Benzimidazole inhibitors of hepatitis C virus NS5B polymerase: Identification of 2-[(4-diarylmethoxy)phenyl]-benzimidazole

Article abstract of DOI:10.1016/j.bmcl.2006.01.032

A series of 1-cycloalkyl-2-phenyl-1H-benzimidazole-5-carboxylic acid derivatives was synthesized and evaluated for inhibitory activity against HCV NS5B RNA-dependent RNA polymerase (RdRp). A SAR study was performed and led to identify the 2-[(4-diarylmeth

Full text of DOI:10.1016/j.bmcl.2006.01.032

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1859–1863
Benzimidazole inhibitors of hepatitis C virus NS5B polymerase:
Identification of 2-[(4-diarylmethoxy)phenyl]-benzimidazole
Tomio Ishida,a Takayoshi Suzuki,a, Shintaro Hirashima,a Kenji Mizutani,a
Atsuhito Yoshida,b Izuru Ando,b Satoru Ikeda,b
Tsuyoshi Adachic and Hiromasa Hashimotoa,*
aChemical Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc,
1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan
bBiological/Pharmacological Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc,
1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan
cPharmaceutical Frontier Research Laboratories, Japan Tobacco Inc, 1-13-2 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan
Received 29 November 2005; revised 29 December 2005; accepted 4 January 2006
Available online 7 February 2006
Abstract—A series of 1-cycloalkyl-2-phenyl-1H-benzimidazole-5-carboxylic acid derivatives was synthesized and evaluated for
inhibitory activity against HCV NS5B RNA-dependent RNA polymerase (RdRp). A SAR study was performed and led to identify
the 2-[(4-diarylmethoxy)phenyl]-benzimidazoles as potent inhibitors. They inhibit subgenomic HCV RNA replication in the replicon
cells at low micromolar concentrations (EC50 as low as 1.1 lM). They are selective against DNA polymerases (IC50 > 10 lM) and
exhibit low cytotoxicity.
Ó 2006 Elsevier Ltd. All rights reserved.
Hepatitis C is an infectious disease caused by hepatitis C
virus (HCV).1 It is estimated that more than 170 million
people in the world are chronically infected.2 About 20%
of the chronically infected people develop cirrhosis.
Moreover, most hepatocellular carcinomas are caused
by HCV infection. The currently available drug for
HCV is a cytokine, IFN-a.3–5 But the problems of
IFN-based therapy are low efficacy and side effects.
Even in the recent combination therapy of pegylated
IFN-a and ribavirin, the sustained viral response rate
is under 50% in the patients infected with the most wide-
spread genotype 1 virus. In spite of the low efficacy, ad-
verse events are seen in almost all patients. Therefore,
there is urgent and strong medical demand for the devel-
opment of new, effective and well-tolerated anti-HCV
drugs.
about 3000 amino acids, which is processed into struc-
tural and non-structural (NS) proteins.3 NS5B RdRp,
one of the NS proteins, plays a central role in the repli-
cation of HCV and therefore is an attractive target for
drug development.3,6 Many groups have ongoing re-
search programs to find inhibitors of NS5B. Several
classes of nucleoside and non-nucleoside inhibitors have
been reported recently.3,6–9 We discovered a new class of
benzimidazoles.7,8 In this paper, we report the synthesis
and SAR studies of a series of 1-cycloalkyl-2-phenyl-
1H-benzimidazole 5-carboxylic acids as NS5B inhibitors
and also report anti-HCV activity of the 2-[(4-diaryl-
methoxy)phenyl]-benzimidazoles in a cell-based assay.
We screened our in-house compound collection to iden-
tify NS5B (genotype 1b) inhibitors and found a benz-
imidazole derivative 1 (Fig. 1) as one of the hit
compounds (47% inhibition at 10 lM). Initial structural
optimization of the amide part of 1 led to a more potent
carboxylic acid 2 (IC50 = 3.2 lM). We selected this com-
pound 2 for further SAR studies and structural
optimization.
HCV is an enveloped single strand RNA virus in the
Flaviviridae family and encodes a polyprotein chain of
Keywords: HCV; NS5B polymerase; Inhibitor; Benzimidazole.
*
Corresponding author. Tel.: +81 72 681 9905; fax: +81 72 681
9725; e-mail: hiromasa.hashimoto@ims.jti.co.jp
The lead compound 2 and its cyclohexyl or cycloheptyl
analogues 13 and 14 were synthesized as shown in
Scheme 1. Ethyl 4-fluoro-3-nitrobenzoate, 3, was reacted
Present address: Graduate School of Pharmaceutical Sciences,
Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya,
Aichi 467-8603, Japan.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.032

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T. Ishida et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1859–1863
of the ester group afforded 33 and 34, respectively. All
the compounds assayed in this article were characterized
O
N
N
X
1
by H NMR, MS and elemental analysis.
O
The benzimidazole 5-carboxylic acids synthesized in this
study were evaluated for their ability to inhibit HCV
NS5B RdRp activity. While there are several genotypes
in HCV, our assay employed genotype 1 enzymes. All
compounds were examined against a genotype 1b en-
zyme lacking C-terminal 47 residues (1b NS5B544),13
and the results are summarized in Tables 1–4. The com-
pounds in Table 4 were also evaluated for another sub-
type 1a enzyme (1a NS5B544), and we examined their
ability to inhibit the replication of subgenomic HCV
RNA in a replicon cell system using a Huh-5-2 cells (a
Huh-7 derived cell line which possesses a 1b HCV repli-
con containing the luciferase reporter gene).14
1
2
X = NH2
X = OH
Figure 1.
with appropriate cycloalkylamine to give the corre-
sponding anilines 4–6. The nitro group was hydrogenat-
ed to afford diaminobenzenes 7–9 which were converted
to the benzimidazoles by acylation with 4-ben-
zyloxybenzoyl chloride and subsequent heating in
AcOH.10 Hydrolysis of ester gave the benzimidazole 5-
carboxylic acids 2, 13 and 14. Benzimidazoles 16 bearing
a substituent on the benzyl group of compound 2 were
prepared from compound 10 (Scheme 1). The benzyl
group was removed by hydrogenolysis to give a phenol
15. Alkylation of the phenol and subsequent hydrolysis
of the ester group afforded compound 16.
We first examined the cyclopentyl ring part and the ef-
fect of substituents on the benzyl moiety of the lead
compound 2. As seen in Table 1, an expansion of the
ring size to cyclohexyl (13) showed 2-fold increased
activity. Further ring expansion to a seven-membered
ring (14) retained the activity. It seems that a six-mem-
bered ring optimally fits to a hydrophobic pocket.15
Introduction of the lipophilic substituents such as Me,
CF3, t-Bu and Cl (16a–c,e) at the para position slightly
increased the potency, while an ionic substituent COOH
(16d) decreased activity (Table 2). Changing the benzene
ring to 4-pyridine (16g) could not improve the activity.
Changing the position of Cl group (16h and i) or intro-
duction of another Cl atom (16j and k) did not afford
further increase in the potency. Thus, an attempt to in-
crease the activity by introducing a substituent on the
benzyl moiety brought an unsatisfactory result. There-
fore, we next tried to seek a more attractive structure
for optimization instead of the benzyl group.
Benzimidazoles 24 were prepared by essentially the same
method as employed in the synthesis of the benzyloxy
version 13 (Scheme 2). Two biphenyl compounds, 25
and 26, were prepared from the corresponding bromide
in two steps by Suzuki coupling11 and following hydro-
lysis as shown in Scheme 3. The diarylmethyl ethers, 33
and 34, were synthesized by using the imidate coupling
route from the diamine 8 as shown in Scheme 4. The dia-
mine 8 was reacted with imidate 29 or 30 to give the
benzimidazole 31 or 32, respectively.12 The imidates
were prepared from the corresponding 4-hydroxybenzo-
nitrile. Alkylation of the phenol group of 31 and 32 with
various diarylmethylhalides and subsequent hydrolysis
EtOOC
NO2
F
EtOOC
NO2
EtOOC
NH2
b
a
NHR
NHR
4 R = cyclopentyl
5 R = cyclohexyl
6 R = cycloheptyl
7 R = cyclopentyl
8 R = cyclohexyl
9 R = cycloheptyl
3
EtOOC
HOOC
N
N
e
c, d
OBn
OBn
N
N
R
R
10 R = cyclopentyl
11 R = cyclohexyl
12 R = cycloheptyl
2
R = cyclopentyl
13 R = cyclohexyl
14 R = cycloheptyl
EtOOC
f
HOOC
g,e
N
N
N
O
N
OH
10
Ar
15
16
Scheme 1. Reagents and conditions: (a) RNH2, K2CO3, DMSO, rt; (b) H2 (1 atom), Pd/C, EtOH; (c) 4-benzyloxybenzoyl chloride, cat. DMAP,
pyridine–CH2Cl2, rt; (d) AcOH, reflux; (e) 4 N aq NaOH, EtOH–THF, reflux; (f) H2 (1 atom), Pd/C, AcOH; (g) ArCH2Br, K2CO3, DMF, 85 °C.

T. Ishida et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1859–1863
1861
OR
OR
X
a or b or c
d
COX
COOR'
COOR'
19
20 X = OH
21 X = Cl
17 X = OH, R' = Me
18 X = F, R' = Et
e
OR
H
N
R'OOC
EtOOC
f
N
N
g
OR
8
O
NH
23 R' = Et
24 R' = H
22
h
Scheme 2. Reagents and conditions: (a) ROH, PPh3, DIAD, THF, 0 °C then rt; (b) RX, NaH, DMF, 0 °C then rt; (c) ROH, K2CO3, DMSO, 130 °C;
(d) 4 N aq NaOH, MeOH–THF, reflux; (e) (COCl)2, CH2Cl2, rt; (f) 21, pyridine, rt; (g) AcOH, reflux; (h) 4 N aq NaOH, EtOH–THF, reflux.
EtOOC
Br
N
N
O
a,b
23g
HOOC
N
O
N
25
EtOOC
N
N
O
a,b
Br
23h
HOOC
N
N
O
26
Scheme 3. Reagents and conditions: (a) PhB(OH)2, cat. Pd(PPh3)4, NaHCO3, DME–H2O, reflux; (b) 4 N aq NaOH, EtOH–THF, reflux.
Several types of ether were examined, and the results are
shown in Table 3. In this series, cyclohexyl group was
employed. Elongation of the alkyl chain from benzyl
to phenylpropyl, phenylpentyl or phenoxyethyl (24a–c)
did not give much influence on the potency. Although
the three-dimensional structure of the binding site of
the compounds investigated in this article is not known,
we estimated that there is enough space around the ben-
zyl moiety and we might be able to use the space to in-
crease the affinity. So, we added another benzene ring on
the benzyl part of 13. Biphenylmethyl (24e and 25) and
diphenylmethyl (33a) derivatives showed 2- to 4-fold in-
crease in potency. Lengthening the methylene chain of
24e to that in 26 showed a similar potency, while a
shorter one in 24f gave a 2-fold decrease. Diphenylethyl
24d slightly reduced the potency compared to diphe-
nylmethyl, 33a. With these results, we selected the diphe-
nylmethyl type of compound 33a for further study.16
A series of diarylmethyl derivatives 33 and 34 listed in
Table 4 was synthesized and evaluated for activity
against the 1b and 1a NS5B enzymes. In general, these
derivatives are potent inhibitors for both enzymes and
more potent against the 1b subtype. SARs against both
enzymes seem to be roughly correlated, although SAR
for 1a enzyme is more flat. Introduction of substituents
such as Me, Cl and F (33b–e) on the diphenylmethyl
part slightly decreased the potency. We considered that
introduction of a substituent on the central phenyl ring
connected to the benzimidazole ring might have an influ-
ence on the potency. Introduction of a fluorine atom
(34a and b) achieved a 2- to 6-fold increase in potency
against the 1b compared with the corresponding parent
compound (33a and d), while no or a slight increase was
seen against 1a. At this point, these compounds were
tested in the replicon assay (Table 5). They efficiently
blocked the replication of subgenomic HCV RNA at

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T. Ishida et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1859–1863
Table 3. In vitro 1b NS5B inhibitory activity of 24–26 and 33a
OH
OH
CN
a
Compound
R
1b IC50 (lM)
a
13
Bn
1.4 0.63
0.80 0.28
1.0 0.31
X
X
24a
24b
24c
24d
24e
24f
25
Ph(CH2)3–
Ph(CH2)5–
PhO(CH2)2–
Ph2CHCH2–
(2-Ph)PhCH2–
(2-Ph)Ph–
HCl
HN
OMe
1.2 0.43
27 X = H
28 X = F
29 X = H
30 X = F
0.98 0.21
0.30 0.091
0.58 0.097
0.39 0.10
0.34 0.040
0.59 0.12
X
EtOOC
(3-Ph)PhCH2–
(2-Ph)PhCH2CH2–
Ph2CH–
N
N
EtOOC
NH2
26
33a
OH
b
N
H
a Values are means SD determined from three separate experiments.
31 X = H
32 X = F
8
Table 4. In vitro 1b NS5B and 1a NS5B inhibitory activity of 33 and
34
X
HOOC
N
N
Compound Ar1, Ar2
1b IC50 (lM) 1a IC50 (lM)
a
a
c, d
O
Ar2
33a
33b
33c
33d
33e
34a
34b
34c
34d
34e
Ph
0.59 0.12
1.2 0.31
1.3 0.05
3.1 0.29
2.4 0.19
1.7 0.40
1.9 0.08
0.8 0.14
0.84 0.11
0.95 0.35
2.2 0.35
Ar1
4-Me-Ph
4-Cl-Ph
4-F-Ph
0.93 0.33
0.80 0.17
0.76 0.18
0.096 0.040
0.30 0.066
0.64 0.030
1.1 0.25
33 X = H
34 X = F
3-F-Ph
Ph
4-F-Ph
Scheme 4. Reagents and conditions: (a) AcCl, MeOH, 0 °C then rt;
(b) 29 or 30, MeOH, reflux; (c) Ar1Ar2CHBr, K2CO3, DMF, 80 °C;
(d) 4 N aq NaOH, EtOH–THF, reflux.
4-(CONMe2)-Ph
3-Pyridyl
3-Pyridyl, Ph
0.27 0.0069 0.80 0.12
Table 1. In vitro 1b NS5B enzyme inhibition activity of 1-cycloalkyl-2-
(4-benzyloxy)phenylbenzimidazole-5-carboxylic acid derivatives
a Values are means SD determined from three separate experiments.
a
Compound
R
1b IC50 (lM)
Table 5. HCV RNA replication inhibitory activity and cytotoxicity on
replicon cell of 33 and 34
2
Cyclopentyl
Cyclohexyl
Cycloheptyl
3.2 0.75
1.4 0.63
1.8 0.34
13
14
Compound
Replicona
Cytotoxicityb
c
c
a Values are means SD determined from three separate experiments.
EC50 (lM)
CC50 (lM)
33a
33b
33c
33d
33e
34a
34b
34c
34d
34e
2.6 0.7
3.7 0.9
3.6 1.1
2.2 0.6
2.8 0.9
1.9 0.5
1.6 0.4
1.2 0.2
2.6 0.1
1.1 0.3
31.9 0.6
23.5 1.0
23.7 1.4
27.7 2.7
24.4 2.3
27.3 1.1
27.2 1.3
>50
Table 2. In vitro 1b NS5B inhibitory activity of 1-cyclopentyl
derivatives 16
a
Compound
Ar
1b IC50 (lM)
2
Ph
3.2 0.75
1.6 0.18
1.8 0.54
1.5 0.42
6.7 0.15
1.2 0.37
2.1 0.98
4.2 1.0
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
4-Me-Ph
4-CF3-Ph
4-t-Bu-Ph
4-(COOH)-Ph
4-Cl-Ph
>50
>50
a Compounds were incubated in Huh-5-2 cell culture for 48 h.
b MTT assay on parallel samples at the same time.
c Values are means SD determined from three separate experiments.
4-OMe-Ph
4-Pyridyl
2-Cl-Ph
2.3 0.78
1.6 0.26
1.7 0.22
1.3 0.19
3-Cl-Ph
3,5-Cl-Ph
3,4-Cl-Ph
the 1b enzyme compared to 34a. However, 34c and e
exhibited a higher cellular potency (EC50 of 1.2 and
1.1 lM, respectively). In addition, these compounds
showed no cytotoxicity up to 50 lM. Compounds 33
and 34 were selective against DNA polymerases a and
b (IC50 > 10 lM).
a Values are means SD determined from three separate experiments.
low micromolar concentrations and showed weak cyto-
toxicity. Although the potency against the 1b enzyme
improved (34a and b), the cellular potency was not as
expected, suggesting that the highly lipophilic property
of these molecules had to be changed. Introduction of
In conclusion, we have synthesized a new class of benz-
imidazole compounds that are potent inhibitors of HCV
NS5B RdRp. Preliminary optimization of this series led
to the identification of the diarylmethoxy derivatives 33
and 34. They efficiently blocked the replication of subge-
nomic HCV RNA in the replicon cells. Further SAR
a
polar substituent, N,N-dimethylamido (34c) or
replacement of the benzene ring with pyridine (34d
and e), showed 3- to 10-fold reduced potency against

T. Ishida et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1859–1863
1863
8. (a) Another group, Boehringer Ingelheim, also indepen-
dently discovered benzimidazole derivatives as NS5B
inhibitors: Beaulieu, P. L.; Fazal, G.; Gillard, J.;
Kukolj, G.; Austel, V. Patent WO-0204425, 2002; (b)
Beaulieu, P. L.; Bo¨s, M.; Bousquet, Y.; Fazal, G.;
Gauthier, J.; Gillard, J.; Goulet, S.; LaPlante, S.;
Poupart, M.-A.; Lefebvre, S.; McKercher, G.; Pellerin,
C.; Austel, V.; Kukolj, G. Bioorg. Med. Chem. Lett.
2004, 14, 119; (c) Beaulieu, P. L.; Bo¨s, M.; Bousquet,
Y.; Deroy, P.; Fazal, G.; Gauthier, J.; Gillard, J.;
Goulet, S.; McKercher, G.; Poupart, M.-A.; Valois, S.;
Kukolj, G. Bioorg. Med. Chem. Lett. 2004, 14, 967; (d)
Beaulieu, P. L.; Bousquet, Y.; Gauthier, J.; Gillard, J.;
Marquis, M.; McKercher, G.; Pellerin, C.; Valois, S.;
Kukolj, G. J. Med. Chem. 2004, 47, 6884.
studies including the effect of substituents other than F
atom on the central phenyl ring are on the way and will
be reported in due course.
Acknowledgments
We thank Mr. Mitsumasa Takahashi and Mr. Eita
Nagao for analytical support. We are grateful to
Mr. Yasushi Niwa and Mr. Masakazu Komatsu for
support running the biological assays.
Supplementary data
9. (a) Recently, structurally related indole derivatives were
reported from several groups: Beaulieu, P. L.; Fazal, G.;
Goulet, S.; Kukolj, G.; Poirier, M.; Tsantrizos, Y. S.;
Jolicoeur, E.; Gillard, J.; Poupart, M.-A; Rancourt, J.
Patent WO-2003010141, 2003; (b) Harper, S.; Pacini, B.;
Avolio, S.; Di Filippo, M.; Migliaccio, G.; Laufer, R.; De
Francesco, R.; Rowley, M.; Narjes, F. J. Med. Chem.
2005, 48, 1314; (c) Oka, T.; Yata, S.; Ikegashira, K.; Noji,
S.; Akaki, T.; Hirashima, S.; Niwa, Y.; Ando, I.; Sato, T.
Patent WO-2005014543, 2005; (d) Harper, S.; Avolio, S.;
Pacini, B.; Di Filippo, M.; Altamura, S.; Tomei, L.;
Paonessa, G.; Di Marco, S.; Carfi, A.; Giuliano, C.;
Padron, J.; Bonelli, F.; Migliaccio, G.; De Francesco, R.;
Laufer, R.; Rowley, M.; Narjes, F. J. Med. Chem. 2005,
48, 4547.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2006.
01.032.
References and notes
1. Lauer, G. M.; Walker, B. D. N. Eng. J. Med. 2001, 345,
41.
2. World Health Organization Weekly Epidemiological Record
2000, 75, 18.
3. (a) Tan, S.-L.; Pause, A.; Shi, Y.; Sonenberg, N. Nat. Rev.
Drug Disc. 2002, 1, 867; (b) Poynard, T.; Yuen, M.-F.;
Ratziu, V.; Lai, C. L. Lancet 2003, 362, 2095.
4. Manns, M. P.; McHutchison, J. G.; Gordon, S. C.; Rustgi,
V. K.; Schiffman, M.; Reindollar, R.; Goodman, Z. D.;
Koury, K.; Ling, M.-H.; Albrecht, J. K. Lancet 2001, 358,
958.
5. Poynard, T.; Macrellin, P.; Lee, S. S.; Niederau, C.;
Minuk, G. S.; Ideo, G.; Bain, V.; Heathcote, J.; Zeuzem,
S.; Trepo, C.; Albrecht, J. Lancet 1998, 352, 1426.
6. NS5B inhibitors are summarized in the following reviews
(a) Gordon, C. P.; Keller, P. A. J. Med. Chem. 2005, 48, 1;
(b) Beaulieu, P. L.; Tsantrizos, Y. S. Curr. Opin. Invest.
Drugs 2004, 5, 838; (c) De Francesco, R.; Tomei, L.;
Altamura, S.; Summa, V.; Migliaccio, G. Antiviral Res.
2003, 58, 1.
10. Kamel, M.; Allam, M. A.; Abou-Zeid, N. Y. Tetrahedron
1967, 23, 1863.
11. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
12. Thomas, P. R.; Tyler, G. J. J. Chem. Soc. 1958, 2197.
13. Adachi, T.; Ago, H.; Habuka, N.; Okuda, K.; Komatsu,
M.; Ikeda, S.; Yatsunami, K. Biochim. Biophys. Acta 2002,
1601, 38.
14. (a) Lohmann, V.; Ko¨rner, F.; Koch, J. O.; Herian, U.;
Theilmann, L.; Bartenschlager, R. Science 1999, 285, 110;
(b) Krieger, N.; Lohmann, V.; Bartenschlager, R. J. Virol.
2001, 75, 4614.
15. The same observation was seen in the case reported by
Boehringer Ingelheim Ltd. See Ref. 8b.
16. The biphenyl type of compounds 24e is also attractive
for further study. The results will be reported in due
course.
7. Hashimoto, H.: Mizutani, K.; Yoshida, A. Patent WO-
0147883, 2001.