A novel class of highly potent irreversible hepatitis C virus NS5B polymerase inhibitors

Article abstract of DOI:10.1021/jm201322r

Starting from indole-based C-3 pyridone HCV NS5B polymerase inhibitor 2, structure-activity relationship (SAR) investigations of the indole N-1 benzyl moiety were performed. This study led to the discovery of irreversible inhibitors with p-fluoro-sulfone- or p-fluoro-nitro-substituted N-1 benzyl groups which achieved breakthrough replicon assay potency (EC₅₀ = 1 nM). The formation of a covalent bond with adjacent cysteine-366 thiol was was proved by mass spectroscopy and X-ray crystal structure studies. The C-5 ethyl C-2 carboxylic acid derivative 47 had an excellent oral area-under-the-curve (AUC) of 18 μM?h (10 mg/kg). Its oral exposure in monkeys and dogs was also very good. The NMR ALARM assay, mass spectroscopy experiments, in vitro counter screening, and toxicology assays demonstrated that the covalent bond formation between compound 47 and the protein was highly selective and specific. The overall excellent profile of 47 made it an interesting candidate for further investigation.

Full text of DOI:10.1021/jm201322r

Article
pubs.acs.org/jmc
A Novel Class of Highly Potent Irreversible Hepatitis C Virus NS5B
Polymerase Inhibitors
Kevin X. Chen,*,† Charles A. Lesburg,‡ Bancha Vibulbhan,† Weiying Yang,† Tin-Yau Chan,†
Srikanth Venkatraman,† Francisco Velazquez,† Qingbei Zeng,† Frank Bennett,† Gopinadhan N. Anilkumar,†
Jose Duca,† Yueheng Jiang,† Patrick Pinto,† Li Wang,† Yuhua Huang,† Oleg Selyutin,† Stephen Gavalas,†
Haiyan Pu,† Sony Agrawal,† Boris Feld,† Hsueh-Cheng Huang,† Cheng Li,† Kuo-Chi Cheng,†
Neng-Yang Shih,† Joseph A. Kozlowski,† Stuart B. Rosenblum,† and F. George Njoroge†
†Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States
‡Drug Design, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States
ABSTRACT: Starting from indole-based C-3 pyridone HCV
NS5B polymerase inhibitor 2, structure−activity relationship
(SAR) investigations of the indole N-1 benzyl moiety were
performed. This study led to the discovery of irreversible in-
hibitors with p-fluoro-sulfone- or p-fluoro-nitro-substituted
N-1 benzyl groups which achieved breakthrough replicon assay
potency (EC50 = 1 nM). The formation of a covalent bond
with adjacent cysteine-366 thiol was was proved by mass spec-
troscopy and X-ray crystal structure studies. The C-5 ethyl C-2
carboxylic acid derivative 47 had an excellent oral area-under-
the-curve (AUC) of 18 μM·h (10 mg/kg). Its oral exposure in
monkeys and dogs was also very good. The NMR ALARM assay, mass spectroscopy experiments, in vitro counter screening, and
toxicology assays demonstrated that the covalent bond formation between compound 47 and the protein was highly selective and
specific. The overall excellent profile of 47 made it an interesting candidate for further investigation.
(NS) proteins located downstream are NS2, NS3, NS4A,
NS4B, NS5A, and NS5B. Because of their essential role in the
replication of HCV virus, intensive research has been focused
on finding drugs which directly target these nonstructural
proteins.5 Exciting progress has been made in these endeavors.5
Recent clinical trials have demonstrated dramatic improve-
ments in SVR rate in patients treated with a direct-acting agent
in combination with standard of care.6 Recent approval of the
two HCV NS3 protease inhibitors boceprevir7 (Victrelis, Merck
& Co.) and telaprevir8 (Incivek, Vertex Pharmaceuticals) by the
FDA have generated great excitement for the new treatments of
HCV infection. Several other NS3 protease inhibitors and a
number of candidates targeting other nonstructural enzymes
are also under development.5
The HCV NS5B gene encodes an RNA-dependent RNA
polymerase (RdRp) whose enzymatic activity is critical to the
replication of the viral RNA genome.9 Replication of the plus-
strand RNA viral genome consists of two steps: synthesis of the
complementary negative-strand RNA using the positive-strand
RNA as a template, and the subsequent synthesis of multiple
positive-strand RNA genome using the minus-strand RNA as a
template. Besides its direct role in the replication of viral RNA
genome, the HCV NS5B polymerase is potentially associated
INTRODUCTION
■
Hepatitis C virus (HCV) has infected an estimated 3% of the
world’s population (over 170 million people).1 The slow
progression and mild symptoms of the HCV infection make it a
stealth epidemic. Most infections progress to a chronic state
that persists for decades and eventually lead to cirrhosis, liver
failure, or liver cancer.2 The new standard of care (SOC) after
recent protease inhibitor approval involves the combination of
a protease inhibitor with pegylated-interferon and the oral
nucleoside antiviral agent ribavirin.3 The outcome of the
treatment is defined by the sustained virologic response (SVR)
or undetectable HCV RNA in serum at the end of treatment
and six-months post treatment. The SVR rate for patients with
the most-difficult-to-treat genotype-1 HCV is about 70% with
the new current therapy.3 Large pill-burden (750−800 mg, three
times a day), relapse (mutations), and discontinuation due to the
presence of side effects with these therapies necessitates the need
for safe and more effective HCV treatments.
Hepatitis C virus was identified more than two decades ago.4
It belongs to the Flaviviridae family of enveloped viruses. It is a
positive-sense single-stranded RNA virus with a single open
frame of ∼9600 nucleosides. The viral genome encodes a
polyprotein of more than 3000 amino acids. The polyprotein is
divided into structural and nonstructural precursor regions. The
structural protein contains the nucleocapsid core protein (C)
and two glycoproteins E1 and E2 (NS1). The nonstructural
Received: October 3, 2011
Published: January 16, 2012
© 2012 American Chemical Society
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with a number of other important functions. It forms a replicase
complex (RC) with other HCV nonstructural proteins (NS3,
NS4A, NS4B, and NS5A) and a number of cellular cofactors
during replication.10 It was also suggested that NS5B might
regulate the function of other HCV enzymes. The crystal
structure of the catalytic domain of N5SB has revealed several
significant features.11 The polymerase shares many common
features of other polymerases with characteristic thumb, finger,
and palm domains analogous with a right-hand. In addition to a
well-conserved active site (located in the palm domain) for
nucleoside binding, mechanistic and structural studies have
revealed the existence of multiple allosteric inhibitor binding
sites.12,13 Binding to these sites interferes with conformational
changes required during RNA synthesis. Thumb pockets I and
II are two allosteric sites thatz are located in thumb domain
while palm site I and II are two pockets located adjacent to the
active site. These thumb and palm region sites have been the
targets for drug development, which makes the NS5B a
particularly drugable target.12,13
this moiety intact while changes were made at other substi-
tutions at the indole core. After considerable effort, new struc-
tures such as compound 2 with much improved potency and
PK profiles emerged as the new lead series.26 The indole core
of inhibitor 2 was substituted by a methyl at C-5 and a cyclo-
propyl acylsulfonamide at C-2. The indole nitrogen was substi-
tuted by a 2′,5′-difluorobenzyl group. A 7-fold improvement in
enzyme potency to an IC50 of 6 nM was observed, probably as a
result of increased hydrophobic interactions with the enzyme
surface from the C-5 and N-1 substituents. More importantly,
this compound demonstrated greatly improved (50-fold) acti-
vity in replicon assay with an EC50 of 90 nM. Compound 2 also
had an improved AUC of 9.1 μM·h in rats following 10 mg/kg
oral dosing. Despite its good overall profile, further improve-
ment in compound 2 is required to have a drug candidate, par-
ticularly in the potency in the cellular assay. Thus, the optimi-
zation of the indole series of inhibitors continued. Herein, we
report our SAR development and the discovery of a novel class
of irreversible highly potent HCV NS5B polymerase inhibitors.
(see Figure 1.)
Extensive efforts directed at inhibiting HCV NS5B polymer-
ase have resulted in numerous drug candidates. They belong to
two distinctive classes of inhibitors: nucleoside active site
inhibitors (NIs) and non-nucleoside allosteric inhibitors
(NNIs).12,13 They target different stages of RNA synthesis.14
NNIs were shown to interfere with steps prior to or during the
initiation of RNA synthesis. In contrast, the triphosphate form
of NIs binds to the active site during elongation. NIs are
analogues of natural substrates of the polymerase that are
incorporated into the growing RNA chain leading to chain
termination. These agents require phosphorylation before being
active. Because NS5B is a highly conserved region of the HCV
genome, NIs have similar activity against all genotypes and a
high genetic barrier to resistance.15 Several NIs have advanced
into clinical trials, including prodrugs valopicitabine (Idenix),
R1626 (Roche), RG-7128 (Pharmasset/Roche), PSI-7977
(Pharmasset), PSI-352938 (Pharmasset), IDX-184 (Idenix),
and INX-189 (Inhibitex).16 NNIs, on the other hand, achieve
NS5B inhibition by binding to one of at least four allosteric
enzyme sites, resulting in conformational changes which alter
the protein-inhibiting catalytic activity of polymerase. A number
of clinical candidates have been identified.12,13 Among them,
HCV-796 (Wyeth/Viropharma),17 filibuvir (Pfizer),18 VX-222
(Vertex/Virochem),19 ANA598 (Anadys),20 BI207127 (Boeh-
ringer Ingelheim),21 and BMS-791325 (Bristol-Myers Squibb)22
have progressed to phase II clinical trials. The rapid development
of resistant mutants has been observed with NNIs because they
bind distantly to the active center of NS5B, and mutations at the
NNI binding site may not necessary lead to impairment of the
enzyme function.
Our early research effort on the optimization of an indole-
based screening hit led to compound 1.23 It had good potency
(IC50 = 53 nM) in an NS5B RdRp enzyme assay.24 It was active
in a cell-based replicon assay25 with an EC50 of 4800 nM). The
X-ray structure of 1 in complex with NS5B indicated that it
bound to a NS5B apoprotein cavity adjacent to the active site at
the 'palm’ site of the protein.23 Further SAR studies were carried
out to improve potency, especially replicon assay potency.
Since compound 1 had poor pharmacokinetic (PK) properties
with very low AUC value when dosed orally in rats, it was also
desirable to improve PK properties through structural modifi-
cations. Because the X-ray structure of 1 also revealed that the
C-3 pyridone carbonyl and N-H formed hydrogen bonds with
the backbone isoleucine-447 residue, it was decided to leave
Figure 1. Lead compounds from early SAR studies.
SYNTHESIS OF HCV NS5B POLYMERASE
INHIBITORS
■
The general synthesis of inhibitors is outlined in Schemes 1
and 2. The ethyl indole-2-carboxylate starting material 3,
with desired substituents at the 4-, 5-, and 6-position, was
either commercially available or could be prepared via known
procedures. The intermediate 3 was iodinated at C-3 with N-
iodosuccinimide (NIS) to give compound 4. The Suzuki−
Miyaura cross-coupling reaction27 between 4 and 2-methoxy-3-
pyridineboronic acid in the presence of a palladium catalyst
afforded product 5. The indole nitrogen was then alkylated to
provide compound 6 through alkylation with a benzylic halide.
The ethyl ester was hydrolyzed to its corresponding carboxylic
acid 7, which, after activation by carbonyl diimidazole (CDI),
was converted to an acylsulfonamide 8 through reaction with a
primary alkylsulfonamide.28 The methoxypyridine 8 was then
demethylated to afford pyridone 9 upon treatment with hydro-
chloric acid. The final compounds were tested in RdRp enzyme
assay and replicon cell-based assay.
Alternatively, the indole-2-acylsulfonamide series of polymer-
ase inhibitors were prepared according to the procedures shown
in Scheme 2. Compound 5 was treated with hydrochloric acid to
provide C-3 pyridone product 10, which was hydrolyzed to its
corresponding C-2 carboxylic acid 11. The acid was activated
with EDC and cyclized intramolecularly to the pyridone to form
the tetracyclic lactone 12. The indole nitrogen was then alkylated
with either a benzylic halide similar to the conversion of 5 to 6,
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a
a
Scheme 1.
Scheme 2.
a
Reaction conditions: (a) NIS, CHCl3; (b) 2-methoxy-3-pyridinebor-
onic acid, PdCl2(dppf)2, K2CO3, 1,2-dimethoxyethane, 50−85% (two
steps); (c) ArCH2Br (or ArCH2Cl), Cs2CO3, DMF, 40−90%; (d)
LiOH, THF/H2O, 60−90%; (e) CDI, then RSO2NH2, DBU, THF,
50−80%; (f) 4 M HCl, dioxane, 30−60%.
a
Reaction conditions: (a) 4 M HCl, dioxane; (b) LiOH, THF/H2O;
(c) EDC, NEt3, DMF, 60−90%; (d) ArCH2Br (or ArCH2Cl),
Cs2CO3, DMF; or ArCH2OH, DIAD, PPh3, THF, 60−90%; (e)
RSO2NH2, DBU, DMF, 30−60%; (f) LiOH, THF/H2O, 20−60%.
or via a Mitsunobu reaction29 with a benzylic alcohol. The result-
ing lactone product 13 was either reacted with a sulfonamide in
the presence of 1,8-diazabicyclocundec-7-ene (DBU) to give the
desired acylsulfonamide analogue 9, or hydrolyzed with lithium
hydroxide to carboxylic acid inhibitor 14. The products 9 and 14
were then evaluated in enzyme and replicon assays to determine
their biological activities.
3′-methyl group. Analogue 17 had the same N-1 4-(2′-amino-
pyridine)methyl moiety as that of compound 1. It maintained
enzyme activity (IC50 = 6 nM) but lost even more cellular
potency (EC50 = 500 nM). Further exploration at the 5′-
substitution was also conducted to search for a group that
was capable of establishing additional interactions with nearby
protein backbone residues. Toward that end, moieties with
different functionalities were examined. A 5′-nitrile group in
compound 18 caused a drop in both enzyme and replicon
potency (IC50 = 9 nM, EC50 = 220 nM) when compared to
5′-nonsubstituted 15. The 5′-primary amide analogue 19 faired
better in the enzyme assay (IC50 = 4 nM) but lost activity in the
replicon assay by 3-fold.
Several analogues with a sulfonyl moiety para to the fluoro
group were also prepared (20 to 22). The compound bearing
a C-5′-methyl sulfone (20) maintained a good IC50 of 6 nM
while losing significant cellular assay activity (EC50 = 580 nM).
The primary sulfonamide derivative 21 did not provide any
improvement, having almost the same IC50 and EC50. When an
additional nitrile group was introduced at C-3′ however,
dramatic changes in potency were observed. The 3′-nitrile-5′-
methyl sulfone analogue 22 not only gave a better IC50 of 3 nM
but also provided a much better EC50 (5 nM) in the replicon
assay. The 100-fold improvement in cellular potency compared
to 20 cannot be easily explained by any extra simple contact(s)
established by the nitrile group, and there was a possibility that
RESULTS AND DISCUSSION
■
Although lead compound 2 had an overall good profile, better
potency was desired for a potential drug development
candidate. Several modifications attempted at the indole core
failed to improve the potency of the resulting inhibitors. We
decided to keep the indole core intact but to make changes at
substituents that attached to various positions of the indole ring
system. Thus, to optimize the indole N-1 substituent, a number
of benzyl type structures with various substitution patterns were
examined. Results from selected examples are shown in Table 1.
Compound 15 had only one fluoro group at the 2′-position
and had slightly better potency in both RdRp enzyme (EC50
=
50 nM) and replicon cell-based assays (EC50 = 50 nM) than
that of the benchmark compound 2 which had two fluoro
substitutions at the 2′,5′-positions. When an additional methyl
was introduced to C-3′ of 15, the activity of compound 16
against enzyme improved even further to an IC50 of 4 nM.
However, its replicon assay potency decreased to an EC50 of
380 nM probably due to some unfavorable interactions from
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a
Table 1. SAR of Substitution at the Benzyl Group at the
Indole Core N-1 Position
Table 2. NS5B Polymerase Inhibitors
a
An asterisk denotes that the C5 substituent was an ethyl group for
these inhibitors.
noncovalent inhibitors in Table 1 and definitely much less potent
than that for potential covalent inhibitor 22. The less electron-
withdrawing trifluoromethyl sulfoxide 24 was, not surprisingly,
even less potent in the replicon assay (EC50 = 3400 nM) than
that of 23. The 2′,3′,6′-trifluoro-5′-methylsulfonylbenzene ana-
logue 25 was also not very potent, although it had a better EC50
(270 nM) than those of 23 and 24. Apparently, the two extra
fluorines were not strong enough electron-withdrawing groups.
Similarly, an additional 4′-chloro substituent in 26 was not
sufficient to make the compound susceptible to covalent bonding.
But both its enzymatic and cellular potency (IC50 = 2 nM, EC50 =
60 nM) improved significantly over other sulfones 23−25, prob-
ably because of favorable interaction(s) with the 4′-substituent.
When the 4′-position was substituted with a cyano group (27),
however, the potency was dramatically improved to an EC50 of 3
nM, even better than that of compound 22. Again, it was specu-
lated that the strong electron-withdrawing sulfone and cyano
groups made the nucleophilic replacement of 2′-F much easier.
The two multisubstituted 5′-sulfonamide or primary amide
analogues 28 and 29 were, as expected, much less potent
inhibitors (EC50 = 1400 and 3300 nM, respectively) due to the
weaker electron-withdrawing ability of sulfonamides, amides
and halogens. The fact that C-5 was substituted by an ethyl
group in compounds 26, 27, and 28 instead of a methyl group
in other compounds might have small impact on the IC50s and
EC50s of these inhibitors, but the difference in potency should
be quite small based on SAR from other series.26
some unusual interaction(s) had occurred when this additional
nitrile group was present. Looking at the structure, one could
make an educated guess from chemistry knowledge and litera-
ture that two strong electron-withdrawing-groups at para-and
ortho-positions could have made the fluoro substituent very
susceptible to attack by an adjacent nucleophile. By checking
out the amino acid residues of the enzyme backbone in the
region to where the benzyl group was bound, cysteine-366
appeared to be within attacking distance. The thiol of cysteine-
366 could replace the fluoro atom to form a covalent irrever-
sible sulfide bond with the benzene ring. Although reversible
covalent bond formation with cysteine-366 has been reported
previously,30 no irreversible inhibitors were ever identified. If
the irreversible sulfide bond formation could be confirmed, a
new class of covalently bonded HCV NS5B polymerase inhib-
itors was then discovered. We decided to conduct further inves-
tigation into this class of compounds.
Exploration of Potential Irreversible Inhibitors. To
explore other phenyl rings that had the potential to form a
covalent bond with cysteine-366, a number of N-1 benzyl
groups with multiple electron-withdrawing groups were studied
(Table 2). Leaving groups other than fluoride, such as chloride,
were also examined. In compound 23, the benzene ring was
substituted by 2′-chloro and strongly electron-withdrawing
5′-trifluoromethyl sulfone. However, with an IC50 of 46 nM and
an EC50 of 900 nM, 23 was even less potent than that for most
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Because the nitro group is known to be one of the strongest
electron-withdrawing groups, we hoped that it would provide
facile covalent bond formation. Indeed, in the presence of the
C-5′ nitro group, significant improvement in potency for the
2′-chloro analogue 30 (EC50 = 16 nM) was observed compared
to 23 and 24. The 5′-nitro-2′-fluoro analogue 31 had an EC50 of
1 nM. Simple hydrophobic interactions are unlikely to explain
the significant difference in potency among the inhibitors in
Tables 1 and 2. Evidence for covalent bond formation from
mass spectroscopy and X-ray crystal structure studies will be
discussed in the later section.
Optimization of Substitutions at C-2 and the
Pyridone Ring. Because of the increased potency of p-nitro-
fluoro-benzyl-substituted compound 31, further investigation of
more compounds with this N-1 substituent was conducted.
Thus, analogues with a series of C-2 moieties were prepared
and tested (Table 3). The C-2 carboxylic acid derivative 32
although it was found to be unstable in PK studies and pos-
sessed no appreciable AUC in rats.
Our early SAR studies indicated that introduction of a methyl
substitution at the indole C-3 pyridone ring could improve
rodent PK, probably due to less metabolism at the pyridone.
A methyl group was introduced to pyridone C-6 of com-
pounds 31−33 to give compounds 37, 38, and 39, respectively.
Unfortunately, acid analogue 37 lost substantial activity in both
enzyme and cellular assays. Although methyl acylsulfonamide
38 was as active as 33 in the enzyme assay, it was 8-fold less
potent in replicon assay. The cyclopropyl acylsulfonamide
analogue 39 also lost potency in both the enzyme assay (by
2-fold) and replicon assay (by 9-fold). The decrease in potency
of in these methyl-substituted pyridone analogues could be
attributed to the unfavorable interactions between methyl and
the protein backbone (see X-ray structure in Figure 2 for
protein surface). On the basis of these results, we decided to
continue to use the C-2 cyclopropyl acylsulfonamide moiety in
further SAR development.
Table 3. C-2 and Pyridone Ring Substituent Modifications
SAR Development of C-5 and C-6 Substituents.
Keeping the N-1, C-2, and C-3 moieties constant, the effects
of varying C-5 and C-6 substituents were studied and the
results are shown in Table 4. Because our previous SAR indi-
Table 4. Optimization of C-5 Substitution and the Effect of
the C6 Substituent
compound
R5
R6
IC50 (nM) (n ≥ 2)
EC50 (nM) (n = 2)
31
40
41
42
43
44
45
46
Me
F
H
H
H
H
H
H
H
F
6
5
4
6
4
6
4
7
1
6
H
6
Cl
5
CF3
t-Bu
Et
2
10
1
Me
4
cated that the C-5-position cannot tolerate large groups, a num-
ber of small and compact substituents were tested in com-
pounds 40−45. Compared to benchmark methyl-substituted
analogue 31, the smaller C-5 substituent such as fluoro
decreased activity in the replicon assay (40, EC50 = 6 nM).
A similar loss of potency was observed in nonsubstituted
compound 41. Although the size of a chloro atom is close
to that of a methyl group, C-5 chloro derivative 42 was still
five times less active than that of 31. The slightly larger
trifluoromethyl group in compound 43 faired better (EC50
=
2 nM) with only a 2-fold loss of potency compared to 31. The
large tert-butyl group in inhibitor 44 was tolerated as evidenced
by the same IC50 of 6 nM as that of compound 31. However,
the fact that a 10-fold loss in cellular assay activity (EC50 of
10 vs 1 nM) was observed could not be easily explained. The
slightly larger ethyl group gave excellent potency in both the
enzyme and replicon assays (IC50 = 4 nM and EC50 = 1 nM,
respectively). In summary, the C-5-position preferred small
maintained the excellent replicon potency (EC50 = 1 nM) of
compound 31. The methyl acylsulfonamide 33 also proved to
be an equally potent inhibitor. Isopropyl acylsulfonamide 35
and acylsulfinamide 36, however, were slightly less potent than
that of 31−33. Dimethyl acylsulfonylurea inhibitor 36, sur-
prisingly, had subnanomolar replicon potency (EC50 = 0.5 nM),
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alkyl substituents such as methyl, ethyl, and trifluoromethyl
groups. To explore the effect of C-6 substitution, a small fluoro
atom was introduced to 31, which resulted in compound 46.
Unfortunately, this substitution caused a 4-fold drop in replicon
potency to an EC50 of 4 nM.
Investigation of PK Properties of Best Compounds.
With the discovery of a number of potent inhibitors with superb
potency, the next step was to evaluate their pharmacokinetic
properties. Thus, a series of potent and representative com-
pounds were selected for rat PK studies, and some results are
shown in Table 5. It was very disappointing to find out that all
Table 6. PK Parameters of Compound 47
compound 47
rat
monkey
dog
a
b
b
AUC(PO) (μM·h)
bioavailability (F)
t1/2 (IV) (h)
18
3.4
11
37%
8.2
30%
1.3
10
95%
1.2
10
CL (mL/min/kg)
8.0
rat liver concn (μM @ 6 h)
0.46
−
−
a
b
10 mg/kg. 3 mg/kg. All administered with 0.4% MC.
3 mg/kg oral dosing, it also exhibited a good AUC of 3.4 μM·h
along with 30% bioavailability. The PK profile was even more
impressive in dogs: an oral AUC of 11 μM·h coupled with 95%
bioavailability when dosed at 3 mg/kg. The half-lives of com-
pound 47 in monkeys and dogs are on the short side (1.3 and
1.2 h, respectively). The clearance rates (CL) were moderate
for 47 in all three species. It had a good liver concentration of
200 ng/g (0.46 μM) in rats 6 h after dosing. It was stable for up
to 3 h in rat, monkey, dog, and human plasma. No inhibition of
CYP P450 enzymes 2D6, 3A4, and 2C9 was observed when
tested up to 30 μM. It was also clean in a P450 enzyme induc-
tion assay at 10 μM. Overall, compound 47 exhibited very favor-
able potency, PK, and selectivity. It was considered as a potential
candidate for further evaluation.
Table 5. Evaluation of Pharmacokinetic Property of
Selective Compounds
X-ray Structure of Compound 47.31 The X-ray crystal
structure of compound 47 bound to HCV NS5B polymerase
was solved (Figure 232). Similar to the X-ray structure of
compound 1 bound to the protein, 47 is bound to the ‘palm’
site of NS5B apoprotein within the active site cavity. The
refined structure showed a bond between the side chain of Cys-
366 and the phenyl ring of the compound with a 1.8 to 1.9 Å
S−C distance. This covalent bond is a result of the side
chain thiol attacking the electron-deficient benzene ring at the
position para to the nitro group with departure of the fluoro
group via a presumed SNAr reaction. The indole core and the
C3 pyridone ring contact the side chain of Met-414 while the
C3 pyridone forms two hydrogen bond interactions with the
backbone of Ile-447 and Tyr-448. The C-2 carboxylic acid does
not make direct interactions with the protein, but it rather
interacts indirectly with amino acid residues Gly-449 and Ser-
556 via hydrogen bonds with bridging water molecules. The
indole C5 ethyl group projects into a constricted tunnel which
extends to the protein surface on the other side of the 'thumb’
subdomain.
Selectivity and Toxicity Concerns about Irreversible
Inhibitors. Although we were very excited by the potency
brought by the covalent bonding with the p-fluoro-nitro-benzyl
moiety, and covalently bonded irreversible inhibitors have been
seen in numerous drugs and drug candidates,33 the reactive
nature of these compounds is always raising an alarm of poten-
tially nonselective bond formations. The potential side
reactions from this nitro-fluoro series of irreversible inhib-
itors was also a concern we felt we needed to address. A NMR
spectroscopy-based assay (ALARM NMR)34 which was
designed to identify thiol reactive compounds was used to
evaluate these inhibitors. In this assay, the highly reactive thiols
in the cysteines of the human La antigen were exploited to
detect nonspecific covalent bonding activity. When compounds
45 and 47 were tested in the ALARM assay, no reactions
between these two compounds and the reactive thiols in the
human La antigen were detected by NMR studies. This indi-
cated that the reactivity of the p-fluoro-nitro-substituted
benzene moiety was not indiscriminately reactive toward any
four C-5 methyl derivatives (31, 33, 36, and 32) with various
C-2 acylsulfonamide or carboxylic acid moieties had very low or
no AUC when dosed orally in rats at 10 mg/kg. The C-6 fluoro-
substituted analogue 46 was slightly better with an AUC of
0.35 μM·h. The C-5 chloro and ethyl compounds (42 and 45,
respectively) did not provide good exposure with AUCs of 0.30
and 0.17 μM·h, respectively, although they were somewhat better
than that of the C-5 methyl compound 31. However, it was very
gratifying to see good oral exposure (18 μM·h) with compound
47, a C-2 carboxylic acid inhibitor. The combination of both
potency and good oral exposure in rats led us to further evaluate
this compound.
Profile of Lead Compound 47. In addition to good oral
exposure in rats at 10 mg/kg, the C-5 ethyl C-2 carboxylic acid
derivative 47 demonstrated a bioavailability of 37% with an IV
half-life of 8.2 h in rats (Table 6). In monkey PK studies with a
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CONCLUSION
■
Starting from lead compound 2, extensive SAR development on
the indole N-1 benzyl moiety was performed. The effort led
to the discovery of irreversible inhibitors of NS5B polymerase
with significant improvements in cellular replicon assay potency.
Some indole-based inhibitors with p-fluoro-sulfone-substituted
N-1 benzyl groups were speculated to be capable of forming a
covalent bond with the cysteine-366 thiol of the NS5B poly-
merase. The compounds with a p-nitro-fluoro-benzyl substituent
at the indole nitrogen were the most potent inhibitors found
which achieved impressive replicon potencies with EC50s down
to 1 nM. The effect of C-3 pyridone substitution and variation of
C-2 moieties were also investigated. While all C-2 carboxylic
acid, acylsulfonamide, and acylsulfonylurea analogues had
superior potency, the methyl substitution at the C-3 pyridone
decreased activity of the corresponding inhibitors. The C-5
SAR investigation demonstrated that small alkyl groups such as
methyl and ethyl groups were preferred. The addition of a C-6
fluoro substituent did not improve either potency or PK profile.
A selected group of the most promising compounds were eva-
luated in rat PK studies. Most of them exhibited disappointing
oral exposure (AUC < 0.4 μM·h when dosed at 10 mg/kg).
However, the C-5 ethyl C-2 carboxylic acid analogue 47 stood
out, having an excellent oral AUC of 18 μM·h at 10 mg/kg.
Further studies proved that 47 also had good oral exposure and
bioavailability in monkeys and dogs. The NMR ALARM assay
and mass spectroscopy experiments demonstrated that com-
pound 47 was highly selective and specific in the formation of a
covalent bond with cystaine-366 thiol. The concerns about the
potential nonselective reactivity and reactive intermediates from
the partially reduced nitro group were largely alleviated by the
negative results in several in vitro counter screens and toxico-
logy assays. The overall profile of 47 in terms of potency, PK,
and safety made it a good candidate for further evaluation.
Figure 2. X-ray structure of compound 47 bound to the palm site of
HCV NS5B polymerase. The compound (yellow) is covalently
attached to the protein (cyan) via a bond between the side chain
sulfur of Cys-366 (depicted as a small sphere) and the 5′-position of
the compound. The electron density for the compound and residue
Cys-366 is best modeled as two alternate conformations and are
included as such in the deposited PDB entry 3TYQ. A single
conformation is shown here for clarity. The compound makes identical
interactions as observed in the complex between inhibitor 1 and
NS5B,23 with a pair of hydrogen bonds from the C3-pyridone group to
the backbone of residues Ile-447 and Tyr-448. Furthermore, the
interaction of the C2-carboxylate group with the protein is mediated
by a pair of water molecules, shown as red spheres. Besides the
backbone atoms of Ile-447 and Tyr-448, all side-chain atoms within
5 Å of the ligand are depicted as sticks.
cysteines present in the protein. Rather, the reaction was
selective and specific toward cysteine-366 which existed in close
proximity. The specificity was also confirmed by mass spectros-
copy studies. When compound 33 was incubated with NS5B
enzyme, only tryptic peptide sequence 346−379 was modified
by changing from 3533 Da to 4012 Da, which was consistent
with covalent bonding with cysteine-366. No other tryptic pep-
tide was found to be modified by 480.0 Da. All these studies
suggested that this class of irreversible inhibitors was highly
selective and specific in the formation of a covalent adduct,
reacting only with cysteine-366. The SAR trends of these irre-
versible inhibitors also indicated that the covalent bonding was
induced by proximity after the inhibitor bound to the enzyme
surface through traditional binding interactions.
Even though there are several marketed drugs that contain
nitro-aromatic moieties, some concerns still exist about the
potential safety issues arising from the metabolites of the nitro-
benzene. The partial reduction of the nitro group could lead to
the formation of nitro-anion radical or a nitroso group, which
could cause cytotoxic effects.35 To address this concern, com-
pound 45 and 47 were tested in the Salmonella/mammalian
microsome Mini-AMES reverse mutation assay. No bacterial
mutagenicity was observed with or without metabolic activa-
tion. Both compounds were also evaluated in the in vitro micro-
nucleus induction assay. No chromosome damage was found
with either 45 or 47. Inhibitor 47 was also found to be clean
in counterscreens of kinases and protease panels. In summary,
the fact that compound 47 was clean in all in vitro assays
discussed above and no toxicity was observed in single dose
animal PK studies demonstrated that no evidence for potential
safety issues related to the aromatic nitro moiety has been
found.
EXPERIMENTAL SECTION
■
General Methods. Reagents and solvents, including anhydrous
solvents, such as THF, dichloromethane, and DMF, were purchased
from Aldrich or other commercial sources and were used without
further purification. Reactions that were moisture sensitive or using
anhydrous solvents were performed under either a nitrogen or an
argon atmosphere. Analytical thin layer chromatography (TLC) was
performed on precoated silica gel plates obtained from Analtech.
Visualization was accomplished with UV light or by staining with
basic KMnO4 solution, ethanolic H2SO4, or Vaughn’s reagent. Some
compounds were purified by flash chromatography either on a glass
column using Merck silica gel 60 (230−400 mesh) or on an ISCO
RediSep disposable silica gel column. More polar compounds were
purified by reverse phase HPLC on a Sunfire C18 preparative column
(50 × 250 mm, 10 μm) running at 30 mL/minute using 20−100%
acetonitrile/water (both as a 0.1% trifluoroacetic acid solution) as
eluent. NMR spectra were recorded at 400 or 500 MHz for 1H and at
100 or 125 MHz for 13C on a Bruker or Varian spectrometer with
CDCl3 or DMSO-d6 as solvent. The chemical shifts are given in ppm,
referenced to the internal TMS or deuterated solvent signal. HPLC,
1
LC-MS, and/or H NMR methods were employed to determine the
purity of all synthesized compounds. All new compounds tested had
purity at or above 95%.
General Procedures for Iodination of 3. The solution of
compound 3 (1 mmol) and N-iodosuccinimide (NIS) (1.05 mmol)
in acetone (15 mL) was stirred at room temperature (rt) for 3 h (h).
Saturated aqueous sodium thiosulfate (5 mL) was added. After the
mixture was stirred for 5 min (min), ethyl acetate (30 mL) and water
(20 mL) were added. Layers were separated. The aqueous solution
was extracted with ethyl acetate (2 × 25 mL). The combined organic
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layer was washed with brine, dried over magnesium sulfate, and con-
centrated in vacuo to give the crude product, which was usually pure
enough to be used in the next reaction directly.
solution (7 mL), and water (7 mL) were added. After the layers were
separated, the aqueous solution was extracted with EtOAc (3 × 22 mL).
The combined organic solution was dried over magnesium sulfate and
concentrated in vacuo to give the crude product in 60−90% yield, which
was used without further purification.
Compound 13 was prepared from compound 12 according to the
same general procedures described above for the preparation of 6 from
5. The crude product was used in the next reaction without further
purification.
General Procedures for Suzuki Coupling between 4 and 2-
Methoxy-3-pyridineboronic Acids. To the suspension of 3-iodoindole
(4, 10.0 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloro-
palladium (PdCl2(dppf)2) (1.0 mmol) in dimethoxyethane (DME)
(120 mL) at rt was added a solution of boronic acid (12.0 mmol) and
potassium carbonate (50 mmol) in water (30 mL). The mixture was
bubbled with argon gas through a frit glass bubbler for 5 min before it
was heated to 90 °C in an oil bath and stirred for 4 h. After being
cooled to rt, the mixture was diluted with water and ethyl acetate
(300 mL each), and the two layers were separated. The aqueous solution
was extracted with ethyl acetate (2 × 200 mL). The combined organic
solution was washed with brine, dried over magnesium sulfate, and
concentrated in vacuo to give the crude product, which was purified by
silica gel flash chromatography using 0−50% ethyl acetate in hexanes
as eluent to give the desired product 5 in 50−85% yield (two steps).
General Procedures for the Preparation of 6. The mixture of 5
(5.0 mmol), arylmethyl bromide (or arylmethyl chloride) (7.5 mmol),
and cesium carbonate (7.5 mmol) in anhydrous dimethylformamide
(DMF) (80 mL) was vigorously stirred at rt for 16 h. It was then
diluted with water and ethyl acetate (200 mL each), and the two layers
were separated. The aqueous solution was extracted with ethyl acetate
(100 mL). The combined organic solution was washed with water
(2 × 300 mL) and brine, dried over magnesium sulfate, and concen-
trated in vacuo to give the crude product, which was purified by silica
gel flash chromatography using 0−60% ethyl acetate in hexanes as
eluent to give the desired product 6 in 40−90% yield.
General Procedures for the Preparation of 7. Compound 6
(1.0 mmol) and lithium hydroxide (4.0 mmol) were dissolved in THF
and water (10 mL each). The solution was then heated to 70 °C in an
oil bath and stirred for 16 h before it was cooled to rt. The solution
was then made acidic (pH ∼ 2) by addition of 1 N hydrochloric acid.
Ethyl acetate was added (30 mL), and the layers were separated. The
aqueous solution was extracted with ethyl acetate (2 × 20 mL). The
combined organic solution was washed with brine, dried over mag-
nesium sulfate, and concentrated in vacuo to give the crude product 7
in 60−90% yield, which was used without further purification.
General Procedures for the Preparation of 8. The mixture of 7
(0.50 mmol) and carbonyl diimidazole (CDI) (0.60 mmol) in an-
hydrous THF (5 mL) was heated to 75 °C in an oil bath and stirred
1.5 h. It was then cooled to rt, alkyl sulfonamide and 1,8-diazabicyclo-
cundec-7-ene (DBU) (1.5 mmol each) were added, and the resulting
mixture was stirred at rt for 18 h. The mixture was then diluted with
water and ethyl acetate (30 mL each), and the two layers were
separated. The aqueous solution was extracted with ethyl acetate (2 ×
15 mL). The combined organic solution was washed with brine, dried
over magnesium sulfate, and concentrated in vacuo to give the crude
product, which was purified by silica gel flash chromatography using
0−80% acetone in dichloromethane as eluent to give the desired
product 8 in 50−80% yield.
Alternative General Method for Preparation of 13 from 12
through a Mitsunobu Reaction. To the mixture of 12 (2.0 mmol),
arylmethyl alcohol (2.8 mmol), and triphenylphosphine (3.8 mmol) in
anhydrous THF (50 mL) was added diisopropyl azodicarboxylate
(DIAD) (3.8 mmol). The resulting mixture was stirred at rt for 16 h.
It was concentrated in vacuo to give the crude product, which was
purified by silica gel flash chromatography using 0−60% ethyl acetate
in hexanes as eluent to give the desired product 13 in 60−90% yield.
General Procedures for the Preparation of 9 from 13. To the
solution of compound 13 (0.2 mmol) and appropriate alkyl primary
sulfonamide (0.4 mmol) in anhydrous THF (5 mL) at rt was added
sodium hydride (30 mg, 1.5 mmol, 60% in mineral oil). The mixture
was heated to 70 °C in an oil bath for 1.5 h. After the mixture was
cooled to rt, EtOAc (30 mL) and water (40 mL) were added. After the
layers were separated, the aqueous solution was extracted with EtOAc
(2 × 40 mL). The combined organic solution was dried over magne-
sium sulfate and concentrated in vacuo to give the crude product in
30−60% yield.
General Procedures for the Preparation of 14. Compound 14
(0.3 mmol) and lithium hydroxide (2.0 mmol) were dissolved in THF
and water (10 mL each). The solution was stirred at rt for 4 h. EtOAc
(40 mL) and 5% aqueous phosphoric acid solution (20 mL) were
added. After the layers were separated, the aqueous solution was
extracted with ethyl acetate (2 × 30 mL). The combined organic solu-
tion was dried over magnesium sulfate and concentrated in vacuo to
give the crude product, which was purified by reverse phase HPLC to
give the desired product (14) in 20−60% yield.
N-(Cyclopropylsulfonyl)-1-[(2,5-difluorophenyl)methyl]-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-5-methyl-1H-indole-2-carbox-
amide (2). Compound 2 was prepared according to the reaction
sequence outlined in Scheme 1 and the general procedures described
above for the preparation of generic compound 9. 1H NMR (500 MHz,
DMSO-d6), 12.79 (s, 1 H), 12.71 (s, 1 H), 7.81 (q, J = 1.9 Hz, 1 H),
7.69 (s, 1 H), 7.54−7.52(m, 1 H), 7.32−7.27 (m, 1 H), 7.21−7.20
(m, 2 H), 7.18−7.13 (m, 1 H), 6.64−6.16 (m, 1 H), 6.60−6.56 (m, 1 H),
5.73 (s, 2 H), 2.97−2.91 (m, 1 H), 2.36 (s, 3 H), 0.97 (d, J = 6.3 Hz,
4 H). 13C NMR (125 MHz, DMSO-d6), 163.5, 161.9, 160.0, 158.0, 157.6,
155.7, 145.0, 136.9, 136.5, 131.2, 128.1, 127.9, 127.2, 123.9, 120.7,
118.0, 117.9, 117.8, 117.7, 116.6, 115.9, 115.8, 111.6, 108.3, 42.4, 31.5,
21.9, 6.3. LRMS calcd for C25H22N3O4F2S (M + H)+, 498.1; found, 498.3.
N-(Cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-
1-[(2-fluorophenyl)methyl]-5-methyl-1H-indole-2-carboxa-
mide (15). Compound 15 was prepared according to the reaction
sequence outlined in Scheme 1 and the general procedures described
above for the preparation of generic compound 9. 1H NMR (500 MHz,
DMSO-d6), 12.80 (s, 1 H), 12.74 (s, 1 H), 7.7 9(d, J = 2.2 Hz, 1 H),
7.69 (t, J = 4.4 Hz, 1 H), 7.52 (d, J = 9.0 Hz, 1 H), 7.33−7.28 (m, 1 H),
7.23−7.18 (m, 3 H), 7.10−7.06 (m, 1 H), 6.87−6.82 (m, 1 H), 6.30
(t, J = 6.6 Hz, 1 H), 5.75 (s, 2 H), 2.93−2.89 (m, 1 H), 2.35 (s, 3 H),
0.96 (d, J = 6.3 Hz, 4 H). 13C NMR (125 MHz, DMSO-d6), 174.0,
172.4, 172.0, 170.1, 166.0, 162.8, 157.4, 155.6, 150.5, 147.5, 141.6, 140.8,
140.0, 138.5, 137.7, 135.9, 131.1, 126.8, 126.6, 122.2, 118.9, 116.5,
78.4, 52.8, 45.7, 42.0, 41.8, 32.4, 16.8. LRMS calcd for C25H23N3O4FS
(M + H)+, 480.1; found, 480.3.
General Procedures for the Preparation of 9. Compound 8
(1.0 mmol) was dissolved in a solution of 4 M hydrogen chloride in p-
dioxane (50 mL) in a pressure glass tube. The solution was then sealed
and heated to 90 °C in an oil bath and stirred for 3 h before it was
cooled to rt. The solution was then concentrated in vacuo to give the
crude product, which was purified by reverse phase HPLC to give the
desired product 9 in 30−60% yield.
Compound 10 was prepared from compound 5 according to the
same procedures described above for the preparation of 9 from
compound 8.
Compound 11 was prepared from compound 10 according to the
same procedures described above for the preparation of 7. The crude
product was used in the next reaction without further purification.
General Procedures for the Preparation of 12. To the solution
of compound 11 (1.0 mmol) in anhydrous DMF (5 mL) at rt was
added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (442 mg,
2.3 mmol) and triethylamine (0.69 mL, 5.0 mmol). The mixture was
stirred overnight (17 h). EtOAc (15 mL), 5% aqueous phosphoric acid
N-(Cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-
1-[(2-fluoro-3-methylphenyl)methyl]-5-methyl-1H-indole-2-
carboxamide (16). Compound 16 was prepared according to the
reaction sequence outlined in Scheme 1 and the general procedures
described above for the preparation of generic compound 9. 1H NMR
(500 MHz, DMSO-d6), 12.79 (s, 1 H), 12.73 (s, 1 H), 7.78 (q, J =
1.8 Hz, 1 H), 7.69 (t, J = 5.8 Hz, 1 H), 7.51 (d, J = 9.0 Hz, 1 H), 7.19−
7.15 (m, 3 H), 6.95 (t, J = 7.6 Hz, 1 H), 6.64 (q, J = 5.0 Hz, 2 H), 5.73
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(s, 2 H), 2.95−2.90 (m, 1 H), 2.35 (s, 3 H), 2.22 (s, 3 H), 0.97 (d, J =
6.6 Hz, 4 H). 13C NMR (125 MHz, DMSO-d6), 174.0, 172.4, 155.6,
147.5, 147.0, 145.4, 142.0, 141.5, 140.0, 138.4, 137.6, 137.4, 136.2,
135.3, 134.4, 131.0, 125.9, 122.2, 118.8, 52.9, 45.9, 42.0, 41.8, 32.4,
25.4, 16.8. LRMS calcd for C26H25N3O4FS (M + H)+, 494.2; found,
494.3.
1-[(2-Amino-4-pyridinyl)methyl]-N-(cyclopropylsulfonyl)-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-5-methyl-1H-indole-2-carbox-
amide (17). Compound 17 was prepared according to the reac-
tion sequence outlined in Scheme 1 and the general procedures
described above for the preparation of generic compound 9. 1H NMR
(500 MHz, DMSO-d6), 13.45 (s, 1 H), 12.84 (s, 2 H), 7.94 (s, 1 H),
7.90 (d, J = 6.6 Hz, 1 H), 7.81 (q, J = 1.9 Hz, 1 H), 7.74 (s, 1 H),
7.45 (d, J = 8.2 Hz, 1 H), 7.24 (s, 2 H), 6.68 (t, J = 6.9 Hz, 1 H), 6.60
(d, J = 7.0 Hz, 1 H), 6.41 (s, 1 H), 5.67 (s, 2 H), 2.98−2.93 (m, 1 H),
2.37 (s, 3 H), 1.01 (d, J = 7.9 Hz, 4 H). 13C NMR (125 MHz, DMSO-
d6), 174.0, 172.1, 167.3, 165.4, 155.8, 147.5, 142.1, 139.9, 139.0, 137.8,
134.1, 131.3, 130.0, 126.2, 122.0, 121.9, 120.4, 119.1, 109.3, 58.6, 42.6,
42.1, 41.8, 33.4, 32.4, 25.3, 16.8. LRMS calcd for C24H24N5O4S
(M + H)+, 478.2; found, 478.3.
6.67−6.62 (m, 1 H), 5.87 (s, 2 H), 3.31 (s, 3 H), 2.93−2.89 (m, 1 H),
2.37 (s, 3 H), 0.97 (s, 4 H). 13C NMR (125 MHz, DMSO-d6), 163.6,
161.8, 161.4, 158.8, 157.0, 152.8, 145.4, 139.5, 137.3, 136.8, 133.1,
131.6, 131.2, 131.1, 128.5, 127.3, 120.8, 116.1, 115.3, 111.9, 111.5,
108.6, 44.0, 43.1, 31.6, 31.3, 21.9, 6.3. LRMS calcd for C27H24N4O6FS2
(M + H)+, 583.1; found, 583.0.
1-[[2-Chloro-5-[(trifluoromethyl)sulfonyl]phenyl]methyl]-N-
(cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-
methyl-1H-indole-2-carboxamide (23). Compound 23 was pre-
pared according to the reaction sequence outlined in Scheme 1 and
the general procedures described above for the preparation of generic
1
compound 9. H NMR (500 MHz, DMSO-d6), 13.03 (s, 1 H), 12.88
(s, 1 H), 8.08−8.02 (m, 2 H), 7.84 (dd, J = 1.6, 1.5 Hz, 1 H), 7.76−
7.74 (m, 1 H), 7.50 (d, J = 8.8 Hz, 1 H), 7.24 (s, 1 H), 7.21 (d, J =
8.8 Hz,1 H), 7.06 (d, J = 1.8 Hz, 1 H), 6.68 (t, J = 6.6 Hz, 1 H), 5.87
(s, 2 H), 2.86−2.81 (m, 1 H), 2.37 (s, 3 H), 0.92 (d, J = 6.0 Hz, 4 H).
13C NMR (125 MHz, DMSO-d6), 163.6, 161.7, 156.9, 145.6, 142.4,
140.1, 137.1, 136.8, 133.0, 131.8, 131.6, 131.1, 129.6, 129.4, 128.6, 127.3,
123.5, 120.9, 116.0, 111.4, 109.8, 108.8, 16.6, 31.5, 21.9, 6.2, 5.1. LRMS
calcd for C26H22N3O6ClF3S2 (M + H)+, 628.0; found, 628.3.
1-[(5-Cyano-2-fluorophenyl)methyl]-N-(cyclopropylsulfon-
yl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-methyl-1H-indole-2-
carboxamide (8). Compound 18 was prepared according to the
reaction sequence outlined in Scheme 1 and the general procedures
described above for the preparation of generic compound 9. 1H NMR
(500 MHz, DMSO-d6), 12.73 (s, 1 H), 12.65 (s, 1 H), 7.90−7.86
(m, 1 H), 7.82 (d, J = 6.4 Hz, 1 H), 7.67 (s, 1 H), 7.56−7.49 (s, 2 H),
7.23−7.20 (m, 3 H), 6.61 (t, J = 6.2 Hz, 1 H), 5.76 (s, 2 H), 2.93−2.87
(m, 1 H), 2.37 (s, 3 H), 0.95 (d, J = 7.6 Hz, 4 H). 13C NMR (125 MHz,
DMSO-d6), 164.1, 163.9, 163.4, 162.1, 161.9, 161.5, 146.4, 144.9, 143.8,
140.1, 139.7, 139.4, 136.9, 136.4, 135.3, 134.8, 133.5, 133.2, 131.4, 129.6,
129.4, 128.2, 127.2, 125.8, 123.8, 123.0, 120.8, 118.8, 118.2, 118.1, 115.9,
111.5, 108.7, 108.3, 107.2, 98.3, 42.2, 35.2, 34.8, 32.9, 31.5, 31.3, 30.1,
29.9, 21.9, 6.3, 5.9, 5.1. LRMS calcd for C26H22N4O4FS (M + H)+,
505.1; found, 505.3.
1-[[5-(Aminocarbonyl)-2-fluorophenyl]methyl]-N-(cyclopro-
pylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-methyl-1H-in-
dole-2-carboxamide (19). Compound 19 was prepared according to
the reaction sequence outlined in Scheme 1 and the general proce-
dures described above for the preparation of generic compound 9.
1H NMR (500 MHz, DMSO-d6), 12.82 (s, 1 H), 12.78 (s, 1 H), 7.89
(s, 1 H), 7.84−7.80 (m, 2 H), 7.70 (s, 1 H), 7.55 (d, J = 7.8 Hz, 2 H),
7.38 (s, 1 H), 7.30 (t, J = 9.3 Hz, 1 H), 7.21−7.18 (m, 2 H), 6.65−6.62
(m, 1 H), 5.76 (s, 2 H), 2.96−2.89 (m, 1 H), 2.36 (s, 3 H), 0.96 (d, J =
6.0 Hz, 4 H). 13C NMR (125 MHz, DMSO-d6), 167.6, 163.5, 161.9,
161.5, 152.5, 145.3, 137.2, 136.6, 131.6, 131.1, 130.0, 129.7, 128.1,
127.2, 125.9, 125.8, 125.2, 123.9, 120.7, 116.2, 116.0, 115.6, 111.7, 108.4,
67.9, 42.7, 35.3, 31.6, 31.3, 30.1, 28.9, 16.0, 24.9, 21.9, 6.3. LRMS calcd
for C26H24N4O5FS (M + H)+, 523.1; found, 523.3.
1-[[5-(Aminosulfonyl)-2-fluorophenyl]methyl]-N-(cyclopro-
pylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-methyl-1H-in-
dole-2-carboxamide (21). Compound 21 was prepared according to
the reaction sequence outlined in Scheme 1 and the general proce-
dures described above for the preparation of generic compound 9.
1H NMR (500 MHz, DMSO-d6), 12.84 (s, 2 H), 7.82−7.78 (m, 2 H),
7.73−7.71 (m, 1 H), 7.54 (d, J = 8.1 Hz, 1 H), 7.49−7.43 (m, 2 H),
7.38 (s, 2 H), 7.21 (d, J = 8.8 Hz, 1 H), 7.19 (s, 1 H), 6.66 (t, J = 8.0
Hz, 1 H), 5.78 (s, 2 H), 2.96−2.91 (m, 1 H), 2.36 (s, 3 H), 0.97 (d, J =
6.1 Hz, 4 H). 13C NMR (125 MHz, DMSO-d6), 163.6, 163.2, 161.8,
161.2, 152.3, 145.4, 141.4, 137.3, 136.7, 131.3, 128.5, 127.5, 127.3,
126.9, 125.8, 120.8, 117.3, 115.7, 111.6, 108.6, 67.9, 42.8, 31.6, 31.3,
30.1, 26.0, 21.9. LRMS calcd for C25H24N4O6FS2 (M + H)+, 559.1;
found, 559.3.
1-[[2-Chloro-5-[(trifluoromethyl)sulfinyl]phenyl]methyl]-N-
(cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-
methyl-1H-indole-2-carboxamide (24). Compound 24 was pre-
pared according to the reaction sequence outlined in Scheme 1 and
the general procedures described above for the preparation of generic
1
compound 9. H NMR (500 MHz, DMSO-d6), 12.93 (s, 1 H), 12.83
(s,1 H), 7.89 (d, J = 1.6 Hz, 1 H), 7.84−7.81 (m, 1 H), 7.73 (s,1 H),
7.43 (d, J = 8.8 Hz, 1 H), 7.23 (s,1 H), 7.18 (d, J = 8.4 Hz, 1 H), 6.91
(s, 1 H), 6.87 (s, 1 H), 6.71−6.63 (m, 1 H), 5.83 (s, 2 H), 2.88−2.80
(m, 1 H), 2.36 (s, 3 H), 1.35 (d, J = 1.6 Hz, 4 H). 13C NMR (125 MHz,
DMSO-d6), 163.6, 152.3, 145.4, 140.0, 138.8, 137.8, 136.7, 135.5, 131.8,
131.5, 129.6, 128.9, 128.3, 127.3, 127.2, 126.7, 125.8, 125.2, 120.9, 111.5,
108.6, 108.5, 46.6, 35.2, 31.5, 31.3, 30.1, 21.9, 61.2. LRMS calcd for
C26H22N3O5ClF3S2 (M + H)+, 612.1; found, 612.3.
1-[[4-Chloro-2-fluoro-5-(methylsulfonyl)phenyl]methyl]-N-
(cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-
methyl-1H-indole-2-carboxamide (26). Compound 26 was pre-
pared according to the reaction sequence outlined in Scheme 1 and
the general procedures described above for the preparation of generic
1
compound 9. H NMR (500 MHz, DMSO-d6), 12.89 (s, 1 H), 12.83
(s, 1 H), 7.87 (d, J = 9.8 Hz, 1 H), 7.81 (d, J = 6.4 Hz, 1 H), 7.76
(d, J = 7.6 Hz, 1 H), 7.72 (s, 1 H), 7.61 (d, J = 8.4 Hz, 1 H), 7.28 (d, J =
8.5 Hz, 1 H), 7.20 (s, 1 H), 6.66 (t, J = 6.7 Hz, 1 H), 5.79 (s, 2 H),
3.31 (s, 3 H), 2.95−2.90 (m, 1 H), 2.67 (q, J = 7.5 Hz, 2 H), 1.18
(t, J = 7.6 Hz, 3 H), 0.99 (d, J = 8.5 Hz, 4 H). 13C NMR (125 MHz,
DMSO-d6), 164.2, 163.5, 162.1, 161.8, 161.4, 157.0, 145.5, 138.1, 137.4,
136.7, 136.3, 135.2, 133.1, 132.1, 129.2, 127.4, 127.1, 126.4, 126.3, 123.6,
120.7, 120.4, 119.5, 116.1, 111.6, 108.7, 43.3, 42.4, 31.6, 29.9, 29.4, 29.0,
17.0, 6.3, 5.9, 5.1. LRMS calcd for C27H26N3O6ClFS2 (M + H)+, 606.1;
found, 606.3.
1-[[4-Cyano-2-fluoro-5-(methylsulfonyl)phenyl]methyl]-N-
(cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-
methyl-1H-indole-2-carboxamide (27). Compound 27 was pre-
pared according to the reaction sequence outlined in Scheme 1 and
the general procedures described above for the preparation of generic
1
compound 9. H NMR (500 MHz, DMSO-d6), 12.90 (s, 1 H), 12.85
(s, 1 H), 8.33 (d, J = 9.4 Hz, 1 H), 7.83 (d, J = 5.7 Hz, 1 H), 7.72
(s, 1 H), 7.67 (d, J = 6.7 Hz, 1 H), 7.62 (d, J = 8.8 Hz, 1 H), 7.29 (d, J =
9.0 Hz, 1 H), 7.22 (s, 1 H), 6.66 (s, 1 H), 5.87 (s, 2 H), 3.32 (s, 3 H),
2.90 (s, 1 H), 2.67 (q, J = 7.5 Hz, 2 H), 1.18 (t, J = 7.3 Hz, 3 H), 0.97
(s, 4 H). 13C NMR (125 MHz, DMSO-d6), 163.4, 163.0, 162.4, 161.4,
161.0, 156.4, 155.6, 147.0, 141.3, 140.4, 139.7, 134.4, 132.5, 132.4, 130.9,
130.0, 128.2, 124.5, 124.3, 122.6, 122.0, 121.0, 114.4, 112.6, 85.4, 69.1,
67.0, 63.0, 44.0, 32.9, 29.8, 29.2, 28.3, 24.7, 22.6, 17.2, 6.1, 5.8, 4.7.
LRMS calcd for C28H26N4O6FS2 (M + H)+, 597.1; found, 597.3
1-[[5-(Aminosulfonyl)-2,4-dichlorophenyl]methyl]-N-(cyclo-
propylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-ethyl-1H-
indole-2-carboxamide (28). Compound 28 was prepared according
to the reaction sequence outlined in Scheme 1 and the general proce-
dures described above for the preparation of generic compound 9.
1H NMR (500 MHz, DMSO-d6), 12.87 (s, 2 H), 7.94 (s, 1 H), 7.83
1-[[3-Cyano-2-fluoro-5-(methylsulfonyl)phenyl]methyl]-N-
(cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-
methyl-1H-indole-2-carboxamide (22). Compound 22 was
prepared according to the reaction sequence outlined in Scheme 1
and the general procedures described above for the preparation of generic
1
compound 9. H NMR (500 MHz, DMSO-d6), 12.89 (s, 1 H), 12.83
(s, 1 H), 8.32 (d, J = 9.5 HZ, 1 H), 7.82 (q, J = 1.6 Hz, 1 H), 7.72
(s, 1 H), 7.62 (q, J = 6.8 Hz, 2 H), 7.24 (d, J = 8.9 Hz, 1 H), 7.22 (s, 1 H),
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Journal of Medicinal Chemistry
Article
(q, J = 1.8 Hz, 1 H), 7.75−7.73 (m, 1 H), 7.62 (s, 2 H), 7.44 (d, J = 8.6
Hz, 1 H), 7.29 (s, 1 H), 7.25 (q, J = 1.7 Hz, 1 H), 7.23 (s, 1 H), 6.69
(t, J = 6.6 Hz, 1 H), 5.72 (s, 2 H), 2.93−2.88 (m, 1 H), 2.67 (q, J = 7.5
Hz, 2 H), 1.18 (t, J = 7.6 Hz, 3 H), 0.98−0.96 (m, 4 H). 13C NMR
(125 MHz, DMSO-d6), 163.6, 161.6, 147.0, 145.5, 141.0, 139.6, 138.1,
137.5, 136.8, 136.4, 136.1, 132.5, 130.7, 128.6, 127.5, 127.3, 123.7,
119.7, 115.9, 111.6, 108.7, 46.6, 31.6, 30.2, 29.1, 16.9, 6.3. LRMS calcd
for C26H25N4O6Cl2S2 (M + H)+, 623.1; found, 624.8.
128.4, 128.0, 127.9, 115.8, 111.5, 108.4, 42.6, 42.0, 35.2, 31.3, 30.1,
22.5, 21.8. LRMS calcd for C23H20N4O6FS (M + H)+, 499.1; found,
499.3.
3-(1,2-Dihydro-2-oxo-3-pyridinyl)-1-[(2-fluoro-5-
nitrophenyl)methyl]-5-methyl-N-[(1-methylethyl)sulfonyl]-1H-
indole-2-carboxamide (34). Compound 34 was prepared according
to the reaction sequence outlined in Scheme 2 and the general
procedures described above for the preparation of generic compound
1
9. H NMR (500 MHz, DMSO-d6), 12.81 (s, 1 H), 12.74 (s, 1 H),
1-[[3-(Aminocarbonyl)-2,5,6-trifluorophenyl]methyl]-N-(cy-
clopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-meth-
yl-1H-indole-2-carboxamide (29). Compound 29 was prepared
according to the reaction sequence outlined in Scheme 1 and the gene-
ral procedures described above for the preparation of generic com-
8.25−8.22 (m, 1 H), 7.84−7.83 (m, 1 H), 7.71 (q, J = 8.8 Hz, 2 H),
7.61−7.54 (m, 2 H), 7.24 (s, 1 H), 7.22 (s, 1 H), 6.65 (t, J = 6.6 Hz, 1 H),
5.81 (s, 2 H), 3.61−3.55 (m, 1 H), 2.37 (s, 3 H), 1.13 (d, J = 6.9 Hz, 6
H). 13C NMR (125 MHz, DMSO-d6), 165.2, 163.6, 163.1, 162.0, 145.4,
144.9, 137.2, 136.7, 131.5, 129.3, 128.5, 128.2, 128.1, 127.2, 126.4, 126.3,
125.1, 123.7, 120.8, 118.1, 117.9, 116.1, 111.5, 108.5, 53.6, 42.7, 31.3, 21.9,
16.1. LRMS calcd for C25H24N4O6FS (M + H)+, 527.1; found, 527.3.
3-(1,2-Dihydro-6-methyl-2-oxo-3-pyridinyl)-1-[(2-fluoro-5-
nitrophenyl)methyl]-5-methyl-1H-indole-2-carboxylic acid
(37). Compound 37 was prepared according to the reaction sequence
outlined in Scheme 2 and the general procedures described above for
the preparation of generic compound 14. 1H NMR (500 MHz,
DMSO-d6), 13.20 (s, 1 H), 11.91 (s, 1 H), 8.24−8.20 (m, 1 H), 7.62−
7.61 (m, 1 H), 7.58−7.47 (m, 3 H), 7.24 (s, 1 H), 7.18 (d, J = 8.5 Hz,
2 H), 6.19 (d, J = 6.9 Hz, 1 H), 5.90 (s, 2 H), 2.36 (s, 3 H), 2.26 (s, 3 H).
13C NMR (125 MHz, DMSO-d6), 175.2, 174.6, 173.4, 155.4, 152.2,
1
pound 9. H NMR (500 MHz, DMSO-d6), 12.67 (s, 1 H), 12.64
(d, J = 5.0 Hz, 1 H), 7.81 (s, 1 H), 7.75 (s, 1 H), 7.73 (q, J = 1.8 Hz, 1 H),
7.70−7.64 (m, 2 H), 7.54 (d, J = 8.5 Hz, 1 H), 7.23 (d, J = 8.6 Hz,
1 H), 7.17 (s, 1 H), 6.59 (t, J = 6.6 Hz, 1 H), 5.86 (s, 2 H), 2.98−2.93
(m, 1 H), 2.35 (s, 3 H), 1.06−0.99 (m, 4 H). 13C NMR (125 MHz,
DMSO-d6), 164.1, 163.3, 162.0, 152.3, 144.8, 137.0, 136.5, 131.1,
129.8, 128.0, 127.2, 125.8, 123.8, 120.8, 118.0, 117.9, 115.9, 111.2,
108.1, 67.9, 37.5, 31.3, 26.0, 21.8, 6.3. LRMS calcd for C26H22N4O5F3S
(M + H)+, 559.1; found, 559.3.
1-[(2-Chloro-5-nitrophenyl)methyl]-N-(-(cyclopropylsulfon-
yl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-5-methyl-1H-indole-2-
carboxamide (30). Compound 30 was prepared according to the
reaction sequence outlined in Scheme 1 and the general procedures
described above for the preparation of generic compound 9. 1H NMR
(500 MHz, DMSO-d6), 12.88 (s, 1 H), 12.77 (s, 1 H), 8.15 (q, J =
2.8 Hz, 1 H), 7.86−7.84 (m, 2 H), 7.72 (s, 1 H), 7.48 (d, J = 8.5 Hz,
1 H), 7.27 (s, 2 H), 7.21 (d, J = 8.6 Hz, 1 H), 6.65 (t, J = 6.6 Hz, 1 H),
5.80 (s, 2 H), 2.88 − 2.82 (m, 1 H), 2.38 (s, 3 H), 0.92 (d, J = 6.2 Hz,
4 H). 13C NMR (125 MHz, DMSO-d6), 164.4, 163.8, 163.5, 161.8,
148.7, 147.5, 145.2, 139.1, 138.8, 137.1, 136.6, 131.8, 131.5, 130.7,
128.5, 127.3, 124.6, 122.8, 120.9, 111.5, 108.4, 46.6, 31.5, 21.9, 6.2.
LRMS calcd for C25H22N4O6ClS (M + H)+, 541.1; found, 541.3.
N-(Cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-
1-[(2-fluoro-5-nitrophenyl)methyl]-5-methyl-1H-indole-2-car-
boxamide (31). Compound 31 was prepared according to the reac-
tion sequence outlined in Scheme 1 and the general procedures de-
150.9, 147.5, 141.2, 139.2, 138.3, 137.7, 135.1, 133.0, 131.4, 128.7, 128.5,
128.3, 121.8, 116.1, 108.5, 78.4, 32.4, 29.8. LRMS calcd for C23H19N3O5F
(M + H)+, C23H18N3O5F: 436.1; found, 436.2.
3-(1,2-Dihydro-6-methyl-2-oxo-3-pyridinyl)-1-[(2-fluoro-5-
nitrophenyl)methyl)methyl]-5-methyl-N-(methylsulfonyl)-1H-
indole-2-carboxamide (38). Compound 38 was prepared according
to the reaction sequence outlined in Scheme 2 and the general
procedures described above for the preparation of generic compound
1
9. H NMR (500 MHz, DMSO-d6), 13.12(s, 1 H), 12.83 (s, 1 H),
8.25−8.22 (m, 1 H), 7.77−7.75 (m, 2 H), 7.58−7.54 (m, 2 H), 7.23−
7.21 (m, 2 H), 6.50 (d, J = 7.2 Hz, 1 H), 5.80 (s, 2 H), 3.27 (s, 3 H),
2.37 (d, J = 1.8 Hz, 6 H). 13C NMR (125 MHz, DMSO-d6), 172.8,
163.6, 162.9, 158.2, 156.1, 155.2, 147.7, 141.8, 139.7, 138.9, 136.9, 135.7,
133.5, 133.4, 133.2, 131.5, 131.3, 130.3, 129.9, 129.8, 129.6, 128.4, 126.4,
123.4, 122.0, 118.7, 66.3, 52.6, 45.8, 32.4, 29.8, 27.2. LRMS calcd for
C24H22N4O6FS (M + H)+, 513.1; found, 513.3.
1
scribed above for the preparation of generic compound 9. H NMR
(500 MHz, DMSO-d6), 12.86 (s, 1 H), 12.76 (s, 1 H), 8.25 −8.22
(m, 1 H), 7.82 (d, J = 7.2 Hz, 1 H), 7.75−7.73 (m, 1 H), 7.70 (s, 1 H),
7.58 (q, J = 7.8 Hz, 2 H), 7.23 (d, J = 6.5 Hz, 2 H), 6.66−6.61 (m,
1 H), 5.82 (s, 2 H), 2.93−2.87 (s, 1 H), 2.37 (s, 3 H), 0.96 (d, J = 5.7
Hz, 4 H). 13C NMR (125 MHz, DMSO-d6), 165.1, 163.5, 163.1, 161.9,
145.2, 144.9, 137.1, 136.6, 131.4, 129.5, 128.3, 128.1, 128.0, 127.3,
126.3, 125.2, 123.8, 120.8, 118.1, 117.9, 115.9, 108.4, 42.6, 31.6, 21.9,
6.3. LRMS calcd for C25H22N4O6FS (M + H)+, 525.1; found, 525.3.
1-[(2-Fluoro-5-nitrophenyl)methyl]-3-(2-hydroxy-3-pyridin-
yl)-5-methyl-1H-indole-2-carboxylic acid (32). Compound 32
was prepared according to the reaction sequence outlined in Scheme 2
and the general procedures described above for the preparation of
generic compound 14. 1H NMR (500 MHz, DMSO-d6), 13.04
(s, 1 H), 11.81 (s, 1 H), 8.23−8.20 (m, 1 H), 7.61 (q, J = 2.85 Hz, 1 H),
7.58−7.53 (m, 3 H), 7.42 (q, J = 1.9 Hz, 1 H), 7.26 (s, 1 H), 7.19−
7.17 (m, 1 H), 6.35 (t, J = 6.6 Hz, 1 H), 5.91 (s, 2 H), 2.37 (s, 3 H).
13C NMR (125 MHz, DMSO-d6), 174.5, 173.5, 172.7, 164.6, 155.4,
151.6, 149.1, 147.4, 145.6, 141.5, 141.3, 139.3, 138.3, 137.6, 136.6,
135.0, 131.3, 128.7, 128.5, 128.3, 121.9, 116.6, 108.9, 87.0, 52.9, 41.8,
36.8, 32.4. LRMS calcd for C22H17N3O5F (M + H)+, 422.1; found,
422.2.
3-(1,2-Dihydro-2-oxo-3-pyridinyl)-1-[(2-fluoro-5-
nitrophenyl)methyl]-5-methyl-N-(methylsulfonyl)-1H-indole-
2-carboxamide (33). Compound 33 was prepared according to the
reaction sequence outlined in Scheme 2 and the general procedures
described above for the preparation of generic compound 9. 1H NMR
(500 MHz, DMSO-d6), 12.83 (s, 1 H), 12.71 (s, 1 H), 8.25−8.22
(m, 1 H), 7.83 (d, J = 6.9 Hz, 1 H), 7.76−7.74 (m, 1 H), 7.68 (s, 1 H),
7.59−7.53 (m, 2 H), 7.24 (s, 2 H), 6.65−6.62 (m, 1 H), 5.80 (s, 2 H),
3.25 (s, 3 H), 2.37 (s, 3 H). 13C NMR (125 MHz, DMSO-d6), 65.1,
163.3, 163.1, 162.2, 155.6, 145.0, 140.1, 137.0, 136.4, 134.2, 131.5,
N-(Cyclopropylsulfonyl)-3-(1,2-dihydro-6-methyl-2-oxo-3-
pyridinyl)-1-[(2-fluoro-5-nitrophenyl)methyl]-5-methyl-1H-in-
dole-2-carboxamide (39). Compound 39 was prepared according
to the reaction sequence outlined in Scheme 2 and the general pro-
cedures described above for the preparation of generic compound
1
9. H NMR (500 MHz, DMSO-d6), 13.12 (s, 1 H), 12.87 (s, 1 H),
8.26−8.22 (m, 1 H), 7.76 (s, 2 H), 7.59−7.55 (m, 2 H), 7.23−7.20
(m, 2 H), 6.50 (s, 1 H), 5.82 (s, 2 H), 2.92 (s, 1 H), 2.36 (s, 6 H), 0.97
(s, 4 H). 13C NMR (125 MHz, DMSO-d6), 164.0, 163.1, 162.0, 147.8,
145.7, 144.9, 137.2, 134.4, 131.4, 129.3, 128.4, 128.1, 128.0, 127.4, 126.4,
125.1, 123.0, 120.9, 119.8, 118.1, 117.9, 116.0, 111.5, 108.3, 45.6, 31.6,
26.1, 25.2, 24.6, 21.8, 19.3, 6.2, 5.8. LRMS calcd for C26H24N4O6FS
(M + H)+, 539.1; found, 539.3.
5-Chloro-N-(cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-
pyridinyl)-1-[(2-fluoro-5-nitrophenyl)methyl]-1H-indole-2-car-
boxamide (42). Compound 42 was prepared according to the reac-
tion sequence outlined in Scheme 2 and the general procedures de-
1
scribed above for the preparation of generic compound 9. H NMR
(500 MHz, DMSO-d6), 12.81 (s, 1 H), 12.64 (s, 1 H), 8.26−8.23
(m, 1 H), 7.81 (q, J = 1.8 Hz, 1 H), 7.79−7.77 (m, 2 H), 7.67 (s, 1 H),
7.56 (t, J = 9.5 Hz, 1 H), 7.47 (d, J = 1.9 Hz, 1 H), 7.41 (q, J=2.2 Hz, 1 H),
6.59 (t, J = 6.8 Hz, 1 H), 5.85 (s, 2 H), 2.92−2.87 (m, 1 H), 0.97 (d, J = 5.9
Hz, 4 H). 13C NMR (125 MHz, DMSO-d6), 165.2, 163.1, 161.6, 152.3,
145.0, 144.8, 136.9, 128.0, 127.6, 127.5, 127.0, 126.6, 126.5, 126.4, 125.8,
125.4, 125.3, 120.9, 118.1, 117.9, 115.7, 113.7, 108.2, 42.8, 35.2, 31.5, 31.3,
22.6, 6.3. LRMS calcd for C24H18N4O6ClFS, 544.1; found, 544.8.
N-(Cyclopropylsulfonyl)-3-(1,2-dihydro-2-oxo-3-pyridinyl)-
1-[(2-fluoro-5-nitrophenyl)methyl]-5-(trifluoromethyl-1H-in-
dole-2-carboxamide (43). Compound 43 was prepared according to
the reaction sequence outlined in Scheme 2 and the general procedures
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Journal of Medicinal Chemistry
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1
described above for the preparation of generic compound 9. H NMR
(500 MHz, DMSO-d6), 12.80 (s, 1 H), 12.60 (s, 1 H), 8.27−8.23 (m, 1 H),
7.97 (d, J = 8.8 Hz, 1 H), 7.84−7.81 (m, 2 H), 7.80 (s, 1 H), 7.69
(q, J = 1.4 Hz, 2 H), 7.56 (t, J = 9.2 Hz, 1 H), 6.59 (t, J = 6.6 Hz, 1 H),
5.9 (s, 2 H), 2.92−2.87 (m, 1 H), 0.97 (d, J = 5.2 Hz, 4 H). 13C NMR
(125 MHz, DMSO-d6), 165.3, 163.2, 163.0, 161.6, 152.3, 145.0, 144.7,
139.7, 136.9, 128.9, 127.4, 127.2, 126.9, 126.6, 126.3, 125.8, 125.5,
124.7, 122.9, 122.3, 119.7, 118.2, 118.0, 117.0, 113.1, 108.0, 42.9, 31.5,
31.3, 30.1, 6.3. LRMS calcd for C25H18N4O6F4S, 578.1; found, 578.8.
3-(1,2-Dihydro-2-oxo-3-pyridinyl)-5-ethyl-1-[(2-fluoro-5-
nitrophenyl)methyl]-1H-indole-2-carboxylic Acid (47). Com-
pound 47 was prepared according to the reaction sequence outlined
in Scheme 2 and the general procedures described above for the prepa-
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1
ration of generic compound 14. H NMR (500 MHz, DMSO-d6),
12.99 (s, 1 H), 11.80 (s, 1 H), 8.23−8.20 (m, 1 H), 7.65 (q, J = 2.9 Hz,
1 H), 7.58−7.54 (m, 3 H), 7.42 (q, J = 1.9 Hz, 1 H), 7.26 (s, 1 H),
7.23 (d, J = 8.4 Hz, 1 H), 6.36 (t, J = 6.9 Hz, 1 H), 5.91 (s, 2 H), 2.67
(q, J = 7.6 Hz, 2 H), 1.18 (t, J = 7.6 Hz, 3 H). 13C NMR (125 MHz,
DMSO-d6), 165.0, 164.0, 163.0, 162.3, 144.9, 141.1, 137.5, 137.1, 135.1,
128.8, 128.6, 127.9, 127.1, 126.8, 126.2, 126.1, 124.7, 124.6, 119.6, 118.4,
118.0, 117.8, 111.5, 106.2, 42.4, 29.1, 17.1. LRMS calcd for C23H19-
N3O5F (M + H)+, 436.1; found, 436.0.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: 732-662-1333. Fax: 908-740-7556. E-mail: kxcn@
yahoo.com.
ABBREVIATIONS USED
■
HCV, hepatitis C virus; PK, pharmacokinetics; SAR, structure−
activity relationship; NS, nonstructural; AUC, area-under-
the-curve; SOC, standard of care; SVR, sustained virologic
response; RdRp, RNA dependent RNA polymerase; RC, replicase
complex; NIs, nucleoside inhibitors; NNIs, non-nucleoside
inhibitors; NIS, N-iodosuccinimide; CDI, carbonyl diimidazole;
DBU, 1,8-diazabicyclocundec-7-ene; DIAD, diisopropyl azodicar-
boxylate; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
CL, clearance rate; CYP, cytochrome P450; hERG, the human
ether-a- go-go-related gene; PXR, pregnane X receptor; TLC,
thin layer chromatography; rt or RT, room temperature; min,
minutes; DME, dimethoxyethane; DMF, dimethylformamide;
DMSO, methyl sulfoxide; THF, tetrahydrofuran
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