Biological evaluation of hepatitis C virus helicase inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00263-3

A small chemical library has been synthesized and assayed for inhibition of HCV helicase activity. This study provides the structure-activity relationship of the reported inhibitors, with emphasis placed on the aminophenylbenzimidazole moiety and the link

Full text of DOI:10.1016/S0960-894X(01)00263-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1647–1650
Biological Evaluation of Hepatitis C Virus Helicase Inhibitors
Chee Wee Phoon,a Poh Yong Ng,b Anthony E. Ting,b Su Ling Yeob and Mui Mui Sima,*
aMedicinal and Combinatorial Chemistry Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609
bCollaborative Antiviral Research Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609
Received 6 November2000; accepted 9 April 2001
Abstract—A small chemical library has been synthesized and assayed for inhibition of HCV helicase activity. This study provides
the structure–activity relationship of the reported inhibitors, with emphasis placed on the aminophenylbenzimidazole moiety and
the linkers. Our data highlight the importance of preserving the aminophenylbenzimidazole core and the hydrophobic linkers for
biological activity. The development of a robust HCV helicase assay is also described. # 2001 ElsevierScience Ltd. All irghts
reserved.
Introduction
containing aminophenylbenzimidazole (1a–g) and ben-
zimidazole-like moieties (2) attached to symmetrical
linkers of different lengths (Fig. 1).5 The IC50’s of these
inhibitors are in the low micromolar range. Vertex
Pharmaceuticals Inc. reported several aminothiadiazo-
liums which also inhibit helicase activity but with lower
potency.6
Hepatitis C virus (HCV) infection is a major health
concern worldwide, with about 3% of the world popu-
lation infected.1 HCV is the principal pathogen of
transfusion-associated non-A non-B hepatitis.2 Infec-
tion with HCV results in acute or chronic hepatitis,
which could lead to liver cirrhosis and hepatocellular
carcinoma.3 There is no vaccine against HCV at pre-
sent. The current therapy using a-interferon alone or in
combination with ribavirin is not highly efficacious.4
Hence, there is an urgent drive towards the discovery of
other therapeutic targets and potent inhibitors for
development into anti-HCV drugs.
The SAR study of the ViroPharma’s inhibitors has not
been described. It is imperative to identify the critical
pharmacophores to aid the design of potent helicase
inhibitors. Reported herein are the results of our pre-
liminary SAR study with emphasis placed on the
The HCV genome encodes a polyprotein which is pro-
cessed into 10 distinct structural and non-structural
(NS) proteins.4 Recently, much attention has been
paid to the NS3 protein which possesses serine pro-
teolytic activity at the N-terminus, helicase and
nucleotide triphosphatase activities at the C-terminus.
The helicase unwinds the nucleic acid duplexes, play-
ing a crucial role during the viral replication cycle.
Intervention of the helicase activity with chemical
ligands has the potential of terminating the HCV pro-
liferation.
There are only a few HCV helicase inhibitors reported so
far. ViroPharma Inc. patented two series of compounds
*Corresponding author. Tel.: +65-874-1443; fax: +65-779-1117;
e-mail: mcbsimmm@imcb.nus.edu.sg
Figure 1. The HCV helicase inhibitors reported by ViroPharma Inc.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00263-3

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C. W. Phoon et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1647–1650
aminophenylbenzimidazole moiety and the linkers. The
development of a robust helicase assay is also described.
Biological Assay
The bacterial expression and purification of HCV NS3
helicase was carried out as reported in the literature.10
The helicase was found to be more than 95% pure as
determined by SDS-PAGE with Coomassie Blue stain-
ing. A double-stranded DNA template was prepared11
and used as the helicase substrate in our assay due to its
ease of handling. The helicase activity was determined
by monitoring the unwinding of the double-stranded
DNA template that resulted in the presence of single-
stranded DNA.12 Within 10 min, over75% of the template
was observed as single-stranded DNA. In the absence of
enzyme, only double-stranded DNA was observed.
Synthesis
The necessity of having benzimidazole was firstly eval-
uated by replacement with benzoxazoles, benzothiazoles
and a pyridinoxazole. Five linkers with aromatic ring,
double bond, and aliphatic chains of various lengths are
selected forthe study. The synthesis of these analogues
is outlined in Scheme 1. Aminophenols and thiophenols
reacted easily with p-aminobenzoic acid in the presence
of polyphosphoric acid to afford the corresponding
aminophenylbenzoxazoles and aminophenylbenzothia-
zoles 5 in 64–84% yields after recrystallization from
MeOH–H2O (Table 1).7
The compounds were assayed in duplicate for helicase
inhibition at a concentration of 25 mg/mL. Fourknown
inhibitors (1a, b, d, and e) were synthesized and included
in the assay as positive controls. They exhibited 75–93%
inhibition at the assay concentration of 25 mg/mL.
Subsequent coupling of aminophenylbenzothiazoles 5
with acid dichlorides furnished the products 6 (Fig. 2)
which precipitated spontaneously. The precipitates were
isolated by centrifugation. In order to assess the con-
tribution of benzene ring within the aminophenylbenz-
imidazole core to biological activity, a few diamides 7
were prepared by reacting the commercially available 2-
aminobenzimidazole with acid dichlorides. We are also
interested in probing whether the bioactivity could be
affected by changes in the linkers. This question was
addressed by preparing the aminophenylbenzimidazole-
derived diureas 8.8
Results and Discussion
Of 66 compounds screened, there was no inhibition with
the majority of the aminophenylbenzoxazoles- and
In addition, other heterocycles such as furfurylamine,
tryptamine, 3-(aminomethyl)pyridine, aminobenzothia-
zoles and aminothiazoles shown in Figure 3 were also
reacted with acid dichlorides to provide the diamides.
Precipitation of products was induced by adding water
in some cases (amines 13, 16, and 18). Higherpuirty
(>95%) was observed in products that precipitated
spontaneously.9 Those products with purity higher than
85% were tested in the bioassay.
Scheme 1. Synthesis of the aminophenylbenzothiazoles 5.
Table 1. The yields of compounds 5a–e
Compound
Z
Y
R1
R2
% Yield
5a
5b
5c
5d
5e
O
S
O
O
S
CH
CH
N
CH
CH
H
H
H
CH3
H
H
H
H
H
CH3
67
84
64
81
Commercially
available
Figure 2. The diamides 6, aminobenzimidazole-derived diamides 7
and aminophenylbenzimidazole-derived diureas 8.

C. W. Phoon et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1647–1650
1649
aminophenylbenzothiazoles-containing compounds 6.
The aminobenzimidazole-derived diamides 7 showed 6–
13% inhibition whereas the aminophenylbenzimidazole-
derived diureas 8 (n=4 and 6) have 20 and 28% inhibi-
tory activity, respectively. Compounds 19 and 20 (Fig.
4) also displayed 14 and 10% inhibition, respectively.
benzene group at the C-2 position of benzimidazole,
and the nature of the linker are essential for inhibitory
activity. Although the exact mechanism of the inhibition
is still not clear, it has been suggested that ViroPharma’s
inhibitors might compete with nucleic acids for the
substrate’s binding site. Thus, the NH group could
possibly interact with the enzyme through hydrogen
bonding while the benzene ring might be interacting
through hydrophobic interaction. It is hoped that the
biological evaluation carried out in this work would
provide further insight to the design of HCV helicase
inhibitors.
There is a drastic decrease of potency after replacing the
benzimidazole moiety (1a–e) with benzoxazole 6a(i, ii,
iv, and v) and benzothiazole 6b(i–v) moieties. Similarly,
the biological activity is also reduced after deletion of
the benzene ring. Inhibition of compounds 7i–iv decrea-
ses tremendously as compared to the ViroPharma’s
inhibitors 1c–f. The linkeralso plays an important role as
replacement of the diamide linkage (1e,g) with diurea
(8i–ii) leads to diminished potency of the inhibitors.
Acknowledgements
The authors would like to thank Dr. Siew Pheng Lim
forproviding the HCV helicase clone and Ms Pei Ying
Lee for the assistance in library synthesis. This work is
supported by the National Science and Technology
Board of Singapore.
The information derived from our SAR study shows
that the NH group within the benzimidazole ring, the
References and Notes
1. World Health Organization Weekly Epidemiological Record
1997, 72, 65.
2. (a) Hagedorn, C. H.; Rice, C. M. In Curr. Top. Microbiol.
& Immunol.; The Hepatitis C Viruses; Springer: Berlin 2000;
Vol. 242. (b) Reding, M. T. Expert Opin. Ther. Pat. 2000, 10,
1201.
3. Hoofnagle, J. H. Hepatology 1997, 26, 15 S.
4. Walker, M. A. DDT 1999, 4, 518.
5. (a) Diana, G. D.; Bailey, T. R. US5633388, 1997. (b) Diana,
G. D.; Bailey, T. R.; Nitz, T. J. WO9736554, 1997.
6. Janetka, J. W.; Ledford, B. E.; Mullican, M. D.
WO0024725, 2000.
7. Representative procedure for the synthesis of compound 6:
To a mixture of aminophenol or aminothiophenol (6.5 mmol)
and p-aminobenzoic acid (7 mmol) was added polyphosphoric
acid (10 g). The mixture was stirred vigorously at 220 ꢀC for4
h, cooled and poured into 10% Na2CO3 solution. The sus-
pension was stirred until gas evolution ceased and then fil-
tered. The solid collected was washed with H2O (3ꢁ50 mL),
and recrystallized from MeOH–H2O to afford the product 5.
To a solution of compound 5 (0.6 mmol) in anhydrous
CH2Cl2 orDMF (5 mL) containing N,N-diisopropylethyl-
amine (0.105 mL, 0.6 mmol) was added acid dichloride (0.2
mmol). The mixture was stirred at room temperature for 18 h.
The suspension was centrifuged and the supernatant was dis-
carded. The pellet was resuspended with CH2Cl2 orDMF–
H2O and centrifuged. This washing procedure was carried out
twice. Product 6 was then dried in vacuo.
Figure 3. Amine building blocks employed in the library synthesis.
Compound 19: 1H NMR (400 MHz, DMSO-d6) d 2.44 (6H,
s), 2.72 (4H, s), 7.32 (2H, d, J=8.3 Hz), 7.77 (4H, d, J=8.6
Hz), 7.88 (2H, d, J=8.3 Hz), 7.89 (2H, s), 8.00 (4H, d, J=8.6
Hz), 10.35 (2H, s); 13C NMR (100 MHz, DMSO-d6) d 20.5,
28.4, 118.6, 121.2, 121.6, 126.9, 127.3, 127.5, 133.8, 134.5,
141.4, 151.2, 170.3; m/z (ESI) C32H27N4O2S2 (M+H)+calcd
563.1575, found 563.1563.
Compound 20: 1H NMR (400 MHz, DMSO-d6) d 1.59 (4H,
brs), 2.43 (4 H, brs), 7.16 (2H, d, J=3.5 Hz), 7.43 (2H, d,
J=3.5 Hz), 12.07 (2H, s); 13C NMR (100 MHz, DMSO-d6) d
23.7, 34.0, 112.7, 137.0, 157.4, 170.5; m/z (ESI) C12H15N4O2S2
(M+H)+ calcd 311.0636, found 311.0646.
Figure 4.

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C. W. Phoon et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1647–1650
8. Compound 8 was purified by preparative TLC (CH2Cl2–
MeOH 7:1, developed twice).
radioisotope was removed using Qiagen nucleotide removal
kit. The radiolabeled OLG 54 was incubated with OLG 55 at
a 1:3 molar ratio and annealed by cooling the mixture from
100 ꢀC to rt gradually.
Compound 8i (n=4): 1H NMR (400 MHz, DMSO-d6) d
1.47 (4H, m), 3.16 (4H, m), 6.45 (2H, brt, J=5.7 Hz), 7.14
(4H, brd, J=5.5 Hz), 7.46 (2H, brs), 7.55 (4H, d, J=8.7 Hz),
7.56 (2H, brs), 8.01 (4H, d, J=8.7 Hz), 8.87 (2H, s), 12.70
(2H, s); 13C NMR (100 MHz, DMSO-d6) d 27.7, 39.2, 111.5,
117.9, 118.9, 121.9, 122.5, 123.1, 127.6, 135.4, 142.8, 144.4,
152.0, 155.6; m/z (ESI) C32H32N8O2 (M+2H)2+ calcd
280.1324, found 280.1325.
12. Helicase assay: To a 6 mL of drug solution (DMSO–H2O
1:5) was added 9 mL of helicase stock (which contains helicase,
5ꢁ assay buffer, and water in a ratio of 1:3:5). The mixture
was left at rt for 3 min before 5 mL of DNA solution (which
contains 1 pmol/mL of radiolabeled double-stranded DNA, 5ꢁ
assay buffer, and water in a ratio of 1:5:19) was introduced.
These make up to a final mixture that contains 0.2 mg of heli-
case and 0.2 pmol of dsDNA. The mixture was incubated at
37 ꢀC for10 min. The helicase activity was stopped by adding
5 mL of 5ꢁ loading buffer. The single- and double-stranded
DNA were separated on a 10% native polyacrylamide gel. The
gel was dried in vacuo and the single- and double-stranded
DNA were detected by autoradiography and quantitated on a
densitometer (Bio-Rad). The percentage of single-stranded
DNA was calculated as the percentage of radioactivity of
the single-stranded DNA from the total radioactivity in
each reaction. Calculation of inhibition is based on the
formula:
Compound 8ii (n=6): 1H NMR (400 MHz, DMSO-d6) d
1.32 (4H, m), 1.44 (4H, m), 3.09 (4H, m), 6.52 (2H, brs), 7.14
(4H, brd, J=4.3 Hz), 7.46 (2H, brs), 7.55 (4H, dd, J=8.7, 2.5
Hz), 7.56 (2H, brs), 8.00 (4H, d, J=8.7 Hz), 8.95 (2H, s),
12.70 (2H, s); 13C NMR (100 MHz, DMSO-d6) d 26.6, 30.1,
39.5, 111.5, 117.9, 118.8, 122.0, 122.6, 122.9, 127.6, 135.3,
142.8, 144.2, 152.0, 155.6; m/z (ESI) C34H36N8O2 (M+2H)2+
calcd 294.1481, found 294.1479.
9. The purities of the compounds were assessed by observa-
1
tion of H NMR spectra.
10. Khu, Y.-L.; Koh, E.; Lim, S. P.; Tan, Y. H.; Brenner, S.;
Lim, S. G.; Hong, W.; Goh, P.-Y. J. Virol. 2001, 75, 205.
11. Preparation of the helicase substrate: Two oligos, OLG 54
(50-GTC AGT TGA GTG GCA GGC GGC ACA CAT TAT
AGT GTC GTA GGC TTC-30) and OLG 55 (50- GTG TGC
CGC CTG CCA CTC AAC TGA CTC AAC TAC TGT CTT
GGG CAT CGG CA-30) were used to prepare a double-
stranded DNA template for the helicase assay. 75 pmol of
OLG54 was radiolabeled with [g-32P]-ATP (100 nCi) using
polynucleotide kinase (NEB) in a final volume of 30 mL. The
labeling reaction was performed at 37 ꢀC for1 h. The unbound
ÈÂ
Ã
É
%ssDNARef ꢂ %ssDNADrug =%ssDNARef ꢁ 100
Composition of 5ꢁ assay buffer: 100 mM Hepes-KOH pH
7.3, 10 mM DTT, 7.5 mM MnCl2, 12.5 mM ATP, 0.5 mg/mL
BSA.
Composition of 5ꢁ loading buffer: 500 mM Tris-Cl pH 7.4,
50 mM EDTA, 0.25% SDS, 0.05% bromophenol blue, 25%
glycerol.
The reference control well contains enzyme, substrate, and
1 mL of DMSO in assay buffer.