2776
G. Anquetin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2773–2776
2. Aminimanizani, A.; Beringer, P.; Jelliffe, R. Clin. Phar-
macokinet. 2001, 40, 169–187.
10. Sanchez, J. P.; Gogliotti, R. D.; Domagala, J. M.;
Gracheck, S. J.; Huband, M. D.; Sesnie, J. A.; Cohen,
M. A.; Shapiro, M. A. J. Med. Chem. 1995, 38, 4478–4487
11. Brighty, K. E. U.S. Patent 5,229,396, 1992.
12. Cecchetti, V.; Fravolini, A.; Palumbo, M.; Sissi, C.;
Tabarrini, O.; Terni, P.; Xin, T. J. Med. Chem. 1996, 39,
4952–4957.
3. Hooper, D. C. Emerg. Infect. Dis. 2001, 7, 337–341.
4. Fichera, M. E.; Roos, D. S. Nature 1997, 390, 407–409.
5. Roos, D. S.; Crawford, M. J.; Donald, R. G.; Kissinger, J.
C.; Klimczak, L. J.; Striepen, B. Curr. Opin. Microbiol.
1999, 2, 426–432.
6. Ralph, S. A.; D’Ombrain, M. C.; McFadden, G. I. Drug
Resist. Updat. 2001, 4, 145–151.
13. Cecchetti, V.; Fravolini, A.; Lorenzini, M. C.; Tabarrini,
O.; Terni, P.; Xin, T. J. Med. Chem. 1996, 39, 436–
445.
7. Gozalbes, R.; Brun-Pascaud, M.; Garcia-Domenech, R.;
Galvez, J.; Girard, P. M.; Doucet, J. P.; Derouin, F.
Antimicrob. Agents Chemother. 2000, 44, 2771–2776.
Briefly, the virulent RH strain of T. gondii was maintained
in mice by intraperitoneal passage every two days. For each
experiment, tachyzoites were collected from the peritoneal
cavity of infected mice then resuspended in physiological
saline. Tissue cultures and drug tests were carried out using
MRC5 fibroblast tissue cultures. Confluent monolayers
prepared in 96-well tissue culture plates were inoculated
with 2000 fresh tachyzoites. After 4 h, drugs at various
concentrations were added into the culture medium and
culture plates were incubated for an additional 72 h. Each
culture plate comprised eight negative control (without T.
gondii) and eight positive control wells (without drug).
After incubation, the plates were examined microscopically
for cytopathic effects and thereafter fixed with cold
methanol for 5 min. Toxoplasma growth was assessed by
enzyme linked immunoassay (ELISA) performed directly
on the fixed cultures using a peroxydase labeled mono-
clonal antibody directed against the SAG-1 surface protein
of T. gondii. After addition of the substrate, spectropho-
tometric readings were performed at a wavelength of
405 nm with blanking on the negative control well. For
each well, the results were expressed as optical density
(OD) values. The effect of each drug at various concentra-
tions was described by plotting the ODvalues as a function
of the logarithm of the concentration and a linear
regression model was used to summarize the concentra-
tion-effect relationship and to determine the IC50.
14. Cecchetti, V.; Tabarrini, O.; Sabatini, S.; Miao, H.;
Filipponi, E.; Fravolini, A. Bioorg. Med. Chem. 1999, 7,
2465–2471.
15. Da Silva, A. D.; De Almeida, M. V.; De Souza, M. V. N.;
Couri, M. R. C. Curr. Med. Chem. 2003, 10, 21–
39.
16. The chemical structure of compounds 1–4 was unambig-
uously established by 1H, 13C-DEPT, HSQC 1H–13C
NMR, ESI-MS. Compound 1 (TFA salt): ESI-MS:
(M+H)þ 374.2, (M+Na)þ 396.2 (in agreement with the
mass calculated for M ¼ C19H20FN3O4). For atom num-
1
bering, see Scheme 1: H NMR (1:4 CD3CN/D2O): 0.93–
0
1.13 (m, 4H, CH2 cyclopropyl), 2.04 (s, 2H, H1 and H5 ),
0
0
2.54 (s, 1H, H6 ), 3.52 (s, 3H, OCH3), 3.59, 3.82 (d, d, 2H,
2
0
0
2H, H2 /H4 exo/endo, JH–H ¼ 9:8 Hz), 4.10 (m, 1H, CH,
cyclopropyl), 7.63 (d, 1H, H5,3JH–F ¼ 13:1 Hz), 8.75 (s,
1H, H2). 13C NMR (DMSO-d6): 9.3 (s, CH2 cPr), 20.9 (s,
0
0
0
C1 and C5 ), 29.9 (s, C6 ), 41.1 (s, CH cPr), 51.1/51.2 (2s,
0
0
C2 and C4 ), 62.4 (s, OCH3), 106.7 (d, C5,
2JC–F ¼ 24:1 Hz), 106.7 (s, C3), 117.4 (q, TFA, CF3,
1JC–F ¼ 299:0 Hz), 119.9 (d, C9, JC–F ¼ 8:0 Hz), 134.5 (s,
4
2
2
C10), 136.1 (d, C7, JC–F ¼ 11:4 Hz), 144.8 (d, C8, JC–F
¼
1
6:9 Hz), 150.8 (s, C2), 154.9 (d, C6, JC–F ¼ 250:1 Hz),
2
158.8 (q, TFA, COOH, JC–F ¼ 31:7 Hz), 166.3 (s, C4),
176.5 (s, COOH). 19F NMR (1:4 CD3CN/D2O): )74.4
(6.3F, TFA), )117.4 (1F, F6).
17. Mahmoudi, N.; Ciceron, L.; Franetich, J.-F.; Farhati, K.;
Silvie, O.; Eling, W.; Sauerwein, R.; Danis, M.; Mazier,
D.; Derouin, F. Antimicrob. Agents Chemother. 2003, 47,
2636–2639.
18. Renau, T. E.; Gage, J. W.; Dever, J. A.; Roland, G. E.;
Joannides, E. T.; Shapiro, M. A.; Sanchez, J. P.;
Gracheck, S. J.; Domagala, J. M.; Jacobs, M. R.;
Reynolds, R. C. Antimicrob. Agents Chemother. 1996,
40, 2363–2368.
8. Khan, A. A.; Araujo, F. G.; Brighty, K. E.; Gootz, T. D.;
Remington, J. S. Antimicrob. Agents Chemother. 1999, 43,
1783–1787.
9. Gozalbes, R.; Brun-Pascaud, M.; Garcia-Domenech, R.;
Galvez, J.; Girard, P. M.; Doucet, J. P.; Derouin, F.
Antimicrob. Agents Chemother. 2000, 44, 2764–2770.