Inhibitory properties of 2-substituent-1H-benzimidazole-4-carboxamide derivatives against enteroviruses

Article abstract of DOI:10.1016/j.bmc.2011.03.007

A series of novel benzimidazole derivatives were designed, synthesized, and evaluated for their activities against four kinds of enteroviruses, that is, Coxsackie virus A16, B3, B6 and Enterovirus 71 in VERO cells. Strong activities against enterovirus replication and low cytotoxicities were observed in these benzimidazoles generally. The most promising compound was (l)-2-(pyridin-2-yl)- N-(2-(4-nitrophenyl)pentan-3-yl)-1H-benzimidazole-4-carboxamide (16), with a high antiviral potency (IC₅₀ = 1.76 μg/mL) and a remarkable selectivity index (328). These compounds were selected for further evaluation as novel enterovirus inhibitors.

Full text of DOI:10.1016/j.bmc.2011.03.007

Bioorganic & Medicinal Chemistry 19 (2011) 2641–2649
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitory properties of 2-substituent-1H-benzimidazole-4-carboxamide
derivatives against enteroviruses
Fei Xue a, Xianjin Luo a, , Chenghao Ye a, , Weidong Ye b, Yue Wang c,
⇑
⇑
a School of Chemistry and Chemical Engineering, Shanghai Jiaotong University, Shanghai 200240, PR China
b Zhejiang Medicine Co., Ltd., Xinchang Pharmaceutical Factory, 59 East Huangcheng Road, Xinchang, Zhejiang 312500, PR China
c State Key Laboratory for Molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, China Center for Disease Control
and Prevention, 100 Yingxin Lane, Xuanwu District, Beijing 100052, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel benzimidazole derivatives were designed, synthesized, and evaluated for their activities
against four kinds of enteroviruses, that is, Coxsackie virus A16, B3, B6 and Enterovirus 71 in VERO cells.
Strong activities against enterovirus replication and low cytotoxicities were observed in these benzimi-
Received 10 January 2011
Revised 3 March 2011
Accepted 4 March 2011
Available online 10 March 2011
dazoles generally. The most promising compound was (
L
)-2-(pyridin-2-yl)-N-(2-(4-nitrophenyl)pentan-
g/mL) and a
3-yl)-1H-benzimidazole-4-carboxamide (16), with a high antiviral potency (IC50 = 1.76
l
remarkable selectivity index (328). These compounds were selected for further evaluation as novel
enterovirus inhibitors.
Keywords:
Benzimidazole-4-carboxamide
Enterovirus
Ó 2011 Elsevier Ltd. All rights reserved.
Picornavirus
Coxsackie virus A16
Coxsackie virus B3
Coxsackie virus B6
Enterovirus 71
1. Introduction
Enteroviruses were a genus of (+)ssRNA viruses associated with
several human and mammalian diseases. Conventionally, enterovi-
Benzimidazole compounds were known as promising biologi-
cally active agents. The fact that 5,6-dimethylbenzimidazole was
a component of natural vitamin B12 had triggered the interest of
researchers toward benzimidazole derivatives. Benzimidazole
compounds had proven abilities to suppress bacterial growth1
and proton pump function.2 Additionally, many antihelminthic
benzimidazoles were used in the veterinary and medical practice.3
Due to the ability to interact with DNA, benzimidazole unit became
an effective precursor for a wide array of drugs targeting DNA and
DNA associated processes. The benzimidazole unit could interact
with DNA in the minor groove to interfere with DNA processing en-
zymes, such as DNA polymerase, RNA polymerase, and topoisome-
rases I and II. For example, bisbenzimidazole derivatives were
strong ligands to inhibit the activity of the ribozyme of Escherichia
coli RnaseP, effecting the maturation of tRNA.4
ruses were classified into polioviruses, Coxsackie A viruses (CVA),
Coxsackie B viruses (CVB), echoviruses, and enteroviruses 68–71
(EV68-71).5 Coxsackie A viruses tended to infect the skin and mu-
cous membranes, causing herpangina, acute hemorrhagic conjunc-
tivitis and hand-foot-and-mouth disease (HFMD). Coxsackie B
viruses tended to infect the heart, pleura, pancreas, and liver, caus-
ing pleurodynia, myocarditis, pericarditis, and hepatitis. As one of
the major causative agents for HFMD, Enterovirus 71 was associ-
ated with severe central nervous system diseases sometimes. In re-
cent years, numerous disease outbreaks worldwide have been
caused by enteroviruses. In the spring of 2008, a large HFMD out-
break caused by EV71 in China resulted in a high aggregation of
fatal cases.6 HFMD outbreaks caused by CVA16 also occurred in
Asia and all over the world.7,8 Currently, as no efficient drug was
found for the clinical treatment of enteroviruses, there is a tremen-
dous clinical need to develop novel classes of antiviral agents for
the treatment of enterovirus infection.
After the host cell was infected, the genome of enterovirus was
translated in a cap-independent manner into a single polyprotein,
and processed by virus-encoded proteases into structural capsid
proteins and nonstructural proteins subsequently. Both kinds of
proteins were mainly involved in the replication of virus.9 Due to
⇑
Corresponding authors. Tel.: +86 021 54745863; fax: +86 021 54741297 (X.L.);
tel.: +86 010 63554474; fax: +86 010 63510565 (Y.W.).
E-mail addresses: luoxianjin@sjtu.edu.cn (X. Luo),
(Y. Wang).
Trainee from Hangzhou Normal University.
euy-tokyo@umin.ac.jp
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.007

2642
F. Xue et al. / Bioorg. Med. Chem. 19 (2011) 2641–2649
Cyclization of 2,3-diaminobenzoic acid (6) with compounds
containing aldehyde group provided the corresponding 2-(substi-
tuent)-1H-benzimidazole-4-carboxylic acids (compounds 7–13)
in about 80% yield respectively. 2-Substituent-1H-benzimidazole-
4-carboxamide derivatives (compounds 14–53) were synthesized
by the amidation of corresponding 2-R1-1H-benzimidazole-
4-carboxylic acids (7–13) and amines under regular EDC/HOBt
conditions.
Figure 1. General structure of synthesized compounds.
3. Result and discussion
the special structure of benzimidazole, specific hydrogen bonded
interactions could be formed with the genome, interfering the
translating process. Therefore, a series of molecules were designed
based on the benzimidazole system to inhibit enterovirus.
In our previous work,10 a series of 2-pyridyl-1H-benzimidazole-
4-carboxamide derivatives were synthesized, screened and inden-
tified as modest inhibitors of CVB3. In view of its novel structural
template, which differed from those of all reported anti-enterovi-
rus agents, we were interested to study further the structure–
activity relationships of the related class of compounds. It was
believed that a lead compound could be found effective against
picornavirus. More benzimidazole derivatives were designed based
on these compounds and their biological activities were tested
(compounds 14–53 with the general structure shown in Fig. 1).
Inhibitory activities of these benzimidazole derivatives were tested
against CVA16, CVB3, CVB6, and EV71. There were no active clini-
cal drugs against enterovirus. Therefore, a relatively effective drug,
that is, ribavirin (RBV),11 was selected as the positive control drug.
These benzimidazole derivatives were found to exhibit good
inhibitory activities against four kinds of enteroviruses. Hopefully,
these 2-substituent-1H-benzimidazole-4-carboxamide derivatives
might be effective against all the enteroviruses, even the entire
pico-RNA-virus family, because of their similar inhibitory
mechanisms.
The potential anti-enterovirus activities and cytotoxicities of
these synthesized benzimidazoles were evaluated in VERO cells
against CVB3, CVA16, CVB6, and EV71. As there were no active clin-
ical drugs against enterovirus and RBV was the recommended clin-
ical antiviral drug in China, the IC50 values of RVB were provided as
comparable data. These compounds were tested in different
batches with various test items respectively. Pyridyl derivatives
(14–30) and furyl derivatives (31–36) were evaluated against
CVB3, and phenyl derivatives (37–53) were evaluated against
CVB3, CVA16, CVB6, and EV71. From the activities of phenyl deriv-
atives (37–53) against different enteroviruses, it was inferred that
if effective against one kind of enterovirus, a compound could be
effective against others. Therefore, activities against Cox A16, Cox
B6, and Ev71 of pyridyl and furyl derivatives were not tested in
our following studies. The results are summarized in Tables 1
and 2. The anti-enterovirus activity of each compound was ex-
pressed as the concentration of compound that achieved 50% inhi-
bition (IC50) of enterovirus growth. The cytotoxicity of each
compound was expressed as the concentration of compound re-
quired to kill 50% (TC50) of the VERO cells. As a major pharmaceu-
tical parameter for possible future clinical development, the
selectivity index (SI) was determined as the ratio of TC50 to IC50
The bioactivity of each compound was evaluated by the combina-
tion of its IC50 and SI.
The antiviral activities of these compounds against CVB3 are
summarized in Tables 1 and 2. As shown in Table 1, compounds
14–30 with moieties of 2-pyridyl, 3-pyridyl and 4-pyridyl at the
R1 position generally exhibited good antiviral potency against
.
2. Chemistry
Starting from 3-nitrophthalic acid (2), the synthetical route of
the 2-substituent-1H-benzimidazole-4-carboxamide derivatives
(14–53) is shown in Scheme 1. The 3-nitrophthalic anhydride (3)
was obtained in 90% yield by the anhybridization of 3-nitrophtha-
lic acid (2) in acetic anhydride.12 Compound 3 was transformed to
3-nitrophthalamic acid (4) by the treatment of aqueous ammo-
nia,10 and compound 4 was converted into 2-amino-3-nitro-ben-
zoic acid (5) by Hofmann rearrangement.13 The nitro group was
reduced with hydrazine hydrate/methanol catalyzed by Raney
nickel, and the obtained 2,3-diaminobenzoic acid (6) was isolated
as bis-hydrochloride salt.10
CVB3 with IC50s of less than 150
IC50 of 1690 M. IC50s of compounds 16, 17, 18, 19, and 23 were
even less than 5 M. As the most potent compound in this subser-
ies, compound 16 (IC50 = 4.06 0.6 M and SI = 328) was 67 times
lM, much better than RVB with
l
l
l
more selective than RVB (SI = 4.9). Assay for the activity against
EV71 of compound 16 had been carried out. It was found that
the activity against EV71 of compound 16 was also significant (this
result is presented in the Supplementary data). IC50s of compounds
17, 18, 19, and 23 (IC50 = 0.459 0.1, 1.63 0.2, 1.76 0.2, and
Scheme 1. Synthetical route of 2-substituent-1H-benzimidazole-4-carboxamide derivatives (14–53). Reagents and conditions: (i) acetic anhydride, reflux; (ii) NH3(aq), 0 °C;
(iii) NaClO, NaOH, H2O, 0–80 °C; (iv) NaOH, CH3OH, Raney Ni, N2H4ÁH2O, reflux; (v) O2, DMF, 80 °C; (vi) EDCÁHCl, HOBt, Et3N, DMF, 0–25 °C.

F. Xue et al. / Bioorg. Med. Chem. 19 (2011) 2641–2649
2643
Table 1
Activity of benzimidazole derivatives against Coxsackie virus B3 in VERO cells
a
b
Compd
R1
R2
TC50
(
lM)
IC50
(lM)
SIc
N
N
N
14
H
224 33
568 84
30.6 4.6
131 19.6
7.3
4.3
15
16
–CH2CH2OH
OH
OH
1332 72
4.06 0.6
328
O2N
F
N
N
17
18
17.9 2.7
54.7 8.2
0.459 0.1
1.63 0.2
38.9
28.4
N
CH3
N
19
17.7 3.2
1.76 0.2
10
S
N
N
N
20
21
H
77.7 11.7
682 102
26 3.9
3
–CH2CH2OH
90.7 13.6
7.5
OH
OH
22
148 18
28.6 4.3
5.2
O2N
F
N
N
23
24
34.2 5.1
54.7 8.2
3.72 0.6
9.73 1.5
9.2
3.2
N
CH3
N
25
26
111 16.6
498 74
16.2 2.4
NTd
6.8
—
S
–CH2CH2OH
N
OH
N
OH
27
123 18.4
30.2 4.5
4.1
O2N
F
28
29
49.8 7.5
111 16.6
12.3 1.8
37.2 5.6
4
3
N
N
CH3
N
N
30
111 16.6
21.3 3.2
5.2
4.9
S
RVB
—
—
8190 1228
1690 254
a
b
c
Cytotoxic concentration required to inhibit VERO cell growth by 50%.
Concentration required to inhibit Coxsackie virus B3 growth by 50%.
Selectivity Index values equaled to TC50/IC50
Not tested.
.
d
3.72 0.6
lM, respectively) were better than compound 16. IC50 of
subseries (IC50s less than 45 lM), but compounds 15, 21, and 26
compound 18 was best in this subseries. However, compounds 17,
18, 19, and 23 had pronounced cytotoxicity (TC50 = 17.9 2.7,
appeared to be less toxic (TC50 = 568 82, 682 102 and
498 74 M, respectively) than most of the other compounds
(TC50 less than 300 M except compound 16). It was indicated that
l
54.7 8.2, 17.7 3.2, and 34.2 5.1
l
M, respectively) resulting in
l
relatively small selectivity indices (SI = 38.9, 28.4, 10, and 9.2,
respectively). Compounds 15 and 21 with hydroxyethyl at
both antiviral activity and cytotoxicity could be reduced due to the
introduction of hydroxyethyl in the R2 position. Generally, 2-pyri-
dyl derivatives were more efficient than 3-pyridyl and 4-pyridyl
derivatives. Structurally, the main difference between 2-pyridyl
the R2 position showed higher IC50
s (IC50 = 131 19.6 and
90.7 13.6 M, respectively) than other compounds in this
l

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F. Xue et al. / Bioorg. Med. Chem. 19 (2011) 2641–2649
Table 2
Activity of benzimidazole derivatives against Coxsackie virus B3 in VERO cells
a
b
Compd
R1
R2
TC50
(l
M)
IC50
(lM)
SIc
O
O
F
31
25 3.8
3.3 0.5
7.5
OH
OH
32
152 22.8
37.7 5.6
4
O2N
O
OH
CH3
33
34
35
162 24.3
75.9 11.4
68.7 10.3
13.4
2
12.1
3
Cl
F
O
O
NO2
NO2
25.3 3.8
22.9 3.4
OH
OH
3
O2N
O
OH
NO2
CH3
36
48.6 7.3
16.2 2.4
3
Cl
F
OCH3
37
38
39
40
41
24.5 3.7
46 6.9
3.5 0.5
17.2 2.6
0.7 0.1
18.4 2.8
9.8 1.5
7
OCH3
OCH3
–CH2CH2CH2OH
2.7
3
OCH3
OCH3
CH2CH3
2.23 0.3
78.3 11.7
37.8 5.7
OCH3
OCH3
CH2CH3
N
Cl
4.3
3.9
OCH3
OCH3
N
H3CO
H3CO
F
OCH3
OCH3
OH
OH
42
9.85 1.5
4.73 0.7
2
OCH3
O2N
H3CO
H3CO
H3CO
H3CO
—
OCH3
43
44
45
–CH2CH2CH2OH
40
6
19.5 2.9
18.7 2.8
17.4 2.6
2.1
1.7
3.8
OCH3
OCH3
CH2CH3
CH2CH3
32.3 4.8
65.2 9.8
OCH3
OCH3
N
Cl
N
OCH3
OCH3
N
46
29 4.3
16.9 2.5
1.7
4.9
N
H
OCH3
RVB
—
8190 1228
1690 254
a
b
c
Cytotoxic concentration required to inhibit VERO cell growth by 50%.
Concentration required to inhibit Coxsackie virus B3 growth by 50%.
Selectivity Index values equaled to TC50/IC50
.

F. Xue et al. / Bioorg. Med. Chem. 19 (2011) 2641–2649
2645
and 3-/4-pyridyl derivatives was that a hydrogen bond could be
formed between H in position 1 of benzimidazole and N in 2-pyr-
idyl, while it was not achieved in 3-pyridyl and 4-pyridyl deriva-
tives. The reason why 2-pyridyl derivatives were more efficiently
than 3-pyridyl and 4-pyridyl compounds might rely on this struc-
tural difference.
compounds had a purity of >95% as assessed by analytical HPLC.
HPLC analyses were conducted on Shimazu Prominence LC-20A
system using a YMC-PACK ODS-A 150 Â 4.6 nm, 5
lm column with
UV 220 and 245 nm detection. The mobile phase consisted of ace-
tonitrile–methanol–water (45:45:10) with the flow rate of 1 mL/
min. 1H NMR spectra of DMSO-d6 solutions were recorded on a
Bruker DPX400 spectrometer. Elementary analyzes were per-
formed on a Vario ELIII instrument within 0.5% of the theoretical
values.
Compounds 31–46 had groups of furyl and phenyl at the R1 posi-
tion. As shown in Table 2, these derivatives were found to have
excellent IC50s (IC50s less than 45
IC50 of 1690 153 M. The 2-fluorophenyl derivatives showed good
IC50s (IC50s of compound 31, 37, and 41 less than 12 M) except
compound 34, with a slightly higher IC50 of 25.3 3.8 M. The chiral
lM), much better than RVB with
l
l
5.1.1. General procedure A: synthesis of 2-substituent-
1H-benzimidazole-4-carboxylic acids (compounds 7–13)
Appropriate aldehyde (10.5 mmol) was added to the solution of
2,3-diaminobenzoic acid 6 (1.52 g, 10 mmol) in DMF (15 mL). The
solution was heated to 80 °C and then stirred for about 120 h. Then,
the solution was cooled to room temperature. The precipitates
were filtered, washed with ethanol and dried.
l
substituent compounds 32, 33, 35, 36, and 42 showed different
activities (IC50 = 37.7 5.6, 13.4 2, 22.9 3.4, 16.2 2.4, and
4.73 0.7
chlorophenyl)-1-hydroxypropan-2-yl derivatives pronounced bet-
ter antiviral activities than the )-1,3-dihydroxy-1-(4-nitro-
lM, respectively). It was found that the (erythro)-1-(4-
(
L
phenyl)propan-2-yl derivatives did. The pentan-3-yl compounds
39 and 44 expressed better IC50s (IC50s = 0.7 0.1 and
5.1.2. General procedure B: synthesis of 2-substituent-
18.7 2.8
l
M, respectively) than the corresponding 3-hydroxypro-
M) and
1H-benzimidazole-4-carboxamide derivatives (compounds 14–53)
A mixture of appropriate 2-substituent-1H-benzimidazole-4-
carboxylic acid (1 mmol), EDCÁHCl (0.29 g, 1.5 mmol), HOBt
(0.20 g, 1.5 mmol) and Et3N (0.15 g, 1.5 mmol) in DMF (10 mL)
was stirred for 1 h at 0 °C. Then appropriate amine (1.05 mmol)
was added to the solution. The solution was heated up to 25 °C,
stirred for 12 h and then evaporated to dryness. The solid was
washed with water and dried. The products were purified on silica
gel column using 10:1 ethyl acetate/ethanol.
pyl compounds 38 and 43 (IC50s = 17.2 2.6 and 19.5 2.9
l
the pentan-3-yl compounds were more toxic than the 3-hydroxy-
propyl compounds (TC50s of compounds 39 and 44 less than
35 lM and TC50s of compounds 38 and 43 more than 35 lM), result-
ing in similar selective indices of compounds 38, 39, 43, and 44
(SI = 2.7, 3, 2.1, and 1.7, respectively). Generally, the antiviral regu-
larity of this series was inconspicuous. However, it could still be
found that furyl derivatives pronounced better anti-CVB3 activities
than 5-nitrofuryl derivatives did, and 3,4-dimethoxyphenyl deriva-
tives performed better than 2,3,4-trimethoxyphenyl derivatives.
Compounds with phenyl group at the R1 position were also
screened against CVA16, CVB6, and EV71. The results are presented
in Supplementary data. IC50s of most of these benzimidazole com-
5.1.3. 2-(Pyridin-2-yl)-1H-benzimidazole-4-carboxylic acid (7)
Compound 7 was synthesized from picolinaldehyde and 2,3-
diaminobenzoic acid 6 using general procedure A as a grayish-
white solid. Yield = 85%. 1H NMR (DMSO, 400 MHz) d: 7.36 (t,
J = 8 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.88
(d, J = 8 Hz, 1H), 8.04 (t, J = 8.4 Hz, 1H), 8.46 (d, J = 8 Hz, 1H), 8.76
(d, J = 7.2 Hz, 1H). Anal. Calcd for C13H9N3O2: C, 65.27; H, 3.77; N,
17.57. Found: C, 65.54; H, 3.68; N, 17.40.
pounds against CVA16 were less than 33
and 49. Compound 48 had relatively moderate activity (IC50
102 15.3 M) and compound 49 was not active. The most selec-
tive compound against CVA16 was compound 37, with SI of 7
and IC50 of 3.5 0.5 M. IC50 of compound 39 against CVA16,
CVB6, and EV71 was the best (IC50 = 0.736 0.1 M), but an insig-
nificant selectivity (SI = 3) was caused by the cytotoxicity
(TC50 = 2.23 0.3 M). Comparing these results with those shown
lM except compounds 48
=
l
l
l
5.1.4. 2-(Pyridin-3-yl)-1H-benzimidazole-4-carboxylic acid (8)
Compound 8 was synthesized from nicotinaldehyde and 2,3-
diaminobenzoic acid 6 using general procedure A as a grayish-
white solid. Yield = 83%. 1H NMR (DMSO, 400 MHz) d: 7.36 (t,
J = 8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 8 Hz, 1H), 7.89 (d,
J = 8.4 Hz, 1H), 8.57 (t, J = 7.2 Hz, 1H), 8.72 (d, J = 8 Hz, 1H), 9.45
(s, 1H). Anal. Calcd for C13H9N3O2: C, 65.27; H, 3.77; N, 17.57.
Found: C, 65.01; H, 3.80; N, 17.62.
l
in Table 2, it was found that the similarity existed among antiviral
activities against CVB3, CVA16, CVB6, and EV71 of a certain com-
pound, indicating that a compound seemed to be effective against
other virus if it was effective against one virus of the same family.
4. Conclusion
In summary, a series of novel benzimidazole analogues based
on 1 was synthesized and assessed for their anti-enterovirus activ-
ities in VERO cells. Most were proved to be potential enterovirus
inhibitors. Compounds 16, 17, 18, and 19 displayed optimal pro-
5.1.5. 2-(Pyridin-4-yl)-1H-benzimidazole-4-carboxylic acid (9)
Compound 9 was synthesized from isonicotinaldehyde and 2,3-
diaminobenzoic acid 6 using general procedure A as a grayish-
white solid. Yield = 83%. 1H NMR (DMSO, 400 MHz) d: 7.36 (t,
J = 8 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 8.27
(d, J = 7.6 Hz, 2H), 8.75 (d, J = 7.6 Hz, 2H). Anal. Calcd for
files against CVB3, with IC50s of 0.4–4
most promising result was observed for compound 16 with a po-
tent antiviral activity (IC50 = 4.06 0.6 M) and an extraordinarily
lM and SIs of 10–328. The
l
C13H9N3O2: C, 65.27; H, 3.77; N, 17.57. Found: C, 65.15; H, 3.82;
high selectivity (SI = 328). Such activity and cytotoxicity profiles
and their ease of preparation made them attractive candidate com-
pounds for further assessment in vivo as anti-enterovirus agents.
N, 17.72.
5.1.6. 2-(Furan-2-yl)-1H-benzimidazole-4-carboxylic acid (10)
Compound 10 was synthesized from furan-2-carbaldehyde and
2,3-diaminobenzoic acid 6 using general procedure A as a grayish-
white solid. Yield = 80%. 1H NMR (DMSO, 400 MHz) d: 6.90 (d,
J = 8 Hz, 1H), 7.26 (t, J = 8 Hz, 1H), 7.32 (t, J = 8.4 Hz, 1H), 7.68 (d,
J = 8 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H). Anal. Calcd for C12H8N2O3: C,
63.16; H, 3.53; N, 12.28. Found: C, 62.97; H, 3.57; N, 12.35.
5. Experimental
5.1. Chemistry
The starting materials and reagents, purchased from commer-
cial suppliers, were used without further purification. All final

2646
F. Xue et al. / Bioorg. Med. Chem. 19 (2011) 2641–2649
5.1.7. 2-(5-Nitrofuran-2-y)-1H-benzimidazole-4-carboxylic acid
(11)
5.1.13. 2-(Pyridin-2-yl)-N-phenyl-1H-benzimidazole-4-
carboxamide (17)
Compound 11 was synthesized from 5-nitrofuran-2-carbalde-
hyde and 2,3-diaminobenzoic acid 6 using general procedure A
as a grayish-white solid. Yield = 82%. 1H NMR (DMSO, 400 MHz)
d: 7.30 (t, J = 8 Hz, 1H), 7.82 (d, J = 8 Hz, 1H), 7.88 (d, J = 8 Hz,
3H). Anal. Calcd for C12H7N3O5: C, 52.76; H, 2.58; N, 15.38. Found:
C, 52.84; H, 2.63; N, 15.27.
Compound 17 was synthesized from aniline and compound 7
using general procedure B as a slightly yellow solid. Yield = 88%.
1H NMR (CDCl3, 400 MHz) d: 7.16 (t, J = 8 Hz, 1H), 7.41–7.47 (m,
4H), 7.65 (d, J = 8 Hz, 1H), 7.92–8.00 (m, 3H), 8.28 (d, J = 7.6 Hz,
1H), 8.46 (d, J = 7.6 Hz, 1H), 8.68 (d, J = 5.6 Hz, 1H), 11.07 (s, 1H),
12.11 (s, 1H). Anal. Calcd for C19H14N4O: C, 72.60; H, 4.49; N,
17.82. Found: C, 72.43; H, 4.47; N, 17.95.
5.1.8. 2-(3,4-Dimethoxyphenyl)-1H-benzimidazole-4-carboxylic
acid (12)
5.1.14. 2-(Pyridin-2-yl)-N-(2-fluorophenyl)-1H-benzimidazole-
4-carboxamide (18)
Compound 12 was synthesized from 3,4-dimethoxybenzalde-
hyde and 2,3-diaminobenzoic acid 6 using general procedure A
as a grayish-white solid. Yield = 81%. 1H NMR (DMSO, 400 MHz)
d: 3.83 (s, 3H), 3.88 (s, 3H), 7.10 (d, J = 8.4 Hz, 1H), 7.28 (t,
J = 7.2 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.85–7.93 (m, 3H), 12.22
(s, 1H), 13.23 (s, 1H). Anal. Calcd for C16H14N2O4: C, 64.42; H,
4.73; N, 9.39. Found: C, 64.35; H, 4.77; N, 9.42.
Compound 18 was synthesized from 2-fluoroaniline and com-
pound 7 using general procedure B as a slightly yellow solid.
Yield = 87%. 1H NMR (DMSO, 400 MHz) d: 7.16 (t, J = 8 Hz, 1H),
7.26 (t, J = 7.6 Hz, 1H), 7.40–7.48 (m, 2H), 7.61 (t, J = 7.6 Hz, 1H),
7.81 (d, J = 7.6 Hz, 1H), 8.01 (d, J = 7.6 Hz, 1H), 8.14 (t, J = 7.6 Hz,
1H), 8.46 (d, J = 8 Hz, 1H), 8.66 (t, J = 8 Hz, 1H), 8.80–8.81 (d,
J = 4.8 Hz, 1H), 12.52 (s, 1H), 13.84 (s, 1H). Anal. Calcd for
C
19H14FN4O: C, 68.67; H, 3.94; N, 16.86. Found: C, 68.75; H, 4.06;
5.1.9. 2-(2,3,4-Trimethoxyphenyl)-1H-benzimidazole-4-
carboxylic acid (13)
N, 16.74.
Compound 13 was synthesized from 2,3,4-dimethoxybenzalde-
hyde and 2,3-diaminobenzoic acid 6 using general procedure A as a
grayish-white solid. Yield = 85%. 1H NMR (DMSO, 400 MHz) d: 3.83
(s, 3H), 3.88 (s, 3H), 3.99 (s, 3H), 7.04 (d, J = 9.2 Hz, 1H), 7.30 (t,
J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 8 Hz, 1H), 8.05
(d, J = 8.8 Hz, 1H), 11.61 (s, 1H), 13.42 (s, 1H). Anal. Calcd for
5.1.15. 2-(Pyridin-2-yl)-N-(4-methylthiazol-2-yl)-1H-
benzimidazole-4-carboxamide (19)
Compound 19 was synthesized from 4-methylthiazol-5-amine
and compound 7 using general procedure B as a slightly yellow so-
lid. Yield = 87%. 1H NMR (DMSO, 400 MHz) d: 2.34 (s, 3H), 6.87 (s,
1H), 7.47 (t, J = 7.6 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.85 (d, J = 8 Hz,
1H), 8.03 (d, J = 8 Hz, 1H), 8.17 (t, J = 8 Hz, 1H), 8.36 (d, J = 8 Hz, 1H),
8.81 (d, J = 4.8 Hz, 1H), 12.22 (s, 1H), 13.89 (s, 1H). Anal. Calcd for
C17H16N2O5: C, 62.19; H, 4.91; N, 8.53. Found: C, 62.40; H, 4.81;
N, 8.47.
C17H13N5OS: C, 60.88; H, 3.91; N, 20.88. Found: C, 60.87; H, 3.92;
N, 20.75.
5.1.10. 2-(Pyridin-2-yl)-1H-benzimidazole-4-carboxamide (14)
Compound 14 was synthesized from ammonium hydroxide and
5.1.16. 2-(Pyridin-3-yl)-1H-benzimidazole-4-carboxamide (20)
Compound 20 was synthesized from ammonium hydroxide and
compound
7 using general procedure B as a white solid.
Yield = 90%. 1H NMR (DMSO, 400 MHz) d: 7.36 (t, J = 8 Hz, 1H),
7.57 (d, J = 6.8 Hz, 1H), 7.72 (d, J = 8 Hz, 1H), 7.82 (d, J = 2.8 Hz,
1H), 7.88 (d, J = 7.6 Hz, 1H), 8.02 (t, J = 8 Hz, 1H), 8.44 (d, J = 8 Hz,
1H), 8.77 (t, J = 4.8 Hz, 1H), 9.27 (d, J = 2 Hz, 1H), 13.59 (s, 1H). Anal.
Calcd for C13H10N4O: C, 65.54; H, 4.23; N, 23.52. Found: C, 65.81; H,
4.21; N, 23.32.
compound
8 using general procedure B as a white solid.
Yield = 90% 1H NMR (DMSO, 400 MHz) d: 7.37 (t, J = 8 Hz, 1H),
7.61 (d, J = 8 Hz, 1H), 7.76–7.80 (m, 2H), 7.89 (d, J = 7.6 Hz, 1H),
8.57 (t, J = 8 Hz, 1H), 8.71 (d, J = 4.4 Hz, 1H), 9.26 (s, 1H), 9.40 (s,
1H), 13.60 (s, 1H). Anal. Calcd for C13H10N4O: C, 65.54; H, 4.23;
N, 23.52. Found: C, 65.55; H, 4.17; N, 23.48.
5.1.11. 2-(Pyridin-2-yl)-N-ethylol-1H-benzimidazole-4-carbox-
amide (15)
5.1.17. 2-(Pyridin-3-yl)-N-ethylol-1H-benzimidazole-4-
carboxamide (21)
Compound 15 was synthesized from 2-aminoethanol and com-
pound 7 using general procedure B as a white solid. Yield = 86%. 1H
NMR (DMSO, 400 MHz) d: 3.54 (q, J = 5 Hz, 2H), 3.65 (q, J = 5.2 Hz,
2H), 4.99 (t, J = 4.8 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.57 (d, J = 8 Hz,
1H), 7.70 (d, J = 9.2 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 8.04 (t, J = 8 Hz,
1H), 8.46 (d, J = 7.6 Hz, 1H), 8.76–8.77 (d, J = 4.8 Hz, 1H), 10.14–
10.16 (t, J = 5.2 Hz, 1H), 13.62 (s, 1H). Anal. Calcd for C15H14N4O2:
C, 63.82; H, 5.00; N, 19.85. Found: C, 63.87; H, 4.89; N, 19.72.
Compound 21 was synthesized from 2-aminoethanol and com-
pound 8 using general procedure B as a white solid. Yield = 89%. 1H
NMR (DMSO, 400 MHz) d: 3.54 (q, J = 5.6 Hz, 2H), 3.64 (q, J = 5.2 Hz,
2H), 4.92–4.84 (t, J = 4.8 Hz, 1H), 7.36 (t, J = 8 Hz, 1H), 7.61 (t,
J = 7.6 Hz, 1H), 7.75 (d, J = 8 Hz, 1H), 7.90 (t, J = 7.6 Hz, 1H), 8.57
(d, J = 7.6 Hz, 1H), 8.72 (d, J = 4.8 Hz, 1H), 9.45 (s, 1H), 10.12 (t,
J = 5.2 Hz, 1H), 13.57 (s, 1H). Anal. Calcd for C15H14N4O2: C,
63.82; H, 5.00; N, 19.85. Found: C, 63.94; H, 4.95; N, 19.74.
5.1.18. (L)-2-(Pyridin-3-yl)-N-(2-(4-nitrophenyl)pentan-3-yl)-
5.1.12. (L)-2-(Pyridin-2-yl)-N-(2-(4-nitrophenyl)pentan-3-yl)-
1H-benzimidazole-4-carboxamide (22)
Compound 22 was synthesized from (
1H-benzimidazole-4-carboxamide (16)
Compound 16 was synthesized from (
L
)-2-amino-1-(4-nitro-
L)-2-amino-1-(4-nitro-
phenyl)propane-1,3-diol and compound 8 using general procedure
B as a white solid. Yield = 82%. 1H NMR (DMSO, 400 MHz) d: 3.58
(q, J = 5.2 Hz, 1H), 3.68 (q, J = 5.2 Hz, 1H), 4.22 (q, J = 4 Hz, 1H),
5.10 (q, J = 4 Hz, 1H), 5.27 (d, J = 3.2 Hz, 1H), 6.30–6.31 (d,
J = 4 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.64 (d, J = 8 Hz, 1H), 7.70–
7.75 (m, 4H), 8.00–8.06 (m, 2H), 8.65 (d, J = 8 Hz, 1H), 8.74 (d,
J = 7.6 Hz, 1H), 9.56 (d, J = 4.8 Hz, 1H), 10.45 (d, J = 8.4 Hz, 1H),
13.87 (s, 1H). Anal. Calcd for C22H19N5O5: C, 60.97; H, 4.42; N,
16.16. Found: C, 60.88; H, 4.39; N, 16.09.
phenyl)propane-1,3-diol and compound 7 using general procedure
B as a white solid. Yield = 85%. 1H NMR (DMSO, 400 MHz) d: 3.58
(q, J = 5.2 Hz, 1H), 3.68 (q, J = 5.2 Hz, 1H), 4.21 (q, J = 5.2 Hz, 1H),
5.07 (q, J = 6.4 Hz, 1H), 5.28 (d, J = 3.2 Hz, 1H), 6.26 (d, J = 4 Hz,
1H), 7.29 (t, J = 7.6 Hz, 1H), 7.58 (d, J = 8 Hz, 1H), 7.67–7.73 (m,
4H), 8.02–8.06 (m, 3H), 8.57 (d, J = 8 Hz, 1H), 8.78 (d, J = 4.8 Hz,
1H), 10.46 (d, J = 8.4 Hz, 1H), 13.62 (s, 1H). Anal. Calcd for
C22H19N5O5: C, 60.97; H, 4.42; N, 16.16. Found: C, 60.92; H, 4.50;
N, 16.07.

F. Xue et al. / Bioorg. Med. Chem. 19 (2011) 2641–2649
2647
5.1.19. 2-(Pyridin-3-yl)-N-phenyl-1H-benzimidazole-4-
carboxamide (23)
5.1.25. 2-(Pyridin-4-yl)-N-(2-fluorophenyl)-1H-benzimidazole-
4-carboxamide (29)
Compound 23 was synthesized from aniline and compound 8
using general procedure B as a slightly yellow solid. Yield = 91%.
1H NMR (DMSO, 400 MHz) d: 7.14 (t, J = 8 Hz, 1H), 7.42–7.48 (m,
3H), 7.68 (d, J = 8 Hz, 1H), 7.84–7.90 (m, 3H), 8.00–8.02 (d,
J = 7.2 Hz, 1H), 8.63 (t, J = 4 Hz, 1H), 8.76–8.77 (d, J = 3.6 Hz, 1H),
9.47 (s, 1H), 12.12 (s, 1H), 13.80 (s, 1H). Anal. Calcd for
Compound 29 was synthesized from 2-fluoroaniline and com-
pound 9 using general procedure B as a slightly yellow solid.
Yield = 75%. 1H NMR (DMSO, 400 MHz) d: 7.16 (t, J = 7.6 Hz, 1H),
7.25 (t, J = 7.6 Hz, 1H), 7.40–7.51 (m, 2H), 7.88 (d, J = 8 Hz, 1H),
8.04 (d, J = 7.6 Hz, 1H), 8.19–8.21 (m, 2H), 8.62–8.66 (t, J = 7.6 Hz,
1H), 8.85 (d, J = 5.6 Hz, 2H), 13.93 (s, 1H). Anal. Calcd for
C19H14N4O: C, 72.60; H, 4.49; N, 17.82. Found: C, 72.84; H, 4.48;
C19H14FN4O: C, 68.67; H, 3.94; N, 16.86. Found: C, 68.62; H, 3.89;
N, 17.74.
N, 16.81.
5.1.26. 2-(Pyridin-4-yl)-N-(4-methylthiazol-2-yl)-1H-
benzimidazole-4-carboxamide (30)
5.1.20. 2-(Pyridin-3-yl)-N-(2-fluorophenyl)-1H-benzimidazole-
4-carboxamide (24)
Compound 30 was synthesized from 4-methylthiazol-5-amine
and compound 9 using general procedure B as a slightly yellow so-
lid. Yield = 82%. 1H NMR (DMSO, 400 MHz) d: 2.34 (s, 3H), 6.87 (s,
1H), 7.50–7.54 (t, J = 8 Hz, 1H), 7.93 (d, J = 8 Hz, 1H), 8.05 (d,
J = 7.2 Hz, 1H), 8.13 (d, J = 5.2 Hz, 2H), 8.88 (d, J = 5.2 Hz, 2H),
13.16 (s, 1H), 14.05 (s, 1H). Anal. Calcd for C17H13N5OS: C, 60.88;
H, 3.91; N, 20.88. Found: C, 60.74; H, 3.85; N, 20.93.
Compound 24 was synthesized from 2-fluoroaniline and com-
pound 8 using general procedure B as a slightly yellow solid.
Yield = 86%. 1H NMR (DMSO, 400 MHz) d: 7.14 (t, J = 7.6 Hz, 1H),
7.24 (d, J = 8 Hz, 1H), 7.38–7.42 (m, 2H), 7.68 (d, J = 7.6 Hz, 1H),
7.86 (d, J = 8 Hz, 1H), 8.02 (d, J = 7.6 Hz, 1H), 8.58–8.67 (m, 3H),
9.50 (d, J = 4 Hz, 1H), 12.52 (s, 1H), 13.82 (s, 1H). Anal. Calcd for
C19H14FN4O: C, 68.67; H, 3.94; N, 16.86. Found: C, 68.78; H, 4.01;
N, 16.77.
5.1.27. 2-(Furan-2-yl)-N-(2-fluorophenyl)-1H-benzimidazole-4-
carboxamide (31)
5.1.21. 2-(Pyridin-3-yl)-N-(4-methylthiazol-2-yl)-1H-
benzimidazole-4-carboxamide (25)
Compound 31 was synthesized from 2-fluoroaniline and com-
pound 10 using general procedure B as a slightly yellow solid.
Yield = 90%. 1H NMR (DMSO, 400 MHz) d: 6.86 (d, J = 7.6 Hz, 1H),
7.16 (t, J = 7.6 Hz, 1H), 7.24 (t, J = 8 Hz, 1H), 7.35 (m, J = 8 Hz, 1H),
7.38–7.41 (m, 2H), 7.43–7.49 (m, 2H), 7.77–7.82 (t, J = 8 Hz, 1H),
8.00 (d, J = 7.6 Hz, 1H), 8.04–8.06 (m, 2H), 8.61 (d, J = 8 Hz, 1H),
13.75 (s, 1H). Anal. Calcd for C18H12FN3O2: C, 67.29; H, 3.76; N,
13.08. Found: C, 67.47; H, 3.64; N, 13.11.
Compound 25 was synthesized from 4-methylthiazol-5-amine
and compound 8 using general procedure B as a slightly yellow so-
lid. Yield = 80%. 1H NMR (DMSO, 400 MHz) d: 2.33 (s, 3H), 6.86 (s,
1H), 7.47 (t, J = 8 Hz, 1H), 7.69 (t, J = 8 Hz, 1H), 7.90 (d, J = 8 Hz, 1H),
8.02 (d, J = 7.6 Hz, 1H), 8.54 (d, J = 8 Hz, 1H), 8.76 (d, J = 3.2 Hz, 1H),
9.43 (s, 1H), 13.29 (s, 1H), 13.83 (s, 1H). Anal. Calcd for
C17H13N5OS: C, 60.88; H, 3.91; N, 20.88. Found: C, 60.75; H, 3.98;
N, 20.79.
5.1.28. (L)-2-(Furan-2-yl)-N-(2-(4-nitrophenyl)pentan-3-yl)-1H-
benzimidazole-4-carboxamide (32)
5.1.22. 2-(Pyridin-4-yl)-N-ethylol-1H-benzimidazole-4-
carboxamide (26)
Compound 32 was synthesized from (L)-2-amino-1-(4-nitro-
phenyl) propane-1,3-diol and compound 10 using general proce-
dure B as a white solid. Yield = 90%. 1H NMR (DMSO, 400 MHz) d:
3.58 (q, J = 5.2 Hz, 1H), 3.66 (q, J = 5.2 Hz, 1H), 4.17 (q, J = 5.2 Hz,
1H), 5.18 (s, 1H), 5.25 (d, J = 8.8 Hz, 1H), 6.12 (d, J = 4.4 Hz, 1H),
6.87 (d, J = 3.6 Hz, 1H), 7.27 (t, J = 8 Hz, 1H), 7.27–7.36 (m, 2H),
7.65–7.76 (m, 4H) 8.05 (d, J = 8 Hz, 2H), 10.18 (d, J = 8.4 Hz, 1H),
13.55 (s, 1H). Anal. Calcd for C21H18N4O6: C, 59.71; H, 4.30; N,
13.26. Found: C, 59.82; H, 4.33; N, 13.17.
Compound 26 was synthesized from 2-aminoethanol and com-
pound 9 using general procedure B as a white solid. Yield = 75%. 1H
NMR (DMSO, 400 MHz) d: 3.54–3.57 (q, J = 5.2 Hz, 2H), 3.64 (q,
J = 5.2 Hz, 2H), 4.97 (t, J = 4.8 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.78
(d, J = 8 Hz, 1H), 7.92 (d, J = 7.6 Hz, 1H), 8.18 (d, J = 7.2 Hz, 2H),
8.80 (s, 1H), 10.10 (t, J = 4.8 Hz, 1H), 13.75 (s, 1H). Anal. Calcd for
C15H14N4O2: C, 63.82; H, 5.00; N, 19.85. Found: C, 63.96; H, 5.02;
N, 19.78.
5.1.29. (Erythro)-2-(furan-2-yl)-N-(1-amino-2-hydroxy-2-(4-
nitrophenyl)ethyl)-1H-benzimidazole-4-carboxamide (33)
Compound 33 was synthesized from (erythro)-2-amino-1-(4-
nitrophenyl) propan-1-ol and compound 10 using general proce-
dure B as a slightly yellow solid. Yield = 91%. 1H NMR (DMSO,
400 MHz) d: 1.04 (d, J = 6.8 Hz, 3H), 1.20 (t, J = 8 Hz, 1H), 4.29 (q,
J = 6 Hz, 1H), 4.83 (d, J = 4.4 Hz, 1H), 5.79 (s, 1H), 6.79 (s, 1H),
7.21 (t, J = 8 Hz, 1H), 7.29–7.47 (m, 6H), 7.65 (d, J = 7.6 Hz, 1H),
7.84 (d, J = 8 Hz, 1H), 8.02 (s, 1H), 10.02 (d, J = 8 Hz, 1H), 13.42 (s,
1H). Anal. Calcd for C21H18ClN3O3: C, 63.72; H, 4.58; N, 10.62.
Found: C, 63.84; H, 4.47; N, 10.57.
5.1.23. (L)-2-(Pyridin-4-yl)-N-(2-(4-nitrophenyl)pentan-3-yl)-
1H-benzimidazole-4-carboxamide (27)
Compound 27 was synthesized from (
L)-2-amino-1-(4-nitro-
phenyl)propane-1,3-diol and compound 9 using general procedure
B as a white solid. Yield = 87%. 1H NMR (DMSO, 400 MHz) d: 3.58
(q, J = 5.2 Hz, 1H), 3.68 (q, J = 5.2 Hz, 1H), 4.22 (q, J = 4.4 Hz, 1H),
5.09 (q, J = 4.4 Hz, 1H), 5.28 (d, J = 3.2 Hz, 1H), 6.32 (d, J = 4 Hz,
1H), 7.35 (t, J = 8 Hz, 1H), 7.71–7.77 (m, 4H), 8.00–8.07 (m, 2H),
8.27–8.29 (m, 2H), 8.80–8.82 (m, 2H), 10.45 (d, J = 8.4 Hz, 1H),
13.78 (s, 1H). Anal. Calcd for C22H19N5O5: C, 60.97; H, 4.42; N,
16.16. Found: C, 60.85; H, 4.47; N, 16.10.
5.1.30. 2-(5-Nitrofuran-2-yl)-N-(2-fluorophenyl)-1H-
benzimidazole-4-carboxamide (34)
5.1.24. 2-(Pyridin-4-yl)-N-phenyl-1H-benzimidazole-4-
carboxamide (28)
Compound 34 was synthesized from 2-fluoroaniline and com-
pound 11 using general procedure B as a slightly yellow solid.
Yield = 92%. 1H NMR (DMSO, 400 MHz) d: 7.15 (t, J = 8 Hz, 1H),
7.24 (t, J = 8 Hz, 1H), 7.38 (t, J = 8 Hz, 1H), 7.50–7.54 (m, 2H), 7.86
(d, J = 8 Hz, 1H), 7.94 (d, J = 3.6 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H),
8.59 (t, J = 8 Hz, 1H), 12.20 (d, J = 2.8 Hz, 1H), 14.25 (s, 1H). Anal.
Calcd for C18H11FN4O4: C, 59.02; H, 3.03; N, 15.30. Found: C,
59.23; H, 2.98; N, 15.27.
Compound 28 was synthesized from aniline and compound 9
using general procedure B as a slightly yellow solid. Yield = 68%.
1H NMR (DMSO, 400 MHz) d: 7.15 (t, J = 7.6 Hz, 1H), 7.41–7.50
(m, 3H), 7.88 (t, J = 8 Hz, 3H), 8.02 (d, J = 7.2 Hz, 1H), 8.21 (d,
J = 5.6 Hz, 2H), 8.84 (d, J = 4.4 Hz, 2H), 12.05 (s, 1H). Anal. Calcd
for C19H14N4O: C, 72.60; H, 4.49; N, 17.82. Found: C, 72.43; H,
4.42; N, 17.92.

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F. Xue et al. / Bioorg. Med. Chem. 19 (2011) 2641–2649
5.1.31. (
L
)-2-(5-Nitrofuran-2-yl)-N-(2-(4-nitrophenyl)pentan-3-
13.23 (s, 1H), 13.51 (s, 1H). Anal. Calcd for C20H16ClN5O3: C,
yl)-1H-benzimidazole-4-carboxamide (35)
Compound 35 was synthesized from (
58.61; H, 3.94; N, 17.09. Found: C, 58.74; H, 3.88; N, 17.15.
L)-2-amino-1-(4-nitro-
phenyl)propane-1,3-diol and compound 11 using general proce-
dure B as a white solid. Yield = 92%. 1H NMR (DMSO, 400 MHz) d:
3.60 (q, J = 5.2 Hz, 1H), 3.66 (q, J = 5.2 Hz, 1H), 4.18 (q, J = 5.2 Hz,
1H), 5.07 (s, 1H), 5.27 (d, J = 2.4 Hz, 1H), 6.16 (d, J = 3.6 Hz, 1H),
7.37 (t, J = 8 Hz, 1H), 7.53 (d, J = 4.4 Hz, 1H), 7.72–7.78 (m, 4H),
8.00 (d, J = 4.4 Hz, 1H), 8.06 (d, J = 9.6 Hz, 3H), 10.05 (d, J = 8.4 Hz,
2H), 14.05 (s, 1H). Anal. Calcd for C21H17N5O8: C, 53.96; H, 3.67;
N, 14.98. Found: C, 54.17; H, 3.56; N, 14.85.
5.1.37. 2-(2,3,4-Trimethoxyphenyl)-N-(2-fluorophenyl)-1H-
benzimidazole-4-carboxamide (41)
Compound 41 was synthesized from 2-fluoroaniline and com-
pound 13 using general procedure B as a slightly yellow solid.
Yield = 75%. 1H NMR (DMSO, 400 MHz) d: 3.85 (s, 3H), 3.91 (s,
3H), 3.99 (s, 3H), 7.09–7.17 (m, 2H), 7.25 (t, J = 7.6 Hz, 1H), 7.36–
7.42 (m, 2H), 7.87 (d, J = 8 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 8.22
(d, J = 8.8 Hz, 1H), 8.66 (t, J = 8 Hz, 1H), 12.58 (s, 1H), 12.59 (s,
1H). Anal. Calcd for C23H20FN3O4: C, 65.55; H, 4.78; N, 9.97. Found:
C, 65.67; H, 4.82; N, 9.85.
5.1.32. (Erythro)-2-(5-nitrofuran-2-yl)-N-(1-amino-2-hydroxy-
2-(4-nitrophenyl)ethyl)-1H-benzimidazole-4-carboxamide (36)
Compound 36 was synthesized from (erythro)-2-amino-1-(4-
nitrophenyl)propan-1-ol and compound 11 using general proce-
dure B as a slightly yellow solid. Yield = 90%. 1H NMR (DMSO,
400 MHz) d: 1.06–1.08 (d, J = 6.4 Hz, 3H), 4.29 (q, J = 4 Hz, 1H),
4.85 (d, J = 3.2 Hz, 1H), 5.73 (d, J = 5.2 Hz, 1H), 7.29 (d, J = 8.4 Hz,
2H), 7.37 (t, J = 8 Hz, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8 Hz,
1H), 7.72 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 4 Hz, 1H), 7.92 (d, J = 8 Hz,
1H), 9.90 (d, J = 2 Hz, 1H), 13.93 (s, 1H). Anal. Calcd for
5.1.38. (
pentan-3-yl)-1H-benzimidazole-4-carboxamide (42)
Compound 42 was synthesized from ( )-2-amino-1-(4-nitro-
L)-2-(2,3,4-Trimethoxyphenyl)-N-(2-(4-nitrophenyl)-
L
phenyl)propane-1,3-diol and compound 13 using general proce-
dure B as a white solid. Yield = 80%. 1H NMR (DMSO, 400 MHz) d:
3.55 (q, J = 6.8 Hz, 1H), 3.67 (q, J = 7.6 Hz, 1H), 3.85 (s, 3H), 3.90
(s, 3H), 4.00 (s, 3H), 4.23 (q, J = 3.6 Hz, 1H), 5.04 (q, J = 2 Hz, 1H),
5.25 (d, J = 4 Hz, 1H), 6.20 (d, J = 4 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H),
7.22 (t, J = 8 Hz, 1H), 7.66–7.74 (m, 4H), 8.05 (d, J = 9.2 Hz, 2H),
8.34 (d, J = 8.8 Hz, 1H), 10.50 (d, J = 8.8 Hz, 1H), 12.40 (s, 1H). Anal.
Calcd for C26H26N4O8: C, 59.77; H, 5.02; N, 10.72. Found: C, 59.92;
H, 5.09; N, 10.61.
C21H17ClN4O5: C, 57.22; H, 3.89; N, 12.71. Found: C, 57.44; H,
3.76; N, 12.65.
5.1.33. 2-(3,4-Dimethoxyphenyl)-N-(2-fluorophenyl)-1H-
benzimidazole-4-carboxamide (37)
Compound 37 was synthesized from 2-fluoroaniline and com-
pound 12 using general procedure B as a slightly yellow solid.
Yield = 76%. 1H NMR (DMSO, 400 MHz) d: 3.86 (s, 3H), 3.93 (s,
3H), 7.14 (t, J = 5.2 Hz, 1H), 7.20–7.27 (m, 2H), 7.37–7.41 (m, 2H),
7.78 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.95–7.98 (m, 2H),
8.69 (t, J = 8 Hz, 1H), 12.68 (s, 1H), 13.42 (s, 1H). Anal. Calcd for
5.1.39. 2-(2,3,4-Trimethoxyphenyl)-N-(3-hydroxypropyl)-1H-
benzimidazole-4-carboxamide (43)
Compound 43 was synthesized from 3-aminopropan-1-ol and
compound 13 using general procedure
B as a white solid.
Yield = 80%. 1H NMR (DMSO, 400 MHz) d: 1.76 (q, J = 6.8 Hz, 2H),
3.51 (q, J = 6.4 Hz, 1H), 3.58 (q, J = 5.2 Hz, 1H), 3.83 (s, 3H), 3.89
(s, 3H), 3.94 (s, 3H), 4.58 (t, J = 5.2 Hz, 1H), 7.04 (d, J = 8.8 Hz,
1H), 7.28 (t, J = 8 Hz, 1H), 7.75 (d, J = 8 Hz, 1H), 7.84 (d, J = 6.8 Hz,
1H), 8.04 (d, J = 8.8 Hz, 1H), 10.00 (t, J = 5.6 Hz, 1H), 12.49 (s, 1H).
Anal. Calcd for C20H23N3O5: C, 62.33; H, 6.01; N, 10.90. Found: C,
62.12; H, 6.11; N, 10.85.
C22H18FN3O3: C, 67.51; H, 4.64; N, 10.74. Found: C, 67.40; H,
4.69; N, 10.82.
5.1.34. 2-(3,4-Dimethoxyphenyl)-N-(3-hydroxypropyl)-1H-
benzimidazole-4-carboxamide (38)
Compound 38 was synthesized from 3-aminopropan-1-ol and
compound 12 using general procedure
B
as
a
white solid.
5.1.40. 2-(2,3,4-Trimethoxyphenyl)-N-(pentan-3-yl)-1H-
benzimidazole-4-carboxamide (44)
Yield = 80%. 1H NMR (DMSO, 400 MHz) d: 1.78 (q, J = 6.4 Hz, 2H),
3.53 (q, J = 5.2 Hz, 2H), 3.63 (q, J = 5.2 Hz, 2H), 3.84 (s, 3H), 3.89
(s, 3H), 4.60 (t, J = 6 Hz, 1H), 7.16 (d, J = 8 Hz, 1H), 7.279 (t,
J = 8 Hz, 1H), 7.67 (d, J = 8 Hz, 1H), 7.81–7.83 (m, 3H), 10.02 (t,
J = 5.2 Hz, 1H), 13.22 (s, 1H). Anal. Calcd for C19H21N3O4: C,
64.21; H, 5.96; N, 11.82. Found: C, 64.38; H, 5.84; N, 11.74.
Compound 44 was synthesized from pentan-3-amine and com-
pound 13 using general procedure B as a yellow solid. Yield = 80%.
1H NMR (DMSO, 400 MHz) d: 0.95 (t, J = 8 Hz, 6H), 1.53–1.69 (m,
4H), 3.83 (s, 3H), 3.88 (s, 3H), 3.94–4.00 (m, 4H), 7.08 (d,
J = 8.8 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.75 (d, J = 8 Hz, 1H), 7.813
(d, J = 7.6 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 9.88 (d, J = 8.4 Hz, 1H),
12.50 (s, 1H). Anal. Calcd for C22H27N3O4: C, 66.48; H, 6.85; N,
10.57. Found: C, 66.54; H, 6.78; N, 10.68.
5.1.35. 2-(3,4-Dimethoxyphenyl)-N-(pentan-3-yl)-1H-
benzimidazole-4-carboxamide (39)
Compound 39 was synthesized from pentan-3-amine and com-
pound 12 using general procedure B as a yellow solid. Yield = 83%.
1H NMR (DMSO, 400 MHz) d: 0.97 (t, J = 8 Hz, 6H), 1.58–1.66 (m,
4H), 3.84 (s, 3H), 3.86 (s, 3H), 3.96 (q, J = 6 Hz, 1H), 7.18 (d,
J = 9.2 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.67 (d, J = 8 Hz, 1H), 7.76–
7.83 (m, 3H), 9.93 (d, J = 8.4 Hz, 1H), 13.24 (s, 1H). Anal. Calcd for
5.1.41. 2-(2,3,4-Trimethoxyphenyl)-N-(5-chloropyrazin-2-yl)-
1H-benzimidazole-4-carboxamide (45)
Compound 45 was synthesized from 5-chloropyrazin-2-amine
and compound 13 using general procedure B as a yellow solid.
Yield = 75%. 1H NMR (DMSO, 400 MHz) d: 3.85 (s, 3H), 3.91 (s,
3H), 3.98 (s, 3H), 7.11 (d, J = 9.2 Hz, 1H), 7.41 (t, J = 8 Hz, 1H),
7.90 (d, J = 8 Hz, 1H), 7.99–8.05 (m, 2H), 8.63 (s, 1H), 9.46 (s, 1H),
12.74 (s, 1H), 12.96 (s, 1H). Anal. Calcd for C21H18ClN5O4: C,
57.34; H, 4.12; N, 15.92. Found: C, 57.16; H, 4.08; N, 16.13.
C21H25N3O3: C, 68.64; H, 6.86; N, 11.44. Found: C, 68.80; H, 6.94;
N, 11.27.
5.1.36. 2-(3,4-Dimethoxyphenyl)-N-(5-chloropyrazin-2-yl)-1H-
benzimidazole-4-carboxamide (40)
Compound 40 was synthesized from 5-chloropyrazin-2-amine
and compound 12 using general procedure B as a yellow solid.
Yield = 75%. 1H NMR (DMSO, 400 MHz) d: 3.86 (s, 3H), 3.93 (s,
3H), 7.21 (d, J = 8 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.82–7.87 (m,
2H), 7.92 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 8.64 (s, 1H), 9.45 (s, 1H),
5.1.42. 2-(2,3,4-Trimethoxyphenyl)-N-(1H-benzimidazol-2-yl)-
1H-benzimidazole-4-carboxamide (46)
Compound 46 was synthesized from 1H-benzimidazol-2-amine
and compound 13 using general procedure B as a slightly yellow
solid. Yield = 69%. 1H NMR (DMSO, 400 MHz) d: 3.84 (s, 3H), 3.88
(s, 3H), 4.02 (s, 3H), 7.05 (d, J = 9.2 Hz, 1H), 7.31–7.37 (m, 3H),

F. Xue et al. / Bioorg. Med. Chem. 19 (2011) 2641–2649
2649
7.46 (d, J = 7.6 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 7.6 Hz,
1H), 7.94 (d, J = 8 Hz, 1H), 8.05 (d, J = 9.2 Hz, 1H), 11.55 (s, 1H),
12.14 (s, 1H), 12.39 (s, 1H). Anal. Calcd for C24H21N5O4: C, 65.00;
H, 4.77; N, 15.79. Found: C, 64.75; H, 4.83; N, 15.87.
(s, 3H), 7.09–7.12 (m, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.89–7.91 (m,
3H), 8.02 (d, J = 7.6 Hz, 2H), 12.34 (s, 1H), 13.28 (s, 1H), 13.52 (s,
1H). Anal. Calcd for C23H19N5O3: C, 66.82; H, 4.63; N, 16.94. Found:
C, 66.74; H, 4.71; N, 17.02.
5.1.43. (L)-2-(3,4-Dimethoxyphenyl)-N-(2-(4-
nitrophenyl)pentan-3-yl)-1H-benzimidazole-4-carboxamide
(47)
5.1.49. 2-(3,4-Dimethoxyphenyl)-N-(3-hydroxypyridin-2-yl)-
1H-benzimidazole-4-carboxamide (53)
Compound 53 was synthesized from 3-hydroxypyridin-2-amine
and compound 12 using general procedure B as a slightly yellow
solid. Yield = 62%. 1H NMR (DMSO, 400 MHz) d: 3.85 (s, 3H), 3.92
(s, 3H), 7.14–7.20 (m, 3H), 7.40 (t, J = 7.6 Hz, 1H), 7.83 (d,
J = 8 Hz, 1H), 7.89–7.91 (m, 2H), 7.96–7.98 (m, 2H), 11.14 (s, 1H),
13.10 (s, 1H), 13.48 (s, 1H). Anal. Calcd for C21H18N4O4: C, 64.61;
H, 4.65; N, 14.35. Found: C, 64.78; H, 4.58; N, 14.40.
Compound 47 was synthesized from (L)-2-amino-1-(4-nitro-
phenyl)propane-1,3-diol and compound 12 using general proce-
dure B as a white solid. Yield = 80%. 1H NMR (DMSO, 400 MHz) d:
3.60 (q, J = 5.2 Hz, 1H), 3.69 (q, J = 5.2 Hz, 1H), 3.85 (s, 3H), 3.88
(s, 3H), 4.23 (q, J = 5.6 Hz, 1H), 5.07 (q, J = 4.8 Hz, 1H), 5.28 (d,
J = 4.4 Hz, 1H), 6.20 (d, J = 4.4 Hz, 1H), 7.16 (d, J = 8.8 Hz, 1H),
7.22 (t, J = 7.6 Hz, 1H), 7.63–7.67 (m, 2H), 7.73 (d, J = 8.4 Hz, 1H),
7.86 (d, J = 8.4 Hz, 1H), 8.03–8.08 (m, 3H), 10.53 (d, J = 8.8 Hz,
1H), 12.21 (s, 1H). Anal. Calcd for C25H24N4O7: C, 60.97; H, 4.91;
N, 11.38. Found: C, 61.16; H, 4.87; N, 11.25.
5.2. Antiviral activity evaluation in vitro
The virus strains of CVA16, CVB3, CVB6, and EV71 were pro-
vided by American Type Culture Collection (ATCC). The positive
control drug, RBV was produced by Hubei Keyi Pharmaceutical Fac-
tory. The newly synthesized benzimidazole compounds (14–53)
were dissolved in DMSO and diluted with the culture medium.
VERO cells were planted in 96-well culture plates. After 24 h the
plates were placed in the corresponding virus bulk for 2 h. Then
the solutions of benzimidazole compounds and RBV were added
in the plates and cell wells and virus wells were set simulta-
neously. When the cytopathic effect (CPE) of virus wells was over
4, the CPE of cell wells was observed. The concentration required
to inhibit virus growth by 50% (IC50) was determined by the
Reed–Muench method.14
5.1.44. 2-(3,4-Dimethoxyphenyl)-N-(1H-imidazol-4-yl)-1H-
benzimidazole-4-carboxamide (48)
Compound 48 was synthesized from 1H-imidazol-4-amine and
compound 12 using general procedure B as a slightly yellow solid.
Yield = 63%. 1H NMR (DMSO, 400 MHz) d: 3.86 (s, 3H), 3.93 (s, 3H),
7.23 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.83 (d, J = 8 Hz, 1H),
7.87 (d, J = 8 Hz, 1H), 7.93 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 8.43 (d,
J = 2.4 Hz, 1H), 8.49 (t, J = 2.8 Hz, 1H), 9.64 (s, 1H), 13.10 (s, 1H),
13.49 (s, 1H). Anal. Calcd for C19H17N5O3: C, 62.80; H, 4.72; N,
19.27. Found: C, 62.63; H, 4.86; N, 19.37.
5.1.45. 2-(3,4-Dimethoxyphenyl)-N-(1H-imidazol-2-yl)-1H-
benzimidazole-4-carboxamide (49)
Acknowledgment
Compound 49 was synthesized from 1H-imidazol-2-amine and
compound 12 using general procedure B as a slightly yellow solid.
Yield = 65%. 1H NMR (DMSO, 400 MHz) d: 3.83 (s, 3H), 3.88 (s, 3H),
7.14–7.21 (m, 2H), 7.29 (t, J = 8 Hz, 1H), 7.40 (s, 1H), 7.68 (d,
J = 7.6 Hz, 1H), 7.78–7.81 (m, 2H), 7.91 (t, J = 6.8 Hz, 1H), 9.34 (s,
1H), 13.27 (s, 1H). Anal. Calcd for C19H17N5O3: C, 62.80; H, 4.72;
N, 19.27. Found: C, 62.69; H, 4.76; N, 19.35.
This work was supported by Shanghai Biomedical Project (No.
09431902400).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.007. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
5.1.46. 2-(3,4-Dimethoxyphenyl)-N-(4,5-dicyano-1H-imidazol-
2-yl)-1H-benzimidazole-4-carboxamide (50)
Compound 50 was synthesized from 4,5-dicyano-1H-imidazol-
2-amine and compound 12 using general procedure B as a slightly
yellow solid. Yield = 60%. 1H NMR (DMSO, 400 MHz) d: 3.83 (s, 3H),
3.88 (s, 3H), 7.15 (d, J = 8.8 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.41 (d,
J = 7.6 Hz, 1H), 7.69 (d, J = 8 Hz, 1H), 7.82 (t, J = 8 Hz, 1H), 7.91 (d,
J = 6.8 Hz, 1H), 9.34 (s, 1H), 13.20 (s, 1H). Anal. Calcd for
References and notes
1. Chassaing, C.; Berger, M.; Heckeroth, A.; Ilg, T.; Jaeger, M.; Kern, C.; Schmid, K.;
Uphoff, M. J. Med. Chem. 2008, 51, 1111.
2. Jain, K. S.; Shah, A. K.; Bariwal, J.; Shelke, S. M.; Kale, A. P.; Jagtap, J. R.; Bhosale,
A. V. Bioorg. Med. Chem. 2007, 15, 1181.
3. Köhler, P. Int. J. Parasitol. 2001, 31, 336.
4. Satz, A. L.; White, C. M.; Beerman, T. A.; Bruice, T. C. Biochemistry 2001, 40,
6465.
C21H15N7O3: C, 61.01; H, 3.66; N, 23.72. Found: C, 61.25; H, 3.57;
N, 23.68.
5. Santti, J.; Harvala, H.; Kinnunen, L.; Hyypiä, T. J. Gen. Virol. 2000, 81, 1361.
6. Zhang, Y.; Zhu, Z.; Yang, W.; Ren, J.; Tan, X.; Wang, Y.; Mao, N.; Xu, S.; Zhu, S.;
Cui, A.; Zhang, Y.; Yan, D.; Li, Q.; Dong, X.; Zhang, J.; Zhao, Y.; Wan, J.; Feng, Z.;
Sun, J.; Wang, S.; Li, D.; Xu, W. Virol. J. 2010, 7, 94.
7. Chen, S. P.; Huang, Y. C.; Li, W. C.; Chiu, C. H.; Huang, C. G.; Tsao, K. C.; Lin, T. Y. J.
Microbiol. Immunol. Infect. 2010, 43, 99.
5.1.47. 2-(3,4-Dimethoxyphenyl)-N-(pyrazin-2-yl)-1H-
benzimidazole-4-carboxamide (51)
Compound 51 was synthesized from pyrazin-2-amine and com-
pound 12 using general procedure B as a slightly yellow solid.
Yield = 60%. 1H NMR (DMSO, 400 MHz) d: 3.83 (s, 3H), 3.88 (s,
3H), 7.10 (d, J = 8.4 Hz, 1H), 7.28 (t, J = 8 Hz, 1H), 7.78 (d,
J = 8.4 Hz, 1H), 7.85 (s, 1H), 7.88–7.93 (m, 3H), 12.27 (s, 1H),
12.55 (s, 1H). Anal. Calcd for C20H17N5O3: C, 63.99; H, 4.56; N,
18.66. Found: C, 63.78; H, 4.62; N, 18.74.
8. Blomqvist, S.; Klemola, P.; Kaijalainen, S.; Paananen, A.; Simonen, M. L.;
Vuorinen, T.; Roivainen, M. J. Clin. Virol. 2010, 48, 49.
9. Merkle, I.; Van Ooij, M. J. M.; Van Kuppeveld, F. J. M.; Glaudemans, D. H. R. F.;
Galama, J. M. D.; Henke, A.; Zell, R.; Melchers, W. J. G. J. Virol. 2002, 76, 9900.
10. Cheng, J.; Xie, J.; Luo, X. Bioorg. Med. Chem. Lett. 2005, 15, 267.
11. Gilbert, B. E.; Knight, V. Antimicrob. Agents Chemother. 1986, 30, 201.
12. Hughes, T. V.; Emanuel, S. L.; O’Grady, H. R.; Connolly, P. J.; Rugg, C.; Fuentes-
Pesquera, A. R.; Karnachi, P.; Alexander, R.; Middleton, S. A. Bioorg. Med. Chem.
Lett. 2008, 18, 5130.
13. Godinez, C. E.; Zepeda, G.; Mortko, C.; Dang, H.; Garcia-Garibay, M. A. J. Org.
Chem. 2004, 69, 1652.
14. Reed, L. J.; Muench, H. Am. J. Hyg. 1938, 27, 493.
5.1.48. 2-(3,4-Dimethoxyphenyl)-N-(1H-benzimidazol-2-yl)-1H-
benzimidazole-4-carboxamide (52)
Compound 52 was synthesized from 1H-benzimidazol-2-amine
and compound 12 using general procedure B as a slightly yellow
solid. Yield = 70%. 1H NMR (DMSO, 400 MHz) d: 3.86 (s, 3H), 3.94