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1577
even inversion of the activity; thus compound 6d actu-
ally stimulates platelet aggregation. Preliminary phar-
macological studies were performed in order to evaluate
the anti-PDE-III activity of compounds 6a, 6b, and 6c.
These values were determined using purified prepara-
tions obtained from ventricular tissue of guinea pigs
according to a previously reported protocol20 and reveal
that, in contrast to other pyridazinones, the anti-platelet
effect observed is not due to the inhibition of the PDE-III
(results not shown).
4. Kitamura, S.; Fukushi, H.; Miyawaki, T.; Kawamura, M.;
Konishi, N. J. Med. Chem. 2001, 44, 2438.
5. Liverton, N. J.; Armstrong, D. J.; Claremon, D. A.; Remy,
D. C.; Baldwing, J. J.; Lynch, R. J.; Zhang, G.; Gould, R. J.
Bioorg. Med. Chem. Lett. 1998, 8, 483.
6. Aguirre, F. V.; Topol, E. J.; Ferguson, J. J.; Anderson, K.;
Blankenship, J. C.; Heuser, R. R.; Sigmon, K.; Tailor, M.;
Gottleb, R.; Hannovich, G.; Rosemberg, M.; Donohue, T. J.;
Weisman, H. F.; Califf, R. M. Circulation 1995, 91, 2882.
7. Simpfendorfer, C.; Kottke-Marchant, K.; Lowrie, M.;
Andres, R. J.; Burns, D. M.; Miller, D. P.; Cove, C. S.;
DeFranco, A. C.; Ellis, S. G.; Moliterno, D. J.; Raymond,
R. E.; Sutton, J. M.; Topol, E. J. Circulation 1997, 96, 76.
8. Chew, D. P.; Bhatt, D. L.; Sapp, S.; Topol, E. J. Circulation
2001, 103, 201.
9. Moos, W. H.; Humblet, C. C.; Sircar, I.; Rithner, C.;
Weishar, R. E.; Bristol, J. A.; McPhail, A. J. J. Med. Chem.
1995, 38, 1990, and references cited therein.
10. Frank, H.; Heinisch, G. Pharmacologically Active Pyr-
idazines. Part 2. In Progress in Medicinal Chemistry; Ellis, G.
P., Luscombe, D. K., Eds.; Elsevier: Amsterdam, 1992; Vol.
29, p 141.
11. Laguna, R.; Rodriguez-Linares, B.; Cano, E.; Estevez, I.;
Ravina, E.; Sotelo, E. Chem. Pharm. Bull. 1997, 45, 1151.
12. Montero-Lastres, A.; Fraiz, N.; Laguna, R.; Cano, E.;
Estevez, I.; Ravina, E. Biol. Pharm. Bull. 1999, 22, 1376.
13. Sotelo, E.; Fraiz, N.; Yanez, M.; Terrades, V.; Laguna,
R.; Cano, E.; Ravina, E. Bioorg. Med. Chem. In press.
14. Sotelo, E.; Ravina, E.; Estevez, I. J. Heterocyclic Chem.
1999, 36, 985.
Studies performed on platelet lysates by protein elec-
trophoresis and western blot with an anti-phosphotyro-
sine antibody show that compounds 6a, 6b, and 6c
increase the tyrosine-phosphorylation profile of some
platelet proteins (results not shown). This fact could be
related to the anti-platelet activity shown by these com-
pounds. The phosphorylation state of any protein is the
result of the balance between phosphorylation/dephos-
phorylation processes. Bearing this point in mind, the
increase in the level of tyrosine-phosphorylation shown
by these compounds could be explained both by an
increase in tyrosine-kinase activity or by an inhibition in
tyrosine-phosphatase activity.
In conclusion, we have developed a palladium-assisted
synthetic approach to 5-alkylidene-6-phenyl-3(2H)-pyri-
dazinones 6a–e, which show promising anti-platelet
activity. Further studies are in progress in our labora-
tory in order to determine in detail both the mechanism
of action and the structural requirements for this series
of anti-platelet agents.
15. Estevez, I.; Coelho, A.; Ravina, E. Synthesis 1999, 9, 1666.
16. Complete details of the synthesis and spectral data will be
published elsewhere in a full paper. All compounds gave
satisfactory microanalytical (C, H, Nꢂ0.4%) and spectral
1
data (300 MHz H NMR and FTIR).
17. 6a: 1H NMR (DMSO-d6) d: 13.20 (bs, 1H), 7.55 (m,
5H+1H), 6.72 (s, 1H), 3.78 (s, 3H), 2.38 (s, 3H). 6b: 1H NMR
(DMSO-d6) d: 13.40 (bs, 1H), 7.60–7.45 (m, 5H+1H), 6.68 (s,
1H), 3.73 (s, 3H), 3.67 (s, 3H). 6d: 1H NMR (DMSO-d6) d:
13.56 (bs, 1H), 7.92 (s, 1H), 7.46 (m, 5H), 7.38 (s, 1H), 4.25 (q,
J=7.15 Hz, 2H), 1.23 (t, J=7.15 Hz, 3H). 6e: 1H NMR
(DMSO-d6) d: 13.63 (bs, 1H), 8.27 (s, 1H), 7.50 (m, 5H), 7.42
(s, 1H).
18. The E-configuration for 6a and 6d was assigned unam-
biguously from the 1H-coupled 13C NMR spectrum, for
example for 6a the vicinal coupling constant of the olefinic
proton with the ketone is considerably larger (13.7 Hz) than
that with the ester (7.1 Hz).
Acknowledgements
This work was supported in part by the Xunta de Gali-
cia (XUGA 20301B93 and 8151389). We also thank the
Agencia Espanola de Cooperacion Iberoamericana for a
grant to E. Sotelo.
References and Notes
1. Sotelo, E.; Centeno, B. N.; Rodrigo, J.; Ravina, E. Tetra-
hedron 2002, 58, 2389.
19. Born, G. V. R. Nature 1962, 194, 927.
2. Davies, M. J.; Thomas, A. C. Br. Heart J. 1985, 53, 363.
3. Fuster, V.; Badimon, L.; Badimon, J. J.; Chesebro, J. H. N.
Engl. J. Med. 1992, 326, 242.
20. Dal Piaz, V.; Giovannoni, M. P.; Castellana, C.; Palacios,
J. M.; Beleta, J.; Domenech, T.; Segarra, V. Eur. J. Med.
Chem. 1998, 33, 789.