Structure-activity relationship (SAR) development and discovery of potent indole-based inhibitors of the Hepatitis C Virus (HCV) NS5B polymerase

Article abstract of DOI:10.1021/jm201258k

Starting with the indole-based C-3 pyridone lead HCV polymerase inhibitor 2, extensive SAR studies were performed at different positions of the indole core. The best C-5 groups were found to be compact and nonpolar moieties and that the C-6 attachments were not affecting potency. Limited N-1 benzyl-type substituent studies indicated that the best substitutions were fluoro or methyl groups at 2′ or 5′ positions of the benzyl group. To improve pharmacokinetic (PK) properties, acylsulfonamides were incorporated as acid isosteres at the C-2 position. Further optimization of the combination at N-1, C-2, C-5, and C-6 resulted in the identification of compound 56, which had an excellent potency in both NS5B enzyme (IC₅₀ = 0.008 μM) and cell-based replicon (EC₅₀ = 0.02 μM) assays and a good oral PK profile with area-under-the curve (AUC) of 14 and 8 μM?h in rats and dogs, respectively. X-ray structure of inhibitor 56 bound to the enzyme was also reported.

Full text of DOI:10.1021/jm201258k

Article
pubs.acs.org/jmc
Structure−Activity Relationship (SAR) Development and Discovery of
Potent Indole-Based Inhibitors of the Hepatitis C Virus (HCV) NS5B
Polymerase
Kevin X. Chen,*,† Bancha Vibulbhan,† Weiying Yang,† Mousumi Sannigrahi,† Francisco Velazquez,†
Tin-Yau Chan,† Srikanth Venkatraman,† Gopinadhan N. Anilkumar,† Qingbei Zeng,† Frank Bennet,†
Yueheng Jiang,† Charles A. Lesburg,‡ Jose Duca,† Patrick Pinto,† Stephen Gavalas,† Yuhua Huang,†
Wanli Wu,† Oleg Selyutin,† 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, NJ 07033, United States
ABSTRACT: Starting with the indole-based C-3 pyridone lead
HCV polymerase inhibitor 2, extensive SAR studies were
performed at different positions of the indole core. The best
C-5 groups were found to be compact and nonpolar moieties
and that the C-6 attachments were not affecting potency.
Limited N-1 benzyl-type substituent studies indicated that the
best substitutions were fluoro or methyl groups at 2′ or 5′
positions of the benzyl group. To improve pharmacokinetic (PK)
properties, acylsulfonamides were incorporated as acid isosteres
at the C-2 position. Further optimization of the combination at N-1, C-2, C-5, and C-6 resulted in the identification of
compound 56, which had an excellent potency in both NS5B enzyme (IC50 = 0.008 μM) and cell-based replicon (EC50 = 0.02
μM) assays and a good oral PK profile with area-under-the curve (AUC) of 14 and 8 μM·h in rats and dogs, respectively. X-ray
structure of inhibitor 56 bound to the enzyme was also reported.
and two glycoproteins E1 and E2 (NS1). The nonstructural
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 directly targeting these nonstructural proteins,
and exciting progress has been made in these endeavors.5
Clinical trials have demonstrated dramatic improvements in
SVR rate in patients treated with a direct acting agent in
combination with pegylated interferon and ribavirin.6 Recent
approval of the two HCV NS3 protease inhibitors boceprevir7
(Merck & Co.) and telaprevir8 (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
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 Currently, there is no anti-HCV vaccine
available. The new standard of care (SOC) after recent protease
inhibitor approval involves the combination of a protease
inhibitor with pegylated α-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 3-
times a day), relapse (mutations), and discontinuation due to
the presence of side effects with these therapies necessitates the
need for new 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)
Received: September 20, 2011
Published: December 13, 2011
© 2011 American Chemical Society
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genome, the HCV NS5B polymerase is potentially associated
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. Binding to these sites interferes with conformational
changes required during RNA synthesis. Thumb pockets I and
II are two allosteric sites that 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
popular targets for drug development, which makes the NS5B a
particularly druggable target.12,13
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 five 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 been 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.
and the benzene ring at N-1 substituent was replaced with 4-(2-
amino)pyridine, as shown in structure 2 (Figure 1).25 The
Figure 1. HCV NS5B polymerase inhibitors early lead structures.
pyridone carbonyl and N−H formed strong hydrogen bonds
with backbone isoleucine-447 residue, and the amino-pyridine
moiety had a hydrogen bonding interaction with serine-367. As
a result of the additional interactions, there was a near 20-fold
improvement in enzyme potency. More importantly, 2
demonstrated activity in the cellular replicon assay with an
EC50 of 4.8 μM. Starting from this new lead compound 2, our
task was to develop inhibitors with potency and PK properties
desirable in a drug candidate. Herein, we report the SAR
development and the discovery of a potent HCV NS5B
polymerase inhibitor which exhibited a good PK profile in rats.
Synthesis of HCV NS5B Polymerase Inhibitors. The
general synthesis of HCV NS5B polymerase inhibitors is
outlined in Schemes 1 and 2. The ethyl indole-2-carboxylate 3,
a
Scheme 1
The indole-based lead compound, 1, was discovered through
screening effort at Schering-Plough Research Institute. It had
only moderate binding activity (IC50 = 0.9 μM) in an RdRp
enzyme binding assay.23 It was inactive in the cell-based
replicon assay24 (EC50 > 100 μM). 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.25 There was an unusual F···H−N hydrogen
bonding between the C-3 fluorophenyl and tyrosine-448.
Starting from this lead compound, extensive SAR investigations
were conducted to improve potency of the indole-based
inhibitors. A breakthrough was achieved when C-3 2-
fluorophenyl group was replaced with a 3-pyridone moiety,
a
Reagents and conditions: (a) NIS, acetone, quant; (b) PdCl2(dppf)2,
K2CO3, 1,2-dimethoxyethane, 50−85%; (c) ArCH2Br or ArCH2Cl,
Cs2CO3, DMF, 40−90%; (d) 4 M HCl, p-dioxane; (e) LiOH, THF/
H2O 30−60% (2 steps).
with desired substituents at 4,5,6-positions, was either
commercially available or could be prepared via known
procedures. Intermediate 3 was iodinated at C-3 with N-
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a
Scheme 2
Table 1. Modifications at C-4, C-5, and C-6 of Indole Core
a
Reagents and conditions: (a) LiOH, THF/H2O; (b) CDI, then
RSO2NH2, DBU, THF, 50−80% (2 steps); (c) 4 M HCl, p-dioxane,
30−50%.
iodosuccinimide (NIS) to give compound 4. The Suzuki−
Miyaura cross-coupling reaction26 between 4 and 2-methoxy-3-
pyridine boronic acid 5 in the presence of a palladium catalyst
afforded product 6 in 50−85% yields from 3. The indole
nitrogen was then alkylated to provide compound 7 in 40−90%
yields through alkylation with a benzylic halide. The
methoxypyridine in 7 was then converted to pyridone product
8 upon treatment with hydrochloric acid. The ethyl ester was
finally hydrolyzed to carboxylic acid 9 with yields of 30−60% in
two steps, which was tested in RdRp enzyme assay and replicon
cell-based assay.
The indole-2-acylsulfonamide series of polymerase inhibitors
were prepared according to the procedures shown in Scheme 2.
Compound 7 was hydrolyzed to its corresponding carboxylic
acid 10. The acid was activated with carbonyl diimidazole
(CDI) and then reacted with a sulfonamide in the presence of
diazabicyclicunderdecane (DBU)27 to give desired acylsulfona-
mide 11 in 50−80% yields from 7. Product 11 was then treated
with hydrochloric acid to provide pyridone final product 12 in
30−50% purified yields. The inhibitors were evaluated in
enzyme and replicon assays to determine their biological
activities.
RESULTS AND DISCUSSIONS
■
Preliminary SAR at Indole C-4, C-5, and C-6 Positions.
With the lead compound 2 exhibiting interesting biological
activity, an effort was taken to improve the potency via
optimization of various substituents at different positions of the
indole core. First, the effect of additional substitution at C-4
and C-6 positions was examined and the SAR is summarized in
Table 1 (compounds 13−15). Starting from compound 2, a
second chloro substituent was introduced at C-4. Unfortu-
nately, the resulting inhibitor 13 had an IC50 of 0.75 μM in an
RdRp enzyme binding assay,23 a 14-fold decrease in activity.
The removal of the C-5 chloro group and introduction of a
larger bromo atom at C-4 gave 14 (IC50 = 4.0 μM) with a 75-
fold loss in activity compared to 2. The detrimental effect of the
C-4 substituents was probably caused by its intervention of
proper orientation of the C-3 pyridone ring for optimal
hydrogen bonding. The investigation was then focused on C-6
modifications. An additional chloro group was introduced to C-
6 of compound 2 to give inhibitor 15. The potency of this
compound remained essentially unchanged at approximately
0.05 μM (IC50). It appeared that the C-6 substituent did not
adversely affect the potency.
The effort was next focused on C-5 optimization (Table 1).
Groups were introduced at this position to evaluate the SAR
effect of the size, polarity, and H-bonding ability of the
substituents. Replacing the chloro atom in 2 with a larger
bromo atom gave rise to compound 16, which had similar
activity (IC50 = 0.047 μM). A large six-membered pyridone
analogue (17) was clearly not tolerated with significantly
decreased activity (IC50 of 7.0 μM). Similar effects were
observed for inhibitors (19, 20, and 22) with other polar
substituents, from the larger methyl sulfone in 19, to medium
sized acetyl group in 20, and to small hydroxyl in 22. The
complete lack of activity of 22 was quite striking. These results
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point to the conclusion that the enzyme surface near the C-5
substituent was highly hydrophobic and that polar function-
alities were not tolerated. Further evidence of a hydrophobic
pocket was provided by the good activity observed with the
cyclopropyl analogue 18 and acetylene analogue 21. Both 18
and 21 had similar potency to that of compound 2. The
breakthrough came from trifluoromethyl and trifluoromethoxy
substituted compounds 23 and 24. These two inhibitors were
three times more potent than the lead compound 2 and served
as the basis for further optimization at other positions of the
indole ring.
SAR Studies of N-1 Aromatic Substituents. Although
compound 24 had improved binding (IC50), it did not possess
a desirable PK profile in rats as exemplified by its negligible oral
AUC value (<0.1 μM·h). Most other compounds with the 2-
aminopyridinemethyl substituent at indole nitrogen (N-1) also
displayed poor PK. This structure element was presumably
responsible for the lack of exposure of these compounds in
vivo. A metabolically more stable moiety was clearly desirable at
the N-1 position. Thus, a number of N-1 benzyl-based
substituents were investigated. Because anilines are known to
have potential toxicity concerns, we decided to screen benzyl
groups with substituents other than a primary amino group.
The results of this SAR study are shown in Table 2 with
representative compounds.
However, simple ortho-methyl or fluoro substituted benzyl
groups provided more potent compounds 27 and 28, both with
an IC50 of 0.009 μM. Additional methyl or fluoro groups at the
para-position in 29 or 30 caused a small drop in activity (IC50 =
0.016 and 0.025 μM, respectively). In contrast, the additional
substitution at either meta-position enhanced the potency as
evidenced in compounds 31 and 32. Both 2′-fluoro-3′-methyl
substituted analogue 31 and the 2′,5′-difluoro structure 32 had
excellent activities (IC50 = 0.005 and 0.004 μM, respectively).
Some polar functionalities such as a primary amide at the 5′-
position (33) were not only tolerated but improved the
potency (IC50 = 0.007 μM) compared to 24. Overall, the N-1
substituent SAR exercise improved the potency by 3−4-fold,
and more importantly, several aromatic moieties that were
more likely to demonstrate good PK properties were
discovered. The 2′,5′-difluorobenzyl group in compound 32
was selected for further SAR investigations.
Further Optimization of C-5 Substitution. After the
discovery of an N-1 benzyl group suitable for continued SAR
development, we believed that it was necessary to re-examine
C-5 substituents. We learned from earlier C-5 SAR studies
(Table 1) that very polar substituents were not suitable for that
position. Thus, a select group of nonpolar aliphatic structures
were evaluated (Table 3). The compounds bearing trifluor-
omethoxy (23) or trifluoromethyl (24) groups were found to
be equally potent in Table 1. In contrast, the compound with a
C-5 trifluoromethoxy group in the 2′,5′-difluorobenzyl series
(34, IC50 = 0.024 μM) was less active than the corresponding
trifluoromethyl analogue (32, IC50 = 0.004 μM). The methoxy
substituted compound 35 fared slightly better (IC50 = 0.015
μM), albeit slightly less active. Replacement with the larger
ethoxy moiety caused a significant drop in activity in analogue
36 (IC50 = 0.079 μM). The four-carbon chain 1-butenyl group,
not surprisingly, gave rise to an even greater loss of potency in
inhibitor 37 (IC50 = 0.15 μM). A few smaller alkyl substituents,
however, afforded interesting compounds with excellent IC50
values. The 5-methyl analogue 38 had very good activity (IC50
= 0.008 μM). The slightly larger ethyl and bromo substituents
provided two inhibitors (39, 40) with IC50s of 0.004 and 0.003
μM, respectively. Similar sized trifluoroethyl analogue 41 was
also quite active, although with slight loss of potency compared
to 32. The bulkier tert-butyl and 1-methylcyclopropyl
substitutions were tolerated (42 and 43) with IC50s of 0.008
and 0.009 μM, respectively, about 2-fold higher than that of 32.
On the basis of the overall profile of the compounds, the
potential liability of the substituents and ease of synthesis, we
decided to focus our future SAR studies on three small alkyl C-
5 substituents that showed excellent potency: methyl, ethyl, and
trifluoromethyl groups.
Table 2. SAR at Indole Core N-1 Aromatic Substituent
C-2-Acylsulfonamide SAR Investigation. Although a
number of C-2 carboxylic acid inhibitors examined in Tables 1,
2, and 3 demonstrated excellent potency in the enzyme assay,
their activities in cellular replicon assay were much less
impressive, with an EC50 typically in the range of 0.1 μM to
greater than 1.0 μM. For example, compound 38 had an EC50
of 1.1 μM. The fact that these carboxylic acids were poorly
soluble might be a factor in hindering them from cellular uptake
because they existed as carboxylate salts under physiological
pH. To improve replicon assay potency and solubility of these
indole derivatives, the carboxylic acid moiety was replaced by
an acylsulfonamide isostere. Thus, a set of simple alkyl
acylsulfonamides were prepared (Table 4) and their potency
in enzyme and cellular replicon assays was determined.
Substitution at different positions of the benzene ring was
explored. The para-methoxy substituted benzyl analogue 25
was almost equally potent (IC50 = 0.025 μM) to compound 24,
while difluoromethoxy analogue 26 was about 3-fold less active.
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Table 3. Optimization of C-5 Substituent
Table 4. C-2-Acylsulfonamide Optimization
inhibitors in Table 4. All four C-5 methyl acylsulfonamides
(44−47) had good to excellent AUCs ranging from 6.8 to 22
μM·h. In the C-5-ethyl series (48−51), however, AUCs varied
more significantly from extremely high for 48 (72 μM·h) to
poor for 50 (0.8 μM·h). Among four more potent compounds
with EC50 < 0.1 μM in the table, a pair of cyclopropyl
acylsulfonamides (47 and 51) demonstrated very good PK with
AUCs of 9.1 and 12 μM·h, respectively. When both potency
and PK factors were considered, the cyclopropyl acylsulfona-
mide was a better C-2 moiety in a given series with the same C-
5 substituent and was used for further SAR studies.
Reoptimization of C-5 and C-6 Substituents. With the
N-1, C-2, and C-3 moieties optimized, the effects of C-5 and C-
6 substituents were then revisited to determine the best
combination for compounds with a C-2 acylsulfonamide.
Preliminary SAR studies in Table 1 indicated that only compact
and nonpolar alkyl groups were tolerated at C-5 position, and
C-6 substitution had little impact on potency. Several compact
and nonpolar substituents were tested at the C-5 and C-6 (51−
60, Table 5). They all had excellent activity against RdRp
enzyme with an IC50 between 0.003 and 0.008 μM. The
difference in replicon assay potency, however, was significant,
with EC50 ranging from 0.02 μM (56) to 0.6 μM (54). Among
the six compounds without C-6 substituents (R6 = H), those
with 1-methylcyclopropyl and trifluoroethyl at C-5 were less
potent than others in the replicon assay (EC50 = 0.6 μM for 54,
and EC50 = 0.23 μM for 55). Both inhibitors with
trifluoromethyl and tert-butyl C-5 groups were slightly more
active in cellular assay, with EC50 values around 0.05 μM. Most
compounds in this group demonstrated good PK in rats with
Compounds 44−47 had a C-5 methyl substituent, while
compounds 48−51 had a C-5 ethyl group. From the C-2
methyl acylsulfonamide (44) to ethyl, isopropyl, and cyclo-
propyl acylsulfonamides (45, 46, and 47, respectively), the
potency against the enzyme remained approximately the same
in a narrow range of 0.006−0.010 μM. The potency in cell-
based replicon assay, however, varied significantly, but they
were all better than the EC50 of compound 38 (1.1 μM). The
methyl sulfonamide analogue (44) was the least potent (EC50 =
0.49 μM), but ethyl, isopropyl, and cyclopropyl sulfonamides
became progressively more potent (EC50 = 0.20, 0.09, 0.09 μM,
for compounds 45, 46, and 47, respectively). Similar trends
were observed in the C-5-ethyl series. From compounds 48 to
51, the enzyme assay activity IC50 improved 2-fold from 0.006
(for 48) to 0.003 μM (for 51). More improvement was
observed in replicon activity in this series from methyl
acylsulfonamide 48 (EC50 = 0.15 μM) to cyclopropyl
acylsulfonamide 51 (EC50 = 0.06 μM).
Along with the efforts to improve the enzyme and cellular
assay potency, the PK profile of the compounds was evaluated
and used as an important criterion to select the best compound
for further investigation. The AUCs in rats after oral dosing (10
mg/kg) was measured to determine the exposure of the
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clearance of the compound in human hepatocytes was low at
<1.0 μL/min/Mcells. The IC50s of compound 56 against the
cytochrome P450 (CYP) 2D6, 3A4, 2C9, and 2C19 enzymes
(concurrent/preincubation) were all greater than 20 μM. The
compound had no P450 3A4 enzyme induction signal at 10 μM
in a human PXR assay. There was also no signal at 10 μM in an
hERG efflux assay. This compound was selected for more
comprehensive studies because of its overall superior profile.
X-ray Structure of Compound 56..28 The X-ray crystal
structure of inhibitor 56 bound to the HCV NS5B polymerase
was solved (Figure 2, Protein Data Bank access code 3TYV).29
Table 5. Optimization of C-5 and C-6 Substituents
Figure 2. X-ray crystal structure of compound 56 bound to HCV
NS5B polymerase.
Similar to the X-ray structure of compound 1 bound to the
enzyme, 56 also binds to the “palm” site of NS5B apoprotein
within the active site cavity. The indole core makes π-stacking
interactions with the benzene ring of protein backbone Tyr-
448. The N-1 benzyl moiety of the inhibitor stacks atop the
thiol side chain of Cys-366. The C-2 acylsulfonamide motif
does not have direct binding with the protein, but it interacts
indirectly with protein residues Ser-556, Gly-449, and Ser-288
through the formation of a hydrogen bonding network with
adjacent water molecules. The indole core and the C-3
pyridone ring have contacts with the side chain of Met-414.
There is a pair of hydrogen bonding interaction between the
pyridone and the protein backbone, one between the pyridone
oxygen and the backbone N−H of Tyr-448, the other between
the pyridone N−H and the backbone carbonyl of Ile-447.
Because the dihedral angle between the pyridone ring and the
indole was not big, any sizable substituent at C-4 position will
change the orientation of the pyridone ring and thus disrupt the
two hydrogen bonds. The indole C-5 methyl and C-6 fluoro
groups reach into a constricted tunnel which extends to protein
surface on the other side of the “thumb” subdomain.
AUCs at or above 5 μM·h, except 55, which had a poor AUC of
0.67 μM·h.
Five analogues with a C-6 substituent (56−60) were
included in Table 5. Among three compounds with a C-5-
methyl group, 6-fluoro analogue 56 was much more potent
(EC50 = 0.02 μM) than 6-chloro and 6-trifluoromethyl
analogues (EC50 of 0.15 and 0.28 μM, respectively). If 6-F
substitution was combined with 5-CF3 or 5-ethyl groups (59
and 60), the results (EC50 = 0.28 and 0.06 μM, respectively)
were not as impressive as that of 56. The rat oral AUC was
poor for compound 58 (0.69 μM·h), good for compound 60
(6.4 μM·h), and excellent for compound 56 (14 μM·h). When
the overall profiles of these compounds were evaluated,
compound 56 had the best combination of potency and PK.
It was selected as a potential candidate for further evaluation.
Profile of Compound 56. With a calculated cLogP of 3.8
and polar-surface-area (PSA) of 100, it is not surprising to
observe that compound 56 has only moderate permeability of
35 nm/s (apical to basolateral) in a Caco-2 cell permeability
assay. When dosed orally in rats at 10 mg/kg, compound 56
had an excellent AUC0−24h of 14 μM·h. It also had a very high
concentration of 5850 ng/g in rat livers 6 h after oral dosing.
The PK of compound 56 in dogs was also robust, with a good
AUC0−24h of 8.0 μM·h in 24 h after 2 mg/kg oral dosing. The
CONCLUSION
■
Starting from compound 2, an indole-based early lead inhibitor
of HCV NS5B polymerase, extensive SAR development on
multiple positions of the indole core was performed. While the
unique and optimized 3-pyridone moiety at C-3 position was
kept constant, substituents at N-1, C-2, C-4, C-5, and C-6
positions were varied systemically to optimize potency in both
enzyme assays and cell-based replicon assays and to improve
PK properties. The C-4 SAR investigation demonstrated that
no substitution was tolerated. The best C-5 groups were found
to be compact and nonpolar moieties. The C-6 substituents, in
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cesium carbonate (7.5 mmol) in anhydrous dimethylformamide
(DMF) (80 mL) was vigorously stirred at rt for 16 h. The solution
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), brine, dried over magnesium sulfate, and
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 (7) in 40−90% yield.
General procedures for the preparation of 8: Compound 7 (1.0
mmol) was dissolved in a solution of 4 M hydrogen chloride in p-
dioxane (50 mL) in a sealed tube. The solution was then 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 usually pure enough to be used in the next reaction.
General procedures for the preparation of 9: Compound 8 (1.0
mmol) and lithium hydroxide (4.0 mmol) was 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 layers were separated. The aqueous
solution was extracted with ethyl acetate (2 × 20 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 reverse phase HPLC to give the desired product (9) in
30−60% yield (2 steps).
general, did not provide potency enhancement except in the C-
5 methyl and C-6 fluoro combination series. Limited benzyl-
type N-1 substituent studies indicated that the best
substitutions were fluoro or methyl groups at 2′ or 5′ positions.
The incorporation of acylsulfonamides as carboxylic acid
isosteres at C-2 position improved PK properties. Further
optimization of the combination at N-1, C-2, C-5, and C-6
positions resulted in the identification of compound 56, which
had excellent enzymatic and cellular assay potencies (IC50
=
0.008 μM, EC90 = 0.02 μM, respectively) and an excellent PK
profile in rats and dogs (AUC = 14 and 8 μM·h, respectively).
An X-ray structure of inhibitor 56 bound to the enzyme was
also reported. The indole core, C-3 pyridone, C-2 acylsulfo-
namide, C-5 methyl, and 6-F all contributed to the high
potency of compound 56. This work demonstrated the
importance of reoptimization after significant changes were
made to other positions of the molecule.
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 mm × 250 mm, 10 μm) running at 30 mL/min 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 1 H 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,
General procedures for the preparation of 10: Compound 10 was
prepared from compound 7 according to the same procedures
described above for the preparation of 9. The crude product was
usually pure enough to be used in the next reaction.
General procedures for the preparation of 11: The mixture of 10
(0.50 mmol) and carbonyl diimidazole (CDI) (0.60 mmol) in
anhydrous THF (5 mL) was heated to 75 °C in an oil bath and stirred
1.5 h. The solution was then cooled to rt, alkyl sulfonamide and DBU
(1.5 mmol each) were added, and the resulting mixture was stirred at
rt for 18 h. It 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
solutions were 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 dichloro-
methane as eluent to give the desired product (11) in 50−80% yield.
General procedures for the preparation of 12: Compound 12 was
prepared from compound 11 according to the same procedures
described above for the preparation of 8. The crude product was
purified by reverse phase HPLC to give the desired product (12) in
30−50% yield.
1-[(2-Amino-4-pyridinyl)methyl]-4,5-dichloro-3-(1,2-dihy-
dro-2-oxo-3-pyridinyl)-1H-indole-2-carboxylic Acid (13). Com-
pound 13 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),
11.36 (s, 2 H), 7.76 (d, J = 5.2 Hz, 1 H), 7.36 (q, J = 1.9 Hz, 1 H), 7.27
(s, 2 H), 7.18 (q, 8.9 Hz, 2 H), 6.28 (d, J = 5.4 Hz, 1 H), 6.14 (s, 2 H),
5.79 (s, 2 H), 5.60 (d, J = 16.8 Hz, 1 H). 13C NMR (125 MHz,
DMSO-d6), 168.9, 163.8, 160.7, 148.5, 147.9, 145.0, 143.3, 135.6,
134.3, 127.5, 111.4, 111.1, 110.9, 110.0, 106.2, 98.7, 67.9, 47.4, 27.8,
19.2. HRMS calcd for C20H15Cl2N4O3 (M + H)+, 429.0521; found,
429.0517.
1
LC-MS, and/or H NMR methods were employed to determine the
purity of the 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 stirring for 5 min
(min), ethyl acetate (30 mL) and water (20 mL) were added. The
layers were then separated. The aqueous solution was extracted with
ethyl acetate (2 × 25 mL). The combined organic layer was washed
with brine, dried over magnesium sulfate, and concentrated in vacuo to
give the crude product, which was usually pure enough to be used in
the next reaction.
General procedures for Suzuki coupling between 4 and 2-methoxy-
3-pyridine boronic acid (5): To the suspension of 3-iodoindole (4,
10.0 mmol) and [1,1′-bis(diphenylphosphino)ferrocene] dichloropal-
ladium (PdCl2(dppf)2) (1.0 mmol) in dimethoxyethane (DME) (120
mL) at rt was added a solution of boronic acid (5) (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 cooling 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 (6) in 50−85% yield.
1-[(2-Amino-4-pyridinyl)methyl]-4-bromo-3-(1,2-dihydro-2-
oxo-3-pyridinyl)-1H-indole-2-carboxylic Acid (14). Compound
14 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),
13.44 (s, 1 H), 11.58 (s, 1 H), 7.90 (d, J = 6.0 Hz, 1 H), 7.76 (s, 2 H),
7.59 (d, J = 8.1 Hz, 1 H), 7.40−7.38 (m, 3 H), 7.24 (t, J = 7.9 Hz, 1
H), 6.58 (d, J = 5.8 Hz, 1 H), 6.30 (s, 1 H), 6.24 (t, J = 6.4 Hz, 1 H),
4.38 (q, J = 18.0 Hz, 2 H). 13C NMR (125 MHz, DMSO-d6), 163.3,
163.0, 156.1, 155.7, 142.1, 139.7, 138.2, 135.3, 128.5, 127.1, 126.9,
General procedures for the preparation of 7: The mixture of 6 (5.0
mmol), arylmethyl bromide (or arylmethyl chloride) (7.5 mmol), and
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Journal of Medicinal Chemistry
Article
126.3, 125.0, 120.8, 116.1, 111.5, 111.2, 109.0, 105.4, 48.2. HRMS
calcd for C20H16N4O3Br (M + H)+, 439.0406; found LRMS, 439.0.
1-[(2-Amino-4-pyridinyl)methyl]-5-bromo-3-(1,2-dihydro-2-
oxo-3-pyridinyl)-1H-indole-2-carboxylic Acid (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),
11.97 (s, 1 H), 8.44 (s, 1 H), 7.74 (s, 1 H), 7.58 (s, 1 H), 7.42 (s, 2 H),
7.18 (s, 2 H), 6.27 (s, 2 H), 6.20 (s, 1 H) 5.80 (s, 2 H), 5.59 (s, 2 H).
13C NMR (125 MHz, DMSO-d6), 165.8, 162.9, 160.7, 149.3, 148.5,
141.6, 135.0, 129.7, 124.4, 123.8, 113.0, 112.4, 111.4, 109.7, 109.5,
106.4, 100.4, 97.0, 96.4, 93.5, 81.5, 74.3, 62.6, 52.1, 48.3, 47.4, 44.1.
HRMS calcd for C20H16N4O3Br (M + H)+, 439.0406; found,
439.0398.
5-Acetyl-1-[(2-amino-4-pyridinyl)methyl]-3-(1,2-dihydro-2-
oxo-3-pyridinyl)-1H-indole-2-carboxylic Acid (20). Compound
20 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),
11.96 (s, 2 H), 8.12 (s, 1 H), 8.00 (s, 2 H), 7.95−7.91 (m, 3 H), 7.66
(d, J = 8.9 Hz, 1 H), 7.57 (d, J = 7.8 Hz, 1 H), 6.56 (d, J = 6.0 Hz, 1
H), 6.40 (d, J = 6.6 Hz, 1 H), 6.36 (s, 1 H), 5.87 (s, 2 H), 2.59 (s, 3
H). 13C NMR (125 MHz, DMSO-d6), 198.1, 163.5, 162.1, 156.4,
155.4, 141.5, 140.6, 137.4, 135.6, 131.5, 129.4, 126.5, 125.7, 125.5,
123.5, 120.3, 111.8, 111.2, 109.4, 106.1, 48.2, 27.6. LRMS calcd for
C22H19N4O4 (M + H)+, 403.1; found: 403.2.
1-[(2-Amino-4-pyridinyl)methyl]-3-(1,2-dihydro-2-oxo-3-
pyridinyl)-5-(trifluoromethyl)-1H-indole-2-carboxylic Acid
(24). Compound 24 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), 13.78 (s, 1 H), 11.90 (s, 1 H), 8.03 (s, 2 H), 7.92 (d, J = 6.7 Hz, 1
H), 7.82 (s, 1 H), 7.80 (s, 1 H), 7.66−7.61 (m, 2 H), 7.47 (q, J = 1.9
Hz,1 H), 6.63 (q, J = 1.3 Hz, 1 H), 6.38−6.35 (m, 2 H), 5.90 (s, 2 H).
13C NMR (125 MHz, DMSO-d6), 163.4, 162.0, 156.8, 155.0, 141.7,
Hz, 1 H), 7.43 (dd, J = 1.90, 1.54 Hz, 1 H), 7.36 (s, 1 H), 7.32−7.28
(m, 1 H), 7.18−7.14 (m, 1 H), 6.54−6.50 (m, 1 H), 6.34 (t, J = 6.6
Hz, 1 H), 5.88 (s, 2 H). 13C NMR (125 MHz, DMSO-d6), 163.7,
162.1, 161.1, 159.1, 158.0, 150.7, 143.8, 141.2, 138.2, 137.2, 128.1,
127.0, 118.1, 118.0, 117.9, 117.8, 116.4, 115.3, 113.6, 113.3, 103.0,
94.5, 67.2, 42.7, 30.0, 24.2. HRMS calcd for C22H14F5N2O4 (M + H)+,
465.0874; found, 465.0868.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-(methoxy)-1H-indole-2-carboxylic Acid (35). Com-
pound 35 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),
12.98 (s, 1 H), 11.79 (s, 1 H), 7.59 (q, J = 2.4 Hz, 1 H), 7.50 (d, J =
9.2 Hz, 1 H), 7.41 (q, J = 3.6 Hz, 1 H), 7.31−7.27 (m, 1 H), 7.16−
7.11 (m, 1 H), 6.98 (q, J = 2.6 Hz, 1 H), 6.86 (d, J = 2.5 Hz, 1 H),
6.37−6.33 (m, 2 H), 5.83 (s, 2 H), 3.73 (s, 3 H). 13C NMR (125
MHz, DMSO-d6), 174.4, 172.8, 166.0, 151.3, 145.4, 144.1, 138.8,
137.6, 137.0, 128.4, 128.2, 127.3, 126.7, 125.6, 125.4, 123.2, 120.8,
116.6, 112.4, 66.7, 52.9. LRMS calcd for C22H17F2N2O4 (M + H)+,
411.1; found, 411.2.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-ethoxy)-1H-indole-2-carboxylic Acid (36). Compound
36 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),
11.76 (s, 1 H), 7.58 (q, J = 2.1 Hz, 1 H), 7.48 (d, J = 9.1 Hz, 1 H), 7.40
(q, J = 2.0 Hz, 1 H), 7.30−7.27 (m, 1 H), 7.15−7.11 (m, 1 H), 6.97
(q, J = 2.6 Hz, 1 H), 6.84 (d, J = 2.1 Hz, 1 H), 6.38−6.33 (m, 2 H),
5.82 (s, 2 H), 3.99−3.95 (m, 2 H), 1.30 (t, J = 6.9 Hz, 3 H). 13C NMR
(125 MHz, DMSO-d6), 174.4, 172.8, 170.4, 168.5, 167.9, 166.0, 165.2,
151.4, 145.4, 144.0, 139.2, 138.8, 137.7, 136.9, 128.4, 128.2, 127.6,
126.8, 126.7, 125.6, 125.4, 123.3, 123.1, 121.0, 120.8, 116.7, 113.4,
74.8, 52.9, 26.1. LRMS calcd for C23H19F2N2O4 (M + H)+, 425.1;
found, 425.1.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-methyl-1H-indole-2-carboxylic Acid (38). Compound
38 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),
13.0 (s, 1 H), 11.8 (s, 1 H), 7.56 (dd, J = 2.2, 1.9 Hz, 1 H), 7.5 (d, J =
8.6 Hz, 1 H), 7.41 (d, J = 6.3 Hz, 1 H), 7.32−7.27 (m, 1 H), 7.24 (s, 1
H), 7.17−7.08 (m, 2 H), 6.37−6.33 (m, 2 H), 5.83 (s, 2 H), 2.36 (s, 3
H). 13C NMR (125 MHz, DMSO-d6), 181.8, 181.4, 177.0, 174.5,
172.8, 152.8, 151.5, 147.3, 145.6, 141.2, 138.2, 137.5, 136.3, 131.2,
128.4, 121.9, 113.7, 113.1, 112.4, 78.4, 66.3, 59.9, 41.8, 36.6, 36.5, 32.4.
LRMS calcd for C22H17F2N2O3 (M + H)+, 395.1; found, 395.0.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-ethyl-1H-indole-2-carboxylic Acid (39). Compound 39
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), 12.97 (s, 1 H),
11.78 (s, 1 H), 7.58 (dd, J = 1.8, 2.2 Hz, 1 H), 7.49 (d, J = 8.5 Hz, 1
H), 7.42 (d, J = 4.8 Hz, 1 H), 7.32−7.27 (m, 1 H), 7.24 (s, 1 H), 7.20
(d, J =8.4 Hz, 1 H), 7.17−7.11 (m, 1 H), 1.18 (t, J =7.6 Hz, 3 H). 13C
NMR (125 MHz, DMSO-d6), 164.0, 162.3, 159.4, 158.0, 157.5, 155.6,
141.0, 137.4, 137.0, 135.0, 128.0, 127.0, 126.6, 119.5, 118.1, 118.0,
117.9, 117.8, 117.7, 116.4, 116.3, 116.2, 116.1, 115.2, 115.0, 106.1,
29.1, 17.1. HRMS calcd for C23H19F2N2O4 (M + H)+, 409.1364;
found, 409.1358.
139.7, 136.9, 135.8, 129.7, 125.1, 124.8, 122.9, 122.6, 122.1, 119.7,
119.5, 119.4, 113.0, 111.2, 109.5, 106.0, 48.2. LRMS calcd for
C21H16F3N4O3 (M + H)+, 429.1; found, 429.0.
1-[(2,4-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-(trifluoromethyl)-1H-indole-2-carboxylic Acid (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), 13.25 (s, 1 H), 11.83 (s, 1 H), 7.84 (d, J = 8.8 Hz, 1 H), 7.75 (s, 1
H), 7.62−7.60 (m, 2 H), 7.44 (dd, J = 1.9, 1.5 Hz,1 H), 7.30−7.25 (m,
1 H), 7.00−6.96 (m, 1 H), 6.81−6.77 (m, 1 H), 6.34 (t, J = 6.6 Hz, 1
H), 5.91 (s, 2 H). 13C NMR (125 MHz, DMSO-d6), 163.6, 162.1,
161.5, 161.4, 159.6, 159.5, 141.4, 139.6, 135.6, 130.3, 130.2, 130.1,
127.0, 126.1, 125.2, 124.8, 122.6, 122.4, 122.3, 122.2, 122.1, 121.7,
119.2, 119.1, 119.0, 113.0, 112.6, 112.5, 112.4, 106.0, 105.1, 104.9,
104.7, 42.4. LRMS calcd for C22H14F5N2O3 (M + H)+, 449.1; found,
449.0.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-(trifluoromethyl)-1H-indole-2-carboxylic Acid (32).
Compound 32 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), 13.28 (s, 1 H), 11.83 (s, 1 H), 7.85 (d, J = 7.5 Hz, 1 H), 7.76 (s, 1
H), 7.64 (s, 2 H), 7.45 (s, 1 H), 7.32−7.38 (m, 1 H), 7.16 (d, J = 2.7
Hz, 1 H), 6.50 (s, 1 H), 6.35 (s, 1 H), 5.93 (s, 2 H). 13C NMR (125
MHz, DMSO-d6), 163.6, 162.1, 159.9, 157.6, 141.4, 139.5, 135.6,
129.8, 128.0, 127.0, 126.1, 121.8, 119.3, 119.2, 118.0, 117.9, 116.6,
116.5, 115.4, 115.3, 115.2, 113.0, 106.0, 42.9. LRMS calcd for
C22H14F5N2O3 (M + H)+, 449.1; found, 449.1.
5-Bromo-1-[(2,5-difluorophenyl)methyl]-3-(1,2-dihydro-2-
oxo-3-pyridinyl)-1H-indole-2-carboxylic Acid (40). Compound
40 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),
13.14 (s, 1 H), 11.80 (s, 1 H), 7.62−7.58 (m, 3 H), 7.45 (dd, J = 1.9,
1.9 Hz, 1 H), 7.42 (dd, J = 1.7, 1.9 Hz, 1 H), 7.32−7.27 (m, 1 H),
7.18−7.14 (m, 1 H), 6.45−6.41 (m, 1 H), 6.33 (t, J = 6.6 Hz, 1 H),
5.86 (s, 2 H). 13C NMR (125 MHz, DMSO-d6), 163.6, 162.1, 160.5,
147.2, 141.4, 136.9, 129.2, 128.5, 128.3, 123.6, 119.3, 119.1, 117.9,
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-(trifluoromethoxy)-1H-indole-2-carboxylic Acid (34).
Compound 34 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), 11.82 (s, 1 H), 7.72 (d, J = 9.2 Hz, 1 H), 7.59 (dd, J = 1.94, 1.77
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117.7, 115.3, 114.3, 114.0, 109.1, 106.2, 55.9, 48.8, 42.6. LRMS calcd
for C21H14F2N2O3Br (M + H)+, 459.0, 461.0; found, 461.3.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-ethyl-N-(methylsulfonyl)-1H-indole-2-carboxamide
(48). Compound 48 was prepared according to the reaction sequence
outlined in Scheme 2 and the general procedures described above for
the preparation of generic compound 12. 1H NMR (500 MHz,
DMSO-d6), 12.77 (s, 1 H), 12.64 (s, 1 H), 7.82 (d, J = 6.4 Hz, 1 H),
7.66 (s, 1 H), 7.53 (d, 8.9 Hz, 1 H), 7.32−7.27 (m, 1 H), 7.25 (s, 1 H),
7.24 (s, 1 H), 7.18−7.13 (m, 1 H), 6.63−6.6 (m, 1 H), 5.7 (s, 2 H),
3.25 (s, 3 H), 2.66 (q, J = 7.5 Hz, 2 H), 1.18 (t, J = 7.4 Hz, 3 H). 13C
NMR (125 MHz, DMSO-d6), 163.4, 159.9, 158.0, 157.6, 155.7, 155.6,
144.7, 137.9, 137.0, 136.4, 127.1, 119.5, 118.0, 117.8, 117.7, 116.7,
116.6, 116.0, 116.4, 115.9, 115.8, 115.7, 111.7, 108.2, 42.5, 42.0, 29.1,
17.0. LRMS calcd for C24H22F2N3O4S (M + H)+, 486.1; found, 486.0.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-ethyl-N-(ethylsulfonyl)-1H-indole-2-carboxamide (49).
Compound 49 was prepared according to the reaction sequence
outlined in Scheme 2 and the general procedures described above for
the preparation of generic compound 12. 1H NMR (500 MHz,
DMSO-d6), 12.72 (s, 2 H), 7.83 (dd, J = 1.7, 1.8 Hz, 1 H), 7.69 (s, 1
H), 7.56 (d, J = 8.5 Hz, 1 H), 7.32−7.27 (m, 1 H), 7.25 (d, J = 8.9 Hz,
1 H), 7.21 (s, 1 H), 7.19−7.14 (m, 1 H), 6.65−6.57 (m, 2 H), 5.72 (s,
1 H), 3.35 (q, J = 7.3 Hz, 2 H), 2.66 (q, J = 7.5 Hz, 2 H), 1.18 (t, J =
7.6 Hz, 3 H), 1.05 (t, J = 7.4 Hz, 3 H). 13C NMR (125 MHz, DMSO-
d6), 163.5, 162.1, 159.9, 158.0, 157.6, 155.7, 145.1, 140.0, 137.9, 137.1,
136.6, 127.2, 119.5, 118.0, 117.9, 117.8, 117.7, 116.7, 116.6, 116.5,
115.8, 115.6, 111.7, 108.4, 47.7, 42.5, 31.3, 29.1, 17.0, 8.3. LRMS calcd
for C25H24F2N3O4S (M + H)+, 500.1; found, 500.0.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-(1,1-dimethylethyl)-1H-indole-2-carboxylic Acid (42).
Compound 42 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), 12.99 (s, 1 H), 11.83 (s, 1 H), 7.58 (q, J = 1.9 Hz, 1 H), 7.50 (d, J
= 8.8 Hz, 1 H), 7.45−7.41 (m, 2 H), 7.36 (s, 1 H), 7.32−7.29 (m, 1
H), 7.17−7.12 (m, 1 H), 6.44−6.40 (m, 1 H), 6.36 (t, J = 6.7 Hz, 1
H), 5.83 (s, 2 H), 1.29 (s, 9 H). 13C NMR (125 MHz, DMSO-d6),
174.5, 172.8, 170.4, 168.5, 168.0. 166.1, 154.7, 151.5, 147.1, 145.5,
139.1, 138.4, 136.9, 135.0, 129.0, 128.5, 128.4, 128.3, 128.2, 126.9,
126.8, 126.7, 125.8, 125.7, 125.6, 125.5, 121.8, 116.6, 52.8, 45.7, 42.8.
LRMS calcd for C25H23F2N2O3 (M + H)+, 437.2; found, 437.3.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-methyl-N-methylsulfonyl)-1H-indole-2-carboxamide
(44). Compound 44 was prepared according to the reaction sequence
outlined in Scheme 2 and the general procedures described above for
the preparation of generic compound 12. 1H NMR (500 MHz,
DMSO-d6), 12.77 (s, 1 H), 12.64 (s, 1 H), 7.82 (d, J = 9.7 Hz, 1 H),
7.66 (s, 1 H), 7.50 (d, J = 8.5 Hz, 1 H), 7.32−7.27 (m, 1 H), 7.23 (s, 1
H), 7.19 (d, J = 8.6 Hz, 1 H), 7.18−7.14 (m, 1 H), 6.60 (s, 2 H), 5.71
(s, 2 H), 3.25 (s, 3 H), 2.36 (s, 3 H). 13C NMR (125 MHz, DMSO-
d6), 159.9, 158.0, 157.6, 155.7, 136.9, 136.4, 131.2, 128.1, 127.2, 125.8,
120.7, 118.0, 117.9, 117.8, 117.7, 116.7, 116.6, 116.5, 116.4, 115.9,
115.7, 111.6, 108.2, 44.0, 42.5, 42.1, 41.0, 31.3, 21.9. HRMS calcd for
C23H20F2N3O4S (M + H)+, 472.1143; found, 472.1137.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-N-(ethylsulfonyl)-5-methyl-1H-indole-2-carboxamide
(45). Compound 45 was prepared according to the reaction sequence
outlined in Scheme 2 and the general procedures described above for
the preparation of generic compound 12. 1H NMR (500 MHz,
DMSO-d6), 12.73 (s, 1 H), 7.82 (d, J = 6.0 Hz, 1 H), 7.68 (s, 1 H),
7.54 (d, J = 9.5 Hz, 1 H), 7.32−7.27 (m, 1 H), 7.21 (s, 2 H), 7.18−
7.13 (m, 1 H), 6.63 (t, J = 6.3 Hz, 1 H), 6.56- 6.52 (m, 1 H), 5.72 (s, 2
H), 3.35 (q, J = 7.3 Hz, 2 H), 2.36 (s, 3 H), 1.05 (t, J = 7.3 Hz, 3 H).
13C NMR (125 MHz, DMSO-d6), 163.5, 162.0, 159.0, 158.0, 157.6,
155.7, 145.1, 140.1, 136.6, 131.3, 128.2, 127.9, 127.2, 120.7, 118.0,
117.9, 117.8, 117.7, 115.8, 115.5, 111.6, 108.3, 52.4, 47.7, 42.4, 31.3,
30.1, 21.9, 8.3. HRMS calcd for C24H22F2N3O4S (M + H)+, 486.1299;
found, 486.1295.
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-ethyl-N-[(1-methylethyl)sulfonyl)-1H-indole-2-carbox-
amide (50). Compound 50 was prepared according to the reaction
sequence outlined in Scheme 2 and the general procedures described
1
above for the preparation of generic compound 12. H NMR (500
MHz, DMSO-d6), 12.79 (s, 1 H), 12.70 (s, 1 H), 7.83 (d, J = 5.4 Hz, 1
H), 7.70 (s, 1 H), 7.57 (d, J = 8.4 Hz, 1 H), 7.32−7.25 (m, 2 H), 7.20
(s, 1 H), 7.18−7.14 (m, 1 H), 6.64 (t, J = 6.6 Hz, 1 H), 6.56−6.53 (m,
1 H), 5.73 (s, 2 H), 3.62−3.57 (m, 1 H), 2.66 (q, J = 7.5 Hz, 2 H),
1.18 (d, J = 7.5 Hz, 3 H), 1.15 (d, J = 6.6 Hz, 6H). 13C NMR (125
MHz, DMSO-d6), 163.6, 162.0, 160.0, 158.0, 157.6, 155.7, 145.3,
137.9, 137.2, 136.7, 128.0, 127.2, 123.8, 119.5, 118.0, 117.9, 117.8,
117.7, 116.7, 116.5, 116.4, 116.0, 115.8, 111.7, 108.5, 53.6, 42.5, 29.1,
17.0, 16.1. LRMS calcd for C26H26F2N3O4S (M + H)+, 514.2; found,
514.1.
N-(Cyclopropylsulfonyl)-1-[(2,5-difluorophenyl)-methyl]-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-5-ethyl-1H-indole-2-carboxa-
mide (51). Compound 51 was prepared according to the reaction
sequence outlined in Scheme 2 and the general procedures described
1-[(2,5-Difluorophenyl)methyl]-3-(1,2-dihydro-2-oxo-3-pyri-
dinyl)-5-methyl-N-[(1-methylethyl)sulfonyl)-1H-indole-2-car-
boxamide (46). Compound 46 was prepared according to the
reaction sequence outlined in Scheme 2 and the general procedures
described above for the preparation of generic compound 12. 1H
NMR (500 MHz, DMSO-d6), 12.78 (s, 1 H), 12.70 (s, 1 H), 7.83 (d, J
= 7.0 Hz, 1 H), 7.70 (s, 1 H), 7.55 (d, J = 8.6 Hz, 1 H), 7.32−7.27 (m,
1 H), 7.22−7.12 (m, 3 H), 6.64 (t, J = 6.60 Hz, 1 H), 6.53−6.49 (m, 1
H), 5.73 (s, 2 H), 3.64−3.56 (m 1 H), 2.36 (s, 3 H), 1.14 (d, J = 6.9
Hz, 6H). 13C NMR (125 MHz, DMSO-d6), 163.6, 162.0, 160.0, 157.6,
155.7, 145.2, 137.1, 136.6, 131.3, 129.4, 128.3, 127.2, 123.8, 120.7,
118.0, 117.9, 117.8, 117.7, 116.6, 116.5, 116.4, 115.9, 115.7, 115.5,
111.6, 108.4, 53.6, 42.4, 21.9, 16.1. LRMS calcd for C25H24F2N3O4S
(M + H)+, 500.1; found, 500.3
1
above for the preparation of generic compound 12. H NMR (500
MHz, DMSO-d6), 12.79 (s, 1 H), 12.71 (s, 1 H), 7.82 (q, J = 1.8 Hz, 1
H), 7.69 (s, 1 H), 7.56 (d, J = 8.6 Hz, 1 H), 7.32−7.15 (m, 4 H),
6.65−6.60 (m, 2 H), 5.73 (s, 2 H), 2.96−2.91 (m, 1 H), 2.68−2.64
(m, 2 H), 1.17 (t, J = 7.5 Hz, 3 H), 0.98 (d, J = 6.3 Hz, 4 H). 13C
NMR (125 MHz, DMSO-d6), 163.5, 161.9, 159.9, 158.0, 157.7, 155.7,
145.0, 137.9, 137.1, 136.6, 129.5, 127.2, 127.1, 123.9, 119.5, 118.0,
117.8, 116.7, 116.5, 115.9, 115.8, 115.7, 111.7, 108.3, 42.3, 31.6, 29.1,
17.0, 6.3. LRMS calcd for C26H24F2N3O4S (M + H)+, 512.1; found,
512.1.
N-(Cyclopropylsulfonyl)-1-[(2,5-difluorophenyl)methyl]-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-5-(trifluoromethyl)-1H-indole-
2-carboxamide (52). Compound 52 was prepared according to the
reaction sequence outlined in Scheme 2 and the general procedures
described above for the preparation of generic compound 12. 1H
NMR (500 MHz, DMSO-d6), 12.78 (s, 2 H), 7.90 (d, J = 8.8 Hz, 1
H), 7.81 (q, J = 2.2 Hz, 1 H), 7.70 (s, 1 H), 7.66 (q, J = 1.6 Hz, 2 H),
7.32−7.27 (m, 1 H), 7.20−7.16 (m, 1 H), 6.71−6.68 (m, 1 H), 6.58 (t,
J = 6.7 Hz, 1 H), 5.80 (s, 2 H), 2.95−2.90 (m, 1 H), 0.99 (s, 2 H),
0.90−0.85 (m, 2 H). 13C NMR (125 MHz, DMSO-d6), 163.6, 163.0,
161.6, 160.0, 155.8, 144.6, 139.5, 136.8, 132.4, 129.5, 126.2, 123.0,
122.1, 119.6, 118.0, 117.9, 116.9, 116.2, 116.0, 113.1, 107.9, 68.3, 42.7,
32.9, 31.5, 30.7, 24.1, 23.2, 11.7, 6.3, 5.8. LRMS calcd for
C25H19F5N3O4S (M + H)+, 552.1; found, 552.0.
N-(Cyclopropylsulfonyl)-1-[(2,5-difluorophenyl)methyl]-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-5-methyl-1H-indole-2-carbox-
amide (47). Compound 47 was prepared according to the reaction
sequence outlined in Scheme 2 and the general procedures described
1
above for the preparation of generic compound 12. H 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 C25H22F2N3O4S (M + H)+, 498.1;
found, 498.3.
762
dx.doi.org/10.1021/jm201258k | J. Med. Chem. 2012, 55, 754−765

Journal of Medicinal Chemistry
Article
N-(Cyclopropylsulfonyl)-1-[(2,5-difluorophenyl)methyl]-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-5-(1,1-dimethylethyl)-1H-in-
dole-2-carboxamide (53). Compound 53 was prepared according to
the reaction sequence outlined in Scheme 2 and the general
procedures described above for the preparation of generic compound
Abbreviations Used
HCV, hepatitis C virus; PK, pharmacokinetics; SAR, structure−
activity relationship; AUC, area-under-the-curve; SOC, stand-
ard 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, diazabicy-
clicunderdecane; CYP, cytochrome P450; hERG, the human
1
12. H NMR (500 MHz, DMSO-d6), 7.83 (q, J = 2.5 Hz, 1 H), 7.70
(d, J = 5.9 Hz, 1 H), 7.57 (d, J = 8.8 Hz, 1 H), 7.49 (q, J = 2.3 Hz, 1
H), 7.32 (d, J = 1.4 Hz, 1 H), 7.29 (q, J = 4.3 Hz, 1 H), 7.20−7.14 (m,
1 H), 6.65 (t, J = 7.4 Hz, 2 H), 5.73 (s, 2 H), 2.98−2.91 (m, 1 H), 1.28
(s, 9H), 0.99 (d, J = 5.7 Hz, 4 H). 13C NMR (125 MHz, DMSO-d6),
174.0, 172.4, 170.4, 168.5, 168.1, 166.2, 155.6, 155.2, 147.2, 139.9,
138.5, 138.3, 137.1, 135.5, 134.3, 128.5, 128.3, 126.9, 126.8, 126.5,
126.3, 122.0, 118.8, 52.8, 45.7, 42.7, 42.0, 16.8. LRMS calcd for
C28H28F2N3O4S (M + H)+, 540.2; found, 540.1.
̀
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
N-(Cyclopropylsulfonyl)-1-[(2,5-difluorophenyl)methyl]-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-5-(1-methylcyclopropyl)-1H-
indole-2-carboxamide (54). Compound 54 was prepared according
to the reaction sequence outlined in Scheme 2 and the general
procedures described above for the preparation of generic compound
ASSOCIATED CONTENT
■
Accession Codes
Protein Data Bank ID 3TYV.
1
12. H NMR (500 MHz, DMSO-d6), 12.81 (s, 1 H), 12.72 (s, 1 H),
AUTHOR INFORMATION
■
7.84 (q, J = 1.6 Hz, 1 H), 7.69 (d, J = 4.7 Hz, 1 H), 7.58 (d, J = 8.9 Hz,
1 H), 7.47−7.45 (m, 1 H), 7.33 (s, 1 H), 7.32−7.29 (m, 1 H), 7.19−
7.15 (m, 1 H), 6.64−6.62 (m, 2 H), 5.74 (s, 2 H), 3.86 (s, 3 H), 2.97−
2.92 (m, 1 H), 1.98 (s, 2 H), 1.74 (d, J = 6.5 Hz, 2 H), 0.98 (d, J = 6.3
Hz, 4 H). 13C NMR (125 MHz, DMSO-d6), 173.9, 172.3, 168.5, 168.1,
166.2, 155.5, 151.1, 148.4, 148.0, 147.1, 146.6, 140.2, 138.3, 137.4,
136.6, 135.1, 134.2, 132.6, 129.3, 128.5, 128.3, 128.0, 126.9, 126.4,
126.2, 122.0, 118.8, 52.9, 42.0, 37.5, 31.3, 27.0, 26.3, 25.6, 16.8. LRMS
calcd for C28H26F2N3O4S (M + H)+, 538.2; found, 538.1.
(N-(Cyclopropylsulfonyl)-1-[(2,5-difluorophenyl)methyl]-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-6-fluoro-5-(methyl)-1H-indole-
2-carboxamide (56). Compound 56 was prepared according to the
reaction sequence outlined in Scheme 2 and the general procedures
described above for the preparation of generic compound 12. 1H
NMR (500 MHz, DMSO-d6), 7.78 (dd, J = 7.2, 2.0 Hz, 1 H), 7.67−
7.65 (m, 1 H), 7.53 (d, J = 10.8 Hz, 1 H), 7.29−7.24 (m, 2 H), 7.18−
7.12 (m, 1 H), 6.61−6.57 (m, 2 H), 5.69 (s, 2 H), 2.93−2.85 (s, 1 H),
2.26 (s, 3 H), 0.95 (d, J = 6.4 Hz, 4 H). 13C NMR (125 MHz, DMSO-
d6), 162.3, 160.7, 158.9, 157.0, 154.8, 144.0, 135.7, 122.5, 122.3, 119.2,
117.0, 116.8, 115.8, 115.6, 115.1, 114.9, 114.7, 107.3, 96.9, 96.7, 42.7,
31.5, 15.6, 15.5. HRMS calcd for C25H21F3N3O4S (M + H)+,
516.1205; found, 516.1200.
Corresponding Author
*Phone: 732-662-1333. Fax: 908-740-7152. E-mail: kxcn@
yahoo.com.
REFERENCES
■
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N-(Cyclopropylsulfonyl)-1-[(2,5-difluorophenyl)methyl]-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-6-fluoro-5-(trifluoromethyl)-
1H-indole-2-carboxamide (59). Compound 59 was prepared
according to the reaction sequence outlined in Scheme 2 and the
general procedures described above for the preparation of generic
compound 12. 1H NMR (500 MHz, DMSO-d6), 12.70 (s, 1 H), 12.56
(s, 1 H), 7.99 (d, J = 12.2 Hz, 1 H), 7.81−7.79 (m, 2 H), 7.66 (d, J =
5.7 Hz, 1 H), 7.33−7.28 (s, 1 H), 7.23−7.17 (m, 1 H), 6.74−6.69 (m,
1 H), 6.56 (t, J = 6.8 Hz, 1 H), 5.77 (s, 2 H), 2.93−2.87 (m, 1 H), 0.98
(d, J = 6.1 Hz, 4 H). 13C NMR (125 MHz, DMSO-d6), 173.4, 171.7,
170.6, 170.4, 169.3, 168.5, 168.3, 167.4, 166.4, 155.1, 150.5, 147.5,
142.0, 137.4, 133.1, 133.0, 132.1, 128.6, 128.5, 128.4, 128.0, 127.3,
126.8, 126.6, 118.3, 110.8, 110.6, 53.6, 42.0, 16.8. LRMS calcd for
C25H18F6N3O4S (M + H)+, 570.1; found, 570.1.
N-(Cyclopropylsulfonyl)-1-[(2,5-difluorophenyl)methyl]-3-
(1,2-dihydro-2-oxo-3-pyridinyl)-5-ethyl-6-fluoro-1H-indole-2-
carboxamide (60). Compound 60 was prepared according to the
reaction sequence outlined in Scheme 2 and the general procedures
described above for the preparation of generic compound 12. 1H
NMR (500 MHz, DMSO-d6), 12.71 (s, 2 H), 7.80 (q, J = 1.9 Hz, 1
H), 7.69 (s, 1 H), 7.55 (d, J = 11.7 Hz, 1 H), 7.32−7.27 (m, 2 H),
7.21−7.14 (m, 1 H), 6.66 (s, 2 H), 5.71 (s, 2 H), 2.95−2.88 (m, 1 H),
2.67 (q, J = 7.4 Hz, 2 H), 1.16 (t, J = 7.2 Hz, 3 H), 0.97 (d, J = 6.9 Hz,
4 H). 13C NMR (125 MHz, DMSO-d6), 173.8, 172.1, 170.4, 170.2,
168.5, 168.2, 166.3, 155.5, 147.8, 147.2, 140.1, 138.2, 137.0, 134.0,
132.3, 128.5, 128.3, 127.1, 126.8, 126.5, 126.3, 118.8, 108.6, 108.4,
53.2, 42.0, 33.6, 26.1, 16.8. LRMS calcd for C26H23F3N3O4S (M + H)+,
530.1; found, 530.0.
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Journal of Medicinal Chemistry
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