml streptomycin (All from GIBCO, Grand Island, NY, USA). Cells were
trypsinated and 10 cells were plated on each well of 96-well cul-
ture plates in RPMI 1640 media. For induction of neuronal differ-
entiation, PC12 cells were cultured in serum-free media (RPMI 1640
media containing 100 units/ml penicillin and 100 mg/ml strepto-
mycin) for 2 days, thereafter their medium changed to above serum
free medium containing NGF (50 ng/ml, Sigma) and continued for 5
days until neurite outgrowth was observed under inverted micro-
scope (Supplementary material) [24]. Differentiated PC12 cells
4
HyperChem7 (Hypercube Inc.) using AM1 semi-empirical method.
For the docking procedure, the pdb structure of 1EVE was taken
from the Brookhaven protein database (http://www.rcsb.org) as a
complex bound with inhibitor E2020 (donepezil). Afterward, the
water molecules and co-crystallized ligand were eliminated from
the protein structure and polar hydrogens were added. The Koll-
man charge was calculated and the protein was saved as pdbqt
format. The structure of the target compounds was prepared us-
ing MarvineSketch 5.8.3, 2012, ChemAxon (http://www.
chemaxon.com) and converted to 3D pdbqt coordinates by
Openbabel version (2.3.1) [19]. The autodock format of protein
was also prepared using Autodock Tools (ver. 1.5.4) [20]. Docking
were pretreated with different concentrations (1, 10 and 100
mM) of
the compounds for 3 h before treatment with H (250 M). The
2
O
2
m
occurrence of apoptosis was established after staining with DAPI,
and cell viability was measured after 24 h by using the MTT assay.

630
A. Asadipour et al. / European Journal of Medicinal Chemistry 70 (2013) 623e630
4
.4.2. Measurement of cell viability with MTT assay
experimental Alzheimer therapeutic, dihydrobenzodioxepine cymserine,
Neurochem. Res. 33 (2008) 745e753.
7] H. Sugimoto, Y. Yamanishi, Y. Iimura, Y. Kawakami, Donepezil hydrochloride
E2020) and other acetylcholinesterase inhibitors, Curr. Med. Chem. 7 (2000)
4
Cells were plated at a density of 10 cells/well in the 96-well
plate, cultured, differentiated and treated as described above.
l of MTT (Sigma) with the concentration of 5 mg/ml was added
to the cell culture media (150 l) and incubated in CO incubator for
.5 h. Thereafter, medium and MTT solution was removed and
[
[
(
10
m
303e339.
8] S. Kavanagh, M. Gaudig, B.B. Van, M. Adami, A. Delgado, C. Guzman,
E. Jedenius, B. Schäuble, Galantamine and behavior in Alzheimer disease:
analysis of four trials, Acta Neurol. Scand. 124 (2011) 302e308.
m
2
3
DMSO (150
m
l) was added into the each well and the formazan
[9] R. Hoerr, M. Noeldner, Ensaculin (KA-672 HCl): a multitransmitter approach to
dementia treatment, CNS Drug Rev. 8 (2002) 143e158.
10] P. Anand, B. Singh, N. Singh, A review on coumarins as acetylcholinesterase
precipitates were dissolved by shaking the plate for 10 min on
shaker with the speed of 120 rpm. Finally optical density (OD) was
evaluated at 560 nm on the ELISA plate reader. Results were
compensated with OD measured in the same conditioned well
without adding MTT [25].
[
inhibitors for Alzheimer’s disease, Bioorg. Med. Chem. 20 (2012) 1175e1180.
[11] C. Dan, P. Ya-fei, L. Chuan-jun, X. Yun-feng, J. Yu-ren, Virtual screening of
acetylcholinesterase inhibitors, in: M. Taha (Ed.), Virtual Screening, InTech
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12] N. Duval, S. Bon, I. Silman, J. Sussman, J. Massoulie, Site-directed mutagenesis
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13] F. Leonetti, M. Catto, O. Nicolotti, L. Pisani, A. Cappa, A. Stefanachi, A. Carotti,
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potent, dual binding site AChE inhibitors, Bioorg. Med. Chem. 16 (2008)
7450e7456.
14] M. Catto, L. Pisani, F. Leonetti, O. Nicolotti, P. Pesce, A. Stefanachi, S. Cellamare,
A. Carotti, Design, synthesis and biological evaluation of coumarin alkyl-
amines as potent and selective dual binding site inhibitors of acetylcholin-
esterase, Bioorg. Med. Chem. 21 (2013) 146e152.
15] M. Alipour, M. Khoobi, A. Foroumadi, H. Nadri, A. Moradi, A. Sakhteman,
M. Ghandi, A. Shafiee, Novel coumarin derivatives bearing N-benzyl pyr-
idinium moiety: potent and dual binding site acetylcholinesterase inhibitors,
Bioorg. Med. Chem. 20 (2012) 7214e7222.
[
4.4.3. DAPI staining to evaluate apoptosis induction
[
2 2
DAPI staining was accomplished to evaluate the effect of H O
on induction of apoptosis. For this purpose the differentiated PC12
cells were cultured in 96 well plate and treated with different
doses of drugs as indicated above and stained with the DNA-
specific fluorochrome 4ʹ,6-diamidino-2-phenylindole dihydro-
chloride (DAPI, Sigma). DAPI was added to the culture medium of
[
[
live cells with the final concentration of 1 mg/ml for 20 min. Because
the cell membrane of apoptotic cell is permeable, DAPI enters into
the cell and stains the nucleus, while normal cell is not permeable
sufficiently and gets lightly stained (Supplementary material) [24].
[
[
16] X. Zhou, X.B. Wang, T. Wang, L.Y. Kong, Design, synthesis, and acetylcholin-
esterase inhibitory activity of novel coumarin analogues, Bioorg. Med. Chem.
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hybrids as acetylcholinesterase inhibitors for the treatment of Alzheimer’s
disease, Bioorg. Med. Chem. Lett. 14 (2004) 4639e4642.
Acknowledgments
This research was supported by grants from the Research
Council of Tehran University of Medical Sciences and the Iran Na-
tional Science Foundation (INSF).
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A. Cavalli, M. Recanatini, Acetylcholinesterase inhibitors for potential use in
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1
1H-indeno-[1,2-b]-quinolin-10-ylamine derivatives, Bioorg. Med. Chem. 8
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(
[
Appendix A. Supplementary data
3
(
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.024.
[20] M.F. Sanner, Python: a programming language for software integration and
development, J. Mol. Graph. Model. 17 (1999) 57e61.
[
[
[
21] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of
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