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 pharmacophoric groups: Importance of a Phe/Arg/NH2 cluster for receptor activation and implications in the design of nonpeptide thrombin receptor mimetics

Article abstract of DOI:10.1021/jm0001525

The novel cyclic analogues cyclo(Phe-Leu-Leu-Arg-εLys-Dap) (1) and cyclo(D-Phe-Leu-Leu-ArgεLys-Dap) (2), which differ only in the absolute conformation of Phe, have been designed and synthesized based upon the minimal peptide sequence Phe-Leu-Leu-Arg which has been found to exhibit biological activity for the thrombin receptor. Compound 1, in which all amino acids have the L-configuration, exhibited higher activity in the rat aorta relaxation and rat longitudinal muscle bioassays compared to compound 2, in which the Phe residue is in the D-configuration. This is attributed to the spatial proximity of the Phe and Arg in compound 1 which does not exist in its diastereomeric compound 2, as is depicted from a combination of NMR studies and computational analysis. Structure - activity studies (SAR) showed that the Phe and Arg side chains along with a primary amino group form an active recognition motif that is augmented by the presence of a second primary amino group in the cyclic peptide. We suggest that a comparable cyclic conformation may be responsible for the interaction of linear TRAPs with the thrombin receptor. The validity of this proposition was tested by the synthesis of four active nonpeptide thrombin receptor mimetics. Substance (S)-N-(6-guanidohexanoyl)-N′-(2-amino-3-phenylpropionyl)piperazine (3), in which the pharmacophoric phenyl, guanidino, and amino groups were incorporated onto a piperazine template, was found to be the most active compared to the other synthesized compounds which lack the amino pharmacophoric group.

Full text of DOI:10.1021/jm0001525

328
J . Med. Chem. 2001, 44, 328-339
Design , Syn th esis, a n d Mod elin g of Novel Cyclic Th r om bin Recep tor -Der ived
P ep tid e An a logu es of th e Ser 42-P h e-Leu -Leu -Ar g46 Motif Sequ en ce w ith F ixed
Con for m a tion s of P h a r m a cop h or ic Gr ou p s: Im p or ta n ce of a P h e/Ar g/NH2
Clu ster for Recep tor Activa tion a n d Im p lica tion s in th e Design of Non p ep tid e
Th r om bin Recep tor Mim etics†
Kostas Alexopoulos,‡ Dimitris Panagiotopoulos,‡ Thomas Mavromoustakos,§ Panagiotis Fatseas,‡
Maria Christina Paredes-Carbajal,| Dieter Mascher,| Stefan Mihailescu, and J ohn Matsoukas*,‡
Department of Chemistry, University of Patras, Patras 26500, Greece, Institute of Organic and Pharmaceutical Chemistry,
National Hellenic Research Foundation, Athens 11635, Greece, Department of Physiology, School of Medicine,
UNAM, Mexico DF 04510, Mexico, and Department of Physiology, Faculty of Medicine, University of Craiova,
Craiova RO-1100, Romania
Received April 3, 2000
The novel cyclic analogues cyclo(Phe-Leu-Leu-Arg-ꢀLys-Dap) (1) and cyclo(D-Phe-Leu-Leu-Arg-
ꢀLys-Dap) (2), which differ only in the absolute conformation of Phe, have been designed and
synthesized based upon the minimal peptide sequence Phe-Leu-Leu-Arg which has been found
to exhibit biological activity for the thrombin receptor. Compound 1, in which all amino acids
have the L-configuration, exhibited higher activity in the rat aorta relaxation and rat
longitudinal muscle bioassays compared to compound 2, in which the Phe residue is in the
D-configuration. This is attributed to the spatial proximity of the Phe and Arg in compound 1
which does not exist in its diastereomeric compound 2, as is depicted from a combination of
NMR studies and computational analysis. Structure-activity studies (SAR) showed that the
Phe and Arg side chains along with a primary amino group form an active recognition motif
that is augmented by the presence of a second primary amino group in the cyclic peptide. We
suggest that a comparable cyclic conformation may be responsible for the interaction of linear
TRAPs with the thrombin receptor. The validity of this proposition was tested by the synthesis
of four active nonpeptide thrombin receptor mimetics. Substance (S)-N-(6-guanidohexanoyl)-
N′-(2-amino-3-phenylpropionyl)piperazine (3), in which the pharmacophoric phenyl, guanidino,
and amino groups were incorporated onto a piperazine template, was found to be the most
active compared to the other synthesized compounds which lack the amino pharmacophoric
group.
In tr od u ction
thrombin mimetic activity in both vascular and non-
vascular smooth muscle preparations.7-9 Based on a
gastric smooth muscle contractile bioassay for SFLL-
RNPNDKYEPF, it has been shown that only the first
five amino acids of this decatetrapeptide (i.e. SFLLR)
are required to exhibit contractile activity and that the
intrinsic activity of the peptide resides in the sequence
FLLR.8-10 Furthermore, structure-activity (SAR) analy-
sis has singled out the key importance of the Phe43 and
Arg46 residues for the biological activity of the receptor-
activating peptides (TRAPs), not only in smooth muscle9
but also in a human platelet assay.11,12 The alanine scan
experiment indicated that residues other than Phe and
Arg were not critical for the biological activity of
SFLLR.9,11
From previous SAR studies,9 it was observed that the
peptide N-propionyl-FLLR was active in the gastric
contractile bioassay, indicating that the primary phar-
macophores of the TRAPs resided in the sequence
FLLR. Further SAR and NMR studies made by us have
indicated the importance of the SFLLR cyclic conforma-
tion wherein the Phe and Arg residues cluster to-
gether.13 Recent studies have also indicated that a
positively charged NH2-terminus of SFLLR is important
for full agonist activity.14,15 On the basis of the above
Thrombin, a multifunctional serine protease gener-
ated at sites of vascular injury, plays a central role in
blood coagulation. Thrombin is also a powerful agonist
for a variety of cellular responses.1,2 Most of these
biological activities of thrombin are mediated through
its specific G-protein-linked functional receptors, which
have been cloned and shown to be present on a variety
of cells such as human platelets and endothelial cells3
and rat vascular smooth muscle cells.4 According to a
novel mechanism of receptor activation, thrombin binds
to its receptor’s N-terminal extension cleaving the
peptide bond between Arg41 and Ser42 within the
sequence LDPR41/S42FLLRNPNDKYEPF.3,5,6 This pro-
teolytic event unmasks a new N-terminal domain that
acts as an anchored ligand to stimulate receptor func-
tion. The synthetic decatetrapeptide SFLLRNPND-
KYEPF which mimics this new N-terminus possesses
† Part of this work is incorporated in the Ph.D. dissertations of K.
Alexopoulos and D. Panagiotopoulos.
* To whom correspondence should be addressed. Tel: +3061 997180.
Fax: +3061 997180 or 997118. E-mail: johnmatsoukas@hotmail.com.
‡ University of Patras.
§ National Hellenic Research Foundation.
| UNAM.
University of Craiova.
10.1021/jm0001525 CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/29/2000

Thrombin Receptor-Derived Peptide Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 329
Sch em e 1. Synthetic Procedure for Cyclic Peptides 1
and 2
information, we proceeded in the synthesis of diaster-
eomeric cyclic hexapeptides containing the active core
tetrapeptide FLLR with Phe in its L- or D-configuration,
as well as Lys and diaminopropionic acid. In particular
we synthesized, using Fmoc methodology and the acid-
sensitive 2-chlorotrityl resin as solid support, the fol-
lowing two cyclic analogues: cyclo(Phe-Leu-Leu-Arg-
ꢀLys-Dap) (1) and cyclo(D-Phe-Leu-Leu-Arg-ꢀLys-Dap)
(2), where Dap ) diaminopropionic acid and ꢀLys
denotes that the ꢀ-amino group of Lys was coupled to
the R-carboxyl group of Arg. In these analogues two free
R-amino groups from Lys and Dap are available, while
the key Phe (L or D) and Arg side chains are constrained
in the same or opposite sides.
The two cyclic compounds were assayed for their
biological activity in the rat aorta relaxation and rat
gastric longitudinal smooth muscle (LM) contraction
assays,16 and their activity was compared with the
activity of the linear TRAP SFLLR-NH2. The cyclic
compound 1 mimicked the relaxant activity of SFLLR-
NH2 in the rat aorta relaxation assay, and its activity
was 100-fold higher compared to the activity of cyclic
compound 2 in which the Phe/Arg side chains are
constrained in opposite sites. In the rat gastric contrac-
tile assay, compound 1 exhibited 8% contraction, while
no contractile effects were observed for compound 2. The
comparable activity of cyclic analogue 1 with that of
SFLLR-NH2 and the substantially higher activity com-
pared to cyclic analogue 2 show the importance of a Phe/
Arg cluster for receptor activation and of a primary
amino group for augmenting activity. The Phe/Arg
relative conformation of the two cyclic analogues was
also assessed using NMR spectroscopy and molecular
dynamics.
To validate the Phe/Arg/NH2 cluster conformational
model, we designed four novel nonpeptide mimetic
substances. These substances were also tested in the
rat aorta relaxation assay and they were found to
stimulate the endothelial thrombin receptor in a con-
centration-dependent manner, showing a similar phar-
macological profile with cyclic compound 1. (S)-N-(6-
Guanidohexanoyl)-N′-(2-amino-3-phenylpropionyl)piper-
azine (3), containing the three pharmacophoric groups,
was the most active compared to the other compounds
which lack the amino pharmacophoric group. The
comparable activity of cyclic TRAP analogue 1 and
nonpeptide TRAP analogue 3 confirms our hypothesis
that a cluster of phenyl, guanidino, and amino groups
is responsible for thrombin receptor triggering and
activation.
resulting linear-protected peptide failed to cyclize,
presumably because of steric hindrance. Using the
2-chlorotrityl resin and mild conditions, it was possible
to cleave the peptide-resin bond and subsequently to
cyclize the desired protected peptide, which was then
deprotected with 75% trifluoroacetic acid in dichlo-
romethane (Scheme 1). For the synthesis of compounds
1 and 2 the δ-amino group of NR-Boc-protected Dap was
coupled to the R-COOH group of NR-Boc (Nꢀ-Fmoc) of
Lys and subsequently the ꢀ-amino group of Nꢀ-Boc-
protected Lys was coupled to the R-COOH group of Arg
in the course of the synthesis of H-Leu-Arg(Pmc)-ꢀLys-
(Boc)-δDap(Boc)-Phe-Leu-OH as outlined in Scheme 1.
For cyclization of the protected peptides of the general
composition H-Leu-Arg-Lys-Dap-Phe-Leu-OH, it was
used either benzotriazol-1-yloxytris(dimethylamino)-
phosphonium hexafluorophosphate (BOP) or 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetraflu-
oroborate (TBTU) and N,N-diisopropylethylamine
(DIPEA) in a solvent of dimethylformamide (DMF). As
monitored using a ninhydrin test of the synthesized
cyclic peptide, resolved by thin-layer chromatography
with a chloroform-methanol (6:1, v/v) solvent system,
cyclization was more efficient using TBTU as a coupling
reagent compared with BOP. The success of the cycliza-
Resu lts
Ch em istr y. SFLLR-NH2 was synthesized and puri-
fied using methods which have been detailed previ-
ously.17 The cyclic analogues, based on the FLLR motif
(compounds 1, 2), were synthesized using the 2-chlo-
rotrityl chloride resin (CLTR)18 that was used previously
for the synthesis of novel cyclic amide-linked analogues
of angiotensin II and III,19 TRAPs,14,20 and myelin basic
21
protein epitope MBP72-85
.
For the successful cycliza-
tion of the precyclic linear peptides, it was necessary to
initiate the synthesis using the Leu2 residue of the
linear Phe1-Leu2-Leu3-Arg4 motif, as outlined in Scheme
1. If Arg was used for the first step of synthesis, the

330 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Alexopoulos et al.
F igu r e 1. Chemical structure of compound 1 [cyclo(Phe-Leu-
Leu-Arg-ꢀLys-Dap)].
F igu r e 2. Chemical structure and functionality of pharma-
cophoric groups of compound 3.
Sch em e 2. Solid-Phase Organic Synthesis of
(S)-N-(6-Guanidohexanoyl)-N′-(2-amino-3-phenyl-
propionyl)piperazine (3)a
was first activated with DIC and HOBt; then it was
coupled with piperazine in DMF and in the presence of
DIEA. Couplings with Fmoc-ꢀAhx-OH (2.5 equiv) were
aided by the use of DIC and HOBt under basic condi-
tions (DIEA) using minimum of DMF. Fmoc group
removals were carried out by treatment with 20%
piperidine/DMF for 30 min. The guanidino group was
incorporated using 1H-pyrazole-1-carboxamide hydro-
chloride in DMF/DIEA22 in the last solid-phase chem-
istry step before cleavage. The resin was cleaved with
10% TFA/CH2Cl2 for 15 min at room temperature. The
crude product was purified by HPLC. The chemical
structure of compound 3 is shown in Figure 2.
The general methodology for the synthesis of N-(6-
guanidohexanoyl)-4-(4-fluorophenylmethylamidoacetyl)-
piperidine (4) is depicted in Scheme 3. First the N-amino
group of isonipecotic acid was Boc-protected, and then
the Boc analogue was coupled with 4-fluorobenzylamine
using DCC and HOBt as coupling reagents. After Boc
deprotection with trifluoroacetic acid, ꢀ-aminohexanoic
acid (ꢀAhx) was incorporated at the free amine salt
aided by the use of DCC and HOBt under basic
conditions (DIEA). Boc deprotection was then followed,
and guanylation of the primary amine using 1H-
pyrazole-1-carboxamide hydrochloride22 afforded com-
pound 4. Most of the synthetic intermediates were
purified by flash chromatography (Merck silica gel 60,
230-400 mesh) and the final product by recrystalliza-
tion (MeOH/acetone/Et2O). The synthesis of N-phenyl-
acetyl-N′-(6-guanidohexanoyl)-1,4-diaminobutane (5) and
N-(4-fluorophenyl)acetyl-N′-(6-guanidohexanoyl)-1,4-di-
aminobutane (6) was carried out by an analogous
procedure as shown in Scheme 4. The chemical struc-
tures of compounds 4-6 are shown in Figure 3.
Bioa ssa y Da ta . The relaxant activities of cyclic
compounds 1 and 2 and nonpeptide compounds 3-6
were compared with that of the amidated linear penta-
peptide SFLLR-NH2 (Figure 4). SFLLR-NH2 represents
the shortest TRAP that exhibits a potency greater than
that of the originally described receptor-activating
tetradecapeptide.3,8-10 The relative potency (REC) for
each compound was determined from the dose-response
curves.10 REC represents the ratio between each of the
concentrations of the six TRAP analogues and the
concentration of SFLLR-NH2 necessary for producing
a
(i) Phe-OH, (CH3)3SiCl, TEA, CH2Cl2; (ii) DIC, HOBt, THF;
(iii) piperazine, DIEA, DMF; (iv) Fmoc-ꢀAhx-OH, DIC, HOBt,
DMF; (v) 20% piperidine in DMF; (vi) 1H-pyrazole-1-carboxamide,
DIEA, DMF; (vii) 10% TFA/CH2Cl2.
tion procedure, as indicated by the lack of a reaction
with ninhydrin, was confirmed by fast atom bombard-
ment (FAB) mass spectrometry and NMR spectroscopy.
The structures of the two peptides were confirmed by
amino acid and FAB analysis (Table 1). The chemical
structure of compound 1 is shown in Figure 1.
The synthesis of the nonpeptide thrombin receptor
analogue 3 (Scheme 2) was accomplished using also
solid-phase methods and the 2-chlotrityl chloride resin
as solid support. Attachment of the amino group of Phe
(L-Phe) to the resin (2.27 mequiv Cl-/g of resin) was
achieved by refluxing using TEA in CH2Cl2 solution and
after protection of the carboxyl group of Phe with (CH3)3-
SiCl. The loading of Phe to the resin was certified by
the Kaiser ninhydrin test. The Phe attached to the resin

Thrombin Receptor-Derived Peptide Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 331
Sch em e 3. Synthetic Procedure for N-(6-Guanido-
hexanoyl)-4-(4-fluorophenylmethylamidoacetyl)piperidine
(4)a
Sch em e 4. Synthetic Procedure for N-Phenylacetyl-
N′-(6-guanidohexanoyl)-1,4-diaminobutane (5, for X ) H)
and N-(4-Fluorophenyl)acetyl-N′-(6-guanidohexanoyl)-
1,4-diaminobutane (6, for X ) F)a
a
(i) DCC, HOBt, CHCl3; (ii) Boc-ꢀAhx-OH, DCC, HOBt, DIEA,
a
(i) Boc2O, dioxane, H2O; (ii) 4-fluorobenzylamine, DCC, HOBt,
CH2Cl2; (iii) 30% TFA/CH2Cl2; (iv) 1H-pyrazole-1-carboxamide,
DIEA, DMF.
CH2Cl2; (iii) 30% TFA/CH2Cl2; (iv) Boc-ꢀAhx-OH, DCC, HOBt,
DIEA, CH2Cl2; (v) 30% TFA/CH2Cl2; (vi) 1H-pyrazole-1-carbox-
amide, DIEA, DMF.
an equal relaxing effect on the isolated aortic rings (REC
) ECTRAP ÷ ECSFLLR-NH2). The REC value of compound
1 was 3.30 ( 0.68 (the compound was 3.3 times less
potent than SFLLR-NH2). In contrast, the REC value of
compound 2 was higher than 500 (the compound was
at least 500 times less active than SFLLR-NH2 and at
least 100 times less active than compound 1). The REC
values of nonpeptide compounds 3-6 were 32.8 ( 7.3,
37.3 ( 4.0, 68 ( 6.8, and 50.6 ( 3.1, respectively (Table
2). These REC values show that compound 3 is the most
active nonpeptide analogue. The relative potency of
compound 3 is 3 times smaller than that of compound
1 and 32.8 times smaller that that of SFLLR-NH2.
In particular, cyclo(Phe-Leu-Leu-Arg-ꢀLys-Dap) in-
duced a concentration-dependent relaxation of the aortic
rings with intact endothelium, precontracted with phen-
ylephrine (1 µM) (Figure 5). This effect became visible
at a concentration of 0.1 µM and reached a maximum
of 54 ( 2% (n ) 6) at a concentration of 0.5 mM. The
relaxing effect was abolished by pretreatment of the
aortic rings with Nω-nitro-L-arginine methyl ester (L-
NAME) (300 µM). Compound 1 also induced contractile
effects in the aortic rings without endothelium, at
concentrations equal to or higher than 0.1 mM (maxi-
mum 8% at 0.05 mM). Cyclo(D-Phe-Leu-Leu-Arg-ꢀLys-
Dap) exerted reduced relaxation, in a concentration-
dependent manner, in which the aortic rings with intact
endothelium were precontracted with phenylephrine (1
µM). The lowest concentration of the drug which induced
relaxation was 10 µM, while the maximal relaxing effect
of 15 ( 3% (n ) 6) was observed at 0.1 mM. The
F igu r e 3. Chemical structures of compounds 4 (top), 5
(bottom; for X ) H), and 6 (bottom; for X ) F).
appearance of this relaxing effect was prevented by
pretreatment with L-NAME (300 µM). No contractile
effects of compound 2 were observed in the aortic rings
without endothelium, even at higher concentrations of
the compound (0.5 mM). The analysis of the dose-
response by two-way repeated measure of analysis of
variance (ANOVA) followed by the Student Newman-
Keuls test revealed that there was a significant differ-
ence in the responses induced by the two thrombin
mimetics at concentrations of 0.1 mM or higher.

332 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Alexopoulos et al.
µM) (Figure 4). Pretreatment of the aortic rings with
L-NAME (300 µM) and experiments with aortic rings
without endothelium showed that the relaxing effects
produced by these compounds, as the ones of substance
3, were both endothelium-dependent and endothelium-
independent. For all these compounds the endothelium-
dependent relaxing effects appeared at a concentration
of 0.1 µM and were blocked by L-NAME (300 µM). The
dose-response curves for the endothelium-dependent
relaxing effects of compounds 3-6, presented in Figure
4, were significantly different when analyzed by two-
way ANOVA (p < 0.01). The REC values (Table 2)
indicate an order of potency 3 > 4 > 6 > 5. The
endothelium-independent effects appeared at concentra-
tions of 10 µM, had a dose-dependent character, and had
values ranging between 1.43-12% for 4, 0.4-5% for 5,
and 3.83-15% for 6.
Figu r e 4. Concentration-dependent relaxing effects of SFLLR-
NH2, cyclo(Phe-Leu-Leu-Arg-ꢀLys-Dap) (1), cyclo(D-Phe-Leu-
Leu-Arg-ꢀLys-Dap) (2), (S)-N-(6-guanidohexanoyl)-N′-(2-amino-
3-phenylpropionyl)piperazine (3), N-(6-guanidohexanoyl)-4-(4-
fluorophenylmethylamidoacetyl)piperidine (4), N-phenylacetyl-
N′-(6-guanidohexanoyl)-1,4-diaminobutane (5), and N-(4-
fluorophenyl)acetyl-N′-(6-guanidohexanoyl)-1,4-diaminobu-
tane (6) upon the phenylephrine-induced contraction of aortic
rings with intact endothelium. Results are expressed as means
( SE (n ) 6).
NMR Stu d ies. In the present study 2D-TOCSY and
1D-NOE experiments in DMSO-d6 for cyclo(Phe-Leu-
Leu-Arg-ꢀLys-Dap) were conducted in an attempt to
relate structure with biological activity. 1H assignment
was achieved by combining information from TOCSY
spectra and 1D-NOE experiments. The 1D-NOE experi-
ments provided also distance information for the side
chain protons of Arg and Phe. For the Phe residue, the
CR proton resonance was readily assigned at δ ) 4.46
ppm through intense TOCSY peaks of the CR proton
with the characteristic Cââ, protons at δ ) 3.02 and 2.82
The above data show that the activity of cyclo(D-Phe-
Leu-Leu-Arg-ꢀLys-Dap) appeared to be lower compared
to that of cyclo(Phe-Leu-Leu-Arg-ꢀLys-Dap), indicating
that an Arg/Phe side chain cluster is essential for
maximum activity. Substances 1 and 2 have respectively
the two pharmacophoric groups Phe and Arg on the
same or different sides of the cyclic backbone. Further-
more, two adjacent primary amino groups provided by
Lys and Dap were introduced in the chemical structure
to confirm previous findings suggesting a combined
action of Phe/Arg/NH2 groups for maximum activity.
Our work illustrates also that cyclic peptides based on
the pentapeptide motif of the active thrombin receptor-
derived sequences containing the two pharmacophores
(Phe and Arg) and an adjacent free amino group exhibit
high contractile and relaxant activity in tissues.
Compound 3 (0.1-300 µM) produced a concentration-
dependent relaxation of the aortic rings with endothe-
lium, precontracted with phenylephrine (1 µM), by 1.5-
59% (n ) 7; Figure 4). Pretreatment of the aortic rings
with L-NAME (300 µM) reduced the maximum relaxing
effect of compound 3 to 26-28%, which represents the
value of the nitric oxide-dependent component of the
relaxation. Compound 3 (0.1-300 µM) also induced the
relaxation of the aortic rings without endothelium
precontracted with phenylephrine (1 µM) by 1.2-31%.
The relaxing effects of compound 3, in aortic rings with
and without endothelium, were reversible through
administration of phenylephrine (1 µM). In aortic rings
with endothelium, but not in the ones without endo-
thelium, repeated administrations of compound 3 re-
duced the amplitude of the phenylephrine-induced
contraction by 23-25%. This effect was reverted by
administration of L-NAME (300 µM). At concentrations
of 10 and 100 µM, compound 3 induced a transient
contraction of 2-3% in the aortic rings without endo-
thelium.
ppm. For the Arg residue, the NHR, CR, Cââ′, Cγγ′, Cδδ′
,
Nꢀ, and Nω proton resonances were readily assigned
through intraresidue TOCSY cross-peaks, based on the
well-established interaction between Arg Cδ and Nꢀ
proton resonances at δ ) 3.05 and 7.98 ppm, respec-
tively. The Phe/Arg side chain proximity in cyclic
peptide 1 was indicated in the 1D-NOE experiments.
Saturation of the Phe ring protons at δ ) 7.28 ppm
resulted in enhancements of the Arg Nꢀ proton at δ )
7.98 ppm (13.4%) and of the Arg Nꢀ protons at δ ) 6.65
ppm (17.2%), indicating this proximity. This NOE
phenomenon was a reversible one. Thus, saturation of
the Arg Nꢀ proton resulted in enhancement of the Phe
ring protons at δ ) 7.28 ppm (9.2%) confirming the Phe/
Arg side chains interaction. Such enhancements were
not observed in cyclic peptide 2 upon saturation of Phe
ring protons and Arg Nꢀ proton.
Com p u ta tion a l An a lysis. To extend the observa-
tions made using NMR, theoretical calculations were
performed. The most striking difference in the biological
activity was between cyclic peptides 1 and 2. As
explained above, this can be attributed to the spatial
orientation of the phenyl ring of the amino acid Phe. In
compound 1 the phenyl ring orients in such a way to
spatially approximate the Arg amino acid. This is not
observed with compound 2 and the phenyl ring orients
away from the Arg amino acid. Therefore, 2 lacks the π
interactions between the Arg and Phe rings. This may
explain adequately the dramatic loss of biological activ-
ity of 2 relative to 1. The low-energy conformations of
the two compounds are shown in Figure 6a. A super-
imposition of compound 1 with the parent compound
SFLLR-NH2, already discussed in previous papers,13,14
shows that the two peptides have their phenyl and
guanidino groups in spatial proximity (rms ) 1 Å) (see
The nonpeptide TRAPs 4-6 (0.1-300 µM) produced
concentration-dependent relaxing effects on aortic rings
with endothelium precontracted with phenylephrine (1

Thrombin Receptor-Derived Peptide Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 333
Ta ble 1. Chemical Data for the Synthesized Cyclic TRAPs
MW
amino acid analysis
a
peptide
Rf (%)
FAB-MS
Phe
Leu
Arg
Lys
1, cyclo(Phe-Leu-Leu-Arg-ꢀLys-Dap)
2, cyclo(D-Phe-Leu-Leu-Arg-ꢀLys-Dap)
0.57
0.56
587
587
0.93
0.90
2.10
2.12
1.00
1.00
0.98
0.94
a
Retention fraction from TLC with BAW.
Ta ble 2. Bioassay Results for Cyclic and Nonpeptide TRAPs
(means ( SE)
extension to reveal a new N-terminus that functions as
a tethered peptide ligand.3 Synthetic TRAPs, comprising
only the first five amino acids (S42FLLR46) of tethered
ligand, have been shown to be sufficient to possess
thrombin mimetic activity.11,12,24 Recent conformational
studies have shown that a cyclic conformation for
SFLLR in which the Phe and Arg residues cluster
together to form a primary pharmacophore motif may
be required for the biological activity of the peptide.13
The above proposed cyclic model was testified since
cyclic derivatives of SFLLR were found to be biologically
active.14,20 To further investigate the bioactive confor-
mation of SFLLR, we proceeded in the synthesis of novel
cyclic TRAP analogues in which two free ꢀ-amino groups
from Lys and Dap would be available and the key Phe
and Arg residues would be constrained in the same or
opposite sides.
compd
rel potency (REC
)
compd
rel potency (REC)
SFLLR-NH2
1
4
5
6
37.3 ( 4.0
68 ( 6.8
50.6 ( 3.1
1
2
3
3.3 ( 0.68
>500
32.8 ( 7.3
F igu r e 5. Concentration-dependent effects of cyclo(Phe-Leu-
Leu-Arg-ꢀLys-Dap) upon isolated aortic rings precontracted
with phenylephrine (PE), 1 µM. After TRAP administration,
the preparations were washed (W) and left to recover for 30
min.
In our SAR survey we tested the conformationally
restricted analogues cyclo(Phe-Leu-Leu-Arg-ꢀLys) (1)
and cyclo(D-Phe-Leu-Leu-Arg-ꢀLys-Dap) (2) in which the
Phe/Arg side chains are respectively on the same or
different sides of the peptide ring in the rat aorta
relaxation and rat gastric longitudinal smooth muscle
contraction assays. In analogue 1, in which the two
pharmacophoric groups cluster together, the activity is
high and comparable to that exerted by linear SFLLR-
NH2. In contrast, in analogue 2, wherein the two
pharmacophoric groups are located at different sites of
the peptide ring, the activity is much lower (100 times
less) indicating the importance of the Phe/Arg cluster
for maximum activity. This activity is augmented by the
presence of the two R-amino groups provided by Lys and
Dap residues. Indeed, the existence of a primary NH2
group has been proved critical for receptor activation
by TRAPs,14,15 while an extra R-amino group probably
enhances the positive charge of the Phe/Arg/NH2 cluster,
thus increasing the activity.
Figure 6b). However, the cyclic peptide mimics only part
of SFLLR-NH2. This may explain their different activity.
A superimposition between active cyclic peptide 1 and
the most active peptide mimetic 3 is shown in Figure
7a. The comparative groups used for superimposition
were: (a) phenyl ring of 1 with phenyl ring of 3; (b)
amide bond between Phe-Dap of 1 with amino group of
3; (c) Arg of 1 with Arg of 3; (d) two amino groups of
Dap in 1 with piperazine of 3. The results show that
the superimposition between peptide 1 and peptidomi-
metic 3 using the above equivalent groups has an rms
of 0.96 Å. This represents a good match and may explain
the comparative activity of 3 relative to 1. The much
less activity of 3 prompts us to design and synthesize
more novel compounds in the future that would better
fit the parent SFLLR-NH2. This information may be
valuable for synthetic chemists who are willing to
attempt this design for synthesis of drugs with a better
biological profile. A superimposition between peptido-
mimetics 3 and 4 (Figure 7b) shows a deviation of 0.24
Å between the matched groups. This deviation is
reflected in the rms value of 1.34 Å when compound 4
is superimposed with cyclic peptide 1. Superimposition
of peptidomimetics 5 and 6 with cyclic peptide 1 showed
rms values of 1.56 Å. The higher activity of 6 relative
to 5 may relate to the CH/π interactions of the phenyl
ring with thrombin receptor. Compound 6 has the
phenyl ring para substituted with fluorine, and this is
well-known to cause an increase of the CH/π interac-
tions with the receptor.23
The bioassays performed in the isolated aortic rings
with intact endothelium, precontracted with phenyl-
ephrine (1 µM), show that both cyclic TRAPs induce
relaxation by releasing nitric oxide (the relaxing effect
was blocked by pretreatment of the isolated vessels with
nitric oxide synthase inhibitor L-NAME). However, the
relaxing effect was significantly lower (100 times less)
for cyclic analogue 2, compared to cyclic analogue 1 at
concentrations of 0.1 mM or higher, which indicates a
lower capacity of the first compound to stimulate the
receptor (lower efficacy or intrinsic activity). Cyclic
compound 1 was also found to exhibit contractile activity
in aortic rings without endothelium, while compound 2
did not exert such activity. Therefore, these results
suggest that the main function of Phe43 is the activation
of the thrombin receptor, whereas its role in binding to
the receptor seems to be less important. A similar
conclusion was reached by Scarborough et al.,24 using
a different technical approach (substitution of Phe43
with other amino acids).
These superimposition studies suggest that peptide
mimetics that show best mimicking of active peptide 1
closely approach its biological activity.
Discu ssion
Thrombin receptor is activated by a novel mechanism
in which thrombin cleaves its receptor’s N-terminal

334 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Alexopoulos et al.
F igu r e 6. (a) Low-energy conformations of cyclo(Phe-Leu-Leu-Arg-ꢀLys-Dap) (left) and cyclo(D-Phe-Leu-Leu-Arg-ꢀLys-Dap) (right).
(b) Superimposition of cyclic peptide 1 with SFLLR-NH2.
Relative activities were anticipated considering the
molecular models of compounds 1 and 2 shown in Figure
6a. In compound 1 (Figure 6a, left) the conformational
model shows that Phe and Arg side chains are in a
spatial close proximity. These two key amino acids are
protruding in a different phase in relation to the alkyl
chains of the two Leu. In the case of compound 2 (Figure
6a, right) the spatial vicinity between Phe and Arg no
longer exists. In this conformation, both alkyl chains of
Leu and Arg are in the same phase while the phenyl
ring of Phe is in an opposite phase. It appears that
distinct conformational preferences can exist between
the two cyclic peptides.
The molecular dynamics approach, in which the
structure was manipulated to fulfill the imposed con-
straints and NOE data, yielded a model with the Phe
and Arg residues on the same side of the cyclic ring of
analogue 1 (Figure 6a). In terms of the cis orientation
of the Phe and Arg side chains, this model is consistent
with previous suggestions in which the two pharma-
cophoric groups are on the same side in TRAPs.13,14,20
Our findings would suggest further that the active
conformation of TRAPs may bring the Arg and Phe
residues into closer proximity in a quasicyclic structure.
Our data appear to argue against other models that
have suggested unstructured or (S)-like conformations
for the TRAPs.25-27
To test the proposed cyclic model, four novel nonpep-
tide thrombin receptor analogues have been designed
rationally28-32 to mimic the active cyclic conformation
adopted by SFLLR. (S)-N-(6-Guanidohexanoyl)-N′-(2-
amino-3-phenylpropionyl)piperazine (3) is carrying the
essential pharmacophoric groups of Phe, Arg, and NH2
incorporated onto the piperazine template (Figure 2).
N-(6-Guanidohexanoyl)-4-(4-fluorophenylmethylamido-
acetyl)piperidine (4), N-phenylacetyl-N′-(6-guanidohex-
anoyl)-1,4-diaminobutane (5), and N-(4-fluorophenyl)-
acetyl-N′-(6-guanidohexanoyl)-1,4-diaminobutane (6) were
built onto two different small bifunctional templates
carrying the essential pharmacophoric groups of Phe

Thrombin Receptor-Derived Peptide Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 335
mimetic. The relaxing effects of the nonpeptide throm-
bin receptor mimetics became visible at concentrations
as low as 1-10 µM. The ED50 values for compounds 3-6
were 17.7, 21.7, 27.3, and 22.1 µM, respectively. The
ED50 values were in accordance with their REC values
(Table 2) showing an order of potency 3 > 4 > 6 > 5.
Superimposition studies showed that compound 3
mimicked the best cyclic compound 1, and this explains
their comparative activities. The activities of the other
synthetic peptide mimetic analogues paralleled the
degree of mimicry of cyclic analogue 1. It appears that
the combination of NMR spectroscopy with computa-
tional analysis can give a plausible explanation of the
observed biological activities for the nonpeptide mimetic
analogues.
Compound 1 is still less active than SFLLR-NH2.
While the two models of the two compounds have in
close proximity both phenyl and Arg moieties, other
structural features are not in spatial vicinity (for
example their hydrophobic parts). This observation
suggests that a novel cyclic peptide compound that
better mimics SFLLR-NH2 is in need. Such a peptide
cyclic compound may generate other classes of peptide
mimetics which in turn have better biological activity.
The present promising biological activities of the syn-
thetic nonpeptide mimetic analogues make this future
effort a reasonable goal.
Con clu sion
Two cyclic analogues of the thrombin receptor peptide
SFLLR containing Lys, Dap, and L- or D-Phe have been
synthesized using TBTU as cyclization reagent, and
their potency has been compared with that of linear
SFLLR-NH2 in the rat aortic smooth muscle relaxation
and contraction assays. The cyclic analogue Phe-Leu-
Leu-Arg-ꢀLys-Dap in which the two pharmacophoric
groups Phe and Arg are on the same side of the peptide
ring showed comparable activity with linear SFLLR-
NH2 and higher activity compared to cyclic analogue
D-Phe-Leu-Leu-Arg-ꢀLys-Dap in which the two groups
are fixed on opposite sides. The key function of Phe43 is
the activation of the thrombin receptor, whereas its role
in binding to the receptor seems to be less important.
These findings confirm the suggestion that a cluster of
the two groups (phenyl, guanidine) together with an
adjacent primary amino group is important for expres-
sion of maximum biological activity by thrombin recep-
tor-derived peptides. A comparable cyclic conformation
may be responsible for the interaction of linear TRAPs
with the thrombin receptor.
This model was used as a basis for the design of
nonpeptide thrombin receptor mimetics. (S)-N-(6-Guan-
idohexanoyl)-N′-(2-amino-3-phenylpropionyl)piperazine
which contains the structural requirements of SFLLR
(Phe/Arg/NH2) on the molecular template of piperazine
was found to be the most active nonpeptide TRAP
analogue in the rat aorta relaxation assay, confirming
our proposal for the interaction of TRAPs with the
thrombin receptor. These findings may open new av-
enues in the design of potent thrombin receptor mimet-
ics that might be efficacious in drug therapy.
F igu r e 7. (a) Superimposition of nonpeptide mimetic 3 with
cyclic peptide 1. (b) Superimposition of nonpeptide mimetic 3
with nonpeptide mimetic 4.
and Arg but missing the NH2 pharmacophoric group.
Compound 4 was built onto a piperidine template
(isonipecotic acid) having attached a guanidino linear
chain of five methyl groups and a phenyl group having
a fluorine atom in the para position (Figure 3). Intro-
duction of a fluorine atom in the para position of a
phenyl group is known to result in an increment of
activation of TRAPs.33 Compounds 5 and 6 were built
onto a linear template (1,4-diaminobutane) having
attached the pharmacophoric groups of Phe and Arg
with the difference in the presence of a fluorine atom
in the para position of the phenyl group in compound 6
(Figure 3). These nonpeptide TRAP mimetics were also
tested for biological activity in perfused aortic rings
(Figure 4).
Compounds 3-6 relaxed in a concentration-dependent
manner the isolated aortic rings with intact endothe-
lium, precontracted with phenylephrine. Generally, the
magnitude of the relaxation induced by a given TRAP
mimetic was higher in preparations with endothelium
than in deendothelized preparations. The two types of
relaxation were very close or equal in the preparations
pretreated with L-NAME, which comprises an additional
way of separating the endothelium-dependent from
endothelium-independent effects for the same TRAP
Exp er im en ta l Section
Abbreviations. Abbreviations used are in accordance with
the rules of the IUPAC-IUB Commission on Biochemical
Nomenclature (Eur. J . Biochem. 1984, 138, 9-37; J . Biol.

336 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Alexopoulos et al.
Chem. 1989, 264, 663-673). Abbreviations: AcOH, acetic acid;
DCM, dichloromethane; Et2O, diethyl ether; TFE, 2,2,2-
trifluoroethanol; TFA, trifluoroacetic acid; TEA, triethylamine;
DMF, N′,N′-dimethylformamide; i-PrOH, 2-propanol; MeOH,
methanol; Boc, (tert-butyloxy)carbonyl; Fmoc, (9-fluorenyl-
methoxy)carbonyl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sul-
fonyl; DMSO, dimethyl sulfoxide; Ahx, aminohexanoic acid;
DIC, N,N′-diisopropylcarbodiimide; DCC, N,N′-dicyclohexyl-
carbodiimide; HOBt, 1-hydroxybenzotriazole; THF, tetrahy-
drofuran; LM, longitudinal muscle; TRAPs, thrombin receptor-
activating peptides; L-NAME, N′-nitro-L-arginine methyl ester;
ANOVA, analysis of variance; ®, poly(styrene)-2-chlorotrityl
resin.
solution was concentrated to a small volume and the final free
cyclic peptide was precipitated as a light yellow amorphous
solid, upon addition of cold diethyl ether (purity g 79%, as
determined by HPLC).
Solid -P h a se Or ga n ic Syn th esis. P r ep a r a tion of HO-
P h e-2-ch lor otr ityl r esin . Phe (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 temperature 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 cooling 2-chlorotrityl chloride resin (1.0 g,
2.27 mequiv of Cl-/g resin) and CH2Cl2 (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 temperature.
P r ep a r a tion of BtO-P h e-2-ch lor otr ityl r esin . 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 and they
were all added to the resin and the mixture was stand 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.
Syn th esis of P ip er a zin e-P h e-2-ch lor otr ityl r esin . BtO-
Phe-resin (500 mg, 0.8 mmol/g) was swelled and saturated with
DMF. Piperazine (86 mg, 1 mmol), dissolved in the minimum
volume of DMF, and DIEA (0.4 mL) were added to the above
resin and the mixture was shaken overnight. 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.
Syn th esis of F m oc-ꢀAh x-p ip er a zin e-P h e-2-ch lor otr ityl
r esin . Piperazine-Phe-resin (0.4 mmol of compound theoreti-
cal) 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.
Solid -P h a se P ep tid e Syn th esis. The synthesis of precyclic
thrombin receptor-derived peptides H-Leu-Arg-ꢀLys-Dap-Phe-
Leu-OH and H-Leu-Arg-ꢀLys-Dap-D-Phe-Leu-OH was accom-
plished by the Fmoc methodology utilizing the 2-chlorotrityl
chloride resin as solid support as reported by Barlos et al.18
Attachment of the first N-Fmoc-amino acid (Leu) to the resin
(1.4-1.6 mequiv Cl-/g resin) was achieved by a simple, fast
and racemization-free reaction using DIPEA in DMF solution
at room temperature. The loading of the first amino acid per
gram of substituted resin was calculated by weight and by a
quantitative photometric Kaiser test. Solid-phase peptide
synthesis was achieved on the 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. 2.5 equiv
of Fmoc-protected amino acid was preactivated with DCC in
the presence of HOBt at 0 °C in each coupling step. The
solution was filtered to remove the remaining DCU and poured
into the vessel. The coupling was maintained for 150 min and
completion of the coupling was verified by the ninhydrin
(Kaizer) test and TLC. A second coupling under the same
conditions was employed in cases of incomplete coupling. The
Fmoc protective groups were removed by incubation in 20%
piperidine in DMF (v/v), for 30 min. The amino acids used in
Fmoc synthesis were: Fmoc-Leu-OH, Fmoc-Phe-OH (or Fmoc-
D-Phe-OH), NR-Boc-Dap(Nâ-Fmoc)-OH, NR-Boc-Lys(Nꢀ-Fmoc)-
OH and NR-Fmoc-Arg(Nω/ω′-Pmc)-OH. After the final coupling-
deprotection step, the obtained peptide resin was dried in
vacuo and then treated with the splitting mixture DCM/AcOH/
TFE (7:1:2, v/v/v; 15 mL/g resin) for 45 min at room temper-
ature to remove the peptide from the resin. The mixture was
filtered off and the resin washed with the splitting mixture
and DCM several times. The solvent was removed on a rotary
evaporator and the obtained oily product precipitated from cold
diethyl ether. The protected precyclic peptide was neutralized
with TEA in DMF and cyclized using TBTU as cyclization
reagent in the presence of DIPEA in DMF solution (10-3 M).
The cyclization process was easily monitored by the ninhydrin
test of the synthesized peptide resolved by TLC. The success-
fully cyclized products were ninhydrin test negative and final
products were identified by FAB-MS and NMR spectroscopy.
Cycliza tion P r oced u r e. P r ep a r a tion of Cyclo(Leu -Ar g-
(P m c)-ꢀLys(Boc)-Da p (Boc)-P h e-Leu ) (1a ). To a solution of
the linear precyclic pentapeptide H-Leu-Arg(Pmc)-ꢀLys(Boc)-
Dap(Boc)-Phe-Leu-OH (0.3 mmol) in DMF (3 mL) was added
TEA (0.3 mmol) and the solution was stirred for 5 min at room
temperature. The neutralized linear protected hexapeptide was
triturated with H2O, filtered off, washed with H2O (2 × 10
mL) and diethyl ether (2 × 10 mL) and dried in vacuo for 16
h. The neutralized linear protected peptide (0.3 mmol) was
dissolved in DMF (15 mL) and added dropwise to a solution
of TBTU (0.9 mmol) and DIPEA (0.3 mL, 1.8 mmol) in DMF
(170 mL) at room temperature, over 45 min. The cyclization
was completed in an additional 30 min, as determined by TLC
and analytical reversed-phase HPLC. The solvent was removed
under reduced pressure affording a light yellow oily residue.
The cyclic protected hexapeptide 1a was precipitated from H2O
and dried in vacuo for 16 h.
(S)-N-(6-Gu a n id oh exa n oyl)-N′-(2-a m in o-3-p h en ylp r o-
p ion yl)p ip er a zin e (3). Fmoc-ꢀAhx-piperazine-Phe-resin (0.4
mmol of compound theoretical) was swelled and saturated with
DMF and then was treated twice with 20% piperidine in DMF
(20 min, 30 min) at room temperature. After removal of excess
reagent the H-ꢀAhx-piperazine-Phe-resin was washed liberally
with DMF and treated with 1H-pyrazole-1-carboxamide hy-
drochloride (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 which was then
triturated with dry ether. The crude product was purified by
preparative HPLC as described below to yield 33 mg (0.05
mmol, 13% overall yield) of a waxy white solid product: Rf
1
0.47 (4:1:1, ButOH:H2O:AcOH); H NMR (DMSO-d6) δ 1.3 (t,
2H), 1.5 (m, 4H), 2.3 (m, 2H), 2.6 (m, 2H), 3.1-3.6 (m, 8H),
3.1 (m, 2H), 3.5 (t, 1H), 6.9-7.4 (brs, 4H), 7.2-7.3 (m, 5H),
7.5 (brs, 1H), 8.3 (brs, 3H); MS/FAB 389 [(M + 1)+ - 2TFA].
P r ep a r a tion of Cyclo(Leu -Ar g-ꢀLys-Da p -P h e-Leu ) ≡
Cyclo(P h e-Leu -Leu -Ar g-ꢀLys-Da p ) (1). The cyclic protected
hexapeptide 1a was treated with 75% TFA in DCM (1 mL/
100 mg peptide) for 4 h at room temperature. The resulting
Syn th esis of N-(6-Gu a n id oh exa n oyl)-4-(4-flu or op h en -
ylm eth yla m id oa cetyl)p ip er id in e (4). N-(ter t-Bu toxyca r -
bon yl)-4-p ip er id in eca r boxylic Acid . A solution of isonipe-
cotic acid (2.6 g, 20 mmol) in a mixture of dioxane (40 mL),

Thrombin Receptor-Derived Peptide Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 337
water (20 mL) and 1 N NaOH (20 mL) was stirred and cooled
in an ice-water bath. Di-tert-butyl pyrocarbonate (4.7 g, 21
mmol) was added and stirring was continued at room tem-
perature for 3 h. The solution was concentrated in vacuo to
about 10-15 mL, cooled in an ice-water bath, covered with a
layer of ethyl acetate (30 mL) and acidified with a dilute
solution of HCl to pH 3-4. The aqueous phase was extracted
with ethyl acetate (2 × 15 mL) and the extracts were pooled,
washed with water (2 × 30 mL), dried over anhydrous Na2-
SO4 and evaporated in vacuo. The crude product was purified
by recrystallization from EtOAc/hexane to give 3.15 g (13.8
mmol, 69%) of a white solid: mp 115-116 °C; Rf 0.58 (10%
MeOH/CHCl3); 1H NMR (DMSO-d6) δ 1.4 (s, 9H), 1.7-1.9 (m,
4H), 2.4 (m, 1H), 2.8 (m, 2H), 3.4 (m, 2H); MS (nominal) 229
(M+), 156 (M+ - (CH3)3CO•); MS (nominal) 229 (M+).
Then, dry ether (10 mL) was added and the product appeared
as gel at the bottom of the flask, while the supernatant liquid
was decanted. The crude product was purified by recrystalli-
zation from MeOH/Acetone/Et2O to give 180 mg (0.46 mmol,
47%) of a waxy white solid: Rf 0.48 (4:1:1, ButOH:H2O:AcOH);
1H NMR (DMSO-d6) δ 1.3-1.6 (m, 6H), 1.4 (m, 2H), 1.7 (m,
2H), 2.3 (t, 2H), 2.4 (m, 1H), 2.7 (dt, 2H), 3.1 (m, 2H), 4.2 (d,
2H), 4.2 (dd, 2H), 6.8-7.5 (brs, 3H), 7.1-7.3 (m, 4H), 7.7 (brs,
1H), 8.4 (t, 1H); MS/FAB 392 (M+ + 1).
Syn th esis of N-P h en yla cetyl-N′-(6-gu a n id oh exa n oyl)-
1,4-d ia m in obu ta n e (5). N-P h en yla cetyl-1,4-d ia m in obu -
ta n e. Phenylacetic acid (820 mg, 6.0 mmol) and HOBt (840
mg, 6.2 mmol) were suspended in CHCl3 (20 mL). DCC (1.28
g, 6.2 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
precold solution of 1,4-diaminobutane (2.64 g, 30 mmol) in 40
mL CHCl3. The reaction was stirred for 3 h at room temper-
ature and then the white suspension (DCU) was filtered and
the filtrate acidified with 2 M HCl (2 × 40 mL). The HCl
extracts were basified to pH ) 9 with 2 M NaOH, extracted
with CHCl3 (4 × 30 mL), dried over anhydrous Na2SO4 and
reduced in vacuo to give a pale orange solid as a crude product.
The crude product was purified by recrystallization from
EtOAc/hexane to give 660 mg (3.2 mmol, 53%) of a white
solid: mp 110-113 °C; Rf 0.45 (4:1:1, ButOH:H2O:AcOH); 1H
NMR (DMSO-d6) δ 1.4 (m, 4H), 2.5 (t, 2H), 3.0 (q, 2H), 3.4 (s,
2H), 7.1-7.3 (m, 5H), 8.0 (brs, 1H); MS (nominal) 206 (M+),
N -(t er t -Bu t oxyca r b on yl)-4-(4-flu or op h e n ylm e t h yl-
a m id oa cetyl)p ip er id in e. N-(tert-Butoxycarbonyl)-4-piperi-
dinecarboxylic acid (1.5 g, 6.5 mmol), 4-fluorobenzylamine (810
mg, 6.5 mmol), HOBt (900 mg, 6.7 mmol) and DIEA (2 mL)
were dissolved in CH2Cl2 (20 mL). The mixture was cooled to
0 °C and DCC (1.4 g, 6.8 mmol) was added. The reaction
solution was stirred at this temperature for ∼30 min and then
at room temperature for 3 h. The suspension (DCU) was
filtered and washed with 50 mL CH2Cl2. The filtrate was
washed with 8% citric acid (2 × 40 mL), 50% saturated
NaHCO3 solution (2 × 40 mL) and H2O (2 × 40 mL), dried
over anhydrous Na2SO4 and concentrated in vacuo to give a
gray solid as a crude product. Flash chromatography (50%
EtOAc/hexane) yielded a white solid (1.55 g, 4.6 mmol, 71%):
177 (M+ - H2NCH•), 115 (M+ - C6H5CH2 ).
•
1
mp 92-93 °C; Rf 0.80 (10% MeOH/CHCl3); H NMR (DMSO-
N-P h en yla cetyl-N′-[6-(ter t-bu toxyca r bon yla m in o)h ex-
a n oyl]-1,4-d ia m in obu ta n e. N-Phenylacetyl-1,4-diaminobu-
tane (600 mg, 2.9 mmol), Boc-ꢀAhx-OH (700 mg, 3.0 mmol),
HOBt (420 mg, 3.1 mmol) and DIEA (1 mL) were dissolved in
20 mL of a (3:1) CH2Cl2/DMF solution. The mixture was cooled
to 0 °C and DCC (640 mg, 3.1 mmol) added. The reaction was
stirred at this temperature for ∼30 min and then at room
temperature for 6 h. The suspension (DCU) was filtered and
washed with 25 mL CH2Cl2. The filtrate was dried over Na2-
SO4 and concentrated in vacuo to give an orange-yellow solid
as a crude product. Flash chromatography (3% MeOH/CHCI3)
yielded a white solid (900 mg, 2.1 mmol, 75%): mp 125-126
d6) δ 1.4 (s, 9H), 1.4 (m, 2H), 1.7 (m, 2H), 2.3 (m, 1H), 2.7 (brt,
2H), 3.9 (d, 2H), 4.2 (d, 2H), 7.1-7.3 (m, 4H), 8.3 (t, 1H); MS
(nominal) 336 (M+).
4-(4-F lu or op h en ylm eth yla m id oa cetyl)p ip er id in e Tr i-
flu or oa cetic Sa lt. The above Boc derivative (2.4 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 white solid (2.1 mmol, 89%): mp 120-121 °C; Rf 0.07
1
(10% MeOH/CHCl3); H NMR (DMSO-d6) δ 1.7-1.9 (m, 4H),
2.4 (m, 1H), 2.9 (dt, 2H), 3.3 (dd, 2H), 4.2 (d, 2H), 7.1-7.3 (m,
4H), 8.4 (t, 1H), 8.6 (brs, 2H); MS (nominal) 236 (M+ - TFA).
°C; Rf 0.83 (4:1:1, ButOH:H2O:AcOH); H NMR (DMSO-d6) δ
1
1.2 (m, 2H), 1.4 (s, 9H), 1.4 (m, 4H), 1.5 (m, 4H), 2.0 (t, 2H),
2.9 (q, 2H), 3.0 (m, 2H), 3.0 (m, 2H), 3.4 (s, 2H), 6.7 (t, 1H),
7.2-7.3 (m, 5H), 7.7 (t, 1H), 8.0 (t, 1H); MS (nominal) 419 (M+),
345 (M+ - (CH3)3CO• + 1).
N-[6-(ter t-Bu t oxyca r b on yla m in o)h exa n oyl]-4-(4-flu o-
r op h en ylm eth yla m id oa cetyl)p ip er id in e. The above salt
(600 mg, 1.9 mmol), Boc-ꢀAhx-OH (460 mg, 2.1 mmol), HOBt
(290 mg, 2.1 mmol) and DIEA (1.5 mL) were dissolved in 18
mL of a 5:1 CH2Cl2/DMF solution. The mixture was cooled to
0 °C and DCC (460 mg, 2.2 mmol) was added. The reaction
was monitored by TLC (5% MeOH:CHCl3) at room tempera-
ture and showed completion after 24 h. The supernatant liquid
was filtered in vacuo, dried over anhydrous Na2SO4 and
concentrated in vacuo to give a gray solid as a crude product.
Flash chromatography (2% MeOH/CHCI3) yielded a white solid
(500 mg, 1.1 mmol, 60%): mp 106-109 °C; Rf 0.57 (10%
MeOH/CHCI3); 1H NMR (DMSO-d6) δ 1.2 (m, 2H), 1.3 (s, 9H),
1.3-1.5 (m, 4H), 1.4 (m, 2H), 1.6 (m, 2H), 2.2 (t, 2H), 2.4 (m,
1H), 2.7 (dt, 2H), 2.9 (m, 2H), 4.2 (d, 2H), 4.2 (dd, 2H), 6.7
(brt, 1H), 7.1-7.3 (m, 4H), 8.3 (t, 1H); MS (nominal) 449 (M+),
376 (M+ - (CH3)3CO•).
N-(6-Am in oh exa n oyl)-4-(4-flu or op h en ylm eth yla m id o-
a cetyl)p ip er id in e Tr iflu or oa cetic Sa lt. The Boc group
removal took place according to general procedure for Boc
deprotection with trifluoroacetic acid. A waxy white solid
resulted (1.00 mmol, 95%): Rf 0.28 (4:1:1, ButOH:H2O:AcOH);
1H NMR (DMSO-d6) δ 1.3-1.6 (m, 6H), 1.4 (m, 2H), 1.7 (m,
2H), 2.2 (t, 2H), 2.4 (m, 1H), 2.7 (dt, 2H), 2.8 (m, 2H), 4.2 (d,
2H), 4.2 (dd, 2H), 7.1-7.3 (m, 4H), 7.7 (brs, 3H), 8.3 (t, 1H);
MS (nominal) 340 (M+ - TFA).
N-P h en yla cetyl-N′-(6-a m in oh exa n oyl)-1,4-d ia m in obu -
ta n e Tr iflu or oa cetic Sa lt. The above Boc analogue (2.0
mmol) was dissolved in 4 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 white solid (1.8 mmol, 91%): mp 107-
1
109 °C; Rf 0.36 (4:1:1, ButOH:H2O:AcOH); H NMR (DMSO-
d6) δ 1.3 (m, 2H), 1.4 (m, 4H), 1.5 (m, 4H), 2.0 (t, 2H), 2.8 (m,
2H), 3.0 (m, 2H), 3.0 (m, 2H), 3.4 (s, 2H), 7.2-7.3 (m, 5H), 7.7
(t, 1H), 8.0 (t, 1H); MS (nominal) 319 (M+ - TFA), 261 (M+
-
•
TFA - H2NCH2CH2CH2 ).
N-P h en yla cetyl-N′-(6-gu a n id oh exa n oyl)-1,4-d ia m in o-
bu ta n e (5). Following the guanylation procedure of synthesis
of compound 4 a white solid of compound 5 was obtained at
58% yield: mp 101-103 °C; Rf 0.44 (4:1:1, ButOH:H2O:AcOH);
1H NMR (DMSO-d6) δ 1.2 (m, 2H), 1.4 (m, 4H), 1.5 (m, 4H),
2.0 (t, 2H), 3.0 (m, 2H), 3.0 (m, 2H), 3.1 (m, 2H), 3.4 (s, 2H),
6.9-7.3 (brs, 3H), 7.0-7.3 (m, 5H), 7.6 (brt, 1H), 7.8 (t, 1H),
8.0 (t, 1H); MS/FAB 362 (M+ + 1).
Syn t h esis of N-(4-F lu or op h en yl)a cet yl-N′-(6-gu a n -
id oh exa n oyl)-1,4-d ia m in obu ta n e (6). N-(4-F lu or op h en -
yl)a cetyl-1,4-d ia m in obu ta n e. The synthesis was similar to
the one applied for N-phenylacetyl-1,4-diaminobutane, except
that 4-fluorophenylacetic acid was used instead of phenylacetic
acid. The crude product was purified by recrystallization from
EtOAc/hexane giving a white solid at 58% yield: mp 109-
N -(6-G u a n id o h e x a n o y l)-4-(4-flu o r o p h e n y lm e t h y l-
a m id oa cetyl)p ip er id in e (4). TFA salt (450 mg, 0.97 mmol),
1H-pyrazole-1-carboxamide hydrochloride (220 mg, 1.5 mmol)
and DIEA (0.7 mL) were dissolved in 2 mL DMF. The mixture
was stirred under nitrogen for ∼24 h at room temperature.
1
114 °C; Rf 0.46 (4:1:1, ButOH:H2O:AcOH); H NMR (DMSO-

338 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Alexopoulos et al.
d6) δ 1.4 (m, 4H), 2.5 (t, 2H), 3.0 (q, 2H), 3.4 (s, 2H), 7.0-7.3
(m, 4H), 8.0 (brs, 1H); MS (nominal) 224 (M+), 195 (M+ - H2-
NCH•).
ring was connected to a Grass FT 03 isometric force transducer
and stretched at a basal tension of 2 g. The contractile activity
of the aortic rings was recorded with a Grass polygraph, model
79, and with a CODAS DATAQ DI-120 computer-based system
(4/s sampling rate). The presence of the endothelium 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
absent if no relaxation was observed.
N-(4-F lu or op h en yl)a cetyl-N′-[6-(ter t-bu toxyca r bon yl-
a m in o)h exa n oyl]-1,4-d ia m in obu ta n e. The synthetic pro-
cedure was similar to the one applied for the nonfluorinated
analogue and gave a white solid at 40% yield: mp 128-129
1
°C; Rf 0.85 (4:1:1, ButOH:H2O:AcOH); H NMR (DMSO-d6) δ
1.3 (m, 2H), 1.4 (s, 9H), 1.4 (m, 4H), 1.5 (m, 4H), 2.0 (t, 2H),
2.9 (q, 2H), 3.0 (m, 2H), 3.0 (m, 2H), 3.4 (s, 2H), 6.7 (t, 1H),
7.0-7.3 (m, 4H), 7.7 (t, 1H), 8.0 (t, 1H); MS (nominal) 437 (M+).
The action of the cyclic and nonpeptide thrombin receptor
mimetics, in aortic smooth muscle and at concentrations of
0.1 nM-0.5 mM, was studied in preparations precontracted
with 1 µM phenylephrine. The administrations of the thrombin
receptor mimetics were separated by 30-min periods of normal
perfusate administration. The relaxing action of the three
thrombin receptor mimetics was also measured after pretreat-
ment with the competitive inhibitor of nitric oxide synthase
L-NAME, 300 µM. All results are expressed as means ( SEM.
The relaxing action of thrombin is expressed as percentage
decrease of the contraction induced by 1 µM phenylephrine.
The dose-response curves for both compounds were analyzed
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 values of compounds 3-6 were
calculated graphically from their dose-response curves.
NMR Exp er im en ts. NMR experiments were carried out
using a Bruker 400-MHz NMR spectrometer. Three milligrams
of thrombin receptor analogue were dissolved in 0.33 mL of
DMSO-d6. The chemical shifts were reported relative to the
undeuterated fraction of the methyl group of DMSO-d6 at 2.50
ppm with respect to TMS. 1D-Spectra were recorded with a
sweep width of 4600 Hz and 32 K (zero-filled to 64 K) data
points and by methods previously described.35-39 1D-NOE
experiments were carried out in the difference mode using
multiple irradiation. The methods used for the TOCSY and
NOE experiments were similar to those previously
described.37-39
N-(4-Flu or op h en yl)a cetyl-N′-(6-a m in oh exa n oyl)-1,4-d i-
a m in obu ta n e Tr iflu or oa cetic Sa lt. Following the general
procedure for Boc deprotection with trifluoroacetic acid, a
white solid was afforded at 93% yield: mp 122-124 °C; Rf 0.38
(4:1:1, ButOH:H2O:AcOH); 1H NMR (DMSO-d6) δ 1.3 (m, 2H),
1.4 (m, 4H), 1.5 (m, 4H), 2.0 (t, 2H), 2.8 (t, 2H), 3.0 (m, 2H),
3.0 (m, 2H), 3.4 (brs, 3H), 3.5 (s, 2H), 7.0-7.3 (m, 4H), 7.8 (t,
1H), 8.0 (t, 1H); MS (nominal) 337 (M+ - TFA), 279 (M+
-
•
TFA - H2NCH2CH2CH2 ).
N-(4-F lu or op h en yl)a cetyl-N′-(6-gu a n id oh exa n oyl)-1,4-
d ia m in obu ta n e (6). Guanylation of the above TFA salt took
place by a procedure analogous to the one applied for the
synthesis of compound 5 and gave a white solid at 61% yield:
1
mp 119-122 °C; Rf 0.46 (4:1:1, ButOH:H2O:AcOH); H NMR
(DMSO-d6) δ 1.3 (m, 2H), 1.4 (m, 4H), 1.5 (m, 4H), 2.0 (t, 2H),
3.0 (m, 2H), 3.0 (m, 2H), 3.1 (m, 2H), 3.4 (s, 2H), 6.9-7.3 (brs,
3H), 7.0-7.3 (m, 4H), 7.6 (brt, 1H), 7.8 (t, 1H), 8.0 (t, 1H); MS
(FAB) 380 (M+ + 1).
P r ep a r a tive Rever sed -P h a se HP LC. Compounds were
purified on a Waters HPLC system equipped with a 600E
system controller and using a Lichrosorb RP-18 reversed-phase
preparative column (250 × 10 mm) with 7-µm packing mate-
rial. Separations were achieved with a stepped linear gradient
of acetonitrile in 0,1% aqueous TFA at a flow rate of 3 mL/
min. The crude peptide material (18 mg) was dissolved in
water (450 mL), clarified by centrifugation and the solution
was injected through a Rheodyne 7125 injector with 500-µL
sample loop. The mobile phases used were A: 0.1% aqueous
TFA and B: 0.1% TFA in acetonitrile, with a linear gradient
from 20%B to 65%B over 60 min (3 mL/min). Fractions were
manually collected at 0.5-min intervals and the elution time
of the major product was 25-30 min. Elution of the peptide
was determined simultaneously from the absorbances at 220,
230, 257 and 280 nm (Waters 996 photodiode array detector).
Fractions determined to be pure (214 nm) by analytical
reversed-phase HPLC (Techsil C18, 250 × 4.6 mm) were
combined and acetonitrile was removed on a rotary evaporator.
After lyophilization the product was stored at -20 °C.
Am in o Acid An a lysis, TLC, a n d F AB-MS. Amino acid
analysis was performed on Beckman G300 high-performance
analyzer. Compositional analysis data were obtained from 6
M HCl hydrosylates (150 °C, 1 h). The purity of products was
established by analytical HPLC reruns and by thin-layer
chromatography (TLC). Two solvent systems were used, 1-bu-
tanol-acetic acid-water (4:1:1) (BAW) and chloroform-
methanol-acetic acid (70:15:15) (CMA). The identity of the
desired products was established by FAB-MS using conditions
previously described.34 FAB spectra were run on a AEI M29
mass spectrometer. 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).
Molecu la r Mod elin g Meth od s. Theoretical calculations
were performed as described previously39,40 using a Silicon
Graphics O2 workstation and Quanta version of MSI. Thus,
structures of peptides SFLLR-NH2 and cyclo(FLLR-ꢀK-Dap)
were built using the Sequence Builder while peptide mimetic
structures used the Molecular Editor. The built structures
were energy-minimized under distance constraints obtained
by 1D-NOE spectroscopy and using the algorithms embedded
in the software. Finally, dynamics was applied to choose the
lowest-energy structures that are compatible with NMR data.
Molecular dynamics was performed at 500 K using 1-, 2-, and
2-ps time frame for heating, equilibration, and simulation
steps. The final structures were superimposed using Rigid
Body that allows only translation and rotation of the molecules
and not changes in their conformations.39,40
Ack n ow led gm en t. This work was supported by
European Community (EC) grants (BIOMED Pro-
gramme No. 920038, BIOMED-PECO No. 930158, CO-
PERNICUS No. 940238), the Ministry of Energy and
Technology of Greece (EΠET II, 115), and the Ministry
of Education (EPEAK). Part of the bioassays were
performed by Dr. Stefan Mihailescu in the Department
of Physiology, School of Medicine, UNAM, Mexico.
Bioa ssa y P r oced u r es. The experimental technique was
described extensively by Paredes et al.16 Briefly, rings of 2 mm
width were obtained from the central part of the thoracic aorta
of adult male Wistar rats. 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
Tyrode buffer (composition in mM: 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 main-
tained at a pH of 7.44 by bubbling with carbogene. Each aortic
Refer en ces
(1) Bar-Shavit, R.; Kahn, A.; Wilner, G.; Fenton, J . Monocyte
chemotaxis: Stimulation by specific exocite region in thrombin.
Science 1983, 220, 728-731.
(2) Fenton, J . Regulation of thrombin generation and functions.
Semin. Thromb. Hemostasis 1988, 14, 234-240.
(3) Vu, T.-K.; Hung, D.; Wheaton, V.; Coughlin, S. Molecular cloning
of a functional thrombin receptor reveals a novel proteolytic
mechanism of receptor activation. Cell 1991, 64, 1057-1068.

Thrombin Receptor-Derived Peptide Analogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 339
(4) Rasmussen, U.; Vouret-Graviari, V.; J allat, 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 Ca2+ mobilization. FEBS
Lett. 1991, 288, 123-128.
group and of a cyclic conformation for induction of experimental
allergic encephalomyelitis. J . Med. Chem. 1999, 42, 1170-1177.
(22) Bernatowicz, M.; Wu, Y.; Matsueda, G. 1H-Pyrazole-1-carbox-
amidine hydrochloride: an attractive reagent for guanylation
of amines and its application to peptide synthesis. J . Org. Chem.
1992, 57, 2497-2502.
(23) Fujita, T.; Nose, T.; Matsushima, A.; Okada, K.; Asai, D.;
Yamauchi, Y.; Shirasu, N.; Honda, T.; Shigehiro, D.; Shimohi-
gashi, Y. Synthesis of a complete set of L-difluorophenylalanines,
L-(F2)Phe, as molecular explorers of the CH/π interaction
between peptide ligand and receptor. Tetrahedron Lett. 2000,
41, 923-927.
(24) 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.
(25) Mathews, I.; Padmanabhan, K.; Ganesh, V.; Tulinsky, A.; Ishii,
M.; Chen, J .; Turck, C.; Coughlin, S.; Fenton, J . 2nd. Crystal-
lographic structures of thrombin complex with thrombin receptor
peptides: existence of expected and novel binding modes.
Biochemistry 1994, 33, 3266-3279.
(26) Smith, K. Trayer, I.; Grand, R. Structure around the cleavage
site in the thrombin receptor determined by NMR spectroscopy.
Biochemistry 1994, 33, 6063-6073.
(27) Bouton, M.; J androt-Perrus, M.; Moog, S.; Cazenave, J .; Guill,
M.; Lanza, F. Thrombin interaction with a recombinant N-
terminal extracellular domain of the thrombin receptor in an
accellular system. Biochem. J . 1995, 305, 635-641.
(5) Coughlin, S. Thrombin receptor structure and function. Thromb.
Haemostasis 1993, 70, 184-187.
(6) Brass, L.; Ahuja, M.; Belmonte, E.; Pizarro, S.; Tarver, A.; Hoxie,
J . The human platelet thrombin receptor. Turning it on and
turning it off. Ann. N. Y. Acad. Sci. 1994, 714, 1-12.
(7) Yang, S.-G.; Laniyonu, A.; Saifeddine, M.; Moore, G.; Hollenberg,
M. Actions of thrombin and thrombin receptor peptide analogues
in gastric and aortic smooth muscle: development of bioassays
for structure activity studies. Life Sci. 1992, 51, 1325-1332.
(8) Hollenberg, M.; Yang, S.-G.; Laniyonu, A.; Moore, G.; Saifeddine,
M. Actions of thrombin receptor polypeptide in gastric smooth
muscle: identification of a core pentapeptide retaining full
thrombin-mimetic intrinsic activity. Mol. Pharmacol. 1992, 42,
186-191.
(9) Hollenberg, M.; Laniyonu, A.; Saifeddine, M.; Moore, G. Role of
the amino- and carboxyl-terminal domains of thrombin receptor-
derived polypeptides in biological activity in vascular endothe-
lium and gastric smooth muscle: Evidence for receptor subtypes.
Mol. Pharmacol. 1993, 43, 921-930.
(10) Laniyonu, A.; Hollenberg, M. Vascular actions of thrombin
receptor-derived polypeptides: structure-activity profiles for
contractile and relaxant effects in rat aorta. Br. J . Pharmacol.
1995, 114, 1680-1686.
(11) 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.
(12) 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.
(13) 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.
(14) 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.
(15) 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.
(16) Paredes-Carbajal, M.; J uarez-Oropeza, M.; Ortiz-Mendoza, C.;
Mascher, D. Effects of acute and chronic estrogenic treatment
on vasomotor responses of aortic rings from ovariectomized rats.
Life Sci. 1995, 57, 474.
(17) Matsoukas, J .; Agelis, G.; Hondrelis, J .; Yamdagni, R.; Wu, Q.;
Ganter, R.; Smith, J .; Moore, D.; Moore, G. Synthesis and
biological activities of angiotensin II, sarilesin and samersin
analogues containing Aze or Pip at position 7. J . Med. Chem.
1993, 33, 904-911.
(28) Alexopoulos, K.; Matsoukas, J .; Tselios, T.; Roumelioti, P.;
Holada, K. A comparative SAR study of thrombin receptor
derived nonpeptide mimetics: Importance of Phenyl/Guanidino
proximity for activity. Amino Acids 1998, 15, 211-220.
(29) Alexopoulos, K.; Fatseas, P.; Melissari, E.; Vlahakos, D.; Smith,
J .; Mavromoustakos, T.; Saifeddine, M.; Moore, G.; Hollenberg
M.; Matsoukas, J . Design and synthesis of Thrombin receptor-
derived non-peptide mimetics utilizing a piperazine scaffold.
Bioorg. Med. Chem. 1999, 7, 1033-1041.
(30) Moore, G.; Smith, J .; Baylis, B.; Matsoukas, J . Design and
pharmacology of peptide mimetics. Adv. Pharmacol. (San Diego)
1995, 6, 91-141.
(31) Giannis, A.; Rubsam, F. Peptidomimetics in drug design. Adv.
Drug Res. 1997, 29, 1-78.
(32) Adang, A.; Hermkens, P.; Linders, J .; Ottenheijm, H.; Staveren,
C. Case histories of peptidomimetics: Progression from peptides
to drugs. J . R. Neth. Chem. Soc. 1994, 113, 63-78.
(33) Fugita, T.; Nose, T.; Nakajima, M.; Inoue, Y.; Casta, T.; Shimo-
higashi, Y. Design and synthesis of para-fluorophenylalanine
amide derivatives as thrombin receptor antagonists. J . Biochem.
1999, 1, 174-179.
(34) Hogg, A. Conversion of mass spectrometers for fast-atom bom-
bardment using easily constructed components. Int. J . Mass
Spectrom. Ion Phys. 1983, 49, 24-34.
(35) Otter, A.; Scot, P.; Kotovych, G. Type I collagene alpha-1 chain
c-teleopeptide: solution structure determined by 600 MHz proton
NMR spectroscopy and implications for its role in collagen
fibrillogenesis. Biochemistry 1988, 27, 3560-3567.
(36) Wuthrich, K. NMR of proteins and nucleic acids; J ohn Willey
and Sons: New York, 1983; pp 117-129.
(37) Matsoukas, J .; Bigham, G.; Zhou, N.; Moore, G. 1NMR studies
of [Des1]angiotensin II conformation by nuclear Overhauser
effect spectroscopy in the rotating frame (ROESY): clustering
of the aromatic rings in dimethyl sulfoxide. Peptides 1990, 11,
359-366.
(18) Barlos, K.; Gatos, D.; Hondrelis, J .; Matsoukas, J .; Moore, G.;
Schafer, W.; Sotiriou, P. Preparation of a new acid-labile resins
of sec-alcohol type and their application in peptide synthesis.
Liebigs Ann. Chem. 1989, 951-955.
(19) Matsoukas, J .; Hondrelis, J .; Agelis, G.; Barlos, K.; Gatos, D.;
Ganter, R.; Moore, D.; Moore, G. Novel synthesis of cyclic amide-
linked analogues of Angiotensins II and III. J . Med. Chem. 1994,
37, 2958-2969.
(20) Panagiotopoulos, D.; Matsoukas, J .; Alexopoulos, K.; Zebeki, A.;
Mavromoustakos, T.; Saifeddine, M.; Hollenberg, M. Synthesis
and activities of cyclic thrombin-receptor-derived peptide ana-
logues of the Ser42-Phe-Leu-Leu-Arg46 motif sequence containing
D-Phe and/or D-Arg. LiPS 1996, 3, 233-240.
(21) Tselios, T.; Probert, L.; Daliani, I.; Matsoukas, E.; Troganis, A.;
Gerothanassis, I.; Mavromoustakos, T.; Moore, G.; Matsoukas,
J . Design and synthesis of a potent cyclic analogue of the myelin
basic protein epitope MBP72-85: Importance of the Ala81 carboxyl
(38) Matsoukas, J .; Yamdagni, R.; Moore, G. 1NMR studies of
sarmersin and [Des1]sarmesin conformation in dimethyl sulfox-
ide by the N- and C-terminal domains. Peptides 1990, 11, 367-
374.
(39) Matsoukas, J .; Hondrelis, J .; Keramida, M.; Mavromoustakos,
T.; Makriyiannis, A.; Yamdagni, R.; Wu, Q.; Moore, G. Role of
the NH2-terminal domain of angiotensin II and [Sar1]angiotensin
II on conformation and activity: NMR evidence for aromatic ring
clustering and peptide backbone-folding compared to [Des1,2,3]-
angiotensin II. J . Biol. Chem. 1994, 269, 5303-5312.
(40) Mavromoustakos, T.; De-Ping, Y.; Theodoropoulou, E.; Makriy-
iannis, A. Studies of the conformational properties of the
cannabimimetic-aminoalkylindole pravadoline using NMR and
molecular modeling. Eur. J . Med. Chem. 1995, 30, 227-234.
J M0001525