II. Novel HCV NS5B polymerase inhibitors: Discovery of indole C2 acyl sulfonamides

Article abstract of DOI:10.1016/j.bmcl.2011.10.041

Development of SAR at the C2 position of indole lead 1, a palm site inhibitor of HCV NS5B polymerase (NS5B IC₅₀ = 0.053 μM, replicon EC₅₀ = 4.8 μM), is described. Initial screening identified an acyl sulfonamide moiety as an isostere for the C2 carboxylic acid group. Further SAR investigation resulted in identification of acyl sufonamide analog 7q (NS5B IC₅₀ = 0.039 μM, replicon EC₅₀ = 0.011 μM) with >100-fold improved replicon activity.

Full text of DOI:10.1016/j.bmcl.2011.10.041

Bioorganic & Medicinal Chemistry Letters 22 (2012) 713–717
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
II. Novel HCV NS5B polymerase inhibitors: Discovery of indole C2 acyl
sulfonamides
⇑
Gopinadhan N. Anilkumar , Oleg Selyutin, Stuart B. Rosenblum, Qingbei Zeng, Yueheng Jiang,
Tin-Yau Chan, Haiyan Pu, Li Wang, Frank Bennett, Kevin X. Chen, Charles A. Lesburg, Jose Duca,
Stephen Gavalas, Yuhua Huang, Patrick Pinto, Mousumi Sannigrahi, Francisco Velazquez,
Srikanth Venkatraman, Bancha Vibulbhan, Sony Agrawal, Eric Ferrari, Chuan-kui Jiang, H.-C. Huang,
Neng-Yang Shih, F. George Njoroge, Joseph A. Kozlowski
Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Development of SAR at the C2 position of indole lead 1, a palm site inhibitor of HCV NS5B polymerase
Received 31 August 2011
Revised 10 October 2011
Accepted 11 October 2011
Available online 20 October 2011
(NS5B IC50 = 0.053
lM, replicon EC50 = 4.8 lM), is described. Initial screening identified an acyl sulfon-
amide moiety as an isostere for the C2 carboxylic acid group. Further SAR investigation resulted in iden-
tification of acyl sufonamide analog 7q (NS5B IC50 = 0.039
improved replicon activity.
l
M, replicon EC50 = 0.011
lM) with >100-fold
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
HCV
NS5B Polymerase
Hepatitis C virus (HCV) is a major health concern affecting over
170 million individuals worldwide1a and is a leading cause of liver
transplants and chronic liver cirrhosis, leading to death from liver
disease in the United States.1b,c The current standard of care is
treatment with a combination of subcutaneous pegylated inter-
feron administration with oral dosing of the cytotoxic nucleoside
drug ribavirin.2 The response rate is >75% for HCV patients with
genotypes 2 and 3 after a 24 week treatment regimen, while geno-
type 1 patients have a response rate of less than 50% after 48 weeks
of treatment.3 Recent FDA approval of Victrelis (boceprevir) has
invigorated interest in small molecule inhibitors of HCV.4 With a
clear opportunity to improve clinical outcomes, and given the side
effects associated with the current standard of care, it is valuable to
discover potent inhibitors of HCV replication that will improve
outcomes and shorten treatment duration.
The HCV NS5B protein is an RNA-dependent RNA polymerase
critical for the synthesis of progeny viral genomes. The crystal
structure of HCV NS5B displayed an overall subdomain architec-
ture similar to other members of the PolI family,5 with a deep ac-
tive site cavity, located at the top of the ‘palm’ subdomain, and
sealed at its base by a unique b-loop. Furthermore, there was an
unexpected interaction identified between the tip of the ‘fingers’
subdomain and the ‘thumb’ subdomain to encircle the presumed
nucleoside triphosphate substrate entry trajectory.
Sequence variation analysis suggests that residues lining the ac-
tive site cavity (‘palm site’) are more conserved than in other
regions, making the palm site an attractive target for inhibition
of the viral polymerase. Clinical efficacy has been demonstrated
with non-nucleoside inhibitors binding at the palm, thumb, and
finger-loop subdomains.6
In a previous Letter,7 we described discovery of a novel class of
indole C2 carboxylic acids as non-nucleoside palm site inhibitors of
HCV NS5B polymerase exemplified by structure 1 (Fig. 1). The un-
ique feature of this class of inhibitors was the bidentate hydrogen
bonding interactions with the polypeptide backbone of Tyr-448
and Ile-447. These dual interactions anchor the inhibitors to the
palm site cavity. The lead compounds in this series exhibited good
NS5B inhibition activities and moderate antiviral properties in the
cell based replicon assay. The lead inhibitors all contained an in-
dole C2 carboxylic acid moiety, which did not make direct interac-
tion with the protein as revealed by X-ray structure. Several
potential sites for interactions with protein backbone prompted
investigation of elaborated carboxylic acid isosteres. Probable acyl-
glucuronide conjugation of an acid functionality was an added
incentive to look for suitable functionality at C2 position.
During the initial screening of C2 functionalities, a C5 chloro
group was chosen since this was well tolerated in the previous
SAR explorations. At the N1 position of the indole either an amino-
pyridylmethyl group or fluoro substituted benzyl moieties were
chosen, as these substitutions exhibited the best activity profile
in earlier studies. The C-linked pyridone was essential at the indole
⇑
Corresponding author.
E-mail address: gopinadhan.anilkumar@Merck.com (G.N. Anilkumar).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.041

714
G. N. Anilkumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 713–717
Table 1
Variation of C2 substitutions
N
O
Cl
O
NH
N
OH
O
R2
Cl
N
R1
N
NH2
1, NS5B IC50 = 0.053 uM
Replicon EC50 = 4.8 uM
R1
R2
IC50
(
lM)
EC50
4.8
(lM)
8
9
Compds
Figure 1. X-ray structure of complex between HCV NS5B and lead indole acid 1
bound in the palm site. The compound (yellow) makes two hydrogen bond
interactions (orange) with the backbone carbonyl oxygen and amide nitrogen of Ile-
447 and Tyr-448, respectively. In addition to these main chain atoms, side chain
atoms within 5 Å of the compound are shown as thin sticks. The C2 acid group
interactions with the protein indirectly via two water molecules labeled W1 and
W2. Hydrogen bond interactions are shown as dotted lines.
O
OH
1
0.053
>20
N
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
N
4a
4b
4c
4d
4e
4f
8.3
NA
0.3
NA
NA
NA
2.2
0.6
N
N
N
N
N
N
N
N
C3 position and anchors the inhibitors to the palm site of the en-
zyme as detailed above. Synthesis of most of the analogs at the
C2 position was accomplished as outlined in Scheme 1. Intermedi-
ate 2 was prepared as described previously.7 Hydrolysis of the C2
ester under basic conditions afforded the advanced intermediate
3 in nearly quantitative yields. Activation with carbonyl diimidaz-
ole and subsequent treatment with alkyl sulfonamides and DBU
afforded C2 acyl sufonamide intermediates. Final synthetic manip-
ulation to yield analogs 4c,i,j involved the demethylation of the
methoxy pyridine moiety by refluxing with 4 N HCl in dioxane.
Similarly, treatment with sulfonyl urea instead of an alkyl sulfon-
amide and subsequent demethylation reaction afforded C2 acyl
sulfamide analog 4g. For the synthesis of cyclic thiadiazine analog
4h, the activated acyl imidazole intermediate was treated with o-
amino phenyl sulfonamide to afford the corresponding phenyl acyl
sulfonamide intermediate. Subsequent cyclization and demethyla-
tion with 4 N HCl afforded C2 thiadiazine analog 4h in modest
yield.
O
O
>20
O
O
S
O
NH
0.018
O
OH
7
O
O
S
O
NH
2.2
The initial screening of the C2 position with cyano (4a), and ester
(4b) moieties showed very weak binding affinities (IC50 >20
compared to the carboxylic acid analog 1 (IC50 = 0.053 M) suggest-
ing the need for a H-bond donor (Table 1). Attempts to prepare the
C2 primary amide from either 4a or 4b failed, as it was readily hydro-
lyzed to the acid 1. Replacement of the C2 acid functionality with an
acyl sulfonamide provided 4c with enhanced binding affinity of
O
S
O
NH
lM)
>20
l
O
O
S
O
NH
NH2
4g
4h
0.029
0.012
IC50 = 0.018 lM and significantly improved replicon activity of
EC50 = 0.3
lM (>10-fold). Extending the acid and acyl sulfonamide
HN
N
S
O
O
NH
R5
O
O
O
O
S
O
N
4i
4j
0.033
0.041
3.0
0.23
2.2
NA
NA
8
HN SO2Me
NH
R1
F
4c,i,j
b,c
O
NH
O
S
O
N
R5
N
O
NH
O
R5
d,c
a
OMe
CO2H
R5
OMe
CO2R
F
F
F
F
F
F
F
F
N
HN SO2NH2
R1
N
O
S
N
R1
4g
O
NH
R1
e,f
4k
4l
2
NH
O
3
R5
H
N
O
S
O
NH2
N
N
2.7
R1
S
O2
4h
H
N
Scheme 1. General synthesis of C2 analogs. Reagents and conditions: (a) aq 1 N
LiOH, THF, reflux; (b) CDI, Me-SO2NH2, DBU, THF, reflux; (c) 4 N HCl/dioxane, 90 °C;
(d) CDI, NH2SO2NH2, DBU, THF, reflux; (e) CDI, o-NH2-PhCONH2, DBU, THF, reflux;
(f) 4 N HCl/dioxane, MeOH, 90 °C.
4m
0.57
N

G. N. Anilkumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 713–717
715
moieties with a methylene linker to enable the inhibitor to achieve
additional interactions in this region was not successful. All corre-
sponding analogs (4d, 4e) showed diminished enzyme activities.
Moreover, the sulfonamide functionality without the carbonyl lin-
ker at C2 showed weak activity (4f). These results pointed toward
the critical importance of a sp2 center at the C2 position for activity.
The acyl sulfamide analog 4g showed good binding activity with a
vs EC50 = 0.7 lM). Thus the C-linked pyridone was chosen as the
optimal C3 functionality for further SAR development with a goal
of achieving another log reduction in the replicon activity.
Further SAR exploration with a variety of C2 acyl sulfonamide
analogs is represented in Table 3. The syntheses of these analogs
were similar to the methyl sulfonamide analog, 4c shown in
Scheme 1. In general alkyl and aryl variations were tolerated at
the C2 acyl sulfonamide moiety. None of these variations (6a–g)
showed a significant improvement in replicon activity including
the extended alkyl analogs with substitutions (6h–i). The fact that
phenyl analog 6c showed comparable activity to methyl analog 4c
reduced cell based activity (EC50 = 2.2 lM). We also explored het-
erocyclic modifications containing a sulfonyl group which could
present the groups in a different spatial orientation. The benzothi-
adiazine analog 4h showed excellent enzyme activity while main-
taining sub-micromolar replicon activity (IC50 = 0.012
lM,
was encouraging (EC50 = 0.4 lM vs EC50 = 0.3 lM). We hypothe-
EC50 = 0.6 M). Since fluoro substituted benzyl groups were well
tolerated at the N1 indole position, additional C2 variations were
targeted in this series. The C2 acyl sulfonamide analogs with 2-fluo-
l
sized phenyl would be a suitable tether to explore functionalities
which could make some new interactions with the protein
backbone.
robenzyl (4i, EC50 = 0.23
lM) and 2,4-difluorobenzyl groups (4j,
EC50 = 2.2 M) at N1, showed comparable enzyme activities while
l
the former displayed better replicon activity. Reverse sulfonamides
(4k, 4l) and benzimidazole (4m) substitutions were among the
many functionalities at the C2 position that were not tolerated.
The initial C2 SAR clearly indicated that the methyl acyl sulfonamide
group was the most preferred moiety at this position. The fact that
acyl sulfonamide can also act like a tether to extend new functional
groups further into in this area prompted exploration of substituted
sulfonamides.
Table 3
Variation of C2 acyl sulfonamides
NH
O
Cl
O
O
S
O
R8
N
R1
HN
In our earlier publication we have identified other heterocycles
such as C-linked and N-linked pyrimidine diones at the indole C3
position which make similar interactions with the protein back-
bone and showed a comparable activity profile to the pyridone.7
It was appropriate to benchmark the C2 acyl sulfonamides with
C3 pyridone to the C3 pyrimidinedione analogs (Table 2). In com-
parison to pyridone derivative 4c, the C-linked pyrimidinedione
analog 5a showed a comparable enzyme activity but a significantly
8
9
Compds
R1
R8
IC50
(lM)
EC50
(lM)
4c
Me
0.018
0.018
0.031
0.016
0.024
0.3
N
NH2
NH2
NH2
NH2
NH2
6a
6b
6c
6d
Et
0.6
1.0
0.4
1.2
N
N
N
N
reduced cell based activity (EC50 = 0.3 lM vs EC50 = 3.0 lM). Simi-
larly the N-linked bicyclic pyrimidinedione derivative 5c also
showed a comparable enzyme activity, but a reduced replicon
activity to the corresponding pyridone analog 5b (EC50 = 0.12
lM
Table 2
Comparison of the C3 heterocycles in C2 acyl sulfonamide series
Ph
Bn
R3
R5
O
O
N
HN
S
O
R1
8
9
Compds
R5
Cl
R1
R3
IC50
(lM)
EC50
0.3
(lM)
6e
6f
0.005
0.013
0.028
0.008
0.005
0.27
0.4
NH
F
F
F
F
F
O
4c
0.018
0.027
N
NH2
NH2
O
HN
NH
5a
Cl
3.0
N
6g
6h
6i
1.4
O
NH
Cl
2.8
5b
5c
CF3
CF3
0.006
0.015
0.12
0.7
O
F
F
H
N
S
O
S
0.47
NH
O
O
N
O

716
G. N. Anilkumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 713–717
Table 4
Table 4 (continued)
Substitutions of C2 phenyl acyl sulfonamides
8
9
Compds
R1
R8
IC50
(lM)
EC50
(lM)
NH
CN
O
O
Cl
7m
0.036
0.032
0.027
0.034
0.039
1.7
N
O
S
N
HN
R8
NH2
NH2
NH2
NH2
NH2
NH2
R1
O
CO2Me
Compds
R1
R8
IC50
(l
M)
EC50
0.4
(lM)
8
9
7n
7o
7p
7q
1.1
0.3
1.9
N
N
N
N
N
6c
0.016
0.022
0.019
0.031
0.043
0.028
0.026
0.049
0.034
0.028
0.036
0.035
0.024
N
O
OMe
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
OMe
7a
7b
7c
7d
7e
7f
0.5
0.6
0.8
1.5
0.7
0.7
1.6
1.9
1.7
1.0
0.4
0.1
N
N
N
N
N
N
N
SO2NH2
Me
O
S
O
O
HN
0.011
F
F
F
O
S
NH
7r
7s
0.02
0.5
Cl
O
S
O
HN
0.018
0.02
F
F
F
Table 4 represents a summary of selected SAR efforts in the phe-
nyl acyl sulfonamide substitutions. Differentially substituted phe-
nyl acyl sulfonamides were synthesized using a similar route as
before (Scheme 1). Various lipophilic mono substitutions on the
phenyl ring such as methoxy, methyl, fluoro and chloro showed
no improvement in the replicon activity (7a–d, EC50 >0.5
Additionally the disubstituted phenyl ring as exemplified in 7e–
h, also resulted in diminished replicon activities (EC50 >0.7 M)
F
F
F
lM).
7g
7h
7i
l
Me
compared to parent phenyl analog 6c. Since lipophilic substitutions
in general were not desirable, we turned our attention to polar
substitutions which are more likely to make additional interactions
N
N
NH2
NH2
H2N
NH
O
Cl
NHSO2Me
O
NH2
O
S
N
HN
7j
O
N
N
N
NH2
NH2
NH2
N
NH2
NH2
7q, NS5B IC50 = 0.039 uM
Replicon EC50 = 0.011 uM
7k
7l
Figure 2. X-ray structure of complex between HCV NS5B and extended acyl
sulfonamide 7q bound in the palm site. As in the previous complex, the compound
is shown in yellow and hydrogen bonds interactions are shown as dotted lines. Side
chain atoms within 5 Å of the compound are shown as thin sticks. Water molecule
W1 remains in the identical position as observed in the complex with 1, and
compound 7q makes indirect interactions with the protein via water W3. There are
direct compound/protein interactions with the side chain atoms of Asp-318 and
Asn-291.10,11
OH

G. N. Anilkumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 713–717
717
Table 5
ated, most of the acyl sulfonamide substitutions did not enhance
replicon activity further. Appropriate polar substitutions in the
extended phenyl acyl sulfonamide series such as 7q
PK profile of lead HCV NS5B inhibitors
7
8
Compds
IC50
(l
M)
EC50
(lM)
Rat AUCc (nM h)
(EC50 = 0.01 lM) and 7s (EC50 = 0.02 lM) showed an additional
4c
4i
7q
7s
0.018
0.033
0.039
0.018
0.3
0.23
0.011
0.02
740
11,400
0
one log improvement in the replicon activity. This work gave rise
to highly potent HCV inhibitors suitable for further development.
Further optimization in this series to identify a HCV NS5B inhibitor
with improved pharmacokinetic properties will be reported in sep-
arate publications.
14
c
PO, Sprague-Dawly rat, 10 mpk, 6 h, 0.4% MC.
References
and also improve cell permeability. Among the different amino
substituted analogs, the ortho and meta substitutions (7i and 7j,
1. (a) Cohen, J. Science 1999, 285, 26; (b) Alter, M. J.; Kruszon-Moran, D.; Nainan,
O. V.; McQuillan, G. M.; Gao, F.; Moyer, L. A.; Kaslow, R. A.; Margolis, H. S. N.
Eng. J. Med. 1999, 341, 556; (c) Cuthbert, J. A. Clin. Microbiol. Rev. 1994, 7, 505;
(d) Monto, A.; Wright, T. L. Semin. Oncol. 2001, 441.
2. (a) Neumann, A. U.; Lam, N. P.; Dahari, H.; Gretch, D. R.; Wiley, T. E.; Layden, T.
J.; Perelson, A. S. Science 1998, 282, 103; (b) Rosen, H. R.; Gretch, D. R. Mol. Med.
Today 1999, 5, 393; (c) Di Bisceglie, A. M.; McHutchison, J.; Rice, C. M.
Hepatology 2002, 35, 224.
EC50 >1
para amino analog (7k, EC50 = 0.4
sponding para hydroxy derivative showed a four fold improvement
in the replicon activity (7l, EC50 = 0.1 M). Other hydrogen bond
l
M) showed decreased replicon activity compared to the
lM). Interestingly the corre-
l
acceptor groups such as cyano and methyl ester at this position
were not tolerated for replicon activity. Surprisingly the meta
3. Zeuzem, S.; Berg, T.; Moeller, B.; Hinrichsen, H.; Mauss, S.; Wedemeyer, H.;
Sarrazin, C.; Hueppe, D.; Zehnter, E.; Manns, M. P. J. Viral Hepat. 2009, 16, 75.
4. Venkatraman, S.; Bogen, S.; Arasappan, A.; Bennet, F.; Chen, K.; Jao, E.; Liu, Y.-T.;
Lovey, R.; Hendreta, 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, T. Y.; Brisson, J.-M.; Prelusky, D.;
Korfmacher, W.; 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. (a) Lesburg, C. A.; Cable, M. B.; Ferrari, E.; Hong, Z.; Mannarino, A. F.; Weber, P.
C. Nat. Struct. Biol. 1999, 6, 937; (b) Bressanelli, S.; Tomei, L.; Roussel, A.; Incitti,
I.; Vitale, R. L.; Mathieu, M.; De Francesco, R.; Rey, F. A. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 13034.
6. (a) Beaulieu, P. L. Expert Opin. Ther. Patents 2009, 19, 145; (b) Ago, H.; Adachi, H.
T.; Yoshida, A.; Yamamoto, A.; Habuka, N.; Yatsunami, K.; Miyano, M. Structure
1999, 7, 1417.
methyl ester compound 7o (EC50 = 0.3
improved replicon activity compared to para isomer 7n
(EC50 = 1.1 M). This prompted further exploration of the meta po-
sition with similar functionalities. The sulfonamide analog 7p
showed weak cell based activity. Unexpectedly a reverse methyl
lM) showed appreciably
l
sulfonamide analog 7q (EC50 = 0.011 lM) showed another log
improvement in the replicon activity (2log improvement com-
pared to the aniline 7j), thus providing highly potent HCV inhibitor
for further evaluation. The importance of meta substitution was
further confirmed by loss of activity of corresponding para isomer
7r. Other optimized N1 substitutions such as fluoro benzyl also
showed excellent replicon activity in this series as exemplified
by 7s (EC50 = 0.02 lM).
The X-ray structure of the most potent analog 7q with NS5B
showed that it resides in the expected location in the palm site (
Fig. 2). Previously observed hydrogen bonding interactions with
the backbone of Ile-447 and Tyr-448 were maintained. Further-
more, the extended C2 acyl sulfonamide group displaced one of
the two water molecules observed in the C2 carboxylic acid struc-
ture which are part of the hydrogen bonding network between the
compound and the protein. The compound (7q) forms a new indi-
rect protein interaction via an additional ordered water molecule.
The phenyl ring of 7q stacks in a face-to-face manner with the
N1 aminopyridine group. The terminal methyl sulfonamide moiety
makes a series of polar interactions with both backbone and side
chain atoms within a preexisting polar sub pocket.
7. Anilkumar, G. N.; Lesburg, C. A.; Selyutin, O.; Rosenblum, S. R.; Zeng, Q.; Jiang,
Y.; Chan, T.-Y.; Pu, H.; Vaccaro, H.; Wang, L.; Bennett, F.; Chen, K. X.; Duca, J.;
Gavalas, S.; Huang, Y.; Pinto, P.; Sannigrahi, M.; Velazquez, F.; Venkatraman, S.;
Vibulbhan, B.; Agrawal, S.; Butkiewicz, N.; Feld, B.; Ferrari, E.; He, Z.; Jiang, C. K.;
Palermo, R. E.; Mcmonagle, P.; Huang, H. C.; Shih, N. Y.; Njoroge, G.; Kozlowski,
J. A. Bioorg. Med. Chem. Lett. 2011, 21, 5336.
8. HCV NS5B polymerase activity was measured in a radiolabeled nucleotide
incorporation assay as described [Cheng 2010], in a reaction buffer containing
20 mM HEPES (pH 7.3), 7.5 mM DTT, 20 units/mL RNasIN, 0.1
UTP, 60
Ci/ml [33P]-CTP supplemented to 20 nM CTP, 10 mM MgCl2, 60 mM
NaCl, 100 g/ml BSA, 100 nM heteropolymer RNA template, 0.25 mM
trinucleotide initiator and 30 nM NS5B ( 21) enzyme. Reaction was allowed
lM GTP, ATP and
l
l
D
to proceed for 150 min at room temperature and terminated by EDTA. The
reaction mixture was washed on Millipore DE81 filter plate and the
incorporated labeled CTP quantitated by Packard TopCount. Compound IC50
values were calculated from experiments with 10 serial twofold dilutions of
the inhibitor in duplicate.
After the SAR for replicon activity at C2 position was established,
attention was turned to the pharmacokinetics of the lead acyl sul-
fonamide series (Table 5). Initial methyl sulfonamide analog 4c
demonstrated moderate exposure in a rat when orally dosed
(AUC = 740 nM h, 10 mpk, 6 h, MC). The change of the N1 substitu-
tion to a fluoro benzyl, compound 4i, had a profound improvement
in the oral exposure (AUC = 11,400 nM h, 10 mpk, 6 h, MC).12
Unfortunately the most active extended sulfonamide analogs 4q
and 4s showed very low oral exposure irrespective of the N1 substi-
tutions presumably due to high PSA and high molecular weight.13
In summary, modifications at the C2 position of indole 1, led to
the discovery of acyl methyl sulfonamide moiety as an isosteric
replacement for the carboxylic acid functionality, with approxi-
mately one log improvement in the replicon activity. While toler-
9. To measure cell-based anti-HCV activity, replicon cells (1b-Con1) were seeded
at 5000 cells/well in 96-well plates one day prior to inhibitor treatment.
Various concentrations of an inhibitor in DMSO were added to the replicon
cells, with the final concentration of DMSO at 0.5% and fetal bovine serum at 5%
in the assay media. Cells were harvested 3 days post dosing. The replicon RNA
level was measured using real-time RT-PCR (Taqman assay) with GAPDH RNA
as endogenous control. EC50 values were calculated from experiments with 10
serial twofold dilutions of the inhibitor in duplicate.
10. Figures 1and 2 were generated using the program PYMOL (The PYMOL Molecular
Graphics System, Version 1.2r1, Schrödinger, LLC).
11. Crystal structures of HCV NS5B in complex with 1 and 7q have been deposited
in the Protein Data Bank with accession numbers 3U4O, 3U4R, respectively.
12. ClogP and PSA measurements of compounds 4c (ClogP = 1.43, polar surface
area = 138 Å2) and 4i (ClogP = 3.39, polar surface area = 100 Å2).
13. ClogP and PSA measurements of compounds 7q (ClogP = 2.25, polar surface
area = 185 Å2) and 7s (ClogP = 4.2, polar surface area = 148 Å2).