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(Scheme 2). Removal of the benzyloxycarbonyl group of
compound 9 was accomplished in 29% yield by catalytic
transfer hydrogenation. The low yield of this step was
attributed to poor solubility of the starting material 9
and loss of material during the HPLC purification of 3.
´ ´
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Ureidopeptide 3 and the protected intermediate 9 were
assayed for HIV-1 protease inhibition. A competitive
binding assay24 was used in which a solution of the
inhibitor in dimethylsulfoxide (DMSO) at various con-
centrations was incubated with HIV-1 protease in a
pH5.52 buffer for 1h at room temperature.25 Inhibitor
and literature substrate (10) were combined and fluores-
cence was measured. Assays showed moderate inhibi-
8. Marastoni, M.; Bazzaro, M.; Salvadori, S.; Bortolotti, F.;
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tion of HIV-1 protease by compound 3 (IC50
=
32.4 0.5lM). In contrast, the protected intermediate
9 showed negligible inhibition. It was found to have a
maximum inhibition around 35%, highlighting that the
free amine of inhibitor 3 is important in binding to
HIV-1 protease.
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2. Conclusion
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The methodology reported here, employing the oxidative
Hofmann rearrangement of C-terminal peptide amides,
affords ureidopeptides using a two-step, one-pot trans-
formation from commercially available starting materi-
als. The reaction tolerates a variety of substrates, amine
nucleophiles and solvents, and the urea linkage produced
was determined to be stable over a wide pH range and to
proteolysis. Replacement of the scissile amide bond of
Leu-enkephalin, a substrate for ACE, with a urea re-
sulted in the ureidopeptide ACE inhibitor 2, which
showed binding to ACE comparable to that of Leu-
enkephalin itself. The ureidopeptide HIV-1 protease
inhibitor 3 further illustrates the ability of ureidopeptides
to inhibit proteases. This methodology provides a flexible
strategy for rapidly synthesizing urea-containing pepti-
domimetic protease inhibitors, providing another tool
for de novo design of inhibitors for proteolytic enzymes.
Current efforts are underway to apply this methodology
to other proteases of therapeutic interest.
16. Guichard, G.; Semetey, V.; Didierjean, C.; Aubry, A.;
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18. See supporting information for details.
19. Spyroudis, S.; Varvoglis, A. J. Chem. Soc., Chem. Com-
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20. PK-ACE (20lL, 2.66lM) in buffer (100mM TrisÆHCl,
300mM NaCl, 10lM ZnCl2, pH8.3 buffer) was incubated
at 37°C with 10lL of a 27.8mM aqueous solution of 2
and diluted to 200lL with buffer. Reactions were analyzed
after 4h by RP-HPLC (VydacÒ C8, 214nm detection,
gradient: 5–95% CH3CN–H2O).
21. Cheung, H. S.; Wang, F. L.; Ondetti, M. A.; Sabo, E. F.;
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Rayner, M. M.; Wong, Y. N.; Chang, C. H.; Weber, P. C.;
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Acknowledgements
We thank the Chmielewski research group at Purdue for
assistance with HIV-1 protease assays.
23. Dreyer, G. B.; Lambert, D. M.; Meek, T. D.; Carr, T. J.;
Tomaszek, T. A., Jr.; Fernandez, A. V.; Bartus, H.;
Cacciavillani, E.; Hassel, A. M.; Minnich, M.; Petteway,
S. R., Jr.; Metcalf, B. W. Biochemistry 1992, 31,
6646–6659.
Supplementary data
Supplementary data associated with this article can be
24. Toth, M. V.; Marshall, G. R. Int. J. Pept. Prot. Res. 1990,
36, 544–550.
25. Compound 3 in DMSO (1nM to 500lM) was incubated
for 1h at 25°C with HIV-1 protease (50nM) in an aqueous
buffer of 2mM KH2PO4, 10% glycerol, 0.1% CHAPS,
1mM EDTA, and 1mM DTT, pH adjusted to 5.52 using
Na2HPO4. Each concentration of inhibitor (60lL) in
protease was plated into 40lL of 150lM substrate 10.
Substrate cleavage was monitored at k = 415nm
(k = 355nm excitation).
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