Substituted tetracyclic indole core derivatives of HCV NS5A inhibitor MK-8742

Article abstract of DOI:10.1016/j.bmcl.2016.08.002

As part of an ongoing effort in NS5A inhibition at Merck we now describe our efforts for introducing substitution around the tetracyclic indole core of MK-8742. Fluoro substitution on the core combined with the fluoro substitutions on the proline ring improved the potency against GT1a Y93H significantly. However, no improvement on GT2b potency was achieved. Limiting the fluoro substitution to C-1 of the tetracyclic indole core had a positive impact on the potency against the resistance associated variants, such as GT1a Y93H and GT2b, and the PK profile as well. Compounds, such as 62, with reduced potency shifts between wild type GT1a to GT2b, GT1a Y93H, and GT1a L31V were identified.

Full text of DOI:10.1016/j.bmcl.2016.08.002

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted tetracyclic indole core derivatives of HCV NS5A inhibitor
MK-8742
a
b
a
a
a
Wensheng Yu a, , Guowei Zhou , Craig A. Coburn , Qingbei Zeng , Ling Tong , Michael P. Dwyer ,
Bin Hu c, Bin Zhong c, Jinglai Hao c, Tao Ji c, Shuai Zan c, Lei Chen a, Robert Mazzola a, Jae-Hun Kim a,
Deyou Sha a, Oleg Selyutin a, Stuart B. Rosenblum a, Brian Lavey a, Anilkumar G. Nair a, Seong Heon Kim a,
Kerry M. Keertikar a, Laura Rokosz d, Sony Agrawal d, Rong Liu e, Ellen Xia d, Ying Zhai d, Stephanie Curry e,
Patricia McMonagle e, Paul Ingravallo e, Ernest Asante-Appiah e, Shiying Chen f, Joseph A. Kozlowski a
⇑
a Department of Medicinal Chemistry, Merck Research Laboratories, 126 E. Lincoln Avenue, Rahway, NJ 07065, USA
b Department of Medicinal Chemistry, Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA
c WuXi AppTec, 288 Fute Zhong Road, Shanghai 200131, China
d Department of In Vitro Pharmacology, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
e Department of Infectious Diseases, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
f Department of PPDM, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of an ongoing effort in NS5A inhibition at Merck we now describe our efforts for introducing sub-
stitution around the tetracyclic indole core of MK-8742. Fluoro substitution on the core combined with
the fluoro substitutions on the proline ring improved the potency against GT1a Y93H significantly.
However, no improvement on GT2b potency was achieved. Limiting the fluoro substitution to C-1 of
the tetracyclic indole core had a positive impact on the potency against the resistance associated variants,
such as GT1a Y93H and GT2b, and the PK profile as well. Compounds, such as 62, with reduced potency
shifts between wild type GT1a to GT2b, GT1a Y93H, and GT1a L31V were identified.
Received 8 June 2016
Revised 30 July 2016
Accepted 1 August 2016
Available online xxxx
Keywords:
HCV NS5A inhibitor
MK-8742
Ó 2016 Elsevier Ltd. All rights reserved.
Elbasvir
HCV infection
HCV DAA
Resistance associated variants
Hepatitis C virus (HCV) is the leading cause of chronic liver dis-
ease and liver transplants in the developed world. It was estimated
that 130–170 million people worldwide are infected with HCV.1
There are seven major genotypes (GT1-7) with a majority of them
having multiple subtypes. However, genotypes 1, 2, and 3 account
for 80–90% of all HCV infections.2 Current HCV therapy has
improved significantly over early approaches and utilizes inter-
feron-free, direct-acting antiviral agents (DAA), with each targeting
a different step in the HCV life cycle. Toward this strategy, three
major categories of drugs have been developed including:
NS3/4A protease inhibitors, non-structural protein 5A (NS5A)
replication complex inhibitors, and NS5B polymerase nucleotide-
like inhibitors.3
The HCV NS5A protein is a multifunctional RNA binding
phosphoprotein essential for HCV replication.4 Early clinical expe-
rience with NS5A inhibitors indicated that they suffered from a low
genetic barrier to resistance and demonstrated weak activity
against non-GT1 and GT1a variants.5 Thus, the need for identifica-
tion of NS5A inhibitors with increased potency against a wider
variety of genotypes as well as NS5A resistance-associated poly-
morphisms became a focus for many groups. To date, five NS5A
inhibitors have been approved for HCV treatment, either alone or
in combination with other DAA’s: daclatasvir (BMS-790052),5,6
ledipasvir (GS-5885),7 ombitasvir (ABT-267),8 elbasvir (MK-
8742),9,10 and velpatasvir (GS-5816).11
Research efforts at Merck identified the tetracyclic indole-based
inhibitor, MK-8742 (elbasvir) shown in Figure 1.9,10 Continued
effort sought to explore the impact of structural changes to the
tetracyclic indole-based NS5A inhibitors, with the aim of
determining if an inhibitor with a ‘flat’ profile could be obtained.
⇑
Corresponding author.
E-mail address: wensheng.yu@merck.com (W. Yu).
http://dx.doi.org/10.1016/j.bmcl.2016.08.002
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yu, W.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.002

2
W. Yu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
core of either MK-8742 or compound 1 (Table 1). The preparation
core
N
O
O
of the 1-F substituted analog 12 is outlined in Scheme 1. 3-Bromo-
5-fluorophenyl acetate 3 was prepared from 3-bromo-5-fluorophe-
nol 2 followed by an AlCl3-promoted rearrangement to give 1-(4-
bromo-2-fluoro-6-hydroxyphenyl)ethan-1-one 4. Fisher indole
synthesis of compound 4 afforded indole 6 in good yield. The ami-
nal carbon was introduced by the reaction of 6 with (dibro-
momethyl)benzene under basic condition to produce tetracyclic
indole 7. Compound 7 was converted to bis-pinacol boronate ester
8 which upon treatment under Suzuki coupling conditions with
N
O
N
H
O
N
O
N
N
H
N
H
O
N
N
H
O
MK-8742
O
R2
O
N
R1
O
O
N
O
N
H
N
N
N
O
tert-butyl
(S)-2-(5-bromo-1H-imidazol-2-yl)pyrrolidine-1-car-
N
H
H
N
H
N
boxylate10 gave the bis-Boc intermediate 9. Treatment of the inter-
mediate 9 under acidic conditions followed by HATU-mediated
O
Z
amide coupling with methoxycarbonyl-L-valine produced a mix-
The "Z" group
Figure 1. Strategy for core substitution of MK-8742 derivatives.
ture of two diastereomers at the aminal position. The mixture of
diastereomers was subjected to chiral SFC separation to yield com-
pound 12 as well as the less active isomer which was not reported
here. The other fluoro-substituted analogs 13–17 and chloro-sub-
stituted analog 19 were prepared by using the same chemistry out-
lined in Scheme 1 starting from the appropriate fluoro or chloro
starting material. The in vitro virologic profiles of the active iso-
mers for the final compounds are reported unless otherwise noted.
The 12-F analog 18 was prepared from dibromo-3-F-indole 11
by following the chemistry outlined in Scheme 1. Compound 11
was prepared from the corresponding dibromo indole analog 10
under Select-Fluor mediated fluorination (Scheme 2). Compound
10 was prepared based on the chemistry outlined in Scheme 1.
The 1-F-12-Cl disubstituted analog 20 was prepared based on the
chemistry outlined in Schemes 1 and 2.
The in vitro antiviral profiles of compounds bearing either a flu-
oro or chloro substitution on the tetracyclic indole core are sum-
marized in Table 1. Introduction of a fluoro to the C-1 position
produced compound 12 which showed good potency against all
the genotypes tested. However, the corresponding 2-F analog 13
showed reduced potency against GT2b and GT3a, as compared to
compound 12. The 4-F analog 14 showed diminished potency
against multiple replicons tested, including genotype 2b, 3a, 4a,
and 1a Y93H. The weaker potency of compounds 13 and 14 sug-
gests that fluoro substitution on C-2 or C-4 positions were not very
well-tolerated. SAR studies on the fluoro substitution at C-8, C-9,
and C-11 positions were done by utilizing compound 1 for the sake
In particular, a ‘‘flat” potency profile is defined by a minimal
potency shift (ꢀ10ꢁ) among genotypes and resistance-associated
variants (RAV’s), such as GT2b (bearing the resistance-associated
methionine at position 31), GT1a Y93H, and GT1a L31V, which
were used as model replicons in our SAR studies. Toward this
end, we recently reported SAR studies directed toward the impact
of variation on the Z-group (Fig.1) with either alkyl, substituted
aryl, or a heteroaryl Z groups.12,13 In addition, efforts to explore
the L-proline-L-valine methyl carbamate region of this inhibitor
class has also been recently reported.14 These early SAR studies
yielded NS5A inhibitors13 with reduced potency shifts from GT1a
to GT2b, GT1a Y93H, and GT1a L31V which had not been observed
in our previous efforts on the core developments.15–17 However,
compounds with ‘‘flat” potency profiles have not been identified
yet. SAR efforts were continued to work toward this goal. Herein,
we describe our results on substitutions around the tetracyclic
indole core (Fig. 1), and the impact of the changes on the potency
against GT2b, GT1a Y93H, and GT1a L31V.
As part of an ongoing SAR studies based on MK-8742, we
wanted to explore the substitutions around the tetracyclic indole
core and examine their impact on the potency against RAV’s
(Fig. 1). The initial efforts explored the impact of introducing fluo-
rine substitution onto various positions of the tetracyclic indole
Table 1
In vitro potency profiles of MK-8742, compounds 1, 12–2018,19
O
O
O
O
N
H
O
O
N
H
N
2
1
11
8
12
N
N
N
N
N
H
N
9
H
4
O
Z
IDa
Substitution on the core
Z
Replicon EC90 (nM)
GT1a
GT1b
GT2a
GT2b
11
76
18
>100
>100
>100
150
—
GT3a
GT4a
GT1a Y93H
GT1a L31V
MK-8742
1
None
None
1-F
2-F
4-F
8-F
9-F
11-F
12-F
1-Cl
1-F-12-Cl
Ph
H
Ph
Ph
Ph
H
H
H
Ph
H
Ph
0.006
0.02
0.005
0.003
0.04
0.1
0.007
>0.8
0.006
0.02
0.003
0.003
0.003
0.01
0.019
0.02
0.15
0.003
0.4
0.1
0.02
—
0.12
1
0.4
1
>10
>10
5
0.016
0.2
0.04
0.02
1
0.2
0.2
—
28
100
34
1
7
0.4
1
36
3
12
13
14
15
16
17
18
19
20
62
>100
139
77
0.007
>0.5
68
—
—
—
0.007
0.05
0.02
0.007
0.03
0.006
0.004
—
0.03
15
125
42
0.3
—
2
0.06
—
0.04
25
94
51
0.7
—
0.4
A hyphen ‘‘—” in the table means not tested.
a
Active isomer at the aminal carbon if apply.
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W. Yu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Br
Br
OH
OH
Br
OAc
Br
OH
O
c
O
a
b
Cl
N
O
O
O
N
H
N
O
a
N
N
H
N
H
F
F
F
O
N
N
H
N
O
2
3
4
19
F
Br
Br
e
d
O
N
Br
O
N
H
N
N
O
O
N
H
H
N
O
F
HO
N
N
H
N
H
5
6
O
N
N
N
H
O
F
O
B
F
Br
21
O
O
O
Br
B
f
N
N
Scheme 3. Synthesis of 21. Reagents and conditions: (a) cyclopropyl boronic acid,
Pd2(dba)3, X-Phos, dioxane, 110 °C, 16 h.
O
O
7
8
according to the synthetic route shown in Scheme 3. Compound 21
was prepared from the corresponding Cl analog 19 under standard
Suzuki coupling conditions with cyclopropyl boronic acid. Com-
pounds 22–24 were prepared from their corresponding chloro ana-
log in an analogous manner to compound 21. Compound 25 was
prepared from compound 1 and benzyl alcohol under Pd/C and
potassium carbonate conditions.
F
Boc
N
N
N
Boc
N
N
H
h, i
g
N
N
H
O
9
The in vitro antiviral profiles of compounds 21–25 are summa-
rized in Table 2. Introduction of a cyclopropyl group to C-1 of com-
pound 1 (compound 21, Table 2) reduced the potency against
GT1a, GT2b, and GT3a, and a similar result was observed for the
corresponding 1-phenyl substituted analog 22. The C-12 cyclo-
propyl analog 23 showed similar potency profile to compound 1
(Table 1), except for a ꢀ10ꢁ decrease in potency against GT3a.
The corresponding 12-phenyl substituted analog 24 was much
weaker against GT1a Y93H than 23. Finally, a 12-benzyl analog
of compound 1 (compound 25, Table 2) was prepared and found
to be more potent than the corresponding phenyl analog 24 against
GT 1a Y93H and GT2b, but still weaker against some of the geno-
types compared to compound 1. These results indicated that a
cyclopropyl, phenyl, or benzyl substitution at either the C-1 or C-
12 position was not preferred.
With the SAR data summarized in Tables 1 and 2 in hand, efforts
were undertaken to combine the 1- or 12-fluorinated tetracyclic
cores with modifications identified in the other regions of the
molecule. The aim of this effort was to identify compounds with
improved potency against the RAV’s, especially GT1a Y93H and
GT2b, which proved to be among the least susceptible RAV’s based
on our previous SAR observations.12–17 As reported previously,
O
O
F
N
O
O
N
H
N
O
N
1
N
H
N
H
O
N
N
H
N
O
12
Scheme 1. Synthesis of 12. Reagents and conditions: (a) AcCl, DIPEA, DCM, rt, 16 h;
(b) AlCl3, DCM, 140 °C, 3 h; (c) 4-bromophenyl hydrazine HCl, AcOH/EtOH (1:10),
reflux, 6 h; (d) polyphosphoric acid, xylenes, 110 °C, 3 h; (e) PhCHBr2, K2CO3, DMF,
100 °C, 16 h; (f) bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane, 90 °C, 16 h; (g)
tert-butyl (S)-2-(5-bromo-1H-imidazol-2-yl)pyrrolidine-1-carboxylate, Pd(dppf)Cl2,
K2CO3, dioxane/H2O (10:1), 90 °C, 16 h; (h) HCl, MeOH, 0.5 h; (i) (methoxycar-
bonyl)-L-valine, BOP, DIPEA, DMF, 10 h; then chiral SFC separation.
of simplified chemistry (15–17). Comparing with the potency pro-
file of compound 1, the 8-F analog 15 was weaker against GT2b and
GT3a as was the case for the 9-F analog 16. In addition, the 11-F
analog 17 was weaker against both GT1a and GT1b. These results
indicated that fluoro substitution at the C-8, C-9, or C-11 positions
of the tetracyclic indole core was not tolerated as well. Gratify-
ingly, the 12-F substituted analog, compound 18, demonstrated
good potency against all genotypes tested, as illustrated in Table 1.
Having explored fluorine substitution around the tetracyclic core,
efforts shifted toward the introduction of chlorine at specific posi-
tions of the core. The 1-Cl substituted analog (19) showed a
potency profile similar to the corresponding 1-H compound 1, indi-
cating that C-1 chloro substitution was tolerated. In addition, 1-F-
12-Cl di-substituted analog (20) was also prepared and it showed
good potency against all the genotypes tested.
replacement of L-proline with the (4R)-F-L-proline group demon-
strated improved potency against GT1a Y93H.15 To follow up on
this finding, several analogs with mix and matched 1- or 12-fluori-
nated core combined with the (4R)-F-L-proline on either left- or
right-hand side were prepared. The preparation of one such analog
32 is outlined in Scheme 4 which relies upon selective manipula-
tion of the chloro bromo tetracyclic intermediate 27. The chemistry
is analogous to the route shown in Scheme 1 and the other fluori-
nated analogs 33–38 were prepared in a similar manner using the
correct fluorinated starting materials according to Scheme 4.
The in vitro antiviral profiles for compounds 32–38 are summa-
Having established that both fluoro- and chloro-substitutions
were tolerated at either the C-1 or C-12 position, we wondered if
additional substitution such as an alkyl or an aryl substitution
could be tolerated at these two positions. Introduction of a cyclo-
propyl group to the C-1 position of compound 1 was accomplished
rized in Table 3. Combination of the 12-F core with (4’R)-F-L-pro-
line (32) demonstrated good potency against all genotypes tested
with 10ꢁ improvement on GT1a Y93H potency compared to the
F
corresponding L-proline analog 18 (Table 1). A similar result was
Br
Br
a
Br
obtained for analog which combined the 12-F core and (400R)-F-
proline (33) with a 10x improvement against GT1a Y93H observed.
Combination of both (40R)-F- -proline and (400R)-F-
-proline with
L-
Br
N
H
N
HO
H
HO
11
L
L
10
the 12-F core afforded compound 34 which showed similar
potency profile as compounds 32 and 33. Combination of the 1-F
Scheme 2. Synthesis of 11. Reagents and conditions: (a) Select-Fluor, DMSO/CH3CN
(1:1), 25 °C, 1 h.
core with either (40R)-F- -proline or (400R)-F-
L L-proline afforded
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4
W. Yu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
In vitro potency profiles of compounds 21–2518,19
O
O
O
R1
O
R12
N
O
N
H
O
O
N
H
N
N
N
N
N
H
N
H
ID
R12
R1
Replicon EC90 (nM)
GT3a
GT1a
GT1b
GT2a
GT2b
GT4a
GT1a Y93H
GT1a L31V
21
22
23
24
25
H
H
c-Pr
Ph
H
H
H
0.7
1
0.03
0.09
0.1
—
—
0.004
0.004
0.01
—
—
0.005
0.01
—
454
108
122
204
28
74
47
10
3
—
—
0.3
0.09
—
890
250
100
5000
187
0.7
1
8
4
9
c-Pr
Ph
Benzyl
—
A hyphen ‘‘—” in the table means not tested.
improved potency against GT1a Y93H. Its GT1a Y93H EC90 was
determined to be in the double digit picomolar range, and is
ꢀ10ꢁ more potent than either compounds 35 or 36 and ꢀ100ꢁ
F
Cl
F
Br
Cl
a
b
N
Br
more potent than the corresponding L-proline analog 12 (Table 1).
O
N
H
The calculated potency shift of 37 from GT1a Y93H to GT1a is only
16-fold, which was unprecedented in our previous effort. For com-
parison, the corresponding analog without the 1-F substitution
(38) was prepared. Compound 38 showed potency similar to either
35 or 36 (against GT1a Y93H) while being ꢀ20ꢁ less potent than
compound 37 (GT1a Y93H). This result clearly demonstrated the
importance of the 1-F substitution of compound 37 for potency
improvement against GT1a Y93H.
HO
26
27
F
F
Cl
Cl
O
O
N
Boc
N
B
c
N
N
N
H
O
O
28
29
The significant potency improvement of compound 37 against
GT1a Y93H was encouraging. However, the GT2b EC90 value of
compound 37 did not meet the goal of a 10x shift requirement.
Efforts were undertaken to modify the other regions of the mole-
cule to improve GT2b potency while retaining the excellent
potency against GT1a Y93H. In a previous report,13 alkoxy substi-
tuted phenyl ‘‘Z” groups on the tetracyclic indole core demon-
strated good potency on all genotypes tested including GT2b.
Analogs (39–45) incorporating these alkoxy substituted phenyl
‘‘Z” groups coupled the 1,40,400-tri-fluoro substitution were pre-
pared according to the chemistry outlined in Scheme 4. The
in vitro antiviral profiles for these compounds are summarized in
Table 4. The 3-MeO-Ph analog 39 showed a similar potency profile
to phenyl analog 37 (Table 3) with EC90 value against GT1a Y93H in
the double digit picomolar range. This result confirmed the syner-
gistic effect of the 1,40,400-tri-fluoro substitution against GT1a
Y93H. Introduction of other alkoxy groups, such as EtO (40),
i-PrO (41), n-BuO (42), or CF3O (43) group to the C-3 position of
the phenyl all demonstrated similar profiles as compound 39 how-
ever with no improvement in potency against GT2b. The introduc-
tion of a tetrahydrofuran-(3R)-oxy group to the C-3 of the phenyl
‘‘Z” group (44) offered a good balance between GT1a Y93H and
GT2b potency, however, at the expense of the potency against
GT1a Y93H, which was >10ꢁ less potent than compound 39. Intro-
ducing a trifluoromethoxy group to the 4-position to the phenyl
‘‘Z” group, 45, showed a similar result to the corresponding 3-sub-
stituted analog 43.
O
B
F
N
Boc
N
O
e
d
N
H
N
O
30
F
Boc
N
N
Boc
N
f, g
N
H
N
N
N
H
O
F
31
O
O
F
N
O
O
N
H
N
O
N
N
H
N
H
O
N
N
H
N
O
F
32
Scheme 4. Synthesis of 32. Reagents and conditions: (a) PhCHBr2, K2CO3, DMF,
100 °C, 16 h; (b) bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane, 90 °C, 16 h; (c)
tert-butyl (S)-2-(5-bromo-1H-imidazol-2-yl)pyrrolidine-1-carboxylate (prepared
based on procedures described in reference 10), Pd(dppf)Cl2, K2CO3, dioxane/H2O
(10:1), 90 °C, 16 h; (d) bis(pinacolato)diboron, Pd2(dba)3, X-Phos, dioxane, 110 °C,
2 h; (e) tert-butyl (2S,4R)-2-(5-bromo-1H-imidazol-2-yl)-4-fluoropyrrolidine-1-
carboxylate (prepared based on procedures described in reference 10), K2CO3,
Having been unable to improve the potency against GT2b with
the 1,40,400-tri-fluoro substitution analogs (39–45) shown in Table 4,
efforts were redirected toward exploration of analogs bearing 1,400-
di-fluoro substitution since compound 36 in Table 3 showed a
more balanced GT1a Y93H and GT2b potency profile. 1,400-Di-flu-
oro substituted analogs 46–51 were prepared based upon the
chemistry outlined in Scheme 3 and the in vitro antiviral profiles
for these derivatives are summarized in Table 5. Not surprisingly,
the 3-MeO-Ph analog 46 showed ꢀ 10-fold loss in potency against
dioxane/H2O (10:1), 90 °C, 16 h; (f) HCl, MeOH, 0.5 h; (g) (methoxycarbonyl)-L-
valine, BOP, DIPEA, DMF, 10 h; then chiral SFC separation.
compounds 35 and 36 both of which showed good potency against
all genotypes tested, with ꢀ10ꢁ improvement on GT1a Y93H com-
pared to the corresponding L-proline analog 12 (Table 1). Very
interestingly, analog 37 which combined the 1-F core with both
(40R)-F- -proline and (400R)-F-
L
L
-proline showed significantly
a
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W. Yu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Table 3
In vitro potency profiles of compounds 32–3818,19
O
O
O
R1
N
H
R12
N
O
O
O
N
H
N
N
N
N
N
N
H
H
R4'
R4''
O
0
00
IDa
R4
R12
R1
R4
Replicon EC90 (nM)
GT3a
GT1a
GT1b
GT2a
GT2b
GT4a
GT1a Y93H
GT1a L31V
32b
33
F
H
F
F
H
F
F
F
F
H
H
H
H
H
H
H
F
F
F
H
F
F
H
F
F
0.005
0.002
0.02
0.003
0.004
0.005
0.01
0.003
0.001
0.005
0.003
0.003
0.003
0.004
0.03
0.003
0.3
0.01
0.04
0.2
—
2
0.2
8
0.3
0.1
0.8
2
0.1
0.04
0.3
0.05
0.07
0.1
2
2
2
4
1
0.08
2
2
1
7
2
2
9
10
33
58
32
16
72
63
34b
35b
36b
37b
38b
F
H
F
0.7
0.2
A hyphen ‘‘—” in the table means not tested.
a
Active isomer at the aminal carbon.
Mixture of two diastereomers at the aminal carbon.
b
Table 4
Table 5
In vitro potency profiles of compounds 39–4518,19
In vitro potency profiles of compounds 46–5118,19
O
O
O
O
O
O
O
O
N
O
N
N
N
F
F
O
O
O
N
H
N
H
N
N
H
N
H
N
N
N
4''
1
N
N
N
N
H
H
H
N
N
H
F
O
F
O
F
R3
R3
R4
R4
IDa
R3
R4
Replicon EC90 (nM)
IDa R3
R4
Replicon EC90 (nM)
GT1a
GT1b
GT2b
GT3a
GT1a
Y93H
GT1a
L31V
GT1a GT1b GT2b GT3a GT1a
Y93H
GT1a
L31V
46
47
48
49
50
51
MeO
EtO
i-PrO
CF3O
CHF2O
H
H
H
H
H
0.002
0.01
0.01
0.02
0.002
0.03
0.007
—
—
—
—
19
17
28
48
22
13
0.1
0.1
0.07
—
0.2
—
0.6
0.4
0.9
1.7
0.3
0.3
0.3
—
0.6
1.8
0.7
—
39
40
41
42
43
MeO
EtO
i-PrO
n-BuO
CF3O
H
H
H
H
H
0.006 0.007 39
—
0.6
1
—
—
0.07
0.2
0.1
0.1
0.2
2
3
4
4
7
0.01
0.01
—
—
69
122
0.008 126
0.006 0.005 52
H
4-CF3O
0.008
O
44b
45
H
0.04
—
—
2
2
1
1
7
O
1
A hyphen ‘‘—” in the table means not tested.
H
CF3O 0.01
35
0.2
—
a
Active isomer at the aminal carbon.
A hyphen ‘‘—” in the table means not tested.
a
Active isomer at the aminal carbon.
Mixture of two diastereomers at the aminal carbon.
with 1-F core (56) is 6ꢁ and 8ꢁ more potent against GT1a Y93H
and GT2b, respectively, than the 1-H analog 57. A similar trend
was observed among the rest of the compounds listed in Table 6
(58–63). From the survey summarized in Table 6, compounds, such
as 62, were identified which possessed potent activity against both
GT2b and GT1a Y93H. Using the EC90 values in Table 6, the calcu-
lated potency shifts for genotypes 2b, 1a Y93H, and 1a L31V from
GT1a were 70ꢁ, 300ꢁ, and 10ꢁ for compound 62, which was pro-
gress toward our 10-fold shift goal.
b
GT1a Y93H than 39; however, no meaningful improvement on
GT2b was observed. Similar SAR trends were observed for com-
pounds 47–51 as was shown for compound 46.
With the knowledge of the SAR from Tables 4 and 5, efforts
were undertaken to combine the fluorine substitution on the C-1
position of the tetracyclic indole core with various ‘‘Z” groups,
Several compounds bearing 1-F substitution on the core were
tested in rat pharmacokinetic (PK) studies and the data are sum-
marized in Table 7. Compound 12 showed good bioavailability
upon oral administration. The PK profile of compound 12 was
improved over similar analogs bearing an unsubstituted tetracyclic
indole core, as reported previously.13 Compound 52 showed a bet-
ter PK profile than the corresponding 1-H analog 53. Compound 62
demonstrated reduced bioavailability relative to 12 and 52.
In conclusion, a systematic SAR study of substitutions on the
tetracyclic core led to the identification of well-tolerated fluorine
substitutions at both the C-1 and C-12 positions. C-1 fluoro
without any substitutions on the L-prolines. For comparison sake,
their corresponding C-1 unsubstituted analogs were also prepared.
Compounds 52–63 were prepared according to the chemistry out-
lined in Scheme 3 and their in vitro antiviral profiles are summa-
rized in Table 6. While the 3-MeO-Ph analog 52 showed a similar
potency profile to its corresponding 1-H analog 53, we observed
a trend that compounds bearing the 1-F substitution demonstrated
better potency on GT1a Y93H and GT2b than the corresponding
1-H analogs. For example, the 3-EtO-Ph analog with 1-F core (54)
is 8ꢁ and 5ꢁ more potent against GT1a Y93H and GT2b, respec-
tively, than its corresponding 1-H analog 55. 3-i-PrO-Ph analog
Please cite this article in press as: Yu, W.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.002

6
W. Yu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 6
002. These data include MOL files and InChiKeys of the most
important compounds described in this article.
In vitro potency profiles of compounds 52–6318,19
O
O
References and notes
O
R1
O
O
N
H
O
O
N
H
N
N
N
N
1. Hajarizadeh, B.; Grebely, J.; Dore, J. G. Nat. Rev. Gastroenterol. Hepatol. 2013, 10,
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Simmonds, P. Hepatology 2014, 59, 318.
N
H
N
N
H
3
3. For a review on DAAs see: (a) Holmes, J.; Thompson, A. Hepat. Med. Evid. Res.
2015, 51; (b) Poordad, F.; Dieterich, D. J. Viral Hepat. 2012, 19, 449; (c) Scheel, T.
K.; Rice, C. M. Nat. Med. 2013, 19, 837.
R2
5
4
4. Sheridan, C. Nat. Biotechnol. 2011, 29, 553.
IDa
R1 R2
Replicon EC90 (nM)
GT2b GT3a GT1a Y93H GT1a L31V
5. Gao, M.; Nettles, R. E.; Belema, M.; Snyder, L. B.; Nguyen, V. N.; Fridell, R. A.;
Serrano-Wu, M. H.; Langley, D. R.; Sun, J.-H.; O’Boyle, D. R., II; Lemm, J. A.;
Wang, C.; Knipe, J. O.; Chien, C.; Colonno, R. J.; Grasela, D. M.; Meanwell, N. A.;
Hamman, L. G. Nature 2010, 465, 96.
GT1a
52
53
54
55
56
57
58
59
60
61
62
63b
F
H
F
3-MeO
3-MeO
3-EtO
0.01
0.005
0.01
0.02
0.01
0.01
0.01
0.04
6
2
8
22
18
145
5
0.07
0.1
0.02
0.2
0.05
0.08
—
6
8
2
16
6
34
3
82
3
0.2
0.2
0.2
0.9
0.2
0.5
0.2
—
0.1
0.3
0.1
0.2
6. Belema, M.; Meanwell, N. A. J. Med. Chem. 2014, 57, 5057.
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Caspi, D. D.; Krishnan, P.; Mistry, N.; Koev, G.; Reisch, T. J.; Mondal, R.; Pilot-
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H
F
H
F
H
F
H
F
3-EtO
3-i-PrO
3-i-PrO
4-MeO
4-MeO
3,5-Di-MeO 0.004 0.6
3,5-Di-MeO 0.004 11
15
—
0.06
0.08
0.1
8
3-Cl-5-MeO 0.01
3-Cl-5-MeO 0.03
0.7
3
H
202
0.8
15
A hyphen ‘‘—” in the table means not tested.
a
Active isomer at the aminal carbon.
Mixture of two diastereomers at the aminal carbon.
10. Kozlowski, J. A.; Rosenblum, S. B.; Coburn, C. A.; Shankar, B. B.; Anilkumar, G.
N.; Chen, L.; Dwyer, M. P.; Jiang, Y.; Keertikar, K. M.; Lavey, B. J.; Seluytin, O. B.;
Tong, L.; Wong, M.; Yang, D.-Y.; Yu, W.; Zhao, G.; Wu, H.; Hu, B.; Zhong, B.; Sun,
F.; Ji, T.; Shen, C.; Rizvi, R.; Zeng, Q. WO 2012041014.
b
11. Younossi, Z. M.; Stepanova, M.; Feld, J.; Zeuzem, S.; Jacobson, I.; Agarwal, K.;
Hezode, C.; Nader, F.; Henry, L.; Hunt, S. J. Hepatol. 2016, 65, 33.
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S. H.; Lavey, B. J.; Zhou, G.; Rizvi, R.; Shankar, B. B.; Zeng, Q.; Chen, L.; Agrawal,
S.; Carr, D.; Rokosz, L.; Liu, R.; Curry, S.; McMonagle, P.; Ingravallo, P.; Lahser,
F.; Asante-Appiah, E.; Nomeir, A.; Kozlowski, J. A. Bioorg. Med. Chem Lett. 2016,
26, 3800.
13. Yu, W.; Coburn, C. A.; Anilkumar, G. N.; Wong, M.; Rosenblum, S. B.; Zhou, G.;
Dwyer, M. P.; Tong, L.; Hu, B.; Zhong, B.; Hao, J.; Ji, T.; Zan, S.; Kim, S. H.; Zeng,
Q.; Seluytin, O.; Chen, L.; Masse, F.; Agrawal, S.; Liu, R.; Xia, E.; Zhai, Y.; Curry,
S.; McMonagle, P.; Ingravallo, P.; Asante-Appiah, E.; Lin, X.; Kozlowski, J. A.
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Table 7
Rat PK profiles of compounds 12, 52, 53, and 6220
12
52
53
62
Dose (mpk)
PO AUC ( M h)
M)
T1/2 (h)
Cl (ml/min/kg)
5
10
5
10
0.75
0.1
5.2
8
l
2.8
0.33
3.6
11
2.1
0.24
5.9
10
0.51
0.1
4.3
11
6
PO Cmax (
l
F (%)
33%
12
3
Male Sprague–Dawley rats were used. Compound was dosed at 2 mg/kg, IV in 60%
PEG200 as well as at indicated doses in P.O. in 0.5% MC (12) or 10% TWEEN (52, 53,
62).
14. Dwyer, M. P.; Keertikar, K. M.; Chen, L.; Tong, L.; Seluytin, O.; Anilkumar, G. N.;
Yu, W.; Zhou, G.; Lavey, B. J.; Yang, D.-Y.; Wong, M.; Kim, S. H.; Coburn, C. A.;
Rosenblum, S. B.; Zeng, Q.; Jiang, Y.; Nomeir, A. A.; Liu, R.; Agrawal, S.; Xia, E.;
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Chen, L.; Mazzola, R.; Caldwell, J.; Tang, H.; Allard, M.; Buckle, R. N.; Gauuan, P.
J. F.; Holst, C. L.; Martin, G. S.; Naicker, K. P.; Vellekoop, S.; Agrawal, S.; Liu, R.;
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structures are fully characterized by LCMS and 1H NMR spectra.
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Skelton, A.; Xia, E.; Ralston, R. Antiviral Res. 2008, 77, 177; (b) Carroll, S. S.;
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substitution on the tetracyclic core was shown to improve the
potency against both GT1a Y93H and GT2b (bearing the naturally
occurring 31 M RAV), and also showed comparable or improved
rat PK profiles over the corresponding 1-H analogs. Combination
of the C-1 fluoro substituted tetracyclic core, with (4R)-F-L-proline
motifs, offered significant potency improvement against GT1a
Y93H, but led to weaker GT2b potency. Compounds with reduced
potency shifts from GT1a to GT2b, GT1a Y93H, and GT1a L31V,
such as compound 62, have been identified. However, these com-
pounds still have not achieved a ‘‘flat” profile, i.e. a potency shift
of ꢀ10-fold. Combinations of these SAR findings with other SAR
observations from other regions of the molecule are under investi-
gation and the results will be reported in separate publications.
Acknowledgement
The authors would like to thank Dr. Jingjun Yin and Dr. Rebecca
Ruck for the generous supply of synthetic intermediates.
20. For the rat PK profile of MK-8742, see reference 9.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.08.
Please cite this article in press as: Yu, W.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.002