Design and synthesis of new dual binding site cholinesterase inhibitors: In vitro inhibition studies with in silico docking

Article abstract of DOI:10.2174/15701808113106660078

Cholinesterases (ChEs) play a vital role in the regulation of cholinergic transmission. The inhibition of ChEs is considered to be involved in increasing acetylcholine level in the brain and thus has been implicated in the treatment of Alzheimer's disease. We have designed and synthesized a series of novel indole derivatives and screened them for inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Most of the tested compounds exhibited inhibitory activity against AChE and BChE. Among them 4f and 6e showed the highest AChE inhibitory activity with IC₅₀ 91.21±0.06 and 68.52±0.04 μM, respectively. However compound 5a exhibited the highest inhibitory activity against BChE (IC₅₀ 55.21±0.12 μM).

Full text of DOI:10.2174/15701808113106660078

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Letters in Drug Design & Discovery, 2014, 11, 331-338
331
Design and Synthesis of New Dual Binding Site Cholinesterase Inhibitors:
in vitro Inhibition Studies with in silico Docking
Muhammad Yara,*, Marek Bajdab, Rana Atif Mehmoodc, Lala Rukh Sidraa, Nisar Ullahd, Lubna
Shahzadia, Muhammad Ashrafe, Tayaba Ismaile, Sohail Anjum Shahzadf, Zulfiqar Ali Khang,
Syed Ali Raza Naqvig and Nasir Mahmoodh
aInterdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore,
54000, Pakistan
bFaculty of Chemistry, University of Warsaw, 02-093 Warsaw, Pasteura 1, Poland and Department of Physicochemical
Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical College, 30-688 Cracow, Medyczna 9, Poland
cDepartment of Chemistry, Government College University, Lahore, 54000, Pakistan
dDepartment of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran, 31261, Saudi Arabia
eDepartment of Biochemistry & Biotechnology, The Islamia University of Bahawalpur, Bahawalpur, 63100, Pakistan
fDepartment of Chemistry, COMSATS Institute of Information Technology, Abbottabad, 22060, Pakistan
gDepartment of Chemistry, Government College University, Faisalabad, 38000, Pakistan
hDepartment of Allied Sciences and Chemical Pathology, University of Health Sciences, Lahore, 54600, Pakistan
Abstract: Cholinesterases (ChEs) play a vital role in the regulation of cholinergic transmission. The inhibition of ChEs is
considered to be involved in increasing acetylcholine level in the brain and thus has been implicated in the treatment of
Alzheimer’s disease. We have designed and synthesized a series of novel indole derivatives and screened them for inhibi-
tion of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Most of the tested compounds exhibited inhibitory
activity against AChE and BChE. Among them 4f and 6e showed the highest AChE inhibitory activity with
IC50 91.21±0.06 and 68.52±0.04 µM, respectively. However compound 5a exhibited the highest inhibitory activity against
BChE (IC50 55.21±0.12 µM).
Keywords: Acetylcholinesterase, Alzheimer’s disease, Butyrylcholinesterase, Hydrazides, Indole derivatives, Molecular dock-
ing, SAR.
INTRODUCTION
performs secondary non-cholinergic functions and co-
localizes with the β-amyloid peptide (Aβ) deposits present in
the brain of Alzheimer’s patients. It has been postulated that
due to the presence of peripheral anionic site (PAS), AChE
may bind amyloid fibrils, stabilize them and induce a con-
formational transition from Aβ into its amyloidogenic form
[7, 8].
Alzheimer’s disease (AD), the most common form of
neurodegenerative senile dementia, is associated with selec-
tive loss of cholinergic neurons and reduced level of acetyl-
choline neurotransmitter. The illness is characterized by
memory deficit and progressive impairment of cognitive
functions [1]. It has been revealed that an estimated 35.6
million people worldwide live with dementia [2]. The cho-
linergic hypothesis postulates that Alzheimer’s is caused by
a decrease in acetylcholine (ACh) level in the brain, leading
to gradual neurodegeneration. In normal brain signaling,
ACh, in turn, is related to preserving and accessing memory,
as well as function [3].
While BChE is primarily found in plasma, liver, and
muscle tissues, its biological function is not fully known.
However, whereas AChE preferentially hydrolyzes acetyl
esters such as acetylcholine, BChE hydrolyzes butyrylcho-
line [9-11].
In order to understand the molecular pathogenesis of AD,
enormous research efforts have been devoted in the past two
decades [12]. The current therapeutic approach exploits the
enhancement of the central cholinergic function [13] to in-
crease the acetylcholine levels in the brain. As a result, vari-
ous cholinergic drugs, such as tacrine, [14] donepezil, [15]
rivastigmine, [16] and more recently galantamine [17] have
been developed to alleviate the symptoms of AD (Fig. 1).
However, due to adverse events, tacrine was discontinued
[18].
Therefore, the mainstays of current pharmacotherapy of
AD are drugs aimed at increasing the acetylcholine level
through the inhibition of enzymes: acetylcholinesterase and
butyrylcholinesterase [4-6]. Studies have shown that AChE
*Address correspondence to this author at the Interdisciplinary Research
Center in Biomedical Materials, COMSATS Institute of Information Tech-
nology, Lahore, 54000, Pakistan; Tel: +92-42-111001007; Ext 829;
Fax: 0092-42-35321090; E-mail:drmyar@ciitlahore.edu.pk
1875-628X/14 $58.00+.00
©2014 Bentham Science Publishers

332 Letters in Drug Design & Discovery, 2014, Vol. 11, No. 3
Yar et al.
O
H3CO
H3CO
NH2
N
N
Donepezil
Tacrine
H3C
H3C
N
O
N
CH3
O
CH3
OCH3
O
H3C
N
HO
H
CH3
Rivastigmine
Galantamine
Fig. (1). Chemical structures of the best known AChE inhibitors.
R
H
H
N
N
O
R= H or Cl
n
N
O
N
n= 5-10
H
R
CH3
N
H
N
H
O
N
R= H or Cl
n
n
O
N
HN
n=3
Fig. (2). Indole group containing dual binding site AChE inhibitors.
The potential effectiveness offered by the above inhibi-
tors, unfortunately, is often limited by the appearance of cen-
tral and peripheral side effects. For example, clinical studies
have shown that tacrine has hepatotoxic liability [19, 20].
Therefore, large diversity of multi-target directed AChE in-
hibitors such as tacrine and nimodipine hybrids have also
been evaluated [21, 22]. In addition, no therapeutic treatment
is available for AD in Down syndrome [23].
Based on the promising nature of the indole analogues,
we have designed and synthesized a series of new indole-
moiety containing compounds (5a-5c, 6a-6e) along with
known (4a-4g) and screened them for inhibition of AChE
and BChE. This communication deals with the synthesis of
these compounds and their biological and docking studies.
MATERIALS AND METHODS
Chemistry
Indole alkaloids are well known due to their wide bio-
logical importance [24, 25] such as inhibitors of AChE [26-
28] and BChE [29, 30]. Compounds containing indole ring
were also found to be the dual binding site AChE inhibitors
(Fig. 2), which in turn has a potential of disease modifying
agents by inhibiting the Aβ peptide due to their binding abil-
ity with both the catalytic and peripheral sites of the enzymes
[28]. Thus there is a great deal of interest in the development
of dual binding site AChE inhibitors in order to control AD
[26].
Indole analogues 2-6 were synthesized from commer-
cially available indole-3-acetic acid 1 as depicted in Scheme
1. Acid catalyzed esterification of 1 in methanol gave ester 2,
which was treated with hydrazine to produce the desired
indole hydrazide 3 in 85% yield. Reaction of hydrazide 3
with a variety of sulfonyl chlorides in a mixture of dichloro-
methane and water produced the desired sulfonohydrazides
6a-6e. Likewise, hydrazide 3 was condensed with acetic an-

New Dual Binding Site Cholinesterase Inhibitors
Letters in Drug Design & Discovery, 2014, Vol. 11, No. 3 333
O
O
OH
MeOH,
OCH3
H2SO4, reflux
N
N
89%
H
H
1
2
85%
NH2NH2, MeOH
O
O
O
R2
O
O
S
R1SO2Cl
NaHCO3
NHNH2
NHNH
R2CO-O-COR2
PhOCOCl
NHNH
R1
O
N
H
CH2Cl2, H2O
N
N
H
3
H
6a = R1 = 4-methylphenyl, 46%
6b = R1 = 2-nitrophenyl, 72%
6c = R1 = 3-nitrophenyl, 83%
6d = R1 = 4-nitrophenyl, 70%
6e = R1 = 4-bromophenyl, 72%
5a = R2 = CH3, 49%
5b = R2 = CF3, 77%
5c = R2 = OPh, 73%
Ref [31]
R2
4a R1 = H, R2 = H
4b R1 = H, R2 = 2-OH
4c R1 = H, R2 = 4-OCH3
4d R1 = H, R2 = 4- CH3
4e R1 = CH3, R2 = H
4f R1 = Ph, R2 = H
N
NH
O
R1
4g R1 = H, R2 = 4-Cl
N
H
56-95 %
Scheme (1). Synthetic protocol of indole derivatives.
4a-g
hydride and trifluoroacetic anhydride to synthesize acetohy-
drazides 5a and 5b respectively (Scheme 1). Similarly the
hydrazine carboxylate 5c was obtained by the reaction of
phenyl chloroformate with hydrazide 3 in 73% yields
(Scheme 1). Compounds (4a-4g) were synthesized according
to the literature procedures [31]. The structures of all new
General Procedure (GP-1) for the Synthesis of (6a-6e)
To a solution of compound 3 (0.2 g, 1.06 mM) in 3 M
aqueous NaHCO3 (2 mL) a solution of the corresponding
sulfonyl chloride (1.16 mM) was added dropwise in 2 mL
CH2Cl2 and the reaction mixture was stirred at room tem-
perature for 4 hours. The precipitated product formed was
filtered and successively washed with dilute HCl and n-
hexane. The residue was purified by recrystallization in
methanol to provide pure 6a-6e.
1
compounds were established with the aid of IR, H-NMR,
13C-NMR, mass spectrometry and elemental analyses.
Material and Instruments
Reagents were purchased from common commercial
suppliers and were used without further purification. Sol-
vents were purified and dried by standard procedures, when
necessary. TLC was performed on silica coated aluminum
N'-(2-(1H-Indol-3-yl)acetyl)-4-methylbenzenesulfonohydr-
azide (6a)
Following the general procedure (GP-1) the compound
6a was obtained as a yellowish brown solid, yield 46%; m.p.
90 °C; Rf (EtOAc: hexane, 2:1) 0.62; IR (KBr) cm-1 3409
1
plates (6F254, 0.2 mm). H-NMR and 13C-NMR spectra
were recorded on Bruker NMR 500 MHz and chemical shifts
were calculated with reference to CDCl3 (7.26). IR spectra
were recorded on a Jasco A-302 IR spectrophotometer. Mass
spectra were recorded on a Varian MAT 312 double focusing
spectrometer, connected to an IBM-AT compatible PC com-
puter system. Elemental analyses were recorded on the Ele-
mentar, Vario micro cube, Germany.
1
(NH), 3211, 3208 (NHNH), 1657 (C=O); H NMR (500
MHz; CD3OD): δ 7.71-6.86 (9H, m, Ar-H), 3.78 (2H, s,
CH2), 2.35 (3H, s, CH3); 13C NMR (125 MHz; CD3OD): δ
172.7 (C=O), 145.4 (C), 142 (C), 138.2 (C), 136 (C), 130.4
(CH), 130.2 (CH), 130.1 (CH), 129.5 (CH), 128.8 (CH),
127.2 (CH), 125.1 (CH), 122.8 (CH), 120.1 (CH), 112.5 (C),
31.9 (CH2), 21.74 (CH3); MS m/z (%) 343 (M+); Anal. calc.

334 Letters in Drug Design & Discovery, 2014, Vol. 11, No. 3
Yar et al.
for C17H17N3O3S: C, 59.46; H, 4.99; N, 12.24; found: C, C,
59.42; H, 4.91; N, 12.19.
HCl to remove unreactive hydrazide. Crystallization from
methanol yielded 5a as purple crystalline solid (0.12 g,
49%). m.p 117 °C; Rf (EtOAc: hexane, 1:1) 0.36; IR (KBr)
cm-1 3401 (NH), 3191, 3188 (NHNH), 1633, 1666 (C=O);
1H NMR (500 MHz; CD3OD): δ 7.59-6.9 ( 5H, m, ArH), 3.7
(2H, s, CH2), 1.9 (3H, s, CH3); 13C NMR (125 MHz;
CD3OD): δ 173.7 (C=O), 172.1 (C=O), 138 (C), 128.5 (C),
124.9 (CH), 122.5 (CH), 119.8 (CH), 119.4 (CH), 112.2
(CH), 108.7 (C), 31.8 (CH2), 20.4 (CH3); MS m/z (%) 231
(M+); Anal. calc. for C13H15N2O2: C, 62.33; H, 5.67; N,
18.17; found: C, 62.32; H, 5.63; N, 18.12.
N'-(2-(1H-Indol-3-yl)acetyl)-2-nitrobenzenesulfonohydra-
zide (6b)
Following the general procedure compound 6b was ob-
tained as an off-white solid; yield 72%; m.p. 254 °C; Rf
(EtOAc) 0.413; IR (KBr) cm-1 3400 (NH), 3201, 3206
(NHNH), 1661 (C=O); 1H NMR (500 MHz; CD3OD): δ 7.9-
6.9 (9H, m, ArH), 3.61 (2H, s, CH2); 13C NMR (125 MHz;
CD3OD): δ 174.2 (C=O), 138.3 (C), 128.7 (C), 125.13 (CH),
122.8 (CH), 122.2 (CH), 121 (CH), 119.6 (CH), 112.5 (CH),
109.3 (C), 32.2 (CH2); MS m/z (%) 374 (M+); Anal. calc. for
C16H14N4O5S: C, 51.33; H, 3.77; N, 14.97; found: C, 51.30;
H, 3.71; N, 14.95.
N'-(2-(1H-indol-3-yl)acetyl)-2,2,2-trifluoroacetohydrazide
(5b)
To a solution of compound 3 (0.2 g, 1.058 mM) in THF
(5 mL) trifluoroacetic anhydride (0.2 mL, 1.164 mM) was
added dropwise and the mixture was stirred at room tem-
perature for 2 hours. The precipitated product was filtered
off and washed successively with NaHCO3 (3 M) and diluted
to obtain 5b (0.23 g, 77%) as light brown solid; m.p. 120 °C;
Rf (EtOAc) 0.74; IR (KBr) cm-1 3399 (NH), 3205, 3201
(NHNH), 1644, 1670 (C=O); 1H NMR (500 MHz; CD3OD):
δ 7.76-6.99 (5H, m, ArH), 3.72 (2H, s, CH2); 13C NMR (125
MHz; CD3OD): δ 174.2 (C=O), 169.73 (C=O), 138.2 (C),
128.7 (C), 125.1 (CH), 122.7 (CH), 120.3 (CH), 119.6 (CH),
112.5 (CH), 109.2 (C), 32.26 (CH2); MS m/z (%) 285 (M+);
Anal. calc. for C12H10F3N3O2: C, 50.53; H, 3.53; N, 14.73;
found: C, 50.49; H, 3.51; F, 19.98; N, 14.69.
N'-(2-(1H-Indol-3-yl)acetyl)-3-nitrobenzenesulfonohydr-
azide (6c)
Following the general procedure compound 6c was ob-
tained as golden yellow solid; yield 83%; m.p. > 350 °C; Rf
(EtOAc: hexane, 7:3) 0.282; IR: (KBr) cm-1 3395 (NH),
3198, 3202 (NHNH), 1649 (C=O); 1H NMR(500 MHz;
CD3OD): δ 8.62-7.01 (9H, m, ArH), 3.65 (2H, s, CH2); 13C
NMR (125 MHz; CD3OD): δ 133.2 (CH), 131.2 (CH), 126
(CH), 122.2 (CH), 119.6 (CH), 33.8 (CH2); MS m/z (%) 374
(M+); Anal. calc. for C16H14N4O5S: C, 51.33; H, 3.77; N,
14.97; found: C, 51.29; H, 3.73; N, 14.91.
N'-(2-(1H-Indol-3-yl)acetyl)-4-nitrobenzenesulfonohydraz-
ide (6d)
Phenyl 2-(2-(1H-indol-3-yl)acetyl)hydrazinecarboxylate (5c)
To a solution of compound 3 (0.2 g, 1.058 mM) in aque-
ous NaHCO3 (3 M, 1 mL) phenyl chloroformate (0.13 mL,
1.058 mM) was added dropwise and the mixture was stirred
at room temperature for 2 hours. The precipitated product
was filtered off and washed with dilute HCl to obtain 5c
(0.24 g, 73%) as a white solid. m.p. 148°C; Rf (EtOAc) 0.64;
IR: (KBr) cm-1 3424 (NH), 3213, 3206 (NHNH), 1652, 1689
Following the general procedure (GP-1) the compound
6d was obtained as a light yellow solid; yield 70%; m.p. 108
°C; Rf (EtOAc) 0.739; IR (KBr) cm-1 3403 (NH), 3225, 3219
(NHNH), 1655 (C=O); 1H NMR (500 MHz; CD3OD): δ 8.2-
6.98 (9H, m, ArH), 3.65 (2H, s, CH2); 13C NMR (125 MHz;
CD3OD): δ 172.8 (C=O), 150.4 (C), 131.6 (C), 130.7 (C),
128.6 (C), 125.3 (CH), 124.9 (CH), 124.6 (CH), 123 (CH),
120.2 (CH), 120 (CH), 112.7 (C), 32 (CH2); MS m/z (EI)
374; Anal. calc. for C16H14N4O5S: C, 51.33; H, 3.77; N,
14.97; found: C, 51.31; H, 3.76; N, 14.94.
1
(C=O); H NMR (500 MHz; CD3OD): δ 7.7-6.9 (10H, m,
ArH), 3.72 (2H, s, CH2); 13C NMR (125 MHz; CD3OD): δ
174.7 (C=O), 157.1 (C=O), 152.5 (C), 138.3 (C), 130.9
(CH), 130.7 (CH), 128.8 (C), 127.0 (CH), 126.9 (CH),125.1
(CH), 122.9 (CH), 122.8 (CH), 120.2 (CH), 119.7 (CH),
112.4 (CH), 108.9 (C), 32.1 (CH2); MS m/z (%) 309 (M+);
Anal. calc. for C17H15N3O3: C, 66.01; H, 4.89; N, 13.58;
found: C, 65.98; H, 4.83; N, 13.55.
N'-(2-(1H-indol-3-yl)acetyl)-4-bromobenzenesulfonohydra-
zide (6e)
Following the general procedure (GP-1) compound 6e
was obtained as light yellow crystalline solid; yield 72 %;
m.p. 88 °C; Rf (EtOAc) 0.869; IR (KBr) cm-1 3415 (NH),
AChE and BChE Assay
1
3217, 3213 (NHNH), 1646 (C=O); H NMR (500 MHz;
The AChE and BChE inhibition activity was performed
according to the method of Ellman [32] with slight modifica-
tions. Total volume of the reaction mixture was 100 µL con-
tained 60 µL Na2HPO4 buffer with concentration of 50 mM
and pH 7.7. A 10 µL test compound (0.5 mM per well) was
added, followed by the addition of 10 µL enzyme (0.005 unit
AChE, 0.5 unit BChE per well, Sigma Inc). The contents
were mixed and pre-read at 405 nm and pre-incubated for
10 min at 37°C. The reaction was initiated by the addition of
10 µL of 0.5 mM per well substrate (acetylthiocholine iodide
or butyrylthiocholine bromide), followed by the addition of
10 µL DTNB (0.5 mM per well). After 30 min of incubation
at 37ºC, absorbance was measured at 405 nm. Synergy HT
CD3OD): δ 7.73-6.9 (9H, m, ArH) 3.62 (2H, s, CH2); 13C
NMR (125 MHz; CD3OD): δ 132.7 (C), 132.4 (C), 130.2
(C), 127.5 (CH), 125.1 (CH), 122.8 (CH), 120.2 (CH), 119.8
(CH), 112.5 (C), 32.8 (CH2); MS m/z (%) 406 (M+); Anal.
calc. for C16H14BrN3O3S: C, 47.07; H, 3.46; Br, N, 10.29;
found: C, 47.03; H, 3.42; Br, N, 10.26.
N'-acetyl-2-(1H-indol-3-yl)acetohydrazide (5a)
To a solution of compound 3 (0.2 g, 1.06 mM) in H2O
(1.6 mL) acetic anhydride (0.1 mL, 1.16 mM) was added and
the mixture was stirred for 2 hours at room temperature. The
precipitated product was filtered off and washed with dilute

New Dual Binding Site Cholinesterase Inhibitors
Letters in Drug Design & Discovery, 2014, Vol. 11, No. 3 335
Table 1. In vitro AChE & BChE Inhibition Activity of Compound 2-6e (Inhibition Percentage and IC50 Values are Means Given
with SEM).
AChE Inhibition
Inhibition (%) at 0.5 mM
BChE Inhibition
Inhibition (%) at 0.5 mM
Entry
Compound
IC50
IC50
(µM)
(µM)
1
2
3
70.61±0.34
69.16±0.25
63.11±0.52
75.50±0.33
75.50±0.56
79.54±0.54
66.86±0.36
89.34±0.91
75.79±0.25
60.52±0.25
75.22±0.41
55.91±0.33
78.11±0.36
46.41±0.21
56.21±0.25
58.62±0.52
90.78±0.32
91.29±1.17
138.51±0.21
152.11±0.07
198.61±0.14
103.11±0.05
109.61±0.11
98.81±0.14
174.51±0.11
91.21±0.06
106.51±0.07
298.61±0.15
152.31±0.07
<400
59.82±0.15
73.95±0.67
79.58±0.42
43.16±0.18
50.22±0.69
43.61±0.55
68.43±0.18
89.51±0.69
30.13±0.58
90.81±0.69
86.64±0.24
39.29±0.25
72.52±0.33
42.61±0.42
81.24±0.52
63.69±0.61
81.02±0.14
82.82±1.09
184.21±0.21
139.21±0.07
86.31±0.14
-
2
3
4a
4
4b
4c
5
<300
6
7
4d
4e
-
146.21±0.08
59.81±0.06
-
8
4f
9
4g
10
5a
55.21±0.12
68.91±0.07
-
11
5b
5c
12
13
6a
121.41±0.16
-
101.21±0.16
-
14
6b
6c
15
<400
78.51±0.21
<400
16
6d
6e
<400
17
68.52±0.04
0.04±0.0001
73.52±0.04
0.85±0.0001
Control
Eserine
(BioTek, USA) 96-well plate reader was used in all experi-
ments. All experiments were carried out with their respective
controls in triplicate. Eserine (0.5 mM per well) was used as
a positive control [33]. The percent inhibition was calculated
by the help of following equation.
removed, and binding site defined as all amino acid residues
within 10 Å from donepezil. The presence of some water
molecules was also taken into account. A standard set of
genetic algorithm with population size 100, number of op-
erations 100 000 and clustering tolerance of 1 Å was applied.
As a result, 20 ligand conformations were obtained and
sorted according to ChemScore function values. Results
were visualized by PyMOL [38].
Control- Test
Inhibtion(%) =
!100
Control
IC50 values (concentration at which there is 50% enzyme
inhibition) of compounds were calculated using EZ–Fit En-
zyme kinetics software (Perella Scientific Inc. Amherst,
USA).
CONCLUSION
The synthesized compounds were screened for their in-
hibitory effect against AChE and BChE. Eserine was used as
control [33] with inhibition percentage of 91.29±1.17 at the
concentration of 0.5 mM. Among the series, compound 6e
was found to be the most active against AChE with an IC50
value of 68.52±0.04 µM (Table 1). The seemingly preferred
interaction of p-bromo benzene sulfonyl group of 6e with
enzyme may arise due to the planner orientation and polarity
of bromo group. Compounds 4f (IC50 91.21±0.06 µM) and
4h (IC50 93.61±0.15 µM) also showed good activity, which
could be attributed to the presence of more rigid structures
and bulky biphenyl and naphthyl groups, which in turn may
have caused greater hydrophobic interactions with enzyme
and hence resulted in comparatively more inhibition.
Docking Studies
Three-dimensional representation of ligand was created
using Corina online tool [34] and saved as pdb file. Using
Sybyl 8.0 [35] Gasteiger-Marsili charges were assigned fol-
lowing check of atom types and protonation of the com-
pounds. Finally, ligand structure was saved in the mol2 for-
mat. Docking was performed to Torpedo californica AChE
from 1EVE crystal complex [36] using Gold 5.1 program
[37]. In the preparatory phase, all histidine residues were
protonated at Nε, hydrogen atoms added, ligand molecules

336 Letters in Drug Design & Discovery, 2014, Vol. 11, No. 3
Yar et al.
Fig. (3). Binding mode of compound 6e with AChE.
Likewise compounds 6c, 5c, 6b and 6d showed weak
against AChE (Table 1). The lower inhibition in case of 6b-
6d is considered to be due to the presence of much polar and
bulky nitro group, which may have prevented effective inter-
action with the enzyme. However, in case of 5c carbamate
moiety might have played role in its lower inhibition. The
overall order of inhibition against AChE was found to be:
pound showed the highest activity against AChE and even
though its IC50 value was in the middle micro-molar range it
was a good starting point for analysis. It was docked to the
active gorge of AChE to find possible binding mode and to
explain why activity was not high enough. In the second step
it was possible to propose structural modifications which
could improve the potency of novel derivatives. The AChE
from 1EVE complex [36] was chosen as target structure ac-
cording to the validation process, described elsewhere [39].
Among reference inhibitors from PDB complexes, donepezil
was the most similar to novel compounds - they were linear
molecules with two aromatic groups at the ends. This con-
firmed that 1EVE structure was a good choice. Docking
studies revealed that compound 6e was bound to both cata-
lytic and peripheral active site (Fig. 3). It is quite important
because dual binding site derivatives can increase choliner-
gic transmission and inhibit AChE-dependent β-amyloid
aggregation. The strength of binding was assessed by Chem-
Score function which adopted value 38.76 for ligand 6e in
comparison with 49.48 for reference compound - donepezil.
It remained in accordance with experimental results because
anti-AChE activity of donepezil is much higher than potency
of compound 6e. The IC50 values for reference and novel
ligand were equal to 31.2 nM [39] and 68.52 µM, respec-
tively. Derivative 6e occurred in conformation with slightly
bent linker. The outermost fragments of molecule interacted
with two tryptophan residues: indole created CH-π interac-
tions with Trp84, and p-bromophenyl formed π-π stacking
interactions with Trp279. The chain should have been a bit
longer to provide better fit to both tryptophan residues. The
tether was engaged in H-bond network due to the presence of
sulfonamide and amide fragments. One of the oxygen atoms
from -SO2- group created hydrogen bond with hydroxyl
group of Tyr121. The second one was a part of H-bond net-
work: S=O ·····H2O (WAT1254) ·····HN-Phe288, and the
carbonyl group formed the following bridge: C=O ·····H2O
(WAT1159) ·····OH-Phe121 but its geometry was poor. It has
seemed that introduction of one or two methylene groups
between nitrogen atoms in the linker could improve the qual-
ity of that bridge and a fit of indole moiety to Trp84, leading
to classical π-π stacking interaction.
6e > 4f > 4h > 4d > 6a > 4g = 5b = 4b = 4c >2 > 3 > 4e
> 4a > 5a > 6d >6c > 5c
In case of BChE inhibition studies, compounds 5a, 4f and
5b were the most active with an IC50 values of 55.21±0.12,
59.81±0.06 and 68.91±0.07 µM, respectively. In case of
compounds 5a and 5b, an amide moiety might have played a
role by providing more rigid structures which in return en-
hanced inhibitory power of these compounds. Compound 5a
was found to be slightly more active than 5b which may be
related to the size and electronic factors of acetyl vs
trifluoroacetyl group. In addition, higher similarity of com-
pound 5a with acetylcholine compared to 5b could also have
played a role in its higher inhibition. The higher activity of
4f could also be attributed due to the presence of biphenyl
ring. Similarly, compounds 6b, 5c, 4d and 4b showed weak
inhibitory activities against BChE enzyme (Table 1).
The overall order of inhibition percentage against BChE
was:
5a > 4f > 5b >6c = 6e> 4a> 3 > 6a> 4e > 6d > 2 > 4c
In the case of AChE inhibition, it was revealed that the
chlorine substitution in the aromatic ring (4g) sharply en-
hanced the inhibition (4a vs 4g, Table 1). Likewise the size
and hydrophobicity of the substituent also played a signifi-
cant role; 4h is a better inhibitor than 4b. Similarly, electron
releasing groups (OH, OCH3) in the aromatic ring also en-
hanced the inhibitory effect, for instance 4b and 4c were
more active compared to 4a (Table 1). These groups help in
to form hydrogen bonding which is quite important for bind-
ing with enzyme.
Among the whole series of indole derivatives, compound
6e was selected for molecular docking studies. This com-

New Dual Binding Site Cholinesterase Inhibitors
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CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flicts of interest.
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ACKNOWLEDGEMENTS
We acknowledge the Higher Education Commission and
Ministry of Science and Technology Pakistan for financial
support. Molecular modeling studies were financially sup-
ported by Polish National Center of Science, Postdoctoral
Research Grant No. DEC-2012/04/S/NZ2/00116.
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Received: July 24, 2013
Revised: August 09, 2013
Accepted: September 16, 2013