R. Siles et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4405–4409
4409
15. All compounds are well characterized by detailed proton,
carbon, and DEPT 135 NMR spectra and X-ray crystal-
lography has also been carried out for three compounds
although the results of these studies are not reported
herein. X-ray structures confirm the stereochemistry as E
for compounds 6, 13, and 21 and thiosemicarbazone
analogs (such as 5, 6, 20–26, and 46–49) are indicated as
single isomers by NMR studies. X-ray crystallographic
data have been deposited with the Cambridge Crystallo-
graphic Data Center for compound 21 and assigned the
deposition number CCDC 606033. It is important to note
that some of the thiosemicarbazone derivatives (such as 4,
7, 8, 13, 14, and 41) appear as mixtures in the NMR. The
major isomer in all cases is presumed to be the E isomer
although this is not confirmed except for 6, 13, and 21.
Furthermore, the issue of thiosemicarbazone derivatives
appearing as two compounds is reported in the literature
and at this point it is not possible to say for certain
whether we are actually seeing E and Z double bond
isomers of these compounds in solution by NMR or if
instead we are observing tautomeric forms of the thio-
semicarbazone functionality,16,17 or simply rotamers.18
Since compound 14 cannot exist as double bond isomers
due to symmetry, it is highly probable that the mixture
observed by NMR is due to tautomeric forms or rotamers.
Despite continued efforts to refine this question, including
low and high temperature NMR, the issue remains for
further study.
16. Raj, S. S. S.; Fun, H.-K.; Zhang, X.-J.; Tian, Y.-P.; Xie,
F.-X.; Ma, J.-L. Acta Crystallogr. Section C 2000, C56,
1238.
17. Fun, H.-K.; Chantrapromma, S.; Suni, V.; Sreekanth, A.;
Sivakumar, S.; Kurup, M. R. P. Acta Crystallogr. 2005,
E61, 1337.
18. Bagrov, F. V.; Vasil’eva, T. V. Russ. J. Org. Chem. 2002,
38, 1309.
19. Palmer, J. T.; Rasnick, D.; Klaus, J. L. PCT, Patent
9523222, 1995.
20. Buu-Hoi, N. P.; Xuong, N. D.; Binon, F. J. Chem. Soc.
Abstr. 1956, 713.
21. Cruzain (0.1 nM) in 100 mM sodium acetate buffer (pH
5.50) containing 5 mM DTT was preincubated for 5 min at
25 °C with inhibitor. The reaction was initiated5–7 by the
addition of 20 lL of substrate, benzyloxycarbonyl-L-phen-
Acknowledgments
The authors wish to express their appreciation to Profes-
sor J. H. McKerrow and Ms. Elizabeth Hansell (Univer-
sity of California, San Francisco) for their generous gift
of the recombinant cruzain enzyme and for helpful dis-
cussions, and to Drs. Kevin K. Klausmeyer and Rodney
Feazell for X-ray crystallography. The Bruker X8
APEX diffractometer was purchased with funds received
from the National Science Foundation Major Research
Instrumentation Program (Grant CHE-0321214). The
authors are most appreciative of the generous financial
support provided by The Robert A. Welch Foundation
(Grant No. AA-1278 to K.G.P.), the Vice-Provost for
Research of Baylor University (M.Z.) and Baylor
University (Research Leave and a Faculty Research
Incentive Program grant to M.L.T.).
References and notes
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13. Siles, R., PhD Dissertation, Baylor University, December,
2005.
ylalanyl-L-arginyl-7-amido-4-methylcoumarin
(Z-Phe-
Arg-AMC, Sigma, Km = 1.1 lM) to the enzyme–inhibitor
mixture at 25 °C to give 300 lL with a final concentration of
10 lM substrate and nine to twenty concentrations of
inhibitor ranging from 0.001 to 50,000 nM. The increase in
fluorescence (excitation at 355 nM and emission at 460 nM)
upon release of 7-amino-4-methylcoumarin was recorded
for 3 min with a spectrofluorimeter (Jobin Yvon-SPEX
Instruments, fluoroMAX-2). Inhibitor stock solutions were
prepared in DMSO, and serial dilutions were made in
DMSO to give a final concentration of 0.7% DMSO in the
assay. Controls were performed using enzyme alone,
substrate alone, and enzyme with DMSO. IC50 values were
determined by non-linear regression analysis using
14. Siles, R.; Chen, S. E.; Zhou, M.; Trawick, M. L.; Pinney,
K. G. Abstract of Papers, 229th National Meeting of the
American Chemical Society, San Diego, CA, American
Chemical Society: Washington, DC, 2005; Abstract
191739.
vo(inh) = Vmax/{1 + 10(X ꢀ log IC )Hillslope} and Prism soft-
50
ware (GraphPad).