3536
S.-H. Chen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3531–3536
Table 5, it is clear that the eletrophilic keto moiety in 4N
is needed for optimal antiviral activity. In contrast,
analogues lacking such ‘keto’ functionality (e.g., 5A and
5B) are devoid of significant antiviral activities.
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Conclusion
Starting from our initial screen hit 4A, we synthesized
and evaluated a number of 4A based a-ketoamides
containing modification(s) at P3, P2, P1 and P10 posi-
tion. The most potent HRV 3C inhibitor identified
through this effort, 4I exhibited impressive activity in
both the enzyme assay (IC50=0.17 mM) and cell based
assay (EC50 ꢀ0.85 mM against HRV-14). Furthermore,
we also found that deletion of the a-keto moiety from 4
resulted in significant loss of antiviral activity (4N vs 5A).
13. c.f. Monn, J. A.; Valli, M. J. J. Org. Chem. 1994, 59, 2773.
The enatiomerically pure material was obtained via chiral
HPLC separation.
Acknowledgements
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We would like to thank Drs. J. Munroe, J. Colacino,
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agement.
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19. P1-Nvaline bearing a-hydroxyacid was prepared from Z-
Nva(H) via its corresponding cyanohydrin intermediate.
20. HRV enzyme assay: All compounds synthesized were tes-
ted for their ability to inhibit HRV 3C protease activity (with
0.1 uM enzyme concentration) using a colorimetric assay with
EALFQ-p-nitroanilide as substrate. All cleavage reactions
were initiated by the addition of 0.4 mM peptide substrate and
monitored for 60 min. Ki values were calculated according to
the following reference: Morrison, J. F. Biochim. Biophys.
Acta. 1969, 185, 269 for tight binding inhibitors. The Prism
program (GraphPad Software) was used for this procedure.
21. Cell-based antiviral assays were performed using confluent
monolayers of HeLa cells infected with either HRV14 or
HRV16 under the conditions described in the following refer-
ence: J. Med. Chem. 1999, 42, 50.
22. XTT cytotoxicity assay: HeLa cells were cultured under
the identical conditions without virus infection but with the
test compounds. Also see: Roehm, N. W.; Rodgers, G. H.;
Hatfield, S. M.; Glasebrook, A. L. J. Immunol. Methods 1991,
142, 257.
23. For a-ketoamide based HCV protease inhibitors, see:
Babine, R.; Chen, S. H.; Lamar, J.; Snyder, N.; Sun, X.;
Tebbe, M.; Victor, F.; Wang, Q. M.; Yip, Y.; Collado, I.;
Garcia-Paredes, C.; Parker, R.; Jin, L.; Guo, D.; Glass, J. PCT
Patent WO 0218369 A2, March 7, 2002. Detailed reports
describing P1 (Nva) racemization under cell culture media and
rat plasma will be published in due time.
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