Synthesis and biological evaluation of 1H-benzimidazol-5-ols as potent HBV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.10.099

A new series of 1-methyl-1H-benzimidazol-5-ol derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activity and cytotoxicity in the HepG2.2.15 cell line. Some of the analogues in this series displayed inhibitory activity superior to lamivudine. Of them, compound 13b was the most potent one, showing an IC₅₀ value of 7.8 μM and a SI value of 13.0. 2010 Published by Elsevier Ltd. All rights reserved.

Full text of DOI:10.1016/j.bmcl.2010.10.099

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7230–7233
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 1H-benzimidazol-5-ols as potent
HBV inhibitors
⇑
Yanfang Zhao, Yajing Liu, Dong Chen, Zengquan Wei, Wenzhao Liu, Ping Gong
Key Lab of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 1-methyl-1H-benzimidazol-5-ol derivatives were synthesized and evaluated for their
anti-hepatitis B virus (HBV) activity and cytotoxicity in the HepG2.2.15 cell line. Some of the analogues
in this series displayed inhibitory activity superior to lamivudine. Of them, compound 13b was the most
Received 8 September 2010
Revised 15 October 2010
Accepted 20 October 2010
Available online 26 October 2010
potent one, showing an IC50 value of 7.8
lM and a SI value of 13.0.
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Keywords:
1-Methyl-1H-benzimidazol-5-ols
Synthesis
Anti-HBV activity in vitro
Hepatitis B virus (HBV) is a major cause of acute and chronic
hepatitis which could lead to liver cirrhosis, liver failure and hepa-
tocellular carcinoma.1 An estimated 360 million people are chron-
ically infected by HBV throughout the world with 0.5–1.2 million
global deaths per year.2 Nowadays, at least two different treatment
options, including interferon and nucleoside analogues such as
lamivudine, adefovir dipivoxil and entecavir are considered as
antiviral therapy for chronic hepatitis B infection.3 Unfortunately,
Figure 1. Modifications of ethyl 5-hydroxy-1H-indole-3-carboxylates
1-methyl-1H-benzimidazol-5-ols B.
A to
interferon-a is effective in only one-third of patients and the treat-
ment is greatly hampered by significant adverse effects.4 The long-
term usage of nucleoside analogues in HBV carriers may eventually
lead to the development of virus drug resistance.5,6 Therefore, the
search for compounds with novel anti-HBV target and mechanism
is still urgently needed.
Nitration of compound 4 with a mixture of nitric acid and sulfuric
acid in chloroform formed compound 5. Compound 6 was obtained
by methylation of compound 5 with dimethyl sulfate in the
presence of NaH in dry DMF. Treatment of compound 6 with
substituted thiophenols 7 gave thioethers 8, followed by reduction
of the nitro group and cyclization in one pot to afford benzimidaz-
oles 9, which were subsequently deprotected in a 2:1 mixture of
acetic acid and hydrochloric acid to afford 10. The target com-
pounds 11a–f were then synthesized via a Mannich reaction of
10 with formaldehyde and different secondary alkylamines.
To efficiently explore the SARs around the 4-position of the
benzimidazole, a modified route introducing an imidazolyl or
2-methylimidazolyl group was developed (Scheme 2). The dimeth-
ylamine derivatives 12 were obtained via a Mannich reaction. And
then treatment of 12 with imidazole or 2-methylimidazole in
1,4-dioxane generated seven N-heteroaromatic derivatives 13a–h
in good yields. With the purpose of investigating the influence of
the oxidation state of the sulfur atom on the anti-HBV activity
and cytotoxicity, we prepared 2-sulfinyl analogues 14a–c and
2-sulfonyl analogues 15a–c (Scheme 3). The spectral data of all
We previously reported that ethyl 5-hydroxy-1H-indole-3-car-
boxylate analogues (A, Fig. 1) exhibited significant antiviral activity
in vitro against HBV.7–10 We have recently reported our efforts to
replace the indole nucleus by quinoline11,12 and hydroxyimi-
dazo[1,2-a]pyridine13 that produced potent HBV inhibitor. As a
continuation of this work, we describe here the synthesis and
structure–activity relationships (SARs) of new 1-methyl-1H-ben-
zimidazol-5-ol derivatives (B, Fig. 1).
1-Methyl-1H-benzimidazol-5-ols 11a–11f were achieved with
an efficient synthetic route (Scheme 1). Protection of the phenolic
hydroxyl group of commercially available 4-nitrophenol 1 with
benzyl chloride in the presence of K2CO3 at 80 °C, followed by
reduction of nitro group, gave 4-(benzyloxy)benzenamine 3, which
was treated with 2-chloroacetyl chloride to yield compound 4.
⇑
Corresponding author. Tel./fax: +86 24 23986429.
E-mail address: gongpinggp@126.com (P. Gong).
0960-894X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.099

Y. Zhao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7230–7233
7231
Scheme 1. Reagents and conditions: (a) Benzyl chloride, K2CO3, DMF, 80 °C, 3 h, 95%; (b) 80% hydrazine hydrate, FeCl3Á6H2O, activated carbon, 95% EtOH, reflux, 5 h, 75%; (c)
2-chloroacetyl chloride, pyridine, CH2Cl2, À10 °C, 0.5 h, rt, 3 h, 82%; (d) HNO3, H2SO4, CHCl3, 10 °C, 2 h, 68%; (e) (CH3)2SO4, NaH, DMF, 0 °C, 0.5 h, rt, 1 h, 86%; (f) KOH, MeOH, rt,
6 h, 70–85%; (g) Fe, NH4Cl (catalytic), AcOH, 95% EtOH, reflux, 6 h, 50–66%; (h) HCl, AcOH, argon, 80 °C, 8 h, 90%; (i) alkyl secondary amine, 37% HCHO, MeOH, rt, 10 h, 65–80%.
Scheme 2. Reagents and conditions: (a) Dimethylamine, 37% HCHO, MeOH, rt, 10 h, 70–80%; (b) imidazole or 2-methylimidazole, HCl, 1,4-dioxane, 80 °C, 2–3 h, 65–75%.
Scheme 3. Reagents and conditions: (a) NaBO3Á4H2O, AcOH, 50 °C, 2–3 h, 68–79%; (b) H2O2, Na2WO4, MeOH, 5 °C, 4–6 h, 70–86%.
the target compounds are in full agreement with the proposed
structures.14
The subseries of compounds 11a–d, 13a, and 13b has different
Mannich side chains on 4-position of the benzimidazole core. Imid-
azolyl derivatives13a and 13b showedremarkable inhibitionof HBV
These synthesized 1-methyl-1H-benzimidazol-5-ols were eval-
uated for their anti-HBV activity and cytotoxicity with the antiviral
drug lamivudine as reference control in HepG2.2.15 cells.15 The re-
sults, expressed as IC50, CC50 and SI, are illustrated in Table 1.
As shown in Table 1, six compounds (13a–e and 13g) demon-
strated more potent inhibition of HBV DNA replication than lami-
DNA replication (IC50 = 9.1 and 7.8 lM, respectively). However,
replacement of the imidazolyl group of 13a (or 13b) with dimethyl-
amino, pyrrolidinyl, morpholinyl, or 4-methyl piperazinyl groups
resulted in a complete loss of inhibitory activity, as seen in com-
pounds 11a–d. This result suggests that the imidazolyl group at
the R2 position played an important role on anti-HBV DNA activity.
Besides, 2-methylimidazol-1-yl analogues showed less toxic than
imidazol-1-yl analogues (13a vs 13b, 13d vs 13e, 13f vs 13g).
The antiviral activity of these compounds appeared to be re-
lated to the electronic effect of the substituents on the phenyl ring
vudine with IC50 values between 7.8 and 44.5 lM. Of them, 13a,
13b and 13c possessed SI from 6.9 to 13.0, which were comparable
to or higher than that of lamivudine (SI = 9.1). In addition, com-
pounds 11d, 11f, 14a–c and 15a–c exhibited moderate to good
inhibitory effect on the secretion of HBsAg.

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Y. Zhao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7230–7233
Table 1
The substituents, cytotoxicity, and anti-HBV activity of target compounds in vitroe
a
Compd
R1
R2
CC50
(lM)
HBsAg
M)
HBV DNA replication
b
IC50
(l
SIc
IC50
(l
M)
SI
d
11a
11b
11c
11d
11e
11f
13a
13b
13c
13d
13e
H
H
H
H
4-F
4-F
H
Dimethylamino
Pyrrolidin-1-yl
Morpholino
4-Methylpiperazin-1-yl
Pyrrolidin-1-yl
4-Methylpiperazin-1-yl
Imidazol-1-yl
2-Methylimidazol-1-yl
Imidazol-1-yl
Imidazol-1-yl
2-Methylimidazol-1-yl
Imidazol-1-yl
2-Methylimidazol-1-yl
2-Methylimidazol-1-yl
Morpholino
245.1 4.8
227.0 4.1
86.9 3.6
209.8 4.7
74.8 3.9
64.5 2.8
76.3 3.3
101.2 3.8
80.1 3.5
60.8 4.6
91.2 3.5
64.5 4.2
163.9 4.4
102.7 3.3
900.5 5.3
556.9 1.8
757.8 4.3
739.9 3.6
622.8 3.4
506.3 3.2
2183.1 4.1
—
—
—
—
—
—
7.8
—
6.0
—
—
—
—
—
—
—
—
2.2
2.9
4.3
2.3
8.9
8.1
—
—
—
—
—
—
—
—
—
—
—
—
—
27.0 3.5
—
10.7 1.4
—
—
—
—
—
—
—
—
9.1 2.0
7.8 1.5
11.6 1.8
32.6 0.9
36.1 2.0
—
44.5 2.1
—
—
—
8.4
H
13.0
6.9
1.9
2.5
—
3.7
—
—
—
—
—
—
4-F
4-Me
4-Me
4-OMe
4-OMe
3-OMe
H
H
4-Me
H
13f
13g
13h
14a
14b
14c
15a
15b
15c
400.6 3.0
189.2 3.2
175.6 2.3
325.7 3.5
70.2 2.8
62.6 1.6
—
Imidazol-1-yl
Imidazol-1-yl
Morpholino
Imidazol-1-yl
—
—
—
—
H
4-Me
Imidazol-1-yl
—
9.1
Lamivudine
240.0 2.2
a
b
c
CC50 is 50% cytotoxic concentration in HepG2.2.15 cells.
IC50 is 50% inhibitory concentration.
Selectivity index (SI: CC50/IC50).
Means no antiviral activity at the concentration lower than its CC50
Data are represented by mean values and standard deviations for three separate experiments.
d
e
.
6. Zoulim, F.; Durantel, D.; Deny, P. Liver Int. 2009, 29, 108.
of 2-position. It has been observed that compounds 13a–c with
electron-withdrawing groups such as F atom or with no substitu-
tion showed more potent inhibitory activity than compounds
13d–h with electron-donating groups. With the conversion of sul-
fur into sulfinyl and sulfonyl group, the cellular toxicity was obvi-
ously reduced, whereas a complete loss in anti-HBV DNA activity
was also noted (13a vs 14b vs 15b, 13d vs 14c vs 15c). 2-Sulfinyl
analogues 14a–c and 2-sulfonyl analogues 15a–c showed moder-
ate activity against HBsAg with IC50 values ranging from 62.6 to
7. Chai, H. F.; Zhao, Y. F.; Zhao, C. S.; Gong, P. Bioorg. Med. Chem. 2006, 14, 911.
8. Zhao, C. S.; Zhao, Y. F.; Chai, H. F.; Gong, P. Bioorg. Med. Chem. 2006, 14, 2552.
9. Zhao, C. S.; Zhao, Y. F.; Chai, H. F.; Gong, P. Chem. Res. Chin. Univ. 2006, 22, 577.
10. Zhao, Y. F.; Feng, R. L.; Liu, Y. J.; Zhang, Y. K.; Gong, P. Chem. Res. Chin. Univ.
2010, 26, 272.
11. Liu, Y. J.; Zhao, Y. F.; Zhai, X.; Feng, X. S.; Wang, J. X.; Gong, P. Bioorg. Med. Chem.
2008, 16, 6522.
12. Liu, Y. J.; Zhao, Y. F.; Zhai, X.; Liu, X. P.; Sun, L. X.; Ren, Y. X.; Gong, P. Arch.
Pharm. 2008, 341, 446.
13. Chen, D.; Liu, Y. J.; Zhang, S. L.; Guo, D. X.; Liu, C. H.; Li, S.; Gong, P. Arch. Pharm.
in press.
14. Spectroscopic data of selected compounds: 11d: mp 140–142 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 2.17 (s, 3H), 2.35 (m, 8H), 3.71 (s, 3H), 3.99 (s, 2H),
4.50 (s, 2H), 6.67 (d, J = 8.6 Hz, 1H), 7.22 (m, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.46 (d,
J = 7.5 Hz), 10.49 (s, 1H). IR (KBr) cmÀ1: 3445.1, 2944.4, 2795.7, 1616.1, 1585.4,
1467.1, 1208.5, 804.7, 741.3. MS (ESI) m/z: 383.0 (M+H). Compound 13a: mp
135–137 °C. 1H NMR (DMSO-d6, 300 MHz) d: 3.74 (s, 3H), 4.56 (s, 2H,), 5.31 (s,
2H), 6.74(s, 1H), 6.81 (d, J = 8.7 Hz, 1H), 6.99 (s, 1H), 7.18–7.37 (m, 4H), 7.48 (d,
J = 7.5 Hz, 2H), 7.60 (s, 1H), 9.44 (s, 1H). IR (KBr) cmÀ1: 3417.9, 2663.6, 1602.0,
1506.5, 1480.5, 1424.0, 1395.1, 1224.6, 1051.7, 803.3, 730.6. MS (ESI) m/z:
351.2 (M+H). Compound 13b: mp 141–143 °C. 1H NMR (DMSO-d6, 300 MHz) d:
2.45 (s, 3H), 3.75 (s, 3H), 4.55 (s, 2H), 5.22 (s, 2H), 6.62 (s, 1H), 6.82 (d,
J = 8.5 Hz, 1H), 6.96 (s, 1H), 7.19–7.33 (m, 4H), 7.47 (d, J = 7.4 Hz, 2H), 9.46 (s,
1H). IR (KBr) cmÀ1: 3427.8, 2924.6, 1594.2, 1499.1, 1418.9, 1383.9, 1269.8,
1147.1, 807.3. MS (ESI) m/z: 365.1 (M+H). Compound 14b: mp 200–202 °C. 1H
NMR (DMSO-d6, 300 MHz) d: 3.64 (s, 3H), 4.61 (d, J = 13.3 Hz, 1H), 4.73 (d,
J = 13.4 Hz, 1H), 5.30 (s, 2H), 6.76 (s, 1H), 6.84 (d, J = 8.7 Hz, 1H), 6.98 (s, 1H),
7.30 (d, J = 8.7 Hz, 1H), 7.51–7.66 (m, 6H), 9.50 (s, 1H). IR (KBr) cmÀ1: 3429.8,
2926.4, 2643.0, 1603.5, 1475.2, 1428.9, 1282.0, 1227.8, 1050.9, 802.8. MS (ESI)
m/z: 367.2 (M+H). Compound 15b: mp 209–211 °C. 1H NMR (DMSO-d6,
300 MHz) d: 3.77 (s, 3H), 5.21 (s, 2H), 5.25 (s, 2H), 6.78 (s, 2H), 6.87 (d,
J = 8.7 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.51 (s, 1H), 7.62 (t, J = 7.6 Hz, 2H), 7.79
(d, J = 7.7 Hz, 3H), 9.53 (s, 1H). IR (KBr) cmÀ1: 3420.1, 2950.9, 1604.2, 1481.8,
1427.0, 1308.5, 1223.4, 1151.9, 1083.0, 803.4, 729.5. MS (ESI) m/z: 383.0
(M+H).
15. In vitro anti-HBV assays: The in vitro anti-HBV activities included the ability to
inhibit the production of HBsAg and HBeAg, and the replication of HBV DNA in
HBV-infected 2.2.15 cells. For the antiviral analyses, confluent cultures of
2.2.15 cells were maintained on 96-well flat-bottomed tissue culture plates in
RPMI 1640 medium with 2% fetal bovine serum. Cultures were treated with
eight consecutive daily doses of the test compounds and lamivudine. The cell
control was set up. Medium was changed daily with fresh test compounds and
positive control. HBV nucleic acid and protein levels were measured eight days
after the first treatment. Extracellular HBV surface (HBsAg) and e (HBeAg)
antigen levels produced from 2.2.15 cells were evaluated by semiquantitative
enzyme immunoassay (EIA) methods using commercial kits (HBsAg, Abbott
Laboratories; HBeAg, Diasorin, Inc) as previously described.16,17 Intracellular
HBV DNA levels were measured by quantitative Southern blot hybridization.
400.6 lM. Moreover, 2-sulfonyl analogues were more active than
the 2-sulfinyl ones (14a vs 15a, 14b vs 15b, and 14c vs 15c). Com-
pounds 11a–15c were also investigated for inhibition of HBeAg
secretion in HepG2.2.15 cells and none of them possessed activity
up to their 50% cytotoxic concentration.
In summary, a series of novel 1-methyl-1H-benzimidazol-5-ol
derivatives were synthesized and assessed for their anti-HBV activ-
ity and cytotoxicity in vitro, using lamivudine as reference control.
Six compounds showed significant inhibition of HBV DNA replica-
tion with IC50 values less than 45
lM. Of them, compound 13b,
with an IC50 value of 7.8 M and a SI value of 13.0, was found to
l
be more potent than lamivudine. The preliminary SAR provided
useful indications for guiding the further design of new 1H-ben-
zimidazol-5-ols as more active HBV inhibitors.
Acknowledgments
This work was supported by a grant from the National Natural
Science Foundation of China (NSFC No. 30672519) and a grant from
the Science and Technology Projects of Shenyang City, China
(No. 081256).
References and notes
1. Ganem, D.; Prince, A. M. N. Engl. J. Med. 2004, 350, 1118.
2. Braz, P. E. J. Infect. Dis. 2008, 12, 355.
3. Dienstag, J. L. N. Engl. J. Med. 2008, 359, 1486.
4. Franco, J.; Saeian, K. J. J. Vasc. Interv. Radiol. 2002, 13, S191.
5. Heathcote, E. J.; Gane, E.; DeMan, R. Hepatology 2007, 46, 861.

Y. Zhao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7230–7233
7233
The IC50 and selected index of the evaluated compounds and lamivudine were
calculated, respectively. Cytotoxicity assay: 2.2.15 cells were grown to
confluence in 96-well flat-bottomed tissue culture plates and treated with
tested compound (in 0.2 mL culture medium/well) as described above.
Untreated control cultures were maintained on each 96-well plate. Toxicity
was determined by measuring neutral red dye uptake, as determined from the
cells’ 510 nm absorbance relative to untreated cells, after the treatment of
consecutive 9 days.
16. Korba, B. E.; Gerin, J. L. Antiviral Res. 1992, 19, 55.
17. Korba, B. E.; Gerin, J. L. Antiviral Res. 1995, 28, 225.