Thrombin receptor (PAR-1) antagonists. Solid-phase synthesis of indole-based peptide mimetics by anchoring to a secondary amide

Article abstract of DOI:10.1016/S0960-894X(01)00378-X

A novel, 10-step, solid-phase method, based on a secondary amide linker, was developed to construct a diverse library of indole-based SFLLR peptide mimetics as thrombin receptor (protease-activated receptor 1, PAR-1) antagonists. The key steps include stepwise reductive alkylation, urea formation, and Mannich reaction. Screening of the library led to a quick development of the SAR and the significant improvement of PAR-1 activity.

Full text of DOI:10.1016/S0960-894X(01)00378-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2105–2109
Thrombin Receptor (PAR-1) Antagonists. Solid-Phase
Synthesis of Indole-Based Peptide Mimetics by
Anchoring to a Secondary Amide
Han-Cheng Zhang,a,* David F. McComsey,a Kimberly B. White,a
Michael F. Addo,a Patricia Andrade-Gordon,a Claudia K. Derian,a
Donna Oksenbergb and Bruce E. Maryanoffa
aDrug Discovery, The R. W. Johnson Pharmaceutical Research Institute, Spring House, PA 19477-0776, USA
bCOR Therapeutics, Inc., South San Francisco, CA 94080, USA
Received 29 March 2001; accepted 16 May 2001
Abstract—A novel, 10-step, solid-phase method, based on a secondary amide linker, was developed to construct a diverse library of
indole-based SFLLR peptide mimetics as thrombin receptor (protease-activated receptor 1, PAR-1) antagonists. The key steps
include stepwise reductive alkylation, urea formation, and Mannich reaction. Screening of the library led to a quick development of
the SAR and the significant improvement of PAR-1 activity. # 2001 Elsevier Science Ltd. All rights reserved.
Thrombin receptors are attractive drug discovery tar-
gets because they mediate a variety of cellular actions of
thrombin, such as platelet aggregation, lymphocyte
mitosis, monocyte chemotaxis, and endothelial cell pro-
liferation.1 The first example of a thrombin receptor
(protease-activated receptor 1, PAR-1) was cloned in
1991 and identified as a member of the superfamily of
seven-transmembrane, G-protein-coupled receptors.2
Thrombin activates PAR-1 by proteolytically cleaving
the N-terminal extracellular domain between Arg-41
and Ser-42 to reveal a new N-terminus containing the
activation motif SFLLRN, which serves as a ‘tethered’
peptide ligand. Three closely related protease-activated
receptors, PAR-2,3 PAR-3,4 and PAR-4,5 have also
been cloned and each possesses a unique agonist peptide
epitope. Since PAR-1 mediates many cellular actions of
thrombin, a PAR-1 antagonist may have therapeutic
potential for treating various disorders, such as throm-
bosis, restenosis, atherosclerosis, inflammation, cancer
metastasis, and stroke.1,6
of potent and selective PAR-1 antagonists has proved to
be very challenging. This situation is partially due to the
unfavorable entropy for an external ligand to compete
with a tethered ligand. Of the PAR-1 antagonists
reported,7b,8,9 several have notable deficiencies,7b,8 such
as weak potency, inability to inhibit thrombin-induced
platelet aggregation consistently, mixed agonist-antago-
nist activity, and/or lack of PAR selectivity. Through a
de novo design approach, we discovered a series of
novel indole-based SFLLR peptide mimetics as potent
and selective PAR-1 antagonists.9 The prototype of this
series, RWJ-53052 (1), inhibited both thrombin- and
SFLLRN-NH2-induced human platelet aggregation
(IC50=2.3 and 0.27 mM, respectively) and bound to
PAR-1 with an IC50 of 2.2 mM. Compound 1 was pre-
pared via a convergent solution-phase method in nine
steps (23% overall yield) from 6-nitroindole and appro-
priate Fmoc-protected amino acids.9 We have devel-
oped parallel solid-phase methods for the synthesis of
analogues of 1, which proved to be much more efficient
than the solution-phase method. This approach allowed
us to rapidly explore the SAR and optimize the potency
of the series. Herein, we report our solid-phase method
that is based on a secondary amide anchor, which led to
the rapid construction of a highly diverse library of
analogues and the identification of RWJ-56110 (34) as a
potent and selective PAR-1 antagonist.9
Despite the extensive structure–function data for PAR-1
agonist peptides related to SFLLRN,7 the identification
*Corresponding author. Tel.: +1-215-628-5988; fax.: +1-215-628-
4985; e-mail: hzhang@prius.jnj.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00378-X

2106
H.-C. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2105–2109
phosphate] and HOBt, and then deprotection again
with piperidine to afford 6.
With the resin-bound dipeptide amine 6 in hand, we
started to investigate the incorporation of the indole
template via a urea linkage, one of the key steps in the
whole synthetic process. 6-Aminoindole intermediate 8
was prepared from 79 via Mannich reaction under stan-
dard solution-phase conditions followed by nitro
reduction (Scheme 2). The urea formation between resin
6 [R=Ph(CH2)3] and 6-aminoindole 8 was attempted by
using a reported procedure.13 Thus, resin 6 was treated
with p-nitrophenyl chloroformate in THF/CH2Cl2 in
the presence of i-Pr2NEt to form a carbamate inter-
mediate and then coupled with amine 8 at 23 ꢀC.
Unfortunately, analysis of cleaved product (with TFA)
from an aliquot of resin indicated that no indole was
incorporated into the molecule; instead, the MS (ES+)
of the product was consistent with a dipeptide isocya-
nate (NH+=761). Reasoning that an isocyanate should
react with indole amine 8 at elevated temperature to
form a urea bond, the reaction temperature was
increased. However, after stirring at 95 ꢀC, no change
was observed by TLC and MS. Further analysis
revealed that the product was peptide hydantoin 10
instead of a dipeptide isocyanate (Scheme 3).
Considering the structural features of RWJ-53052, the
guanidine moiety, originally believed to be important
for PAR-1 activity, appears to be an appropriate point
for resin attachment. However, there was no resin
available at the time for anchoring the guanidine
group.10 The only other group suitable for resin attach-
ment is the terminal amide. Thus, we attempted to
attach this secondary amide NH to an amide-type resin
and then construct a dipeptide segment, followed by
introducing the indole scaffold. Rink amide-type resins
have been widely used as an amide linker for the solid-
phase synthesis of small organic molecules, for example,
in the construction of indoles.11 However, application
of this resin for anchoring a secondary amide, such as in
1, has been limited due to steric hindrance. Thus, we
used the Sieber amide resin, in which the amino group is
less sterically hindered than in Rink amide-type resins.
The N-arylalkyl or N-alkyl group at the C-terminus was
introduced via a stepwise reductive alkylation process.12
Sieber resin 2 (0.37 mmol/g, Novabiochem) was depro-
tected with 20% piperidine in DMF and then treated
with excess of hydrocinnamaldehyde (3a) in DMF/
HOAc (100:1) to form an imine (Scheme 1). The excess
of aldehyde was washed out before adding the reducing
agent NaBH4 to avoid formation of dialkylated pro-
duct. The resulting resin-bound hindered secondary
amine, 4a, was successfully coupled with Fmoc-
Arg(Pmc)-OH (Pmc, 2,2,5,7,8-pentamethylchroman-6-
sulfonyl) by using HATU [O-(7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate] and
HOAt (1-hydroxy-7-azabenzotriazole). Fmoc-echo ana-
To avoid undesired hydantoin 10, dipeptide 6 must be
removed from exposure to p-nitrophenyl chloroformate.
Therefore, we attempted to generate the p-nitrophenyl
carbamate intermediate with indole amine 8. Dis-
appointingly, treatment of 8 with p-nitrophenyl chloro-
formate caused the decomposition of Mannich base of
8. To overcome this problem, indole amine 9, prepared
from 7 via nitro reduction, was used and an effort was
made to introduce the Mannich base on the solid phase
after urea formation. Thus, the urea was produced by
treating 9 with 0.83 equiv of p-nitrophenyl chloro-
formate in the presence of i-Pr2NEt at ꢁ20 ꢀC for 5 min
and then coupled with resin-bound dipeptide amine 6
(0.24 equiv). The reaction mixture was allowed to warm
to 23 ꢀC and then stirred for 3 h. TLC and MS analysis
on the cleaved material indicated that the desired urea
11 was the major product along with a small amount of
peptide hydantoin 10 (less than 10%).
lysis (at 290 nm)12 of the coupled product
5
[R=Ph(CH2)3] indicated a good loading level of 0.28
mmol/g. Deprotection of the Fmoc group in 5 with
piperidine was followed by coupling reaction with
Fmoc-Tyr(Me)-OH in the presence of HBTU [O-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
Our initial test of the Mannich reaction on resin 11
turned out to be problematic. Under the common
Mannich conditions used in solution phase (pyrrolidine/
Scheme 1. (a) Piperidine, DMF; (b) aldehyde (3), DMF/HOAc (100:1, for Sieber resin), or CH(OMe)3 (for Tentagel S AM resin); (c) NaBH4, DMF/
MeOH (10:3, for Sieber resin), or NaB(OAc)3H, ClCH2CH2Cl/HOAc (100:1, for Tentagel S AM resin); (d) Fmoc-Arg(Pmc)-OH, HATU, HOAt, i-
Pr2NEt, DMF; (e) Fmoc-Tyr(Me)-OH, HBTU, HOBt, i-Pr2NEt, DMF.

H.-C. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2105–2109
2107
Scheme 2. (f) Pyrrolidine, HCHO, HOAc; (g) Me2NNH2, FeCl3ꢂ 6H2O, charcoal, MeOH, reflux.
as seen with 4a and 4b to give resin 5 (R=Bn) with a
good loading level of 0.20 mmol/g, as determined by
Fmoc-echo at 290 nm.12
By using resins 4a–c and subsequent parallel solid-phase
chemistry, as described above,15 we constructed a
library of indole-based peptide mimetics bearing diverse
substituents around the indole scaffold (R, R1, R2, R3,
A1 and A2, Table 1). Over 200 individual analogues
have been prepared. Typically, the products displayed
75–85% purity as determined by reverse-phase HPLC
and this purity was sufficient for biological evaluation
and SAR interpretation. Representative compounds
were purified and retested and their activities were con-
sistent with those of the crude products.
Scheme 3. (h) p-NO2C6H4OCOCl, i-Pr2NEt, THF/CH2Cl2.
HCHO/HOAc), the reaction of 11 was very sluggish,
even at elevated temperature (80 ꢀC). Reaction condi-
tion studies led to identification of 1,4-dioxane/HOAc
(4:1) as co-solvent which allowed the Mannich reaction
of resin 11 to proceed smoothly at 25–50 ꢀC (Scheme 4).
One-pot Pmc deprotection and resin cleavage was con-
ducted by using TFA/i-Pr3SiH/1,2-ethanedithiol/H2O
(90:1:4.5:4.5) at 23 ꢀC. The resulting crude cleaved pro-
duct was triturated with Et2O to afford the desired pro-
duct 12 [R=Ph(CH2)3] in 37% overall yield (based on
the loading level of resin 4a) and about 80% purity by
reverse-phase HPLC.
Compounds prepared via the solid-phase methods were
tested for inhibition of platelet aggregation induced by
thrombin, SFLLRN-NH2, collagen, and U46619
(thromboxane mimetic). We also examined their com-
petitive binding versus [3H]-S-(p-F-Phe)-homoarginine-
l-homoarginine-KY-NH2 to PAR-1 on the membranes
of CHRF-288-11 cells.9 The representative compounds
with their in vitro biological data are presented in Table
1. Replacement of benzyl of R in 1 with 3-phenylpropyl
(12) or cyclohexylmethyl (18) resulted in a 2- to 4-fold
decrease of potency against thrombin-induced gel-fil-
tered platelet aggregation, though similar binding affi-
nities to PAR-1 were observed for all three
compounds.16 Substitution of 4-F of R1 with 2-Cl (13),
2-Me (15 and 28) or 2,6-diCl (26) led to more potent
analogues. However, a larger group, such as CO2Me
(22) or OCF3 (23 and 24), could not be tolerated at the
4-position. Pyrrolidine (R2R3N) was generally preferred
over piperidine (17 and 19), diethylamine (16 and 20) or
azetidine (25). A further improvement of potency was
achieved by substituting Tyr(Me) of A1 in 26 with 3,4-
diF-Phe (31). We also replaced the guanidine moiety in
31 with a primary amine (32). This guanidine group was
We then tested the reaction (2!4, Scheme 1) by using
other aldehydes. Stepwise reductive alkylation of the
Sieber resin with cyclohexanecarboxaldehyde (3b) was
as effective as with 3a to give resin 4b. However, the
chemistry was less successful when benzaldehyde (3c)
was used, where reductive alkylation of the Sieber resin
was incomplete under various reaction conditions. Ten-
tagel S AM resin was then identified to be a better
anchor for introducing a benzyl group. Thus, Tentagel S
AM resin (0.23 mmol/g, Rapp Polymere) was treated
with benzaldehyde in trimethylorthoformate14 over-
night and then the excess of aldehyde was removed by
washing. The resulting imine was treated immediately
with NaB(OAc)3H in ClCH2CH2Cl/HOAc to afford
resin 4c. Coupling of 4c with Fmoc-Arg(Pmc)-OH in
the presence of HATU and HOAt proceeded smoothly
Scheme 4. (i) p-NO2C6H4OCOCl, i-Pr2NEt, CH2Cl2; (j) pyrrolidine, HCHO, 1,4-dioxane/HOAc (4:1); (k) TFA/i-Pr3SiH/HSCH2CH2SH/H2O
(90:1:4.5:4.5).

2108
H.-C. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2105–2109
originally believed to be important for PAR-1 activity17
but caused undesirable cardiovascular effects in an
anesthetized guinea pig hemodynamic model. The Orn
analogue 32 was equally potent as 31 in thrombin- and
SFLLRN-NH2-induced gel-filtered platelet aggre-
gation, as well as PAR-1 binding assays, and showed an
improved cardiovascular hemodynamic profile over 31.
However, 32 showed a decreased selectivity over col-
lagen. Systematic changes of the aminoalkyl side chain
length in 32 led to 34 (RWJ-56110), a potent and selec-
tive PAR-1 antagonist.9 Given the successful replace-
ment of the guanidine with a primary amine, a trityl
resin-based solid-phase method was then developed for
the further synthesis of RWJ-56110 analogues.9 Mod-
ification of the indole template of RWJ-56110 to an
indazole resulted in RWJ-58259,18 a second-generation,
indazole-based PAR-1 antagonist, which serves as a
suitable agent for in vivo assessment of the therapeutic
potential of PAR-1 antagonists.
We have developed a novel, efficient secondary amide-
linked resin-based solid-phase method for construction
of a diverse library of indole-based peptide mimetics as
PAR-1 antagonists. Screening of the library led to a
quick development of the SAR of this peptide mimetic
series, a significant improvement in potency over the
prototype RWJ-53052, and the identification of RWJ-
56110 as a potent and selective PAR-1 antagonist.
Table 1. Platelet aggregation and PAR-1 binding IC50 values (mM)a
Gel-filtered platelet aggregationb
Compd
R1
R2R3N
A1
A2
R
Thrombin
SFLLRN-NH2
Collagen
U46619
Bindingc
1
4-F
4-F
2-Cl
3-Me
2-Me
2-Me
2-Me
4-F
4-F
4-F
3-Me
4-CO2Me
4-OCF3
4-OCF3
4-F
2,6-diCl
4-F
2-Me
(CH2)4N
(CH2)4N
(CH2)4N
Et2N
(CH2)4N
Et2N
(CH2)5N
(CH2)4N
(CH2)5N
Et2N
(CH2)5N
(CH2)4N
(CH2)4N
(CH2)5N
(CH2)3N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
(CH2)4N
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
Tyr(Me)
4-NO2-Phe
4-NO2-Phe
4-CN-Phe
4-CN-Phe
3,4-DiF-Phe
3,4-DiF-Phe
3,4-DiF-Phe
3,4-DiF-Phe
3,4-DiF-Phe
3,4-DiF-Phe
3,4-DiF-Phe
3,4-DiF-Phe
3,4-DiF-Phe
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Arg
Ornf
Lys
Dbug
Dprh
His
Bn
Phpd
Php
Php
Php
Php
Php
Chme
Chm
Chm
Chm
Chm
Chm
Chm
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
2.32+0.11
8.5ꢃ1.3
2.6
0.27ꢃ0.01
0.6ꢃ0.2
0.14
IA
16.1ꢃ2.5
12.0
10.2
11.9
IA
21.4ꢃ5.6
2.2ꢃ0.5
1.3
0.38
2.6
1.0
2.5
0.73
2.6
2.3
3.1
3.2
3.5
2.5
5.6
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
44.4
1.3
1.0
3.0
4.8
0.21
0.20
0.23
0.31
IA
44.2
IA
IA
17.0
13.0
4.4
0.64
37.6ꢃ22.6
26.1ꢃ10.9
25.8ꢃ7.6
27.5ꢃ7.8
IA
40.4ꢃ19.1
33.1ꢃ13.9
IA
12.5
26.2ꢃ15.7
23.8ꢃ6.8
11.1ꢃ4.7
11.7ꢃ5.0
38.7
40.3ꢃ10.3
36.5ꢃ10.6
9.7
1.0ꢃ0.1
0.9ꢃ0.4
0.9ꢃ0.4
IA
4.8ꢃ0.5
6.4ꢃ0.5
0.4ꢃ0.2
0.30ꢃ0.09
0.13
39.5ꢃ13.6
36.3ꢃ11.2
IA
33.0ꢃ7.7
43.5ꢃ23.7
IA
3.3
1.3ꢃ0.00
0.90ꢃ0.34
46.3ꢃ28.6
IA
IA
IA
IA
IA
IA
IA
41.9
IA
IA
IA
IA
6.1
1.3
16.5
20.6
1.1
0.8
0.15
0.6ꢃ0.5
2-Me
0.62
0.48
25.9
IA
6.4
5.3
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
0.07
0.57ꢃ0.21
0.6ꢃ0.2
0.7ꢃ0.3
0.34ꢃ0.04
0.9ꢃ0.2
0.46
0.18ꢃ0.21
0.1ꢃ0.05
0.1ꢃ0.02
0.16ꢃ0.05
0.5ꢃ0.3
0.2ꢃ0.01
0.3ꢃ0.1
2.0ꢃ1.8
0.4ꢃ0.1
IA
15.7ꢃ9.9
0.82ꢃ0.34
0.5ꢃ0.3
0.3ꢃ0.00
0.44ꢃ0.21
0.92
1.3
0.24
1.8
11.2
21.3
IA
Bn
Bn
Bn
Bn
Bn
Bn
36.9
34.0
IA
IA
Citi
1.9
7.9ꢃ7.4
Gln
Met
IA
IA
10.7ꢃ10.1
IA
aResults are expressed as meanꢃSEM (nꢄ 2; n=1 for values without error limits). IA denotes inactivity @ 50 mM of test compound.
bConcentrations of agonists for aggregation studies: a-thrombin, 0.15 nM; SFLLRN-NH2, 2 mM; collagen, 3 mg/mL; U46619, 0.3 mM.
cThrombin receptor (PAR-1) binding assay; ligand: [3H]-S-(p-F-Phe)-homoarginine-l-homoarginine-KY-NH2, 10 nM (Kd=15 nM).
dPhp, 3-phenylpropyl.
eChm, cyclohexylmethyl.
fOrn, ornithine.
gDbu, 2,4-diaminobutyric acid.
hDpr, 2,3-diaminopropionic acid.
iCit, citrulline.

H.-C. Zhang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2105–2109
2109
Acknowledgements
McComsey, D. F.; Hecker, L. R.; Andrade-Gordon, P.; Addo,
M. F.; Maryanoff, B. E. Bioorg. Med. Chem. Lett. 1999, 9,
255. (e) Hoekstra, W. J.; Hulshizer, B. L.; McComsey, D. F.;
Andrade-Gordon, P.; Kauffman, J. A.; Addo, M. F.; Oksen-
berg, D.; Scarborough, R. M.; Maryanoff, B. E. Bioorg. Med.
Chem. Lett. 1998, 8, 1649. (f) Bernatowicz, M. S.; Klimas,
C. E.; Hartl, K. S.; Peluso, M.; Allegretto, N. J.; Seiler, S. M.
J. Med. Chem. 1996, 39, 4879. (g) Seiler, S. M.; Peluso, M.;
Michel, I. M.; Goldenberg, H., II; Fenton, J. W.; Riexinger,
D.; Natarajan, S. Biochem. Pharmacol. 1995, 49, 519. (h) Lin-
dahl, A. H.; Scarborough, R. M.; Naughton, M. A.; Harker,
L. A.; Hanson, S. R. Thromb. Haemost. 1993, 69, 1196.
9. 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. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12257.
10. (a) For solid-phase linkers recently developed for attach-
ing guanidine group, see: Zhong, H. M.; Greco, M. N.; Mar-
yanoff, B. E. J. Org. Chem. 1997, 62, 9326. (b) Josey, J. A.;
Tarlton, C. A.; Payne, C. E. Tetrahedron Lett. 1998, 39, 5899.
(c) Edwards, P. D.; Mauger, R. C.; Cottrell, K.; Morris, F. X.;
Pine, K. K.; Sylvester, M. A.; Scott, C. W.; Furlong, S. T.
Bioorg. Med. Chem. Lett. 2000, 10, 2291.
The authors thank Louis Fitzpatrick and Jack Kauff-
man for excellent technical assistance, and Drs. William
Hoekstra, Michael Greco, Robert Scarborough, and
Ralph Rivero for helpful discussions.
References and Notes
1. (a) Brass, L. F. Thromb. Haemost. 1995, 74, 499. (b) Ogle-
tree, M. L.; Natajaran, S.; Seiler, S. M. Perspect. Drug Discov.
Des. 1994, 1, 527. (c) Coughlin, S. R. Thromb. Haemost. 1993,
70, 184. (d) Ray, A.; Hegde, L. G.; Gupta, J. B. Thromb. Res.
1997, 87, 37.
2. Vu, T.-K. H.; Hung, D. T.; Wheaton, V. I.; Coughlin, S. R.
Cell 1991, 64, 1057.
3. Nystedt, S.; Emilsson, K.; Wahlestedt, C.; Sundelin, J.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 9208.
4. Ishihara, H.; Connolly, A. J.; Zeng, D.; Kahn, M. L.;
Zheng, Y. W.; Timmons, C.; Tram, T.; Coughlin, S. R. Nature
1997, 386, 502.
5. (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. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6642. (b) Kahn,
M. L.; Zheng, Y.-W.; Huang, W.; Bigornia, V.; Zeng, D.;
Moff, S.; Farese, R. V., Jr.; Tam, C.; Coughlin, S. R. Nature
1998, 394, 690.
6. (a) Coughlin, S. R. Trends Cardiovasc. Med. 1994, 4, 77. (b)
Dennington, P. M.; Berndt, M. C. Clin. Exp. Pharmacol.
Physiol. 1994, 21, 349. (c) Van Obberghen-Schilling, E.;
Chambard, J.-C.; Vouret-Craviari, V.; Chen, Y.-H.; Grall, D.;
11. (a) Zhang, H.-C.; Maryanoff, B. E. J. Org. Chem. 1997,
62, 1804. (b) Zhang, H.-C.; Brumfield, K. K.; Maryanoff, B. E.
Tetrahedron Lett. 1997, 38, 2439.
12. Chan, W. C.; White, P. D.; Beythien, J.; Steinauer, R. J.
Chem. Soc., Chem. Commun. 1995, 1475.
13. Hutchins, S. M.; Chapman, K. T. Tetrahedron Lett. 1994,
35, 4055. Hutchins, S. M.; Chapman, K. T. Tetrahedron Lett.
1995, 36, 2583.
14. Look, G. C.; Murphy, M. M.; Campbell, D. A.; Gallop,
M. A. Tetrahedron Lett. 1995, 36, 2937.
15. Other 6-aminoindole intermediates with different R1
groups were prepared in the same way as was 9, from 6-
nitroindole.
16. For some compounds in Table 1, inconsistencies were
observed between receptor binding and functional activity in
platelet aggregation. The reason for this is not yet understood.
However, other laboratories have observed similar incon-
sistencies with their PAR-1 antagonists.8b,f
17. Representative guanidine-containing PAR-1 antagonists
from our series, including 1, 14, and 31 in this paper, were
tested in a chromogenic assay for thrombin inhibition and
showed no activity at 100 mM, thereby excluding a direct
enzyme-based mechanism of inhibition.
Pouysse
´
gur, J. Eur. J. Med. Chem. 1995, 30 (Suppl.), 117.
7. (a) Scarborough, R. M.; Naughton, M. A.; Teng, W.;
Hung, D. T.; Rose, J. W.; Vu, T.-K.; Wheaton, V. I.; Turck,
C. W.; Coughlin, S. R. J. Biol. Chem. 1992, 267, 13146. (b)
Seiler, S. M.; Peluso, M.; Tuttle, J. G.; Pryor, K.; Klimas, C.;
Matsueda, G. R.; Bernatowicz, M. S. Mol. Pharmacol. 1996,
49, 190. (c) Feng, D. M.; Veber, D. F.; Connolly, T. F.; Con-
dra, C.; Tang, M.-J.; Nutt, R. F. J. Med. Chem. 1995, 38,
4125. (d) Natarajan, S.; Reixinger, D.; Peluso, M.; Seiler, S. M.
Int. J. Pept. Protein Res. 1995, 45, 145.
8. (a) Alexopoulos, K.; Panagiotopoulos, D.; Mavromousta-
kos, T.; Fatseas, P.; Paredes-Carbajal, M. C.; Mascher, D.;
Mihailescu, S.; Matsoukas, J. J. Med. Chem. 2001, 44, 328. (b)
Ahn, H.-S.; Arik, L.; Boykow, G.; Burnett, D. A.; Caplen,
M. A.; Czarniecki, M.; Domalski, M. S.; Foster, C.; Manna,
M.; Stamford, A. W.; Wu, Y. Bioorg. Med. Chem. Lett. 1999,
9, 2073. (c) Fujita, T.; Nakajima, M.; Inoue, Y.; Nose, T.;
Shimohigashi, Y. Bioorg. Med. Chem. Lett. 1999, 9, 1351. (d)
18. Zhang, H.-C.; Derian, C. K.; Andrade-Gordon, P.;
Hoekstra, W. J.; McComsey, D. F.; White, K. B.; Poulter,
B. L.; Addo, M. F.; Cheung, W.-M.; Damiano, B. P.; Oksen-
berg, D.; Reynolds, E. E.; Pandey, A.; Scarborough, R. M.;
Maryanoff, B. E. J. Med. Chem. 2001, 44, 1021.