Identification of 1-isopropylsulfonyl-2-amine benzimidazoles as a new class of inhibitors of hepatitis B virus

Article abstract of DOI:10.1016/j.ejmech.2007.03.005

A series of 1-isopropylsulfonyl-2-amine benzimidazole derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activity and cytotoxicity in the HepG2.2.15 cell line. In general, these derivatives are potent HBV inhibitors (IC

Full text of DOI:10.1016/j.ejmech.2007.03.005

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European Journal of Medicinal Chemistry 42 (2007) 1358e1364
http://www.elsevier.com/locate/ejmech
Original article
Identification of 1-isopropylsulfonyl-2-amine benzimidazoles
as a new class of inhibitors of hepatitis B virus
Yun-Fei Li b,1, Gui-Feng Wang b,1, Yu Luo a,b, Wei-Gang Huang b,
Wei Tang b, Chun-Lan Feng b, Li-Ping Shi b, Yu-Dan Ren b,
Jian-Ping Zuo b, , Wei Lu
*
a,**
a Institute of Medicinal Chemistry, Shanghai Key Laboratory of Green Chemistry and Chemical Process, Department of Chemistry,
East China Normal University, 3663 North Zhongshan Road, Shanghai, Shanghai 200062, China
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, SIBS, Chinese Academy of Sciences, 555 Zuchongzhi Road,
Shanghai 201203, China
Received 28 September 2006; received in revised form 24 January 2007; accepted 12 March 2007
Available online 31 March 2007
Abstract
A series of 1-isopropylsulfonyl-2-amine benzimidazole derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV)
activity and cytotoxicity in the HepG2.2.15 cell line. In general, these derivatives are potent HBV inhibitors (IC50 < 4 mM) with high selectivity
indices (SIs > 40). Compounds 5bee, g, j, and 9a were among the most prominent compounds, with IC50s of 0.70e2.0 mM and SIs of 41e274.
The potent anti-HBV activity and safety profiles of the most promising compounds 5d and j (IC50s ¼ 0.70 mM, SIs > 120) demonstrate the
potential of this series of benzimidazoles for the development of new anti-HBV drugs.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Benzimidazoles; Synthesis; Anti-HBV activity
1. Introduction
therapeutic agent developed for HBV carries since the early
1980s, suffers from low cure rate (only effective for 30e
40% patients under this treatment) and serious side effects
[4]. The form of i.v. administration and expensive price limit
its application. Lamivudine and adefovir dipivoxil are two
nucleoside analogues with excellent activity and oral bio-
availability. The mechanism of their functions is believed
to mainly act by inhibition of HBV polymerase activity re-
sulting in quantitative decrease in viral replication with
few side effects in most patients [5]. However, lamivudine
has the potential of inducing drug resistant HBV after 6e12
months of therapy, and causes an associated risk of increase
of viremia during the therapy in HBV patients. Further, after
cessation of this treatment, relapse rates are reported high:
long-lasting virological and biochemical responses have
been maintained in approximately 20% of patients, and
a 74% relapse rate has been reported at 12 months after ther-
apy withdrawal [4,6]. Adefovir dipivoxil has proven to be
Hepatitis B virus (HBV) infection is the world’s ninth
leading cause of death, and responsible for both acute and
chronic hepatitis [1]. An estimated 350e400 million people
are chronically infected by HBV throughout the world with
0.5e1.2 million global deaths per year. Chronic HBV infec-
tion can lead to cirrhosis, liver failure, and hepatocellular
carcinoma [2,3]. Only a few agents have been approved
for the clinical treatment of HBV infections: a-interferon
(IFN-a), lamivudine (3-TC), adefovir dipivoxil, and enteca-
vir (Fig. 1). Interferon-a, an immunomodulator and the first
* Corresponding author. Tel./fax: þ86 21 50806701.
** Corresponding author. Tel./fax: þ86 21 62602475.
E-mail addresses: jpzuo@mail.shcnc.ac.cn (J.-P. Zuo), wlu@chem.ecnu.
edu.cn (W. Lu).
1
These authors contributed equally to this work.
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.03.005

Y.-F. Li et al. / European Journal of Medicinal Chemistry 42 (2007) 1358e1364
1359
NH2
In an ongoing effort to identify such compounds [11], we con-
tinuously screen our in-house collection of compounds for active
leads. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]imidazol-
6-yl)-3-(trifluoromethyl)benzenesulfonamide (1, Fig. 1), with
an IC50 of 4 mM in inhibiting HBV DNA replication and selectiv-
ity index (SI) of 27 in vitro, was identified from our sample col-
lection. To date, 1-isopropylsulfonyl-2-amine benzimidazole
derivatives have not been reported to exhibit activity against
HBV, even though a set of structurally related analogues has
been reported to have potent broad spectrum antiviral activity
against both rhinoviruses and enteroviruses [12e14].
Herein, we chose compound 1 as a new lead compound and
developed a series of related benzimidazoles, which were
mainly modified at positions 5 and 6 on the fused phenyl
ring of the benzimidazole core, with significant anti-HBV
activities.
N
N
N
NH2
N
N
O
N
O
O
O
P
O
O
O
O
H
HO
S
O
Lamivudine (3-TC)
O
Adefovir dipivoxil
O
3
4
7
N
HN
5
6
N
O
S
O
2
NH2
N
H2N
N
N
S
1
N
H
O
O
HO
CF3
HO
1
Entecavir
Fig. 1.
2. Chemistry
effective in the treatment of infections with lamivudine-resis-
tant HBV in vitro and in vivo [7]. It might be used as a ther-
apeutic option in view of its good tolerability and few
resistant HBV mutants. However, similar to lamivudine, re-
lapse rates are also high because the nucleoside analogues
cannot eradicate the covalently closed circular (ccc) DNA
pool of HBV. Entecavir, which was approved by the FDA
in 2005 for the treatment of chronic hepatitis B infection,
was found to be more potent than lamivudine and effective
to lamivudine-resistant HBV mutant [8,9]. However, it may
exert its effects via the same biological mechanism and en-
counter the same problems as lamivudine and adefovir dipi-
voxil because of their similar chemical structures.
Although the combination therapy of lamivudine with ade-
fovir dipivoxil, or antiviral drugs together with immunomodu-
lators such as IFN-a might be more effective and safer in the
therapeutic process [4], it is still limited [10]. Therefore, there
is an urgent need for the development of novel classes of anti-
HBV agents with new molecular structures and optimal phar-
macological profiles for the chemotherapy of HBV infection.
The syntheses of this class of benzimidazoles are shown in
Schemes 1 and 2. Treatment of commercially available 2-ami-
nobenzimidazole with isopropylsulfonyl chloride in the pres-
ence of NaOH in a mixture of acetonitrile and water led to
the formation of compound 2 [15]. Nitration of 2 with 67% ni-
tric acid in concentrated sulfuric acid afforded a mixture of 6-
nitro (3) and 5-nitro (7) analogues. After recrystallization the
mixture from ethanol twice, the major product 6-nitro com-
pound 3 was obtained as yellow crystals in 65% yield [16].
Compound 3 was subsequently reduced with 10% Pd/C under
hydrogen atmosphere in acidic conditions, to give the corre-
sponding 6-amine derivative 4. Treatment of compound 4
with various benzoyl, benzenesulfonyl and benzyl chlorides
furnished derivatives 5aek.
The isomeric position 5 benzimidazole derivatives were
prepared by another route (Scheme 2). Starting from 4-ni-
tro-1,2-phenylenediamine, benzimidazole 6 was synthesized
in good yield. N-Sulfonylation of the free benzimidazole ni-
trogen of 6 with isopropylsulfonyl chloride in the presence
of K2CO3 in acetone at room temperature produced the
N
N
N
N
N
N
N
N
N
c
d
a
b
NH2
NH2
NH2
NH2
NH2
X
N
H
O2N
N
H
H2N
SO2
SO2
SO2
SO2
R
2
3
4
5a-i
f
e
O
N
N
NH2
N
N
H
NH2
SO2
N
SO2
N
O
5k
5j
Scheme 1. Synthetic pathway to substituted 1-(isopropylsulfonyl)-2,6-diamine benzimidazole analogs 5aek. Reagents and conditions: (a) (CH3)2CHSO2Cl,
CH3CN/H2O, NaOH, rt; (b) 67% HNO3, concd. H2SO4, 0 ꢀC to rt; (c) 10% Pd/C, H2, 2 N HCl, rt; (d) R-benzoyl or R-benzenesulfonyl chlorides, dry pyridine,
rt; (e) 3-methoxybenzyl chloride, Na2CO3, EtOH, reflux; (f) benzyl chloride, NaHCO3, EtOH/H2O, reflux.

1360
Y.-F. Li et al. / European Journal of Medicinal Chemistry 42 (2007) 1358e1364
H2N
O2N
H
N
N
N
N
N
N
NH2
NH2
b
c
d
a
N
N
NH2
NH2
NH2
X
R
NH2
N
H
O2N
O2N
SO2
SO2
SO2
6
7
8
9a-c
Scheme 2. Synthetic pathway to substituted 1-(isopropylsulfonyl)-2,5-diamine benzimidazole analogs 9aec. Reagents and conditions: (a) BrCN, H2O, reflux; (b)
(CH3)2CHSO2Cl, acetone, K2CO3, rt; (c) 10% Pd/C, H2, 2 N HCl, rt; (d) R-benzoyl or R-benzenesulfonyl chlorides, dry pyridine, rt.
5-nitro derivative 7 with a minor amount of the 6-nitro an-
alogue 3. Compound 7 was obtained as a pure product after
crystallization of the product mixture from acetonitrile and
recrystallization from methanol subsequently. Compound 7
was then reduced to afford the corresponding 5-amine deriv-
ative 8. Derivatives 9aec were prepared by the reaction of
compound 8 with appropriate benzoyl and benzenesulfonyl
chlorides.
As we investigated the substituents on the benzoyl ring, sev-
eral derivatives with substituents of different electronic, steric,
lipophilic, and substituted position characters on the phenyl
were prepared (Scheme 1, 5aef ). We found that the unsubsti-
tuted derivative 5a had only a weak antiviral activity
(IC50 ¼ 33.7 mM) and was significantly less potent than the
substituted benzoyl derivatives 5bef. This result suggests that
the substituent on the benzoyl ring is an important feature in con-
ferring relatively potent inhibitory activity. Among derivatives
5bef, the halide substituted analogues 5bee demonstrated
IC50 values between 0.7 and 1.2 mM, and were more active
than the methyl substituted analogue 5f (IC50 ¼ 4.2 mM).
Changing the position of the F group from ortho position (5b,
IC50 ¼ 0.94 mM, SI ¼ 51) to para position (5c, IC50 ¼ 1.2 mM,
SI ¼ 56) could not improve the inhibitory activity, while intro-
duction of another F atom at the ortho position (5d,
IC50 ¼ 0.70 mM, SI ¼ 274) slightly increased the potency. En-
larging the bulk of the substituent from F (5b) to Cl (5e,
IC50 ¼ 0.82 mM, SI ¼ 107) led to increase in both antiviral po-
tency and SI. The bis(fluoro) derivative 5d displayed more po-
tent antiviral activity (IC50 ¼ 0.70 mM) than other halide
substituted analogues (5b, c, and e) and a 48-fold enhancement
in antiviral potency relative to the unsubstituted benzoyl deriv-
ative 5a, and demonstrated the best SI (274). Compound 5d
emerged as the optimal compound in this series.
3. Results and discussion
The synthesized benzimidazole analogues were evaluated
for their anti-HBV activity and cytotoxicity with the
antiviral drugs lamivudine and adefovir as reference controls
in HepG2.2.15 cells, and the results are summarized in
Table 1.
Table 1
Anti-HBV activity and cytotoxicity of 1-isopropylsulfonyl-2-amine benzimid-
azole analogues in vitro
N
N
H
N
NH2
N
N
X
X
R
NH2
N
H
SO2
To identify the effects of substituting the benzoyl moiety
with benzenesulfonyl group, three benzenesulfonamide deriv-
atives were prepared (Scheme 1, 5gei). The 6-benzenesulfona-
mide derivative 5g (IC50 ¼ 1.5 mM, SI ¼ 41) showed anti-HBV
potency comparable to that of the 4-F benzoyl derivative 5c
(IC50 ¼ 1.2 mM, SI ¼ 56) and adefovir (IC50 ¼ 1.7 mM, SI ¼ 34).
An electron-donating methyl group at the 4-position (5h) and an
electron-withdrawing nitro group at the 3-position (5i) were exam-
ined and they led to decrease in both the IC50 and SI values. As for
the benzyl analogues, the bis(3-methoxybenzyl) derivative 5j
(IC50 ¼ 0.7 mM, SI ¼ 123) exhibited similar antiviral potency
to that of the bis(fluoro) derivative 5d. However, compound
5k, which bears no substituent on the phenyl, proved to be vir-
tually inactive.
With the purpose of investigating the anti-HBV effects of
regioisomers, we prepared three 5-position substituted deriva-
tives (Scheme 2, 9aec). As seen in Table 1, the 5-benzoyl de-
rivative 9a exhibited a potency that was nearly 17-fold greater
than its corresponding 6-benzoyl derivative 5a. No significant
toxicities were observed for 9a at the highest tested concentra-
tion of 500 mM, which led to a high selectivity index (>250).
However, compounds 9b and c were less potent than their
6-position isomers.
SO2
R
5a-i
9a-c
Compds.
X
R
IC50 (mM)a
CC50 (mM)b
SIc
5a
5b
CO
CO
CO
CO
CO
CO
SO2
SO2
SO2
e
H
33.7
0.94
1.2
>500
48
67
>15
2-F
4-F
51
56
5c
5d
2,6-DiF
2-Cl
4-CH3
H
0.70
0.82
4.2
192
88
274
107
>119
41
5e
5f
5g
>500
62
80
1.5
4.8
5h
5i
4-CH3
3-NO2
e
17
5
10.5
0.70
NA
2.0
57
86
5j
5k
123
e
e
e
98
>500
34
9a
9b
CO
CO
SO2
e
H
4-F
>250
9
3.7
9c
Lamivudine
Adefovir
4-CH3
e
16
151
>1000
57
9
0.38
1.7
>2632
34
e
e
NA, not active.
a
Concentrations of compounds achieving 50% inhibition of cytoplasmic
HBV DNA synthesis.
b
Concentrations of compounds required for 50% extinction of HepG2.2.15
cells.
c
Selectivity index (SI) was determined as the CC50/IC50 value.

Y.-F. Li et al. / European Journal of Medicinal Chemistry 42 (2007) 1358e1364
1361
4. Conclusions
organic extracts were washed with brine, dried over anhydrous
MgSO4 and concentrated to afford 4 (600 mg, 99% yield) as
1
a brown solid; H NMR (400 MHz, CDCl3) d 7.15 (d, 1H,
In conclusion, a series of 1-isopropylsulfonyl-2-amine
benzimidazole derivatives based on compound 1 were synthe-
sized and assessed for their anti-HBV activity and cytotoxicity
in vitro, using lamivudine and adefovir as reference controls.
Most of them proved to be potential HBV inhibitors
(IC50 < 4 mM) with high selectivity indices (SI > 40). Com-
pounds 5bee, g, j, and 9a showed outstanding anti-HBV po-
tency with IC50s of 0.70e2.0 mM and SIs of 41e274. The
most promising compounds 5d and j were selected for further
assessment in vivo. Further studies of structureeactivity rela-
tionships and mechanisms of action of this new class of anti-
HBV agents are going on in our group.
J ¼ 8.4 Hz), 7.00 (s, 1H), 6.61 (dd, 1H, J ¼ 8.4, 2.2 Hz),
3.65 (m, 1H), 1.41 (d, 6H, J ¼ 6.9 Hz); 13C NMR
(400 MHz, CDCl3)
d 151.09, 141.59, 133.94, 132.13,
117.10, 112.75, 99.54, 55.67, 16.01 (2C); MS (EI) m/z 254
(Mþ), 147, 120, 57.
5.1.3. 6-Nitro-1H-benzo[d]imidazol-2-amine (6)
A
suspension of 4-nitro-o-phenylenediamine (1.4 g,
9.1 mmol) in a solution of BrCN (0.97 g, 9.2 mmol) in water
(30 mL) was refluxed for 7 h, cooled, and neutralized with
25% NH4OH to pH 10e11. The formed precipitate was fil-
tered, washed with water and dried to give the title compound
5. Experimental
1
as a yellow solid (1.5 g, 92% yield); H NMR (300 MHz,
DMSO-d6) d 7.95 (d, 1H, J ¼ 2.4 Hz), 7.89 (dd, 1H, J ¼ 8.7,
5.1. Chemistry
2.4 Hz), 7.20 (d, 1H, J ¼ 8.4 Hz), 7.01 (br s, 2H).
1H NMR spectral data were recorded in DMSO-d6, or
CDCl3 on Varian Mercury 400 or 300 NMR spectrometer
and 13C NMR spectral data were recorded in DMSO-d6, or
CDCl3 on Varian Mercury 400 NMR spectrometer. Chemical
shifts (d) are reported in parts per million (ppm). Data are re-
ported as follows: chemical shift, multiplicity (br s ¼ broad
singlet, d ¼ doublet, dd ¼ doublet of doublet, dt ¼ doublet of
triplet, m ¼ multiplet, s ¼ singlet and t ¼ triplet), coupling
constants (Hz), integration. Low-resolution mass spectra
(MS) and high-resolution mass spectra (HRMS) were recorded
at an ionizing voltage of 70 eV on a Finnigan/MAT95 spec-
trometer. Pyridine was distilled from CaH2. Column chroma-
tography was carried out on silica gel (200e300 mesh).
5.1.4. 1-(Isopropylsulfonyl)-5-nitro-1H-benzo[d]imidazol-
2-amine (7)
To a reaction mixture containing 6 (2.0 g, 11.2 mmol) and
K2CO3 (1.6 g, 11.6 mmol) in acetone (70 mL) was added
dropwise 2-propanesulfonyl chloride (1.6 g, 11.2 mmol). The
reaction mixture was stirred at room temperature for 4 h and
treated with 1 N HCl (360 mL). The resulting mixture was
stirred for 1 h at room temperature and filtered. The filtrate
was neutralized to pH 6. The formed precipitate was filtered,
crystallized from acetonitrile and recrystallized from methanol
subsequently to give compound 7 as yellow crystals (0.66 g,
1
20% yield); H NMR (300 MHz, DMSO-d6) d 8.01 (d, 1H,
5.1.1. 1-(Isopropylsulfonyl)-6-nitro-1H-benzo[d]imidazol-
2-amine (3)
J ¼ 2.1 Hz), 7.97 (dd, 1H, J ¼ 8.4, 2.1 Hz), 7.68 (d, 1H,
J ¼ 8.7 Hz), 7.39 (br s, 2H), 3.97 (m, 1H), 1.31 (d, 6H,
J ¼ 6.6 Hz); MS (EI) m/z 284 (Mþ), 178, 148, 132, 105, 77.
To a solution of 2 (6.0 g, 25.1 mmol) in 80 mL of concen-
trated sulfuric acid, which was cooled to 0 ꢀC, was added
dropwise 67% nitric acid (18.0 mL, 27.6 mmol). After that,
the reaction mixture was stirred at room temperature for 3 h
and then poured into crushed ice slowly. The resulting mixture
was neutralized to pH 8 with 25% NH4OH. The formed pre-
cipitate was filtered, washed with water, and dried to give
the crude product, which was recrystallized from EtOH twice
to give the title compound as yellow crystals (4.6 g, 65%
yield); 1H NMR (400 MHz, DMSO-d6) d 8.25 (d, 1H,
J ¼ 2.0 Hz), 8.10 (dd, 1H, J ¼ 8.8, 2.4 Hz), 7.68 (br s, 2H),
7.34 (d, 1H, J ¼ 9.2 Hz), 4.00 (m, 1H), 1.29 (d, 6H,
J ¼ 6.8 Hz); 13C NMR (400 MHz, DMSO-d6) d 156.36,
148.83, 140.34, 130.91, 121.21, 115.19, 107.67, 56.12, 15.58
(2C); MS (EI) m/z 284 (Mþ), 178, 149, 57.
5.1.5. 1-(Isopropylsulfonyl)-1H-benzo[d]imidazole-2,5-
diamine (8)
The product was obtained from 7 using a procedure similar
to that described for the preparation of 4 as a brown solid.
Yield ¼ 99%; 1H NMR (300 MHz, CDCl3) d 7.34 (d, 1H,
J ¼ 8.4 Hz), 6.69 (d, 1H, J ¼ 2.4 Hz), 6.45 (dd, 1H, J ¼ 8.4,
2.0 Hz), 5.78 (br s, 2H), 3.60 (m, 1H), 1.36 (d, 6H,
J ¼ 6.8 Hz); MS (EI) m/z 254 (Mþ), 147, 120, 105.
5.2. General procedure for the preparation of derivatives
5aei and 9aec
5.1.2. 1-(Isopropylsulfonyl)-1H-benzo[d]imidazole-2,6-
diamine (4)
Compound 3 (680 mg, 2.4 mmol) was hydrogenated over
10% Pd/C (70 mg) in 2 N HCl (50 mL) at room temperature
overnight. The reaction mixture was filtered, neutralized
with 25% NH4OH, and extracted with ethyl acetate. The
A stirred solution of compound 4 or compound 8 (1 equiv)
in dry pyridine (12 mL/mmol) was treated in one portion with
the appropriate acyl chloride (1 equiv). The reaction mixture
was stirred at room temperature overnight and poured into wa-
ter (30 mL/mmol). The resulting precipitate was filtered and
dried to obtain the product.

1362
Y.-F. Li et al. / European Journal of Medicinal Chemistry 42 (2007) 1358e1364
5.2.1. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-6-yl)benzamide (5a)
Compound 5a was prepared from 4 and benzoyl chloride as
(400 MHz, DMSO-d6) d 164.98, 152.60, 141.39, 138.41,
132.74, 132.15, 131.07, 128.87 (2C), 127.63 (2C), 117.50,
115.46, 105.22, 55.44, 21.01, 15.65 (2C); MS (EI) m/z 372
(Mþ), 266, 119, 79; HRMS (EI): cal. for C18H20N4O3S
372.1256, found 372.1256.
1
a white solid. Yield ¼ 85%; H NMR (300 MHz, DMSO-d6)
d 10.26 (s, 1H), 8.11 (d, 1H, J ¼ 2.1 Hz), 7.95 (d, 2H,
J ¼ 7.8 Hz), 7.61e7.49 (m, 4H), 7.21 (d, 1H, J ¼ 9.0 Hz),
3.84 (m, 1H), 1.29 (d, 6H, J ¼ 6.9 Hz); MS (EI) m/z 358
(Mþ), 252, 105, 77; HRMS (EI): cal. for C17H18N4O3S
358.1100, found 358.1108.
5.2.7. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-6-yl)benzenesulfonamide (5g)
Compound 5g was prepared from 4 and benzenesulfonyl
1
chloride as a white solid. Yield ¼ 81%; H NMR (300 MHz,
5.2.2. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-6-yl)-2-fluorobenzamide (5b)
Compound 5b was prepared from 4 and 2-fluorobenzoyl
DMSO-d6) d 7.65e7.48 (m, 5H), 7.22 (d, 1H, J ¼ 2.1 Hz),
7.09 (d, 1H, J ¼ 8.7 Hz), 6.89 (dd, 1H, J ¼ 8.4, 2.1 Hz), 3.56
(m, 1H), 1.15 (d, 6H, J ¼ 6.6 Hz); MS (EI) m/z 394 (Mþ),
253, 147, 79, 52; HRMS (EI): cal. for C16H18N4O4S2
394.0769, found 394.0768.
1
chloride as a white solid. Yield ¼ 82%; H NMR (300 MHz,
CDCl3) d 10.40 (s, 1H), 8.10 (d, 1H, J ¼ 1.8 Hz), 7.69e7.48
(m, 3H), 7.37e7.30 (m, 2H), 7.20 (d, 1H, J ¼ 8.7 Hz), 6.88
(br s, 2H), 3.84 (m, 1H), 1.28 (d, 6H, J ¼ 6.6 Hz); MS (EI)
m/z 376 (Mþ), 270, 123; HRMS (EI): cal. for C17H17FN4O3S
376.1005, found 376.0997.
5.2.8. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-6-yl)-4-methylbenzenesulfonamide (5h)
Compound 5h was prepared from 4 and tosyl chloride as
1
a white solid. Yield ¼ 85%; H NMR (300 MHz, DMSO-d6)
5.2.3. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-6-yl)-4-fluorobenzamide (5c)
Compound 5c was prepared from 4 and 4-fluorobenzoyl
d 7.53 (d, 2H, J ¼ 8.1 Hz), 7.29 (d, 2H, J ¼ 8.4 Hz), 7.25 (s,
1H), 7.08 (d, 1H, J ¼ 8.4 Hz), 6.88 (m, 2H), 3.62 (m, 1H),
2.31 (s, 3H), 1.15 (d, 6H, J ¼ 6.9 Hz); 13C NMR (400 MHz,
1
chloride as a white solid. Yield ¼ 81%; H NMR (400 MHz,
DMSO-d6) d 152.71, 142.96, 139.48, 136.32, 131.34,
CDCl3) d 10.27 (s, 1H), 8.07e8.01 (m, 3H), 7.55 (dd, 1H,
J ¼ 8.8, 2.0 Hz), 7.37e7.32 (m, 2H), 7.19 (d, 1H,
J ¼ 8.7 Hz), 3.82 (m, 1H), 1.27 (d, 6H, J ¼ 6.4 Hz); MS (EI)
m/z 376 (Mþ), 270, 123, 79; HRMS (EI): cal. for
C17H17FN4O3S 376.1005, found 376.1004.
130.40, 129.43 (2C), 126.80 (2C), 119.40, 115.88, 106.76,
55.64, 20.92, 15.54 (2C); MS (EI) m/z 408 (Mþ), 253, 147,
91; HRMS (EI): cal. for C17H20N4O4S2 408.0926, found
408.0920.
5.2.9. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-6-yl)-3-nitrobenzenesulfonamide (5i)
5.2.4. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-6-yl)-2,6-difluorobenzamide (5d)
Compound 5i was prepared from 4 and 3-nitrobenzenesul-
fonyl chloride as yellow crystals. Yield ¼ 85%; 1H NMR
(400 MHz, DMSO-d6) d 10.2 (s, 1H), 8.45e8.41 (m, 2H),
8.04 (d, 1H, J ¼ 7.9 Hz), 7.83 (t, 1H, J ¼ 8.1 Hz), 7.26 (s,
1H), 7.12 (d, 1H, J ¼ 8.3 Hz), 6.92e6.89 (m, 3H), 3.65 (m,
1H), 1.16 (d, 6H, J ¼ 6.6 Hz); MS (EI) m/z 439 (Mþ), 253,
147, 119; HRMS (EI): cal. for C16H17N5O6S2 439.0620, found
439.0626.
Compound 5d was prepared from 4 and 2,6-difluoroben-
zoyl chloride as a white solid. Yield ¼ 80%; 1H NMR
(300 MHz, CDCl3) d 10.79 (s, 1H), 8.04 (s, 1H), 7.56 (m,
1H), 7.46 (dt, 1H, J ¼ 8.4, 1.8 Hz), 7.23 (m, 3H), 3.83 (m,
1H), 1.27 (d, 6H, J ¼ 6.9 Hz); MS (EI) m/z 394 (Mþ), 288,
141; HRMS (EI): cal. for C17H16F2N4O3S 394.0911, found
394.0897.
5.2.5. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-6-yl)-2-chlorobenzamide (5e)
Compound 5e was prepared from 4 and 2-chlorobenzoyl
5.2.10. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-5-yl)benzamide (9a)
Compound 9a was prepared from 8 and benzoyl chloride as
an off-white solid. Yield ¼ 88%; 1H NMR (300 MHz, CDCl3)
d 10.23 (s, 1H), 7.95 (m, 2H), 7.74 (s, 1H), 7.59e7.34 (m, 5H),
3.84 (m, 1H), 1.26 (d, 6H, J ¼ 6.6 Hz); MS (EI) m/z 358 (Mþ),
251, 105, 77; HRMS (EI): cal. for C17H18N4O3S 358.1100,
found 358.1107.
1
chloride as a white solid. Yield ¼ 79%; H NMR (400 MHz,
CDCl3) d 8.04 (d, 1H, J ¼ 1.8 Hz), 7.98 (s, 1H), 7.79 (dd,
1H, J ¼ 7.4, 1.9 Hz), 7.48e7.34 (m, 5H), 5.74 (br s, 2H),
3.72 (m, 1H), 1.42 (d, 6H, J ¼ 6.9 Hz); MS (EI) m/z 392
(Mþ), 286, 139; HRMS (EI): cal. for C17H17ClN4O3S
392.0710, found 392.0714.
5.2.11. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-5-yl)-4-fluorobenzamide (9b)
5.2.6. N-(2-Amino-1-(isopropylsulfonyl)-1H-benzo[d]-
imidazol-6-yl)-4-methylbenzamide (5f)
Compound 5f was prepared from 4 and 4-methylbenzoyl
Compound 9b was prepared from 8 and 4-fluorobenzoyl
chloride as an off-white solid. Yield ¼ 55%; 1H NMR
(300 MHz, CDCl3) d 10.22 (s, 1H), 8.06e8.02 (m, 2H), 7.74
(s, 1H), 7.46e7.34 (m, 4H), 3.85 (m, 1H), 1.25 (d, 6H,
J ¼ 6.6 Hz); 13C NMR (400 MHz, DMSO-d6) d 164.25,
164.00 (JC,F ¼ 247.1 Hz), 153.14, 142.42, 135.89, 131.47,
1
chloride as a white solid. Yield ¼ 88%; H NMR (300 MHz,
CDCl3) d 8.01 (s, 1H), 7.85 (s, 1H), 7.78 (d, 2H,
J ¼ 8.1 Hz), 7.41e7.28 (m, 4H), 5.67 (br s, 2H), 3.70 (m,
1H), 2.43 (s, 3H), 1.42 (d, 6H, J ¼ 6.6 Hz); 13C NMR

Y.-F. Li et al. / European Journal of Medicinal Chemistry 42 (2007) 1358e1364
1363
130.32 (2C), 127.53, 115.30 (2C), 113.37, 111.49, 108.49,
55.40, 15.62 (2C); MS (EI) m/z 376 (Mþ), 269, 123, 83;
HRMS (EI): cal. for C17H17FN4O3S 376.1005, found
376.0996.
compounds in minimal essential medium (MEM) supple-
mented with 10% fetal bovine serum. Fresh MEM with the
same concentration of compounds was replaced on day 4,
and the supernatants were harvested on day 8, then the extra-
cellular (virion) HBV DNA was measured by real time fluores-
cent PCR.
5.2.12. N-(2-Amino-1-(isopropylsulfonyl)-1H-
benzo[d]imidazol-5-yl)-4-methylbenzenesulfonamide (9c)
Compound 9c was prepared from 8 and tosyl chloride as
5.5.2. Toxicity measurements
1
a white solid. Yield ¼ 73%; H NMR (300 MHz, DMSO-d6)
Cytotoxicity of compound was assessed by the MTT assay
as previously described [19]. Briefly, HepG2.2.15 cells were
cultured in triplicate of 96-well tissue culture plates for 8
days with various doses of test compounds. The cells with me-
dia 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% N,N-dimethyl-
formamide, pH ¼ 7.2. O.D. values were read at 570 nm 6 h
later and the cell death percent was calculated.
d 7.60 (d, 2H, J ¼ 8.1 Hz), 7.32 (m, 3H), 6.96 (s, 1H), 6.77
(dd, 1H, J ¼ 8.7, 2.1 Hz), 3.82 (m, 1H), 2.31 (s, 3H), 1.23
(d, 6H, J ¼ 6.9 Hz); MS (EI) m/z 408 (Mþ), 301, 253, 149,
91; HRMS (EI): cal. for C17H20N4O4S2 408.0926, found
408.0942.
5.3. 1-(Isopropylsulfonyl)-N6,N6-bis(3-methoxybenzyl)-
1H-benzo[d]imidazole-2,6-diamine[N-6] (5j)
A mixture of 4 (137 mg, 0.54 mmol) and anhydrous
sodium carbonate (80 mg, 075 mmol) in EtOH (5 mL) was
heated to reflux. 3-Methoxybenzyl chloride (248 mg,
1.58 mmol) was added and the resulting mixture was refluxed
for 18 h. The reaction mixture was filtered while hot and
washed with EtOH. The combined mixture was filtered to
give 5j (169 mg, 63% yield) as a pale yellow solid; 1H
NMR (300 MHz, CDCl3) d 7.25e7.14 (m, 3H), 6.95e6.71
(m, 8H), 5.52 (br s, 2H), 4.62 (s, 4H), 3.75 (s, 6H), 3.18
(m, 1H), 1.15 (d, 6H, J ¼ 6.9 Hz); MS (EI) m/z 494 (Mþ),
387, 267, 121; HRMS (EI): cal. for C26H30N4O4S 494.1988,
found 494.1986.
5.5.3. Real time fluorescent PCR
The supernatants of HepG2.2.15 cells were collected from
8 days’ culture after the compounds were added. The HBV
DNA in the supernatants was quantified by using fluorescent
PCR [11]. Briefly, 50 mL of the supernatants were added
into the extraction buffer, boiled for 10 min and centrifuged
for 5 min, and then proper aliquots were used for the fluores-
cent PCR. PCR primers were: P1: 50-ATCCTGCTGC
TATGCCTCATCTT-30, P2: 50-ACAGTGGGGAAAGCCCTA
CGAA-30. The probe was 50-TGGCTAGTTTACTAGTGC
CATTTTG-30. PCR reaction was run at MJ Research PTC-
200, and results were analyzed by software OpticonMonitor
v2.01.
5.4. N6-Benzyl-1-(isopropylsulfonyl)-1H-
[N-6]
benzo[d]imidazole-2,6-diamine
(5k)
Acknowledgements
To a solution of 4 (222 mg, 0.87 mmol) in EtOH (5 mL)
were added sodium bicarbonate (15 mg, 0.18 mmol) and
two drops of water. To this ice cooled mixture was added
dropwise a solution of benzyl chloride (22 mg, 0.17 mmol)
in EtOH (1 mL). The resulting mixture was stirred at room
temperature for 0.5 h and then refluxed for 2.5 h, cooled to
room temperature, filtered, and concentrated in vacuo. The
residue was subjected to silica gel column chromatography
(CHCl3) to afford compound 5k (53 mg, 91% yield) as an or-
This work was financially supported by the grants of
Shanghai Science and Technology Committee (No.
03DZ19228 and No. 05DZ19331).
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