1892 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Letters
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benzylmercaptan at the C-2 carbon (16, Figure 2). An
attack at the C-3 carbon would result in a chemical shift
of the C-3 hydrogen at 3.59 ppm (17, Figure 2) as
predicted by the additivity rules21 as used in Chem-
Draw. There was essentially no further change in the
spectrum after 48 h, which indicates that probably a
stable thioether adduct had formed. The dienyl deriva-
tive showed no change in its spectrum when reacted
with benzylmercaptan for 48 h. The NMR shift predic-
tions by ChemDraw are generally not infallible; how-
ever, the greater reactivity of acrylates, the lack of
inhibitory potency of compounds such as the dienyl
derivative, and the NMR data supports our hypothesis
of a C-2 thioalkylation.
Aza-peptide Michael acceptors designed with the
appropriate P1 amino acids are potent and specific
inhibitors for clan CD cysteine proteases. The second-
order inhibition rate constants are as high as 3 280 000
M-1 s-1. The inhibitors with the appropriate peptide
sequence for the targeted enzyme do not show any cross
reactivity with clan CA cysteine proteases such as
papain, cathepsin B, and calpain. There is also little to
no cross reactivity toward the other members of clan
CD cysteine proteases. Hence, the aza-peptide Michael
acceptor design is clearly specific for clan CD cysteine
proteases. Currently, we are trying to refine the Michael
acceptor inhibitor design22 in the P′ portion to obtain
greater specificity, and we plan to test the caspase
specific inhibitors with all other caspases.
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(17) Thornberry, N. A.; Rano, T. A.; Peterson, E. P.; Rasper, D. M.;
Timkey, T.; Garcia-Calvo, M.; Houtzager, V. M.; Nordstrom, P.
A.; Roy, S.; Vaillancourt, J . P.; Chapman, K. T.; Nicholson, D.
W. A combinatorial approach defines specificities of members
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established for key mediators of apoptosis. J . Biol. Chem. 1997,
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(18) Wei, Y.; Fox, T.; Chambers, S. P.; Sintchak, J .; Coll, J . T.; Golec,
J . M.; Swenson, L.; Wilson, K. P.; Charifson, P. S. The structures
of caspases-1, -3, -7 and -8 reveal the basis for substrate and
inhibitor selectivity. Chem. Biol. 2000, 7, 423-432.
(19) Caffrey, C. R.; Mathieu, M. A.; Gaffney, A. M.; Salter, J . P.; Sajid,
M.; Lucas, K. D.; Franklin, C.; Bogyo, M.; McKerrow, J . H.
Identification of a cDNA encoding an active asparaginyl en-
dopeptidase of Schistosoma mansoni and its expression in Pichia
Pastoris. FEBS Lett. 2000, 466, 244-248.
(20) Freidig, A. P.; Verhaar, H. J . M.; Hermens, J . L. M. Comparing
the potency of chemicals with multiple modes of action in aquatic
toxicology: Acute toxicity due to narcosis versus reactive toxicity
of acrylic compounds. Environ. Sci., Technol. 1999, 33, 3038-
3043.
(21) Buergin-Schaller, R.; Arnold, C.; Pretsch, E. New parameters
for predicting 1H NMR chemical shifts of protons attached to
carbon atoms. Anal. Chim. Acta 1995, 312, 95-105.
(22) At least two other Michael acceptors (Ruprintrivir and CRA-
3316) are currently in clinical trials: (a) Graul, A.; Castaner, J .
AG-7088: anti-rhinovirus drug, HRV 3C protease inhibitor.
Drugs Fut. 2000, 25, 9-15. (b) Matthews, D. A.; Dragovich, P.
S.; Webber, S. E.; Fuhrman, S. A.; Patick, A. K.; Zalman, L. S.;
Hendrickson, T. F.; Love, R. A.; Prins, T. J .; Marakovits, J . T.;
Zhou, R.; Tikhe, J .; Ford, C. E.; Meador, J . W.; Ferre, R. A.;
Brown, E. L.; Binford, S. L.; Brothers, M. A.; DeLisle, D. M.;
Worland, S. T. Structure-assisted design of mechanism-based
irreversible inhibitors of human rhinovirus 3C protease with
potent antiviral activity against multiple rhinovirus serotypes.
Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 11000-11007.
Ack n ow led gm en t. The work was supported by
grants from the National Institute of General Medical
Sciences (GM61964), the NIH (AI053247) and the
Sandler Family Support Foundation. J an Potempa
acknowledges a grant from the Committee of Scientific
Research (KBN, Poland, 3 PO4A 002 24). M.G. and B.R.
acknowledge the Graduate Assistance in Areas of
National Need (GAANN) fellowship from the U.S.
Department of Education. K.J . acknowledges a fellow-
ship from the Center for the Study of Women, Science,
and Technology (WST) at Georgia Tech, and J . A.
acknowledges a fellowship from the Molecular Design
Institute under prime contract from the Office of Naval
Research.
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
dures, final product characterization, and enzyme assays. This
material is available free of charge via the Internet at http://
pubs.acs.org
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