6-(Azaindol-2-yl)pyridine-3-sulfonamides as potent and selective inhibitors targeting hepatitis C virus NS4B

Article abstract of DOI:10.1016/j.bmcl.2014.12.093

A structure-activity relationship investigation of various 6-(azaindol-2-yl)pyridine-3-sulfonamides using the HCV replicon cell culture assay led to the identification of a potent series of 7-azaindoles that target the hepatitis C virus NS4B. Compound 2ac, identified via further optimization of the series, has excellent potency against the HCV 1b replicon with an EC₅₀ of 2 nM and a selectivity index of >5000 with respect to cellular GAPDH RNA. Compound 2ac also has excellent oral plasma exposure levels in rats, dogs and monkeys and has a favorable liver to plasma distribution profile in rats.

Full text of DOI:10.1016/j.bmcl.2014.12.093

Accepted Manuscript
6-(Azaindol-2-yl)pyridine-3-sulfonamides as potent and selective inhibitors tar-
geting hepatitis C virus NS4B
Guangming Chen, Hongyu Ren, Nanjing Zhang, William Lennox, Anthony
Turpoff, Steven Paget, Chunshi Li, Neil Almstead, F. George Njoroge,
Zhengxian Gu, Jason Graci, Stephen P. Jung, Joseph Colacino, Fred Lahser, Xin
Zhao, Marla Weetall, Amin Nomeir, Gary M. Karp
PII:
S0960-894X(14)01401-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.12.093
BMCL 22338
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 November 2014
23 December 2014
29 December 2014
Please cite this article as: Chen, G., Ren, H., Zhang, N., Lennox, W., Turpoff, A., Paget, S., Li, C., Almstead, N.,
George Njoroge, F., Gu, Z., Graci, J., Jung, S.P., Colacino, J., Lahser, F., Zhao, X., Weetall, M., Nomeir, A., Karp,
G.M., 6-(Azaindol-2-yl)pyridine-3-sulfonamides as potent and selective inhibitors targeting hepatitis C virus NS4B,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2014.12.093
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6-(Azaindol-2-yl)pyridine-3-sulfonamides as potent and selective inhibitors
targeting hepatitis C virus NS4B
Guangming Chen a*, Hongyu Ren a, Nanjing Zhang a, William Lennox a, Anthony Turpoff a, Steven Paget
a, Chunshi Li a, Neil Almstead a, F. George Njoroge b, Zhengxian Gu a, Jason Graci a, Stephen P. Jung a,
Joseph Colacino a, Fred Lahser b, Xin Zhao a, Marla Weetall a, Amin Nomeir b, Gary M. Karp a
aPTC Therapeutics, Inc., 100 Corporate Court, South Plainfield, NJ 07080, USA
bMerck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
ABSTRACT
A Structure−activity relationship investigation of various 6-(azaindol-2-yl)pyridine-3-sulfonamides using the HCV replicon cell culture assay led to the
identification of a potent series of 7-azaindoles that target the hepatitis C virus NS4B. Compound 2ac, identified via further optimization of the series,
has excellent potency against the HCV 1b replicon with an EC50 of 2 nM and a selectivity index of >5000 with respect to cellular GAPDH RNA.
Compound 2ac also has excellent oral plasma exposure levels in rats, dogs and monkeys and has a favorable liver to plasma distribution profile in rats.
Keywords:
HCV inhibitors
Replicon
6-(Azaindol-2-yl)pyridine-3-sulfonamides
Structure−activity relationship
NS4B
Hepatitis C virus (HCV) is an enveloped, single-stranded positive sense 9.4 kb RNA virus belonging to the Hepacivirus genus of the
Flaviviridae family. HCV infects an estimated 150 million people worldwide and an estimated 3-4 million people in the United States.1
Approximately 80% of HCV infections develop into chronic hepatitis that can ultimately lead to liver fibrosis, cirrhosis and
hepatocellular carcinoma, and is the leading reason for liver transplantation in the United States.2 In the U.S. alone, up to 10,000 deaths
per year are attributed to hepatitis C related liver disease. In recent years, significant progress has been made in the area of HCV therapy.
Three direct acting antiviral agents targeting the HCV protease; Incivek™ (telaprevir)3, Victrelis™ (boceprevir)4, and Olysio™
(simeprevir)5, were granted FDA approval in the last few years. In 2013, the first drug targeting the viral RNA polymerase, Sovaldi™
(sofosbuvir)6 was granted FDA approval. Harvoni®, a single tablet combination of sofosbuvir and ledipasvir was approved in 2014 for
the treatment for patients with genotype 1 chronic hepatitis C. This all-oral, interferon-free therapy was shown to improve the sustained
virologic response to >94% in clinical trials. Sofosbuvir has also been approved as part of the first all-oral dual therapy for patients
infected with HCV genotype 2 or 3.6-10 However, there is still a continued medical need in finding agents that act on novel HCV targets,
so that in combination with the current or future standard of care, the emergence of resistance and the rebound of viremia after cessation
of treatment can be curtailed.11-13
In addition to newer compounds targeting the NS3 protease and NS5B RNA polymerase, several compounds targeting other viral
proteins, without an obvious enzymatic activity, e.g., NS5A, are in preclinical or clinical development.14 The non-structural membrane-
bound protein NS4B,15 a 27-kDa integral membrane protein, plays an essential role in HCV replication and has recently emerged as a
potential drug target for the treatment of chronic HCV infection.16 Several novel chemotypes targeting HCV NS4B have been disclosed
in recent publications.17
Previously,17c, 17d we reported the identification of a series of 6-(indol-2-yl)pyridine-3-sulfonamides as novel inhibitors of HCV RNA
replication that target HCV NS4B. Compound 117d (Fig. 1) is highly potent and selective with an EC50 of 7 nM against the HCV 1b
replicon and a selectivity index of 1300 with respect to its effect on levels of cellular GAPDH RNA. The combination of the 5-F and 6-
OCHF2 substituents in the indole phenyl ring of 1, resulting from chemical optimization of the DMPK and safety profile, provided
reduced metabolic oxidative arene oxide formation and a significant decrease in glutathione (GSH) adduct formation. In order to further
expand the chemical space and explore novel scaffolds with favorable pharmaceutical properties, we envisioned replacing the indole
core with various azaindole moieties resulting in compounds 2 (Fig. 1). The presence of additional nitrogen atoms in the azaindole ring
would lead to reduced electronic density thus rendering it less prone to oxidative metabolism. Further, the presence of additional
nitrogen atoms in the arene moiety, could, depending on the substitution pattern, eliminate the potential for glutathione adduct
formation. Herein, we report on the identification of several novel azaindoles that target HCV NS4B.
We first surveyed various azaindoles by walking the aza N around the six-membered ring. Based on our previous experience in the
indole series whereby the combination of N-cyclobutyl and (S)-N-(1,1,1-trifluoropropan-2-yl)pyridine-3-sulfonamide groups generally
provided compounds with the highest potency,17c we prepared analogs in several azaindole series containing these key moieties in
combination with selected substituents on the pyridine ring of the azaindole core. The general synthetic strategies utilized for the
syntheses of 2 as outlined in Scheme 1 have been described previously.17d

Commercially available 5-methoxy-, 6-methyl-, and 6-chloro-4-azaindoles (3a−c) were alkylated at the N-1 position with cyclobutyl
bromide in the presence of Cs2CO3 in DMF, followed by cyanation at C-3 with chlorosulfonyl isocyanate in a mixture of DMF and
acetonitrile providing 1-cyclobutyl-3-cyano-4-azaindoles 5a−c (Scheme 2). The transformations of 5a−c to 2a−c were realized through
Stille coupling chemistry as shown in path C in Scheme 1.
The 6-methyl-5-azaindole 3d was prepared starting from readily available 2,5-dimethylpyridine 9 (Scheme 3). Compound 9 was
oxidized to the N-oxide 10 by m-CPBA, followed by nitration with fuming nitric acid to provide nitropyridine N-oxide 11. Batcho-
Leimgruber indole synthesis furnished 12 in 3 steps with a 52% yield. Iron reduction of 12 in acetic acid provided the 6-methyl-5-
azaindole intermediate 3d which was elaborated to 5d via N-alkylation and cyanation. Compound 5d was converted to 2d using the
Stille coupling strategy described above (Scheme 1). Utilizing similar synthetic methodology, compound 2e was prepared from
commercially available 6-chloro-5-azaindole.
6-Azaindole analogs 2f and 2g were prepared from commercially available 5-chloro- and 5-methyl-6-azaindole, respectively,
according to the chemistry described in Scheme 1 via the Stille coupling strategy.
5,7-Diazaindole intermediates 5h and 5i were prepared from commercially available 5-bromo-2,4-dichloropyrimidine 13 (Scheme 4).
Reaction of 13 with cyclobutylamine provided 14, which was converted to 15 in methanol in the presence of K2CO3. Compound 15 then
underwent Sonagashira coupling with trimethylsilylacetylene, followed by desilylation with K2CO3 in methanol to furnish 16. CuI
promoted cyclization of 16 to 4h was accomplished by heating a DMF solution in the presence of Barton base at 140 °C. Demethylation
of 4h with HBr in acetic acid at 100 °C gave a phenol intermediate, which was treated with sodium difluoroacetate in DMF in the
presence of Cs2CO3 to yield 4i. Cyanation of 4h and 4i with chlorosulfonyl isocyanate in a mixture of DMF and acetonitrile (1:1)
provided 5h and 5i, respectively. Compounds 2h and 2i were then prepared via the Stille coupling strategy utilizing 5h and 5i (Scheme
1).
Utilizing the Stille coupling methodology described in Scheme 1, several 7-azaindole analogs were synthesized from commercially
available azaindole starting materials. The 5-methoxy-7-azaindole analogs 2j, 2r, and 2s were prepared from readily available 5-
methoxy-7-azaindole. The 5-chloro-7-azaindole analogs 2l, 2x, and 2y were prepared starting from 5-chloro-7-azaindole. Compounds
2k, 2m, 2n, 2o, 2ab, 2ac, and 2ad were prepared from 6-methoxy-7-azaindole, 6-chloro-7-azaindole, 5-methyl-7-azaindole, 6-methyl-
7-azaindole, 5-fluoro-7-azaindole, 5-trifluoromethyl-7-azaindole and 7-azaindole, respectively.
Additional 7-azaindole analogs were prepared from the versatile 5-bromo-7-azaindole 17 (Scheme 5). Compound 17 was converted
to 5-bromo-1-cyclobutyl-3-cyano-7-azaindole 18 in two steps (Scheme 5). Palladium catalyzed boronate formation followed by Oxone®
oxidation provided the 5-hydoxy intermediate 19. Alkylation of 19 with alkylating reagents furnished 5-alkoxy intermediates 5t−v.
Freonation of 19 gave the 5-difluoromethoxy intermediate 5w. Negishi coupling of 18 with ethyl- or cyclopropylzinc halides, prepared
in situ from the corresponding Grignard reagents and ZnCl2, in the presence of catalytic PdCl2(dppf) provided 5z and 5aa. The target
compounds 2t−v and 2aa were synthesized through a Suzuki coupling strategy; compound 2w was prepared via a Stille coupling
strategy (Scheme 1).
These compounds were then evaluated for inhibition of HCV subgenomic RNA replication using an Huh-7 derived cell line.18
Results for the initial set of analogs indicated that the most potent compounds are derived from the 7-azaindole series (Table 1),
particularly those compounds substituted at the 5-position of the 7-azaindole ring. Compounds 2j, 2l, and 2n exhibit replicon EC50
values <30 nM and have >330 fold selectivity with respect to their effect on levels of cellular GAPDH RNA. The corresponding 6-
substituted 7-azaindole analogs showed decreased activity against the replicon. The activity of the 6-OMe (2k) and 6-Cl (2m) analogs
against the replicon decreased 35- and 41-fold, respectively, compared to the activity of the corresponding 5-subsituted analogs 2j and
2l. The 6-Me analog 2o showed a ~7-fold decrease in activity against the replicon compared to the 5-Me analog 2n. The 4-azaindole
sulfonamides 2a−c showed moderate to good replicon activity with the 6-Me (2b) and 6-Cl (2c) analogs exhibiting EC50 values of 119
and 94 nM, respectively. The 5-OMe analog (2a) was less potent (EC50 = 230 nM). Direct comparison of the 4-azaindole and 7-
azaindole series shows the significant effect imparted by the azaindole substituent on activity against the replicon. 5-OMe substitution
favors the 7-azaindole 2j over the 4-azaindole 2a by ~8-fold. In contrast, 6-Cl substitution favors the 4-azaindole 2c over the 7-
azaindole 2m by ~7-fold. 6-Me substitution results in comparable activity against the replicon between the 4- (2b) and 7-azaindole (2o)
analogs with EC50 values of 119 and 87 nM, respectively.
A comparison of methyl and chloro substituted 5-aza and 6-azaindole analogs revealed that the activity of the 5-azaindole analogs
against the replicon is about 2−7 fold more potent than that of the 6-azaindole analogs. As an example, the 5-Me-6-azaindole analog 2g
inhibits HCV RNA replication with an EC50 of 1,400 nM, compared to the 6-Me-5-azaindole analog 2d, which has an EC50 of 230 nM.
The 5,7-diazaindoles 2h and 2i showed modest activity against the replicon, with EC50 values of 240 and 970 nM, respectively.
Having determined from our initial survey that the 5-substituted 7-azaindole series provided analogs with the most potent activity,
we further evaluated this series. Reinvestigation of the azaindole 2-position revealed SAR that was consistent with the indole series
reported previously.17c Replacement of the 2’-pyridyl ring with either a phenyl or 2’-pyrimidyl group was disadvantageous for potency
against the replicon (Table 2). Compared to the 2’-pyridinyl compound 2n, the replicon EC50 values for the 2-phenyl compound 2p and
the 2’-pyrimidyl compound 2q decreased more than 6-fold and 12-fold, respectively.
Next, we conducted a comprehensive SAR study around the 7-azaindole core by varying R1 (C-5 position) and R2 (N-1 position) and
the results are summarized in Table 3.
We initially varied R1 substitution while maintaining R2 = c-Bu. In general, small lipophilic R1 groups are favored, a finding
consistent with that reported for indole series.17c,d Trifluoromethyl substitution at C-5 (2ac) provided the most potent compound (EC50
=
2 nM). Small alkyl groups are also well tolerated at the 5-position. In addition to the 5-Me analog 2n (EC50 = 13 nM), the 5-Et (2z), and
5-c-Pr (2aa) analogs were also very potent with replicon EC50 values of 15 and 40 nM, respectively. Halogens such as F and Cl were
also well-tolerated. The replicon EC50 values for the 5-F analog 2ab and 5-Cl analog 2l were 46 and 17 nM, respectively. In comparison,

the unsubstituted analog 2ad was less active (EC50 = 217 nM). In addition to the 5-OMe analog 2j (EC50 = 27 nM), several additional 5-
alkoxy analogs were evaluated. Increasing the size of the alkoxy group to 5-OEt (2t), 5-O-n-Pr (2u), and 5-O-i-Pr (2v) resulted in
decreased potency with replicon EC50 values of 200, 2150 and 7600 nM, respectively. The 5-difluoromethoxy analog 2w, however,
demonstrated good activity (EC50 = 50 nM).
The effect of the R2 group on activity was evaluated in two series (5-OMe and 5-Cl). In the 5-OMe series, the N-c-butyl analog 2j
was 5-fold more potent than the N-c-pentyl analog 2s and 10-fold more potent than the N-c-PrMe analog 2r. In the 5-chloro series, the
N-c-butyl analog 2l was nearly 2-fold more potent than either the N-c-PrMe 2y or the N-c-pentyl analog 2x. The SAR trend for the R2
group in the 7-azaindole series is consistent with the observations in the indole series in which the c-Bu group provided the most potent
activity.17c
Compounds with replicon EC50 values less than 100 nM were evaluated in a pharmacokinetic screen for plasma drug levels (AUC0-6h
in the rat after oral administration of a single 10 mg/kg dose in 0.4% hydroxypropyl methylcellulose (Table 3). In general, good to
excellent drug exposure levels were observed. Among the nine compounds evaluated, the plasma AUC0-6h ranged from 2389 nM·h to
10,164 nM·h. For all compounds evaluated, the plasma concentration multiples over the in vitro replicon EC50 were more than 20-fold
at 6 h. The potent 5-CF3 analog (2ac, EC50 = 2 nM) had excellent exposure in the rat (AUC0~6h = 5833 nM·h) and plasma concentration
multiples of >500 over the replicon EC50 at 6 h. These compounds also showed very favorable drug distribution between liver and
plasma. Among the compounds evaluated, liver to plasma ratios at 6 h after a 10 mg/kg oral dose ranged from 6−44-fold. This is
important as the primary target organ for HCV is the liver.
)
To further assess the potential utility of compounds from this series, 2j, 2l, 2n, 2y, and 2ac were evaluated for plasma drug levels in
beagle dogs and cynomolgus monkeys after oral administration at doses of 2 and 3 mg/kg, respectively (Table 4). Data for compound 1
are also included in Table 4 for comparison. At these doses, oral exposure (AUC0-24h) was higher in dogs than in monkeys. The 5-CF3
analog (2ac) had the highest exposure in both dogs and monkeys (AUC0-24h = 8162 and 5440 nM·h, respectively). In contrast, the 5-Me
(2n) and 5-Cl (2y) analogs had lower overall exposures in both species and were 9- and 7-fold lower in dog than in monkeys,
respectively. Plasma concentration multiples over the in vitro replicon EC50 at 8 h were also determined in these species. The 5-CF3
analog (2ac) had the highest plasma exposure multiples in dogs and monkeys (125- and 105-fold above the replicon EC50 at 8h,
respectively.) Compared to lead compound 1 (EC50 = 7 nM), 2ac (EC50 = 2 nM) showed improved replicon potency and higher plasma
exposure multiples over the in vitro replicon EC50 in rats, dogs and monkeys.
At concentrations up to 10,000 nM, Compound 2j, a representative example from the series, did not inhibit the HCV NS3 protease or
NS5B polymerase in vitro (data not shown). Selection of resistant HCV genotype 1b replicons was performed by serial passage in the
presence of 2j at concentrations of 1X, 10X, and 30X the in vitro replicon EC90 (90, 900, and 2700 nM, respectively).17h Resistant
replicons were isolated, sequenced and found to have mutations in the coding sequence in the HCV NS4B, in particular resulting in the
amino acid substitutions F98L, F98C, and V105M. The F98C substitution was identified as the predominant mutation selected at 30X
the replicon EC90 (found in 15 of 16 clones sequenced). Replicons engineered to contain each of these mutations alone were found to be
25-fold (F98C) and 17-fold (V105M) resistant to 2j. The same amino acid substitutions in NS4B were previously found to confer
60−70-fold resistance to 6-(indol-2-yl)pyridine-3-sulfonamides.17c, 17d
In summary, we have identified several novel azaindole sulfonamide chemotypes that potently and selectively inhibit the replication
of the HCV 1b replicon. SAR investigations of various azaindole cores revealed that 5-substituted 7-azaindole sulfonamides provide
compounds with the most potent activity against the replicon and that these compounds target HCV NS4B. These compounds are
highly selective with no apparent effect on cellular GAPDH RNA levels. Several compounds have demonstrated excellent drug
exposure levels in rats with favorable liver to plasma ratios. These efforts led to the identification of 2ac, a highly potent analog (EC50
2 nM) with excellent oral exposure in rats, dogs and monkeys.
=
Acknowledgements
The authors would like to thank Shirley Yeh for analytical support.
References and notes:
1. (a) Alter, M. J. World J. Gastroenterol. 2007, 13, 2436; (b) Kim, A. Y.; Timm, J. Expert Rev. Anti-infect. Ther. 2008, 6, 463; (c) Almasio, P. L.;
Ingrassia, D.; Vergara, B.; Filosto, S. Expert Rev. Anti-infect. Ther. 2008, 6, 775; (d) Hepatitis C fact sheet No. 164, April 2014, www.who.int.
2. Ghobrial, R. M.; Steadman, R.; Gornbein, J.; Lassman, C; Holt, C. D.; Chen, P.; Farmer, D. G.; Yersiz, H.; Danino, N.; Collisson, E.; Baquarizo, A.;
Han, S. S.; Saab, S.; Goldstein, L. I.; Donovan, J. A.; Esrason, K.; Busuttil, R. W. Ann. Surgery, 2001, 234, 384.
3. Lin, K.; Perni, R. B.; Kwong, A. D.; Lin, C. Antimicrob. Agents Chemother. 2006, 50, 1813.
4. Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu, Y.-T.; Lovey, R.; Hendrata, S.; Huang, Y.; Pan, W.; Parekh, T.;
Pinto, P.; Popov, V.; Pike, R.; Ruan, S.; Santhanam, B.; Vibulbhan, B.; Wu, W.; Yang, W.; Kong, J.; Liang, X.; Wong, J.; Liu, R.; Butkiewicz, N.;
Chase, R.; Hart, A.; Agrawal, S.; Ingravallo, P.; Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.; Prongay, A.; Madison, V.; Broske, L.;
Cui, X.; Cheng, K.-C.; Hsieh, Y.; Brisson, J.-M.; Prelusky, D.; Korfmacher, A.; White, R.; Bogdanowich-Knipp, S.; Pavlovsky, A.; Bradley, P.;
Saksena, A. K.; Ganguly, A.; Piwinski, J.; Girijavallabhan, V.; Njoroge, F. G. J. Med. Chem. 2006, 49, 6074.
5. Rosenquist, A.; Samuelsson, B.; Johansson, P.-O.; Cummings, M. D.; Lenz, O.; Raboisson, P.; Simmen, K.; Vendeville, S.; de Kock, H.; Nilsson, M;
Horvath, A.; Kalmeijer, R.; de la Rosa, G.; Beumont-Mauviel, M. J. Med. Chem. 2014, 57, 1673.
6. (a) Gane, E. J.; Stedman, C. A.; Hyland, R. H.; Ding, X.; Svarovskaia, E.; Symonds, W. T.; Hindes, R. G.; Berrey, M. M. N. Engl. J. Med. 2013, 368,
34; (b) Koff, R. S. Aliment. Pharmacol. Ther. 2014, 39, 478.
7. McHutchison, J. G.; Lawitz, E. J.; Shiffman, M. L.; Muir, A. J.; Galler, G. W.; McCone, J.; Nyberg, L. M.; Lee, W. M.; Ghalib, R. H.; Schiff, E. R.;
Galati, J. S.; Bacon, B. R.; Davis, M. N.; Mukhopadhyay, P.; Koury, K.; Noviello, S.; Pedicone, L. D.; Brass, C. A.; Albrecht, J. K.; Sulkowski, M. S.
N. Engl. J. Med. 2009, 361, 580.
8. Nainan, O. V.; Alter, M. J.; Kruszon-Moran, D.; Gao, F.-X.; Xia, G.; McQuillan, G.; Margolis, H. S. Gastroenterology, 2006, 131, 478.

9. Asselah, T.; Marcellin, P. Liver International 2011, 68.
10. Sheridan, C. Nature Biotechnol. 2011, 29, 553.
11. Halfon, P.; Locarnini, S. J. Hepatol. 2011, 55, 192.
12. Lee, L. Y.; Tong, C. Y. W.; Wong, T.; Wilkinson, M. Int. J. Clin. Pract. 2012, 66, 342.
13. Sarrazin, C.; Zeuzem, S. Gastroenterology, 2010, 138, 447.
14. (a) Schinazi, R.; Halfon, P.; Marcellin, P.; Asselah, T., Liver International 2014, 69; (b) Scheel, T. K. H.; Rice, C. M. Nature Med. 2013, 19, 837.
15. (a) Sklan, E. H.; Glenn, J. S. Hepatitis C Viruses: Genomes and Molecular Biology; Tan, S. L., Ed.; Horizon Bioscience: Norfolk, UK, 2006; Chapter
8. (b) Einav, S.; Dvory-Sobol, H.; Gehrig, E.; Glenn, J. S. J. Infect. Dis. 2010, 202, 65.
16. (a) Dvory-Sobol, H.; Pang, P. S.; Glenn, J. S. Viruses 2010, 2, 2481. (b) Gouttenoire, J.; Penin, F.; Moradpour, D. Rev. Med. Virol. 2010, 20, 117. (c)
Rai, R.; Deval, J. Antiviral Res. 2011, 90, 93.
17. (a) Shotwell, J. B.; Baskaran, S.; Chong, P.; Creech, K. L.; Crosby, R. M.; Dickson, H.; Fang, J.; Garrido, D.; Mathis, A.; Maung, J.; Parks, D. J.;
Pouliot, J. J.; Price, D. J.; Rai, R.; Seal, J. W., III; Schmitz, U.; Tai, V. W. F.; Thomson, M.; Xie, M.; Xiong, Z. Z.; Peat, A. J. ACS Med. Chem. Lett.
2012, 3, 565. (b) Miller, J. F.; Chong, P. Y.; Shotwell, J. B.; Catalano, J. G.; Tai, V. W.-F.; Fang, J.; Banka, A. L.; Roberts, C. D.; Youngman, M.;
Zhang, H.; Xiong, Z.; Mathis, A.; Pouliot, J. J.; Hamatake, R. K.; Price, D. J.; Seal, J. W.; Stroup, L. L.; Creech, K. L.; Carballo, L. H.; Todd, D.;
Spaltenstein, A.; Furst, S. M.; Hong, Z.; Peat, A. J. Med. Chem. 2014, 57, 2107. (c) Zhang, X.; Zhang, N.; Chen, G.; Turpoff, A.; Ren, H.; Takasugi,
J.; Morrill, C.; Zhu, J.; Li, C.; Lennox, W.; Paget, S.; Liu, Y.; Almstead, N.; Njoroge, F. G.; Gu, Z.; Komatsu, T.; Clausen, V.; Espiritu, C.; Graci, J.;
Colacino, J.; Lahser, F.; Risher, N.; Weetall, M.; Nomeir, A.; Karp, G. M. Bioorg. Med. Chem. Lett. 2013, 23, 3947. (d) Zhang, N.; Zhang, X.; Zhu,
J.; Turpoff, A.; Chen, G.; Morrill, C.; Huang, S.; Lennox, W.; Kakarla, R.; Liu, R.; Li, C.; Ren, H.; Almstead, N.; Venkatraman, S.; Njoroge,, F. G.;
Gu, Z.; Clausen, V.; Graci, J.; Jung, S. P.; Zheng, Y.; Colacino, J. M.; Lahser, F.; Sheedy, J.; Mollin, A.; Weetall, M.; Nomeir, A.; Karp, G. M. J.
Med. Chem. 2014, 57, 2121. (e) Kakarla, R.; Liu, J.; Naduthambi, D.; Chang, W.; Mosley, R. T.; Bao, D.; Micolochick Steuer, H. M.; Keilman, M.;
Bansal, S.; Lam, A. M.; Seibel, W.; Neilson, S.; Furman, P. A.; Sofia, M. J. J. Med. Chem. 2014, 57, 2136. (f) Phillips, B.; Cai, R.; Delaney, W.; Du,
Z.; Ji, M.; Jin, H.; Lee, J.; Li, J.; Niedziela-Majka, A.; Mish, M.; Pyun, H.-J.; Saugier, J.; Tirunagari, N.; Wang, J.; Yang, H.; Wu, Q.; Sheng, C.;
Zonte, C. J. Med. Chem. 2014, 57, 2161. (g) Dufner-Beattie, J.; O’Guin, A.; O’Guin, S.; Briley, A.; Wang, B.; Balsarotti, J.; Roth, R.; Starkey, G.;
Slomczynska, U.; Noueiry, A.; Olivo, P. D.; Rice, C. M. Antimicrob. Agents Chemother. 2014, 58, 3399. (h) Gu, Z.; Graci, J. D.; Lahser, F. C.;
Breslin, J. J.; Jung, S. P.; Crona, J. H.; McMonagle, P; Xia, E.; Liu, S.; Karp, G.; Zhu, J.; Huang, S.; Nomeir, A.; Weetall, M.; Almstead, N. G.; Peltz,
S. W.; Tong, X.; Ralston, R.; Colacino, J. M. Antimicrob. Agents Chemother. 2013, 57, 3250.
18. Lohmann, V.; Körner, F.; Koch, J.-O.; Herian, U.; Theilmann, L.; Bartenschlager R. Science 1999, 285, 110.
19. EC50 values are the average of at least two independent determinations. Huh7 cells harboring a genotype 1b HCV bicistronic replicon (Con1) were
plated at 5000 cells/well in 96 well plates. Compounds were added to the plates with a final DMSO concentration of 0.5% and plates were incubated
at 37°C. Cells were harvested 3 days post dosing and replicon RNA and GAPDH RNA as an endogenous control for selectivity, were quantified by
real time RT-PCR.

Figure 1: Structures of novel HCV inhibitors
Scheme 1. Reagents and conditions: (a) R1X, Cs2CO3, DMF, rt to 100 °C; (b) chlorosulfonyl isocyanate, CH3CN/DMF, –20 °C
to 50 °C; (c) B(OiPr)3, LDA, THF, –78 °C; (d) 8, PdCl2(dppf), aqueous K2CO3, CH3CN, 60 °C; (e) B(OiPr)3, LDA, THF, –78 °C
then 8, PdCl2(dppf), aqueous K2CO3, DMF, 80 °C; (f) LDA, Bu3SnI, THF, –78 °C to 0 °C; (g) 8, Pd(PPh3)4 CuI, dioxane, 100
°C.
Scheme 2. Reagents and conditions: (a) c-BuBr, Cs2CO3, DMF, 90 °C, 48 h; (g) chlorosulfonyl isocyanate, CH3CN/DMF, –20
°C to rt.
Scheme 3. Reagents and conditions: (a) m-CPBA, CHCl3, 60 °C, overnight, 38%; (b) fuming HNO3 (90%), conc. H2SO4, 100 °C,
2 h, 91%; (c) DMF·DMA, DMF, 95 °C, 1 h; (d) 10% Pd/C, EtOH, rt, 3 h; (e) Fe, AcOH, 5 h, 52% over three steps; (f) c-BuBr,
Cs2CO3, DMF, 90 °C, 48 h; (g) chlorosulfonyl isocyanate, CH3CN/DMF, –20 °C to rt.
Scheme 4. Reagents and conditions: (a) c-BuNH2, DIEA, THF, 0 °C to rt; (b) K2CO3, MeOH, 60 °C, 8 h, 100% over two steps;
(c) trimethylsilylacetylene, PdCl2(dppf), CuI, Et3N, THF, 70 °C, 24 h; (d) K2CO3, MeOH, rt, 5 h, 79% over two steps; (e) CuI,
Barton base, DMF, 140 °C, 3 h, 82%; (f) HBr, AcOH, 100 °C, overnight, 77%; (g) ClF2CCO2Na, DMF, Cs2CO3, 75 °C, 2 h,
24%; (h) chlorosulfonyl isocyanate, CH3CN/DMF (1:1), 30−58%.
Scheme 5. Reagents and conditions: (a) c-BuBr, Cs2CO3, DMF, 60 °C; (b) chlorosulfonyl isocyanate, CH3CN/DMF (1:1), –20
°C, 80% over two steps; (c) bis(pinacolato)diborane, PdCl2(dppf), KOAc, dioxane, 80 °C, 3h, then Oxone®; (d) RMgBr, ZnCl2,
THF, PdCl2(dppf), –78 to 50 °C; (e) alkyl iodide or ClF2CCO2Na, DMF, Cs2CO3, 50 to 100 °C.

Fig. 1

CN
R1
B(OH)2
N
path A
N
c
d
R2
8
7
CN
CN
X
Y
(S)CF3
NH
O
S
a
e
b
R1
R1
R1
N
R1
N
N
N
5
8
N
R2
N
R2
N
N
O
H
path B
R2
f
3
2
4
g
CN
8
R1
SnBu3
N
path C
N
R2
(S)
CF3
NH
O
S
X
Y
6
Cl
O
8a; X=CH, Y=N
8b; X=CH, Y=CH
8c; X=N, Y=N
Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Table 1. Activity of selected 6-(azaindol-2-yl)pyridine-3-sulfonamides
HCV replicon
1b EC50a (nM)
230
GAPDH
IC50a (nM)
8900
Compd
2a
R
Azaindole
5-OMe
6-Me
6-Cl
4-
2b
2c
4-
119
94
>10000
2500
4-
2d
2e
6-Me
6-Cl
5-
230
260
530
1400
240
970
27
>10000
1310
5-
2f
5-Cl
6-
>10000
>10000
>10000
>10000
>9000
9700
2g
5-Me
6-OMe
6-OCHF2
5-OMe
6-OMe
5-Cl
6-
2h
2i
5,7-di
5,7-di
7-
2j
2k
2l
7-
960
17
7-
>10000
6300
2m
2n
2o
6-Cl
7-
700
13
5-Me
6-Me
7-
>10000
>10000
7-
87
a See Ref. 19 for assay conditions.

Table 2. Effect of various arene sulfonamides on replicon replication
HCV replicon 1b
EC50a (nM)
GAPDH
IC50a (µM)
Compd X, Y
2n
2p
2q
N, CH
13
>10000
>10000
>10000
CH, CH
N, N
87
160
a See Ref. 19 for assay conditions.

Table 3. Replicon inhibition and rat PK of selected 6-(7’-azaindol-2-yl)pyridine-3-sulfonamides
Rat PK (PO)b Plasma
Liver /
plasmae (6
h)
HCV replicon
GAPDH
Compd R1
R2
AUC0-6h
(nM·h)
(6 h) /
d
EC50
1b EC50a (nM) IC50a (nM)
2j
MeO
c-Bu
27
>9000
7606
n.d.c
30
14
2r
MeO
MeO
EtO
c-PrCH2
c-Pent
c-Bu
300
150
200
2150
7600
50
7900
n.d. c
n.d. c
n.d. c
n.d. c
n.d. c
36
n.d. c
n.d. c
n.d. c
n.d. c
n.d. c
7
2s
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>5160
n.d. c
n.d. c
n.d. c
n.d.
2t
2u
2v
2w
2l
n-PrO
i-PrO
c-Bu
c-Bu
F2HCO c-Bu
9365
2389
n.d. c
6844
6925
2628
10164
8779
5833
n.d. c
Cl
c-Bu
17
22
23
2x
2y
2n
2z
Cl
c- PrCH2 39
n.d. c
n.d. c
Cl
c- Pent
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
c-Bu
27
13
15
40
46
2
40
19
Me
Et
55
9
27
13
2aa
2ab
2ac
2ad
c-Pr
F
>10000
>10000
>10000
>10000
32
11
23
6
CF3
503
n.d. c
44
H
217
n.d. c
a See Ref. 19 for assay conditions.
b Compounds were lyophilized from acetonitrile/water (1:1) and administered as a suspension in 0.4% hydroxypropyl methylcellulose to male Sprague Dawley
rats (n = 2) PO at a dose of 10 mg/kg. Plasma samples were obtained from the rats for LC-MS/MS analysis at 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h post-dose.
c Not determined.
d Ratio of the plasma concentration measured at 6 h post-dose to the replicon EC50 value.
e Ratio of the liver concentration to the plasma concentration measured at 6 h post-dose.

Table 4. Pharmacokinetic profiles of selected compounds
Dog PK (PO,
Plasma concentration (6 h or 8 h) /
EC50
HCV
Compd replicon 1b AUC0-6h
Rat PK (PO)b
Monkey PK
(PO, 3 mpk)d
AUC0-24h (nM·h)
2 mpk)c
AUC0-24h
(nM·h)
EC50a (nM) (nM·h)
Monkeyf (8
Rate (6 h) Dogf (8 h)
h)
1
7
13412
7606
2389
6925
6844
5833
6858
4177
4081
5670
2067
8162
7568
2277
1035
641
277
30
33
6
46
4
2j
27
17
13
27
2
2l
22
12
14
2
2
2n
2y
2ac
52
1
290
40
0.3
105
5440
506
125
a See Ref. 19 for assay conditions.
b Compounds were lyophilized from acetonitrile/water (1:1) and administered as a suspension in 0.4% hydroxypropyl methylcellulose to male Sprague Dawley
rats (n = 2) PO at a dose of 10 mg/kg. Plasma samples were obtained from the rats for LC-MS/MS analysis at 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h post-dose.
c Compounds were lyophilized from acetonitrile / water (1:1) and administered as a suspension in 0.4% hydroxypropyl methylcellulose to male beagle dogs (n =
2) PO at a dose of 2 mg/kg. Plasma samples were obtained from the dogs for LC-MS/MS analysis at 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h post-dose.
d Compounds were lyophilized from acetonitrile/water (1:1) and administered as a suspension in 0.4% hydroxypropyl methylcellulose to male cynomolgus
monkeys (n = 2) PO at a dose of 3 mg/kg. Plasma samples were obtained from the cynomolgus monkeys for LC-MS/MS analysis at 30 min, 1 h, 2 h, 4 h, 8 h, and
24 h post-dose.
e Ratio of the plasma concentration measured at 6 h post-dose to the replicon EC50 value.
f
Ratio of the plasma concentration measured at 8 h post-dose to the replicon EC50 value.

Graphical abstract