Pyridine carboxamides: Potent palm site inhibitors of HCV NS5B polymerase

Article abstract of DOI:10.1021/ml100128h

Pyridine carboxamide-based inhibitors of the hepatitis C virus (HCV) NS5B polymerase were diversified and optimized to a variety of topologically related scaffolds. In particular, the 2-methyl nicotinic acid scaffold was developed into inhibitors with improved biochemical (IC₅₀-GT1b = 0.014 μM) and cell-based HCV replicon potency (EC₅₀-GT1b = 0.7 μM). Biophysical and biochemical characterization identified this novel series of compounds as palm site binders to HCV polymerase.

Full text of DOI:10.1021/ml100128h

pubs.acs.org/acsmedchemlett
Pyridine Carboxamides: Potent Palm Site Inhibitors of
HCV NS5B Polymerase
Cliff C. Cheng,*,† Xiaohua Huang,† Gerald W. Shipps, Jr.,† Yu-Sen Wang,†
Daniel F. Wyss,† Kyle A. Soucy,† Chuan-kui Jiang,‡ Sony Agrawal,‡
Eric Ferrari,‡ Zhiqing He,‡ and H.-C. Huang*,‡
†Department of Lead Discovery Chemistry, Merck Research Laboratories, 320 Bent Street, Cambridge, Massachusetts 02141,
and ‡Department of Infectious Diseases, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033
ABSTRACT Pyridine carboxamide-based inhibitors of the hepatitis C virus (HCV)
NS5B polymerase were diversified and optimized to a variety of topologically related
scaffolds. In particular, the 2-methyl nicotinic acid scaffold was developed into
inhibitors with improved biochemical (IC50-GT1b = 0.014 μM) and cell-based HCV
replicon potency (EC50-GT1b = 0.7 μM). Biophysical and biochemical characte-
rization identified this novel series of compounds as palm site binders to HCV
polymerase.
KEYWORDS Hepatitis C virus, HCV NS5B polymerase, palm site inhibitors, direct-
acting antiviral (DAA), automated ligand identification system (ALIS), 2D 15N-HSQC
hronic infection by the hepatitis C virus (HCV) afflicts
an estimated 2-3% of the world population and
represents a serious health issue.1 Currently, it is
for label-free, high-throughput screening of mixture-based
combinatorial libraries.15 The screen yielded several classes
of compounds, one of which was recently reported in detail.16
Here, we describe another distinct series that is based on a
nicotinamide-based scaffold and inhibits through binding at
the palm site of NS5B.
C
treated with a combination of pegylated interferon-R and
ribavirin, which results in sustained virologic response (SVR)
in only approximately 40-50% of patients infected with
genotype 1 virus.2 With major advances in our understand-
ing of this pathogen that came from the identification of
essential viral functions,3 the development of subgenomic
replicons allowing HCV RNA replication in Huh-7 cells,4 and
the ability to replicate specific viral strains in the laboratory,5
several direct-acting antivirals (DAA) against HCV have now
achieved encouraging results in phase 1 to 3 clinical trials.6
A major target of DAA against HCV is the viral nonstructural
protein 5B (NS5B), which possesses RNA-dependent RNA
polymerase (RdRp) activity essential for viral genome replica-
tion. Severalpotent, selective, structurallydistinctnon-nucleo-
side inhibitors of NS5B, binding at palm sites 1 and 2 and
thumb sites 1 and 2, have demonstrated clinical proof of
concept.7 Clinical efficacy was first demonstrated with HCV-
796, which targets palm site 2,8,9 although safety signals
precluded further development of this compound. Several
palm site 1 (e.g., ABT-33310 and ANA59811 ), thumb site 1 (MK-
N-(4-Isopropyl-2-(4-(pyrazin-2-yl)piperazin-1-yl)phenyl)-
nicotinamide 1 (Figure 1), a hit from the initial screen, was
a modestly potent compound in the biochemical assay but
did not appreciably inhibit HCV RNA replication in the cell-
based replicon assay. The decline in potency when the i-Pr
group of 1 was replaced with the similar c-Pr group (2) or a
Br (4) group suggested that this position was very sensitive
to changes and important for activity. Synthesis of com-
pounds in the nicotinic carboxamide series began with
oxidation of 2-bromo-4-isopropylaniline (5) as outlined in
Scheme 1. We therefore surveyed the substitution patterns
on nicotinic acid of 1 and other varieties of scaffolds (Table 1)
to provide compounds 10-45 with a substantial improve-
ment in potency, quickly establishing structure-activity re-
lationship (SAR) and improving upon activity in both the
biochemical and the cell-based replicon assays.
Utilizing 1 as a starting point, we began the optimization
process by investigating the substitution pattern on the nico-
tinic acid and other scaffolds (Table 1). Attempts to substitute
the pyridine ring with polar substituents (10-12) resulted in
lesser potency relative to monofluorinated (13) nicotinic or
2-aminonicotinic (14) analogues. 2-Trifluoromethyl analogue
(15) retained potency, while the 6-trifluoromethyl (16) and
12
3281 ), and thumb site 2 binders (e.g., PF-868554/filibuvir13
and VX-22214) have since entered clinical testing. However, as
monotherapywith DAA isexpectedto engenderdevelopment
of viral resistance, new small-molecule inhibitors of HCV
remain a focus of intense drug discovery efforts in search of
candidate components of combination therapy.
A medicinal chemistry effort to optimize small molecule
inhibitors of NS5B was initiated following high-throughput
screening of NS5B(Δ21)-genotype 1b (Con1) enzyme using
our proprietary Automated Ligand Identification System (ALIS)
platform, an affinity selection-mass spectrometry platform
Received Date: June 4, 2010
Accepted Date: July 28, 2010
Published on Web Date: August 17, 2010
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4-trifluoromethyl (17) nicotinic analogues were weak inhibi-
tors. The addition ofa bulky group to the pyridine ring of R2 as
in the 2-methyl vinyl (18) and phenyl (19) analogues resulted
in a loss of potency. The isomeric pyridines 20 and 21 also
were weaker inhibitors than the starting 1.
or small alkyl substituent had a positive effect on potency
(61 and 62) relative to parent compound (1), as did py-
rimidine replacements (63 and 64) and substituted pyrimi-
dines (65 and 66). The intrinsic potency was improved
moderately; however, despite measurable cell-based effects
indicating compound penetration, it was not possible to
differentiate anti-HCV replicon activity from overlapping
cytotoxicity.
To continue the investigation of the SAR at R3, we prepared
several analogues incorporating the 2-methylpyridyl group
(Scheme 2), which as exemplified by compound (40) en-
hanced both intrinsic potency and replicon activity with a
∼10-fold cytotoxicity window. We changed to the more
potent 4-pyrimidine analogue combined with 2-methyl nico-
tinic carboxamide (70) as a benchmark (Table 3) and found
that whereas pyrazolopyrimidine analogue (71) and 2-meth-
ylthiomethylpyrimidine (72) provided a modest improve-
ment in enzyme potency without gaining cellular activity,
thienopyrimidine (73) afforded a 7-fold enhancement in
enzyme potency (IC50 = 14 nM) and yielded submicromolar
replicon activity (EC50 = 0.7 μM) with a >20-fold cytotoxi-
city window, representing a potency gain of 200- and 30-fold
in biochemical and cell-based HCVreplicon activities, respec-
tively, from the initial hit (1).
Three experimental approaches were employed to char-
acterize the binding site of the pyridine carboxamide inhibitor
series. First, we conducted an analysis of inhibitor-induced
chemical shift changes in 2D 15N-HSQC NMR spectra of 15N-
labeled HCV NS5B polymerase. Because chemical shift as-
signments of NS5B are not readily obtained due to its large
size (64 kDa), peaks of residues within interesting subsites
of NS5B were mapped using various NS5B binders whose
binding sites were previously known from X-ray crystal struc-
tures. Such chemical shift perturbation maps were then used
to infer the binding sites of newly discovered HCV NS5B inhi-
bitor series. As detailed in the Supporting Information, the
thienopyrimidine inhibitor (73) induced chemical shift per-
turbation patterns similar to those of a benzofuran inhibitor,
HCV-796, a known palm site 2 binder,17 suggesting that the
two inhibitors share an overlapping but not identical binding
site in the palm domain. By contrast, a distinctly different
chemical shift perturbation pattern was induced by a dihy-
dropyranone derivative previously shown to bind at thumb
site 2,18,19 indicating that the pyridine carboxamides are
unlikely to be thumb site binders. Limited aqueous solubi-
lity of a benzothiadiazine analogue, A-848837, precluded
Attempts to replace nicotinic acid with pyrimidine (22),
pyridazine (23), or pyrazine (24) analogues or to replace with
saturated heterocycles (25-27) resulted in less potent com-
pounds as well as with H-bond donors, for example, 3-methyl-
pyrazole (34) and aza-indole (35), relative to five-membered
ring heterocyclic analogues (28-33). Surprisingly, replace-
ment of the 5-methylisoxazole (31) analogue with a 3-meth-
ylisoxazole (36) resulted in a notable decrease in activity.
A breakthrough was uncovered upon difluorination of the
phenyl ring. Whereas the 2-methyl-3,6-difluoro (37) and 3,5-
difluoro-4-methyl analogues (38) were uniformly less active
than the 2-methyl-3,5-difluoro analogue (39), the 2-methyl-
3,5-difluoro derivative (39), with an IC50 = 540 nM, dis-
played a remarkable 6-fold improvement in potency relative
to 1. At this point, we incorporated the 2-methyl substitution
on the nicotinic acid (40) as well as some five-membered
ring heterocyclic analogues (41) and were pleased to observe
a similar effect. Compounds 40 and 41 inhibited NS5B with
an IC50 = 520 and 580 nM, respectively. No significant pote-
ncy improvements were observed by introducing methyl
group in different positions (42 and 43) or by replacing
methyl with a cyclopropyl substituent (44) or with a quino-
line analogue (45).
The SAR of N-substitution (R3) is presented in Table 2, and
the chemistry route is outlined in Scheme 2. BOC-protected
piperazine (48), amides (51 and 52), urea (53), and sulfon-
amide (54) substitutions led to analogues with greatly
diminished activity relative to 1, but the original activity
was restored by attaching five-membered heterocycles
(55-58). Replacement of a pyrazine with a 2-pyridine
(59) or addition of a phenyl substituent (60) also caused a
notable decrease in activity. The addition of a straight chain
Figure 1. Initial nicotinamide-based NS5B inhibitors.
Scheme 1. Synthesis of 4-Isopropyl-2-(4-(pyrazin-2-yl)piperazin-1-yl)aniline and Amidationa
a Reagent and conditions: (a) m-CPBA, toluene, reflux. (b) 2-(Piperazin-1-yl)pyrazine, DIEA, DMF, 200 °C for 20 min in microwave. (c) Zn, CaCl2, EtOH,
4 h. (d) Carboxylic acid, HATU, DIEA, DMF.
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Table 1. Scaffold Replacements in Nicotinic Carboxamide Analogues of Compound 9 at R2
a Values are means of duplicate experiments on two separate weightings. NS5B enzyme, replicon, and cytotoxicity assay protocols are described in
the Supporting Information. b ND, not determined. c Dashed lines, anti-HCV replicon activity masked by overlapping cellular CC50
.
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Scheme 2a
a Reagents and conditions: (a) tert-Butyl piperazine-1-carboxylate, DIEA, DMF, 200 °C for 20 min in the microwave. (b) H2, 10% Pd/C, MeOH.
(c) Nicotinic acid or 2-methyl niconic acid, HATU, DIEA, DMF. (d) TFA. (e) R3X, DIEA, DMF, 200 °C for 20 min in the microwave.
Table 2. SAR Development of Compound 50 at R3
a Values are means of duplicate experiments on two separate weightings. NS5B enzyme, replicon, and cytotoxicity assay protocols are described in
the Supporting Information. b ND, not determined. c Dashed lines, anti-HCV replicon activity masked by overlapping cellular CC50
.
evaluation of this known palm site 1 binder20 using the bio-
NMR approach.
convergent lines in such a Yonetani-Theorell plot, representing
additive or synergistic inhibition. As shown in Figure 2A, a
mutually exclusive pattern of inhibition was clearly evident
when a pyridine carboxamide compound (73) was added in
the presence of the palm site 2 binder HCV-796, similar to that
seen with the palm site 1 binder A-848837 (data not shown). In
contrast, an additive pattern of inhibition was observed when 73
was titrated with TS2, a known thumb site 2 binder (Figure 2B),
or with an indole derivative previously shown to bind at thumb
site 1 (data not shown).22 Results from the inhibitor competition
We then performed kinetic competition with inhibitors of
known binding sites and analyzed by the method first described
by Yonetani-Theorell.21 Compounds that share identical or over-
lapping binding sites will yield a mutually exclusive inhibition
pattern, indicated by a series of parallel lines in a plot of the
reciprocal of enzyme velocity (V0/Vi) versus the concentration of
a titrated inhibitor. Compounds that bind independently at
distinct sites of the target enzyme will exhibit nonparallel,
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Table 3. Further SAR Development of Compound 69 at R3
Figure 2. Yonetani-Theorell plots of inhibition of HCV NS5B
polymerase by a combination of inhibitors. The NS5B polymerase
activity was measured as described23 (see the Supporting Informa-
tion) in the presence of various concentrations of 73 and (A) 10,
20, 80, or 160 nM HCV-796; (B) 12.5, 50, or 100 nM TS2.
a Values are means of duplicate experiments on two separate weight-
ings. NS5B enzyme, replicon, and cytotoxicity assay protocols are
described in the Supporting Information.
than for the Con1 NS5B enzyme form.17 Unlike HCV-796, how-
ever, which is not affected by the G554D mutation and only
minimally by the M414T mutation at palm site 1, the pyridine
carboxamides appeared much more susceptible to these mu-
tant variants. These results suggest that the binding pocket of
the pyridine carboxamides, while overlapping with that of HCV-
796, likely extends further into palm site 1 than the benzofuran
derivative, consistent with the overlapping yet distinct 2D bio-
NMR chemical shift perturbation patterns observed between
compound 73 and HCV-796. The resistance profile of the
pyridine carboxamides is, however, distinct from that of the
palm site 1 binder benzothiadiazine analogue A-848837, which
exhibited little or no potency shift against the C316N/Y, 1b-BK,
or M414T variants but far more impacted by G554D than 71 or
73. It should be noted that other benzothiadiazine analogues
have been reported to lose significant potency against M414T, a
major palm site 1 resistance locus.20 Also in contrast to HCV-796
and A-848837, which were equipotent against the 1a enzyme,
the pyridine carboxamide inhibitors lost >100-fold activity
against a genotype 1a enzyme (Table 4) and were inactive up
to 10 μM against genotypes 2, 3, and 4 (data not shown). It thus
appears that the mechanism of action of pyridine carboxamide
inhibitors is more sensitive to perturbations in their binding site
than that of HCV-796 or A-848837. The activity data on enzyme
mutant variants and genotypes further support findings from
bio-NMR and inhibitor mutual exclusivity analyses; together,
they reveal a distinct binding mode of this series of pyridine
analysis thus strengthen the conclusion from bio-NMR data that
the pyridine carboxamides share an overlapping binding site
with HCV-796 in the palm domain of the polymerase. Further-
more, these biochemical studies suggest that compounds in this
novel series may be useful in combination with inhibitors
targeting other polymerase binding sites and result in at least
additive inhibition of HCV RNA replication.
A third approach to characterize the inhibition mode of this
novel compound class involves evaluation of its activity against
several polymerase mutant variants and genotypes and com-
parison with the profile of known palm site binders HCV-796
and A-848837. As shown in Table 4, the inhibitory potency of
pyridine carboxamides was reduced by resistance variants
selected by palm sites 1 and 2, but not thumb sites 1 and 2,
binders. Resistance mutations P495L (at thumb site 1) and
M423T (at thumb site 2) did not affect the activity of compounds
71, 73, HCV-796, or A-848837, consistent with the expected
profile of compounds that do not bind at these sites. On the
other hand, the inhibitor activity was affected by polymorphism
at the palm site 2 residue 316, a major resistance locus for HCV-
796.8 A similar potency loss was observed for both pyridine
carboxamides and HCV-796, with a moderate shift against a
C316N variant and a much greater loss against C316Y. A
significant shift was also observed for both compound classes
against a 1b-BKvariant, which contains N316. The shift for HCV-
796 was in accord with a previous study of its binding kinetics
that reported a ∼10-fold higher equilibrium constant for the BK
Table 4. Activity against HCV NS5B Enzyme Variants
fold shift over 1b-Con1
1b-Con1 IC50
Con1 C316N
Con1 C316Y
Con1M414T
Con1 G554D
Con1M423T
Con1 P495L
1b- 1a-
BK H77
compound
(μM)
(PS2)
(PS2)
(PS1)
(PS1)
(TS2)
(TS1)
71
0.032
0.014
0.04
4ꢀ
3ꢀ
3ꢀ
1ꢀ
>300ꢀ
>300ꢀ
50ꢀ
27ꢀ
21ꢀ
1.5ꢀ
1ꢀ
8ꢀ
1ꢀ
1ꢀ
1ꢀ
1ꢀ
1ꢀ
1ꢀ
1ꢀ
1ꢀ
27ꢀ 270ꢀ
14ꢀ 140ꢀ
6ꢀ 1ꢀ
73
4ꢀ
HCV-796
A-848837
1ꢀ
100ꢀ
0.003
3ꢀ
1ꢀ 1ꢀ
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carboxamide NS5B inhibitors that overlaps yet differs from that
of HCV-796 in the palm domain of the HCV polymerase.
We also sought to determine if the mechanism of action of
this novel series was through inhibition of NS5B-catalyzed RNA
synthesis at the initiation or elongation step. As shown in the
Supporting Information, while compound 73 was active against
primer-initiated RNA synthesis involving multiple rounds of
initiation and elongation, in a single-cycle elongation assay16,23
it did not inhibit the elongation of preformed NS5B enzyme-
primer-template complexes, similar to HCV-796 and
A-848837 but unlike a chain terminator 30-dCTP. The pyridine
carboxamides thus appeared to act as inhibitors of initiation of
replication, as observed with other palm site binders to NS5B.
In summary, we described a novel series of HCV NS5B
polymerase inhibitors based on the nicotinic carboxamide
scaffold. Beginning with relatively weak hits from the
ALIS platform, we were able to provide significant potency
improvements in enzymatic activity and in a cell-based rep-
licon system of HCV RNA replication, yielding HCV polymer-
ase inhibitors with low nanomolar potency against genotype
1b NS5B enzyme that are submicromolar inhibitors of
the GT-1b HCV replicon. Mechanistic insights gained by 2D
bio-NMR, inhibitor exclusivity analysis, and enzyme mutant
variant characterizations identified a distinct binding mode for
this new class of NS5B inhibitors in the palm domain of the HCV
polymerase. Significant future lead optimization efforts will be
required to generate inhibitors with broadened genotype
spectrum and suitable pharmacokinetics and safety character-
istics as clinical development candidates against HCV.
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SUPPORTING INFORMATION AVAILABLE Experimental
synthetic procedures, spectroscopic characterization, assay proto-
cols, inhibitor mechanism of action, and 2D bio-NMR data and
discussion. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. E-mail: cliff.cheng@merck.com(C.C.C.) or hsueh-cheng.
huang@merck.com (H.-C.H.).
ACKNOWLEDGMENT We thank Dr. Xianshu Yang for perform-
ing the initial ALIS screening, Dr. Michael Ziebell for HR LC/MS
analyses, Michael Starks for analytical support, and Patricia McMo-
nagle for generating NS5B enzyme constructs.
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