1024 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 7
Letters
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Acad. Sci. U.S.A. 1994, 91, 9208-9212.
(11) Ishihara, H.; Connolly, A. J .; Zeng, D.; Kahn, M. L.; Zheng, Y.
W.; Timmons, C.; Tram, T.; Coughlin, S. R. Protease-Activated
Receptor 3 Is a Second Thrombin Receptor in Humans. Nature
1997, 386, 502-506.
(12) (a) Xu, W.-F.; Andersen, H.; Whitmore, T. E.; Presnell, S. R.;
Yee, D. P.; Ching, A.; Gilbert, T.; Davie, E. W.; Foster, D. C.
Cloning and Characterization of Human Protease-Activated
Receptor 4. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6642-6646.
(b) Kahn, M. L.; Zheng, Y.-W.; Huang, W.; Bigornia, V.; Zeng,
D.; Moff, S.; Farese, R. V., J r.; Tam, C.; Coughlin, S. R. A Dual
Thrombin Receptor System for Platelet Activation. Nature 1998,
394, 690-694.
these assays, 15 was completely effective, even at high
thrombin concentrations (up to 200 nM; results not
shown), which suggests the inherent potential of 15 to
treat the vascular diseases. Since thrombin has been
implicated in the proliferative and inflammatory events
associated with restenosis, we evaluated 15 in a vas-
cular restenosis model involving balloon angioplasty in
rats. Significant reduction of neointimal thickness (45
( 5 µm in treated animals vs 77 ( 5 µm in controls, p
< 0.05) was observed by administering 15 perivascu-
larly, basically, by implanting 10 mg of 15 in a hydrogel
formulation.26,27 This favorable outcome clearly dem-
onstrates an important role for PAR-1 in vascular
injury.
(13) Coughlin, S. R. Thrombin Receptor Function and Cardiovascular
Disease. Trends Cardiovasc. Med. 1994, 4, 77-83.
(14) Dennington, P. M.; Berndt, M. C. The Thrombin Receptor. Clin.
Exp. Pharmacol. Physiol. 1994, 21, 349-358.
Con clu sion . We have discovered a novel series of
indole-based SFLLR peptide mimetics as potent, selec-
tive PAR-1 antagonists and optimized the series for in
vivo studies by altering the guanidine-containing side
chain and introducing an indazole template. Indazole-
based PAR-1 antagonist 15 has improved in vivo efficacy
and cardiovascular safety and served as a pharmaco-
logical tool for assessing the therapeutic potential of a
PAR-1 antagonist in disease models. Although 15 was
not particularly effective in two guinea pig thrombosis
models, probably because of the presence of PAR-4 on
guinea pig platelets, it was effective in reducing rest-
enosis in a rat balloon angioplasty model. Consequently,
a selective PAR-1 antagonist could be beneficial for
treating restenosis attendant to arterial injury, such as
that following balloon angioplasty.
(15) Van Obberghen-Schilling, E.; Chambard, J .-C.; Vouret-Craviari,
V.; Chen, Y.-H., Grall, D.; Pouysse´gur, J . The Thrombin Recep-
tor: Activation and Coupling to Mitogenic Signaling Systems.
Eur. J . Med. Chem. 1995, 30 (Suppl.), 117-130.
(16) McComsey, D. F.; Hecker, L. R.; Andrade-Gordon, P.; Addo, M.
F.; Maryanoff, B. E. Macrocyclic Hexapeptide Analogues of the
Thrombin Receptor (PAR-1) Activation Motif SFLLRN. Bioorg.
Med. Chem. Lett. 1999, 9, 255-260.
(17) Fujita, T.; Nakajima, M.; Inoue, Y.; Nose, T.; Shimohigashi, Y.
A Novel Molecular Design of Thrombin Receptor Antagonist.
Bioorg. Med. Chem. Lett. 1999, 9, 1351-1356.
(18) Ahn, H.-S.; Arik, L.; Boykow, G.; Burnett, D. A.; Caplen, M. A.;
Czarniecki, M.; Domalski, M. S.; Foster, C.; Manna, M.; Stam-
ford, A. W.; Wu, Y. Structure-Activity Relationships of Pyrrolo-
quinazolines as Thrombin Receptor Antagonists. Bioorg. Med.
Chem. Lett. 1999, 9, 2073-2078.
(19) Hoekstra, W. J .; Hulshizer, B. L.; McComsey, D. F.; Andrade-
Gordon, P.; Kauffman, J . A.; Addo, M. F.; Oksenberg, D.;
Scarborough, R. M.; Maryanoff, B. E. Thrombin Receptor (PAR-
1) Antagonists. Heterocycle-Based Peptidomimetics of the SFLLR
Agonist Motif. Bioorg. Med. Chem. Lett. 1998, 8, 1649-1654.
(20) Bernatowicz, M. S.; Klimas, C. E.; Hartl, K. S.; Peluso, M.;
Allegretto, N. J .; Seiler, S. M. Development of Potent Thrombin
Receptor Antagonist Peptides. J . Med. Chem. 1996, 39, 4879-
4887.
(21) Seiler, S. M.; Peluso, M.; Michel, I. M.; Goldenberg, H.; Fenton,
J . W., II; Riexinger, D.; Natarajan, S. Inhibition of Thrombin
and SFLLR-Peptide Stimulation of Platelet Aggregation, Phos-
pholipase A2 and Na+/H+ Exchange by a Thrombin Receptor
Antagonist. Biochem. Pharmacol. 1995, 49, 519-528.
(22) Lindahl, A. H.; Scarborough, R. M.; Naughton, M. A.; Harker,
L. A.; Hanson, S. R. Antithrombotic Effect of a Thrombin
Receptor Antagonist Peptide in Baboons. Thromb. Haemostasis
1993, 69, 1196.
Ack n ow led gm en t. We thank J ohn Longo for excel-
lent technical assistance and Dr. Michael Greco for
helpful discussions. We thank Drs. Harold Almond and
Mary-Pat Beavers for computational studies.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails and analytical data. This material is available free of
Refer en ces a n d Notes
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(23) Bernatowicz et al.20 described peptide compounds with high
PAR-1 affinity and potent antagonist activity in platelet ag-
gregation induced by SFLLRNP-NH2. However, these antago-
nists were considerably less potent, and less consistent, in
interfering with the action of thrombin, the endogenous PAR-1
agonist. Similarly, Ahn et al. reported18 on a series of pyrrolo-
quinazolines as PAR-1 antagonists with high PAR-1 binding
affinity and potent inhibition of platelet aggregation induced by
a ha-TRAP (PAR-1 agonist peptide). However, these compounds
were just weakly active in thrombin-induced platelet aggrega-
tion.
(24) Andrade-Gordon, P.; Maryanoff, B. E.; Derian, C. K.; Zhang, H.-
C.; Addo, M. F.; Darrow, A. L.; Eckardt, A. J .; Hoekstra, W. J .;
McComsey, D. F.; Oksenberg, D.; Reynolds, E. E.; Santulli, R.
J .; Scarborough, R. M.; Smith, C. E.; White, K. B. Design,
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(25) Unpublished results to be published separately.
(26) Andrade-Gordon, P.; Derian, C. K.; Maryanoff, B. E.; Zhang, H.-
C.; Addo, M. F.; Cheung, W.-M.; Damiano, B. P.; D’Andrea, M.
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Antagonist of Protease-Activated Receptor-1 (PAR-1) Attenuates
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(27) This mode of drug administration (sustained local delivery) was
necessary because of the lengthy time frame of the vascular
injury restenosis experiments (14 days) and the short intrave-
nous residency time of 15.
(9) Natarajan, S.; Reixinger, D.; Peluso, M.; Seiler, S. M. Tethered
Ligand Derived Pentapeptide Agonists of Thrombin Receptor:
A Study of Side Chain Requirements for Human Platelet
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