Identification of N,N-disubstituted phenylalanines as a novel class of inhibitors of hepatitis C NS5B polymerase

Article abstract of DOI:10.1021/jm0340400

The HCV NS5B RNA dependent RNA polymerase plays an essential role in viral replication. The discovery of a novel class of inhibitors based on an N,N-disubstituted phenylalanine scaffold and structure-activity relationships studies to improve potency are described.

Full text of DOI:10.1021/jm0340400

J . Med. Chem. 2003, 46, 1283-1285
1283
side triphosphate as well as other low molecular weight
inhibitors are shown in Figure 1. The crystal structure
of the NS5B polymerase has also been independently
resolved by three groups,6 and the enzyme has the
characteristic right-handed “fingers-palm-thumb” do-
main of polymerases. The active site that resides in the
palm region contains the conserved GDD motif of
polymerases and is partially enclosed by the fingers and
thumb domains. An NS5B polymerase inhibitor (J TK-
002/J TK-003) from J apan Tobacco Inc.6b has been
reported to be undergoing phase II clinical trials.7 The
structure of this compound has not been disclosed, but
it may be structurally related to J TI (Figure 1).
Id en tifica tion of N,N-Disu bstitu ted
P h en yla la n in es a s a Novel Cla ss of
In h ibitor s of Hep a titis C NS5B
P olym er a se
Laval Chan,* T. J agadeeswar Reddy, Me´lanie Proulx,
Sanjoy K. Das, Oswy Pereira, Wuyi Wang,
Arshad Siddiqui, Constantin G. Yannopoulos,
Carl Poisson, Nathalie Turcotte, Alexandre Drouin,
M. Hicham Alaoui-Ismaili, Richard Bethell,
Martine Hamel, Lucille L’Heureux,
Darius Bilimoria, and Nghe Nguyen-Ba
To identify NS5B inhibitors, an assay using a full-
length recombinant polymerase expressed from bacu-
lovirus-infected Sf9 insect cells was developed. A Flash-
Plate scintillation proximity assay was used to measure
the incorporation of radiolabeled UTP in a polyrA/
biotinylated-oligo dT template-primer captured on the
surface of streptavidin-coated microtiter plate.8 Our
screening campaign resulted in the identification of a
novel phenylalanine derivative 1 with an IC50 of 5.7 µM.
Shire BioChem Inc., 275 Armand-Frappier Boulevard,
Laval, Quebec H7V 4A7, Canada
Received February 17, 2003
Abstr a ct: The HCV NS5B RNA dependent RNA polymerase
plays an essential role in viral replication. The discovery of a
novel class of inhibitors based on an N,N-disubstituted phe-
nylalanine scaffold and structure-activity relationships stud-
ies to improve potency are described.
In tr od u ction . Infection by the hepatitis C virus
(HCV) has been recognized as one of the leading causes
of liver impairment such as cirrhosis and hepatocellular
carcinoma. It is estimated that 3% of the world’s
population or about 170 million people are seropositive,
and there is compelling evidence that within 10-20
years, progression to cirrhosis and hepatocellular car-
cinoma will occur in about 20% and 5%, respectively, of
those afflicted.1 Other factors such as alcohol consump-
tion can also be important determinants for disease
progression.2 Currently, HCV infection is one of the
leading causes of liver transplantation in the U.S. and
it is estimated that 9000 deaths per year result from
HCV infection. The recommended treatment, interferon
R-2b (or a poly(ethylene glycol) conjugate) in combina-
tion with ribavirin, provides a sustained response in
only a portion of the treated patients, and furthermore,
side effects can be severe.1 The lack of an effective and
well-tolerated treatment has therefore spurred intense
research efforts to develop anti-HCV agents.
HCV is a 9.6 kb positive strand RNA virus of the
flaviviridae, genus Hepacivirus, and it contains a single
open reading frame coding for a ∼3000 amino acid
polyprotein, which is further processed into various
structural (core, E1, and E2) and nonstructural (NS2,
NS3, NS4A, NS4B, NS5A, and NS5B) viral proteins by
host and viral proteases.2 The NS3 chymotrypsin-like
protease and the NS5B RNA dependent RNA poly-
merase are probably the most studied targets for anti-
HCV therapy because they are crucial for viral repli-
cation.3 Extensive structure-activity relationship studies
(SAR) of several classes of NS3 protease inhibitors have
been reported.4 Various institutions and pharmaceutical
companies, including ours, have disclosed patents de-
scribing structurally diverse low molecular weight
inhibitors of NS5B polymerase.5 Examples of a nucleo-
This lead compound provided a good starting point for
our optimization study because it was also found to be
selective against human DNA polymerases. The IC50 for
R, â, and γ polymerases was found to be greater than
50 µM.
Syn th esis.9 All the compounds described in this
study were prepared according to one of the three routes
depicted in Scheme 1. Reaction of the amino acid (as
the tert-butyl ester) with a suitable aldehyde under
reductive amination conditions yielded an N-alkylated
amino acid that was then transformed to a tertiary
amide followed by acid deprotection to give the desired
compounds (method A). An alternative approach was
to first install the benzamide moiety followed by ab-
straction of the amide proton with sodium hydride in
DMF and alkylation with a suitable electrophile (method
B). Last, alkylation of a N-MTR10 protected amino acid
followed by release of the amine with TFA/EtSMe,
amidation and basic hydrolysis of the methyl ester
(method C) was also used to provide the desired
compounds. This last method was used to ascertain that
alkylation of the amide moiety provided the requisite
N-alkylation instead of O-alkylation. The biaryl unit in
21 was constructed using standard Suzuki conditions.
Discu ssion . From preliminary structure-activity
analysis of inhibition of NS5B polymerase, it became
apparent that the potential for optimization resided
primarily in the N-benzylic portion of the phenylalanine
derivative. For example, evaluation of the bromo deriva-
tives 2, 3, and 4, which were to be used as palladium
cross coupling intermediates, showed that NS5B inhibi-
* To whom correspondence should be addressed. Phone: (450) 978-
7802. Fax: (450) 978-7777. E-mail lchan@ca.shire.com.
10.1021/jm0340400 CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/08/2003

1284 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Letters
Ta ble 1. HCV NS5B Polymerase Inhibitory Activity of
N-Substituted Phenylalanine Analoguesa
F igu r e 1. HCV NS5B Polymerase Inhibitors.
Sch em e 1. Synthesis of Compounds 2-21a
a
IC50 values were determined singly from dose-response curves
using 11 concentrations for each compound. Curves were fitted to
data points using nonlinear regression analysis, and IC50 values
were interpolated from the resulting curves using GraphPad Prism
software, version 2.0 (GraphPad Software, Inc., San Diego, CA).
A positive control was included as an internal standard in each
set of experiments, and results were considered accurate only when
the IC50 value of the positive control was within 0.45 ( 0.16 µM.
a
Reagents. Method A: (a) Aldehyde, NaCNBH3, ZnCl2, MeOH;
(b) acid chloride, N-methylmorpholine, DMAP, CH2Cl2; (c) TFA/
CH2Cl2. Method B: (a) 2,4-dichlorobenzoyl chloride, NEt3, CH2Cl2;
(b) alkyl bromide, NaH, DMF; (c) TFA/CH2Cl2. Method C: (a) NaH,
alkyl bromide, DMF, 0 °C; (b) TFA/CH2Cl2, EtSMe; (c) acid
chloride, NEt3, DMAP, CH2Cl2; (d) LiOH, THF/MeOH/water.
Suzuki reaction: boronic acid, Pd(PPh3)4, DME, Na2CO3(aq), 60
°C.
substituents at the meta position such as methyl 12 and
trifluoromethyl 13 are tolerated and provided analogues
that inhibited NS5B polymerase with IC50 values of
about 1.8 µM. The most potent compounds identified in
this study were the m-nitro-14 and m-cyano-15. Both
had IC50 values of about 0.7 µM and retained selectivity
tion was retained in these analogues and the meta
isomer, which had an IC50 of 3.5 µM, was virtually
equipotent to screening lead 1 (Table 1). Changing the
phenylalanine moiety to glycine analogue 5 significantly
decreased activity, indicating a requirement for a
hydrophobic group at this part of the molecule. It
appears that there may be preference for the L-amino
acid because the D-phenylalanine analogue 6 was slightly
less active with an IC50 of 8.6 µM. All subsequent
analogues were therefore prepared using L-phenylala-
nine as the starting material. The monochlorobenza-
mides 7 (para) and 8 (ortho) were less active than their
2,4-dichloro counterpart, suggesting that chloro groups
at both these positions are important.11 On the basis of
these findings, the 2,4-dichlorobenzamide together with
the L-phenylalanine moieties were retained and a
systematic exploration of the N-benzylic part of the
molecule was initiated with emphasis on the meta
position.
against human DNA polymerases R, â, and γ (IC50
>
50 µM). The meta preference was again demonstrated
because the o-cyano and p-cyano analogues 16 and 17
were almost 10-fold less potent than the meta counter-
parts. Interestingly, the 3-pyridyl N-oxide 18 had an
IC50 of 2.7 µM, whereas the 3-pyridyl derivative 19 was
4-fold less potent, again underlying the necessity of
having a substituent at the 3 position. Finally, bioisos-
teric replacement of the phenyl ring with a thiophene
provided analogues that retained some inhibition. For
example, the 5-bromothiophene 20 and benzofuran
analogue 21 had IC50 values of 3.1 and 8.6 µM, respec-
tively, indicating that there is probably more scope for
this type of modification. We have also evaluated one
of the most potent inhibitors (J TI) described by J apan
Tobacco Inc. In our assays, J TI displayed an IC50 value
of 0.62 µM,12 which is similar to the IC50 values of our
best analogues 14 and 15.
A 2-fold decrease in activity was observed in the
unsubstituted phenyl 9 compared to m-bromo-3, under-
lying the importance of having a substituent at this
position. Both the m-chloro-10 and the fluoro derivatives
11 were also active with 10 (IC50 of 1.4 µM), being 4-fold
more potent than the screening lead 1. Other small
Con clu sion . We have identified a novel class of
potent HCV NS5B polymerase inhibitors that are also
selective against human DNA polymerases. The phe-
nylalanine analogues 14 and 15 have NS5B inhibitory

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1285
2001. (b) Gardelli, C.; Giuliano, C.; Harper, S.; Koch, U.; Narjes,
F.; Ontoria, O.; J esus, M.; Poma, M.; Ponzi, S.; Stansfield, I.;
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Yoshida, A. Preparation of Heterocyclic Compounds as Remedies
for Hepatitis C. Patent WO0147883, 2001; Patent EP1162196,
2001.
activities that compare favorably with an inhibitor
reported by J apan Tobacco Inc. This study has delin-
eated the structural requirements for potency, and it
suggests that small substituents at the meta position
of the N-benzyl group are optimum. In addition, crystal
structures of 13 and 21 bound to HCV NS5B polymerase
have been generated to a resolution of 1.9 and 2.9 Å,
respectively. The binding site is the same for both
compounds and is located in a region about 30 Å from
the active site.13 Further optimization and mechanistic
and structural studies to elucidate the mode of action
of this series are ongoing.14
(6) (a) Lesburg, C. A.; Cable, M. B.; Ferrari, E.; Hong, Z.; Mannarino,
A. F.; Weber, P. C. Crystal Structure of the RNA-Dependent
RNA Polymerase from Hepatitis
C Virus Reveals a Fully
Encircled Active Site. Nat. Struct. Biol. 1999, 6, 937-943. (b)
Ago, H.; Adachi, T.; Yoshida, A.; Yamamoto, M.; Habuka, N.;
Yatsunami, K.; Miyano, M. Crystal Structure of the RNA-
Dependent RNA Polymerase of Hepatitis C Virus. Structure
(London) 1999, 7, 1417-1426. (c) Bressanelli, S.; Tomei, L.;
Roussel, A.; Incitti, I.; Vitale, R. L.; Mathieu, M.; De Francesco,
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Polymerase of Hepatitis C Virus. Proc. Natl. Acad. Sci. U.S.A.
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Ack n ow led gm en t. We thank J ean Duchaine for
providing the human polymerase R, â, and γ inhibition
data and The´re`se Godbout for her assistance in the
preparation of this manuscript.
(7) Information is reported at www.jti.co.jp.
(8) Earnshaw, D. L.; Pope, A. J . FlashPlate Scintillation Proximity
Assays for Characterization and Screening of DNA Polymerase,
Primase, and Helicase Activities. J . Biomol. Screening 2001, 6,
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Su p p or tin g In for m a tion Ava ila ble: General synthetic
procedures, H NMR, HPLC and HRMS data. This material
is available free of charge via the Internet at http://pubs.
acs.org.
1
(9) All the compounds in this study were characterized by 1H NMR
and MS, and purity was assessed by HPLC. The 1H NMR spectra
of the compounds also indicated the presence of numerous
rotamers.
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