Discovery of Ruzasvir (MK-8408): A Potent, Pan-Genotype HCV NS5A Inhibitor with Optimized Activity against Common Resistance-Associated Polymorphisms

Article abstract of DOI:10.1021/acs.jmedchem.6b01310

We describe the research that led to the discovery of compound 40 (ruzasvir, MK-8408), a pan-genotypic HCV nonstructural protein 5A (NS5A) inhibitor with a "flat" GT1 mutant profile. This NS5A inhibitor contains a unique tetracyclic indole core while maintaining the imidazole-proline-valine Moc motifs of our previous NS5A inhibitors. Compound 40 is currently in early clinical trials and is under evaluation as part of an all-oral DAA regimen for the treatment of chronic HCV infection.

Full text of DOI:10.1021/acs.jmedchem.6b01310

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Article
The Discovery of Ruzasvir (MK-8408): A Potent, Pan-
genotype HCV NS5A Inhibitor with Optimized Activity
Against Common Resistance-Associated Polymorphisms
Ling Tong, Wensheng Yu, Lei Chen, Oleg Selyutin, Michael P. Dwyer, Anilkumar Gopinadhan Nair,
Robert Mazzola, Jae-Hun Kim, Deyou Sha, Jingjun Yin, Rebecca Tamra Ruck, Ian W. Davies, Bin
Hu, Bin Zhong, Jinglai Hao, Tao Ji, Shuai Zan, Rong Liu, Sony Agrawal, Ellen Xia, Stephanie Curry,
Patricia Mcmonagle, Karin Bystol, Frederick Lahser, Donna Carr, Laura Rokosz, Paul Ingravallo,
Shiying Chen, Kung-I Feng, Mark Cartwright, Ernest Asante-Appiah, and Joseph A. Kozlowski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01310 • Publication Date (Web): 03 Nov 2016
Downloaded from http://pubs.acs.org on November 7, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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The Discovery of Ruzasvir (MKꢀ8408): A Potent,
Panꢀgenotype HCV NS5A Inhibitor with Optimized
Activity Against Common ResistanceꢀAssociated
Polymorphisms
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a
a
a
a
a
a
Ling Tong ,* Wensheng Yu , Lei Chen , Oleg Selyutin , Michael P. Dwyer , Anilkumar G. Nair ,
a
a
a
b
b
a,b
Robert Mazzola , Jae-Hun Kim , Deyou Sha , Jingjun Yin , Rebecca T. Ruck , Ian W. Davies ,
g
g
g
g
g
c
c
c
Bin Hu , Bin Zhong , Jinglai Hao , Tao Ji , Shuai Zan ,Rong Liu , Sony Agrawal , Ellen Xia ,
c
c
c
c
c
Stephanie Curry , Patricia McMonagle , Karin Bystol , Frederick Lahser , Donna Carr , Laura
c
c
d
e
f
Rokosz , Paul Ingravallo , Shiying Chen , Kung-I Feng , Mark Cartwright , Ernest Asante-
c
a
Appiah , and Joseph A. Kozlowski
a
c
d
Department of Medicinal Chemistry, Department of Discovery Biology, Department of
f
Pharmacokinetics , Department of Drug Safety, Merck & Co., Inc., 2000 Galloping Hill Rd.,
Kenilworth, NJ 07033, USA
b
e
Department of Process and Analytical Chemistry, Department of Discovery Pharmaceutic
Science, Merck & Co., Inc., 126 E. Lincoln Ave. Rahway, NJ 07065, USA
g
Department of Medicinal Chemistry, WuXi AppTec, Shanghai, 200131 China
*Ling.Tong@merck.com
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ABSTRACT: We describe the research that led to the discovery of compound 40 (ruzasvir, MKꢀ
8408), a panꢀgenotypic HCV nonstructural protein 5A (NS5A) inhibitor with a “flat” GT1
mutant profile. This NS5A inhibitor contains a unique tetracyclic indole core, while maintaining
the imidazoleꢀprolineꢀvaline Moc motifs of our previous NS5A inhibitors. Compound 40 is
currently in early clinical trials and is under evaluation as part of an allꢀoral DAA regimen for
the treatment of chronic HCV infection.
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INTRODUCTION
Hepatitis C virus (HCV) infection has become the most common bloodꢀborn infection in
1
developed countries. Recent estimates of disease burden show an increase in seroprevalence
over the period of 15 years (between 1990 and 2005) to ~2.8% of the global population, more
2
than 185 million infections worldwide. HCV infection is initially asymptomatic; as the duration
of chronic infection increases, symptoms become manifested as increased liver disease
3
including fibrosis, cirrhosis (compensated and decompensated) and hepatocellular carcinoma.
Advances in treatment regimens have resulted in significantly improved outcomes for many
patients. However, not every patient becomes cured, hence, there remains a medical need in the
3, 4
treatment of these patient population.
The challenge in treating HCV originates from the diversity present in the virus where it
5
is classified into seven major genotypes, with more than 50 subtypes known. Early treatment
options such as interferon and ribavirin were poorly tolerated and showed limited efficacy due to
the emergence of resistanceꢀassociated variants (RAVs). Over time, a consensus emerged that a
better tolerated, more efficacious treatment was needed and that the diversity of HCV infection
might best be addressed through administration of a combination of several directꢀacting
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antiviral agents (DAAs), each targeting a different step in the replication cycle of the virus. This
approach provides a chance to overcome poor treatment outcomes by allowing the combination
of multiple mechanisms to suppress the emergence of RAVs escaping from any single
mechanism. This approach has become the current treatment paradigm and has led to the
improvement in patient outcomes. From this strategy, three major categories of drugs have been
developed including NS3/4A protease inhibitors, NS5A replication complex inhibitors and
NS5B polymerase nucleotide and nonꢀnucleotide inhibitors. The ideal profile for each
component of the DAA regimen includes high potency, high barrier to resistance, panꢀgenotype
activity, few drugꢀdrug interactions (DDIs), minimal toxicity, and a pharmacokinetic profile that
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allows onceꢀdaily oral dosing.
An area of high interest for Merck has been the identification and development of potent
6
ꢀ11
NS5A inhibitors to be used as part of an all oral DAA regimen.
The exact mechanism of
NS5A inhibition remains unclear and is poorly understood; however, NS5A plays a critical role
1
2
in viral ribonucleic acid (RNA) synthesis and virion assembly. As a therapeutic target, NS5A
has been validated clinically, where multiple inhibitors were shown to be well tolerated and to
1
3ꢀ15
produce rapid and robust viral load declines in patients.
Today NS5A inhibitors have
become an integral part of DAA regimens with five NS5A inhibitors currently used in clinical
13
16
practice for HCV treatment including daclatasvir (BMSꢀ790052), ledipasvir (GSꢀ5885),
1
7
6
18
ombitasvir (ABTꢀ267), elbasvir (MKꢀ8742, 1) and velpatasvir (GSꢀ5816).
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In a continued effort to develop NS5A inhibitors, we asked the question: is it possible to
identify an inhibitor with potent activity against all genotypes and a minimal potency shift from
the wild type virus to previously identified RAVs, selected by existing NS5A inhibitors,
particularly in the most prevalent GT1a? We use the term “flat compound” to describe such a
panꢀgenotype inhibitor with a minimal potency loss (~10ꢀfold) between the wildꢀtype virus and
clinically relevant polymorphisms. In addition, the desired flat compound should also maintain
sufficiently good pharmacokinetic parameters to support onceꢀdaily dosing and suitable
pharmaceutical properties in order to enable a fixedꢀdose combination (FDC) with partner
DAAs. We now describe the structural modifications to the core structure of 1 that led to the
1
9
discovery of MKꢀ8408 (40), also known as ruzasvir, a potent NS5A inhibitor with a flat in vitro
profile. Compound 40 is currently undergoing clinical testing as part of an allꢀoral, interferonꢀ
free DAA regimen for the treatment of chronic HCV infection.
RESULT AND DISCUSSION
From our experience in the development of NS5A inhibitors, we observed that the GT 1a
RAV 1aY93H consistently conferred a high level of resistance, across a range of different
6ꢀ11
molecules. However, we also observed that molecules with improved inhibition of
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GT1a_Y93H often resulted in similar or better inhibition of other GT1 variants. We
hypothesized that gaining the ability to better inhibit this RAV might provide a path toward the
flat profile we desired and therefore GT1a_Y93H was selected as a part of the screening set for
SAR optimization. Concurrent with the selection of GT1a_Y93H, GT1a_L31V was also picked
for optimization as it offered diversity in a mutation at a different location in the protein.
GT2b(31M) was also added to the screening set as it represented a clinically important nonꢀ
genotype 1 example. Inclusion of GT3a and GT4a offered verification of performance against
additional genotypes. Acknowledging the risk associated with defining SAR with a focus on
GT1a_Y93H, GT1a_L31V and 2b, where the SAR may not translate to other genotypes and
RAVs, we proceeded with the strategy and decided to validate it by periodically checking the full
virologic profile of key compounds. The discussion herein is restricted to replicon EC values
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unless otherwise noted. The stereochemistry at the aminal position for compound 40 was
determined as (S), which was more potent than the corresponding (R) configuration. Compounds
reported in this paper were separated and assayed as single enantiomers with the potency for the
most active isomer reported, unless otherwise noted.
For the purposes of this discussion, we arbitrarily designated the aminalꢀphenyl group as
the ‘Zꢀgroup’, Table 1. From our earlier results we observed sensitivity of the mutant profiles
7, 8
toward changes at the Zꢀgroup, as illustrated by compound 2, Table 1. Numbers in
parenthesis were potency shift of mutants relative to GT1a, which was used as a measurement of
flatness of the profile. We also observed that improvement in the mutant profiles was achievable
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through addition of a fluorine atom at R shown by comparison of compound 3 versus
1
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compound 4. The combination of a chromane Z group and a fluorine atom at R could give a
1
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modest reduction in the shift in potency against key mutants to < 200 fold shown by compound
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5
, Table 1.
Table 1. Potency and shift comparison of 1 to 5
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O
O
O
O
N
R1
O
O
H
N
O
N
N
H
N
N
N
N
N
Z
Replicon EC nM (foldꢀshift†)
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No.
Z
R1
H
GT1a
GT1a Y93H
28
GT2b(31M)
1
2
0.006
11
H
0.004(1X)
8(1900X)
1(300X)
3
4
H
F
0.02(1X)
0.01(1X)
16(800X)
2(200X)
22(1100X)
8(800X)
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F
0.003(1X)
0.5(167X)
0.3(100X)
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†
Foldꢀshift is relative to the WT activity, set = 1.
While these modifications did not produce NS5A inhibitors with the “flat” profile that we
desired, it suggested a path forward on how we might obtain an NS5A inhibitor with a flat
profile. While substitutions on the phenyl group of the tetracyclic indole core had been
8
described previously, and improvements in the ”flatness” of the inhibitors were observed, we
did not feel that this approach by itself would lead to a flat compound. Thus, we looked for
wider variations in the Zꢀgroup, and upon review of the previous SAR, noticed that the isosteric
replacement of the phenyl ring with a thiophene ring had not been examined, so compound 6 was
prepared. We observed improved inhibitory activities in GT1a_Y93H over compound 1, as
shown in Table 2. Additionally, compound 6 demonstrated a balanced potency shift profile
towards both GT1a_Y93H (100X) and 2b (267X) (Table 2). Verification of this trend was
observed with compound 7, which displayed a similar profile to compound 6, giving us
confidence that substitution on the thiophene ring could be beneficial. The addition of simple
alkyl groups such as ethyl and cyclopropyl (compounds 8 and 10) onto the thiophene ring further
improved GT2b potency to subnano molar range. The addition of a R fluorine atom
1
demonstrated significant improvement on GT 1a_Y93H potency for the matched pairs
(
compounds 9 and 11). In both cases where the additional fluorine atom is located at R position,
1
the potency shift against key mutant GT1a_Y93H were below 50ꢀfold (Table 2). We were
encouraged by these results and additional efforts were undertaken to explore a broader
substitution patterns on the thiophene ring while retaining the R fluoro group.
1
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Table 2. Key mutant profile of compounds 6 to 11.
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O
O
O
O
N
H
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N
O
N
H
N
N
N
N
N
Z
O
Replicon EC nM (foldꢀshift†)
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0
No.
Z
R1
F
GT1a
GT1a Y93H
3 (100X)
GT2b (31M)
8 (267X)
a
6
0.03 (1X)
a
7
H
H
F
0.02 (1X)
0.005 (1X)
0.003 (1X)
0.003 (1X)
4 (200X)
3.4 (680X)
0.06 (20X)
1.2 (400X)
1 (50X)
8
9
0.25 (50X)
0.90 (300X)
0.35 (117X)
1
0
H
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F
0.003 (1X)
0.11(37X)
0.16 (200X)
a
†
Foldꢀshift is relative to the WT activity, set = 1; Denotes mixture of diastereomers at the aminal carbon.
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Table 3: In vitro virologic profile of substituted thiophene analogs
Replicon EC (nM)
9
0
GT1a
L31V
GT1a
†
No.
Z
X
H
H
GT1a
0.006
0.03
GT2b
GT3a
0.12
GT4a
0.016
n.d.
Y93H
1
1.0
28
11
a
6
0.76
3.4
8.2
0.016
9
H
H
0.003
0.003
0.067
0.02
0.06
0.90
0.16
0.002
0.007
0.003
0.009
1
1
1
2
0.11
H
H
H
0.002
0.002
0.13
0.06
0.59
0.26
1.74
3.7
0.013
0.007
0.008
0.01
0.003
n.d.
13
14
0.88
1.60
<0.002
0.028
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H
H
0.012
0.002
1.74
0.01
70
>33
4.7
0.32
1
1
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6
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8
0.13
0.52
0.001
0.003
0
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H
H
0.005
0.003
0.013
0.097
0.069
0.31
0.44
0.31
0.009
0.007
0.005
0.003
1
9
H
H
0.002
0.001
0.016
0.022
0.14
0.10
0.26
0.65
0.007
0.007
0.005
0.004
2
2
0
1
H
H
0.003
0.002
0.011
0.02
0.18
0.13
0.43
0.004
0.007
0.003
0.003
b
2
2
0.084
c
2
3
H
H
<0.002
0.014
0.087
0.035
2.38
0.47
3.40
0.45
0.013
0.12
0.004
24
0.024
2
5
H
0.005
0.049
0.22
0.54
0.016
0.009
2
2
6
7
H
H
0.002
0.005
0.009
0.029
1.70
0.17
0.88
0.68
0.004
0.012
0.002
0.014
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9
2
8
H
F
0.002
0.01
n.d.
0.74
1.1
0.68
3.4
0.008
0.19
0.003
n.d.
d
2
9
0.53
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
3
0
1
H
H
0.003
0.005
0.003
0.027
0.26
0.31
0.24
0.61
0.007
0.020
0.005
0.008
a
b
c
d
Mixture of diastereomers; Single diastereomer; EC value; R = H; † The replicon bears methionine at position
5
0
1
31
Table 3 summarizes the SAR results for substituted thiophene derivatives. The
attachment point of the thiophene ring to the aminal carbon was examined with the 2ꢀthiophene
6) and 3ꢀthiophene (12) analogs, with both possessing very similar profiles. The lack of a
(
preferred regiochemistry around the thiophene ring was again supported by the cyclopropylꢀ
substituted analogs 11 and 13, which had similar activities. Given the lack of differentiation for
2ꢀ versus 3ꢀattachment of the thiophene ring and a preference toward the 2,5ꢀsubstitution pattern,
2
0
for potential metabolic stability, the 2ꢀposition was selected as the attachment point to the
aminalꢀcarbon for the further investigation of substituting different positions of the thiophene
ring.
Moving the cyclopropyl group to the 3ꢀ and 4ꢀposition of the ring (14 and 15) did reveal
potency differences in comparison to compound 11 with compound 15 showing a very dramatic
loss in activity. Next, a series of substituents were installed in the 5ꢀpositon ranging from alkyl,
branched alkyl, fluorinated alkyl, as well as heteroatom containing groups substitutions (9, 16ꢀ
25) shown in Table 3. These substituents were all wellꢀtolerated demonstrating comparable
wildꢀtype and mutant profiles with the exception of 23 which was found to be weaker at both
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
GT1a_Y93H and GT2b(31M). Unfortunately, none of these analogs demonstrated an
improvement in the overall “flatness” of the profile compared to compound 11. A second
substituent was introduced on the thiophene ring to assess if it would impact inhibitory activity
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(26 ꢀ28). These derivatives were very similar in potency and showed no meaningful
improvement over the monoꢀsubstituted compound 11. Several 4,5ꢀfused bicyclic thiophenes
were prepared including benzothiophene 29 ( R = H and Rꢀfluoroꢀproline on the right hand side)
1
to explore the impact of fusing an aryl group onto the thiophene ring. While compound 29
demonstrated modest improvement on mutant profiles, it did show decreased potency on GT3a
when compared to the unsubstituted thiophene analog 6 as well as related 2,5ꢀdisubstituted
thiophene analogs. By comparison, the saturated fused bicyclic thiophene compounds 30 and 31
had profiles comparable to 11.
The SAR data summarized in Table 3 demonstrated that introduction of a substituted
thiophene group into the tetracyclic indole core has a surprisingly positive effect on flattening
variant profiles. Many of the compounds in this series exhibited potent in vitro profiles
including high potency toward key variants, which were difficult to achieve in the Z = phenyl
6
ꢀ11
series. While the data summarized in Table 3 represented significant improvement in key
mutant profiles, the goal of flatness was not reached and further optimization was explored.
Inspired by these results, we carried out further variation of 5ꢀmembered heterocycles at the Z
group, preparing both thiophene variants as well as those lacking the sulfur atom of thiophene.
Table 4 summarizes the virologic profiles of these additional fiveꢀmembered heterocycle
derivatives.
Table 4. In vitro profile of heterocycles Z group.
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9
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22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Replicon EC ( nM)
90
GT1a
GT1a
Y93H
†
No.
Z
GT1a
0.003
0.06
GT2b
0.16
14.1
1.1
GT3a GT4a
0.007 0.009
L31V
1
3
1
2
0.02
0.11
8.5
n.d.
0.25
0.18
n.d.
33
0.007
0.46
1.8
0.03
3
4
0.004
0.002
0.042
0.056
0.32
0.42
1.0
0.025 0.008
35
0.92
0.019
0.18
n.d.
n.d.
3
3
6
7
0.016
0.003
n.d.
7.4
>1.7
0.72
0.14
0.36
0.047 0.012
38
39
0.002
0.005
n.d.
2.6
1.0
4.6
0.073
n.d.
n.d.
n.d.
0.017
1.63
4
4
0
1
0.003
0.006
0.016
n.d.
0.067
1.0
0.036 0.007 0.007
2.47
0.079
n.d.
†
The replicon bears methionine at position 31.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Replacement of the thiophene ring of 11, with a 5ꢀsubstituted oxazole, yielded compound
2 which possessed a much weaker potency profile. Addition of a cyclopropyl group to the 2ꢀ
3
position of the oxazole (33) improved the potency profile compared to the 2ꢀH analog but was
not comparable to the thiophene derivative. While this trend was consistent with observations
made in the thiophene series, the key mutant GT1a_Y93H potency was weaker by 10ꢀfold with
the oxazole scaffold compared to 11. Benzoxazole analog 34 demonstrated further potency
improvement on the GT1a_Y93H to 0.32 nM but at the expense of the 2b profile which was ~
10X weaker than compound 11. Isooxazole analog 35 exhibited a promising potency profile as
all of the key mutant potencies were in subꢀnanomolar range while the most shifted genotype
was GT2b (460X). Thiadiazole 36 showed a poor GT1a_Y93H mutant profile which could be
improved by incorporation of the 1,2,3ꢀthiadiazole instead to give 37, which was better tolerated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Table 4). The thiazole analogs, 38ꢀ40, demonstrated that the position of the nitrogen within the
heterocycle, as well as the placement of the cyclopropyl group can modulate the mutant profiles
significantly. By placing the nitrogen atom further away from the aminal position, as in
compound 40, the virologic profile was significantly improved compared to the other
regioisomeric thiazole variants, 38 and 39. The verification of the importance of the cyclopropyl
substitution on the thiazole ring in 40 was demonstrated by the parental thiazole compound 41,
which showed significantly reduced inhibitory activies against key mutants GT1a_Y93H and
GT2b(31M).
Having shown the potential value of the Z = cyclopropyl thiazole ring, and the position of
the nitrogen in the thiazole ring, it remained to determine if further improvements in the
virologic profile of 40 could be obtained. Table 5 summarizes the virologic profile of thiazole
analogs 40, 42-50, where the cyclopropyl group has been modified. Adding a methyl group onto
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the cyclopropyl group gave 42 which showed weak GT2b(31M) potency compared to the
cyclopropyl derivative 40. Similarly, opening up the cyclopropyl ring to give the nꢀpropyl
compound 44, or changing the cyclopropyl to an ethyl (43) or to an isopropyl, compound 45,
resulted in slightly weaker activity vs. GT2b(31M). Changing the cyclopropyl to a trifluoro ethyl
again resulted in slightly weaker activity in GT2b(31M), but the difluoromethyl analog 47 was
comparable to 40. Introduction of polar functionality (48 and 49) was tolerated with both
compounds demonstrating good activity across genotypes. Interestingly, additional substitution
on the thiazole ring, as found in compound 50, proved to be detrimental to both GT1a_Y93H and
GT2b(31M) activity. With the identification of a number of compounds with excellent in vitro
profiles that were very similar to one another (Tables 4 and 5), a number of analogs were taken
into broader mutant profiling and PK experiments to aid in differentiation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 5. SAR of substituted thiazole series
Replicon EC (nM)
9
0
GT1a
L31V
GT1a
†
No.
Z
GT1a
0.003
GT2b
GT3a
GT4a
0.007
Y93H
4
0
0.016
0.067
0.036 0.007
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
4
2
3
0.017
0.004
0.065
0.048
0.38
0.14
0.68
0.046
0.027
0.011
0.097 0.009
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
44
0.001
0.002
0.02
0.088
0.05
0.14
0.002
0.01
4
5
0.022
0.14
0.22
0.006
0.015
0.004
4
4
6
7
0.005
0.002
0.050
0.003
0.18
0.013
0.002
0.056
0.069 0.004
0.068 0.007
4
8
0.002
0.015
0.035
0.004
49
50
0.002
0.004
0.017
0.36
0.04
12.5
0.12
>1.7
0.007
0.034
0.003
0.009
†
The replicon bears methionine at position 31
On the basis of structural diversity, in vitro potency, and apparent flatness of mutant
profiles vs. wildꢀtype, a small group of compounds was selected for pharmacokinetic evaluation
in both rat and dog, as shown in Table 6. In rat, the thiophene and thiazole derivatives exhibited
moderate oral exposure and bioavailability. However, the oral exposure of total compound in
dog was generally higher than the rat and similarly the bioavailability was also better than the
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9
rat. Among these compounds, 9 showed the highest oral exposure in dog (13.8 µM.hr). Further
analysis of the in vitro profiles of these compounds, with respect to flatness, is described in
Table 7.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Table 6. Inꢀvivo pharmacokinetic parameters of selected compounds in rat and dog
No.
9
11
40
44
1
S
N
Z group
Rat:
b
IV
CLp
8
11
7
13
4.7
16
(mL/min/Kg)
t (h)
6.4
4.3
1/2
PO
AUC µM.h
0.7(10)
1.1(10)
0.4(10)
0.8(10)
(dose, mpk)
C24h (nM)
17
3
16
5
3
4
4
7
%
(F)
Dog:
c
IV
CLp
1.7
15
1.5
10
1.6
4
1.8
(
mL/min/Kg)
t (h)
11.5
1/2
PO
AUC(0ꢀt)
µ
M.h (dose,
13.8(5)
10.6(5)
134
12.9(5)
11(5)
mpk)
C24h (nM)
%(F)
172
21
43
22
76.7
23
11
a
b
c
See experimental section for details; 2 mpk; 1 mpk.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The values shown in Table 7 are the fold shift, or the ratio of the mutant or genotype
EC divided by the 1a wild type potency which would provide a measure of “flatness” of the
90
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
profile. Our goal was to identify inhibitors with ~10ꢀfold of shift of wildꢀtype virus against
GT1a clinically relevant mutants and other genotypes as previously mentioned. At the first
glance, all of the compounds in Table 7 showed significant improvements in fold shift, in key
mutants and genotypes. Happily, all of the selected compounds listed were shifted <200X. In
particular, compounds 9 and 40 demonstrated the least shift in GT1a_Y93H, approximately 20X.
Moreover, compound 40 exhibited ~12X shift in genotype 2b.
Table 7. Flatness comparison of selected thiophene and thiazole derivatives
No.
9
11
40
44
1
S
N
Z group
1
aL31V/1a
22X
20X
300X
1X
3X
37X
53X
2X
5X
22X
12X
2X
20X
88X
1
aY93H/1a
b/1a
2
140X
12X
10X
3
4
a/1a
a/1a
1X
3X
2X
In an effort to determine if the virologic profiles of the NS5A inhibitors described thus
far could be further improved, we applied lessons that we learned in previous studies. We had
observed that introduction of a fused cyclopropyl proline moiety to the left hand side of the
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molecule, in certain cases, could improve both the 2b potency and the rat oral exposure. In
applying this learning to the current series described above, a couple of these analogs, bearing
either a thiazole or thiophene Z group, were studied and their virologic profiles are summarized
in Table 8. In most examples, fusing a cyclopropyl group to the leftꢀside proline gave a slight
potency loss towards GT2b(31M) (52ꢀ57). However, compound 51, bearing the cyclopropyl
thiazole Z group, showed a mutant and genotype profile similar to, or better than, that of
compound 40. Further evaluation of 51 in rat PK did not real an overall improvement over 40
(Table 9).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 8. Proline modification on thiazole/thiophene series
Replicon EC ( nM)
9
0
GT1a
L31V
GT1a
†
Z
GT1a
0.006
0.003
0.005
GT2b
GT3a GT4a
Y93H
5
5
1
2
0.007
0.008
0.022
0.021
0.076 0.011 0.006
0.028
0.040
0.23
0.21
0.006 0.006
0.012 0.008
53
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
5
5
4
5
6
0.005
0.005
0.013
0.007
0.021
0.02
0.14
0.18
0.009 0.012
0.009 0.005
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.004
0.006
0.012
0.015
0.030
0.20
0.30
0.67
0.007 0.005
0.012 0.005
57
† The replicon bears methionine at position 31
a
Table 9. Rat PK profile for compound 51
IV
P.O
CLp
Dose
t1/2
(h)
Dose
AUC(0ꢀt)
C24
No.
%(F)
(
mL/min/
Kg)
(mpk)
(mpk)
(µM.h)
(nM)
5
1
2
3.9
12
10
0.52
1.9
4
a
See experimental section for details.
Figure 1. Compound 40
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9
10
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12
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15
16
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18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Table 10. Rhesus monkey PK profile for compound 40
IV
P.O
Dose AUC(0ꢀt)
Dose
t1/2
(h)
CLp
(mL/min/Kg)
2.2
C24
No.
%(F)
10
(mpk)
(mpk)
5
(µM.h)
(nM)
35
1
10.2
3.9
28
a
See experimental section for details.
Table 11. In vitro potency profiles of compound 40 vs. 1 against GT1ꢀ6 and key GT1 variants
a
a
EC nM
EC nM
90
90
Replicon
Replicon
Compound
Compound
40
1
1
40
0
.006
0.003
0.009
GT2a
GT2b
GT3a
GT4a
GT5a
GT6
0.019
11
0.001
0.036
0.006
0.011
0.001
0.007
GT1b
Y93H
GT1a
Q30R
L31V
Y93C
0.89
0
.003
.011
0.12
0.006
2.5
1
0
0.016
0.002
0.017
0.016
0.002
1.1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
8
0.067
GT6d
0.008
0.027
Y93H
a
See experimental section for details.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
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0
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7
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9
0
Although many of the NS5A inhibitors described within show surprisingly and
significantly improved in vitro profiles, with minimal potency shifts, many of them did not differ
greatly from one another. We selected 40 for further study, based on its replicon potency
profiles, which were flat or nearly flat, and its demonstrated improvements on key mutants
GT1a_Y93H, L31V and GT2b(31M). A summary of the pharmacokinetic parameters of
compound 40 in rhesus monkey is presented in Table 10.
CONCLUSION
In summary, building from the knowledge base of our existing NS5A program and
applying rationale SAR development to the tetracyclic indole based template, we discovered
compounds with significantly improved in vitro virologic profiles. Multiple compounds with
either “flat” or “nearly flat” profiles were identified. In particular, compound 40 was selected for
preclinical and later clinical evaluation; compound 40 is now known as ruzasvir. The in vitro
profile of this compound (Table 11) represents an example of an HCV NS5A inhibitor with a
“flat” GT1, panꢀgenotypic profile. Ruzasvir (40) is currently in clinical trials as part of an allꢀ
oral DAA regimen for the treatment of chronic HCV infection. The full characterization of
ruzasvir (40) will be communicated in future publications.
CHEMISTRY
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2
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7
8
9
Figure 2. Common intermediates
10
11
12
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23
24
25
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38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthesis of common intermediates 69 and 70
o
o
o
Reagents and conditions: (a) AcOH/EtOH/40 C to reflux, 6 h; (b) PPA/xylene, 100 C, 2 h; (c) Zn/TFA, 70ꢀ76 C,
1
7 h.
Scheme 2. General synthetic approach to thiophene/thiazole analogs
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9
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0
o
Reagents and conditions: (a) TFA/CH CN, 25 C, 6 h; (b) DDQ, toluene, reflux, 2 h; (c) bis(pinacolato)diboron,
3
o
o
KOAc, Pd(dppf)Cl , dioxane, 100 C, overnight; (d) 58, Na CO , Pd(dppf)Cl , THF/H O, 80 C, overnight; (e) 59,
K CO , Pd(dppf)Cl , THF/H O, 80 C, overnight; (f) HCl in dioxane/CH Cl , 20 C, 1 h; (g) HATU, MocꢀLꢀvaline,
DIPEA, DMF, 0 C, 40 min; (h) pyridine/PCl /CH Cl , ꢀ10 C, 0.5 h; (i) Cs CO /DMSO, 100 C, 1 h; (j) Chiral SFC
2
2
3
2
2
o
o
2
3
2
2
2
2
o
o
o
5
2
2
2
3
separation.
Scheme 3. Synthesis of compounds 6 and 11.
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40
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59
60
o
Reagents and conditions: (a) cyclopropyl boronic acid, Pd (dba) , XꢀPhos, K CO /dioxane, 110 C, 16 h, 46% for 82
and 18% for 6. (b) chiral SFC separation.
2
3
2
3
1
9, 21,22
Final compounds were prepared according to published procedures.
The absolute
configuration of the aminal carbon for compound 40 is unambiguously assigned to be “S” by
2
3
single crystal Xꢀray analysis. The remaining stereo centers present in the final molecules were
derived from the commercially available starting materials.
The intermediate cores 69 and 70 were synthesized according to the chemistry shown in
2
1
Scheme 1. Treatment of ketone 62 with hydrazine 63 or 64 afforded the intermediate
hydrazones (65 and 66) which could be cyclized to afford indole derivatives 67 and 68.
Reduction with zinc in TFA afforded the indoline derivatives 69 and 70. The indole based
tetracyclic core 74 (Scheme 2) was synthesized either through cyclization of indoline derivative
7
1 with various thiophene aldehydes 72 followed by oxidation of the indoline ring, or through
cyclization of dichloromethyl thiophene 76 with intermediate such as 67. The diꢀbromo
compound 74 was converted to the corresponding bisꢀboronic ester 77. 77 could undergo a
double Suzuki coupling reaction with either the imidazole proline intermediate 58 to afford the
racemic form of the final compound 78, or with the Bocꢀprotected imidazole proline intermediate
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9 followed by global deprotection of the Boc groups and HATU promoted double amide
coupling with MocꢀLꢀvaline to get to 78. 78 was subjected to chiral separation to afford the final
products (8ꢀ35 in Tables 2 to 4 were synthesized with the same approach). In the case of
compound 6, compound 81 was synthesized followed the route illustrated in Scheme 3.
Advance intermediate 81 was subjected to a Suzuki coupling with cyclopropyl boronic acid
shown in Scheme 3. Analog 82 (racemate) was obtained as the major product and 81 was deꢀ
halogenated to afford 6 as a side product. Chiral SFC separation afforded compound 11.
Thiazole analogs 38ꢀ50, and hetercyclic analogs 32ꢀ37 were prepared following the same
approach shown in Scheme 2. A representative example of synthetic approach to 40 was shown
in Scheme 4.
0
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0
Scheme 4. Synthesis of compound 40
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F
F
Br
Br
N
S
N
N
S
Br
a
b
c
Br
N
Br
O
NH
O
OH
B
S
HO
8
3
84
H
85
OH
S
N
6
9
10
11
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40
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86
F
F
O
B
Br
O
O
Br
O
B
N
N
d
e
O
O
f
S
S
N
N
8
7
88
N
F
N
(S) N
(S)
S)
N
N
H
O
O
NH
g
(Z)
(S)
N
O
O
40
NH
O
O
(
HN
O
S
N
89
o
Reagents and conditions: (a) Pd(OAc) , Cs CO ,nꢀBuPdCl , toluene/H O, 100 C, overnight,
2
2
3
2
2
o
o
3
2%; (b) nꢀBuLi, DMF, ꢀ78 C, 3 h, 49% (c) TFA/CH CN, 25 C, 6 h, 68%; (d) DDQ, toluene,
3
o
reflux, 2 h, 82%; (e) bis(pinacolato)diboron, KOAc, Pd(dppf)Cl , dioxane, 110 C, overnight,
2
o
8
2%; (f) 58, Na CO , Pd(dppf)Cl , THF/H O, 95 C, overnight. (g) SFC chiral separation, 10%
2 3 2 2
over two steps.
2
ꢀBromothiazole (83) was subjected to Suzuki coupling with cyclopropyl boronic acid to
afford 84, which was converted to aldehyde 85. Acidꢀcatalyzed cyclization of 85 with 69
afforded 86 which was treated with DDQ to afford indole analog 87. The dibromo compound 87
was converted to the bisꢀboronic ester 88 which was subjected to double Suzuki coupling with
Mocꢀvaline fragment 58 to afford 89. The racemate mixture of aminal diastereomers was
subjected to SFC chiral separation to provide compound 40.
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