The Synthesis and evaluation of a novel class of (E)-3-(1-cyclohexyl-1H- pyrazol-3-yl)-2-methylacrylic acid Hepatitis C virus polymerase NS5B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.03.086

Herein we report the identification and evaluation of a novel series of (E)-3-(1-cyclohexyl-1H-pyrazol-3-yl)-2-methylacrylic acid derivatives identified from a deannulation study performed on the reported benzimidazole NS5B inhibitor, 1. This resulted in

Full text of DOI:10.1016/j.bmcl.2011.03.086

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2869–2872
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The Synthesis and evaluation of a novel class of (E)-3-(1-cyclohexyl-1H-pyrazol-
3-yl)-2-methylacrylic acid Hepatitis C virus polymerase NS5B inhibitors
Scott W. Martin a, , Peter Glunz c, , Brett R. Beno b, Carl Bergstrom a, Jeffrey L. Romine a, E. Scott Priestley c,
Makenzie Newman c, Min Gao d, Susan Roberts d, Karen Rigat d, Robert Fridell d, Dike Qiu d, Galina Knobloh d,
Ying-Kai Wang d
⇑
⇑
a Department of Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway Wallingford, CT 06492, United States
b Computer-Assisted Drug Design Department, Applied Biotechnology, Bristol-Myers Squibb Research and Development, 5 Research Parkway Wallingford, CT 06492, United States
c Department of Chemistry, Bristol-Myers Squibb Research and Development, 311 Penington-Rocky Hill Rd. Hopewell, NJ 08534, United States
d Department of Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway Wallingford, CT 06492, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report the identification and evaluation of a novel series of (E)-3-(1-cyclohexyl-1H-pyrazol-3-
yl)-2-methylacrylic acid derivatives identified from a deannulation study performed on the reported
benzimidazole NS5B inhibitor, 1. This resulted in the identification of (E)-3-(2-(4-((40-cyano-4-(4-
hydroxypiperidine-1-carbonyl)biphenyl-2-yl)methoxy)phenyl)-1-cyclohexyl-1H-imidazol-4-yl)-2-methyl-
acrylic acid (11) as a potent inhibitor of NS5B. Potential pathways for the further optimization of this
series are suggested.
Received 3 February 2011
Revised 18 March 2011
Accepted 22 March 2011
Available online 30 March 2011
Keywords:
Hepatitis
Polymerase
NS5B
Ó 2011 Elsevier Ltd. All rights reserved.
Inhibitors
Pyrazoles
Hepatitis C virus (HCV) is estimated to have chronically infected
160 million people worldwide with 5.3 million cases reported in
the United States.1 Infection with HCV can result in the develop-
ment of a chronic condition that frequently progresses to cirrhosis
and hepatocellular carcinoma.2,3 The current optimal treatment in-
came interested in evaluating novel derivatives of 1, and examined
a number of possible structural modifications to this compound.
One of our more productive approaches explored a deannula-
tion strategy in which we excised a single carbon atom from the
benzimidazole to generate the acrylate 2, as shown in Figure 1.
This compound displayed an approximate three-fold reduction in
potency. Replacement of the imidazole heterocycle in 2 with an
isomeric pyrazole, as shown in analog 3, was associated with a fur-
ther loss of activity. Surprisingly however, the positional pyrazole
isomer 4 was essentially equipotent with the original lead 1, and
led us to evaluate the series of pyrazol-3-yl-2-methylacrylic acids
shown in Table 1.
volves extended administration with PEG-interferon-a and ribavi-
rin,4,5 although a number of advanced clinical studies employing
direct acting antivirals (DAA) may be on the verge of changing cur-
rent optimal thearapy.5b,c The current regime suffers from limited
efficacy against 1a and 1b HCV genotypes and is associated with an
array of severe side effects. This latter issue leads to treatment dis-
continuation in a significant proportion of patients.5a,6 Correspond-
ingly, there exists a clear and urgent medical need for improved
therapeutic agents. HCV non-structural protein 5B (NS5B), an
RNA-dependent RNA-polymerase (RdRp) has long been considered
an attractive target for drug discovery due to its essential role in
viral replication.5a,7 In addition to a number of reported active site
inhibitors, multiple allosteric inhibitors have been identified that
act at four distinct binding sites,5b,8[Pockets 1–4]. Analogs of com-
Scheme 1 outlines the synthetic route used to access pyrazoles
4 through 14. Compounds 1,9 2 and 313 were prepared as previ-
ously reported.
Diketoester 1515 was condensed with cyclohexyl hydrazine in
refluxing ethanol to generate the pyrazole 16. Reduction with lith-
ium aluminum hydride gave an alcohol which, upon subsequent
oxidation with manganese(IV)oxide, provided an aldehyde that
could be reacted with the in situ-generated Wittig reagent derived
from 1-[(ethoxycarbonyl)ethyl]triphenylphosphonium bromide.
The resultant methacrylate 18 was debenzylated upon exposure
to boron trichloride, and subsequent alkylation with the benzyl
bromide 209b gave key intermediate 21. Compound 21 allowed
pound 19 have been reported to bind in pocket I of NS5B,8,10,11
a
site that has been clinically validated.5b,12 Correspondingly, we be-
⇑
Corresponding authors.
E-mail address: scott.martin@bms.com (S.W. Martin).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.086

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S. W. Martin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2869–2872
Figure 1. Literature disclosed NS5B allosteric inhibitor 19, and related deannulated analogs 2, 3 and 4 displaying NS5B inhibitory activity.13
Table 1
subsequent Suzuki reaction (e.g., 21 to 23). Target compound 7
was obtained from 24 after ester hydrolysis.
Inhibition of HCV NS5B genotype 1b enzyme;13 structures corresponding to Figure 1
and Figure 4
As stated above, we evaluated a number of isomeric diazole
cores in our deannulation approach to the synthesis of novel ana-
logs of compound 1. Of these, pyrazole 4 was found to be approx-
imately four-fold more active than analog 2, and ten-fold more
potent than its isomeric counterpart 3 (Table 1). These observa-
tions may be accounted for based on variations in the binding
interactions between each of the ligands’ carboxylate moieties
and Arg503 of NS5B, as suggested by molecular modeling studies.
For example, the superposition of analogs 2, 3 and 4 with com-
pound B, as shown in Figure 2, (from published NS5B/Compound B
co-crystal structure17) demonstrates that as the distances between
the carboxylate oxygen atoms and the Arg503 guanidine nitrogen
atoms increase, potency decreases.
R1
R2
IC50
(
lM)
a
Compd
Ar
1b
2b
3b
4
5
6
7
8
9
10
11
12
13
14
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-CN-phenyl
4-CN-phenyl
4-CN-phenyl
5-Acetyl-thiophen-2-yl
5-Acetyl-thiophen-2-yl
5-Acetyl-thiophen-2-yl
CH3NH
CH3NH
CH3NH
CH3NH
0.64 ( 0.15)
1.62 ( 0.26)
4.03 (n = 1)
0.38 (n = 1)
0.46 ( 0.31)
0.44 ( 0.12)
0.65 ( 0.14)
1.03 ( 0.13)
0.79 ( 0.11)
0.82 ( 0.60)
0.34 ( 0.06)
2.29 ( 0.03)
2.7 ( 1.2)
H
H
H
Br
H
H
H
H
H
H
H
(CH3)2N
4-OH-piperidine
4-OH-piperidine
NHCH2CH2 OH
CH3NH
(CH3)2N
4-OH-piperidine
CH3NH
(CH3)2N
Morpholine
5.2 ( 1.2)
We concluded that differences in the bond lengths and angles in
the pyrazole and imidazole rings translate to different positioning
of the carboxylate moieties relative to the cyclohexyl group that
serves to anchor these compounds in the polymerase, and thus
impact the strength of the interactions between the acid function-
alities of the ligand and Arg503. An obvious alternative explanation
is that the less potent diazoles described here could be expected to
experience allylic strain25 when adopting the probable bioactive
conformation, see Figure 3.
Pursuing our initial rationale for the observed enhanced activity
of analog 4, we prepared the series of derivatives shown in Table 1,
based on the generic structure depicted in Figure 4.
Significant structure-activity relationship (SAR) of the biaryl
side chain of the benzimidazole reference compound 1 has been
established in a previous report.9b From the above Table 1 it can
be seen that the combination of an N-methyl, N,N-dimethyl, or
piperidin-4-ol amide appended to either a 4-cyano or 4-chloro
biaryl as shown in analogs 1–7 and 9–11, respectively, generally
a
Values are means of a minimum of two test occasions run in duplicate, standard
deviation is given in parentheses, values with n = 1 are single test occasions run in
duplicate.
b
Structures corresponding to Figure 1.
for diversification at two vectors: the aryl side chain through Suzu-
ki coupling, and amide formation to 23 upon selectively unmasking
the t-butyl carboxylate. Hydrolysis of the acrylate ester led to com-
pounds 4–6 and 8–14 as shown in Table 1, where R2 = H. Function-
alization at R2 was achieved through the bromo pyrazole 17
obtained upon bromination of 16 with bromine in acetic acid. Bro-
mide 17 was further transformed into acrylate 19 via chemistry
analogous to that used in the preparation of 18. A more convergent
approach was employed in the synthesis of compound 7 (Scheme
1). Debenzylation of 19 followed by Mitsunobu alkylation with
the biaryl derivative 2216 directly gave 24 and thus eliminated pos-
sible chemoselectivity concerns related to using a dibromide in a

S. W. Martin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2869–2872
2871
Scheme 1. Reagents and conditions: (a) ethanol reflux; (b) for R2 = Br, Br2, AcOH, 0 °C to rt; (c) LiAlH4, THF, 0 °C to rt; (d) MnO2, DME, reflux; (e) [1-
(ethoxycarbonyl)ethyl]triphenylphosphonium bromide, n-BuLi, THF, 0 °C to rt; (f) BCl3, CH2Cl2, ꢀ40 °C; (g) Cs2CO3, DMF, rt; (h) Ar-B(OH)2, (Ph3P)4Pd, THF, satd aqueous
NaHCO3, reflux 18 h, or Ar-B(OH)2, (Ph3P)4Pd, 1,4-dioxane, satd aqueous NaHCO3, microwave 120 °C, 10 min. (i) TFA, CH2Cl2, rt; (j) TBTU, DMF, amine, rt; (k) DIAD, Ph3P, THF,
0 °C to rt; (l) NaOH, THF, 60 °C.
results in sub-micromolar potency against NS5B. This SAR parallels
findings reported in the series typified by compound 1, where
introduction of a small electron withdrawing group and inclusion
of a carbonyl moiety in the 4-position of the biaryl side chain en-
hanced intrinsic potency and whole cell activity while modulating
protein binding and cellular toxicity.9b Compound 11 displayed
similar potency to the parent benzimidazole 1 in both the enzyme
NS5B assay,13 and in a subsequent genotype 1b replicon assay14
(compound 11 EC50 = 0.4 0.2
n = 1). Moreover, compound 11 demonstrated a ten-fold loss in
activity (EC50 = 4.0 M) against the mutant P495A replicon system,
lM vs compound 1 EC50 of 0.3 lM
l
strongly supporting the assumption that this analog binds in pock-
et 1. Compound 11 was determined to be relatively non-toxic
(CC50 = 25
lM) and had a therapeutic index of >60. In addition,
the closely related analog 5 (replicon EC50 = 1.9 1.3
lM) was
shown to have good bioavailability in rat when dosed orally at
2 mg/Kg, (F = 69%, T1/2 = 1.3 0.7 h), thus demonstrating the poten-
tial of pyrazol-3-yl-2-methylacrylic acids to possess favorable
absorption properties.
Compounds 12–14, in which the distal phenyl moiety of the
biphenyl group is replaced with a thiophene isostere, and compound
8, that incorporates an extended 2-hydroxyethyl functionality in
the amide group, generally displayed a moderate loss of activity.
As can be seen with compound 7, substitution at position C4
(R2 = Br) afforded a compound with comparable activity in both
Figure 2. Interaction of core carboxylate moieties with NS5B ARG503. Geometries
of truncated core molecules 2–4 optimized with DFT at the B3LYP/6–31G⁄⁄ level
using Q-Chem.19–24 See Ref. 18.

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S. W. Martin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2869–2872
Figure 3. Preferred conformations of pyrazole and imidazole inhibitor classes.
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A
future disclosure will detail our research investigating this region.
In conclusion, we have described a novel series of (E)-3-(1-
cyclohexyl-1H-pyrazol-3-yl)-2-methylacrylic acid NS5B inhibitors
identified from a deannulation study performed on the known
benzimidazole inhibitor 1. The relative potencies of a series of iso-
meric 2-methylacrylate appended heterocycles could be rational-
ized with regard to molecular modeling predictions of variations
in the strengths of the interactions of the ligands’ carboxyl moie-
ties with Arg503 of NS5B. Subsequent elucidation of an initial
structure–activity relationship of the pyrazole 4 identified com-
pounds with potencies in both enzyme and replicon assays, com-
parable to the parent chemotype 1. Data from resistance studies
strongly suggested these compounds bind in Pocket 1. Finally, in
analog 7, we have identified paths for the further optimization of
this compound class.
16. Procedure in Supplementary data.
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replaced with hydrogen atoms are shown superimposed with B. Coordinates
for the model of B and ARG503 were extracted directly from the PDB entry
(2BRK) for the NS5B/A complex. Superpositions were performed via RMS fitting
2–4 to B. All non-hydrogen atoms in the cyclohexyl and pendant phenyl rings
and the central five-membered ring to which they are connected were used for
the RMS fitting procedure. The color of carbon atoms in each molecule
corresponds to that of the entry for that molecule in the inset table. Image
created with Maestro™.
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Acknowledgments
The corresponding authors acknowledge Dr. Carl Bergstrom for
his significant help in reviewing this manuscript.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.086.
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