Discovery of BMS-961955, an allosteric inhibitor of the hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2017.06.024

The synthesis, structure-activity relationship (SAR) data, and further optimization of the metabolic stability and pharmacokinetic (PK) properties for a previously disclosed class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors are described. These efforts led to the discovery of BMS-961955 as a viable contingency backup to beclabuvir which was recently approved in Japan for the treatment of HCV as part of a three drug, single pill combination marketed as Ximency^TM.

Full text of DOI:10.1016/j.bmcl.2017.06.024

Accepted Manuscript
Discovery of BMS-961955, an Allosteric Inhibitor of the Hepatitis C Virus
NS5B Polymerase
Barbara Zhizhen Zheng, Stanley V. D'Andrea, Umesh Hanumegowda, Jay O.
Knipe, Kathy Mosure, Xiaoliang Zhuo, Julie A. Lemm, Mengping Liu, Karen
L. Rigat, Ying-Kai Wang, Hua Fang, Chris Poronsky, Jingfang Cutrone, Dauh-
Rurng Wu, Pirama Nayagam Arunachalam, T.J. Balapragalathan, Arunachalam
Arumugam, Arvind Mathur, Nicholas A. Meanwell, Min Gao, Susan B. Roberts,
John F. Kadow
PII:
S0960-894X(17)30626-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.024
BMCL 25058
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 May 2017
6 June 2017
7 June 2017
Please cite this article as: Zheng, B.Z., D'Andrea, S.V., Hanumegowda, U., Knipe, J.O., Mosure, K., Zhuo, X.,
Lemm, J.A., Liu, M., Rigat, K.L., Wang, Y-K., Fang, H., Poronsky, C., Cutrone, J., Wu, D-R., Arunachalam, P.N.,
Balapragalathan, T.J., Arumugam, A., Mathur, A., Meanwell, N.A., Gao, M., Roberts, S.B., Kadow, J.F., Discovery
of BMS-961955, an Allosteric Inhibitor of the Hepatitis C Virus NS5B Polymerase, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.024
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Discovery of BMS-961955, an Allosteric Inhibitor of the Hepatitis C
Virus NS5B Polymerase
Barbara Zhizhen Zheng,* a Stanley V. D’Andrea,a Umesh Hanumegowda,b Jay O. Knipe,b
Kathy Mosure,b Xiaoliang Zhuo,b Julie A. Lemm,c Mengping Liu, c Karen L. Rigat,c Ying-Kai
Wang,d Hua Fang,d Chris Poronsky,b Jingfang Cutrone,b Dauh-Rurng Wu,e Pirama Nayagam
Arunachalam,f T. J. Balapragalathan,f Arunachalam Arumugam,f Arvind Mathur,e
Nicholas A. Meanwell,a Min Gao,c Susan B. Roberts,c and John F. Kadowa
aDepartment of Discovery Chemistry and Molecular Technologies, Bristol-Myers Squibb Research and
Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States
bDepartment of Preclinical Candidate Optimization, Bristol-Myers Squibb Research and Development, 5 Research
Parkway, Wallingford, Connecticut 06492, United States
cDepartment of Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford,
Connecticut 06492, United States
dDepartment of Lead Evaluation, Bristol-Myers Squibb Research and Development, 5 Research Parkway,
Wallingford, Connecticut 06492, United States
eDepartment of Discovery Synthesis, Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ
08543, United States
fBiocon Bristol-Myers Squibb R&D Center, Biocon Park, Bommasandra IV phase, Jigani Link Road, Bengaluru –
560099, India.
Abstract: The synthesis, structure-activity relationship (SAR) data, and further optimization of
the metabolic stability and pharmacokinetic (PK) properties for a previously disclosed class of
cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors are described. These
efforts led to the discovery of BMS-961955 as a viable contingency backup to beclabuvir which
was recently approved in Japan for the treatment of HCV as part of a three drug, single pill
combination marketed as XimencyTM.
Keywords: HCV NS5B, Polymerase, Cyclopropyl-fused indolobenzazepine, Metabolic
stability, Direct-acting antiviral agent, Antiviral agent

Recent estimates suggest that hepatitis C virus (HCV) infects approximately 130-170
million people or 2.8% of the world’s population.1, 2 While primary infection with HCV is often
asymptomatic, most HCV infections progress to a chronic state that can slowly progress for
decades, eventually causing liver cirrhosis, liver failure or hepatocellular carcinoma.3, 4 There are
more than 700,000 deaths per year from HCV-related diseases.5
The development of direct acting antiviral agents (DAAs) against HCV is made more
complicated by both its heterogeneity, as evidenced by the recent classification of seven major
genotypes (GTs) with several subtypes within each group, and its rapid rate of replication by a
highly error-prone NS5B polymerase.6 While GT 1 HCV predominates in North America and
Europe, infections by other GTs such as 2 and 3 are also prevalent in significant numbers and
may predominate in other geographic areas.7 Historically, GT 1 HCV has been considered the
most difficult to cure. For a considerable span of time, the standard of care for treatment of HCV
infection was a combination of pegylated α-interferon (PEG-IFN) and ribavirin (RBV).
However, the sustained viral response (SVR) to this therapy was limited to between 40% and
60% in patients, depending on the infecting genotype, with some of the poorer response rates
associated with GT 1 HCV infections.8 Moreover, interferon-based therapies were poorly
tolerated, associated with side effects such as fatigue and flu-like symptoms which often
significantly reduced adherence or even discouraged the initiation of therapy.9 The first DAAs
against HCV that were approved for clinical use, telaprevir and boceprevir, targeted the HCV
NS3 protease.10, 11 Combination of one of these two protease inhibitors with PEG-IFN and RBV
improved efficacy as measured by SVRs after the end of treatment of 68-75% in GT 1 patients;
unfortunately, these new therapies required frequent dosing and more significantly were
burdened with significant additional side effects, a profile that eventually led to their withdrawal
from the market.12, 13 The heterogeneity and rapid mutation rate of HCV strongly suggested that
the development of PEG-IFN-free DAA-only regimens would require combinations of
compounds acting by diverse mechanisms.14
The NS5B polymerase, a viral RNA-dependent RNA polymerase, is essential for HCV
replication.15 We have pursued NS5B inhibitors as potential partners to be used in combination
therapy with mechanistically distinct inhibitors. Beclabuvir (BMS-791325, 1) in Figure 1, which
binds to the polymerase at the thumb 1 domain16, 17 is an allosteric inhibitor of NS5B that was
recently approved in Japan as a part of fixed-dose combination with two mechanistically distinct

DAAs, the NS3 protease inhibitor asunaprevir and the NS5A inhibitor daclatasvir.18 After the
discovery of 1, additional contingency backup molecules were targeted to ensure the ultimate
success of the program in the event of any unforeseen issues that might arise during its clinical
development.
Figure 1. The structures of compound 1 and 2.
Biotransformation studies with 1 (see scheme 1) had indicated that CYP 3A4-mediated
oxidative demethylation of the N, N-dimethylsulfamide moiety was the primary path of
metabolism and produced the equipotent mono-desmethyl derivative 2 as the major metabolite.16
Sulfonamide 2 was further oxidized by CYP 3A4 or CYP 2C19 to form the O-demethylated
product 4 which was shown to produce metabolite 5, the product of subsequent glucuronidation.
Scheme 1. Biotransformation studies with compound 1.

The initial campaign to generate a contingency backup to 1 centered on preventing the
generation of the primary metabolite 2 which proceeded at a reasonable rate as reflected by the
t1/2 values in in vitro microsomal stability studies in human liver microsomes (HLM) of 46.1
minutes and cynomolgous monkey liver microsomes (CLM) of 20.8 minutes. Compounds in
which this major pathway of metabolism was blocked were more stable in liver microsomes.
Specifically, as reported previously, when the left-hand N,N-dimethylsulfamide was replaced
with the metabolically more stable isopropyl acylsulfonamide, the resulting molecule 3 in figure
2, demonstrated a much enhanced t1/2 in both HLM (>200 minutes) and CLM (87.5 min.).17
However, this initially promising compound was advanced directly into pre-IND toxicity studies
because of its structural similarity to 1 but ultimately was not a suitable backup due to
unexpected findings of an unknown mechanism.17
Figure 2. The structure of compound 3.
Therefore our objective was to identify a metabolically more stable analog of 1 which
was structurally differentiated from 3 so that it might avoid the toxicological issues seen with
that compound. It was felt that two structural changes would be preferential in order to avoid the
toxicity issues associated with 3. Addressing the metabolism of the aryl methoxy would provide
additional structural and metabolic differentiation, thereby serving a dual purpose. Thus, changes
in this region of the molecule were pursued in combination with efforts to avoid the oxidative N-
demethylation of the sulfamide moiety of 1. In order to block O-demethylative metabolism,
replacement of the methoxy group of 1 with a fluoro atom was examined in conjunction with an
exploration of the SAR associated with acylsulfamide and acylsulfonamide replacements.
The success of this approach was not assured since earlier studies of fluoro-substituted
molecules had revealed an erosion of potency toward GT 1a and 1b viruses in both the enzyme

inhibition assay and in cellular replicons when compared to the corresponding methoxy analogs.
As shown by the comparison of 6 and 7 in Figure 3, the EC50 values for 7, with and without 40%
human serum, against GT 1b were between 2- and 4.5-fold higher than that of the corresponding
methoxy analogue 6. The origin and SAR of the morpholine amides in the context of the
methoxy series that includes 6 has been previously disclosed.16 Against the backdrop of this SAR
point, optimization of the fluorophenyl series in the more potent cyclopropyl indolobenzazepine
core was pursued.
Figure 3. The structures of compound 6 and 7.
The synthesis of the fluoroaryl acylsulfonamides and sulfamides are shown in Scheme 2.
Scheme 2. The synthesis of the fluoroaryl acylsulfonamides and sulfamides.

Baylis-Hillman reaction of 2-bromo-5-fluorobenzaldehyde (24) with tert-butyl acrylate
(25) yielded 26, which was acetylated and subsequently coupled with 3-cyclohexyl-1H-indole-6-
carboxylate (28) to generate bromide 29. An intramolecular Heck reaction of 29 produced the
diester 30 which was subjected to a Corey-Chaykovsky cyclopropanation reaction to afford 31 as
a racemic mixture, which was resolved using SFC chromatography to isolate the desired (R, S)
enantiomer16 32 which was isolated as the first eluent. The tert-butyl ester protecting group of
32 was removed using TFA and the resulting acid 34 coupled with bicyclic amine 35 to give
amide 36. Saponification of the methyl ester of 36 provided the corresponding acid 37 which was
activated with CDI and imidazole and then coupled with a variety of sulfonamides to yield the
acylsulfonamide series 38. Sulfamide analogues 39 were prepared in a similar manner via the
coupling of acid 37 with a variety of sulfamides.
To gauge the potential for realizing the target profile and the impact of the fluoro
substituent in the context of the more optimized background, the analog of 1 in which the
methoxy group was replaced with a fluoro atom was prepared along with several close sulfamide
analogs. The potency of this series of compounds was well maintained when compared to 1 with
analogues 8-11 showing similar potency to the prototype (Table 1). However, the metabolic
stability of 8 in CLM was similar to 1 while 9 and 10 were poorer. The metabolic stabilities of
these compounds in HLM were comparable or only modestly better than 1 leading to the
conclusion that the sulfamides in the fluoro series were not significantly differentiated with
respect to metabolic stability. The azetidinyl sulfamide 11 demonstrated improved metabolic
stability in HLM (>120 minutes) and CLM (43 minutes) but this compound displayed poor
absorption after oral dosing to rats (see table 2), a result that was consistent with what has been
observed previously for the sulfamide analogue in the methoxy series (Table 2).17

Table 1: Potency and metabolic stability of fluorinated analogues and their methoxy
comparators.
GT-1b
replicon
EC50
GT-1a
replicon
EC50
GT-1b
enzyme
IC50
CLM
t/1/2 (min)
HLM
t/1/2 (min)
Compound
R1
R2
(nM)
(nM)
(nM)
N
OMe
OMe
F
6
10
5
4
4
5
20.4
5.0
20.8
87.5
27
46.1
> 200
75
1
3
8
N
N
F
7.1
4.5
9.9
51.5
9
H
F
F
F
F
F
14.9
5.3
17
6
6.2
3.0
11
5
11.7
43
61
10
11
12
13
14
N
N
> 120
> 120
> 120
> 120
59
50
6
6.5
46
F
F
F
23
7.9
7.9
10
6.5
4.3
47
38
> 120
> 120
> 120
15
16
17
4.6
65.5
F
F
F
22
10.5
7.0
9.7
6.4
4.0
35
48
>120
>120
>120
18
19
20
F
>120

F
F
F
9.2
5.2
25
3.0
2.9
10
36
>120
>120
21
22
23
42.5
Having established that a fluorophenyl ring was capable of providing targeted antiviral
potency in the sulfamide series, the acylsulfonamide analogues were explored in a similar
structural context. Previous SAR studies in the methoxy series had shown that small groups were
tolerated on the acylsulfonamide moiety19 and the series of compounds 12-23 was prepared and
evaluated. Overall, the compounds displayed excellent potency in antiviral replicon assays with
the majority demonstrating EC50 values toward GT 1a/1b HCV replicons of <10 nM. The
metabolic stability of these acylsulfonamides were evaluated in HLM and CLM with the result
that they were generally quite stable in HLM, with compounds 11-22 in Table 1 displaying t1/2
values of >120 minutes. The majority of the acylsulfonamides also displayed enhanced metabolic
stability in CLM, with t1/2 values ranging from 35 to >120 minutes. Compounds 12, 15, 18, 19
and 23 were deprioritized due to double digit nM potency in one of the initial replicon assays
assessing GT 1b inhibitory potency. The tert-butyl acylsulfonamide 20 exhibited the best
metabolic stability in both HLM (t1/2 >120 minutes) and CLM (t1/2 >120 minutes). The potency of
20 was very similar to compounds 1 and 3 and this compound was selected for PK evaluation in
a snapshot rat screening study conducted over 6 hours. However, 20 showed low exposure after
oral dosing (AUC =1.3 µM.h; F = 8%, 6 h) which was attributed to poor absorption (PAMPA
permeability = 135 nm/s at pH 5.5 and 238 nm/s at pH 7.4).
The acylsulfonamides with replicon EC50 values toward GT 1a/1b of <10 nM were
advanced into additional in vitro profiling and rat PK studies with the exception of 14 and 16
which were discarded due to a potential risk for CYP induction as predicted by a PXR
transactivation signal in an in vitro screen (Table 2). Cyclopropyl acylsulfonamide 13 was a
potent antiviral agent, displaying EC50 values of 6 nM and 5 nM against GT 1b and 1a replicons,
respectively, and showed no potential for CYP induction in the PXR transactivation assay (EC50
>50 M). However, this compound exhibited high clearance (41 mL/min/kg) and insufficient
exposure following oral administration in a rat PK study. Compounds 21 and 22 showed targeted

potency and a clean PXR profile; however, these compounds did not offer an advantage in
metabolic stability in CLM when compared to 17 so they were not profiled further in favor of
compounds that did. From this survey, 17 (BMS-961955) emerged as the best overall candidate
to move into PK studies in higher species.
Table 2: PK parameters and PXR trans-activation activity associated with acylsulfonamide- and
N, N-dimethylsulfamide-based HCV NS5B inhibitors.
Structure
R
Antiviral activity
Rat PK*
PXR
EC50
(M)
GT 1b
replicon
EC50
GT 1a
replicon
EC50
Compound
Cl
(mL/min
/kg)
AUC0-24h
(µM•h)
t1/2
(h)
% F
(nM)
(nM)
6
4
> 50
> 50
2.4
43
65
1
N
10
5.3
6
4
14.5
3
12 (rat
screen)
N
3.0
5
0.5 (0-6h)
1.508
19
11
13
14
16
17
20
50
41
42
4.2
5.4
23
6
6.5
6.5
4.3
4.0
2.78
8.33
16.7
16.7
0.857
13.6
7.9
7.9
7.0
10
12.38
6.2
52
8
12 (rat
screen)
1.3 (0-6h)
*IV dose: 5mg/kg, PO dose: 10 mg/kg

The methyl-substituted cyclopropyl acylsulfonamide 17 exhibited potency comparable to
both 1 and 3 against GT 1a and 1b replicons with EC50 values of 4 nM and 7 nM, respectively.
The EC50 value of 17 toward GT 1a increased by 8.2-fold in the presence of 40% human serum,
indicative of a minimal effect.20 Compound 17 potently and specifically inhibited the HCV
NS5B polymerase derived from HCV GT 1, and GTs 3-6, with IC50 values of 4 nM (1a), 5 nM
(1b), 2 nM (3a), 44 nM (4a), 15 nM (5a) and 124 nM (6a) while GT 2 NS5B was found to be less
susceptible with IC50 values of 597 nM (2a) and 212 nM (2b).21 Compound 17 displayed
significantly enhanced metabolic stability in CLM and HLM when compared to 1 and it was not
an inhibitor of recombinant CYP isoforms in HLM. In addition, no evidence of CYP induction
was observed in human hepatocytes. Compound 17 exhibited high permeability in a PAMPA
assay with Pc values of 523 nm/s at pH 5.5 and 590 nm/s at pH 7.4 and also displayed excellent
PK properties in the rat, dog and cynomolgus monkey (Table 3). Clearance was low to moderate
across the species while the volume of distribution and half-life values were moderate and
bioavailability ranged from 48-84%. The predicted human dose based on the replicon potency
and 3-species PK was 70 mg once a day (QD). As a result of its excellent potency, clean
preclinical profile and excellent QD dosing potential, 17 was advanced into a two week
toxicology study in rats.22 No significant liabilities or issues were identified with only minimal
findings at adequate exposure margins. A two week toxicology study in dogs was used to
determine that 17 had a toxicology safety profile that supported advancement and clearly
differentiated this compound from 3. However, further development of 17 was not pursued based
on the successful continued development of beclabuvir.23, 24, 25, 26

Table 3: Rat, dog and cynomolgus monkey PK data for 17.
AUC (0-
Dose
Vs
(L/kg)
24h)
Species
Rat
Route
t (h)
Cl (mL/min/kg)
% F
½
(mg/kg)
(M•h)
IV
5
12.3  6.2  1.7
12.4  0.3
10.0  2.5
3.9  1.4
PO
10
52
84
12.2 
25.7  4.3
IV
1
3
0.8  0.2
0.6  0.1
3.1  1.3
1.1
Dog
PO
62.1 5.8
IV
1
3
2.3  0.2 8.7  3.4
3.1 0.3
10.5  0.5
Monkey
PO
48
The treatment landscape for patients with chronic hepatitis C infection has changed
significantly over the last 5 years. The rapid evolution of clinical data has demonstrated that
treatment with PEG-IFN-free combinations of DAAs14 achieves high rates of SVR with shorter
treatment durations. By 2016, the standard of care for HCV infection had evolved to consist of
all oral therapies. The oral DAAs approved for use in the U.S. include: DaklinzaTM (daclatasvir),
Sovaldi® (sofosbuvir), Olysio® (simeprevir), Harvoni (a fixed-dose combination of ledipasvir
and sofosbuvir), TechnivieTM (ombitasvir/paritaprevir/ritonavir), Viekira Pak® (ombitasvir-
paritaprevir-ritonavir and dasabuvir), ZepatierTM (the combination of elbasvir and grazoprevir)
and Epclusa® (the combination of sofosbuvir and velpatasvir). Several other pan-genotype
inhibiting regimens are being reviewed for possible approval including glecaprevir/pibrentasvir
and a single tablet regimen that combines sofosbuvir, velpatasvir and voxilaprevir. Notably, both
active-site27, 28 and allosteric inhibitors29, 30 of the HCV NS5B polymerase are currently present

in approved regimens. Since it is believed that the design and realization of polymerase
inhibitors from this chemotype that act at thumb pocket 1 and are potent inhibitors of GT2 HCV
would be challenging, compounds 1 and 17 represent the most optimal molecules realized in our
efforts vs. this target. Further efforts aimed at identifying pan genotype inhibitors at an alternate
allosteric site will be disclosed in the future.
In conclusion, after the discovery of compound 17, preclinical profiling showed that the
compound was a metabolically-stable HCV NS5B thumb site 1 inhibitor with a low projected
human dose for the potential treatment of GT 1 HCV viruses and the compound was accepted as
a candidate for development. Inhibitor 17 has two regions of structural differentiation from the
beclabuvir (1), both located at sites of metabolic modification. However, the ultimately
successful progression of 1 in clinical development eventually obviated further advancement of
17.
Acknowledgements
The authors would like to thank Bang-Chi Chen, Jiangqing Li, and Daniel Smith from the
Department of Discovery Synthesis and Subramaniam Raj, Chidananda, MG., and Shabana
Ansar from BMS Biocon for their contributions toward the scaling up of BMT-961955 (17). The
authors would also like to thank the Bioanalytical group for their experimental support.
*Author for correspondence: Zhizhen Zheng@bms.com; Tel.: 203-677-5818
Current address: ViiV Healthcare, 5 Research Parkway, Wallingford, CT 06410.
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19. Unpublished results.
20. The human serum assay was performed essentially the same as described in the standard
GT 1a replicon assay in the presence of 4% fetal bovine serum supplemented with an
additional 40% human serum.
21. IC50 Human Pol. Enzyme assay , ,  = >25 M; IC50 BVDV Pol. Enzyme assay >25
M; EC50 BVDV replicon cell assay = 10 M; EC50 Dengue replicon cell assay = 7 M;
EC50 HIV-2 = 2 M; Cytotoxicity (Huh-7, Vero, MT2 cells) CC50 = 2.9-11.8 M
22. The doses for the two week rat toxicity studies were 10, 30, 100 and 300 mg/kg/day.
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Graphical abstract
Discovery of BMS-961955, an Allosteric
Inhibitor of the Hepatitis C Virus NS5B
Polymerase
Leave this area blank for abstract info.
Barbara Zhizhen Zheng,*a Stanley V. D’Andrea,a Umesh Hanumegowda,b Jay O. Knipe,b Kathy Mosure,b Xiaoliang
Zhuo,b Julie A. Lemm,c Mengping Liu,c Karen L. Rigat,c Ying-Kai Wang,d Hua Fang,d Chris Poronsky,b Jingfang Cutrone,b
Dauh-Rurng Wu,e Pirama Nayagam Arunachalam,f T. J. Balapragalathan,f Arunachalam Arumugam,f Arvind Mathur,e
Nicholas A. Meanwell,a Min Gao,c Susan B. Roberts,c and John F. Kadowa