Design and synthesis of novel benzimidazole derivatives as inhibitors of hepatitis B virus

Article abstract of DOI:10.1016/j.bmc.2010.05.076

A series of novel benzimidazole derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activity and cytotoxicity in the HepG2.2.15 cell line. The preliminary SAR was discussed. Compound 12a, with IC₅₀ <0.41 μM and SI >81.2, was the most promising compound and selected as the benchmark compound for further optimization.

Full text of DOI:10.1016/j.bmc.2010.05.076

Bioorganic & Medicinal Chemistry 18 (2010) 5048–5055
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel benzimidazole derivatives as inhibitors
of hepatitis B virus
a,
Yu Luo a, Jia-Ping Yao a, Li Yang b, Chun-Lan Feng b, Wei Tang b, Gui-Feng Wang b, Jian-Pin Zuo b, , Wei Lu
*
*
a Department of Chemistry, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, PR China
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, 555 Zu Chongzhi Road, Shanghai 201203, 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 synthesized and evaluated for their anti-hepatitis B
virus (HBV) activity and cytotoxicity in the HepG2.2.15 cell line. The preliminary SAR was discussed.
Received 17 April 2010
Revised 26 May 2010
Accepted 27 May 2010
Available online 1 June 2010
Compound 12a, with IC50 <0.41
lM and SI >81.2, was the most promising compound and selected as
the benchmark compound for further optimization.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Benzimidazoles
Anti-HBV activity
1. Introduction
ous study involving benzimidazole derivative 1a was screened
which exhibited moderate anti-HBV activity. Our previous study
involving benzimidazole derivative 1a indicated that replacement
of sulfonyl groups with alkyl substituents in N-1 position could
dramatically increase anti-viral activities and selectivity (exempli-
fied by compound 1b and 1c) (Fig. 2).19 However, further research
indicated that the two compounds had very low water-solubility as
well as low oral bioavailability. For these reasons, further studies
were initiated in order to find new benzimidazole derivatives with
better biological profiles.
Hepatitis B virus (HBV) infection frequently results in both
acute and chronic hepatitis and remains a significant health prob-
lem worldwide.1 Chronic HBV infection will often lead to viral per-
sistence in the absence of a strong antibody or cellular immune
response. The number of chronic HBV-infected people is estimated
to be more than 400 million worldwide, with about 2 million peo-
ple carriers of hepatitis B surface antigen (HBsAg). HBV is also asso-
ciated with a high risk of developing cirrhosis and hepatocellular
carcinoma, with roughly 4 million deaths from the resulting cirrho-
sis and hepatocellular carcinoma every year.2,3
In
2, which was previously reported by Garuti et al.20 was identified
as an anti-HBV inhibitor with an IC50 of 1.37 M and selectivity
a recent screening, another benzimidazole derivative
The drugs available for the clinical treatment of hepatitis B could
be categorized as interferons and nucleotides analogues. Although,
l
index of 131.8. This compound with pyridine moiety exhibited bet-
ter water-solubility and oral bioavailability than the previous
counterparts. Besides, some previous literatures reported that
benzimidazole analogues with carboxyl group in position 5 exhib-
ited good antiviral activities.21–23 Moreover, a recent report sug-
gested N-methyl-2-pyrrol in place of 2-pyridyl moiety exhibited
good or better antiviral activity.24
In the light of the above reports and studies, compound 2 was
chosen as the new benchmark compound for further optimization.
And our strategies for structure modification were as following
(Fig. 3): (1) replacement of sulfonyl groups with alkyl groups in
N-1 position; (2) introduction of hydrophilic groups into the
phenyl ring to probe its effects on the anti-HBV activity; (3) inves-
tigation of the different alkyl linker between the benzimidazole
moiety and the pyridine; (4) replacement of 2-pyridyl group with
N-methyl-2-pyrrolly moiety to probe the effect on the anti-HBV
activities. In this paper, we report a series of novel benzimidazole
derivatives and their anti-HBV activity.
a-interferon is effective in treating chronic HBV, the side effects
are also serious, such as influenza-like symptoms, depression and
insomnia.4 The nucleoside analogues such as lamivudine (LAM),
adefovir (ADV) and entecavir have the advantages of oral adminis-
tration and fewer side effects (Fig. 1). Some defects (such as exacer-
bations of hepatitis, limited efficacy and high relapse rate) are still
existing.5–7 To search for new anti-HBV reagents with novel mecha-
nisms, several kinds of non-nucleotide anti-HBV compounds have
been investigated recently.8–14
Benzimidazole derivatives are of wide interest because of their
diverse biological activity and clinical applications.15–18 Our previ-
* Corresponding authors. Tel./fax: +86 50806701 (J.-P.Z.); tel./fax: +86 21
62602475 (W.L.).
E-mail addresses: jpzuo@mail.shcnc.ac.cn (J.-P. Zuo), wlu@chem.ecnu.edu.cn (W.
Lu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.076

Y. Luo et al. / Bioorg. Med. Chem. 18 (2010) 5048–5055
5049
NH2
NH2
NH2
N
N
N
N
N
N
N
N
O
N
H
N
S
H2N
HO
O
O
PO(OH)2
O
PO(OH)2
PMEA
Lamivudine
PMEDAP
NH2
O
N
N
N
N
N
HN
O
P
N
O
O
O
H2N
N
O
O
O
O
HO
OH
adefovir dipivoxyl
entecavir
Figure 1. The structures of major antiviral agents for HBV.
O
Cl
Cl
N
N
N
O
O
N
N
N
S
1b
O
O
O
O
Cl
N
N
O2N
1a
N
Cl
O
1c
Figure 2. Compounds 1a, 1b and 1c.
3
4
R'
N
5
6
N
2
alkyl
N
N
alkyl
N
N
SO2
1
7
N
N
O2N
N
2
alkyl
Figure 3.
condensation with propanedioic acid and hydrogenation in an
overall yield of 49%. The base fragments 8a–c were prepared
according to the reported methods.25–27 Thereafter, compound
8a–c was condensed with different aromatic acids (compounds 5,
7, 9 and 10) using EDAc, and subsequently refluxed in acetic acid
to produce the target molecule (Scheme 1).28,29
Derivatives with substituents in position 5 was synthesized
from intermediates 16a,b, which were prepared from 4-fluoro-3-
nitro-benzoic acid methyl ester (15) according to reported
methods.30,31 Thus, compounds 16a,b were transformed into benz-
imidazole compounds 17a,b using abovementioned methods. The
2. Results and discussion
2.1. Chemistry
The synthesis of one subseries of benzimidazole derivatives
were illustrated in Scheme 1. Two acid fragments (compounds 5
and 7) were first prepared from pyridine-2-aldehyde (3) and
N-methyl-2-pyrrolecarboxaldehyde (6), respectively. Thus, pyri-
dine-2-aldehyde (3) was subjected to Wittig–Horner reaction,
hydrogenation and subsequent hydrolysis to give compound 5 in
81% overall yield. The pyrrol compound 7 was prepared via

5050
Y. Luo et al. / Bioorg. Med. Chem. 18 (2010) 5048–5055
CH3
O
b
CH3
COOH
COOH
a
+
H3CO
OCH3
OCH3
N
CH3
P
O
N
CH3
O
N
N
O
5
3
6
7
4
NH2
NH2
N
H
8a
N
H
8b
NH2
N
N
N
N
N
H3C
N
H
8c
N
c
R
11a-c
13a-c
12a-c
R
N
CH3
N
N
N
N
R
CH3
HOOC
HOOC
N
HOOC
N
N
R
9
14a-c
5
HOOC
a R = Bn
b R = cyclohexyl
N
CH3
N
7
10
c
R = isopropyl
Scheme 1. Reagents and conditions: (a) (i) NaH, THF; (ii) Pd/C, H2, MeOH; (iii) HCl, reflux; (b) (i) malonic acid, piperidine, pyridine, reflux; (ii) Pd/C, H2, MeOH; (c) (i) DMAP,
EDAc, CH2Cl2; (ii) AcOH, reflux.
esters 17a,b were hydrolyzed smoothly to give acids 18a,b
(Scheme 2). Finally, acid 18a was condensed with amines to afford
two amides 19a,b in good yields (Scheme 3).
O
a
N
N
N
R1
19a R1 = H
19b
18a
N
R1 = CH3
2.2. In vitro studies
Bn
19a,b
All the synthesized benzimidazole derivatives were evaluated
for their anti-HBV activity and cytotoxicity in HepG 2.2.15 cells,
with the antiviral drug lamivudine as reference control.
Scheme 3. Reagents and conditions: (a) amine hydrochloride, DMAP, EDAc, CH2Cl2.
To identify the effects of the substituents in N-1 position as well
as replaced 2-pyrrolyl counterparts, two subseries of derivatives
11a–c and 12a–c were synthesized and assessed for anti-HBV
activity (Table 1). Compounds 11a–c with different alkyl substitu-
ents on position 1 were first prepared and the pharmaceutical pro-
files were tested. The derivatives 11b and 11c exhibited moderate
antiviral activities, compared with the reference drug lamivudine,
suggesting N-1 position might be compatible with a broad range
of substituents of different steric characteristics.
Afterwards, the effects of alkyl linker between the benzimid-
azole and pyridyl moiety were investigated (Table 2). For com-
pounds 13a–c, the length of the alkyl linker was reduced from
ethylene to methylene. Obviously, their anti-HBV activity was dra-
matically reduced. In addition, compounds 14a–c with one methyl
group on the ethylene chain also exhibited low anti-HBV activity.
The above results suggested that both length and flexibility of
the alkyl linker were important to the antiviral activity. Thus,
derivatives with relatively good activities should bear a flexible
two-carbon alkyl linker.
However, it was interesting to note that 2-pyrrolyl compound
12a (IC50 = 0.41
lM, SI = 81.2) showed dramatically increased anti-
To identify the effects of different polarized substituents on po-
sition 5, six benzimidazole compounds (17–19) were synthesized.
However, most of them exhibited poor antiviral activities. The es-
ter substituent (17a) with less hydrophilic property indicated
much higher antiviral activity than other carboxylic derivatives
viral activity. Although the anti-HBV activity of compound 12a
(IC50 = 0.41
l
M) was slightly less potent than the reference control
M), its cytotoxicity (CC50 = 33.3 M) was over six
M), resulting in nearly three
times higher selectivity than lamivudine.
(IC50 = 0.16
l
l
times less than lamivudine (CC50 = 5
l
H3COOC
NO2
ref. 30,31
H3COOC
N
H3COOC
NH2
a
N
R
N
N
R
F
17a,b
H
15
16a,b
HOOC
b
N
N
N
a R = Bn
b R = methyl
R
18a,b
Scheme 2. Reagents and conditions: (a) (i) DMAP, EDAc, CH2Cl2; (ii) AcOH, reflux; (b) 6 mol/L HCl, 60 °C.

Y. Luo et al. / Bioorg. Med. Chem. 18 (2010) 5048–5055
5051
Table 1
Table 3
Anti-HBV activity and cytotoxicity of analogues 11a–c and 12a–c in vitro
Anti-HBV Activity and Cytotoxicity of Analogues 17a,b, 18a,b and 19a,b in vitro
HOOC
N
N
H3COOC
N
N
N
N
N
N
H3C
N
N
N
N
R
17a,b
19a,b
R
R
R
18a,b
11a-c
12a-c
O
a
b
Compound
R
CC50
33.3
(lM)
IC50
(lM)
SIc
—
N
N
N
N
R1
11a
NCd
Bn
11b
11cf
>100
>100
6.6
8.6
>15.2
>11.6
–CH(CH3)2
–CH(CH3)2
a
b
Compound
R
R1
CC50
(l
M)
IC50
2.1
(
l
M)
SIc
12a
33.3
<0.41
81.2
17a
17b
—
—
>100
>100
>47.6
12b
12.5
>11.1
1.1
–CH3
NAd
—
—
12c
Lamivudine
>100
5
NAe
0.16
—
31.3
18a
—
>100
>100
18b
19a
19b
–CH3
—
—
—
H
–CH3
>100
>100
>100
NA
NA
>100
—
—
‘—‘ The relevant SI cannot be calculated.
a
Concentrations of compounds required for 50% extinction of HepG 2.2.15 cells.
Concentrations of compounds achieving 50% inhibition of cytoplasmic HBV-
b
DNA synthesis.
‘—‘ The relevant SI cannot be calculate.
c
Selectivity index (SI) was determined as the CC50/IC50 value.
a
Concentrations of compounds required for 50% extinction of HepG 2.2.15 cells.
Concentrations of compounds achieving 50% inhibition of cytoplasmic HBV-
d
e
f
IC50 can not be calculated.
b
Not active.
DNA synthesis.
The oily free base 11c was converted to its hydrochloride salt to afford a solid.
c
Selectivity index (SI) was determined as the CC50/IC50 value.
Not active.
d
Table 2
Anti-HBV activity and cytotoxicity of analogues 13a–c and 14a–c in vitro
17a showed potent antiviral activities (IC50 <0.41
lM and IC50 =
2.1 M, respectively) and high selectivity (SI >81.2 and SI = 47.6,
l
respectively). Further studies of SAR based on 12a are going on
in our group.
N
N
N
N
N
R
CH3
N
R
4. Materials and methods
4.1. General
13a-c
14a-c
SIc
—
a
b
Compound
R
CC50
33.3
(
l
M)
IC50
NA
(lM)
1H NMR spectral data were recorded in DMSO-d6, CDCl3 and
D2O on Varian Mercury 500 NMR spectrometer and 13C NMR data
were recorded in DMSO-d6, CDCl3 and D2O on Varian Mercury 100
NMR spectrometer. Chemical shifts (d) are reported in parts per
million (ppm), and the signals are described as s (singlet), d (dou-
blet), t (triplet), q (quarter), m (multiple), and dd (doublet of dou-
blet). Coupling constants (J values) are given in Hz. Low-resolution
mass spectra (MS) and high-resolution mass spectra (HRMS) were
recorded at an ionizing voltage of 70 eV on a Agilent/5973 N spec-
trometer and Waters Micromass GCT. Column chromatography
was carried out on silica gel (300 mesh). Compounds 9 and 10 were
purchased from Aldrich. All reactions were monitored using thin-
layer chromatography (TLC) on silica gel plates.
13a32
13b
13c
33.3
>18.2
NAd
—
—
–CH(CH3)2
–CH(CH3)2
>100
33.3
14a
NA
—
14b
14c
33.3
NA
—
—
>100
>100
‘—‘ The relevant SI cannot be calculated.
a
Concentrations of compounds required for 50% extinction of HepG 2.2.15 cells.
Concentrations of compounds achieving 50% inhibition of cytoplasmic HBV-
b
DNA synthesis.
c
Selectivity index (SI) was determined as the CC50/IC50 value.
Not active.
4.2. Synthesis
d
4.2.1. Preparation of 2-methyl-3-(pyridin-2-yl)propanoic
acid (5)
(18a,b and 19a,b), suggesting that lipophilic property was an
important determinant for the anti-HBV activity (Table 3).
To a suspension of NaH (7 g, 0.17 mol) in dry THF at 0 °C was
added dropwise methyl 2-(dimethoxyphosphoryl)propanoate
(13 g, 0.06 mol) and stirred for 1 h. Then picolinaldehyde (5.5 mL,
0.06 mol) was dropped into the mixture and stirred at room tem-
perature for 6 h. After the reaction was completed, the mixture
was poured into water (100 mL) and extracted with dichlorometh-
ane (10 mL ꢀ 3). The organic layer was separated and washed with
saturated brine (10 mL ꢀ 2), and dried over Na2SO4 and evaporated
3. Conclusion
In summary, a series of novel benzimidazole analogues were
prepared and assessed for their anti-HBV activity and cytotoxicity
in vitro. The preliminary SAR was discussed. Compounds 12a and

5052
Y. Luo et al. / Bioorg. Med. Chem. 18 (2010) 5048–5055
to dryness to give a residue, which was hydrogenated (1 atm.) on
10% Pd/C (0.8 g) in methanol for 7 h. The Pd/C was filtrated off
and the organic phase was concentrated under reduced pressure
to give a pale yellow residue. The residue was suspended in
6 mol/L HCl (40 mL) and heated at 60 °C for 5 h. The mixture was
treated with aqueous 6 mol/L NaOH (40 mL). The resulting solution
was extracted with ethyl acetate (10 mL ꢀ 3). The organic portions
were combined, dried (MgSO4) and filtered. The solution was con-
centrated to give compound 5 (8.1 g, 81%) as a white solid, mp
100–101 °C. 1H NMR (DMSO-d6): d 8.43–8.45 (d, J = 4.5 Hz, 1H),
7.54–7.57 (m, 1H), 6.99–7.15 (m, 2H), 3.15–3.19 (m, 1H), 3.01–
3.05 (m, 1H), 2.79–2.83 (m, 1H), 1.02 (d, J = 7 Hz, 3H). LRMS (EI):
m/z 137 [M]+. HRMS (EI): calcd for C29H11NO2 165.0790, found
165.0789.
153.86, 149.27, 143.22, 136.46, 133.65, 123.36, 121.39, 121.37,
121.21, 119.34, 111.87, 55.88, 36.05, 31.16, 27.41, 25.97, 25.27.
LRMS (EI): m/z 305.1 [M]+. HRMS (EI): calcd for C20H23N3 305.1892,
found 305.1890.
4.2.3.3. 1-Isopropyl-2-[2-(pyridin-2-yl)-ethyl]-1H-benzoimidaz-
ole (11c). The crude product was purified through a silica gel col-
umn (ethyl acetate/petroleum ether = 3:1) to afford light a yellow
oily free base (60 mg, 30% yield). 1H NMR (DMSO-d6): d 8.71 (d,
J = 4.5 Hz, 1H), 8.25–8.26 (m, 1H), 8.13 (d, J = 7.5 Hz, 1H), 7.80–
7.83 (t, J = 15.5 Hz, 2H), 7.69–7.70 (m, 1H), 7.53–7.57 (m, 2H),
5.14–5.19 (m, 1H), 3.75–3.78 (t, J = 15 Hz, 2H), 3.57–3.60 (t,
J = 15 Hz, 2H), 1.65 (d, J = 7 Hz, 6 H). LRMS (EI): m/z 265 [M]+.
A solution of the oily free base in CH2Cl2 was purged into the
hydrochloride gas for 0.5 h, following it was deposited and filtrated
to afford a light a yellow solid (69 mg), mp 63–64 °C. 1H NMR
4.2.2. Preparation of 3-(1-methyl-1H-pyrrol-2-yl)propanoic
acid (7)
(D2O):
d 8.64–8.65 (m, 1H), 8.39 (d, J = 4 Hz, 1H), 7.96 (d,
A mixture of malonic acid (20 g, 0.2 mol), 1-methyl-1H-pyrrole-
2-carbaldehyde (10.8 g, 0.1 mol) and piperidine (1.2 mL, 0.01 mol)
in pyridine (30 mL) was heated under N2 at 90 °C for 3 h then
cooled to room temperature and poured into water. The resulting
aqueous mixture was neutralized with 2 mol/L HCl (25 mL). The
resulting precipitate was collected by filtration to give yellow solid
(7.5 g, 50%). The yellow solid (7.5 g, 0.05 mol) was hydrogenated
(1 atm.) on 10% Pd/C (0.7 g) in methanol for 2 h at room tempera-
ture. Then Pd/C was filtrated off and the organic phase was concen-
trated to dryness to give 7.4 g of compound 7 as a yellow solid (49%
yield), mp 87–89 °C (lit.33 mp 80–82 °C). 1H NMR (CDCl3): d 6.57–
6.58 (t, 1H), 6.06–6.07 (t, 1H), 5.91–5.92 (m, 1H), 3.56 (s, 3H), 2.88
(t, J = 15.6 Hz, 2H), 2.72 (t, J = 15.6 Hz, 2H). LRMS (EI): m/z 153 [M]+.
J = 3.5 Hz, 1H), 7.84–7.85 (m, 2H), 7.68–7.69 (m, 1H), 7.53 (d,
J = 9.5 Hz, 2H), 4.97–5.00 (m, 1H), 3.70–3.73 (m, 2H), 3.58–3.60
(m, 2H), 1.65 (d, J = 6.5 Hz, 6H). 13C NMR (D2O): d 153.40, 150.01,
148.20, 142.52, 131.29, 130.83, 127.95, 127.18, 126.69, 126.66,
115.42, 114.95, 51.90, 30.86, 25.26, 20.84. HRMS (EI): calcd for
C17H20N3Cl 301.1346, found 301.1360.
4.2.4. General procedure for the preparation of compounds
12a–c
Compounds 12a–c were synthesized from compounds 8a–c and
7 using the procedure for the preparation of 11a–c.
4.2.4.1. 1-Benzyl-2-(2-(1-methyl-1H-pyrrol-2-yl)ethyl)-1H-ben-
zoimidazole (12a). The crude product was purified through a sil-
ica gel column (ethyl acetate/petroleum ether = 1:2) to afford a
white solid (143 mg, 60% yield), mp 97–98 °C. 1H NMR (CDCl3): d
7.79 (d, J = 7.5 Hz, 1H), 7.22–7.30 (m, 6H), 7.01 (d, J = 6.5 Hz, 2H),
6.53–6.54 (t, J = 4 Hz, 1H), 6.04–6.05 (t, J = 6 Hz, 1H), 5.88–8.89 (t,
J = 3.5 Hz, 1H), 5.23 (s, 2H), 3.43 (s, 3H), 3.13 (s, 4H). 13C NMR
(CDCl3): d 154.39, 142.60, 135.95, 131.81, 129.01, 127.89, 126.11,
122.49, 122.12, 121.54, 119.33, 109.45, 106.76, 105.72, 46.59,
33.51, 27.45, 24.24. MS (ESI): m/z 316.2 [M+1]+. HRMS (ESI): calcd
for C21H22N3H [M+H]+ 316.1812, found 316.1808.
4.2.3. General procedure for the preparation of compounds
11a–c
A mixture of compounds 8a–c (0.70 mmol) and 9 (1.5 mmol)
was stirred overnight in dry CH2Cl2 at room temperature, with
4-(dimethylamino) pyridine (0.15 mmol) as the catalyst and
1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride
(2.2 mmol) as condensation agent. The resulting solution was
poured into saturated NH4Cl. Then it partitioned between chloro-
form and water. The organic layer was washed with water and
brine, dried over Na2SO4, and concentrated in vacuum.
The immediate product was directly poured into dry acetic acid
and was refluxed for about 5 h. The mixture was neutralized to pH
7 with saturated Na2CO3. The precipitate was filtered, washed with
water, and dried to give the compound 11a–c.
4.2.4.2. 1-Cyclohexyl-2-[2-(1-methyl-1H-pyrrol-2-yl)ethyl]-1H-
benzoimidazole (12b). The crude product was purified through
a silica gel column (ethyl acetate/petroleum ether = 1:2.5) to afford
a yellow solid (145 mg, 62.5% yield), mp 67–68 °C. 1H NMR
(CDCl3): d 7.73–7.75 (m, 1H), 7.52–7.54 (m, 1H), 7.18–7.21 (m,
2H), 6.55–6.56 (t, J = 4 Hz, 1H), 6.08–6.09 (t, J = 6 Hz, 1H), 6.00–
6.01 (t, J = 3 Hz, 1H), 4.12–4.16 (m, 1H), 3.53 (s, 3H), 3.17–3.20
(m, 4H), 2.21–2.24 (m, 2H), 1.94–2.04 (m, 2H), 1.79–1.85 (m,
3H), 1.41–1.43 (m, 2H), 1.25–1.38 (m, 1H). 13C NMR (CDCl3): d
153.50, 143.02, 133.66, 131.89, 121.59, 121.52, 121.38, 119.37,
111.81, 106.77, 105.70, 105.55, 56.25, 33.61, 31.25, 30.94, 28.14,
26.07, 25.26, 24.57. LRMS (EI): m/z 307 [M]+. HRMS (EI): calcd for
4.2.3.1. 1-Benzyl-2-[2-(pyridin-2-yl)-ethyl]-1H-benzoimidazole
(11a). The crude product was recrystallized from ethyl acetate
and petroleum ether to afford a yellow solid (95 mg, 40% yield),
mp 87–88 °C. 1H NMR (CDCl3): d 8.50 (d, J = 5 Hz, 1H), 7.76 (d,
J = 8 Hz, 1H), 7.54 (d, J = 2 Hz, 1H), 7.22–7.26 (m, 4H), 7.16–7.18
(m, 3H), 7.09–7.11 (m, 1H), 6.99–7.01 (m, 1H), 5.32 (s, 2H), 3.43
(t, J = 15 Hz, 2H), 3.33(t, J = 15 Hz, 2H). 13C NMR (CDCl3): d
160.09, 154.63, 149.20 142.68, 136.35, 135.92, 135.29, 128.83,
127.68, 126.12, 123.28, 122.22, 121.91, 121.37, 119.19, 109.55,
46.70, 35.54, 26.75. LRMS (EI): m/z 313.2 [M]+. HRMS (EI): calcd
for C21H19N3 313.1579, found 313.1580.
C20H25N3 307.2049, found 307.2048.
4.2.4.3. 1-Isopropyl-2-(2-(1-methyl-1H-pyrrol-2-yl)ethyl)-1H-ben-
zoimidazole (12c). The crude product was purified through a sil-
ica gel column (ethyl acetate/petroleum ether = 1:1) to afford a
yellow solid (94 mg, 46.5% yield), mp 42–43 °C. 1H NMR (CDCl3):
d 7.74–7.76 (m, 1H), 7.50–7.51 (m, 1H), 7.20–7.22 (m, 2H), 6.56–
6.57 (t, J = 4 Hz, 1H), 6.08–6.09 (t, J = 6 Hz, 1H), 5.99–6.00 (m,
1H), 4.64–4.69 (m, 1H), 3.54 (s, 3H), 3.17–3.21 (m, 4H), 1.60 (d,
J = 7 Hz, 6H). 13C NMR (CDCl3): d 153.36, 143.17, 133.35, 131.89,
121.67, 121.53, 121.44, 119.46, 111.43, 106.74, 105.53, 60.33,
47.73, 33.63, 27.83, 24.47, 21.33. LRMS (EI): m/z 267 [M]+. HRMS
(EI): calcd for C17H21N3 267.1735, found 267.1738.
4.2.3.2. 1-Cyclohexyl-2-[2-(pyridin-2-yl)-ethyl]-1H-benzoimidaz-
ole (11b). The crude product was recrystallized from ethyl acetate
and petroleum ether to afford a yellow solid (79 mg, 34% yield), mp
120–121 °C. 1H NMR (CDCl3): d 8.57 (d, J = 4 Hz, 1H), 7.73–7.74 (t,
J = 8.5 Hz, 1H), 7.56–7.72 (m, 1H), 7.51–7.53 (m, 1H), 7.13–7.20 (m,
4H), 4.24–4.26 (m, 1H), 3.41–3.45 (m, 2H), 3.35–3.39 (m, 2H),
2.17–2.22 (m, 2H), 1.91–1.94 (m, 2H), 1.74–1.80 (m, 3H), 1.39–
1.45 (m, 2H), 1.29–1.39 (m, 1H). 13C NMR (CDCl3): d 160.31,

Y. Luo et al. / Bioorg. Med. Chem. 18 (2010) 5048–5055
5053
4.2.5. General procedure for the preparation of compounds
13a–c
Compounds 13a–c were synthesized from compounds 8a–c and
10 using the procedure for the preparation of 11a–c.
1H), 1.76–1.83 (m, 2H), 1.64–1.66 (m, 1H), 1.53–1.57 (m, 3H),
1.46–1.47 (m, 3H), 1.40–1.42 (m, 1H). 13C NMR (DMSO-d6): d
154.86, 153.33, 144.06, 143.52, 131.22, 129.90, 127.02, 125.73,
125.24, 124.84, 115.17, 114.39, 57.35, 38.09, 30.69, 30.08, 29.92,
24.84, 24.80, 24.06, 18.70. LRMS (EI): m/z 319 [M]+. HRMS (EI):
calcd for C21H25N3 319.2050, found 319.2048.
4.2.5.1.
1-Benzyl-2-(pyridin-2-yl-methyl)-1H-benzoimidazole
(13a). The crude product can be used directly without further
purification as a yellow solid (91 mg, 40% yield), mp 137–138 °C.
1H NMR (DMSO-d6): d 8.48–8.49 (m, 1H), 7.85–7.93 (m, 1H),
7.72–7.84 (m, 1H), 7.67–7.70 (m, 2H), 7.49–7.57 (m, 2H), 7.31–
7.41 (m, 1H), 7.18–7.31 (m, 5H), 5.89 (s, 2H), 5.03 (s, 2H). 13C
4.2.6.3. 1-Isopropyl-2-[1-(pyridin-2-yl)propan-2-yl]-1H-benzo-
imidazole (14c). The crude product was purified through a silica
gel column (ethyl acetate/petroleum ether = 1:1) to afford 14c as a
red solid (85 mg, 40% yield), mp 140–141 °C. 1H NMR (DMSO-d6): d
8.66 (d, J = 4.5 Hz, 1H), 8.22–8.23 (m, 1H), 8.12 (d, J = 8 Hz, 1H),
7.94 (d, J = 7.5 Hz, 1H), 7.76 (d, J = 7.5 Hz, 1H), 7.67–7.68 (m, 1H),
7.52–7.58 (m, 2H), 5.21–5.26 (m, 1H), 4.34–4.40 (m, 1H), 3.76–
3.81 (m, 1H), 3.54–3.59 (m, 1H), 1.64 (d, J = 7 Hz, 3H), 1.55–1.56
(d, J = 7 Hz, 3H), 1.49–1.50 (d, J = 7 Hz, 3H). 13C NMR (DMSO-d6):
d 154.62, 153.34, 144.07, 143.64, 131.21, 129.66, 127.05, 125.81,
125.37, 124.88, 114.81, 114.45, 50.12, 30.78, 20.53, 20.43, 18.44.
LRMS (EI): m/z 279 [M]+. HRMS (EI): calcd for C18H21N3 279.1735,
found 279.1732.
NMR (DMSO-d6):
d 150.72, 147.25, 147.02, 134.12, 131.99,
130.95, 128.70, 128.62, 128.04, 127.10, 126.82, 126.07, 125.73,
125.38, 123.94, 114.62, 113.16, 47.94, 32.02. LRMS (EI): m/z 299
[M]+. HRMS (EI): calcd for C20H17N3 299.1422, found 299.1426.
4.2.5.2. 1-Cyclohexyl-2-(pyridin-2-yl-methyl)-1H-benzoimidaz-
ole (13b). The crude product was purified through a silica gel col-
umn (ethyl acetate/petroleum ether = 1:1) to afford 13b as a light
yellow solid (86 mg, 39% yield), mp: 180–181 °C. 1H NMR
(DMSO-d6): d 8.49–8.50 (m, 1H), 8.17–8.19 (m, 1H), 7.94–7.97
(m, 1H), 7.84–7.85 (m, 1H), 7.71–7.73 (m, 1H), 7.53–7.59 (m,
2H), 7.40–7.42 (m, 1H), 5.00 (s, 2H), 4.58 (m, 1H), 2.14–2.21 (m,
2H), 1.78–1.80 (m, 2H), 1.70–1.73 (m, 2H), 1.61–1.63 (m, 1H),
1.28–1.41 (m, 3H). 13C NMR (DMSO-d6): d 152.73, 150.09, 148.02,
139.35, 130.73, 130.08, 125.77, 125.40, 124.72, 123.63, 115.03,
114.48, 57.71, 32.85, 29.58, 24.94, 24.01. LRMS (EI): m/z 291
[M]+. HRMS (EI): calcd for C19H21N3 291.1735, found 291.1736.
4.2.7. General procedure for the preparation of compounds 17a
and 17b
Compounds 17a and 17b were synthesized from compounds
16a,b, respectively, and compound 9 using the procedure for the
preparation of 11a–d.
4.2.7.1. Methyl 1-benzyl-2-(2-(pyridin-2-yl)ethyl)-1H-benzoimi-
dazole-5-carboxylate (17a). The crude product was purified
through a silica gel column (ethyl acetate/petroleum ether = 3:1)
to afford 17a as a pale yellow solid (118 mg, 42% yield), mp 122–
123 °C. 1H NMR (CDCl3): d 8.47–8.50 (m, 1H), 7.91–7.93 (m, 1H),
7.53–7.55 (m, 1H), 7.26–7.27 (m, 3H), 7.17–7.21 (m, 2H), 7.09–
7.11 (m, 1H), 6.98–7.00 (m, 2H), 5.34 (s, 2H), 3.93 (s, 3H), 3.40–
3.44 (t, J = 18.7 Hz, 2H), 3.33–3.36 (t, J = 18.7 Hz, 2H); 13C NMR
(CDCl3): d 167.66, 159.87, 156.59, 149.25, 142.33, 138.63, 136.45,
135.45, 129.01, 127.97, 126.15, 124.26, 124.03, 123.40, 121.57,
121.51, 109.26, 52.01, 46.99, 35.32, 26.76. LRMS (EI): m/z 371
[M]+. HRMS (EI): calcd for C23H21N3O2 371.1634, found 371.1635.
4.2.5.3. 1-Isopropyl-2-(pyridin-2-yl-methyl)-1H-benzoimidazole
(13c). The crude product was purified through a silica gel column
(ethyl acetate/petroleum ether = 1:2) to afford 13c as a light yellow
solid (78 mg, 41% yield), mp 139–140 °C. 1H NMR (CDCl3): d 8.53
(d, J = 4 Hz, 1H), 7.75–7.77 (m, 1H), 7.57–7.58 (m, 1H), 7.49–7.50
(m, 1H), 7.15–7.26 (m, 4H), 4.86–4.92 (m, 1H), 4.53 (s, 2H), 1.45
(d, J = 7 Hz, 6H). 13C NMR (CDCl3): d 156.77, 151.42, 149.05,
143.10, 136.71, 133.28, 123.22, 121.84, 121.79, 121.44, 119.60,
111.69, 48.16, 37.80, 20.77. LRMS (EI): m/z 251 [M]+. HRMS (EI):
calcd for C16H17N3 251.1422, found 251.1423.
4.2.6. General procedure for the preparation of compounds
14a–c
Compounds 14a–c were synthesized from compounds 8a–c and
5 using the procedure for the preparation of 11a–c.
4.2.7.2. Methyl 1-methyl-2-[2-(pyridin-2-yl)ethyl]-1H-benzoim-
idazole-5-carboxylate (17b). The crude product was purified
through a silica gel column (ethyl acetate/methanol = 30:1) to af-
ford 17b as a yellow solid (100 mg, 45% yield), mp 111–112 °C.
1H NMR (CDCl3): d 8.55 (d, J = 5 Hz, 1H), 8.44–8.45 (m, 1H), 7.97–
7.99 (m, 1H), 7.57–7.59 (m, 1H), 7.277.29 (m, 1H), 7.16–7.20 (m,
1H), 7.14–7.15 (m, 1H), 3.94 (s, 3H), 3.68 (s, 3H), 3.37–3.43 (m,
4H). 13C NMR (CDCl3): d 174.02, 167.67, 159.70, 156.39, 149.05,
141.80, 138.77, 136.74, 124.04, 123.79, 123.44, 121.65, 121.22,
108.66, 51.98, 35.35, 29.83, 26.79. LRMS (EI): m/z 295 [M]+. HRMS
(EI): calcd for C17H17N3O2 295.1321, found 295.1319.
4.2.6.1. 1-Benzyl-2-[1-(pyridin-2-yl)propan-2-yl]-1H-benzoimi-
dazole (14a). The crude product can be used directly without fur-
ther purification as a yellow solid (101 mg, 41% yield), mp 131–
132 °C. 1H NMR (DMSO-d6): d 8.60 (d, J = 5 Hz, 1H), 8.20–8.21 (d,
J = 7 Hz, 1H), 7.81–7.83 (m, 2H), 7.72–7.74 (m, 1H), 7.67–7.68 (m,
1H), 7.49–7.55 (m, 2H), 7.19–7.31 (m, 3H), 7.16–7.18 (m, 2H),
5.80–5.92 (m, 2H), 4.31–4.35 (m, 1H), 3.81–3.86 (m, 1H), 3.53–
3.60 (m, 1H), 1.32 (d, J = 7 Hz, 3H). 13C NMR (DMSO-d6): d
155.75, 153.01, 144.20, 143.45, 134.65, 131.89, 131.12, 128.95,
128.16, 126.96, 126.86, 126.14, 125.79, 124.90, 114.48, 113.18,
47.68, 37.67, 30.76, 18.48. LRMS (EI): m/z 327 [M]+. HRMS (EI):
calcd for C22H21N3 327.1735, found 327.1731.
4.2.8. General procedure for the preparation of compounds 18a
and 18b
Compound 17a or 17b (0.32 mmol) was suspended in 6 mol/L
HCl (5 mL) and heated at 60 °C for 5 h. The mixture was treated
with aqueous 6 mol/L NaOH (5 mL). The formed precipitate was fil-
tered, washed with water, and dried to give the target compound.
4.2.6.2. 1-Cyclohexyl-2-[1-(pyridin-2-yl)propan-2-yl]-1H-benzo-
imidazole (14b). The crude product was purified through a silica
gel column (ethyl acetate/petroleum ether = 1:1) to afford 14b as a
yellow solid (104 mg, 43% yield), mp 153–154 °C. 1H NMR (DMSO-
d6): d 8.65–8.66 (m, 1H), 8.21–8.22 (m, 1H), 8.12 (d, J = 8 Hz, 1H),
7.80 (d, J = 8 Hz, 1H), 7.75–7.76 (m, 1H), 7.66–7.67 (m, 1H), 7.47–
7.54 (m, 2H), 4.74–4.79 (m, 1H), 4.49–4.50 (m, 1H), 3.74–3.78
(m, 1H), 3.55–3.59 (m, 1H), 2.14–2.20 (m, 2H), 1.92–1.95 (m,
4.2.8.1. 1-Benzyl-2-[2-(pyridin-2-yl)ethyl]-1H-benzoimidazole-
5-carboxylic acid (18a). The crude product can be used directly
without further purification as a white solid (94 mg, 83% yield),
mp 198–199 °C. 1H NMR (DMSO-d6): d 8.46–8.47 (m, 1H), 8.16–
8.17 (m, 1H), 7.79 (d, J = 7.5 Hz, 1H), 7.67 (s, 1H), 7.53 (d,
J = 7.5 Hz, 1H), 7.26–7.30 (m, 4H), 7.19–7.20 (m, 1H), 7.08–7.09
(d, J = 5.5 Hz, 2H), 5.50 (s, 2H), 3.29–3.31 (m, 4H). 13C NMR

5054
Y. Luo et al. / Bioorg. Med. Chem. 18 (2010) 5048–5055
(DMSO-d6): d 171.64, 159.87, 156.69, 148.89, 141.83, 140.56,
140.16, 138.55, 136.61, 136.40, 128.77, 127.52, 127.34, 126.44,
123.23, 122.98, 121.45, 120.21, 109.95, 46.14, 34.35, 26.02. LRMS
(EI): m/z 357 [M]+. HRMS (EI): calcd for C22H19N3O2 357.1477,
found 357.1478.
cellular (virion) HBV-DNA were measured by real time fluorescent
PCR.
4.3.2. Toxicity measurements
Cytotoxicity of compounds was assessed by the MTT assay as
previously described.34–36 Briefly, HepG2.2.15 cells were cultured
in triplicate of 96-well tissue culture plates for 8 days with various
doses of tested compounds. The cells with media alone were used
as controls. MTT (5 g/L) reagent was added 4 h before the end of
culture, and then cells were lysed with 10% sodium dodecyl sulfate
(SDS), 50% DMF, pH 7.2. O.D. values were read at 570 nm 6 h later
and the cell death percent was calculated.
4.2.8.2. 1-Methyl-2-[2-(pyridin-2-yl)ethyl]-1H-benzoimidazole-
5-carboxylic acid (18b). The crude product can be used directly
without further purification as a yellow solid (89 mg, 99% yield),
mp 220–221 °C. 1H NMR (DMSO-d6): d 8.70–8.71 (d, J = 4.5 Hz,
1H), 8.29–8.30 (m, 1H), 8.24–8.25 (d, J = 1Hz, 1H), 8.03–8.05 (m,
1H), 7.90–7.94 (m, 2H), 7.72 (d, J = 6 Hz, 1H), 3.99 (s, 3H), 3.66–
3.70 (m, 4H). 13C NMR (DMSO-d6): d 166.66, 154.70, 144.28,
142.95, 135.97, 132.18, 127.57, 126.61, 125.57, 124.72, 116.20,
112.39, 100.08, 31.35, 29.93, 24.76. LRMS (EI): m/z 281 [M]+. HRMS
(EI): calcd for C16H15N3O2 281.1164, found 281.1167.
4.3.3. Real time fluorescent PCR
The supernatants of HepG2.2.15 cells were collected from
8 days culture after the compounds added. The HBV-DNA in the
supernatants was quantified by using fluorescent PCR. Briefly,
4.2.9. General procedure for the preparation of compounds 19a
and 19b
50 lL of the supernatants were added into the extraction buffer,
boiled for 10 min and centrifuged for 5 min, and then proper ali-
quots were used for the fluorescent PCR. PCR primers were:
P1: 50-ATCCTGCTGCTATGCCTCATCTT-30,
To a solution of compound 18a (0.32 mmol) in dry CH2Cl2 was
added methylamine hydrochloride or dimethylamine hydrochlo-
ride (0.6 mmol), with 4-(dimethylamino) pyridine (0.06 mmol) as
the catalyst and 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide
hydrochloride (0.96 mmol) as condensation agent. The resulting
solution was stirred overnight at room temperature and poured
into saturated NH4Cl. Then it partitioned between chloroform
and water. The organic layer was washed with water and brine,
dried over Na2SO4, and concentrated in vacuum.
P2: 50-ACAGTGGGGAAAGCCCTACGAA-30.
The probe was 50-TGGCTAGTTTACTAGTGCCATTTTG-30. PCR
reaction was run at MJ Research PTC-200, and results were ana-
lyzed by software OpticonMonitor v2.01.
Acknowledgments
The authors would like to thank State Key Laboratory of Drug
Research, Shanghai Institute of Medicinal Chemistry, Chinese
Academy of Sciences for carrying out the antiviral screening of
the newly synthesized compounds. This work was supported by
the grants of National Basic Research Program of China (No.
30701033), the grants of Shanghai Natural Science Foundation
(09ZR1438100) and the Open Projects of State Key Lab of Drug Re-
search of the Chinese Academy of Sciences (SIMM0909 KF-08).
4.2.9.1. 1-Benzyl-N-methyl-2-(2-(pyridin- 2-yl)ethyl)-1H-benzo-
imidazole-5-carboxamide (19a). The crude product was purified
through a silica gel column (ethyl acetate/methanol = 10:1) to af-
ford 19a as a pale yellow solid (107 mg, 91.3% yield), mp 83–
84 °C. 1H NMR (CDCl3): d 8.46–8.47 (m, 1H), 8.10–8.11 (m, 1H),
7.67–7.69 (m, 1H), 7.49–7.52 (m, 1H), 7.22–7.25 (m, 3H), 7.96–
7.16 (m, 3H), 6.94–6.95 (m, 2H), 6.47–6,48 (m, 1H), 5.29 (s, 2H),
3.31–3.38 (m, 4H), 2.98 (d, J = 4.5 Hz, 3H). 13C NMR (CDCl3): d
168.72, 159.85, 149.23, 142.29, 136.45, 135.49, 128.98, 127.94,
126.14, 123.37, 121.96, 121.53, 117.71, 109.66, 94.93, 46.98,
35.36, 26.91, 26.75. LRMS (EI): m/z 370 [M]+. HRMS (EI): calcd for
C23H22N4O 370.1794, found 370.1801.
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benzoimidazole-5-carboxamide (19b). The crude product was
purified through silica gel column (ethyl acetate/metha-
1-Benzyl-N,N-dimethyl-2-(2-(pyridin-2-yl)ethyl)-1H-
a
nol = 20:1) to afford 19b as a white solid (114 mg, 93% yield), mp
92–93 °C. 1H NMR (CDCl3): d 8.50 (d, J = 4 Hz, 1H), 7.80–7.81 (m,
1H), 7.54–7.55 (m, 1H), 7.29–7.31 (m, 1H), 7.24–7.26 (m, 4H),
7.20 (s, 1H), 7.17–7.18 (m, 1H), 7.15–7.16 (m, 1H), 6.97–6.99 (m,
2H), 5.32 (s, 2H), 3.42 (t, J = 15 Hz, 2H), 3.34 (t, J = 15 Hz, 2H),
3.12 (d, J = 35 Hz, 3H), 3.05 (s, 3H). 13C NMR (CDCl3): d 172.12,
159.94, 156.02, 149.23, 142.03, 136.42, 136.13, 135.62, 130.18,
128.96, 127.89, 126.17, 123.33, 122.08, 121.46, 118.34, 109.72,
46.93, 35.47, 26.77. LRMS (EI): m/z 384 [M]+. HRMS (EI): calcd for
C24H24N4O 384.1950, found 384.1953.
4.3. Biological assays
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day 4, and the supernatants were harvested at day 8, then the extra-

Y. Luo et al. / Bioorg. Med. Chem. 18 (2010) 5048–5055
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