Inhibitors of hepatitis C virus polymerase: Synthesis and characterization of novel 2-oxy-6-fluoro-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-benzamides

Article abstract of DOI:10.1016/j.bmcl.2010.02.054

SAR exploration from an initial hit, (S)-N-(2-cyclohexenylethyl)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)benzamide (1), identified using our proprietary automated ligand identification system (ALIS),¹ has led to a novel series of selective hepatitis C virus (HCV) NS5B polymerase inhibitors with improved in vitro potency as exemplified by (S)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)-N-isopentyl-N-methylbenzamidecarboxamide (41) (IC₅₀ = 0.5 μM). The crystal structure of an analogue (44) was solved and provided rationalization of the SAR of this series, which binds in a distinct manner in the palm domain of NS5B, consistent with biochemical analysis using enzyme mutant variants. These data warrant further lead optimization efforts on this novel series of non-nucleoside inhibitors targeting the HCV polymerase.

Full text of DOI:10.1016/j.bmcl.2010.02.054

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2119–2124
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors of hepatitis C virus polymerase: Synthesis and characterization
of novel 2-oxy-6-fluoro-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-benzamides
a
a,
Cliff C. Cheng a, , Gerald W. Shipps Jr. , Zhiwei Yang , Noriyuki Kawahata a,à, Charles A. Lesburg b,
José S. Duca b, Jamie Bandouveres a, Jack D. Bracken a, Chuan-kui Jiang c, Sony Agrawal c, Eric Ferrari c,
H.-C. Huang c
*
a Department of Lead Discovery, Merck Research Laboratories, 320 Bent Street, Cambridge, MA 02141, United States
b Department of Drug Design, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
c Department of Infectious Diseases, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
SAR exploration from an initial hit, (S)-N-(2-cyclohexenylethyl)-2-fluoro-6-(2-(1-hydroxy-3-phenylpro-
pan-2-ylamino)-2-oxoethoxy)benzamide (1), identified using our proprietary automated ligand identifi-
cation system (ALIS),1 has led to a novel series of selective hepatitis C virus (HCV) NS5B polymerase
inhibitors with improved in vitro potency as exemplified by (S)-2-fluoro-6-(2-(1-hydroxy-3-phenylpro-
Received 14 October 2009
Revised 11 February 2010
Accepted 11 February 2010
Available online 18 February 2010
pan-2-ylamino)-2-oxoethoxy)-N-isopentyl-N-methylbenzamidecarboxamide (41) (IC50 = 0.5 lM). The
crystal structure of an analogue (44) was solved and provided rationalization of the SAR of this series,
which binds in a distinct manner in the palm domain of NS5B, consistent with biochemical analysis using
enzyme mutant variants. These data warrant further lead optimization efforts on this novel series of non-
nucleoside inhibitors targeting the HCV polymerase.
Keywords:
HCV NS5B polymerase
Hepatitis C virus
Palm site inhibitors
Replicon
Ó 2010 Elsevier Ltd. All rights reserved.
Antiviral
ALIS: automated ligand identification
system
Structure based drug design (SBDD)
Hepatitis C virus (HCV) was characterized in 1989 as the organ-
ism responsible for non-A non-B hepatitis.2 Because of an esti-
mated 170 million people worldwide chronically infected with
HCV, this virus represents a global public health problem. Approx-
imately 80% of patients infected with HCV develop chronic hepati-
tis, which can lead to liver cirrhosis and hepatocellular carcinoma.3
Current standard-of-care of combination therapy with pegylated
cation of viral RNA genome, and exhibits the classic ‘right-hand’
configuration as other polymerases, with palm, thumb and finger
subdomains.5 Intense drug discovery efforts have been directed to-
ward the identification of potent and selective inhibitors of NS5B.
Clinical proof-of-concept of NS5B polymerase inhibitors has been
achieved with both nucleosides and non-nucleosides.6 Among
nucleoside analogues, R7128 (Pharmasset/Roche)7 is currently
the most advanced, followed by more recent inhibitors with im-
proved in vitro potency such as IDX184 (Idenix)8 and PSI-7851
(Pharmasset).9 Clinical efficacy of a non-nucleoside inhibitor was
first demonstrated with HCV-796 (ViroPharma/Wyeth) targeting
the palm site,10 although further development of this compound
was discontinued due to safety findings. Several structurally dis-
tinct palm site binders, however, have advanced into early clinical
trials with encouraging results, for example, ABT-333 (Abbott)11
and ANA598 (Anadys).12 Compounds targeting other subdomains
of NS5B are also undergoing clinical testing, such as thumb site
binders PF-868554/filibuvir (Pfizer)13 and VX-222/VCH-222 (Ver-
tex),14 and finger-loop site binder MK-3281 (Merck).15
interferon-a and the nucleoside analogue ribavirin results in sus-
tained virologic response (SVR) in only approximately 40–50% of
patients with genotype 1.4 In contrast, SVR is achieved for greater
than 80% of patients infected with genotypes 2 and 3 viruses. Thus,
the greatest unmet medical need for HCV therapy is the improve-
ment of SVR in genotype 1 patients.
The nonstructural protein 5B (NS5B) of HCV possesses RNA-
dependent RNA polymerase (RdRp) activity essential for the repli-
* Corresponding author.
E-mail address: cccheng@alum.mit.edu (C.C. Cheng).
Present address: Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008,
United States.
In a continuing quest to discover novel HCV NS5B inhibitors, we
initiated a hit characterization chemistry effort following high-
throughput screening of a library of small molecules against
NS5B using our proprietary ALIS (Automated Ligand Identification
à
Present address: Aileron Therapeutics, Inc., 840 Memorial Dr., Cambridge MA
02139, United States.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.054

2120
C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2119–2124
3R2RN
R1O
O
1. R1
=
H, R2 = H, R3 = 2-cyclohexenylethyl , IC50 = 4,300 nM
-
O
(S)
O
F
N
H
2. R1 = -CH3, R2 = H, R3 = 2-cyclohexenylethyl , IC50 = > 20,000 nM
(S)
HO
HO
HN
O
O
NH
O
O
(R)
O
F
O
F
N
H
N
H
3. IC50 = > 20000 nM
4. IC50 = > 20000 nM
Figure 1. 2-Oxy-6-fluoro-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-benzamide-based NS5B hits identified from ALIS screening.
System) platform.1 We describe herein the discovery and charac-
terizations of potent and selective 2-oxy-6-fluoro-N-((S)-1-
hydroxy-3-phenylpropan-2-yl)-benzamide-based NS5B inhibitors
that were identified from mixture-based optimization of ALIS hits.
We report our early mixture-based hit characterization efforts,
using parallel synthesis and structure based drug design (SBDD),
which led to the identification of submicromolar inhibitors of
NS5B polymerase activity. Using X-ray crystallography and com-
puter modeling we show an overlapping but distinct binding mode
of this series of compounds from other palm site NNIs, consistent
with biochemical characterization using enzyme mutants.
inhibition of enzyme activity.16 Further evidence of specificity
was inferred when the two pendant amines of compound 1 were
transposed as in compound 4 and a complete loss of potency
resulted. These ligand–protein interactions were subsequently
revealed upon the solution of the X-ray crystal structure of a rep-
resentative complex described in detail below.
Preparation of key intermediates and final compounds in the
2-oxy-6-fluoro-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-benzamide
series began with methylation of 2-fluoro-6-hydroxybenzoic acid
(5) as outlined in Scheme 1. Scaffolds such as 10 provided immedi-
ate access to SAR via hypothesis-driven ideas as well as more inclu-
sive mixture-based synthetic libraries. Mixture-based libraries
were analyzed using ALIS and provided both binary binding data
as well as qualitative rank ordering.1 Re-synthesis of the most
promising hits was subsequently performed using high-through-
put synthetic methods. Most notably a variety of amines (Table 1)
was combined with the acid (10) to provide compounds 38–42
with a notable improvement in potency, quickly providing us with
submicromolar inhibitors of NS5B.
A hit characterization chemistry effort to find small molecule
inhibitors targeting NS5B was initiated following high-throughput
screening of NS5B(D21)-genotype 1b (Con1) enzyme using the
ALIS platform, an affinity selection-mass spectrometry platform
for label-free, high-throughput screening of mixture-based combi-
natorial libraries. This screen revealed several classes of inhibitors,
of which one (based on (S)-N-(2-cyclohexenylethyl)-2-fluoro-6-(2-
(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)benzamide
(1), Fig. 1) is described herein. Interestingly, the lack of potency in
the methoxy (2) and (R)-enantiomer (3) analogues suggested that
the stereochemistry of the 1-hydroxy-3-phenylpropan-2-ylamide
and hydrogen bond interactions with NS5B were important for
Utilizing 1 as a starting point, we began the in vitro SAR charac-
terizations by investigating the benzamides on the 6-fluorobenzoic
acid (Table 1). From the early mixture-based optimization it was
observed that the (S)-2-amino-3-phenylpropan-1-ol motif partici-
O
OH
O
O
O
O
O
c
a
HO
HO
F
HO
F
O
7
F
b
O
6
5
HO
O
O
O
O
O
9
O
d
e
O
F
O
F
N
H
8
NR1R2
F
HO
O
OH
HO
O
O
O
f
O
F
O
N
H
N
H
10
11
Scheme 1. Synthesis of (S)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)benzoic acid and amidation. Reagents and conditions: (a) 2 equiv TMSCH2N2,
MeOH, 0 °C; (b) 1 equiv tert-butyl bromoacetate, 1.5 equiv K2CO3, DMF, rt, 18 h; (c) 9:1 TFA:H2O, 2 h; (d) (S)-2-amino-3-phenylpropan-1-ol, HATU, DIEA, DMF, 12 h; (e) 1
.5 equiv 1 N NaOH(aq), 1:1 MeOH–H2O, then 1 N HCl(aq), 4 h; (f) NHR1R2, 1.5 equiv 1,1’-carbonyldiimidazole, 1.5 equiv DIEA, NMP, 80 °C.

C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2119–2124
2121
M)
Table 1
Table 1 (continued)
SAR development of amides derived from intermediate 10
NR1R2
NS5B IC50
4.3
(l
a,16
Compd
Compd
NR1R2
NS5B IC50
>20
(lM)
a,16
O
O
33
O
12
N
H
N
O
13
14
>20
>20
34
4.6
N
H
O
O
N
O
35
36
N
8
6
N
H
O
O
N
H
15
16
>20
>20
N
N
37
38
17
N
H
O
O
N
>20
17
>20
N
H
N
39
40
6
6
O
N
H
18
19
>20
>20
N
N
OH
N
H
41
42
0.47
7
N
O
S
20
>20
N
H
21
22
>20
>20
N
N
N
43
44
45
46
47
48
2.3
0.8
0.47
2.7
2.2
2.6
N
N
N
N
23
24
2.3
2.6
N
N
N
25
26
4
a
Values are means of duplicate experiments on two separate weightings.
N
pated in an essential H-bond network, confirmed by X-ray crystal
structure with compound 44 (Fig. 2). Attempts to replace the cyclo-
hexene ring with a saturated cyclohexane (12), phenyl (13) or with
an H-bond accepting methoxy substituted (14) ring to de-couple or
increase interactions with the adjacent Tyr-452 side chain were
not tolerated and resulted in a significant loss of potency. Replace-
ment of the cyclohexene ring with alkyl chains (15–18) to reduce
possible steric interactions near the protein surface, add an H-bond
donor (19) or a polar group (20) resulted in significant loss of po-
tency. After further examination of the crystal structure and
employing modeling techniques we observed that the adjacent
Trp-550 was ꢀ4.5 Å away from the N of the benzamide, suggesting
that small substituents may be tolerated. Compounds (21 and 22)
were made to test this hypothesis and unfortunately were inactive
10
N
Cl
N
27
1.9
O
O
28
29
7.7
20
N
N
up to 20 lM. Surprisingly, both 4-methylcyclohexyl (23) and cyclo-
30
31
>20
20
heptyl (24) benzamides were tolerated. This may be due to the
flexibility of NS5B protein at the binding site. Polar substituents
on pyrrolidines (28–30) resulted in significant loss in potency rel-
ative to more hydrophobic substituents on the pyrrolidine amides
(25–27) and affinity for NS5B may be due to the interactions with
Trp-550. Since tertiary amides were somewhat tolerated we
sought to explore tertiary amides analogues with linear alkyl
chains (35–48). These groups provided good improvement in
N
N
N
32
0.43
N
potency,
whereas
(R)-N-methyl-1-phenylethanamide
(32),

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C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2119–2124
Figure 2. The complex containing HCV NS5B (green) and compound 44 (yellow) was superimposed with published structures containing binders at Palm Site 1 (cyan; PDB
2giq17) and Palm Site 2 (brown; PDB 3fqk18). Inhibitors are shown in stick representation with a transparent surface. Panel A (left) is a view into the active site cavity with the
fingers subdomain to the right, the thumb subdomain to the left, and the palm subdomain at the bottom of the image. Asp-318 and Asp-319 do not move substantially among
these three structures while Cys-366 shifts to form Palm Site 2. Viewing the Palm Site binders from the left side yields the view in Panel B (right). Compound 44 resides in a
novel location denoted Palm Site 3, formed by a number of conformational changes, including a major shift Tyr-448 at the tip of the b-loop and a number of residues on the C-
terminal portion of NS5B. Residues which undergo the largest rearrangements upon compound 44 binding are labeled, and arrows indicate the direction of the changes. This
figure was prepared using PyMOL.19
N-methyl-3-methylbutylamide (41), and N-ethyl-butylamide (45)
demonstrated submicromolar activity.
group with the same bound water molecule as well as with the
backbone carbonyl oxygen of Tyr-195. The remaining interactions
are largely lipophilic and involve both aromatic and aliphatic pro-
tein side chains.
Compound 44 was found to bind within the active site cavity of
NS5B near the top of the palm subdomain. Although proximal to
the locations of a number of other published NS5B inhibitors,6
compound 44 binds in a distinct manner between the b-loop (res-
idues 443–454) and the C-terminal tail region (551–555) in an
almost completely enclosed cavity formed via significant shifts of
both of those protein regions relative to other NS5B ligand com-
plexes. This novel binding site is denoted Palm Site 3. Figure 2
illustrates the comparison of protein conformations and binding
modes of compound 44 with Palm Site 1 (PDB code 2giq17) and
Palm Site 2 (PDB code 3fqk18) binders.
Specific interactions between 44 and NS5B are detailed in Fig-
ure 3. An intramolecular hydrogen bond is observed between the
carbonyl oxygen of the terminal amide and the nitrogen atom of
the central amide. Intermolecular hydrogen bonds are observed
from the carbonyl oxygen of the central amide both to a bound
water molecule and to the side chain amide of Asn-316. Additional
hydrogen bonding interactions are observed between the hydroxyl
The binding mode of this compound is consistent with the ob-
served SAR: while there is sufficient volume to accommodate the
methoxy compound 2, such an analogue juxtaposes the methoxy
oxygen with the carbonyl oxygen of Tyr-195, leading to an unfa-
vorable electrostatic mismatch. The (S) stereoisomer is essential
in order to position the hydroxyl group within the hydrogen bond-
ing network centered on the bound water molecule described
above. Inversion of the chirality (3) leads to a polarity mismatch
by placing the terminal benzyl group in a hydrophilic environment
and the hydroxyl group in the lipophilic pocket between the side
chains of Leu-384 and Met-414. Swapping of the terminal substit-
uents (4) is sterically incompatible with the binding mode ob-
served for compound 44.
With respect to the SAR of amide substitution, the binding cav-
ity presents a larger volume at the ‘cis’ side of the amide nitrogen.
This observation accounts for the preference for N,N-disubstitution
on the terminal amide to avoid the energetic penalty of a mono-
substituted cis-amide.
Consistent with the binding interactions revealed in the X-ray
crystal structure, the inhibitory potency of 44 was impacted by
resistance mutations selected by known Palm Sites 1 and 2 bind-
ers. As shown in Table 2, there was an approximately 10-fold in-
crease in IC50 against
a genotype 1b (Con1) NS5B variant
containing a M414T change, and an even greater loss of potency
against the C316Y (ꢀ100-fold) or G554D (ꢀ150-fold) variant. How-
ever, compound activity was unaffected by the C316N change. As
expected, resistance mutations at the thumb site (M423T) or fin-
ger-loop site (P495L) did not reduce potency of this series of inhib-
itors (data not shown). The resistance profile of this class of
inhibitors thus overlaps but is distinct from the previously de-
scribed Palm Site
1
binder benzothiadiazine analogue
A-84883721 or the Palm Site 2 binder benzofuran derivative HCV-
796.10,18 Activity of neither compound was much affected by the
M414T mutation, even though other inhibitors from the benzothi-
adiazine series have shown significant reduction in potency
against this resistance locus. In contrast to compound 44, HCV-
796 activity was not changed by the G554D variant, which
impacted A-848837 (ꢀ100-fold potency loss), consistent with
Figure 3. The crystal structure of NS5B (green) complexed with compound 44
(yellow). The intramolecular hydrogen bond is shown in orange. The intermolecular
hydrogen bond from the inhibitor’s hydroxyl group to the bound water molecule
(labeled WAT) is shown in blue. The hydrogen bond network surrounding this
water is shown in black. The coordinates have been deposited in the Protein Data
Bank20 with accession code 3lkh. This figure was prepared using PyMOL.19

C. C. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2119–2124
2123
Table 2
Activity versus HCV NS5B enzyme variants
Compd
1b-Con1-IC50
,
l
Ma
Con1-M414T IC50
,
a l
M
Con1-G554D IC50
,
a l
M
Con1-C316Y IC50
,
a l
M
Con1-C316N IC50
,
a l
M
1a IC50, M
a l
44
HCV-796
A-848837
0.8
0.04
0.003
10
0.06
0.004
120
0.05
0.3
90
2
0.008
0.9
0.12
0.004
43
0.04
0.003
a
Values are means of duplicate experiments on two separate weightings.
previous reports. However, HCV-796 activity was influenced by the
polymorphism at residue 316, from a threefold increase in IC50
against the N316 variant to a 50-fold increase against the Y316
variant versus its Con1 (C316) value. By comparison, compound
44 was sensitive to the C316Y but not the C316 N change, whereas
A-848837 was unaffected by this resistance site.
With respect to the genotype spectrum of this series, compound
44 lost significant activity (50-fold) against a genotype 1a enzyme,
and was not active up to 200 lM against genotypes 2, 3, and 4
into the mechanism of inhibition and rationalization of the SAR.
Biochemical characterizations have further elucidated the mecha-
nism of inhibition of this new class of NS5B inhibitors. It is believed
that further structural modifications such as rigidification may
improve cell penetration and yield compounds with anti-HCV
replicon activity. Future lead optimization efforts focusing on
broadened genotype coverage and suitable pharmacokinetics and
ancillary properties may lead to the identification of a clinical
candidate for the treatment of hepatitis C.
(data not shown). In contrast, HCV-796 and A-848837 demon-
strated equal potency against 1a and 1b genotypes; HCV-796 in
particular has shown broad activity across multiple genotypes.10
To further investigate the mechanism of action of this novel
series of NS5B inhibitors, we performed a single-round elongation
assay as described previously16 to determine if these compounds
inhibit at the initiation or the elongation step. As shown in Figure
Acknowledgments
We thank Dr. Xianshu Yang for performing the initial ALIS
screening, Michael Starks for analytical support, Zhiqing He and
Patricia McMonagle for generating the NS5B enzyme constructs.
4, while compound 44 was fully active (IC50 = 0.6 lM) against
primer-initiated RNA synthesis involving multiple cycles of initia-
References and notes
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(IC50 >200 lM) in inhibiting the elongation of preformed NS5B
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l
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phenylpropan-2-yl)-benzamide-based scaffold. Beginning with
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olar range by combining mixture-based optimization with
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0
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-20
-3
-2
-1
0
1
2
3
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Cmpd 44 Log [µM]
Figure 4. Mechanism of action of compound 44. NS5B activity assay measured
multiple rounds of initiation and primer-extension, whereas the elongation assay
measured the burst phase activity consisting of single-round extension of enzyme–
primer–template after preincubation.16
14. Cooper, C.; Larouche, R.; Bourgault, B.; Chauret, N.; Proulx, L. J. Hepatol. 2009,
50, S342.

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HCV NS5B polymerase activity was measured in a radiolabeled nucleotide
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incorporation assay as follows: 50
7.3), 7.5 mM DTT, 20 units/mL RNasIN, 1
33P]-CTP supplemented to 20 nM CTP, 10 mM MgCl2, 60 mM NaCl, 100
BSA, 100 nM heteropolymer RNA template, 0.25 mM trinucleotide initiator and
30 nM NS5B ( 21) enzyme were incubated at room temperature for 2 h. Assay
was terminated by the addition of 50 L 500 mM EDTA. The reaction mixture
l
L reactions containing 20 mM HEPES (pH
M GTP, ATP and UTP, 60 Ci/mL
g/mL
l
l
l
[
D
l
was transferred to Millipore DE81 filter plate and the incorporation of labeled
CTP was determined by Packard TopCount. For primer-initiated RNA synthesis,
the trinucleotide initiator was replaced by 100 nM of an 11-mer RNA primer
complementary to the 30-end of the RNA template. Single-round elongation
reaction was performed at room temperature for 15 min using enzyme, primer
and template complex preincubated at room temperature for 22 h. Compound
IC50 values were calculated from experiments with 10 serial twofold dilutions
of the inhibitor in duplicate.
22. Liu, Y.; Jiang, W. W.; Pratt, J.; Rockway, T.; Harris, K.; Vasavanonda, S.; Tripathi,
R.; Pithawalla, R.; Kati, W. M. Biochemistry 2006, 45, 11312.