Novel 2-Arylbenzimidazole derivatives as multi-targeting agents to treat Alzheimer’s disease

Article abstract of DOI:10.1007/s00044-017-1874-1

This study describes the synthesis, pharmacological evaluation, including acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) inhibition, amyloid beta (Aβ) antiaggregation, and neuroprotective effects, as well as molecular modeling of novel 2-(4-subs

Full text of DOI:10.1007/s00044-017-1874-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1874-1
ORIGINAL RESEARCH
Novel 2-Arylbenzimidazole derivatives as multi-targeting agents to
treat Alzheimer’s disease
Oya Unsal-Tan1 Keriman Ozadali-Sari1 Beyza Ayazgok2
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Tuba Tüylü Küçükkılınç2 Ayla Balkan1
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Received: 21 November 2016 / Accepted: 10 March 2017
© Springer Science+Business Media New York 2017
Abstract This study describes the synthesis, pharmacolo-
gical evaluation, including acetylcholinesterase (AChE)/
butyrylcholinesterase (BChE) inhibition, amyloid beta (Aβ)
antiaggregation, and neuroprotective effects, as well as
molecular modeling of novel 2-(4-substituted phenyl)-1H-
benzimidazole derivatives. These derivatives were synthe-
sized by cyclization of o-phenylenediamines with sodium
hydroxy(4-substituted phenyl)methanesulfonate salts. In
vitro studies indicated that the most of the target compounds
showed remarkable inhibitory activity against BChE (IC50:
13.60–95.44 µM). Among them, 3d and 3g-i also exhibited
high selectivity (SI ≥ 35.7) for BChE with IC50 values
39.56, 13.60, 14.45, and 15.15 µM, respectively. According
to the molecular modeling studies, it may be assumed that
the compounds are able to reach the catalytic site of BChE
but not that of AChE. The compounds showing BChE
inhibitory effects were subsequently examined for their Aβ-
antiaggregating and neuroprotective activities. Among the
compounds, 3d inhibited the Aβ1–40 aggregation and
demonstrated significant neuroprotection against H2O2-
induced and Aβ1–40-induced cell death. Collectively, com-
pound 3d showed the best multifunctional activity (BChE;
IC50 = 39.56 µM, SI > 126; Aβ self-mediated aggregation;
67.78% at 100 μM; H2O2-induced cytotoxicity with cell
viability of 98% and Aβ1-40-induced cytotoxicity with cell
viability of 127%). All these results suggested that 2-(4-(4-
methylpiperidin-1-yl)phenyl)-1H-benzo[d]imidazole (com-
pound 3d) could be a promising multi-target lead candidate
against Alzheimer’s disease.
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Keywords 2-Arylbenzimidazole Alzheimer’s disease
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Butyrylcholinesterase Aβ aggregation Neuroprotection
Introduction
Alzheimer’s disease (AD) is a progressive and irreversible
neurodegenerative brain disorder and the most common
type of dementia. It is known that some factors seem to play
a significant role in the pathology of AD, such as low levels
of acetylcholine (ACh), β-amyloid (Aβ) aggregation, tau
protein phosphorylation, increased oxidative stress and
neuroinflammation of the central nervous system. Two main
mechanisms have been proposed for the pathogenesis of the
disease, a cholinergic hypothesis and amyloid cascade
hypothesis (Oddo et al. 2003; Taylor et al. 2002; Cummings
2004; Anand and Singh 2013; Manev et al. 2011).
The cholinergic hypothesis is based on reduced ACh
levels in the brain of AD patients, which leads to a decline
in cognitive skills and memory. Cholinesterase (ChE)
inhibitors such as galantamine, rivastigmine, and donepezil
are commonly used to increase the ACh levels and improve
cholinergic functions. However, the efficacy of all these
inhibitors is clinically limited (Anand and Singh 2013;
Yiannopoulou and Papageorgiou 2013). In the healthy
brain, acetylcholinesterase (AChE) is the main enzyme
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1874-1) contains supplementary material,
which is available to authorized users.
* Oya Unsal-Tan
oyaunsal@hacettepe.edu.tr
1
Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Hacettepe University, Ankara, Turkey
2
Faculty of Pharmacy, Department of Biochemistry, Hacettepe
University, Ankara, Turkey

Med Chem Res
responsible for ACh hydrolysis, which terminates neuro-
transmission. Butyrylcholinesterase (BChE) is a scavenging
enzyme, which substitutes for AChE when necessary. In the
AD brain, the BChE activity rises, while that of AChE
remains unchanged or declines (Perry et al. 1978; Giacobini
2003).
A randomized controlled trial of 998 patients has shown
that the dual ChE inhibitor rivastigmine is more beneficial
than the selective AChE inhibitor donepezil (Bullock et al.
2005). BChE knockout studies showed unchanged cogni-
tive properties (Holmes et al. 2005) and a decrease in
fibrillar Aβ (Reid and Darvesh 2015; Darvesh and Reid
2016). Thus, inhibition of BChE may be important for
treatment of AD by raising ACh levels and decreasing
fibrillar Aβ in the brain.
The amyloid hypothesis states that the assembly of Aβ
into oligomers and fibrils is one of the hallmarks in the
progression of AD, leading to neuronal cell death and sec-
ondary events such as neuroinflammation which contributes
to the neurodegeneration (Sun et al. 2015; Huang et al.
2015). Despite the effort during last two decades, researchers
have not been successful either in the clinical trials for anti
Aβ drugs or in the explanation of amyloid mechanism in AD
(Karran et al. 2011). But there is no doubt that AD patho-
genesis is strongly related to the abnormal accumulation and
aggregation Aβ. Thus, targeting Aβ is still thought to be a
pivotal strategy for AD treatment (Gouras et al. 2015; Selkoe
and Hardy 2016; Mullard 2017).
Benzimidazoles have many useful therapeutic activities,
such as antiviral, antihistaminic, anticancer, antiulcer, anti-
hypertensive, antidiabetic, antifungal, antimicrobial, and
antiinflammatory (Yadav and Ganguly 2015; Bansal and
Silakari 2012; Secci et al. 2012). Despite being a privileged
scaffold in medicinal chemistry, benzimidazoles have not
been considered for their ChE inhibitory activities, with
only a few studies published in recent years (Alpan et al.
2013; Zhu et al. 2013; Yoon et al. 2013).
Although there is no evidence that the benzimidazoles
possess any inhibitory effect on Aβ aggregates, some
reports emphasized that benzimidazole derivatives con-
taining amine side chains (Fig. 1) can be used as amyloid
imaging probes due to their high binding affinity to Aβ
aggregates and high uptake into the brain (Cui et al. 2011;
Harada et al. 2014).
Fig. 1 Benzimidazole derivatives with high-binding affinity to Aβ
aggregates
in one neuroprotective structure. For this purpose, we
designed, synthesized, and biologically evaluated some 2-
phenylbenzimidazole derivatives carrying aliphatic/aromatic
amine side chains as MTDLs for AD treatment. The biolo-
gical evaluation of the benzimidazoles included AChE/BChE
inhibition, self-induced Aβ aggregation, and neuroprotection.
Materials and methods
Chemistry
Melting points were determined with a Thomas—Hoover
capillary melting point apparatus (Thomas Scientific, Phi-
ladelphia, PA, USA) and were not corrected. Attenuated
total reflection (ATR)—Fourier transform IR (FTIR) spectra
were obtained using a MIRacle ATR accessory (Pike
Technologies, Fitchburg, WI, USA) in conjunction with a
Spectrum BX FTIR spectrometer (Perkin Elmer, USA) and
were reported in cm−1 1H-NMR and 13C-NMR spectra
.
(Dimethyl sulfoxide (DMSO)-d6) were recorded on a Var-
ian Mercury 400 FT NMR spectrophotometer using tetra-
methylsilane as an internal reference (chemical shift
represented in δ p.p.m.). ESI—MS spectra were measured
on a micromass ZQ-4000 single—quadruple mass spectro-
meter. Elemental analyses (C, H, and N) were performed on
Leco CHNS 932 analyzer (Leco, St. Joseph, MI, USA).
General procedure for the preparation of 4-substituted-
benzaldehydes (1a–e)
4-Fluorobenzaldehyde and appropriate amine derivatives
were reacted in the presence of potassium carbonate in
DMSO as mentioned in the literature (Meciarova et al.
2003).
A multitarget-directed ligand (MTDL) strategy has been
developed for the treatment of disorders with complex
pathological mechanisms, such as AD. Since the exact
neuropathogenesis of AD is not fully known, selecting
biological targets for AD is difficult. However, among
others, ChE and Aβ are considered the most significant
targets for the development of novel MTDLs today.
The aim of the present study was to obtain novel MTDLs
by combining BChE and Aβ aggregation inhibitory activities
General procedure for the preparation of sodium hydroxy
(4-substituted phenyl)methanesulfonate salts (2a–e)
Substituted benzaldehydes (0.04 mol) and sodium bisulfite
(0.04 mol), dissolved in 20 mL of an ethanol and water

Med Chem Res
mixture, were stirred for 1 h at room temperature. The
formed sodium hydroxy(4-substituted phenyl)methane-
sulfonate salts were obtained by filtration.
119.1, 121.4, 125.8, 127.5, 129.0, 140.1, 151.8, 151.9. ESI-
MS (m/z); 390.17 [M + Na]+, 368.18 [M + H]+; Anal.
Calcd. for C25H25N3: C, 81.71; H, 6.86; N, 11.43. Found:
C, 81.69; H, 7.12; N, 11.30.
General procedure for the preparation of 2-(4-
substitutedphenyl)-1H-benzimidazole derivatives (3a–j)
5-Methoxy-2-(4-(1H-1,2,4-triazol-1-yl)phenyl)-1H-benzo
[d]imidazole (3f) Yellow solid; yield 86%; m.p. 148 °C;
IR (cm−1); 3111, 1633, 1513, 1453; 1H-NMR (DMSO, 400
MHz); 3.82 (3H; s; OCH3), 6.87–6.89 (1H; dd; 6-HAr, J:
8.8/2), 7.10 (1H; d; 4-HAr, J:2), 7.53 (1H; d; 7-HAr, J:8.8),
8.07 (2H; d; 3′-HAr and 5′-HAr, J:8.8), 8.29–8.31 (3H; m;
triazole, 2′-HAr and 6′-HAr), 9.40 (1H; s; triazole); 13C-
NMR (DMSO, 100 MHz); 55.5 (CH3), 112.3, 119.7, 127.6,
128.8, 137.4, 142.5, 149.5, 152.6, 156.2. ESI-MS (m/z);
314.39 [M + Na]+, 292.40 [M + H]+; Anal. Calcd. for
C16H13N5O: C, 65.97; H, 4.50; N, 24.04. Found: C, 65.71;
H, 4.56; N, 23.88.
The crude sodium hydroxy(4-substituted phenyl)methane-
sulfonate salts (0.01 mol) and o-phenylenediamine (0.01
mol) were refluxed in 30 mL of dimethyl formamide for
2–4 h. After monitoring by thin-layer chromatography, the
mixture was cooled to room temperature and poured into ice
water. The obtained 1H-benzimidazoles were filtered and
recrystallized from methanol/water.
2-(4-(1H-1,2,4-Triazol-1-yl)phenyl)-1H-benzo[d]imidazole
(3a) M.p. 105 °C (m.p. 102–104 °C) (Jadhav et al. 2009).
2-(4-(1H-Imidazol-1-yl)phenyl)-1H-benzo[d]imidazole
(3b) M.p. 215 °C. (Yu et al. 2013)
5-Methoxy-2-(4-(1H-imidazol-1-yl)phenyl)-1H-benzo[d]
imidazole (3g) Yellow solid; yield 82%; m.p. 173 °C; IR
(cm−1); 3357, 3108, 1636, 1611, 1515, 1479; 1H-NMR
(DMSO, 400 MHz); 3.84 (3H; s; OCH3), 6.96–6.99 (1H;
dd; 6-HAr, J: 8.8/2.4), 7.16 (1H; d; 4-HAr, J:2.4), 7.61 (1H;
d; 7-HAr, J:8.8), 7.74 (1H; s; imidazole), 8.04 (2H; d; 3′-
HAr and 5′-HAr, J:8.4), 8.27 (1H; s; imidazole), 8.45 (2H;
d; 2′-HAr and 6′-HAr, J:8.4), 9.45 (1H; s; imidazole); 13C-
NMR (DMSO, 100 MHz); 55.6 (CH3), 96.8, 113.6, 115.7,
119.6, 121.7, 123.9, 127.5, 128.3, 131.4, 134.9, 136.9,
148.3, 156.8. ESI-MS (m/z); 313.39 [M + Na]+, 291.40 [M
+ H]+; Anal. Calcd. for C17H14N4O: C, 70.33; H, 4.86; N,
19.30. Found: C, 70.09; H, 5.11; N, 19.43.
2-(4-(1H-Benzo[d]imidazol-1-yl)phenyl)-1H-benzo[d]imi-
dazole (3c) M.p.196 °C (m.p. 199–200 °C) (Alp et al.
2014).
2-(4-(4-Methyllpiperidin-1-yl)phenyl)-1H-benzo[d]imida-
zole (3d) Yellow solid; yield 76%; m.p. 183 °C; IR
1
(cm−1); 2924, 1604, 1506, 1461; H-NMR (DMSO, 400
MHz); 0.93 (3H; d; CH3, J: 6.8), 1.13–1.23 (2H; qd;
NCH2CH2ax
,
J: 12.6/3.6), 1.56–1.61 (1H; m;
NCH2CH2CH), 1.69 (2H; dbr; NCH2CH2eq, J: 12.8), 2.82
(2H; td; NCH2ax, J: 12.4/2.4), 3.92 (2H; dbr; NCH2eq, J:
12.8), 7.11 (2H; d; 3′-HAr and 5′-HAr, J: 9.2), 7.29–7.26
(2H; m; 4-HAr and 7-HAr), 7.61–7.59 (2H; m; 5-HAr and
6-HAr), 8.00 (2H; d; 2′-HAr and 6′-HAr, J: 8.8); 13C-NMR
(DMSO, 100 MHz); 21.7 (CH3), 30.3 (CH), 33.1 (CH2),
47.4 (CH2), 113.9, 114.3, 114.9, 123.0, 128.3, 136.2, 150.9,
152.5. ESI-MS (m/z); 314.23 [M + Na]+, 292.26 [M +
H]+; Anal. Calcd. for C19H21N3: C, 78.32; H, 7.26; N,
14.42. Found: C, 77.99; H, 7.32; N, 14.11.
5-Methoxy-2-(4-(1H-benzo[d]imidazol-1-yl)phenyl)-1H-
benzo[d]imidazole (3h) Yellow solid; yield 80%; m.p.
185 °C; IR (cm−1); 3064, 1639, 1606, 1494, 1454; 1H-
NMR (DMSO, 400 MHz); 3.86 (3H; s; OCH3), 6.98–7.01
(1H; dd; 6-HAr, J: 8.8/2.4), 7.19 (1H; d; 4-HAr, J:2.4),
7.35–7.43 (2H; m; 5-Hbenzimidazole and 6-Hbenzimida-
zole), 7.63 (1H; d; 7-HAr, J:8.8), 7.78 (1H; dd; 7-Hbenzi-
midazole, J: 6.8/1.6), 7.83 (1H; dd; 4-Hbenzimidazole, J:
6.8/1.6), 7.98 (2H; d; 3′-HAr and 5′-HAr, J: 8.4), 8.38 (2H;
d; 2′-HAr and 6′-HAr, J: 9.2), 8.78 (1H; s; 2-Hbenzimida-
zole); 13C-NMR (DMSO, 100 MHz); 55.6 (CH3), 94.3,
97.1, 110.9, 113.0, 115.8, 119.8, 122.8, 123.8, 123.9,
127.4, 128.1, 132.6, 137.3, 137.6, 143.0, 143.3, 149.1,
156.6. ESI-MS (m/z); 363.39 [M + Na]+, 341.41 [M +
H]+; Anal. Calcd. for C21H16N4O: C, 74.10; H, 4.74; N,
16.46. Found: C, 73.95; H, 5.02; N, 16.33.
2-(4-(4-Benzylpiperidin-1-yl)phenyl)-1H-benzo[d]imida-
zole (3e) Yellow solid; yield 65%; m.p. 187 °C; IR
1
(cm−1); 2923, 1607, 1497, 1439; H-NMR (DMSO, 400
MHz); 1.22–1.32 (2H; qd; NCH2CH2ax, J: 12/3.6), 1.65
(2H; brd; NCH2CH2eq, J: 14), 1.70–1.75 (1H; m;
NCH2CH2CH), 2.54 (2H; d; CH2Ph, J: 7.2), 2.72 (2H; td;
NCH2ax, J: 11.4/1.2), 3.84 (2H; brd; NCH2eq, J: 12.8), 7.04
(2H; d; 3′-HAr and 5′-HAr, J: 9.2), 7.12–7.15 (2H; m; 4-
HAr and 7-HAr), 7.17–7.20 (2H; m; Ar), 7.28–7.31 (3H; m;
Ar), 7.50–7.53 (2H; m; 5-HAr and 6-HAr), 7.98 (2H; d; 2′-
HAr and 6′-HAr, J: 9.2); 13C-NMR (DMSO, 100 MHz);
31.1 (CH2), 37.3 (CH), 42.2 (CH2), 47.7 (CH2), 114.7,
5-Methoxy-2-(4-(4-methyllpiperidin-1-yl)phenyl)-1H-
benzo[d]imidazole (3i) Yellow solid; yield 77%; m.p.
156 °C; IR (cm−1); 2923, 1606, 1504, 1454; 1H-NMR
(DMSO, 400 MHz); 0.93 (3H; d; CH3, J: 6.8), 1.18–1.25

Med Chem Res
(2H; qd; NCH2CH2ax, J: 12.4/3.6), 1.56–1.58 (1H; m;
NCH2CH2CH), 1.69 (2H; brd; NCH2CH2eq, J: 12.4), 2.76
(2H; td; NCH2ax, J: 12.4/2.4), 3.79 (3H; s; OCH3), 3.84
(2H; brd; NCH2eq, J: 12.8), 6.78–6.81 (1H; dd; 6-HAr, J:
8.4/2.4), 7.02–7.06 (3H; m; 3′-HAr, 5′-HAr and 4-HAr),
7.41 (1H; d; 7-HAr, J:8.8), 7.94 (2H; d; 2′-HAr and 6′-HAr,
J: 8.8); 13C-NMR (DMSO, 100 MHz); 21.7 (CH3), 30.3
(CH), 33.2 (CH2), 47.8 (CH2), 55.4 (CH3), 110.9, 114.7,
118.5, 127.4, 151.4, 151.8, 155.6. ESI-MS (m/z); 344.47
[M + Na]+, 322.50 [M + H]+; Anal. Calcd. for
C20H23N3O: C, 74.74; H, 7.21; N, 13.07. Found: C, 75.02;
H, 7.18; N, 13.41.
Amyloid peptide preparation
Aβ1–40 protein fragment (A1075, Sigma—Aldrich) was
dissolved in phosphate-buffered saline (PBS), pH 7.4, at a
concentration of 50 µM and incubated at 37 °C for seven
days to induce peptide aggregation.
Inhibition of Aβ1–40 aggregation
Aβ1–40-destabilizing effects were determined for the
compounds with IC50 values lower than 100 µM for both
ChEs. Aβ1–40 (10 µL) was added to the assay medium
containing 0.01 M NaCl in 0.05 M potassium phosphate
buffer, pH 7.4, and incubated at 37 °C for 48 h in the
absence and presence of the inhibitors (100 μM). The
incubated Aβ1–40 (100 µL) was mixed with 50 μL of thio-
flavin T (ThT; 200 μM) in 50 mM glycine—NaOH buffer
(pH 8.5). Inhibition of Aβ aggregation was evaluated by the
decrease of ThT fluorescence intensity at the excitation
wavelength of 448 nm and emission wavelength of 490 nm
using an RF 5301 PC spectrofluorophotometer. Rifampicin
and donepezil (100 μM) were tested as reference
compounds.
5-Methoxy-2-(4-(4-benzylpiperidin-1-yl)phenyl)-1H-benzo
[d]imidazole (3j) Yellow solid; yield 79%; m.p. 143 °C;
IR (cm−1); 2928, 1635, 1605, 1506, 1475; 1H-NMR
(DMSO, 400 MHz); 1.21–1.30 (2H; qd; NCH2CH2ax, J:
12/3.2), 1.66 (2H; brd; NCH2CH2eq, J: 12.8), 1.75–1.80
(1H; m; NCH2CH2CH), 2.54 (2H; d; CH2Ph, J: 7.2), 2.80
(2H; t; NCH2ax, J: 12), 3.83 (3H; s; OCH3), 3.92 (2H; brd;
NCH2eq, J: 12.8), 6.92–6.95 (1H; dd; 6-HAr, J: 8.8/2.4),
7.07–7.09 (3H; m; Ar and 4-HAr), 7.18–7.20 (3H; m; Ar
and 3′-HAr and 5′-HAr), 7.27–7.31 (2H; m; Ar), 7.51 (1H;
d; 7-HAr, J:8.8), 8.01 (2H; d; 2′-HAr and 6′-HAr, J: 9.2);
13C-NMR (DMSO, 100 MHz); 31.5 (CH2), 37.8 (CH), 42.6
(CH2), 47.8 (CH2), 56.1 (CH3), 97.2, 113.1, 114.8, 115.1,
126.3, 128.6, 128.7, 129.5, 136.5, 140.6, 150.6, 153.0,
157.0. ESI-MS (m/z); 420.50 [M + Na]+, 398.51 [M +
H]+; Anal. Calcd. for C26H27N3O: C, 78.56; H, 6.85; N,
10.57. Found: C, 78.32; H, 7.10; N, 10.50.
Cell culture
Fetal bovine serum (FBS; S181G-500) and 200 mM L-
glutamine (17-605E) for cell culture were obtained from
Biowest and Lonza, respectively. HyClone 0.25% 1 ×
trypsin (SV30031.01) and HyClone PBS (SH30256.01)
were purchased from Thermo Scientific. DMSO (67-68-5)
was obtained from Merck. SH-SY5Y human neuroblastoma
cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% FBS, 1% L-glutamine,
and 1% antibiotic mix. Cells were maintained at 37 °C in a
humidified atmosphere containing 5% CO2. Non-adherent
cells were removed, and adherent cells were passaged in a
fresh medium every three days. Neuronal differentiation
was induced by the treatment with 10 µM retinoic acid for
10 days (Dwane et al. 2013).
Biological assays
Cholinesterase inhibitory activity
Acetylcholinesterase human recombinant (C1682), butyr-
ylcholinesterase from equine serum (C1057), acet-
ylthiocholine iodide (A5751), and S-butyrylthiocholine
iodide (B3253) were obtained from Sigma—Aldrich, and
5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was from Cal-
biochem (Los Angeles, CA, USA). IC50 values of the novel
benzimidazole derivatives were determined by the Ellman’s
assay (Ellman et al. 1961). Reactions were initiated by the
addition of an enzyme into a medium containing the sub-
strate (0.05–0.4 mM) and 0.125 mM DTNB in 100 mM 3-
(N-morpholino)propanesulfonic acid buffer, pH 8.0, at
25 °C and monitored spectrophotometrically at 412 nm in a
UV-visible 1700 Shimadzu PC spectrophotometer. Dose-
response curves were plotted using the GraphPad Prism
5 software. An enzyme kinetic assay was performed for
compound 3g at different butyrylthiocholine (BTC) con-
centrations (0.1–0.5 mM). Donepezil—HCl (Sigma) was
tested as a reference compound.
Determination of cell viability by MTT assay
Cell viability was determined using a thiazolyl blue tetra-
zolium bromide [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide] (MTT) assay (Mosmann 1983).
Differentiated SH-SY5Y cells were seeded into 96-well
plates at 5000 cells per well and cultured in DMEM sup-
plemented with 10% FBS at 37 °C, 5% CO2. After the
treatment with a 10 µM concentration of each novel com-
pound, the plate was incubated for 48 h. After the incuba-
tion, 10 µL of the MTT reagent (5 mg/mL) was added to
each well. The formazan crystals formed were dissolved by

Med Chem Res
the addition of 100 µL of DMSO. Absorbance values were
measured at 690 and 570 nm using a Biotek Power Wave
XS microplate reader. Cell viability was determined as
below:
Viability ¼
Average OD of treated wells ꢀ Average OD of blank wells
Average OD of control wells ꢀ Average OD of blank wells
ꢁ 100
Scheme 1 Synthesis of the target compounds
Cytotoxicity of compounds
Differentiated SH-SY5Y cells (5000 cells/well) were treated
with 10 µM compounds for 48 h, and cytotoxic effects of
the compounds were evaluated by the MTT reduction assay.
Results and discussion
Chemistry
H2O2-induced and Aβ1–40-induced cytotoxicity
In this study, ten 2-(4-substituted phenyl)-1H-benzimida-
zole derivatives were studied, of which three (3a–c) had
been described in the literature (Jadhav et al. 2009; Yu et al.
2013; Alp et al. 2014). Compounds 3a–j were obtained
by following the synthetic route outlined in Scheme 1.
First, benzaldehydes with aliphatic/aromatic amine moieties
(1a–e) were synthesized with 45–57 % yield in accordance
with a method described in the literature (Meciarova et al.
2003). The intermediates, sodium hydroxy(4-substituted
phenyl)methanesulfonate salts (2a–e), were obtained with
85–95 % yield by reacting the mentioned benzaldehydes with
sodium bisulfite. The target compounds (3a–j) were gained
with 65–86 % yield by treating sodium hydroxy(4-sub-
stituted phenyl)methanesulfonate salts (2a–e) with appro-
priate o-phenylenediamines (Alpan et al. 2007). The spectral
For the evaluation of neuroprotective effects of the com-
pounds against the H2O2-induced and Aβ1–40-induced cell
death, mitochondrial reduction (MTT) experiments were
performed. Differentiated SH-SY5Y cells (5000 cells/well)
were treated with 10 µM compounds for 3 h prior to 250 µM
H2O2 or 10 µM Aβ1–40 treatment and then incubated for an
additional 24 h. The MTT reduction assay was performed to
evaluate cell viability (Kim et al. 2000; Loo et al. 1993).
Molecular modeling studies
Docking studies were performed with the Molecular
Operating Environment (MOE) version 2015.1001 software
available from Chemical Computing Group, Inc. (Montreal,
Canada, http://www.chemcomp.com) using the crystal
structures of human AChE and BChE (Protein Data Bank
IDs: 4EY7 (Cheung et al. 2012) and 1P0I (Nicolet et al.
2003), respectively).
After all water and non-protein atoms were removed, the
errors in the proteins were corrected by the Structure Pre-
paration process in MOE. Active sites of the enzymes were
generated using the Site Finder application. Compound 3g
was constructed using the MOE builder tool, and the energy
was minimized using the Merck molecular force field
(MMFF94x, gradient: 0.05 kcal mol−1 Å−1). Docking stu-
dies for compound 3g with 4EY7 and 1P0I were performed
using the default Triangle Matcher placement method.
GBVI/WSA dG, a force field-based scoring function, which
estimates the free energy of binding of the ligand from a
given pose, was used to rank the final poses. Then the poses
with the lowest S score were selected for the enzymes.
1
methods (IR, H/13C-NMR, ESI–MS), and elemental analy-
sis were used to elucidate the structures of the compounds
that had not been described in the literature (3d–j).
In the IR spectra of (3d–j), the bands were seen around
1490–1510 cm−1 due to –C=N– stretching. It was very
difficult to distinguish the C–H stretching vibrations
(around 3300–3100 cm−1) from the broad –NH stretching
frequencies (around 3300–2800 cm−1).
1
In the H NMR spectra, for compounds 3d and 3e, the
protons on the benzimidazole ring appeared as two multiple
peaks at around 7.15 (4-H and 7-H) and 7.55 (5-H and 6-H)
p.p.m. For compounds 3f–j, the signals belonging to 6-H, 4-
H, 7-H and methoxy protons on the benzimidazole ring
were observed at around 6.90 (dd, J = 8.8, 2.4 Hz), 7.15 (d,
J = 2.4 Hz), 7.55 (d, J = 8.8 Hz) and 3.82 p.p.m., respec-
tively. In the 13C-NMR spectra, the peaks between
148–151 p.p.m. were assigned to the carbon atoms at the 2
position of benzimidazole ring of the target compounds.
The signals appeared at around 55 and 156 p.p.m. in the

Med Chem Res
Table 1 The physicochemical parameters and in vitro AChE/BChE enzyme inhibition data for the compounds
Compounds
R1
R2
H
PSAa
59.39
LogPa
MW
H-Bond
Don/Accep.
IC50 (µM)b SE BChE AChE
Selectivity
for BChEc
3a
1.80
261.28
2/4
>100
>100
3b
3c
H
H
46.50
46.50
3.06
4.71
260.30
310.36
2/3
2/3
41.35 1.14
74.21 1.20
67.23 1.11
302 1.67
1.6
4
3d
3e
3f
H
31.92
31.92
68.62
4.52
5.89
1.79
291.40
367.50
291.31
2/2
2/2
2/5
39.56 1.18
>100
>5000
>126
H
>100
OCH3
95.44 1.23
471.40 1.24
4.9
3g
3h
OCH3
OCH3
55.73
55.73
3.06
4.70
290.32
340.39
2/4
2/4
13.60 1.11
14.45 1.31
485.70 1.03
1051 1.10
35.7
72.7
3i
3j
OCH3
OCH3
41.15
41.15
4.52
5.89
321.42
397.52
2/3
2/3
15.15 1.25
51.20 1.15
4.94 1.04
736.10 1.19
>1000
48.5
>19
Donepezil
6.16 1.03d
a
PSA and LogP calculated using MOE 2015.1001 (Molecular Operating Environment, Chemical Computing Group)
IC50 values of compounds represent the concentration that caused 50% enzyme activity loss
Selectivity for BChE is defined as IC50(AChE)/IC50(BChE)
b
c
d
IC50 (nM) SE
spectra of compound 3f–j, showed the presence of the
Cholinesterase inhibitory activity
methoxy group at the 5 position of benzimidazole ring.
Furthermore, the structures of all the target compounds were
confirmed by the peaks belonging to [M + Na]+ and [M +
H]+ seen in the ESI mass spectra.
Inhibitory activities of 3a–j against human recombinant
AChE and equine BChE were measured according to the
Ellman method. For comparison purposes, donepezil was

Med Chem Res
Table 2 Inhibition of self-mediated Aβ1–40 aggregation and
cytotoxicity of the compounds
Compounds
Aβ1–40 aggregation inhibition
(%) SEM
Cell viability (%)
3a
nd
nd
3b
>100
146
154
135
nd
3c
>100
3d
67.78 0.16
nd
Fig. 2 Dixon plot for non competitive inhibition of BChE by com-
pound 3g, BTC, r2 values for the trendlines > 0.95
3e
3f
>100
172
229
185
211
169
100
106
105
3g
>100
used as a reference compound. The IC50 values of 3a–j and
selectivity indices (SIs) of some compounds for BChE over
AChE are summarized in Table 1.
Although, most of the target compounds showed
remarkable selective activities for BChE over AChE, none
of them exhibited a higher affinity for both AChE and
BChE than that of donepezil (IC50 = 6.16 nM for human
AChE and 4.94 μM for equine BChE), except that 3b
showed balanced inhibition of both ChEs.
3h
92.35 0.90
75.82 0.99
68.39 0.15
100
3i
3j
Controla
Rifampicin
Donepezil
a
48.40 0.30
83.38 0.04
Control of each assay was individual as non treated.
nd not determined.
Among the target compounds, 3d and 3g–j were found
as the most potent (IC50 = 13.60–39.56 µM) and selective
inhibitors of BChE (SI ≥ 35.7) in this series. Surprisingly,
the triazole moiety as a bioisostere of the imidazole moiety
decreased the BChE inhibitory activity, as observed for 3a
standard compound, rifampicin (48.40%). Furthermore, it
was determined that 3d, 3i, and 3j (67.78, 75.82, and
68.39%, respectively) showed more potent activity on Aβ
aggregation than donepezil (83.38%).
(IC50 > 100 µM) vs. 3b (IC50 = 41.35 µM) and 3f (IC50
=
95.44 µM) vs. 3g (IC50 = 13.60 µM). On the other hand,
compounds with the 4-methyl piperidine moiety (3d and 3i)
showed interesting potency and selectivity profiles. This
significant potency was lost by the replacement of the
methyl group with the benzyl group, as observed for 3d
Cytotoxicity
No evidence of cytotoxicity to SH-SY5Y cells exposed to a
10 μM concentration of the test compounds was observed
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) assay. The percentage of growth
of the cells is reported in Table 2.
(IC50 = 39.56 µM) vs. 3e (IC50 > 100 µM) and 3i (IC50
=
15.15 µM) vs. 3j (IC50 = 51.20 µM). Moreover, introduc-
tion of a methoxy group to the benzimidazole ring sig-
nificantly increased inhibitory activity and selectivity of the
compounds against BChE, except 3d and its counterpart 3i.
In the BChE inhibition studies, 5-methoxy-2-(4-(1H-
imidazol-1-yl)phenyl)-1H-benzo[d]imidazole 3g was found
to be the most active (IC50 = 13.60 µM). For this reason, 3g
was selected for further kinetic analysis. The Dixon plot of
3g for BChE indicated the inhibition type as non competi-
tive (Fig. 2) with Ki values 20.79 3.22 µM (r2 = 0.9751).
Neuroprotection
The MTT assay was performed to evaluate the neuropro-
tective effects of the compounds against H2O2-induced and
Aβ1–40-induced cell death in SH-SY5Y cells by using
rifampicin and donepezil as reference compounds.
When the results of H2O2-induced cytotoxicity were
examined, it was seen that protective effects of the tested
compounds were significantly higher than that of donepezil.
Among them, 3b and 3d displayed more protective effect
against H2O2-induced cytotoxicity at a concentration of
250 µM than rifampicin and donepezil (Fig. 3a).
Aβ1–40-induced cytotoxicity results showed that 3c and
3d have the highest protective effect against Aβ1–40-induced
cytotoxicity at a concentration of 10 µM (Fig. 3b). Fur-
thermore, the protective effects of the tested compounds
were higher than those of rifampicin (except 3i and 3j) and
donepezil.
Inhibition of self-mediated Aβ1–40 aggregation
The compounds with BChE inhibitory activity (IC50 < 100
µM) were further examined for their Aβ-antiaggregating
activity and neuroprotective properties. The compounds
were tested for their ability to inhibit self-mediated Aβ1–40
aggregation using the ThT fluorescence assay. Rifampicin
and donepezil were used as reference compounds. The
results are summarized in Table 2. Based on the results,
none of the compounds was found as effective as the

Med Chem Res
Fig. 3 Neuroprotective effect of
compounds on H2O2-induced
and Aβ1-40-induced cytotoxicity
in SH-SY5Y cells. a Cells were
treated with 250 µM H2O2 in the
presence or absence of
compounds (10 µM) at 24 hours.
b Cells were treated with 10 µM
Aβ1-40 in the presence or
absence of compounds (10 µM)
at 24 hours. The values are the
mean values SEM from at
least three independent
experiments. * p < 0.05
Fig. 4 a Representation of the binding mode of 3g (purple) in the
BChE active site. The catalytic triad (Ser198, His438, and Glu325) is
shown in blue, the anionic site (Trp82, Tyr128, and Phe329) is shown
in green, and the peripheral anionic site (Tyr332 and Asp70) is shown
in yellow. b Representation of the binding mode of 3g (purple) in the
AChE active site. The catalytic triad (Ser203, His447, and Glu202) is
shown in blue, the anionic site (Trp86 and Tyr337) is shown in green,
and the peripheral anionic site (Trp286 and Tyr341) is shown in yellow
(color figure online)
Molecular modeling
(one of the catalytic triad residues). Besides, a CH—pi
interaction, which can be classified as a weak hydrogen
bond (Brandl et al. 2001), occurred between Phe329 and the
benzimidazole ring in the anionic site. The determined
orientation and interactions might be responsible for the
inhibitory activity of 3g.
From the docking studies with AChE, it was seen that 3g
did not pass the gorge to reach the catalytic or anionic site.
In addition, there was no appropriate interaction between
AChE and 3g (Fig. 4b). It can be inferred that these com-
pounds cannot inhibit AChE because AChE has a narrow
gorge, and the compounds do not have the conformational
flexibility to pass this narrow gorge.
To predict the drug-likeness of the designed compounds,
the Lipinski’s rule of five was calculated. All the com-
pounds conformed to the rule of five, except 3e and 3j
(Table 1). The passage through the blood—brain barrier
(BBB) is a major issue for central nervous system-acting
drugs. It has been hypothesized that the polar surface area
(PSA) of a compound that passes the BBB should be
typically less than 70 Å2 (Muchmore et al. 2010; Pajouhesh
and Lenz 2005). The calculated PSA values of the designed
compounds were found to be between 31 to 68 Å2
(Table 1). This finding may suggest that, according to this
hypothesis, the compounds are able to pass the BBB.
Compared to AChE, BChE, in which two phenylalanine
residues are replaced with smaller amino acids, valine and
leucine, allows larger molecules to get in. The docking
studies revealed that the most active compound, 3g, is
accommodated in the catalytic site of the BChE active
pocket (Fig. 4a). A hydrogen bond is formed between the
methoxy moiety of 3g and the hydroxyl group of Ser198
Conclusion
In conclusion, a series of 2-phenylbenzimidazoles have
been designed, synthesized, and evaluated for their ChE
inhibitory activites in this study. The target compounds (3a–j)
were obtained by cyclization of o-phenylenediamines with

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sodium hydroxy(4-substituted phenyl)methanesulfonate
salts. In vitro studies indicated that the most of the target
compounds showed remarkable inhibitory activity against
BChE (IC50: 13.60–95.44 µM). Among them, 3d and 3g–i
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significant neuroprotection against H2O2-induced and
Aβ1–40-induced cell death. Collectively, compound 3d
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=
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