Structure–activity relationships of proline modifications around the tetracyclic-indole class of NS5A inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.08.097

We describe the impact of proline modifications, in our tetracyclic-indole based series of nonstructural protein 5A (NS5A) inhibitors, to their replicon profiles. This work identified NS5A inhibitors with an improved and flattened resistance profiles.

Full text of DOI:10.1016/j.bmcl.2016.08.097

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of proline modifications around
the tetracyclic-indole class of NS5A inhibitors
a
a
a
a
a
Ling Tong a, , Wensheng Yu , Craig A. Coburn , Lei Chen , Oleg Selyutin , Qingbei Zeng ,
Michael P. Dwyer a, Anilkumar G. Nair a, Bandarpalle B. Shankar a, Seong Heon Kim a, De-Yi Yang a,
Stuart B. Rosenblum a, Rebecca T. Ruck a, Ian W. Davies a, Bin Hu b, Bin Zhong b, Jinglai Hao b, Tao Ji b,
Shuai Zan b, Rong Liu a, Sony Agrawal a, Donna Carr a, Stephanie Curry a, Patricia McMonagle a, Karin Bystol a,
Frederick Lahser a, Paul Ingravallo a, Shiying Chen a, Ernest Asante-Appiah a, Joseph A. Kozlowski a
⇑
a Merck & Co., Inc., 126 E. Lincoln Ave., Rahway, NJ 07065, USA
b Department of Medicinal Chemistry, WuXi AppTec, Shanghai 200131, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the impact of proline modifications, in our tetracyclic-indole based series of nonstructural
protein 5A (NS5A) inhibitors, to their replicon profiles. This work identified NS5A inhibitors with an
improved and flattened resistance profiles.
Received 15 August 2016
Accepted 31 August 2016
Available online xxxx
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
HCV NS5A inhibitor
Elbasvir
MK-8742
Direct-acting antiviral agents
HCV infection treatment
Indole based tetracyclic core
Hepatitis C virus (HCV) is currently the leading cause of chronic
liver disease and has become the most common blood-borne infec-
tion in developed countries.1 Because HCV infection is silent in its
early stages, most individuals are not aware they are carrying the
virus, unless they undergo screening for it. If left untreated HCV
can progress to the point where it results in debilitating liver dis-
eases such as cirrhosis or liver cancer1 and ultimately liver trans-
plantation.2 HCV displays a high degree of genetic diversity and
can be classified into seven genotypes (GT1–7) and more than 50
subtypes.3 Additionally, recombinant forms of the virus have been
identified, however their importance has yet to be established.3
Early therapy for HCV involved the use of pegylated-interferon
and ribavirin, which was limited by both efficacy and tolerability.4
A detailed characterization of the HCV lifecycle was important to
advancing treatments for HCV as it allowed researchers to directly
target specific steps for intervention in HCV replication. These
agents are now known as direct-acting antiviral agents (DAAs).5,6
In particular, it is the combination of several DAAs, each targeting
changed the outcomes for HCV patients and allowed for inter-
feron-free regimens, with significantly improved rates of sustained
virologic response (SVR).7
Nonstructural protein 5A (NS5A) is a viral phosphoprotein that
is essential for the replication of HCV. NS5A interacts with a num-
ber of host proteins involved in numerous cellular processes
including apoptosis, cell-cycling, innate immunity, and membrane
biogenesis that can be subverted to favor viral replication. NS5A
plays a critical role in viral ribonucleic acid (RNA) synthesis and
virus assembly,8 though its exact mechanistic role in the HCV life
cycle remains enigmatic.9 The potential utility7,10 of an NS5A inhi-
bitor in the treatment of HCV has been demonstrated clinically11
and currently four NS5A inhibitors are in use for the treatment of
HCV including daclatasvir (DCV),12 ledipasvir (LDV),13 ombitasvir
(OBV),14 and elbasvir15(EBR), Figure 1.
We sought to determine if it is possible to identify an NS5A
inhibitor, with potent inhibitory activity against all genotypes
and clinically-relevant resistance-associated variants (RAVs). In
particular, our goal was to identify a ‘flat’ compound, with flat as
defined by a minimal potency shift (ꢀ10ꢁ) between the wild-type
virus and clinically relevant variants. Our previous efforts16–19
indicated that structural variations could influence the relative
a
different mechanism, into one regimen that has radically
⇑
Corresponding author.
E-mail address: ling.tong@merck.com (L. Tong).
http://dx.doi.org/10.1016/j.bmcl.2016.08.097
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tong, L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.097

2
L. Tong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Boc
N
R7
N
O
O
Br
O
Boc
N
Boc
N
O
N
N
b
O
a
N
H
H3CO
OCH3
N
H
N
N
N
H
N
X
N
N
N
X
N
X
H
H
H
H
3a
O
3b
3c
Z group
Figure 1. Elbasvir (MK-8742).
Scheme 1. Reagents and conditions: (a) Glyoxal, 7 N NH3 in MeOH, rt; (b) NBS, THF
then Na2SO3, EtOH/H2O, reflux.
which were prepared according to literature procedures,20 with
glyoxal in the presence of 7 M ammonia in MeOH afforded the imi-
dazole 3b. Bromination with NBS yielded the intermediate
dibromo adduct which was treated under reductive conditions
employing Na2SO3 to obtain bromide 3c which was ready for
coupling.
A general preparation of symmetric proline analogs 2–6 and
9–15 is shown in Scheme 2. Utilizing a Fischer indole synthesis
protocol,20–22 5-bromoacetophenone 3d was condensed with
p-bromophenylhydrazine under acidic conditions to form the
intermediate hydrazone which was treated at high temperature
activity of various genotype and mutant activities and thus the
‘flatness’ of the in vitro profile could also vary. We had previously
identified compound 1 (Table 1), and thought it was relevant to
achieving the goal outlined above as it showed surprisingly good,
low nanomolar, potency on the key RAVs GT1a_L31V and
GT1a_Y93H,18 and effectively flattened the GT1 mutant ratio. We
therefore used compound 1 as a starting point for further opti-
mization and as a baseline from which to explore the impact of
variations to the proline rings on the flatness of the in vitro profiles.
The preparation of the requisite bromoimidazole fragments 3c
is illustrated in Scheme 1.20–22 Condensation of aldehydes 3a,
Table 1
In vitro potency for compounds 1–8 in replicons
O
O
O
O
N
H3CO
N
R7
OCH3
N
H
N
H
N
N
N
H
P1
P2
N
N
H
O
X
23
No.
P1
P2
Replicon, EC90 (nM)
GT1a L31V
X
R7
F
GT1a
0.01
GT1a Y93H
5.76
GT2by
5.72
GT3a
0.07
N
N
1
OMe
H
0.20
1.72
5.74
2a
3b
H
H
ND
ND
64
75
6.14
51.5
N
N
N
N
H
158
129
N
N
4
5
OMe
OMe
F
F
0.015
0.017
0.19
0.04
0.79
8.87
90.7
5.74
5.52
0.71
O
O
O
O
N
N
N
N
6
OMe
F
2.67
24.5
3.86
322
30.0
O
O
N
N
N
7c
8
H
F
F
0.020
0.002
21.0
0.90
>100
26.1
337
>10
N
OMe
51.8
0.92
ND: not determined.
y
The replicon bears methionine at position 31.
Single diastereomer A.
Single diastereomer B.
a
b
c
Mixture of diastereomers at aminal carbon.
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L. Tong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
F
Br
Br
OH
O
F
Br
Br
N
a
b
Br
O
N
H
F
HO
3d
3e
3f
O
Boc
N
N
F
c
X
N
N
H
N
HN
NBoc
O
X
3h
O
Moc-Val
F
N
N
X
SFC separation
Moc-Val
d,e
N
active isomer
N
H
N
HN
N
2-10
O
X
3i
O
Scheme 2. Reagents and conditions: (a) (1) p-Bromophenylhydrazine, AcOH, EtOH, reflux; 65% then (2) PPA, 110 °C; (b) 3-methoxybenzaldehyde, p-toluenesulfonic acid,
o-xylene, 170 °C, 15 h.; (c) bis(pinacolato)diboron, KOAc, PdCl2(dppf), dioxane, 110 °C then bromoimidazole 3c, 1 M Na2CO3, Pd(dppf)Cl2, THF, 90 °C; (d) 4 N HCl, MeOH; (e)
Moc-L-valine, HATU, DIPEA, DMF.
with polyphosphoric acid (PPA) to afford indole 3e. Treatment of
3e with 3-methoxybenzaldehyde, p-toluenesulfonic acid in o-
xylene at 170 °C gave 3f. Treatment of dibromide intermediate 3f
with two equivalents of bis(pinacolato)diboron in the presence of
KOAc and Pd(dppf)Cl2 afforded the intermediate bis-pinacol boro-
nic ester, the key coupling partner. Reaction of this core under pal-
ladium catalysis with two equivalents of bromoimidazole
intermediates 3c afforded the bis Boc adducts 3h. Global deprotec-
tion with HCl followed by HATU promoted double amide coupling
(S)-isomer 5, but was weaker versus GT2b and GT3a. While neither
compound 4 or 5 displayed an overall profile that was improved as
compared to 1, it did demonstrate that both genotype and mutant
potencies were sensitive to changes at the 4-position. The prefer-
ence of 4-proline to 3-proline was evident in comparison between
compounds 4 and 6. We also investigated the impact of fusing the
3,4 positions with a cyclopropyl group. This modification proved
to be detrimental to potency as exemplified by compound 7. Bridg-
ing the 3 and 5 positions on the proline, with a second ring, as shown
by compound 8 also failed to demonstrate meaningful improve-
ment. Overall, the data in Table 1 suggested the methoxy proline
at the 4-position was preferred, but in comparison to 1, we did not
see an improvement in the overall profile.
We next expanded the diversity of proline variants at the 4-
position of the proline ring, with a focus on improving the
GT1a_Y93H activity. We were encouraged by the improvement
that compound 4 showed versus this mutant and wanted to see
if we could broaden its activity profile to also cover GT1a_L31V,
GT2b and GT3a. With this objective in mind we maintained the
beta-configuration for the substituents described in Table 2, ana-
logs 9–15, with the variations made simultaneously to both proline
rings (P1 = P2). The 4-fluoro analog 9 improved the GT1a_Y93H
potency into the picomolar range, but it also showed a detrimental
effect in GT2b potency. The difluoromethoxy derivative 10 was less
potent in GT1a_Y93H, when compared with its methoxy counter-
part 4 and was also weaker in GT2b and GT3a (10 vs 4). Spiro-fused
rings were also examined, as exemplified by 11 and 12. Compound
11 exhibited potency roughly comparable to 1, while 12 demon-
strated an improvement in GT1a_Y93H (0.44 nM for 12 and
5.76 nM for 1). The fused-cyclopropyl proline analog 13 demon-
strated improved activity for GT1a_Y93H, but exhibited weaker
potency in GT1a_L31V, when compared to 1. Increasing the size
of the ring fused to the proline had a detrimental effect on the
in vitro profiles as illustrated by 14 and 15.
reactions with Moc-L-valine afforded the final compounds as mix-
tures of two epimeric aminal compounds.22 The mixtures were
subjected to SFC chiral separation to afford compounds as the sin-
gle, more potent diastereomer of the pair. Unless otherwise noted,
the more active diastereomer is reported in the SAR tables.
The synthesis of mixed proline analogs begins as in Scheme 3.
Using a Fisher indole synthesis, the 5-bromoacetophenone 11a
was converted to indole 11b. This material was cyclized under
the standard conditions to afford 11c which was ready for selective
functionalization of the bromide. Intermediate 11c was subjected
to the borylation/Suzuki coupling protocol used previously utiliz-
ing Pd(dppf)Cl2 as catalyst to afford the mono-coupled material
which after HCl deprotection and capping yielded chloride 11d.
With compound 11d in hand, treatment with Pd2(dba)3 in the
presence of bis(pinacolato)diboron and KOAc afforded the interme-
diate boronate which was treated directly with bromoimidazole 3c
to afford the Boc-protected intermediate. Treatment with HCl fol-
lowed by capping afforded compound 11e which underwent SFC
chiral separation to afford final compounds 7–8, 16–21, 22–25
and 27 shown in Tables 1 and 3–6. Compounds in present commu-
nication were synthesized according to previously published
patent proccedures.21,22
Our first intent was to have a general scan for tolerability of
groups and preferred substitution patterns, on the proline rings.
Table 1 summarizes the data for this investigation. For this purpose,
prolines bearing a methyl at the 5 position were examined first.
Compounds 2 and 3, which were diastereomers of methyl group,
both showed lower potency across the board. Interestingly, differ-
ences in the relative activity of the individual diastereomers were
observed, with compound 2 exhibiting a moderately better profile
than compound 3. Activity at the 4 position of the proline ring was
examined with a methoxy substitution. The (R)-methoxy isomer 4
was superior in GT1a_Y93H potency compared to the corresponding
Although the proline analogs 9 and 13 (Table 2) demonstrated
improved GT1a_Y93H potency, their GT2b potency was slightly
lower. Therefore, we sought to investigate the impact of indepen-
dent substitution of either the left or the right proline, on potency.
Table 3 summarizes data for this investigation. The combination of
a 4-fluoro-proline on the left with an unsubstituted proline on the
right, compound 16, showed a profile similar to compound 17,
which possessed the reversed combination of prolines. Both
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L. Tong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
F
Cl
F
Cl
OH
O
Cl
Br
N
a
b
Br
O
N
H
F
11a
HO
11b
11c
O
F
Moc-Val
N
F
N
Cl
N
c,d,e
P1
N
Moc-Val
f, g, h
N
H
Moc-Val
N
N
HN
N
N
HN
O
P2
O
P2
O
11d
O
11e
SFC separation
9-15
Scheme 3. Reagents and conditions: (a) (1). p-Bromophenylhydrazine, AcOH, EtOH, reflux; then (2) PPA, 110 °C; (b) 3-methoxybenzaldehyde, p-toluenesulfonic acid,
o-xylene, 170 °C, 15 h.; (c) bis(pinacolato)diboron, KOAc, PdCl2(dppf)2, dioxane, 110 °C then bromoimidazole 3c, 1 M Na2CO3, Pd(dppf)Cl2, THF, 90 °C; (d) 4 N HCl, MeOH; (e)
Moc-L-valine, HATU, DIPEA, DMF; (f) bis(pinacolato)diboron, KOAc, Pd2(dba)3, dioxane, 120 °C, bromoimidazole 3c, 1 M Na2CO3, THF, 90 °C; (g) 4 N HCl, MeOH; (h) Moc-L-
valine, HATU, DIPEA, DMF.
Table 2
In vitro profile of compounds 9–15 in replicons
O
O
O
O
F
O
N
H
H3CO
OCH3
N
H
N
N
N
N
N
H
P1
P2
N
N
H
Compounds 1, 9-15
OMe
23
No.
Proline
P1 = P2
Genotype, EC90 (nM)
GT1a Y93H
GT1a
GT1a L31V
1.59
GT2by
39.0
GT3a
ND
N
9
0.006
0.021
0.011
0.065
5.27
23.7
F
N
10
11
ND
55.6
38.8
12.9
0.16
F
O
F
N
0.050
N
12a
13
0.018
0.005
0.017
0.20
0.44
0.51
52.6
114
22.5
58
15.0
0.17
0.29
O
O
N
1.72
0.078
N
14
F
F
N
15
0.18
0.51
37.6
21
ND
ND: not determined.
y
The replicon bears methionine at position 31.
Mixture of diastereomers at aminal carbon.
a
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L. Tong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Table 3
In vitro profile of compounds with mixed prolines 16–21 in replicons
O
O
O
O
F
O
N
H
H3CO
OCH3
N
H
N
N
N
N
N
P1
P2
N
H
N
H
Compounds 16-21
OMe
23
No.
P1
P2
Replicon, EC90 nM
GT1a Y93H
GT1a
0.01
GT1a L31V
0.20
GT2by
5.72
GT3a
0.07
N
N
1
5.76
1.81
N
N
16
0.010
0.29
0.34
33.6
18.8
ND
F
N
N
N
17
<0.002
0.55
0.13
F
N
N
18
19
0.007
0.026
0.084
0.36
1.64
10.9
19.4
49.3
0.15
ND
N
N
N
N
20
21
0.032
0.30
0.92
13.6
5.15
91.4
52.2
391
ND
ND
F
F
N
Si
ND: not determined.
y
The replicon bears methionine at position 31.
Table 4
In vitro profile of compounds 22 and 23
Moc-Val
N
R7
N
Moc-Val
N
N
N
H
N
H
N
Z
O
Compounds 22 and 23
23
No.
Z
R7
Replicon, EC90 (nM)
GT1a Y93H
GT1a
0.01
GT1a L31V
GT2by
0.20
(20ꢁ)
5.76
(230ꢁ)
5.72
1
F
(570ꢁ)
O
O
0.42
(26ꢁ)
3.17
(200ꢁ)
16.3
(1000)
22
23
F
0.016
0.013
0.13
(10ꢁ)
6.45
(500ꢁ)
4.66
(360ꢁ)
H
y
The replicon bears methionine at position 31; numbers in parentheses are fold-shift relative to the GT1a potency, set = 1.
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6
L. Tong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 5
In vitro profile of compounds 24–25 in replicons
Moc-Val
N
R7
O
N
Moc-Val
N
N
N
H
N
H
N
Z
F
Compounds 24 and 25
23
No.
Z
R7
Replicon, EC90 (nM)
GT1a Y93H
GT1a
0.01
GT1a L31V
GT2by
0.20
(20ꢁ)
5.76
(230ꢁ)
5.72
1
F
(570ꢁ)
O
O
2.17
(362ꢁ)
1.40
(233ꢁ)
63.6
(10600ꢁ)
24
25
F
0.006
<0.02
0.81
(>41ꢁ)
19.8
(>990ꢁ)
60.8
(>3040ꢁ)
H
y
The replicon bears methionine at position 31; numbers in parentheses are fold-shift relative to the GT1a potency, set = 1.
Table 6
In vitro profile of compounds 26 and 27 in replicons
O
O
O
O
N
H
F
H3CO
OCH3
N
H
N
N
N
A
N
N
N
H
N
H
O
O
Compounds 26 and 27
23
No.
A
Replicon, EC90 nM
GT1a
GT1a L31V
GT1a Y93H
GT2by
0.0025
(1ꢁ)
0.48
(160ꢁ)
0.31
(103ꢁ)
N
26
27
0.003
0.004
0.007
(2ꢁ)
0.12
(30ꢁ)
0.38
(95ꢁ)
N
y
The replicon bears methionine at position 31; numbers in parentheses are fold-shift relative to the GT1a potency, set = 1.
derivatives showed moderate improvement in GT1a_Y93H potency
when compared to 1 while both were weaker in comparison to the
bis-fluoro proline 9. By contrast, there seemed to be a preference
for the fused cyclopropyl proline on the left side (18) over the right
side (19), but interestingly 18 showed improved activity for
GT1a_L31V potency, as compared with the bis-cyclopropyl proline
13 (0.084 nM for 18 vs 1.72 nM for 13). However, compounds 1
and 18 were similar in overall mutant profile with 18 trending
slightly better in GT1a_Y93H. The combination of cyclopropyl-pro-
line (left) and fluoro-proline (right) yielded compound 20. This
combination did not show added benefit to the potency profile
when compared with 1. A silyl-proline had previously demon-
strated advantages in PK properties in our earlier studies and thus
was introduced, compound 21.24 However, incorporation of this
motif coupled with the 4-fluoroproline in compound 21 showed
reduced inhibitory activity across all genotypes.
Although the proline modifications in Table 3 showed unpre-
dictable differences in mutant profile compared to compound 1,
we decided to further investigate the fused cyclopropyl proline
variant on the left to see if it could provide a path toward a flatter
compound. For this analysis we chose to look at the shift in
potency versus the wild type virus as well as the absolute value
of the replicon activity. The SAR work was conducted by exploring
the combination of different Z-groups, with and without the R7 flu-
orine substitution. The results are summarized in Table 4, with the
values in parentheses being the potency shift of variants versus
genotype 1a activity. In general, these modifications yielded com-
pounds with in vitro profiles similar to each other.
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L. Tong et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
7
Table 7
Pharmacokinetic parameters of compounds 23 and 27 in rat
No.
IV
PO
Dose (mpk)
t1/2 (h)
Cl (ml/min/kg)
Dose (mpk)
AUC (
l
M.h)
Cmax (
l
M)
F%
23
27
2
2
4.7
4.6
6
11.4
10
10
3.64
0.63
0.63
0.073
13
4
Vehicle: IV (60% PEG200); PO (10% TWEEN).
Hamann, L. G. Nature 2010, 465, 96; (b) Nettles, R. E.; Gao, M.; Bifano, M.;
Chung, E.; Persson, A.; Marbury, T. C.; Goldwater, R.; DeMicco, M. P.;
Rodriguez-Torres, M.; Vutikullird, A.; Fuentes, E.; Lawitz, E.; Lopez-Talavera,
J. C.; Grasela, D. M. Hepatology 1956, 2011, 54.
When conducting a similar investigation, but this time using
the fluoro-proline variant on the right, (Table 5), similar results
were obtained, where 24 and 25 were comparable to 1 for
GT1a_L31V and GT1a_Y93H, but weaker for GT2b.
From a previous SAR study, we observed that a chroman ring
system for the Z group could result in improved mutant profiles,
as illustrated by compound 26.18 When the cyclopropyl proline
on the left side was combined with this Z group, compound 27,
we observed a more balanced profile (Table 6). More specifically,
the potency shift for GT1a_Y93H was 30ꢁ versus the 160ꢁ for reg-
ular proline analog 26. Also, the potency shifts for GT1a_L31V and
GT2b were maintained in a range similar to 26.
Owing to the most balanced mutant profiles, compounds 23 and
27 were selected for exploratory rat PK studies with the results
summarized in Table 7. Compound 23 demonstrated acceptable
oral exposure and bioavailability in the rat. However, compound
27 showed lower exposure and reduced bioavailability. Both com-
pounds showed similar half-life measurements.
We found the replicon profile of compound 1 to be sensitive to
proline ring substitutions, some of which produced analogs with
improved in vitro profiles. More specifically, the incorporation of
either a cyclopropyl-fused proline, or a fluoro-substituted proline
provided increased potency versus the GT1a_Y93H RAV. When
proline modifications were combined with other structural fea-
tures, e.g., Z-group changes, inhibitors with improved resistance
profiles were identified. These data indicate the unpredictable
impact that various structural combinations can have on the
in vitro profiles. Compounds 23 and 27 both demonstrate mutant
profiles with smaller potency shifts against GT1a, potentially pro-
viding a path forward toward the identification of an NS5A inhibi-
tor with a ‘flat’ profile. Additional efforts toward the optimization
of activity against common resistance-associated polymorphisms
will be reported in future publications.
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Please cite this article in press as: Tong, L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.097