196
Z. Mincheva et al. / Bioorg. Med. Chem. 12 (2004) 191–197
5.3.6. 2-[(Dihydroisoxazol[4,5-c]succinimide)-3-yl)]ethyl-
1
3. (a) Jomaa, H.; Wiesner, J.; Sandrebrand, S.; Altincicek,
B.; Weidemeyer, C.; Hintz, M.; Turbachova, I.; Eberl, M.;
Zeidler, J.; Lichtenthaler, H. K.; Soldati, D.; Beck, E.
Science 1999, 285, 1573. (b) Kuzuyama, T.; Shimizu, T.;
Takahashi, S.; Seto, H. Tetrahedron Lett. 1998, 39, 7913.
4. Altincicek, B.; Hintz, M.; Sanderbrand, S.; Wiesner, J.;
Beck, E.; Jomaa, H. FEMS Microbiol. Lett. 2000, 190,
329.
5. Missinou, M. A.; Bormann, S.; Schindler, A.; Issifou, S.;
Adegnika, A. A.; Matsiegui, P. B.; Binder, R.; Lell, B.;
Wiesner, J.; Baranek, T.; Jomaa, H.; Kremser, P. G.
Lancet 2002, 360, 1941.
6. (a) Kozikowski, A. P. Acc. Chem. Res. 1984, 17, 410. (b)
Confalone, P. N.; Ko, S. S. Tetrahedron. Lett. 1984, 25,
947. (c) Corral, J. M.; Gordaliza, M.; Castro, M. A.;
Vazquez, L. L.; Gravalos, D. G.; Broughton, H. B.;
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7. (a) Confalone, P. N.; Jin, F.; Mousa, S. A. Bioorg. Med.
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Costello, T. D.; Valvis, I.; Ma, P.; Kakuffman, S.; Ward,
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T. M.; Pinto, D. J.; Emmett, G.; Sze, Y. Y.; Liu, J.;
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phosphonic acid (8a). Yellow oil; yield: 83%. H NMR
(D2O) d 2.01 (2H, m, PCH2), 2.60(2H, m, CH2CH2P),
4.52 (1H, d, J=8.7 Hz, CH), 5.36 (1H, d, J=9.0Hz,
CH). 13C NMR (D2O) d 20.39 ðPCH2CH2Þ, 21.50, 24.24
(JPC=137.9 Hz, PCH2), 58.61 ðCHÞ, 80.94 ðCHÞ,
155.90, 156.21 (JPC=15.6 Hz, C¼N), 174.56, 177.00
ðC¼OÞ. MS (IC with NH3) m/z 249 (M +1). Anal.
C7H9N2O6P (C, H, N).
5.3.7. 2-[N-Phenyl(dihydroisoxazol[4,5-c]succinimide)-3-
1
yl)]ethylphosphonic acid (8b). Yellow oil; yield: 80%. H
NMR (D2O) d 1.58 (2H, m, PCH2), 2.51 (2H, m,
CH2CH2P), 4.34 (1H, d, J=9.0Hz, CH), 5.00 (1H, d,
J=9.0Hz, CH), 7.21–7.37 (5H, m, arom. H). 13C NMR
(D2O)
d
22.46
ðPCH2CH2Þ,
25.11,
27.68
(JPC=129.3 Hz, PCH2), 83.29 ðCHÞ, 122.59, 126.57,
129.68, 136.70 ðCaromÞ, 159.58, 159.94 (JPC=18.1 Hz,
C¼N), 169.24, 176.82 ðC¼OÞ. MS (IC with NH3) m/z
325 (M+1). Anal. C13H13N2O6P (C, H, N).
5.4. Biological evaluation
5.4.1. Plant material. Periwinkle (C. roseus [L.] G. Don)
cells suspensions (line C20) were subcultured every 7
days (dilution rate 1:10) in the B5 Gamborg et al.’s med-
ium26 containing 58 mM sucrose and 4.5 mM 2,4-
dichlorophenoxacetic acid (2,4-D).27 They were grown in
250mL Erlenmeyer flasks (with 50mL culture medium)
on a rotary shaker (100 rpm) at 24 ꢀC, in darkness.28
9. Patchett, A. A.; Nargund, R. P. Annu. Rep. Med. Chem.
2000, 35, 289.
10. Contin, A.; Van Der Heijden, R.; Lefeber, A. W. M.;
Verpoorte, R. FEBS Lett. 1998, 434, 413.
11. Chahed, K.; Oudin, A.; Guivarc’h, N.; Hamdi, S.;
Che
Biochem. 2000, 38, 559.
12. Veau, B.; Courtois, M.; Oudin, A.; Che
´
nieux, J. C.; Rideau, M.; Clastre, M. Plant Physiol.
5.4.2. Treatments. Periwinkle cells were subcultured in a
MIA-inducing medium (i.e., 2,4-D free B5 medium to
which 4.5 mM zeatin were added at day 3).29 The mol-
ecules to be tested were added at day 3 to the medium
(final concentrations 50, 75, 100 and 125 mM). The cells
were harvested at day 7 (vacuum filtration) and freeze
dried for ajmalicine and dry mass quantitations.
´
nieux, J. C.;
Rideau, M.; Clastre, M. Biochim. Biophys. Acta 2000,
1517, 159.
13. (a) Torssell, K. B. G. In Nitrile Oxide, Nitrones and
Nitronates in Organic Synthesis; Feuer, H., Ed.; VCH:
New York, 1988. (b) P. Caramella; P. Grunager. In 1,3-
Dipolar Cycloaddition Chemistry; Padwa, A., Ed.; John
Wiley & Sons: New York, 1984; p 291. (c) Gothelf, K. V.;
Jorgensen, K. A. Chem. Rev. 1998, 98, 863. (d) Adams,
J. P.; Box, D. S. J. Chem. Soc., Perkin Trans. 1 1999, 749.
14. Whitney, R. A.; Nicholas, E. S. Tetrahedron Lett. 1981,
35, 3371.
5.4.3. Alkaloid quantitation. MIA were extracted from
25 mg freeze dried cells with methanol. Ajmalicine, the
chosen marker of alkaloid accumulation, was quantified
by spectrofluorodensitometry28 (TLC scanner 3,
Camag, lem: 365 nm).
15. Li, C.; Yuan, C. Synthesis 1993, 471.
16. (a) Liu, K.-C.; Shelton, B. R.; Howe, R. K. J. Org. Chem.
1980, 45, 3916. (b) Casero, F.; Cipolla, L.; Lay, L.; Nico-
tra, F.; Panza, L.; Russo, G. J. Org. Chem. 1996, 61, 3428.
17. Larsen, K. E.; Torssell, K. B. G. Tetrahedron 1984, 40,
2985.
18. Kim, B. H. Synth. Commun. 1987, 17, 1199.
19. Shang, Y. J.; Wang, Y. G. Tetrahedron Lett. 2002, 43,
2247.
Acknowledgements
We would like to express our appreciation to the ‘Le
STUDIUM’ (Recherche et Accueil Scientifique Inter-
national, Orle
research associate position for Mincheva, Z . This work
was also supported by Conseil Regional (Region Centre).
´
ans, France) that supported the 1-year
20. Kamiya, T.; Hashimoto, M.; Hemmi, K.; Takeno, H.
Japan Patent 4,206,156, 1978.
´
´
21. McKenna, C. E.; Melvin, T. H.; Cheung, N. H.;
McKenna, M. C. Tetrahedron Lett. 1977, 18, 155.
22. Perrin, D. D.; Armarego, W. L. F.; Perrin, D. R. Puri-
fication of Laboratory Chemical; Pergamon: Oxford,
1986.
23. (a) Cates, L. A.; Jones, G. S.; Good, D. J.; Tsai, H. Y.-
L.H.; Li, V.-S. J. Med. Chem. 1980, 23, 300. (b) Varlet,
J. M.; Fabre, G.; Sauveur, F.; Collignon, N. Tetrahedron
1981, 37, 1377.
References and notes
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