Synthesis, characterization, and molecular docking analysis of novel benzimidazole derivatives as cholinesterase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2013.06.008

Two series of novel acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors containing benzimidazole core structure were synthesized by a four-step reaction pathway starting from 4-fluoro-3-nitrobenzoic acid as the basic compound. The structure of the novel benzimidazoles was characterized and confirmed by the elemental and mass spectral analyses as well as ¹H NMR spectroscopic data. Of the 34 novel synthesized compounds, three benzimidazoles revealed AChE inhibition with IC₅₀ < 10 lM. The highest inhibitory activity (IC50 = 5.12 lM for AChE and IC50 = 8.63 lM for BChE) corresponds to the compound 5IIc (ethyl 1-(3-(1H-imidazol-1-yl)propyl)-2- (4-nitrophenyl)-1H-benzo[d]imidazole-5-carboxylate). The relationship between lipophilicity and the chemical structures as well as their limited structure-activity relationship was discussed.

Full text of DOI:10.1016/j.bioorg.2013.06.008

Bioorganic Chemistry 49 (2013) 33–39
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, characterization, and molecular docking analysis of novel
benzimidazole derivatives as cholinesterase inhibitors
Yeong Keng Yoon a, , Mohamed Ashraf Ali a,b,c, Ang Chee Wei a, Tan Soo Choon a, Kooi-Yeong Khaw d,
⇑
Vikneswaran Murugaiyah d, Hasnah Osman e, Vijay H. Masand f
a Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, 11800 Penang, Malaysia
b New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy College, Alwar, Rajasthan 301 030, India
c New Drug Discovery Research, Department of Medicinal Chemistry, Sunrise University, Alwar, Rajasthan 301 030, India
d Department of Pharmacology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, 11800 Penang, Malaysia
e Department of Organic Chemistry, School of Chemical Sciences, Universiti Sains Malaysia, Minden, 11800 Penang, Malaysia
f Department of Chemistry, Vidya Bharati College, Amravati, Maharashtra 444 602, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 February 2013
Available online 4 July 2013
Two series of novel acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors containing
benzimidazole core structure were synthesized by a four-step reaction pathway starting from 4-fluoro-
3-nitrobenzoic acid as the basic compound. The structure of the novel benzimidazoles was characterized
and confirmed by the elemental and mass spectral analyses as well as 1H NMR spectroscopic data. Of the
Keywords:
Benzimidazoles
Acetylcholinesterase
Butyrylcholinesterase
Alzheimer’s Disease
34 novel synthesized compounds, three benzimidazoles revealed AChE inhibition with IC50 < 10
lM. The
highest inhibitory activity (IC50 = 5.12 M for AChE and IC50 = 8.63 M for BChE) corresponds to the com-
l
l
pound 5IIc (ethyl 1-(3-(1H-imidazol-1-yl)propyl)-2-(4-nitrophenyl)-1H-benzo[d]imidazole-5-carboxylate).
The relationship between lipophilicity and the chemical structures as well as their limited structure–
activity relationship was discussed.
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
affinity toward a variety of enzymes and protein receptors [7,8].
Furthermore, since benzimidazole derivatives have been widely
Alzheimer’s Disease (AD), discovered by Alois Alzheimer in
1907, is described as a degenerative disease of the central nervous
system (CNS) characterized especially by premature senile mental
deterioration. Although the disease was once considered rare, it is
now established as the leading cause of dementia and affects
approximately 24% of people aged 85 years and above worldwide
[1]. Despite all the major efforts, AD remains not curable although
recent advances in drug therapy have challenged this view [2,3].
In the last decades, the treatment for AD has been based on the
‘‘cholinergic hypothesis,’’ that it is associated with an impairment in
cholinergic transmission [4]. This led to the suggestion that cholin-
esterase (ChE) inhibitors would probably stop a deficit in acetyl-
choline levels and help to reverse the memory impairments
characteristic of the disease [5]. Four pharmaceutical drugs,
namely donepenzil, rivastigmine, tacrine, and galantamine, are ap-
plied clinically as cholinesterase inhibitors. We were surprised that
not much effort has been put into utilizing benzimidazoles in
developing new AD drugs [6]. The benzimidazole nucleus is of sig-
nificant importance in medicinal chemistry research due to high
used in other areas such as anti-hypertensive [9], anti-viral [10],
and anti-mycobacterial [11] agents, their pharmacokinetics are
well understood. Therefore, they represent a good starting point
to develop new drugs.
Based on the limited option of drugs available for the treatment
of AD, we set our sights to use benzimidazole as lead compound to
develop new drugs to combat AD. In this paper, we describe the
synthesis and cholinesterase inhibition activity of 34 novel benz-
imidazole derivatives.
2. Results
The synthesis scheme is a four-step pathway leading to the for-
mation of a variety of benzimidazole derivatives. Esterification of
4-fluoro-3-nitro benzoic acid in ethanol by reflux, with concen-
trated sulfuric acid as catalyst yielded the ethyl ester 1 in 75%
yield. The ethyl benzoate 1 was then treated with 2-aminoethanol
and N,N-Diisopropylethylamine (DIPEA) in dry dichloromethane at
room temperature to yield amino compound 2, which was reduced
to the amine 3 using ammonium formate and 10% Pd/C. This phen-
ylenediamine 3 was found to be stable at room temperature, unlike
⇑
Corresponding author. Fax: +60 4 6569796.
E-mail address: kyyeong@gmail.com (Y.K. Yoon).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.06.008

34
Y.K. Yoon et al. / Bioorganic Chemistry 49 (2013) 33–39
Fig. 1. ORTEP diagram of 5Ii.
other phenylenediamine derivatives that were reported to decom-
pose over a short period of time at room temperature [12]. The sta-
bility of the amino derivative 3 is possibly due to the substitution
at 5-position which somewhat reduced the reactivity of the amino
group. The structure of 3-amino ethyl benzoate 3 was confirmed by
1H NMR and LC–MS analysis.
The phenylenediamine 3 was then refluxed with various substi-
tuted bisulfite adduct of aromatic aldehydes 4 in DMF overnight to
afford benzimidazole derivatives 5 in moderate to good yields (43–
90%). The structure and purity of the novel benzimidazoles were
confirmed by chromatographic and spectroscopic analysis and fur-
ther unambiguously ascertained by single X-ray crystallographic
analysis (Figs. 1 and 2) [13,14]. Proton NMR assignment for 5Ig
(Fig. 3) and 5IIc (Fig. 4) was shown as representation for other
compounds in the 5I series and 5II series, respectively. Among
the literature reports available for the synthesis of benzimidazoles
by the reaction of phenylenediamine with acid chloride [15], alde-
hyde [16], and acid [17], we found that access into benzimidazole
derivatives via this metabisulfite route is efficient, environmental
friendly and afforded good to excellent yield of the benzimidazoles.
The formation of the novel benzimidazole derivatives is proposed
and summarized in Scheme 1.
A total of 34 novel benzimidazole derivatives were synthesized
and assayed for their cholinesterase inhibition potency by Ellman’s
method [18]. Results for their acetylcholinesterase (AChE) inhibi-
tion potentials are shown in Table 1. The five most potent com-
pounds were then selected to undergo further testing for
butyrylcholinesterase (BChE) inhibition activites. Table 2 showed
their IC50 values for AChE and BChE inhibitions as well as the
logP/ClogP values. Donepezil and rivastigmine were used as refer-
ence in the assays.
A simple structure–activity relationship analysis showed that
the AChE inhibition potency is closely related to the substitution
of the 2-position of the benzimidazole core. Comparing the two
series of synthesized compounds (5I series and 5II series), it can
be concluded that compounds from the 5II series in general gave
better AChE inhibition activity. In our effort to improve the activity,
further modification of the 4-substitution on the sodium bisulfite
adduct was then carried out. We synthesized compounds with a
wide range of substitution comprising electron-donating as well
Fig. 2. ORTEP diagram of 5IIl.

Y.K. Yoon et al. / Bioorganic Chemistry 49 (2013) 33–39
35
Fig. 3. Proton NMR assignment for 5Ig.
as electron-withdrawing groups. Across the board, we observed
that electron-withdrawing substituents at 4-position in the phenyl
ring are important for good activities as shown by 5IIc (–NO2), 5IIg
(–CF3) and 5IIn (–COOH). All three of them,together with 5In and
5IIo, gave AChE inhibition activities with IC50 values of less than
ortho- and meta-substitution. These results implied the steric dif-
ferences in the active sites of both enzymes [19].
All five compounds were more potent than rivastigmine, how-
ever, relatively less potent on AChE as compared to Donepezil.
Quite interestingly, these new compounds also have a similarly
strong inhibition toward BChE as compared to AChE which implied
that they are dual cholinergic inhibitors. This could be an added
advantage as BChE may represent an important therapeutic target
for AD.
20
lM. The best inhibition was achieved by 5IIc with IC50 of
8.63
lM. These five compounds were selected for further testing
against butyrylcholinesterase (BChE). All of them showed better
inhibition activity against BChE compared to AChE as shown in Ta-
ble 2 with the best IC50 shown by 5IIc (5.12
l
M). Furthermore,
Molecular docking analysis was performed to get better insight
into mechanism of action of benzimidazoles. The active site of
AChE is located at the base of ꢀ20 Å deep and narrow gorge from
the surface of the enzyme and is composed by two subsites: (1)
catalytic esteratic site (CES) and (2) peripheral anionic site (PAS).
The active site consists of Gln71-Tyr-Val-Asp-Thr-Leu76, Gly82-
Thr-Glu84, Trp86-Asn-Pro88, Tyr121, Leu130, Tyr133, Glu199,
their logP values are also in good range for prediction of drug activ-
ity and tolerable toxicity. The slight preference for BChE inhibition
could be due to the 4-position substitution on the phenyl ring of
the benzimidazole derivatives. Recently published results have
indicated that AChE preferspara- and meta-substitution than
ortho-substitution, whereas BChE prefers para-substitution over
Fig. 4. Proton NMR assignment for 5IIc.

36
Y.K. Yoon et al. / Bioorganic Chemistry 49 (2013) 33–39
COOH
COOCH2CH3
COOCH2CH3
COOCH2CH3
R1-NH2
C2H5OH
H2SO4
HCOONH4
Pd/C
DIPEA, DCM
overnight, RT
NO2
NO2
NO2
NH
NH2
NH
F
F
R1
R1
1
2
3
HO
R2
4
SO3Na
DMF
90 o
C
O
O
N
N
R2
R1
5
Scheme 1. Protocol for synthesis of titled compounds.
Table 1
Inhibition of AChE by the synthesized compounds.
Compound
R1
R2
AChE inhibition (%) at 10 lM
5Ia
5Ib
5Ic
5Id
5Ie
5If
5Ig
5Ih
5Ii
5Ij
5Ik
5Il
5Im
5In
5Io
5Ip
5Iq
5IIa
5IIb
5IIc
5IId
5IIe
5IIf
5IIg
5IIh
5IIi
5IIj
5IIk
5IIl
5IIm
5IIn
5IIo
5IIp
5IIq
Rivastigmine
Donepezil
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
4-Ethylmorpholine
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
N-propylimidazole
–
Phenyl
4-Bromophenyl
4-Nitrophenyl
18.54
21.99
38.82
26.39
23.37
14.23
29.74
15.17
22.15
18.26
15.88
27.84
16.08
39.72
29.31
19.12
17.21
24.26
25.68
55.73
28.39
26.26
20.77
53.07
19.78
17.45
23.53
18.33
23.14
22.19
39.40
58.65
23.41
19.68
34.12
98.91
4-Hydroxy-3-methoxyphenyl
4-Trifluoromethoxyphenyl
4-Dimethylaminophenyl
4-Trifluoromethylphenyl
1-(4-(Piperidine-1-yl)phenyl)
1-(4-(Morpholine-1-yl)phenyl)
5-[(4-Fluorophenyl)-pyridine]
4-Methoxyphenyl
Benzo[d][1,3]dioxole
1-(4-Methoxyphenyl)-4-oxo-3-phenylazetidine-2-carbaldehyde
4-Carboxyphenyl
4-Hydroxyphenyl
4-Chlorophenyl
2,4-Dihydroxyphenyl
Phenyl
4-Bromophenyl
4-Nitrophenyl
4-Hydroxy-3-methoxyphenyl
4-Trifluoromethoxyphenyl
4-Dimethylaminophenyl
4-Trifluoromethylphenyl
1-(4-(Piperidine-1-yl)phenyl)
1-(4-(Morpholine-1-yl)phenyl)
5-[(4-Fluorophenyl)-pyridine]
4-Methoxyphenyl
Benzo[d][1,3]dioxole
1-(4-Methoxyphenyl)-4-oxo-3-phenylazetidine-2-carbaldehyde
4-Carboxyphenyl
4-Hydroxyphenyl
4-Chlorophenyl
2,4-Dihydroxyphenyl
–
–
–
Ser200, Glu202-Ser-Ala204, Trp279, Trp286, Phe295, Phe297,
Glu327, Phe330, Glu334, Tyr337-Phe338, Tyr341, Trp439,
His447-Gly-Tyr449, Ile451. The mechanism of acetylcholine
(ACh) hydrolysis involves trapping of ACh to PAS for better cata-
lytic efficiency of the process followed by transfer of ACh to active
site.
All the molecules were docked in the active site of AChE (pdb-
1GQR, X-ray resolution = 2.20 Å). The receptor and the drug candi-
dates were optimized before actual docking in Schrodinger Suite
2011 using standard procedure of the software. For simplicity, as
a representative, we report the docking pose for the most active
compound 5IIc only.

Y.K. Yoon et al. / Bioorganic Chemistry 49 (2013) 33–39
37
Table 2
potentially be a good alternative to AD patients not responding
to selective AChE inhibitors.
Encourage by the positive results, we have reported here, fur-
ther modification on the 2-substituted position on the benzimid-
azole core as well as 4-position on the bisulfite adducts is
currently in progress in our laboratory.
In summary, we have described the molecular design, synthesis,
and cholinesterase inhibition activities of 34 novel benzimidazole
derivatives. The preliminary bioassay data and cytotoxicity test
show that some of the compounds possess moderately good AChE
and BChE inhibition. The present work demonstrates that replace-
ment of 2-position substitution on the benzimidazole core as well
as 4-position on the phenyl ring moiety could potentially result in
new compounds with excellent cholinesterase inhibition activity.
IC50 values activities toward AChE and BChE for selected compounds and their logP
values.
Compounds
AChE
inhibition
BChE
inhibition
IC50 lM
Selectivity
Index (SI) for
AChE
Lipophilicity
logP/ClogP
IC50
l
M
5In
19.57
8.63
9.74
16.38
9.20
18.08
5.12
6.59
11.44
6.81
22.00
7.95
0.92
0.59
0.68
0.70
0.74
0.44
265
2.42/1.749
2.50/4.030
3.61/5.168
2.25/4.092
2.30/3.812
–
5IIc
5IIg
5IIn
5IIo
Rivastigmine 50.20
Donepezil 0.03
–
The docking analysis reveals that the benzimidazoles interact
with receptor primarily due to hydrophobic and mild polar interac-
tions. They are situated near to peripheral anionic site at the en-
trance of the gorge with prominent interactions with Tyr70,
Asp276, Trp279, Asn280, Leu282, Ser286, and Gly335. It appears
that similar to Donepezil, benzimidazoles also target the gorge
connecting the active site with the surface of the enzyme, the
benzimidazole moiety interacting with Trp279, and the phenyl
moiety at 2-position is responsible for interaction with Ser286
and Gly335. The interaction between phenyl moiety (at 2-position)
and the amino acids ser286 and Gly335 are prominent when an
electron-withdrawing group (EWG) is present at para-position of
phenyl ring. The alkyl chain at R1 provides additional flexibility
for interaction with the receptor, bringing the heterocyclic ring clo-
ser to Leu282 (Fig. 5).
4. Materials and methods
4.1. Chemistry
All chemicals were supplied by Sigma–Aldrich (USA) and Merck
Chemicals (Germany). Thin layer chromatography (TLC) using sil-
ica gel G was performed in the solvent system chloroform–metha-
nol (9:1). The spots were located under short (254 nm)/long
(365 nm) UV light. Elemental analyses were performed on Perkin
Elmer 2400 Series II CHN Elemental Analyzer and were within
0.4% of the calculated values. 1H and 13C NMR were performed
on Bruker Avance 300/500 (1H: 300 MHz/500 MHz, 13C: 75 MHz/
125 MHz) spectrometer in CDCl3 using TMS as internal standard.
Mass spectra were recorded on Varian 320-MS TQ LC/MS using
ESI. Crystal structure analysis was carried out using Bruker SMART
APEXII CCD area-detector diffractometer.
3. Discussion
4.1.1. Procedure for the preparation of ethyl-4-fluoro-3-nitrobenzoate
(1)
Over the years, there has not been a unanimous conclusion on
the role of butyrylcholinesterase in AD. There were numerous re-
cent reports which have shown that BChE level increases in AD pa-
tients’ central nervous system [20–22]. Furthermore, for AD
patients below 75 years old who were not responding to Donepezil
(selective AChE inhibitor), most of them showed improvement
when they were treated with Rivastigmine (dual AChE and BChE
inhibitor) [23]. Although inhibition of BChE may cause potentiating
side effects [24], these patients only showed some minor effects of
nausea and vomiting. Therefore, BChE may represent an important
therapeutic target for AD, and dual AChE and BChE inhibitors such
as the novel benzimidazole derivatives described herein could
4-Fluoro-3-nitrobenzoic acid (5 g, 27 mmol) was refluxed in
ethanol (50 mL) and concentrated H2SO4 (2 mL) for 8 h. After com-
pletion of reaction (as evident from TLC), the solvent was evapo-
rated under reduced pressure. The aqueous layer was extracted
with ethyl acetate (25 mL ꢁ 3). The organic layer was dried over
Na2SO4 and concentrated under reduced pressure to yield 1 as
cream-colored powder (75%).
4.1.2. General procedure for the preparation of 4-(2-
substitutedamino)-3-nitro-ethyl benzoate (2)
Ethyl-4-fluoro-3-nitrobenzoate, 1 (0.5 g, 2.34 mmol), amine [I:
4-(2-Aminoethyl)morpholine; II: N-(3-Aminopropyl)imidazole]
(2.58 mmol), and N,N-Diisopropylethylamine, DIPEA (0.49 mL,
2.78 mmol) were mixed in dichloromethane (10 mL). The reaction
mixture was stirred overnight at room temperature. After comple-
tion of reaction (as evident from TLC), the reaction mixture was
washed with water (10 mL ꢁ 2) followed by 10% Na2CO3 solution
(10 mL). The organic layer was dried over Na2SO4 and concentrated
under reduced pressure to afford 2 as brown oil/yellow solid (80–
95%).
4.1.3. General procedure for the preparation of ethyl-3-amino-4-(2-
substituted amino)benzoate (3)
4-(2-Substituted amino)-3-nitro-ethyl benzoate, 2 (1 mmol),
ammonium formate (3 mmol), and Pd/C (50 mg) were mixed in
ethanol (10 mL). The reaction mixture was refluxed until comple-
tion (solution turned colorless). The reaction mixture was then fil-
tered through Celite 545. The filtrate was evaporated under
reduced pressure. It was resuspended in ethyl acetate (20 mL),
washed with water (10 mL ꢁ 2), dried over Na2SO4, and evaporated
to dryness to yield 3 (60%).
Fig. 5. Compound 5IIc docked in first active site.

38
Y.K. Yoon et al. / Bioorganic Chemistry 49 (2013) 33–39
4.1.4. General procedure for the preparation of sodium bisulfite
addcuts of 4-substituted benzaldehyde (4a–4q)
[M+H]+. Anal. Calcd for C23H25N3O5: C, 65.19%; H, 6.03%; N,
9.94%. Found: C, 65.24%; H, 6.13%; N, 10.00%.
Appropriate benzaldehyde (10 mmol) was dissolved in ethanol
(20 mL). Sodium metabisulfite (15 mmol) in 5 mL water was added
in portion over 5 min. The reaction mixture was stirred at room
temperature for 1 h and subsequently stirred at 4 °C overnight.
The precipitate formed was filtered and dried to afford sodium
bisulfite adducts (55–90%).
4.2. Biology
Acetylthiocholine iodide (ATCl), acetylcholinesterase (AChE,
from electric eel), butyrylcholinesterase (BChE, from equine ser-
um), S-butyrylthiocholine chloride, and 5,50-Dithiobis(2-nitroben-
zoic acid) (Ellman’s reagent, DTNB) were purchased from Sigma–
Aldrich (USA).
4.1.5. General procedure for the preparation of 2-substituted
benzimidazole derivatives (5Ia–5Iq and 5IIa–5IIq)
Ethyl-3-amino-4-(2-substituted amino)benzoate, 3 (1 mmol),
and various sodium bisulfite adducts, 4 (1.5 mmol), were dissolved
in DMF (5 mL). The reaction mixture was stirred at 90 °C under N2
atmosphere for 24–48 h. After completion of reaction (evident by
TLC), the reaction mixture was diluted in ethyl acetate (25 mL)
and washed with water (10 mL ꢁ 3). The organic layer was col-
lected, dried over Na2SO4, and evaporated under reduced pressure
to afford compounds 5 in 43–90% yields.
4.2.1. In vitro cholinesterase inhibition assay
Cholinesterase enzyme inhibitory potential of the test samples
was determined following Ellman’s method with slight modifica-
tions. Briefly, test samples and Donepenzil were prepared in DMSO
at the initial concentration of 1 mg/mL. The final concentration of
DMSO in reaction mixture was 1%. At this concentration, DMSO
has no inhibitory effect on both acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) enzymes as was determined in sepa-
rate prior experiments.
Data for 5IIc. Yield: 77%; 1H NMR (300 MHz, CDCl3): d 1H NMR:
1.44 (3H, t, J = 7.2 Hz), 2.20–2.40 (2H, m), 3.95 (2H, t, J = 6.6 Hz),
4.29 (2H, t, J = 6.6 Hz), 4.43 (2H, q, J = 7.2 Hz), 6.82 (1H, s), 7.12
(1H, s), 7.29 (1H, d, J = 8.4 Hz), 7.41 (1H, s), 7.58 (2H, d,
J = 8.4 Hz), 8.10 (1H, dd, J = 1.5 Hz, 8.4 Hz), 8.24 (2H, d, J = 8.4 Hz),
8.58 (1H, s). 13C NMR: 14.42, 30.89, 41.87, 43.76, 61.13, 109.30,
118.32, 122.81, 125.89, 126.11, 126.17, 129.45, 132.54, 133.27,
137.02, 138.44, 142.70, 150.01, 153.29, 166.79. ESI-MS: m/z 420.2
[M+H]+. Anal. Calcd for C22H21N5O4: C, 63.00%; H, 5.05%; N,
16.70%. Found: C, 63.19%; H, 5.10%; N, 16.53%.
For acetylcholinesterase (AChE) inhibitory assay, 140
0.1 M sodium phosphate buffer (pH 8) was added to a 96 wells
microplate followed by 20 of test samples and 20 of
0.09 units/mL AChE enzyme. Then, 10
L of 10 mM 5,50-Dithi-
obis(2-nitrobenzoic acid) (DTNB) was added into each well fol-
lowed by 10 of 14 mM of acetylthiocholine iodide. After
lL of
lL
lL
l
lL
30 min of incubation, absorbance of the colored end product was
measured using Tecan Infinite 200 Pro Microplate Spectrophotom-
eter at 412 nm. Each test was conducted in triplicate.
For butyrylcholinesterase (BChE) inhibitory assay, the same
procedures were applied as AChE except for the use of enzyme
and substrate, which were BChE from equine serum and S-butyryl-
thiocholine chloride.
Data for 5IIg. Yield: 90%; 1H NMR (500 MHz, CDCl3): d 1H NMR:
1.43 (3H, t, J = 7.0 Hz), 2.20–2.30 (2H, m), 3.92 (2H, t, J = 6.5 Hz),
4.27 (2H, t, J = 7.5 Hz), 4.43 (2H, q, 7.0 Hz), 6.76 (1H, s), 7.09 (1H,
s), 7.28 (1H, d, J = 8.5 Hz), 7.39 (1H, s), 7.77 (2H, d, J = 8 Hz), 7.80
(2H, d, J = 8 Hz), 8.09 (1H, dd, J = 1.5 Hz, 8.5 Hz), 8.56 (1H, s). 13C
NMR: 14.42, 30.89, 41.93, 43.81, 61.12, 109.30, 118.29, 122.78,
125.24, 125.85, 126.10, 126.21, 129.54, 130.43, 132.49, 133.32,
137.01, 138.37, 142.67, 153.33, 166.79. ESI-MS: m/z 444.2
[M+H]+. Anal. Calcd for C23H21N4O2F3: C, 62.44%; H, 4.78%; N,
12.88%. Found: C, 62.44%; H, 4.78%; N, 12.89%.
Acknowledgments
The authors wish to express their gratitude and appreciation to
Pharmacogenetics and Novel Therapeutics Research Cluster, Insti-
tute for Research in Molecular Medicine, University Science Malay-
sia, Penang for supporting this work. This work was funded
through Research Grant No. RUC (1001/PSK/8620012) and HiCoE
research Grant No. (311.CIPPM.4401005).
Data for 5IIn. Yield: 80%; 1H NMR (300 MHz, CDCl3): d 1H NMR:
1.45 (3H, t, J = 7.2 Hz), 2.20–2.40 (2H, m), 3.93 (2H, t, J = 6.6 Hz),
4.27 (2H, t, J = 6.6 Hz), 4.44 (2H, q, J = 7.2 Hz), 6.79 (1H, s), 7.12
(1H, s), 7.28 (1H, d, J = 8.4 Hz), 7.38 (1H, s), 7.52 (2H, d,
J = 8.4 Hz), 7.68 (2H, d, J = 8.4 Hz), 8.09 (1H, dd, J = 1.5 Hz, 8.4 Hz),
8.58 (1H, s). 13C NMR: 14.48, 29.03, 41.99, 43.85, 61.55, 109.98,
112.67, 124.13, 125.09, 128.73, 129.13, 129.96, 130.17, 131.11,
134.95, 137.66, 143.84, 151.47, 167.84, 169.11. ESI-MS: m/z
419.3 [M+H]+. Anal. Calcd for C23H22N4O4: C, 66.02%; H, 5.30%; N,
13.39%. Found: C, 66.06%; H, 5.32%; N, 13.35%.
The authors hereby declare there is no conflict of interests.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.bio-
Data for 5IIo. Yield: 76%; 1H NMR (300 MHz, CDCl3): d 1H NMR:
1.45 (3H, t, J = 7.0 Hz), 2.20–2.30 (2H, m), 3.88 (2H, t, J = 6.5 Hz),
4.30 (2H, t, J = 7.5 Hz), 4.43 (2H, q, J = 7.0 Hz), 6.79 (1H, s), 7.07
(1H, s), 7.28 (1H, d, J = 8.5 Hz), 7.39 (1H, s), 7.77 (2H, d, J = 8 Hz),
7.80 (2H, d, J = 8 Hz), 8.09 (1H, dd, J = 1.5 Hz, 8.5 Hz), 8.56 (1H, s).
13C NMR: 14.62, 29.05, 42.01, 43.83, 62.02, 107.83, 109.87,
110.24, 111.06, 123.07, 126.59, 131.18, 137.76, 151.74, 153.40,
157.67, 167.89. ESI-MS: m/z 391.2 [M+H]+. Anal. Calcd for
org.2013.06.008. These data include MOL files and InChiKeys of
the most important compounds described in this article.
References
[1] K. Blennow, M.J. de Leon, H. Zetterberg, Alzheimer’s disease, Lancet 368 (2006)
387–403.
[2] B.M. McGleenon, K.B. Dynan, A.P. Passmore, Acetylcholinesterase inhibitors in
Alzheimer’s disease, Br. J. Clin. Pharmacol. 48 (4) (1999) 471–480.
[3] T. Mohamed, P.P.N. Rao, Alzheimer’s disease: emerging trends in small
molecule therapies, Curr. Med. Chem. 18 (28) (2011) 4299–4320.
[4] S.B. Dunnett, H.C. Fibiger, Role of forebrain cholinergic systems in learning and
memory: relevance to the cognitive deficits of aging and Alzheimer’s
dementia, Prog. Brain Res. 98 (1993) 413–420.
[5] M. Weinstock, Possible role of the cholinergic system and disease models, J.
Neural Transm. Suppl. 49 (1997) 93–102.
[6] V. Pejchal, S. Stepankova, Z. Padelkova, A. Imramovsky, J. Jampilek, 1,3-
Substituted imidazolidine-2,4,5-triones: synthesis and inhibition of
cholinergic enzymes, Molecules 16 (2011) 7565–7582.
C22H22N4O3: C, 67.68%; H, 5.68%; N, 14.35%. Found: C, 67.66%; H,
5.62%; N, 14.50%.
Data for 5In. Yield: 89%; 1H NMR (300 MHz, CDCl3): d 1H NMR:
1.44 (3H, t, J = 7.2 Hz), 2.68 (4H, t, J = 4.8 Hz), 3.01 (2H, t, J = 4.8 Hz),
3.79 (4H, t, J = 4.8 Hz), 4.46 (2H, q, J = 7.2 Hz), 4.64 (2H, t,
J = 4.8 Hz), 7.50 (1H, d, J = 9 Hz), 7.82 (2H, d, J = 9 Hz), 8.10–8.20
(3H, m), 8.60 (1H, s). 13C NMR: 14.46, 42.78, 53.84, 57.49, 60.48,
66.60, 110.05, 112.51, 124.13, 125.09, 128.73, 129.13, 129.96,
130.17, 134.95, 143.85, 167.81, 169.04. ESI-MS: m/z 424.2
[7] M. Mader, A. de Dios, C. Shih, B.D. Anderson, Imidazolyl benzimidazoles and
imidazo[4,5-b]pyridines as potent p38a MAP kinase inhibitors with excellent

Y.K. Yoon et al. / Bioorganic Chemistry 49 (2013) 33–39
39
in vivo antiinflammatory properties, Bioorg. Med. Chem. Lett. 18 (2008) 179–
183.
H37Rv inhibitors as a potential treatment for tuberculosis, Bioorg. Med. Chem.
Lett. 18 (2008) 6244–6247.
[8] B.E. Evans, K.E. Rittle, M.G. Bock, R.M. Dipardo, R.M. Freidinger, W.L. Whitter,
G.F. Lundell, D.F. Veber, S. Anderson, Methods for drug discovery: development
of potent, selective, orally effective cholecystokinin antagonists, J. Med. Chem.
42 (1988) 2235–2246.
[17] T. Roth, M.L. Morningstar, P.L. Boyer, S.H. Hughes, R.W. Buckheit, C.J. Michejda,
Synthesis and biological activity of novel nonnucleoside inhibitors of HIV-1
reverse transcriptase. 2-Aryl-substituted benzimidazoles, J. Med. Chem. 40
(1997) 4199–4207.
[9] J.R. Kumar, J. Jawahar, D.P. Pathak, Synthesis of benzimidazole derivatives: as
anti-hypertensive agents, E-Journal Chem. 3 (13) (2006) 278–285.
[10] G. Komazin, R.G. Ptak, B.T. Emmer, L.B. Townsend, J.C. Drach, Resistance of
[18] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone, A new and rapid
colorimetric determination of acetylcholinesterase activities, Biochem.
Pharmacol. 7 (1961) 88–95.
human cytomegalovirus to D- and L-ribosyl benzimidazoles as
identify potential targets for antiviral drugs, Nucleosides, Nucleotides Nucleic
Acids 22 (2003) 1725–1727.
a
tool to
[19] S.Y. Chiou, C.F. Huang, M.T. Hwang, G. Lin, Comparison of active sites of
butyrylcholinesterase and acetylcholinesterase based on inhibition by
geometric isomers of benzene-di-N-substituted carbamates, J. Biochem. Mol.
Toxicol. 5 (2009) 303–308.
[20] N.H. Greig, D.K. Lahiri, K. Sambamurthi, Butyrylcholinesterase: an important
new target in Alzheimer’s disease therapy, Int. Psychogeriatr. 14 (2002) 77–91.
[21] M.A. Ali, R. Ismail, S.C. Tan, R.S. Kumar, M. Asad, A.I. Almansour, K.Y. Yeong,
C.W. Ang, K. Elumalai, S. Pandian, Alzheimer diseases: substituted spiro [2.30]
oxindolespiro [3.200]-5, 6-dimethoxy-indane-100-one-indolizine analogue as
inhibitors of acetylcholinesterase, Med. Chem. 2 (2012) 1–4.
[11] M. Pieroni, S.M. Tipparaju, S. Lun, Y. Song, A.W. Sturm, W.R. Bishai, A.P.
Kozikowski, Pyrido[1,2-a]benzimidazole-based agents active against
tuberculosis (TB), multidrug-resistant (MDR) TB and extensively drug-
resistant (XDR) TB, ChemMedChem 6 (2011) 334–342.
[12] C. Kus, Synthesis of new substituted 6-(morpholin-4-yl)-1H-benzimidazole
derivatives, Turk. J. Chem. 26 (2002) 559–564.
[13] K.Y. Yeong, M.A. Ali, R. Ismail, M.M. Rosli, H.K. Fun, Ethyl 1-[2-(morpholin-4-
yl)ethyl]-2-[4-(morpholin-4-yl)phenyl]-1H-1,3-benzimidazole-5-carboxylate,
Acta Crystallogr. E E67 (2011) o1772.
[22] P. Szymanski, M. Markowicz, E. Mikiciuk-Olasik, Synthesis and biological
activity of derivatives of tetrahydroacridine as acetylcholinesterase inhibitors,
Bioorg. Chem. 39 (2011) 138–142.
[14] K.Y. Yeong, M.A. Ali, S.C. Tan, S.I.K. Asik, I.A. Razak, Ethyl 2-(1,3-benzodioxol-5-
yl)-1-[3-(1H-imidazol-1-yl)propyl]-1H-benzimidazole-5-carboxylate,
Crystallogr. E E68 (2012) o247.
Acta
[23] S. Auriacombe, J.J. Pere, Y. Loria-Kanza, B. Vellas, Efficacy and safety of
rivastigmine in patients with Alzheimer’s disease who failed to benefit from
treatment with donepenzil, Curr. Med. Res. Opin. 18 (3) (2002) 129–138.
[24] G. Kryger, I. Silman, J.L. Sussman, Structure of acetylcholinesterase complexed
with E2020 (Aricept): implications for the design for the new anti-Alzheimer
drugs, Structure 7 (1999) 297–307.
[15] W. Zeller, A.S. Kiselyov, J. Singh, Regiospecific synthesis of 3-alkyl-4-
hydroxybenzimidazoles as intermediates for an expedient approach to
potent EP3 receptor antagonists, Tetrahedron Lett. 51 (2010) 1380–1382.
[16] C. Gill, G. Jadhav, M. Shaikh, R. Kale, A. Ghawalkar, D. Nagargoje, M. Shiradkar,
Clubbed [1,2,3] triazoles by fluorine benzimidazole: a novel approach to