Discovery of a nonpeptidic small molecule antagonist of the human platelet thrombin receptor (PAR-1)

Article abstract of DOI:10.1016/S0960-894X(01)00745-4

The synthesis and biological evaluation of a series of nonpeptidic small molecule antagonists of the human platelet thrombin receptor (PAR-1) are described. Optimization of the 5-amino-3-arylisoxazole lead resulted in an approximate 100-fold increase in potency. The most potent of these compounds (54) inhibits platelet activation with IC₅₀s of 90 nM against the thrombin receptor agonist peptide (TRAP) and 510 nM against thrombin as the agonist. Further, antagonist 54 fully blocks platelet aggregation stimulated by 1 nM thrombin for 10 min.

Full text of DOI:10.1016/S0960-894X(01)00745-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 319–323
Discovery of a Nonpeptidic Small Molecule Antagonist of the
Human Platelet Thrombin Receptor (PAR-1)
Philippe G. Nantermet,a,* James C. Barrow,a George F. Lundell,a Janetta M. Pellicore,a
Kenneth E. Rittle,a MaryBeth Young,a Roger M. Freidinger,a Thomas M. Connolly,b
Cindra Condra,b Jerzy Karczewski,b Rodney A. Bednar,b Stanley L. Gaul,b
Robert J. Gould,b Kris Prendergastc and Harold G. Selnicka
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Molecular Design and Diversity, Merck Research Laboratories,
West Point, PA 19486, USA
Received 17 September 2001; accepted 2 November 2001
Abstract—The synthesis and biological evaluation of a series of nonpeptidic small molecule antagonists of the human platelet
thrombin receptor (PAR-1) are described. Optimization of the 5-amino-3-arylisoxazole lead resulted in an approximate 100-fold
increase in potency. The most potent of these compounds (54) inhibits platelet activation with IC50s of 90 nM against the thrombin
receptor agonist peptide (TRAP) and 510 nM against thrombin as the agonist. Further, antagonist 54 fully blocks platelet aggre-
gation stimulated by 1 nM thrombin for 10 min. # 2002 Published by Elsevier Science Ltd.
Thrombin plays a key role not only in hemostasis, but
also in the activation of a variety of cell types.1 While
the role of this protease in blood coagulation is well
defined, the mechanism underlying thrombin’s actions
upon cells remained unclear until recently. The cloning
of a platelet thrombin receptor (PAR-1)2 has provided
some insight into the mechanism of thrombin activation
of cells.3 The thrombin receptor belongs to a family of
seven transmembrane domain G-protein-coupled recep-
tors which are activated via a novel mechanism
involving proteolytic modification of the extracellular
N-terminus. In the case of PAR-1, thrombin cleaves the
extracellular domain of the receptor at the Arg41-Ser42
site to expose a new truncated N-terminus bearing the
peptide recognition motif SFLLRN, which binds intra-
molecularly to an undefined extracellular site and acts
as a tethered ligand (agonist) for the receptor. Synthetic
peptides that mimic this motif, such as the thrombin
receptor activating peptide (TRAP, SFLLR-NH2) have
complete PAR-1 agonist properties independent of
thrombin activation thus confirming the role of the
tethered SFLLRN motif as an activating ligand. Three
other receptors that function via a similar mechanism—
PAR-2, PAR-3, and PAR-4—have been identified sub-
sequently.4
Selective blockade of the intramolecular activation step
of the thrombin receptor should result in moderation of
platelet activation and aggregation without interfering
with the enzymatic activity of thrombin in the coagula-
tion cascade. Therefore, an antagonist of the thrombin
receptor may represent a safer antiplatelet agent for the
treatment of thrombotic disorders. Thrombin is also
known to activate a variety of cell types involved in
inflammatory and proliferative disorders presumably
via the thrombin receptor; thus suggesting additional
potential therapeutic uses5 of a PAR-1 antagonist.
Several reports of PAR-1 antagonists derived from the
TRAP peptide6 as well as nonpeptidic antagonists7 have
been disclosed recently. Earlier reports from these
laboratories8 have described a series of small molecule
thrombin receptor antagonists exemplified by 1 (Fig. 1).
Directed screening9 based on 1 resulted in the identifi-
cation of isoxazole 210 as a structurally novel lead of
moderate potency (TRAP IC50=9 mM). Reported
herein is a summary of our progress in optimizing 2.
*Corresponding author. Tel.:+1-215-652-0945; fax:+1-215-652-3971;
e-mail: philippe_nantermet@merck.com
0960-894X/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0960-894X(01)00745-4

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P. G. Nantermet et al. / Bioorg. Med. Chem. Lett. 12 (2002) 319–323
Scheme 1. (a) POCl3, Et3N, 80 ꢀC; (b) N-3-aminopropylpiperidine
neat, 80 ꢀC; (c) NaH, RCH2X, DMF, 0 ꢀC to rt.
Scheme 3. (a) Acryloyl chloride; (b) azepine; (c) LiAlH4, THF or
BH3–THF, THF; (d) NaH, diethlycarbonate; (e) nBuLi, EtO2CCH2-
CO2H; (f) NH2OH HCl, EtOH; (g) POCl3; (h) 12, nBuLi, Et2O, 0 ꢀC.
Scheme 2. (a) ArCH2NH2; (b) ArCOCl, Et3N, CH2Cl2; (c) LiAlH4,
THF; (d) (i) NaH, ethyl bromoacetate, DMF; (ii) LiAlH4, THF; (iii)
MeSO2Cl, Et3N, CH2Cl2; (e) NaH, 1-chloro-3-iodopropane, DMF; (f)
NaH, 1,4-dibromobutane, DMF; (g) R1R2NH.
New compounds were tested for their ability to inhibit
the activation of platelets by either 3 uM TRAP
(EC50=1 mM) or 1 nM thrombin (EC50=0.6 mM) by
measuring the release of 3H-serotonin from activated
platelets.6a Selected compounds were also examined in a
binding assay for their ability to displace a radiolabeled
peptide.11
Initial investigations involved the independent modi-
fication of both substituents on the exocyclic amino
group. Chlorination of isoxazolone 3 provided 4, which
was reacted with N-3-aminopropylpiperidine and fur-
ther alkylated with alkyl or benzyl bromides to afford
derivatives of type 5, as shown in Scheme 1.
The first area of investigation centered around the
N-benzyl substituent of lead isoxazole 2 (Table 1).
Modification of the piperidine-bearing substituent was
accomplished as described in Scheme 2. Intermediates
of type 6 could be obtained from chloroisoxazole 4 and
benzyl amines or via acylation/reduction of aminoisox-
azole 7. Tethers carrying potential leaving groups were
then installed to afford intermediates of type 8. Nucleo-
philic displacement with various amines provided deri-
vatives of general structure 9.
While alkyl replacements are not tolerated (16 and 17),
up to a 10-fold increase in potency over lead compound
2 can be obtained by selected substitution of the
N-benzyl substituent. The best groups were 1-naphthyl
(20) and 3,4-methylenedioxy (22) with IC50s of 0.5 and 0.6
mM, respectively, when TRAP was used as the agonist.
Modifications of the tertiary amine side chain were then
investigated. Removal of the amine (23) resulted in
complete loss of activity. Variation of ring size indicated
a marked preference for piperidine (26) and azepine (27)
derivatives (Table 2).
Optimization of the isoxazole aryl substituent was
accomplished according to Scheme 3. Acylation of
piperonyl amine 10 with acryloyl chloride followed by
Michael addition of azepine and reduction lead to the
preparation of 12. Acetophenones and arylbenzoyl
chlorides could be converted to b-keto esters of type 13.
Cycloaddition of hydroxylamine and subsequent
chlorination afforded chloroisoxazoles 14. Lithiation of
Table 1. N-Benzyl modifications
o
amine 12 in Et2O at 0 C followed by addition of 14
provided the target compounds 15.
RCompd IC
(mM)a
RCompd IC
3-OMe–Ph
1-Naphthyl
2-Naphthyl
3,4-(OCH2O)–Ph
50 (mM)a
50
Ph
n-Pr
Cyclohexyl
3-Cl–Ph
2
16
17
18
9
>40
>10
1
19
20
21
22
1.6
0.5
2
0.6
aValues represent inhibition of secretion of radiolabeled serotonin
from washed human platelets stimulated by 3 mM TRAP peptide
(n>2, standard error of the mean <10%).
Figure 1. From trisubstituted urea to aminoisoxazole PARantagonists.

P. G. Nantermet et al. / Bioorg. Med. Chem. Lett. 12 (2002) 319–323
321
The optimal tether length was demonstrated to be three
methylenes (29–31, Fig. 2).
and 43). para-Substitution was beneficial with methyl,
methoxy and thiomethyl (34, 37, and 47). Substitution
with chloro or fluoro (40 and 42), phenyl (46) or polar
residues such as methyl sulfoxide (48) and sulfone (49)
were not tolerated. When compared to other 4-halogen
derivatives the 4-iodo derivative 50 surprisingly
appeared as one of the most active antagonists with an
with IC50 of 0.09 mM with TRAP used as the agonist.
The final area of investigation centered around the
3-aryl substituent on the isoxazole (Table 3). Substitu-
tion at the ortho position was detrimental to activity as
seen with compounds 32, 35, and 38. meta-Substitution
had a positive effect on the potency of these isoxazole
PAR-1 antagonists. Except for strong electron-with-
drawing groups (44) and larger groups (45), potency
could be maintained or slightly improved (33, 36, 39, 41,
Several compounds demonstrating promising activity
when TRAP was used as the agonist were examined in
the more physiologically relevant thrombin (1 nM)
stimulated secretion assay. Interesting differences
appeared in the SARdepending on whether TRAP or
thrombin was used as agonist. While meta-substitution
was generally beneficial to the activity of these
antagonists against thrombin as an agonist, para-sub-
stitution was found to be detrimental to activity with
Table 2. Side-chain modifications
RCompd IC
50 (mM)a
RCompd IC
50 (mM)a
Cyclohexylb
N-Azetidine
23
24
>10
>10
N-Piperidine
N-Azepine
26
27
1.6
0.5
N-Pyrrolidine 25
3.3 N-Homoazepine 28
>10
aValues represent inhibition of secretion of radiolabeled serotonin
from washed human platelets stimulated by 3 mM TRAP peptide
(n>2, standard error of the mean <10%).
bThe 3-methoxy phenyl group is replaced by 1-naphthyl in this case.
Figure 2. Tether length modifications.
Table 3. 3-Ph modifications
RCAomP pICd
TR
(mM)a,c
Thrombin IC50 (mM)b,c
Binding IC50 (mM)d
50
2-Me
3-Me
4-Me
2-OMe
3-OMe
4-OMe
2-Cl
3-Cl
4-Cl
3-F
4-F
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
3.22
0.12
0.14
2.22
0.13
0.13
2.99
0.27
1.24
0.14
1.31
0.35
0.83
6.42
4.44
0.43
3.7
nd
nd
60% @ 4 mM
3.2
nd
2.24
60% @ 10 mM
nd
0.89
0.29
2.21
0.26
0.25
nd
1.16
0.99
0.77
nd
0.91
4.25
1.18
>10
1.97
>10
nd
nd
>4
nd
nd
3-CN
0.6
3-NO2
3-Ph
4-Ph
4-SMe
4-SOMe
4-SO2Me
4-I
3,4-F2
3,4-(OMe)2
3-F-4-OMe
3,5-F2
3,5-(OMe)2
1.02
nd
nd
nd
nd
3.2
0.09
0.5
nd
0.88
2.4
4.67
2.14
0.51
1.2
0.47
2.14
1.2
nd
0.15
0.52
0.4
0.07
0.09
0.13
aValues represent inhibition of secretion of radiolabeled serotonin from washed human platelets stimulated by 3 mM TRAP peptide.
bValues represent inhibition of secretion of radiolabeled serotonin from washed human platelets stimulated by 1 nM thrombin.
cn>2, standard error of the mean < 10%.
dValues represent displacement of a specific radiolabeled peptide11 (n>3, standard error of the mean < 10%).
nd=not determined.

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P. G. Nantermet et al. / Bioorg. Med. Chem. Lett. 12 (2002) 319–323
and functional assays with such compounds suggest that
they indeed behave as selective antagonists of the
thrombin receptor PAR-1, competing with the binding
of the tethered ligand to its receptor site rather than
interfering with the enzymatic activity of thrombin.
Acknowledgements
The authors thank Mary Becker for help in the pre-
paration of this manuscript and J. Murphy, S. Pitzen-
berger, S. Varga, A. Coddington, C. Ross, H. Ramjit,
K. Anderson, P. Ciecko and M. Zrada for analytical
support.
Figure 3. Inhibition of platelet aggregation induced by 1 nM thrombin
and measured by change in transmittance, using various concentration
of 54 (0, 0.4, 1, 4, 10 mM).
References and Notes
the exception of the 4-iodo derivative 50. Combination
of the previous results lead us to the synthesis of di-
substituted antagonists 51–55. Disubstitution at the 3,4-
position resulted in compounds with good activity
against TRAP but not thrombin-stimulated platelet
secretion. The diminished activity of compounds such as
53 against thrombin as the agonist could be due to
various factors. The off-rate of these compounds may be
too fast, allowing for the tethered ligand to bind and
activate platelets. Involvement of PAR-4 on platelets is
also a complicating feature although it usually requires
a much higher thrombin concentration for activation.4
Finally, the compounds may exhibit different binding
affinities for the cleaved (thrombin activated) and
uncleaved (TRAP activated) receptors. Further studies
are required to define the importance of these and other
potential factors.
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In contrast to the previous results, the 3,5-difluoro ana-
logue 54 demonstrates significant potency against both
TRAP and 1 nM thrombin stimulated platelet secretion
(thrombin IC50=0.51 mM, n=10).
Compound 54 is not an inhibitor of thrombin
catalytic activity (Ki>10 mM), nor does it display
any inhibition of platelet aggregation induced by
ADP or collagen at concentrations up to 20 mM. As
observed with most other tested compounds, the bind-
ing affinity of 54 for the receptor, as measured by dis-
placement of a specific radiolabeled peptide,11 is in
agreement with the functional data (Table 3). Taken
together these results suggest that 54 is acting via selec-
tive antagonism of PAR-1 and competing with the
intramolecular ligand.
To further evaluate its potential as an antiplatelet agent,
54 was tested for its ability to block human platelet
aggregation induced by 1 nM thrombin.12 As shown in
Figure 3, antagonist 54 fully inhibits platelet aggre-
gation stimulated by 1 nM thrombin for the length of
the experiment (10 min) at 4 mM, and for 5–6 min at 1
mm, which represents an improvement over our pre-
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In conclusion, aminoisoxazole derivatives such as 54 are
potent inhibitors of platelet activation. In vitro binding

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