Structure-activity relationship (SAR) optimization of 6-(Indol-2-yl) pyridine-3-sulfonamides: Identification of potent, selective, and orally bioavailable small molecules targeting hepatitis C (HCV) NS4B

Article abstract of DOI:10.1021/jm401621g

A novel, potent, and orally bioavailable inhibitor of hepatitis C RNA replication targeting NS4B, compound 4t (PTC725), has been identified through chemical optimization of the 6-(indol-2-yl)pyridine-3-sulfonamide 2 to improve DMPK and safety properties.

Full text of DOI:10.1021/jm401621g

Article
pubs.acs.org/jmc
Structure−Activity Relationship (SAR) Optimization of 6‑(Indol-2-
yl)pyridine-3-sulfonamides: Identification of Potent, Selective, and
Orally Bioavailable Small Molecules Targeting Hepatitis C (HCV)
NS4B
Nanjing Zhang,†,* Xiaoyan Zhang,† Jin Zhu,† Anthony Turpoff,† Guangming Chen,† Christie Morrill,†
Song Huang,† William Lennox,† Ramesh Kakarla,† Ronggang Liu,† Chunshi Li,† Hongyu Ren,†
Neil Almstead,† Srikanth Venkatraman,‡ F. George Njoroge,‡ Zhengxian Gu,† Valerie Clausen,†
Jason Graci,† Stephen P. Jung,† Yingcong Zheng,† Joseph M. Colacino,† Fred Lahser,‡ Josephine Sheedy,†
Anna Mollin,† Marla Weetall,† Amin Nomeir,‡ and Gary M. Karp†
†PTC Therapeutics, Inc., 100 Corporate Court, South Plainfield, New Jersey 07080, United States
‡Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States
S
* Supporting Information
ABSTRACT: A novel, potent, and orally bioavailable inhibitor of hepatitis C RNA
replication targeting NS4B, compound 4t (PTC725), has been identified through chemical
optimization of the 6-(indol-2-yl)pyridine-3-sulfonamide 2 to improve DMPK and safety
properties. The focus of the SAR investigations has been to identify the optimal combination
of substituents at the indole N-1, C-5, and C-6 positions and the sulfonamide group to limit
the potential for in vivo oxidative metabolism and to achieve an acceptable pharmacokinetic
profile. Compound 4t has excellent potency against the HCV 1b replicon, with an EC50 = 2
nM and a selectivity index of >5000 with respect to cellular GAPDH. Compound 4t has an
overall favorable pharmacokinetic profile with oral bioavailability values of 62%, 78%, and
18% in rats, dogs, and monkeys, respectively, as well as favorable tissue distribution properties with a liver to plasma exposure
ratio of 25 in rats.
kDa integral membrane protein which plays an essential role in
HCV replication, however, has remained relatively unexploited
as a target for chemotherapeutic intervention.12 Recently,
imidazopyridines targeting HCV NS4B have been disclosed.13
We recently reported on two novel series of inhibitors of
HCV RNA replication, the N-(4′-(indol-2-yl)phenyl)-
sulfonamides14 exemplified by 1 and the 6-(indol-2-yl)-
pyridine-3-sulfonamides15 exemplified by 2 (Figure 1).
Optimization in the 6-(indol-2-yl)pyridine-3-sulfonamide series
led to highly potent and selective inhibitors of HCV RNA
replication that target HCV NS4B, of which compound 2
showed highly potent activity (EC50 = 4 nM) against the HCV
1b replicon16 as well as excellent pharmacokinetic properties in
rats. However, safety evaluation in rats identified two potential
liabilities: extensive N-dealkylation of the sulfonamide moiety,
presumably generating 1,3-difluoroacetone,17 and significant
metabolism of the indole ring, leading to the formation of
several glutathione (GSH) adducts. GSH adducts are indicative
of the formation of reactive intermediates which might be
associated with toxicity.18 Furthermore, generation of these
INTRODUCTION
■
Hepatitis C virus (HCV) infects an estimated 150 million
people worldwide, with about 4 million afflicted in the United
States.1 HCV infections typically progress to a chronic state,
often resulting in fibrosis, cirrhosis, and hepatocellular
carcinoma, and is the leading cause for liver transplantation
in the U.S.2 There are at least six HCV genotypes, of which
genotype 1 is the most prevalent in the U.S. and has been
particularly difficult to treat.3 The new standard of care for
patients infected with genotype 1a or 1b is a combination of the
recently approved HCV protease inhibitor telaprevir or
boceprevir with pegylated interferon and the antiviral agent
ribavirin, increasing the sustained virologic response (SVR) to
about 70%.4 However, the high mutation rate of HCV and the
rapid emergence of drug resistance necessitate the continued
discovery and development of drugs with novel mechanisms of
action.5
The HCV genome is a positive sense single-stranded RNA of
approximately 9.6 kb that encodes a polyprotein precursor
which is processed to individual structural (C, E1, E2, and p7)
and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B)
proteins by host and viral proteases.6 The recent development
of direct-acting antiviral agents7 has been mainly focused on the
viral proteins NS3,8 NS5A,9 and the RNA-dependent RNA
polymerase NS5B.10 The nonstructural protein NS4B,11 a 27
Special Issue: HCV Therapies
Received: October 18, 2013
© XXXX American Chemical Society
A
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Figure 1. Novel HCV inhibitors and their activity against the HCV 1b replicon.
a
Scheme 1
a
Conditions: (a) chlorosulfonyl isocyanate, DMF, −78 to −10 °C; (b) R1X, Cs2CO3, DMF, 100 °C; (c) B(Oi-Pr)3, LDA, THF, −78 °C; (d) 9,
PdCl2dppf, aq K2CO3, acetonitrile, 60 °C; (e) B(Oi-Pr)3, LDA, THF, −78 °C then 9, PdCl2dppf, aq K2CO3, DMF, 80 °C; (f) LDA, Bu3SnI, THF,
−78 to 0 °C; (g) 9, Pd(PPh3)4, CuI, DMF, 100 °C.
adducts may result in further GSH depletion in HCV patients,
who are already depleted in GSH as a result of their disease and
are thus especially susceptible to other drug-mediated liver
injuries.19
Herein, we describe our work to optimize the in vivo DMPK
and safety profile of this novel class of 6-(indol-2-yl)pyridine-3-
sulfonamides, which led to the identification of 4t (PTC725),20
a potent, selective, and orally bioavailable compound targeting
HCV NS4B currently under preclinical development.
9. When the Suzuki coupling strategies in both paths A and B
were unsuccessful, a Stille coupling strategy was utilized as
depicted in path C in which a stannane intermediate 10 was
isolated and used in subsequent Stille coupling reactions.
Several intermediates 7 were prepared via functional group
transformation. As depicted in Scheme 2, 6-methoxyindoles
11a and 11b, readily prepared from the corresponding indoles
5 through the sequence in Scheme 1, were demethylated to
give the 6-hydroxyindoles 12a and 12b and further transformed
to 7a and 7m by freonation with chlorodifluoromethane. The
6-bromoindole 13 was readily converted to the 6-ethylindole 7g
and 6-cyclopropylindole 7h through Negishi or Suzuki coupling
reactions. Palladium catalyzed borylation of 13 followed by
oxidation with potassium peroxymonosulfate provided the 6-
hydroxyindole 7i.
Additional final compounds 3 were also accessed through
direct derivatization and functional group transformations
(Scheme 3). Compound 3a, prepared by the sequence shown
in Scheme 1, was brominated at the indole 5-position to give
14. Bromine−lithium exchange of 14 with n-BuLi followed by
treatment with triisopropyl borate and subsequent oxidation
with potassium peroxymonosulfate provided the 5-OH
analogue 3c. Negishi coupling of 14 with the corresponding
zinc reagent provided the 5-Me analogue 3d and 5-Et analogue
3e.
CHEMISTRY
■
The general synthesis of compounds 3 is outlined in Scheme 1.
The starting indoles 5 with appropriate substitutions at the 4-,
5-, and 6- positions were either commercially available or
prepared via multistep synthesis from commercial starting
materials (see Supporting Information). Cyanation at the
indole 3-position with chlorosulfonyl isocyanate provided
intermediates 6, which were alkylated with appropriate alkyl
halides at the indole N-1 position to give compounds 7. The
principal strategy to prepare the final targets 3 was to couple 7
with 2-chloropyridin-5-yl sulfonamide 9 prepared from 2-
chloropyridin-5-yl sulfonyl chloride and (S)-1,1,1-trifluorome-
thylisopropylamine. Depending on the substituents on the
indole ring, three coupling paths were developed. Path A
utilizes a boronic acid intermediate 8, which was subjected to
Suzuki coupling with 9 to furnish the target compounds 3. In
cases where the boronic acid intermediate 8 was not sufficiently
stable or could not be easily isolated, a one-pot process (path
B) was then used in which compounds 7 were converted into a
boronate complex intermediate and, without isolation,
subsequently subjected to Suzuki coupling with sulfonamide
Starting from the 6-OH analogue 3i, methylation at the 6-
OH group provided the 6-OMe analogue 3k. The 6-triflate
compound 15 was obtained by treatment of 3i with triflic
anhydride, which was subsequently converted to the 6-SMe
analogue 3l through a palladium catalyzed coupling reaction
with sodium thiomethoxide.
B
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a
mides 18. Base induced cleavage of the Boc group followed by
cyanation of 19 at the indole 3-position with chlorosulfonyl
isocyanate provided intermediates 20. Copper catalyzed N-
arylation of intermediates 20 at the indole N-1 position with
various commercially available aryl halides provided the final
targets 4.
Scheme 2
All synthesized compounds were evaluated for activity
against the genotype 1b HCV replicon and selectivity with
respect to cellular GAPDH mRNA in replicon-containing Huh-
7 cells, as described previously.15 Briefly, quantitative RT-PCR
was performed to quantify the amount of intracellular HCV
RNA and the concentration of a compound inhibiting HCV
RNA replication by 50% (EC50) is indicated. In parallel,
endogenous GAPDH mRNA was quantified as a control to
determine selectivity. Pharmacokinetic properties of highly
potent compounds were evaluated in rats to assess the potential
of the compounds for development as orally administered anti-
HCV drugs. Compounds were lyophilized from acetonitrile/
water (1:1) and administered PO to male Sprague−Dawley rats
at a dose of 10 mg/kg as a suspension of 0.4% hydroxypropyl
methylcellulose. Both the area under the plasma concen-
tration−time curve from time zero to 6 h after administration
(AUC0−6h) and plasma concentration multiples at 6 h over the
replicon EC50 were evaluated. Compounds with the best
combination of potency and oral exposure in rats were selected
for further evaluation.
a
Conditions: (a) BBr3, CH2Cl2, −20 °C; (b) EtSH, AlCl3, CH2Cl2, 0
°C; (c) CHF2Cl, NaOH, i-PrOH, 75 °C; (d) Et2Zn, PdCl2dppf,
dioxane, 100 °C; (e) c-PrB(OH)2, Pd2(dba)3, t-Bu3PHBF4, KF, THF,
30 °C; (f) PdCl2dppf, B2Pin2, KOAc, dioxane, 80 °C then potassium
peroxymonosulfate, acetone.
The general synthesis of N-aryl analogues 4 is outlined in
Scheme 4. Protection of indoles 5 with Boc2O provided
intermediates 16. LDA treatment of a mixture of 16 and
triisopropyl borate at −78 °C followed by acidic aqueous
workup furnished boronic acids 17, which was coupled with the
sulfonamide 9 to provide the 6-(indol-2-yl)pyridine-3-sulfona-
RESULTS AND DISCUSSION
■
SAR and Pharmacokinetic Studies. The goal of our
optimization effort was to identify compounds with activity
against the HCV replicon comparable to that of the lead
a
Scheme 3
a
Conditions: (a) Br2, HOAc, 95 °C; (b) MeLi, n-BuLi, B(Oi-Pr)3, THF, then potassium peroxymonosulfate, −78 °C to rt; (c) MeZnCl, PdCl2dppf,
THF, 40 °C; (d) Et2Zn, PdCl2dppf, dioxane, 100 °C; (e) MeI, K2CO3, 40 °C; (f) Tf2O, pyridine, CH2Cl2, 0 °C to rt; (g) NaSMe, Pd2(dba)3,
xantphos, dioxane, 100 °C.
C
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a
Scheme 4
a
Conditions: (a) Boc2O, DMAP, CH2Cl2; (b) B(Oi-Pr)3, LDA, THF, −78 °C to rt; (c) 9, Pd2(dba)3, t-Bu3PHBF4, KF, THF, 60 °C; (d) pyrrolidine,
CH3CN, 50 °C; (e) chlorosulfonyl isocyanate, DMF/CH3CN (1:1), 0 °C; (f) ArX, CuI, K2CO3, DMF, 110 °C.
compound 2 but having improved in vivo DMPK and safety
profiles. During the identification of 2, we demonstrated that
the metabolic stability of the 6-(indol-2-yl)pyridine-3-sulfona-
mide series could be significantly improved by incorporating
fluorine(s) or other electron-withdrawing groups into the
amine moiety of the sulfonamide or by utilizing amines lacking
an α-proton such as tert-butyl type amines.15 In particular, the
N-(S)-1,1,1-trifluoroisopropyl sulfonamide group in 3a pro-
vided similar activity against the replicon but with improved
microsome stability compared to that of the N-1,3-difluor-
oisopropyl sulfonamide 215 and, very importantly, lacking
known liabilities associated with the potential N-dealkylated
metabolite(s)21 derived from 2. Thus it became the group of
choice for further optimization.
Table 1. SAR of Substitution on the Indole Core
replicon 1b EC50
(nM)
GAPDH IC50
compd R4
R5
R6
(nM)
3a
3b
3c
3d
3e
3f
H
H
H
H
H
H
H
H
H
H
H
H
F
H
CHF2O
CHF2O
CHF2O
CHF2O
CHF2O
Me
2
7
9600
9100
F
OH
Me
Et
F
63
>10000
10000
108
1400
7
In the process of metabolite characterization of [3H]-2, we
identified several GSH adducts formed through metabolic
activation of the indole in vivo, presumably through transient
epoxide formation on the electron rich indole phenyl ring (data
not shown). However, these GSH conjugates could not be
identified in vitro in either rat hepatocytes or human or rat liver
microsomes or S9 fortified with NADPH and GSH, indicating
in this particular case there was no predictive in vitro system to
evaluate metabolism via the GSH pathway. Consequently, our
strategy was to introduce substituents to the indole phenyl ring
that would eliminate two adjacent unsubstituted carbons with
the goal of reducing metabolic oxidative arene oxide formation
and thus block or reduce GSH adduct formation of these
transiently produced reactive intermediates.
Starting from compound 3a, we evaluated a number of
substituents on the indole phenyl ring, particularly at the 4- and
5- positions in conjunction with 6-substituents, which eliminate
the presence of two contiguous unsubstituted carbons on the
indole phenyl ring. First, a variety of substituents of different
size and lipophilicity were investigated at the indole 5-position
in combination with 6-OCHF2 substitution. As shown in Table
1, the size of the substituent has a significant impact on replicon
activity. The 5-F substitution was well tolerated, with
compound 3b providing the best activity (EC50 = 7 nM),
slightly less potent than the parent compound 3a. Other small
substituents, e.g., 5-Me (3d) and 5-OH (3c), led to a 54- and
32-fold decrease in activity, respectively. The larger 5-Et
substituent (3e) resulted in a >600-fold decrease in activity
compared to that of 3a.
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
3g
3h
3i
F
Et
4
F
c-Pr
28
F
HO
410
460
74
3j
F
Cl
3k
3l
F
MeO
SMe
F
35
3m
3n
H
H
CHF2O
Me
18
F
153
small lipophilic groups were preferred. In addition to the 6-
OCHF2 (3b), small alkyl groups such as 6-Me (3f) and 6-Et
(3g) showed single-digit nanomolar activity, with 3g as the
most potent (EC50 = 4 nM). Decreased activity was observed
for c-Pr (3h), OMe (3k), and SMe (3l), with replicon EC50s in
the range of 28−74 nM. A further decrease in activity was
observed for the 6-Cl (3j) and 6-OH (3i) analogues, with
EC50s of 460 and 410 nM, respectively. The 6-OMe (3k),
though of similar size but differing in electronic properties and
lipophilicity from the OCHF2 group (3b), decreased activity by
more than 10-fold.
Compounds substituted at the indole C-4 and C-6 positions
also do not present two contiguous H-bearing carbon atoms
and thus would also be expected to have a reduced potential for
GSH adduct formation. At the indole 4-position, selected fluoro
substitution was also evaluated. In combination with a 6-
OCHF2 group, compound 3m was 2-fold less active (EC50 = 18
nM) than 3b and 9-fold less active than 3a. However, when
combined with a 6-Me group (3n), the activity was reduced by
more than 20-fold (3n vs 3f).
Having identified the F- group as the optimal substituent at
the indole 5-position, we next re-evaluated substitutions at the
indole 6-position. Consistent with our previous findings,15
D
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Table 2. Pharmacokinetic Properties of Selected Compounds
a
b
compd
replicon 1b EC50 (nM)
rat PK (PO) AUC0−6h (nM·h)
plasma fold over EC50 at 6 h
liver/plasma ratio
3b
3f
7
7
4
13400
1100
300
277
24
11.4
29.1
30.6
3g
18
a
The compounds were lyophilized from acetonitrile/water (1:1) and dosed PO as an oral suspension at 10 mg/kg in 0.4% hydroxypropyl
b
methylcellulose to male Sprague−Dawley rats (n = 2). Determined at 6 h.
The potent 5,6-disubstituted analogues 3b, 3f, and 3g were
assessed for oral exposure in rats (Table 2). After oral
administration at 10 mg/kg, compound 3b bearing 5-F, 6-
OCHF2 substitution showed excellent plasma exposure (13400
nM·h) and high plasma concentration multiples over the
replicon EC50 (277-fold) at 6 h. This is far superior to both
compound 3f with 5-F, 6-Me substitution (1100 nM·h), and 3g
with 5-F, 6-Et substitution (300 nM·h) which have plasma
exposure ratios at 6 h < 25-fold their respective EC50s. In
addition, because the liver is the primary target organ of HCV,
it is desirable for HCV drugs to partition efficiently into the
liver. In this regard, these compounds had very favorable liver
to plasma ratios (10−30-fold).
Compound 3b was further evaluated based on its excellent
activity against the replicon and high oral exposure in rats. In
vivo metabolite characterization of [3H]-3b was evaluated in
bile duct cannulated rats to assess the potential for in vivo GSH
conjugation. After a 5 mg/kg dose, a single GSH adduct
accounting for 2.5% of the dose was observed. This compared
favorably with the metabolism of [3H]-2, in which several GSH
adducts accounting for 17−20% of the dose were observed,
indicating that the approach to reduce the level of GSH adducts
by incorporating 5-F substitution worked as expected. The
metabolite 3o derived from the oxidative N-dealkylation of the
sulfonamide group in 3b (Figure 2) was present in significant
quantities in the bile. In plasma, 3o was the major metabolite.
Importantly, no known toxicities have been reported for 1,1,1-
trifluoroacetone,21 the presumed metabolite formed upon
oxidative N-dealkylation of 3b.
Comprehensive PK studies were also conducted in rats, dogs,
and monkeys. Compound 3b had excellent exposure in all three
species, with AUC0−inf values of 24790−59900 nM·h when
dosed orally at 10 mg/kg in rats and 5 mg/kg in dogs and
monkeys. Compound 3b had oral bioavailability values of 49%,
91%, and 35% in rats, dogs, and monkeys, respectively.
However, the IV clearance of 3b is low in rats (3.0 mL/min/
kg) and monkeys (2.2 mL/min/kg) and particularly low in
dogs (0.3 mL/min/kg). The IV half-lives for rats, monkeys, and
dogs are 11, 22, and 149 h, respectively. The low clearance and
long half-life in large animal species was of some concern.
We turned our attention to the subset of compounds with 5-
F, 6-alkyl substitution such as 3f and 3g, which demonstrated
excellent replicon activity and whereby the 6-alkyl group could
potentially function as an additional metabolic site to increase
clearance. However, as shown in Table 2, the oral exposures of
3f and 3g were modest and required further improvement.
In the course of optimization of compound 3a, one direction
we have taken was to replace the indole N-alkyl groups with
heteroaryl groups in order to expand the chemical space and
improve potency and DMPK properties. Starting from
compound 3a, we replaced the indole N-cyclobutyl substituent
with a pyrimidine ring (4a) whereby the potency and selectivity
were retained and the oral exposure in rats (AUC0−6h = 20120
nM·h) was significantly improved (Table 3). Moreover, when
the 6-OCHF2 group was replaced by a 6-methyl substituent, the
N-2′-pyrimidine analogue 4b retained excellent potency (EC50
= 3 nM). Thus we decided to pursue analogues containing both
5-F, 6-alkyl substitution and N-aryl moieties. We were gratified
to find that compound 4d, obtained from introduction of a F
group to the 5-position of 4b, showed only slightly reduced
activity (EC50 = 6 nM) but had excellent oral exposure
Figure 2. The major plasma metabolite of 3b in rats.
Table 3. SAR of R Substitution on Replicon Activity and Rat PK
a
compd
R
replicon 1b EC50 (nM)
GAPDH IC50 (nM)
Rat PK (PO) AUC0−6h (nM·h)
4a
4b
4c
4d
6-OCHF2
6-Me
7
3
>10000
>10000
>10000
>10000
20120
b
nd
5-F, 6-OCHF2
5-F, 6-Me
16
6
3809
8910
a
Compounds were lyophilized from acetonitrile/water (1:1) and administered to male Sprague−Dawley rats (n = 2) PO at a dose of 10 mg/kg as a
b
suspension in 0.4% hydroxypropyl methylcellulose. Not determined.
E
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Table 4. Replicon Activity and Rat PK of 5-F-6-Alkyl-N-arylindoles
a
b
All compounds had GAPDH mRNA IC50s >10000 nM, the highest concentration tested. Compounds were lyophilized from acetonitrile/water
(1:1) and administered to male Sprague−Dawley rats (n = 2) PO at a dose of 10 mg/kg as a suspension in 0.4% hydroxypropyl methylcellulose.
c
Compounds were lyophilized from acetonitrile/water (1:1) and dosed PO at 10 mg/kg as a suspension in 0.5% hydroxypropyl methylcellulose and
d
0.1% Tween 80 to male Sprague−Dawley rats (n = 3). Not determined.
(AUC0−6h = 8910 μM·h). The PK of the N-2′-pyrimidinyl
analogue 4d compared very favorably with the N-cyclobutyl
analogue 3f. Interestingly, when a F group was introduced to
the 5-position of compound 4a, the oral exposure of the
resulting analogue 4c decreased significantly while the potency
was slightly reduced as well.
We then undertook a comprehensive investigation of N-aryl
analogues in the 5-F, 6-alkyl series. Starting from the readily
F
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available building block 5-F, 6-Me indole, we initially explored a
series of 5-F, 6-Me indole analogues substituted with a wide
variety of heteroaryl rings attached to the indole nitrogen (4d−
4s). As shown in Table 4, replacement of the 2′-pyrimidinyl
ring with a variety of heteroaryl rings provided a series of
compounds (4e−4m) with activity against the replicon at low
nanomolar concentrations (EC50 ≤ 20 nM). The investigated
heteroaryls include electron deficient rings such as pyridines
(4e−4g), pyrazines (4h), and pyridazines (4i), and electron
rich moieties such as thiazoles (4j−4l) and thiophenes (4m),
indicating that a diverse set of N-heteroaryls can be tolerated
and can potentially be used as “handles” to modulate PK or
other properties. The activity of the N-pyridyl derivatives 4e−
4g was not affected by the attachment position of the pyridine
ring to the indole nitrogen. The 2′- (4e), 3′- (4f), and 4′-
pyridyl (4g) analogues had similar potency, with EC50 values
ranging from 11 to 18 nM. A greater difference in activity was
observed for compounds substituted with a thiazole ring.
Activity decreased in the order of 2′-(4j), 5′-(4l), and 4′-
thiazolyl (4k), with replicon EC50 values of 3, 7, and 20 nM,
respectively. The 4′-thiazole analogue 4k had the highest oral
exposure (AUC0−6h = 10270 nM·h). However, when the
plasma concentration at 6 h was normalized to potency, the 2′-
thiazole 4j had the highest concentration multiples over
replicon EC50 (96-fold). The 2′-thienyl analogue 4m was very
potent, with a replicon EC50 = 2 nM but had only modest oral
exposure. Overall, the 2′-pyrimidinyl analogue 4d provided an
optimal balance of replicon potency (EC50 = 6 nM) and oral
exposure (AUC0−6h = 8910 nM·h), with plasma concentration
multiples over the replicon EC50 of more than 200-fold at 6 h.
Next, we examined the effect of substituents on the
heteroaryl rings attached to the indole N-1. Introducing a
fluorine atom to the 2′-pyrimidinyl ring at the distal (5′)
position (4o) was well tolerated (EC50 = 4 nM). Replacing the
5′-F with 5′-Me (4n) resulted in an 8-fold reduction in activity.
Introducing a fluorine atom to the 2′-pyridyl ring at the distal
5′- position (4p) led to a slight improvement in activity (EC50
= 8 nM) compared to that of the parent 4e. Replacing the 5′-F
substituent on the 2′-pyridyl ring with 5′-Me (4q) reduced
activity (EC50 = 25 nM). Addition of a fluorine atom at the 3′-
position of the 2′-pyridyl ring (4r) conferred improved activity
(EC50 = 3 nM) compared to that of the parent 2′-pyridyl
analogue 4e (EC50 = 18 nM), while a larger substituent, e.g., 3′-
Me (4s), reduced the activity significantly (EC50 = 400 nM),
suggesting that the torsion angle between the indole and the N-
aryl ring may play an important role in replicon activity. In
addition to conferring potent activity against the replicon, the
small, electronegative fluorine atom in 4o, 4p, and 4r also
imparts good oral exposures, with AUC values >3000 nM·h and
high plasma concentration multiples, particularly for 4o and 4r
with 187- and 194-fold over their respective replicon EC50s at 6
h.
equal or improved activity compared to that of the
corresponding 5-fluoro-6-methyl analogues. Introducing a
fluorine atom at the 5′-positon of the 2′-pyrimidine (4y)
retained the replicon activity compared to 4t, whereas
substituting with a methyl group at the same position (4z)
resulted in decreased activity, a similar SAR trend as observed
in the 5-fluoro-6-methyl indole series. The 5′-F-2′-pyridinyl
indole 4aa showed decreased activity compared to that of the
parent 2′-pyridyl compound 4u in contrast to the analogous
modification in the 5-fluoro-6-methyl series (4p and 4e), where
introduction of the fluorine led to a small increase in activity.
A limited exploration of the 5-fluoro-6-c-propyl indoles
(4bb−4dd) was also conducted. Although the 5-F, 6-c-Pr
indole 4bb containing a 2′-pyrimidine moiety was equipotent
to the corresponding 5-F, 6-Me analogue 4d, the oral exposure
was reduced significantly. The 2′-pyridine analogues 4cc and
4dd had reduced activity compared to that of 4bb.
Several compounds with an optimal balance between activity
against the replicon (EC50 < 10 nM) and good oral exposure in
rats (AUC0−6h > 3000 nM·h) with plasma concentration
multiples over the replicon EC50 at 6 h of more than 50-fold
were identified (Table 4). These compounds were selected for
further pharmaceutical profiling, including evaluation of their
potential for drug−drug interactions as these drugs will likely
be coadministered with other medications. Such evaluation
included characterizing the compounds for their ability to
inhibit or induce the major drug metabolizing enzymes. The
inhibition of cytochrome P450 enzymes (CYPs) was evaluated
by determining the activity of the compounds against CYPs
2D6, 3A4, and 2C9 in human liver microsomes. The results
revealed essentially no inhibition, with IC50 values >10 μM, the
highest concentration tested (Table 5).
Table 5. Profile of Selected Compounds
human hepatocyte CYP3A4
a
induction (% activation)
CYP inhibition
(3A4, 2D6,
2C9) IC50
(μM)
CYP 3A4
PXR (%
activation,
10 μM)
mRNA
expression
compd
activity
4d
>10
36
7% (3 μM)
39.8% (3 μM)
41.5% (10 μM)
3% (10 μM)
b
b
4o
4r
4t
>10
>10
>10
96
59
37
nd
nd
b
b
nd
nd
6% (3 μM)
9% (5 μM)
9.8% (3 μM)
15.4% (5 μM)
b
b
4u
4y
>10
>10
54
71
nd
nd
b
b
nd
nd
a
b
Percent induction relative to rifampicin, ref 22. Not determined.
The CYP induction was initially evaluated at 10 μM in a PXR
(pregnane X receptor) reporter gene assay. PXR is a nuclear
receptor that when activated increases the transcription and
therefore the synthesis and activity of several drug metabolizing
enzymes and transporters including CYP 3A4, an important
phase I oxidative enzyme responsible for the metabolism of
many drugs.22 Therefore, compounds with significant PXR
activation have the potential to induce drug−drug interactions.
Compounds that showed a strong signal (>40% of positive
control rifampicin) were deprioritized. Compounds with a
borderline signal (30−40%) were further evaluated for
induction in cryopreserved cultured human hepatocytes in
which the activity and mRNA expression levels of CYP3A4
Having investigated various indole N-heteroaryl rings in the
5-fluoro-6-methyl series, we next examined a limited set of 5-
fluoro-6-ethyl indole analogues with selected heteroaryl groups
attached to the indole nitrogen (4t−4aa). Replacement of the
5-F, 6-Me substitution (4b) with 5-F, 6-Et (4t) afforded one of
the most potent analogues in this series. Compound 4t (EC50
=
2 nM) is 3-fold more potent than 4b while maintaining similar
oral exposure in rats (AUC0−6h = 8164 nM·h). At 6 h, the
plasma concentration is more than 340-fold over the replicon
EC50. Replacement of the 2′-pyrimidinyl ring in 4t with other
heteroaryl rings (4u−4x) generally provided analogues with
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were evaluated following a 3-day incubation with test
compound at several concentrations.
half-life (IV) in all species, ranging from 7.5 to 9 h. The
systemic clearance (CL) is higher in rats and monkeys (∼10−
11 mL/min/kg) than in dogs (4.6 mL/min/kg), which
compares favorably to the earlier lead 3b.
Because HCV primarily targets the liver, it is desirable that
drugs have distribution properties that favor exposure in that
organ. We measured the concentration of 4t in plasma, liver,
and brain at multiple time points over 48 h following a 10 mg/
kg oral dose in rats. The mean drug concentration in liver was
25-fold higher than in plasma (based on AUC values). For
comparison, the concentration in brain was ∼5-fold lower than
in plasma, indicating limited brain uptake thereby reducing the
risk of central nervous system toxicity.
Four of the six selected compounds were found to be
transactivators of PXR. The fluoro-substituted N-2′-pyrimidinyl
indole analogues 4o and 4y and the N-(2′-pyridinyl) indoles 4r
and 4u showed significant PXR induction (>50% induction
relative to rifampicin), while the PXR induction for the 2′-
pyrimidinyl analogues 4d and 4t showed a borderline signal
(35−40%). In a follow-up study using cultured cryopreserved
human hepatocytes, both compounds 4d and 4t showed <10%
induction of CYP 3A4 at the highest concentration tested.
However, compound 4d was found to increase CYP3A4 mRNA
expression levels by ∼40% with respect to rifampicin, whereas
compound 4t showed much lower activation (∼10%) at the
same tested concentration (3 μM). Therefore, compound 4t
was advanced for further evaluation.
Metabolite formation of [3H]-4t was evaluated in bile duct
cannulated rats, dogs, and monkeys to assess the metabolic
profile and potential for in vivo GSH conjugation. Compound
4t was metabolized in rats, dogs, and monkeys. Similar to 3b, a
single GSH conjugate accounting for 2% of the dose was
observed in rat bile. No GSH adducts were identified in dog or
monkey bile. Three primary metabolites of 4t were detected in
rat plasma (Figure 3): 4ee, the metabolite derived from the
oxidative N-dealkylation of the sulfonamide group, 4ff, the
metabolite derived from hydroxylation of the ethyl group, and
4gg, the metabolite derived from both N-dealkylation and
hydroxylation. The structures of these metabolites were
confirmed by independent synthesis. The observation of two
hydroxylated metabolites 4ff and 4gg validated our approach of
using the 6-alkyl group as a metabolic handle for increased
clearance. Among the three metabolites, only the hydroxylated
metabolite 4ff retains modest replicon activity. The metabolites
4ee and 4gg, resulting from sulfonamide N-dealkylation, had
dramatically reduced replicon activity. These metabolites were
rapidly cleared from plasma and were found in varying amounts
in rats, dogs, and monkeys. In addition, compounds 4ee−4gg
were observed in rat, dog, and human cryopreserved
hepatocytes, indicating that no human specific metabolites of
4t were identified.
De novo selection of replicon resistance against 4t resulted in
the identification of mutations encoding three amino acid
substitutions in the HCV nonstructural protein 4B (NS4B)
region, F98L, F98C, and V105M.20 The NS4B substitutions
were separately engineered into the Con1 HCV replicon and
each conferred ≥60-fold resistance to 4t. A mutation encoding
the amino acid substitution H94R in NS4B was also identified,
conferring 16-fold resistance. Previous results from our group
and others identified similar mutations in NS4B using other
anti-HCV compounds,13,20 indicating that 4t targets HCV
NS4B.
Profile of the Lead Compound. The pharmacokinetic
profiles of compound 4t in rats, dogs, and monkeys were
evaluated and are summarized in Table 6.20 When dosed orally
Table 6. Pharmacokinetic Profiles of 4t Following Single
a
Dose Administration to Rats, Dogs, and Monkeys
b
c
d
parameters
rat
dog
monkey
AUC0−∞ (PO, nM·h)
Cmax (PO, nM)
20000
2400
10.1
7.5
29000
4000
4.6
3100
540
11.0
8.0
CL (IV) (mL/min/kg)
T1/2 (IV) (h)
8.9
Vdss, (L/kg)
3.9
2.1
3.8
F (%)
62
78
18
plasma/(8 h)/EC50
liver/plasma/brain ratio
325
460
50
e
e
25/1/0.22
nd
nd
a
b
Selected data taken from ref 20. Compound 4t was lyophilized from
acetonitrile/water (1/1) and administered PO to fed male Sprague−
Dawley rats (n = 3) at a dose of 10 mg/kg as a suspension in 0.4%
hydroxypropyl methylcellulose. It was administered IV at a dose of 6
mg/kg as a solution in N-methyl pyrrolidinone/PEG300/propylene
c
glycol (10:40:50). Administered to fed beagle dogs (n = 3) PO at a
dose of 5 mg/kg as a suspension in 0.4% hydroxypropyl
methylcellulose. It was administered IV at a dose of 2.5 mg/kg as a
solution in N-methyl pyrrolidinone/PEG300/propylene glycol
d
(5:50:45). Administered to fed cynomolgous monkeys (n = 3) PO
at a dose of 5 mg/kg as a suspension in 0.4% hydroxypropyl
methylcellulose. It was administered IV at a dose of 2.5 mg/kg as a
solution in N-methyl pyrrolidinone/PEG300/propylene glycol
In addition to the activity against the genotype 1b replicon,
4t also inhibited the replication of the genotype 1a H77S full-
length genome with an EC50 of 7 nM.20,23 Further, 4d had only
modest activity against genotype 2a infectious JFH-1 virus in
Huh-7 cells with an EC50 ∼2200 nM. However, JFH-1 naturally
contains a leucine at position 98 of NS4B that we identified as
conferring resistance to 4t in genotype 1b. Thus, genotype 2
would be expected to be less susceptible to this compound.20
With a favorable overall profile, 4t was evaluated in a non-
GLP 14-day rat safety study and found to be well-tolerated at
doses up to 2000 mg/kg/day.
e
(5:40:55). Not determined.
in rats at 10 mg/kg, 4t had an area under the concentration−
time curve from time zero extrapolated to infinity (AUC0−∞) of
20000 nM·h, with an oral bioavailability of 62% and achieved a
plasma concentration of 325-fold over the replicon EC50 at 8 h.
When dosed orally to dogs at 5 mg/kg, 4t had an AUC0−∞ of
29000 nM·h, with an oral bioavailability of 78% and a plasma
concentration of 460-fold over the replicon EC50 at 8 h. At an
oral dose of 5 mg/kg in monkeys, compound 4t had moderate
exposure with an AUC0−∞ of 3100 nM·h and 18%
bioavailability. Compound 4t possesses a moderate terminal
CONCLUSION
■
Starting from lead compound 2, we conducted extensive SAR
development to improve the DMPK properties and safety
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Figure 3. Primary metabolites of 4t in rats and their activity against the HCV 1b replicon.
1
6-Bromo-5-fluoro-1H-indole-3-carbonitrile (13). Yield 100%. H
NMR (500 MHz, DMSO-d6) δ 12.39 (br s, 1H), 8.36 (d, J = 2.84 Hz,
1H), 7.88 (d, J = 5.99 Hz, 1H), 7.66 (d, J = 8.83 Hz, 1H).
profile and identified a novel series of N-heteroaryl-6-(indol-2-
yl)pyridine-3-sulfonamides. Chemical optimization was focused
primarily on identifying the optimal heteroaryl moieties at the
indole N-1 position and defining the optimal substitution
pattern on the indole benzenoid ring to limit the potential for
in vivo oxidative metabolism including the formation of GSH
adducts. The SAR investigation culminated in identifying
compounds with an optimal balance of replicon potency and
favorable PK properties including acceptable drug clearance.
Several compounds were evaluated for their potential to induce
drug−drug interactions, and compound 4t was selected for
further evaluation. Compound 4t exhibits potent activity in the
cell-based HCV 1b replicon (EC50 = 2 nM), with more than
5000-fold selectivity with respect to cellular GAPDH mRNA.
Compound 4t has a favorable pharmacokinetic profile in rats,
dogs, and monkeys and has favorable tissue distribution, with a
liver to plasma exposure ratio of 25 in rats. It has an excellent
safety profile, being well tolerated at doses up to 2000 mg/kg/
day in a 14-day pilot rat safety study. The overall profile of 4t
makes it a good candidate for preclinical development.
4-Fluoro-6-methyl-1H-indole-3-carbonitrile (6n). Yield 82%. MS
m/z 174.5 [M + H]+. H NMR (500 MHz, DMSO-d6) δ 12.33 (br s,
1
1H), 8.23 (d, J = 3.0 Hz, 1H), 7.18 (s, 1H), 6.88 (d, J = 11.5 Hz, 1H),
2.42 (s, 3H).
General Procedure for the N-Alkylation of Indole-3-carbonitrile 7
(GP-B). To a solution of indole-3-carbonitrile 6 (92 mmol, 1.0 equiv)
in DMF (20 mL) was added cesium carbonate (76 g, 233 mmol, 2.5
equiv) and the corresponding alkyl halide (112 mmol, 1.2 equiv). The
mixture was stirred at 100 °C overnight, then cooled to room
temperature and poured into ice water (500 mL). The precipitate was
collected by filtration, washed with water and ethyl ether, and dried in
a vacuum oven at 50 °C to give the corresponding N-alkyl-indole-3-
carbonitrile 7.
1-Cyclobutyl-6-methoxy-1H-indole-3-carbonitrile (11a). This
compound was prepared from 6-methoxy-1H-indole by following
the general procedures GP-A and GP-B (86% yield over 2 steps). MS
m/z 227.1 [M + H]+. 1H NMR (500 MHz, acetone-d6) δ 8.13 (s, 1H),
7.55 (d, J = 8.5 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 6.96 (dd, J = 8.5, 2.0
Hz, 1H), 5.07 (pentet, J = 8.5 Hz, 1H), 4.15 (s, 3H), 2.65 (m, 2H),
2.52 (m, 2H), 1.98 (m, 2H).
1-Cyclobutyl-6-hydroxy-1H-indole-3-carbonitrile (12a). To a
solution of 11a (100 g, 442 mmol) in CH2Cl2 (700 mL) at −20 °C
was added BBr3 (300 g, 1.2 mol) dropwise over 2.5 h. The mixture was
then poured into ice water (2 L), neutralized to pH 7 with 5 N
aqueous sodium hydroxide, and stirred overnight at room temperature.
The remaining organic solvent was evaporated, and the resulting
suspension was filtered. The solid was washed with water (2 × 200
mL), then 1:1 hexane/ethyl ether (2 × 125 mL), and was dried in a
stream of nitrogen to give the product 12 as a beige powder (90.0 g,
EXPERIMENTAL SECTION
■
Chemistry. Unless otherwise noted, commercial reagents and
solvents were used without further purification. Air or moisture
sensitive reactions were performed under either a nitrogen or argon
atmosphere. Flash chromatography was performed using silica gel with
standard techniques or with silica gel cartridges on an ISCO
Combiflash chromatography instrument. NMR spectra were recorded
1
at 500 MHz for H on a Bruker NMR spectrometer. The chemical
shifts are given in ppm referenced to the deuterated solvent signal.
Coupling constants (J) are recorded in hertz (Hz). LC-MS analyses
were performed on a Waters Acquity UPLC/MS system with an
analytical C18 column, and compounds were detected by UV at 254
nm. All final compounds with reported biological data were
determined to be >95% pure based on LC/MS and NMR unless
otherwise noted.
General Procedure for the Cyanation of Indole 5 to Indole-3-
carbonitrile 6 (GP-A). Into a solution of indole 5 (219 mmol, 1.0
equiv) in DMF (100 mL) cooled in a dry ice−acetone bath was added
dropwise chlorosulfonyl isocyanate (22.8 g, 262 mmol, 1.2 equiv)
while keeping the temperature below −20 °C. After the addition, the
temperature was allowed to slowly rise to −10 °C. The mixture was
stirred at that temperature for 1.5 h before being poured into ice−
water. The mixture was stirred and filtered. The solid was washed with
water, dried, and collected to give the corresponding indole-3-
carbonitrile 6. The following compounds were prepared following this
procedure:
1
96% yield). MS m/z 213.1 [M + H]+. H NMR (500 MHz, acetone-
d6) δ 8.41 (s, 1H), 8.05 (s, 1H), 7.48 (d, J = 8.5 Hz, 1H), 6.99 (d, J =
2.0 Hz, 1H), 6.90 (dd, J = 8.5, 2.0 Hz, 1H), 4.96 (pentet, J = 8.5 Hz,
1H), 2.64 (m, 2H), 2.52 (m, 2H), 1.98 (m, 2H).
1-Cyclobutyl-6-(difluoromethoxy)-1H-indole-3-carbonitrile (7a).
To a solution of 12a (31.7 g, 149.5 mmol) in 160 mL of 2-propanol
at 75 °C was bubbled CClF2H gas via a needle while four portions of
20% aqueous NaOH were added via an addition funnel at 30 min
intervals (40 mL × 4). After 2 h of total reaction time, the mixture was
cooled to room temperature and the organic layer was separated and
concentrated. The aqueous layer was extracted with ethyl acetate, and
the combined organic phases were washed with brine, dried over
MgSO4, filtered, and concentrated. The residue was dissolved in 4:1
hexane/CH2Cl2 (800 mL), stirred with basic alumina (80 g, pH 9.5),
and then filtered and concentrated to yield compound 7a as a white
1
solid (28.3 g, 72% yield). MS m/z 263.1 [M + H]+. H NMR (500
MHz, CDCl3) δ 7.76 (s, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.18 (d, J = 2.0
Hz, 1H), 7.12 (dd, J = 8.5, 2.0 Hz, 1H), 6.55 (t, J = 75 Hz, 1H), 4.82
(pentet, J = 8.0 Hz, 1H), 2.66 (m, 2H), 2.47 (m, 2H), 2.01 (m, 2H).
1-Cyclobutyl-6-(difluoromethoxy)-5-fluoro-1H-indole-3-carboni-
trile (7b). This compound was prepared from 6b by following the
6-Difluoromethoxy-5-fluoro-1H-indole-3-carbonitrile (6b). Yield
1
100%. MS m/z 227.0 [M + H]+. H NMR (500 MHz, DMSO-d6) δ
12.4 (br s, 1H), 8.36 (d, J = 3.0 Hz, 1H), 7.67 (d, J = 10.5 Hz, 1H),
7.53 (d, J = 6.5 Hz, 1H), 7.27 (t, J = 75 Hz, 1H).
5-Fluoro-6-methyl-1H-indole-3-carbonitrile (6f). Yield 100%. H
1
1
general procedure GP-B (77% yield). H NMR (500 MHz, CDCl3) δ
7.78 (s, 1H), 7.52 (d, J = 9.77 Hz, 1H), 7.26 (d, J = 6.30 Hz, 1H), 6.58
(t, J = 73.5 Hz, 1H), 4.77−4.85 (m, 1H), 2.62−2.70 (m, 2H), 2.39−
2.49 (m, 2H), 1.98−2.07 (m, 2H).
NMR (500 MHz, CDCl3) δ 8.45−8.68 (br s, 1H), 7.69 (d, J = 3.15
Hz, 1H), 7.39 (d, J = 9.46 Hz, 1H), 7.26 (d, J = 6.32 Hz, 1H), 2.40 (d,
J = 1.89 Hz, 3H).
I
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1-Cyclobutyl-5-fluoro-6-methyl-1H-indole-3-carbonitrile (7f).
This compound was prepared from 6f by following the general
procedure GP-B (92% yield). 1H NMR (500 MHz, CDCl3) δ 7.68 (s,
1H), 7.35 (d, J = 9.77 Hz, 1H), 7.17 (d, J = 6.31 Hz, 1H), 4.76−4.85
(m, 1H), 2.64 (dt, J = 3.63, 5.60 Hz, 2H), 2.37−2.48 (m, 5H), 1.96−
2.04 (m, 2H).
6-Bromo-1-cyclobutyl-5-fluoro-1H-indole-3-carbonitrile (13).
This compound was prepared from 6-bromo-5-fluoro-1H-indole by
following the general procedures GP-A and GP-B (90% yield over 2
steps). 1H NMR (500 MHz, CDCl3) δ 7.75 (s, 1H), 7.60 (d, J = 5.36
Hz, 1H), 7.50 (d, J = 8.51 Hz, 1H), 4.81 (s, 1H), 2.61−2.72 (m, 2H),
2.35−2.52 (m, 2H), 1.96−2.10 (m, 2H)
following the general procedures GP-A and GP-B (100% yield over 2
steps). MS m/z 245.3 [M + H]+. 1H NMR (500 MHz, CDCl3) δ 7.61
(s, 1H), 6.58−6.68 (m, 2H), 4.70−4.82 (m, 1H), 3.87 (s, 3H), 2.57−
2.71 (m, 2H), 2.43 (dt, J = 2.52, 9.62 Hz, 2H), 1.95−2.07 (m, 2H).
1-Cyclobutyl-4-fluoro-6-hydroxy-1H-indole-3-carbonitrile (12b).
To a cooled (0 °C) solution of 11b (0.6 g, 2.5 mmol) in
dichloromethane (20 mL) was added ethanethiol (2.0 mL), followed
by the addition of aluminum chloride (1.0 g, 7.5 mmol). The reaction
mixture was stirred at 0 °C for 1 h. The reaction was quenched by
addition of water, and the resulting solids were filtered and washed
with water, then dried to provide the title compound as a white solid
1
(0.55 g, 95% yield). MS m/z 231.3 [M + H]+. H NMR (500 MHz,
1-Cyclobutyl-6-ethyl-5-fluoro-1H-indole-3-carbonitrile (7g). To a
solution of 13 (1.3 g, 4.4 mmol) in dioxane (4 mL) under a nitrogen
atmosphere was added PdCl2dppf−dichloromethane complex (0.07 g,
0.09 mmol), followed by addition of a solution of Et2Zn in heptane
(8.0 mL, 1.0 M, 8.0 mmol) at room temperature. The mixture was
heated to 100 °C and stirred for 1 h and then cooled down to 0 °C,
into which saturated sodium bicarbonate (∼25 mL) was added
dropwise. The mixture was then treated with water and extracted with
dichloromethane. The dichloromethane layer was dried over sodium
sulfate and evaporated. The resulting oil was passed through a silica
plug and eluted with 10% ethyl acetate in hexane. The eluent was
evaporated to give the title compound as a white solid (1.0 g, 89%).
acetone-d6) δ 8.65 (s, 1H), 7.94 (s, 1H), 6.69 (d, J = 1.58 Hz, 1H),
6.47 (dd, J = 1.89, 11.98 Hz, 1H), 4.72−4.87 (m, 1H), 2.29−2.54 (m,
4H), 1.75−1.87 (m, 2H).
1-Cyclobutyl-6-(difluoromethoxy)-4-fluoro-1H-indole-3-carboni-
trile (7m). This compound was prepared from 12b in a similar manner
1
to that described for 7a (62% yield). MS m/z 281.1 [M + H]+. H
NMR (500 MHz, CDCl3) δ 7.65 (s, 1H), 6.89 (d, J = 1.58 Hz, 1H),
6.74 (dd, J = 1.50, 10.0 Hz, 1H), 6.45 (t, J = 70.0 Hz, 1H), 4.68−4.75
(m, 1H), 2.55−2.59 (m, 2H), 2.38−2.33 (m, 2H), 1.92−1.96 (m, 2H).
1-Cyclobutyl-4-fluoro-6-methyl-1H-indole-3-carbonitrile (7n).
This compound was prepared from 6n following the general
1
procedure GP-B (88% yield). MS m/z 229.7 [M + H]+. H NMR
1
MS m/z 243.3 [M + H]+. H NMR (500 MHz, CDCl3) δ 7.70 (s,
(500 MHz, CDCl3) δ 8.67 (s, 1H), 7.74 (s, 1H), 7.32 (d, J = 11.5 Hz,
1H), 5.52 (pentet, J = 8.5 Hz, 1H), 3.11 (m, 2H), 3.02 (m, 2H), 2.93
(s, 3H), 2.43 (m, 2H).
1H), 7.37 (d, J = 9.50 Hz, 1H), 7.17 (d, J = 6.00 Hz, 1H), 4.82−4.85
(m, 1H), 2.79 (q, J = 8.00 Hz, 2H), 2.63−2.67 (m, 2H), 2.42−2.46
(m, 2H), 2.00−2.03 (m, 2H), 1.29 (t, J = 8.00 Hz, 3H).
General Procedure for the Coupling of 7 with Sulfonamide 9 to
Prepare 3. Path A (GP-C1). (i) Boronic acid preparation: To a
solution of N-alkyl-indole-3-carbonitrile 7 (8.5 mmol) and triisopropyl
borate (15.2 mmol) in THF (35 mL) at −78 °C was added LDA (1.5
M in cyclohexane, 7.3 mL, 11 mmol). The mixture was stirred at −78
°C for 30 min, then quenched with ice water (60 mL) and stirred for
15 min without cooling. The mixture was extracted with 1/1 hexane/
ethyl acetate (20 mL). The aqueous layer was acidified with 2 N
aqueous HCl to pH 5 and then extracted with CH2Cl2 (60 mL). The
organic layer was dried over Na2SO4, filtered, and concentrated to give
the corresponding boronic acid 8. The purity was estimated by UPLC,
and the crude boronic acid was used without further purification. (ii)
Suzuki coupling: A mixture of the boronic acid 8 prepared as above
(1.15 mmol), 6-chloro-pyridine-3-sulfonamide 9 (0.17 g, 0.63 mmol),
tri-tert-butylphosphonium tetrafluoroborate (20 mg, 0.069 mmol),
Pd2(dba)3 (32 mg, 0.035 mmol), and potassium fluoride (0.4 g, 6.9
mmol) in THF (4.0 mL) was stirred at 40 °C overnight under an Ar
atmosphere. The solvent was then evaporated, and the residue was
purified by silica gel flash chromatography to provide the
corresponding target compound 3.
Path B (GP-C2). To a solution of N-alkyl-indole-3-carbonitrile 7
(0.75 mmol) and triisopropyl borate (1.0 mmol) in THF (2.0 mL) at
−78 °C was added dropwise a solution of LDA (1.5 M in cyclohexane,
0.6 mL, 0.9 mmol). After stirring at this temperature for 30 min, acetic
acid (50 μL, 0.9 mmol) was added. The mixture was then warmed to
room temperature, and the solvent was removed under a stream of
nitrogen. To the residue was added 6-chloro-pyridine-3-sulfonamide 9
(0.6 mmol), PdCl2dppf−dichloromethane complex (25 mg, 0.03
mmol), 2 M aqueous K2CO3 (1.0 mL, 2.0 mmol), and acetonitrile (2.0
mL). The mixture was stirred for 15 h at room temperature, then
diluted with H2O and extracted into EtOAc. The organic layer was
dried over Na2SO4, filtered, and concentrated under vacuum.
Purification by silica gel chromatography provided the corresponding
target compound 3.
1-Cyclobutyl-6-cyclopropyl-5-fluoro-1H-indole-3-carbonitrile
(7h). To a nitrogen flushed flask was added sequentiallyl 6-bromo-1-
cyclobutyl-1H-indole-3-carbonitrile 13 (23 g, 83.6 mmol), cyclo-
propylboronic acid (11.35 g, 125 mmol), Pd2(dba)3 (1.91 g, 2.1
mmol), tri-tert-butylphosphonium tetrafluoroborate (1.46 g, 5.0
mmol), potassium fluoride (29 g, 24.6 mmol), and THF (500 mL).
The mixture was stirred overnight at 30 °C. The mixture was then
filtered through Celite, concentrated, and purified by silica gel flash
chromatography to yield a product containing 1% 3-cyano-1-N-
cyclobutylindole, which was suspended in boiling hexanes (500 mL)
for 10 min and filtered. The purified solid was collected and dried to
1
give the title product as a white solid (78% yield). H NMR (500
MHz, CDCl3) δ 7.67 (s, 1H), 7.33 (d, J = 9.77 Hz, 1H), 6.92 (d, J =
5.99 Hz, 1H), 4.75−4.85 (m, 1H), 2.57−2.68 (m, 2H), 2.40 (dquin, J
= 2.68, 9.58 Hz, 2H), 2.13−2.21 (m, 1H), 1.95−2.04 (m, 2H), 1.00−
1.06 (m, 2H), 0.70−0.76 (m, 2H).
1-Cyclobutyl-5-fluoro-6-hydroxy-1H-indole-3-carbonitrile (7i). A
mixture of 13 (29.9 g, 102 mmol), bis(pinacolato)diboron (33.7 g, 133
mmol), PdCl2dppf−CH2Cl2 complex (4.16 g, 5.1 mmol), and
potassium acetate (30 g, 306 mmol) in dioxane (300 mL) was stirred
at 80 °C overnight. The mixture was then cooled to room
temperature, treated with ethyl acetate (200 mL), and filtered through
a silica Celite pad. The filtrate was concentrated, dissolved in acetone
(300 mL), and cooled to 0 °C, into which was added a slurry of oxone
(125.5 g, 204 mmol) in water (300 mL). The mixture was stirred at
room temperature for 15 min, diluted with ethyl acetate, and
separated. The aqueous layer was extracted with ethyl acetate. The
organic extractions were combined and washed with satd aqueous
NaHSO3 and brine, dried over Na2SO4, and concentrated. The residue
was purified on silica gel to provide 7i as a light-gray solid (19.2 g,
1
82%). MS m/z 229.1 [M − H]−. H NMR (500 MHz, acetone-d6) δ
8.11 (s, 1H), 7.36 (d, J = 10.72 Hz, 1H), 7.18 (d, J = 7.25 Hz, 1H),
4.90−5.05 (m, 1H), 2.80 (s, 1H), 2.58−2.68 (m, 2H), 2.45−2.58 (m,
2H), 1.90−2.02 (m, 2H).
Path C (GP-C3). (i) Stannane preparation: To a solution of N-alkyl-
indole-3-carbonitrile 7 (2.4 mmol), tri-n-butylstannyl iodide (860 μL,
3.1 mmol) in THF (7.0 mL) at −78 °C was added dropwise a solution
of LDA (1.5 M in cyclohexane, 2.6 mL, 3.8 mmol). The mixture was
allowed to warm to room temperature and then stirred for an
additional 3 h. The mixture was then concentrated, and the crude
residue was purified by silica gel chromatography to yield the
corresponding 2-(tributylstannyl)-indole-3-carbonitrile 10 as a clear oil
(90−100% yield). (ii) A mixture of the indole-2-stannane 10 (0.4
6-Chloro-1-cyclobutyl-5-fluoro-1H-indole-3-carbonitrile (7j). This
compound was prepared from 5j by following the general procedures
GP-A and GP-B (92% yield over 2 steps). 1H NMR (500 MHz,
CDCl3) δ 7.75 (s, 1H), 7.50 (d, J = 8.51 Hz, 1H), 7.44 (d, J = 5.67 Hz,
1H), 4.76−4.85 (m, 1H), 2.66 (d, J = 3.47 Hz, 2H), 2.39−2.51 (m,
2H), 1.98−2.08 (m, 2H).
1-Cyclobutyl-4-fluoro-6-methoxy-1H-indole-3-carbonitrile (11b).
This compound was prepared from 4-fluoro-6-methoxy-1H-indole by
J
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mmol), 6-chloro-pyridine-3-sulfonamide 9 (0.4 mmol), Pd(PPh3)4 (45
mg, 0.039 mmol), and CuI (18 mg, 0.094 mmol) in dioxane (0.8 mL)
was heated at 100 °C for 1 h. The reaction mixture was then diluted
with EtOAc and filtered through Celite. The filtrate was concentrated
under vacuum and purified by silica gel chromatography to provide the
corresponding target compound 3.
(3e). To a mixture of 14 (0.15 g, 0.27 mmol) and PdCl2dppf (10 mg,
0.013 mmol) in dioxane (2.0 mL) under a nitrogen atmosphere was
added a solution of diethylzinc in heptane (1.0 M × 1.0 mL, 1.0
mmol). The mixture was stirred at 100 °C for 3 h, then cooled and
carefully quenched with methanol. The mixture was diluted with ethyl
acetate and then washed with 2N HCl and brine. The organic layer
was dried over sodium sulfate and evaporated, and the residue was
purified by reverse phase HPLC to give 3e (85 mg, 58%). MS m/z
543.0 [M + H]+. 1H NMR (500 MHz, CDCl3) δ 9.21 (d, J = 2.21 Hz,
1H), 8.33 (dd, J = 2.05, 8.04 Hz, 1H), 8.02 (d, J = 8.20 Hz, 1H), 7.66
(s, 1H), 7.45 (s, 1H), 6.56 (t, J = 73.5 Hz, 1H), 5.33 (t, J = 8.67 Hz,
1H), 5.09 (d, J = 9.46 Hz, 1H), 4.15 (td, J = 6.98, 9.38 Hz, 1H), 2.82
(q, J = 7.57 Hz, 2H), 2.49−2.60 (m, 2H), 2.37−2.46 (m, 2H), 1.83−
1.96 (m, 2H), 1.45 (d, J = 6.94 Hz, 3H), 1.28−1.33 (m, 3H).
(S)-6-(3-Cyano-1-cyclobutyl-6-(difluoromethoxy)-1H-indol-2-yl)-
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (3a). This
compound was prepared from 7a and 9 by following the general
procedure GP-C3 (43% yield); mp 159−162 °C. MS m/z 515.4 [M +
1
H]+. H NMR (500 MHz, CDCl3) δ 9.23 (d, J = 2.0 Hz, 1H), 8.34
(dd, J = 8.0, 2.5 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.5 Hz,
1H), 7.49 (s, 1H), 7.17 (dd, J = 8.5, 2.0 Hz, 1H), 6.59 (t, J = 76 Hz,
1H), 5.35 (pentet, J = 8.5 Hz, 1H), 4.98 (br s, 1H), 4.17 (m, 1H), 2.57
(pentet, J = 10.0 Hz, 2H), 2.43 (m, 2H), 1.93 (m, 2H), 1.47 (d, J = 7.0
Hz, 3H).
(S)-6-(3-Cyano-1-cyclobutyl-5-fluoro-6-methyl-1H-indol-2-yl)-N-
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (3f). This com-
pound was prepared from 7f and 9 by following the general procedure
(S)-6-(3-Cyano-1-cyclobutyl-6-(difluoromethoxy)-5-fluoro-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(3b). This compound was prepared from 7b and 9 following the
general procedure GP-C1 (56% yield); mp 172−175 °C. MS m/z
533.2 [M + H]+. 1H NMR (500 MHz, DMSO-d6) δ 9.21 (dd, J = 0.90,
2.50 Hz, 1H), 9.01 (br s, 1H), 8.49 (dd, J = 2.21, 8.20 Hz, 1H), 8.12
(dd, J = 0.79, 8.35 Hz, 1H), 7.80(d, J = 9.80 Hz, 1H), 7.78 (d, J = 6.70
Hz, 1H), 7.34(t, J = 73.2 Hz, 1H), 5.17−5.26 (m, 1H), 4.28−4.40 (m,
1H), 2.32 (m, 4H), 1.73 (m, 2H), 1.11 (d, J = 6.94 Hz, 3H).
(S)-6-(5-Bromo-3-cyano-1-cyclobutyl-6-(difluoromethoxy)-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(14). A mixture of 3a (2.2 g, 4.3 mmol) and bromine (0.72 g, 4.5
mmol) in acetic acid (20 mL) was stirred at 95 °C for 6 h and then
poured into ice−water. The mixture was neutralized with NaOH to
pH ∼6 and filtered. The resulting solid was washed with water, dried,
and then purified by silica gel flash chromatography with ethyl acetate
in hexanes (5 to 25% gradient) to give 14 (0.92 g, 36%). MS m/z
1
GP-C1 (68% yield); mp 218−219 °C. MS m/z 481.2 [M + H]+. H
NMR (500 MHz, CDCl3) δ 9.21 (br s, 1H), 8.32 (dd, J = 2.21, 8.20
Hz, 1H), 8.01 (d, J = 8.20 Hz, 1H), 7.49 (d, J = 6.31 Hz, 1H), 7.41 (d,
J = 9.14 Hz, 1H), 5.28−5.37 (m, 1H), 5.04 (d, J = 9.14 Hz, 1H), 4.14−
4.20 (m, 1H), 2.54−2.65 (m, 2H), 2.46 (d, J = 1.89 Hz, 3H), 2.36−
2.45 (m, 2H), 1.83−1.97 (m, 2H), 1.45 (d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-1-cyclobutyl-6-ethyl-5-fluoro-1H-indol-2-yl)-N-
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (3g). This com-
pound was prepared from 7g and 9 by following the general procedure
1
GP-C1 (58% yield); mp 205−206 °C. MS m/z 495.2 [M + H]+. H
NMR (500 MHz, CDCl3) δ 9.13 (d, J = 1.89 Hz, 1H), 8.19−8.30 (m,
1H), 7.93 (dd, J = 0.63, 8.20 Hz, 1H), 7.38−7.45 (m, 1H), 7.34 (d, J =
9.46 Hz, 1H), 5.18−5.32 (m, 1H), 4.81−4.90 (m, 1H), 3.99−4.15 (m,
1H), 2.76 (q, J = 7.60 Hz, 2H), 2.43−2.58 (m, 2H), 2.29−2.39 (m,
2H), 1.75−1.90 (m, 2H), 1.38 (d, J = 7.25 Hz, 3H), 1.25 (t, J = 7.60
Hz, 3H).
1
592.9/594.9 [M + H]+. H NMR (500 MHz, CDCl3) δ 9.22 (dd, J =
(S)-6-(3-Cyano-1-cyclobutyl-6-cyclopropyl-5-fluoro-1H-indol-2-
yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (3h). This
compound was prepared from 7h and 9 by following the general
0.79, 2.36 Hz, 1H), 8.35 (dd, J = 2.52, 8.20 Hz, 1H), 8.07 (s, 1H), 8.02
(dd, J = 0.79, 8.35 Hz, 1H), 7.60 (s, 1H), 6.59 (t, J = 73.5 Hz, 1H),
5.28−5.37 (m, 1H), 4.94 (d, J = 9.46 Hz, 1H), 4.12−4.21 (m, 1H),
2.37−2.55 (m, 4H), 1.84−1.95 (m, 2H), 1.47 (d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-1-cyclobutyl-6-(difluoromethoxy)-5-hydroxy-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(3c). To a solution of 14 (0.3 g, 0.5 mmol) in THF (2.0 mL) at −78
°C was added a solution of MeLi in THF (1.5 M × 0.33 mL, 0.5
mmol) followed by a solution of n-BuLi in hexane (1.6 M × 0.38 mL,
0.6 mmol) after 5 min. B(Oi-Pr)3 was added, and the temperature was
allowed to rise to room temperature. The reaction was then quenched
with saturated ammonium chloride and extracted with ethyl acetate.
The organic layer was separated, dried over sodium sulfate, and
evaporated. The residue was purified by silica gel flash chromatography
with ethyl acetate in dichloromethane (0 to 30% gradient) to provide
1
procedure GP-C1 (34 mg, 31%). MS m/z 507.4 [M + H]+. H NMR
(500 MHz, CDCl3) δ 9.20 (d, J = 1.90 Hz, 1H), 8.32 (dd, J = 2.50,
8.20 Hz, 1H), 8.00 (d, J = 8.20 Hz, 1H), 7.41 (d, J = 9.80 Hz, 1H),
7.23 (d, J = 6.00 Hz, 1H), 5.27−5.35 (m, 1H), 5.08 (d, J = 9.50 Hz,
1H), 4.12−4.19 (m, 1H), 2.46−2.58 (m, 2H), 2.36−2.45 (m, 2H),
2.19−2.25 (m, 1H), 1.84−1.93 (m, 2H), 1.45 (d, J = 6.94 Hz, 3H),
1.06−1.12 (m, 2H), 0.75−0.80 (m, 2H).
(S)-6-(3-Cyano-1-cyclobutyl-5-fluoro-6-hydroxy-1H-indol-2-yl)-N-
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (3i). This com-
pound was prepared from 7i and 9 by following the general procedure
1
GP-C2 (97% yield); mp 194−196 °C. MS m/z 483.3 [M + H]+. H
NMR (500 MHz, DMSO-d6) δ 10.15 (s, 1H), 9.10 (d, J = 1.5 Hz,
1H), 8.92 (br s, 1H), 8.38 (dd, J = 2.52, 8.20 Hz, 1H), 7.98 (dd, J =
0.63, 8.20 Hz, 1H), 7.43 (d, J = 10.72 Hz, 1H), 7.27 (d, J = 7.25 Hz,
1H), 5.13 (t, J = 8.51 Hz, 1H), 4.27 (br s, 1H), 2.27−2.38 (m, 2H),
2.27−2.16 (m, 2H), 1.63−1.77 (m, 2H), 1.03 (d, J = 6.62 Hz, 3H).
(S)-6-(6-Chloro-3-cyano-1-cyclobutyl-5-fluoro-1H-indol-2-yl)-N-
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (3j). This com-
pound was prepared from 7j and 9 by following the general procedure
1
3c (28 mg, 10%). MS m/z 530.9 [M + H]+. H NMR (500 MHz,
CDCl3) δ 9.21 (dd, J = 0.60, 2.21 Hz, 1H), 8.33 (dd, J = 2.21, 8.20 Hz,
1H), 7.98 (dd, J = 0.60, 8.20 Hz, 1H), 7.44 (s, 1H), 7.31 (s, 1H), 6.63
(t, J = 73.8 Hz, 1H), 6.38 (br s, 1H), 5.70 (d, J = 9.40 Hz, 1H), 5.23−
5.30 (m, 1H), 4.11−4.18 (m, 1H), 2.44−2.52 (m, 2H), 2.35−2.43 (m,
2H), 1.79−1.91 (m, 2H), 1.42 (d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-1-cyclobutyl-6-(difluoromethoxy)-5-methyl-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(3d). Into a mixture of 14 (0.3 g, 0.54 mmol) and PdCl2dppf (40 mg,
0.054 mmol) in THF (2.0 mL) under a nitrogen atmosphere was
added a solution of MeZnCl (2.0 M × 0.6 mL, 1.2 mmol). The
mixture was then stirred at 40 °C for 20 h. The reaction was quenched
with saturated NH4Cl and extracted with ethyl acetate. The organic
layers were combined, washed with brine, dried, and purified by
reverse phase HPLC to give 3d (66 mg, 23%). MS m/z 529.2 [M +
H]+. 1H NMR (500 MHz, CDCl3) δ 9.21 (dd, J = 0.63, 2.52 Hz, 1H),
8.33 (dd, J = 2.52, 8.20 Hz, 1H), 8.02 (dd, J = 0.90, 8.20 Hz, 1H), 7.64
(s, 1H), 7.45 (s, 1H), 6.57 (t, J = 73.8, 1H), 5.29−5.37 (m, 1H), 5.12
(d, J = 9.46 Hz, 1H), 4.11−4.20 (m, 1H), 2.49−2.60 (m, 2H), 2.37−
2.46 (m, 5H), 1.82−1.95 (m, 2H), 1.45 (d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-1-cyclobutyl-6-(difluoromethoxy)-5-ethyl-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
1
GP-C1 (52%). MS m/z 501.0/503.0 [M + H]+. H NMR (500 MHz,
CDCl3) δ 9.22 (dd, J = 0.70, 8.20 Hz, 1H), 8.34 (dd, J = 2.20, 8.20 Hz,
1H), 8.02 (dd, J = 1.00, 8.20 Hz, 1H), 7.74 (d, J = 6.00 Hz, 1H), 7.56
(d, J = 8.20 Hz, 1H), 5.28−5.36 (m, 1H), 4.93 (d, J = 9.40 Hz, 1H),
4.14−4.20 (m, 1H), 2.49−2.59 (m, 2H), 2.39−2.46 (m, 2H), 1.85−
1.97 (m, 2H), 1.47 (d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-1-cyclobutyl-5-fluoro-6-methoxy-1H-indol-2-yl)-
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (3k). A mix-
ture of 3i (0.2 g, 0.41 mmol), MeI (65 mg, 0.46 mmol), and aqueous
NaOH (6.25 M × 0.13 mL, 0.82 mmol) in THF was stirred at 40 °C
overnight. The mixture was purified by reverse phase HPLC to provide
1
3k (3.0 mg, 1%). MS m/z 497.4 [M + H]+. H NMR (500 MHz,
CDCl3) δ 9.16 (dd, J = 0.79, 2.36 Hz, 1H), 8.23−8.32 (dd, J = 2.5, 8.2
Hz, 1H), 7.97−8.05 (d, J = 8.2 Hz, 1H), 7.49 (d, J = 9.77 Hz, 1H),
7.34 (d, J = 7.25 Hz, 1H), 5.27−5.39 (m, 1H), 4.71−4.84 (m, 1H),
K
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2.94 (d, J = 0.63 Hz, 3H), 2.55−2.68 (m, 2H), 2.33−2.48 (m, 2H),
1.82−1.97 (m, 3H), 1.40 (d, J = 7.25 Hz, 3H).
evaporated, and the residue was partitioned between ethyl acetate and
HCl (1N). The aqueous phase was further extracted with ethyl acetate.
The combined organics were washed with 1N HCl and brine and then
dried over Na2SO4 and concentrated to give a crude product which
was purified by silica gel flash chromatography using ethyl acetate in
hexanes as eluent to give the desired product 19.
General Procedure for the Preparation of 20. To a solution of 19
(0.53 mmol) in a mixture of DMF (0.5 mL) and acetonitrile (0.5 mL)
was added chlorosulfonyl isocyanate dropwise at −50 °C. The reaction
mixture was kept stirring at 0 °C for 0.5 h and then quenched with ice,
poured into water, and then stirred at room temperature for an
additional 0.5 h. The resulting solid was collected by filtration, washed
sequentially with water, hexane, and CH2Cl2, and air-dried to give 19,
which was usually pure enough to be used in the next step without
further purification.
General Procedure for the Preparation of 4. A mixture of 20 (0.5
mmol), aryl halide (1.0 mmol), potassium carbonate (1.5 mmol), and
copper(I) iodide (20 mg, 0.1 mmol) were mixed with DMF (2.5 mL).
The system was evacuated and replaced with an argon atmosphere.
The reaction mixture was stirred at 110 °C overnight, then cooled to 0
°C, diluted with ethyl acetate, and neutralized with 1N HCl. The ethyl
acetate layer was washed with water and brine, dried over Na2SO4, and
filtered through silica gel pad and evaporated. The resulting residue
was purified by silica gel flash chromatography using ethyl acetate in
hexanes as eluent to give the desired product 4.
(S)-6-(3-Cyano-1-cyclobutyl-5-fluoro-6-(methylthio)-1H-indol-2-
yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (3l). To a
solution of 3i (1.1 g, 2.5 mmol) and pyridine (1.0 mL, 12.5 mmol) in
CH2Cl2 (25 mL) was added triflic anhydride (0.5 mL, 3.0 mmol)
dropwise at 0 °C. The mixture was stirred at room temperature for 2 h,
then treated with ether (50 mL), washed with 1N HCl and brine, and
then dried and evaporated to give the crude triflate 15, which was used
in the next step without further purification.
A mixture of the triflate 15 obtained above (90 mg, 0.16 mmol),
sodium thiomethoxide (13 mg, 0.18 mmol), Pd2(dba)3 (3.7 mg, 0.004
mmol), xantphos (4.6 mg, 0.008 mmol), and DIPEA (41 mg, 0.32
mmol) in dioxane (2.0 mL) was stirred at 100 °C overnight under an
Ar atmosphere. The mixture was then cooled, treated with water, and
extracted with ethyl acetate. The combined organic layers were dried
over sodium sulfate and evaporated. The residue was purified by silica
gel chromatography to provide 3l (88 mg, 100%); mp 201−203 °C.
MS m/z 513.6 [M + H]+. 1H NMR (500 MHz, CDCl3) δ 9.21 (dd, J =
0.6, 2.2 Hz, 1H), 8.33 (dd, J = 2.2, 8.2 Hz, 1H), 8.01 (dd, J = 0.9, 8.2
Hz, 1H), 7.58 (d, J = 6.0 Hz, 1H), 7.46 (d, J = 9.2 Hz, 1H), 5.39−5.31
(m, 1H), 5.26 (d, J = 9.4 Hz, 1H), 4.18−4.13 (m, 1H), 2.56 (m, 5H),
2.38−2.49 (m, 2H), 1.86−1.95 (m, 2H), 1.44 (d, J = 6.9 Hz, 3H).
(S)-6-(3-Cyano-1-cyclobutyl-6-(difluoromethoxy)-4-fluoro-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(3m). This compound was prepared from 7m and 9 following the
general procedure GP-C1 (27% yield); mp 210−212 °C. MS m/z
533.1 [M + H]+. 1H NMR (500 MHz, acetone-d6) δ 9.01 (d, J = 1.58
Hz, 1H), 8.13 (dd, J = 2.52, 8.20 Hz, 1H), 7.83 (dd, J = 0.95, 8.20 Hz,
1H), 6.67 (d, J = 8.20 Hz, 1H), 6.36 (t, J = 70.0 Hz, 1H), 5.01−5.16
(m, 1H), 4.65−4.77 (m, 1H), 3.87−4.07 (m, 1H), 2.08−2.36 (m, 5H),
1.64−1.73 (m, 2H), 1.25 (d, J = 6.94 Hz, 3H).
The following compounds were prepared according to the reaction
sequence outlined in Scheme 4 and the general procedure described as
above.
(S)-6-(3-Cyano-6-(difluoromethoxy)-1-(pyrimidin-2-yl)-1H-indol-
2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4a).
Melting point 186−189 °C. MS m/z 539.3 [M + H]+. 1H NMR
(500 MHz, CDCl3) δ 8.83 (d, J = 2.2 Hz, 1H), 8.68 (d, J = 4.6 Hz,
2H), 8.31 (dd, J = 2.2, 8.2 Hz, 1H), 8.11 (d, J = 8.2 Hz, 1H), 7.99 (d, J
= 2.2 Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.30 (t, J = 4.5 Hz, 1H), 7.26
(d, J = 1.9 Hz, 1H), 6.59 (t, J = 73.8 Hz, 1H), 4.84 (d, J = 9.5 Hz, 1H),
4.06−4.12 (m, 1H), 1.44 (d, J = 6.9 Hz, 3H).
(S)-6-(3-Cyano-1-cyclobutyl-4-fluoro-6-methyl-1H-indol-2-yl)-N-
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (3n). This com-
pound was prepared from 7n and 9 by following the general procedure
1
GP-C3 (50% yield); mp 205−210 °C. MS m/z 481.4 [M + H]+. H
NMR (500 MHz, CDCl3) δ 9.22 (s, 1H), 8.34 (d, J = 8.0 Hz, 1H),
8.04 (d, J = 8.5 Hz, 1H), 7.26 (s, 1H), 6.84 (d, J = 9.0 Hz, 1H), 5.29
(pentet, J = 8.5 Hz, 1H), 5.04 (d, J = 9.5 Hz, 1H), 4.16 (m, 1H), 2.60
(m, 2H), 2.53 (s, 3H), 2.40 (m, 2H), 1.89 (m, 2H), 1.45 (d, J = 7.0
Hz, 3H).
(S)-6-(3-Cyano-6-methyl-1-(pyrimidin-2-yl)-1H-indol-2-yl)-N-
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4b). Melting
1
point 195−198 °C. MS m/z 487.2 [M + H]+. H NMR (500 MHz,
CDCl3) δ 8.80 (d, J = 2.52 Hz, 1H), 8.69 (d, J = 4.7 Hz, 2H), 8.28 (dd,
J = 2.36, 8.35 Hz, 1H), 8.12 (dd, J = 0.79, 8.35 Hz, 1H), 7.90 (d, J =
0.63 Hz, 1H), 7.75 (d, J = 8.20 Hz, 1H), 7.28 (t, J = 4.7 Hz, 1H), 7.27
(d, J = 8.2 Hz, 1H), 5.08 (d, J = 9.4 Hz, 1H), 4.02−4.10 (m, 1H), 2.53
(s, 3H), 1.41 (d, J = 7.25 Hz, 3H).
General Procedure for the Preparation of 16. To a solution of 5
(1.25 g, 5.3 mmol) in CH2Cl2 (10 mL) were added di-tert-butyl
dicarbonate (1.5 g, 7.0 mmol) and DMAP (13 mg, 0.1 mmol). The
reaction mixture was kept stirring for 2 h at room temperature and
then partitioned between CH2Cl2 and water. The organic layer was
washed with water and brine and dried over Na2SO4 and evaporated.
The resulting white solid was usually pure enough to be used in the
next step without further purification.
General Procedure for the Preparation of 17. Into a solution of 16
(3.5 mmol) and tri-isopropyl borate (1.2 mL, 5.25 mmol) in THF (7.0
mL) at −78 °C was added LDA (1.5 M in cyclohexane, 2.8 mL, 4.2
mmol) under nitrogen. The mixture was stirred at −78 °C for 15 min
and then for 2 h at 0 °C. The reaction was quenched by slow addition
of acetic acid (0.24 mL, 4.2 mmol), followed by the removal of
solvents by rotary evaporation. The resulting boronic acid 17 was used
in the next step without further purification.
General Procedure for the Preparation of 18. Into a solution of
the boronic acid 17 prepared as above, sulfonamide 9 (0.91 g, 3.15
mmol), and PdCl2dppf (0.23 g, 0.31 mmol) in acetonitrile (21 mL)
was added a solution of potassium carbonate (2.0 M, 7.0 mL, 14
mmol). The mixture was stirred at 50 °C overnight, cooled to room
temperature, and then quenched by addition of HCl (1 N) to pH ∼5.
The mixture was then extracted with ethyl acetate. The combined
organics were dried over Na2SO4 and concentrated to give a crude
product which was usually used in the next step without further
purification.
(S)-6-(3-Cyano-6-(difluoromethoxy)-5-fluoro-1-(pyrimidin-2-yl)-
1H-indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
1
(4c). MS m/z 556.9 [M + H]+. H NMR (500 MHz, CDCl3) δ 8.80
(br s, 1H), 8.69 (d, J = 4.7 Hz, 2H), 8.28 (dd, J = 2.5, 8.5 Hz, 1H),
8.13 (d, J = 6.6 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 9.1 Hz,
1H), 7.32 (t, J = 4.7 Hz, 1H), 6.63 (t, J = 73.4 Hz, 1H), 4.54−4.63 (m,
1H), 1.39 (d, J = 7.25 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(pyrimidin-2-yl)-1H-indol-2-
yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4d). MS
1
m/z 505.1 [M + H]+. H NMR (500 MHz, CDCl3) δ 8.81 (dd, J =
0.6, 2.5 Hz, 1H), 8.68 (d, J = 4.7 Hz, 2H), 8.29 (dd, J = 2.5, 8.5 Hz,
1H), 8.11 (dd, J = 0.6, 8.5 Hz, 1H), 7.96 (d, J = 6.3 Hz, 1H), 7.47 (d, J
= 8.8 Hz, 1H), 7.29 (t, J = 4.7 Hz, 1H), 4.89 (d, J = 9.1 Hz, 1H),
4.06−4.11 (m, 1H), 2.44 (d, J = 1.5 Hz, 3H), 1.42 (d, J = 7.0 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(pyridin-2-yl)-1H-indol-2-yl)-
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4e). Melting
1
point 160−164 °C. MS m/z 504.8 [M + H]+. H NMR (500 MHz,
acetone-d6) δ 8.88 (dd, J = 0.9, 2.2 Hz, 1H), 8.49−8.52 (m, 1H), 8.39
(dd, J = 2.6, 8.2 Hz, 1H), 8.02 (dt, J = 1.9, 7.7 Hz, 1H), 7.97 (dd, J =
0.8, 8.3 Hz, 1H), 7.66 (br s, 1H), 7.48−7.53 (m, 4H), 4.22−4.32 (m,
1H), 2.38 (d, J = 1.6 Hz, 3H), 1.25 (d, J = 6.9 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(pyridin-3-yl)-1H-indol-2-yl)-
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4f). Melting
1
General Procedure for the Preparation of 19. A mixture of 18
prepared as above and pyrrolidine (1.5 mL, 18 mmol) in acetonitrile
(15 mL) was stirred at 50 °C for 2 h. The solvents were then
point 217−221 °C. MS m/z 503.5 [M + H]+. H NMR (500 MHz,
DMSO-d6) δ 8.89 (dd, J = 0.79, 2.36 Hz, 2H), 8.67−8.73 (m, 1H),
8.61 (d, J = 2.21 Hz, 1H), 8.35 (dd, J = 2.36, 8.35 Hz, 1H), 7.91−7.96
L
dx.doi.org/10.1021/jm401621g | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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(m, 1H), 7.88 (dd, J = 0.79, 8.35 Hz, 1H), 7.65 (d, J = 9.14 Hz, 1H),
7.56−7.61 (m, 1H), 7.28 (d, J = 5.99 Hz, 1H), 4.19−4.32 (m, 1H),
2.34 (d, J = 1.58 Hz, 3H), 1.02 (d, J = 6.94 Hz, 3H).
4.23−4.34 (m, 1H), 2.40 (d, J = 1.89 Hz, 3H), 1.27 (d, J = 6.94 Hz,
3H).
(S)-6-(3-Cyano-5-fluoro-1-(5-fluoropyridin-2-yl)-6-methyl-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(4p). Melting point 239−241 °C. MS m/z 512.8 [M + H]+. 1H NMR
(500 MHz, CDCl3) δ 8.79 (dd, J = 1.0, 2.5 Hz, 1H), 8.34 (d, J = 2.5
Hz, 1H), 8.24 (dd, J = 2.5, 8.2 Hz, 1H), 8.11 (dd, J = 0.9, 8.5 Hz, 1H),
7.58−7.63 (m, 1H), 7.50 (d, J = 8.9 Hz, 1H), 7.31−7.34 (m, 1H), 7.21
(d, J = 6.3 Hz, 1H), 4.86 (d, J = 9.5 Hz, 1H), 4.04−4.10 (m, 1H), 2.39
(d, J = 1.5 Hz, 3H), 1.42 (d, J = 6.9 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(pyridin-4-yl)-1H-indol-2-yl)-
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4g). Melting
1
point 250−255 °C. MS m/z 504.5 [M + H]+. H NMR (500 MHz,
acetone-d6) δ 8.91−8.94 (m, 1H), 8.74 (d, J = 5.36 Hz, 2H), 8.41 (dd,
J = 2.36, 8.35 Hz, 1H), 8.01 (dd, J = 0.79, 8.35 Hz, 1H), 7.64−7.70 (m,
1H), 7.54 (d, J = 9.14 Hz, 1H), 7.39−7.45 (m, 3H), 4.23−4.34 (m,
1H), 2.39 (dd, J = 0.63, 2.21 Hz, 3H), 1.25 (d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(pyrazin-2-yl)-1H-indol-2-yl)-
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4h). Melting
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(5-methylpyridin-2-yl)-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(4q). Melting point 250−254 °C. MS m/z 517.5 [M + H]+. 1H NMR
(500 MHz, acetone-d6) δ 8.90 (dd, J = 1.0, 2.2 Hz, 1H), 8.37 (dd, J =
3.5, 8.2 Hz, 1H), 8.33−8.35 (m, 1H), 7.92 (dd, J = 0.9, 8.5 Hz, 1H),
7.81−7.85 (m, 1H), 7.58−7.66 (br s, 1H), 7.50 (d, J = 9.5 Hz, 1H),
7.49 (d, J = 5.9 Hz, 1H), 7.40 (d, J = 8.51 Hz, 1H), 4.24−4.32 (m,
1H), 2.42 (s, 3H), 2.38 (d, J = 2.2 Hz, 3H), 1.25 (d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-1-(3-fluoropyridin-2-yl)-6-methyl-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
1
point 220−223 °C. MS m/z 505.4 [M + H]+. H NMR (500 MHz,
CDCl3) δ 8.71 (dd, J = 0.79, 2.05 Hz, 1H), 8.67 (d, J = 2.52 Hz, 1H),
8.65 (d, J = 0.95 Hz, 1H), 8.51 (dd, J = 1.26, 2.52 Hz, 1H), 8.23−8.30
(m, 2H), 7.52 (d, J = 8.83 Hz, 1H), 7.27 (d, J = 5.40 Hz, 1H), 4.96 (d,
J = 9.46 Hz, 1H), 4.01−4.10 (m, 1H), 2.41 (d, J = 1.89 Hz, 3H), 1.41
(d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(pyridazin-3-yl)-1H-indol-2-
yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4i). Melt-
ing point 245−250 °C. MS m/z 504.5 [M + H]+. 1H NMR (500 MHz,
DMSO-d6) δ 9.36 (dd, J = 2.21, 4.10 Hz, 1H), 8.89 (br s, 1H), 8.76−
8.80 (m, 1H), 8.41 (dd, J = 2.36, 8.35 Hz, 1H), 8.10−8.15 (m, 1H),
7.94−8.00 (m, 2H), 7.69 (d, J = 9.14 Hz, 1H), 7.52 (d, J = 5.99 Hz,
1H), 4.17−4.32 (m, 1H), 2.36 (d, J = 1.58 Hz, 3H), 1.03 (d, J = 6.94
Hz, 3H).
1
(4r). Melting point 193−198 °C. MS m/z 521.5 [M + H]+. H NMR
(500 MHz, acetone-d6) δ 8.78 (dd, J = 0.9, 2.2 Hz, 1H), 8.46 (dd, J =
2.5, 8.5 Hz, 1H), 8.41 (td, J = 0.9, 4.7 Hz, 1H), 8.24 (dd, J = 0.9, 8.5
Hz, 1H), 7.88−7.94 (m, 1H), 7.68−7.72 (m, 1H), 7.64 (br s, 1H),
7.56 (d, J = 9.2 Hz, 1H), 7.37 (d, J = 6.0 Hz, 1H), 4.22−4.32 (m, 1H),
2.38 (d, J = 1.89 Hz, 3H), 1.23 (d, J = 7.25 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(3-methylpyridin-2-yl)-1H-
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(thiazol-2-yl)-1H-indol-2-yl)-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4j). Melting
1
(4s). Melting point 219−221 °C. MS m/z 518.2 [M + H]+. H NMR
1
point 203−208 °C. MS m/z 509.5 [M + H]+. H NMR (500 MHz,
(500 MHz, CDCl3) δ 8.71 (d, J = 1.9 Hz, 1H), 8.38 (br s, 1H), 8.17
(dd, J = 2.2, 8.5 Hz, 1H), 8.12 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 7.2 Hz,
1H), 7.51 (d, J = 9.2 Hz, 1H), 7.38−7.42 (m, 1H), 6.78 (d, J = 6.0 Hz,
1H), 4.88 (d, J = 9.4 Hz, 1H), 3.99−4.06 (m, 1H), 2.35 (d, J = 2.2 Hz,
3H), 2.04 (d, J = 3.15 Hz, 3H), 1.38 (d, J = 7.0 Hz, 3H).
acetone-d6) δ 8.99 (dd, J = 0.79, 2.36 Hz, 1H), 8.46 (dd, J = 2.21, 8.20
Hz, 1H), 8.09 (dd, J = 0.79, 8.35 Hz, 1H), 7.79−7.83 (m, 2H), 7.70
(br s, 1H), 7.63 (d, J = 6.31 Hz, 1H), 7.54 (d, J = 9.14 Hz, 1H), 4.26−
4.36 (m, 1H), 2.42 (d, J = 2.21 Hz, 3H), 1.26 (d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(thiazol-4-yl)-1H-indol-2-yl)-
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4k). Melting
(S)-6-(3-Cyano-6-ethyl-5-fluoro-1-(pyrimidin-2-yl)-1H-indol-2-yl)-
1
point 184−189 °C. MS m/z 509.5 [M + H]+. H NMR (500 MHz,
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4t). Melting
1
point 195−198 °C. MS m/z 519.1 [M + H]+. H NMR (500 MHz,
DMSO-d6) δ 9.17 (d, J = 2.2 Hz, 1H), 8.96 (dd, J = 0.9, 2.2 Hz, 1H),
8.88−8.94 (m, 1H), 8.33 (dd, J = 2.2, 8.5 Hz, 1H), 8.02 (d, J = 2.21
Hz, 1H), 7.75 (dd, J = 0.79, 8.35 Hz, 1H), 7.63 (d, J = 9.46 Hz, 1H),
7.42 (d, J = 5.3 Hz, 1H), 4.19−4.34 (m, 1H), 2.37 (d, J = 1.58 Hz,
3H), 1.05 (d, J = 6.94 Hz, 3H).
CDCl3) δ 8.81 (dd, J = 0.9, 2.2 Hz, 1H), 8.69 (d, J = 4.73 Hz, 2H),
8.29 (dd, J = 2.5, 8.2 Hz, 1H), 8.11 (dd, J = 0.95, 8.51 Hz, 1H), 7.97
(d, J = 6.3 Hz, 1H), 7.48 (d, J = 9.46 Hz, 1H), 7.30 (t, J = 4.89 Hz,
1H), 4.90 (d, J = 9.8 Hz, 1H), 4.04−4.12 (m, 1H), 2.83 (q, J = 7.5 Hz,
2H), 1.42 (d, J = 6.94 Hz, 3H), 1.30 (t, J = 7.57 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(thiazol-5-yl)-1H-indol-2-yl)-
(S)-6-(3-Cyano-6-ethyl-5-fluoro-1-(pyridin-2-yl)-1H-indol-2-yl)-N-
N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4l). Melting
1
point 234−238 °C. MS m/z 509.5 [M + H]+. H NMR (500 MHz,
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4u). Melting
1
point 205−208 °C. MS m/z 517.8 [M + H]+. H NMR (500 MHz,
DMSO-d6) δ 9.25 (d, J = 0.95 Hz, 1H), 9.01 (dd, J = 0.9, 2.2 Hz, 1H),
8.94 (d, J = 8.6 Hz, 1H), 8.37 (dd, J = 2.2, 8.2 Hz, 1H), 8.13 (d, J =
0.63 Hz, 1H), 7.91 (dd, J = 1.0, 8.2 Hz, 1H), 7.64 (d, J = 9.1 Hz, 1H),
7.35 (d, J = 6.0 Hz, 1H), 4.23−4.34 (m, 1H), 2.37 (d, J = 1.58 Hz,
3H), 1.04 (d, J = 6.94 Hz, 3H).
CDCl3) δ 8.79−8.81 (m, 1H), 8.50−8.52 (m, 1H), 8.21 (dd, J = 2.5,
8.5 Hz, 1H), 8.02 (dd, J = 1.0, 8.5 Hz, 1H), 7.89 (dt, J = 1.9, 7.6 Hz,
1H), 7.50 (d, J = 9.5 Hz, 1H), 7.41 (ddd, J = 1.0, 4.8, 7.3 Hz, 1H),
7.28−7.32 (m, 2H), 4.85 (d, J = 9.4 Hz, 1H), 4.01−4.09 (m, 1H), 2.78
(t, J = 7.6 Hz, 2H), 1.41 (d, J = 6.9 Hz, 3H), 1.25 (t, J = 7.6 Hz, 3H).
(S)-6-(3-Cyano-6-ethyl-5-fluoro-1-(pyridin-4-yl)-1H-indol-2-yl)-N-
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4v). Melting
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(thiophen-2-yl)-1H-indol-2-
yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4m). MS
m/z 508.5 [M + H]+. 1H NMR (500 MHz, DMSO-d6) δ 9.05 (dd, J =
0.95, 2.52 Hz, 1H), 8.92 (d, J = 8.5 Hz, 1H), 8.31 (dd, J = 2.36, 8.35
Hz, 1H), 7.75 (dd, J = 0.79, 8.35 Hz, 1H), 7.69 (dd, J = 1.42, 5.52 Hz,
1H), 7.62 (d, J = 9.46 Hz, 1H), 7.35 (dd, J = 1.58, 3.78 Hz, 1H), 7.32
(d, J = 5.99 Hz, 1H), 7.15 (dd, J = 3.78, 5.67 Hz, 1H), 4.23−4.34 (m,
1H), 2.36 (d, J = 1.58 Hz, 3H), 1.04 (d, J = 6.94 Hz, 3H).
(S)-6-(3-Cyano-5-fluoro-6-methyl-1-(5-methylpyrimidin-2-yl)-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(4n). Melting point 179−184 °C. MS m/z 518.5 [M + H]+. 1H NMR
(500 MHz, acetone-d6) δ 8.87 (dd, J = 0.79, 2.36 Hz, 1H), 8.63 (d, J =
0.95 Hz, 2H), 8.44 (dd, J = 2.36, 8.35 Hz, 1H), 8.09 (dd, J = 0.79, 8.35
Hz, 1H), 8.02 (d, J = 7.20 Hz, 1H), 7.68 (d, J = 9.14 Hz, 1H), 7.47 (d,
J = 9.14 Hz, 1H), 4.23−4.35 (m, 1H), 2.40 (d, J = 1.95 Hz, 3H), 2.38
(s, 3H), 1.27 (d, J = 6.94 Hz, 3H).
1
point 209−215 °C. MS m/z 518.3 [M + H]+. H NMR (500 MHz,
acetone-d6) δ 8.94 (dd, J = 0.79, 2.36 Hz, 1H), 8.78 (br s, 2H), 8.42
(dd, J = 2.6, 8.2 Hz, 1H), 8.02 (dd, J = 1.0, 8.5 Hz, 1H), 7.68 (d, J =
8.8 Hz, 1H), 7.55 (d, J = 9.46 Hz, 1H), 7.48 (br s, 2H), 7.43 (d, J =
5.99 Hz, 1H), 4.24−4.34 (m, 1H), 2.80 (q, J = 7.5 Hz, 2H), 1.25 (d, J
= 7.2 Hz, 3H), 1.23 (t, J = 7.5 Hz, 3H).
(S)-6-(3-Cyano-6-ethyl-5-fluoro-1-(pyrazin-2-yl)-1H-indol-2-yl)-N-
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4w). Melting
1
point 225−229 °C. MS m/z 519.0 [M + H]+. H NMR (500 MHz,
acetone-d6) δ 8.82 (dd, J = 0.79, 2.36 Hz, 1H), 8.79 (d, J = 1.89 Hz,
1H), 8.75 (d, J = 2.6 Hz, 1H), 8.62 (dd, J = 1.42, 2.68 Hz, 1H), 8.48
(dd, J = 2.6, 8.6 Hz, 1H), 8.24 (dd, J = 0.9, 8.5 Hz, 1H), 7.64−7.69 (br
s, 1H), 7.60 (d, J = 5.67 Hz, 1H), 7.57 (d, J = 9.46 Hz, 1H), 4.25−4.32
(m, 1H), 2.81 (q, J = 7.5 Hz, 2H), 1.21−1.27 (m, 6H).
(S)-6-(3-Cyano-5-fluoro-1-(5-fluoropyrimidin-2-yl)-6-methyl-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(4o). Melting point 201−203 °C. MS m/z 507.1 [M + H]+. 1H NMR
(500 MHz, acetone-d6) δ 8.87 (dd, J = 0.95, 2.52 Hz, 1H), 8.81 (s,
2H), 8.47 (dd, J = 2.6, 8.6 Hz, 1H), 8.17 (dd, J = 1.0, 8.2 Hz, 1H),
7.94−7.99 (m, 1H), 7.70−7.76 (m, 1H), 7.48 (d, J = 9.14 Hz, 1H),
(S)-6-(3-Cyano-6-ethyl-5-fluoro-1-(thiazol-2-yl)-1H-indol-2-yl)-N-
(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4x). Melting
1
point 187−191 °C. MS m/z 523.5 [M + H]+. H NMR (500 MHz,
acetone-d6) δ 8.99 (dd, J = 0.79, 2.36 Hz, 1H), 8.46 (dd, J = 2.5, 8.2
Hz, 1H), 8.09 (dd, J = 1.0, 8.2 Hz, 1H), 7.80 (s, 2H), 7.70 (br s, 1H),
M
dx.doi.org/10.1021/jm401621g | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7.64 (d, J = 5.67 Hz, 1H), 7.54 (d, J = 9.46 Hz, 1H), 4.26−4.36 (m,
1H), 2.81 (q, J = 7.57 Hz, 2H), 1.26 (d, J = 7.0 Hz, 3H), 1.25 (t, J =
7.2 Hz, 3H).
ASSOCIATED CONTENT
■
S
* Supporting Information
Syntheses for indoles 5b and 5n and sulfonamides 9. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(S)-6-(3-Cyano-6-ethyl-5-fluoro-1-(5-fluoropyrimidin-2-yl)-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
1
(4y). MS m/z 537.0 [M + H]+. H NMR (500 MHz, acetone-d6) δ
8.87 (dd, J = 0.9, 2.5 Hz, 1H), 8.83 (s, 2H), 8.48 (dd, J = 2.5, 8.2 Hz,
1H), 8.19 (dd, J = 0.9, 8.2 Hz, 1H), 8.01 (d, J = 6.3 Hz, 1H), 7.64−
7.69 (m, 1H), 7.54 (d, J = 9.77 Hz, 1H), 4.26−4.33 (m, 1H), 2.82 (q, J
= 7.5 Hz, 2H), 1.24−1.28 (m, 6H).
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +1-9089129238. E-mail: nzhang@ptcbio.com.
Notes
(S)-6-(3-Cyano-6-ethyl-5-fluoro-1-(5-methylpyrimidin-2-yl)-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
1
(4z). Melting point 201−206 °C. MS m/z 533.2 [M + H]+. H NMR
The authors declare no competing financial interest.
(500 MHz, acetone-d6) δ 8.87 (dd, J = 0.63, 2.21 Hz, 1H), 8.65 (d, J =
0.63 Hz, 2H), 8.45 (dd, J = 2.21, 8.20 Hz, 1H), 8.09−8.12 (m, 1H),
8.07 (d, J = 6.62 Hz, 1H), 7.62−7.69 (br s, 1H), 7.52 (d, J = 9.46 Hz,
1H), 4.25−4.34 (m, 1H), 2.82 (q, J = 7.57 Hz, 2H), 2.39 (s, 3H),
1.24−1.30 (m, 6H).
ACKNOWLEDGMENTS
■
We thank Dr. Takashi Komatsu, Shirley Yeh, and Jane Yang
(PTC Therapeutics, Inc.) for generating data reported in this
manuscript. We also thank Drs. Alan Bass, John Piwinski, and
Malcolm MacCoss (Merck Research Laboratories) for their
helpful discussions during the course of this work.
(S)-6-(3-Cyano-6-ethyl-5-fluoro-1-(5-fluoropyridin-2-yl)-1H-indol-
2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4aa).
Melting point 210−212 °C. MS m/z 535.8 [M + H]+. 1H NMR
(500 MHz, CDCl3) δ 8.79 (dd, J = 1.0, 2.5 Hz, 1H), 8.35 (d, J = 2.5
Hz, 1H), 8.24 (dd, J = 2.5, 8.2 Hz, 1H), 8.10 (dd, J = 0.9, 8.2 Hz, 1H),
7.59−7.63 (m, 1H), 7.50 (d, J = 9.4 Hz, 1H), 7.32−7.35 (m, 1H), 7.20
(d, J = 5.4 Hz, 1H), 4.83 (d, J = 9.5 Hz, 1H), 4.04−4.10 (m, 1H), 2.78
(1, J = 7.5 Hz, 2H), 1.42 (d, J = 7.0 Hz, 3H), 1.25 (t, J = 7.5 Hz, 3H).
(S)-6-(3-Cyano-6-cyclopropyl-5-fluoro-1-(pyrimidin-2-yl)-1H-
indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(4bb). Melting point 236−240 °C. MS m/z 531.2 [M + H]+. 1H NMR
(500 MHz, CDCl3) δ 8.88 (dd, J = 0.9, 2.2 Hz, 1H), 8.68 (d, J = 4.7
Hz, 2H), 8.28 (dd, J = 2.6, 8.5 Hz, 1H), 8.10 (dd, J = 0.9, 8.5 Hz, 1H),
7.71 (d, J = 6.0 Hz, 1H), 7.47 (d, J = 9.1 Hz, 1H), 7.29 (t, J = 4.7 Hz,
1H), 4.88 (d, J = 9.4 Hz, 1H), 4.04−4.12 (m, 1H), 2.19−2.25 (m,
1H), 1.42 (d, J = 6.94 Hz, 3H), 1.02−1.09 (m, 2H), 0.76−0.81 (m,
2H).
(S)-6-(3-Cyano-6-cyclopropyl-5-fluoro-1-(pyridin-2-yl)-1H-indol-
2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide (4cc).
Melting point 220−223 °C. MS m/z 530.1 [M + H]+. 1H NMR
(500 MHz, CDCl3) δ 8.79 (dd, J = 1.0, 2.5 Hz, 1H), 8.49−8.52 (m,
1H), 8.20 (dd, J = 2.5, 8.5 Hz, 1H), 8.00 (dd, J = 0.9, 8.5 Hz, 1H), 7.88
(dt, J = 1.9, 7.6 Hz, 1H), 7.49 (d, J = 9.4 Hz, 1H), 7.38−7.42 (ddd, J =
0.9, 4.7, 7.5 Hz, 1H), 7.24−7.27 (m, 1H), 7.02 (d, J = 5.9 Hz, 1H),
4.85 (d, J = 9.7 Hz, 1H), 4.00−4.09 (m, 1H), 2.14−2.21 (m, 1H), 1.40
(d, J = 7.25 Hz, 3H), 0.99−1.04 (m, 2H), 0.68−0.72 (m, 2H).
(S)-6-(3-Cyano-6-cyclopropyl-5-fluoro-1-(5-fluoropyridin-2-yl)-
1H-indol-2-yl)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide
(4dd). Melting point 240−242 °C. MS m/z 548.2 [M + H]+. 1H NMR
(500 MHz, CDCl3) δ 8.78 (d, J = 2.5 Hz, 1H), 8.34 (d, J = 2.84 Hz,
1H), 8.23 (dd, J = 2.5, 8.5 Hz, 1H), 8.09 (dd, J = 0.9, 8.2 Hz, 1H),
7.58−7.63 (m, 1H), 7.50 (d, J = 9.46 Hz, 1H), 7.30 (dd, J = 3.5, 8.8
Hz, 1H), 6.94 (d, J = 5.7 Hz, 1H), 4.84 (d, J = 9.8 Hz, 1H), 4.03−4.10
(m, 1H), 2.15−2.22 (m, 1H), 1.42 (d, J = 6.94 Hz, 3H), 0.99−1.06
(m, 2H), 0.66−0.72 (m, 2H).
Biological Assays. HCV Replicon Assay. Huh-7 cells harboring a
genotype 1b HCV bicistronic replicon (Con1)16 were plated at 5000
cells/well in 96-well plates. Compounds were added to the plates with
a final concentration of 0.5% DMSO, and plates were incubated at 37
°C. At the end of compound treatment, the cells were lysed with Cell-
to-cDNA lysis buffer (Ambion, Austin, TX). RT-PCR was carried out
using TaqMan One-Step RT-PCR Master Mix reagents kit (Applied
Biosystems) with HCV primers (sense S66 [ACGCAGAAAGCGTC-
TAGCCAT] and antisense A165 [TACTCACCGGTTCCGCAGA])
and probe (5′-6FAM-TCCTGGAGGCTGCACGACACTCAT-3′
TAMRA) at 100 μM. The effect of the compound on the amount
of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate
dehydrogenase) mRNA as a control was determined by using
Predeveloped TaqMan Assay reagents (Applied Biosystems, Foster
City, CA). Both HCV replicon and GAPDH RNAs were amplified in
the same well using ABI 7900HT (Applied Biosystems). EC50 values
are reported as the average of at least two independent determinations.
ABBREVIATIONS USED
■
B2Pin2, bis(pinacolato)diboron; GSH, glutathione; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; NS, nonstructural;
NS4B, nonstructural protein 4B; RT-PCR, real-time polymer-
ase chain reaction
REFERENCES
■
(1) (a) Hepatitis C Fact Sheet No. 164; World Health Organization:
Geneva, July 2012; www.who.int. (b) Alter, M. J.; Kruszon-Moran, D.;
Nainan, O. V.; McQuillan, G. M.; Gao, F.; Moyer, L. A.; Kaslow, R. A.;
Margolis, H. S. The prevalence of hepatitis C virus infection in the
United States, 1988 through 1994. N. Engl. J. Med. 1999, 341, 556−
562.
(2) Shepard, C. W.; Finelli, L.; Alter, M. J. Global epidemiology of
hepatitis C virus infection. Lancet Infect. Dis. 2005, 5, 558−567.
(3) Zein, N. N. Clinical significance of hepatitis C virus genotypes.
Clin. Microbiol. Rev. 2000, 13, 223−235.
(4) Sheridan, C. New Merck and Vertex drugs raise standard of care
in hepatitis C. Nature Biotechnol. 2011, 29, 553−554.
(5) Rong, L.; Dahari, H.; Ribeiro, R. M.; Perelson, A. S. Rapid
emergence of protease inhibitor resistance in hepatitis C virus. Sci.
Transl. Med. 2010, 2, 30ra32.
(6) (a) Kato, N. Genome of human hepatitis C virus (HCV): gene
organization, sequence diversity, and variation. Microb. Comp.
Genomics 2000, 5, 129−151. (b) Simmonds, P.; Bukh, J.; Combet,
C.; Deleage, G.; Enomoto, N.; Feinstone, S.; Halfon, P.; Inchauspe, G.;
Kuiken, C.; Maertens, G.; Mizokami, M.; Murphy, D. G.; Okamoto,
H.; Pawlotsky, J. M.; Penin, F.; Sablon, E.; Shin, I. T.; Stuyver, L. J.;
Thiel, H. J.; Viazov, S.; Weiner, A. J.; Widell, A. Consensus proposals
for a unified system of nomenclature of hepatitis C virus genotypes.
Hepatology 2005, 42, 962−973.
(7) Kiser, J. J.; Flexner, C. Direct-acting antiviral agents for hepatitis
C virus infection. Annu. Rev. Pharmacol. Toxicol. 2013, 53, 427−449.
(8) (a) Chen, K. X.; Njoroge, F. G. A review of HCV protease
inhibitors. Curr. Opin. Invest. Drugs 2009, 10, 821−837. (b) Marks, K.
M.; Jacobson, I. M. The first wave: HCV NS3 protease inhibitors
telaprevir and boceprevir. Antiviral Ther. 2012, 17, 1119−1131.
(9) (a) 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.; Hamann, L. G.
Chemical genetics strategy identifies an HCV NS5A inhibitor with a
potent clinical effect. Nature 2010, 465, 96−100. (b) Belda, O.;
Targett-Adams, P. Small molecule inhibitors of the hepatitis C virus-
encoded NS5A protein. Virus Res. 2012, 170, 1−14. (c) Suk-Fong, L.
N
dx.doi.org/10.1021/jm401621g | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
A. HCV NS5A inhibitors in development. Clin. Liver Dis. 2013, 17,
111−121.
lymphocytic concentrations and with the activity of the liver disease.
Am. J. Gastroenterol. 1996, 91, 2569−2573.
(10) (a) Watkins, W. J.; Ray, A. S.; Chong, L. S. HCV NS5B
polymerase inhibitors. Curr. Opin. Drug Discovery Dev. 2010, 13, 441−
465. (b) Sofia, M. J.; Chang, W.; Furman, P. A.; Ralph T. Mosley, R.
T.; Bruce S. Ross, B. S. Nucleoside, nucleotide, and non-nucleoside
inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymer-
ase. J. Med. Chem. 2012, 55, 2481−2531.
(20) Gu, Z.; Graci, J. D.; Lahser, F. C.; Breslin, J. J.; Jung, S. P.;
Crona, J. H; McMonagle, P.; Xia, E.; Liu, S.; Karp, G.; Zhu, J.; Huang,
S.; Nomeir, A.; Weetall, M.; Almstead, N. G.; Peltz, S. W.; Tong, X.;
Ralston, R.; Colacino, J. M. Identification of PTC725, an orally
bioavailable small molecule that selectively targets the hepatitis C virus
NS4B protein. Antimicrob. Agents Chemother. 2013, 57, 3250−3261.
(21) Biological activity of 1,1,1-trifluoroacetone, see: (a) Allen, K. N.;
Abeles, R. H. Inhibition kinetics of acetylcholinesterase with
fluoromethyl ketones. Biochemistry 1989, 28, 8466−8473. (b) Allen,
K. N.; Abeles, R. H. Inhibition of pig liver esterase by trifluoromethyl
ketones: modulators of the catalytic reaction alter inhibition kinetics.
Biochemistry 1989, 28, 135−140. (c) Hammock, B. D.; Wing, K. D.;
McLaughlin, J.; Lovell, V. M.; Sparks, T. C. Trifluoromethylketones as
possible transition state analog inhibitors of juvenile hormone esterase.
Pestic. Biochem. Physiol. 1982, 17, 76−88. (d) Brodbeck, U.;
Schweikert, K.; Gentinetta, R.; Rottenberg, M. Fluorinated aldehydes
and ketones acting as quasi-substrate inhibitors of acetylcholinesterase.
Biochim. Biophys. Acta 1979, 567, 357−369.
(11) (a) Sklan, E. H.; Glenn, J. S. HCV NS4B: From obscurity to
central stage. In Hepatitis C Viruses: Genomes and Molecular Biology;
Tan, S. L., Ed.; Horizon Bioscience: Norfolk, UK, 2006; Chapter 8.
(b) Einav, S.; Sobol, H. D.; Gehrig, E.; Glenn, J. S. The hepatitis C
virus (HCV) NS4B RNA binding inhibitor clemizole is highly
synergistic with HCV protease inhibitors. J. Infect. Dis. 2010, 202,
65−74.
(12) (a) Dvory-Sobol, H.; Pang, P. S.; Glenn, J. S. The future of HCV
therapy: NS4B as an antiviral target. Viruses 2010, 2, 2481−2492.
(b) Gouttenoire, J.; Penin, F.; Moradpour, D. Hepatitis C virus
nonstructural protein 4B: a journey into unexplored territory. Rev.
Med. Virol. 2010, 20, 117−129. (c) Rai, R.; Deval, J. New
opportunities in anti-hepatitis C virus drug discovery: Targeting
NS4B. Antiviral Res. 2011, 90, 93−101.
(13) (a) Shotwell, J. B.; Baskaran, S.; Chong, P.; Creech, K. L.;
Crosby, R. M.; Dickson, H.; Fang, J.; Garrido, D.; Mathis, A.; Maung,
J.; Parks, D. J.; Pouliot, J. J.; Price, D. J.; Rai, R.; Seal, J. W., III;
Schmitz, U.; Tai, V. W. F.; Thomson, M.; Xie, M.; Xiong, Z. Z.; Peat,
A. J. Imidazo[1,2-a]pyridines that directly interact with hepatitis C
NS4B: Initial preclinical characterization. ACS Med. Chem. Lett. 2012,
3, 565−569. (b) Miller, J. F.; Chong, P. Y.; Shotwell, J. B.; Catalano, J.
G.; Tai, V. W.-F.; Fang, J. M.; Banka, A. L.; Roberts, C. D.; Youngman,
M. K.; Zhang, H.; Xiong, Z.; Mathis, A.; Pouliot, J. J.; Hamatake, R. K.;
Price, D. J.; Seal, J. W.; Stroup, L. L.; Creech, K. L.; Carballo, L. H.;
Todd, D.; Spaltenstein, A.; Furst, S. M.; Hong, Z.; Peat, A. J. Hepatitis
C replication inhibitors that target the viral NS4B protein. J. Med.
Chem. 2013, DOI: 10.1021/jm400125h.
(22) (a) Moore, J. T.; Kliewer, S. A. Use of the nuclear receptor PXR
to predict drug interactions. Toxicology 2000, 153, 1−10. (b) Gao, Y.-
D.; Olson, S. H.; Balkovec, J. M.; Zhu, Y.; Royo, I.; Yabut, J.; Evers, R.;
Tan, E. Y.; Tang, W.; Hartley, D. P.; Mosley, R. T. Attenuating
pregnane X receptor (PXR) activation: a molecular modeling
approach. Xenobiotica 2007, 37 (2), 124−138.
(23) Yi, M.; Villanueva, R. A.; Thomas, D. L.; Wakita, T.; Lemon, S.
M. Production of infectious genotype 1a hepatitis C virus (Hutchinson
strain) in cultured human hepatoma cells. Proc. Natl. Acad. Sci. U. S. A.
2006, 103, 2310−2315.
(14) Chen, G.; Ren, H.; Turpoff, A.; Arefolov, A.; Wilde, R.;
Takasugi, J.; Khan, A.; Almstead, N.; Gu, Z.; Komatsu, T.; Freund, C.;
Breslin, J.; Colacino, J.; Hedrick, J.; Weetall, M.; Karp, G. M. Discovery
of N-(4′-(indol-2-yl)phenyl) sulfonamides as novel inhibitors of HCV
replication. Bioorg. Med. Chem. Lett. 2013, 23, 3942−3946.
(15) Zhang, X.; Zhang, N.; Chen, G.; Turpoff, A.; Ren, H.; Takasugi,
J.; Morrill, C.; Zhu, J.; Li, C.; Lennox, W.; Paget, S.; Liu, Y.; Almstead,
N.; Njoroge, F. G.; Gu, Z.; Komatsu, T.; Clausen, V.; Espiritu, C.;
Graci, J.; Colacino, J.; Lahser, F.; Risher, N.; Weetall, M.; Nomeir, A.;
Karp, G. M. Discovery of novel HCV inhibitors: synthesis and
biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting
hepatitis C virus NS4B. Bioorg. Med. Chem. Lett. 2013, 23, 3947−3953.
(16) Lohmann, V.; Korner, F.; Koch, J.-O.; Herian, U.; Theilmann,
̈
L.; Bartenschlager, R. Replication of subgenomic hepatitis C virus
RNAs in a hepatoma cell line. Science 1999, 285, 110−113.
(17) Biological activity of 1,3-difluoroacetone, see: (a) Menon, K. I.;
Feldwick, M. G.; Noakes, P. S.; Mead, R. J. The mode of toxic action
of the pesticide Gliftor: the metabolism of 1,3-difluoroacetone to
(−)-erythro-fluorocitrate. J. Biochem. Mol. Toxicol. 2001, 15, 47−54.
(b) Feldwick, M. G.; Noakes, P. S.; Prause, U.; Mead, R. J.; Kostyniak,
P. J. The biochemical toxicology of 1,3-difluoro-2-propanol, the major
ingredient of the pesticide gliftor: the potential of 4-methylpyrazole as
an antidote. J. Biochem. Mol. Toxicol. 1998, 12, 41−52.
(18) Toxicology of glutathione adducts, see: (a) Walsh, J. S.; Miwa,
G. T. Bioactivation of drugs: risk and drug design. Annu. Rev.
Pharmacol. Toxicol. 2011, 51, 145−167. (b) Park, B. K.; Laverty, H.;
Srivastava, A.; Antoine, D. J.; Naisbitt, D.; Williams, D. P. Drug
bioactivation and protein adduct formation in the pathogenesis of
drug-induced toxicity. Chem. Biol. Interact. 2011, 192, 30−36.
(19) HCV patients may have depleted glutathione levels, see:
Barbaro, G.; di Lorenzo, G.; Soldini, M.; Parrotto, S.; Bellomo, G.;
Belloni, G.; Grisorio, B.; Barbarini, G. Hepatic glutathione deficiency
in chronic hepatitis C: quantitative evaluation in patients who are HIV
positive and HIV negative and correlations with plasmatic and
O
dx.doi.org/10.1021/jm401621g | J. Med. Chem. XXXX, XXX, XXX−XXX