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replicon assay without inherent cytotoxicity. One of
such promising compounds, 6a, also demonstrated
favorable drug exposure in liver and plasma upon oral
dosing. In view of these data, we believe that further
optimization of 6a could lead to a novel class of pepti-
dyl a-ketoamides with therapeutic potential.
11. a-Ketoacids: PCT Patent: WO/99/64442, 10/6/1998.
12. (a) a-Ketoamides: Bennett, J. M.; Campbell, A. D.;
Campbell, A. J.; Carr, M. G.; Dunsdon, R. M.; Greening,
J. R.; Hurst, D. N.; Jennings, N. S.; Jones, P. S.; Jordan,
S.; Kay, P. B.; O’Brien, M. A.; King-Underwood, J.;
Raynham, T. M.; Wilkinson, C. S.; Wilkinson, T. C. I.;
Wilson, F. X. Bioorg. Med. Chem. Lett. 2001, 11, 355. (b)
Han, W.; Hu, Z.; Jiang, X.; Decicco, C. P. Bioorg. Med.
Chem. Lett. 2000, 10, 711. (c) PCT Patent (BI): WO/99/
07733, 11/8/1997. (d) Tung, R.; Harbeson, S. L.;
Deininger, D. D.; Murcko, M. A.; Bhisetti, G. R.; Farmer,
L. J. US Patent (Vertex): 6,265,380 B1, 7/24/2001.
13. PCT Patent (Eli Lilly): WO/02/18369 A2, Mar. 7, 2002.
14. Prime site inhibitors: Ingallinella, P.; Fattori, D.; Alta-
mura, S.; Steinkuler, C.; Koch, U.; Cirero, D.; Bazzo, R.;
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15. HOAT/DCC(DIC) was used to minimize racemization
occurred at a-carbon during peptide coupling; cf.: Car-
pino, L. A.; El-Faham, A. Tetrahedron 1999, 55, 6813.
16. For the synthesis of racemic form of compound II, see:
Monn, J. A.; Valli, M. J. J. Org. Chem. 1994, 59, 2773.
The enantiomerically pure version of compound II was
obtained via chiral HPLC separation.
Acknowledgements
We shall thank the chemists at Vertex Pharmaceuticals
for sharing their expertise in connection to HCV pro-
tease inhibitor design with us. J. Catlow and J. Eckstein
are acknowledged for the efforts in conducting the in
vivo exposure studies. We are also indebted to Drs. J.
Audia, C. Lopez, and G. Cassell for helpful discussions
and encouragement.
References and notes
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17. P1-Nva a-hydroxyacid was prepared from Z-Val(H) via
its corresponding cyanohydrin intermediate.
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19. HCV NS3 protease domain was expressed and purified as
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custom synthesized by American Peptide Com (CA,
USA). Cleavage of EDVVAbuC-p-nitroanilide (500 uM)
substrate by purified HCV NS3 protease (0.5 uM) was
performed at 30 ꢃC in the buffer containing 30 uM NS4A
fragment, 46 mM Hepes, pH 8.0, 92 mM NaCl, 18% gly-
cerol, 5 mM DTT, and 7.5% DMSO in the absence or
presence of the testing compound. The compounds of this
invention were tested for their ability to inhibit HCV NS3
protease activity using a spectrophotometric assay with
EDVVAbuC-p-nitroanilide as substrate. All cleavage reac-
tions were run in a 96-well microtiter plate and monitored
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, Km and
Vmax=Km were generated under the conditions described
above. Ki values were calculated from rate versus (inhi-
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strate, by a nonlinear least squares fit of the data to the
equation of Morrison for tight binding competitive inhibi-
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The Prism program (GraphPad Software) was used for this
procedure.
8. (a) Boronic acids: Attwood, M. R.; Bennett, J. M.; Camp-
bell, A. D.; Canning, G . G . M.; Carr, M. G .; Conway, E.;
Dunsdon, R. M.; Greening, J. R.; Jones, P. S.; Kay, P. B.;
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including elastase, chymotrypsin, trypsin, kallikrein, plas-
min, thrombin, Factor Xa, and cathepsins B, G, and L
were selected. Assays were performed using the conditions
substrates suggested by the manufacturer.
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23. Four-step sequence overall yields for 4a: 51%; 4b: 70%;
5a: 65%; 5b: 71%; 5c: 52%; 6a: 51%; 6b: 59%; 6c: 20%;
6d: 31%; 7b: 44%; 7c: 23%; 7d: 32%; 8a: 42%; 8c: 44%.