Discovery of novel HCV inhibitors: Synthesis and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus NS4B

Article abstract of DOI:10.1016/j.bmcl.2013.04.049

A novel series of 6-(indol-2-yl)pyridine-3-sulfonamides was prepared and evaluated for their ability to inhibit HCV RNA replication in the HCV replicon cell culture assay. Preliminary optimization of this series furnished compounds with low nanomolar potency against the HCV genotype 1b replicon. Among these, compound 8c has identified as a potent HCV replicon inhibitor (EC₅₀ = 4 nM) with a selectivity index with respect to cellular GAPDH of more than 2500. Further, compound 8c had a good pharmacokinetic profile in rats with an IV half-life of 6 h and oral bioavailability (F) of 62%. Selection of HCV replicon resistance identified an amino acid substitution in HCV NS4B that confers resistance to these compounds. These compounds hold promise as a new chemotype with anti-HCV activity mediated through an underexploited viral target.

Full text of DOI:10.1016/j.bmcl.2013.04.049

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3947–3953
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel HCV inhibitors: Synthesis and biological activity
of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus
NS4B
a
a
a
a
a
Xiaoyan Zhang a, , Nanjing Zhang , Guangming Chen , Anthony Turpoff , Hongyu Ren , James Takasugi ,
Christie Morrill a, Jin Zhu a, Chunshi Li a, William Lennox a, Steven Paget a, Yalei Liu a, Neil Almstead a,
F. George Njoroge b, Zhengxian Gu a, Takashi Komatsu a, Valerie Clausen a, Christine Espiritu a,
Jason Graci a, Joseph Colacino a, Fred Lahser b, Nicole Risher a, Marla Weetall a, Amin Nomeir b, Gary M. Karp a
⇑
a PTC Therapeutics, 100 Corporate Court, South Plainfield, NJ 07080, USA
b 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:
Available online 30 April 2013
A novel series of 6-(indol-2-yl)pyridine-3-sulfonamides was prepared and evaluated for their ability to
inhibit HCV RNA replication in the HCV replicon cell culture assay. Preliminary optimization of this series
furnished compounds with low nanomolar potency against the HCV genotype 1b replicon. Among these,
compound 8c has identified as a potent HCV replicon inhibitor (EC50 = 4 nM) with a selectivity index with
respect to cellular GAPDH of more than 2500. Further, compound 8c had a good pharmacokinetic profile
in rats with an IV half-life of 6 h and oral bioavailability (F) of 62%. Selection of HCV replicon resistance
identified an amino acid substitution in HCV NS4B that confers resistance to these compounds. These
compounds hold promise as a new chemotype with anti-HCV activity mediated through an underexploit-
ed viral target.
Keywords:
HCV inhibitors
Replicon
6-(Indol-2-yl)pyridine-3-sulfonamides
NS4B
Ó 2013 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is a major cause of acute hepatitis and
chronic liver disease, including fibrosis, cirrhosis and liver cancer.1
Globally, up to 3% of the world’s population is chronically infected
with HCV and 3–4 million people are newly infected each year.2
There is no vaccine available to prevent hepatitis C. In the past dec-
ade, the standard of care for the treatment of chronic hepatitis C
has been based exclusively on the combination of pegylated inter-
feron and ribavirin, a combination which is ineffective in genotype
1 patients (<50% sustained virologic response), contraindicated in
some patients, or associated with severe side effects.3 A new era
of therapy for HCV infection has emerged with the approval of
the first direct acting antiviral agents, the HCV protease inhibitors
Incivek™ (telaprevir) and Victrelis™ (boceprevir). These agents
prevent HCV replication by inhibiting the activity of the HCV pro-
tease NS3/4A.4 Results from clinical trials have demonstrated that
boceprevir or telaprevir in combination with pegylated interferon
and ribavirin significantly improve sustained virologic response
rates, both in treatment-naïve patients and in prior relapsers and
nonresponders.5 However, the ideal treatment regimen for HCV
infection, in which interferon is spared or eliminated, has not yet
been realized, and the drawbacks of current HCV therapy necessi-
tate the development of more effective anti-HCV agents.
Besides protease inhibitors, a number of other HCV inhibitors
are in preclinical or clinical development targeting viral proteins
such as NS5A6a and the RNA-dependent RNA polymerase NS5B.6b,6c
Recently, the HCV NS4B protein has emerged as an unexploited
target for direct acting antivirals.7 NS4B is a 27-kDa integral mem-
brane protein that has a number of described functions but is
thought to act primarily as a scaffold for assembly at the host
endoplasmic reticulum of viral replication complexes that are re-
quired for HCV RNA replication.8
We recently described the discovery and chemical optimization
of N-(40-(indol-2-yl)phenyl)sulfonamides as novel inhibitors of the
HCV 1b replicon.9 We have identified the sulfonamide moiety at-
tached to the phenyl ring (e.g., compound 1, Fig. 1) as a preferred
pharmacophore leading to submicromolar potency in the replicon
cell-based assay. As part of the continuing optimization of this no-
vel series, we reversed the arrangement of the sulfonamide group
attached to the phenyl ring in compound 1, placing the sulfur atom
adjacent to the aromatic ring. This approach resulted in the identi-
fication of 2a as a potent HCV replicon inhibitor (EC50 = 100 nM).
This compound is about fourfold more potent than compound 1.
Although very potent, 2a has poor aqueous solubility and is highly
metabolized in the presence of human liver microsomes. Oral
⇑
Corresponding author. Tel.: +1 9089129239; fax: +1 9082220567.
E-mail address: szhang@ptcbio.com (X. Zhang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.049

3948
X. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3947–3953
CN
CN
CN
O
S
NH
O
S
O
O
S
O
O
F
F
F
NH
NH
N
N
N
F
O
F
O
N
F
O
3a
EC50 (1b) = 33 nM
6-(Indol-2-yl)pyridine-3-sulfonamides
1
2a
EC50 (1b) = 100 nM
4-(Indol-2-yl)benzene sulfonamides
EC50 (1b) = 390 nM
N-(4'-(Indol-2-yl)phenyl)sulfonamides
Figure 1. Structures of representative compounds and activity against the HCV 1b replicon.
administration of a single 10 mg/kg dose of 2a to rats resulted in
the formation of a major metabolite corresponding to a loss of 42
mass units suggesting that the major metabolite 2b was formed
by oxidative N-dealkylation of 2a (Fig. 2). The structure of the
metabolite was confirmed by synthesis of the primary sulfonamide
2b. The metabolite 2b was found to be significantly less active than
the parent (2a) with an EC50 of 4700 nM in the replicon assay.
These results provided us with a starting point to optimize further
the antiviral activity as well as metabolic and pharmacokinetic
properties of this class of compounds. In response to the limita-
tions of compound 2a, a small number of arene sulfonamides were
investigated by replacing the phenyl ring with nitrogen-containing
heteroaryl rings. We found that (indol-2-yl)pyridine 3a (Fig. 1)
provided the most potent activity compared to other arene substi-
tution patterns (compounds 4–6 in Table 1). We therefore chose to
explore further the effect of substitutions using the 6-(indol-2-
yl)pyridine-3-sulfonamide as a core structure. The preparation of
these compounds and an initial assessment of their biological
properties are the subject of this article.
of the boronic acid 9b, generated from the key intermediate 9a,
with the corresponding aryl halides 10–13. The synthesis of 9a
with various R1 and R2 substitutions on the indole ring was de-
scribed previously.9
The synthetic approaches to the intermediate aryl chlorides 10
and 12 and aryl bromides 11 and 13 utilized in the Suzuki coupling
described in Scheme 1 are illustrated in Scheme 2. Diazotization of
commercially available 5-amino-2-chloropyridine followed by
chlorosulfonylation gave the sulfonyl chloride 14b using a modi-
fied procedure reported previously.10 Condensation of 14a (com-
mercially available) or 14b with various amines, in the presence
of pyridine at 0 °C afforded the corresponding sulfonamide deriva-
tives 10. The pyridine building block 11, in which the sulfonamide
group is ortho to the pyridine nitrogen, was prepared by converting
2-chloro-5-bromopyridine to the corresponding mercaptopyridine
15. Compound 15 was then oxidized in situ followed by amination.
Sulfonylation of 2-aminopyrimidine gave an intermediate sulfonic
acid 16,11 which was then converted to the desired 2-chloropyrim-
idine-5-sulfonamide 12 via sulfonyl chloride 17. The synthesis of
5-bromopyrimidine-2-sulfonamide 13 entails the conversion of
5-bromo-2-chloropyrimidine to the corresponding mercaptopy-
rimidine 18, which was then elaborated to the desired sulfonamide
The general synthetic route to prepare the (indol-2-yl)arene
sulfonamides 2–8 is outlined in Scheme 1. The target compounds
were prepared through a Suzuki coupling strategy by treatment
(a)
10
2a (Parent Dosed)
2b (Metabolite Detected)
1
0.1
0.01
0.001
0
2
4
6
Time (Hours Post Dose)
(b)
CN
CN
O
S
O
O
S
O
F
F
NH2
NH
oxidative N-dealkylation
N
N
F
O
F
O
2b
EC50 (1b) = 4,700 nM
2a
EC50 (1b) = 100 nM
Figure 2. (a) Plasma concentrations of 2a and metabolite 2b following administration of a 10 mg/kg oral dose of 2a in Sprague–Dawley rats (n = 3). (b) Major metabolic
pathway of 2a in rats.

X. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3947–3953
3949
Table 1
Effect of various arene sulfonamides on replicon activity, selectivity and metabolic stability
CN
F
Ar
R
N
F
O
1-6
a
b
Indole
Indole
Ar
Compd
R
HCV replicon 1b EC50 (nM)a
390
GAPDH IC50 (nM)
HLM, CLint (mL/min/kg)
O
HN
S
1
>10,000
5000
<1
O
4
O
S
O
Indole
Indole
Indole
2a
3a
4
100
33
204
51
NH
6
O
S
O
>10,000
>10,000
>10,000
>10,000
N
NH
NH
NH
NH
5
O
S
O
560
240
10,000
297
24
N
N
N
O
S
O
Indole
Indole
5
N
N
O
S
O
6
n.d.c
a
b
c
See Ref. 19 for assay conditions.
See Ref. 20 for assay conditions.
Not determined.
CN
CN
CN
a
b
R1
R1
B(OH)2
R1
SO2NHR3
Ar
N
N
N
R2
R2
R2
9a
9b
2-8
Scheme 1. Reagents and conditions: (a) LDA, THF, B(OMe)3, ꢀ78 °C; (b) aryl halides (10–13), Pd2(dba)3, tBu3PHBF4; KF, THF.
13 under conditions similar to those utilized in the preparation of
intermediate 11.
hydrochloride 29 using the same method as described for the prep-
aration of 23.
Amines 21, 23, 24, and 29 were prepared as outlined in
Scheme 3. 1,3-Difluoropropan-2-amine 21 was prepared from
commercially available 1,3-difluoro-2-propanol in four steps. 1,3-
Difluoro-2-propanol was first transformed into tosylate 19 and
then treated with sodium azide to produce azide 20. Reduction
of azide 20 through hydrogenation, followed by treatment with
HCl provided the desired amine 21 as the hydrochloride salt. 1-
(Trifluoromethyl)cyclopropanamine 23 was prepared from the cor-
responding 1-trifluoromethylcyclopropane-1-carboxylic acid by a
modified Curtius rearrangement with diphenyl phosphorazidate
(DPPA) and triethylamine in tert-butyl alcohol with subsequent
acidic cleavage of the Boc moiety.12 Trifluoro-2-methylpropan-2-
amine 24 was prepared by treatment of 2,2,2-trifluoroacetamide
with methylcerium chloride, generated in situ from cerium chlo-
ride and methyllithium. The product was easily isolated as the
hydrochloride salt in high yield.13 3-Methylenecyclobutanecarbo-
nitrile was converted into 3,3-difluorocyclobutanecarboxylic acid
27 in three steps according to the procedure reported previously.14
Compound 27 was then converted to 3,3-difluorocyclobutanamine
The chiral amine (S)-1,1,1-trifluoropropan-2-amine 34 was ob-
tained by asymmetric synthesis from 1,1,1-trifluoropropan-2-one
and (R)-1-phenylethanamine by a modification of the method of
Soloshonok et al.15 as shown in Scheme 4. The chiral Schiff base
30 was prepared by direct condensation of 1,1,1-trifluoropropan-
2-one and (R)-1-phenylethanamine. Isomerization of 30 to 31
was achieved in a solution of DBU at 60 °C, followed by hydrolysis
under acidic conditions to give the desired chiral amine 32 in mod-
erate yield with 92% enantiomeric excess (ee). The optical purity of
32 was further improved by salt formation of 32 with D-tartaric
acid. Crystallization of the diastereomer-rich mixture provided tar-
taric acid salt 33 with >99% ee, which was then converted into the
hydrochloride salt of 34 in high yield with retention of high enan-
tiomeric purity. The (R)-enantiomer of 34 was obtained from (S)-1-
phenylethanamine following the same procedure. The optical pur-
ity of 34 and its enantiomer was determined by chiral HPLC anal-
ysis of the N-(3,5-dinitrobenzoyl) derivative and the absolute
configuration was confirmed by comparison with an authentic
sample.16

3950
X. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3947–3953
O
S
O
a, b
c
Cl
SO2NHR3
Cl
Br
NH2
Cl
Cl
X
X
X
14a X = CH (commercially available)
14b X = N
X = N
10a X = CH
10b X = N
d
e, f
Cl
Br
Cl
SO2NHiPr
Br
SH
N
N
N
11
17
15
O
S
O
O
N
N
N
N
N
N
N
N
i
g
h
HO
S
OH
Cl
SO2NHiPr
12
H2N
Cl
O
16
N
N
N
N
N
j, k
e, f
SO2NHiPr
Br
Cl
Br
SH
Br
N
13
18
Scheme 2. Reagents and conditions: (a) NaNO2, HCl (concd), 0 °C, 82%; (b) SOCl2, H2O, CuCl, 70%; (c) R3NH2, pyridine, 0 °C. (d) NaSH, DMF, 70 °C; (e) NaOCl, HCl; (f) isopropyl
amine; (g) H2SO4, 180 °C, 48 h; (h) PCl5, 180 °C, 4 h, 24% for two steps; (i) isopropyl amine, triethylamine, THF, ꢀ78 °C, 0.5 h, 94%; (j) thiourea; (k) NaOH (1 N), 85% for two
steps.
F
F
F
F
F
F
F
F
a
b
c, d
HCl.NH2
TsO
HO
N3
21
19
20
COOH
NH2.HCl
NHBoc
CF3
e
f
CF3
CF3
22
23
O
g, h
NH2.HCl
CF3
24
F3C
NH2
O
N
k
N
N
H
N
O
NH2.HCl
OH
i
j
f
e
O
F
F
F
F
O
F
F
F
F
28
26
27
29
25
Scheme 3. Reagents and conditions: (a) TsCl, pyridine, 91%; (b) NaN3, DMSO, 70 °C, 5 h; (c) H2, Pd/C (10%), EtOAc, 3 h; (d) HCl, 78% from 19; (e) DPPA, Et3N, t-BuOH, 80 °C, o/n,
84–97%; (f) HCl (1 N), 110 °C, 2 h, 86%; (g) CeCl3, MeLi, THF, ꢀ78 °C to rt; (h) HCl, 75% for two steps; (i) RuCl3, NaIO4, CH2Cl2, MeCN, H2O; 91%; (j) DAST, CH2Cl2, 0 °C to rt, 99%;
(k) NaOH, MeOH, H2O, 88%.
Me
Ph
The compounds described in this study were evaluated for inhi-
bition of HCV subgenomic RNA replication using an Huh-7 derived
cell line.17 The metabolic stability of these compounds in human
and rat liver microsomes was also evaluated to predict their
in vivo metabolic stability.18 Initial efforts were focused on evalu-
ating the heteroarenes attached to the 2-position of the indole ring
while retaining the N-isopropyl sulfonamide group on the hetero-
arenes (Table 1). Replacement of the phenyl ring (2a) with a pyri-
dine ring (3a) not only improved potency against the replicon by
more than threefold, but also improved selectivity with respect
to cellular GAPDH. The isomeric 5-(indol-2-yl)pyridine 4 was 17-
fold less potent than the 6-(indol-2-yl)pyridine 3a in the replicon
assay (EC50 = 560 vs 33 nM). Adding an additional nitrogen to the
arene (leading to pyrimidine 5) was also detrimental to activity
compared to 3a. Furthermore, 5-(indo-2-yl)pyrimidine 6, the reg-
ioisomer of pyrimidine 5, had diminished activity against the
Me
O
Me
b
N
(R)
Ph
a
N
+
(R)
Ph
Me
H2N
F3C
(S)
F3C
O
Me
30
Me
F3C
31
c, d
OH
(S)
OH
NH2.HCl
NH2
NH2. HO
(S)
OH
e
f, g
O
(S)
F3C
Me
(S)
F3C
Me
(S)
F3C
Me
34
33
32
Scheme 4. Reagents and conditions: (a) p-TsOH (5%), Si(OEt)4 (0.5 equiv), 2-Me-
THF, reflux, 16 h; (b) DBU (1.5 equiv), 60 °C, 20 h; (c) HCl (concd), MTBE, rt, 13 h; (d)
NaOH (50%), H2O, reflux, then distillation; 58% for 4 steps, 92% ee; (e) D-tartaric acid,
MeOH, reflux, then recrystallization; 90%, 99% ee; (f) NaOH (50%), H2O, reflux, then
distillation, 87%; (g) HCl (6 M), rt, 1 h, 99% , >99% ee.

X. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3947–3953
3951
replicon, indicating the precise requirement for the substitution
pattern of the link between the indole ring and sulfonamide group.
Even though all compounds described in Table 1 were substituted
with an N-isopropyl sulfonamide group, comparison between com-
pounds 3a and 5 and the lead compound 2a revealed that an arene
modification could result in significant improvement in vitro met-
abolic stability.
and metabolic stability. Results from selected examples are shown
in Table 3.
Introducing hydrophilic groups to the terminus of the isopropyl
group (e.g., 1,3-dihydroxyisopropyl, 8d) led to a significant loss of
activity (EC50 = 940 nM) compared to compound 3d. However,
replacement of the isopropyl group in 3d with the more lipophilic
1,3-difluoroisopropyl group (sulfonamide 8c) led to a modest in-
crease in activity (EC50 = 4 nM). The same level of potency ob-
served for compound 8c was also obtained with the 6-
cyclopropyl analog 8o (EC50 = 4 nM). Several mono-, di-, and trif-
luoroalkyl sulfonamides were then investigated. Among these fluo-
roalkyl substituted analogs, compounds 8h and 8r, containing the
(S)-trifluoroisopropyl sulfonamide group (EC50 = 2 and 12 nM,
respectively), also provided similar replicon activity compared to
compounds 8c and 8o. However, the corresponding (R)-trifluoro-
isopropyl enantiomers, 8g and 8q were 30- and 10-fold less potent,
respectively, than the corresponding (S)-enantiomers 8h and 8r,
indicating a stereochemical bias towards activity. Removal of the
methyl group (trifluoroethyl analog 8l) led to a further sixfold drop
in activity compared to that of the (R)-trifluoroisopropyl analog 8g.
Complete removal of the sulfonamide alkyl chain (primary sulfon-
amide 8n) resulted in a further threefold decrease in activity
(EC50 = 1300 nM).
Importantly, we also found that structural modifications to the
sulfonamide moiety (R3) have a pronounced effect on metabolic
stability (Table 3). Based on our understanding of the metabolic
pathway leading to 2a, we hypothesized that introducing elec-
tron-withdrawing groups on the alkyl chain (e.g., fluorine) would
suppress oxidative N-dealkylation. As expected, when the isopropyl
group in 3d was replaced by alkyl groups bearing strong electron-
withdrawing substituents such as trifluoromethyl (8g, 8h, 8i, 8j,
8k and 8l) and cyano (8m), the metabolic stability was improved.
Sulfonamide substituents containing tert-butyl type groups, which
Maintaining the 6-(indol-2-yl)pyridine core structure in 3a, we
next examined variation of the substituents R1 and R2 attached to
the indole ring (Table 2), based on our earlier studies9 indicating
that substituent modification on the indole could dramatically im-
pact replicon activity. For example, we observed that 6-difluoro-
methoxy substitution on the indole ring, as compared to other
functional groups, generally provided compounds with increased
activity against the replicon. Therefore, we initially screened 6-dif-
luoromethoxyindole analogs substituted with simple alkyl groups
on the indole nitrogen. Potent inhibition of the replicon (EC50
<100 nM) was maintained regardless of the size of the alkyl chain.
Furthermore, compounds 3b–3f had a moderate reduction in
metabolism as compared to the lead compound 3a. When the 6-
difluoromethoxy group in 3d (EC50 = 12 nM) was replaced by a 6-
cyclopropyl group in 7a (EC50 = 21 nM), replicon potency was only
slightly reduced. However, the metabolic stability of 7a was im-
proved compared to that of 3d. Moving substituents from the 6-po-
sition of the indole to the 5-position (3d vs 7f, 7a vs 7h, 3g vs 7g),
typically resulted in a more than 10-fold decrease in activity. Com-
pared to the difluoromethoxy substituted compounds 3d and 7f,
the trifluoromethoxy analogs 3g and 7g have improved metabolic
stability but at the expense of reduced replicon activity. Halogen
substitution could be tolerated at the 5-position of the indole as
evidenced by the 5-F (7i) and 5-Cl (7j) analogs which had EC50 val-
ues of 26 and 28 nM, respectively.
SAR studies of the R1 substituent indicated that substitution
with 6-difluoromethoxy and 6-cyclopropyl groups were preferred
as they represent a good balance between activity and metabolic
stability. We then explored the combination of these R1 substitu-
ents with various sulfonamide groups (R3), while maintaining the
R2 group as cyclobutyl, to study further improvements to potency
do not possess an abstractable a-hydrogen on the carbon adjacent
to the sulfonamide nitrogen atom, are typically more resistant to
oxidative N-dealkylation. The t-butyl sulfonamide 8a was metabol-
ically stable. The most active analogs containing the 1,3-difluoro-
isopropyl (8c and 8o) or (S)-trifluoroisopropyl sulfonamides (8h)
Table 2
Effect of R1 and R2 groups on activity and metabolic stability of selected compounds
CN
O
5
R1
S
O
NH
6
N
N
R2
3a-3g, 7a-7j
R1
R2
HCV replicon 1b EC50 (nM)
GAPDH IC50 (nM)a
HLM, CLint (mL/min/kg)
a
b
Compd
3a
3b
3c
3d
3e
3f
3g
7a
7b
7c
7d
7e
7f
6-OCHF2
6-OCHF2
6-OCHF2
6-OCHF2
6-OCHF2
6-OCHF2
6-OCF3
6-c-Pr
c-PrCH2
n-Pr
i-Pr
33
72
61
12
15
22
67
21
>10,000
>10,000
>10,000
9200
>10,000
>10,000
>10,000
9600
>10,000
>10,000
>10,000
>10,000
7100
>10,000
>10,000
>10,000
>10,000
51
40
20
35
23
16
6
12
24
48
54
53
48
1
c-Bu
c-Pentyl
c-Hexyl
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
6-c-Bu
550
13
6-CHF2
6-OCH2CHF2
6-i-PrS
5-OCHF2
5-OCF3
5-c-Pr
1900
1310
540
1100
580
26
7g
7h
7i
<1
121
50
5-F
5-Cl
7j
28
a
See Ref. 19 for assay conditions.
See Ref. 20 for assay conditions.
b

3952
X. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3947–3953
Table 3
Effect of R1 and R3 groups on activity and metabolic stability
CN
R3
NH
O
S
R1
O
N
N
3d,7a, 8a-8r
R1
R3
HCV replicon 1b EC50 (nM)
GAPDH IC50 (nM)
HLM, CLint (mL/min/kg)
a
a
b
Compd
3d
–OCHF2
12
9200
9600
9500
9000
35
7a
8a
8b
c-Pr
21
27
23
12
–OCHF2
–OCHF2
<1
(S)
104
F
8c
–OCHF2
4
>10,000
10
F
OH
8d
8e
8f
–OCHF2
–OCHF2
–OCHF2
940
>10,000
5200
<1
OH
F
520
274
38
F
F
2700
9500
(R)
8g
8h
8i
–OCHF2
–OCHF2
–OCHF2
67
2
>10,000
9800
<1
<1
<1
CF3
(S)
CF3
180
>10,000
CF3
8j
–OCHF2
30
>10,000
<1
CF3
CF3
8k
8l
–OCHF2
–OCHF2
55
>10,000
>10,000
<1
8
CF3
420
8mc
8n
–OCHF2
–OCHF2
220
>10,000
>10,000
<1
<1
CN
H
1300
F
8o
c-Pr
4
>10,000
22
F
8p
8q
8r
c-Pr
c-Pr
c-Pr
74
>10,000
>10,000
>10,000
<1
15
49
CF3
(R)
125
12
CF3
(S)
CF3
a
b
c
See Ref. 19 for assay conditions.
See Ref. 20 for assay conditions.
Racemic.
exhibited a good balance between activity against the replicon and
metabolic stability.
Encouraged by the above results, we evaluated the pharmacoki-
netic properties of selected compounds in the rat after oral admin-
exposure of these compounds correlated well with in vitro metab-
olism. Further, 8c had moderate IV clearance of 13 mL/min/kg and
oral bioavailability of 62% in the rat (Table 5).
Selected compounds including 8c were tested against isolated
HCV enzymes (HCV polymerase, protease, and helicase) and had
no significant inhibition of any of these enzymes at concentrations
up to 10,000 nM (data not shown). To investigate further the
mechanism of action of these compounds, we selected for resis-
tance by passaging HCV replicon cells in the presence of 8c at
istration of
methylcellulose (Table 4). The N-isopropyl sulfonamide analogs
3d and 7a had only moderate oral exposure (Cmax and AUC0–6 h
a single 10 mg/kg dose in 0.4% hydroxypropyl
)
in rat. By comparison, the 1,3-difluoroisopropyl sulfonamides 8c
and 8o had a profound improvement in oral exposure. The oral

X. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3947–3953
3953
Table 4
Replicon activity, metabolic stability and rat PK of selected compounds
a
b
b
c
c
c
Compd Replicon 1ba EC50
(nM)
Replicon 1ba EC90
(nM)
GAPDH IC50
(nM)a
HLM, CLint (mL/min/ RLM, CLint (mL/min/
AUC0–6 h
(nM h)
TMax
(h)
CMax (lg/
mL)
kg)
kg)
3d
7a
8c
8o
12
21
4
60
180
30
9200
9600
>10,000
>10,000
35
12
10
22
99
15
2
579
964
3418
6204
2
2
3
2
0.053
0.087
0.348
0.759
4
40
<1
a
b
c
See Ref. 19 for assay conditions.
See Ref. 20 for assay conditions.
Compounds were lyophilized from acetonitrile/water (1:1) and administered PO as a suspension at a dose of 10 mg/kg in 0.4% hydroxypropyl methylcellulose to male
Sprague–Dawley rats (n = 2). Plasma samples were obtained from the rats for analysis at 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h post-dose.
Table 5
In vitro and in vivo Rat DMPK profile for 8ca
Cmax (PO) (
l
g/mL)
AUC0– (nM h) PO / IV
t1/2 (IV) h
CL (IV) (mL/min/kg)
13
Vss (L/kg)
F (%)
62
1
0.673
15990/12,990
6.0
4.7
a
Dosed PO as a suspension at 10 mg/kg in 0.4% hydroxypropyl methylcellulose; administered IV as a solution at a dose of 5 mg/kg in N-methyl pyrrolidinone/PEG300/
propylene glycol (10:50:40) to male Sprague–Dawley rats.
Boparai, N.; DiNubile, M. J.; Sniukiene, V.; Brass, C. A.; Albrecht, J. K.;
Bronowicki, J.-P. N. Eng. J. Med. 2011, 364, 1195.
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2X, 20X, and 60X the replicon EC90 (60, 600, and 1800 nM, respec-
tively). Resistant replicons were isolated and sequenced, revealing
a number of mutations leading to amino acid substitutions in the
HCV NS4B protein (data not shown). One of these, a phenylalanine
to cysteine substitution at position 98 of NS4B (F98C), was engi-
neered into the wild type replicon. The resulting replicon was se-
quenced to confirm the presence of the engineered mutation and
was shown to be >70-fold less sensitive than the wild type replicon
to 8c. A leucine substitution at this amino acid position (F98L) has
been reported to confer resistance to a series of imidazo[1,2-a]pyr-
idine HCV inhibitors targeting NS4B.18
In summary, we have described the identification and develop-
ment of a novel series of 6-(indol-2-yl)pyridine-3-sulfonamides
that target the HCV NS4B. Preliminary optimization of this series
furnished compounds with highly potent and selective activity
against the HCV replicon. The sulfonamide group was found to
be susceptible to metabolism and modification of both the arene
and sulfonamide moieties resulted in the identification of com-
pounds with improved oral exposure, leading to the discovery of
8c, a highly potent analog with excellent pharmacokinetic proper-
ties in the rat. Further SAR analyses and mechanistic studies of this
novel class of HCV inhibitors will be reported in a subsequent
communication.
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Acknowledgments
The authors would like to thank Shirley Yeh and Jane Yang for
analytical support.
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19. EC50 values are the average of at least two independent determinations. Huh7
cells harboring a genotype 1b HCV bicistronic replicon (Con1) were plated at
5000 cells/well in 96 well plates. Compounds were added to the plates with a
final DMSO concentration of 0.5% and plates were incubated at 37 °C. Cells
were harvested 3 days post dosing and replicon RNA, and GAPDH RNA as an
endogenous control for selectivity, were quantified by real time RT-PCR.
20. Compounds were incubated at a concentration of 1 lM with pooled human or
Sprague–Dawley liver microsomes (0.5 mg/mL protein concentration) for 0, 10,
20, 30 and 60 min, in the presence of 1 mM EDTA, 3 mM MgCl2, 1.3 mM NADP,
3.3 mM
D-glucose 6-phosphate and 1 unit/mL of glucose 6-phosphate
dehydrogenase. Incubated samples were centrifuged at 2000 rpm and the
supernatants were analyzed by LC/MS.