J. A. Pfefferkorn et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3338–3343
3343
Table 6. Effect of compound 47 on time to the formation of occlusive
thrombus in rat ferric chloride injury model of arterial thrombosis
6. Hechler, B.; Nonne, C.; Roh, E. J.; Cattaneo, M.;
Cazenave, J.-P.; Lanza, F.; Jacobson, K. A.; Gachet, C.
J. Pharmacol. Exp. Ther. 2006, 316, 556.
IV dose
Occlusion
incidence
Time to
occlusion
(min)
7. P2Y1 antagonists similar to structure 4 have been recently
reported in patent application, see: Chao, H. J.; Tuerdi,
H.; Herpin, T.; Roberge, J. Y.; Liu, Y.; Lawrence, M. R.;
Rehfuss, R. P.; Clark, C. G.; Qiao, J. X.; Gungor, T.;
Lam, P. Y. S.; Wang, T. C.; Ruel, R.; L’Heureux, A. L.;
Thibeault, C.; Bouthillier, G.; Schnur, WO 20051133511.
8. DeRuiter, J.; Carter, D. A.; Arledge, W. S.; Sullivan, P. J.
J. Het. Chem. 1987, 24, 149.
Bolus
(mg/kg)
+
Infusion
(lg/kg/min)
Vehicle
47
47
—
—
16
33
65
2/2
3/5
3/6
1/5
18
30
35
50
0.4
0.8
1.6
47
9. Farr, R. N.; Alabaster, R. J.; Chung, J. Y. L.; Craig, B.;
Edwards, J. S.; Gibson, A. W.; Ho, G.-J.; Humphrey, G.
R.; Johnson, S. A.; Grabowski, E. J. J. Tetrahedron
Asymm. 2003, 14, 353.
10. Duffy, K. J.; Darcey, M. G.; Delorme, E.; Dillon, S. B.;
Eppley, D. F.; Erickson-Miller, C.; Giampa, L.; Hopson,
C. B.; Huang, Y.; Keenan, R. M.; Lamb, P.; Leong, L.;
Liu, N.; Miller, S. G.; Price, A. T.; Rosen, J.; Shah, R.;
Shaw, T. N.; Smith, H.; Stark, K. C.; Tian, S.-S.; Tyree,
C.; Wiggall, K. J.; Zhang, L.; Luengo, J. I. J. Med. Chem.
2001, 44, 3730.
(bolus + infusion) resulted in a dose dependent decrease
in both the incidence of occlusive thrombus formation
and the average time to occlusion.
We have described the discovery and optimization of a
series of P2Y1 antagonists. These efforts resulted in the
identification of analogs with nanomolar affinity for
the P2Y1 receptor and micromolar potency for inhibit-
ing platelet aggregation. Evaluation of a representative
analog in a rat ferric chloride arterial thrombosis mod-
el revealed it to be effective at preventing thrombus
formation suggesting that the current series of antago-
nists may have potential as a novel antithrombotic
therapy.
11. Geronikaki, A.; Babaev, E.; Dearden, J.; Dahaen, W.;
Filimonov, D.; Galaeva, I.; Krajneva, V.; Lagunin, A.;
Macaev, F.; Molodavkin, G.; Poroikov, V.; Pogrebnoi, S.;
Saluotin, V.; Stepanchikova, A.; Stingaci, E.; Tkach, N.;
Vlad, L.; Voronina, T. Bioorg. Med. Chem. 2004, 12, 6559.
12. Analogs evaluated in biological assays had analytical
purity P95% as determined by 1H NMR, HPLC and MS.
13. The P2Y1 binding assay was conducted using a modifica-
tion of the previously reported methods. [b-33 P]-2-thio-
methyl adenosine diphosphate was utilized as the
radioligand. Human astrocytoma cells (1321N1) stably
expressing the hP2Y1 were used to prepare membrane
preparations of the receptor. A typical procedure was as
follows: Binding of 0.3 nM [b-33P]-2-thiomethyl ADP to
platelet P2Y1 receptor in 1321N1 cell membrane was
carried out in triplicate at 25 °C in a 96-well plate.
Nonspecific binding was defined as binding in the presence
of 1 lM of 2-Me-S-ADP. A 1.0 lL aliquot of DMSO
containing a test compound was diluted to the final assay
concentrations (0.1 nM to 1 mM) and placed into a 96-
well plate. A volume of 60 lL of Incubation buffer
containing 0.3 nM [b-33P]-2-thiomethyl ADP and 40 lL
of hP2Y1 expressing 1321N1 cell membranes prep (0.6lg/
well) was added to each well. After a 1.0 h incubation, the
contents of the wells were filtered and washed with a
Tomtec Cell Harvester through UniFilter 96, GF/B plates.
A 45 lL aliquot of scintillation cocktail (Ultima-Flo-M)
was added into each well and the plates were counted on a
Trilux Microbeta Counter. Utilizing the assay as described
above, we found benchmark compound MRS2500 (2) to
have Ki = 1.1 0.3 nM for the P2Y1 receptor which was
consistent with the published value of Ki = 0.78 nM (Ref.
5b). For a description of the 132N1 cell line, see: (a) Patel,
K.; Barnes, A.; Camacho, J.; Paterson, C.; Boughtflower,
R.; Cousens, D.; Marshall, F. Eur. J. Pharmacol. 2001,
430, 203; for representative examples of other previously
reported P2Y1 binding assays, see: (b) Waldo, G. L.;
Harden, T. K. Mol. Pharm. 2004, 56, 426, and Ref. 5f.
14. (a) Gould, W. R.; Baxi, S. M.; Schroeder, R.; Peng, Y. W.;
Leadley, R. J.; Peterson, J. T.; Perrin, L. A. J. Thromb.
Haemost. 2005, 3, 733; (b) Sbrana, S.; Pina, F. D.; Rizza,
A.; Buffa, M.; De Filippis, R.; Gianetti, J.; Clerico, A.
Cytometry B 2008, 74B, 30.
References and notes
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Vermylen, J.; Hoylaerts, M. F. Curr. Pharm. Des. 2006,
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2. For a review, see: Plosker, G. L.; Lyseng-Williamson, K.
A. Drugs 2007, 67, 613.
3. For a review, see: Husted, S. Eur. Heart J. Supp. 2007, 9,
D20.
4. (a) Fabre, J.-E.; Nguyen, M.; Latuor, A.; Keifer, J. A.;
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5. For MRS 2179, see: (a) Camaioni, E.; Boyer, J. L.;
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P2Y1 antagonists, see: (c) Nandanan, E.; Camaioni, E.;
Jang, S.-Y.; Kim, Y.-C.; Cristalli, G.; Herdewijn, P.;
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Herdewijn, P.; Harden, T. K.; Boyer, J. L.; Jacobson, K.
A. J. Med. Chem. 2000, 43, 829; (e) Raboisson, P.;
Baurand, A.; Cazenave, J.-P.; Gachet, C.; Retat, M.;
Spiess, B.; Bourguignon, J.-J. J. Med. Chem. 2002, 45, 962;
(f) Mathieu, R.; Baurand, A.; Schmitt, M.; Gachet, C.;
Bourguignon, J.-J. Bioorg. Med. Chem. Lett. 2004, 12,
1769, and references therein.
15. Kurz, K. D.; Main, B. W.; Sandusky, G. E. Thromb. Res.
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