Design and synthesis of novel biologically active thrombin receptor non-peptide mimetics based on the pharmacophoric cluster Phe/Arg/NH2 of the Ser42-Phe-Leu-Leu-Arg46 motif sequence: Platelet aggregation and relaxant activities

Article abstract of DOI:10.1021/jm031080v

The identification of the thrombin receptor has promoted the interest for the development of new therapeutic agents capable of selectively inhibiting unwanted biological effects of thrombin on various cell types. In this study we have designed and synthesized two series of new thrombin receptor antagonists based on the thrombin receptor motif sequence S42FLLR46, one possessing two (Phe/Arg) pharmacophoric groups and the other possessing three (Phe/Arg/NH ₂). N-(6-Guanidohexanoyl)-N′-(phenylacetyl)piperazine (1), N-(phenylacetyl)-4-(6-guanidohexanoyl-amidomethyl)piperidine (2), and N-(phenylacetyl)-3-(6-guanidohexanoylamido)pyrrolidine (3) (group A) carry the two pharmacophoric side chains of Phe and Arg residues incorporated on three different templates (piperazine, 4-aminomethylpiperidine, and 3-aminopyrrolidine). Compounds with three pharmacophoric groups (group B) were built similarly to group A using the same templates with the addition of an extra methylamino group leading to (S)-N-(6-guanidohexanoyl)-N′ -(2-amino-3-phenylpropionyl)piperazine (4), (S)-N-(2-amino-3-phenylpropionyl)-4-(6-guanidohexanoylamidomethyl)piperidine (5), and (S)-N-(2-amino-3-phenylpropionyl)-3-(6-guanidohexanoylamido) pyrrolidine (6). Compounds were able to inhibit thrombin-induced human platelet activation even at low concentrations. In particular, among compounds in group A, compound 3 was found to be the most powerful thrombin receptor activation inhibitor, showing an IC₅₀ of approximately 0.11 mM on platelet aggregation assay. Among compounds in group B, compound 4 was the most powerful to inhibit thrombin-induced platelet aggregation, showing an IC₅₀ of approximately 0.09 mM. All compounds were also found to act as agonists in the rat aorta relaxation assay. Interestingly, the order of potency of these compounds as agonists of the endothelial thrombin receptor was the inverse of the order of potency of the same compounds as antagonists of the platelet thrombin receptor. Such compounds that are causing vasodilation while simultaneously inhibiting platelet aggregation would be very useful in preventing the installation of atherosclerotic lesions and deserve further investigation as potential drugs for treating cardiovascular diseases. The above findings coupled with computational analysis molecular dynamics experiments support also our hypothesis that a cluster of phenyl, guanidino, and amino groups is responsible for thrombin receptor triggering and activation.

Full text of DOI:10.1021/jm031080v

3338
J. Med. Chem. 2004, 47, 3338-3352
Articles
Design and Synthesis of Novel Biologically Active Thrombin Receptor
Non-Peptide Mimetics Based on the Pharmacophoric Cluster Phe/Arg/NH2 of
the Ser42-Phe-Leu-Leu-Arg46 Motif Sequence: Platelet Aggregation and Relaxant
Activities
Kostas Alexopoulos,*,†,‡ Panagiotis Fatseas,§ Euthemia Melissari,§ Demetrios Vlahakos,§ Panagiota Roumelioti,†
Thomas Mavromoustakos,# Stefan Mihailescu,| Maria Christina Paredes-Carbajal, Dieter Mascher, and
John Matsoukas*,†
Department of Chemistry, University of Patras, Patras 26110, Greece, Blood Department, Agios Andreas Hospital,
Patras 26335, Greece, Onassis Cardiac Surgery Center, 356 Sygrou Avenue, Athens 17674, Greece, National Hellenic Research
Foundation, Institute of Organic and Pharmaceutical Chemistry, Vas. Constantinou 48 Avenue, Athens 11635, Greece,
Department of Physiology, Faculty of Medicine, University of Craiova, Craiova, RO-1100, Romania, and Department of
Physiology, School of Medicine, Universidad Nacional Autonoma de Mexico, Mexico DF, 04510, Mexico
Received October 27, 2003
The identification of the thrombin receptor has promoted the interest for the development of
new therapeutic agents capable of selectively inhibiting unwanted biological effects of thrombin
on various cell types. In this study we have designed and synthesized two series of new thrombin
receptor antagonists based on the thrombin receptor motif sequence S42FLLR46, one possessing
two (Phe/Arg) pharmacophoric groups and the other possessing three (Phe/Arg/NH2). N-(6-
Guanidohexanoyl)-N′-(phenylacetyl)piperazine (1), N-(phenylacetyl)-4-(6-guanidohexanoyl-
amidomethyl)piperidine (2), and N-(phenylacetyl)-3-(6-guanidohexanoylamido)pyrrolidine (3)
(group A) carry the two pharmacophoric side chains of Phe and Arg residues incorporated on
three different templates (piperazine, 4-aminomethylpiperidine, and 3-aminopyrrolidine).
Compounds with three pharmacophoric groups (group B) were built similarly to group A using
the same templates with the addition of an extra methylamino group leading to (S)-N-(6-
guanidohexanoyl)-N′-(2-amino-3-phenylpropionyl)piperazine (4), (S)-N-(2-amino-3-phenylpro-
pionyl)-4-(6-guanidohexanoylamidomethyl)piperidine (5), and (S)-N-(2-amino-3-phenylpropionyl)-
3-(6-guanidohexanoylamido)pyrrolidine (6). Compounds were able to inhibit thrombin-induced
human platelet activation even at low concentrations. In particular, among compounds in group
A, compound 3 was found to be the most powerful thrombin receptor activation inhibitor,
showing an IC50 of approximately 0.11 mM on platelet aggregation assay. Among compounds
in group B, compound 4 was the most powerful to inhibit thrombin-induced platelet aggregation,
showing an IC50 of approximately 0.09 mM. All compounds were also found to act as agonists
in the rat aorta relaxation assay. Interestingly, the order of potency of these compounds as
agonists of the endothelial thrombin receptor was the inverse of the order of potency of the
same compounds as antagonists of the platelet thrombin receptor. Such compounds that are
causing vasodilation while simultaneously inhibiting platelet aggregation would be very useful
in preventing the installation of atherosclerotic lesions and deserve further investigation as
potential drugs for treating cardiovascular diseases. The above findings coupled with
computational analysis molecular dynamics experiments support also our hypothesis that a
cluster of phenyl, guanidino, and amino groups is responsible for thrombin receptor triggering
and activation.
Introduction
Thrombin is a multifunctional serine protease that
plays a central role in thrombosis and hemostasis, as
well as in atherosclerotic and inflammatory diseases.1-3
Thrombin acts on a specific receptor, a member of the
seven transmembrane receptor family,4 and causes a
limited proteolytic cleavage, leading to the formation of
a new N-terminus capable of activating the receptor via
intramolecular interaction.5-8 The identification of such
proteolytically activated tethered-ligand thrombin re-
ceptor sequences on a variety of cell types (platelets,
vascular smooth muscle cells, endothelial cells, fibro-
blasts) has increased our understanding on how throm-
bin signals are mediated within the cells5,9-10 and
* To whom correspondence should be addressed. For K.A.: phone,
30 2610 227073; fax, 30 2610 227075; e-mail, kostalexopoulos@
yahoo.com. For J.M.: phone and fax, 30 2610 997180; e-mail,
johnmatsoukas@hotmail.com.
† University of Patras.
‡ Agios Andreas Hospital.
§ Onassis Cardiac Surgery Center.
# Institute of Organic and Pharmaceutical Chemistry.
| University of Craiova.
Universidad Nacional Autonoma de Mexico.
10.1021/jm031080v CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/19/2004

Thrombin Receptor Mimetics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3339
Scheme 1. Thrombin Receptor Non-Peptide Mimetics
stimulated our interest in synthesizing new thrombin
receptor antagonists.11
solid-phase organic synthesis. In particular we have
synthesized and investigated the following compounds:
N-(6-guanidohexanoyl)-N′-(phenylacetyl)piperazine (1),
N-(phenylacetyl)-4-(6-guanidohexanoylamidomethyl)-
piperidine (2), N-(phenylacetyl)-3-(6-guanidohexanoyl-
amido)pyrrolidine (3), (S)-N-(6-guanidohexanoyl)-N′-(2-
amino-3-phenylpropionyl)piperazine (4), (S)-N-(2-amino-
3-phenylpropionyl)-4-(6-guanidohexanoylamidometh-
yl)piperidine (5), and (S)-N-(2-amino-3-phenylpropio-
nyl)-3-(6-guanidohexanoylamido)pyrrolidine (6). These
compounds carrying the essential pharmacophoric groups
of Phe, Arg and of Phe, Arg, NH2 incorporated onto three
different small, bifunctional templates (piperazine, 4-ami-
nomethylpiperidine, and 3-aminopyrrolidine) (Scheme
1) were subsequently tested for biological activity in
platelet-rich plasma and perfused aortic rings. Data
show strong potency in both assays, with compound 4
being the most active rendering these compounds
potential substances for further investigation and pos-
sible development in the cardiovascular field. The
conformation of the most active non-peptide antagonist
in the platelet aggregation (compound 4) was assessed
using simulation and modeling methods in keeping with
our previous studies.17,20,21,23
About a decade ago, synthetic thrombin receptor
activating peptides (TRAPs) comprising only the first
five amino acids (S42FLLR46) of tethered ligand have
been shown to be sufficient to mimic human receptor
activation by thrombin.12-15 Structure-activity rela-
tionships (SAR) and amino acid substitutions coupled
with NMR studies and molecular modeling have deter-
mined the specific role of each amino acid in the native
SFLLR sequence.12,16,17 Early studies also indicated that
a positively charged NH2-terminus is important for full
agonist activity.18-20 By using cyclic TRAPs that we
have designed and synthesized in our laboratories, we
demonstrated that the Phe/Arg relative conformation
is an important recognition motif in triggering the
receptor, suggesting that a comparable cyclic conforma-
tion may be responsible for the interaction of linear
TRAPs with the thrombin receptor.20-22 Furthermore,
we have demonstrated that this active motif is aug-
mented by the presence of a primary amino group in
the cyclic peptides.20,21
In this study we have designed and synthesized novel
small-size non-peptide TRAP mimetics based on our
SFLLR conformational model17,20,21 using solution- and

3340 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Alexopoulos et al.
Scheme 2. Synthetic Procedure for N-(Phenylacetyl)-4-(6-guanidohexanoylamidomethyl)piperidine (2)a
a Reagents: (i) DCC, HOBt, CHCl3; (ii) Boc-ꢀ-Ahx-OH, DCC, HOBt, DIEA, CH2Cl2; (iii) 30% TFA/CH2Cl2; (iv) 1H-pyrazole-1-carboxamide,
DIEA, DMF.
Results
Chemistry. Compound 1 of group A has been syn-
using solid-phase methods and the 2-chlotrityl chloride
resin as solid support (Scheme 4). Briefly, attachment
of the amino group of phenylalanine (L-Phe) to the resin
(2.27 mequiv of Cl-/g of resin) was achieved by refluxing
using TEA in CH2Cl2 solution and after protection of
the carboxyl group of Phe with (CH3)3SiCl. The loading
of Phe to the resin was certified by the Kaiser ninhydrin
test. The carboxyl group of Phe attached to the resin
through the amino group was first activated with DIC
and HOBt and then divided among two reaction vessels
where it was coupled with two different diamines (4-
aminomethylpiperidine and 3-aminopyrrolidine) in DMF
and in the presence of DIEA. Couplings with Fmoc-ꢀ-
aminohexanoic acid (2.5 equiv) were aided by the use
of DIC and HOBt under basic conditions (DIEA) using
a minimum of DMF. Fmoc group removals were carried
out by treatment with 20% piperidine/DMF for 30 min.
The amino group was converted to the guanidino group
by using 1H-pyrazole-1-carboxamide hydrochloride in
DMF/DIEA in the last solid-phase chemistry step before
cleavage. The resin was treated with 10% TFA/CH2Cl2
for 15 min at room temperature to provide crude
products 5 and 6, which were purified by HPLC.
thesized by methods previously described,24 while syn-
thesis of compound 2 was accomplished as is shown in
Scheme 2. First, 4-aminomethylpiperidine was reacted
with phenylacetic acid (2:1 molar excess of diamine to
carboxylic acid) using DCC and HOBt as coupling
reagents. Only one of the two possible regioisomers, 7a
and not 7b (see Scheme 6), was obtained as identified
by NMR and HPLC analysis. Boc-ꢀ-aminohexanoic acid
was then incorporated in the free primary amino group
of piperidine, aided by the use of DCC and HOBt under
basic conditions (DIEA). Boc deprotection was ac-
complished with trifluoroacetic acid, giving the free
amine salt. Guanylation of the primary amine using 1H-
pyrazole-1-carboxamide hydrochloride afforded com-
pound 2, which was purified by recrystallization (MeOH/
acetone/Et2O). Compound 3 was synthesized in a similar
way starting from 3-aminopyrrolidine (Scheme 3). Again,
only one of the two possible regioisomers 8a and 8b (see
Scheme 6) was obtained after the first alkylation step
and identified by NMR and HPLC analysis to be isomer
8a.
To determine definitely the stereochemistry of com-
pounds 2, 3, 5, and 6, we resorted to selective chemical
reactions differentiating primary and secondary amine
Compound 4 has been synthesized by methods re-
cently described,20 while compounds 5 and 6 were
synthesized by an analogous procedure accomplished

Thrombin Receptor Mimetics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3341
Scheme 3. Synthetic Procedure for N-(Phenylacetyl)-3-(6-guanidohexanoylamido)pyrrolidine (3)a
a Reagents: (i) DCC, HOBt, CHCl3; (ii) Boc-ꢀ-Ahx-OH, DCC, HOBt, DIEA, CH2Cl2; (iii) 30% TFA/CH2Cl2; (iv) 1H-pyrazole-1-carboxamide,
DIEA, DMF.
groups. Thus, we repeated the first acylation step with
phenylacetic acid using as starting material the primary
amine protected N-[1-(4,4-dimethyl-2,6-dioxocyclohex-
ylidene)ethyl] (Dde) derivatives of 4-aminomethylpip-
eridine and 3-aminopyrrolidine. The selectivity of the
Dde protecting group towards primary amines has been
demonstrated by other groups25 and is presumably a
function of the stabilization provided by a strong in-
tramolecular hydrogen bond (NH δ ) 13-14 ppm) which
is not possible with secondary amines (Scheme 5). Thus,
the two diamines 4-aminomethylpiperidine and 3-ami-
nopyrrolidine reacted readily with 2-acetyldimedone to
afford the corresponding primary amine protected Dde
derivatives. Phenylacetic acid was then coupled to the
above Dde-protected diamines, using DCC and HOBt
as activating reagents. Hydrazinolysis of the N-Dde
afforded the free primary amine analogues 7a and 8a
(Scheme 5), identified also by 1H NMR and HPLC
analysis.
high yields of free primary amine derivatives. N-
acylation selectivity is due to the higher nucleophilicity
of the secondary nitrogen in these molecules.
Bioassay Data. Non-peptide TRAP mimetics were
tested on human platelet-rich plasma in order to
determine whether such compounds could affect throm-
bin receptor activation on the platelet surface. By the
use of this assay, it was found that all tested compounds
were able to inhibit thrombin-induced platelet aggrega-
tion in a dose-dependent manner. As shown in Figure
1, non-peptide mimetics, compounds 1 and 3 of group
A, were able to inhibit thrombin-induced platelet ag-
gregation at a concentration of thrombin as high as 0.3
IU/mL. In contrast, the inhibitory effect of compound 2
was demonstrated when thrombin was used at a con-
centration less than 0.25 IU/mL while the effect was
lost at higher (0.3 IU/mL) concentrations. Compound 3
was clearly the most effective among the three com-
pounds of group A, with an IC50 of 0.11 mM at a
thrombin concentration of 0.20 IU/mL (Table 1). As
shown in Figure 2, compound 5 of group B showed a
biphasic response. At lower concentrations, it not only
failed to inhibit thrombin-induced platelet aggregation
but also increased it, whereas at higher concentrations
it showed clearly an inhibitory effect. Compound 4 was
the most effective inhibitory compound (IC50 ) 0.09 mM
for a thrombin concentration of 0.25 IU/mL) followed
by compound 6.
Furthermore, using as standards the compounds 7a
1
and 8a, we compared their H NMR spectra with the
relative spectra of the synthesized compounds 7 and 8
and found them to be identical. The TLC and HPLC
profiles of each pair 7-7a and 8-8a were also identical.
The above described method provides an alkylation
procedure of 4-aminomethylpiperidine and 3-amino-
pyrrolidine (2-fold or more molar excess of diamine to
carboxylic acid), which in short reaction time leads to

3342 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Alexopoulos et al.
Scheme 4. Solid-Phase Organic Synthesis of Compounds 5 and 6 of Group Ba
a Reagents: (i) Phe-OH, (CH3)3SiCl, TEA, CH2Cl2; (ii) DIC, HOBt, THF; (iii) 4-aminomethylpiperidine, DIEA, DMF; (iv) 3-aminopyr-
rolidine, DIEA, DMF; (v) Fmoc-ꢀ-Ahx-OH, DIC, HOBt, DMF; (vi) 20% piperidine in DMF; (vii) 1H-pyrazole-1-carboxamide, DIEA, DMF;
(viii) 10% TFA/CH2Cl2.
As shown in Table 1, the addition of an amino group
improved the inhibitory effect of non-peptide mimetic
4 with a piperazine template but failed to improve and
in fact worsened the inhibitory effect of the other two
non-peptide mimetics 5 and 6. None of the tested
compounds were able by itself to produce any ap-
preciable platelet aggregation when incubated at vari-
ous concentrations with stirred platelet-rich plasma. In
addition, none of the tested compounds were able to
cleave the chromogenic substrate S-2238, indicating that
these compounds lack thrombin enzymatic activity.26,27
1-6 were 0.19, 0.06, 17.3, 38.2, 13.6, and 44, respec-
tively (Table 2).
As shown in Figure 3, all compounds in group A
induced a concentration-dependent relaxation of the
aortic rings. In particular, compound 1 produced a
concentration-dependent relaxation of the aortic rings
precontracted with 1 µM phenylephrine. The maximal
relaxing effect (81%) was obtained at a concentration
of 100 µM. The relaxing effect of compound 1 was
completely abolished by pretreatment of the aortic rings
with L-NAME (300 µM). Moreover, in aortic rings
without endothelium, compound 1 was without relaxing
effect. This proves that the relaxing effects of compound
1 in aortic rings are mediated by nitric oxide release.
Compound 2 relaxed the aortic rings precontracted with
1 µM phenylephrine with a maximal efficacy (91.4%)
at 100 µM. The relaxing effects of compound 2 were
absent in aortic rings pretreated with 300 µM L-NAME
or with lacking of endothelium. Compound 3 relaxed the
aortic rings precontracted with phenylephrine in a
concentration-dependent manner, with a maximal ef-
ficacy (37.5%) at 100 µM. The relaxing effects of
compound 3 were abolished by removing the endothe-
The relaxant activities of compounds 1-6 were com-
pared with that of the amidated linear pentapeptide
SFLLR-NH2. SFLLR-NH2 represents the shortest
receptor-activating peptide that exhibits a potency
greater than that of the originally described receptor-
activating tetradecapeptide.5 The relative potency (REC
)
for each compound was determined from the dose-
response curves. REC represents the ratio of each of the
concentrations of the six TRAP analogues to the con-
centration of SFLLR-NH2 necessary for producing an
equal relaxing effect on the isolated aortic rings (REC
ECTRAP/ECSFLLR-NH2). The REC values for compounds
)

Thrombin Receptor Mimetics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3343
Scheme 5. (a) Synthesis of N-Phenylacetyl-4-aminomethylpiperidine (7a) and (b) Synthesis of
N-Phenylacetyl-3-aminopyrrolidine (8a)a
a Reagents and conditions: (i) EtOH, room temp, 2 h; (ii) phenylacetic acid, DCC, HOBt, DIEA, CH2Cl2; (iii) 2% v/v methanolic hydrazine
solution, 30 min.
lium or by pretreatment with 300 µM L-NAME. Com-
pound’s 3 efficacy and potency were significantly lower
than the ones of compound 1 and 2, which did not differ
structurally significantly from each other.
compound 5 (0.1-100 µM) was thus found to be from
0% to 25%. Compound 6 (0.1-100 µM) induced a
concentration-dependent relaxation of the aortic rings
with endothelium precontracted with 1 µΜ phenyl-
ephrine, by 1.8-40.7%. Pretreatment of these aortic
rings with L-NAME induced the disappearance of the
relaxing response. At concentrations of 10 and 100 µM,
compound 6 induced a transient contraction of 2-3%
in the aortic rings without endothelium. A comparison
of the dose-response curves of the non-peptide TRAPs,
performed by two-way repeated measures of ANOVA
analysis, revealed that compounds from group A had
statistically significant higher effects than compounds
from group B.
All compounds in group B were also found to stimu-
late the endothelial thrombin receptor in a concen-
tration-dependent matter as seen in Figure 3. The
biological activity of compound 4 has been previously
described.20 Compound 5 (0.1-100 µM) induced a
concentration-dependent relaxation of the aortic rings
with endothelium precontracted with 1 µΜ phenyl-
ephrine, by 1.3-42.7%. Pretreatment of these aortic
rings with Nω-nitro-L-arginine (L-NAME) reduced the
maximal relaxing response to 17-18%. In the aortic
rings without endothelium, compound 5 (0.1-100 µM)
also induced a concentration-dependent relaxation by
2.3-14%. The nitric oxide dependent relaxing effect of
Molecular Modeling. In a previous publication17 the
molecular modeling of SFLLR was built on the basis of
NMR data. These data revealed that SFLLR probably

3344 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Scheme 6. Regioisomers of Compounds 7a,b and 8a,b
Alexopoulos et al.
favors cyclic conformation characterized by reduced
mobility of the Arg and Phe side chain residues, which
have been shown to be essential for the biological
activity of the pentapeptide. On the basis of the syn-
thesis of novel potent cyclic analogues and the fact that
the amide SFLLR-NH2 with the higher biological
potency assumes a predominately cyclic conformation,
we have suggested that probably cyclic conformation of
SFLLR represents its bioactive conformation. To further
analyze the proposed model, we studied the super-
imposition properties of the synthesized compounds and
SFLLR. From the superimposition studies, compound
4 gave the best results. Therefore, we will focus on this
structure because this was also of the most biological
significance. The starting structure of compound 4 was
built to be extended with the phenyl and guanidino
groups positioned far away. This structure was mini-
mized using a combination of steepest descent and
Newton-Ramphson algorithms. The alkyl chain was
turned in a way such that the phenyl ring established
a spatial vicinity with the guanidino group. To this turn
the presence of the carbonyl group was mainly contrib-
uted, which favors electrostatic interaction with the
positively charged guanidino one. To further explore the
conformational space that compound 4 can adopt, a
short molecular dynamics experiment was performed at
the high temperature of 1000 K to allow high flexibility
of the alkyl chain and thus to adopt a great variety of
possible conformers.
The simulated high-energy structures were further
minimized using ꢀ ) 45 to simulate a receptor environ-
ment and 2000 iterations using the steepest descent
algorithm. This intermediate dielectric constant simu-
lates an amphipathic environment which is the most
putative one to represent a receptor environment. Three
family structures were generated from the 100 mini-
mized structures. The lowest energy structures derived
from these family structures differ in energy only by
1-3 kcal/mol. The major characteristic of these struc-
tures is the high flexibility of the alkyl chain. Thus, the
alkyl chain either can be in an extended form or can
turn toward the phenyl ring. By this turning, the ꢀ-NH
of the guanidino group approaches the carbonyl group
and stabilizes the molecule by an electrostatic inter-
action. The guanidino group also comes in the vicinity
of the phenyl ring. The observed conformers in the
dynamic experiment with a proximity of the phenyl
rings to the guanidino group predominate (constitute
85% of the simulated ones) and seem to represent the
bioactive conformer of compound 4. The final structure
was derived by imposing distance constraints between
the phenyl and guanidino groups and further subjected
to minimization procedures using the steepest descent
and Newton-Raphson algorithms. The approach of the
guanidino group toward the phenyl ring would be even
more stable if the amino group was not positively
charged. However, in a receptor environment the amino
group may interact with a carbonyl group as is sug-
gested in a recent paper, which deals with potent
inhibitors incorporating a phenyl group as a peptide
mimetic and aminopyridines as guanidine substi-
tutes.28,29 Such an interaction will allow the guanidino
group to approach even closer the phenyl ring and
stabilize interactions of ꢀ-NH of the guanidino group to
the carbonyl group (Figure 4).
Subsequently, the proposed bioactive conformer among
the low-energy structures derived from molecular dy-
namics experiment was superimposed with SFLLR
using rigid-body superimposition. During the superim-
position the phenyl and guanidino groups of SFLLR
were matched with the corresponding ones of compound
4 (Figure 5), showing rms distances of only 0.15 Å
between the matched groups. This indicates that the
proposed bioactive conformer of compound 4 can mimic
the two requirements demanded by SFLLR to exert its
biological activity. Moreover, other molecular charac-
teristics were also matched. The -NH2 group of 4 was
in spatial proximity with serine -NH2 and the serine-
phenylananine amide bond. The nitrogen of piperazine
and the carbonyl groups in the vicinity of -NH2 for
compound 4 are matched with the amide bond between
phenylananine and leucine. These superimpositions
resulted in an increase of rms to 1.5 Å.
Discussion
Thrombin interacts with the cells through a specific
G-protein-coupled receptor that has been cloned and
sequenced.5 Receptor activation occurs when thrombin
cleaves the N-terminus between Arg41 and Ser42, expos-
ing a new amino terminus that functions as a tethered
peptide ligand.1,5 In support of this model, peptides of
only five amino acids (SFLLR) have been shown to
mimic the effects of thrombin on platelets13 and smooth
muscle,30 and antibodies directed against this domain
have been shown to inhibit the effects of thrombin.31
Furthermore, it has been recently shown that replace-

Thrombin Receptor Mimetics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3345
Table 1. Effect of Thrombin Receptor Non-Peptide Mimetics
on Human Platelet Aggregation in Vitro
IC50 (mM)
SFLLR
control
0.20 IU/mL
TRAP
0.25 IU/mL
TRAP
0.30 IU/mL
TRAP
1
2
3
4
5
6
0.46
0.14
0.11
0.12
>1.0
0.42
0.51
>0.25
0.12
0.44
>0.25
0.21
0.09
0.24
>1.0
0.34
0.87
0.39
active topology. The described compounds have been
designed rationally on purpose to mimic the active cyclic
conformation adopted by SFLLR,32,33 because they
contain key pharmacophoric groups of Phe, Arg, and the
primary NH2 group. The guanidino group is at the end
of the linear lipophilic chain of five methylene groups,
since it has been found through SAR that such a long
alkyl chain gives the advantage to these molecules to
maximize hydrophobic interaction as it happens with
the leucine side chain of SFLLR.23
SFLLR mimetics were found to be able to prevent
thrombin-induced platelet aggregation. All three com-
pounds in group A inhibited thrombin-induced platelet
activation when thrombin was used at low concentration
(up to 0.2 IU/mL or 2 nM) which requires thrombin’s
anion-binding exosite and occurs via activation of the
thrombin tethered-ligand receptor.34,35 However, at
higher concentrations of thrombin (up to 0.3 IU/mL or
3 nM), where platelet activation was achieved indepen-
dent of thrombin’s exosite and mediated by a moderate
affinity (KD ) 10 nM) binding site,34 thrombin could
overcome such inhibition for both compounds 1 and 2
but not for compound 3. Thus, at higher concentrations
of thrombin, induced platelet activation was suppressed
effectively with compound 3 but not with compounds 1
and 2.
In group B, platelet activation with low concentrations
of thrombin was almost totally prevented with the three
SFLLR mimetics of group B at a relatively high con-
centration (0.5-1 mM) for both compounds 5 and 6 but
not for compound 4. Compound 4 inhibited permanently
thrombin-induced platelet activation even at a 4-fold
smaller concentration (0.125 mM) compared to the other
two SFLLR mimetics 5 and 6. At even higher concen-
trations (up to 0.3 IU/mL), thrombin could partly
overcome all three SFLLR inhibitory activities when
SFLLR mimetics were used at low concentrations. By
contrast, higher concentrations of SFLLR mimetics (up
to 1 mM for compounds 5 and 6 and 0.5 mM for
compound 4) completely inhibited thrombin-induced
platelet activation even when thrombin was used up to
3 nM. Therefore, compounds 5 and 6 have limited
efficacy in suppressing a thrombin-stimulated platelet
activation when used up to 0.25 mM even though
compound 5 up to 0.25 mM enhanced weak platelet
activation. Compound 4 at 0.25-0.5 mM caused a
complete and permanent blockage of thrombin-induced
platelet activation.
Figure 1. Effect of group A non-peptide thrombin mimetics
on inhibition of thrombin-induced platelet aggregation in
human PRP after activation with different concentrations of
thrombin.
ment of the aliphatic residues of the active peptide
SFLLR with small bifunctional rings retains the activity
only when the pharmacophoric groups of Phe and Arg
remain in place.20
In this work, the structural requirements of the native
pentapeptide SFLLR have been incorporated onto dif-
ferent molecular scaffolds providing structure rigidity
(piperazine), flexibility (4-aminomethylpiperidine and
3-aminopyrolidine), and in a particular spatial order
that is in agreement with the previously defined bio-
SFLLR mimetics were also tested for biological activ-
ity in isolated aortic rings with intact endothelium
precontracted with phenylephrine and were found ca-
pable of producing a dose-dependent relaxation at
concentrations as low as 1-10 µΜ. Of note, nitric oxide

3346 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Alexopoulos et al.
Table 2. Bioassay Relaxation Results for Non-Peptide TRAPs
(Mean ( SE) in Isolated Aortic Rings (Smooth Muscle ED50
)
compound
relative potency (REC)
SFLLR-NH2
1
1
2
3
4
5
6
0.19
0.06
17.3
38.2
13.6
24.0
Figure 3. Concentration-dependent relaxing effects of group
A (compounds 1-3) and group B (compounds 4-6) SFLLR
mimetics and SFLLR-NH2 on the phenylephrine-induced
contraction of aortic rings with intact endothelium. Results
are expressed as the mean ( SE (n ) 6).
Figure 4. Low-energy conformers of compound 4 derived after
short molecular dynamics coupled with minimization algo-
rithms.
Figure 2. Effect of group B non-peptide thrombin mimetics
on inhibition of thrombin-induced platelet aggregation in
human PRP after activation with different concentrations of
thrombin.
The results obtained in the isolated aortic smooth
muscle bioassay indicate that the presence of piperidine
or piperazine groups in the structure of the thrombin
receptor analogues of group A (compounds 2 or 1,
respectively) increases their potency as agonists of the
endothelial thrombin receptor up to 0.1-0.5 µM, whereas
their substitution with the pyrrolidine group (compound
3) decreases the agonistic potency by approximately 10
times. The REC of compounds 1 and 2 were therefore
significantly lower compared to that of compound 3, and
the order of potency of compounds from group A was
Co2 > Co1 > Co3. The maximal relaxing effects of
synthase blocker L-NAME inhibited SFLLR-induced
aortic relaxation to a different extent. Generally, the
magnitude of the relaxation induced by a given SFLLR
mimetic was higher in preparations with endothelium
than in de-endothelized preparations. The two types of
relaxation were very close or equal in the preparations
pretreated with L-NAME, and this provides an ad-
ditional way of separating the endothelium-dependent
effects from the endothelium-independent effects for the
same SFLLR mimetic.

Thrombin Receptor Mimetics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3347
ment. Compound 6 with a reasonably strong inhibitory
effect in platelet aggregation and simultaneous moder-
ate relaxant activity in rat aorta assay is also a suitable
candidate for further development.
All compounds were readily soluble in both platelet
aggregation and rat aorta relaxation assays. In the
platelet aggregation assay the concentrations of the
compounds used in the experiments were identified
between a range of lower and higher concentrations to
evaluate their capacity to inhibit thrombin-induced
platelet activation in vitro. These concentrations, how-
ever, did not affect platelet activation by other agonists
such as collagen, ADP, ristocetin, and arachidonic acid,
indicating that the concentrations used act directly and
therefore a non-target-related effect is excluded. Ad-
ditionally, compounds 1 and 4 inhibited platelet activa-
tion caused by the amide SFLLR-NH2, 2.5 µM (IC50
)
0.78 and 0.37 mM, respectively), indicative that these
compounds act directly toward thrombin receptor PAR-
1. Regarding the rat aorta relaxation assay, there are
few chances that the response obtained in aortic bio-
assays at the highest concentration (100 µM) was
nonspecific because of the fact that the largest part of
the relaxing effects (70-80%) was already obtained at
the next lowest concentration (10 µM). On the other
hand, it is known that TRAPs are active at high
micromolar concentrations.11 Moreover, the specificity
of the effect is proved by the dramatic changes in
activity of the compounds induced by slight changes in
their structures (presence or absence of the amino
group).
Superimposition studies based on NMR/NOE con-
straints showed that compound 4 mimicked most the
cyclic conformation of SFLLR and this may explain its
biological activity. A pharmacological profile for drug
therapy requires a strong inhibitory effect in platelet
aggregation with simultaneous muscle vasodilatation.
Figure 5. Superimposition of compound 4 with SFLLR. The
major characteristic of this superimposition is the excellent
matching between the phenyl and their guanidino groups.
compounds 1 and 2 were significantly higher (by 2-3
times) compared to compound 3 but different from each
other.
The introduction of an amino group in the structure
of the SFLLR mimetics 1-3 reduced significantly their
potency and efficacy in perfused aortic rings. Thus, the
REC values of compounds 1-3 were significantly lower
than the ones of compounds 4-6, respectively. The
maximal relaxing effects of compounds 4-6 were sig-
nificantly smaller than the ones of compounds 1 and 2
but not from that of compound 3.
The association between an amino group and a
piperazine group in the structure of compound 4 from
group B of thrombin analogues resulted in the lowest
agonistic potency in the aortic smooth muscle bioassay.
The highest potency from compounds in group B was
observed for compound 5 containing the piperidine
group. The order of potency of thrombin analogues from
group B was Co5 > Co6 > Co4.
Conclusion
This study describes the synthesis and activities for
rationally designed thrombin receptor mimetics bearing
Phe/Arg and Phe/Arg/NH2 pharmacophoric groups on
three different templates (piperazine, 4-aminomethyl-
piperidine, and 3-aminopyrrolidine). All of the synthe-
sized compounds were found to be active in the rat aorta
relaxation assay, and the platelet aggregation study
indicated that those bearing an extra amino group were
more active as inhibitors in platelet aggregation. Com-
pound 2 was found to have significant NO relaxing
properties, whereas compound 4 was found to be the
most powerful inhibitor of thrombin-induced platelet
thrombin receptor activation. The use of piperazine as
a template in compound 4 appears to offer the molecule
more rigidity, with the guanidino, phenyl, and amino
groups in the vicinity, required for the interaction with
the thrombin receptor. In contrast, the use of 4-ami-
nomethylpiperidine and the 3-aminopyrrolidine as tem-
plates in compounds 5 and 6 provides for a flexible
conformation that allows inhibition of thrombin receptor
activation only at higher concentrations. These findings
further support the suggestion that a cluster of two
groups (phenyl, guanidine) together with an adjacent
primary amino group is important for the expression of
maximum biological activity. Such compounds are po-
By comparison of activities, the order of potency of
thrombin receptor analogues as agonists of the endo-
thelial thrombin receptor was the inverse of the order
of potency of the same compounds as blockers of the
platelet thrombin receptor. Thus, in group A the order
of potency of thrombin receptor analogues as agonists
of the endothelial receptor was Co2 > Co1 > Co3,
whereas as antagonists in platelet aggregation, the
order of potency was inverted: Co3 > Co1 > Co2. In
group B the order of agonistic potency of thrombin
analogues as agonists of the endothelial receptor was
Co5 > Co6 > Co4, whereas as antagonists in platelet
aggregation, the order of potency was Co4 > Co6 > Co5.
This indicates the existence of a certain degree of
functional analogy between the endothelial and platelet
thrombin receptor although the present and previous
studies indicate that thrombin analogues exhibit a very
low activity (if any) as agonists of platelet thrombin
receptors.
Compounds 4 and 3 with three (Phe/Arg/NH2) and
two (Phe/Arg) pharmacophoric groups, respectively,
which show the highest inhibitory effects in platelet
aggregation while preserving agonist activity in the rat
aorta assay, are the most promising for further develop-

3348 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Alexopoulos et al.
drous Na2SO4, and reduced in vacuo to give 1.4 g (6.0 mmol,
90%) of a white waxy solid product: Rf ) 0.35 (4:1:1, ButOH/
H2O/AcOH); 1H NMR (CD3OD) δ 0.95 (dm, 2H), 1.61 (m, 1H),
1.72 (dd, 2H), 2.48 (d, 2H), 2.82 (dm, 2H), 3.78 (s, 2H), 4.29
(dd, 2H), 7.21-7.33 (m, 5H); MS (nominal) m/z 204 (M+), 91
(C6H5CH2+); MS (accurate) m/z M+ calculated 204.1263, M+
found 204.1260.
a.2. N-(Phenylacetyl)-4-[(6-(tert-butoxycarbonylami-
no)hexanoylamidomethyl)]piperidine. N-Phenylacetyl-4-
aminomethylpiperidine (460 mg, 2.0 mmol), Boc-ꢀ-Ahx-OH
(486 mg, 2.1 mmol), HOBt (310 mg, 2.3 mmol), and DIEA (1
mL) were dissolved in 15 mL of CH2Cl2. The mixture was
cooled to 0 °C, and DCC (475 mg, 2.3 mmol) was added. The
reaction mixture was monitored by TLC (5% MeOH/CHCl3)
and showed completion after 12 h. The supernatant liquid was
filtered in vacuo, dried over anhydrous Na2SO4, and concen-
trated in vacuo to give an orange-yellow solid as a crude
product. Flash chromatography (2% MeOH/CHCI3) yielded a
waxy white solid (535 mg, 1.2 mmol, 60%): Rf ) 0.74 (15%
MeOH/CHCI3); 1H NMR (DMSO-d6) δ 0.9 (m, 2H), 1.2 (m, 2H),
1.3 (s, 9H), 1.3-1.5 (m, 4H), 1.5 (m, 2H), 1.6 (m, 3H), 2.0 (t,
2H), 2.7 (dt, 2H), 2.9 (m, 2H), 3.7 (s, 2H), 4.2 (dd, 2H), 6.7
(brs, 1H), 7.2-7.3 (m, 5H), 7.7 (brs, 1H); MS (nominal) m/z
446 (M+).
tential thrombin receptor antagonists and might be
efficacious in drug therapy.
Experimental Section
I. Methods. Chemistry. The 1H NMR spectra were re-
corded on a Bruker ACF-300 spectrometer operating at 7.05
T. All NMR spectra were obtained in dilute DMSO-d6, CDCl3,
or CD3OD solutions. All chemical shifts were referenced to
tetramethylsilane (TMS). Mass spectra were obtained on FAB
instrumentation operating on a AEI M29 mass spectrometer
and on a TSQ 7000 spectrometer (ESIMS). The FAB gun was
run at 1 mA discharge current at 8 kV. The FAB matrix used
was a mixture of dithiothreitol/dithioerythritol (6:1) (Cleland
Matrix). The accurate M+ of the synthesized compounds was
obtained on a Kratos Profile HV-3 mass spectrometer operat-
ing with a mass range of 0-1200 amu at 4 kV and resolution
variable from nominal mass (600) to accurate mass (10 000).
The ESIMS spectra were run on a TSQ 7000 spectrometer
(electrospray mode) by direct infusion. A solution of the sample
(1 µg/1µL) in methanol was introduced in the ESI probe at a
flow rate of 5 µL/min with a Harvard syringe. The capillary
temperature was at 80 °C, and the sheath gas was at 45 units
while the spray needle voltage was +4.5 KV. Most of the
compounds involved in the solution synthesis were purified
by flash chromatography using Merck silica gel 60 (230-400
mesh). Solid-phase organic synthesis was achieved on the
2-chlorotrityl chloride resin using a manually handled reaction
vessel (2 cm2 × 12 cm) equipped with a porous G filter (size 2)
and a tap connected with a water aspirator. Preparative HPLC
was performed with a Waters system equipped with a 600E
system controller using a Nucleosil C-18 reversed-phase semi-
preparative column (250 mm × 10 mm) with 7 µm packing
material. Solvents used for HPLC elution were (A) water
containing 0.1% TFA and (B) acetonitrile containing 0.1% TFA.
Separations were achieved with a stepped linear gradient of
solvent A going to 50% solvent A in solvent B over 60 min at
a flow rate of 3 mL/min. The crude product (20 mg) was
dissolved in methanol (450 µL), and this solution was injected
using a Waters U6K injector with a 2.0 mL sample loop.
Fractions were manually collected at 0.5 min intervals; the
elution time of the major product was 8-10 min. Eluted
product was determined simultaneously from the absorbances
at 254 and 230 nm (Waters 996 photodiole array detector).
Fractions containing the major product peak were pooled, and
the acetonitrile was removed using a rotary evaporator. After
lyophilization, the product was stored at -20 °C.
a.3. N-(Phenylacetyl)-4-(6-aminohexanoylamidometh-
yl)piperidine Trifluoroacetic Salt. The above Boc deriva-
tive (1.0 mmol) was dissolved in 3 mL of a 30% TFA/CH2Cl2
solution, and the mixture was stirred for 1 h. The solution was
evaporated to dryness in vacuo and the residue was triturated
with dry ether, giving a colorless oil (0.97 mmol, 97%): Rf )
1
0.09 (15% MeOH/CHCI3); H NMR (DMSO-d6) δ 0.9 (m, 2H),
1.2 (m, 2H), 1.4-1.6 (m, 4H), 1.6 (m, 3H), 2.0 (t, 2H), 2.7 (dt,
2H), 2.8 (m, 2H), 2.9 (m, 2H), 3.7 (s, 2H), 4.2 (dd, 2H), 7.2-7.3
(m, 5H), 7.7 (brs, 3H), 7.8 (t, 1H); MS (nominal) m/z 346 (M+
- tfa).
a.4. N-(Phenylacetyl)-4-(6-guanidohexanoylamido-
methyl)piperidine (2). TFA-salt of N-(phenylocetyl)-4-(6-
aminohexanoylamidomethyl)piperidine (260 mg, 0.64 mmol),
1H-pyrazole-1-carboxamide hydrochloride (130 mg, 0.9 mmol),
and DIEA (0.5 mL) were dissolved in 1.5 mL of DMF. The
mixture was stirred under nitrogen for ∼24 h at room
temperature. Then, dry ether (15 mL) was added and the
product appeared as a gel at the bottom of the flask. The
supernatant liquid was decanted. The crude product was
purified by recrystallization from MeOH/acetone/Et2O to give
174 mg (0.45 mmol, 70%) of a white solid: Rf ) 0.13 (15%
MeOH/CHCI3); 1H NMR (DMSO-d6) δ 0.9 (m, 2H), 1.3 (m, 2H),
1.5 (m, 4H), 1.6 (m, 3H), 2.1 (t, 2H), 2.7 (dt, 2H), 2.9 (m, 2H),
3.1 (m, 2H), 3.7 (s, 2H), 4.2 (dd, 2H), 6.9-7.5 (brs, 3H), 7.2-
7.3 (m, 5H), 7.8 (brs, 1H), 7.9 (t, 1H); MS (nominal) m/z 387
(M+)
b. Synthesis of N-(Phenylacetyl)-3-(6-guanidohexan-
oylamido)pyrrolidine (3). b.1. N-Phenylacetyl-3-ami-
nopyrrolidine (8). Compound 8 was synthesized using the
same procedure that was followed for the synthesis of com-
pound 7, and it was obtained in 87% yield as a white waxy
solid: Rf ) 0.37 (4:1:1, ButOH/H2O/AcOH); 1H NMR (CD3OD)
δ 1.96-2.17 (m, 1H), 2.27-2.46 (m, 1H), 3.32 (brs, 2H), 3.54-
3.94 (m, 5H), 3.74 (s, 2H), 7.24-7.35 (m, 5H); MS (nominal)
m/z 204 (M+), 91 (C6H5CH2+); MS (accurate) m/z M+ calcd
204.1263, M+ found 204.1260.
The purity of each intermediate and final product was
assessed by analytical HPLC returns (Nucleosil C-18, 250 mm
× 4 mm) and thin layer chromatography (TLC). Analytical
HPLC was done by the procedure described above. TLC was
performed using Merck precoated silica gel 60 F-254 glass
plates in an n-butanol/acetic acid/water (4:1:1) (BAW) solvent
system. Visualization was accomplished by using a combina-
tion of UV lamp and ninhydrin spray. All synthetic reagents
were purchased from the Aldrich-Sigma Chemical Company
and were used without purification. 2-Chlorotrityl chloride
resin was supplied by CBL Patras. Solvents were analytical
reagent grade or higher purity and were used as supplied. A
Buchi RE 111 rotavapor was utilized for the removal of the
solvents in vacuo. Melting point determinations were per-
formed on a Buchi 530 melting point apparatus.
b.2. N-(Phenylacetyl)-3-[(6-(tert-butoxycarbonylami-
no)hexanoylamido)]pyrrolidine. The same procedure de-
scribed above for the synthesis of the relative piperidine
analogue was followed. The product was obtained pure in 65%
yield as a waxy white solid after flash chromatography (2%
MeOH/CHCl3): Rf ) 0.66 (10% MeOH/CHCI3); 1H NMR
(DMSO-d6) δ 1.2 (m, 2H), 1.3 (s, 9H), 1.3 (m, 2H), 1.5 (m, 2H),
1.6-2.1 (m, 2H), 2.0 (t, 2H), 2.9 (m, 2H), 3.1-3.7 (m, 4H), 3.7
(s, 2H), 4.1-4.3 (m, 1H), 6.7 (brs, 1H), 7.2-7.3 (m, 5H), 8.0 (t,
1H); MS (nominal) m/z 417 (M+), 344 [(M+ + 1) - (CH3)3CO•)].
a. Synthesis of N-(Phenylacetyl)-4-(6-guanidohexan-
oylamidomethyl)piperidine (2). a.1. N-Phenylacetyl-4-
aminomethylpiperidine (7). Phenylacetic acid (950 mg, 6.7
mmol) and HOBt (920 mg, 6.8 mmol) were suspended in CHCl3
(20 mL). DCC (1.4 g, 6.8 mmol) was then added followed by
DMF (2 mL). The mixture was stirred for ∼30 min at room
temperature to give a white suspension. The mixture was
transferred slowly to a cold solution of 4-aminomethylpiperi-
dine (1.47 g, 13 mmol) in 30 mL of CHCl3. The reaction mixture
was stirred for 3 h at room temperature, the white suspension
(DCU) was filtered, and the filtrate was acidified with 2 M
HCl (3 × 30 mL). The HCl extracts were basified with 2 M
NaOH, extracted with CHCl3 (4 × 50 mL), dried over anhy-
b.3. N-(Phenylacetyl)-3-(6-aminohexanoylamido)pyr-
rolidine Trifluoroacetic Salt. After deprotection of the
above Boc derivative with 30% TFA/CH2Cl2 solution, a color-

Thrombin Receptor Mimetics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3349
less oil was obtained in 95% yield: Rf ) 0.38 (4:1:1, ButOH/
H2O/AcOH); 1H NMR (CDCl3) δ 1.2 (m, 2H), 1.3-1.6 (m, 4H),
1.7-2.1 (m, 2H), 2.0 (t, 2H), 2.8 (m, 2H), 3.2-3.6 (m, 4H), 3.6
(s, 2H), 4.1-4.3 (m, 1H), 7.2-7.3 (m, 5H), 7.7 (brs, 3H), 8.0 (t,
1H); MS (nominal) m/z 317 (M+ - tfa).
b.4. N-(Phenylacetyl)-3-(6-guanidohexanoylamido)py-
rrolidine (3). The compound was prepared with the same
guanylation procedure that was followed for the synthesis of
compound 2. The crude product was purified by recrystalliza-
tion from MeOH/acetone/Et2O to give a white solid in 79%
yield: Rf ) 0.42 (4:1:1, ButOH/H2O/AcOH); 1H NMR (DMSO-
d6) δ 1.2 (m, 2H), 1.4-1.5 (m, 4H), 1.6-2.1 (m, 2H), 2.1 (t,
2H), 3.1 (m, 2H), 3.1-3.7 (m, 4H), 3.6 (s, 2H), 4.1-4.3 (m, 1H),
6.9-7.4 (brs, 3H), 7.2-7.3 (m, 5H), 7.7 (brt, 1H), 8.1 (t, 1H);
MS (nominal) m/z 360 (M+ + 1).
c. Solid-Phase Organic Synthesis. c.1. Preparation of
HO-Phe-2-chlorotrityl Resin. Phenylalanine (375 mg, 2.27
mmol) and (CH3)3SiCl (0.86 mL, 2.50 mmol) were dissolved in
CH2Cl2 (8 mL), and the mixture was stirred at room temper-
ature for 30 min. TEA (0.72 mL, 5.20 mmol) was then added
dropwise to the solution, and the mixture was refluxed for 20
min in a water bath. After the mixture was cooled, 2-chloro-
trityl chloride resin (1.0 g, 2.27 mequiv of Cl-/g resin) and CH2-
Cl2 (7 mL) were added and refluxing of the mixture was
followed for 1 h. The reaction was continued under stirring
for another 24 h at room temperature. The HO-Phe resin was
filtered, subsequently was washed with CH2Cl2 (5 × 10 mL),
CH2Cl2/CH3OH/DIEA (85:10:5) (3 × 10 mL), CH2Cl2 (2 × 10
mL), DMF (2 × 10 mL), DMF/H2O (2 × 10 mL), H2O (2 × 10
mL), DMF (2 × 10 mL), i-PrOH (3 × 10 mL), and n-hexane (5
× 10 mL) and then dried in vacuo for 24 h at room tempera-
ture.
c.2. Preparation of BtO-Phe-2-chlorotrityl Resin. HO-
Phe resin (1.37 g, 2.27 mmol theoretical) was washed with THF
(2 × 10 mL). HOBt (600 mg, 4.50 mmol) and DIC (0.7 mL,
4.50 mmol) were dissolved in the minimum volume of THF,
they were all added to the resin, and the mixture stood for 24
h. The BtO-Phe resin was filtered, subsequently washed with
DMF (3 × 10 mL), i-PrOH (2 × 10 mL), DMF (2 × 10 mL),
i-PrOH (2 × 10 mL), and n-hexane (3 × 10 mL) and then dried
in vacuo for 24 h at room temperature.
c.3. Synthesis of 4-Aminomethylpiperidine-Phe-2-
chlorotrityl Resin. BtO-Phe resin (500 mg, 0.8 mmol/g) was
swelled and saturated with DMF. 4-Aminomethylpiperidine
(114 mg, 1 mmol) was dissolved in the minimum volume of
DMF, DIEA (0.4 mL) was added to the above resin, and the
mixture was shaken for 8 h. The resin was then filtered and
subsequently was washed with DMF (3 × 10 mL), i-PrOH (2
× 10 mL), DMF (2 × 10 mL), i-PrOH (2 × 10 mL), and
n-hexane (3 × 10 mL) and then was dried in vacuo for 24 h at
room temperature.
c.4. Synthesis of 3-Aminopyrrolidine-Phe-2-chlorotri-
tyl Resin. A procedure similar to the one described above was
followed except 3-aminopyrrolidine was used instead of 4-ami-
nomethylpiperidine.
c.5. Synthesis of Fmoc-ꢀ-Ahx-4-(aminomethyl)piperi-
dine-Phe-2-chlorotrityl Resin. 4-Aminomethylpiperidine-
Phe resin (0.4 mmol of compound theoretical) was swelled and
saturated with DMF. DIC (0.23 mL, 1.5 mmol) was added to
a solution of Fmoc-ꢀ-Ahx-OH (350 mg, 1.0 mmol) and HOBt
(200 mg, 1.5 mmol) in DMF, and the mixture was poured into
the above resin. The reactants were shaken for 24 h, and then
the resin was filtered and subsequently was washed with DMF
(3 × 10 mL), i-PrOH (2 × 10 mL), DMF (2 × 10 mL), i-PrOH
(2 × 10 mL), and n-hexane (3 × 10 mL) and then was dried in
vacuo for 24 h at room temperature.
twice with 20% piperidine in DMF (20 min, 30 min) at room
temperature. After removal of excess reagent, the H-ꢀ-Ahx-4-
aminomethylpiperidine-Phe resin was washed liberally with
DMF and treated with 1H-pyrazole-1-carboxamide hydrochlo-
ride (730 mg, 5 mmol) and DIEA (1 mL) diluted to 1.5 mL
with DMF and the reaction was allowed to proceed at 47 °C
for 3 h. The resulting guanylated resin was washed with DMF
(3 × 10 mL), i-PrOH (2 × 10 mL), DMF (2 × 10 mL), i-PrOH
(2 × 10 mL), and n-hexane (3 × 10 mL), was dried in vacuo,
and then was treated twice with 10% TFA in CH2Cl2 for 15
min. Solvent and excess of TFA were removed under reduced
pressure, affording a light-yellow solid that was then triturated
with dry ether. The crude product was purified by preparative
HPLC as described in the Experimental Section to yield 146
mg (0.35 mmol, 35% overall yield) of a white waxy solid
product: Rf ) 0.46 (4:1:1, ButOH/H2O/AcOH); 1H NMR
(DMSO-d6) δ 0.9 (m, 2H), 1.3 (m, 2H), 1.5 (m, 4H), 1.6 (m, 3H),
2.3 (t, 2H), 2.6 (m, 2H), 2.6 (d of t, 2H), 3.0 (m, 2H), 3.1 (m,
2H), 3.8 (m, 1H), 4.1 (d of d, 2H), 6.9-7.4 (br s, 4H), 7.2-7.3
(m, 5H), 7.6 (t, 1H), 8.3 (br s, 3H), 8.4 (t, 1H); MS/FAB 417
[(M + 1)+ - 2TFA], 357 [(M + 1)+ - 2TFA - H2NC(NH)NH2].
c.8. (S)-N-(2-Amino-3-phenylpropionyl)-3-(6-guanidohex-
anoylamido)pyrrolidine (6). The procedure described for the
synthesis of 5 was followed for the synthesis of 6, giving after
preparative HPLC purification a white waxy solid (17% overall
yield): Rf ) 0.42 (4:1:1, ButOH/H2O/AcOH); 1H NMR (DMSO-
d6) δ 1.3 (m, 2H), 1.4-1.5 (m, 4H), 1.6-2.1 (m, 2H), 2.2 (m,
2H), 2.6 (m, 2H), 3.1 (m, 2H), 3.1-3.7 (m, 4H), 3.8 (m, 1H),
4.1-4.3 (m, 1H), 6.9-7.4 (br s, 4H), 7.3-7.4 (m, 5H), 7.6 (br s,
1H), 8.3 (br s, 3H), 8.6 (m, 1H); MS/FAB m/z 389 [(M + 1)+
2TFA], 329 [(M + 1)+ - 2TFA - H2NC(NH)NH2].
-
d. Synthesis of N-Phenylacetyl-4-aminomethylpiperi-
dine (7a). 4-[1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-
ethylamino]methylpiperidine. 4-Aminomethylpiperidine
(460 mg, 4.0 mmol) and 2-acetyldimedone (810 mg, 4.5 mmol)
were dissolved in absolute ethanol (20 mL), and the solution
was stirred for 12 h at room temperature. The solvent was
rotary-evaporated, and the yellowish residue was triturated
with dry ether. The crude product was purified by recrystal-
lization from MeOH/ether to give a pale-yellow solid (840 mg,
3.0 mmol, 75%): mp 104-107 °C; Rf ) 0.20 (4:1:1, ButOH/
H2O/AcOH); 1H NMR (CDCl3) δ 1.04 (s, 6H), 1.24 (m, 2H), 1.76
(m, 1H), 1.79 (d, 2H), 2.36 (s, 2H), 2.39 (s, 2H), 2.56 (s, 3H),
2.62 (t, 2H), 3.13 (d, 2H), 3.28 (t, 2H), 13.6 (s, 1H); MS
(nominal) m/z 279 (M+ + 1).
d.1. N-Phenylacetyl-4-[1-(4,4-dimethyl-2,6-dioxocyclo-
hexylidene)ethylamino]methylpiperidine. The above Dde-
protected 4-aminomethylpiperidine (430 mg, 1.55 mmol),
phenylacetic acid (211 mg, 1.55 mmol), HOBt (230 mg, 1.70
mmol), and DIEA (0.5 mL) were dissolved in 15 mL of CH2-
Cl2. The mixture was cooled to 0 °C, and DCC (350 mg, 1.70
mmol) was added. The reaction mixture was then stirred at
room temperature for 18 h. The suspension (DCU) was filtered
and washed with 25 mL of CH2Cl2. The filtrate was subse-
quently concentrated in vacuo to give a waxy yellowish solid
as a crude product. Flash chromatography (1.5% MeOH/
CHCI3) yielded a waxy white solid (376 mg, 0.95 mmol, 61%):
1
Rf ) 0.76 (4:1:1, ButOH/H2O/AcOH); H NMR (CDCl3) δ 1.04
(s, 6H), 1.05 (dm 2H), 1.79 (dd, 2H), 1.83 (m, 1H), 2.35 (s, 2H),
2.40 (s, 2H), 2.54 (s, 2H), 2.77 (dt, 2H), 3.24 (dm, 2H), 3.75 (s,
2H), 4.36 (dd, 2H), 7.24-7.35 (m, 5H), 13.6 (s, 1H); MS
(nominal) m/z 397 (M+ + 1).
d.2. N-Phenylacetyl-4-aminomethylpiperidine (7a). The
previous amide (150 mg, 0.38 mmol) was dissolved in 10 mL
of 2% v/v methanolic hydrazine solution. The reaction was
monitored by TLC (10% MeOH/CHCl3) and showed completion
after ∼30 min. The excess of hydrazine was removed by rotary
evaporation. The remaining white solid was dissolved in CHCl3
(20 mL) and then washed with 2 M HCl (2 × 15 mL). The HCl
extracts were basified with 2 M NaOH to pH 9, extracted with
CHCl3 (3 × 20 mL), dried over anhydrous Na2SO4, and reduced
in vacuo to give 75 mg (3.2 mmol, 85%) of a colorless oil as a
crude product. The crude product was purified by preparative
HPLC to yield 60 mg (0.26 mmol, 68%) of a waxy white solid:
c.6. Synthesis of Fmoc-ꢀ-Ahx-3-aminopyrrolidine-Phe-
2-chlorotrityl Resin. The procedure described above was
followed for the coupling of Fmoc-ꢀ-Ahx-OH to 3-aminopyrro-
lidine-Phe resin.
c.7. (S)-N-(2-Amino-3-phenylpropionyl)-4-(6-guanidohex-
anoylamidomethyl)piperidine (5). Fmoc-ꢀ-Ahx-4-amino-
methylpiperidine-Phe resin (0.4 mmol of compound theoretical)
was swelled and saturated with DMF and then was treated

3350 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Alexopoulos et al.
Rf ) 0.35 (4:1:1, ButOH/H2O/AcOH); 1H NMR (CD3OD) δ 1.02
(dm, 2H), 1.74 (dd, 2H), 1.86 (m, 1H), 2.78 (d, 2H), 2.85 (dm,
2H), 3.31 (brt, 2H), 3.78 (s, 2H), 4.32 (dd, 2H), 7.21-7.33 (m,
5H); MS (nominal) m/z 233 (M+ + 1).
in suspension (PRP) taken as 0%. To evaluate the compounds
under investigation on platelet activation through the throm-
bin receptor, the PRP samples were incubated under stirring
with increasing concentrations (0.05-1 mM) of each compound
for at least 4 min prior to the addition of thrombin. Each study
was completed within 1 h after blood collection. The response
of platelet activation by thrombin varies tremendously between
blood samples withdrawn from different blood donors. Each
series of experiments was carried out using a different healthy
blood donor.
b. Amidolytic Assay. The possibility that these compounds
were direct thrombin inhibitors was investigated by an ami-
dolytic assay using the chromogenic substrate S-2238 (D-Phe-
Arg-pNA) (Chromogenix, Sweden) for thrombin. In a mi-
croelisa system (CERES 900C, Bio-Tec), the light emission was
measured at 405 nm, blanking on air. The standard kinetics
curve concerning the dissociation of the chromogenic substrate
caused by thrombin was obtained. To determine whether the
compounds under investigation influence the amidolytic capac-
ity of thrombin, low and high concentrations of these com-
pounds were preincubated with thrombin or the chromogenic
substrate and the corresponding curve was compared with the
standard one.
c. Rat Aorta Relaxation Assay. The experimental tech-
nique was described extensively elsewhere.37 Briefly, rings of
2 mm width were obtained from the central part of the thoracic
aorta of adult male Wistar. Two rings were used in each
experiment, one with intact endothelium and the other with
the endothelium mechanically removed. Both rings were
placed in a horizontal organ chamber (0.5 mL volume) and
continuously superfused at a rate of 1 mL/min, with a modified
Tyrobe buffer (composition in mM/L: NaCl, 137; KCl, 2.7;
MgCl2, 0.69; NaHCO3, 11.9; NaH2PO4, 0.4; CaCl2, 1.8; glucose,
10). The preheated (37 °C) perfusate was oxygenated and
maintained at pH 7.44 by bubbling with carbogene. Each aortic
ring was connected to a Grass FT 03 isometric force transducer
and stretched with a basal tension of 2 g. The contractile
activity of the aortic rings was recorded with a Grass poly-
graph, model 79, and with a CODAS DATAQ DI-120 computer-
based system (4/s sampling rate). The presence of the endo-
thelium in both aortic rings was checked 1 h after isolation
by measuring the relaxing action of 1 µM carbachol upon the
contraction induced by 1 µM phenylephrine. The endothelium
was considered intact if carbachol induced a relaxation of 90%
or higher and was considered absent if no relaxation was
observed.
The action of the TRAP mimetic compounds 4-6 (0.1-0.5
nM), upon the aortic smooth muscle was studied in prepara-
tions precontracted with 1 µM phenylephrine. Thrombin
mimetics were applied 30 min after normal perfusate admin-
istration. The relaxing action of the two thrombin mimetics
was also measured after pretreatment with 300 µM of the
competitive inhibitor of nitric oxide synthase, Nω-nitro-L-
arginine methyl ester (L-NAME).
All results are expressed as the mean ( SEM. The relaxing
action of the thrombin mimetics is expressed as a percentage
decrease of the contraction induced by 1 µM phenylephrine.
The dose-response curves for all compounds were compared
using two-way repeated measures of ANOVA, followed by the
Student Newman-Keuls test in the case of significant differ-
ences (p < 0.05). The ED50 of the various compounds was
calculated graphically from the dose-response curve.
III. Molecular Modeling. Theoretical calculations were
performed on a Silicon Graphics computer using QUANTA
from Molecular Simulations. The three-dimensional structure
of compound 4 was built and was subjected to steepest descent
(SD) and Newton-Raphson algorithms using an energy toler-
ance 0.01 kcal mol-1 Å-1 in order to reach a local minimum
conformer. To the obtained local minimum conformer, the
molecular dynamics experiment was performed at 1000 K
using 1 ps for heating, equilibration, and stimulation steps
using ꢀ ) 45. One-hundred structures from the simulated ones
were minimized using 2000 iteration steps and the SD
algorithm. The obtained low-energy structures were divided
e. Synthesis of N-Phenylacetyl-3-aminopyrrolidine
(8a). e.1. 3-[1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-
ethylamino]pyrrolidine. 3-Aminopyrrolidine dihydrochlo-
ride (636 mg, 4.0 mmol), 2-acetyldimedone (810 mg, 4.5 mmol),
and DIEA (1.4 mL) were dissolved in absolute ethanol (20 mL),
and the solution was stirred for 12 h at room temperature.
The solvent was rotary-evaporated, and the yellowish residue
was triturated with dry ether. The crude product was purified
by recrystallization from MeOH/ether to give a white solid (870
mg, 3.5 mmol, 87%): mp 206-208 °C; Rf ) 0.26 (4:1:1, ButOH/
1
H2O/AcOH); H NMR (CDCl3) δ 1.04 (s, 6H), 2.35 (dm, 2H),
2.37 (s, 4H), 2.61 (s, 3H), 3.43 (dq, 2H), 3.57 (m, 1H), 4.23 (dq,
2H), 13.8 (s, 1H); MS (nominal) m/z 251 (M+ + 1).
e.2. N-Phenylacetyl-3-[1-(4,4-dimethyl-2,6-dioxocyclo-
hexylidene)ethylamino]pyrrolidine. The above Dde-pro-
tected 4-aminopyrrolidine (500 mg, 2.0 mmol), phenylacetic
acid (280 mg, 2.05 mmol), HOBt (285 mg, 2.1 mmol), and DIEA
(1 mL) were dissolved in 15 mL of CH2Cl2. The mixture was
cooled to 0 °C, and DCC (435 mg, 2.1 mmol) was added. The
reaction mixture was then stirred at room temperature for 18
h. The suspension (DCU) was filtered and washed with 25 mL
of CH2Cl2. The filtrate was subsequently concentrated in vacuo
to give a waxy yellowish solid as a crude product. Flash
chromatography (1.5% MeOH/CHCI3) yielded a waxy white
solid (650 mg, 1.76 mmol, 88%): Rf ) 0.80 (4:1:1, ButOH/H2O/
AcOH); 1H NMR (CDCl3) δ 1.04 (s, 6H), 2.23 (dm, 2H), 2.36 (s,
2H), 2.40 (s, 2H), 2.61 (s, 3H), 3.44-3.91 (m, 5H), 3.69 (s, 2H),
7.25-7.36 (m, 5H), 13.8 (s, 1H); MS (nominal) m/z 369 (M+
1).
+
e.3. N-Phenylacetyl-3-aminopyrrolidine (8a). The above
amide (70 mg, 0.19 mmol) was dissolved in 5 mL of 2% v/v
methanolic hydrazine solution. The reaction was monitored
by TLC (10% MeOH/CHCl3) and showed completion after ∼30
min. The excess of hydrazine was removed by rotary evapora-
tion. The remaining white solid was dissolved in CHCl3 (20
mL) and then washed with 2 M HCl (2 × 15 mL). The HCl
extracts were basified with 2 M NaOH to pH 9, extracted with
CHCl3 (3 × 20 mL), dried over anhydrous Na2SO4, and reduced
in vacuo to give 35 mg (3.2 mmol, 90%) of a colorless oil as a
crude product. The crude product was purified by preparative
HPLC to yield 28 mg (0.14 mmol, 72%) of a waxy white solid:
Rf ) 0.37 (4:1:1, ButOH/H2O/AcOH); 1H NMR (CD3OD) δ 2.07
(m, 1H), 2.37 (m, 1H), 3.22 (m, 2H), 3.54-3.93 (m, 5H), 3.73
(s, 2H), 7.24-7.35 (m, 5H); MS (nominal) m/z 205 (M+ + 1).
II. Biological Activity. a. Platelet Aggregation. Venous
blood (30 mL) for the preparation of platelet-rich plasma (PRP)
was withdrawn between 8 and 11 a.m. from healthy volunteers
who had not taken any medication for the previous 2 weeks.
Blood samples were subsequently centrifuged at 800 rpm at
room temperature for 8 min. The supernatant platelet-rich
plasma (PRP) was counted by a Coulter counter (Coulter Corp.)
and was diluted with platelet-poor plasma (PPP) to the desired
final concentration (2 × 108 platelets/mL).
Platelet aggregation was determined by the optical method
in a four-channel platelet aggregometer (PAP-4, Bio/Data).
Samples of platelet suspensions (450 µL) under stirring at 1100
rpm were activated at 37 °C with 50 µL of increasing
concentrations (0.125-0.3 IU/mL) of bovine thrombin (Chro-
mogenix, Sweden). Higher concentrations of thrombin could
not be used because fibrin clot formation interfered with the
measurement of aggregation on platelet-rich plasma.36 On the
other hand, less than 0.2 IU/mL thrombin concentration did
not constantly cause proper platelet aggregation; therefore,
platelet activation was carried out with a thrombin concentra-
tion greater than 0.2 IU/mL.
Light transmittance was measured and recorded for 4 min,
at which time platelets had reached a maximal response to
thrombin. Results were expressed as the percent change in
light transmittance, with the light transmittance of plasma
alone (PPP) taken as 100% and that of plasma with platelets

Thrombin Receptor Mimetics
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3351
Graviari, V.; Jallat, S.; Schlesinger, Y.; Pages, G.; Pavirani, A.;
Lecocq, J. P.; Pouyssegur, J.; Van Obberghen-Schilling, E. cDNA
cloning and expression of a human R-thrombin receptor coupled
to Ca+2 mobilization. FEBS Lett. 1991, 288, 123-128.
into three family structures using the dihedral angle criterion
(60°) (Rmax ) 101.6, Rmin ) 0). The lowest energy conformers
from each family were considered as representative conform-
ers. These structures were used for superimposition with the
template, the cyclic conformer of SFLLR. The best superim-
posed of those were further optimized through SD and NR
minimization algorithms. The details of the building of the
SFLLR structure was given in a previous publication.17 The
superimposition of the four pairs of structures was performed
using a rigid-body fit to the target method of molecular
similarity software. Rigid-body fitting translates and rotates
working structures to minimize the rms of the fit to the target
structure.
(11) Ahn, H.-S.; Chackalamannil, S. Nonpeptide thrombin receptor
antagonists. Drugs Future 2001, 26, 1065-1085.
(12) Scarborough, R.; Naughton, M.; Teng, W.; Hung, D.; Rose, J.;
Vu, T. K.; Wheaton, V.; Turck, C.; Coughlin, S. Tethered ligand
agonist peptides: Structural requirements for thrombin receptor
activation reveal mechanism of proteolytic unmasking of agonist
function. J. Biol. Chem. 1992, 267, 13146-13149.
(13) Vassallo, R.; Kieber-Emmons, T.; Cichowski, K.; Brass, L.
Structure-function relationships in the activation of platelets
thrombin receptors by receptor-derived peptides. J. Biol. Chem.
1992, 267, 6081-6085.
(14) Chao, B.; Kalkunte, S.; Maraganore, J.; Stone, S. Essential
groups in synthetic agonist peptides for activation of the platelet
thrombin receptor. Biochemistry 1992, 31, 6175-6178.
(15) Hui, K.; Jakubowski, J.; Wyss, V.; Angleton, E. Minimal
sequence requirement of thrombin receptor agonist peptide.
Biochem. Biophys. Res. Commun. 1992, 184, 790-796.
(16) Natarajan, S.; Riexinger, D.; Cambardella, M.; Seiler, S. “Teth-
ered ligand” derived pentapeptide agonists of thrombin recep-
tor: A study of side chain requirements for platelet aggregation.
Int. J. Pept. Protein Res. 1995, 45, 145-151.
Acknowledgment. This work was supported by the
Ministry of Energy and Technology of Greece (ΕΠΕΤ
ΙΙ, 115), and the Ministry of Education (EPEAK). Some
of the bioassays were performed by Dr. Stefan Mi-
hailescu in the Department of Physiology, School of
Medicine, Universidad Nacional Autonoma de Mexico,
and by Doctor E. Melissari and Professor D. Vlahakos
at Onasis Cardiac Institute in Athens.
(17) Matsoukas, J.; Hollenberg, M.; Mavromoustakos, T.; Panagioto-
poulos, D.; Alexopoulos, K.; Yamdagni, R.; Wu, Q.; Moore, G.
Conformational analysis of the thrombin receptor agonist pep-
tides SFLLR and SFLLR-NH2 by NMR: Evidence for cyclic
bioactive conformation. J. Protein Chem. 1997, 16, 113-131.
(18) Coller, B.; Ward, P.; Ceruso, M.; Scudder, L.; Springer, K.; Kutok,
J.; Prestwich, G. Thrombin receptor activating peptides; impor-
tance of the N-terminal serine and its ionization state as judged
by pH dependence, nuclear magnetic resonance spectroscopy and
cleavage by aminopeptidase. M. Biochemistry 1992, 31, 11713-
11722.
(19) Adrade-Gordon, P.; Maryanoff, B.; Derian, C.; Zhang, H.-C.;
Addo, M.; Darrow, A.; Eckardt, A.; Hoekstra, W.; McComsey,
D.; Oksenberg, D.; Reynolds, E.; Santulli, R.; Scarborough, R.;
Smith, C.; White, K. Design, synthesis, and biological charac-
terization of a peptide-mimetic antagonist for a tethered-ligand
receptor. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12257-12262.
(20) Alexopoulos, K.; Panagiotopoulos, D.; Mavromoustakos, T.;
Fatseas, P.; Mihailescu, S.; Paredes-Carbajal, M.; Mascher, D.;
Matsoukas, J. Design, synthesis and modeling of novel cyclic
thrombin-receptor-derived peptide analogues of the Ser42-Phe-
Leu-Leu-Arg46 motif sequence with fixed conformations of phar-
macophoric groups: Importance of a Phe/Arg/NH2 cluster for
receptor activation and implications in the design of non-peptide
thrombin receptor mimetics J. Med. Chem. 2001, 44, 328-339.
(21) Matsoukas, J.; Panagiotopoulos, D.; Keramida, M.; Mavro-
moustakos, T.; Yamdagni, R.; Qiao, W.; Moore, G.; Saifeddine,
M.; Hollenberg, M. Synthesis and contractile activities of cyclic
thrombin receptor-derived peptide analogues with a Phe-Leu-
Leu-Arg motif: Importance of the Phe/Arg relative conformation
and the primary amino group for activity. J. Med. Chem. 1996,
39, 3585-3591.
Appendix
Abbreviations. Abbreviations used are in accordance
with the rules of IUPAC-IUB Commission on Biochemi-
cal Nomenclature: Eur. J. Biochem. 1984, 9-37; J. Biol.
Chem. 1989, 264, 663-673. AcOH, acetic acid; TFA,
trifluoroacetic acid; TEA, triethylamine; DIEA, diiso-
propylethylamine; DMF, N,N′-dimethylformamide; i-
PrOH, 2-propanol; MeOH, methanol; EtOH, ethanol;
Fmoc (9-fluorenylmethoxy)carbonyl; DMSO, dimethyl
sulfoxide; DIC, N,N′,diisopropylcarbodiimide; HOBt,
1-hydroxybenzotriazole; THF, tetrahydrofuran; Et2O,
diethyl ether; Boc, (tert-butyloxy)carbonyl; Fmoc, (9-
fluorenylmethoxy) carbonyl; Ahx, aminohexanoic acid;
DCC, N,N′-dicyclohexylcarbodiimide; Dde, 1-(4,4-di-
methyl-2,6-dioxocyclohexylidene)ethyl; TRAPs, throm-
bin receptor activating peptides; FAB, fast atom bom-
bardment; L-NAME, Nω-nitro-L-arginine methyl ester;
ANOVA, analysis of variance; rms, root mean square;
PRP, platelet-rich plasma; PPP, platelet-poor plasma.
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