Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1317
Supporting Information Available: Protocols for bio-
logical assays, synthetic procedures, and spectral data for
5-22. This material is available free of charge via the Internet
indole nitrogen has little impact on enzyme affinity. In
contrast, the nature of the aryl substituent at the C2
position of the indole proved crucial for intrinsic potency.
Substituents at the ortho position of the phenyl ring
were detrimental to activity (15, Table 2), but gains
could be made through introduction of functionality at
the meta or especially para positions. Small, lipophilic
substituents proved optimal in this regard, with 17-
21 showing between a 3- and 10-fold improvement over
the unsubstituted 2-phenylindole 13. Improved cell-
based activity was also achieved, albeit at times to a
lesser extent. 21 highlights an extreme case, with poorer
activity than 13 being observed in the cell despite 10-
fold higher enzyme affinity. A likely explanation is that
introduction of lipophilic functionality causes higher
binding to serum proteins present in the assay media,
the consequence being a reduced intracellular concen-
tration of the inhibitor. In line with this hypothesis,
compounds in this series showed potent inhibition of
HCV replication when the cell-based assay was per-
formed in the absence of serum proteins (e.g., 17, EC50
) 0.13 µM; 21 showed 85% inhibition at 0.3 µM). It is
apparent that a challenge for future work in this area
is the alignment of high enzyme affinity with efficacy
in a physiologically relevant cell-based assay system.
Nonetheless, application of the SAR described in Table
2 in the more active morpholineacetamide series pro-
vided compounds such as 22 (EC50 ) 0.3 µM) that
already show an improvement in inhibition of HCV RNA
replication over 1 (EC50 ) 0.5 µM) despite lower mo-
lecular weight and reduced structural complexity. Im-
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portantly, no cytotoxicity was observed for compounds
of this type, the CC50 values for compounds 13-22 being
above 50 µM.
We have described here the development of a series
of indole-N-acetamides that are potent allosteric inhibi-
tors of the HCV NS5B enzyme. Preliminary optimiza-
tion of this series furnished compounds that show
submicromolar blockade of subgenomic HCV RNA rep-
lication in HUH-7 cells. These results, together with the
encouraging profile shown by the initial lead 13 in in
vivo pharmacokinetic studies and in vitro counter-
screens, highlight the potential of this series for the
development of new anti-HCV agents.
Acknowledgment. We thank Sergio Altamura and
Licia Tomei for enzymology; Fabrizio Fiore and Odalys
Gonzalez-Paz for PK analysis; Marina Taliani, Mark
Jairaj, and Claudio Giuliano for metabolism studies;
Fabio Bonelli, Alan Bishop, and Maria Verdirame for
analytical work; Dr. Dennis Dean (Merck & Co., Rah-
(20) The log P of an indole is typically 0.5-1 log unit higher than
that of the corresponding benzimidazole.
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3
way, NJ) for provision of H-13; and Nadia Gennari,
Monica Bisbocci, and Ottavia Cecchetti for biological
testing. This work was funded in part by a grant from
the MIUR.
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