Synthesis, in vitro and in silico NS5B polymerase inhibitory activity of benzimidazole derivatives

Article abstract of DOI:10.2174/157340612801216120

Hepatitis C virus (HCV) NS5B polymerase is the key replicating protein of the virus and thus an attractive target for drug development. Here we report on the synthesis and biological evaluation of a new series of benzimidazole derivatives as HCV NS5B inhi

Full text of DOI:10.2174/157340612801216120

Medicinal Chemistry, 2012, 8, 629-635
629
Synthesis, In Vitro and In Silico NS5B Polymerase Inhibitory Activity of
Benzimidazole Derivatives
Vaishali M. Patil1*, Gurukumar K. R.2, Maksim Chudayeu2, Satya Prakash Gupta3, Subeer
Samanta4, Neeraj Masand5 and Neerja Kaushik-Basu2*
1Medicinal Chemistry Research Lab, School of Pharmacy, Bharat Institute of Technology, Partapur, Meerut-250 103
(UP) India
2Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue,
Newark, NJ 07103, USA
3Department of Applied Sciences, Meerut Institute of Engineering and Technology, Meerut-250 005 (UP) India
4Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi -835 215 (Jharkhand) India
5Department of Pharmacy, Lala Lajpat Rai Memorial Medical College, Meerut-250 001(UP) India
Abstract: Hepatitis C virus (HCV) NS5B polymerase is the key replicating protein of the virus and thus an attractive tar-
get for drug development. Here we report on the synthesis and biological evaluation of a new series of benzimidazole de-
rivatives as HCV NS5B inhibitors. This yielded compound 6b and 6d bearing 2-(2-benzyloxy)phenyl and 2-(4-
methylbenzyloxy)phenyl moieties, respectively, as promising leads. Binding mode of compound 6d in allosteric pocket
(AP)-1 of NS5B will form the basis for future structure-activity relationship optimization.
Keywords: HCV, NS5B polymerase, Benzimidazole derivatives, RNA-dependent RNA polymerase.
INTRODUCTION
HCV NS5B has been extensively characterized both bio-
chemically and structurally. Similar to other polymerase, the
66 kDa NS5B exhibits the characteristic “right hand” archi-
tecture with fingers, thumb and palm domains [7,9,10]. Ra-
tional drug-design approach has heavily relied on NS5B
crystallographic analysis for designing and developing NS5B
inhibitors, broadly classified as nucleoside or non-nucleoside
inhibitors (NI or NNI, respectively). The former represent
rNTP substrate mimics which cause chain termination upon
incorporation into a growing RNA chain and thus function as
competitive inhibitors [7, 9-14]. By contrast, NNIs consist of
diverse small molecule scaffolds which bind to one of the
five distinct allosteric pocktes (AP) on NS5B and inhibit at
the initiation phase of the polymerase reaction [10, 14]. Al-
though several small molecule inhibitors of NS5B have been
identified in recent years with promising in vitro activities,
they have proved unsatisfactory in clinical trials due to toxic-
ity related issues or emergence of resistant mutant HCV [7,
9-15]. Therefore, development of novel and efficacious anti-
viral therapeutics targeting HCV are highly warranted.
Hepatitis C virus (HCV) is the principal etiological agent
of chronic hepatitis C infection [1, 2], affecting 170-200 mil-
lion people worldwide [2, 3]. Nearly 80% of the infected
individuals develop chronic liver disease, 5-20% of which
progresses to liver cirrhosis and hepatocellular carcinoma [3,
4]. The current standard of treatment against HCVꢀinfectionꢀ
consists of combination therapy with pegylated interferon
and ribavirin. This treatment, however, is limited in efficacy
against the most prevalent HCV genotype (1a/b) and is also
associatedꢀwith significant side effects, thus limiting patient
compliance. In industrialized nations, HCV infection has
become the major cause of orthotopic liver transplants [3, 5].
Viral polymerases are key enzymes of the viral replica-
tion machinery and as such represent significant targets for
therapeutic intervention. Viral polymerase inhibitors have
been clinically validated against human immunodeficiency
virus (HIV), cytomegalovirus (CMV), herpes simplex virus
(HSV), and hepatitis B virus (HBV), among others. In con-
text of HCV, its non-structural protein 5B (NS5B) harbors
the RNA-dependent RNA polymerase (RdRp) activity, cru-
cial for replicating the viral RNA genome [6-8]. Further,
HCV NS5B has no functional equivalent in the host and thus
represents a promising and attractive target for development
of novel anti-HCV agents [9, 10].
Based on QSAR models, we recently predicted that non-
nucleosides such as benzimidazole-coumarin conjugates (1),
thiouracil derivatives (2), and pyrimidine nucleosides (3 and
4) (Fig. 1) have common structural fragments [16-18]. Mul-
tiple regression analysis on the benzimidazole-coumarin de-
rivatives (1, Fig. 1) [16] revealed the following equation:
*Address correspondence to these authors at the Medicinal Chemistry Re-
search Lab, School of Pharmacy, Bharat Institute of Technology, Partapur,
Meerut-250 103 (UP) India; Tel: +91-0-9412703881; Fax: +91-121-
2761846 (V.M.P.); Department of Biochemistry and Molecular Biology,
UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark,
NJ 07103, USA; Tel: +1-973-9728653; Fax: +1-973-9725594 (N.K-B.);
E-mails: vaishuwise@gmail.com (V.M.P.), kaushik@umdnj.edu (N.K-B.)
log(1/EC50) = 0.415(±0.104)ClogP + 3.232(±0.463)
2
n = 18, r = 0.904, rcv = 0.77, s = 0.22, F1,17 = 71.29
(8.40) (1)
1875-6638/12 $58.00+.00
© 2012 Bentham Science Publishers

630 Medicinal Chemistry, 2012, Vol. 8, No. 4
Patil et al.
SR2
NH
R3
R1
R2
N
N
O
O
O
N
H
R1
O
R4
CN
2
1
R2
N
R1
R2
N
N
N
N
HO
N
R1
R3
O
N
OH
HO
3
4
Fig. (1). Structures of benzimidazoles (1), thiouracil (2), pyrrolopyrimidine nucleosides (3), and 6-hydrazinopurine 2ꢀ-methyl ribonucleosides
(4) showing the common structural fragment.
Table 1. Physicochemical Properties of the Synthesized Compounds (5a-i, 6a-d).
Compd no.
Mol Wt
MP (oC)
Mol Formula
clogP
Elemental Analysis (Calcd/Obsd)
5a
5b
5c
5d
5e
5f
263
230
271
195
237
236
300
334
314
264
328
362
314
295
234
370
273
278
241
350
393
374
241
350
392
374
C13H8Cl2N2
C13H8F2N2
C18H13N3
5.00
3.91
4.26
2.32
3.94
5.09
4.89
5.61
5.39
5.64
5.43
6.15
5.93
C, 59.34/59.30; H, 3.06/3.11; N,10.65/10.64; Cl,26.95/26.96
C, 67.82/67.79; H, 3.50/3.60; F, 16.51/16.46; N, 12.17/12.19
C, 79.68/79.64; H, 4.83/4.81; N, 15.49/15.43
C12H9N3
C, 73.83/73.87; H, 4.65/4.62; N, 21.52/21.51
C15H15N3
C, 75.92/75.90; H, 6.37/6.34; N,17.71/17.66
C16H16 N2
C, 81.29/81.31; H, 6.82/6.80; N, 11.85/11.87
5g
5h
5i
C20H16N2O
C20H15ClN2O
C21H18N2O
C18H20N2
C, 79.98/79.95; H, 5.37/5.33; N,9.33/ 9.36; O, 5.33/5.35
C,71.75/71.76; H, 4.52/4.51; Cl, 10.59/10.61; N, 8.37/8.40; O, 4.78/4.81
C, 80.23/80.21; H, 5.77/5.79; N, 8.91/8.95; O, 5.09/5.10
C, 81.78/81.73; H, 7.63/7.61; N, 10.60/10.50
6a
6b
6c
6d
C22H20N2O
C22H19ClN2O
C23H22N2O
C, 80.46/80.45; H, 6.14/6.24; N, 8.53/8.59; O, 4.87/4.85
C, 72.82/72.80; H,5.28/5.38; Cl, 9.77/9.67; N, 7.72/7.75; O, 4.41/4.43
C, 80.67/80.62; H, 6.48/6.50; N, 8.18/8.28; O, 4.67/4.62
where EC50 refers to the molar concentration of the drug
leading to 50% inhibition of HCV RNA replication and
ClogP to calculated hydrophobicity of the molecule derived
from the utilization of ChemDraw version 8.0 software. In
eq. (1), n is the number of data points, r is the correlation
potential towards biological activity [17]. Based on these
analyses, we synthesized new benzimidazole scaffolds with
variation in steric fields and hydrophobicity and investigated
their structural requirements for NS5B inhibition. Herein we
describe the synthesis, activity investigations, and molecular
modeling analysis of these compounds.
2
coefficient, rcv is the square of the cross-validated correla-
tion coefficient obtained from leave-one-out (LOO) jack-
knife procedure, s is the standard deviation, F is the F-ratio
between the variances of calculated and observed activities,
and the data within the parentheses with ± sign are 95% con-
fidence intervals. The figure within the parenthesis for F is
the standard F-value at 99% confidence level. This analysis
suggested that the activity of the compounds were significant
function of the hydrophobicity of the molecules. In another
study, a model generated by k Nearest Neighbor-Molecular
Field Analysis (kNN-MFA) of the same set of benzimida-
zole-coumarin derivatives had revealed contribution of steric
MATERIALS AND METHODS
Synthetic Methods and Spectroscopic Details
All reactions were carried out by using microwave irra-
diation techniques. All reactions were monitored by thin-
layer chromatography on 0.25 mm silica gel plates (60GF-
254) and visualized with UV light, or iodine vapor. Melting
points were determined using digital display melting point
apparatus (uncorrected) (Table 1). Infrared spectra were

Synthesis, In Vitro and In Silico NS5B Polymerase Inhibitory
Medicinal Chemistry, 2012, Vol. 8, No. 4 631
1
measured on a Shimadzu FTIR-8400S using KBr plate. H
NMR spectra were determined on a Brucker 300 MHz spec-
trometer using TMS as an internal standard.
3037, 2979, 2918, 2853, 1584, 1498, 1458, 1377, 1320, 761;
1H NMR (DMSO-d6, ppm): ꢀ 2.33-2.55 (s, 3H), 5.89-5.97 (s,
2H), 6.86-6.95 (dd, 4H), 6.98-7.14 (m, 4H), 7.17-7.25 (m,
4H), 9.01 (s, 1H).
2-(alkoxyaryl)-1H-benzimidazoles (5a-i): A mixture of
1,2-phenylenediamine (0.0313 mol), 1.01 equivalents of ap-
propriate aldehyde, and 1.01 equivalents of sodium metabi-
sulfite was mixed and introduced in an open Erlenmeyer
flask. The mixture was irradiated in a microwave oven for
24–60 s. After irradiation, the mixture was poured onto cold
water. The precipitate was collected by filtration, washed
with water, dried, and recrystallized.
1-ethyl-2-(alkoxyaryl)-1H-benzimidazoles (6a-d): were
obtained by using an excess of alkylating agent iodoethane.
1-ethyl-2-(4-isopropylphenyl)-1H-benzo[d]imidazole
1
(6a): cream solid, Yield: 74%, (ethanol); H NMR (DMSO-
d6, ppm): ꢀ1.24 (d, 6H), 1.67 (t, 3H), 3.14-3.20 (m, 1H), 3.78
(m, 2H), 7.11-7.14 (dd, 4H), 7.56 (m, 4H), 9.87-9.89 (s, 1H);
2-(2-(benzyloxy)phenyl)-1-ethyl-1H-benzo[d]imidazole
2-(3,4-dichlorophenyl)-1H-benzo[d]imidazole (5a):
white solid, Yield: 62%, (methanol). IR (KBr, cm-1): 3065,
3049, 2918, 1586, 1572, 1494, 1477, 1387, 1320, 1073, 880,
1
(6b): red solid, Yield: 69%, (methanol); H NMR (DMSO-
d6, ppm): ꢀ1.65 (t, 3H), 3.89-3.93 (m, 2H), 5.27-5.33 (s, 2H),
6.62-6.74 (m, 4H), 7.05 (d, 2H), 7.56 (m, 4H), 7.77-7.89 (d,
3H), 9.71 (s, 1H).
1
793; H NMR (300 MHz, DMSO-d6, ppm): ꢀ 6.21 (dd, 2H),
6.89-6.96 (dd, 2H), 7.27-7.79 (m, 2H), 7.98 (s, 1H), 9.55 (s,
2-(2-(4-chlorobenzyloxy)phenyl)-1-ethyl-1H-benzo[d]
imidazole (6c): brown solid, Yield: 56% (methanol); 1H
NMR (DMSO-d6, ppm): ꢀ 1.70-1.73 (t, 3H), 3.90-3.94 (m,
2H), 5.91 (s, 2H), 6.58-6.77 (d, 4H), 7.42-7.44 (m, 2H),
7.58-7.60 (d, 2H), 9.88 (s, 1H).
1H).
2-(3,4-difluorophenyl)-1H-benzo[d]imidazole
(5b):
white solid, Yield: 67%, (methanol). IR (KBr, cm-1): 3078,
2977, 2921, 2894, 1602, 1514, 1433, 1406, 1384, 1323,
1091, 842; 1H NMR (DMSO-d6, ppm): ꢀ 6.38 (dd, 2H), 7.61
(m, 2H), 7.82 (d, 2H), 7.95 (d, 2H), 8.17 (s, 1H), 9.35-9.73
(s, 1H).
2-(2-(4-methylbenzyloxy)phenyl)-1-ethyl-1H-benzo[d]
imidazole (6d): cream solid, Yield: 87%, (methanol: water,
8:2); 1H NMR (DMSO-d6, ppm): ꢀ1.69 (t, 3H), 2.46-2.49 (s,
3H), 3.78 (q, 2H), 5.96-5.99 (s, 2H), 6.80-6.93 (dd, 4H),
7.13.-7.24 (m, 4H), 7.30-7.35 (m, 4H), 9.21 (s, 1H).
2-(4-(pyridine-2-yl)phenyl)-1H-benzo[d]imidazole (5c):
white solid, Yield: 58%, (methanol). IR (KBr, cm-1): 3098,
2998, 2934, 2887, 1610, 1517, 1487, 1434, 1398, 1102, 798;
1H NMR (DMSO-d6, ppm):ꢀ 6.50 (d, 2H), 6.86 (m, 2H),
7.25-7.68 (dd, 6H), 7.79-8.25 (m, 2H), 9.55-9.73 (s, 1H).
Biological Evaluation
In Vitro Screening of Inhibitors The anti-NS5B activity
of the candidate compounds was evaluated in vitro by the
NS5B RNA dependent RNA polymerase (RdRp) inhibition
assay as described previously [19, 20]. This reaction utilized
poly rA/U12 as the template-primer (TP) and recombinant
HCV NS5BCꢁ21 (genotype 1b) carrying N-terminal His-tag
and C-terminal 21-amino acid deletion, as the source of en-
zyme. Aurintricarboxylic acid (ATA), a validated NS5B
inhibitor previously identified by us was included as a posi-
tive reference control [20]. To identify candidates belonging
to a wider range of structural scaffolds, preliminary screen-
ing was conducted at a concentration of 100 ꢂM for each
compound. Reactions were initiated by the addition of 1.0
mM MnCl2 and terminated by the addition of ice-cold 10%
(v/v) trichloroacetic acid (TCA) containing 0.5 mM pyro-
phosphate. The amount of radioactive UTP incorporated into
nascent RNA was subjected to trichloroacetic acid (TCA)
precipitation, spotted on a GF-B filter and quantified on a
liquid scintillation counter (Packard). Activity of NS5B in
the absence of the inhibitor was set at 100% and that in the
presence of the inhibitor was calculated relative to this con-
trol.
2-(pyridine-4-yl)-1H-benzo[d]imidazole (5d): white
solid, Yield: 48%, (ethanol). IR (KBr, cm-1): 3099, 3055,
1
2975, 2921, 2869, 1587, 1523, 1473, 1438, 1325, 750; H
NMR (DMSO-d6, ppm):ꢀ 6.93-7.01 (d, 2H), 7.26-7.45 (m,
2H), 7.72-7.93 (m, 2H), 8.39 (d, 2H), 9.45 (s, 1H).
4-(1H-benzo[d]imidazol-2-yl)-N,N-dimethylbenzena-
mine (5e): white solid, Yield: 59%, (methanol). IR (KBr,
cm-1): 3072, 3028, 2975, 2931, 1583, 1528, 1479, 1402,
1379, 1093, 837; 1H NMR (DMSO-d6, ppm): ꢀ 1.17 (s, 6H),
6.44-7.16 (m, 4H), 7.51-7.69 (d, 2H), 7.80-8.12 (d, 2H),
10.98 (s, 1H).
2-(4-isopropylphenyl)-1H-benzo[d]imidazole (5f): white
solid, Yield: 73%, (methanol). IR (KBr, cm-1): 3121, 3046,
2983, 2920, 2893, 2858, 1590, 1515, 1436, 1406, 1321, 827;
1H NMR (DMSO-d6, ppm): ꢀ 1.29 (d, 6H), 3.00-3.17 (m,
1H), 7.03-7.24 (dd, 4H), 7.76 (m, 4H), 9.84-9.94 (s, 1H).
2-(2-(benzyloxy)phenyl)1H-benzo[d]imidazole (5g):
white solid, Yield: 55%, (ethanol). IR (KBr, cm-1): 3099,
3064, 2958, 2929, 2891, 1602, 1514, 1461, 1440, 1367,
1
1247; H NMR (DMSO-d6, ppm): ꢀ 5.17-5.43 (s, 2H), 6.52-
6.75 (m, 4H), 7.15 (d, 2H), 7.52 (m, 4H), 7.74-7.89 (d, 3H),
9.81 (s, 1H).
Molecular Docking
2-(2-(4-chlorobenzyloxy)phenyl)1H-benzo[d]imidazole
(5h): white solid, Yield: 70%, (methanol). IR (KBr, cm-1):
3113, 3071, 2987, 2920, 2885, 1594, 1523, 1492, 1080, 757;
1H NMR (DMSO-d6, ppm): ꢀ 5.93-5.95 (s, 2H), 6.56-6.78
(d, 4H), 7.32-7.35 (m, 2H), 7.55-7.58 (d, 2H), 9.58 (s, 1H).
Crystal structures of NS5B polymerase in complex with
tetracyclic indole (PDB ID: 2dxs) [21], representing AP-1
pocket was used in the present study to perform the entire
virtual screening operation.
The docking approach was validated by determining ‘the
lowest energy pose’ (binding conformation) predicted by the
FlexX [22], versus the experimental binding mode as deter-
2-(2-(4-methylbenzyloxy)phenyl)1H-benzo[d]imidazole
(5i): white solid, Yield: 69%, (ethanol). IR (KBr, cm-1):

632 Medicinal Chemistry, 2012, Vol. 8, No. 4
Patil et al.
NH2
+
N
O
Ar
Na2S2O5
Ar
+
N
H
H
NH2
5a-i
5a; Ar = 3,4-diClPh
5b; Ar = 3,4-diFPh
5f; Ar = i-pr Ph
5g; Ar = 2-benzoxyPh
5c; Ar = 4-(2-pyridyl)Ph 5h; Ar = 2-(4-Cl-benzoxy)Ph
5d; Ar = 4-pyridyl
5i; Ar = 2-(4-Me-benzoxy)Ph
5e; Ar = N,N-diMePh
N
N
N
CH3CH2I
K2CO3/Acetone
Ar
Ar
N
H
6a-d
CH3
6a; Ar = i-pr Ph
6b; Ar = 2-benzoxyPh
6c; Ar = 2-(4-Cl-benzoxy)Ph
6d; Ar = 2-(4-Br-benzoxy)Ph
Scheme 1. Synthesis of compounds 5a-i and 6a-d.
mined by X-ray crystallography. Towards, this end, we re-
moved the afore-mentioned crystallographic bound inhibi-
tors from their binding sites, minimized and then re-docked
them into their respective binding sites, on HCV NS5B po-
lymerase. To establish the specificity of the FlexX method-
ology, the dataset was docked into the binding pocket, AP-1
of NS5B.
in an inactive compound. Introduction of 2-pyridine at the
para-position of the phenyl ring (5c) as well as changing the
benzene ring to heterocyclic ring like 4-pyridine (5d) also
did not improve the activity of the compounds.
Further, neither benzoxy substituent at the ortho position
of the phenyl ring (5g) nor substitution of Cl, or Me on the
benzoxy part (5h, 5i), contribute in any positive way towards
HCV NS5B polymerase inhibition. To improve the activity
of this series of compounds, we introduced the alkyl group
on the benzimidazole nucleus. All the alkylated derivatives
thus obtained, except 6c, exhibited 2-fold increase in activity
(6a, 6b, 6d). Compounds 6b and 6d were found to be the
most active with 24.2% inhibition, thus suggesting the im-
portance of N-alkylation with 2-benzoxyphenyl substituent at
the benzimidazole ring.
RESULTS AND DISCUSSION
Chemistry
Synthesis of benzimidazole derivatives was carried out as
described in Scheme 1. For compounds 5a-i, the synthesis
was initiated with 1,2-phenylenediamine and substituted
aldehydes, using microwave-assisted synthesis procedure.
Compounds 5f-i were subsequently alkylated with io-
doethane to give good yield. All the compounds gave satis-
factory elemental analysis (Table 1). IR and 1H NMR spectra
were consistent with the assigned structures.
The phenyl ring at C-2 position was found to be more ac-
tive than the six-membered heterocycle such as 4-pyridine.
Furthermore, replacement of the phenyl substituent by a six-
membered heterocyclic moiety resulted in decreased inhibi-
tory activity. The bulky substituents at the C-2 position of
the benzimidazole ring resulted in compounds with good
inhibitory potency. It is evident from the 3D kNN-MFA
model that steric fields have some influence on HCV NS5B
polymerase inhibition [17]. Based on this model, the NS5B
inhibitory activity of the set of compounds is predicted, and
for compounds 6b and 6d the in vitro results coincide with
the predicted one. Similarly, eq. (1) was used to predict the
activity based on ClogP of each compound.
HCV NS5B Polymerase Inhibition and Analysis of SAR
With the objective of identifying novel HCV NS5B in-
hibitors, we explored the anti-NS5B activities of the synthe-
sized benzimidazole derivatives employing recombinant
HCV NS5B (genotype 1b) and in vitro NS5B RdRp inhibi-
tion assay, as described previously [19, 20]. Preliminary
screening was conducted at 100 ꢀM compound concentra-
tion.
Of the thirteen benzimidazole derivatives, eight com-
pounds exhibited 5-30% inhibition of HCV NS5B at 100
micromolar (ꢀM) concentrations, while the remaining five
had no effect. A systematic analysis of the substitutions on
C2 and N3 positions of compounds 5a-i and 6a-d is listed in
Table 2. The lack of NS5B inhibition by compounds, 5b, 5g,
5h and 5i appears to be correlated with the unsubstituted N
of the benimidazole ring. Similarly, compounds 5a-i, having
no substitution at N, exhibited weaker inhibition compared
to compounds 6a-d having N-ethyl substitution. Introduction
of lipophilic substituents such as Cl or isopropyl on the
phenyl ring slightly increased their activity (5a, 5f). On the
other hand, the presence of 3,4-difluoro group (5b) resulted
Molecular Modeling
The FlexX docking program is an automated, fast method
for posing ligands into protein binding sites, based on incre-
mental construction algorithm [22-24]. Molecular docking
computations were carried by running the FlexX module on
CPU Intel(R) Xeon CPU 3.06 GHz, RAM Memory 2 GB,
under the OS Enterprise Linux 3.0.
The X-ray crystal structure of NS5B polymerase in com-
plex with JTP_1000 (PDB ID: 2dxs) [21], obtained from the
RCSB Protein Data Bank (PDB), was used to dock the con-
formational library of each ligand. Multiple low energy bind-
ing modes were observed for all the docked compounds. The

Synthesis, In Vitro and In Silico NS5B Polymerase Inhibitory
Medicinal Chemistry, 2012, Vol. 8, No. 4 633
Table 2. Binding Energy, Predicted activity as per eq. (1) and kNN MFA Model Described in Text and Experimental HCV NS5B
Inhibitory Activity of 2-(alkoxyaryl)1H-benzimidazoles (5a-i) and 1-ethyl-2-(alkoxyaryl)1H-benzimidazoles (6a-d)
Predicted
Activity as per
Eq (1)
Predicted
Activity Using
kNN MFA
FlexX Binding
Score
%
Compd no.
Inhibitiona
(kcal/mol)
5a
5b
5c
5d
5e
5f
5.307
4.855
4.999
4.195
4.867
5.344
5.261
5.560
5.469
5.573
5.485
5.784
5.693
5.884
5.723
5.934
6.023
5.478
6.129
5.738
6.112
5.098
6.463
6.241
5.938
6.472
-25.66
-25.11
-25.43
-25.46
-27.10
-26.43
-20.13
-24.17
-22.34
-21.56
-27.11
-18.34
-27.10
7.1
0
6.2
7.5
14.6
10.9
0
5g
5h
5i
0
0
6a
6b
6c
6d
14.6
22.4
0
24.2
aPercent inhibition of HCV NS5B RdRp activity was determined at 100 ꢀM concentration of the indicated compound. The data represents an average of two independent measure-
ments in duplicate. NS5B RdRp activity in the absence of the inhibitor was taken as 100 percent after subtraction of residual background activity.
with the lowest docking energy in the most populated cluster
was selected as the possible ‘active’ conformation against
the 2dxs active site. In the present study, all compounds were
successfully docked into the 2dxs site. Docking poses with
the lowest binding energy shows more negative score for
tighter binding at the receptor site (Table 2).
Binding modes of all the benzimidazole analogues (5a-i,
6a-d) were fairly in good agreement with the structure-
activity discussion as described above. These binding inter-
actions were found similar to the previously studied binding
mode of benzimidazole derivative [25] with residues identi-
fied as His428, Pro495, Val494, Leu392, Ala395, and
Phe429. The benzimidazole ring forms hydrophobic contacts
with residues Val37, Ala395, Thr399, Leu492, and Cys146.
The 2-(4-Me-benzoxy)phenyl substituent attached to C-2
position of the benzimidazole ring is located in a deep hy-
drophobic pocket formed by His428, Leu392, Leu425,
Phe429, Pro495, Pro496, Val499, and Arg503. It is notewor-
thy to mention that on one side of the phenyl ring there is
sufficient room to accommodate a large hydrophobic sub-
stituent. The 4-Me-benzoxy substituent fits nicely into a nar-
row hydrophobic pocket formed by His428, Pro495, Leu497,
Val499, and Arg503. The ethyl group at N-3 position of the
benzimidazole ring shows binding interactions with Leu489,
Gly493, and Val494. The binding mode of the most active
compound 6d is displayed in Fig. (2).
Fig. (2). Docked conformation of compound 6d within allosteric
pocket (AP)-1 of HCV NS5B (PDB ID: 2dxs). Active site amino
acid residues are represented as wire, while the inhibitor is shown
as stick model.
It is important to note that the active site of PDB ID:
2dxs by the crystallographic bound inhibitor versus com-
pound 6d overlap each other (Fig. 3). This suggests that the
benzimidazole nucleus successfully interacts at the active
docked conformations were then used to analyze the binding
interactions. By default, FlexX produces 30 docked struc-
tures for each benzimidazole derivative. The conformation

634 Medicinal Chemistry, 2012, Vol. 8, No. 4
Patil et al.
DECLARATION OF INTEREST
The authors declare no conflict of interest. The authors
alone are responsible for the writing and content of this pa-
per.
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site but the lack of bulky substitutions at positions other than
N1 may contribute negatively towards their activity. Further,
the activity predicted by eq. (1) and the kNN MFA results
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exceptions (5b, 5g, 5h, 5i, 6c) (Table 2). Inhibitory activity
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role. The kNN-MFA results concluded towards variation of
steric bulk at position-1, and -2 but some analogues failed to
produce inhibition. Various substituents at the C-2 position
of the benzimidazole ring may be inactive due to potential
steric clash and the lack of hydrogen bonding at the receptor
site. These findings points towards the significance of sub-
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CONCLUSION
The study reports the effects of N-alkylation and phenyl
substitution on the benzimidazole ring for their anti-HCV
activity. Preliminary screening of this series of compounds
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1H-benzo[d]imidazole (6b) and 2-(2-(4-methylbenzyloxy)
phenyl)-1-ethyl-1H-benzo[d]imidazole (6d) as promising
NS5B inhibitor leads. Docking analysis of compound 6d in
AP-1 of NS5B provided insight for the rational design of
novel inhibitors of HCV NS5B. Further SAR modifications
including the effect of other substituents on the benzimida-
zole ring are on the way and will be reported in due course.
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ACKNOWLEDGEMENTS
Support to V.M.P. in the form of resources from Meerut
Institute of Engineering & Technology, Bharat Institute of
Technology, Meerut, India is gratefully acknowledged. HCV
NS5B inhibition studies were supported by UMDNJ Founda-
tion grant support to N.K-B.
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Synthesis, In Vitro and In Silico NS5B Polymerase Inhibitory
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Germany. www.biosolveit.de.
Received: November 15, 2011
Revised: March 29, 2012
Accepted: March 30, 2012