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cooled to 0 ꢀC for 24 h. The product was ®ltered and crystal-
lized with water±ethanol. The products were characterized by
1H, 13C, and 31P NMR spectrometry and mass spectra. The
purity obtained was >99% in all cases.
16. Rogers, M. J.; Watts, D. J.; Russell, R. G. G. Cancer
1997, 80, 1652.
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33. Drug screening. Biological assays on epimastigotes were
performed as previously described.34 T. cruzi epimastigotes (Y
strain) were grown in 20mL screw-cap tubes at 28 ꢀC in a
liquid medium containing brain±heart infusion (37 g/L),
hemin chlorohydrate (20mg/L) (dissolved in 50% triethano-
lamine) and 10% newborn calf serum. The initial inoculum
contained 2±3Â106 cells/mL (as determined by counting in a
Neubauer chamber) in a ®nal volume of 1 mL. The con-
centration of cells was determined by measuring the absor-
bance of the culture medium containing parasites at 600 nm
against a blank with culture medium alone. Each drug was
tested at four dierent concentrations (1, 5, 10, and 20 mg/mL)
each one in quadruplicate. Drugs were dissolved in ethanol. A
control without drug was done with each group that was tes-
ted.
24. Bergstrom, J. D.; Bostedor, R. G.; Masarachia, P. J.; Reszka,
A. A.; Rodan, G. Arch. Biochem. Biophys. 2000, 373, 231.
25. Cromartie, T. H.; Fisher, K. J.; Grossman, J. N. Pest
Biochem. Physiol 1999, 63, 114.
26. Grove, J. E.; Brown, R. J.; Watts, D. J. J. Bone Miner.
Res. 2000, 15, 971.
27. Fisher, J. E.; Rogers, M. J.; Halasy, J. M.; Luckman, S. P.;
Hughes, D. E.; Masarachia, P. J.; Wesolowski, G.; Russell,
R. G. G.; Rodan, G. A.; Reszka, A. A. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 133.
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Elhalem, E.; Rodriguez, J. B. J. Med. Chem. 2000, 43, 1826.
30. Cinque, G. M.; Szajnman, S. H.; Zhong, L.; Docampo,
R.; Rodriguez, J. B.; Gros, E. G. J. Med. Chem. 1998, 41,
1540.
31. Kieczykowski, G. R.; Jobson, R. B.; Melillo, D. G.;
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32. General procedure for the preparation of bisphosphonates
(mono sodium salt). To a ¯ame dried 100 mL three neck ¯ask
having an addition funnel and a re¯ux condenser through
which was circulated water at 0 ꢀC was added the carboxylic
acid (38 mmol), PO3H3 acid (3.2 g, 38 mmol), and anhydrous
benzenesulfonic acid (15 g) under argon atmosphere. The
reaction mixture was heated to 65 ꢀC, then PCl3 (7 mL, 80
mmol) was added dropwise with vigorous stirring. The reac-
tion was stirred at 65 ꢀC for 16 h. The reaction was allowed to
cool to rt. Cold water (60mL) was added and the reaction was
stirred at 100 ꢀC for 5 h. The reaction was cooled to rt and the
pH was adjust to 4.3 with a 50% aqueous NaOH solution.
Ethanol (20mL) was added, and the resulting mixture was
To calculate percent inhibition, the following formula was
used: 100À(ÁAdÂ100)/ÁAc=percent inhibition, where ÁAc
and ÁAd are the dierences in the absorbance of control cul-
tures and drug-treated cultures, respectively, at the beginning
and at the end of the experiment. The maximum amount of
solvent used (1% ethanol) did not have any signi®cant eect
on the epimastigotes growth. The values of IC50 were esti-
mated by linear and polynomial regression.
Experiments on the intracellular form of the parasite were
conducted on T. cruzi-infected L6E9 myoblasts (Y strain) as
described before.35 L6E9 myoblasts were exposed to 2000 rads
of gamma radiation and plated on 75 cm2 ¯asks at a density of
1.2Â107 cells/¯ask in DMEM containing 20% fetal calf serum
in a total volume of 10mL. After 24 h of incubation at 35 ꢀC,
the cells were exposed to a suspension of 5Â107 trypomasti-
gotes/¯ask in DMEM containing 20% fetal calf serum for 2 h,
then cultures were washed twice with Dulbecco' PBS and the
culture medium was replaced. Dierent concentration of drugs
were added to the cultures that were labeled with 1.0 mCi of [5-
6-3H]uracil and incubated for an additional 72 h. Incorpora-
tion of [3H]uracil was measured, the percent inhibition of
[3H]uracil incorporation (parasite proliferation) was calculated
employing the following formula: inhibition percent=[(AÀB/
A]Â100, where A and B are the mean counts per minute of
infected control-treated myoblasts and infected drug-treated
myoblasts, respectively.
34. Schvartzapel, A. J.; Zhong, L.; Docampo, R.; Rodriguez,
J. B.; Gros, E. G. J. Med. Chem. 1997, 40, 2314.
35. Yan, W.; Moreno, S. N. J. J. Immunol. Methods 1998,
220, 123.
36. Soares, M. J.; de Souza, W. Parasitol. Res. 1991, 77, 461.
37. Rogers, M. J.; Xiong, X.; Ji, X.; Monkkonen, J.; Russell,
È
È
G. G.; Williamson, M. P.; Ebetino, F. H.; Watts, D. J. Pharm.
Res. 1997, 14, 625.