Synthesis and anti-Hepatitis B virus activity of a novel class of thiazolylbenzimidazole derivatives

Article abstract of DOI:10.1002/ardp.201000167

Recently, heterocyclic benzimidazole derivatives have been investigated and validated as a promising class of antiviral agents. In this paper, a series of novel thiazolylbenzimidazole derivatives was synthesized and evaluated for their anti-hepatitis B vi

Full text of DOI:10.1002/ardp.201000167

78
Arch. Pharm. Chem. Life Sci. 2011, 2, 78–83
Full Paper
Synthesis and Anti-Hepatitis B Virus Activity of a Novel
Class of Thiazolylbenzimidazole Derivatives
Yu Luo1, Jia-Ping Yao1, Li Yang2, Chun-Lan Feng2, Wei Tang2, Gui-Feng Wang2, Jian-Pin Zuo2,
and Wei Lu1,3
1 Department of Chemistry, East China Normal University, Shanghai, P.R. China
2 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai, P.R. China
3 Institute of Drug Discovery and Development, East China Normal University, Shanghai, P.R. China
Recently, heterocyclic benzimidazole derivatives have been investigated and validated as a promising
class of antiviral agents. In this paper, a series of novel thiazolylbenzimidazole derivatives was
synthesized and evaluated for their anti-hepatitis B virus (HBV) activity and cytotoxicity on the
HepG2.2.15 cell line. Afterwards, the preliminary structure–activity relationship (SAR) was
discussed. Compound 8b, with IC50 ¼ 1.1 mM and SI > 90.9, was the most promising compound
and could be selected as a benchmark compound for further investigation.
Keywords: Anti-HBV / Synthesis / Thiazolylbenzimidazole
Received: June 6, 2010; Revised: September 4, 2010; Accepted: September 14, 2010
DOI 10.1002/ardp.201000167
Introduction
Benzimidazole derivatives are of wide interest because of
their diverse biological activities, such as anticancer [12], anti-
viral [10, 11], antihistaminic [13] and antifungal [14] activities.
Recently, a large body of work reported that a set of hetero-
cyclic benzimidazole analogues with the generic structure of
compound 1 showed promising anti-HCV activity (Fig. 2)
[15–18]. However, there is very scarce data on the anti-HBV
activity for these heterocyclic benzimidazole compounds.
Our previous study has indicated that some benzimidazole
derivatives could exhibit potent anti-HBV activity with very
low cytotoxicity [10, 11]. In the light of the above reports and
our continued interest in the study of anti-HBV activity of
benzimidazole compounds, a library of two structurally
related thiazolylbenzimidazole derivatives (compounds 2a
and 2b) was designed and prepared (Fig. 2). Their anti-HBV
activity as well as the cytotoxity on HepG 2.2.15 cells was
evaluated. Herein, we report the details of our investigation
of this class of thiazolylbenzimidazole compounds.
Hepatitis B virus (HBV) infection is one of the leading causes
of lethal infectious diseases worldwide. There are approxi-
mately 400 million people with chronic HBV infection, with a
global death toll of 4 million per year. It can often result in
both acute and chronic hepatitis. For these reasons, intensive
research has focused on treatment during the past three
decades [1]. Currently, only a few drugs are available for
the clinical treatment of HBV infection, including a-inter-
feron (IFN-a) and a series of nucleoside analogues such as
lamivudine (3-TC), adefovir dipivoxyl, and entecavir (Fig. 1). In
spite of tremendous progress in the treatment of HBV infec-
tion, these nucleotide drugs are generally plagued with
limitations such as exacerbations of hepatitis, limited effi-
cacy and high relapse rate [2]. Recently, non-nucleotide anti-
HBV compounds have intrigued many chemists to explore
other kinds of antiviral agents, which might have novel
mechanisms of action [3–11].
Results and discussion
Chemistry
Correspondence: Prof. Wei Lu, East China Normal University, 3663
North Zhongshan Road, Shanghai 200062, P.R. China.
E-mail: wlu@chem.ecnu.edu.cn
All analogues were synthesized from starting materials 4a
and 4b, which were prepared from 4-fluoro-3-nitro-benzoic
acid methyl ester (3) according to reported methods [19].
Fax: þ86 21 62602475
Prof. Jian-Pin Zuo, contributed equally. e-mail: jpzuo@mail.shcnc.ac.cn
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Arch. Pharm. Chem. Life Sci. 2011, 2, 78–83
Anti-HBV Activity of Novel Thiazolylbenzimidazole Derivatives
79
NH2
O
N
N
NH2
N
N
HN
O
P
O
N
N
O
O
O
N
H2N
N
O
N
S
O
O
O
HO
H
HO
O
OH
Lamivudine (3-TC)
adefovir dipivoxyl
entecavir
Figure 1. The structures of some major anti-HBV agents
O
N
S
R2
O
N
R1
N
N
N
R1 = alkyl
R2 = hydroxyl, alkoxy or
amine
R2
2a
2b
5
Heterocycle
1
O
S
R1
1
N
N
R2
N
R1
Figure 2. Design of novel thiazolylbenzimidazole derivatives as anti-HBV agents
Condensation of intermediate 4a and 4b with thiazole-4-
carboxylic acid yielded the thiazolylbenzimidazoles 5a and
5b, respectively. The esters 5a and 5b were then hydrolyzed
smoothly to give acids 6a and 6b, respectively, followed by
coupling with methylamine or dimethylamine to afford
amides 7a, 7b and 8a, 8b (Scheme 1). Compounds 5a, 5b to 8a,
8b constituted the first subseries of thiazolylbenzimidazole
analogue 2a.
Thiazolylmethylbenzimidazoles 9a, 9b, 10a, and 10b (com-
pounds 2) were prepared via initial condensation of inter-
S
N
H3COOC
N
H3COOC
NH2
H3COOC
NO
F
2ref 19
S
HOOC
a
R
N
N
R
N
H
4a,b
3
5a,b
O
N
N
H
S
N
N
HOOC
N
R
S
N
7a,b
c
a R = Bn
b R = Me
b
N
R
O
N
N
N
S
6a,b
N
R
8a,b
Reagents and conditions: (a) (i) DMAP (0.2 equiv.), EDC (3 equiv.), CH2Cl2; (ii) AcOH, reflux, 5 h; (b) 6 M HCl,
60°C; (c) amine, DMAP (0.2 equiv.), EDC (3 equiv.), CH2Cl2.
Scheme 1. Synthesis of compound 2a
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Y. Luo et al.
Arch. Pharm. Chem. Life Sci. 2011, 2, 78–83
S
S
S
N
N
H
COOC
H COOC
3
N
HOOC
3
NH
N
N
N
N
COOH
a
2
b
NH
R
a R = Bn
b R = Me
R
4a,b
R
9a,b
10a,b
Reagents and conditions: (a) (i) DMAP (0.2 equiv.), EDC (3 equiv.), CH2Cl2; (ii) AcOH, reflux, 5 h; (b) 6 M HCl.
Scheme 2. Synthesis of compound 2b
Table 1. In-vitro anti-HBV activity and cytotoxicity of analogues 5a,
5b to 8a, 8b
mediates 4a and 4b with (2-methyl-thiazol-4-yl)-acetic acid
(Scheme 2) to afford analogues 9a and 9b, respectively.
Esters 9a and 9b were hydrolyzed under acidic condition
to produce 10a and 10b, respectively.
O
N
N
S
R
2
In-vitro studies
N
All the synthesized benzimidazole derivatives were evaluated
for their anti-HBV activity and cytotoxity on HepG 2.2.15
cells, with the antiviral drug lamivudine as reference control.
To investigate the effect of the substituents on the N-1 and
C-5 positions, eight compounds were prepared and evaluated
for anti-HBV activity. As shown in Table 1, all these com-
pounds exhibit low cytotoxicity (CC50 ꢀ 33.3 mM) against
HepG 2.2.15 cell, while their antiviral activity is quite differ-
ent. Compounds 5a, 5b and 6a, 6b with ester or carboxyl
moiety on C-5 position exhibit no antiviral activity, while
their amide counterparts (7a, 7b and 8a, 8b) demonstrate
good anti-HBV activity (IC50 values between 1.1 and 6.1 mM).
Among them, compound 8b exhibits the most promising
biological profile with CC50 > 100 mM and IC50 ¼ 1.1 mM.
Although its anti-HBV activity is less potent than lamivudine,
its cytotoxicity is even much lower than the reference con-
trol, resulting in nearly three times higher selectivity
(SI > 90.9). The above results suggest that the C-5 position
is very pivotal for the anti-HBV activity. It is also worth to note
that the different substituent (benzyl or methyl) on the N-1
position exhibit limited difference for the antiviral activity.
This result may indicate that the N-1 position played less
important role for the anti-HBV activity.
R
1
2a
CC50 (mM)a IC50 (mM)b
Compound
R1
R2
SIc
5a
5b
6a
6b
7a
7b
8a
8b
Bn
Me
Bn
Me
Bn
Me
Bn
Me
-OCH3
-OCH3
-OH
>100
>100
>100
>100
33.3
>100
33.3
>100
5
NAd
NA
NA
NA
2.1
4.5
6.1
1.1
0.16
–
–
–
-OH
–
-NHCH3
-NHCH3
-N(CH3)2
-N(CH3)2
15.9
>22.2
5.5
>90.9
31.3
Lamivudine
a Concentrations of compounds required for 50% extinction of
HepG 2.2.15 cells. The values were reproduced in three exper-
iments
b Concentrations of compounds achieving 50% inhibition of
cytoplasmic HBV-DNA synthesis. The values were reproduced
in three experiments
c Selectivity index (SI) was determined as the CC50/IC50 value
d Not active. ‘‘–’’ The relevant SI cannot be calculated.
To identify the effects of the length of carbon chain
between benzimidazole and thiazole moieties, four
thiabendazole derivatives were evaluated (Table 2). Low
cytotoxicity in vitro. The preliminary structure–activity
relationship (SAR) was discussed. Compound 8b showed good
antiviral activities and high selectivity. Further studies of SAR
based on 8b are ongoing in our group.
cytotoxicity is also observed for these compounds (CC50
ꢀ
33.3 mM). In contrast to the first subseries of compounds 5a,
5b and 6a, 6b, most of this subseries of ester and carboxyl acid
compounds (9a, 9b and 10b) demonstrate good antiviral
activity, with IC50 from 2.8 to 3.6 mM, suggesting that a
longer carbon chain could contribute to the antiviral
activity.
Experimental
Chemistry
1H-NMR spectral data were recorded in DMSO-d6 or CDCl3 on
Varian Mercury 500 NMR spectrometer and 13C-NMR data were
recorded in DMSO-d6 or CDCl3 on Varian Mercury 400 NMR
spectrometer. Low-resolution mass spectra (MS) and high-
In summary, a series of novel benzimidazole analogues was
prepared and assessed for their anti-HBV activity and
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Arch. Pharm. Chem. Life Sci. 2011, 2, 78–83
Anti-HBV Activity of Novel Thiazolylbenzimidazole Derivatives
81
Table 2. In-vitro anti-HBV activity and cytotoxicity of analogues 9a,
9b and 10a, 10b
Methyl 1-methyl-2-(thiazol-4-yl)-1H-benzoimidazole-
5-carboxylate 5b
The crude product was purified through a silica gel column
(ethyl acetate/petroleum ether, 1:1) to afford a light yellow solid.
Yield: 84 mg, 51%, m.p.: 175–1768C; 1H-NMR (CDCl3) d: 8.95 (d,
1H, J ¼ 2.0 Hz), 8.51 (s, 1H), 8.36 (d, 1H, J ¼ 2.0 Hz), 8.05 (m, 1H),
7.44 (d, 1H, J ¼ 8.5 Hz), 4.25 (s, 3H), 3.95 (s, 3H); 13C-NMR (CDCl3)
d: 167.63, 153.00, 148.81, 147.60, 142.44, 139.76, 124.91, 124.60,
122.10, 121.95, 109.46, 52.08, 32.45; EI-MS (m/z): 273 [M]þ; HRMS
(EI): Calcd. for C13H11N3O2S [M]þ 273.0572, found 273.0569.
O
S
N
N
R
2
N
R
2b
1
General procedure for the preparation of compounds 6a,
6b and 10a, 10b
Compound
R1
R2
CC50 (mM)a
IC50 (mM)b
SIc
Compounds 5a, 5b or 9a, 9b (0.40 mmol) were suspended in 6 M
HCl (5 mL) and heated at 608C for 5 h. The mixture was treated
with aqueous 6 M NaOH (5 mL). The formed precipitate was
filtered, washed with water, and dried to give the target com-
pound, which is pure enough for in-vitro studies.
9a
9b
10a
10b
Bn
Me
Bn
Me
-OCH3
-OCH3
-OH
33.3
>100
>100
>100
5
3.1
2.8
>100
3.6
0.16
10.7
>35.7
–
>27.8
31.3
-OH
Lamivudine
a Concentrations of compounds required for 50% extinction of
HepG 2.2.15 cells. The values were reproduced in three exper-
iments
b Concentrations of compounds achieving 50% inhibition of
cytoplasmic HBV-DNA synthesis. The values were reproduced
in three experiments
1-Benzyl-2-(thiazol-4-yl)-1H-benzoimidazole-5-carboxylic
acid 6a
Yield: 119 mg, 89%, m.p.: 265–2668C; H-NMR (DMSO-d6) d: 9.35
(d, 1H, J ¼ 2.0 Hz), 8.66 (d, 1H, J ¼ 2.0 Hz), 8.27 (s, 1H), 7.87 (d,
1H, J ¼ 8.5 Hz), 7.66 (d, 1H, J ¼ 8.5 Hz), 7.26 (m, 2H), 7.22 (m, 1H),
7.12 (d, 2H, J ¼ 7.5 Hz), 6.15 (s, 2H); 13C-NMR (DMSO-d6) d: 167.43,
155.98, 147.85, 145.10, 138.22, 136.67, 128.64, 128.01, 127.53,
126.66, 125.95, 125.10, 124.76, 124.57, 120.09, 115.23, 111.43,
48.05; EI-MS (m/z): 335 [M]þ; HRMS (EI): Calcd. for C19H15N3O2S
[M]þ 335.0728, found 335.0729.
1
c Selectivity index (SI) was determined as the CC50/IC50 value. ‘‘–’’
The relevant SI cannot be calculated.
resolution mass spectra (HRMS) were recorded at an ionizing
voltage of 70 eV on an Agilent/5973N spectrometer and Waters
Micromass GCT.
1-Methyl-2-(thiazol-4-yl)-1H-benzoimidazole-5-carboxylic
acid 6b
Yield: 87 mg, 84%, m.p.: 269–2708C; 1H-NMR (DMSO-d6) d: 9.43 (d,
1H, J ¼ 2.0 Hz), 8.74 (s, 1H), 8.27 (d, 1H, J ¼ 2.0 Hz), 8.00 (d, 1H,
J ¼ 8.5 Hz), 7.86 (d, 1H, J ¼ 8.5 Hz), 4.24 (s, 3H); 13C-NMR (DMSO-
d6) d: 166.88, 156.69, 146.38, 141.73, 137.35, 134.76, 127.49,
127.41, 125.56, 117.50, 112.26, 33.16; EI-MS (m/z): 259 [M]þ;
HRMS (EI): Calcd. for C12H9N3O2S [M]þ 259.0415, found 259.0412.
General procedure for the preparation of compounds 5a,
5b and 9a, 9b
A
mixture of compounds 4a or 4b (0.60 mmol), DMAP
(0.12 mmol), thiazole-4-carboxylic acid or (2-methyl-thiazol-4-
yl)-acetic acid (1.2 mmol), and EDC (1.8 mmol) in dry CH2Cl2
was stirred overnight at room temperature. The resulting
solution was washed with saturated NH4Cl solution and brine,
dried over Na2SO4, and concentrated in vacuum to give a residue,
which was directly poured into acetic acid and was refluxed for
about 5 h. The mixture was neutralized to pH 7 with saturated
Na2CO3 solution to afford a precipitate, which was filtered,
washed with water, and dried to give the crude compounds
5a, 5b or 9a, 9b, respectively. The crude compound was purified
by column chromatography.
General procedure for the preparation of compounds 7a,
7b and 8a, 8b
A solution of compounds 6a, 6b (0.4 mmol), methylamine or
dimethylamine hydrochloride (0.80 mmol), DMAP (0.08 mmol)
and EDC (1.2 mmol) in dry CH2Cl2 was stirred overnight at room
temperature. The mixture was washed with saturated NH4Cl and
brine, dried over Na2SO4, and concentrated in vacuum to give the
target compound.
Methyl 1-benzyl-2-(thiazol-4-yl)-1H-benzoimidazole-
5-carboxylate 5a
1-Benzyl-N-methyl-2-(thiazol-4-yl)-1H-benzoimidazole-
5-carboxamide 7a
The crude product was purified through a silica gel column
(ethyl acetate/petroleum ether, 1:1) to afford a light yellow solid.
Yield: 117 mg, 56%, m.p.: 197–1988C; 1H-NMR (CDCl3) d: 8.90 (d,
1H, J ¼ 2.0 Hz), 8.53 (d, 1H, J ¼ 2.0 Hz), 8.39 (s, 1H), 7.97 (m, 1H),
7.32 (d, 1H, J ¼ 8.5 Hz), 7.25 (m, 2H), 7.13 (m, 2H), 6.11 (s, 2H),
3.94 (s, 1H); 13C-NMR (CDCl3) d: 167.48, 153.12, 147.31, 142.61,
139.17, 136.56, 129.12, 128.73, 127.66, 127.45, 127.10, 126.65,
125.09, 124.75, 122.21, 122.10, 110.29, 52.04, 48.78; EI-MS (m/z):
349 [M]þ; HRMS (EI): Calcd. for C19H15N3O2S [M]þ 349.0888, found
349.0885.
The product was pure enough without further purification.
Yield: 135 mg, 97%, m.p.: 206–2078C; 1H-NMR (CDCl3) d: 8.90
(d, 1H, J ¼ 2.0 Hz), 8.35 (d, 2H, J ¼ 2.0 Hz), 8.15 (s, 1H), 7.75
(m, 1H), 7.35 (m, 1H), 7.25 (m, 2H), 7.13 (m, 2H), 6.18 (s, 1H),
6.10 (s, 2H), 3.06 (d, 3H); 13C-NMR (CDCl3) d: 168.57, 153.14,
148.39, 147.34, 142.65, 138.04, 136.60, 129.80, 128.73, 127.66,
127.10, 126.64, 122.73, 122.05, 120.11, 118.22, 110.70, 48.75,
26.95; EI-MS (m/z): 348 [M]þ; HRMS (EI): Calcd. for C19H16N4OS
[M]þ 348.1045, found 348.1048.
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Arch. Pharm. Chem. Life Sci. 2011, 2, 78–83
8.33 (d, 1H, J ¼ 7.0 Hz), 8.00 (d, 1H, J ¼ 9.0 Hz), 7.73 (d, 1H,
J ¼ 7.0 Hz), 7.50 (d, 1H, J ¼ 15.5 Hz), 7.28 (t, 3H, J ¼ 19.0 Hz),
7.09 (d, 2H, J ¼ 6.0 Hz), 5.80 (s, 2H), 4.78 (s, 2H), 2.54 (s, 3H); 13C-
NMR (DMSO-d6) d: 166.56, 166.30, 153.52, 145.93, 139.45, 134.95,
133.95, 128.58, 128.39, 127.90, 127.45, 126.77, 126.21, 125.78,
118.06, 116.26, 113.08, 55.90, 27.67, 18.44; EI-MS (m/z): 363 [M]þ;
HRMS (EI): Calcd. for C20H17N3O2S [M]þ 363.1041, found 363.1044.
N,1-Dimethyl-2-(thiazol-4-yl)-1H-benzoimidazole-
5-carboxamide 7b
The crude product was recrystallized from ethyl acetate and
petroleum ether to afford a yellow solid. Yield: 77 mg, 71%,
m.p.: 206–2078C; 1H-NMR (CDCl3) d: 8.95 (d, 1H, J ¼ 2.0 Hz),
8.34 (d, 1H, J ¼ 2.0 Hz), 8.13 (d, 1H, J ¼ 1.0 Hz), 7.86 (m, 1H),
7.46 (d, 1H, J ¼ 8.5 Hz), 6.24 (br, 1H), 4.25 (s, 3H), 3.06 (d, 3H,
J ¼ 5.0 Hz); 13C-NMR (CDCl3) d: 168.64, 153.00, 148.57, 147.52,
138.57, 129.56, 122.62, 121.62, 121.81, 118.08, 109.82, 32.36,
26.95; EI-MS (m/z): 272 [M]þ; HRMS (EI): Calcd. for C13H12N4OS
[M]þ 272.0732, found 272.0728.
1-Methyl-2-[(2-methylthiazol-4-yl)methyl]-
1H-benzoimidazole-5-carboxylic acid 10b
The product was pure enough without further purification.
Yield: 103 mg, 90%, m.p.: 248–2498C; 1H-NMR (DMSO-d6) d:
8.30 (s, 1H), 8.12 (dd, 1H, J ¼ 8.7 Hz), 8.02 (dd, 1H, J ¼ 8.7 Hz),
7.60 (s, 1H), 4.73 (s, 2H), 4.02 (s, 3H), 2.61 (s, 3H); 13C-NMR (DMSO-
d6) d: 166.82, 166.35, 153.27, 145.84, 135.25, 130.10, 128.51,
126.20, 117.87, 115.64, 112.97, 31.62, 27.41, 18.61; EI-MS (m/z):
287 [M]þ; HRMS (EI): Calcd. for C14H13N3O2S [M]þ 287.0728, found
287.0731.
1-Benzyl-N,N-dimethyl-2-(thiazol-4-yl)-1H-
benzoimidazole-5-carboxamide 8a
The product was pure enough without further purification.
Yield: 90 mg, 62%, m.p.: 146–1478C; 1H-NMR (CDCl3) d: 8.90 (d,
1H, J ¼ 2.0 Hz), 8.35 (s, 1H), 7.85 (s, 1H), 7.34 (m, 2H), 7.24 (m, 3H),
7.14 (m, 2H), 6.10 (s, 2H), 3.14 (s, 6H); 13C-NMR (CDCl3) d: 171.96,
153.07, 148.08, 147.48, 142.33, 136.71, 130.95, 128.71, 127.61,
126.68, 126.25, 125.44, 122.96, 121.85, 118.77, 115.38, 110.81,
48.70, 38.45, 38.21; EI-MS (m/z): 362 [M]þ; HRMS (EI): Calcd. for
C20H18N4OS [M]þ 362.1201, found 362.1212.
Biological assays
Cell culture and antiviral assays
N,N,1-Trimethyl-2-(thiazol-4-yl)-1H-benzoimidazole-
5-carboxamide 8b
Details of the design of the antiviral procedure and the
growth conditions for HepG2.2.15 cells have been previously
described [20–22]. Briefly, confluent cultures in 96-well tissue
culture plates were treated with various doses of antiviral
compounds in minimal essential medium (MEM) supple-
mented with 10% fetal bovine serum. Fresh MEM with the
same concentration of compounds was replaced at day 4, and
the supernatants were harvested at day 8, then the extra-
cellular (virion) HBV-DNA were measured by real time fluor-
escent PCR.
The crude product was recrystallized from ethyl acetate and
petroleum ether to afford a white solid. Yield: 113 mg. HPLC
purity: >99%, m.p.: 158–1598C; 1H-NMR (CDCl3) d: 8.95 (d, 1H,
J ¼ 2.0 Hz), 8.33 (d, 1H, J ¼ 2.0 Hz), 7.46 (m, 2H), 4.25 (s, 3H), 3.15
(s, 6H); 13C NMR (CDCl3) d: 172.05, 152.93, 148.28, 147.69, 142.09,
137.30, 130.74, 122.84, 121.60, 118.76, 109.88, 38.75, 38.68,
32.30; EI-MS (m/z): 286 [M]þ; HRMS (EI): Calcd. for C14H14N4OS
[M]þ 286.0888, found 286.0879.
Methyl 1-benzyl-2-[(2-methylthiazol-4-yl)methyl]-
1H-benzoimidazole-5- carboxylate 9a
The crude product was purified through a silica gel column (ethyl
acetate/petroleum ether, 1:1) to afford a white solid. Yield:
154 mg, 68%, m.p.: 126–1278C; 1H-NMR (CDCl3) d: 8.48 (m, 1H),
7.95 (m, 1H), 7.25 (m, 4H), 6.96 (t, 2H, J ¼ 7.0 Hz), 6.89 (s, 1H), 5.50
(s, 2H), 4.39 (s, 2H), 3.93 (s, 3H), 2.59 (s, 3H); 13C-NMR (CDCl3) d:
167.60, 166.18, 154.34, 150.14, 142.29, 138.92, 138.01, 135.52,
128.89, 127.90, 126.20, 124.50, 124.34, 121.99, 119.53, 115.45,
115.02, 52.02, 47.61, 29.69, 19.02; EI-MS (m/z): 377 [M]þ; HRMS
(EI): Calcd. for C21H19N3O2S [M]þ 377.1198, found 377.1195.
Toxicity measurements
Cytotoxicity of compound was assessed by the MTT assay as
previously described [20–22]. 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 lyzed 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.
Methyl 1-methyl-2-[(2-methylthiazol-4-yl)methyl]-
1H-benzoimidazole-5-carboxylate 9b
The product was pure enough without further purification. Yield:
108 mg, 60%, m.p.: 160–1618C; 1H-NMR (CDCl3) d: 8.44 (d, 1H,
J ¼ 8.0 Hz), 8.00 (m, 1H), 7.34 (d, 1H, J ¼ 8.0 Hz), 6.88 (s, 1H), 4.44
(s, 2H), 3.94 (s, 3H), 3.81 (s, 3H), 2.68 (s, 3H); 13C-NMR (CDCl3) d: 167.68,
166.30, 154.11, 150.37, 142.17, 139.21, 124.27, 124.07, 121.87,
115.17, 108.84, 52.01, 30.53, 24.34, 19.12; EI-MS (m/z): 301 [M]þ;
HRMS (EI): Calcd. for C15H15N3O2S [M]þ 301.0885, found 301.0886.
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, 50 mL of the supernatants were added to the
extraction buffer, boiled for 10 min and centrifuged for
5 min, and then proper aliquots were used for the fluorescent
PCR. PCR primers were:
Benzyl-2-[(2-methylthiazol-4-yl)methyl]-
1H-benzoimidazole-5-carboxylic acid 10a
The product was pure enough without further purification.
Yield: 139 mg, 96%, m.p.: 244–2458C; 1H-NMR (DMSO-d6) d:
P1: 5’-ATCCTGCTGCTATGCCTCATCTT-3’
P2: 5’-ACAGTGGGGAAAGCCCTACGAA-3’
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 2, 78–83
Anti-HBV Activity of Novel Thiazolylbenzimidazole Derivatives
83
[8] J. S. Lee, H. S. Shim, Y. K. Park, et al., Bioorg. Med. Chem. Lett.
2002, 12, 2715–2717.
The probe was 5’-TGGCTAGTTTACTAGTGCCATTTTG-3’. PCR
reaction was run at MJ Research PTC-200, and results were
analyzed by software OpticonMonitor v2.01.
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4794.
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 Research of the Chinese Academy of Sciences (SIMM0909 KF-08).
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The authors have declared no conflict of interest.
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ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com