Novel coumarin derivatives bearing N-benzyl pyridinium moiety: Potent and dual binding site acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.08.052

A novel series of coumarin derivatives linked to benzyl pyridinium group were synthesized and biologically evaluated as inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The enzyme inhibitory activity of synthesized compounds was measured using colorimetric Ellman's method. It was revealed that compounds 3e, 3h, 3l, 3r and 3s have shown higher activity compared with donepezil hydrochloride as standard drug. Most of the compounds in these series had nanomolar range IC₅₀ in which compound 3r (IC₅₀ = 0.11 nM) was the most active compound against acetylcholinesterase enzyme.

Full text of DOI:10.1016/j.bmc.2012.08.052

Bioorganic & Medicinal Chemistry 20 (2012) 7214–7222
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel coumarin derivatives bearing N-benzyl pyridinium moiety:
Potent and dual binding site acetylcholinesterase inhibitors
Masoumeh Alipour a, Mehdi Khoobi b, Alireza Foroumadi b, Hamid Nadri c, Alireza Moradi c,
Amirhossein Sakhteman c, Mehdi Ghandi a, Abbas Shafiee b,
⇑
a School of Chemistry, University College of Science, University of Tehran, PO Box 14155-6455, Tehran, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
c Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd 8915173143, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2012
Revised 26 August 2012
Accepted 27 August 2012
Available online 7 September 2012
A novel series of coumarin derivatives linked to benzyl pyridinium group were synthesized and biolog-
ically evaluated as inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The
enzyme inhibitory activity of synthesized compounds was measured using colorimetric Ellman’s method.
It was revealed that compounds 3e, 3h, 3l, 3r and 3s have shown higher activity compared with donepezil
hydrochloride as standard drug. Most of the compounds in these series had nanomolar range IC50 in
which compound 3r (IC50 = 0.11 nM) was the most active compound against acetylcholinesterase
enzyme.
Keywords:
Coumarin
Benzylpyridinium
Actylcholinesterase inhibitor
Ellman’s method
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
of fibrils.16 According to these findings, designing the compounds
able to interact with both active site and PAS of AChE would be a
Alzheimer’s disease (AD), a progressive and degenerative disor-
der of the brain, is supposed to be the most common cause of
dementia among the aged people. Approximately, 24 million peo-
ple worldwide suffer from this disease. It was demonstrated that
AD is mainly caused by the loss of cholinergic activity in some
parts of the brain such as hippocampus which are related to learn-
ing and cognition.1,2 Thus, increasing the synaptic level of acetyl-
choline (ACh) by means of acetylcholinesterase (AChE) inhibition
could be helpful to alleviate AD’s symptoms.3–7
In addition, some evidence suggests that butyrylcholinesterase
(BuChE) levels are unchanged or even rise in advanced AD, while
the activity of the acetylcholinesterase is decreased in certain area
of the brain.8–10 As a matter of fact, the inhibition of BuChE can
raise ACh levels.11,12 Consequently, dual inhibition of AChE/BuChE
may be beneficial to improve AD symptoms without remarkable
side effects.13
new therapeutic approach for effective management of AD’s symp-
toms.17,18 Coumarins, a groups of naturally occurring substances in
many plants exhibit a wide range of biological activities such as
anti-inflammatory,19 anti-tumor,20 hepatoprotective,21 anti-HIV-
1,22 antiviral,23 antifungal,24 antimicrobial,25 anti-oxidant,26 and
antidepressant effects.27 Moreover, the potent anticholinesterase
activity of many synthetic coumarins was revealed similar to that
observed in the natural compounds.28–31
Many of these efforts were conducted by connecting the cou-
marin scaffold, via different spacers, to the structures with high
affinity towards the catalytic site of the enzyme.32
Moreover, benzylpyridinium salts (Fig. 1) have been repre-
sented an excellent acetylcholinesterase inhibitory in several re-
ports.33 In our previous work we have reported synthesis and
anticholinesterase activity of new benzofuranone derivatives bear-
ing benzyl pyridinium moiety (Fig. 1, X@O).33a Observing a dra-
matic decrease in anticholinesterase activity of the compounds
lacking the benzyl pyridinium moiety, proved that this part is nec-
essarily required for superior activity which was in accord with the
previously reported data by Iimura.33b
Based on these findings and in pursuit of our previous study on
coumarin scaffold,34 we have presented a novel series of coumarin
derivatives that were attached to benzyl pyridinium group through
alpha beta unsaturated carbonyl linker (Fig. 2). An outstanding fea-
ture of this linker is the conformational restriction caused by the
Besides, it was revealed that AChE augments the neurotoxic ef-
fect of amyloid beta through accelerating the formation of beta
amyloid deposits in the brain. Therefore, the role of amyloid beta
in beginning and progression of AD is a matter of debate.14,15 It
is well known that the enzyme interacts with beta amyloid
through peripheral anionic site (PAS) and promotes the formation
⇑
Corresponding author. Tel.: +98 21 66406757; fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.052

M. Alipour et al. / Bioorg. Med. Chem. 20 (2012) 7214–7222
7215
2.2. Acetylcholinesterase and butyrylcholinesterase inhibition
and preliminary SAR studies
O
R'
X
N
The target compounds were assayed for their inhibitory activity
against AChE from Electrophorus electricus (eel) and BuChE from
equine serum using the previously reported method and Donepezil
hydrochloride was taken as the positive control.33a According to
the enzyme inhibition data, as depicted in Table 1, most of the target
compounds showed potent AChE inhibition activity with IC50 values
within pico and micro molar ranges. Among the synthesized com-
pounds, 3r (IC50 = 0.11 nM) showed superior activity in comparison
to the reference drug, Donepezil hydrochloride (IC50 = 14 nM). By
contrast, compound 3h gave the most potent inhibition for BuChE
(IC50 = 125 nM). Compound 3e exhibited the most selectivity for
AChE. It was also revealed that anticholinesterase activity of the tar-
get compounds depends largely to the steric and electronic features
of the substituents. For example, a fourfold reduction in the activity
was observed by the movement of fluoro from position 2 in com-
pound 3r to position 4 in compound 3s (IC50 = 0.46 nM). This fact
supported by the docking studies. According to computational and
experimental studies, it is suggested that combination of steric and
electronic features can determine the activity of this series of com-
pounds. Based on docking studies, presence of fluoro at orto position
R
X= O, CH2
X
Figure 1. Some potent anticholinesterase structures bearing benzyl pyridinium
moiety.
conjugated double bond, which makes it possible to study the sub-
stituent modifications regardless of any conformational alterations
in the linker.
2. Results and discussion
2.1. Chemistry
Compounds 3 were synthesized via the route outlined in
Scheme 1. In the first step, 3-acetylcomarin derivatives 6 were syn-
thesized using a commercial 2-hydroxybenzaldehydes 4 and ethyl
acetoacetate 5 in the presence of catalytic amount of piperidine
according to the previous reported procedure.30 In the next step
3-acetylcomarins were transformed into the key intermediate 7
through condensation with pyridine-4-carbaldehyde under micro-
wave irradiation with good to excellent yields (64–77%). For prep-
aration of the latter compounds several conditions were screened
(p-TsOH in toluene, piperidine in ethanol and piperidine in n-buta-
nol both under thermal and microwave conditions) but only in the
presence of n-butanol as solvent and piperidine as catalyst under
microwave irradiation the best result was obtained. The aldol
products 7 were only in E-configuration as thermodynamically fa-
vored structures. The coupling constant of 15–16 Hz between the
olefinic protons clearly showed that the compound 7 possess the
E-configuration. The target compounds 3 were easily prepared
through the addition of proper benzyl bromide or chloride deriva-
tives to compounds 7 in dry acetonitrile under reflux condition. On
cooling, the precipitates was filtered and washed with diethyl
ether.
can disrupt the p–p stacking interactions via rotation of the phenyl
ring as in compound 3d. Movement of fluoro to position meta as in
compound 3e has therefore resulted in better activity due to proper
stacking of the phenyl ring with Trp 84. On the other hand the activ-
ity would slightly diminish when the flouro shifts to the para posi-
tion. It is assumed that the steric hindrance of substituents with
amino acids at the bottom of AChE gorge may be responsible for this
effect. Furthermore the activity of target compounds is very sensi-
tive to the size of the substituent at para position. Thus compounds
such as 3p and 3q having bulky groups at para position showed
weaker activity. It is worth noting that in case of 3a and 3b, removing
6-bromo substiuent in coumarin ring, led to a large decrease in the
activity of the target compounds (IC50 = 540; 760 nM, respectively).
To study the effects of more hydrophilic substituents on coumarin
ring, some compounds bearing methoxy group on 8 position of cou-
marin ring were synthesized (3c–q). As it could be observed from Ta-
ble 1, the size of the substituent in position 2, regardless of its
electronic properties, is important in order to show considerable
activity. Larger substituents have therefore exhibited better activi-
ties in comparison to those with the smaller size. The anticholines-
terase activity was in the order: CH3 > Cl > F according to the Van
der Waals radii 200, 180 and 135 pm, respectively (IC50 = 3l: 0.16,
3h: 0.46, 3d: 26 nM). In addition, the data showed that compounds
with more electron-withdrawing substituent on position 3 of benzyl
moiety exhibited higher activity in the order of 3e (F, 0.47 nM) > 3o
(CN, 76 nM) > 3i (Cl, 86 nM) > 3m (CH3, 800 nM). Finally, insertion of
second substituent at any position of benzyl moiety dramatically de-
creased the activity of the resulted compound (3g, 3j, 3k).
O
X
R
O
N
O
O
1
O
2
R
O
2.3. Antioxidant activity assay
Linker
Two different methods; DPPH (1,1-diphenyl-1-picrylhydrazyl)
and FRAP (ferric reducing antioxidant power) assays were used
to evaluate target compounds for their antioxidant efficacy. The
antioxidant assay data were summarized in Table 2. According to
the data none of the tested compounds showed significant antiox-
idant ability in both methods compared with reference
compounds.
O
R
N
Ar
O
O
2.4. Molecular docking studies
3a-s
In an attempt to study the binding mode for the interaction
Figure 2. Design strategy of the target compounds.
of the target compounds within gorge of AChE from Torpedo

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M. Alipour et al. / Bioorg. Med. Chem. 20 (2012) 7214–7222
H
O
b
O
O
O
O
O
O
O
H
N
+
O
N
a
OH
O
O
R
R
R
7
4
5
6
Only E-configuration
R= H, 3-OMe, 5-Br
O
β
b
5
4
R'
X
a
R'
6
7
α
N
O
O
R
8
c
1
X
3a-s
Scheme 1. Reagents and conditions: (a) piperidine, 50 °C; (b) piperidine, MW, 30 min; (c) benzyl halide derivatives, CH3CN, reflux, 2–4 h.
Table 1
AChE and BuChE inhibition data and their selectivity for synthesized compounds 3a–s
O
R
N
Ar
O
O
3a-s
Compound
R
Ar
IC50 AChEa,b (nM)
IC50 BuChEa,b (nM)
Selectivity for AChEd
1.57
F
F
3a
H
540
848
F
3b
3c
H
760
NDc
910
NDc
1.2
8-OMe
NDc
3d
8-OMe
26
329
12.6
F
3e
3f
8-OMe
8-OMe
0.47
472
2100
931
4468
1.97
F
F
3g
8-OMe
330
685
2.1
O2N
Cl
3h
3i
8-OMe
8-OMe
8-OMe
8-OMe
0.46
86
125
272
6.5
Cl
Cl
Cl
560
Cl
3j
120
440
6800
5790
57
3k
13.6
Cl

M. Alipour et al. / Bioorg. Med. Chem. 20 (2012) 7214–7222
7217
Table 1 (continued)
Compound
R
Ar
IC50 AChEa,b (nM)
IC50 BuChEa,b (nM)
Selectivity for AChEd
H3C
3l
8-OMe
0.16
342
2137
CH3
3m
3n
3o
8-OMe
8-OMe
8-OMe
800
11,200
27,000
1000
14
CH3
3650
76
7.4
13.1
CN
Cl
3p
3q
8-OMe
8-OMe
1600
1470
15,100
971
9.4
0.7
OCH3
F
3r
6-Br
6-Br
0.11
489
4445
F
3s
0.46
14
494
1074
384
Donepezil
5380
a
The concentration of inhibitor required to produce 50% enzyme inhibition.
Data are means of triplicate independent experiments.
Data was not determined.
b
c
d
Selectivity for AChE = IC50 (BuChE)/IC50 (AChE).
Table 2
The antioxidant activity of target compounds
Compounds
DPPH IC50
(l
g/ml)
FRAP value
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
3.6
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
<150
1890
430
Figure 3. Superimposition of 3r (blue) and donepezil (magenta) in the gorge of
TcAChE.
agreement with the previously reported data.37 Additionally, the
three p–p stacking interactions between the phenyl group of ben-
3q
3r
3s
zyl moiety and coumarin with the indole ring of catalytic anionic
site (CAS) Trp84 and Trp279 (PAS), respectively, and pyridine ring
with phenyl ring of Phe330 seems to stabilize the orientation of the
molecule in the gorge of TcAChE. The latter feature allowed for one
hydrogen bonding interaction between the oxygen of coumarin
carbonyl group with nitrogen of Phe288. Therefore, ligand 3r is a
dual PAS and CAS binding inhibitor of AChE that is in good agree-
ment with our rational design.
Ascorbic acid
BHTa
—
a
Butylated hydroxytoluene.
californica (TcAChE) active site, molecular docking was conducted
using autodock vina.36 Among the synthesized series of com-
pounds, 3r, the most potent compound, was subjected to docking
studies. Superposition of the best pose of 3r and the co-crystallized
Donepezil in the active site revealed that its binding mode at the
gorge of TcAChE resembled very much to that of Donepezil (Fig. 3).
Subsequently, compound 3r was analyzed in order to investi-
gate the possible interactions with amino acid residues on the ac-
3. Conclusions
A new series of coumarin derivatives linked to benzyl pyridini-
um group has been synthesized and evaluated for AChE and BuChE
inhibitory activity. Acetylcholinesterase activity of these com-
pounds was sensitive to substitiuent steric and electronic proper-
ties at position 2 and 3 of the benzyl moiety, respectively.
Among them compound 3r (IC50 = 0.11 nM) was identified as the
tive site of the enzyme. As depicted in Figures 4 and 5,
p-cation
interaction of the positively charged nitrogen with Tyr334, was
playing an important role in the penetration of the ligand into ac-
tive site as well as in the inhibition process. This finding was in

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M. Alipour et al. / Bioorg. Med. Chem. 20 (2012) 7214–7222
Figure 4. 2D representation of binding mode of 3r and amino acid residues in the
gorge of TcAChE created by poseview.
Figure 5. Docking of 3r in the active site of of TcAChE. Hydrogen atoms are not
shown for clarity.
most potent AChE inhibitor. Modeling studies showed similar
modes for the interaction of the compound and donepezil a well
known potent inhibitor of the enzyme. According to computational
and experimental studies, it is suggested that combination of steric
and electronic features can determine the activity of this series of
compounds. Based on docking studies, presence of fluoro at orto
chemical shifts (d) and coupling constants (J) are expressed in parts
per million and hertz, respectively. Elemental analyses were car-
ried out by a CHN-Rapid Heraeus elemental analyzer. The results
of elemental analyses (C, H, N) were within 0.4% of the calculated
values.
position can disrupt the
p–p stacking interactions via rotation of
the phenyl ring as in compound 3d. This finding is largely depen-
dent to electronic features and size of the substituents. Conse-
quently, it can be concluded that, regardless of the electronic
features, substitution at para position is not normally favored
(i.e. 3p,3q,3n, 3k etc).
4.2. Representative procedure for the preparation of 3-acetyl
coumarin derivatives 6
To a rapid stirring cold mixture of 2-hydroxy benzaldehyde
derivatives 4 (0.2 mol) and ethyl acetoacetate 5 (0.2 mol), piperi-
dine (2 ml) was added. The mixture was allowed to warm to
50 °C. Progress of the reaction was monitored by TLC. After com-
pletion of the reaction, the solid was filtered off and subsequently
washed with ethanol and recrystallized from water/ethanol
(30:70).35
4. Experimental section
4.1. General
All commercially available reagents were purchased from
Merck AG, Aldrich or Acros Organics and used without further
purification. Column chromatography was carried out on silica
gel (70–230 mesh). TLC was conducted on silica gel 250 micron,
F254 plates. For the synthesis of compounds 7 the experiments
were performed using a microwave oven (ETHOS 1600, Milestone)
with a power of 600 W specially designed for an organic synthesis
and modified with a condenser and mechanical stirrer. Melting
points were measured on a Kofler hot stage apparatus and are
uncorrected. The IR spectra were taken using Nicolet FT-IR Magna
550 spectrographs (KBr disks). Mass spectra of the products were
obtained with an HP (Agilent technologies) 5937 Mass Selective
Detector. 1H NMR spectra were recorded on a Bruker 400 or
500 MHz NMR instruments. The atoms numbering of the target
compounds used for 1H NMR data are depicted in Scheme 1. The
4.3. Representative procedure for the preparation of (E)-3-(3-
(pyridin-4-yl)acryloyl)-2H-chromen-2-one drivatives (7)
To a mixture of 3-acetyl coumarin drivatives 6 (0.12 mmol) and
pyridine-4-carbaldehyde (0.12 mmol) in 3 ml n-butanol were
added catalytic amount of piperidine (2 drops). The mixture was
irradiated with microwaves at 150 °C for 30 min. After completion
of the reactions, mixture was cooled and the precipitated solid was
filtered off and washed with ethanol. The organic phase was evap-
orated and the residue was purified by silica gel column chroma-
tography using petroleum ether/ethyl acetate (7:3) as the mobile
phase to give the pure product.

M. Alipour et al. / Bioorg. Med. Chem. 20 (2012) 7214–7222
7219
4.3.1. (E)-3-(3-(Pyridin-4-yl)acryloyl)-2H-chromen-2-one (7a)
215 °C, IR t
max/cmꢀ1 (KBr): 1724, 1675 (C@O), 1H NMR (DMSO-
Yield 77%, yellow solid, mp 110–112 °C, IR
t
max/cmꢀ1 (KBr):
d6, 400 MHz), 5.90 (s, 2H, –CH2N+), 7.28–7.32 (m, 2H, H2,6 phenyl),
1720, 1668 (C@O), 1H NMR (DMSO-d6, 400 MHz), 7.45 (t, 1H, H6
coumarin, J = 8.0 Hz), 7.51 (d, 1H, H8 coumarin, J = 8.0 Hz), 7.69–
7.44–7.53 (m, 3H, H6,7,8 coumarin), 7.69–7.73 (m, 2H, H3,5 phenyl),
7.81 (d, 1H, H vinylic, J = 16.4 Hz), 8.01 (d, 1H, H5 coumarin,
a
7.73 (m, 3H, Hb-pyridine and H vinylic), 7.77 (t, 1H, H7 coumarin,
J = 7.2 Hz), 8.15 (d, 1H, Hb vinylic, J = 16.4 Hz), 8.49 (d, 2H, Hb-pyr-
idine, J = 6.4 Hz), 8.86 (s, 1H, H4 coumarin), 9.32 (d, 2H, Ha-pyri-
dine, J = 6.4 Hz). Anal. Calcd for C24H17ClFNO3: C, 68.33; H, 4.06;
N, 3.32; Found: C, 68.24; H, 4.32; N, 3.65.
a
J = 8.0 Hz), 7.86 (d, 1H, Hb vinylic, J = 16.4 Hz), 7.96 (d, 1H, H5 cou-
marin, J = 8.0 Hz), 8.67 (d, 2H, Ha-pyridine, J = 4.6 Hz), 8.73 (s, 1H,
H4 coumarin). Anal. Calcd for C17H11NO3: C, 73.64; H, 4.00; N,
5.05. Found: C, 73.28; H, 4.22; N, 5.17.
4.4.3. (E)-1-Benzyl-4-(3-(8-methoxy-2-oxo-2H-chromen-3-yl)-
3-oxoprop-1-enyl)pyridinium chloride (3c)
4.3.2. (E)-8-Methoxy-3-(3-(pyridin-4-yl)acryloyl)-2H-chromen-
2-one (7b)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and benzyl chloride (1.2 mmol, 0.252 g), for 2 h, product
Yield 72%, yellow solid, mp 103–105 °C, IR
t
max/cmꢀ1 (KBr):
1719, 1664 (C@O), 1H NMR (DMSO-d6, 400 MHz), 3.73 (s, 3H,
3c was obtained, yield 75%, yellow solid, mp 255–257 °C, IR tmax/
OMe), 7.21–7.35 (m, 3H, H5,6,7 coumarin) 7.82 (d, 1H, H vinylic,
cmꢀ1 (KBr): 1720, 1679 (C@O), 1H NMR (DMSO-d6, 400 MHz),
a
J = 16.3 Hz), 7.88 (d, 2H, Hb-pyridine, J = 4.6 Hz), 8.12 (d, 1H, Hb
vinylic, J = 16.3 Hz), 8.74 (s, 1H, H4 coumarin), 8.82 (d, 2H, Ha-
pyridine, J = 4.6 Hz). Anal. Calcd for C18H13NO4: C, 70.35; H, 4.26;
N, 4.56. Found: C, 70.18; H, 4.41; N, 4.33.
3.95 (s, 3H, OMe), 5.88 (s, 2H, –CH2N+), 7.37–7.58 (m, 8H, H5,6,7
coumarin and 5H phenyl), 7.82 (d, 1H, H vinylic, J = 15.6 Hz),
a
8.14 (d, 1H, Hb vinylic, J = 15.6 Hz), 8.48 (d, 2H, Hb-pyridine,
J = 6.4 Hz), 8.82 (s, 1H, H4 coumarin), 9.31 (d, 2H, Ha-pyridine,
J = 6.4 Hz). EI-MS m/z (%) 398 (M+, 15), 397 (M+ꢀ1, 52), 307 (74),
278 (26), 251 (28), 203 (33), 91 (100), 65 (31). Anal. Calcd for
4.3.3. (E)-6-Bromo-3-(3-(pyridin-4-yl)acryloyl)-2H-chromen-2-
one 7c)
C25H20ClNO4: C, 69.20; H, 4.65; N, 3.23. Found: C, 68.94; H, 4.73;
N, 3.49.
Yield 64%, yellow solid, mp 114–116 °C, IR
t
max/cmꢀ1 (KBr):
1710, 1656 (C@O), 1H NMR (DMSO-d6, 400 MHz), 7.14 (d, 1H, H8
coumarin, J = 8.7 Hz), 7.29 (d, 1H, H7 coumarin, J = 8.7 Hz), 7.67
4.4.4. (E)-1-(2-Fluorobenzyl)-4-(3-(8-methoxy-2-oxo-2H-
chromen-3-yl)-3-oxoprop-1-enyl) pyridinium bromide (3d)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 2-fluorobenzyl chloride (1.2 mmol, 0.173 g), for 3 h,
product 3d was obtained, yield 75%, yellow solid, mp 215–
(d, 1H, H vinylic, J = 15.5 Hz), 7.88 (s, 1H, H5 coumarin), 8.00 (m,
a
3H, Hb-pyridine and Hb vinylic), 8.71 (s, 1H, H4 coumarin), 8.83
(d, 2H, Ha-pyridine, J = 4.6 Hz). Anal. Calcd for C17H10BrNO3: C,
57.33; H, 2.83; N, 3.93. Found: C, 57.61; H, 2.52; N, 3.70.
217 °C, IR t
max/cmꢀ1 (KBr): 1723, 1664 (C@O), 1H NMR (DMSO-
4.4. General procedure for synthesis of pyridinium halide
derivatives 3a–s
d6, 500 MHz), 3.94 (s, 3H, OMe), 5.97 (s, 2H, –CH2N+), 7.32–7.39
(m, 3H, H7 coumarin and H4,6 phenyl), 7.46–7.53 (m, 3H, H5,6 cou-
marin and H5 phenyl), 7.63–7.65 (m, 1H, H3 phenyl), 7.83 (d, 1H,
Dry acetonitrile (7 ml) was added to (E)-3-(3-(pyridin-4-
yl)acryloyl)-2H-chromen-2-one drivatives 7 (1 equiv), and the
mixture was dissolved by heating under reflux. Then appropriate
benzyl halides (1.2 equiv) and a catalytic amount of KI were added.
After heating under reflux for 2–4 h, it was left for cooling to room
temperature. The precipitated solids were separated by filtration,
washed with diethyl ether and dried. The resulting products were
further purified if needed by flash chromatography employing
chloroform/methanol (99:1) as the mobile phase to afford com-
pounds 3a–s.
H vinylic, J = 13.9 Hz), 8.14 (d, 1H, Hb vinylic, J = 13.9 Hz), 8.47
a
(d, 2H, Hb-pyridine, J = 6.4 Hz), 8.82 (s, 1H, H4 coumarin), 9.21 (d,
2H, Ha-pyridine, J = 6.4 Hz). 13C NMR (DMSO-d6, 125 MHz) 56.2,
57.3, 115.8, 116.0, 116.8, 118.7, 121.2, 121.6, 124.5, 125.0, 125.2,
126.4, 131.4, 132.0, 134.6, 136.2, 143.9, 145.4, 146.3, 148.5,
150.8, 157.9, 161.4, 186.9. EI-MS m/z (%) 416 (M+, 11), 415
(M+ꢀ1, 50), 307 (83), 278 (35), 203 (65), 109 (100). Anal. Calcd
for C25H19BrFNO4: C, 60.50; H, 3.86; N, 2.82. Found: C, 60.37; H,
3.75; N, 2.94.
4.4.5. (E)-1-(3-Fluorobenzyl)-4-(3-(8-methoxy-2-oxo-2H-
chromen-3-yl)-3-oxoprop-1-enyl)pyridinium chloride (3e)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 3-fluorobenzyl chloride (1.2 mmol, 0.173 g), for 4 h,
product 3e was obtained, yield 80%, yellow solid, mp 222–224 °C,
4.4.1. (E)-1-(2-Fluorobenzyl)-4-(3-oxo-3-(2-oxo-2H-chromen-3-
yl)prop-1-enyl)pyridinium chloride (3a)
Following Section 4.4, from compound 7a (R = H, 1 mmol,
0.277 g) and 2-fluorobenzyl chloride (1.2 mmol, 0.173 g), for 3 h,
product 3a was obtained, yield 72%, yellow solid, mp 246–248 °C,
IR
t
max/cmꢀ1 (KBr): 1726, 1661 (C@O), 1H NMR (DMSO-d6,
IR
t
max/cmꢀ1 (KBr): 1721, 1666 (C@O), 1H NMR (DMSO-d6,
400 MHz), 3.99 (s, 3H, OMe), 5.83 (s, 2H, –CH2N+), 7.28 (t, 1H, H5
500 MHz), 5.95 (s, 2H, –CH2N+), 7.32–7.36 (m, 2H, H4,6 phenyl),
7.47 (t, 1H, H6 coumarin, J = 7.3 Hz), 7.50–7.54 (m, 2H, H8 coumarin
and H3 phenyl), 7.62 (t, 1H, H5 phenyl, J = 7.3 Hz), 7.80 (t, 1H, H7
phenyl, J = 7.3 Hz), 7.38–7.55 (m, 6H, H5,6,7 coumarin and H phe-
nyl2,4,6), 7.82 (d, 1H, H vinylic, J = 16.4 Hz), 8.12 (d, 1H, Hb vinylic,
a
J = 16.4 Hz), 8.46 (d, 2H, Hb-pyridine, J = 6.4 Hz), 8.78 (s, 1H, H4
coumarin), 9.24 (d, 2H, Ha-pyridine, J = 6.4 Hz). Anal. Calcd for
coumarin, J = 7.3 Hz), 7.83 (d, 1H, H vinylic, J = 16.0 Hz), 7.99 (d,
a
1H, H5 coumarin, J = 7.3 Hz), 8.13 (d, 1H, Hb vinylic, J = 16.0 Hz),
8.46 (d, 2H, Hb-pyridine, J = 6.0 Hz), 8.82 (s, 1H, H4 coumarin),
9.18 (d, 2H, Ha-pyridine, J = 6.0 Hz). EI-MS m/z (%) 386 (M+, 11),
385 (M+ꢀ1, 39), 363 (26), 277 (78), 248 (47), 220 (32), 173 (89),
109 (100), 83 (67). Anal. Calcd for C24H17ClFNO3: C, 68.33; H,
4.06; N, 3.32. Found: C, 68.54; H, 3.82; N, 3.61.
C25H19ClFNO4: C, 66.45; H, 4.24; N, 3.10. Found: C, 66.71; H,
4.58; N, 2.86. EI-MS m/z (%) 416 (M+, 3), 415 (M+-1, 8), 308 (81),
307 (63), 203 (88), 109 (100), 83 (86).
4.4.6. (E)-1-(4-Fluorobenzyl)-4-(3-(8-methoxy-2-oxo-2H-
chromen-3-yl)-3-oxoprop-1-enyl)pyridinium chloride (7f)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 4-fluorobenzyl chloride (1.2 mmol, 0.173 g), for 2 h,
product 3f was obtained, yield 85%, yellow solid, mp 262–264 °C,
4.4.2. (E)-1-(4-Fluorobenzyl)-4-(3-oxo-3-(2-oxo-2H-chromen-3-
yl)prop-1-enyl)pyridinium chloride (3b)
Following Section 4.4, from compound 7a (R = H, 1 mmol,
0.277 g) and 4-fluorobenzyl chloride (1.2 mmol, 0.173 g), for 2 h,
product 3b was obtained, yield 90%, yellow solid, mp 213–
IR
400 MHz), 3.91 (s, 3H, OMe), 5.77 (s, 2H, –CH2N+), 7.20–7.24 (m,
2H, H vinylic and H7 coumarin), 7.35–7.38 (m, 2H, H2,6 phenyl),
t
max/cmꢀ1 (KBr): 1726, 1675 (C@O), 1H NMR (DMSO-d6,
a

7220
M. Alipour et al. / Bioorg. Med. Chem. 20 (2012) 7214–7222
7.44–7.47 (m, 2H, H5,6 coumarin), 7.52–7.56 (m, 3H, Hb vinylic and
H3,5 phenyl), 8.12 (d, 2H, Hb-pyridine, J = 6.4 Hz), 8.57 (s, 1H, H4
coumarin), 9.12 (d, 2H, Ha-pyridine, J = 6.4 Hz). EI-MS m/z (%) 416
(M+, 3), 415 (M+ꢀ1, 9), 307 (91), 278 (30), 251 (31), 203 (35),
109 (100). Anal. Calcd for C25H19ClFNO4: C, 66.45; H, 4.24; N,
3.10. Found: C, 66.68; H, 4.18; N, 3.35.
142 (33), 125 (27). Anal. Calcd for C25H18Cl3NO4: C, 59.72; H,
3.61; N, 2.79. Found: C, 59.94; H, 3.39; N, 2.55.
4.4.11. (E)-1-(3,4-Dichlorobenzyl)-4-(3-(8-methoxy-2-oxo-2H-
chromen-3-yl)-3-oxoprop-1-enyl)pyridinium chloride (3k)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 3,4-dichlorobenzyl chloride (1.2 mmol, 0.234 g), for
3 h, product 3k was obtained, yield 79%, brown solid, mp 238–
4.4.7. (E)-1-(2-Fluoro-6-nitrobenzyl)-4-(3-(8-methoxy-2-oxo-
2H-chromen-3-yl)-3-oxoprop-1-enyl)pyridinium chloride (3g)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 2-fluoro-6-nitrobenzyl chloride (1.2 mmol, 0.227 g),
for 2 h, product 3g was obtained, yield 70%, yellow solid, mp
240 °C, IR t
max/cmꢀ1 (KBr): 1727, 1663 (C@O), 1H NMR (DMSO-
d6, 400 MHz), 3.99 (s, 3H, OMe), 5.85 (s, 2H, –CH2N+), 7.45 (d, 1H,
H7 coumarin, J = 8.4 Hz), 7.52–7.64 (m, 3H, H5,6 phenyl and H6 cou-
marin), 7.79 (d, 1H, H5 coumarin, J = 8.4 Hz), 7.86 (d, 1H, H vinylic,
a
t
max/cmꢀ1 (KBr): 1728, 1679 (C@O), 1H NMR
J = 15.2 Hz), 8.01 (s, 1H, H2 phenyl), 8.17 (d, 1H, Hb vinylic,
J = 15.2 Hz), 8.50 (d, 2H, Hb-pyridine, J = 6.2 Hz), 8.84 (s, 1H, H4
coumarin), 9.29 (d, 2H, Ha-pyridine, J = 6.2 Hz). EI-MS m/z (%) 470
(M++4, 1), 468 (M++2, 6), 466 (M+, 9), 307 (40), 278 (15), 251
(14), 203 (30), 163 (32), 161(94), 159 (100). Anal. Calcd for
223–225 °C, IR
(DMSO-d6, 400 MHz), 3.96 (s, 3H, OMe), 6.20 (s, 2H, –CH2N+),
7.41 (t, 1H, H6 coumarin, J = 7.8 Hz), 7.49 (d, 1H, H7 coumarin,
J = 7.8 Hz), 7.53 (d, 1H, H5 coumarin, J = 7.8 Hz), 7.83–7.91 (m,
3H, H vinylic and H4,5 phenyl), 8.11–8.15 (m, 2H, H3 phenyl and
a
C25H18Cl3NO4: C, 59.72; H, 3.61; N, 2.79. Found: C, 59.45; H,
Hb vinylic), 8.43 (d, 2H, Hb-pyridine, J = 6.4 Hz), 8.79 (s, 1H, H4 cou-
marin), 9.12 (d, 2H, Ha-pyridine, J = 6.4 Hz). EI-MS m/z (%) 461 (M+,
2), 307 (90), 278 (62), 251 (24), 203 (55), 154 (67), 132 (100), 107
(69). Anal. Calcd for C25H18ClFN2O6: C, 60.43; H, 3.65; N, 5.64.
Found: C, 60.26; H, 3.93; N, 5.77.
3.76; N, 2.51.
4.4.12. (E)-4-(3-(8-Methoxy-2-oxo-2H-chromen-3-yl)-3-
oxoprop-1-enyl)-1-(2-methylbenzyl)pyridinium chloride (3l)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 2-methylbenzyl chloride (1.2 mmol, 0.168 g), for 2 h,
product 3l was obtained, yield 90%, yellow solid, mp 241–243 °C,
4.4.8. (E)-1-(2-Chlorobenzyl)-4-(3-(8-methoxy-2-oxo-2H-
chromen-3-yl)-3-oxoprop-1-enyl) pyridinium chloride (3h)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 2-chlorobenzyl chloride (1.2 mmol, 0.193 g), for 2 h,
product 3h was obtained, yield 73%, yellow solid, mp 222–
IR
t
max/cmꢀ1 (KBr): 1727, 1664 (C@O), 1H NMR (DMSO-d6,
400 MHz), 2.32 (s, 3H, CH3), 3.95 (s, 3H, OMe), 5.92 (s, 2H,
–CH2N+), 7.18 (d, 1H, H3 phenyl, J = 7.0 Hz), 7.27–7.41 (m, 4H,
H6,7 coumarin and H4,5 phenyl), 7.48 (d, 1H, H6 phenyl,
224 °C, IR
d6, 400 MHz), 3.95 (s, 3H, OMe), 6.00 (s, 2H, –CH2N+), 7.37–7.60
t
max/cmꢀ1 (KBr): 1725, 1678 (C@O), 1H NMR (DMSO-
J = 7.0 Hz), 7.53 (d, 1H, H5 coumarin, J = 7.2 Hz), 7.85 (d, 1H, H
a
vinylic, J = 15.6 Hz), 8.15 (d, 1H, Hb vinylic, J = 15.6 Hz), 8.47 (d,
2H, Hb-pyridine, J = 6.4 Hz), 8.82 (s, 1H, H4 coumarin), 9.11 (d,
2H, Ha-pyridine, J = 6.4 Hz). Anal. Calcd for C26H22ClNO4: C,
69.72; H, 4.95; N, 3.13. Found: C, 69.51; H, 5.18; N, 3.32.
(m, 7H, H5,6,7 coumarin and 4H phenyl), 7.84 (d, 1H, H vinylic,
a
J = 16.4 Hz), 8.15 (d, 1H, Hb vinylic, J = 16.4 Hz), 8.48 (d, 2H, Hb-
pyridine, J = 6.4 Hz), 8.82 (s, 1H, H4 coumarin), 9.18 (d, 2H, Ha-
pyridine, J = 6.4 Hz). EI-MS m/z (%) 434 (M++2, 1), 432 (M+, 3),
396 (41), 307 (67), 278 (22), 251 (21), 203 (22), 125 (100), 91
(96). Anal. Calcd for C25H19Cl2NO4: C, 64.11; H, 4.09; N, 2.99.
Found: C, 64.36; H, 4.24; N, 3.21.
4.4.13. (E)-4-(3-(8-Methoxy-2-oxo-2H-chromen-3-yl)-3-
oxoprop-1-enyl)-1-(3-methylbenzyl)pyridinium chloride (3m)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 3-methylbenzyl chloride (1.2 mmol, 0.168 g), for 3 h,
product 3m was obtained, yield 92%, yellow solid, mp
4.4.9. (E)-1-(3-Chlorobenzyl)-4-(3-(8-methoxy-2-oxo-2H-
chromen-3-yl)-3-oxoprop-1-enyl)pyridinium chloride (3i)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and (3-chlorobenzyl chloride (1.2 mmol, 0.193 g), for 3 h,
product 3i was obtained, yield 80%, yellow solid, mp 213–215 °C,
223–226 °C, IR
t
max/cmꢀ1 (KBr): 1719, 1674 (C@O), 1H NMR
(DMSO-d6, 400 MHz), 2.31 (s, 3H, CH3), 3.95 (s, 3H, OMe), 5.90 (s,
2H, –CH2N+), 7.24 (s, 1H, H2 phenyl), 7.33–7.53 (m, 6H, H5,6,7 cou-
marin, H4,5,6 phenyl), 7.82 (d, 1H, H vinylic, J = 15.6 Hz), 8.12 (d,
a
IR
t
max/cmꢀ1 (KBr): 1726, 1679 (C@O), 1H NMR (DMSO-d6,
1H, Hb vinylic, J = 15.6 Hz), 8.45 (d, 2H, Hb-pyridine, J = 6.4 Hz),
8.81 (s, 1H, H4 coumarin), 9.25 (d, 2H, Ha-pyridine, J = 6.4 Hz). EI-
MS m/z (%) 412 (M+, 2), 307 (23), 278 (13), 251 (10), 203 (8), 127
(13), 105 (100), 77 (24). Anal. Calcd for C26H22ClNO4: C, 69.72; H,
4.95; N, 3.13. Found: C, 69.55; H, 4.71; N, 3.45.
400 MHz), 3.95 (s, 3H, OMe), 5.85 (s, 2H, –CH2N+), 7.38–7.61 (m,
6H, H5,6,7 coumarin and H4,5,6 phenyl), 7.72 (s, 1H, H2 phenyl),
7.82 (d, 1H, H vinylic, J = 16.4 Hz), 8.12 (d, 1H, Hb vinylic,
a
J = 16.4 Hz), 8.46 (d, 2H, Hb-pyridine, J = 6.4 Hz), 8.79 (s, 1H, H4
coumarin), 9.25 (d, 2H, Ha-pyridine, J = 6.4 Hz). Anal. Calcd for
C
25H19Cl2NO4: C, 64.11; H, 4.09; N, 2.99. Found: C, 64.33; H,
4.4.14. (E)-4-(3-(8-Methoxy-2-oxo-2H-chromen-3-yl)-3-
oxoprop-1-enyl)-1-(4-methylbenzyl)pyridinium chloride (3n)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 4-methylbenzyl chloride (1.2 mmol, 0.168 g), for 2 h,
product 3n was obtained, yield 95%, yellow solid, mp 250–
4.20; N, 2.78.
4.4.10. (E)-1-(2,3-Dichlorobenzyl)-4-(3-(8-methoxy-2-oxo-2H-
chromen-3-yl)-3-oxoprop-1-enyl) pyridinium chloride (3j)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 2,3-dichlorobenzyl chloride (1.2 mmol, 0.234 g), for
4 h, product 3j was obtained, yield 75%, brown solid, mp 240–
252 °C, IR
t
max/cmꢀ1 (KBr): 1715, 1678 (C@O), 1H NMR
(DMSO-d6, 400 MHz), 2.31 (s, 3H, CH3), 3.95 (s, 3H, OMe), 5.78 (s,
2H, –CH2N+), 7.26 (d, 2H, H2,6 phenyl, J = 8.0 Hz), 7.37.7.52 (m,
242 °C, IR t
max/cmꢀ1 (KBr): 1725, 1664 (C@O), 1H NMR (DMSO-
5H, H5,6,7 coumarin and H3,5 phenyl), 7.82 (d, 1H, H vinylic,
a
d6, 400 MHz), 3.96 (s, 3H, OMe), 6.15 (s, 2H, –CH2N+), 7.41 (d, 1H,
H7 coumarin, J = 9.0 Hz), 7.46–7.54 (m, 4H, H5,6 coumarin and
J = 16.2 Hz), 8.10 (d, 1H, Hb vinylic, J = 16.2 Hz), 8.43 (d, 2H, Hb-
pyridine, J = 6.4 Hz), 8.77 (s, 1H, H4 coumarin), 9.20 (d, 2H, Ha-
pyridine, J = 6.4 Hz). 13C NMR (DMSO-d6, 100 MHz) d: 20.6, 56.2,
95.3, 116.8, 118.7, 121.6, 124.5, 125.0, 126.4, 128.7, 129.7, 131.1,
134.4, 136.2, 138.9, 142.0, 144.9, 146.3, 148.4, 150.4, 158.0,
186.9. Anal. Calcd for C26H22ClNO4: C, 69.72; H, 4.95; N, 3.13.
Found: C, 69.61; H, 4.77; N, 3.31.
H5,6 phenyl), 7.77 (d, 1H, H4 phenyl, J = 7.2 Hz), 7.85 (d, 1H, H
a
vinylic, J = 15.2 Hz), 8.16 (d, 1H, Hb vinylic, J = 15.2 Hz), 8.50 (d,
2H, Hb-pyridine, J = 6.4 Hz), 8.83 (s, 1H, H4 coumarin), 9.18 (d,
2H, Ha-pyridine, J = 6.4 Hz). EI-MS m/z (%) 471 (M++4, 1), 469
(M++2, 6), 467 (M+, 9), 396 (15), 307 (47), 203 (31), 159 (100),

M. Alipour et al. / Bioorg. Med. Chem. 20 (2012) 7214–7222
7221
4.4.15. (E)-1-(3-Cyanobenzyl)-4-(3-(8-methoxy-2-oxo-2H-
chromen-3-yl)-3-oxoprop-1-enyl)pyridinium bromide (3o)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 3-cyanobenzyl chloride (1.2 mmol, 0.181 g), for 3 h,
product 3o was obtained, Yield 83%, Yellow solid, mp 262–
4.4.19. (E)-4-(3-(6-Bromo-2-oxo-2H-chromen-3-yl)-3-oxoprop-
1-enyl)-1-(4-fluorobenzyl)pyridinium chloride (3s)
Following Section 4.4, from compound 7c (R = 6-Br, 1 mmol,
0.356 g) and 4-fluorobenzyl chloride (1.2 mmol, 0.173 g), for 4 h,
product 3s was obtained, yield 86%, yellow solid, mp 252–254 °C,
264 °C; IR
t
max/cmꢀ1 (KBr): 1718, 1677 (C@O), 1H NMR (DMSO-
IR t
max/cmꢀ1 (KBr): 1745, 1663 (C@O), 1H NMR (DMSO-d6,
d6, 400 MHz), 3.95 (s, 3H, OMe), 5.93 (s, 2H, –CH2N+), 7.39 (t, 1H,
H5 phenyl, J = 6.6 Hz), 7.47 (d, 1H, H7 coumarin, J = 7.5 Hz), 7.52
(d, 1H, H5 coumarin, J = 7.5 Hz), 7.67 (t, 1H, H6 coumarin,
400 MHz) 5.86 (s, 2H, –CH2N+), 7.28–7.33 (m, 2H, H2,6 phenyl),
7.51 (d, 1H, H8 coumarin, J =8.8 Hz), 7.66–7.70 (m, 2H, H3,5 phenyl),
7.83 (d, 1H, H vinylic, J = 15.6 Hz), 7.94 (dd, 1H, H7 coumarin,
a
J = 7.5 Hz), 7.82 (d, 1H, H vinylic, J = 16.8 Hz), 7.89–7.93 (m, 2H,
J = 2.4, 8.8 Hz), 8.10 (d, 1H, Hb vinylic, J = 15.6 Hz), 8.25 (d, 1H, H5
coumarin, J = 2.4 Hz), 8.47 (d, 2H, Hb-pyridine, J = 6.8 Hz), 8.78 (s,
1H, H4 coumarin), 9.28 (d, 2H, Ha-pyridine, J = 6.8 Hz). EI-MS m/z
(%) 466 (M++2, 27), 464 (M+, 30), 357 (61), 356 (51), 355 (51),
248 (40), 109 (100), 83 (45). Anal. Calcd for C24H16BrClFNO3 C,
57.57; H, 3.22; N, 2.80. Found: C, 57.39; H, 3.42; N, 2.47.
a
H4,6 phenyl), 8.10 (s, 1H, H2 phenyl), 8.13 (d, 1H, Hb vinylic,
J = 16.8 Hz), 8.47 (d, 2H, Hb-pyridine, J = 6.4 Hz), 8.79 (s, 1H, H4
coumarin), 9.27 (d, 2H, Ha-pyridine, J = 6.4 Hz). EI-MS m/z (%) 423
(M+, 6), 422 (M+ꢀ1, 19), 307 (100), 278 (47), 251 (46), 203 (46),
116 (65). Anal. Calcd for C26H19BrN2O4: C, 62.04; H, 3.80; N, 5.57.
Found: C, 62.31; H, 3.69; N, 5.78.
4.5. Molecular docking studies
4.4.16. (E)-1-(4-(Chloromethyl)benzyl)-4-(3-(8-methoxy-2-oxo-
2H-chromen-3-yl)-3-oxoprop-1-enyl)pyridinium chloride (3p)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 1,4-bis(chloromethyl)benzene (1.2 mmol, 0.578 g), for
4 h, product 3p was obtained, yield 72%, brown solid, mp 222–
For docking purpose, the pdb structure of 3I6Z was retrieved
from the Brookhaven protein database38 as a complex bound with
inhibitor N-sacharinohexyl-galantamine. Then, the water mole-
cules and co-crystallized ligand were removed from the protein.
The structure of target compound was constructed by Marvine-
Sketch which was converted to 3D structure using Openbabel ver-
sion (2.3.1). Finally the pdbqt formats (The format is used by
docking software) of the protein and ligand were prepared using
Autodock Tools (ver. 1.5.4)39 using default parameters. Docking
was carried out using Autodock vina (ver. 1.1.1) with default
parameters and exhaustiveness was set at 100. The docking site
was defined by establishing a box at geometrical center of the na-
tive ligand present in the above mentioned PDB structure with the
dimensions of 36, 34, 56, that covering all binding sites occurred in
the active site of the enzyme. The grid spacing of 0.375 Å was used
and the coordinates x, y, z for the center of grid box were 1.75, 63.1,
67.11, respectively. Finally the lowest energy conformations were
selected for analyzing the interactions between the AChE and
inhibitor. The results were shown using PosViewWeb 1.97.040
and Chimera 1.6.41
224 °C, IR t
max/cmꢀ1 (KBr): 1716, 1681 (C@O), 1H NMR (DMSO-
d6, 400 MHz), 3.95 (s, 3H, OMe), 4.77 (s, 2H, –CH2Cl), 5.87 (s, 2H,
–CH2N+), 7.38–7.55 (m, 7H, H5,6,7 coumarin and 4H phenyl), 7.81
(d, 1H, H vinylic, J = 16.4 Hz), 8.12 (d, 1H, Hb vinylic, J = 16.4 Hz),
a
8.45 (d, 2H, Hb-pyridine, J = 6.4 Hz), 8.78 (s, 1H, H4 coumarin),
9.23 (d, 2H, Ha-pyridine, J = 6.4 Hz). Anal. Calcd for C26H21Cl2NO4:
C, 64.74; H, 4.39; N, 2.90. Found: C, 64.43; H, 4.48; N, 2.57.
4.4.17. (E)-4-(3-(8-Methoxy-2-oxo-2H-chromen-3-yl)-3-
oxoprop-1-enyl)-1-(4-methoxybenzyl)pyridinium chloride (3q)
Following Section 4.4, from compound 7b (R = 8-OMe, 1 mmol,
0.307 g) and 4-methoxybenzyl chloride (1.2 mmol, 0.187 g), for
2 h, product 3q was obtained, yield 80%, brown solid, mp 193–
195 °C, IR t
max/cmꢀ1 (KBr): 1727, 1677 (C@O), 1H NMR (DMSO-
d6, 400 MHz), 3.76 (s, 3H, OMe), 3.95 (s, 3H, OMe coumarin), 5.89
(s, 2H, –CH2N+), 7.00 (d, 2H, H3,5 phenyl, J = 8.4 Hz), 7.40 (t, 1H,
H6 coumarin, J = 8.0 Hz), 7.48 (d, 1H, H7 coumarin, J = 8.0 Hz),
7.53 (d, 1H, H5 coumarin, J = 8.0 Hz), 7.56 (d, 2H, H2,6 phenyl,
4.6. AChE and BuChE inhibition assay
J = 8.4 Hz), 7.81 (d, 1H, H vinylic, J = 16.4 Hz), 8.12 (d, 1H, Hb
a
vinylic, J = 16.4 Hz), 8.45 (d, 2H, Hb-pyridine, J = 5.8 Hz), 8.81 (s,
1H, H4 coumarin), 9.26 (d, 2H, Ha-pyridine, J = 5.8 Hz). EI-MS m/z
(%) 428 (M+, 9), 427 (M+ꢀ1, 31), 307 (47), 203 (54), 121 (100),
106 (65), 77 (65). Anal. Calcd for C26H22ClNO5: C, 67.31; H, 4.78;
N, 3.02. Found: C, 67.01; H, 5.12; N, 3.34.
Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized
powder, from electric eel, 1000 unit), butylcholinesterase (BuChE,
E.C. 3.1.1.8, from equine serum), butylthiocholine iodode (BTC),
was purchased from Sigma–Aldrich. 5,50-Dithiobis-(2-nitrobenzoic
acid), potassium dihydrogen phosphate, dipotassium hydrogen
phosphate, potassium hydroxide, sodium hydrogen carbonate,
and acetylthiocholine iodide were obtained from Fluka. The stock
solutions of tested compounds were prepared in a mixture of
1 ml DMSO and 9 ml methanol followed by dilution in 0.1 M
KH2PO4/K2HPO4 buffer (pH 8.0) to obtain final assay concentra-
tions. The previously reported method was applied to evaluate en-
zyme inhibition activity of tested compounds. All solution
temperatures were adjusted to 25 °C prior to use. Five different
concentrations of each compound were tested in triplicate to ob-
tain 20% to 80% inhibition of AChE and BuChE activity. The assay
4.4.18. (E)-4-(3-(6-Bromo-2-oxo-2H-chromen-3-yl)-3-oxoprop-
1-enyl)-1-(2-fluorobenzyl)pyridinium chloride (3r)
Following Section 4.4, from compound 7c (R = 6-Br, 1 mmol,
0.356 g) and 2-fluorobenzyl chloride (1.2 mmol, 0.173 g), for 4 h,
product 3r was obtained, yield 78%, brown solid, mp 218–220 °C,
IR
t
max/cmꢀ1 (KBr): 1752, 1664 (C@O), 1H NMR (DMSO-d6,
400 MHz), 5.98 (s, 2H, –CH2N+), 7.30–7.35 (m, 2H, H4,6 phenyl),
7.49–7.54 (m, 2H, H8 coumarin and H3 phenyl), 7.64 (t, 1H, H5 phe-
nyl, J = 7.4 Hz), 7.84 (d, 1H, H vinylic, J = 15.6 Hz), 7.94 (dd, 1H, H7
a
coumarin, J = 2.4, 9.2 Hz), 8.11 (d, 1H, Hb vinylic, J = 15.6 Hz), 8.25
(d, 1H, H5 coumarin, J = 2.4 Hz), 8.48 (d, 2H, Hb-pyridine,
J = 6.4 Hz), 8.80 (s, 1H, H4 coumarin), 9.22 (d, 2H, Ha-pyridine,
J = 6.4 Hz). 13C NMR (DMSO-d6, 100 MHz) d: 57.6, 115.9, 116.1,
116.6, 118.7, 120.2, 125.3, 125.5, 126.5, 131.5, 132.2, 132.6,
134.5, 136.6, 136.9, 144.6, 145.5, 146.9, 150.8, 153.7, 157.9,
159.5, 161.5, 186.9. EI-MS m/z (%) 466 (M++2, 9), 464 (M+, 11),
357 (15), 248 (12), 109 (100), 83 (18). Anal. Calcd for
medium contained 3 ml of 0.1 M phosphate buffer pH 8.0, 100
of 0.01 M 5,50-dithio-bis(2-nitrobenzoic acid), 100
l of 2.5 unit/
ml enzyme solution (AChE, E.C. 3.1.1.7, Type V-S, lyophilized pow-
der, from electric eel) (Sigma Chemical). 100 l of each tested com-
pounds, were added to the assay medium and incubated at 25 °C
for 15 min followed by adding 20 l of substrate (acetylthiocholine
iodide). After that the rate of absorbance change was measured at
412 nm for 6 minutes. The blank reading solution was used to jus-
tify non-enzymatic hydrolysis of substrate during the assay. The
ll
l
l
l
C24H16BrClFNO3: C, 57.57; H, 3.22; N, 2.80. Found: C, 57.44; H,
3.51; N, 2.97.
blank solution contained 3 ml buffer, 200 ll water, 100 ll DTNB

7222
M. Alipour et al. / Bioorg. Med. Chem. 20 (2012) 7214–7222
2. Selkoe, D. J. Physiol. Rev. 2001, 81, 741.
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zyme without the inhibitor is processed following the same proto-
col. The rate of the substrate enzymatic hydrolysis was calculated,
and inhibition percent of the test compounds was determined. The
IC50 values were determined graphically from inhibition curves
(log inhibitor concentration Vs percent of inhibition). Spectropho-
tometric measurements were performed on a UV-2100 Rayleigh
Double Beam Spectrophotometer. The same method was used for
BuChE inhibition assay.
4.7. Antioxidant activity
4.7.1. FRAP assay
The modified method was taken to evaluate the antioxidant
activity of the synthesized compounds using the FRAP assay.42
300 mM acetate buffer (3.1 g C2H3NaO2ꢁ3H2O and 16 ml C2H4O2),
pH 3.6 and 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in
40 mM hydrochloric acid and 20 mM ferric chloride hexahydrate
solution were mixed. The temperature of the solution was raised
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to 37 °C before the assay. After that 300 ll of the compound stock
solution was added thereto in the dark and the change of absor-
bance was monitored at the 593 nm. The concentration of the re-
duced ferrous ion (Fe2+
)
in the solution as colored ferrous
tripyridyltriazine complex was measured. Results are expressed
in M ferrous/g dry mass according to the plotted standard curve
l
of ferrous sulfate. Ascorbic acid and BHT were used as references.
4.7.2. DPPH (1,1-diphenyl-2-picrylhydrazyl) assay
To assess the scavenging ability of the compounds the reported
DPPH method was applied.43 The target compounds were tested
within the range of 0–25 lg/ml in methanol. To 2.5 ml of tested
compound in 5 different concentrations, 1 ml of 0.3 mM DPPH eth-
anol solution was added. In the meantime 1 ml of methanol was
added and the solution was allowed to react for 30 min in the dark
at room temperature. The change of the absorbance was read at
518 nm. The reading blank consisted of 2.5 ml of tested compound
and 1 ml of methanol meanwhile the mixture of 1 ml DPPH and
2.5 ml of methanol was used as negative control. The percent of
the antioxidant activity was calculated as follow:% Inhibi-
tion=[(ABꢀAA)/AB] ꢂ 100. Where: AB: absorption of blank sample,
AA: absorption of tested samples. The IC50 value was calculated
and compared with ascorbic acid as reference.
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This work was financially supported by Grants from the Re-
search Council of Tehran University of Medical Sciences and INSF
(Iran National Science Foundation). Molecular graphics and analy-
ses 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, with sup-
port from the National Institutes of Health (National Center for Re-
search Resources grant 2P41RR001081, National Institute of
General Medical Sciences grant 9P41GM103311).
References and notes
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