Indolinone-based acetylcholinesterase inhibitors: Synthesis, biological activity and molecular modeling

Article abstract of DOI:10.1016/j.ejmech.2014.01.017

A series of indolinone-based compounds bearing benzylpyridinium moiety was designed as dual-binding inhibitors of acetylcholinesterase (AChE). The target compounds 3a-u were synthesized by condensation of oxindole and pyridin-4-carbalehyde, and subsequent N-benzylation. The anti-cholinesterase activity evaluation of synthesized compounds revealed that most of them had very potent inhibitory activity against AChE, superior to standard drug donepezil. Particularly, 2-chlorobenzyl derivative 3c was the most potent compound against AChE with IC₅₀ value of 0.44 nM, being 32-fold more potent than donepezil. Also, most of compounds were more potent than standard drug donepezil against butyrylcholinesterase (BuChE). Docking study revealed that the hydrophobic aromatic part (indoline) of representative compound 3c binds to the PAS and the N-benzylpyridinium residue binds to the CAS of AChE.

Full text of DOI:10.1016/j.ejmech.2014.01.017

Accepted Manuscript
Indolinone-based acetylcholinesterase inhibitors: synthesis, biological activity and
molecular modeling
Hamidreza Akrami, Bibi Fatemeh Mirjalili, Mehdi Khoobi, Hamid Nadri, Alireza Moradi,
Amirhossein Sakhteman, Saeed Emami, Alireza Foroumadi, Abbas Shafiee
PII:
S0223-5234(14)00048-8
10.1016/j.ejmech.2014.01.017
EJMECH 6670
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 August 2013
Revised Date: 7 January 2014
Accepted Date: 9 January 2014
Please cite this article as: H. Akrami, B.F. Mirjalili, M. Khoobi, H. Nadri, A. Moradi, A. Sakhteman, S.
Emami, A. Foroumadi, A. Shafiee, Indolinone-based acetylcholinesterase inhibitors: synthesis, biological
activity and molecular modeling, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.01.017.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Indolinone-based acetylcholinesterase inhibitors: synthesis, biological activity and
molecular modeling
Hamidreza Akrami, Bibi Fatemeh Mirjalili, Mehdi Khoobi, Hamid Nadri, Alireza Moradi,
Amirhossein Sakhteman, Saeed Emami, Alireza Foroumadi, Abbas Shafiee
Cl
Cl
N
H
O
N
H
3c
Indolinone-based compounds bearing benzylpyridinium moiety was designed as dual-binding
inhibitors of AChE. 2-Chlorobenzyl derivative 3c (IC = 0.44 nM) was 32-fold more potent
5
0
than donepezil as reference drug.

ACCEPTED MANUSCRIPT
Indolinone-based acetylcholinesterase inhibitors: synthesis, biological
activity and molecular modeling
a
a
b
c
Hamidreza Akrami , Bibi Fatemeh Mirjalili , Mehdi Khoobi , Hamid Nadri , Alireza
c
c
d
b
b,
Moradi , Amirhossein Sakhteman , Saeed Emami , Alireza Foroumadi , Abbas Shafiee *
a
Department of Chemistry, College of Science, Yazd University, Yazd, PO Box 8915813149,
Islamic Republic of 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, Iran
d
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty
of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Abstract: A series of indolinone-based compounds bearing benzylpyridinium moiety was
designed as dual-binding inhibitors of acetylcholinesterase (AChE). The target compounds
3a-u were synthesized by condensation of oxindole and pyridin-4-carbalehyde, and
subsequent N-benzylation. The anti-cholinesterase activity evaluation of synthesized
compounds revealed that most of them had very potent inhibitory activity against AChE,
superior to standard drug donepezil. Particularly, 2-chlorobenzyl derivative 3c was the most
potent compound against AChE with IC value of 0.44 nM, being 32-fold more potent than
5
0
donepezil. Also, most of compounds were more potent than standard drug donepezil against
butyrylcholinesterase (BuChE). Docking study revealed that the hydrophobic aromatic part
(indoline) of representative compound 3c binds to the PAS and the N-benzylpyridinium
residue binds to the CAS of AChE.
Keywords: Alzheimer’s disease, Acetylcholinesterase, Indolinone, Oxindole, Docking study
*Corresponding author. Tel: +98 21 66406757; Fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
1

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1. Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive
decline in memory, learning and cognitive functions. Currently, it is estimated that AD
affects about 36 million people worldwide and expecting to reach 66 million by 2030 [1]. AD
is involved with a loss of presynaptic cholinergic function in the areas of the brain related to
memory and learning. This neurological disorder is also associated with the presence of
amyloid β-peptide (Aβ) deposits and neurofibrillary tangles in the brain [2]. The
enhancement of cholinergic neurotransmission by preserving acetylcholine (ACh) levels
would be an effective way to overcome the occurrence, symptoms and progression of AD
[
3,4]. Accordingly, the inhibition of acetylcholinesterase (AChE) which is responsible for the
metabolic breakdown of ACh has been regarded as one of the most promising approaches [5].
Therefore, anti-AChE drugs such as donepezil, rivastigmine, galanthamine and tacrine, were
developed for treatment of AD [6]. Among the anti-AChE drugs, tacrine is associated with
hepatotoxicity thus it is rarely used [7]. On the other hand, donepezil and rivastigmine which
are commonly used in the early-to-moderate stages of AD often present adverse effects and
are not completely effective [8]. However, clinical trial studies revealed that galantamine
shows promising pharmacological profile and clinically relevant neuroprotective effects in
AD [9]. Therefore, design of more effective anti-AChE drugs with low side effects and better
pharmacokinetics properties is an urgent need in the field of AD pharmacotherapy.
Previous studies on the structure and function of AChE revealed that this enzyme has two
binding sites; catalytic anionic site (CAS) and peripheral anionic site (PAS) [10]. It was
proposed that PAS could promote the deposition and aggregation of Aβ in the brain [11].
Accordingly, the multi-binding inhibitors which can inhibit catalytic activity of AChE and
perturb the self-assembly of Aβ could be more effective agents for the management of AD
[
12]. For example, the dual-binding mode of donepezil with AChE has been demonstrated by
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X-ray crystallography and docking studies. While the hydrophobic aromatic part (5,6-
dimethoxyindan-1-one) of donepezil binds to the PAS, the N-benzyl piperidine residue binds
to the CAS of AChE [13]. Accordingly, it was found that the presence of functionalized
amine group such as benzyl piperidine, benzylamino, phenylpiperazine, and anilino moieties
contribute to inhibitor activity by interacting with the catalytic site of the AChE. On the other
hand, a ligand that is rich in aromatic groups, may engage to favorable stacking interactions
with PAS [15]. Numbers of aromatic and heteroaromatic rings were found in AChE inhibitors
as PAS binding scaffolds [16,17]. Recently, we introduced benzofuranone-based AChE-
inhibitors containing benzylpyridinium moiety (Fig. 1) [18a]. In continuation of our previous
efforts in order to find new AChE inhibitors [18], in this work we describe indolinone-based
compounds bearing benzylpyridinium moiety as dual-binding inhibitors of AChE.
2. Results and discussion
2.1. Chemistry
The oxindole derivatives 3a-u were synthesized via the route outlined in Scheme 1. In the
first step, (E)-3-(pyridin-4-ylmethylene)indolin-2-one (2) were synthesized using oxindole
(1) and pyridin-4-carbalehyde in the presence of para-toluene solfunic acid (PTSA) as a
catalyst. Several acid catalysts and solvents were screened for this reaction but the best
results were obtained in the presence of PTSA in refluxing toluene. In this reaction, the (E)-
isomer was the major product which further crystallized from acetonitrile to obtain the pure
(E)-2. The chemical shift of the vinylic proton could be used to assign the configuration of
product. In the (E)-geometry of compound 2, the vinylic proton would be shifted downfield
due to deshielding effect of carbonyl group [19]. According to the literature reports, the
vinylic hydrogen in (E)-isomers is appeared at 7.6–8.0 ppm [20]. The target compounds 3a-u
were easily prepared by the reaction of proper benzyl bromide or chloride with compound
3

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(E)-2. Accordingly, the reaction mixture was stirred in dry acetonitirile without catalyst, at
60–70 ºC for 6–24 h. On cooling, the product was precipitated as a solid which was
separated, washed with diethyl ether or n-hexane and recrystallized from ethanol-water. The
1
H NMR data of final compounds 3 revealed that the (E)-geometry of compounds have been
preserved based on the downfield chemical shifts of vinilic proton (δ > 7.9 ppm).
2
.2. Inhibitory activity against AChE and BuChE
The IC values of test compounds against AChE reveled that compounds 3b, 3c, 3e, 3g, 3i-
5
0
m showed very potent inhibitory activity (IC values = 0.44–12.8 nM) superior to standard
5
0
drug donepezil. Among them, 2-chlorobenzyl derivative 3c was the most potent compound
against AChE, with IC value of 0.44 nM. This compounds was about 32-fold more potent
5
0
than donepezil. Moreover, 2-fluoro and 2-bromo analogues (compounds 3b and 3e,
respectively) with IC50 values ≤1.46 nM showed high activity against AChE. The
comparison of un-substituted compound 3a with ortho- or meta-substituted analogues 3b-m
demonstrated that introduction of halo, methyl and methoxy group at 2- or 3-position of N-
benzyl pendent residue significantly improved the anti-AChE activity. The 2-chloro
substituent had the most impact on the AChE inhibition of designed compounds. In contrast,
introduction of different substituents on the para-position of benzyl group diminished the
inhibitory activity against AChE (3n-r vs. 3a). As shown by compound 3r, the 4-nitro group
more significantly decreased the activity. Interestingly, the insertion of second chlorine atom
on ortho or meta positions of 4-chlorobenzyl derivative 3o resulted in more potent
compounds 3s and 3t. While, in the case of 2- or 3-chlorobenzyl derivatives (compounds 3c
or 3i, respectively), introduction of second halogen decreased the anti-AChE activity as
observed with compounds 3g, 3h, 3s and 3t. The displacement of halogen atom (Br, Cl and
4

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F) on benzyl group dramatically affects the anti-AChE activity. The order of activity was as
follow: 2-halo > 3-halo > 4-halo.
The observed IC values of target compounds against BuChE revealed that all compounds
5
0
with the exception of 3q and 3r were more potent than standard drug donepezil. The anti-
BuChE activity of the most potent compound 3d was 6 times higher than that of donepezil.
Most of substituted benzyl compounds were more potent than unsubstituted analogue 3a
against BuChE. These results showed that substitution on benzyl group had often positive
effect on anti-BuChE avtivity. The highest activity was observed with 2-methyl analogue.
However, 3-fluoro, 4-methoxy and 4-nitro substituents decreased the inhibitory activity
against BuChE.
As calculated in Table 1, the most active compound against AChE (compound 3c) showed
very high selectivity for this enzyme (SI = 3113). Moreover, other potent compounds 3b and
3e had high selectivity for AChE (SI > 842).
2.3. Docking studies
In order to gain functional and structural insight into the binding mode of the compounds,
molecular docking simulation was performed using Autodock Vina software. To confirm the
validity of used docking parameters, the co-crystallized ligand E2020 was re-docked into the
active site of AChE and the RMSD value (0.87 Å) guaranteed the validity of the docking
procedure (Fig. 2). Subsequently, the most active compound 3c was docked using the
optimized parameters. Regarding the docking studies, three types of interactions;
hydrophobic interaction, hydrophobic π-π interaction and π-cation interaction were involved
in the attachment of ligand to the active site of the enzyme. The quaternary nitrogen of the
pyridinium ring facilitates ligand recognition through binding to mid-gorge recognition site
comprising Phe330. The ligand is anchored at the bottom of active site through a π-cation
5

ACCEPTED MANUSCRIPT
interaction with Phe330 along with a π-π interaction with Trp84 in the catalytic anionic site
(CAS). In addition, the indole moiety was well fitted in the hydrophobic pocket composed by
Tyr70, Tyr121 and Trp279 in the peripheral anionic site (PAS) (Fig. 3). These key
interactions are similar to those of well-known AChE inhibitors complexed with the enzyme.
To get better insight to the concluded SAR, the best pose of both more and less active
compounds (3c and 3r, respectively) were also overlaid and shown in Fig. 4. As illustrated in
Figure 4, the binding modes are similar in a way that two π-π interactions were observed with
Trp84 and Phe330. However, substitution on the para position of compound 3r was not
tolerated due to the steric hindrance with the residues in the bottom of active site (CS). To get
rid of the steric hindrance, the orientation of (4-nitrobenzyl)pyridinium fragment of
compound 3r was changed, leading to weak π-π interactions. These finding could explain the
lower potency of 4-substituted benzylpyridinium compounds.
2.4. Kinetics study
To gain further insight to the mechanism of action of these series of compounds on AChE, a
kinetics study was performed on the most active compound 3c. Graphical analysis of
reciprocal Lineweaver–Burk plot revealed that this compound has shown mixed-type
inhibition on AChE (Fig. 5). The type of inhibition is in agreement with the proposed binding
mode of these compounds in the active-site gorge of AChE. Plotting of the slopes versus
concentration of 3c gave an estimation of inhibition constant, Ki of 1.14 nM (Fig. 6).
2.5. ADMET prediction
The ADMET (absorption, distribution, metabolism, excretion and toxicity) properties of the
target compounds were predicted using admetSAR web-based application [21]. The predicted
ADMET data were included in the Supplementary Material. Based on the predicted values
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for BBB penetration, all compounds might be able to pass through blood brain barrier and
penetrate into the CNS and therefore, are considered as CNS active compounds. Moreover,
all the compounds may not show either acute toxicity according to the calculated LC values
5
0
nor mutagenic effect with respect to the AMES test data.
3. Conclusion
In conclusion, we have described indolinone-based compounds bearing benzylpyridinium
moiety as dual-binding inhibitors of AChE. The target compounds 3a-u were synthesized by
condensation of oxindole (1) and pyridin-4-carbalehyde to obtain (E)-3-(pyridin-4-
ylmethylene)indolin-2-one (2), and subsequent N-benzylation with benzyl halides. The anti-
cholinesterase activity evalauation of synthesized compounds revealed that most of them had
very potent inhibitory activity against AChE, superior to standard drug donepezil.
Particularly, 2-chlorobenzyl derivative 3c was the most potent compound against AChE, with
IC value of 0.44 nM. This compounds was about 32-fold more potent than donepezil. Also,
5
0
all compounds with the exception of 3q and 3r were more potent than standard drug
donepezil against BuChE. Docking study revealed that the hydrophobic aromatic part
(indoline) of representative compound 3c binds to the PAS and the N-benzylpyridinium
residue binds to the CAS of AChE. Kinetics study with compound 3c demonstrated that this
compound has mixed-type inhibition on AChE. The favorable in silico ADMET properties of
designed compounds with in vitro anti-cholinesterase potential of compounds make them as
new lead compounds for further optimization in the field of AD pharmacotherapy.
4. Experimental protocols
4.1. Chemistry
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All commercially available reagents were purchased from Merck AG or Aldrich and used
without further purification. Melting points were measured on the Buchi Melting point B-
540. FT-IR spectra were run on a Bruker, Eqinox 55 spectrometer (KBr disks). Mass spectra
of the products were obtained with an HP (Agilent technologies) 5937 Mass Selective
1
Detector. H NMR spectra were recorded on a Bruker 500 MHz NMR instrument. Elemental
analyses were carried out by a CHN-Rapid Heraeus elemental analyzer. The results of
elemental analyses (C, H, N) were within ±0.4% of the calculated values.
4.1.1. Synthesis of (E)-3-(pyridin-4-ylmethylene)indolin-2-one (2)
A mixture of oxindole (1, 1 mmol), pyridine-4-carbaldehyde (1 mmol), and PTSA (1 mmol)
was refluxed in dry toluene for 3 h. After completion of the reaction (monitored by TLC), the
solvent was evaporated under vacuum. Then, sodium carbonate solution 30% (15 mL) was
added and extracted with chloroform (3×15 mL). The organic phase was dried (Na SO ) and
2
4
the solvent was evaporated under vacuum to obtain yellow solid. The solid was recrystallized
from acetonitrile to afford pure product 2. Yellow solid; yield 78%; mp 182-184 ºC; IR (KBr,
-1
1
cm ): 3417, 1712, 1615, 1597; H NMR (500 MHz, DMSO-d ) δ: 8.76 (d, J = 5.9 Hz, 2H, H-
6
a), 8.45 (s, 1H, NH), 7.70 (s, 1H, H-vinylic), 7.52 (d, J = 5.8 Hz, 2H, H-b), 7.46 (d, J = 7.7
Hz, 1H, H-4), 7.28-7.25 (m, 1H, H-6), 6.91 (d, J = 7.7 Hz, 1H, H-7), 6.91-6.87 (m, 1H, H-5).
Anal. Calcd for C H N O (222.24): C, 75.66; H, 4.54; N, 12.60. Found: C, 75.31; H, 4.74;
1
4
10
2
N, 12.39.
4.1.2. General procedure for the preparation of compounds 3a-u
To a mixture of (E)-3-(pyridin-4-ylmethylene)indolin-2-one (2, 1 mmol) in dry acetonitrile (5
mL), proper benzyl bromide or chloride (1.5 mmol) was added and the mixture was stirred at
60-70 ºC for 6-24 h. Then, the mixture was cooled and the precipitated solid was filtrated off
8

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and washed with diethyl ether or n-hexane. The product was recrystallized from ethanol-
water (1:1) to give pure compounds 3a-u.
4.1.2.1. (E)-1-(benzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium bromide (3a)
-1
1
Red solid; yield 92%; mp 224-226 ºC; IR (KBr, cm ): 3430, 1696, 1634; H NMR (500
MHz, DMSO-d ) δ: 10.97 (s, 1H, NH), 9.27 (d, J = 5.3 Hz, 2H, H-a), 8.73 (d, J = 5.4 Hz, 2H,
6
H-b), 7.99 (s, 1H, H-vinylic), 7.77 (d, J = 7.2 Hz, 1H, H-4), 7.60-7.45 (m, Ar H2,3,4,5), 7.32
(t, J = 7.3 Hz, 1H, H-6), 7.05 (t, J = 7.4, 1H, H-5), 6.87 (d, J = 7.4 Hz, 1H, H-7), 5.88 (s, 2H,
+
13
-
CH N ). C NMR (DMSO-d , 125 MHz) δ: 166.3, 149.4, 144.1, 142.7, 135.4, 134.3, 131.9,
2 6
+
1
2
29.3, 129.2, 128.7, 128.6, 128.2, 123.1, 121.8, 121.7, 110.2, 62.8. MS m/z (%) 313 (M , 4),
22 (100), 194 (29), 166 (15), 144 (74), 91 (97), 65 (16), 51 (14). Anal. Calcd for
C H BrN O (393.28): C, 64.13; H, 4.36; N, 7.12. Found: C, 64.50; H, 4.15; N, 7.37.
21
17
2
4.1.2.2. (E)-1-(2-fluorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3b)
-1
1
Red solid; yield 91%; mp 234-236 ºC; IR (KBr, cm ): 3191, 1696, 1643; H NMR (500
MHz, DMSO-d ) δ: 10.92 (s, 1H, NH), 9.15 (br s, 2H, H-a), 8.73 (br s, 2H, H-b), 7.97 (s, 1H,
6
H-vinylic), 7.77 (br s, 1H, H-4), 7.64 (br s, 1H, Ar H ), 7.54 (br s, 1H, Ar H ), 7.34 (br s, 3H,
6
4
+
13
Ar H , H-6), 7.06 (br s, 1H, H-5), 6.88 (br s, 1H, H-7), 6.02 (s, 2H, -CH N ). C NMR
3
,5
2
(
DMSO-d , 125 MHz) δ: 166.3, 149.8, 144.3, 142.7, 135.5, 132.1, 132.09, 132.02, 131.5,
6
128.7, 128.4, 125.3, 123.1, 121.9, 121.8, 121.2, 121.1, 116.1, 115.9, 110.2, 57.5. Anal. Calcd
for C H ClFN O (366.82): C, 68.76; H, 4.40; N, 7.64. Found: C, 68.52; H, 4.12; N, 7.95.
2
1
16
2
4.1.2.3. (E)-1-(2-chlorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3c)
-1
1
Red solid; yield 90%; mp 225-227 ºC; IR (KBr, cm ): 3396, 1690, 1632; H NMR (500
MHz, DMSO-d ) δ: 10.95 (s, 1H, NH), 9.12 (d, J = 6.8 Hz, 2H, H-a), 8.75 (d, J = 6.8 Hz, 2H,
6
9

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H-b), 8.00 (s, 1H, H-vinylic), 7.78 (d, J = 7.5 Hz, 1H, H-4), 7.61 (d, J =7.7 Hz, 1H, Ar H3),
7.54-7.48 (m, 3H, Ar H4,5,6), 7.35 (t, J = 7.5 Hz, 1H, H-6), 7.06 (t, J = 7.5 Hz, 1H, H-5), 6.88
+
13
(
d, J = 7.5 Hz, 1H, H-7), 5.99 (s, 2H, -CH N ). C NMR (DMSO-d , 125 MHz) δ: 166.3,
2 6
1
49.9, 144.5, 142.8, 135.6, 133.3, 132.0, 131.6, 131.4, 131.3 130.1, 128.6, 128.4, 128.1,
23.2, 121.9, 121.8, 110.2, 60.8. Anal. Calcd for C H Cl N O (383.27): C, 65.81; H, 4.21;
1
21
16
2
2
N, 7.38. Found: C, 65.60; H, 4.43; N, 7.70.
4.1.2.4. (E)-1-(2-methylbenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3d)
-1
1
Red solid; yield 88%; mp 214-217 ºC; IR (KBr, cm ): 3418, 1700, 1634; H NMR (500
MHz, DMSO-d ) δ: 11.00 (s, 1H, NH), 9.09. (d, J = 5.5 Hz, 2H, H-a), 8.74 (d, J = 5.5 Hz,
6
2H, H-b), 8.02 (s, 1H, H-vinylic), 7.78 (d, J = 7.2 Hz, 1H, H-4), 7.32-7.28 (m, 4H, Ar
H3,4,5,6), 7.20 (t, J = 7.2 Hz, 1H, H-6), 7.04 (t, J = 7.2 Hz, 1H, H-5), 6.87 (d, J = 7.2, 1H, H-
+
13
7
1
1
), 5.93 (s, 2H, -CH N ), 2.32 (s, 3H, -CH ). C NMR (DMSO-d , 125 MHz) δ: 166.3,
2 3 6
49.6, 144.3, 142.7, 135.4, 132.3, 131.9, 130.9, 129.3, 129.2, 128.6, 127.8, 126.7, 123.2,
+
21.9, 121.8, 110.2, 61.0, 18.8. MS m/z (%) 327 (M , 4), 222 (94), 194 (28), 144 (62), 139
(
60), 105 (100), 77 (26), 51 (28). Anal. Calcd for C H ClN O (362.85): C, 72.82; H, 5.28;
22 19 2
N, 7.72. Found: C, 72.60; H, 5.55; N, 72.91.
4.1.2.5. (E)-1-(2-bromobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3e)
-1
1
Red solid; yield 91%; mp 150-152 ºC; IR (KBr, cm ): 3406, 1702, 1633; H NMR (500
MHz, DMSO-d ) δ: 10.95 (s, 1H, NH), 9.10 (d, J = 6.2 Hz, 2H, H-a), 8.76 (d, J = 6.2 Hz, 2H,
6
H-b), 8.01 (s, 1H, H-vinylic), 7.78-7.75 (m, 2H, Ar H , H-4), 7.53 (t, J = 7.2 Hz, 1H, Ar H ),
3
5
7
.46-7.43 (m, 2H, Ar H ), 7.34 (t, J = 7.5 Hz, 1H, H-6), 7.06 (t, J = 7.5 Hz, 1H, H-5), 6.88
4,6
+
13
(
d, J = 7.5 Hz, 1H, H-7), 5.97 (s, 2H, -CH N ). C NMR (DMSO-d , 125 MHz) δ: 166.3,
2 6
1
49.9, 144.6, 142.8, 137.0, 135.6, 133.4, 133.03, 132.07, 131.5, 128.7, 128.6, 128.5, 123.5,
1
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ACCEPTED MANUSCRIPT
1
23.2, 121.9, 121.8, 110.2, 62.9. Anal. Calcd for C H BrClN O (427.72): C, 58.97; H, 3.77;
21
16
2
N, 6.55. Found: C, 58.66; H, 4.11; N, 6.80.
4.1.2.6. (E)-1-(2-nitrobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium bromide (3f)
-1
1
Red solid; yield 89%; mp 271-273 ºC; IR (KBr, cm ): 3404, 1707, 1634, 1525, 1343; H
NMR (500 MHz, DMSO-d ) δ: 10.93 (s, 1H, NH), 9.08. (d, J = 6.3 Hz, 2H, H-a), 8.78 (d, J =
6
6
.3 Hz, 2H, H-b), 8.29 (s, 1H, H-vinylic), 7.88-7.85 (m, 2H, Ar H , H-4), 7.79 (br s, 2H, Ar
3
H ), 7.36-7.31 (m, 3H, Ar H , H-6), 7.07 (t, J = 7.8 Hz, 1H, H-5), 6.89 (d, J = 7.8 Hz, 1H,
3
,5
4,6
+
H-7), 6.21 (s, 2H, -CH N ). Anal. Calcd for C H BrN O (438.27): C, 57.55; H, 3.68; N,
2
21 16
3
3
9.59. Found: C, 57.32; H, 3.32; N, 9.80.
4.1.2.7. (E)-1-(2-chloro-6-fluorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium
chloride (3g)
-1
1
Red solid; yield 92%; mp 261-263 ºC; IR (KBr, cm ): 3360, 1695, 1632; H NMR (500
MHz, DMSO-d ) δ: 10.97 (s, 1H, NH), 9.05 (d, J = 5.7 Hz, 2H, H-a), 8.72 (d, J = 5.7 Hz, 2H,
6
H-b), 8.00 (s, 1H, H-vinylic), 7.76 (d, J = 7.5 Hz, 1H, H-4), 7.3-7.61 (m, J = 7 Hz, 1H, Ar
H ), 7.51 (d, J = 7.9, 1H, Ar H ), 7.44 (t, J = 7.9 Hz, 1H, Ar H ), 7.33 (t, J = 7.5 Hz, 1H, H-
5
3
4
+
6
), 7.04 (t, J = 7.5 Hz, 1H, H-5), 6.88 (d, J = 7.5 Hz, 1H, H-7), 6.04 (s, 2H, -CH N ). MS m/z
2
+
+
(
%) 368 (M +2, 4), 366 (M , 11), 222 (100), 221 (48), 194 (29), 143 (84), 144 (62), 107 (24),
8
9 (12), 51 (12). Anal. Calcd for C H Cl FN O (401.26): C, 62.86; H, 3.77; N, 6.98. Found:
21 15 2 2
C, 62.58; H, 3.97; N, 7.26.
4.1.2.8. (E)-1-(2,6-dichlorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride
(3h)
1
1

ACCEPTED MANUSCRIPT
-1
1
Red solid; yield 89%; mp 187-189 ºC; IR (KBr, cm ): 3372, 1686, 1631; H NMR (500
MHz, DMSO-d ) δ: 10.92 (s, 1H, NH), 9.00 (d, J = 5.9 Hz, 2H, H-a), 8.72 (d, J = 5.9 Hz, 2H,
6
H-b), 7.98 (s, 1H, H-vinylic), 7.77 (d, J = 7.7 Hz, 1H, H-4), 7.70 (d, J = 7.3 Hz, 2H, Ar H ),
3
,5
7
7
3
.66-7.63 (m, 1H, Ar H ), 7.39-7.35 (m, 1H, H-6), 7.06 (t, J = 7.7 Hz, 1H, H-5), 6.84 (d, J =
4
+
.7 Hz, 1H, H-7), 6.13 (s, 2H, -CH N ). Anal. Calcd for C H Cl N O (417.72): C, 60.38; H,
2
21 15
3
2
.62; N, 6.71. Found: C, 60.62; H, 3.85; N, 6.35.
4.1.2.9. (E)-1-(3-chlorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium bromide (3i)
-1
1
Red solid; yield 91%; mp 225-227 ºC; IR (KBr, cm ): 3175, 1695, 1633; H NMR (500
MHz, DMSO-d ) δ: 10.87 (s, 1H, NH), 9.20 (d, J = 6.6 Hz, 2H, H-a), 8.72 (d, J = 6.6 Hz, 2H,
6
H-b), 7.95 (s, 1H, H-vinylic), 7.75 (d, J = 7.5 Hz, 1H, H-4), 7.73 (s, 1H, Ar H ), 7.55-7.48
2
(m, 3H, Ar H4,5,6), 7.33 (t, J = 7.5 Hz, 1H, H-6), 7.05 (t, J = 7.5 Hz, 1H, H-5), 6.85 (d, J = 7.5
+
13
Hz, 1H, H-7), 5.84 (s, 2H, -CH N ). C NMR (DMSO-d , 125 MHz) δ: 166.3, 149.7, 144.2,
2
6
1
1
1
3
42.7, 136.3, 135.4, 133.6, 132.03, 131.1, 129.3, 128.9, 128.7, 128.5, 127.6, 123.1, 121.9,
+
+
21.8, 110.1, 62.04. MS m/z (%) 349 (M +2, 6), 347 (M , 17), 222 (100), 194 (27), 166 (20),
44 (60), 125 (90), 89 (40), 51 (20). Anal. Calcd for C H BrClN O (427.72): C, 58.97; H,
2
1
16
2
.77; N, 6.55. Found: C, 58.75; H, 3.95; N, 6.85.
4.1.2.10. (E)-1-(3-bromobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3j)
-1
1
Red solid; yield 86%; mp 183-185 ºC; IR (KBr, cm ): 3395, 1702, 1633; H NMR (500
MHz, DMSO-d ) δ: 10.97 (s, 1H, NH), 9.27 (d, J = 6.5 Hz, 2H, H-a), 8.73 (d, J = 6.5 Hz, 2H,
6
H-b), 7.98 (s, 1H, H-vinylic), 7.90 (s, 1H, Ar H ), 7.76 (d, J = 7.5 Hz, 1H, H-4), 7.64-7.60
2
(
m, 2H, Ar H ), 7.40 (t, J = 7.5 Hz, 1H, Ar H ), 7.32 (t, J = 7.5 Hz, 1H, H-6), 7.03 (t, J = 7.5
4,6 5
+
Hz, 1H, H-5), 6.86 (d, J = 7.5 Hz, 1H, H-7), 5.87 (s, 2H, -CH N ). Anal. Calcd for
2
C H BrClN O (427.72): C, 58.97; H, 3.77; N, 6.55. Found: C, 58.60; H, 3.54; N, 6.86.
21
16
2
1
2

ACCEPTED MANUSCRIPT
4.1.2.11. (E)-1-(3-methylbenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3k)
-1
1
Red solid; yield 85%; mp 225-227 ºC; IR (KBr, cm ): 3419, 1706, 1633; H NMR (500
MHz, DMSO-d ) δ: 10.94 (s, 1H, NH), 9.22 (d, J = 6.5 Hz, 2H, H-a), 8.71 (d, J = 6.5 Hz, 2H,
6
H-b), 7.97 (s, 1H, H-vinylic), 7.75 (d, J = 5.5 Hz, 1H, H-4), 7.39 (s, 1H, Ar H ), 7.35-7.32
2
(m, 3H, Ar H4,5,6), 7.24 (br s, 1H, H-6), 7.03 (t, J = 5.5 Hz, 1H, H-5), 6.86 (d, J = 5.5 Hz, 1H,
+
13
H-7), 5.80 (s, 2H, -CH N ), 2.31 (s, 3H, -CH ). C NMR (DMSO-d , 125 MHz) δ: 166.3,
2
3
6
1
1
5
49.5, 144.09, 142.7, 138.6, 135.3, 134.2, 131.9, 129.3, 129.1, 128.7, 128.5, 127.8, 125.9,
23.1, 121.88, 121.81, 110.2, 62.8, 20.8. Anal. Calcd for C H ClN O (362.85): C, 72.82; H,
2
2
19
2
.28; N, 7.72. Found: C, 73.02; H, 5.54; N, 7.89.
4.1.2.12. (E)-1-(3-methoxylbenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride
(3l)
-1
1
Red solid; yield 90%; mp 223-225 ºC; IR (KBr, cm ): 3417, 1709, 1633; H NMR (500
MHz, DMSO-d ) δ: 11.02 (s, 1H, NH), 9.31 (d, J = 5.3 Hz, 2H, H-a), 8.72 (d, J = 5.3 Hz, 2H,
6
H-b), 8.01 (s, 1H, H-vinylic), 7.76 (d, J = 7.0 Hz, 1H , H-4), 7.60 (s, 1H, Ar H ), 7.38-7.26
2
(m, 3H, Ar H4,5,6), 7.16 (t, J = 7.0 Hz, 1H, H-6), 7.02-6.99 (m, 1H, H-5), 6.87 (d, J =7.0 Hz,
+
13
1
1
1
6
H, H-7), 5.84 (s, 2H, -CH N ), 3.76 (s, 3H, -OCH ). C NMR (DMSO-d , 125 MHz) δ:
2 3 6
66.2, 149.5, 144.1, 142.7, 135.6, 135.4, 133.4, 131.9, 130.4, 128.6, 128.4, 127.7, 123.7,
23.2, 121.9, 121.7, 121.4, 110.2, 62.6, 55.2. Anal. Calcd for C H ClN O (378.85): C,
2
2
19
2
2
9.75; H, 5.05; N, 7.39. Found: C, 69.50; H, 5.27; N, 7.75.
4.1.2.13. (E)-1-(3-fluorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3m)
-1
1
Red solid; yield 95%; mp 226-228 ºC; IR (KBr, cm ): 3428, 1695, 1633; H NMR (500
MHz, DMSO-d ) δ: 10.95 (s, 1H, NH), 9.28 (d, J = 6.8 Hz, 2H, H-a), 8.73 (d, J = 6.8 Hz, 2H,
6
1
3

ACCEPTED MANUSCRIPT
H-b), 7.98 (s, 1H, H-vinylic), 7.76 (d, J = 6.9 Hz, 1H, H-4), 7.54-7.50 (m, 2H, Ar H ), 7.46-
2
,4
7
.44 (m, 1H, Ar H ), 7.35-7.32 (m, 1H, Ar H ), 7.28 (t, J = 6.9 Hz, 1H, H-6), 7.04 (t, J = 6.9
6 5
+
Hz, 1H, H-5), 6.86 (d, J = 6.9 Hz, 1H, H-7), 5.89 (s, 2H, -CH N ). Anal. Calcd for
2
C H ClFN O (366.82): C, 68.76; H, 4.40; N, 7.64. Found: C, 68.52; H, 4.67; N, 7.35.
21
16
2
4.1.2.14. (E)-1-(4-fluorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3n)
-1
1
Red solid; yield 94%; mp 140-142 ºC; IR (KBr, cm ): 3339, 1699, 1634; H NMR (500
MHz, DMSO-d ) δ: 10.90 (s, 1H, NH), 9.20 (d, J = 6.2 Hz, 2H, H-a), 8.72 (d, J = 6.2 Hz, 2H,
6
H-b), 7.95 (s, 1H, H-vinylic), 7.75 (d, J = 7.0 Hz, 1H, H-4), 7.67 (br s, 2H, Ar H ), 7.33 (br
3
,5
+
s, 3H, Ar H , H-6), 7.06 (br s, 1H, H-5), 6.86 (d, J = 7.0 Hz, 1H, H-7), 5.83 (s, 2H, -CH N ).
2
,6
2
+
MS m/z (%) 331 (M , 20), 222 (100), 194 (29), 166 (18), 144 (83), 109 (100), 89 (20), 83
(
22), 51 (24). Anal. Calcd for C H ClFN O (366.82): C, 68.76; H, 4.40; N, 7.64. Found: C,
21 16 2
68.96; H, 4.62; N, 7.47.
4.1.2.15. (E)-1-(4-chlorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3o)
-1
1
Red solid; yield 91%; mp 184-186 ºC; IR (KBr, cm ): 3389, 1699, 1632; H NMR (500
MHz, DMSO-d ) δ: 10.91 (s, 1H, NH), 9.19 (d, J = 6.3 Hz, 2H, H-a), 8.72 (d, J = 6.3 Hz, 2H,
6
H-b), 7.96 (s, 1H, H-vinylic), 7.76 (d, J = 7.6 Hz, 1H , H-4), 7.61 (d, J = 8.5 Hz, 2H, Ar H ),
2
,6
7
.55 (d, J = 8.5 Hz, 2H, Ar H ), 7.32-7.35 (m, 1H, H-6), 7.06 (t, J = 7.6 Hz, 1H, H-5), 6.87
3,5
+
(
d, J = 7.6 Hz, 1H, H-7), 5.84 (s, 2H, -CH N ). Anal. Calcd for C H Cl N O (383.27): C,
2
21 16
2
2
65.81; H, 4.21; N, 7.31 Found: C, 66.07; H, 4.48; N, 7.45.
4.1.2.16. (E)-1-(4-bromobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3p)
-1
1
Red solid; yield 90%; mp 148-150 ºC; IR (KBr, cm ): 3394, 1701, 1633; H NMR (500
MHz, DMSO-d ) δ: 10.93 (s, 1H, NH), 9.21 (d, J = 6.3 Hz, 2H, H-a), 8.72 (d, J = 6.3 Hz, 2H,
6
1
4

ACCEPTED MANUSCRIPT
H-b), 7.97 (s, 1H, H-vinylic), 7.76 (d, J = 7.3 Hz, 1H, H-4), 7.68 (d, J = 7.8 Hz, 2H, Ar H ),
3
,5
7
6
.54 (d, J = 7.8 Hz, 2H, Ar H ), 7.33 (t, J = 7.3 Hz, 1H, H-6), 7.06 (t, J = 7.3 Hz, 1H, H-5),
2,6
+
.87 (d, J = 7.3 Hz, 1H, H-7), 5.84 (s, 2H, -CH N ). Anal. Calcd for C H BrClN O
2
21 16
2
(427.72): C, 58.97; H, 3.77; N, 6.55; Found: C, 59.25; H, 3.66; N, 6.80.
4.1.2.17. (E)-1-(4-methoxybenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride
(3q)
-1
1
Red solid; yield 91%; mp 103-105 ºC; IR (KBr, cm ): 3400, 1699, 1633; H NMR (500
MHz, DMSO-d ) δ: 11.00 (s, 1H, NH), 9.26 (d, J = 6.2 Hz, 2H, H-a), 8.71 (d, J = 6.2 Hz, 2H,
6
H-b), 7.99 (s, 1H, H-vinylic), 7.76 (d, J = 7.8 Hz, 1H , H-4), 7.58 (d, J = 8.4 Hz, 2H, Ar H ),
2
,6
7
.05-6.95 (m, 3H, H-6, Ar H ), 6.91 (t, J = 7.8 Hz, 1H, H-5), 6.87 (d, J = 7.8 Hz, 1H, H-7),
3,5
+
13
5
.80 (s, 2H, -CH N ), 3.76 (s, 3H, -OCH ). C NMR (DMSO-d , 125 MHz) δ: 166.3, 149.4,
2 3 6
1
43.8, 142.7, 135.3, 131.9, 130.9, 130.7, 130.3, 128.6, 127.7, 126.2, 123.2, 121.9, 121.8,
10.2, 62.4, 55.2. Anal. Calcd for C H ClN O (378.85): C, 69.75; H, 5.05; N, 7.39. Found:
1
2
2
19
2
2
C, 69.95; H, 5.40; N, 7.05.
4.1.2.18. (E)-1-(4-nitrobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride (3r)
-1
1
Red solid; yield 89%; mp 150-152 ºC; IR (KBr, cm ): 3394, 1700, 1633; H NMR (500
MHz, DMSO-d ) δ: 10.93 (s, 1H, NH), 9.26 (d, J = 6.3 Hz, 2H, H-a), 8.77 (d, J = 6.3 Hz, 2H,
6
H-b), 7.98 (s, 1H, H-vinylic), 7.72-7.83 (m, 4H, Ar H2,3,5,6), 7.62 (d, J =7.5 Hz, 1H, H-4),
7.33 (br s, 1H, H-6), 7.04 (br s, 1H, H-5), 6.86 (d, J = 7.5 Hz, 1H, H-7), 6.03 (s, 2H, -
+
CH N ). Anal. Calcd for C H ClN O (393.82): C, 64.05; H, 4.09; N, 10.67; Found: C,
2
21 16
3
3
64.31; H, 4.21; N, 10.39.
1
5

ACCEPTED MANUSCRIPT
4.1.2.19. (E)-1-(3,4-dichlorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride
(3s)
-1
1
Red solid; yield 90%; mp 235-237 ºC; IR (KBr, cm ): 3387, 1711, 1634; H NMR (500
MHz, DMSO-d ) δ: 10.65 (s, 1H, NH), 8.70 (d, J = 5.4 Hz, 2H, H-a), 7.61 (d, J = 5.4 Hz, 2H,
6
H-b), 7.54 (s, 2H, Ar H , H-vinylic), 7.34 (d, J = 7.7 Hz, 2H, Ar-H ), 7.23-7.26 (m, 2H, H-
2
5,6
+
4
,6), 6.87 (d, J = 7.7 Hz, 1H, H-7), 6.82 (t, J = 7.7 Hz, 1H, H-5), 5.82 (s, 2H, -CH N ). Anal.
2
Calcd for C H Cl N O (417.72): C, 60.38; H, 3.62; N, 6.71; Found: C, 60.18; H, 3.95; N,
2
1
15
3
2
6.49.
4.1.2.20. (E)-1-(2,4-dichlorobenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium chloride
(3t)
-1
1
Red solid; yield 94%; mp 171-173 ºC; IR (KBr, cm ): 3421, 1701, 1633; H NMR (500
MHz, DMSO-d ) δ: 10.95 (s, 1H, NH), 9.10 (d, J = 5.2 Hz, 2H, H-a), 8.74 (d, J = 5.2 Hz, 2H,
6
H-b), 7.99 (s, 1H, H-vinylic), 7.81-7.78 (m, 2H, H-4, Ar H ), 7.63-7.59 (m, 2H, Ar H ),
3
5,6
7.32-7.35 (m, 1H, H-6), 7.05 (t, J = 6.8 Hz, 1H, H-5), 6.87 (d, J = 6.8, 1H, H-7), 5.97 (s, 2H,
+
+
+
+
-
CH N ). MS m/z (%) 385 (M +4, 3), 383 (M +2, 19), 381 (M , 30), 222 (100), 194 (56),
2
1
66 (24), 159 (95), 144 (75), 89 (38), 63 (34), 51 (30). Anal. Calcd for C H Cl N O
21 15 3 2
(417.72): C, 60.38; H, 3.62; N, 6.71; Found: C, 60.18; H, 3.45; N, 6.91.
4.1.2.21. (E)-1-(4-chloromethylbenzyl)-4-((2-oxoindolin-3-ylidene)methyl)pyridinium
chloride (3u)
-1
1
Red solid; yield 92%; mp 121-123 ºC; IR (KBr, cm ): 3371, 1699, 1632; H NMR (500
MHz, DMSO-d ) δ: 10.95 (s, 1H, NH), 9.22 (d, J = 6.3 Hz, 2H, H-a), 8.69 (d, J = 6.3 Hz, 2H,
6
H-b), 7.96 (s, 1H, H-vinylic), 7.75 (d, J = 7.5 Hz, 1H, H-4), 7.56 (d, J = 8.5 Hz, 2H, Ar H ),
3
,5
7
.32 (t, J = 7.5 Hz, 1H, H-6), 7.04-6.99 (m, 3H, H-5, Ar H ), 6.85 (d, J = 7.5 Hz, 1H, H-7),
2,6
1
6

ACCEPTED MANUSCRIPT
+
13
5
.77 (s, 2H, -CH N ), 3.75 (s, 2H, -CH Cl). C NMR (DMSO-d , 125 MHz) δ: 166.3, 159.9,
2
2
6
1
49.4, 143.8, 142.7, 135.2, 131.9, 130.7, 128.6, 128.5, 126.1, 123.1, 121.8, 121.7, 114.5,
10.1, 62.4, 55.2. Anal. Calcd for C H Cl N O (397.30): C, 66.51; H, 4.57; N, 7.05. Found:
1
2
2
18
2
2
C, 66.80; H, 4.25; N, 7.34.
4.2. In vitro inhibition studies on AChE and BuChE
The spectrophotometric method of Ellman [22] was used to assess the inhibitory activity of
the target compounds toward AChE and BuChE. Acetylcholinesterase (AChE, E.C. 3.1.1.7,
Type V-S, lyophilized powder, from electric eel, 1000 unit), butyrylcholinesterase (BuChE,
E.C. 3.1.1.8, from equine serum), and butyrylthiocholine iodide (BTC) were purchased from
Sigma–Aldrich. 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB), potassium dihydrogen
phosphate, dipotassium hydrogen phosphate, potassium hydroxide, sodium hydrogen
carbonate, and acetylthiocholine iodide were obtained from Fluka. The reaction took place in
a final mixture of 3 mL phosphate buffer (0.1M, pH = 8.0), 100 µL of DTNB solution, 100
ꢀ
L of 2.5 unit/mL enzyme solution (AChE) and 100 ꢀL of each tested compounds. The above
mixture was pre-incubated for 10 min prior to adding 20 ꢀL of substrate (acetylthiocholine
iodide). Changes in absorbance were detected at 412 nm for 6 minutes and the IC values
5
0
were determined graphically from inhibition curves. Five different concentrations for each
compound were tested to obtain the range of 20% to 80% enzyme inhibition. All experiments
were performed in triplicate at 25°C on a UV Unico Double Beam Spectrophotometer. The
same method was also taken for BuChE inhibition assay.
4.3. Kinetics study
To obtain estimates of the inhibition constant K and inhibition model of compounds,
i
reciprocal plots of 1/v versus 1/[s] were constructed at different concentrations of the
1
7

ACCEPTED MANUSCRIPT
substrate acetylthiocholine (0.14-0.69 mM) by using Ellman’s method. The experiments were
performed as same as enzyme inhibition assay in triplicate. The rate of enzyme activity was
measured in the presence of different concentrations of inhibitor and without inhibitor for
proposed concentrations of substrate. For each experiment after adding acetylthiocholine as
substrate, progress curves were monitored at 420 nm for 2 min. Then the double reciprocal
plots (1/v vs. 1/[s]) were constructed using the slopes of progress curves to obtain the type of
inhibition. Slopes of the reciprocal plots were then plotted against the concentration of
inhibitor, to evaluate K data. Data analysis was performed with Microsoft Excel 2003.
i
4.4. Molecular modeling
The crystal structure of acetylcholinesterase complexed with E2020 (code ID: 1EVE) was
obtained from the Protein Data Bank. Then, the water molecules and inhibitor were removed.
Further preparation of protein was performed by Autodock Tools (ver 1.5.4) [23] using
default parameters and finally saved as pdbqt format. The 2D structures of ligands were
generated using MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and
then converted to 3D and pdbqt format by Openbabel (ver. 2.3.1) [24]. Docking studies were
carried out using the Autodock Vina (ver. 1.1.1) program [25]. The search space was defined
as a box with following parameters: size_x=40, size_y=40, size_z=40 which centered on the
geometrical center of co-crystallized ligand using these parameters: center_x=2.023,
center_y=63.295, center_z=67.062. The exhaustiveness was set to 80 and other parameters
were left as default values. Finally, the most favorable conformations based on the free
energy of binding were selected for analyzing the interactions between the AChE and
inhibitor. All the 3D models are depicted using the Chimera 1.6 software [26].
Acknowledgments
1
8

ACCEPTED MANUSCRIPT
This research was supported by grants from the research council of Tehran University of
Medical Sciences and the Iran National Science Foundation (INSF). Molecular graphics and
analyses were performed with the UCSF Chimera package. Chimera is developed by the 25
Resource for Biocomputing, Visualization, and Informatics at the University of California,
San Francisco.
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Captions:
Figure 1. Structures of donepezil hydrochloride as a well-known anti-AChE drug,
benzofuranone-based compounds reported as AChE-inhibitors and indolinone-based
compound as newly designed AChE-inhibitors.
Figure 2. Comparison of the co-crystallized ligand E2020 (red) and its calculated pose (blue)
by docking simulation in the active site of AChE. The catalytic site (CS), catalytic anionic
subsite (CAS) and peripheral anionic site (PAS) were shown in the receptor.
Figure 3. Representation of the binding mode of the most active compound 3c in the active
site of AChE.
Figure 4. Overlay of the best docked pose of 3c (green) and 3r (orange) in the active site of
AChE. The catalytic site (CS) and peripheral anionic site (PAS) were also shown for more
clarification.
Figure 5. Lineweaver–Burk plots of inhibition kinetics of 3c: reciprocals of enzyme activity
(eelAChE) vs. reciprocals of substrate (acetylthiocholine) concentration in the presence of
different concentrations of inhibitor 3c.
Figure 6. The plot of Lineweaver–Burk plots slopes vs. inhibitor concentrations
Scheme 1. Synthesis of (E)-1-benzyl-4-((2-oxoindolin-3-ylidene)methyl)pyridinium halide
derivatives 3a-u. Reagents and conditions: (i) pyridine-4-carboxaldehyde, PTSA, toluene,
reflux (ii) benzyl halide derivatives, acetonitrile, 60-70 ºC.
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Table 1. Chemical structure data, AChE and BuChE inhibitory activities of compounds 3a-u.
X
R
N
H
O
N
H
a
Compound
R
X
IC (nM)
IC (nM)
Selectivity for
5
0
50
b
AChE
BuChE
AChE
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
H
Br
Cl
Cl
Cl
Cl
Br
Cl
Cl
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
47.10 ± 0.41
4300 ± 298
91.3
3416
3113
57.9
842.5
7.5
2-F
1.25 ± 0.017
0.44 ± 0.006
15.3 ± 0.29
1.46 ± 0.013
214 ± 1.87
4.1 ± 0.036
17 ± 0.15
4270 ± 295
1370 ± 94
887 ± 61
2-Cl
2-CH3
2-Br
1230 ± 85
1600 ± 111
2000 ± 138
1500 ± 104
1350 ± 94
1740 ± 121
1700 ± 118
2800 ± 194
4500 ± 312
3100 ± 215
2200 ± 152
1900 ± 132
7900 ± 547
7500 ± 520
3300 ± 227
2500 ± 174
1000 ± 69
5380 ± 340
2-NO2
2-Cl,6-F
2,6-Cl2
3-Cl
g
h
i
487.8
88.2
275.5
169
4.9 ± 0.043
10.3 ± 0.09
5.2 ± 0.045
6.6 ± 0.058
12.8 ± 0.11
677 ± 5.9
j
3-Br
k
l
3-CH3
3-OCH3
3-F
327
424.2
351.5
4.58
3.73
2.9
m
n
o
p
q
r
s
4-F
4-Cl
590 ± 5.15
653.4 ± 5.7
677 ± 5.91
744 ± 6.49
29.4 ± 0.26
257.6 ± 2.25
103.4 ± 0.90
14 ± 0.8
4-Br
4-OCH3
4-NO2
3,4-Cl2
2,4-Cl2
11.67
10
112.2
9.7
t
u
4-CH Cl
2
9.67
384.3
Donepezil
a
Data are expressed as Mean ± S.E. of at least three different experiments.
Selectivity for AChE = IC (BuChE)/IC (AChE).
b
50
50
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O
O
R'
O
N
N
H
H CO
3
X
Cl
RO
H CO
3
Donepezil
hydrochloride
Benzofuranone-based
compounds
X
R
N
H
O
N
H
Indolinone-based
compounds
Figure 1. Structures of donepezil hydrochloride as a well-known anti-AChE drug,
benzofuranone-based compounds reported as AChE-inhibitors and indolinone-based
compound as newly designed AChE-inhibitors.
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Figure 2. Comparison of the co-crystallized ligand E2020 (red) and its calculated pose (blue)
by docking simulation in the active site of AChE. The catalytic site (CS), catalytic anionic
subsite (CAS) and peripheral anionic site (PAS) were shown in the receptor.
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Figure 3. Representation of the binding mode of the most active compound 3c in the active
site of AChE.
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Figure 4. Overlay of the best docked pose of 3c (green) and 3r (orange) in the active site of
AChE. The catalytic site (CS) and peripheral anionic site (PAS) were also shown for more
clarification.
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Figure 5. Lineweaver–Burk plots of inhibition kinetics of 3c: reciprocals of enzyme activity
(eelAChE) vs. reciprocals of substrate (acetylthiocholine) concentration in the presence of
different concentrations of inhibitor 3c.
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8