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B. Planty et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1735–1739
Table 3
Antagonist activity results for analog series V of compound 1
O
X
R
Ar
N
Series V
Compds
Ar
R
X
% Antag. at 10
l
M*
25
26
27
2,6-Difluorophenyl
2,6-Difluorophenyl
2-Chlorophenyl
4-Fluorobenzylamino
4-Fluorobenzylamino
4-Fluorophenylamino
H
F (cis)
F (cis)
0
25
85
N
N
28
29
2-Chlorophenyl
2-Cyanophenyl
F (cis)
F (cis)
23
40
F
4-Fluorobenzylamino
N
N
30
2-Cyanophenyl
2-Cyanophenyl
F (cis)
68
33
F
F
N
N
31
F (trans)
*
Inhibition of calcium release induced by 1 lM of SFLLR-NH2. Data are expressed as mean SEM of at least two independent determinations performed in triplicate.
easily prepared from piperidin-4-one (Scheme 5). Fluoride analogs
26–32 were prepared from the same starting material using the
three-step sequence already published to introduce the fluorine
atom.9
analogs were more potent than trans analogs as shown with com-
pounds 30 and 31.
The pharmacological properties of the best compounds in each
series were further evaluated (Table 4). A concentration–response
study using the same model as in the screening allowed the dis-
crimination of partially versus fully competitive antagonists. In
the case of full antagonist the pA2 was determined. Anti-aggregant
properties were assessed using an SFLLR-induced human platelet
aggregation model,2 and their antithrombotic potential was deter-
mined through an arteriovenous extra-corporal shunt model in
anesthetized rats.4 The best results were obtained with the last
two series of analogs. Even though some cyclobutene-1,2-dione
analogs behaved as strong PAR1 antagonists (compounds 11 and
12), no anti-aggregant properties were detectable in our in vitro
human platelet model. Conversely, pyridine analog 24 revealed a
very comparable pharmacological profile with respect to com-
pound 1, and piperidine analog 30 displayed significant improved
activity in the rat antithrombotic model.
Determination of all compound potencies was based on the
inhibition of fluorescent calcium release induced by the PAR1
selective agonist peptide (SFLLR-NH2) in CHO cells (FLIPR).10 Start-
ing with the left-hand portion of 1, fusing the phenyl and the al-
kene functions of the cinnamoyl motif (Table 1, Series I) resulted
in inactive compounds, whereas more success was obtained with
the cyclobutene-1,2-dione analogs (Table 1, Series II). Indeed, very
potent antagonist of PAR1, such as compounds 7, 11 and 12, could
be obtained after careful tuning of the substitution pattern on the
cinnamoyl aromatic with the ligand on the piperazine ring. In the
pyridines series (Table 2), more potent antagonists of PAR1 where
obtained when the pyridine nitrogen was located in the meta posi-
tion from the carbonyl function (Series IV). Again in this series, it
was difficult to find general rules for structure–activity relation-
ship. Therefore, it was crucial for every ligand R on the pyridine
ring to explore many substitution patterns on the cinnamoyl aro-
matic, in order to properly determine their potential. In the piper-
idine series (Table 3), a small improvement of the antagonist
potency was obtained through the introduction of a fluorine atom
as shown with compounds 25 and 26. Moreover, in all instances cis
In conclusion, two new series of PAR1 receptor antagonists
have been identified. In each series, one analog showed good
in vitro anti-aggregant properties combined with potent in vivo
antithrombotic activity in a rat pharmacodynamic model. Both
compounds were selected for more extensive biological
evaluation.
References and notes
Table 4
1. (a) Coughlin, S. R. Nature 2000, 407, 258; (b) Macfarlane, S. R.; Seatter, M. J.;
Kanke, T.; Hunter, G. D.; Plevin, R. Pharmacol. Rev. 2001, 53, 245; (c) Steinberg,
S. F. Mol. Pharmacol. 2005, 67, 2.
2. Derian, C. K.; Santulli, R. J.; Tomko, K. A.; Haertlein, B. J.; Andrade-Gordon, P.
Thromb. Res. 1995, 78, 505.
3. Perez, M.; Lamothe, M.; Maraval, C.; Mirabel, E.; Loubat, C.; Planty, B.; Horn, C.;
Michaux, J.; Letienne, R.; Pignier, C. B. A.; Nadal-Wollbold, F.; Cussac, D.; De
Vries, L.; Le Grand, B. J. Med. Chem. 2009, 52, 5826.
4. Létienne, R.; Leparq-Panissié, A.; Bocquet, A.; Calmettes, Y.; Culié, C.; Le Grand,
B. Thromb. Res., in press.
Antagonist activity results for analogs series V of compound 1
Human plateletsc
(pKb)
AV Shuntd
(%)
b
Compds % Antag. at
pA2
10
l
Ma
1
7
93
85
97
84
97
84
85
68
6.42
Partial
6.41
6.37
Partial
6.03
5.52
—
4
4
—
5.05
4
5.68
28
—
—
—
—
28
—
53
11
12
21
24
27
30
5. Kinney, W. A.; Lee, N. E.; Garrison, D. T.; Podlesny, E. J.; Simmonds, J. T.;
Bramlett, D.; Notvest, R. R.; Kowal, D. M.; Tasse, R. P. J. Med. Chem. 1992, 35,
4720.
6.41
6.23
6. (a) Reed, M. W.; Pollart, D. J.; Perri, S. T.; Folland, L. D.; Moore, H. W. J. Org.
Chem. 1988, 53, 2477; (b) Liebeskind, L. S.; Fengl, R. W.; Wirtz, K. R.; Shawe, T. T.
J. Org. Chem. 1988, 53, 2482.
7. Gilbert, A. M.; Antane, M. M.; Argentieri, T. M.; Butera, J. A.; Francisco, G. D.;
Freeden, C.; Gundersen, E. G.; Graceffa, R. F.; Herbst, D.; Hirth, B. H.; Lennox, J.
a,bInhibition of calcium release induced by 1
l
M of SFLLR-NH2: % at 10
lM or pA2.
c
Inhibition of SFLLR-induced human platelet aggregation.
% Increase of the occlusion time in an arteriovenous shunt in rat at 1.25 mg/kg
d
iv.