Discovery of Chromane Containing Hepatitis C Virus (HCV) NS5A Inhibitors with Improved Potency against Resistance-Associated Variants

Article abstract of DOI:10.1021/acs.jmedchem.6b01234

The discovery of potent and pan-genotypic HCV NS5A inhibitors faces many challenges including the significant diversity among genotypes, substantial potency shift conferred on some key resistance-associated variants, inconsistent SARs between different genotypes and mutants, and the lacking of models of inhibitor/protein complexes for rational inhibitor design. As part of ongoing efforts on HCV NS5A inhibition at Merck, we now describe the discovery of a novel series of chromane containing NS5A inhibitors. SAR studies around the "Z" group of the tetracyclic indole scaffold explored fused bicyclic rings as alternates to the phenyl group of elbasvir (1, MK-8742) and identified novel chromane and 2,3-dihydrobenzofuran derivatives as "Z" group replacements offered good potency across all genotypes. This effort, incorporating the C-1 fluoro substitution at the tetracyclic indole core, led to the discovery of a new series of NS5A inhibitors, such as compounds 14 and 25-28, with significantly improved potency against resistance-associated variants, such as GT2b, GT1a Y93H, and GT1a L31V. Compound 14 also showed reasonable PK exposures in preclinical species (rat and dog).

Full text of DOI:10.1021/acs.jmedchem.6b01234

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Article
Discovery of chromane containing hepatitis C virus (HCV) NS5A
inhibitors with improved potency against resistance-associated variants
Wensheng Yu, Ling Tong, Bin Hu, Bin Zhong, Jinglai Hao, Tao Ji, Shuai Zan, Craig Alan Coburn,
Oleg Selyutin, Lei Chen, Laura Rokosz, Sony Agrawal, Rong Liu, Stephanie Curry, Patricia
Mcmonagle, Paul Ingravallo, Ernest Asante-Appiah, Shiying Chen, and Joseph A. Kozlowski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01234 • Publication Date (Web): 28 Oct 2016
Downloaded from http://pubs.acs.org on October 30, 2016
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Discovery of chromane containing hepatitis C virus (HCV) NS5A
inhibitors with improved potency against resistance-associated variants
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Wensheng Yua,*, Ling Tonga, Bin Hub, Bin Zhongb, Jinglai Haob, Tao Jib, Shuai Zanb, Craig A.
Coburnc, Oleg Selyutina, Lei Chena, Laura Rokoszd, Sony Agrawald, Rong Liue, Stephanie Currye,
Patricia McMonaglee, Paul Ingravalloe, Ernest AsanteꢀAppiahe, Shiying Chenf, Joseph A. Kozlowskia
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aDepartment of Medicinal Chemistry, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway NJ 07065, USA
bWuXi AppTec, 288 Fute Zhong Road, Shanghai, 200131, China
cDepartment of Medicinal Chemistry, Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA
dDepartment of In Vitro Pharmacology, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
eDepartment of Infectious Diseases, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
fDepartment of PPDM, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
ABSTRACT: The discovery of potent and panꢀgenotypic HCV NS5A inhibitors faces many
challenges including the significant diversity among genotypes, substantial potency shift conferred on
some key resistanceꢀassociated variants, inconsistent SARs between different genotypes and mutants,
and the lacking of models of inhibitor/protein complexes for rational inhibitor design. As part of
ongoing efforts on HCV NS5A inhibition at Merck, we now describe the discovery of a novel series of
chromane containing NS5A inhibitors. SAR studies around the “Z” group of the tetracyclic indole
scaffold explored fused bicyclic rings as alternates to the phenyl group of elbasvir (1, MKꢀ8742) and
identified novel chromane and 2,3ꢀdihydrobenzofuran derivatives as “Z” group replacements offered
good potency across all genotypes. This effort, incorporating the Cꢀ1 fluoro substitution at the
tetracyclic indole core, led to the discovery of a new series of NS5A inhibitors, such as compound 14
and 25-28, with significantly improved potency against resistanceꢀassociated variants, such as GT2b,
GT1a Y93H, and GT1a L31V. Compound 14 also showed reasonable PK exposures in preclinical
species (rat and dog).
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INTRODUCTION
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Hepatitis C virus (HCV) infection comprises both acute and chronic forms that result from
contact with infected blood in most cases. Approximately 20% of infections are spontaneously cleared
within 6 months with no symptoms; however, the majority lead to chronic infections with dire
consequences over a period of 25ꢀ30 years.1 The chronic infection often leads to liver diseases
including fibrosis, cirrhosis, and ultimately hepatocellular carcinoma. It is estimated that 130ꢀ170
million people worldwide are infected with HCV, with 9 million in the United States and western
Europe.2,3 Currently 40ꢀ50% of all liver transplants in the United States are due to HCV infection.4
Globally approximate 500,000 individuals die every year from HCV related liver diseases.2 Until
2013, treatment of HCV infection was focused on interferon/ribavirin based therapy and was limited by
both efficacy (~50% cure rate) and tolerability.5 Addition of HCV protease inhibitors, such as telaprevir
or boceprevir, to the therapy improved cure rates to 70~80% for genotype 1 patients,5 but tolerability
issues, such as fluꢀlike symptoms, still remained due to the retention of interferon. Current HCV
therapy with interferonꢀfree, all oral directꢀacting antiviral agents (DAAs) has significantly improved
on efficacy, treatment duration, and adverse effects. Each DAA targets at one of the HCV nonstructural
(NS) proteins that are important in replication including the NS3/4A protease, NS5A phosphoprotein,6ꢀ
8 and NS5B polymerase.
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NS5A has no known enzymatic activity, but it is essential for HCV RNA replication7 and virus
assembly.9 NS5A inhibition is therefore an important component in regimens for the treatment of HCV
infection. However, early NS5A inhibitors suffered from a low genetic barrier to resistance and
demonstrated weak activity against nonꢀGT1 and GT1a resistanceꢀassociated variants (RAVs).10
Subsequent inhibitors have improved activity against RAVs; however, the need for nextꢀgeneration
NS5A inhibitors with increased potency against all genotypes as well as NS5A RAVs are needed.11 To
date, five NS5A inhibitors have been approved for HCV treatment, either alone or in combination with
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other DAAs: daclatasvir (BMSꢀ790052),10,12 ledipasvir (GSꢀ5885),13 ombitasvir (ABTꢀ267),14 elbasvir
(1, MKꢀ8742),15,16 and velpatasvir (GSꢀ5816).17
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HCV has seven major genotypes (GT1ꢀ7, GT1ꢀ3 account for 70ꢀ90% of all HCV infections
depending on the region), with multiple subtypes identified for each.18ꢀ20 Optimization of the potency
profiles of NS5A inhibitors against broad genotypes and mutants faces unique challenges owing to this
significant diversity among genotypes and the substantial fold of losses in potency conferred by several
key clinicallyꢀrelevant RAVs. The absence of robust biochemical assays and models of inhibitor/NS5A
protein complexes provided additional challenges to the medicinal chemistry efforts. SAR studies were
therefore primarily driven by potency data generated in replicon cells. SARs may not transfer between
different genotypes/mutants and often SARs improving the potency against one genotype/mutant may
hurt the potency against others. Previous research efforts at Merck have identified the tetracyclic
indoleꢀbased inhibitor elbasvir (1) shown in Figure 1.15,16 Continued efforts sought to explore the
impact of structural changes to compound 1, with the aim of determining if an inhibitor with a ‘flat’
potency profile could be obtained. In particular, a “flat” potency profile is defined by a minimal
potency shift (~10x) between GT1a and other genotypes and RAVs, such as GT2b (bearing the
resistanceꢀassociated methionine at position 31), GT1a Y93H, and GT1a L31V, which were the least
sensitive RAVs of compound 1. Due to the lack of Xꢀray structures of inhibitor/protein complexes,
rational designs of NS5A inhibitors are not possible. Our strategy was to explore every region of the
structure of compound 1 and identify SARs that could improve the potency against GT2b, GT1a
Y93H, and GT1a L31V together, and maintain the good potency against the other genotypes and
mutants already achieved by compound 1. Towards this end, we have reported SAR studies directed
toward the “core”,21ꢀ24 “Z” group,25,26 “cap”,27 and proline28 modifications (Figure 2). SAR studies
around the “Z = aryl groups”26 yielded NS5A inhibitors with reduced potency shifts from GT1a to
GT2b, GT1a Y93H, and GT1a L31V which had not been observed in our efforts around the alternative
“core”, “Z = alkyl group”,25 “cap”, and proline modifications, clearly indicating the value of “Z =
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aromatic group” plays in the potency profiles of the tetracyclic indole based NS5A inhibitors. In a
different study, we identified another important SAR that the Cꢀ1 fluoro substitution on the tetracyclic
indole core could improve the potency against both GT2b and GT1a Y93H simultaneously.29 These
key SAR achievements overcame some of the challenges we mentioned above and set the foundation
and direction for our future efforts. Although compounds with “flat” or near “flat” potency profiles
have not been identified, we continued our efforts around the “Z group” modifications with the Cꢀ1
fluoro substituted tetracyclic indole core (Figure 2), and these efforts have led to the discovery of more
potent HCV NS5A inhibitors with significantly reduced potency shift against NS5A RAVs. In this
report, we disclose a novel series of chromane containing NS5A inhibitors with near “flat” potency
profiles as well as reasonable PK exposure in both rat and dog.
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O
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O
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Daclatasvir (BMSꢀ790052)
Ombitasvir (ABTꢀ267)
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O
N
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H
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H
Ledipasvir (GSꢀ5885)
Elbasvir (MKꢀ8742)
1
Figure 1. Approved HCV NS5A inhibitors.
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Figure 2. “Z group” modification strategy using the Cꢀ1 fluoro substituted tetracyclic indole core.
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RESULTS AND DISCUSSION
During our previous SAR studies, efforts trying to differentiate the metaꢀ and paraꢀsubstitution
on the phenyl group of compound 1 led to the conclusion that substitutions at both positions were
tolerated.26 For example, both compounds 2a and 2b (Figure 3) showed good potency against all
genotypes tested (Table 1), indicating good tolerance of biaryl rings in this region. With these data in
hand, we sought to explore the impact of 3,4ꢀfused bicyclic groups, as “Z groups”, with the goal of
improving the potency against RAVs (Figure 3).
Figure 3. Biꢀphenyl substituted aminal derivatives of compound 1 and strategy for fused bicyclic group substitutions.
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Table 1. In vitro potency profiles of compounds 1ꢀ2b.
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2
3
4
Replicon EC90 (nM)
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6
7
8
ID
1
GT1a GT1b
GT2a
0.019
0.004
GT2b GT3a
GT4a
0.016
0.02
GT1a Y93H
GT1a L31V
0.006
0.005
0.006
0.006
11
3.8
2.6
0.12
0.3
28
21
1
1
2aa
2ba
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< 0.02 < 0.02 < 0.2
< 0.2 < 0.02
4.6
< 2
aMixture of diastereomers at the aminal carbon.
Compounds with different fused bicyclic rings as “Z group” were prepared and their in vitro
potency profiles against selected replicons are summarized in Table 2. As anticipated, naphthalenꢀ2ꢀyl
derivative 3 showed good potency against all genotypes tested. In contrast, naphthalenꢀ1ꢀyl derivative
4, with a second phenyl ring fused at the ortho and meta position, showed reduced potency against both
GT2b and GT1a Y93H, confirming the limited space around the ortho position of the phenyl group that
was also noticed previously.26 Introducing a nitrogen to the distal phenyl ring of the naphthalenꢀ2ꢀyl
group led to the quinolinꢀ7ꢀyl analog 5, which was similar in activity to compound 3, except for a ~10x
loss in GT3a potency. Fused alkyl rings were also introduced between the meta and para positions of
the phenyl group. Both the 2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl derivative 6 and the 5,6,7,8ꢀ
tetrahydronaphthalenꢀ2ꢀyl derivative 7 showed good potency against all genotypes tested, with their
potency profiles similar to compound 3.
Table 2. Bicyclic ring substituted aminal derivatives of compound 1
Replicon EC90 (nM)
IDa
Z
GT1a
GT2b
GT3a
GT1a Y93H
GT1a L31V
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1
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0.02
0.01
1
0.04
0.1
7
0.2
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31
32
0.09
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0.01
0.004
0.005
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3
2
0.3
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2
2
0.2
ntb
nt
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0.01
0.01
aActive isomer at the aminal carbon. bnt = not tested.
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Encouraged by the data in Table 2, we incorporated a SAR identified from earlier studies into
the fused Zꢀgroups shown above. Previously, we determined that alkoxy substitutions on either the
metaꢀ or paraꢀposition of the phenyl group could improve the potency against GT2b and GT1a Y93H.26
Thus, we incorporated oxygen atom(s) into the distal ring of the fused “Z groups” found in compounds
6 and 7. Incorporating one oxygen atom into the cyclopentyl ring of compound 6 yielded derivatives 8
and 9 (Table 3), with both compounds showing potency similar to compound 6. Benzo[d][1,3]dioxole
derivative 10 lost 10ꢀfold of its potency against GT3a than compound 9. Interestingly, introducing a
2,2ꢀdifluoro substitution to the benzo[d][1,3]dioxole moiety brought the potency against GT3a back,
and compound 11 is similar in activity to compound 9 against all the genotypes tested. Introducing one
oxygen atom to the cyclohexyl group of compound 7 produced chromane or isochromane derivatives
12ꢀ14. While compounds 12 and 13 showed similar potency to compound 7, compound 14 showed
improved potency against GT2b, GT1a Y93H, and GT1a L31V, with all EC90 values obtained in the
subꢀnM range. This provided the impetus to introduce other bicyclic groups with more structure
diversity. Compound 15, which contains a 2Hꢀchromenꢀ2ꢀone moiety, was 10x weaker in potency
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against GT1a Y93H than 14, and so was the 2Hꢀbenzo[b][1,4]oxazinꢀ3(4H)ꢀone derivative 16.
Introducing a methyl group to the amide nitrogen of 16 produced 17, which was more potent against
GT2b, GT3a, and GT1a Y93H than 16.
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Table 3. Oxygenatedꢀbicyclic ring substituted aminal derivatives of compound 1
Replicon EC90 (nM)
IDa
Z
GT1a
0.003
GT2b
2
GT3a
0.02
GT1a Y93H
5
GT1a L31V
ntb
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9c
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12
13
14
0.02
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3
0.1
1
6
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0.2
0.5
nt
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0.004
0.003
0.003
0.003
5
0.1
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7
0.02
0.02
0.01
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nt
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0.1
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nt
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1
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0.01
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0.4
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nt
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b
c
aActive isomer at the aminal carbon unless noted separately. nt = not tested. Mixture of diastereomers at the aminal
carbon.
Given the encouraging profile of compounds such as 14, we explored minor modifications
around chromane and 2,3ꢀdihydrobenzofuran moieties, aiming to further improve the potency and
block any potential metabolism. Compounds with substituted chromane or 2,3ꢀdihydrobenzofuran were
prepared and their in vitro potency profiles are summarized in Table 4. Introducing a methyl group to
the Cꢀ2 position of compound 12 produced two diastereomers, compounds 18 and 19, which are
similar in activity to 12, and so was the corresponding gemꢀdiꢀMe analog 20. Introducing a fused
cyclopropyl and a methyl group to 12 led to compound 21, which showed potency similar to 12 as
well. It seems that methyl substitutions around the chromane moiety of compound 12 had minimum
effect on the potency; we turned our attention to the modification of compound 14. A similar SAR was
observed for methylations at the Cꢀ2 position of the chromane moiety of 14. Cꢀ2 monoꢀmethylation
derivatives 22 and 23, as well as the corresponding gemꢀdimethyl derivative 24 all showed similar or
slightly weaker potency profiles than 14. However, the 2,2ꢀdimethylꢀ2,3ꢀdihydrobenzofuran derivative
25 showed improved potency against GT2a and GT1a Y93H, as compared to compound 24. The Cꢀ4
methylated chromane derivatives (compounds 26 and 27) of 14 may have slightly better potency
against GT1a Y93H than 14, even though the differences are within the assay variation. This potency
improvement against GT1a Y93H was also observed for the corresponding spiroꢀcyclopropyl analog
28, which was 5x more potent against GT1a Y93H than 14. Replacing the spiroꢀcyclopropyl group of
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compound 28 with a diꢀfluoro group leads to compound 29, which is similar in activity to compound
28, except a 7x loss in activity against GT1a Y93H.
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8
Table 4. Substituted chromane and 2,3ꢀdihydrobenzofuran containing NS5A inhibitors
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Replicon EC90 (nM)
IDa
18b
19b
20
Z
GT1a GT1b GT2b GT3a
GT4a
nt
GT1a Y93H
2
GT1a L31V
nt
0.005
0.004
0.005
Ntc
nt
7
7
2
0.02
0.01
0.03
nt
nt
3
nt
nt
nt
0.9
21d
0.005
nt
5
0.03
nt
1
nt
0.003 0.003
0.007 0.005
0.003 0.005
0.3
0.7
0.9
2
0.01
0.009
0.01
0.002
0.007
0.005
nt
0.5
0.9
0.7
1
0.02
0.04
0.02
nt
14
22b
23b
24
0.005
nt
0.02
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0.003 0.004
0.004 0.002
0.5
0.2
0.005
0.006
0.003
0.003
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0.2
0.02
25
26b
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0.007
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0.004 0.002
0.005 0.004
0.2
0.2
0.01
0.01
0.003
0.006
0.2
0.1
0.01
0.006
0.002 0.002
0.2
0.007
0.002
0.7
0.01
29
aActive isomer at the aminal carbon. bAbsolute stereochemistry at the chromane ring assigned arbitrarily. cnt = not tested.
dMixture of diastereomers at the chromane ring.
Using the EC90 values in Table 4, the calculated potency shifts from GT1a for selected
compounds are listed in Table 5. Compound 14 showed < 10ꢀfold of shift for genotypes 1b, 3a, 4a, and
L31V. Its potency shift for GT2b and GT1a Y93H from GT1a are 100x and 167x, respectively.
Compound 25 showed similar potency shift to 14. The potency shift of compounds 26 and 27 against
both GT2b and GT1a Y93H were improved to 50x. Compound 28 showed the most “flat” potency
profile among these compounds with potency shifts against GT2b and GT1a Y93H of 40x and 20x,
respectively.
Table 5. Calculated potency shifts from GT1a EC90 for compounds 14 and 25ꢀ28.
Calculated potency shifts relative to GT1a EC90
ID
GT1a GT1b
GT2b
1800x
100x
GT3a
20x
3x
GT4a
GT1a Y93H
GT1a L31V
167x
1x
1x
1x
1x
3x
4700x
1
1x
167x
7x
14
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1x
1x
1x
1x
1x
1x
1x
1x
167x
50x
50x
40x
2x
2x
3x
2x
1x
1x
1x
1x
67x
50x
50x
20x
7x
2x
3x
1x
25
26
27
28
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
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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
With the encouraging potency improvement against RAVs as shown in Table 5, we turned our
attention to the pharmacokinetic (PK) profiles of these compounds. The rat or dog PK profiles of
selected compounds are listed in Table 6. Compound 14 showed good po (oral) AUC (area under
curve), low total clearance, reasonable halfꢀlife and C24h in both preclinical species. Although its
bioavailability in rat is low, it was higher (16%) in dog. Due to its subꢀnM EC90 values against all the
genotypes tested, we believe the PK profile of compound 14 is acceptable. Compounds 25, which has a
2,2ꢀdimethylꢀ2,3ꢀdihydrobenzofuran moiety, showed reduced exposure in rat relative to compound 14.
Compounds 28, which has a spiroꢀcyclopropyl substituent at the Cꢀ4 of the chromane moiety, showed
much reduced AUC (po), Cmax, and C24h than Compound 14, potentially due to the increased total
clearance. Compound 14 was tested in an oral single rising dose PK study (Figure 4), and the results
are summarized in Table 7. In this study, compound 14 showed a good linear doseꢀexposure
relationship between 10 mpk and 100 mpk. However, when the dose was increased to 300 mpk, no
additional improvement on the exposure of 14 was observed, indicating the exposure reached a plateau
between 100ꢀ300 mpk. We also noticed a 3ꢀfold difference between the AUC (po) at 10 mpk obtained
in the screening PK study (Table 6) and the single rising dose PK study (Table 7). Since the protocols
of both PK studies were the same, this exposure variation was potentially caused by the salt form
difference of the samples used in the PK studies.
Table 6. Pharmacokinetic profiles of compounds 14, 25, and 28a,b
IV
PO
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Journal of Medicinal Chemistry
AUC
(µM.h)
1.35
Dose
T1/2
(h)
CL
Dose
(mpk)
10
Cmax
(nM)
86
C24h
(nM)
17
1
2
3
4
5
6
7
8
ID
Species
F%
(mpk)
(mL/min/Kg)
14c
25c
28c
14d
Rat
Rat
Rat
Dog
2
2
2
1
5.8
8
7
5
3
8.4
10
0.71
58
9
8.5
15
2
10
0.24
29
1
2
9
10
11
12
11.4e
5
6.92
1224
51
16
aMale Wistar Han rats or Beagle dogs were used. Compound was dosed IV in 60% PEG200 and PO in 10% TWEEN. bFor
the PK profile of compound 1, see reference 15. c2TFA salt d2HCl salt. eIV mean residence time
13
14
15
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19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
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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
.
.
Table 7. Rat oral single rising dose PK profile of compound 14a,b
AUC
(µM.h)
0.42
5.9
Cmax
(µM)
0.04
0.35
0.40
Dose
(mpk)
10
Tmax
(h)
MRT
(h)
5.0
5.0
8.0
8.5
13
100
300
4.9
11
aMale Wistar Han rats were used. Compound was doses in 10% TWEEN. b2HCl salt.
Figure 4. Rat oral single rising dose PK study of compound 14. (PO1: 10 mpk; PO2: 100 mpk; PO3: 300 mpk.)
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1
2
3
4
5
6
7
8
9
Potency profiles of compounds 14 and 28 against additional GT1a and GT1b mutants are
summarized in Table 8. As NS5A inhibitors are likely to be developed in combination with other
DAAs such as NS3 inhibitors, we investigated the impact of compounds 14 and 28 on NS3 RAVs. As
anticipated, compounds 14 and 28 retained their potency and were not shifted against prototypical
GT1a NS3 RAVs, such as GT1a R155K and GT1a V36M+R155K double substitution and GT1b
D168Y, suggesting an absence of cross resistance. Both compounds showed a relatively larger potency
shift (300~400x) against GT1a Q30D and compound 28 also showed a relatively larger potency shift
(300x) against GT1a Y93N.
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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. In vitro potency profiles of compounds 14 and 24 against selected GT1a and GT1b mutants
14
28
EC90 (nM)
0.003
Genotypes
EC90 (nM) Shifta
Shifta
1x
1a
0.003
0.02
1x
7x
1a Q30R
1a Q30D
1a R155K
1a R155K
:V36M
0.03
10x
400x
ꢀ
0.9
300x
1x
1.2
0.0001
ntb
0.001
1x
nt
ꢀ
1a Y93N
1b Y93H
1b L31V
1b D168Y
0.2
67x
23x
2x
0.9
0.02
0.005
nt
300x
7x
2x
ꢀ
0.07
0.005
0.002
1x
aShift calculated from GT1a EC90 value. bnt = not tested.
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Journal of Medicinal Chemistry
The in vitro cell toxicity of compounds 1 and 14 were assessed by using Huh7 cells in a Clickꢀit
1
2
3
4
5
EdU plus cell count assay. CC50 values of both compounds 1 and 14 were determined to be >50 µM,
indicating that, for both compounds, no cell toxicity was observed in this assay.
6
7
8
9
Recently numerous models have been developed to explain subsets of SAR data with
conflicting results.30,31 Effort has been initiated in Merck trying to explain the potency improvement
achieved by these chromane containing tetracyclic indole based HCV NS5A inhibitors, as well as other
potent HCV NS5A inhibitors we identified, either by using any of these existing models or by building
a new model. The results will be reported in due course.
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11
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14
15
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23
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25
26
27
28
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30
31
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35
36
37
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60
CONCLUSION
In spite of the challenges faced by the discovery of panꢀgenotypic HCV NS5A inhibitors caused
by the significant diversity among genotypes/mutants and the lacking of inhibitor/protein complexes Xꢀ
ray structures for rational inhibitor design, we have discovered potent and panꢀgenotypic NS5A
inhibitors with near “flat” potency profiles as well as reasonable PK exposure. SAR studies around the
fused bicyclic groups at the aminal carbon, denoted as the “Z groups,” with the Cꢀ1 fluoro substituted
tetracyclic indole core led to the discovery of novel chromane and 2,3ꢀdihydrobenzofuran containing
HCV NS5A inhibitors with significantly improved potency against NS5A RAVs, such as GT2b, GT1a
Y93H, and GT1a L31V. Modifications on the chromane moiety, such as methylation or
cyclopropylation on the Cꢀ4 carbon, further improved the potency against GT1a Y93H. Compounds
with near “flat” potency profiles, such as compounds 14 and 25ꢀ28, have been identified. Among these
compounds, compound 14 stands out due to its reasonable PK profiles in both rat and dog. Its
bioavailability in rat is relatively low, but its bioavailability in dog is higher and its exposure in rat is
proportional with increasing doses up to 100 mpk. Compound 14 also showed good potency profiles
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against a select panel of GT1a and GT1b mutants as well as NS3 RAVs. No cell toxicity was observed
1
2
3
4
for compound 14 in an in vitro cell toxicity assessment assay using Huh7 cells.
5
6
7
8
9
CHEMISTRY
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14
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23
24
25
26
27
28
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30
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35
36
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43
44
45
46
47
48
49
50
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55
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60
Compounds with different “Z group” were prepared based on the chemistry outlined in Scheme
1. Acetylation of 3ꢀbromoꢀ5ꢀfluorophenol 30 gave 3ꢀBromoꢀ5ꢀfluorophenyl acetate 31 which
underwent an AlCl3ꢀpromoted rearrangement to give 1ꢀ(4ꢀbromoꢀ2ꢀfluoroꢀ6ꢀhydroxyphenyl)ethanꢀ1ꢀ
one 32. Fisher indole synthesis of compound 32 afforded indole 34 which was subsequently reduced to
indoline 35 under zinc/TFA conditions. Treatment of 35 with an aldehyde 36a-x under acidic
conditions induced ring cyclization to afford the tetracyclic indoline intermediate which was oxidized
to indole 37a-x under DDQ condition16. 37a-x was then converted to bisꢀpinacol boronate 38a-x which
underwent Suzuki coupling reaction with tertꢀbutyl (S)ꢀ2ꢀ(5ꢀbromoꢀ1Hꢀimidazolꢀ2ꢀyl)pyrrolidineꢀ1ꢀ
carboxylate (39)16 to afford bisꢀBoc intermediate 40a-x. Treatment of 40a-x under acidic conditions
afforded diamine 41a-x. The (methoxycarbonyl)ꢀLꢀvaline group was installed under BOPꢀmediated
amide coupling conditions. The mixture of diastereomers was separated by chiral SFC to yield the
individual compounds for biological evaluation where indicated. The data for the more active isomers,
which was determined to be the Sꢀisomer in the case of compound 1, are reported unless noted
separately.
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Journal of Medicinal Chemistry
Br
OH
Br
OAc
Br
OH
Br
OH
Br
1
i
ii
iii
iv
O
N
2
3
4
5
6
7
N
H
F
F
F
F
30
31
32
33
F
F
F
Br
Br
Br
v
vi
vii
Br
Br
Br
N
8
9
N
N
O
O
H
H
HO
HO
Z
Z
H
10
11
12
13
36a-x
34
35
37a-x
N
Boc
N
F
O
B
Br
Boc
N
F
N
N
Boc
N
O
O
N
ix
O
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
H
N
B
N
39
H
N
N
H
O
O
viii
Z
Z
38a-x
40a-x
F
O
N
O
F
H
N
N
O
x
O
N
N
N
H
N
O
N
H
H
N
N
N
N
N
O
N
H
N
H
H
N
O
H
O
Z
Z
41a-x
3-29
Z =
1
N
1
1
1
1
1
1
O
a
b
c
i
d
j
e
k
f
l
1
1
1
1
1
O
O
O
O
F
F
O
O
O
O
g
h
n
O
O
1
1
1
1
1
1
O
O
N
N
H
O
O
O
O
O
O
O
m
o
t
p
u
q
v
r
O
1
O
O
O
1
1
1
1
1
F
F
s1
s2
w1
O
1
1
O
w2
x
Scheme 1 Preparation of compounds with different “Z groups”
Reagents and conditions: (i) AcCl, DIPEA, DCM, rt, 16 h; (ii) AlCl3, DCM, 0 C, 30 min; then concentrated, 140 C, 3 h; (iii) 4ꢀ
o
o
o
o
bromophenyl hydrazine HCl, AcOH/EtOH (1:10), reflux, 6 h; (iv) Polyphosphoric acid, xylenes, 110 C, 3 h; (v) Zn, TFA, 75 C, 24 h,
(vi) ZCHO, TFA, CH3CN, 25 C, 6 h; then DDQ, toluene, 115 oC; (vii) Bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane, 90 C, 16
o
o
o
h; (viii) 39, Pd(dppf)Cl2, K2CO3, dioxane/H2O (10:1), 90 C, 16 h; (ix) HCl, MeOH, 0.5 h. (x) (methoxycarbonyl)ꢀLꢀvaline, BOP,
DIPEA, DMF, 10 h; then chiral SFC separation.
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1
2
3
4
5
6
7
8
9
Aldehydes 36a-x were either purchased or prepared. Chromaneꢀ7ꢀcarbaldehyde (36l) was
prepared based on the chemistry outlined in Scheme 2. 1,4ꢀDibromoꢀ2ꢀfluorobenzene (42) was treated
with ethylene glycol and tꢀBuOK afforded alcohol 43; which was converted to bromide 44 under PBr3
conditions. Treatment of compound 44 with one equivalent of nꢀBuLi promoted the ring closure of the
chromane. Without isolation of the intermediate, addition of a 2nd equivalent of nꢀBuLi followed by
DMF afforded chromaneꢀ7ꢀcarbaldehyde (36l).
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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
Scheme 2 Preparation of aldehyde 36l.
Reagents and conditions: (i) Ethylene glycol, NMP, tꢀBuOK, 88%; (ii) PBr3, 100 oC, 2 h, 98%; (iii) nꢀBuLi, THF, ꢀ78 oC, 30 min, then nꢀ
BuLi, DMF, ꢀ78 oC to 25 oC, 1 h, 47%.
The preparation of 2,2ꢀDimethylchromaneꢀ6ꢀcarbaldehyde (36q) was outlined in Scheme 3.
PIFA promoted ring closure of acid 45 led to lactone 46, which was converted to compound 47 under
MeLi conditions. Lithiumꢀhalogen exchange of 47 followed by addition of DMF afforded the desired
aldehyde 36q. The preparation of spiro[chromaneꢀ4,1'ꢀcyclopropane]ꢀ7ꢀcarbaldehyde (36t) was
outlined in Scheme 4. 4ꢀBromoꢀ2ꢀhydroxybenzonitrile (48) was treated with MeMgBr to produce
methyl ketone 49, which was condensed with N, Nꢀdimethylformamide to give compound 50. Acid
promoted ring closure of 50 afforded 51, which was reduced, under DIBAlꢀH conditions, to give
ketone 52. Wittig reaction of 52 and methyltriphenylphosphonium bromide led to olefin 53.
Cyclopropylation of 53 lead to compound 54 which was converted to aldehyde 36t under the nꢀ
BuLi/DMF conditions.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
Scheme 3 Preparation of aldehyde 36q.
7
o
Reagents and conditions: (i) [Bis(trifluoroacetoxy)iodo]benzene (PIFA), TFA, BF3—Et2O, 40ꢀ60 oC, 48 h, 25%; (ii) MeLi, THF, 0 C, 16
8
9
h, 34%; (iii) nꢀBuLi, THF, DMF, ꢀ78 oC, 1 h, 69%.
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11
12
13
14
15
16
17
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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
Scheme 4 Preparation of aldehyde 36t.
Reagents and conditions: (i) MeMgBr, THF, ꢀ78 oC, 1h; then HCl, water, 25 oC, 24h, 73%; (ii) N, Nꢀdimethylformamide, toluene, reflux,
16 h, 75%; (iii) HCl, DCM, reflux, 2 h, 98%; (iv) DIBAL, THF, ꢀ78 oC, 1 h, 64%; (v) Methyltriphenylphosphonium bromide, nꢀBuLi, 25
oC, 1 h, 42%; (vi) CH2I2, ZnEt2, DCM, TFA, 0 oC, then 53, 25 oC, 16 h, 45%; (vii) nꢀBuLi, THF, DMF, ꢀ78 oC, 1 h, 46%.
The preparation of 36w1 and 36w2 were outline in Scheme 5. Wittig reaction of 55 produced
56 which was then reduced, under Ru catalyzed hydrogenation, to give ester 57. TsOH promoted ring
closure of 57 afforded lactone 58, which was reduced to semiꢀketal 59 under DIBAlꢀH condition.
Treatment of 59 with MeMgBr produced compound 60. Intraꢀmolecular Mitsunobu reaction of
compound 60 afforded the racemic ring closure product, which was resolved by chiral SFC to give
enantiomers 61 and 62. Both 61 and 62 were converted to the corresponding aldehydes 36w1 and 36w2
under the nꢀBuLiꢀDMF conditions. The stereochemistry of 36w1 and 36w2 were assigned arbitrarily.
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5
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7
8
9
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14
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17
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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
Scheme 5 Preparation of aldehydes 36w1 and 36w2.
Reagents and conditions: (i) Ph3=CHCO2Et, DCM, 88%; (ii) Ru(PPh3)3Cl, MeOH, H2, 25 oC, 16 h; (iii) TsOH ·H2O, Toluene, 88%; (iv)
DIBAlꢀH, ꢀ78 oC, 2 h, 71%; (v) MeMgBr, 25 oC, 2 h, 61%; (vi) PPh3, DIAD, DCM, 0 oC, 1 h, 72%, then chiral SFC separation; (vii) 61,
nꢀBuLi, THF, ꢀ78 oC, DMF, 1 h, afforded 36w1 in 87%. 36w2 was obtained from 62 in the same manner.
For the preparation of additional chromane aldehyde derivatives, please see the experimental
section.
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Journal of Medicinal Chemistry
1
2
3
Experimental
4
5
6
7
8
9
Reagents were purchased from commercial suppliers and were used without further purification
unless otherwise specified. Normal phase column chromatography was carried out using prepacked
silica gel cartridges for use on the Isco CombiFlash Companion. Analytical thin layer chromatography
(TLC) visualization was performed using 254 nm wavelength ultraviolet light. Preparative HPLC
purifications (reverseꢀphase) were performed using a Gilson 215 Liquid Handler typically with a
Phenomenex Luna C18(2) 100×21.2mm 10 micron column. A 15 min run (14 mL/min, 10% to 90%
MeCN/H2O, with 0.1%TFA) with UV detection at 254 nm or 210 nm was typically used.
SFC separations were carried out on a Thar SFC instrumentation. Column was specified in each
separation; mobile phase A, CO2, mobile phase B, MeOH, back pressure 100 bar, flow rate 25ꢀ50
mL/min, detection at 210−350 nm.
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36
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43
44
45
46
47
48
49
50
51
52
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58
59
60
Nuclear magnetic resonance spectra were recorded on a Varian 400 or 600 MHz spectrometer.
Residual solvent signal was used as internal standard for chemical shift assignments. Spectra were
taken in the indicated solvent at ambient temperature, and the chemical shifts are reported in parts per
million (ppm (δ)) relative to the lock of the solvent used. Resonance patterns are recorded with the
following notations: br (broad), s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).
The purity of the final compounds was determined based on LCMS data acquired 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.
All final compounds were >95% pure by this method. HRMS analysis was carried on Waters Acquity
UPLC and Waters Xevo G2 QTof. Column: Acquity UPLC BEH C18 1.7um, flow rate 0.8 mL/min,
Mobile phase A: ammonium formate solution, 5mM in water + 0.05% ammonium Hydroxide, Mobile
phase B: 0.025% ammonium hydroxide in acetonitrile, Gradient: 5% to 98% B in A.
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1
2
3
4
Virology Assay
5
6
7
8
The tabulated activity data are mean values of at least two experiments, and in general, the
replicon assay exhibits a maximum of 3ꢀfold variation. Detailed replicon biological assays were
described previously.30,31 Briefly, the resistanceꢀassociated variants in NS5A were each introduced into
GT 1aꢀH77, GT 1bꢀCon1, or chimeric replicons. In transient assays, the replicon containing the variant
was transfected via electroporation into a Huh7 derived cell line. The percent inhibition of HCV
replicon replication as determined by decreased luciferase signal was calculated for each compound
concentration, and the EC90 values were calculated as described above.
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60
PK Assessment
Rat and dog PK studies (24 h): Compounds were dosed in overnight fasted rats and dogs via IV
and PO administration. Two animals were included in each dosing group. The IV dose was
administered in 60% PEG200 at 2 mg/kg in rats and 1 mg/kg in dogs whereas oral dose was
administrated in 10% Tween at 10 mg/kg in rats and 5 mg/kg in dogs. Blood was collected up to 24
hours and plasma was obtained by centrifugation of the blood. Compound concentrations in plasma
were detected by LC/MS analysis.
Mammalian Cytotoxicity Assay
The Clickꢀit EdU Alexa Fluor 488 HCS assay kit ( Life technologies, C10351) was used to
assess the potential cytotoxicity in mammalian cells using a modified version of the manufacturer’s
protocol. For the assay, Huh7 cells were seeded at 1,200 per well of a 384ꢀwell polyꢀDꢀlysine coated
plates (Greiner, 781946) in 25 µl of culture medium (Life Technologies) and treated with a 20ꢀpoint
twofold dilution series of compounds. Cells and compounds are incubated for 72 hours, with Edu
added for the last 18ꢀ20 hours. The plates were then fixed, permeabilized and stained with Hoechst as
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per manufacturer’s protocol. Alexa Fluor 488 dye was added to the EdU by way of the clickꢀit reaction
as described in the manufacturer’s protocol. Plates were scanned on the Acumen eX3 (TTP LabTech).
Hoechst was detected using the 405 laser with the 420ꢀ500nm bandpass filter, and EdUꢀAF488 was
detected using the 488 laser with the 500ꢀ530 nm bandpass filter.
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Huh7 cells containing HCV replicon were grown based on literature method.34 The
authentication of the cell line is accomplished by the ability to grow in selective culture conditions with
the antibiotic G418. Controls are included in each assay run to confirm the functioning of the assay.
Cells are routinely checked for mycoplasma by using the LONZA kit, MycoAlert Assay.
Compounds 3ꢀ13, 15ꢀ29 were prepared in similar methods as compound 14.
Methyl
[(1S)ꢀ1ꢀ({(2S)ꢀ2ꢀ[5ꢀ(1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ[(methoxycarbonyl)amino]ꢀ3ꢀ
methylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}ꢀ6ꢀnaphthalenꢀ2ꢀylindolo[1,2ꢀc][1,3]benzoxazinꢀ3ꢀ
yl)ꢀ1Hꢀimidazolꢀ2ꢀyl]pyrrolidinꢀ1ꢀyl}carbonyl)ꢀ2ꢀmethylpropyl]carbamate (3). The more active isomer
1
at the aminal carbon and the absolute stereochemistry at the aminal carbon was not defined. HꢀNMR
(400MHz, CD3OD) δ: 7.99 (s, 1 H), 7.82ꢀ7.74 (m, 3H), 7.67 (m, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.46ꢀ7.41 (m, 3H),
7.40ꢀ7.20 (m, 6H), 5.24ꢀ 5.14 (m, 2 H), 4.23ꢀ 4.18 (m, 2 H), 4.06 (m, 2 H), 3.86 (m, 2 H), 3.63 (s, 6 H), 2.53ꢀ 2.47 (m,
2 H), 2.26ꢀ 2.01 (m, 8 H), 0.93ꢀ0.84 (m, 12 H). MS (ESI) m/z calcd for C53H57FN9O7 [M+H]+, 950.4;
found, 950.5.
methyl
[(1S)ꢀ1ꢀ({(2S)ꢀ2ꢀ[5ꢀ(1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ[(methoxycarbonyl)amino]ꢀ3ꢀ
methylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}ꢀ6ꢀnaphthalenꢀ1ꢀylindolo[1,2ꢀc][1,3]benzoxazinꢀ3ꢀ
yl)ꢀ1Hꢀimidazolꢀ2ꢀyl]pyrrolidinꢀ1ꢀyl}carbonyl)ꢀ2ꢀmethylpropyl]carbamate (4). The more active isomer
1
at the aminal carbon and the absolute stereochemistry at the aminal carbon was not defined. HꢀNMR
(400MHz, CD3OD) δ: δ 8.63 (d, J = 8.4 Hz, 1H), 8.36 (s, 1 H), 8.06 (s, 1 H), 7.90 (d, J = 8.0 Hz, 1H),
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7.82 (d, J = 8.0 Hz, 1H), 7.71 (s, 1 H), 7.68 (m, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 1.6 Hz, 1H),
7.29ꢀ7.23 (m, 3H), 7.11 (1, 3H), 7.03 (s, 1H), 6.13 (d, J = 7.2 Hz, 1H), 5.23ꢀ 5.06 (m, 2 H), 4.20 (d, J =
7.2 Hz, 1H), 4.11 (d, J = 7.2 Hz, 1H), 4.13ꢀ 4.00 (m, 2 H), 3.86ꢀ3.77 (m, 2 H), 3.60 (s, 6 H), 2.56ꢀ 2.47
(m, 2 H), 2.24ꢀ 1.92 (m, 8 H), 0.91ꢀ0.76 (m, 12 H). MS (ESI) m/z calcd for C53H57FN9O7 [M+H]+,
950.4; found, 950.5.
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methyl
[(1S)ꢀ1ꢀ({(2S)ꢀ2ꢀ[5ꢀ(1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ[(methoxycarbonyl)amino]ꢀ3ꢀ
methylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}ꢀ6ꢀquinolinꢀ7ꢀylindolo[1,2ꢀc][1,3]benzoxazinꢀ3ꢀyl)ꢀ
1Hꢀimidazolꢀ2ꢀyl]pyrrolidinꢀ1ꢀyl}carbonyl)ꢀ2ꢀmethylpropyl]carbamate (5). The more active isomer at
1
the aminal carbon and the absolute stereochemistry at the aminal carbon was not defined. HꢀNMR
(400MHz, CD3OD) δ: 8.76 (d, J =1.2 Hz, 1 H), 8.38 (d, J =8 Hz, 1 H), 8.11 (s, 1 H), 8.00ꢀ 7.98 (m, 2
H), 7.86 (s, 1 H), 7.77 (m, 1 H), 7.64 (s, 1 H), 7.57ꢀ 7.55 (s, 1 H), 7.34ꢀ 7.29 (m, 3H), 7.23 (s, 1 H),
5.25ꢀ 5.13 (m, 2 H), 4.22ꢀ 4.06 (m, 4 H), 3.88ꢀ 3.77 (m, 2 H), 3.64ꢀ 6.63 (m, 6 H), 2.59ꢀ 2.46 (m, 2 H),
2.26ꢀ 1.95 (m, 8 H), 0.97ꢀ0.83 (m, 12 H). MS (ESI) m/z calcd for C52H56FN10O7 [M+H]+, 951.4;
found, 951.4.
Methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[6ꢀ(2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)ꢀ1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ
[(methoxycarbonyl)amino]ꢀ3ꢀmethylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}indolo[1,2ꢀ
c][1,3]benzoxazinꢀ3ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀmethylpropyl]carbamate
(6). The more active isomer at the aminal carbon and the absolute stereochemistry at the aminal carbon
1
was not defined. HꢀNMR (400MHz, CD3OD) δ: 8.03 (s, 1 H), 7.86 (s, 1 H), 7.73 (s, 1 H), 7.55 (s, 1
H), 7.46 (dd, J =8.0, 1.2 Hz, 1 H), 7.32ꢀ7.21 (m, 4 H), 7.10 (d, J =7.6 Hz, 1 H), 6.86 (s, 1 H), 6.78 (d, J
=7.6 Hz, 1 H), 5.25ꢀ 5.15 (m, 2 H), 4.20 (m, 2 H), 4.10 (m, 2 H), 3.86ꢀ 3.82 (m, 2 H), 3.64 (s, 6 H),
2.82ꢀ 2.74 (m, 4 H), 2.56ꢀ 2.49 (m, 2 H), 2.26ꢀ 1.97 (m, 10 H), 0.92ꢀ0.89 (m, 12 H). MS (ESI) m/z
calcd for C52H59FN9O7 [M+H]+, 940.4; found, 940.5.
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methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ[(methoxycarbonyl)amino]ꢀ3ꢀ
methylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}ꢀ6ꢀ(5,6,7,8ꢀtetrahydronaphthalenꢀ2ꢀyl)indolo[1,2ꢀ
c][1,3]benzoxazinꢀ3ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀmethylpropyl]carbamate (7).
The more active isomer at the aminal carbon and the absolute stereochemistry at the aminal carbon was
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not defined. HꢀNMR (400MHz, CD3OD) δ: 8.01 (s, 1 H), 7.87 (s, 1 H), 7.74 (s, 1 H), 7.44 (m, 2 H),
7.29ꢀ 7.20 (m, 4 H), 6.95 (d, J =8.0 Hz, 1 H), 6.75ꢀ 6.69 (m, 2 H), 5.25ꢀ 5.17 (m, 2 H), 4.22 (m, 2 H),
4.09 (m, 2 H), 3.86 (m, 2 H), 3.65 (s, 6 H), 2.68ꢀ 2.48 (m, 7 H), 2.28ꢀ 2.03 (m, 8 H), 1.72 (m, 4 H),
0.94 (m, 12 H). MS (ESI) m/z calcd for C53H61FN9O7 [M+H]+, 954.4; found, 954.5.
methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[6ꢀ(1,3ꢀdihydroꢀ2ꢀbenzofuranꢀ5ꢀyl)ꢀ1ꢀfluoroꢀ3ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ
[(methoxycarbonyl)amino]ꢀ3ꢀmethylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}indolo[1,2ꢀ
c][1,3]benzoxazinꢀ10ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀmethylpropyl]carbamate(8).
The more active isomer at the aminal carbon and the absolute stereochemistry at the aminal carbon was
not defined. 1HꢀNMR (400MHz, CD3OD) δ: 8.02 (s, 1 H), 7.77 ꢀ 7.74 (m, 2 H), 7.64 (s, 1 H), 7.47 (m,
1 H), 7.33 ꢀ 7.29 (m, 2 H), 7.25 ꢀ 7.20 (m, 1 H), 7.18ꢀ 6.92 (m, 2 H), 6.95 (m, 2 H), 5.24 (m, 2 H), 4.96
ꢀ 4.87 (m, 4 H), 4.20 (t, J =8 Hz, 2 H), 4.08 (m, 2 H), 3.84 (t, J =7.6 Hz, 2 H), 3.63 (s, 6 H), 2.56 – 2.48
(m, 2 H), 2.25 ꢀ 2.20 (m, 8 H), 0.96 ꢀ 0.86 (m, 12 H). MS (ESI) m/z calcd for C51H57FN9O8 [M+H]+,
942.4; found, 942.5.
methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[6ꢀ(2,3ꢀdihydroꢀ1ꢀbenzofuranꢀ6ꢀyl)ꢀ1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ
[(methoxycarbonyl)amino]ꢀ3ꢀmethylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}indolo[1,2ꢀ
c][1,3]benzoxazinꢀ3ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀmethylpropyl]carbamate (9).
Mixture of diastereomers at the aminal carbon. 1HꢀNMR (400MHz, CD3OD) δ: 8.02 (s, 1 H), 7.83 (s, 1
H), 7.71 (s, 1 H), 7.51ꢀ7.33 (m, 2 H), 7.25ꢀ7.12 (m, 4 H), 7.06 (d, J =8 Hz, 1 H), 6.42 – 6.37 (m, 2 H),
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5.25 ꢀ 5.22 (m, 2 H), 4.45 (t, J =8.8 Hz, 2 H), 4.21 (m, 2 H), 4.06 (m, 2 H), 3.83 (m, 2 H), 3.64 (s, 6 H),
3.09 (t, J =8.8 Hz, 2 H), 2.52 (m, 2 H), 2.30 ꢀ 2.00 (m, 8 H), 0.96 ꢀ 0.80 (m, 12 H). MS (ESI) m/z calcd
for C51H57FN9O8 [M+H]+, 942.4; found, 942.4.
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methyl [(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[6ꢀ(1,3ꢀbenzodioxolꢀ5ꢀyl)ꢀ1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ
[(methoxycarbonyl)amino]ꢀ3ꢀmethylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}indolo[1,2ꢀ
c][1,3]benzoxazinꢀ3ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀmethylpropyl]carbamate(10).
The more active isomer at the aminal carbon and the absolute stereochemistry at the aminal carbon was
1
not defined. HꢀNMR (400MHz, CD3OD) δ: 7.98 (s, 1 H), 7.82 (s, 1H), 7.73 (s, 1 H), 7.49ꢀ7.46 (m, 2
H), 7.33ꢀ7.25 (m, 3 H), 7.19 (s, 1 H), 6.68 (d, J =8 Hz, 1 H), 6.58 (d, J =1.2 Hz, 1 H), 6.38 (d, J =8 Hz,
1 H), 5.89 (s, 2 H), 5.25ꢀ5.15 (m, 2 H), 4.23ꢀ4.19 (t, J =8 Hz, 2 H), 4.09ꢀ4.07 (m, 2 H), 3.87ꢀ3.83 (m, 2
H), 3.64 (s, 6 H), 2.56ꢀ2.47 (m, 2 H), 2.26ꢀ2.00 (m, 8 H), 0.97ꢀ0.87 (m, 12 H). MS (ESI) m/z calcd for
C50H55FN9O9 [M+H]+, 944.4; found, 944.4.
methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[6ꢀ(2,2ꢀdifluoroꢀ1,3ꢀbenzodioxolꢀ5ꢀyl)ꢀ1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ
[(methoxycarbonyl)amino]ꢀ3ꢀmethylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}indolo[1,2ꢀ
c][1,3]benzoxazinꢀ3ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀmethylpropyl]carbamate (11).
The more active isomer at the aminal carbon and the absolute stereochemistry at the aminal carbon was
1
not defined. HꢀNMR (400MHz, CD3OD) δ: 8.05 (s, 1 H), 7.88 (s, 1H), 7.76 (s, 1 H), 7.70 (s, 1 H),
7.53 (dd, d= 8.0Hz, 1.2 HZ, 1 H), 7.42ꢀ7.22 (m, 4 H), 7.06 (d, J =8.4 Hz, 1 H), 6.98 (d, J =1.2 Hz, 1 H),
6.60 (dd, d= 8.4 Hz, 1.2 HZ, 1 H), 5.25ꢀ5.15 (m, 2 H), 4.23ꢀ4.19 (m, 2 H), 4.09ꢀ4.07 (m, 2 H), 3.87ꢀ
3.83 (m, 2 H), 3.64 (s, 6 H), 2.55ꢀ2.47 (m, 2 H), 2.26ꢀ2.02 (m, 8 H), 0.97ꢀ0.86 (m, 12 H). MS (ESI) m/z
calcd for C50H53F3N9O9 [M+H]+, 980.4; found, 980.4.
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methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[6ꢀ(3,4ꢀdihydroꢀ2Hꢀchromenꢀ6ꢀyl)ꢀ1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ
[(methoxycarbonyl)amino]ꢀ3ꢀmethylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}indolo[1,2ꢀ
c][1,3]benzoxazinꢀ3ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀmethylpropyl]carbamate (12).
The more active isomer at the aminal carbon and the absolute stereochemistry at the aminal carbon was
not defined. 1HꢀNMR (400MHz, CD3OD) δ: 8.00 (s, 1 H), 7.87 (s, 1 H), 7.72 (s, 1 H), 7.33 ꢀ 7.30 (m, 2
H), 7.31 (m, 1 H), 7.19 ꢀ 7.14 (m, 3 H), 6.73 (m, 2 H), 6.64 (m, 1 H), 5.24 ꢀ 5.16 (m, 2 H), 4.20 (t, J =
8.4 Hz, 2 H), 4.12 ꢀ 4.07 (m, 4 H), 3.93 ꢀ 3.76 (m, 2 H), 3.63 (s, 6 H), 2.61 (m, 2 H), 2.59 ꢀ 2.49 (m, 2
H), 2.31 ꢀ 2.21 (m, 2 H), 2.24 ꢀ 2.14 (m, 4 H), 2.06 (m, 2 H), 1.89 (m, 2 H), 0.99 – 0.89 (m, 12 H). MS
(ESI) m/z calcd for C52H59FN9O8 [M+H]+, 956.4; found, 956.5.
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methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[6ꢀ(3,4ꢀdihydroꢀ1Hꢀisochromenꢀ7ꢀyl)ꢀ1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ
[(methoxycarbonyl)amino]ꢀ3ꢀmethylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}indolo[1,2ꢀ
c][1,3]benzoxazinꢀ3ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀmethylpropyl]carbamate (13).
The more active isomer at the aminal carbon and the absolute stereochemistry at the aminal carbon was
1
not defined. HꢀNMR (400MHz, CD3OD) δ: 7.93 (s, 1 H), 7.82 (s, 1 H), 7.70 (s, 1 H), 7.44 (s, 1 H),
7.30 ꢀ 7.11 (m, 3 H), 7.00 (J =7.6 Hz,1 H), 6.71 (m, 2 H), 5.25 – 5.18 (m, 2 H), 4.58 ꢀ 4.53(m, 2 H),
4.24 ꢀ 4.20 (m, 2 H), 4.07 (m, 2 H), 3.90 – 3.88 (m, 4 H), 3.82 (s, 6 H), 2.72 (s, 2 H), 2.53 (m, 2 H),
2.25 – 2.00 (m, 9 H), 0.96 – 0.86 (m, 12 H). MS (ESI) m/z calcd for C52H59FN9O8 [M+H]+, 956.4;
found, 956.5.
Dimethyl
((2S,2'S)ꢀ((2S,2'S)ꢀ((6ꢀ(chromanꢀ7ꢀyl)ꢀ1ꢀfluoroꢀ6Hꢀbenzo[5,6][1,3]oxazino[3,4ꢀa]indoleꢀ
3,10ꢀdiyl)bis(1Hꢀimidazoleꢀ5,2ꢀdiyl))bis(pyrrolidineꢀ2,1ꢀdiyl))bis(3ꢀmethylꢀ1ꢀoxobutaneꢀ1,2ꢀ
diyl))dicarbamate (14). To a mixture of 41l (355 mg, 0.45 mmol), (methoxycarbonyl)ꢀLꢀvaline (0.99
mmol) and DIPEA (3.6 mmol) in DMF (5 mL) was added BOP (1.0 mmol). The resulting mixture was
stirred at room temperature until LCMS indicated 41l was consumed up. The mixture was purified by
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PreꢀHPLC to give a mixture of diastereomers of compound 14 and its other isomer (210 mg, 48.8 %
yield). MS (ESI) m/z (M+H+): 956.5. The mixture were separated by chiral SFC to give 14 (69 mg,
33%) and its other diastereomer . SFC condition: Chiralpak ASꢀH, 250×20 mm, 5 µm, Mobile phase:
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40% of EtOH (0.05%DEA) in CO2, Flow rate: 50 mL/min , wavelength: 220 nm. Compound 14: Hꢀ
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NMR (400MHz, MeOHꢀd4) δ: 7.92 (s, 1 H), 7.81 (s, 1 H), 7.69 (s, 1 H), 7.41 ꢀ 7.46 (m, 2 H), 7.25 ꢀ
7.28 (m, 2 H), 7.14 (m, 1 H), 7.08 ꢀ 7.09 (m, 1 H), 6.88 ꢀ 6.90 (m, 1 H), 6.40 ꢀ 6.42 (m, 1 H), 6.29 (s, 1
H), 5.17 ꢀ 5.23 (m, 2 H), 4.20 ꢀ 4.24 (m, 2 H), 4.00 ꢀ 4.08 (m, 4 H), 3.85 ꢀ 3.89 (m, 2 H), 3.63 (s, 6 H),
2.62 ꢀ 2.64 (m, 2 H), 2.48 ꢀ 2.57 (m, 2 H), 2.21 ꢀ 2.32 (m, 2 H), 2.05 ꢀ 2.17 (m, 6 H), 0.86 ꢀ 0.96 (m, 12
13
H). CꢀNMR (600MHz, MeOHꢀd4) δ: 174.3, 174.2, 161.4, 159.7, 159.5, 156.4, 152.4, 152.3, 150.7,
149.5, 136.9, 136.4, 135.7, 131.3, 130.6, 128.3, 125.5, 122.0, 120.7, 119.4, 119.2, 117.2, 115.7, 114.8,
111.9, 111.6, 107.9, 107.8, 103.8, 85.3, 67.5, 59.9, 55.2, 55.0, 52.8, 32.5, 32.4, 31.1, 26.3, 25.5, 23.1,
19.9, 18.2. HRMS (ESI+) m/z calcd for C52H59FN9O8 [M+H]+, 956.4471; found, 956.4495.
Methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[1ꢀfluoroꢀ10ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ[(methoxycarbonyl)amino]ꢀ3ꢀ
methylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}ꢀ6ꢀ(2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)indolo[1,2ꢀ
c][1,3]benzoxazinꢀ3ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀmethylpropyl]carbamate (15).
The more active isomer at the aminal carbon. The absolute stereochemistry at the aminal carbon was
not defined. MS (ESI) m/z calcd for C52H55FN9O9 [M+H]+, 968.4; found, 968.7.
methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[1ꢀfluoroꢀ3ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ[(methoxycarbonyl)amino]ꢀ3ꢀ
methylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}ꢀ6ꢀ(3ꢀoxoꢀ3,4ꢀdihydroꢀ2Hꢀ1,4ꢀbenzoxazinꢀ7ꢀ
yl)indolo[1,2ꢀc][1,3]benzoxazinꢀ10ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀ
methylpropyl]carbamate (16). The more active isomer at the aminal carbon. The absolute
1
stereochemistry at the aminal carbon was not defined. HꢀNMR (400MHz, CD3OD) δ: 8.00 (s, 1H),
7.84 (s, 1H), 7.74 (s, 1H), 7.53−7.31 (m, 2H), 7.24−7.17 (m, 2H), 6.78−6.76 (m, 1H), 6.59−6.56 (m,
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Journal of Medicinal Chemistry
2H), 5.26−5.18 (m, 2H), 4.47 (s, 2H), 4.25−4.21 (m, 2H), 4.09 (s, 2H), 3.90−3.88 (m, 2H), 3.65 (s,
6H), 2.65−2.54 (m, 2H), 2.10−2.05 (m, 8H), 2.10−2.05 (m, 8H), 0.95−0.88 (m, 12H). MS (ESI) m/z
calcd for C51H56FN10O9 [M+H]+, 971.4; found, 971.4.
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methyl [(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[1ꢀfluoroꢀ3ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ[(methoxycarbonyl)amino]ꢀ3ꢀ
methylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}ꢀ6ꢀ(4ꢀmethylꢀ3ꢀoxoꢀ3,4ꢀdihydroꢀ2Hꢀ1,4ꢀbenzoxazinꢀ
7ꢀyl)indolo[1,2ꢀc][1,3]benzoxazinꢀ10ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀ
methylpropyl]carbamate (17). The more active isomer at the aminal carbon and the absolute
1
stereochemistry at the aminal carbon was not defined. HꢀNMR (400MHz, CD3OD) δ: 7.82−7.72 (m,
2H), 7.34−7.12 (m, 6H), 7.05−6.78 (m, 2H), 6.55−6.49 (m, 2H), 5.16−5.09 (m, 2H), 4.60 (s, 2H),
4.37−4.20 (m, 4H), 4.09−3.76 (m, 4H), 3.45−3.28 (m, 6H), 3.09 (s, 3H), 2.26−2.00 (m, 8H), 1.27−1.12
(m, 6H), 0.93−0.89 (m, 6H). MS (ESI) m/z calcd for C52H58FN10O9 [M+H]+, 985.4; found, 985.4.
methyl
[(1S)ꢀ1ꢀ{[(2S)ꢀ2ꢀ{5ꢀ[1ꢀfluoroꢀ3ꢀ{2ꢀ[(2S)ꢀ1ꢀ{(2S)ꢀ2ꢀ[(methoxycarbonyl)amino]ꢀ3ꢀ
methylbutanoyl}pyrrolidinꢀ2ꢀyl]ꢀ1Hꢀimidazolꢀ5ꢀyl}ꢀ6ꢀ(2ꢀmethylꢀ3,4ꢀdihydroꢀ2Hꢀchromenꢀ6ꢀ
yl)indolo[1,2ꢀc][1,3]benzoxazinꢀ10ꢀyl]ꢀ1Hꢀimidazolꢀ2ꢀyl}pyrrolidinꢀ1ꢀyl]carbonyl}ꢀ2ꢀ
methylpropyl]carbamate (18). Compounds 18, 19, and other two less potent diastereomers were
separated by SFC. Conditions: Chiralpak ASꢀH 250×20 mm, 5 µm, Mobile phase: 40% of EtOH
1
(0.05% DEA) in CO2, Flow rate: 50 mL/min, wavelength: 220 nm. HꢀNMR (400MHz, CD3OD) δ:
7.95 (s, 1H), 7.78 (s, 1H), 7.71 (s, 1H), 7.43−7.28 (m, 3H), 7.17−7.13 (m, 3H), 6.76−6.71 (m, 2H),
6.62−6.59 (m, 1H), 5.24−5.14 (m, 2H), 4.22−4.19 (m, 2H), 4.07−4.02 (m, 3H), 3.87−3.85 (m, 2H),3.68
(s, 6H), 2.67−2.46 (m, 4H), 2.23−1.91 (m, 9H), 1.51−1.59 (m, 1H), 1.29 (d, J=6.4 Hz, 3H), 0.92−0.86
(m, 12H). MS (ESI) m/z calcd for C53H61FN9O8 [M+H]+, 970.5; found, 970.7.
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