1H-benzimidazole derivatives as butyrylcholinesterase inhibitors: synthesis and molecular modeling studies

Article abstract of DOI:10.1007/s00044-016-1648-1

A series of N-{2-[2-(1H-benzimidazole-2-yl)phenoxy]ethyl} substituted amine derivatives were synthesized and tested for their cholinesterase inhibitor activity. Acetylcholinesterase and butyrylcholinesterase inhibitor activities were evaluated in vitro by using Ellman’s method. According to the activity results, all of the compounds displayed moderate acetylcholinesterase inhibitory activity and most of the compounds displayed remarkable butyrylcholinesterase inhibitory activity. Compound 3d was the most active compound in the series and also a selective butyrylcholinesterase inhibitor. Molecular docking studies and molecular dynamic simulations were also carried out.

Full text of DOI:10.1007/s00044-016-1648-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1648-1
ORIGINAL RESEARCH
1H-benzimidazole derivatives as butyrylcholinesterase inhibitors:
synthesis and molecular modeling studies
Gunes Coban1 Luca Carlino2 Ayse Hande Tarikogullari1 Sülünay Parlar1
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Görkem Sarıkaya1 Vildan Alptüzün1 Ayşe Selcen Alpan1 Hasan Semih Güneş1
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Erçin Erciyas1
Received: 15 July 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
Abstract A series of N-{2-[2-(1H-benzimidazole-2-yl)
phenoxy]ethyl} substituted amine derivatives were synthe-
sized and tested for their cholinesterase inhibitor activity.
Acetylcholinesterase and butyrylcholinesterase inhibitor
activities were evaluated in vitro by using Ellman’s method.
According to the activity results, all of the compounds
displayed moderate acetylcholinesterase inhibitory activity
and most of the compounds displayed remarkable butyr-
ylcholinesterase inhibitory activity. Compound 3d was the
most active compound in the series and also a selective
butyrylcholinesterase inhibitor. Molecular docking studies
and molecular dynamic simulations were also carried out.
BTC
BuChE
butyrylthiocholine iodide
butyrylcholinesterase
BuChEI butyrylcholinesterase inhibitor
CAS
ChE
ChEI
DMF
DMSO
DTNB
HMBC
HSQC
MD
catalytic active site
cholinesterase
cholinesterase inhibitor
dimethyl formamide
dimethylsulfoxide
5,5-dithiobis(2-nitrobenzoic acid)
Heteronuclear Multiple Bond Correlation
Heteronuclear Single Quantum Correlation
molecular dynamics
RMSD
TLC
VMD
root main square deviation
thin-layer chromatography
visual molecular dynamics
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Keywords Alzheimer’s disease 1H-Benzimidazole
Acetylcholinesterase inhibitor Butyrylcholinesterase
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inhibitor Ellman’s method Molecular modeling
Abbreviations
AD
ACh
Alzheimer’s disease
acetylcholine
Introduction
AChE
AChEI
ATC
acetylcholinesterase
acetylcholinesterase inhibitor
acetylthiocholine iodide
Alzheimer’s disease (AD), the most common cause of
dementia in elder, is a progressive neurodegenerative
disorder characterized by loss of memory and cognition.
5.2 million people in the United States are suffering from
AD and the number of patients increases every year,
worldwide (Han and Mook-Jung, 2014; Thies and Bleiler,
2013). This disease is characterized by formation of cortical
amyloid plaques and neurofibrillary tangles and also
reduced cholinergic neurotransmission in the brain
(Morrison and Hof, 1997). The potential involvement of
acetylcholinesterase (AChE) in the formation and deposi-
tion of extracellular plaques in the human brain of AD
patients has been revealed. So far, there is no cure for AD.
The main strategy for AD is based on the cholinergic
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1648-1) contains supplementary material,
which is available to authorized users.
* Ayşe Selcen Alpan
selcen.alpan@ege.edu.tr
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ege University, Bornova 35100 Izmir, Turkey
2
Department of Pharmaceutical Chemistry, Institute of Pharmacy,
Martin-Luther University of Halle-Wittenberg, Halle/Saale 06120,
Germany

Med Chem Res
hypothesis, which relies on the repair of cognitive and
memory deteriorations by elevating the reduced acetylcho-
line (ACh) (Pan et al., 2014). For this purpose, cholines-
terase inhibitors (ChEI) have been used to treat the
cognitive and functional symptoms of AD (Weinstock,
1999).
AChE and butyrylcholinesterase (BuChE) are the two
types of cholinesterase enzyme. They both play important
roles in the regulation of ACh levels and suggested to have
a role in the development and progression of AD (Greig
et al., 2001). In AD, AChE activity decreases, whereas
BuChE activity increases. It is reported in the literature that
the elevated BuChE may act as a compensatory mechanism
for ACh hydrolysis and ACh regulation may become
increasingly dependent on BuChE as AD progresses (Gia-
cobini, 2004; Greig et al., 2002). Due to these findings, it
can easily be proposed that BuChE inhibition is also sig-
nificant for AD treatment.
The difference between BuChE and AChE’s active gorge
can be demonstrated from the considerably more space
present in the gorge cavity of BuChE than that present in
AChE. Some of the aromatic residues in AChE are replaced
by smaller aliphatic or polar amino acids inside the gorge of
BuChE (Galdeano et al., 2010). Compound’s selectivity
might be due to the fact that the two ChEs differ by the
presence of extent of subdomains within the gorge domains
(mid-gorge aromatic recognition site), peripheral anionic
site, and acyl-binding pocket, even if both ChEs show a
similar overall structure (65 % amino sequence homology)
and both active sites (CAS) are located inside of a ~20 Å
deep gorge cavity (Brus et al., 2014; Nicolet et al., 2003;
Cheung et al., 2012).
In our previous study, 2-phenylsubstituted-1H-
benzimidazole was chosen as a scaffold and basic cyclic,
acyclic amino residues were incorporated to this scaffold
with ethoxy chain. 5-methyl and 5-chloro benzimidazole
derivatives were also synthesized as well as the non-
substituted analogs (Alpan et al., 2013). The results indi-
cated that most of the compounds displayed moderate to
good AChE inhibition and some of them showed weak to
moderate BuChE inhibition. Thus, these activity results
revealed selective AChE inhibitors in some of the
compounds.
Material and methods
Chemistry
Melting points were determined with Electrothermal
IA9100 melting point apparatus (Electrothermal, Essex,
UK). The infrared (IR) spectra of the compounds were
monitored as potassium bromide pellets on Jasco FT/IR-430
(Jasco, Tokyo, Japan) and Perkin-Elmer Spectrum 100 FT-
IR spectrometers (PerkinElmer, Waltham, MA, USA). The
nuclear magnetic resonance (NMR) spectra (400 MHz for
1H and 13C) were recorded on AS400 Mercury Plus NMR
Varian (Varian Inc., Palo Alto, CA, USA) using CDCl3.
Chemical shifts were measured in parts per million (δ). The
J values were given in Hz. Mass spectra were taken on a
Thermo Quantum Access Max connected with TSQ quan-
tum high performance liquid chromatography (Thermo
Fisher Scientific, San Jose, CA, USA), using ESI (+)
method, with C-18 column. Elemental analyses were per-
formed by Leco TruSpec Micro CHNS (Leco, St. Joseph,
MI, USA) and were within 0.4 % of the theoretical values.
Compounds 1a, 1c, 2b, and 3e are reported with fractional
mol of water, compounds 2a, 3a, 3b, and 3c are reported
with fractional mol of ethyl acetate, and compound 3d is
reported with fractional mol of methanol. Analytical thin-
layer chromatography (TLC) was run on Merck silica gel
plates (Kieselgel 60F254) with detection by ultraviolet (UV)
light; column chromatography was performed using Merck
silica gel 60 (63–200 mm diameter) and compounds 1b, 1c,
1d, 1e, 2b, 2c, 2d, and 2e were eluted with benzene:ethyl
acetate:methanol:ammonia (10:2.5:0.5:0.002) and the rest
of the synthesized compounds were eluted with toluene:
ethyl acetate:methanol:ammonia (10:2.5:0.5:0.002). All
starting materials and reagents were of high-grade com-
mercial products.
General procedure for the synthesis of the benzimidazole
derivatives
Compounds 1b, 1c, 1d, 1e, 2b, 2c, 2d, and 2e are reported
derivatives, but corresponding scientific reference data were
expanded with elemental analysis (supplementary data)
(Coban et al., 2009).
In this study, 2-phenylsubstituted-1H-benzimidazole
scaffold was conserved, while the position of the ethoxy
side chain was moved from para to ortho to obtain more
spherical molecules in order to fit in the active gorge of
BuChE and improve butyrylcholinesterase inhibitor
(BuChEI) activity. Docking studies and molecular dynam-
ics (MD) simulations were carried out to explain the ChEI
activity in terms of binding interactions. The reason of the
fluctuation between the BuChEI activity results were pro-
posed and discussed by molecular modeling techniques.
The preparation of the substituted benzimidazoles is
outlined in Scheme 1 (Hein et al., 1957; Nagai et al., 1973).
2-(1H-benzimidazole-2-yl)phenol, 2-(5-methyl-1H-benzi-
midazole-2-yl)phenol, and 2-(5-chloro-1H-benzimidazole-
2-yl)phenol were synthesized by reacting o-phenylenedia-
mine and sodium hydroxy(2-hydroxyphenyl) methane sul-
fonate salt as described (Coban et al., 2009). After reacting
0.005 mol of 2-(1H-benzimidazole-2-yl)phenol derivatives
and 0.01 mol of appropriate N-(2-chloroethyl) substituted
amine with 0.015 mol of sodium hydroxide in 10 mL

Med Chem Res
Scheme 1 The synthesis path-
way of compounds 1a–e, 2a–e,
and 3a–e
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 purified by column chro-
matography [benzene:ethyl acetate:methanol:ammonia
(10:2.5:0.5:0.002)] and then crystallized with an appropriate
solvent.
NMR (400 MHz, CDCl3) δ 2.47 (3H, s, H-1″′ or H-2″′),
2.48 (3H, s, H-1″′ or H-2″′), 2.85 (2H, t, J = 4.8 Hz, H-2″),
4.30 (2H, t, J = 4.8 Hz, H-1″), 7.01 (1H, dd, J = 8.0/0.8 Hz,
H-3′), 7.14 (1H, td, J = 7.6/0.8 Hz, H-5′), 7.21 (1H, d, J =
8.0 Hz, H-6), 7.36 (1H, brs, H-7), 7.40 (1H, t, J = 7.8/0.8
Hz, H-4′), 7.69–7.77 (1H, m, H-4), 8.47 (1H, d, J = 8.0/0.8
Hz, H-6′) 13.07 (1H, s, N-H) ppm. 13C NMR (100 MHz,
CDCl3) δ 44.6 (C-1″′), 57.9 (C-2″), 65.9 (C-1″), 114.2 (C-
3′), 118.8 (C-1′), 122.3 (C-5′), 130.3 (C-6′), 131.1 (C-4′),
156.4 (C-2′) ppm. Anal. Calcd. for: C17H18ClN3O
0.05C4H8O2: C, 64.52; H, 5.79; N, 13.12. Found: C, 64.73;
H, 5.94; N, 12.88.
Structural data of the benzimidazole derivatives
2-[2-(1H-benzo[d]imidazol-2-yl)phenoxy]-N,N-dimethy-
lethanamine (1a) White crystalline (MetOH–H2O). Recry-
stallized from methanol-water (1:1). Yield: 8.52 %; mp
90 °C; IR νmaks (KBr) cm−1: 3066, 2942, 2774, 1621, 1582,
1468, 1439, 1277, 1245, 1123, 1030, 737. LS MS/MS
(ESI+) m/z 282 [M+H, 100]. 1H NMR (400 MHz, CDCl3) δ
2.48 (6H, s, H-1″′, H-2″′), 2.84 (2H, t, J = 5.4 Hz, H-2″),
4.28 (2H, t, J = 5.2 Hz, H-1″), 7.03 (1H, d, J = 8.4 Hz, H-
3′), 7.15 (1H, td, J = 7.8/0.8 Hz, H-5′), 7.22–7.24 (2H, m,
H-5, H-6), 7.35–7.40 (2H, m, H-4′, H-7), 7.81 (1H, brs, H-
4), 8.53 (1H, dd, J = 8.0/2.0 Hz, H-6′) ppm. 13C NMR (100
MHz, CDCl3) δ 44.6 (C-1″′), 57.9 (C-2″), 65.9 (C-1″),
114.2 (C-3′), 120.1 (C-1′), 122.0 (C-5′), 122.2 (C-5, C-6),
130.2 (C-6′), 130.7 (C-7a), 150.1 (C-2), 156.3 (C-2′) ppm.
Anal. Calcd. for: C17H19N3O 0.1H2O: C, 72.11; H, 6.83; N,
14.84. Found: C, 72.13; H, 6.62; N, 14.66.
N,N-dimethyl-2-{2-[5-methyl-1H-benzo[d]imidazol-2-yl]
phenoxy}ethanamine (3a) Oil. Yield: 13.74 %. IR νmaks
(KBr) cm−1: 3171, 2938, 2774, 1629, 1584, 1467, 1273,
1250, 1124, 1040, 802, 762, 749. LS MS/MS (ESI+) m/z
1
296 [M+H, 100]. H NMR (400 MHz, CDCl3) δ 2.48 (6H,
s, H-1″′, H-2″′), 2.49 (3H, s, Ar–CH3), 2.85 (2H, t, J = 5.2
Hz, H-2″), 4.29 (2H, t, J = 5.4 Hz, H-1″), 7.04 (1H, d, J =
7.2 Hz, H-3′), 7.06 (1H, d, J = 8.2 Hz, H-6), 7.14 (1H, td,
J = 7.6/1.2 Hz, H-5′), 7.35–7.42 (2H, m, H-4′, H-7), 7.52
(1H, brs, H-4), 8.52 (1H, dd, J = 8.0/2.0 Hz, H-6′) ppm. 13
C
NMR (100 MHz, CDCl3) δ 21.8 (Ar–CH3), 44.6 (C-1″′),
57.9 (C-2″), 65.9 (C-1″), 114.1 (C-3′), 120.2 (C-1′), 122.2
(C-5′), 123.6 (C-6), 130.1 (C-6′), 130.5 (C-4′), 149.8 (C-2),
156.2 (C-2′) ppm. Anal. Calcd. for: C18H21N3O
0.3C4H8O2: C, 71.66; H, 7.33; N, 13.06. Found: C, 71.91;
H, 7.40; N, 12.90.
2-[2-(5-chloro-1H-benzo[d]imidazol-2-yl)phenoxy]-N,N-
dimethylethanamine (2a) White crystalline (EtOAc). Re-
crystallized from ethyl acetate. Yield: 12.84 %; mp 124 °C.
IR νmaks (KBr) cm−1: 3200, 2941, 2822, 2773, 1602, 1580,
1462, 1280, 1245, 1121, 1038, 749. LS MS/MS (ESI+)
N,N-diethyl-2-[2-(5-methyl-1H-benzo[d]imidazol-2-yl)phe-
noxy]ethanamine (3b) Oil. Yield: 17.32 %. IR νmaks (KBr)
cm−1: 3188, 2970, 2925, 2853, 1627, 1602, 1585, 1466,
1
316 [M+H, 100], 317 [M+1+H, 21], 318 [M+2+H, 25]. H

Med Chem Res
1272, 1242, 1040, 806, 751. LS MS/MS (ESI+) m/z 324[M
+H, 100]. H NMR (400 MHz, CDCl3) δ 1.14 (6H, t, J =
Recrystallized from methanol-water (1:1). Yield: 12.48 %;
mp 142 °C. IR νmaks (KBr) cm−1: 3355, 2953, 2855, 1625,
1583, 1454, 1275, 1245, 1117, 1036, 767, 750. LS MS/MS
(ESI+) m/z 338 [M+H, 100]. 1H NMR (400 MHz, CDCl3) δ
2.50 (3H, s, Ar–CH3), 2.65 (4H, t, J = 4.4 Hz, H-2″′, H-6″
′), 2.92 (2H, t, J = 5.2 Hz, H-2″), 3.83 (4H, t, J = 4.4 Hz, H-
3″′, H-5″′), 4.31 (2H, t, J = 5.6 Hz, H-1″), 7.00 (1H, d, J =
8.0 Hz, H-3′), 7.09 (1H, d, J = 8.4 Hz, H-6), 7.14 (1H, t,
J = 7.6 Hz, H-5′), 7.36–7.39 (2H, m, H-4′, H-7), 7.62–7.71
(1H, m, H-4), 8.55 (1H, d, J = 7.8 Hz, H-6′), 11.60–11.69
(1H, m, N–H) ppm. 13C NMR (100 MHz, CDCl3) δ 21.8
(Ar–CH3), 53.2 (C-2″), 57.3 (C-3″′, C-5″′), 63.7 (C-1″),
66.8 (C-2″′, C-6″′), 112.6 (C-3′), 119.1 (C-1′), 121.9 (C-
5′), 123.9 (C-6), 130.1 (C-6′), 130.8 (C-4′), 149.9 (C-2),
155.9 (C-2′) ppm. Anal. Calcd. for: C20H23N3O2 0.2H2O:
C, 70.44; H, 6.92; N, 12.32. Found: C, 70.74; H, 6.73; N,
12.00.
1
7.0 Hz, H-2″′), 2.49 (3H, s, Ar–CH3), 2.80 (4H, q, J = 7.2
Hz, H-1″′), 2.95 (2H, t, J = 5.2 Hz, H-2″), 4.29 (2H, t, J =
5.2 Hz, H-1″), 7.03–7.07 (2H, m, H-3′, H-6), 7.13 (1H, td,
J = 6.8/0.8 Hz, H-5′), 7.37 (2H, m, H-4′, H-7), 7.57 (1H,
brs, H-4), 8.52 (1H, dd, J = 7.8/1.6 Hz, H-6′) ppm. 13C
NMR (100 MHz, CDCl3) δ 11.0 (C-2″′), 21.8 (Ar–CH3),
46.4 (C-1″′), 51.3 (C-2″), 65.6 (C-1″), 113.8 (C-3′), 119.9
(C-1′), 122.1 (C-5′), 123.7 (C-6), 130.1 (C-6′), 130.6 (C-
4′), 149.8 (C-2), 156.2 (C-2′) ppm. Anal. Calcd. for:
C20H25N3O 0.5C4H8O2: C, 71.90; H, 7.95; N, 11.43.
Found: C, 71.72; H, 7.74; N, 11.34.
5-methyl-2-{2-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-1H-
benzo[d]imidazole (3c) Oil. Yield: 13.53 %. IR νmaks (KBr)
cm−1: 3044, 2962, 2880, 1628, 1603, 1582, 1461, 1278,
1246, 1045, 810, 761, 751. LS MS/MS (ESI+) m/z 322
[M+H, 100]. 1H NMR (400 MHz, CDCl3) δ 1.88–1.91
(4H, m, H-3″′, H-4″′), 2.49 (3H, s, Ar–CH3), 2.73 (4H, t,
J = 5.6 Hz, H-2″′, H-5″′), 3.00 (2H, t, J = 5.2 Hz, H-2″), 4.28
(2H, t, J = 5.2 Hz, H-1″), 6.99 (1H, d, J = 8.4 Hz, H-3′), 7.06
(1H, dd, J = 8.0/1.2 Hz, H-6), 7.12 (1H, td, J = 7.2/1.0 Hz,
H-5′), 7.33–7.38 (2H, m, H-4′, H-7), 7.49 (1H, brs, H-4),
8.47 (1H, dd, J = 7.6/1.6 Hz, H-6′) ppm. 13C NMR (100
MHz, CDCl3) δ 21.8 (Ar–CH3), 23.8 (C-3″′, C-4″′), 53.3 (C-
2″′, C-5″′), 55.0 (C-2″), 67.1 (C-1″), 113.7 (C-3′), 120.0 (C-
1′), 122.0 (C-5′), 123.6 (C-6), 130.2 (C-6′), 130.6 (C-4′),
149.9 (C-2), 156.4 (C-2′) ppm. Anal. Calcd. for: C20H23N3O
0.5C4H8O2: C, 72.30; H, 7.45; N, 11.50. Found: C, 72.11; H,
7.07; N, 11.77.
Biological activity assays
AChE E.C.3.1.1.7 (Type VI-S, from electric eel) and
BuChE E.C. 3.1.1.8, from equine serum, were purchased
from Sigma-Aldrich (Steinheim, Germany). 5,5′-dithiobis
(2-nitrobenzoic acid) (DTNB)-Ellman’s reagent, buffer
compounds (potassium dihydrogen phosphate, potassium
hydroxide), sodium hydrogen carbonate, and acet-
ylthiocholine iodide (ATC) used as a substrate were
obtained from Fluka (Buchs, Switzerland). Spectro-
photometric measurements were performed on a Shimadzu
UV/160-A spectrophotometer.
AChE/BuChE activity assay
5-methyl-2-{2-[2-(piperidin-1-yl)ethoxy]phenyl}-1H-benzo
[d]imidazole (3d) White crystalline (MetOH–H2O).
Recrystallized from methanol-water (1:1). Yield: 11.67 %;
mp 108 °C. IR νmaks (KBr) cm−1: 3274, 2930, 2766, 1627,
1602, 1584, 1462, 1271, 1243, 1121, 1049, 767, 752. LS
Enzyme activity was investigated using a slightly modified
colorimetric method of Ellman et al. (1961). As the product
of the enzymatic hydrolysis, the thiocholine does not pos-
sess a significant chromophore for UV detection, the eva-
luation of enzyme activity was performed using a specific
chromogenic reagent, DTNB.
Stock solutions of the inhibitor compounds were pre-
pared in 2 % dimethylsulfoxide (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
10–3 and 10–8, 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 (2.5 units/mL, 100 μL)
and inhibitor solution (100 μL) were added into a cuvette
containing the phosphate buffer (3.0 mL, 0.1 M; pH = 8.0).
After 5 min of incubation, required aliquots of the DTNB
solution (0.01 M, 100 mL) and of the ATC (0.075 M, 20 μL)
1
MS/MS (ESI+) m/z 336 [M+H, 100]. H NMR (400 MHz,
CDCl3) δ 1.52 (2H, t, J = 4.8Hz, H-4″′), 1.69 (4H, p, J =
5.6 Hz, H-3″′, H-5″′), 2.50 (3H, s, Ar–CH3), 2.55 (4H, s, H-
2″′, H-6″′), 2.82 (2H, t, J = 5.6 Hz, H-2″), 4.27 (2H, t, J =
5.6 Hz, H-1″), 6.98 (1H, d, J = 8.4 Hz, H-3′), 7.08 (1H, d, J
= 7.6 Hz, H-6), 7.12 (1H, td, J = 7.6/0.8 Hz, H-5′),
7.32–7.43 (2H, m, H-4′, H-7), 7.61–7.71 (1H, m, H-4), 8.52
(1H, dd, J = 7.6/1.6 Hz, H-6′) ppm. 13C NMR (100 MHz,
CDCl3) δ 21.9 (Ar–CH3), 24.3 (C-4″′), 25.7 (C-3″′, C-5″′),
54.1 (C-2″), 57.4 (C-2″′, C-6″′), 64.5 (C-1″), 112.9 (C-3′),
119.5 (C-1′), 121.8 (C-5′), 123.7 (C-6), 130.2 (C-6′), 130.7
(C-4′), 150.0 (C-2), 156.1 (C-2′) ppm. Anal. Calcd. for:
C21H25N3O 0.25CH4O: C, 74.31; H, 7.63; N, 12.23. Found:
C, 74.56; H, 7.71; N, 11.84.
4-{2-[2-(5-methyl-1H-benzo[d]imidazol-2-yl)phenoxy]
ethyl}morpholine (3e) White crystalline (MetOH–H2O).

Med Chem Res
were added. After a rapid and immediate mixing, the
absorption was measured at 412 nm. In vitro BuChE assay
was similar with the method described above (Kapková
et al., 2013).
constrained on its starting geometries, while the solvent and
the protein were relaxed again with 800 iterations steepest
descent gradient and 800 iterations conjugate gradient
methods. In the last relaxation step, all constrains were
removed and the whole system was minimized for 1000
iterations using steepest descent gradient and 1000 itera-
tions using conjugate gradient methods. Following the
relaxation steps every system generated was then gradually
heated until 300 K within 300 ps of MD using positional
restraint for the atoms belonging to the solute (10 Kcal/mol/
Å). The solvent and the counterions were allowed to move
freely. To equilibrate the temperature, a constant volume
periodic boundary was set on the systems by Langevin
dynamics setting a collision frequency of 1.0 ps−1 (Pastor
et al., 1988). After the temperature was equilibrated, every
system previously generated was subjected to pressure
equilibration of 1 bar for 300 ps in constant pressure peri-
odic boundary by an isotropic pressure scaling method
employing a pressure relaxation time of 2.0 ps, while the
temperature was kept set on 300 K by the Langevin
dynamics. In this step the solute was constrained with a
positional restrains like in the heating step.
Docking studies
The eqBuChE model generated was subjected to energy
minimization using AMBER99 force field already imple-
mented in MOE2013.08 software (Molecular Operating
Environment Chemical Computing Group Inc., Canada)
(Molecular Operating Environment (MOE) 2013.08, 2006).
All the ligands were drawn using MOE2013.08, then they
were 3D protonated and the partial charges were added
using MMFF94× force field. Finally they were minimized
using MMFF94× force field within MOE2013. All the
docking results were carried out using GOLD5.2 software
with default settings (Jones et al., 1997). A sphere of 20 Å
around the imidazole group of His438 was selected as
binding site, and for every ligand 100 poses were retrained.
To score the poses generated, GoldScore and ChemScore
were the selected scoring functions.
After an equilibration step in which the positional con-
strains were gradually removed (at 300 K temperature and
1 bar pressure), the systems generated were subjected to 40
ns MD simulation. For all the equilibration and the free MD
steps, 8 Å was the cutoff selected for the short-range non-
bonded interactions in combination with the Particle Mesh
Ewald option (Darden et al., 1993), while the length of the
hydrogen atoms covalently bounded to heteroatoms were
kept constant through shake method (Hornak et al., 2006).
Coordination’s and energy parameters were saved every
100 iterations during the relaxation steps, every 400 itera-
tions during the temperature and pressure equilibration
steps, and every 4000 iterations during the MD simulations.
Visual molecular dynamics (VMD) was used for visuali-
zation of the trajectories, whereas PYMOL [The PYMOL
Molecular Graphics System, Version 1.5.0.4 Schrödinger,
LLC] was used for the preparation of the figures (Humphrey
et al., 1996).
MD simulations
All the MD simulations presented here have been obtained
with AMBER12 (Case et al., 2012). The eqBuChE model
was protonated and parameterized using ff99SB force field,
whereas all the small compounds were parameterized with
Gaff force field using the selected docking poses as starting
geometries (Case et al., 2012; Wang et al., 2004). The atom
types and partial charges of the ligands were added using
antechamber module, whereas the missing Gaff force field
parameters were generated using parmchk module, both
implemented in AMBER suite. The complexes were then
generated with Leap module from AMBER. Subsequently,
every complex, formed by the protein and small ligand, was
neutralized adding six Cl-counterions and solvated in
octahedral box using TIP3P water molecules leaving at least
12.0 Å between the solute atoms and the border of the box
(~17,062 water molecules were added for every complex).
Several systems were generated, according to the hypoth-
eses of binding found. Every complex was formed by
~59,691 atoms (Case et al., 2012; Jorgensen et al., 1983).
Finally the MDs were conducted using sander program
implemented in AMBER suite (Case et al., 2012).
Results and discussion
Chemistry
In order to remove bad close contacts, every starting
system was initially relaxed in three steps. During the first
step, counterions and water molecules were relaxed for 800
iterations using steepest descent gradient and for 800
iterations using conjugate gradient, while the solute was
constrained with a positional restraint by a force of 10 Kcal/
mol/Å. During the second step, only the ligand was
1H-benzimidazole derivatives were synthesized by the
reported methods in the literature (Scheme 1) (Coban et al.,
2009).
The structures of title compounds were verified by
1
spectral (IR, H NMR, 13C NMR, and ESI-Mass) and ele-
mental analysis. In the IR spectra of the title compounds,
the characteristic frequencies for benzimidazole derivatives

Med Chem Res
and aryl-alkylethers were observed between 2400–3300
cm−1 and 1030–1250 cm−1, respectively (Hesse et al.,
1997). The assessment of the chemical shifts in H NMR
nitrobenzoicacid), known as 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. The activity was determined by measuring
the increase in absorbance at 412 nm at 5 min intervals at
20 °C. IC50 values of the title compounds are summarized in
Table 1. According to the biological activity results, all
compounds exhibited moderate inhibitory activity against
AChE and most of the compounds displayed good inhibi-
tory activities against BuChE. Moreover most of the com-
pounds possessed selectivity against BuChE.
Regarding BuChEI activity, compounds bearing diethyl
group, pyrrolidine, and piperidine rings on terminal nitro-
gen displayed high inhibition, whereas compounds bearing
dimethyl group and morpholine ring gave poor inhibition
values. When IC50 values of the compounds bearing diethyl
substituents were compared to its cyclized analog pyr-
olidine, similar IC50 values were determined, but when 5-C
ring was replaced with 6-C ring (piperidine), the inhibition
increased. On the contrary, a sharp decrease in BuChE
inhibition was observed with the alteration of the piperidine
ring to its isostere, i.e., morpholine. When each group (1, 2,
3) was evaluated within themselves, the lowest activity was
observed in the derivatives with dimethyl groups at terminal
nitrogen (1a, 2a, 3a IC50 = 7.02, 100, 56.31 µM, respec-
tively). Additionally, substitutions at 5-position of the
benzimidazole ring did not provide a significant change in
the biological activity.
1
spectra demonstrated that aromatic and aliphatic protons
were observed in the expected regions confirming the sub-
stitution patterns. The aromatic proton signals of the side
chain at 2-position were observed within prospective che-
mical shift values and divisions, while the some of the
protons 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-tautomerization (Sridharan et al., 2005). NMR spectral
results were assigned by using the 2D HSQC and HMBC
techniques. The mass spectra of the title compounds were
recorded in respect to the positive ion mode electrospray
ionization (ESI+) technique. The [M+H]+ ions of title
compounds compromised with the calculated molecular
weights. The definitions are inserted in the abbreviation part
but we couldn't set the line format. The visual of the
abbreviation part must be checked.
Biological study
ChEI activities of the target compounds were tested by
spectrophotometric method of Ellman et al. and tacrine was
used as the reference compound (Ellman et al., 1961).
Evaluation of the enzyme activity was performed using a
In respect to AChE activity results, all compounds dis-
played inhibitory activity with IC50 values ranging from
specific
chromogenic
reagent,
5,5′-dithiobis(2-
Table 1 In vitro inhibition of
AChE/BuChE and calculated
log P vaules
IC50 SEM (µM)a
AChE
BChE
Selectivity BChE/AChE
Log Pb
1a
1b
1c
14.03 0.38
15.35 0.41
9.40 0.20
14.44 0.31
8.79 0.36
19.35 0.50
11.35 0.54
10.52 0.23
13.22 0.27
12.30 0.29
13.32 0.42
14.58 0.49
14.73 0.21
14.87 2.82
12.07 0.29
0.075 0.02
7.02 0.51
1.09 0.08
1.02 0.04
0.37 0.02
3.98 0.42
4100
2
3.11
3.80
3.65
4.09
2.68
3.74
4.24
4.27
4.72
3.31
3.45
4.13
3.98
4.42
3.01
–
14.08
9.22
38.92
2.21
1d
1e
2a
2b
2c
0.66 0.06
0.90 0.10
0.83 0.05
19.04 0.47
56.31 2.90
0.69 0.01
0.56 0.03
0.23 0.01
19.51 0.35
0.0098 0.0002
17.20
11.69
15.93
0.65
2d
2e
3a
3b
3c
0.23
21.13
26.30
64.65
0.62
3d
3e
Tacrine
a
–
Data are means standard error of the mean of triplicate independent experiments
b
Log P calculated using MOE 2013.08

Med Chem Res
8.79 to 19.35 µM. These results indicated that the positional
change from para to ortho lead to a decrease in the inhibi-
tory activity compared to our previous study.
Log P values of all the benzimidazole derivatives were
calculated by using MOE 2013.08 program and were
between 2.68 and 4.72. The compounds with log P values
of approximately 4 exhibited the best inhibitory activity
against BuChE. These values support the idea that all of the
compounds might penetrate blood brain barrier.
Overall, compound 3d with IC50 value of 0.23 µM pre-
sented a noteworthy BuChE inhibitory activity compared to
the reference, tacrine (IC50 = 0.0098 µM) and is reported as
the most selective BuChE inhibitor compound in the series
(BuChE/AChE = 64.65).
Computational studies
Docking studies and MD simulations were conducted in
order to predict the interactions of the title compounds
inside eqBuChE and to describe the selectivity for BuChE
over AChE (Alpan et al., 2013). From the lack of eqBuChE
X-ray crystal structure, a homology model was initially
generated. The model was obtained through SWISS-
MODEL server using a default automatized pipeline
(Arnold et al., 2006; Benkert et al., 2011). The three-
dimensional geometries of eqBuChE were obtained after
primary sequences alignment with huBuChE X-ray crystal
structure (2PM8.pdb) that shows a primary sequence iden-
tity of 89.79 % with eqBuChE.
Fig. 1 Proposed binding mode for compound 3d inside eqBuChE
model. The active compound is showed as sticks and colored cyan.
The most involved residues are named using three letters code and
showed as yellow sticks. The hydrogen interactions are represented as
black dashed lines, while only polar hydrogen atoms belonging to the
ligand are showed for clarity
every compound) with the side chain of Trp82 and a
putative hydrogen bond interaction between the benzimi-
dazole nitrogen (when protonated) and the carbonyl group
of His438 backbone (Figs. 1 and 2). Moreover all the
compounds are showing a positively charged nitrogen atom
that can find an intermolecular interaction with the non-
protonated nitrogen atom of the benzimidazole group
(Fig. 1).
Consequently, all the compounds belonging to the data
set were processed to docking studies. Several hypotheses
of binding were selected according to GoldScore scoring
function and through visual inspection.
The MD of the putative binding mode for compound 3d
revealed high stability for the whole MD length (42 ns),
suggesting conserved interactions inside gorge cavity of
eqBuChE protein (supporting information). The average
root main square deviation (RMSD) value of the ligand
increases to ~2.60 Å in the first part of the MD without
losing the key interactions found through docking studies
when compared to the starting conformation. After ~27 ns
the ligand is moving back to its starting geometries showing
a RMSD value of ~1.80 Å, when compared to its starting
conformation, until the end of the MD. The average RMSD
value of the protein reaches the steady state around 1.80 Å,
suggesting small deviation from the starting binding geo-
metries within 42 ns of MD (supporting information).
Furthermore in order to describe the reduced activity of
some compounds present in the database, also 3a and 3e
derivatives were processed to MD simulations following the
geometries of binding of the putative binding mode found
(supporting information). According to the MD simulations
conducted, the weak activity of compounds 3a and 3e, with
an IC50 of 19.51 µM and 56.31 µM, respectively, may be
In order to choose the putative binding mode among the
results obtained through docking technique, for the most
active derivative (3d), several MD simulations were carried
out according to its most promising hypotheses of binding.
Only one binding mode showed high stability inside the
binding cavity of the enzyme without losing the key inter-
actions found by the docking (Fig. 1). For this reason it was
selected as the putative one (the MD simulations of the
hypotheses of binding discarded are reported in the sup-
porting information).
According to the binding hypothesis selected, the new
benzimidazole derivatives interact inside eqBuChE binding
cavity in the same region occupied by tacrine co-crystalized
with huBuChE (4BDS.pdb) (Fig. 2). Here the compounds
find interactions in the middle of the active-site gorge and
partially to the CAS (Fig. 2) (Alpan et al., 2013). The main
protein–ligand interactions are reproduced when compared
to tacrine-binding interactions inside huBuChE crystal
structure (Galdeano et al., 2010; Nachon et al., 2013). A
π–π aromatic stack of the benzimidazole group (present in

Med Chem Res
Fig. 2 a External surface with gorge cavity of eqBuChE showing the
proposed binding mode for 3d, 2d, 1d, 3c, 2c, 3b, and 2b active
compounds superimposed with the binding mode showed by tacrine
inside huBuChE crystal structure (4BDS.pdb). 3d compound is
showed as cyan sticks, while 2d, 1d, 3c, 2c, 3b, 2b, and tacrine are
showed as pale green, yellow, orange, pale cyan, light orange, light
blue, and green sticks, respectively. The proteins surface is showed
gray, while the most involved residues are colored as yellow sticks.
The protein is showed as yellow ribbons. b Proposed binding mode for
3d active compound inside gorge cavity of eqBuChE superimposed
with the binding mode showed by tacrine inside huBuChE crystal
structure (4BDS.pdb). 3d is colored as cyan sticks, tacrine is showed
as green sticks, while the involved residues are named using three
letters code and colored as yellow sticks. Protein–ligand hydrogen
interactions are represented as black dashed lines, while hydrogen
atoms are omitted for clarity
due to their higher instability inside the eqBuChE protein
(supporting information).
p-[aminoethoxy]phenylbenzimidazole derivatives to o-
[aminoethoxy]phenylbenzimidazole derivatives, the activity
of the title compounds switches from AChE to BuChE
(Alpan et al., 2013). Also, different substitutions both
on the benzimidazole ring did not exhibit any noticeably
change on ChE inhibitor activity potency. The activity shift
to BuChE is reasonable since in literature is already
demonstrated how compounds may adopt different
geometries of binding inside AChE and BuChE due to
volume differences inside their respectively active-site
gorge cavities (Galdeano et al., 2010; Silva et al., 2013;
Bautista-Aguilera et al., 2014). Figure 3 clearly shows
the steric clashes that can occur with the side chain of
Tyr337 (Ala328 in eqBuChE) when the active compounds
interact inside huAChE gorge cavity adopting the geome-
tries of binding found inside BuChE. Moreover the flex-
ibility of the groups bounded to the amino terminal group
linked to the ethoxyphenyl spacer at position 2 of the
benzimidazole ring it modifies the activity of the com-
pounds when tested on BuChE. Here, bulky, nonflexible,
and a polar substitutions are favoured. Through the mod-
eling studies described, synthesis of new promising deri-
vatives is in progress in order to obtain less flexible
derivatives able to strongly interact inside the active-site
gorge cavities of BuChE.
The activity differences among the three series of the title
compounds present in the data set might also be partially
explained with the computational models obtained. Indeed,
substitutions at position 5 of the benzimidazole ring fill up a
hydrophobic subpocket (Tyr128, Tyr114, Gly116, and
Thr120) of eqBuChE active-site gorge cavity (Figs. 1
and 2).
Finally the selectivity showed by this new data set for
BuChE over AChE is explained from the considerably more
space present in the gorge cavity of BuChE than that present
in AChE. This reflect the fact that 6 out of 14 aromatic
residues present in the huAChE gorge cavity (Tyr72,
Tyr124, Trp286, Phe295, Phe297, and Tyr337) are replaced
by smaller aliphatic or polar amino acids inside the gorge of
huBuChE [Asn68, Gln119, Ala277 (Val277 inside
eqBuChE), Leu286, Val288, and Ala328] (Galdeano et al.,
2010) (Fig. 3).
Conclusion
It can be concluded that according to the studies conducted
here and in the previous article, substitution from

Med Chem Res
(Supplementary Figure S1). The starting conformation
geometries of the ligands were obtained from the docking
studies, while the protein was obtained through homology
modeling using huBuChE crystal structure (2PM8.pdb) as a
template. After that, the complexes protein–ligand were
generated with AMBER12 and subjected to MD simula-
tions. In Supplementary Figure S1 are represented 42 ns
RMSD of three promising binding modes found through
docking studies. From the RMSD plot, it is clear that only
one putative binding mode (red trace) was able to keep the
starting geometries of binding inside eqBuCHE model for
42 ns and for this reason it was selected as a putative
binding mode (Supplementary Figure S1).
Moreover, MD simulations also allowed describing the
activity for some of the less active compounds present in the
database (Supplementary Figure S2). Compounds 3a and 3e
were also simulated inside eqBuChE according to the
binding mode found. In Supplementary Figure S2 is
represented the RMSD plot of the two moderate active
compounds present in the database and compared to the
RMSD of 3d active compound. The purple trace is coupled
to the 3a, the dark green trace is coupled to 3e, while the red
trace is still representing the 3d movements inside
eqBuChE. According to the binding mode found, com-
pounds 3a and 3e are not able to show the key interactions
inside gorge cavity of eqBuChE for neither 20 ns MD
simulation, which are in accordance to their low-activity
values. In case of compound 3a, its reduced activity might
be due to the lack of bulky groups on the terminal nitrogen
(o-dimethylaminoethoxyphenyl substituent). The dimethyl
group on the terminal nitrogen highly decreases the
hydrophobic contacts to the gorge cavity resulting in more
flexible compound when compared to more active inhibitors
present in the data set and causes huge movements inside
the gorge cavity of eqBuChE promoting loss of the starting
key interactions (Supplementary Figure S2). The RMSD
plot regarding 3a is increasing until ~6 Å when compared to
the starting conformation before finding the steady state in
which the key interactions with eqBuChE are disrupted.
Fig. 3 Superimposition of huAchE crystal structure (4EY7.pdb) with
eqBuChE model showing inside the gorge cavity the proposed binding
mode of 3d active compound and tacrine according to the geometries
of binding inside huBuChE crystal structure (4BDS.pdb). 3d is
showed as cyan sticks, while tacrine is showed as green sticks. The
involved residues regarding eqBuChE are showed as yellow sticks and
named using three letters code. The involved residues concerning
huAchE are showed as purple sticks and named using three letters
code. For Tyr337 and Ala328 also the surface is showed as purple and
yellow, respectively. Protein–ligand interactions are showed as black
dashed lines, while hydrogen atoms are omitted for clarity
Supporting information
Docking results
The
docking
studies
were
conducted
with
GOLD5.2 software using GoldScore and ChemScore as
scoring functions. Several poses were considered as puta-
tive binding modes from the docking studies carried out.
Most of them were discarded according to the score values,
while some of them were discarded after their instability
was confirmed through MD simulations. Since ChemScore
was not able to find a reasonable binding mode for all the
compounds present in the data set, only the GoldScore
values and their absolute ranking position for the docked
compounds according the putative binding mode are
reported in Supplementary Table S1.
Acknowledgments This study was partially supported by TUBI-
TAK (Turkish Scientific and Technical Research Council) (Project
number: 213S027). We appreciate the support of Professor Dr.
Wolfgang Sippl for molecular modeling studies. We also thank the
Medicinal Chemistry Department of Martin Luther University Institute
of Pharmacy and also Pharmaceutical Sciences Research Centre
(FABAL) of Ege University Faculty of Pharmacy for equipmental
support.
MD simulations
Compliance with ethical standards
The MD simulations carried out allowed discriminating
putative binding modes among several-selected hypothesis
Conflict of interest The authors declare that they have no conflict of
of
binding
obtained
through
docking
studies
interest.

Med Chem Res
Hein DW, Alheim RJ, Leavitt JJ (1957) The use of polyphosphoric
acid in the synthesis of 2-aryl- and 2-alkyl-substituted benzimi-
dazoles, benzoxazoles and benzothiazoles. J Am Chem Soc 79
(2):427–429
Hesse M, Meier H, Zeeh B (1997) Spectroscopic methods in organic
chemistry, 1st edn. Georg Thieme Verlag, New York
Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C
(2006) Comparison of multiple Amber force fields and develop-
ment of improved protein backbone parameters. Proteins
65:712–725
Humphrey W, Dalke A, Schulten K (1996) VMD—visual molecular
dynamics. J Mol Graph 14:33–38
Jones G, Willett P, Glen RC, Leach R, Taylor R (1997) Development
and validation of a genetic algorithm for flexible docking. J Mol
Biol 267:727–748
Jorgensen WL, Chandrasekhar J, Madura JD (1983) Comparison of
simple potential functions for simulating liquid water. J Chem
Phys 79:926–935
Kapková P, Stiefl N, Süríg U, Engels B, Baumann K, Holzgrabe U
(2013) Synthesis, biological activity and docking studies of new
acetylcholinesterase inhibitors of bispyrdinium type. Arch Pharm
(Weinheim) 336:523–540
Molecular Operating Environment (MOE) 2013.08. (2006) Chemical
Computing Group Inc., Montreal, Quebec, Canada
Morrison JH, Hof PR (1997) Life and death of neurons in the aging
brain. Science 278:412–419
Nachon F, Carletti E, Ronco C, Trovaslet M, Nicolet Y, Jean L,
Renard PY (2013) Crystal structures of human cholinesterases in
complex with huprine W and tacrine: elements of specificity for
anti-Alzheimer’s drugs targeting acetyl- and butyryl-
cholinesterase. Biochem J 453:393–399
References
Alpan AS, Parlar S, Carlino L, Tarikogullari AH, Alptüzün V, Güneş
HS (2013) Synthesis, biological activity and molecular modeling
studies on 1H-benzimidazole derivatives as acetylcholinesterase
inhibitors. Bioorg Med Chem 21:4928–4937
Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL
workspace:
a web-based environment for protein structure
homology modelling. Bioinformatics 22:195–201
Bautista-Aguilera OM, Esteban G, Bolea I, Nikolic K, Agbaba B,
Moraleda I, Iriepa I, Samadi A, Soriano E, Unzeta M, Marco-
Contelles J (2014) Design, synthesis, pharmacological evaluation,
QSAR analysis, molecular modeling and ADMET of novel
donepezil-indolyl hybrids as multipotent cholinesterase/mono-
amine oxidase inhibitors for the potential treatment of Alzhei-
mer’s disease. Eur J Med Chem 75:82–95
Benkert P, Biasini M, Schwede T (2011) Toward the estimation of the
absolute quality of individual protein structure models. Bioin-
formatics 27:343–350
Brus B, Košak U, Turk S, Pišlar A, Coquelle N, Kos J, Stojan J,
Colletier J, Gobec S (2014) Discovery, biological evaluation and
crystal structure of
a novel nanomolar selective butyr-
ylcholinesterase inhibitor. J Med Chem 57(19):8167–8179
Case DA, Darden TA, Cheatham TE, Simmerling CL, Wang J, Duke
RE, Luo R, Walker RC, Zhang W, Merz KM, Roberts B,
Hayik S, Roitberg A, Seabra G, Swails J, Goetz AW,
Kolossv�ry I, Wong KF, Paesani F, Vanicek J, Wolf RM, Liu J,
Wu X, Brozell SR, Steinbrecher T, Gohlke H, Cai Q, Ye X,
Wang J, Hsieh MJ, Cui G, Roe DR, Mathews DH, Seetin MG,
Salomon-Ferrer R, Sagui C, Babin V, Luchko T, Gusarov S,
Kovalenko A, Kollman PA (2012) Amber 12. University of
California, San Francisco
Cheung J, Rudolph MJ, Burshteyn F, Cassidy MS, Gary EN, Love J,
Franklin MC, Height J (2012) Structures of human acet-
ylcholinesterase in complex with pharmacologically important
ligands. J Med Chem 55:10282–10286
Coban G, Zencir S, Zupko´ I, Re´thy B, Gunes HS, Topcu Z (2009)
Synthesis and biological activity evaluation of 1H-benzimida-
zoles via mammalian DNA topoisomerase I and cytostaticity
assays. Eur J Med Chem 44:2280–2285
Darden T, York D, Pedersen L (1993) Particle mesh Ewald: an N⋅log
(N) method for Ewald sums in large systems. J Chem Phys
98:10089–10092
Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new
and rapid colorimetric determination of acetylcholinesterase
activity. Biochem Pharmacol 7:88–90
Galdeano C, Viayna E, Arroyo P, Bidon-Chanal A, Blas JR, Muñoz-
Torrero D, Luque FJ (2010) Structural determinants of the mul-
tifunctional profile of dual binding site acetylcholinesterase
inhibitors as anti-Alzheimer agents. Curr Pharm Des
16:2818–2836
Nagai T, Fukushima Y, Kuroda T, Shimizu H, Sekiguchi S, Matsu H
(1973) Rearrangement of 2-aryloxybenzazoles. Bull Chem Soc
Jpn 46:2600–2602
Nicolet Y, Lockridge O, Masson P, Fontecilla-Camps JC, Nachon F
(2003) Crystal structure of human butyrylcholinesterase and of its
complexes with substrate and products.
J
Biol Chem
278:41141–41147
Pan L, Wang X, Xie S, Li S, Kong L (2014) Multitarget-directed
resveratrol derivatives: anti-cholinesterases, anti-β-amyloid
aggregation and monoamine oxidase inhibition properties
against Alzheimer’s disease. Med Chem Commun 5:609–616
Pastor RW, Brooks BR, Szabo A (1988) An analysis of the accuracy
of Langevin and molecular dynamics algorithms. Mol Phys
65:1409–1419
Silva D, Chioua M, Samadi A, Agostinho P, Garção P, Lajarín-Cuesta
R, de los Ríos C, Iriepa I, Moraleda I, Gonzalez-Lafuente L,
Mendes E (2013) Synthesis, pharmacological assessment, and
molecular modeling of acetylcholinesterase/butyrylcholinesterase
inhibitors: effect against amyloid-β-induced neurotoxicity. ACS
Chem Neurosci 4(4):547–565
Sridharan V, Saravanan S, Muthusubramanian S, Sivasubramanian S
(2005) NMR investigation of hydrogen bonding and 1,3-tauto-
merism in 2-(2-hydroxy-5-substituted-aryl)benzimidazoles. Magn
Reson Chem 43:551–556
Thies W, Bleiler L (2013) Alzheimer’s association. 2013 Alzheimer’s
disease facts and figures. Alzheimers Dement 9:208–245
Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004)
Development and testing of a general amber force field. J Comput
Chem 25:1157–1174
Giacobini E (2004) Cholinesterase inhibitors: new roles and ther-
apeutic alternatives. Pharmacol Res 50:433–440
Greig NH, Lahiri DK, Sambamurti K (2002) Butyrylcholinesterase: an
important new target in Alzheimer’s disease therapy. Int Psy-
chogeriatr 14(Suppl. 1):77–91
Greig NH, Utsuki T, Yu Q, Zhu X, Holloway HW, Perry T, Lee B,
Ingram DK, Lahiri DK (2001) A new therapeutic target in Alz-
heimer’s disease treatment: attention to butyrylcholinesterase.
Curr Med Res Opin 17:159–165
Weinstock M (1999) Selectivity of cholinesterase inhibition: clinical
implications for the treatment of Alzheimer’s disease. CNS Drugs
12:307–323
Han S, Mook-Jung I (2014) Diverse molecular targets for therapeutic
strategies in Alzheimer’s disease.
893–902
J Korean Med Sci 29: