Synthesis, biological activity and molecular modeling studies on 1H-benzimidazole derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.06.065

A series of N-{2-[4-(1H-benzimidazole-2-yl)phenoxy]ethyl}substituted amine derivatives were designed to assess cholinesterase inhibitor activities. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitor activities were evaluated in vitro by using Ellman's method. It was discovered that most of the compounds displayed AChE and/or BuChE inhibitor activity and few compounds were selective against AChE/BuChE. Compound 3c and 3e were the most active compounds in the series against eeAChE and hAChE, respectively. Molecular docking studies and molecular dynamics simulations were also carried out.

Full text of DOI:10.1016/j.bmc.2013.06.065

Bioorganic & Medicinal Chemistry 21 (2013) 4928–4937
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological activity and molecular modeling studies
on 1H-benzimidazole derivatives as acetylcholinesterase inhibitors
Ayßse Selcen Alpan a, Sülünay Parlar a, Luca Carlino b, Ayse Hande Tarikogullari a, Vildan Alptüzün a,
,
⇑
Hasan Semih Güneßs a
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University, 35100 Bornova, Izmir, Turkey
b Department of Pharmaceutical Chemistry, Martin-Luther University of Halle-Wittenberg, 06120 Halle/Saale, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-{2-[4-(1H-benzimidazole-2-yl)phenoxy]ethyl}substituted amine derivatives were designed
to assess cholinesterase inhibitor activities. Acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE) inhibitor activities were evaluated in vitro by using Ellman’s method. It was discovered that
most of the compounds displayed AChE and/or BuChE inhibitor activity and few compounds were selec-
tive against AChE/BuChE. Compound 3c and 3e were the most active compounds in the series against
eeAChE and hAChE, respectively. Molecular docking studies and molecular dynamics simulations were
also carried out.
Received 9 May 2013
Revised 26 June 2013
Accepted 27 June 2013
Available online 10 July 2013
Keywords:
1H-Benzimidazole
Acetylcholinesterase inhibitor
Butyrylcholinesterase inhibitor
Molecular modeling
Synthesis
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
peripheral anionic site (PAS).9 Therefore, some dual-binding inhib-
itors binding simultaneously to both the catalytic and peripheral
Acetylcholinesterase (AChE) is a key enzyme involved in the
termination of nerve signals through the hydrolysis of acetylcho-
line. It is a target of drug development to combat the neuromuscu-
lar disorders such as myasthenia gravis, glaucoma and Alzheimer’s
disease (AD).1 AChE has been recently targeted in the treatment of
AD, a progressive neurodegenerative disorder characterized by
b-amyloid plaques, neurofibrillary tangles and loss of central cho-
linergic function.2,3 A deficiency in cholinergic neurotransmission
is considered to be one of the major causes of memory impair-
ments in the patients with AD.4,5 One of the effective approaches
for enhancing the cholinergic transmission is to use the acetylcho-
linesterase inhibitors (AChEI). Recent researches have revealed
that several AChEI’s not only facilitate cholinergic transmission
but also interfere with the synthesis, deposition and aggregation
of toxic b-amyloid (Ab).6–8 This action is associated with a specific
motif located near the entrance of the active-site gorge termed as
sites of AChE are the targets of AD.10,11
In recent years, a series of crystal structures of Torpedo californi-
ca AChE (TcAChE) and human AChE (hAChE) have been reported.
AChE structures indicate
a deep, narrow hydrophobic gorge
approximately 20 Å long. The bottom of the gorge which is called
the catalytic active site (CAS) is assigned to the Ser-His-Glu cata-
lytic triad (Ser200-His440-Glu327 in TcAChE and Ser203-His447-
Glu334 in hAChE). In the middle of the TcAChE gorge, there are 5
amino acid residues, including Trp84, Tyr130, Glu199, Phe330
and Tyr442 (Trp86, Tyr133, Glu202 and Tyr449 in hAChE) which
comprise the anionic binding site. Moreover, the interaction be-
tween highly potent inhibitors such as tacrine and donepezil, and
the enzyme is characterized by cation–p interactions between
the protonated nitrogens and tryptophan (Trp84 in TcAChE) and
phenylalanine (Phe330 in TcAChE, Tyr337 in hAChE) residues. At
the entrance of the TcAChE gorge, there is a PAS, including Tyr70,
Asp72, Tyr121, Glu278, Trp279 and Tyr334 residues (Tyr72,
Asp74, Tyr124, Glu285, Trp286, and Tyr341, respectively in
hAChE).12
Only recently hAChE in complex with several well-known
inhibitors has been crystalized. The new crystal structures allowed
defining new binding hot spots inside AChE active site gorge. The
comparison of hAChE crystal structure (4EY7) with TcAChE crystal
structure (1EVE) in complex with donepezil reveals one key
Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; AD,
Alzheimer’s disease; AChEI, acetylcholinesterase inhibitors; Ab, b-amyloid; PAS,
peripheral anionic site; eeAChE, Electrophorus electricus AChE; TcAChE, Torpedo
californica AChE; hAChE, human AChE; CAS, catalytic active site; MD, molecular
dynamics; RMSD, root mean square deviation; DTNB, 5,50-dithiobis(2-nitrobenzoic
acid); ATC, acetylthiocholine iodide.
⇑
Corresponding author. Tel.: +90 232 339 9931x116; fax: +90 232 388 5258.
E-mail address: vildan.alptuzun@ege.edu.tr (V. Alptüzün).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.065

A. S. Alpan et al. / Bioorg. Med. Chem. 21 (2013) 4928–4937
4929
Figure 1. Binding mode comparison between donepezil binding inside TcAChE (1EVE pdb code) and hAChE (4EY7 pdb code). Donepezil is represented as cyan stick; the most
involved residues are named and represented as green sticks for the TcAChE and yellow sticks for the hAChE; water molecules are represented as red sphere, while hydrogen
bond interactions are represented as black dashed lines.
difference. Inside hAChE crystal structure, the piperidine ring is al-
most flipped over, allowing a hydrogen bond water molecule med-
iated between the N15 of donepezil and Tyr341 and Tyr337
(Phe330 and Tyr334 in TcAChE) of hAChE binding site (Fig. 1).12
On the other hand, the crystal structure of TcAChE shows a similar
hydrogen bond interaction with a water molecule mediated, be-
tween N15 of donepezil and Tyr121 side chain (Tyr124 in
hAChE).13
Basic side chains and a free amino group are an integral part of
the structure of several reported acetylcholinesterase inhibitors
and it is known that this nitrogen atom plays an important role
in enzyme–compound interaction.14 Besides, many aromatic struc-
compounds were realized in three steps. Sodium hydroxy
(4-hydroxyphenyl)methane sulfonate salt furnished by treating
4-hydroxybenzaldehyde with sodium bisulfide solution was re-
acted with nonsubstituted or 5-substituted o-phenylenediamine
analogues to obtain 4-(1H-benzimidazole-2-yl)phenol derivatives
(1–3).21 Finally, aminoalkylhalogenates were added to the inter-
mediates to achieve the final compounds. The structures of the
final compounds were confirmed by IR, 1H NMR, 13C NMR and
HRMS spectral methods. The compounds were screened for their
acetylcholinesterase and butyrylcholinesterase inhibitor activity.
Compound 1a and 3b are registered as CASRN 47160-86-1 and
CASRN 959081-12-0, respectively, but corresponding scientific
reference data were not available.
tures have potential hydrophobic and
p–p interactions with the
aromatic residues both on the entrance and inside of the enzyme
gorge in AChE. In the search of new potent acetylcholinesterase
inhibitors, it is important that many heterocyclic rings show signif-
icant inhibitor activity including AD drugs such as tacrine, donepe-
zil and galantamine. Most heterocyclic systems have been used as
a skeleton to discover new compounds with AChEI activity.15–17
Also some imidazole derivatives play a role in discovering novel
candidates having AChEI activity.18–20 Moreover, benzimidazole
scaffold is the ring isoster of indanone pharmacophore of donepezil
which is one of the most important AChEI.
In the light of these findings, 2-phenylsubstitue-1H-benzimid-
azole was chosen as a scaffold and basic cyclic, acyclic amino res-
idues were incorporated to this scaffold with ethoxy chain. All final
compounds were tested and evaluated against cholinesterases. In
order to predict the binding modes of the new active inhibitors
molecular docking studies were carried out by using GOLD 5.1 and
GLIDE docking software’s. While to check the stability of the ligands
inside the enzymes, molecular dynamic simulations (MDs) were
conducted by using AMBER 11 software.
In the IR spectra, N–H stretching band was observed at 3418–
3422 cmꢀ1 for some of the final compounds. It can be speculated
that the compounds which do not have N–H stretching band might
perform a hydrogen bond which was seen as multiple bands in
C–H stretching area between 2830 and 3083 cmꢀ1 (bands arising
from N–H—N type hydrogen bonds for benzimidazole ring is
explained by Preston).22 C@C and C@N stretching bands at
1611–1613 cmꢀ1 and 1491–1508 cmꢀ1 corresponding to benze-
noid ring, asymmetric and symmetric C–O–C stretching bands at
1247–1263 cmꢀ1 and 1031–1068 cmꢀ1 were confirmative values
with the literature survey.23 The assessment of the chemical shifts
in 1H NMR spectrum demonstrated that the aromatic and aliphatic
protons were observed in the expected regions with expected
multiplicities confirming the substitution patterns. 1H NMR data
of the nonsubstituted benzimidazole compounds (1a–1f) showed
a 1,2-disubstituted benzene system at the A ring of the benzimid-
azole nucleus and a 1,4-disubstituted benzene system due to the
p-hydroxyphenyl substituent at 2-position of ring B. The aromatic
proton signals of p-hydroxyphenyl substituent at 2-position were
observed within prospective chemical shift values and divisions
while the hydrogen atoms of 1H-benzimidazole ring at 4- and
7-positions were not detected at the prospective divisions. The lack
of H atom signals on 1H NMR spectra and the lack of the signals
belonging to 3a, 4, 7 and 7a C atoms in 13C NMR spectra are
noteworthy as it may suggest a proton exchange due to 1,3-tauto-
merization.24 13C NMR spectral results were assigned by using the
2D HSQC and HMBC techniques.
2. Results and discussion
2.1. Chemistry
In this study, we synthesized twenty benzimidazole derivatives
bearing different substitutions at 2 and 5 positions of which four-
teen of them are original (Scheme 1). The syntheses of the final

4930
A. S. Alpan et al. / Bioorg. Med. Chem. 21 (2013) 4928–4937
Comp.
1
1a
R
R1
OH
CH
SO3Na
H
-
H
-N(CH3)2
-N(C2H5)2
-N(CH(CH3)2)2
-Pyrrolidine
-Piperidine
-Morpholine
-
CHO
a
b
1
H
+
NaHSO3
1c
1d
1e
1f
2
H
HO
H
HO
H
OH
CH
H
R
NH2
+
NH2
CH3
R
N
SO3Na
2a
2b
2d
2e
2f
3
3a
3b
3c
3d
3e
3f
CH3 -N(CH3)2
CH3 -N(C2H5)2
CH3 -Pyrrolidine
CH3 -Piperidine
CH3 -Morpholine
OH
b
HO
N
H
1-3
R1
Cl
-
4
3
6'
5'
3'
'' 2''
1
R
5
6
Cl -N(CH3)2
N
c
2
1'
4'
R1
Cl -N(C2H5)2
Cl -N(CH(CH3)2)2
Cl -Pyrrolidine
Cl -Piperidine
Cl -Morpholine
. HCl
A
B
O
1-3
+
Cl
N
1
A
2'
H
7
1a-3f
a: ethanol-water, stirred for 1 h at r.t. b: DMF, refluxed for 2 h;
c: NaOH in ethanol, refluxed for 2 h.
Scheme 1. The synthesis pathway and the structures of the compounds.
2.2. Biological activity
On the other hand, inhibitor activity was increased when side
chains bearing nitrogen atom were added to the p-hydroxyphenol
Inhibitor potencies of the final compounds against AChE and
BuChE were evaluated by the spectroscopic method as described.25
This test is based on the reaction of 5,50-dithiobis(2-nitrobenzoic-
acid), known as DTNB or Ellman’s reagent, with the sulfhydryl
group of acetylthiocholine or butyrylthiocholine which result as a
yellow-colored product, that is, 2-nitro-5-thiobenzoic acid.
Changes of absorbance recorded at 412 nm determine the activity
of the tested compounds. The ChEI activity results are summarized
in Table 1.
derivatives (with IC50 values ranging from 14.6 to 0.58 lM) except
for the compounds with morpholine ring (1f, 2f, 3f). Due to the bio-
logical activity results, electron withdrawing and donating groups
in 5-substituted benzimidazole derivatives bearing side chains
were more active than the nonsubstituted counterparts but neither
the electron withdrawing nor the donating groups were more fa-
vored so it can be speculated that electronic factors do not orient
the activity.
In respect to hAChE activity results, 5-substitutedbenzimidazole
derivatives generally displayed better activity than nonsubstituted
counterparts as in eeAChE results. Compounds 2d and 2e with IC50
According to eeAChE activity results, some of the compounds
displayed high AChEI activity as shown in Table 1. Compounds
2e, 3c and 3e with IC50 values of 0.84; 0.58; 0.61
presented a noteworthy anti-AChE activity compared to the refer-
ence compound, tacrine (IC50 = 0.075 M). Also, compound 3e
l
M, respectively,
values of 0.40; 0.39 lM, respectively, possessed activity results
close to the reference compound’s inhibitor potency. The most ac-
tive compound is compound 3e bearing chlorine atom at 5-posi-
tion of the benzimidazole ring and piperidine on the side chain.
l
showed a remarkable selective behavior against AChE. p-Hydroxy-
phenol derivatives with no side chain (1–3) exhibited low activity.
Moreover compound 3e with an IC50(hAChE) value of 0.13 lM is
Table 1
In vitro inhibition of AChE/BuChE and calculated logP values
IC50 SEM (
BChE
l
M)a
eeAChE
hAChE
SelectivityBChE/eeAChE
LogPb
1
17.33 0.27
13.12 0.81
15.64 3.92
1.19 0.08
3.05 0.31
1.08 0.06
18.79 1.98
15.93 0.78
5.06 1.59
2.34 0.11
1.34 0.08
0.84 0.08
>100
14.59 1.13
2.68 4.41
3.50 0.22
0.58 0.06
3.24 0.17
0.61 0.07
11.05 0.87
0.075 0.02
>100
10.68 2.30
>100
23.42 1.70
2.13 0.62
1.03 0.10
>100
4.59 7.34
5.87 1971
5.06 16.68
4.11 0.25
23.05 24.55
2.45 0.05
>100
4.67 3.39
>100
6.48 1.88
3.24 26.40
1.37 0.07
>100
8.52 1896
>100
>100
7.44 1.51
3.42 0.15
>100
0.3
0.4
0.3
3.5
7.6
2.3
3.0
3.1
3.8
4.7
3.7
4.1
2.7
3.3
3.5
4.1
4.0
4.4
3.0
3.6
3.7
4.4
5.4
4.3
4.7
3.3
1a
1b
1c
1d
1e
1f
2
>100
0.3
2a
2b
2d
2e
2f
12.34 2.37
50.98 3.92
0.40 0.05
0.39 0.04
12.92 1.18
>100
8.02 0.61
8.75 1.19
3.68 0.39
1.40 0.33
0.13 0.03
9.73 0.70
0.52 0.09
2.8
2.4
1.6
3
0.6
3a
3b
3c
3d
3e
3f
12.8
1.1
>100
0.0098 0.0002
Tacrine
a
Data are means standard error of the mean of triplicate independent experiments.
LogP calculated using MOE 2011.10 (Molecular Operating Environment, Chemical Computing Group).
b

A. S. Alpan et al. / Bioorg. Med. Chem. 21 (2013) 4928–4937
4931
more active against hAChE compared to the reference compound
tacrine [IC50(hAChE) = 0.52 M]. Unlike ee activity results, the
pounds was able to overlap the same position showed by
donepezil inside both crystal structures. Regarding TcAChE
(PDB code 1EVE) the selected compounds are showing a hydro-
gen bond water molecule mediated with the side chain of
Tyr121, while the benzimidazole moiety can interacts with the
l
inhibitor potency of the tested compounds was higher when only
the nitrogen atom is a member of a cyclic ring other than the mor-
pholine ring. This exception can be speculated that electron with-
drawing from nitrogen to oxygen atom by inductive effect may be
the cause of this situation.
residues Trp279 and Tyr334 through
p–p interactions in the
peripheral pocket. The amino acid residues Trp279 and Tyr334
are known to be located in the PAS. Compounds 3c, 3e and 2e
are thought to interact with the PAS of TcAChE (Fig. 2). Concern-
ing hAChE, the selected compounds are interacting through a
hydrogen bond water molecule mediated with the side chains
of Tyr341 and Tyr337. While the benzimidazole moiety might
Among the tested compounds, it can be concluded that inhibi-
tor activity of the compounds against AChE is generally stronger
than of BuChE. However, most of the compounds with no side
chain display less AChE inhibitor activity then BuChE. Differences
in AChE and BuChE activities may be resulted from the several
structural differences between the hydrophobic gorges of active
center in these enzymes. At the base of the gorge in AChE, the bind-
ing of the substrate is represented by two phenylalanine molecules
whose aromatic residues protrude into the gorge. In BuChE, these
molecules are replaced by two smaller amino acid molecules valine
and leucine. This conformational change creates a larger space
within the deepest area of the gorge of BuChE.12,26 Lower BuChE
inhibitor potency of compounds may be related to the larger active
site gorge of BuChE in respect to AChE.
interacts through
p–p interactions with Trp286 and Tyr341 res-
idues, like in the previous case (Fig. 2).
2.4. Molecular dynamics simulations
In order to understand the difference activity of compound 3c
(Table 1) inside hAChE and TcAChE the docked solution of the
AChE-compound 3c complex was subjected to molecular dynamics
to assess the stability of the compound predicted binding mode in-
side Tc and human enzyme.
In the case of compound 3c inside 1EVE crystal structure the
docked conformation is stable during the 20 ns simulation. The
inhibitor interactions (compound 3c) observed in the starting
structures (obtained by GOLD 5.1) were generally maintained during
the MD simulations. The average root mean square deviation
(RMSD) value of the ligand relative to the initial structure is around
ꢁ0.8 Å in the first 10 ns of the MD, while in the second part of the
MD the average RMSD increases until ꢁ2.3 Å without losing the
key interactions and staying stable for the rest of the MD (Fig. 3).
The average RMSD value of the protein is around ꢁ1.5 Å for the
whole MD.
Concerning the molecular dynamic of compound 3c inside 4EY7
crystal structure, the docked conformation after 1 ns loses the
interactions observed in the putative binding mode. The average
RMSD value of the ligand with respect to the initial structure
shows an increasing point around 2 ns from ꢁ0.8 Å to ꢁ4 Å, due
to a rearrangement of the structure inside the active site caused
by the loss of the initial interactions (Fig. 3).
Log P values of the synthesized compounds were calculated by
using MOE 2011.10 program. These predicted log P values 2.7–5.4
support the idea that all of the compounds might penetrate blood
brain barrier.
2.3. Docking studies
In this study, molecular docking study was performed using
GOLD 5.1 and GLIDE to predict the interaction mode and selectivity
of these derivatives for AChE (Supplementary data). The X-ray
crystal structures of the TcAChE and hAChE in complex with
donepezil (PDB code 1EVE and 4EY7, respectively) were selected
to build the starting model of AChE. Compound 3c, 3e and 2e were
chosen for molecular modeling as the most active compounds in
the series (Table 1).
As shown in Figure 2, (compound 2e is not represented be-
cause the docking studies reproduced the same geometries with
compound 3e inside both enzymes (Supplementary data)) the
protonated nitrogen atom on the side chain of the selected com-
Figure 2. Binding mode comparison between compound 3c and 3e inside TcAChE (1EVE pdb code) and hAChE (4EY7 pdb code). Compound 3c is represented as purple sticks;
compound 3e is represented as orange sticks; the most involved residues are named and showed as green sticks in TcAChE and yellow sticks in hAChE; water molecules are
represented as red sphere while hydrogen bond interactions are represented as black dashed lines.

4932
A. S. Alpan et al. / Bioorg. Med. Chem. 21 (2013) 4928–4937
Figure 3. Comparison of the root mean square deviation (RMSD) plots of AChE-compound 3c complexes. Top is plotted the RMSD of compound 3c (red) inside TcAChE (black,
1EVE pdb code). Bottom is plotted the RMSD of compound 3c (red) inside hACHE (black 4EY7 pdb code).
13C NMR (100 MHz, CDCl3) d ppm: 46.0 (C-1000), 58.4 (C-200), 66.1
3. Experimental
(C-100), 115.2 (C-30, C-50), 122.9 (C-5, C-6), 128.4 (C-20, C-60), 152.1
(C-2), 160.5 (C-40); HRMS (ESI+) calcd for C17H19N3O [M+H+]
3.1. Chemistry
282.1606, found 282.1607.
3.1.1. Material and methods
3.1.2.2. 2-[4-(1H-Benzo[d]imidazol-2-yl)phenoxy]-N,N-diethyle-
thanamine, 1b. : 3418,
Yield 28%; mp: 202 °C;27 IR (KBr)
3054, 2968, 2871, 1613, 1501, 1257, 1041, 844, 744 cmꢀ1 1H
All melting points were determined with a capillary melting
points apparatus (Buchi 510, BUCHI, Flawil, Switzerland). The IR
spectra of compounds were monitored as potassium bromide pel-
lets (FT/IR-430, JASCO, Tokyo, Japan). The NMR spectra (400 MHz
for 1H and 100 MHz for 13C) were recorded in the deuterated sol-
vent indicated with chemical shifts reported in d units downfield
from tetramethylsilane (TMS). Coupling constants (J) are reported
in hertz (Hz) (AS 400 Mercury Plus NMR Varian, Palo Alto, USA).
HRMS was recorded on a HPLC-TOF Waters Micromass LCT Premier
XE (Milford, MA, USA) mass spectrometer using ESI. Analytical
thin-layer chromatography (TLC) was run on Merck silica gel plates
(Kieselgel 60F254) with detection by UV light (254 nm). All starting
materials and reagents were high-grade commercial products.
m
;
NMR (400 MHz, CDCl3) d ppm: 1.07 (6H, t, J = 7.0, H-2000), 2.65
(4H, q, J = 7.0 Hz, H-1000), 2.89 (2H, t, J = 5.9 Hz, H-200), 4.07 (2H, t,
J = 5.9 Hz, H-100), 6.91 (2H, d, J = 8.9 Hz, H-30, H-50), 7.21 (2H, dd,
J = 3.1, 5.9 Hz, H-5, H-6), 7.59 (2H, br s, H-4, H-7), 8.00 (2H, d,
J = 8.6 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) d ppm: 11.9 (C-
2000), 48.0 (C-1000), 51.9 (C-200), 66.9 (C-100), 115.3 (C-30, C-50), 122.7
(C-10), 122.8 (C-5, C-6), 128.4 (C-20, C-60), 152.3 (C-2), 160.7 (C-
40); HRMS (ESI+) calcd for C19H23N3O [M+H+] 310.1919, found
310.1929.
3.1.2.3. N-{2-[4-(1H-Benzo[d]imidazol-2-yl)phenoxy]ethyl}-N-
isopropylpropan-2-amin, 1c.
(KBr) : 3418, 3065, 2965, 2871, 1612, 1500, 1256, 1067, 835,
747 cmꢀ1 1H NMR (400 MHz, CDCl3)
ppm: 1.06 (12H, d,
Yield 15%; mp: 191 °C; IR
3.1.2. Synthesis of N-{2-[4-(1H-benzimidazole-2-yl)
phenoxy]ethyl}substitutedamine derivatives 1a–3f
m
;
d
4-(1H-Benzimidazole-2-yl)phenol, 4-(5-methyl-1H-benzimid-
azole-2-yl)phenol and 4-(5-chloro-1H-benzimidazole-2-yl)phenol
were synthesized by reacting o-phenylenediamine and sodium
hydroxy(4-hydroxyphenyl)methane sulfonate salt as described
(1–3).21 After reacting 0.005 moles of 2-(1H-benzimidazole-2-
yl)phenol derivatives and 0.01 moles of A (Scheme 1) with 0.015
moles of sodium hydroxide in 10 mL ethanol, the mixture was
refluxed at 95 °C for 2 h in an oil bath. Ethanol was evaporated
and the residue was extracted with ether. The compounds were
obtained by preparative TLC (CHCl3/C6H6/CH3OH/25% NH4OH solu-
tion, 5:1:0.5:0.05) and crystallization with methanol/water.
J = 6.6 Hz, H-1000, H-3000), 2.85 (2H, H-200), 3.08 (2H, t, J = 6.6 Hz, H-
2000), 3.96 (2H, t, H-100), 6.94 (2H, d, J = 8.9 Hz, H-30, H-50), 7.23
(2H, dd, J = 3.1, 6.2 Hz, H-5, H-6), 7.61 (2H, br s, H-4, H-7), 7.98
(2H, d, J = 8.6 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) d ppm:
21.0 (C-1000, C-3000), 44.6 (C-200), 50.1 (C-2000), 69.4 (C-100), 115.2 (C-
30, C-50), 122.5 (C-10), 122.9 (C-5, C-6), 128.3 (C-20, C-60), 152.1
(C-2), 160.8 (C-40); HRMS (ESI+) calcd for C21H27N3O [M+H+]
338.2232, found 338.2235.
3.1.2.4. 2-{4-[2-(Pyrrolidin-1-yl)ethoxy]phenyl}-1H-benzo[d]-
imidazole, 1d.
Yield 11%; mp: 195 °C; IR (KBr)
m
: 3053,
2928, 2877, 1613, 1504, 1262, 1048, 839, 747 cmꢀ1
;
1H NMR
3.1.2.1. 2-[4-(1H-Benzo[d]imidazol-2-yl)phenoxy]-N,N-dimeth-
(400 MHz, CDCl3) d ppm: 1.80 (4H, t, J = 6.6 Hz, H-3000, H-4000), 2.65
(4H, t, J = 5.5 Hz, H-2000, H-5000), 2.91 (2H, t, J = 5.6 Hz, H-200), 4.10
(2H, t, J = 5.6 Hz, H-100), 6.87 (2H, d, J = 8.8 Hz, H-30, H-50), 7.20
(2H, dd, J = 3.2, 6 Hz, H-5, H-6), 7.58 (2H, dd, J = 3.2, 6 Hz, H-4,
H-7), 7.96 (2H, d, J = 8.8 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3)
d ppm: 23.7 (C-3000, C-4000), 54.9 (C-2000, C-5000), 55.2 (C-200), 67.1 (C-100),
ylethanamine, 1a.
Yield 32%; mp: 212 °C; IR (KBr)
m
: 3061,
1H NMR
d
ppm: 2.36 (6H, s, H-1000), 2.77 (2H, t,
2973, 2851, 1611, 1497, 1247, 1042, 834, 745 cmꢀ1
;
(400 MHz, CDCl3)
J = 5.2 Hz, H-200), 4.09 (2H, t, J = 5.2 Hz, H-100), 6.92 (2H, d,
J = 8.8 Hz, H-30, H-50), 7.22 (2H, dd, J = 3.2, 6.4 Hz, H-5, H-6), 7.60
(2H, dd, J = 3.2, 6 Hz, H-4, H-7), 7.98 (2H, d, J = 9.2 Hz, H-20, H-60);

A. S. Alpan et al. / Bioorg. Med. Chem. 21 (2013) 4928–4937
4933
115.2 (C-30, C-50), 122.8 (C-5, C-6), 122.9 (C-10), 128.4 (C-20, C-60),
152.3 (C-2), 160.5 (C-40); HRMS (ESI+) calcd for C19H21N3O
[M+H+] 308.1763, found 308.1771.
J = 7.4 Hz, H-7), 7.94 (2H, d, J = 8.6 Hz, H-20, H-60); 13C NMR
(100 MHz, CDCl3) d ppm: 21.9 (Ar-CH3), 23.7 (C-3000, C-4000), 55.0
(C-2000, C-5000), 55.1 (C-200), 67.2 (C-100), 115.2 (C-30, C-50), 123.0 (C-
10), 124.3 (C-6), 128.2 (C-20, C-60), 132.7 (C-5), 151.7 (C-2), 160.4
(C-40); HRMS (ESI+) calcd for C20H23N3O [M+H+] 322.1919, found
322.1928.
3.1.2.5.
imidazole, 1e.
2932, 2849, 1612, 1501, 1258, 1031, 837, 745 cmꢀ1
2-{4-[2-(Piperidin-1-yl)ethoxy]phenyl}-1H-benzo[d]-
Yield 16%; mp: 200 °C; IR (KBr) : 3058,
1H NMR
m
;
(400 MHz, CDCl3) d ppm: 1.37 (2H, m, H-4000), 1.55 (4H, m, H-3000,
H-5000), 2.47 (4H, br s, H-2000, H-6000), 2.72 (2H, t, J = 5.6 Hz, H-200),
4.04 (2H, t, J = 5.6 Hz, H-100), 6.81 (2H, d, J = 8.4 Hz, H-30, H-50),
7.14 (2H, dd, J = 2.8, 5.2, Hz, H-5, H-6), 7.52 (2H, dd, J = 3.2,
6.4 Hz, H-4, H-7), 7.91 (2H, d, J = 8.8 Hz, H-20, H-60); 13C NMR
(100 MHz, CDCl3) d ppm: 24.2 (C-4000), 25.9 (C-3000, C-5000), 55.3 (C-
2000, C-6000), 58.0 (C-200), 66.0 (C-100), 115.2 (C-30, C-50), 122.8 (C-5,
C-6), 128.4 (C-20, C-60), 152.2 (C-2), 160.5 (C-40); HRMS (ESI+) calcd
for C20H23N3O [M+H+] 322.1919, found 322.1911.
3.1.2.10.
1H-benzo[d]imidazole, 2e.
3419, 3048, 2932, 2853, 1612, 1508, 1256, 1033, 837,
740 cmꢀ1 1H NMR (400 MHz, CDCl3)
ppm: 1.50 (2H, d,
5(6)-Methyl-2-{4-[2-(piperidin-1-yl)ethoxy]phenyl}-
Yield 20%; mp: 141 °C; IR (KBr)
m:
;
d
J = 4.0 Hz, H-4000), 1.69 (4H, d, J = 4.8 Hz, H-3000, H-5000), 2.47 (3H, s,
Ar-CH3), 2.63 (4H, br s, H-2000, H-6000), 2.87 (2H, t, J = 5.5 Hz, H-200),
4.19 (2H, t, J = 5.2 Hz, H-100), 6.93 (2H, d, J = 8.8 Hz, H-30, H-50),
7.07 (1H, d, J = 8.2 Hz, H-6), 7.42 (1H, s, H-4), 7.54 (1H, d,
J = 8.0 Hz, H-7), 8.02 (2H, d, J = 8.6 Hz, H-20, H-60); 13C NMR
(100 MHz, CDCl3) d ppm: 21.9 (Ar-CH3), 24.2 (C-4000), 25.9 (C-3000,
C-5000), 55.3 (C-2000, C-6000), 58.0 (C-200), 66.1 (C-100), 115.2 (C-30, C-
50), 123.0 (C-10), 124.3 (C-6), 128.2 (C-20, C-60), 132.6 (C-5), 151.8
(C-2), 160.4 (C-40); HRMS (ESI+) calcd for C21H25N3O [M+H+]
336.2076, found 336.2076.
3.1.2.6. 4-{2-[4-(1H-Benzo[d]imidazol-2-yl)phenoxy]ethyl}mor-
pholine, 1f.
Yield 24%; mp: 185 °C; IR (KBr)
m
: 3422, 3053,
2965, 2857, 1611, 1499, 1251, 1068, 836, 748 cmꢀ1
;
1H NMR
(400 MHz, CDCl3) d ppm: 3.64 (4H, t, J = 4.5 Hz, H-2000, H-6000), 2.77
(2H, t, J = 5.7 Hz, H-200), 2.54 (4H, t, J = 4.5 Hz, H-3000, H-5000), 4.23
(2H, t, J = 5.7 Hz, H-100), 7.17 (2H, d, J = 8.7 Hz, H-30, H-50), 7.25
(2H, m, H-5, H-6), 7.55 (1H, d, J = 6.9 Hz, H-7⁄), 7.67 (1H, d,
J = 7 Hz, H-4⁄), 8.17 (2H, d, J = 8.7 Hz, H-20, H-60); 13C NMR
(100 MHz, CDCl3) d ppm: 67.5 (C-2000, C-6000), 58.2 (C-200), 66.8 (C-
10⁄0), 54.8 (C-3000, C-5000), 112.6 (C-4⁄), 116.5 (C-30, C-50), 120.1 (C-
7 ), 123.1 (C-5#), 123.7 (C-6#), 124.3 (C-10), 129.7 (C-20, C-60),
3.1.2.11. 4-{2-[4-(5(6)-Methyl-1H-benzo[d]imidazol-2-yl)phen-
oxy]ethyl}morpholine, 2f.
m
Yield 33%; mp: 105 °C; IR (KBr)
: 3051, 2951, 2866, 1612, 1507, 1258, 1049, 840, 740 cmꢀ1
;
1H
NMR (400 MHz, CDCl3) d ppm: 2.43 (3H, s, Ar-CH3), 3.73 (4H, t,
J = 4.5 Hz, H-2000,H-6000), 2.79 (2H, t, J = 5.6 Hz, H-200), 2.57 (4H, t,
J = 4.6 Hz, H-3000, H-5000), 4.08 (2H, t, J = 5.6 Hz, H-100), 6.87 (2H, d,
J = 8.8 Hz, H-30, H-50), 7.04 (1H, dd, J = 1.1, 8.2 Hz, H-6), 7.35 (1H,
s, H-4), 7.47 (1H, d, J = 8.2 Hz, H-7), 8.03 (2H, d, J = 8.8 Hz, H-20,
H-60); 13C NMR (100 MHz, CDCl3) d ppm: 21.9 (Ar-CH3), 67.1 (C-
2000, C-6000), 57.8 (C-200), 66.1 (C-100), 54.3 (C-3000, C-5000), 115.2 (C-30,
C-50), 122.8 (C-10), 124.5 (C-6), 128.4 (C-20, C-60), 132.9 (C-5),
151.8 (C-2), 160.4 (C-40); HRMS (ESI+) calcd for C20H23N3O2
[M+H+] 338.1869, found 338.1869.
U
U
U
136.7 (C-3a ), 145.6 (C-7a ), 153.1 (C-2), 161.7 (C-40)⁄,#, Inter-
changeable; HRMS (ESI+) calcd for C19H21N3O2 [M+H+] 324.1712,
found 324.1711.
3.1.2.7. N,N-Dimethyl-2-[4-(5(6)-methyl-1H-benzo[d]imidazol-
2-yl)phenoxy]ethanamine, 2a.
Yield 18%; mp: 134 °C; IR
(KBr)
m
:
3039, 2971, 2882, 1613, 1498, 1262, 1046, 837,
792 cmꢀ1
;
1H NMR (400 MHz, CDCl3) d ppm: 2.30 (6H, s, H-1000),
2.40 (3H, s, Ar-CH3), 2.72 (2H, t, J = 5.5 Hz, H-200), 4.03 (2H, t,
J = 5.5 Hz, H-100), 6.90 (2H, d, J = 8.9 Hz, H-30, H-50), 6.98 (1H, dd,
J = 0.9, 8.2 Hz, H-6), 7.30 (1H, s, H-4), 7.41 (1H, d, J = 8.2 Hz, H-7),
7.90 (2H, d, J = 8.8 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) d
ppm: 21.9 (Ar-CH3), 46.0 (C-1000), 58.3 (C-200), 66.1 (C-100), 115.1
(C-30, C-50), 123.0 (C-10), 124.3 (C-6), 128.3 (C-20, C-60), 132.7 (C-
5), 151.8 (C-2), 160.4 (C-40); HRMS (ESI+) calcd for C18H21N3O
[M+H+] 296.1793, found 296.1771.
3.1.2.12. 2-[4-(5(6)-Chloro-1H-benzo[d]imidazol-2-yl)phenoxy]-
N,N-dimethylethanamine, 3a.
Yield 25%; mp: 115 °C; IR
(KBr)
m
: 2974, 2830, 1612, 1499, 1263, 1046, 837, 742 cmꢀ1
;
1H
NMR (400 MHz, CDCl3) d ppm: 2.37 (6H, s, H-1000), 2.78 (2H, t,
J = 5.5 Hz, H-200), 4.11 (2H, t, J = 5.5 Hz, H-100), 6.94 (2H, d, J = 9 Hz,
H-30, H-50), 7.19 (1H, dd, J = 2, 8.6 Hz, H-6), 7.49 (1H, d, J = 8.6 Hz,
H-7), 7.56 (1H, s, H-4), 7.93 (2H, d, J = 8.6 Hz, H-20, H-60); 13C
NMR (100 MHz, CDCl3) d ppm: 46.0 (C-1000), 58.3 (C-200), 66.2 (C-
100), 115.3 (C-30, C-50), 122.4 (C-10), 123.4 (C-6), 128.4 (C-20, C-60),
153.1 (C-2), 160.8 (C-40); HRMS (ESI+) calcd for C17H18ClN3O;
[M+H+] 316.1217, found 316.1212.
3.1.2.8. N,N-Diethyl-2-[4-(5(6)-methyl-1H-benzo[d]imidazol-2-
yl)phenoxy]ethanamine, 2b.
Yield 12%; mp: 155 °C; IR
(KBr)
m
: 3048, 2969, 1613, 1495, 1259, 1044, 838, 741 cmꢀ1
;
1H
NMR (400 MHz, CDCl3) d ppm: 1.05 (6H, t, J = 7.0 Hz, H-2000), 2.43
(3H, s, Ar-CH3), 2.63 (4H, q, J = 7.0 Hz, H-1000), 2.86 (2H, t,
J = 6.2 Hz, H-200), 4.05 (2H, t, J = 6.2 Hz, H-100), 6.91 (2H, d,
J = 8.6 Hz, H-30, H-50), 7.03 (1H, dd, J = 1.6, 8.2 Hz, H-6), 7.34 (1H,
br s, H-4⁄), 7.48 (1H, br s, H-7⁄), 7.97 (2H, d, J = 8.6 Hz, H-20, H-60)
3.1.2.13. 2-[4-(5(6)-Chloro-1H-benzo[d]imidazol-2-yl)phenoxy]-
N,N-diethylethanamine, 3b.
m
Yield 13%; mp: 142 °C; IR (KBr)
: 2966, 1613, 1498, 1257, 837, 739 cmꢀ1
;
1H NMR (400 MHz,
CDCl3) d ppm: 1.06 (6H, t, J = 6.8 Hz, H-2000), 2.63 (4H, q, J = 6.8 Hz,
H-1000), 2.85 (2H, t, J = 6 Hz, H-200), 4.02 (2H, t, J = 6 Hz, H-100), 6.84
(2H, d, J = 8 Hz, H-30, H-50), 7.14 (1H, d, J = 8.8 Hz, H-6), 7.42 (1H,
d, J = 7.6 Hz, H-7), 7.48 (1H, br s, H-4), 7.91 (2H, d, J = 8 Hz, H-20,
13
⁄Interchangeable; C NMR (100 MHz, CDCl3) d ppm: 12.0 (C-2000),
21.7 (Ar-CH3), 48.0 (C-1000), 51.9 (C-200), 66.9 (C-100), 115.3 (C-30, C-
50), 122.9 (C-10), 124.3 (C-6), 128.3 (C-20, C-60), 152.0 (C-2), 160.6
(C-40); HRMS (ESI+) calcd for C20H25N3O [M+H+] 324.2076, found
324.2085.
13
H-60); C NMR (100 MHz, CDCl3) d ppm: 11.9 (C-2000), 47.9 (C-1000),
51.9 (C-200), 66.8 (C-100), 115.2 (C-30, C-50), 122.2 (C-10), 123.3 (C-
6), 128.4 (C-20, C-60), 128.5 (C-5), 153.3 (C-2), 160.8 (C-40); HRMS
(ESI+) calcd for C19H22ClN3O; [M+H+] 344.1530, found 344.1535.
3.1.2.9.
1H-benzo[d]imidazole, 2d.
: 3418, 2969, 2877, 1613, 1496, 1261, 1048, 839, 741 cmꢀ1
5(6)-Methyl-2-{4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-
Yield 11%; mp: 122 °C; IR (KBr)
1H
3.1.2.14. N-{2-[4-(5(6)-Chloro-1H-benzo[d]imidazol-2-yl)phen-
m
;
oxy]ethyl}-N-isopropylpropan-2-amine, 3c.
161 °C; IR (KBr) : 2965, 2876, 1612, 1495, 1256, 1057, 835,
742 cmꢀ1
Yield 11%; mp:
NMR (400 MHz, CDCl3) d ppm: 1.82 (4H, br s, H-3000, H-4000), 2.46
(3H, s, Ar-CH3), 2.68 (4H, br s, H-2000, H-5000), 2.94 (2H, t, J = 5.9 Hz,
H-200), 4.15 (2H, t, J = 5.9 Hz, H-100), 6.94 (2H, d, J = 8.6 Hz, H-30, H-
50), 7.05 (1H, d, J = 8.2 Hz, H-6), 7.37 (1H, s, H-4), 7.50 (1H, d,
m
;
1H NMR (400 MHz, CDCl3) d ppm: 1.08 (12H, d,
J = 6 Hz, H-1000, H-3000), 2.87 (2H, t, J = 6.8 Hz, H-200), 3.10 (2H, t,
J = 6 Hz, H-2000), 3.96 (2H, t, J = 6.8 Hz, H-100), 6.90 (2H, d, J = 8 Hz,

4934
A. S. Alpan et al. / Bioorg. Med. Chem. 21 (2013) 4928–4937
H-30, H-50), 7.17 (1H, d, J = 8.4 Hz, H-6), 7.47 (1H, br s, H-7), 7.54
(1H, s, H-4), 7.94 (2H, d, J = 8.4 Hz, H-20, H-60); 13C NMR
(100 MHz, CDCl3) d ppm: 20.9 (C-1000, C-3000), 44.7 (C-200), 50.6 (C-
2000), 68.4 (C-100), 115.2 (C-30, C-50), 122.1 (C-10), 123.3 (C-6), 128.3
(C-5), 128.5 (C-20, C-60), 152.3 (C-2), 160.9 (C-40); HRMS (ESI+) calcd
for C21H26ClN3O; [M+H+] 372.1843, found 372.1850.
Stock solutions of the inhibitor compounds were prepared in 2%
DMSO, which were diluted with aqueous assay medium to a final
content of organic solvent always lower than 0.2%. The enzyme
activity was determined in the presence of at least five different
concentrations of an inhibitor, generally between 10ꢀ3 and 10ꢀ8
,
in order to obtain the inhibition of AChE or BuChE activity between
0% and 100%. Each concentration was assayed in triplicate. Prior to
use, all solutions were adjusted to 20 °C. Enzyme solution
3.1.2.15. 5(6)-Chloro-2-{4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-
1H-benzo[d]imidazole, 3d.
Yield 14%; mp: 117 °C; IR (KBr)
(2.5 units/mL, 100 lL) and inhibitor solution (100 lL) were added
m
: 2962, 2830, 1612, 1498, 1263, 1056, 838, 742 cmꢀ1
;
1H NMR
into a cuvette containing the phosphate buffer (3.0 mL, 0.1 M;
pH = 8.0). After 5 min incubation, required aliquots of the DTNB
solution (0.01 M, 100 mL) and of the acetylthiocholine/butyrylthi-
(400 MHz, CDCl3) d ppm: 1.77 (4H, br s, H-3000, H-4000), 2.62 (4H, br
s, H-2000, H-5000), 2.90 (2H, t, J = 5.9 Hz, H-200), 4.09 (2H, t, J = 5.9 Hz,
H-100), 6.84 (2H, d, J = 8.6 Hz, H-30, H-50), 7.15 (1H, dd, J = 2.0,
8.6 Hz, H-6), 7.44 (1H, br s, H-7), 7.50 (1H, br s, H-4), 7.90 (2H, d,
J = 8.6 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) d ppm: 23.7 (C-
3000, C-4000), 54.9 (C-2000, C-5000), 55.2 (C-200), 67.3 (C-100), 115.2 (C-30,
C-50), 122.4 (C-10), 123.2 (C-6), 128.3 (C-5), 128.5 (C-20, C-60),
153.5 (C-2), 160.8 (C-40); HRMS (ESI+) calcd for C19H20ClN3O;
[M+H+] 342.1373, found 342.1373.
ocholine iodide (0.075 M, 20 lL) were added. After a rapid and
immediate mixing, the absorption was measured at 412 nm. In vi-
tro BuChE assay was similar with the method described above.28
3.3. Molecular docking study
3.3.1. Ligand docking
The crystal structures of Donepezil in complex with AChE (pdb
codes: 1EVE and 4EY7 resolved at 2.5 Å and 2.35 Å, respectively)
were taken from the Protein Data Bank. Heteroatoms and water
molecules (excluded the water molecule involved in the binding
mode) in the PDB file were removed and hydrogen atoms were
added to the protein by using MOE 2011.10.29 Prior to the docking
calculations, an energy minimization using the AMBER99 force
field was performed on the enzymes. All the compounds were built
and protonated using the protonate 3D protocol and energy mini-
mized using the MMFF94 force field in MOE 2011.10. Docking of
the ligands was carried out using the GOLD 5.1 and GLIDE program
with default settings.30,31 A sphere of 20 Å around the carbonyl
group of Glu199 (Glu202 in hAChE) was defined as the binding site
for the ligand docking and 50 confirmations was allowed. The
Goldscore and Glidescore standard precision (sp) were calculated
and analyzed (Supplementary data).
3.1.2.16.
1H-benzo[d]imidazole, 3e.
2930, 1611, 1494, 1256, 1056, 835, 740 cmꢀ1
;
5(6)-Chloro-2-{4-[2-(piperidin-1-yl)ethoxy]phenyl}-
Yield 15%; mp: 134 °C; IR (KBr)
1H NMR
m:
(400 MHz, CDCl3) d ppm: 1.44 (2H, m, H-4000), 1.60 (4H, m, H-3000,
H-5000), 2.52 (4H, m, H-2000, H-6000), 2.78 (2H, t, J = 5.9 Hz, H-200),
4.13 (2H, t, J = 5.9 Hz, H-100), 6.94 (2H, d, J = 9 Hz, H-30, H-50), 7.19
(1H, dd, J = 2, 8.6 Hz, H-6), 7.52 (2H, br s, H-4, H-7), 7.93 (2H, d,
J = 9 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) d ppm: 24.4 (C-
4000), 26.1 (C-3000, C-5000), 55.3 (C-2000, C-6000), 58.1 (C-200), 66.4 (C-100),
115.4 (C-30, C-50), 122.2 (C-10), 123.4 (C-6), 128.4 (C-20, C-60),
161.0 (C-40); HRMS (ESI+) calcd for C20H22ClN3O; [M+H+]
356.1530, found 356.1530.
3.1.2.17. 4-{2-[4-(5(6)-Chloro-1H-benzo[d]imidazol-2-yl)phen-
oxy]ethyl}morpholine, 3f.
Yield 27%; mp: 186 °C; IR (KBr)
m
: 3083, 2933, 2868, 1612, 1491, 1252, 1051, 837, 737 cmꢀ1
;
1H
NMR (400 MHz, CDCl3) d ppm: 3.71 (4H, t, J = 4.7 Hz, H-2000,
H-6000), 2.79 (2H, t, J = 5.9 Hz, H-200), 2.57 (4H, t, J = 4.7 Hz, H-3000,
H-5000), 4.08 (2H, t, J = 5.9 Hz, H-100), 6.86 (2H, d, J = 8.6 Hz, H-30,
H-50), 7.13 (1H, dd, J = 2.0, 8.4 Hz, H-6), 7.41 (1H, d, J = 8.6 Hz,
H-7), 7.46 (1H, d, J = 1.2 Hz, H-4), 7.97 (2H, d, J = 8.6 Hz, H-20,
3.3.2. Molecular dynamics simulations
A molecular dynamics simulation was carried out for AChE
complexed with compound 3c using AMBER 11.0 program.32 Protein
and ligands were parameterized using AMBER ff9933 and general AM-
BER force field (GAFF),34 respectively. The most reasonable ranked
solution from our docking studies was chosen as the initial struc-
ture of the AChE-compound 3c complex. Hydrogen atoms were
added to the protein and complex systems were neutralized with
9 Na+ counterions in the case of TcAChE (1EVE crystal structure)
and with 8 Na+ counterions in the case of hAChE (4EY7 crystal
structure) by using the xLeap module of AMBER 11.0. The structures
were solvated in an octahedral box with TIP3P35 water molecules
leaving at least 15 Å between the solute atoms and the borders
of the box. The fully solvated and neutralized systems were sub-
jected to energy minimization with the sander module of the Am-
ber11 package. Following minimization, the systems were
gradually heated from 50 to 300 K with positional restraints (force
constant: 10 kcal/mol/Å) on protein–ligand complex (including the
involved water molecule) over a period of 0.30 ns allowing water
molecules and ions to move freely. 8 Å cutoff for the short-range
nonbonded interactions was used in combination with the particle
mesh Ewald option36 using a grid spacing of ꢁ0.9 Å to account for
long-range electrostatic interactions. The Settle algorithm37 was
used to constrain bond vibrations involving hydrogen atoms. Dur-
ing additional 0.30 ns the positional restraints were gradually re-
duced to allow finally unrestrained MD simulation of all atoms
over a subsequent equilibration time of 2 ns. Further 20 ns free
MD simulations were carried using these equilibrated structures
as a start structures. VMD38 was used for visualization of trajecto-
ries, while PYMOL [The PYMOL Molecular Graphics System, Version
13
H-60); C NMR (100 MHz, CDCl3) d ppm: 67.0 (C-2000, C-6000), 57.7
(C-200), 66.1 (C-100), 54.3 (C-3000, C-5000), 114.8 (C-7), 115.3 (C-30,
C-50), 115.9 (C-4), 122.3 (C-10), 123.4 (C-6), 128.4 (C-5), 128.6
(C-20, C-60), 153.4 (C-2), 160.8 (C-40); HRMS (ESI+) calcd for
C
19H20ClN3O2; [M+H+] 358.1322, found 358.1334.
3.2. Biological activity assays
3.2.1. Material and methods
Acetylcholinesterase E.C.3.1.1.7., (Type VI-S, from electric eel and
recombinant human enzyme)—and butylcholinesterase E.C. 3.1.1.8,
from equine serum were purchased from Sigma–Aldrich (Steinheim,
Germany). 5,50-Dithiobis(2-nitrobenzoic acid) (DTNB)-Ellman’s re-
agent, buffer compounds (potassium dihydrogen phosphate, potas-
sium hydroxide), sodium hydrogen carbonate and acetylthiocholine
iodide (ATC) used as a substrate were obtained from Fluka (Buchs,
Switzerland). Spectrophotometric measurements were performed
on a Shimadzu UV/160-A Spectrophotometer.
3.2.2. Acetylcholinesterase/butyrylcholinesterase activity assay
Enzyme activity was investigated using a slightly modified col-
orimetric method of Ellman et al.25 As the product of the enzymatic
hydrolysis, the thiocholine, does not possess a significant chromo-
phore for UV detection, the evaluation of enzyme activity was per-
formed using a specific chromogenic reagent, DTNB.

A. S. Alpan et al. / Bioorg. Med. Chem. 21 (2013) 4928–4937
4935
Table 2
1.5.0.4 Schrödinger, LLC] was used for the preparation of the
figures.
Docking score results for compound 3e, 3c and 2e, inside TcAChE (pdb 1EVE) and
hAChE (pdb 4EY7), using Glide score and Gold score. Inside brackets there are the
absolute ranking positions for the pose selected as putative binding pose
3.4. LogP predictions
Compounds
1EVE
4EY7
Goldscore
Glidescore
Goldscore
Glidescore
LogP values of the compounds were calculated using MOE
2011.10.
3e
3c
2e
48.36 (42)
50.19 (19)
47.74 (39)
—
—
—
57.38 (22)
40.21 (43)
62.55 (7)
ꢀ8.95 (6)
ꢀ8.94 (9)
ꢀ8.86 (1)
4. Conclusion
A series of 1H-benzimidazole derivatives were synthesized and
assayed for their cholinesterase inhibitor activity. Among them,
nineteen derivatives were found to inhibit eeAChE enzyme in
micromolar range and compound 3c and 3e displayed potent
Table 3
Docking RMSD values of compound 2e inside both crystal structures, using 3e as
reference ligand
inhibitor activity with an IC50 value of 0.58
respectively (Table 1). In hAChE compound 2d, 2e and 3e showed
higher inhibitor activity with an IC50 value of 0.40 M, 0.39
and 0.13 M, respectively, while compound 3c displayed a reduced
IC50 value of 3.68 M (Table 1).
lM and 0.61 lM,
Compounds
1EVE
4EY7
GOLD
GLIDE
GOLD
GLIDE
l
lM
l
3e
2e
0
0
—
0
0
0.627
0.793
0.264
l
Through docking studies was showed that the selected active
inhibitors (3c, 3e and 2e) were able to reproduce the same binding
mode adapted by donepezil inside both crystal structures: hAChE
and TcAChE (Figs. 2 and 3). Goldscore scoring function properly
scored all the tested compounds according to their difference of
activity inside TcAChE and hAChE (Supplementary data). Moreover,
in order to assess the stability of compound 3c, and understand the
inhibitor activity reduction of this derivative, molecular dynamic
simulations were carried out. The simulations showed that when
compound 3c is interacting inside hAChE the flexibility of diisopro-
pylamine moiety present in the ligand structure (Scheme 1) create
steric clashed inside the gorge cavity. These clashes cause a rear-
rangement (Supplementary data) of the structure, and the initial
geometries that characterize the binding mode of compound 3c in-
side 4EY7 crystal structure (Fig. 2) were definitely lost. This might
be the reason of the reduced inhibitor activity of compound 3c
when tested on hAChE. On the other hand, compound 3e does
not lose these interactions for the presence in the structure of a less
flexible and less bulky piperidine group. While the higher activity
of donepezil, compared to compound 3c, 3e and 2e, is due to the
2e inside both crystal structures, using 3e as reference ligand
(Table 3).
5.2. Molecular dynamics
5.2.1. Molecular dynamics of Donepezil inside TcAChE and
hAChE
The starting geometries for AChE–Donepezil complex were ob-
tained from the crystal structures downloaded from the Protein
Data Bank (1EVE pdb for TcAChE and 4EY7 for hAChE). Here re-
ported is plotted the root mean square deviation values (RMSD),
relative to the initial complexes, of donepezil inside 1EVE crystal
structure (top plot) and inside 4EY7 crystal structure (bottom plot).
From the plot is possible to note that in both cases the ligand is
quite stable inside the protein, and does not lose the starting
geometries. In TcAChE (1EVE) crystal structure donezepil structure
is quite flexible (red line) in the first 10 ns of MD with the RMSD
value that reach ꢁ2 Å. After 10 ns the average RMSD value of
donepezil decreases until ꢁ1.5 Å, staying stable for the rest of
the MD. Also in hAChE (4EY7) crystal structure donepezil does
not lose the starting geometries. The RMSD value of donepezil
(red line) increases until ꢁ1.5 Å around the second ns of the MD.
After ꢁ8 ns the RMSD decrease until ꢁ0.8 Å staying stable for the
rest of the MD (Fig. 4).
p-benzyl ring present in the structure that interacts through
p–p
interactions with Trp84 (Trp86 in 4EY7 crystal structure) located
deep inside in the gorge cavity.
It can be concluded that a lipophilic atom or group substitution
on 5-position of benzimidazole scaffold and introduction of a not
high flexible side chain in p-hydroxyphenol derivatives enhance
the activity in eeAChE and hAChE. In the light of the molecular
modeling study results, further studies are in progress to achieve
derivatives having extended side chain for more effective interac-
tion with PAS site.
5.2.2. Compound 3c diisopropylamine group flexibility inside
TcAChE and hAChE
For the molecular dynamic simulations of compound 3c the
starting coordinates of TcAChE-compound 3c complex using
1EVE crystal structure were obtained from GOLD docking result,
while the starting coordinates of hAChE-compound 3c complex
using 4EY7 crystal structure were obtained from GLIDE docking re-
sult. Here reported is plotted the root mean square deviation
(RMSD) values, relative to the initial structure, of compound 3c ter-
minal moiety inside 1EVE crystal structure (black) and 4EY7 crystal
structure (red). How is possible to appreciate from the picture, in-
side 4EY7 crystal structure, the diisopropylamine group is more
flexible (the average RMSD value is around ꢁ3.2 Å) when com-
pared to the flexibility of the same moiety inside 1EVE crystal
structure (the average RMSD value is around ꢁ1.8 Å). The higher
flexibility of this moiety inside 4EY7 is responsible for the instabil-
ity of the binding mode hypothesizes and for this reason it may
5. Supporting information
5.1. Docking results
Docking studies were conducted with GOLD and GLIDE software’s
while the scoring functions selected were Goldscore and Glide-
score standard precision. GOLD program was able to well allocate
the selected ligands (according to the putative binding mode
hypothesized) inside both crystal structures, while GLIDE program
properly allocates the selected ligands (according to the putative
binding mode hypothesized) only inside 4EY7 crystal structure.
Here reported are the scoring values and the absolute ranking po-
sition (inside brackets) of the ligand poses selected as putative
binding mode (Table 2) and docking RMSD values of compound

4936
A. S. Alpan et al. / Bioorg. Med. Chem. 21 (2013) 4928–4937
Figure 4. RMSD fluctuation plots of 22 ns molecular dynamic for Donepezil inside TcAChE (pdb 1EVE, top plot) and hAChE (pdb 4EY7, bottom plot). The RMSD fluctuations
concerning Donepezil are colored red, while the RMSD fluctuations regarding the receptor are colored black.
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