Discovery of MK-6169, a Potent Pan-Genotype Hepatitis C Virus NS5A Inhibitor with Optimized Activity against Common Resistance-Associated Substitutions

Article abstract of DOI:10.1021/acs.jmedchem.7b01927

We describe the discovery of MK-6169, a potent and pan-genotype hepatitis C virus NS5A inhibitor with optimized activity against common resistance-associated substitutions. SAR studies around the combination of changes to both the valine and aminal carbon region of elbasvir led to the discovery of a series of compounds with substantially improved potency against common resistance-associated substitutions in the major genotypes, as well as good pharmacokinetics in both rat and dog. Through further optimization of key leads from this effort, MK-6169 (21) was discovered as a preclinical candidate for further development.

Full text of DOI:10.1021/acs.jmedchem.7b01927

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The Discovery of MK-6169, A Potent Pan-genotype
Hepatitis C Virus NS5A Inhibitor with Optimized Activity
Against Common Resistance-Associated Substitutions
Wensheng Yu, Ling Tong, Oleg Selyutin, Lei Chen, Bin Hu, Bin Zhong, Jinglai Hao, Tao Ji,
Shuai Zan, Jingjun Yin, Rebecca Tamra Ruck, Stephanie Curry, Patricia McMonagle, Sony
Agrawal, Laura Rokosz, Donna Carr, Paul Ingravallo, Karin Bystol, Frederick Lahser, Rong Liu,
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.7b01927 • Publication Date (Web): 21 Apr 2018
Downloaded from http://pubs.acs.org on April 21, 2018
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The Discovery of MK-6169, A Potent Pan-genotype Hepatitis C Virus NS5A Inhibitor with
Optimized Activity Against Common Resistance-Associated Substitutions
a,*
a
a
a
b
b
b
b
Wensheng Yu , Ling Tong , Oleg Selyutin , Lei Chen , Bin Hu , Bin Zhong , Jinglai Hao , Tao Ji ,
b
c
c
d
d
e
Shuai Zan , Jingjun Yin , Rebecca T. Ruck , Stephanie Curry , Patricia McMonagle , Sony Agrawal ,
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Laura Rokosz , Donna Carr , Paul Ingravallo , Karin Bystol , Frederick Lahser , Rong Liu , Shiying
f
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h
d
a
Chen , KungꢀI Feng , Mark Cartwright , Ernest AsanteꢀAppiah , and Joseph A. Kozlowski
a
d
e
Department of Medicinal Chemistry, Department of Infectious Diseases, Department of In Vitro
f
h
Pharmacology, Department of PPDM, Department of Drug Safety, Merck & Co., Inc., 2000
Galloping Hill Road, Kenilworth, NJ 07033, USA
c
g
Department of Process Research & Development, Department of Discovery Pharmaceutical Sciences,
Merck & Co., Inc., 126 E. Lincoln Avenue, Rahway, NJ 07065, USA
b
WuXi AppTec, 288 Fute Zhong Road, Shanghai, 200131, China
ABSTRACT: We describe the discovery of MKꢀ6169, a potent and panꢀgenotype hepatitis C virus
NS5A inhibitor with optimized activity against common resistanceꢀassociated substitutions. SAR
studies around the combination of changes to both the valine and aminal carbon region of elbasvir lead
to the discovery of a series of compounds with substantially improved potency against common
resistanceꢀassociated substitutions in the major genotypes, as well as good pharmacokinetics in both rat
and dog. Through further optimization of key leads from this effort, MKꢀ6169 (21) was discovered as
a preclinical candidate for further development.
INTRODUCTION
In the recently published Global Hepatitis Report 2017 from the World Health Organization, it
was estimated that 71 million people worldwide were chronically infected with hepatitis C virus (HCV)
1
as of 2015, representing approximately 1% of the global population. Studies have suggested that the
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incidence of HCV infection has decreased since the second half of the 20th century; however, there
were still 1.75 million new infections in 2015, mainly due to unsafe healthꢀcare practices (including
2
unsafe healthcare injections) and injection drug use. Without treatment, chronic HCV infection could
lead to severe liver damage and lifeꢀthreatening disease. For example, the risk of cirrhosis of the liver
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among patients with chronic HCV infection is between 15–30% within 20 years. In 2015, there were
1
.34 million deaths worldwide attributable to viral hepatitis, with 30% a result of complications of
4
chronic HCV infection, mostly from cirrhosis and hepatocellular carcinoma. The treatment for HCV
infection has evolved significantly during the last decade. Early treatment involved interferon
injections and use of ribavirin for a long treatment duration. The regimen suffered from low sustained
virologic response (SVR) rates (40~65%) and many debilitating side effects. Addition of firstꢀ
generation HCV nonꢀstructural (NS) 3/4A protease inhibitors (boceprevir or telaprevir) to the
interferon/ribavirin standardꢀofꢀcare improved the SVR rate to 65~85% but the side effects of
interferon remained. Current treatment for HCV infection involves interferonꢀfree all oral directꢀacting
5
ꢀ7
antiviral agent (DAA) combinations chosen from three different categories: NS3/4A protease
inhibitors, nonꢀstructural protein 5A (NS5A) replication complex inhibitors, and nucleoside or nonꢀ
nucleoside NS5B polymerase inhibitors.
HCV NS5A protein is a multifunctional RNA binding protein that interacts with several host
7
,8
proteins and is essential for HCV replication. The protein has no enzymatic activity or a known
human orthologue from which a function can be inferred. A detailed understanding of how NS5A
directly influences HCV replication is lacking and therefore the mechanism of inhibitors remain
9
,10
unclear. While crystal structures of the Nꢀterminal region of the protein have been reported,
the
noted differences in the dimer orientation and the absence of an inhibitorꢀprotein complex render a
structureꢀbased rational drug design approach pretty difficult. In the absence of enzymatic activity for
NS5A, structureꢀactivity relationships (SARs) for inhibitors have been driven by cellꢀbased replicon
activities. First generation NS5A inhibitors showed a low resistance barrier to genotype 1 subtype a
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(GT1a) as well as weak activity against several other genotypes. Thus, the need for identification of
more potent NS5A inhibitors against a wider variety of genotypes as well as NS5A resistance
mutations particularly in the prevalent GT1a became a focus for many groups. Six HCV NS5A
inhibitors have been approved for chronic HCV treatment, in combination with other DAA’s:
1
1,12
13
14
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daclatasvir (BMSꢀ790052),
ledipasvir (GSꢀ5885), ombitasvir (ABTꢀ267), elbasvir (MKꢀ
15,16
17
18
8
742).
velpatasvir (GSꢀ5816), and pibrentasvir (ABTꢀ530).
1
9ꢀ21
Our efforts in the research on HCV NS5A inhibitors have progressed over time.
Multiple
have been identified. MKꢀ
742 (elbasvir, Figure 1) is one of the two components in Zepatier which has gained FDA approval for
1
5
22
15,16
NS5A inhibitors, including MKꢀ4882, MKꢀ8325, and MKꢀ8742,
8
the treatment of GT1 and GT4 chronic HCV infection. Through our continued effort in optimization of
NS5A inhibitors, we aim to identify an inhibitor with a ‘flat’ potency profile, which exhibits less than a
1
0ꢀfold potency reduction from the wild type genotype 1a (GT1a) virus relative to other genotypes and
resistanceꢀassociated substitutions (RASs). Since no Xꢀray structures of inhibitor and protein
complexes are available to facilitate a structureꢀbased inhibitor design approach, we plan to modify
every part of the MKꢀ8742 molecule to identify SAR that could improve the potency against the least
sensitive RASs, such as GT1a Y93H and GT2b L31M (a naturally resistant substitution). Our
experience has shown that activity vs. these variants could often lead to improved flatness across a
broad range over genotypes and mutations. Previously, we reported SAR studies towards an improved
2
3,24
25
26
27ꢀ30
“
core”,
“cap”, proline, and “Z” group region (Figure 2).
Key findings from SAR studies
included potency improvements against GT2b L31M through introducing a tetrahydroꢀ2Hꢀpyran (THP)
2
5
group in the “cap” region, potency improvements against both GT1a Y93H and GT2b L31M through
2
4
introducing a fluorine substitution at the Cꢀ1 position of the tetracyclic indole core, or a relatively
28
28
29
large aryl Z group, such as the 4ꢀcyclopropylphenyl, 4ꢀdiphenyl, and 7’ꢀchromane. More recently,
substituted thiazole and thiophene as “Z” groups were also identified to improve the potency against
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NS5A RASs. Through these efforts we identified MKꢀ8408 (ruzasvir).
Herein, we disclose the
discovery of an additional panꢀgenotype HCV NS5A inhibitor, preclinical candidate MKꢀ6169.
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Figure 1. HCV NS5A inhibitors approved by FDA
Figure 2. Strategy for the modification of MKꢀ8742 molecule.
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RESULTS AND DISCUSSION
As reported previously, we found that replacing the isopropyl group of the valine “cap” of
elbasvir with a (S)ꢀTHF group was able to improve the activity on GT2b L31M while maintaining
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potency against the other NS5A RASs. We therefore wanted to incorporate this SAR with the “Z”
groups identified more recently in an attempt to improve the activity on GT1a Y93H and GT2b L31M.
Thus, the (S)ꢀTHP group was initially incorporated on the leftꢀhandꢀside of the molecule with a range
of “Z” groups; the in vitro potencies (replicon EC ) are summarized in Table 2. Incorporation of the
90
(S)ꢀTHP “cap” with chromane as “Z” group (1ꢀ2) showed a 10x improvement against GT1a Y93H and
2
9
GT2b L31M vs. the corresponding bisꢀvaline analogs. Based on the EC90 values in Table 2, we
observed a reduced shift in potency of the wild type GT1a vs. GT1a Y93H and GT2b L31M which for
1
is 9X and 4X, respectively. A similar result was observed for 3 whose “Z” group is 4ꢀmethylꢀ3,4ꢀ
dihydroꢀ2Hꢀbenzo[b][1,4]oxazine. Encouraged by these data, a series of compounds with substituted
thiophene moieties as the “Z” group (4ꢀ12) was prepared. The substituents on the thiophene included
methyl (4), ethyl (5), nꢀpropyl (6), isoꢀpropyl (7), cyclopropyl (8), diꢀFꢀmethyl (9), and triꢀFꢀmethyl
(10). Analogs with benzothiophene (11) and 5,6ꢀdihydroꢀ4Hꢀcyclopenta[b]thiophene (12) were also
prepared. All these compounds showed a potency shift less than 10X from GT1a wild type to the other
genotypes and mutants tested, except 11 whose “Z” group is a benzothiophenyl group. Compound 11
showed a slightly larger potency shift (20X) against GT1a Y93H. Compounds with the (S)ꢀTHP on the
right hand side of the molecules were also prepared (13ꢀ14) and their in vitro potencies are summarized
in Table 3. Both compounds, with the “Z” group as either chromane or 4ꢀmethylꢀ3,4ꢀdihydroꢀ2Hꢀ
benzo[b][1,4]oxazine, showed single or double digit pM EC values for all the genotypes and mutants
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tested. It was interesting to observe that compound 14 showed 15ꢀfold potency loss against GT1a
relative to compound 3 in which the THP group is on the leftꢀhand side.
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Table 1. In vitro potency profiles of Elbasvir.
a
Replicon EC (nM)
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0
b
ID
GT1a
GT1b
0.006
GT2a
0.019
GT2b L31M
11
GT3a
0.12
GT4a
0.016
GT1a Y93H
28
GT1a L31V
1
Elbasvir
0.006
0
1
2
3
4
5
6
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8
9
0
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9
0
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a
EC is the concentration of inhibitor that reduces the effective response by 90%.
90
b
see abbreviation for the full name of genotype and mutant names.
Table 2. NS5A inhibitors with a (S)ꢀTHP group on the leftꢀhand side
Replicon EC (nM)
90
ID
Z
GT1a GT1b
0.004 0.006
GT2b L31M
0.019
GT3a
0.008
GT4a
0.007
GT1a Y93H
0.035
GT1a L31V
0.005
1
2
0.005 0.003
0.005 0.004
1.069
0.069
0.009
0.038
0.006
0.009
0.356
0.047
0.007
0.006
3
4
5
6
7
8
0.003 0.006
0.004 0.003
0.002 0.006
0.004 0.002
0.004 0.005
0.024
0.020
0.012
0.036
0.014
0.006
0.005
0.005
0.003
0.008
0.004
0.003
0.002
0.004
0.005
0.019
0.008
0.009
0.010
0.018
0.010
0.003
0.003
0.003
0.006
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0.004 0.006
0.003 0.005
0.016
0.010
0.010
0.006
0.004
0.003
0.015
0.023
0.005
0.005
1
0
1
1
0.005 0.003
0.005 0.005
0.057
0.064
0.011
0.022
0.004
0.004
0.111
0.039
0.008
0.013
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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4
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6
0
1
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9
0
1
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9
0
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12
Table 3. NS5A inhibitors with a (S)ꢀTHP group on the rightꢀhand side
Replicon EC (nM)
90
ID
Z
GT1a GT1b
0.002 0.003
GT2b L31M
0.005
GT3a
0.003
GT4a
GT1a Y93H
0.008
GT1a L31V
0.001
a
1
3
0.002
0.009
b
1
4
0.071 0.007
0.054
0.017
0.073
nt
a
EC value, which is the concentration of inhibitor that reduces the effective response by 50%.
50
b
nt = not tested.
The rat and dog PK profiles of selected compounds in Table 3 are summarized in Table 4.
Unfortunately, all compounds tested exhibited low exposure in rat with their AUC normally less than 1
M.h at 10 mpk oral dosing. The oral bioavailability of these compounds is quite low as well. This is
ꢀ
expected due to their high plasma protein binding and low permeability. Compound 3 displayed a
relatively higher exposure with oral AUC = 1.23 ꢀM.h at 10 mpk, but its bioavailability was only
1
.5%. Compound 3 had a low PK exposure in dog as well. Surprisingly, both compounds 4 and 8
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showed good dog PK exposure even though their rat PK exposures were low. The single rising dose rat
PK study of 8 is summarized in Table 5, which revealed a linear increase in exposure from 10 to 100
mpk whereas a less than doseꢀproportional increase in exposure was observed from 100 to 300 mpk.
a
0
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9
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Table 4. Pharmacokinetic profiles of 1, 3-5, 7, 8, 13, and 14 in rat or dog
iv
po
Cmax
ID
Species
T1/2
h)
CL
Dose
AUC
(ꢀM.h)
0.13
1.23
0.31
0.53
0.46
0.29
0.05
0.10
0.14
1.32
2.61
C24
F%
(
(mL/min/Kg) (mpk)
(nM)
0.032
0.162
0.031
0.048
0.050
0.034
0.010
0.016
0.022
0.23
(nM)
0.001
0.005
0.002
0.005
0.002
0.002
0.001
0.001
0.002
0.004
0.022
1
3
4
5
7
8
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
dog
dog
dog
3.2
5.5
4.2
5.3
6.1
4.0
1.9
3.8
6.1
4.5
4.2
11
2
10
10
10
10
10
10
10
10
5
0.8
1.5
4.1
3.9
3.4
3.1
0.5
0.8
1.1
6.2
11.0
24
12
13
18
19
13
8.6
4.1
3.6
1
1
3
4
3
4
8
5
5
0.46
a
Fasted male Wistar Han rats or Beagle dogs were used. IV dose was formulated with 60% PEG200
and the compounds were dosed at 2 mg/kg in rat or 1 mg/kg in dog. P.O. dose was formulated with
10% TWEEN and the compounds were dosed as indicated in the table.
a
Table 5. Rat single rising dose PK profile of 8
Dose
mpk)
PO AUC
(ꢀM.h)
0.14
C
max
C
24h
Tmax
(h)
3
MRT
(h)
(
(ꢀM)
(ꢀM)
1
0
0.02
0.001
7.9
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3
00
00
1.64
3.99
0.20
0.36
0.011
0.032
4
8.3
9.9
1
2
3
4
4
a
Fed male Wistar Han rats were used. Compound was dosed at indicated doses P.O. in 10% TWEEN.
5
6
7
8
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
As reported previously, the bisꢀvaline analog with chromane as the “Z group” showed
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
2
9
reasonable exposure in rat with AUC (po) at 10 mpk being 1.65 ꢀM.h. However, replacing the
isopropyl group of the valine moiety with a tetrahydroꢀ2Hꢀpyran (THP) group, either in the leftꢀhand
side (1) or rightꢀhand side (13), resulted in a significant drop of PK exposure in rat. Therefore the THP
group was believed to be responsible for the poor rat PK of compounds in Table 4. A metabolite study
on a similar compound not shown in this publication revealed that a possible hydroxylation occurred
on the carbon next to the oxygen that led to the ring opening of the THP group. To prevent this
potential metabolic pathway, analogs with a variety of αꢀsubstituted THP groups were synthesized and
their replicon EC data are listed in Table 6. It should be noted that compounds in Table 6 have
9
0
multiple diastereomers. All diastereomers were separated by chiral SFC, but only one or two
diastereomers with the best potency are reported here. Their absolute stereochemistry was either
introduced from the starting material with known stereochemistry, determined by VCD with
confidence, or assigned arbitrarily. Please see the experimental section for details. Both (R)ꢀ or (S)ꢀ2ꢀ
methylꢀTHP analogs (15ꢀ16) exhibited good activity on the genotypes tested, and similar potency
profiles were observed for both cisꢀ and transꢀ2,6ꢀdimethyl THP analogs (17ꢀ18). However, cisꢀ2,6ꢀ
diethyl THP analog 19 exhibited reduced potency against all the genotypes tested, especially on GT2b
L31M with a ~100ꢀfold loss in potency as compared with the corresponding cisꢀ2,6ꢀdimethyl THP
analog 17, indicating that a larger substituent was not tolerated. 2,2ꢀDiꢀmethyl substituted THP analogs
(20ꢀ21) as well as the 2ꢀspiroꢀcyclopropyl THP analog (22) all displayed good potency against all the
genotypes tested. Additionally, the 8ꢀoxabicyclo[3.2.1]octane substituted analog 23 and the 2,2,6,6ꢀ
tetramethylꢀTHP analog 24 also displayed good potency on the genotypes tested.
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2
3
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5
6
7
8
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
Table 6. NS5A inhibitors with a substitutedꢀTHP group
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
Replicon EC (nM)
90
ID
A
GT1a
0.004
GT1b
0.004
GT2b L31M
0.014
GT3a
GT4a
GT1a Y93H
0.009
GT1a L31V
0.003
1
1
5
6
0.007
0.010
0.005
0.004
0.004
0.017
0.007
0.004
0.004
0.023
0.025
0.012
2.701
0.008
0.008
0.007
0.062
0.007
0.007
0.007
nt
0.013
0.027
0.015
0.642
0.002
0.005
0.004
nt
17
1
1
8
9
a
nt
20
0.004
0.004
0.007
0.004
0.027
0.018
0.009
0.001
0.007
0.006
0.018
0.033
0.002
0.004
2
1
22
23
0.006
0.005
0.004
0.004
0.036
0.019
0.008
0.012
0.004
0.008
0.049
0.028
0.007
0.006
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2
3
4
24
0.004
0.002
0.073
0.007
0.004
0.047
0.004
a
5
nt = not tested.
6
7
8
9
The PK parameters of all compounds except 19 in Table 6 are summarized in Table 7. Both
monoꢀmethyl substituted analogs 15 and 16 showed low PK exposure in rat at 10 mpk oral dosing. The
2,6ꢀdimethyl substituted analogs 17 and 18 had higher exposures in rat than 15 and 16. Interestingly,
while one diastereomer (20) of the 2,2ꢀdiꢀmethyl substituted THP analogs displayed low rat PK
exposure, one of the other diastereomers (21) exhibited a much improved PK exposure in rat with AUC
(po) at 10 mpk being 1.65 ꢀM.h. It also showed improved C24h relative to the other compounds. The
rat PK exposure of the 2ꢀspiroꢀcyclopropyl THP analog (22) was low, and so were the rat PK
exposures of 23 and 24. The dog PK profiles of 17, 18, and 21 were also examined. All of them
displayed good dog PK exposures at 5 mpk oral dosing. They also had low clearance and relatively
higher bioavailability than in rat. Rat single rising dose PK study of compound 17, 18, and 21 are
summarized in Table 8. All three compounds showed less than doseꢀproportional increase in exposure
from 10 to 300 mpk.
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
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
a
Table 7. Pharmacokinetic profiles of 15-18 and 20-24 in rat or dog
iv
po
ID
Species
Dose
T1/2
(h)
4.7
5.5
5.1
3.6
5.2
CL
Dose
AUC
(ꢀM.h)
0.21
C
max
C
24
F%
(
mpk)
(mL/min/Kg) (mpk)
(ꢀM)
0.025
0.021
0.067
0.119
0.025
(ꢀM)
0.001
0.001
0.003
0.004
0.001
1
1
1
5
6
7
Rat
Rat
Rat
Rat
Rat
2
2
2
2
2
11
11
11
11
11
10
10
10
10
10
1.4
1.3
5.9
6.3
1.6
0.19
0.92
18
20
0.93
0.24
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2
2
2
1
1
2
1
2
3
4
7
8
1
Rat
Rat
Rat
Rat
dog
dog
dog
2
2
2
2
1
1
1
5.4
5.7
3.9
7.7
7.2
11.3
5.8
7
5
10
10
10
10
5
1.65
0.58
0.33
0.70
2.42
5.05
4.19
0.183
0.057
0.024
0.076
0.33
0.011
0.004
0.003
0.005
0.011
0.087
0.029
6.6
1.9
1
2
3
4
5
6
7
8
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
13
7
2.5
3.1
3.0
2.6
2.9
8.5
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
5
0.64
18.7
14.9
5
0.66
a
Fasted male Wistar Han rats or Beagle dogs were used. IV dose was formulated with 60% PEG200.
P.O. dose was formulated with 10% TWEEN.
a
Table 8. Rat single rising dose PK profiles of 17, 18, 21
Dose
(mpk)
10
AUC
C
max
C
24h
Tmax MRT
ID
(ꢀM.h) (ꢀM)
(ꢀM)
(h)
4
(h)
9.0
17
0.33
1.95
4.08
0.31
2.07
5.58
0.95
7.82
14.7
0.08 0.005
0.22 0.027
0.39 0.040
0.05 0.001
0.24 0.009
0.51 0.029
0.15 0.003
0.76 0.072
1.06 0.164
1
1
2
7
8
1
100
2
3
00
6
11
1
0
3
6.0
7.6
9.3
6.5
12.4
10.7
100
00
3
3
7
1
0
3.0
2.0
5.0
100
00
3
a
Fed male Wistar Han rats were used. Compounds were formulated with 10% TWEEN and dosed
orally.
The 2,2ꢀdimethylꢀTHP isomer employed in 21 was used as a test to determine its impact in
combination with other “Z groups” that had previously been identified to show good potency.
Compounds 25ꢀ35 have the dimethyl ꢀTHP cap on the leftꢀhand side and their replicon EC data are
9
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2
3
4
5
6
7
8
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
listed in Table 9. Not surprisingly, most of them showed “flat” potency profile against all genotypes
and mutants tested, with the exception of 26, 30, 33, and 35 which exhibited slightly reduced potency
against GT1a Y93H with the corresponding EC values in the triple digit pM range. Two compounds
9
0
(
36, 37) with the same 2,2ꢀdimethylꢀTHP isomer on the rightꢀhand side were also prepared. Their EC90
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
data are listed in Table 10. Both compounds were pretty potent against the genotypes and mutants been
tested, except 36 exhibited slightly reduced potency in GT1a, GT2b L31M, and GT1a Y93H.
Table 9. NS5A inhibitors with a 2,2ꢀdimethylꢀTHP group on the leftꢀhand side
Replicon EC (nM)
90
ID
Z
GT1a GT1b
0.003 0.005
GT2b L31M
0.017
GT3a
0.007
GT4a
0.005
GT1a Y93H
0.048
GT1a L31V
0.004
2
5
a
2
6
0.005 0.007
0.102
0.008
0.008
0.348
0.005
2
2
7
8
0.005 0.006
0.010 0.009
0.028
0.084
0.009
0.016
0.007
0.011
0.038
0.078
0.003
0.006
2
9
0.004 0.005
0.005 0.006
0.006 0.005
0.038
0.043
0.041
0.006
0.009
0.006
0.006
0.006
0.004
0.036
0.117
0.017
0.004
0.006
0.005
30
3
1
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3
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5
6
7
8
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
3
3
2
3
0.004 0.005
0.006 0.006
0.006 0.006
0.005 0.004
0.015
0.019
0.015
0.174
0.008
0.038
0.015
0.013
0.006
0.007
0.007
0.007
0.018
0.329
0.029
0.590
0.006
0.004
0.006
34
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
b
3
5
nt
a
Single chromane isomer, absolute stereochemistry assigned arbitrarily.
nt = not tested.
b
Table 10. NS5A inhibitors with a 2,2ꢀdimethylꢀTHP group on the rightꢀhand side
Replicon EC (nM)
90
ID
Z
GT1a GT1b GT2b L31M GT3a
GT4a
0.106
GT1a Y93H
0.155
GT1a L31V
0.046
3
3
6
7
0.033 0.067
0.006 0.005
0.349
0.031
0.082
0.013
0.004
0.036
0.001
The rat PK profile of selected compounds in both Table 9 and 10 are summarized in Table 11.
While the majority of these compounds showed a moderate to low PK exposure in rat, 30 had
comparable PK exposure in rat as 21, as well as reasonable bioavailability.
Table 11. Pharmacokinetic profiles of compounds 25, 27, 29-32, 34, 36, and 37 in rat
ID
iv
po
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T1/2
h)
CL
AUC
(ꢀM.h)
0.08
0.78
0.80
1.90
0.05
0.08
0.51
0.23
0.45
C
max
C
24
1
2
3
4
5
6
7
8
9
F%
(
(mL/min/Kg)
(ꢀM)
0.01
0.11
0.10
0.29
0.01
0.02
0.06
0.02
0.04
(ꢀM)
0.001
0.004
0.004
0.006
0.001
0.001
0.004
0.002
0.004
2
2
2
3
3
3
3
3
5
7
9
0
1
2
4
6
3.8
5.2
9.6
4.3
5.3
4.5
4.0
5.9
6.0
25
8
1.2
3.5
2.7
10.2
0.8
1.0
2.8
1.2
2.0
5
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
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
9
29
21
9
9
37
9
a
Fasted male Wistar Han rats were used. IV dose was formulated with 60% PEG200 and the
compounds were dosed at 2 mg/kg. P.O. dose was formulated with 10% TWEEN and the compounds
were dosed at 10 mg/kg.
With all the potency and PK data in hand, 21 emerged as a lead with both “flat” potency and
acceptable PK profile in both rat and dog. Comparing with other compounds, 21 shows the best C24h in
both rat and dog. To reduce the HCV resistance, it is important to achieve adequate C24h in human to
cover the EC values for as many HCV NS5A genotypes and RASs as possible. Compound 21 has the
9
0
best potential to achieve that in relatively low dose with QD dosing. Its potency profile against
additional HCV NS5A genotypes and RASs are summarized in Table 12. Compound 21 showed <10X
shift on a majority of the genotypes and mutants tested, other than several mutants, such as GT3a
Y93H and GT3a L31V, with the corresponding EC values still in the single digit nM range. As NS5A
90
inhibitors are often developed in combination with other DAAs such as NS3 inhibitors, we tested the
potency of 21 on NS3 RASs. As anticipated, compound 21 retained its potency and showed good
potency on prototypical GT1a NS3 RASs such as GT1a R155K and GT1a V36M+R155K double
substitution, as well as prototypical GT1b NS3 RASs such as GT1b A156T and GT1b D168Y. These
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2
3
4
5
6
7
8
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
data suggested that a cross resistance is unlikely. The potency profile of 21 is among the most flat
profiles we have achieved so far.
The in vitro cytotoxicity potential of compound 21 was tested in Huhꢀ7 cells up to a maximum
concentration of 25 ꢀM by monitoring GAPDH mRNA levels. No cytotoxicity was observed.
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 12. In vitro potency profile of compound 21
EC90
nM)
EC90
(nM)
EC90
(nM)
a
Replicons
Shift
Replicons
Shift Replicons
Shift
(
1
1
1
1
1
A
0.0042
0.00014
0.00034
0.18
ꢀ
1B
0.0044
0.0040
0.026
1X
1X
6X
1X
2B L31M
2B (A146S)
2B (M31V)
3A
0.018
0.0034
0.019
0.0066
0.29
4X
1X
b
c
A (A92P)
<1X 1B (A156T)
<1X 1B (A92K)
43X 1B (A92T)
A (K24R)
5X
A (L31M:Y93C)
A (L31V)
0.0039
0.0018
2X
b
0.0043
1X
1B (D168Y)
<1X 3A (L31F)
15X 3A (A30K)
69X
1
B
1
A (M28K)
0.42
100X
0.065
0.018
4X
(L28M:Y93H)
1A (M28T)
1A (Q30R)
0.0020
0.12
<1X 1B (L31I)
28X 1B (L31M)
0.021
5X
3A (A30T)
0.0036
3.2
1X
0.0016
<1X 3A (L31V)
45X 3A (Y93H)
762X
1
B
c
1
A (R155K)
0.0019
<1X
0.19
2.4
571X
(
L31M:Y93H)
c
1
1
1
1
A (R155K:V36M)
A (R81W)
A (Y93H)
0.0025
0.00032
0.033
<1X 1B (L31V)
<1X 2A
0.0058
0.0027
0.00050
0.069
1X
4A
0.0060
0.072
2X
17X
1X
<1X 4A (L30H)
<1X 4A (P32L)
16X 4A (L31V)
6X
8X
2A (F28L)
0.0044
0.0077
A (Y93N)
0.069
16X 2A (F28Y)
<1X 2A (L31M)
2X
1
A R81S
0.00033
0.025
a
Shift from GT1a EC value
9
0
b
The single amino acid code is indicated for all substitutions e.g. 1A (A92P) refers to a genotype 1a
substitution where an alanine at position 92 is replaced with a proline
c
HCV NS3 RAS
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The structure of compound 21 has been fully determined. The stereochemistry of the six chiral
centers either originated from known chiral starting materials or was determined by Vibrational
Circular Dichroism (VCD) spectroscopy with confidence (see experimental section for details). Given
its superior potency and pharmacokinetic properties, compound 21 was recommended as a preclinical
candidate for further development.
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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2
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0
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8
9
0
CONCLUSION
We incorporated (S)ꢀTHP amino acid “cap” with varying substituents at the aminal carbon (the
“
Z” group) and identified HCV NS5A inhibitors with significantly improved potency against the least
sensitive RASs of elbasvir, such as GT1a Y93H and GT2b L31M. These leads also showed an
excellent in vitro profile in many other genotypes and mutants. Some of the compounds we identified
possess “flat” potency profiles, with EC values against a majority of the mutants tested within a 10ꢀ
9
0
fold range of the EC value for GT1a H77 (wild type). Compounds with an unsubstituted (S)ꢀTHP
9
0
amino acid “cap” exhibited low PK exposure in rat, likely attributable to oxidative metabolism. Small
alkyl substituted (S)ꢀTHP replacements for the isopropyl group of valine showed improved PK
exposure in both rat and dog. Among them, the compound with the (S,R)ꢀ2,2ꢀdimethylꢀTHP amino
acid “cap” (compound 21) had the best PK profile in both rat and dog, and an increased exposureꢀdose
response up to 300 mpk in rat as well. Compound 21 was recommended as a preclinical candidate
(
MK-6169) for further development.
CHEMISTRY
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6
The chemistry for the preparation of compounds 1ꢀ12 is outlined in Scheme 1. The preparation of
1
6
compound 38 was described previously. Aldehydes 39a-l were either purchased or prepared based on
30ꢀ32
known procedures.
Treatment of 38 with aldehydes 39a-l under acidic condition to give the
tetracyclic indoline intermediates which were in turn oxidized to the corresponding racemic indoles.
These compounds were separated by chiral SFC to give 40a-l, representing the enantiomers that give
the more potent final compounds. The stereochemistry of the aminal carbons in 40a-l was not
determined, but we believe it’s reasonable to assume it is “S” since in previous studies we have
determined that the “S” isomers at the aminal carbon of the tetracyclic indole cores always give the
0
1
2
3
4
5
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9
0
1
2
3
4
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9
0
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8
9
0
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2
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5
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9
0
1
2
3
4
5
6
7
8
9
0
1
5,30
more potent final compounds including MKꢀ8742 and MKꢀ8408.
Compounds 40a-l were
converted to the corresponding pinnacol boronate esters 41a-l which upon treatment under Suzuki
coupling conditions with 42 afforded intermediates 43a-l. Compound 42 was prepared based on
1
6
published procedures. Treatment of 43a-l under Pd (dba) /XꢀPhos conditions afforded pinnacol
2
3
16
boronate esters 44a-l that coupled with 45 to give 46a-l. The Boc groups of 46a-l were removed and
a methoxycarbonylꢀLꢀvaline moiety was introduced to give compounds 1ꢀ12. Compound 13 and 14
were also prepared in a similar method by switching the valine and (S)ꢀTHP “caps” in the synthesis.
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0
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2
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4
5
6
7
8
9
0
Scheme 1 Preparation of compounds 1ꢀ12
o
o
Reagents and conditions: (i) TFA, CH CN, 25 C, 6 h; (ii) DDQ, toluene, 115 C, 2 h, then chiral SFC separation; (iii)
3
o
o
Bis(pinacolato)diboron, Pd(dppf)Cl , KOAc, dioxane, 90 C, 16 h; (iv) 42, Pd(dppf)Cl , K CO , dioxane/H O (10:1), 90 C,
2
2
2
3
2
o
1
6 h; (v) Bis(pinacolato)diboron, Pd (dba) , XꢀPhos, dioxane, 110 C, 2 h; (vi) 45, Pd(dppf)Cl , K CO , dioxane/H O
2 3 2 2 3 2
o
(
10:1), 90 C, 16 h; (vii) HCl, MeOH, 0.5 h. (viii) (S)ꢀ2ꢀ((methoxycarbonyl)amino)ꢀ2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)acetic
acid, BOP, DIPEA, DMF, 10 h.
The preparation of 52a-h was outlined in Scheme 2. Ketone intermediates 47a-j were either
purchased from commercial resources or prepared (Schemes 4ꢀ7). Treatment of 47a-h with 48 under
DBU conditions gave condensed products 49a-h, which were reduced by hydrogen under Pd/C to
afford amine intermediates 50a-h. Compounds 50a-h were hydrolyzed to give racemic amino acids
5
1a-h, to which a methyl carbamate functionality was introduced and the diastereomers were separated
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5
6
by chiral SFC to provide 52a-h, representing the diastereomers which lead to the most potent final
compounds. The stereochemistry of the R group on the amino acid was not determined except 52 g.
We believe it is reasonable to assume they have the “S” configuration since in previous studies we have
determined that (S)ꢀ2ꢀaminoꢀ2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)acetic acid always lead to the more potent
25
0
1
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9
0
final compounds than (R)ꢀ2ꢀaminoꢀ2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)acetic acid. The stereochemistry of
the chiral centers on the THP groups were either introduced via starting materials (see Schemes 4ꢀ7) or
assigned arbitrarily, except 52g, whose stereochemistry were fully determined by VCD with
confidence (see experimental section).
Scheme 2 Preparation of compounds 52a-h
o
o
Reagents and conditions: (i) DBU, CH Cl , 25 C, 5 h; (ii) Pd/C (10%), H (35 psi), MeOH, 25 C, 8 h; (iii) LiOH, MeOH,
2
2
2
o
o
H O, 25 C, 15 h; (iv) methyl chloroformate, LiOH, H O, 25 C, 3 h; then chiral SFC separation.
2
2
Compounds 52i and 52j were prepared by a different synthesis outlined in Scheme 3. Ketone
4
7i-j were reduced to the corresponding alcohols and then converted to tosylates 53i-j. Replacement of
the tosyl groups of 53i-j by the enolate generated from compound 54 afforded compounds 55i-j, which
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5
6
were deprotected under acidic conditions to provide amine 56i-j, which were subsequently converted to
methyl carbamates 57i-j and further hydrolyzed to give acids 52i-j.
0
1
2
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0
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8
9
0
1
2
3
4
5
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8
9
0
Scheme 3 Preparation of compounds 52i-j
o
o
Reagents and conditions: (i) Lꢀselectride, THF, ꢀ78 to 25 C, 12 h, then NaOH, H O; (ii) TsCl, pyridine, DCM, 25 C, 12 h;
2
o
o
o
(
iii) 54, LiHMDS, toluene, 100 C, 4 h; (iv) HCl, THF, 25 C, 2 h; (v) methyl chloroformate, DCM, Et N, 25 C, 12 h; (vi)
3
o
Pd/C (10%), H (40 psi), MeOH, 25 C, 12 h.
2
The preparation of (R)ꢀ2ꢀmethyltetrahydroꢀ4Hꢀpyranꢀ4ꢀone (47a) is described in Scheme 4.
Commercially available methyl (R)ꢀ3ꢀhydroxybutanoate (58) was protected with a TBS group to give
compound 59, which was converted to Weinreb amide 60. Addition of a vinyl group to compound 60
gave α,β−unsaturated ketone 61, which was deprotected and provided ketone 47a. (S)ꢀ2ꢀ
methyltetrahydroꢀ4Hꢀpyranꢀ4ꢀone (47b) was prepared in a similar manner starting with commercially
available methyl (S)ꢀ3ꢀhydroxybutanoate.
TBSO
O
OH
O
TBSO
O
i
ii
O
N
(R)
O
O
59
60
58
O
(R)
TBSO
O
iii
iv
O
61
47a
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Scheme 4 Preparation of compounds 47a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
2
2
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
Reagents and conditions: (i) TBSCl, imidazole, EtOAc, 0 to 25 C, 12 h, 95%; (ii) N,Oꢀdimethyl hydroxylamine, iꢀPrMgCl,
o
o
o
THF, ꢀ20 C, 2 h, 72%; (iii) vinyl magnesium bromide, THF, 0 to 5 C, 1 h, 79%; (iv) Amberlyst 15 resin, CHCl , 25 C, 15
3
h, used without purification.
0
1
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9
0
The preparation of cisꢀ2,6ꢀdimethyltetrahydroꢀ4Hꢀpyranꢀ4ꢀone (47c) is described in Scheme 5.
Reduction of compound 62 led to cisꢀalcohol 63, which was oxidized to ketone 47c. The preparation of
ketone 47h is described in Scheme 6. Cyclopropylation of 64 was achieved under Ti(iꢀPrO) /EtMgBr
4
conditions to afford compound 65, which was protected with a TBS group. The dimethyl acetal also
fell off under the TBSOTf condition and aldehyde 66 was obtained. Addition of a vinyl group to the
aldehyde 66 led to alcohol 67, which was oxidized to α,β−unsaturated ketone 68. Deprotection of the
TBS group led to the formation of desired ketone 47h. Ketone 47i was prepared in a twoꢀstep synthesis
(Scheme 7). Reaction between furan and 1,1,3,3ꢀtetrabromopropanꢀ2ꢀone under Zn/(EtO) B conditions
3
gave diꢀbromo ketone 69, which was reduced to the desired ketone 47i under Pd catalyzed
hydrogenation.
Scheme 5 Preparation of compounds 47c
o
Reagents and conditions: (i) Pd/C (10%), H (50 psi), EtOH, 35 C, 17 h, 99%; (ii) TEMPO, NaHCO , KBr, NaClO, DCM,
2
3
o
H O, 25 C, 5 h, 99%.
2
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9
0
Scheme 6 Preparation of compounds 47h
o
Reagents and conditions: (i) Ti(iꢀPrO) , EtMgBr, THF/Et O (1:1), 0 to 25 C, 12 h, 89%; (ii) TBSOTf, 2,6ꢀlutidine, DCM,
4
2
o
o
o
1
0 C, 1 h, 41%; (iii) vinyl magnesium bromide, THF, ꢀ78 C, 0.5 h, 41%; (iv) IBX, CH CN, 80 C, 2 h, 50%; (v)
3
Amberlyst 15 resin, DCM, reflux, 4 h, used without purification.
Scheme 7 Preparation of compounds 47i
o
o
Reagents and conditions: (i) 1,1,3,3ꢀtetrabromopropanꢀ2ꢀone, Zn, (EtO) B, THF, 25 C, 17 h; (ii) Zn, CuCl, MeOH, 25 C,
3
1
9 h, 30% for two steps.
With 52a-j in hand, compounds 15ꢀ24 were synthesized as described in Scheme 8. The
preparation of 75 from 38 and 39h was similar to the chemistry described in Scheme 1. Compound 75
was resolved by chiral SFC. The active isomer, which was identified by carrying both isomers to the
final compounds and comparing their replicon activity, was deprotected to give amine 76. The
substituted THP “cap” was introduced by BOP mediated amide coupling reaction to provide final
compounds 15ꢀ24
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9
0
1
2
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4
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8
9
0
Scheme 8 Preparation of compounds 15-24
o
Reagents and conditions: (i) TFA, CH CN, 25 C, 6 h, 81%; (ii) DDQ, toluene, reflux, 2 h, 88%; (iii)
3
o
Bis(pinacolato)diboron, Pd(dppf)Cl , KOAc, dioxane, 90 C, 16 h, 88%; (iv) 45, Pd(dppf)Cl , K CO , dioxane/H O (10:1),
2
2
2
3
2
o
o
9
0 C, 16 h, 77%; (v) Bis(pinacolato)diboron, Pd (dba) , XꢀPhos, dioxane, 110 C, 2 h, 89%; (vi) 72, Pd(dppf)Cl , K CO ,
2 3 2 2 3
o
dioxane/H O (10:1), 90 C, 16 h, 74%; then chiral SFC separation, 26%. (vii) HCl, MeOH, 2 h, 98%. (viii) 52a-j, BOP,
2
DIPEA, DMF, 10 h.
Compounds 25ꢀ37 were synthesized by similar methods as described in Scheme 1 by replacing the
unsubstituted (S)ꢀTHP “cap” with 52g.
EXPERIMENTAL SECTION
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3
Chemicals purchased from commercial suppliers were used as received unless specified. Silica
gel column chromatography was carried out on ISCO CombiFlash Companion. Prepacked silica gel
cartridges were used. TLC (thin layer chromatography) visualization was performed under 254 nm
ultraviolet light. Reverseꢀphase preparative HPLC purifications were done on a Gilson 215 Liquid
Handler with Unpoint software, typically with a Sun Fire Prep C18 OBD 5 am 19 × 50 mm column.
The following method was typically used: Run time: 15 min. flow rate: 14 mL/min. Mobile phase: 10%
to 90% CH CN/H O, with 0.1%TFA. UV detection at 254 nm or 210 nm was typically used. SFC
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
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5
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5
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0
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7
8
9
0
3
2
separations were normally done on a TharSFC instrumentation. Columns and separation conditions
were specified in each separation.
Nuclear magnetic resonance spectra were obtained on a Varian 400 MHz, 500 MHz or Bruker
400 MHz spectrometers. Spectra were taken at ambient temperature. Chemical shifts were assigned by
using residual solvent signal as internal standard and are reported in parts per million (ppm).
Resonance patterns are reported by using the following notations: br (broad), s (singlet), d (doublet), t
(
triplet), q (quartet), and m (multiplet).
The purity of all final compounds were determined to be >95% according to the LCMS data
obtained on Agilent 1200 Series HPLC equipped with DAD & 6110 single quadrupole MSD & ELSD
with Agilent TCꢀC18, 50×2.1mm, 5ꢁm for acid methods, Waters XꢀBridge ShieldRP18, 50x2.1mm
5ꢁm for basic methods. HRMS analysis was performed on Waters Acquity UPLC, Column: Acquity
UPLC BEH C18 1.7 ꢀm, and Waters Xevo G2 Qtof with ionization mode as positive ESI+ for all
compounds except 21. The HRMS analysis of 21 was carried on Thermo LTQ obitrap mass
spectrometer with ionization mode as positive ESI+ and HPLC system consisting of a Phenomenex
Kinetex C18 column, 150 x 4.6 mm, 2.6 micron particle size. The flow rate was 1.0 ml/min. The
column temperature was 30º C. The mobile phase was water and acetonitrile with 0.1% formic acid
added to each. A linear gradient from 20% to 90% acetonitrile in 8 minutes was used.
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6
Virology assay
3
1,32,34,35
Detailed assay method were published previously.
All EC or EC data are average values of
50 90
at least two measurements, with a maximum variation of 3ꢀfold.
0
1
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0
PK Assessment
Rat and dog PK studies were done in 24 hour period. Overnight fasted or fed rats and dogs were used.
Dose was administered either by IV or PO with two animals in each dosing group. Plasma was
obtained by centrifugation of the blood collected. Compound concentrations in plasma were
determined by LC/MS analysis.
3
1ꢀ33
Aldehyde 39a was prepared based on published procedures.
Aldehydes 39b-c were purchased from
commercial resources. Aldehyde 39d-l were either purchased from commercial resources or prepared
29,31ꢀ33
based on published procedures
Methyl
((S)-2-((S)-2-(5-((S)-6-(chroman-7-yl)-1-fluoro-3-(2-((S)-1-((methoxycarbonyl)-L-
valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)-6H-benzo[5,6][1,3]oxazino[3,4-a]indol-10-yl)-1H-
imidazol-2-yl)pyrrolidin-1-yl)-2-oxo-1-(tetrahydro-2H-pyran-4-yl)ethyl)carbamate
(1).
1
Compound 1 was prepared based on the same procedures as compound 4. HꢀNMR (400MHz,
CD OD) δ: 7.933 (s, 1 H), 7.81 (s, 1 H), 7.68 (s, 1 H), 7.74ꢀ7.08 (m, 6 H), 6.88 (d, J=8.0 Hz, 1 H), 6.41
3
(d, J=8.4 Hz, 1 H), 6.29 (s, 1H), 5.23ꢀ5.18 (m, 2 H), 4.29 (d, 7.6 Hz, 1 H), 4.21 (d, J=6.4 Hz, 1 H),
4.08ꢀ3.88 (m, 8 H), 3.64 (d, J=20 H, 6 H), 3.38ꢀ3.31 (m, 2 H), 2.65ꢀ2.53 (m, 2 H), 2.51ꢀ2.50 (m, 2 H),
2
.26ꢀ2.02 (m, 8 H), 1.83ꢀ1.85 (m, 2 H), 1.55ꢀ1.35 (m, 4 H), 0.90 (dd, J=6.8, 20 Hz, 6 H). HRMS
+
(ESI+) m/z calcd for C54H61FN9O9 [M+H] , 998.4576; found, 998.4587.
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Methyl
((S)-2-((S)-2-(5-((S)-6-(chroman-6-yl)-1-fluoro-3-(2-((S)-1-((methoxycarbonyl)-L-
valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)-6H-benzo[5,6][1,3]oxazino[3,4-a]indol-10-yl)-1H-
imidazol-2-yl)pyrrolidin-1-yl)-2-oxo-1-(tetrahydro-2H-pyran-4-yl)ethyl)carbamate
(2).
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Compound 2 was prepared based on the same procedures as compound 4. HꢀNMR (400MHz,
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CD OD) δ: 8.00 (s, 1 H), 7.88 (s,1 H), 7.72 (s, 1 H), 7.45 (d, J=4.8 Hz, 2 H), 7.32 (d, J=11.2 Hz, 1 H),
3
7
.25ꢀ7.21 (m, 3 H), 6.74 (d, J=7.2 Hz, 2 H), 6.61 (d, J=9.2 Hz, 1 H), 5.24ꢀ5.16 (m, 2 H), 4.26 (d, J=8.0
Hz, 1 H), 4.19 (d, J=7.6 Hz, 1 H), 4.10ꢀ4.07 (m, 4 H), 3.93ꢀ3.82 (m, 4 H), 3.64 (s, 6 H), 3.40ꢀ3.30 (m, 2
H), 2.61ꢀ2.51 (m, 4 H), 2.40ꢀ2.13 (m, 8 H), 2.03ꢀ1.87 (m, 2 H), 1.56ꢀ1.27 (m, 4 H), 0.90ꢀ0.85 (m, 6 H).
+
HRMS (ESI+) m/z calcd for C54H61FN9O9 [M+H] , 998.4576; found, 998.4586.
Methyl ((S)-2-((S)-2-(5-((S)-1-fluoro-3-(2-((S)-1-((methoxycarbonyl)-L-valyl)pyrrolidin-2-yl)-1H-
imidazol-5-yl)-6-(4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-6H-
benzo[5,6][1,3]oxazino[3,4-a]indol-10-yl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-2-oxo-1-(tetrahydro-
2
H-pyran-4-yl)ethyl)carbamate (3). Compound 3 was prepared based on the same procedures as
1
compound 4. HꢀNMR (400MHz, CD OD) δ: 7.86ꢀ7.80 (m, 1 H), 7.75ꢀ7.74 (m, 1 H), 7.60ꢀ7.59 (m, 1
3
H), 7.32ꢀ6.89 (m, 6 H), 6.49ꢀ6.36 (m, 3 H), 5.22ꢀ5.15 (m, 2 H), 4.28 (d, J=7.2 Hz, 1 H), 4.20 (d, J=6.8
Hz, 1 H), 4.08 (s, 4 H), 3.87ꢀ3.85 (m, 4 H), 3.62 (s, 6 H), 3.35ꢀ3.30 (m, 2 H), 3.13 (s, 2 H), 2.73 (d,
J=3.2 Hz, 3 H), 2.49ꢀ2.47 (m, 2 H), 2.22ꢀ2.03 (m, 8 H), 1.55ꢀ1.33 (m, 4 H), 0.92ꢀ0.85 (m, 6 H). HRMS
+
(ESI+) m/z calcd for C54H62FN10O9 [M+H] , 1013.4685; found, 1013.4699.
Methyl ((S)-2-((S)-2-(5-((S)-1-fluoro-3-(2-((S)-1-((methoxycarbonyl)-L-valyl)pyrrolidin-2-yl)-1H-
imidazol-5-yl)-6-(5-methylthiophen-2-yl)-6H-benzo[5,6][1,3]oxazino[3,4-a]indol-10-yl)-1H-
imidazol-2-yl)pyrrolidin-1-yl)-2-oxo-1-(tetrahydro-2H-pyran-4-yl)ethyl)carbamate (4). To
a
solution of the compound 46d (2.1 g, 2.32 mmol) in dry dioxane (8 mL) was added HClꢀdioxane (4M,
6 mL) through syringe and stirred at 25 °C for 2 hours, then concentrated and dried under high vacuum
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to give HCl salt of methyl ((S)ꢀ2ꢀ((S)ꢀ2ꢀ(5ꢀ((S)ꢀ1ꢀfluoroꢀ6ꢀ(5ꢀmethylthiophenꢀ2ꢀyl)ꢀ3ꢀ(2ꢀ((S)ꢀ
pyrrolidinꢀ2ꢀyl)ꢀ1Hꢀimidazolꢀ4ꢀyl)ꢀ6Hꢀbenzo[5,6][1,3]oxazino[3,4ꢀa]indolꢀ10ꢀyl)ꢀ1Hꢀimidazolꢀ2ꢀ
yl)pyrrolidinꢀ1ꢀyl)ꢀ2ꢀoxoꢀ1ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)carbamate (1.87g, 98%). LC/MS Calcd.
+
For [M+H] C43H45FN8O5S: 805.3; found 805.4. To a mixture of the methyl ((S)ꢀ2ꢀ((S)ꢀ2ꢀ(5ꢀ((S)ꢀ1ꢀ
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fluoroꢀ6ꢀ(5ꢀmethylthiophenꢀ2ꢀyl)ꢀ3ꢀ(2ꢀ((S)ꢀpyrrolidinꢀ2ꢀyl)ꢀ1Hꢀimidazolꢀ4ꢀyl)ꢀ6Hꢀ
benzo[5,6][1,3]oxazino[3,4ꢀa]indolꢀ10ꢀyl)ꢀ1Hꢀimidazolꢀ2ꢀyl)pyrrolidinꢀ1ꢀyl)ꢀ2ꢀoxoꢀ1ꢀ(tetrahydroꢀ2Hꢀ
pyranꢀ4ꢀyl)ethyl)carbamate (500 mg, 0.62 mmol), (methoxycarbonyl)ꢀLꢀvaline (108.5 mg, 0.62 mmol)
and DIPEA (160 mg, 1.24 mmol) in DMF (3 mL) was added HATU (235.6 mg, 0.62 mmol). The
resulting mixture was stirred at 25 °C for 2 hours before the solution was applied to HPLC purification
1
to afford the desired compound 4 (340 mg, 57%). H NMR (MeOD) δ: 8.02 (d, J=4 Hz, 1H), 7.94 (s,
1H), 7.83 (s, 1H), 7.76 (d, J=4 Hz, 1H), 7.52 (s, 2 H), 7.40ꢀ7.39 (m, 1 H), 7.32ꢀ7.31 (m, 1 H), 7.20 (s, 1
H), 6.52ꢀ6.49 (m, 2 H), 5.24ꢀ5.20 (m, 2 H), 4.29ꢀ4.27 (m, 2 H), 4.20ꢀ4.10 (m, 2 H), 3.97ꢀ3.90 (m, 4 H),
3
0
.86 (s, 6 H), 3.55ꢀ3.45 (m, 2 H), 2.60ꢀ2.52 (m, 2 H), 2.32ꢀ2.01 (m, 11 H), 1.62ꢀ1.31 (m, 4 H), 0.92ꢀ
+
.85 (m, 6 H). HRMS (ESI+) m/z calcd for C50H57FN9O8S [M+H] , 962.4035; found, 962.4055.
Methyl ((S)-2-((S)-2-(5-((S)-6-(5-ethylthiophen-2-yl)-1-fluoro-3-(2-((S)-1-((methoxycarbonyl)-L-
valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)-6H-benzo[5,6][1,3]oxazino[3,4-a]indol-10-yl)-1H-
imidazol-2-yl)pyrrolidin-1-yl)-2-oxo-1-(tetrahydro-2H-pyran-4-yl)ethyl)carbamate
(5).
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Compound 5 was prepared based on the same procedures as compound 4. HꢀNMR (400MHz,
CD OD) δ: 7.99 (s, 1 H), 7.88 (s, 1 H), 7.81 (s, 1 H), 7.73 (s, 1 H), 7.50ꢀ7.46 (m, 2 H), 7.37ꢀ7.34 (m, 1
3
H), 7.28 (s, 1 H), 7.16 (s, 1 H), 6.55ꢀ6.49 (m, 2 H), 5.24ꢀ5.16 (m, 4 H), 4.27 (d, J=8.4 Hz, 1 H), 4.20 (d,
J=7.2 Hz, 1 H), 4.18ꢀ4.10 (m, 2 H), 4.07ꢀ3.70 (m, 5 H), 3.63 (s, 6 H), 3.40ꢀ3.29 (m, 2 H), 2.70ꢀ2.65 (m,
2
H), 2.60ꢀ2.53 (m, 2 H), 2.35ꢀ1.85 (m, 8 H), 1.65ꢀ1.34 (m, 4 H), 1.13 (t, J=7.6 Hz, 3 H), 0.92ꢀ0.86
+
(
m, 6 H). HRMS (ESI+) m/z calcd for C51H59FN9O8S [M+H] , 976.4191; found, 976.4222.
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Methyl ((S)-2-((S)-2-(5-((S)-1-fluoro-3-(2-((S)-1-((methoxycarbonyl)-L-valyl)pyrrolidin-2-yl)-1H-
imidazol-5-yl)-6-(5-propylthiophen-2-yl)-6H-benzo[5,6][1,3]oxazino[3,4-a]indol-10-yl)-1H-
imidazol-2-yl)pyrrolidin-1-yl)-2-oxo-1-(tetrahydro-2H-pyran-4-yl)ethyl)carbamate
(6).
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Compound 6 was prepared based on the same procedures as compound 4. HꢀNMR (400MHz,
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9
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CD OD) δ: 8.01 (s, 1 H), 7.92 (s, 1 H), 7.82 (s, 1 H), 7.75 (s, 1 H), 7.51ꢀ7.49 (m, 2 H), 7.39ꢀ7.31 (m, 2
3
H), 7.20 (s, 1 H), 6.56ꢀ6.52 (m, 2 H), 5.24ꢀ5.20 (m, 2 H), 4.29ꢀ4.20 (m, 2 H), 4.13ꢀ4.10 (m, 2 H), 3.94ꢀ
3
.66 (m, 4 H), 3.65 (s, 5 H), 3.56ꢀ3.41 (m, 3 H), 2.66ꢀ2.64 (m, 2 H), 2.62ꢀ2.55 (m, 2 H), 2.28ꢀ2.26 (m,
2 H), 2.18ꢀ2.16 (m, 4 H), 2.05ꢀ2.03 (m, 2 H), 1.59ꢀ1.53 (m, 3 H), 1.51ꢀ1.34 (m, 3 H), 0.97ꢀ0.84 (m, 9
+
H). HRMS (ESI+) m/z calcd for C52H61FN9O8S [M+H] , 990.4348; found, 990.4362.
Methyl ((S)-2-((S)-2-(5-((S)-1-fluoro-6-(5-isopropylthiophen-2-yl)-3-(2-((S)-1-((methoxycarbonyl)-
L-valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)-6H-benzo[5,6][1,3]oxazino[3,4-a]indol-10-yl)-1H-
imidazol-2-yl)pyrrolidin-1-yl)-2-oxo-1-(tetrahydro-2H-pyran-4-yl)ethyl)carbamate
(7).
1
Compound 7 was prepared based on the same procedures as compound 4. HꢀNMR (400MHz,
CD OD) δ: 8.01 (s, 1 H), 7.91 (s, 1 H), 7.83 (s, 1 H), 7.76 (s, 1 H), 7.55ꢀ7.53 (m, 2 H), 7.50ꢀ7.41 (m, 1
3
H), 7.38ꢀ7.30 (m, 1 H), 7.19ꢀ7.18 (m, 1 H), 6.61ꢀ6.53 (m, 2 H), 5.26ꢀ5.21 (m, 2 H), 4.32ꢀ4.24 (m, 2 H),
4
.13ꢀ4.12 (m, 2 H), 3.96ꢀ3.88 (m, 4 H), 3.68 (s, 5 H), 3.43ꢀ3.32 (m, 2 H), 3.05ꢀ3.02 (m, 1 H), 2.58ꢀ2.55
(m, 2 H), 2.30ꢀ2.05 (m, 9 H), 1.65ꢀ1.56 (m, 1 H), 1.55ꢀ1.38 (m, 3 H), 1.20ꢀ1.19 (m, 6 H), 0.99ꢀ0.91 (m,
+
6
H). HRMS (ESI+) m/z calcd for C52H61FN9O8S [M+H] , 990.4348; found, 990.4361.
Methyl
(methoxycarbonyl)-L-valyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)-6H-benzo[5,6][1,3]oxazino[3,4-
a]indol-10-yl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-2-oxo-1-(tetrahydro-2H-pyran-4-
((S)-2-((S)-2-(5-((S)-6-(5-cyclopropylthiophen-2-yl)-1-fluoro-3-(2-((S)-1-
(
1
yl)ethyl)carbamate (8). Compound 8 was prepared based on the same procedures as compound 4. Hꢀ
NMR (400MHz, CD OD) δ: 8.01 (s, 1 H), 7.93 (s, 1 H), 7.81 (s, 1 H), 7.75 (s, 1 H), 7.52ꢀ7.48 (m, 2
3
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