Development of potent thrombin receptor antagonist peptides

Article abstract of DOI:10.1021/jm960455s

A peptide-based structure-activity study is reported leading to the discovery of novel potent thrombin receptor antagonists. Systematic substitution of nonproteogenic amine acids for the second and third residues of the human thrombin receptor 'tethered ligand' sequence (SFLLR) led to a series of agonists with enhanced potency. The most potent pentapeptide agonist identified was Ser-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH₂, 9 (EC₅₀ ~ 0.04 μM for stimulation of human platelet aggregation, ~10-fold more potent than the natural pentapeptide). Systematic substitution of the NH₂- terminal Ser in 9 with neutral hydrophobic NH₂-acyl groups led to partial agonists and eventually antagonists with unprecedented potency (greater than 1000-fold increase over the previously reported antagonist 3- mercaptopropionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-NH₂). In the series of NH₂-acyl tetrapeptide antagonists, N-transcinnamoyl-p-fluoroPhe-p- guanidinoPhe-Leu-Arg-NH₂, 41 (BMS-197525), was identified as the tightest binding (IC₅₀ ~ 8 nM) and most potent with an IC₅₀ ~ 0.20 μM for inhibition of SFLLRNP-NH₂-stimulated platelet aggregation. Systematic single substitutions in 41 indicated that, in addition to the NH₂-terminal acyl group, the side chains at the second and third positions were also responsible for important and specific receptor interactions. The p-fluoroPhe and p-guanidinoPhe residues in the second and third positions of 41 were observed to be optimal in both the agonist and antagonist series. In the case of antagonists, however, an appropriately positioned positively charged group (i.e., protonated base) at the third residue was required. In contrast, such a substitution was not required for potent agonist activity. An even more potent antagonist resulted when 41 was extended at the C-terminus by a single Arg residue giving rise to analog 90 (BMS-200261) which had an IC₅₀ ~ 20 nM for inhibition of SFLLRNP-NH₂-stimulated platelet aggregation. When the C-terminal Arg of 90 was replaced by an Orn-(N(δ)-propionyl) residue, the resulting antagonist 91 (BMS-200661) was suitable for use in radioligand binding assays (K(d) = 10-30 nM). Antagonist activity observed for selected compounds was verified through secondary assays in that these analogs prevented SFLLRNP-NH₂-stimulated GTPase activity in platelet membranes and Ca²⁺ mobilization in cultured human smooth muscle cells and mouse fibroblasts. Furthermore, this inhibition occurred at concentrations that had no effect on thrombin catalytic activity indicating a specific activity attributable to receptor binding and not enzyme inhibition.

Full text of DOI:10.1021/jm960455s

J . Med. Chem. 1996, 39, 4879-4887
4879
Develop m en t of P oten t Th r om bin Recep tor An ta gon ist P ep tid es
,
†
†
‡
‡
‡
Michael S. Bernatowicz,* Clifford E. Klimas, Karen S. Hartl, Marianne Peluso, Nick J . Allegretto, and
Steven M. Seiler
‡
Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New J ersey 08543
X
Received J une 24, 1996
A peptide-based structure-activity study is reported leading to the discovery of novel potent
thrombin receptor antagonists. Systematic substitution of nonproteogenic amino acids for the
second and third residues of the human thrombin receptor “tethered ligand” sequence (SFLLR)
led to a series of agonists with enhanced potency. The most potent pentapeptide agonist
identified was Ser-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH
of human platelet aggregation, ∼10-fold more potent than the natural pentapeptide).
Systematic substitution of the NH -terminal Ser in 9 with neutral hydrophobic NH -acyl groups
led to partial agonists and eventually antagonists with unprecedented potency (greater than
000-fold increase over the previously reported antagonist 3-mercaptopropionyl-Phe-Cha-Cha-
Arg-Lys-Pro-Asn-Asp-Lys-NH ). In the series of NH -acyl tetrapeptide antagonists, N-trans-
cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH , 41 (BMS-197525), was identified as the
tightest binding (IC50 ∼ 8 nM) and most potent with an IC50 ∼ 0.20 µM for inhibition of
SFLLRNP-NH -stimulated platelet aggregation. Systematic single substitutions in 41 indicated
that, in addition to the NH -terminal acyl group, the side chains at the second and third
2
, 9 (EC50 ∼ 0.04 µM for stimulation
2
2
1
2
2
2
2
2
positions were also responsible for important and specific receptor interactions. The p-fluoroPhe
and p-guanidinoPhe residues in the second and third positions of 41 were observed to be optimal
in both the agonist and antagonist series. In the case of antagonists, however, an appropriately
positioned positively charged group (i.e., protonated base) at the third residue was required.
In contrast, such a substitution was not required for potent agonist activity. An even more
potent antagonist resulted when 41 was extended at the C-terminus by a single Arg residue
giving rise to analog 90 (BMS-200261) which had an IC50 ∼ 20 nM for inhibition of SFLLRNP-
2
NH -stimulated platelet aggregation. When the C-terminal Arg of 90 was replaced by an Orn-
δ
(
N -propionyl) residue, the resulting antagonist 91 (BMS-200661) was suitable for use in
radioligand binding assays (K ) 10-30 nM). Antagonist activity observed for selected
compounds was verified through secondary assays in that these analogs prevented SFLLRNP-
d
2
+
2
NH -stimulated GTPase activity in platelet membranes and Ca mobilization in cultured
human smooth muscle cells and mouse fibroblasts. Furthermore, this inhibition occurred at
concentrations that had no effect on thrombin catalytic activity indicating a specific activity
attributable to receptor binding and not enzyme inhibition.
In tr od u ction
observable when such cells are treated with thrombin,
and many of these actions can be mimicked by synthetic
peptides corresponding to the N-terminal tethered
ligand sequence. The identification of the thrombin
receptor in relevant cell types, together with the real-
ization that many of the pathophysiological actions of
thrombin on these cells appear to be at least in part
mediated by the thrombin receptor, suggests a role for
this receptor in the pathological processes of thrombosis,
inflammation, atherosclerosis, and fibroproliferative
Thrombin has been shown to have a variety of cellular
actions that are mediated by proteolytic activation of a
specific cell surface receptor known as the thrombin
receptor (or “tethered ligand” receptor). The thrombin
receptor is characterized by seven membrane-spanning
domains1 and is a member of the superfamily of G-
2
protein-coupled receptors. Activation of the receptor
occurs by thrombin cleavage of an extracellular N-
terminal domain thereby exposing a new NH2-terminus
which acts as a “tethered ligand” that intramolecularly
binds to an appropriate site contained in the receptor
structure.1 By virtue of this structural rearrangement,
the receptor becomes activated giving rise to various
observable G-protein-coupled signal transduction path-
ways.4
1
,5-11
disorders.
The development of thrombin receptor antagonists
that may have value as therapeutic agents by specific
inhibition of the cellular actions of thrombin (as opposed
,3
to its actions on clotting proteins) has been recog-
4,12
nized,
but such an agent has not thus far been
The proteolytically activated thrombin receptor has
realized. Although limited peptide-based antagonists
have been developed by analogy to the NH2-terminal
1
,5-7
been cloned
and shown to be present on numerous
1
different cell types including human platelets, endot-
12-14
tethered ligand peptide of the thrombin receptor,
helial cells,6 fibroblasts, vascular smooth muscle,
,8
5
7,9
these compounds suffer serious limitations, specifically,
lack of potency, partial agonist activity, and/or specif-
1
0,11
and cardiac myocytes.
A variety of responses are
4
a
icity.
†
Department of Peptide and Protein Research.
Department of Pharmacology.
Abstract published in Advance ACS Abstracts, November 15, 1996.
‡
An important objective of the work described in this
report was to develop sufficiently potent, specific, and
X
S0022-2623(96)00455-4 CCC: $12.00 © 1996 American Chemical Society

4
880 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Bernatowicz et al.
F igu r e 1. Structures and abbreviations used for nonproteogenic amino acid residues examined in the course of this study.
stable thrombin receptor antagonists that could be used
for evaluation of their therapeutic potential.
This structure-activity excercise was expected to
provide important information pertaining to (1) the
elucidation of receptor binding mechanism(s), (2) the
design and development of structural probes (e.g.,
photoaffinity labels), (3) the identification of peptides
suitable for use to begin “computationally directed”
screening of compound libraries for novel ligand types,
(4) the development of novel antagonist-based binding
assays (e.g., using an antagonist radioligand) for screen-
ing purposes, and (5) a basis for the design and develop-
ment of novel nonpeptide antagonists. These objectives
have at least been partially realized as a result of this
study.
In the abscence of precise structural information for
the receptor, an “analog approach” was taken to an-
tagonist development which involved cycles of system-
atic synthesis and testing. Previous studies of various
3
,12,13,15-22
thrombin receptor ligand peptides,
a reported peptide-based structure-activity relationship
SAR) in which almost all of the natural amino acids
including
(
were substituted, one at a time, for each residue of the
natural biologically active “tethered ligand” sequence
(SFLLR-NH2, 2), provided a basic understanding of the
peptide ligand side chain structural requirements and
their relative tolerances with regard to their interactions
with the human thrombin receptor.2 Although very few
of these single amino acid substitutions produced a
significant increase in agonist potency, an important
finding was that substitution of the nonproteogenic 3-(2-
naphthyl)alanine residue for leucine at position 3 of the
natural sequence produced by far the most potent
1
Resu lts a n d Discu ssion
Agon ist Op tim iza tion . Early on, the strategy em-
ployed here was to optimize agonist activity and then
convert these to antagonists, if possible, by appropriate
NH2-terminal structural modification. Previous studies
suggested that the use of nonproteogenic amino acids
merited further exploration. Specifically, replacements
with optimized unnatural residues, either singly or
multiply in appropriate combination, might lead to
further enhancements in binding and potency for known
agonists. Furthermore analogs with nonproteogenic
amino acid residues could have enhanced stability to
proteases, an advantage if the peptides are to be studied
in a biological setting. Figure 1 provides structures and
agonist (∼4-fold > 2) for human platelet aggregation
found in that study.21 A separate independent study
indicated that substitution of p-fluorophenylalanine for
phenylalanine at position 2 in SFLLRNP-NH2 resulted
in a 4-5-fold increase in activity as demonstrated by
an assay measuring phosphoinositide turnover in hu-
2
0
man epithelial-like SH-EP cells. The analog approach
taken here was largely based upon additional substitu-
tion(s) with untested nonproteogenic amino acids.

Potent Thrombin Receptor Antagonist Peptides
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4881
Ta ble 3. Effect of Preliminary N-Terminal Substitutions
Ta ble 1. Effect of Substitutions in the 2-Position of
SFLLR-NH2
peptide
mean EC50 (µM) (N)a
peptide
, SF(f)LLR-NH2
, SFLLR-NH2 (human sequence)
, SF(OCH3)LLR-NH2
, SF(I)LLR-NH2
, SF(NH2)LLR-NH2
, S-Tic-LLR-NH2
, SF(Gn)LLR-NH2
mean EC50 (µM) (N)a
1
5, Ac-âAF(f)A(N-2)LR-NH2
16, Ac-âAFLLR-NH2
17, âAFLLR-NH2
18, Thz-FLLR-NH2
19, Ac-Thz-FLLR-NH2
0.27 ( 0.03 (3)
1.80 ( 0.87 (3)
11.7 ( 4.51 (3)
46.0 ( 14.0 (3)
>300 (3)
1
2
3
4
5
6
7
8
0.13 ( 0.05 (3)
0.40 ( 0.14 (3)
0.72 ( 0.15 (3)
9.27 ( 5.97 (3)
18.00 ( 0.00 (3)
110 ( 78.1 (3)
>300 (3)
a
See Table 1.
, S-hF-LLR-NH2
>300 (3)
ments for activity. The des-Arg tetrapeptide analog of
9 (SF(f)F(Gn)L-NH2, 14, BMS-194021) was synthesized
and found to be an agonist with an EC50 of 0.28 ( 0.08
µM (N ) 7). This is the most potent tetrapeptide
agonist thus far published and more potent than the
natural pentapeptide 2. In contrast, the natural tet-
rapeptide SFLL-NH2 was >400 times less potent than
pentapeptide SFLLR-NH2. This shows that peptides
with appropriate side chains as small as four residues
can possess full activity and that five residues are not
required as was previously thought.
a
Determined by platelet aggregation assay; EC50 ) concentra-
tion required to stimulate 50% of maximum platelet aggregation,
N ) number of determinations. For amino acid abbreviations, see
Figure 1.
Ta ble 2. Effect of Substitutions at the 3-Position of Peptide 1
peptide
mean EC50 (µM) (N)a
1
8
9
1
1
1
1
, SF(f)F(Gn)LR-NH2
0, SF(f)F(f)LR-NH2
1, SF(f)A(N-2)LR-NH2
2, SF(f)A(N-1)LR-NH2
3, SF(f)-Tic-LR-NH2
0.04 ( 0.02 (3)
0.10 ( 0.03 (3)
0.08 ( 0.04 (3)
0.16 ( 0.06 (3)
0.47 ( 0.19 (6)
It is also noteworthy that the conformational con-
straint imposed by a Tic residue in the third position of
the peptide chain in analog 13 was relatively well
tolerated, providing an analog nearly equal in potency
to the natural sequence 2 but nonetheless substantially
less potent than the other analogs of the series (Table
a
See Table 1.
abbreviations for the various nonproteogenic residues
examined in the course of this study.
Table 1 summarizes the results of platelet aggrega-
tion assays on a series of peptide agonists in which the
Phe residue at position 2 of the human sequence was
replaced by various aromatic side chain-containing
analogs. These data show that the binding pocket for
the second side chain is exquisitely sensitive to size,
conformational constraint, and electronic distribution.
Of all the substitutions made in this series, only the
p-fluoroPhe residue provided an enhancement in po-
tency over the wild type human sequence 2. The
magnitude of the observed enhancement is in good
agreement with previous reports for similar pep-
2
).
P a r tia l Agon ist/An ta gon ist Discover y. Antago-
nists with modest potency had been reported which were
based on the agonist structure where the Ser was
replaced by a 3-mercaptopropionyl (Mpa) group1
which suggested that antagonists might be achieved by
substitution of Ser with neutral hydrophobic N-acyl
groups. Initially derivatives of âAla and (R)-thiazoli-
dine-4-carboxylic acid (Thz) were synthesized as near
isosteric and conformationally constrained replacements
for the Mpa group, respectively. The results are pre-
sented in Table 3. Very interestingly N-acetyl-âAla
peptides 15 and 16, although less potent than their Ser-
containing counterparts, were found to be reasonably
potent agonists. This result was somewhat surprising
because, prior to this discovery, only peptides capable
of providing a positively charged NH2-terminus (i.e., a
protonated amino group) were found to have appreciable
agonist activity. Curiously, free amine peptide 17 was
actually found to be less active in stimulation of platelet
aggregation than its N-acetyl counterpart 16 (Table 3).
This prompted additional biological evaluation including
2,13
2
0,22
tides.
It is clear from this and previously reported
2
1
data that the Phe binding site is very specific and that
the p-fluoroPhe residue may represent a very nearly
optimum substitution at position 2.
Table 2 summarizes the results of platelet aggrega-
tion assays on a series analogs with dual substitutions:
the “optimized” p-fluoroPhe in position 2 and various
residues at position 3. Substitution with both the
p-fluoroPhe and 2-naphthylAla residues in peptide 11
produced an agonist with slightly enhanced potency
(
EC50 of 0.08 ( 0.04 µM) over that observed for either
substitution alone (for reference, SFA(N-2)LR-NH2 had
2
1,23
an EC50 of 0.13 ( 0.07 µM).21
examination by GTPase assay.
Some peptides were
capable of stimulating only a fraction of the total
GTPase activity attainable by saturating levels of
agonist peptide SFLLRNP-NH2. For example, peptides
15, 16, and 18 were identified as partial agonists
possessing 21%, 50-70%, and 33% intrinsic potency,
respectively. This prompted further evaluation of NH2-
terminal substitutions in 9. In the process, additional
partial agonists and novel pure antagonists were identi-
fied. The antagonists were characterized by their ability
The p-guanidinoPhe replacement in the third position
of peptide 9 was designed based on previous observa-
tions that a Phe substitution produced a peptide slightly
greater in potency (∼1.4-fold) than 2 and that an Arg
substitution produced a somewhat greater (∼1.7-fold)
2
1
enhancement. The p-guanidinoPhe residue was thus
conceived as a hybrid combining potentially important
structural aspects of the activity-enhancing residues:
Phe, Arg, and 2-naphthylAla; this substitution in 9
produced the most potent peptide of this series with full
agonist activity. As a result peptide 9 was utilized as
a reasonable basis structure for further structural
elaboration aimed at a crossover to antagonist activity,
an approach that proved to be successful.
3
to inhibit [ H]SFFLRR-NH2 binding to platelet mem-
2
4
branes, inability to stimulate platelet aggregation at
concentrations >300 µM, and dose dependent inhibition
of peptide agonist (or thrombin)-induced platelet ag-
gregation, as well as their inability to stimulate the
GTPase even at very high concentration. Tables 4 and
5 summarize these results.
The results obtained for peptide 9 also suggested
further investigation of minimum structural require-

4
882 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Bernatowicz et al.
Ta ble 4. Effect of N-Terminal Variations in Early Generation
Antagonist Peptides: X-F(f)F(Gn)LR-NH2
group of 41 were well tolerated. NH2-Terminal struc-
tures giving rise to antagonists with binding assay IC50
values <50 nM are given in Figure 2. These results
indicate a high degree of structural specificity for the
receptor binding site interacting with the NH2-termini
of these analogs and an important functional role for
this interaction dictating the type of activity observed
for a particular analog (agonist vs partial agonist vs
antagonist). The data also suggest that the phenyl ring
in the cinnamoyl group in 41 binds with a preferred
conformation (twisted out of the plane of the adjacent
olefin), as more rigid but reasonably conservative ana-
logs 53 and 54 demonstrate dramatic reductions in
binding and potency.
a
b
peptide
X
IC50 (µM) (N)
IC50 (µM) (N)
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
(2-thiophene)acetyl
N-acetyl-2-aminobenzoyl 1.00 ( 0.31 (3)
2-oxo(2-thiophene)acetyl 0.22 ( 0.08 (3)
0.26 (2)
32 ( 23 (4)
ND
47 ( 32 (4)
74 ( 38 (4)
94 ( 37 (3)
193 ( 23 (3)
40 ( 19 (4)
68 ( 17 (3)
>160 (4)
35 ( 11 (4)
54 ( 8 (4)
85 ( 26 (4)
9.9 ( 6.5 (3)
(3-thiophene)acetyl
phenylacetyl
(2-thiophene)sulfonyl
(3-fluorophenyl)acetyl
(4-fluorophenyl)acetyl
3-pyridylacetyl
(2-fluorophenyl)acetyl
(3-indole)acetyl
cyclopentylacetyl
2-oxo(3-indole)acetyl
3-indoloyl
0.31 ( 0.18 (3)
0.68 ( 0.42 (3)
8.95 (2)
0.16 ( 0.05 (3)
0.35 ( 0.16 (3)
0.95 ( 0.34 (3)
0.56 ( 0.14 (3)
0.50 ( 0.10 (3)
0.37 ( 0.17 (3)
0.13 ( 0.08 (3)
0.019 ( 0.011 (3) 2.0 ( 0.7 (3)
0.11 (2) 2.9 ( 1.1 (3)
(3-chlorophenyl)acetyl
Another specific receptor interaction important for
both binding and antagonist function was recognized by
substituting the p-guanidinoPhe residue of 41 with
various aromatic, neutral, or basic residues (Tables
a
IC50 ) concentration required to inhibit 50% of tritiated
b
agonist (SFFLRR-NH2, at 25 nM) binding. IC50 ) concentration
required for 50% inhibition of agonist (SFLLRNP-NH2, at 3 µM)-
induced platelet aggregation.
7
-9). Here again, the lead analog 41 remained the
Ta ble 5. Effect of N-Terminal Structure (X) on Peptides
tightest binding and most potent antagonist. These
data indicate a requirement for a positively charged
group (protonated amine or guanidine) in order to obtain
tight binding antagonist activity. Replacement of the
guanidino group of 41 with hydrogen (analog 69)
produced a very significant reduction in binding and,
remarkably, a crossover to some agonist activity. Ap-
propriate positioning of the charge with respect to the
rest of peptide as well as the size of the charged group
are also important factors influencing binding and
function. For example, Lys, homoArg, and N,N ′-tet-
ramethylhomoArg relacements (analogs 79-81) gave
rise to partial agonist activity, while replacement with
Arg (76) was better tolerated resulting in an antagonist
with slightly reduced affinity and potency. These data,
taken together with the SAR obtained for NH2-terminal
variants, suggest that the arylguanidine of 41 plays an
important role in orienting or positioning the the NH2-
terminal cinnamoyl group for an optimum antagonist
generating interaction with the receptor.
(X-F(f)F(Gn)LR-NH2) Identified as Partial Agonists
a
b
peptide
X
IC50 (µM) (N)
EC50 (µM) (N)
3
5
N-acetyl-4-
0.27 ( 0.16 (3)
8.7 (3)
aminobutyryl
2-thiopheneoyl
3-thiopheneoyl
3-furanoyl
2-indoloyl
4-chlorobenzoyl
3
3
3
3
4
6
7
8
9
0
0.025 ( 0.014 (3) 0.10 ( 0.07 (4)
c
0.024 ( 0.008 (3) PA
c
0.029 ( 0.004 (3) PA
0.024 ( 0.003 (3) 1.8 (2)
0.026 (2)
3.7 (2)
a
IC50 ) concentration required to inhibit 50% of tritiated
b
agonist (SFFLRR-NH2, at 25 nM) binding. EC50 ) concentration
crequired for stimulation of 50% maximum platelet aggregation.
PA ) partial agonists for which maximum platelet aggregation
was not experimentally achieved.
In the course of generating and evaluating the data
(Tables 4 and 5), simple generalizations became pos-
sible. Whenever a relatively small aryl ring system was
directly attached to the carbonyl group of the NH2-
terminal amide, partial agonists resulted. Inclusion of
a spacer group such as a methylene or additional
carbonyl group between the aryl ring and the amide
carbonyl resulted in antagonists. From this data it was
hypothesized that antagonists that provided a structure
capable of extending greater distances from the NH2-
terminal amide, possibly with some conformational
constraint, could result in enhanced binding by virtue
of additional favorable interactions with the receptor
that are unavailable to shorter NH2-termini. To test
this possibility an NH2-terminal trans-cinnamoyl group,
containing a conformationally restrictive and chain-
extending olefinic linker between an aryl ring and the
N-terminal amide group, was incorporated into an
analog in this series (peptide 41). Upon its biological
evaluation, peptide 41 (Table 6) was found to be the
tightest binding and most potent NH2-acyl tetrapeptide
antagonist thus far discovered. The antagonist activity
of compound 41 was confirmed through additional
activity assays (for more details, see Antagonist Valida-
tion section). Table 6 summarizes data obtained for 41
and related NH2-acyl tetrapeptide analogs, many of
which were also found to be reasonably potent antago-
nists.
Effect of P ep tid e Len gth /An ta gon ist Ra d ioli-
ga n d Develop m en t. The effects of C-terminal dele-
tions and additions or replacements in anlogs of lead
peptide 41 were also examined (Tables 10 and 11). Very
interestingly, binding at the sub-micromolar level was
retained in a molecule as small as the NH2-acyl dipep-
tide analog 83, and the potency of this dipeptide was
comparable to that of a number of NH2-acyl tetrapeptide
antagonists (Table 4) having less optimal NH2-termini.
Such results suggest that it may be possible to prepare
relatively small molecule peptidomimetic thrombin
receptor antagonists with useful potency. When the
CO2H-terminal Arg was deleted from 41 to give analog
8
4, significant reduction in both binding and potency
are observed but reasonable antagonist activity is
retained. The contribution of the CO2H-terminal Arg
of 41 to binding and potency is nonetheless substantial.
,15-18,22
SAR reported for peptide agonists3
suggested
that the binding and potency of 41 may be enhanced
by the inclusion of an additional CO2H-terminal residue;
furthermore, if an Orn residue could be tolerated at this
position, acylation of its side chain amine with a
radiolabled acylating agent might provide a useful
radioligand for development of antagonist-based binding
assays. The results obtained for anlogs with CO2H-
terminal replacements or extensions are given in Tables
An ta gon ist Develop m en t. Despite the relatively
large number of NH2-acyl group replacements for the
trans-cinnamoyl moiety in 41 (Tables 4-6), only con-
servative modifications of the NH2-terminal cinnamoyl

Potent Thrombin Receptor Antagonist Peptides
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4883
Ta ble 6. Effect of N-Terminal Structure (X) on N-Acyl Tetrapeptide Series: X-F(f)F(Gn)LR-NH2
a
b
peptide
X
IC50 (µM) (N)
IC50 (µM) (N)
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
trans-cinnamoyl
0.0078 ( 0.0033 (3)
0.023 ( 0.014 (3)
0.020 ( 0.003 (3)
0.099 ( 0.006 (3)
0.034 ( 0.008 (3)
0.040 ( 0.023 (3)
0.046 ( 0.014 (3)
0.041 ( 0.020 (3)
0.042 ( 0.007 (3)
0.021 ( 0.003 (3)
1.63 ( 0.67 (3)
0.20 ( 0.07 (4)
0.28 ( 0.20 (6)
0.09 ( 0.04 (3)
0.24 ( 0.06 (6)
0.95 ( 0.83 (5)
0.39 ( 0.13 (3)
0.43 ( 0.25 (5)
0.08 ( 0.04 (4)
1.19 ( 0.56 (5)
0.26 ( 0.08 (4)
3.87 ( 1.94 (3)
0.88 ( 0.44 (4)
3.00 ( 1.31 (3)
21.7 ( 14.5 (3)
3.93 ( 1.68 (3)
1.63 ( 0.25 (3)
4.67 ( 1.92 (3)
2.73 ( 0.58 (3)
0.69 ( 0.16 (3)
PA f
3-phenyl-2-propynoyl
p-fluoro-trans-cinnamoyl
p-chloro-trans-cinnamoyl
p-methyl-trans-cinnamoyl
p-methoxy-trans-cinnamoyl
4-biphenyloyl
m-chloro-trans-cinnamoyl
3-phenylpropionyl
phenoxyacetyl
c
d
1-naphthylacetyl
3-(2-thiophene)-trans-acryloyl
(()-trans-3-phenylcyclopropanoyl
3-coumarinoyl
4-phenylbutyryl
p-amino-trans-cinnamoyl
2-naphthylacetyl
p-hydroxy-trans-cinnamoyl
(thiophenoxy)acetyl
trans-2-trans-4-hexadienoyl
trans-2-octenoyl
0.023 ( 0.003 (3)
2.32 ( 0.93 (3)
4.33 ( 0.15 (3)
1.26 ( 1.00 (3)
0.061 ( 0.013 (3)
0.295 ( 0.057 (3)
0.062 ( 0.015 (3)
0.019 ( 0.007 (3)
0.024 ( 0.009 (3)
0.035 ( 0.013 (3)
0.023 ( 0.006 (3)
0.043 ( 0.001 (3)
1.53 ( 0.15 (3)
e
7.60 ( 9.02 (3)
0.53 ( 0.27 (3)
8.33 ( 6.81 (3)
66.7 ( 11.6 (3)
R-fluorocinnamoyl
R-methylcinnamoyl
R-phenylcinnamoyl
a
IC50 ) concentration required to inhibit 50% of tritiated agonist (SFFLRR-NH2, at 25 nM) binding. b IC50 ) concentration required
c-e
for 50% inhibition of agonist (SFLLRNP-NH2, at 3 µM)-induced platelet aggregation.
These peptides displayed well-resolved mixed
c
d
e
f
pharmacology with agonist (platelet aggregation) activity EC50’s of 18 µM (N ) 2), 120 µM (N ) 2), and 90 µM (N ) 1). PA ) partial
agonist for which maximum platelet aggregation was not experimentally achieved.
Ta ble 8. Effects of Hydrophophilic Neutral or Weakly Basic
Aromatic Replacements (X) for the p-GuanidinoPhe Residue in
Analogs of 41: trans-Cinnamoyl-F(f)-X-LR-NH2
a
b
peptide
X
IC50 (µM) (N)
EC50 (µM) (N)
7
7
7
7
1
2
3
4
citrulline
His
â-(3-pyridyl)Ala
p-aminoPhe
1.04 ( 0.06 (4)
0.47 ( 0.11 (3)
0.34 ( 0.02 (3)
0.37 ( 0.03 (3)
67 (2)
c
35 ( 22 (4)
c
116 ( 100 (4)
60
a
IC50 ) concentration required to inhibit 50% of tritiated
agonist (SFFLRR-NH2, at 25 nM) binding. b EC50 ) concentration
required for stimulation of 50% maximum platelet aggregation.
Concentration required for 50% inhibition (i.e., IC50) of agonist
c
(SFLLRNP-NH2, at 3 µM)-induced platelet aggregation.
Ta ble 9. Effects of Aliphatic Basic (i.e., Positively Charged)
Residue Replacements (X) for the p-GuanidinoPhe Residue in
Analogs of 41: trans-Cinnamoyl-F(f)-X-LR-NH2
a
b
peptide
X
IC50 (µM) (N)
IC50 (µM) (N)
7
7
7
7
7
8
8
5
6
7
8
9
0
1
Orn
Arg
TMR
Gbu
Lys
hR
TMhR
TMGbu
0.17 ( 0.08 (3)
0.028 ( 0.016 (3)
0.32 ( 0.13 (3)
0.028 ( 0.007 (3)
0.039 ( 0.002 (3)
0.012 ( 0.002 (3)
0.13 ( 0.03 (3)
0.34 ( 0.18 (3)
8.70 ( 3.63 (4)
1.13 ( 0.67 (3)
16 (2)
F igu r e 2. Best tolerated (IC50 < 50 nM, binding) N-terminal
structures in antagonist analogs of 41.
8.0 (1)
Ta ble 7. Effects of Hydrophobic Aromatic Replacements (X)
for the p-GuanidinoPhe Residue in Analogs of 41:
trans-Cinnamoyl-F(f)-X-LR-NH2
c
18 (1)
c
0.5 (1)
90 (1)
c
a
b
peptide
X
IC50 (µM) (N)
IC50 (µM) (N)
82
140 (1)
a
6
6
6
6
6
7
5
6
7
8
9
0
homoPhe
0.63 ( 0.026 (3)
7.55 ( 10.4 (4)
4.71 ( 1.0 (3)
1.18 ( 0.13 (3)
1.12 ( 0.33 (4)
>300 (2)
>300 (2)
>300 (2)
>300 (2)c
17 (2)
IC50 ) concentration required to inhibit 50% of tritiated
b
p-nitroPhe
p-chloroPhe
p-methoxyPhe
Phe
2 50
agonist (SFFLRR-NH , at 25 nM) binding. IC ) concentration
required for 50% inhibition of agonist (SFLLRNP-NH , at 3 µM)-
2
induced platelet aggregation. c These peptides displayed agonist
(or partial agonist) activity with the specified EC50 values.
â-(2-naphthyl)Ala 0.246 ( 0.018 (3)
5 (1)c
a
IC50 ) concentration required to inhibit 50% of tritiated
residue of 88 was acetylated (89) or propionylated (91),
a slight reduction in binding (<3-fold) was observed, but
these analogs were still more potent at inhibiting
platelet aggregation than 41. Given these results,
peptide 91 was targeted for radioligand synthesis. After
propionylation of 88 in solution with N-succinimidyl-
b
agonist (SFFLRR-NH2, at 25 nM) binding. IC50 ) concentration
required for 50% inhibition of agonist (SFLLRNP-NH2, at 3 µM)-
induced platelet aggregation. These peptides displayed agonist
c
activity with the specified EC50 values.
1
1 and 12. Addition of a basic residue (Orn or Arg in
3
analogs 88 and 90, respectively) resulted in analogs with
marginally improved binding but a more significant
positive impact on antagonist potency (about a 5-10-
fold enhancement over 41) was observed. When the Orn
[2,3- H]propionate (Amersham), HPLC-purified tritiated
91 was obtained with a specific activity of 80 Ci/mmol
and a radiochemical purity of >98% (P. Egli, unpub-
lished results). Using this tritiated 91, an antagonist-

4
884 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Bernatowicz et al.
Ta ble 10. Effects of C-Terminal Truncation in Analogs of 41:
trans-Cinnamoyl-F(f)-F(Gn)-X
stimulated by 3 µM SFLLRNP-NH2 with an IC50 of 0.2
(
0.07 µM (N ) 4) and caused concentration dependent
a
b
peptide
X
IC50 (µM) (N)
IC50 (µM) (N)
rightward shifts in the concentration of agonist required
for platelet aggregation (Figure 3), indicating that the
compound is a competitive antagonist for SFLLRNP-
NH2 activation of platelets. The inhibition of platelet
aggregation gave a Schild slope of 1.02 and a pA2 of 7.26
8
8
8
3
4
5
NH2
Leu-NH2
Orn-NH2
0.78 ( 0.20 (3)
0.11 ( 0.09 (3)
0.87 ( 0.13 (3)
64 (2)
4.05 ( 2.66 (4)
141 (2)
a,b
See Table 9.
(
N ) 2). This inhibition was specific to the thrombin
Ta ble 11. Effects of C-Terminal Replacements and Additions
to Analogs of 41: trans-Cinnamoyl-F(f)-F(Gn)L-X
receptor in that platelet aggregation stimulated by ADP
and U-46619, a thromboxane receptor agonist, was
unaffected. Peptide 41 also inhibited thrombin-induced
platelet aggregation, but its potency against SFLLRNP-
NH2 was greater than against thrombin. The IC50 for
inhibition of thrombin-induced platelet aggregation also
depended on the thrombin concentration and the indi-
vidual platelet donor. This result is consistent with
enzymatic cleavage of the receptor generating a tethered
ligand having a very high effective local concentration,
thereby reducing the likelihood of inhibition by a
competitive antagonist at less than saturating concen-
trations.
a
b
peptide
X
IC50 (µM) (N)
IC50 (µM) (N)
8
8
8
8
9
9
6
7
8
9
0
1
Orn-NH
Orn(acetyl)-NH
Arg-Orn-NH
Arg-Orn(acetyl)-NH
Arg-Arg-NH
2
0.017 ( 0.003 (3) 0.19 ( 0.09 (3)
0.044 ( 0.002 (3) 0.25 ( 0.22 (3)
0.0066 ( 0.002 (3) 0.047 ( 0.018 (4)
0.0166 ( 0.006 (3) 0.040 ( 0.020 (4)
0.0075 ( 0.001 (3) 0.021 ( 0.004 (3)
0.0219 ( 0.004 (3) 0.085 ( 0.025 (4)
2
2
2
2
c
Arg-Orn(pro) -NH
2
a,b
See Table 9. c pro ) propionyl.
based binding assay was developed (S. M. Seiler,
unpublished results) showing competitive binding with
various agonists and antagonists, as well as distinctly
different kinetics of binding compared to the normal
agonist radioligand. Those studies will be detailed
separately.
Antagonist 41 also inhibited SFLLRNP-NH2 and
thrombin-stimulated membrane GTPase activity in
platelet and CHRF-288 cell membranes. The inhibition
Results of replacing the NH2-terminal trans-cin-
namoyl group in the most potent analog 90 with some
of the best tolerated replacements identified previously
in the NH2-acyl tetrapeptide series (Table 6, Figure 2)
are given in Table 12. In this case, the NH2-terminal
of SFLLRNP-NH -stimulated platelet membrane GT-
Pase is shown in Figure 4. In human aortic smooth
2
muscle cells (HASMs), most of the intracellular Ca2
mobilization observed with SFLLRNP-NH2 was pre-
vented by 41 (Figure 5). As for the platelet aggregation
studies, a higher concentration of 41 was required to
inhibit HASM activation by thrombin compared to
2
+
3
-phenyl-2-propynoyl group was found to provide the
tightest binding (IC50 ∼ 4 nM) antagonist 94 (BMS-
01516) thus far discovered, but it was comparable in
2
SFLLRNP-NH . Analog 41 also inhibited SFLLRNP-
NH -stimulated Ca mobilization in mouse Swiss 3T3
2
+
platelet aggregation inhibition potency to its trans-
cinnamoyl counterpart 90. Thus, in addition to a
positively charged residue (e.g., p-guanidinoPhe) at
position 3 possibly having some role in positioning the
critical NH2-terminus at the receptor binding site, such
NH2-terminal positioning may also be influenced by
interactions sequentially more remote (i.e., at the more
CO2H-terminal side chains of positions 5 and 6). Over-
all the results reported here for CO2H-terminally ex-
tended antagonists are consistent with and parallel
those observed for peptide agonists for which optimal
2
cells (data not shown). These and other studies indicate
that the antagonists reported here inhibit the thrombin
receptor in at least several different species and tissues.
Furthermore 41 was found not to inhibit thrombin
2
5
proteolytic activity (K ) 52 µM for inhibition of
i
thrombin by 41) at levels where antagonist responses
were observed showing that its biological activity was
thrombin receptor specific.
Su m m a r y
2
2
potency was observed at the length of hexapeptides.
Beginning with existing SAR obtained for thrombin
receptor agonist peptides and preparing novel analogs
by systematically substituting residues with nonpro-
teogenic amino acids, first singly and then in combina-
tion, it was possible to derive the most potent agonist
pentapeptide, SF(f)F(Gn)LR-NH2 (9), reported to date.
Single substitution of the NH2-terminal Ser in agonist
9 with a wide variety of neutral hydrophobic NH2-acyl
groups led to the identification and discovery of novel
partial agonists and antagonists. The most potent NH2-
acyl tetrapeptide antagonist derived from 9 was trans-
cinnamoyl-F(f)F(Gn)LR-NH2 (41) which exhibited spe-
cific binding to the human thrombin receptor at
unprecedented single-digit nanomolar levels (>1000-fold
enhancement over reported peptide antagonists) and
inhibited agonist-induced platelet aggregation with an
IC50 potency of ∼0.2 µM. Data obtained for NH2-
terminal acyl group substitution analogs of 41 suggest
a specific and important interaction for the trans-
cinnamoyl moiety of 41. Further SAR by systematic
substitutions of the p-fluoroPhe and p-guanidinoPhe
residues of 41 did not lead to improved binding or
antagonist potency indicating important and specific
Effect of 2-P osition Su bstitu tion s in An ta go-
n ists. Because the p-fluorophenyl side chain repre-
sented an optimal substitution at the 2-position in the
peptide agonists, this specific and important interaction
was further probed in the tight binding NH2-acyl
tetrapeptide antagonist series by various systematic
substitutions. The results are presented in Table 13.
As observed for agonist peptides, the p-fluorophenyl side
chain provides an optimal and apparently specific and
important interaction. Conservative substitution with
a phenyl side chain in analog 97 was tolerated but
resulted in significant losses in binding (∼3-fold) and
potency (>8-fold). Furthermore a single replacement
of the p-fluorophenyl side chain of 41 by a hydrogen (Gly
analog 96) produced a peptide that was almost devoid
of activity. Clearly a marked preference for a hydro-
phobic aromatic residue has been demonstrated at this
position.
An ta gon ist Va lid a tion . Because of the unprec-
edented binding and antagonist activity observed for
peptide 41, additional biological characterization was
carried out. Peptide 41 blocked platelet aggregation

Potent Thrombin Receptor Antagonist Peptides
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4885
Ta ble 12. Effects of Single (X) or Dual (X, Z) Replacement in Analogs of 90: X-F(f)-F(Gn)-LR-Z-NH2
a
b
peptide
X
Z
IC50 (µM) (N)
IC50 (µM) (N)
9
9
9
9
2
3
4
5
trans-4-fluorocinnamoyl
3-phenylpropionyl
3-phenyl-2-propynoyl
trans-4-fluorocinnamoyl
Arg
Arg
Arg
0.019 ( 0.007 (3)
0.009 ( 0.001 (3)
0.004 ( 0.001 (3)
0.025 ( 0.005 (3)
0.05 ( 0.01 (3)
0.25 ( 0.12 (3)
0.040 ( 0.019 (3)
0.060 ( 0.010 (3)
Orn(acetyl)
a,b
See Table 9.
Ta ble 13. Effects of Substitutions (X) for p-FluoroPhe in
Analogs of 41: trans-Cinnamoyl-X-F(Gn)LR-NH2
a
b
peptide
X
IC50 (µM) (N)
IC50 (µM) (N)
9
9
9
9
6
7
8
9
Gly
Phe
Cha
Tha
.30
>100 (2)
0.024 ( 0.007 (4)
0.093 ( 0.032 (4)
0.042 ( 0.011 (4)
3.44 ( 1.14 (4)
1.73 ( 0.46 (3)
8.73 ( 4.95 (3)
3.31 ( 0.57 (3)
c
c
c
1
00
PyrA
>55 (3)
a,b
See Table 9. c See Figure 1 for structures and abbreviations.
2
F igu r e 4. Inhibition of SFLLRNP-NH -stimulated platelet
membrane GTPase activity by peptide 41. Platelet membrane
GTPase was measured with (O) and without (b) 20 µM
2
SFLLRNP-NH . Thrombin receptor-stimulated GTPase was
2
3
determined as described previously. This experiment is an
example of three yielding similar results.
2
F igu r e 3. Inhibition of SFLLRNP-NH -stimulated human
platelet aggregation by peptide 41. Platelet aggregation was
measured using gel-filtered human platelets in a microtiter
2
1,35
turbidity assay as previously described.
preincubated with the indicated concentrations of 41 prior to
stimulation by SFLLRNP-NH . This experiment is an example
The platelets were
2
of several yielding similar results.
F igu r e 5. Inhibition of SFLLRNP-NH
2
-stimulated Ca2
+
mobilization in cultured human aortic smooth muscle (HASM)
cells. The cells were loaded with the fura-2 Ca2 fluorescent
indicator by incubation with fura-2 AM. The loaded cells were
washed and preincubated with the indicated concentrations
interactions with the receptor at those peptide sites in
addition to those identified at the NH2-terminus. Also
noteworthy is an apparent requirement for an ap-
propriately positioned positively charged group (i.e.,
protonatable base) to mimic the guanidinophenyl side
chain of 41 if tight binding and potent antagonism are
to be realized. In general the antagonist SAR thus
obtained parallels that obtained for agonists. These
observations, combined with the ability of peptide
agonists and antagonists to compete for receptor bind-
ing, suggest at least partially common modes of binding
for both types with distinct and functional differences
residing mainly at the NH2-termini that dictate what
type of activity (GTPase coupling or uncoupling, i.e.,
agonist or antagonist) is observed for a particular
structure. Furthermore it appears that the critical NH2-
terminal interaction is at least in part influenced by
interactions downstream with more CO2H-terminal side
chains. Thus, extension of 41 by inclusion of an ad-
ditional Arg residue at the CO2H-terminus gave the
most potent antagonist 90 with similar receptor binding
affinity to 41 but with a significant increase (∼10-fold,
IC50 ∼ 20 nM) in potency. When the NH2-terminal
+
2+
of peptide 41. Ca mobilization was induced by addition of
10 µM SFLLRNP-NH (arrow). Fluorescence (excitation at 340
2
and 380 nm with emission at 505 nm) was measured.
trans-cinnamoyl group of 90 was replaced with a
3
-phenyl-2-propynoyl group, the tightest binding an-
tagonist 94 (IC50 ∼ 4 nM) resulted, which had potency
comparable to 90. Given the specificity, unprecedented
binding affinity, and potency for the thrombin receptor
antagonists disclosed here, it is likely that these, or
specifically designed derivatives of these, will serve as
valuable tools for elucidation of thrombin receptor
structure, function, and pharmacology and serve as a
starting point for new drug discovery. Indeed, a practi-
cally accessible tritiated antagonist (91) has been shown
to be a useful radioligand suitable for use in binding
assays.
Exp er im en ta l Section
Ma ter ia ls. Boc-Leu-OH, Boc-Arg(Tos)-OH, and p-methyl-
benzhydrylamino-polystyrene resins (1% DVB cross-linked,

4
886 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Bernatowicz et al.
0
.56 and 0.36 mequiv/g) were purchased from Peninsula
1H NMR data for lead antagonist tetrapeptide 41 is rep-
Laboratories (Belmont, CA). All nonproteogenic Boc-amino
acids were from Bachem Bioscience (Philadelphia, PA) except
Boc-Phe(p-N,N ′-bis-Cbz-guanidine)-OH which was prepared
resentative: The spectrum (600 MHz) was obtained on a 3 mM
solution of 41 in D
(ppm) (peak positions for exchangeable amide NH’s were
determined with H O as solvent) are reported relative to
residual HDO in solvent which resonates 5.02 ppm downfield
from TSP. NMR: δ 0.89 (s, 3H, Leu CH ), 0.955 (s, 3H, Leu
), 1.48 (m, 1H, Leu γ CH), 1.635 (m, 3H, Leu γ CH, Leu â
), 1.675 (m, 2H, Arg γ CH ), 1.785 (m, 1H, Arg âCH ), 1.88
), 2.915 (m, 1H, Phe(Gn) âCH ), 3.02 (m, 2H,
2
O (pH 6.6) at 3 °C, and peak positions in δ
by reaction of Boc-Phe(p-NH
2
)-OH with N,N ′-bis-Cbz-1-gua-
2
2
6
27
nylpyrazole as described previously. Trifluoroacetic acid
was from Eastman Kodak (Rochester, NY), diisopropylethyl-
amine was from Fluka (Buchs, Switzerland), and BOP and
HBTU reagents were from Midwest Biotech (Fishers, IN).
Coupling reagent solution, 1 M DCC in NMP, was from
Applied Biosystems (Foster City, CA). Carboxylic acids used
for N-terminal acylations, 2-thiophenesulfonyl chloride, and
anhydrous anisole were from Aldrich (Milwaukee, WI). Syn-
3
CH
CH
3
2
2
2
(m, 1H, Arg âCH
2
2
Phe(fluoro) âCH ), 3.13 (m, 1H, Phe(Gn) âCH ), 3.20 (m, 2H,
2
2
Arg δ CH ), 4.25 (m, 1H, Arg R CH), 4.29 (m, 1H, Leu R CH),
2
4.59 (m, 1H, Phe(Gn) R CH), 4.63 (m, 1H, Phe(fluoro) R CH),
6.57 (d, 1H, J ) 15.3 Hz, cinnamoyl R vinyl), 6.68 (d, 1H,
C-terminal NH), 6.96 (d, 1H, C-terminal NH), 7.05 (m, 2H,
aryl), 7.095 (m, 2H, aryl), 7.21 (m, 2H, aryl), 7.245 (m, 2H,
aryl), 7.27 (m, 1H, Arg ꢀ NH), 7.45 (d, 1H, cinnamoyl â vinyl
overlapped with 7.49, m, 3H, cinnamoyl aryls), 7.66 (m, 2H,
aryl), 8.22 (d, 1H, J ) 6.8 Hz, Leu NH), 8.33 (d, 1H, J ) 7.8
Hz, Phe(Gn) R NH), 8.42 (d, 1H, J ) 7.0 Hz, Arg R NH), 8.50
(d, 1H, J ) 7.6 Hz, Phe(fluoro) NH).
2 2
thesis solvents (CH Cl , DMF, diethyl ether) were obtained
from Fisher Scientific (Pittsburgh, PA). Hydrogen fluoride was
from Matheson (East Rutherford, NJ ). All commercial chemi-
cals used were of the highest quality available (AR grade or
better).
P ep tid e Syn th esis. All peptides were prepared manually
using standard solid phase synthesis techniques and the Boc/
benzyl protection strategy. Syntheses were performed starting
from 0.08-0.10 mmol of p-methylbenzhydrylamino-poly-
styrene resin. Boc group removals were carried out by
Biologica l Assa ys. Assays for human platelet aggregation
21,35
21,23
activity,
platelet membrane GTPase activity,
and recep-
and a
treatment with TFA/CH
was performed by two brief washes with 5% DIEA in CH
Couplings were performed using 4 equiv of Boc-amino acid (or
equiv for nonproteogenic Boc-amino acids) with equivalent
2
Cl
2
(1:1) for 15 min. Neutralization
3
tor binding assays using 20-25 nM [ H] SFFLRR-NH
2
2
Cl .
2
platelet membrane preparation were performed as described
24
elsewhere.
3
Abbr evia tion s. Abbreviations for amino acids and nomen-
clature of peptide structures not specifically stated in the text
follow the recommendations of the IUPAC-IUB Commission
on Biochemical Nomenclature (J . Biol. Chem. 1971, 247, 997).
Other abbreviations not given in the text are as follows: Boc
2
8
amounts of BOP reagent and DIEA in minimum DMF and
were monitored for completion using the Kaiser ninhydrin
2
9
2
test. NH -terminal acylations by carboxylic acids in general
were conducted similarly (i.e., BOP/DIEA) except that 10 equiv
were used. In some cases, where BOP-derived intermediates
precipitated during preactivation, carboxylic acids were coupled
)
tert-butyloxycarbonyl; BOP ) (1H-benzotriazol-1-yloxy)tris-
(
dimethylamino)phosphonium hexafluorophosphate; Cbz )
2
using 1 M DCC in NMP. In the case of the NH -terminal
benzyloxycarbonyl; DCC ) N,N ′-dicyclohexylcarbodiimide;
DIEA ) diisopropylethylamine; DMF ) dimethylformamide;
DVB ) divinylbenzene; FAB ) fast atom bombardment; Fmoc
phenylpropioloyl group (analogs 42, 94), N-acylation was
conducted using the N-hydroxysuccinimide ester of phenyl-
propiolic acid (prepared by reaction of phenylpropiolic acid with
DCC and N-hydroxysuccinimide in THF at 4 °C, 2 h, then at
)
[(9-fluorenylmethyl)oxy]carbonyl, HBTU ) 2-(1H-benzot-
riazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
room temperature, 2 h) in CH
arginine residues were introduced starting with Boc-Orn-
Fmoc)-OH, and after usual peptide chain assembly followed
2 2
Cl /NMP. In some cases,
NMP ) N-methylpyrrolidinone; SAR ) structure-activity
relationship; Tos ) tosyl (p-toluenesulfonyl); TSP ) (trimeth-
(
2
ylsilyl)[2,2,3,3- H
4
]propionate.
by specific cleavage of the side chain Fmoc protection (10%
diethylamine in DMF, 10 min) from the protected peptidyl-
resin, the guanidino group was incorporated using 1H-pyra-
Ack n ow led gm en t. The authors are grateful to Drs.
S. Natarajan and G. R. Matsueda for their interesting
and helpful discussions and advice as well as critical
review of this manuscript. The authors also thank BMS
Analytical R&D for mass spectrometry analysis of the
peptides, Drs. J . K. Rinehart and P. Egli for synthesis
of tritiated antagonist 91, and Dr. K. Constantine for
3
0
zole-1-carboxamidine hydrochloride in DMF/DIEA in the last
solid phase chemistry step before final HF deprotection and
cleavage. Peptides containing N,N ′-tetramethylguanidine
R
side chains were prepared similarly from the appropriate N -
ω
Boc-N -Fmoc-amino acid using HBTU/DIEA (10 equiv each)
in minimal DMF (3 h, room temperature) to incorporate the
tetramethylguanidino group.31 Peptides with NH
2
-acetyl and
-propionyl side chains were also prepared by this strategy
i.e., via orthogonal Fmoc protection of side chain amines
1
NH
2
H NMR spectra.
(
which, after deprotection, were acylated using standard re-
agents while still resin-bound). Completed protected peptidyl-
resins were cleaved and deprotected using HF containing 5%
anisole at 4 °C for 1 h. After HF removal in vacuo, the
products were washed several times with diethyl ether and
extracted with several portions into 20-30 mL of 5% HOAc
in water. The entire solution of crude product thus obtained
was purified by loading directly onto a preparative HPLC
system with a C-18 column using a linear gradient of increas-
ing acetonitrile in water containing 0.1% TFA for elution as
previously detailed. Fractions shown by HPLC to be >95%
pure were pooled and lyophilized to provide, with a few
exceptions, 25-45 mg of peptide products (ca. 30-50% overall
yield) as white powder TFA salts that in general were >98%
pure as determined by HPLC (YMC C18 column, detection at
Refer en ces
(
1) Vu, T.-K. H.; Hung, D. T.; Wheaton, V. I.; Coughlin, S. R.
Molecular Cloning of a Functional Thrombin Receptor Reveals
a Novel Proteolytic Mechanism of Receptor Activation. Cell 1991,
6
4, 1057-1068.
(2) Schwartz, T. W. Locating Ligand-binding Sites in 7TM Receptors
by Protein Engineering. Current Opin. Biotechnol. 1994, 5, 434-
444.
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