High-affinity thrombin receptor (PAR-1) ligands: A new generation of indole-based peptide mimetic antagonists with a basic amine at the C-terminus

Article abstract of DOI:10.1016/S0960-894X(03)00325-1

A new generation of indole-based peptide mimetics, bearing a basic amine at the C-terminus, was developed by the agency of two complementary, multistep, trityl resin-based approaches. Thus, we obtained several high-affinity thrombin receptor (PAR-1) ligands, such as 32 and 34. Compounds 32 and 34 were found to bind to PAR-1 with excellent affinity (IC₅₀=25 and 35 nM, respectively) and to effectively block platelet aggregation induced by SFLLRN-NH₂ (TRAP-6) and α-thrombin.

Full text of DOI:10.1016/S0960-894X(03)00325-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2199–2203
High-Affinity Thrombin Receptor (PAR-1) Ligands: A New
Generation ofIndole-Based Peptide Mimetic Antagonists
with a Basic Amine at the C-terminus
Han-Cheng Zhang,* Kimberly B. White, David F. McComsey,
Michael F. Addo, Patricia Andrade-Gordon, Claudia K. Derian,
Donna Oksenbergy and Bruce E. Maryanoff*
Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, Spring House, PA 19477-0776, USA
Received 9 January 2003; revised 20 March 2003; accepted 21 March 2003
Abstract—A new generation of indole-based peptide mimetics, bearing a basic amine at the C-terminus, was developed by the
agency of two complementary, multistep, trityl resin-based approaches. Thus, we obtained several high-affinity thrombin receptor
(PAR-1) ligands, such as 32 and 34. Compounds 32 and 34 were found to bind to PAR-1 with excellent affinity (IC50=25 and 35
nM, respectively) and to effectively block platelet aggregation induced by SFLLRN-NH2 (TRAP-6) and a-thrombin.
# 2003 Elsevier Science Ltd. All rights reserved.
Traditionally, serine proteases have been viewed as
agents responsible for the degradation of proteins and
the formation of peptide messengers. However, more
recently, serine proteases such as thrombin, trypsin, and
tryptase have been found to serve as cellular signalling
molecules by cleaving and activating so-called protease-
activated receptors (PARs). PARs represent a unique
subtype of 7-transmembrane G-protein-coupled recep-
tor in that they are proteolytically modified to expose a
new extracellular N-terminus, which acts as a tethered
activating ligand.1
induced platelet aggregation and moderate affinity to
PAR-1.7aꢀe In seeking to further improve PAR-1
antagonist potency, we have now explored other solid-
phase approaches to analogues that were difficult to access
via the previous methods. Herein, we disclose a new series
of indole-based peptide mimetics, with a basic amine at
the C-terminus, which were prepared via a versatile, trityl
resin-based, solid-phase parallel synthesis. Several of these
compounds have greatly improved binding affinity to
PAR-1, and effectively block platelet aggregation induced
by both SFLLRN-NH2 (TRAP-6) and a-thrombin.
Presently, four receptors of this class, PAR-1,2 PAR-2,3
PAR-3,4 and PAR-45 are known. PAR-1 is cleaved and
activated by a-thrombin, and thereby mediates most of
the cellular actions of a-thrombin. Thus, considerable
effort has been exerted in the search for potent, selective
PAR-1 antagonists, which have therapeutic potential
especially as antiplatelet drugs.6,7 We have described a
novel series of indole-based peptide mimetics as PAR-1
antagonists, with good potency against thrombin-
In our previous compound-optimization studies,7b we
attached a secondary amide onto a Sieber or Tentagel S
AM resin, then constructed a dipeptide segment fol-
lowed by introduction of the indole scaffold. Thus, we
constructed a library of peptide mimetic PAR-1
antagonists that led to advanced lead RWJ-56110 (1, see
Table 1).7b However, that approach presented some
limitations. First, modification of the C-terminus of the
dipeptide (determined later to be important for PAR-1
potency) was highly limited due to the difficulty of
reductively aminating the resins, and the constraint of
introducing the R6 group (see general structure in Table
1) at the outset of a 10-step process. Second, the final
cleaved products typically had only 75–85% purity, and
the loadings of the Sieber or Tentagel S AM resins were
relatively low. To address these issues, we sought an
*Corresponding authors. Tel.: +1-215-628-5988; fax: +1-215-628-
4985; e-mail: hzhang@prdus.jnj.com (H.-C. Zhang); tel.: +1-215-628-
5530; fax: +1-215-628-4985; e-mail: bmaryano@prdus.jnj.com (B.E.
Maryanoff).
yEmployed at COR Therapeutics, Inc. (now part of Millennium
Pharmaceuticals).
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00325-1

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H.-C. Zhang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2199–2203
Table 1. PAR-1 binding and platelet aggregation IC50 values (mM)a
Compd
Pb
R4
R5
R6
Receptor bindingc
Gel-filtered platelet aggregationd
TRAP-6
Thrombin
Collagen
U46619
1
6
6
6
6
6
6
6
6
6
4
7
6
6
6
6
6
6
6
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
3,4-DiF-Bn
4-Cl-Bn
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)3
H2N(CH2)2
4-Im-CH2
Bn
4-MeOBn
Ph(CH2)2
2-Fur-CH2
2-Thi-CH2
2-Py-CH2
2-Py-(CH2)2
EtNH(CH2)2
1-Pyr-(CH2)2
1-Pyr-(CH2)2
1-Pyr-(CH2)2
1-Pyr-(CH2)2
H2N(CH2)3
H2N(CH2)3
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
0.44ꢂ0.21
0.48ꢂ0.05
0.12
0.16ꢂ0.05
0.1ꢂ0.01
0.16
0.07ꢂ0.03
0.16ꢂ0.02
0.19ꢂ0.02
0.15ꢂ0.01
0.13
0.34ꢂ0.04
0.7ꢂ0.1
IA
6.7
3.0
7.9
7.0
8.4
6.6
IA
IA
IA
IA
16
IA
6.4
7.6
IA
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
0.81ꢂ0.09
0.78ꢂ0.02
0.8ꢂ0.06
1.1ꢂ0.18
0.88ꢂ0.18
0.41ꢂ0.21
0.46ꢂ0.22
40ꢂ5
0.16
0.23
0.33
0.5
IA
19.8
10.3
13.7
IA
IA
IA
22ꢂ3
17.3
18.6
18.6
24ꢂ10
IA
0.09ꢂ0.02
0.07
2.0
0.05ꢂ0.00
16
4.3
22
0.31
23ꢂ4
0.06
0.05
0.25ꢂ0.1
0.2ꢂ0.02
0.41ꢂ0.1
1.3ꢂ0.2
0.43ꢂ0.001
0.13ꢂ0.01
0.06
0.025ꢂ0.004
0.07ꢂ0.04
0.035ꢂ0.004
11
30ꢂ4
4-Cl-Bn
0.07ꢂ0.01
0.06ꢂ0.02
0.07ꢂ0.02
0.05ꢂ0.01
0.08ꢂ0.02
3,4-DiF-Bn
3,4-DiF-Bn
2-F-Bn
MeSCH2
27
MeS(CH2)2
4-Py-CH2
2-Thi-CH2
0.22ꢂ0.07
4.7ꢂ1
IA
16.5
0.13
0.27ꢂ0.12
3,4-DiF-Bn
IA
aResults are expressed as meanꢂSEM (nꢃ2; n=1 for values without error limits). IA denotes inactivity @ 50 mM of test compound. Abbreviations
for R4, R5 and R6: Bn, benzyl. Fur, furyl. Thi, thienyl. Py, pyridyl. Pyr, pyrrolidinyl. Im, imidazolyl.
bPdenotes the position of indole ring attaching to urea linkage.
cThrombin receptor (PAR-1) binding assay; ligand: [3H]-S-(p-F-Phe)-homoarginine-L-homoarginine-KY-NH2, 10 nM (Kd=15 nM).
dConcentrations of agonists for aggregation studies: TRAP-6, 2 mM; a-thrombin, 0.15 nM; collagen, 3 mg/mL; U46619, 0.3 mM.
alternative resin-attachment point and designed a new
synthetic sequence that permits the introduction of
maximum diversity at the C-terminus. Given that a pri-
mary amine side chain is well tolerated at the R5 posi-
tion, such as for RWJ-56110,7b we chose a 2-Cl-trityl
resin,8 which has high loading, is compatible with our
multistep reaction conditions, and is easily cleaved, to
anchor the key amine side chain.
treated with piperidine to give the corresponding resin-
bound dipeptide 6.
For the other desired segment, various nitroindoles 7
were N-alkylated with substituted benzyl bromides in
the presence of cesium carbonate and the products were
ꢁ
reduced with Me2NNH2, FeCl3 6H2O, and charcoal in
MeOH to afford (4-7)-aminoindoles 8 (Scheme 2). In a
key step of the solid-phase process, aminoindole 8 was
Fmoc-protected 2,4-diaminobutyric acid or ornithine
was coupled with allyl alcohol in the presence of
HOBt/DCC followed by removal of the Boc group
with TFA/CH2Cl2 to give the allyl protected com-
pound 3 (Scheme 1). The amine 3 was loaded onto the
2-Cl-trityl resin (1.5 mmol/g, Advanced ChemTech) in
the presence of i-Pr2NEt followed by cleavage of the
Fmoc group to give 4, which was coupled with Fmoc-
protected amino acid 5 using HBTU/HOBt and then
Scheme 2. (a) R1-C6H4CH2-Br, Cs2CO3, DMF; (b) Me2NNH2,
.
FeCl3 6H2O, charcoal, MeOH, reflux.
Scheme 1. (a) Allyl alcohol, DCC, HOBt, MeCN; (b) TFA, CH2Cl2; (c) 2-Cl-Trityl Resin, i-Pr2NEt, CH2Cl2-DMF; (d) Piperidine, DMF; (e) HBTU,
HOBt, i-Pr2NEt, DMF.

H.-C. Zhang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2199–2203
2201
Scheme 3. (a) p-NO2C6H4OCOCl, i-Pr2NEt, CH2Cl2, ꢀ20 ꢄC to 23 ꢄC; (b) R2R3NH, HCHO, 1,4-dioxane-HOAc (4:1); (c) Pd(PPh3)4, dimedone,
THF; (d) R6NH2 (12), HOBt, DIC, DMF, 45 ꢄC; (e) TFA, CH2Cl2, anisole.
converted with p-nitrophenyl chloroformate and
i-Pr2NEt at ꢀ20 ꢄC to a p-nitrophenyl carbamate inter-
mediate, which was coupled with resin-bound dipeptide
amine 6 to cleanly afford urea 9 (Scheme 3). Mannich
reaction of resin 9 with an amine and formaldehyde
using 1,4-dioxane–HOAc (4:1) proceeded smoothly at
23 ꢄC to provide 10. Removal of allyl protecting group
in 10 by using Pd(PPh3)4/dimedone/THF afforded the
key resin-bound precursor 11 for introducing diverse
functionalities at the C-terminus. Unfortunately, the
coupling of resin 11 with amine 12 under common cou-
pling conditions, such as DCC/HOBt in DMF, resulted
in incomplete reaction, and other reagents, such as
HBTU/HOBt, BOP-Cl/NMM, or HATU/HOAt, were
unsatisfactory too. Complete coupling was ultimately
achieved by using DIC/HOBt in DMF at 45 ꢄC. The
coupled products were then released from the resin by
treatment with 30% TFA in CH2Cl2 to give target
compounds 13 in ca. 35–40% overall yield from resin 4.
provided target compounds in adequate amounts
(starting with higher loading resin) with purities >85%,
as determined by reversed-phase HPLC.
Biological evaluation (vide infra) of the compounds
revealed that replacement of the benzyl group at the C-
terminus in 1 with a basic functionality, such as an
amine, could significantly improve the binding affinity
to PAR-1 and the potency in TRAP-6-induced platelet
aggregation. Given that a basic amine group is already
present at the C-terminus in these new analogues, we
wondered if the basic residue at R5 position, originally
believed to be critical for PAR-1 activity, would still be
needed. Therefore, we developed a complementary 2-Cl-
trityl resin-based solid-phase method (Scheme 4) to fur-
ther optimize the series, by modifying R5 position in 17
with various functionalities. Mono-attachment of
1,2-diaminoethane or 1,3-diaminopropane onto the
2-Cl-trityl resin provided resin-bound amine 14, which
was coupled with Fmoc-protected amino acid 15 in the
presence of HBTU/HOBt in DMF to afford 16. Follow-
ing the procedures described in Schemes 1–3, 16 was
deprotected with piperidine, coupled with another Fmoc-
protected amino acid 5, deprotected again with piper-
idine, condensed with aminoindole 8 via a urea linkage,
By using this 7-step solid-phase method, analogues of
RWJ-56110 (1) were constructed, with diverse modifi-
cations at the C-terminus (R6 in 13). This method
turned out to be more robust than our earlier one that
employed the Sieber or Tentagel S AM resin;7b thus, it
Scheme 4. (a) DMF; (b) HBTU, HOBt, i-Pr2NEt, DMF; (c) Piperidine, DMF; (d) 5, HBTU, HOBt, i-Pr2NEt, DMF; (e) 8, p-NO2C6H4OCOCl,
i-Pr2NEt, CH2Cl2, ꢀ20 ꢄC to 23 ꢄC; (f) R2R3NH, HCHO, 1,4-dioxane-HOAc (4:1); (g) TFA, CH2Cl2, anisole.

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H.-C. Zhang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2199–2203
reacted with an amine and formaldehyde to form a
Mannich base, and cleaved from the resin with TFA/
CH2Cl2 to provide the desired targets, 17. Similar to the
approach described in Schemes 1–3, this solid-phase
synthesis provided products in good yields (50–60%
overall from resin 14) with excellent purity (>85%).
peptide mimetics, bearing a basic amine at the C-termi-
nus, that have high affinity for the thrombin receptor
(PAR-1). The ability of these compounds to block
TRAP-6-induced platelet aggregation generally corre-
lated well with PAR-1 affinity, though the potency
against thrombin-induced platelet aggregation did not
correlate as well, perhaps, because of competition with
the tethered ligand. These high-affinity PAR-1 ligands
should serve as useful tools to further address the
physiological roles of PAR-1 and its family members.
Compounds prepared via the solid-phase methods were
tested for competitive binding versus [3H]-S-(p-F-Phe)-
homoarginine-L-homoarginine-KY-NH2 to PAR-1 on
the membranes of CHRF-288-11 cells.7e We also exam-
ined their inhibition of platelet aggregation induced by
TRAP-6, a-thrombin, collagen, and U46619 (throm-
boxane mimetic). Representative compounds with in
vitro biological data are presented in Table 1.
Acknowledgements
We thank Brenda Poulter and Jack Kauffman for
excellent technical assistance, and Drs. William Hoek-
stra, Michael Greco, Bruce Damiano and Robert Scar-
borough for helpful discussions.
Replacement of the benzyl group at the C-terminal (R6
position) in RWJ-56110 with substituted benzyl (18),
phenethyl (19), and various heteroarylmethyl groups,
such as furylmethyl (20), thienylmethyl (21), pyr-
idylmethyl (22), did not significantly affect PAR-1
binding affinity, nor activity in platelet aggregation
induced by thrombin and TRAP-6; however, selectivity
over collagen and U46619 was generally reduced. Con-
tinued screening of the functionality at the C-terminus
of RWJ-56110 led to the identification of a new series of
amino-containing analogues with a significant improve-
ment of PAR-1 binding affinity, as well as potency in
platelet aggregation.9 For example, 25, an analogue of 1
with the benzyl group at the C-terminus replaced by a
pyrrolidinylethyl group, bound to PAR-1 with excellent
affinity (IC50=70 nM), and inhibited platelet aggrega-
tion induced by TRAP-6 and thrombin with IC50 values
of 50 and 460 nM, respectively. Interestingly, shifting
the urea linkage in 25 from the indole 6-position to the
indole 4-position (26) or 7-position (27)10 resulted in a
dramatic loss of potency in both binding and platelet
aggregation assays. Thus, the orientation between the
dipeptide segment, Mannich base, and benzyl group on
the indole nitrogen is critical for PAR-1 activity, which
is consistent with our ‘three-point model’7e for PAR-1
antagonist design. With an aminoethyl or aminopropyl
group attached to the C-terminus, a basic residue at R5
position is no longer required for potent PAR-1 antag-
onism. In fact, after surveying many functional groups,
both basic or nonbasic, a side chain containing a SMe
or thienyl group turned out to be optimal at the R5
position, in combination with an aminoethyl group at
the C-terminus. For example, 31, where R5 is a
CH2SMe group, had IC50 values of 60 nM each in
PAR-1 binding and TRAP-6-induced platelet aggrega-
tion. Extending the side chain by one carbon further
increased the potency: 32 had an IC50 value of 25 nM,
which is the highest PAR-1 binding affinity observed for
any compound from our peptide mimetic PAR-1
antagonist series. High affinity (IC50=35 nM) was also
observed with a 2-thienylmethyl group at the R5 posi-
tion (34). All of these compounds also effectively
blocked platelet aggregation induced by TRAP-6 and
thrombin.
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9. The parallel improvement of potency against thrombin-
induced platelet aggregation was not seen with these new
analogues. The reason for the difference is not yet understood
but may be related to the tether ligand situation or additional
ligand-receptor interactions when thrombin is used as an ago-
nist. All PAR-1 antagonists reported from other laboratories
have generally not demonstrated parallel activities between
PAR-1 receptor binding, TRAP-6-induced platelet aggrega-
tion, and thrombin-induced platelet aggregation.
10. 5-Position urea attachment was also found to be unfavor-
able in a different series (ref 6c).