348 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Letters
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M.; Steinkuehler, C.; Tomei, L.; De Francesco, R.; Kuo, L. C.;
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Colarusso, S.; De Francesco, R.; Matassa, V. G.; Sollazzo, M.
Inhibition of the Hepatitis C virus NS3/4A Protease: The crystal
structures of two protease-inhibitor complexes. J . Biol. Chem.
2000, 275, 7152-7157.
(9) Koch, U.; Biasiol, G.; Brunetti, M.; Fattori, D.; Pallaoro, M.;
Steinkuehler, C. Role of Charged Residues in the Catalytic
Mechanism of Hepatitis C Virus NS3 Protease: Electrostatic
Precollision Guidance and Transition-State Stabilization. Bio-
chemistry 2001, 40, 631-640.
(10) Steinkuehler, C.; Biasiol, G.; Brunetti, M.; Urbani, A.; Koch, U.;
Cortese, R.; Pessi, A.; De Francesco, R. Product Inhibition of the
Hepatitis C Virus NS3 Protease. Biochemistry 1998, 37, 8899-
8905.
(11) Ingallinella, P.; Altamura, S.; Bianchi, E.; Taliani, M.; Ingenito,
R.; Cortese, R.; Francesco, R. D.; Steinkuehler, C.; Pessi, A.
Potent Peptide Inhibitors of Human Hepatitis C Virus NS3
Protease Are Obtained by Optimizing the Cleavage Products.
Biochemistry 1998, 37, 8906-8914.
(12) Llinas-Brunet, M.; Bailey, M.; Fazal, G.; Ghiro, E.; Gorys, V.;
Goulet, S.; Halmos, T.; Maurice, R.; Poirier, M.; Poupart, M. A.;
Rancourt, J .; Thibeault, D.; Wernic, D.; Lamarre, D. Highly
potent and selective peptide-based inhibitors of the hepatitis C
virus serine protease: towards smaller inhibitors. Bioorg. Med.
Chem. Lett. 2000, 10, 2267-2270.
(13) Narjes, F.; Brunetti, M.; Colarusso, S.; Gerlach, B.; Koch, U.;
Biasiol, G.; Fattori, D.; De Francesco, R.; Matassa, V. G.;
Steinkuehler, C. R-Ketoacids Are Potent Slow Binding Inhibitors
of the Hepatitis C Virus NS3 Protease. Biochemistry 2000, 39,
1849-1861.
(14) Narjes, F.; Koehler, K. F.; Koch, U.; Gerlach, B.; Colarusso, S.;
Steinkuhler, C.; Brunetti, M.; Altamura, S.; De Francesco, R.;
Matassa, V. G. A designed P1 cysteine mimetic for covalent and
noncovalent inhibitors of HCV NS3 protease. Bioorg. Med. Chem.
Lett. 2002, 12, 701-704.
(15) Colarusso, S.; Gerlach, B.; Koch, U.; Muraglia, E.; Conte, I.;
Stansfield, I.; Matassa, V. G.; Narjes, F. Evolution, Synthesis
and SAR of Tripeptide R-Ketoacid Inhibitors of the Hepatitis C
virus NS3/NS4A Serine Protease. Bioorg. Med. Chem. Lett. 2002,
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tions are maintained. The model also shows that
introduction of the carboxylate into the para position
as in 19 is most favored due to close contacts with the
charged groups of both Lys136 and Arg109. Introduction
of the carboxylate into the ortho-position places it into
an apolar environment where the energy penalty for
desolvation is detrimental for activity. Compared to the
substrate, the amide function of the phenethyl amide
is slightly shifted out of the active site into the direction
of Lys136 (Figure 1), and we speculate that this shift
may increase the reaction barrier sufficiently to sup-
press cleavage.
In summary, a new series of reversible, competitive
inhibitors of HCV NS3 protease has been developed,
which are characterized by the presence of a phenethyl
amide group in the P1-prime position. While tripeptide
ketoacid 2 (IC50 0.46 µM) is more potent than phenethyl
amides 18 or 19, these inhibitors lack the electrophilic
carbonyl and explore novel interactions in the prime side
of the NS3/4A protease, which renders them highly
specific. The SAR, mutagenesis data, and molecular
modeling suggest a precise binding arrangement for the
phenethyl amide ring against the lipophilic chain of
lysine-136.
Ack n ow led gm en t. The authors thank Mirko Bru-
netti, Gabriella Biasiol, Sergio Serafini and Mauro
Cerretani for determination of the IC50 and Ki values
for NS3 and for elastase. The authors also would like
to thank Dr. R. Kim (MRL) for suggestions about the
use of cysteine in solid-phase synthesis.
Su p p or tin g In for m a tion Ava ila ble: Experimental sec-
tion including information on synthesis and biological evalu-
ation for compounds 3a , 4c, 5b, 11e, 12d , 17, 18, 19. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(16) Landro, J . A.; Raybuck, S. A.; Luong, Y. P. C.; O’Malley, E. T.;
Harbeson, S. L.; Morgenstern, K. A.; Rao, G.; Livingston, D. J .
Mechanistic Role of an NS4A Peptide Cofactor with the Trun-
cated NS3 Protease of Hepatitis C Virus: Elucidation of the
NS4A Stimulatory Effect via Kinetic Analysis and Inhibitor
Mapping. Biochemistry 1997, 36, 9340-9348.
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