Design, synthesis and biological evaluation of non-peptide PAR1 thrombin receptor antagonists based on small bifunctional templates: Arginine and phenylalanine side chain groups are keys for receptor activity

Article abstract of DOI:10.1007/s00726-009-0306-z

In the present study, we report the synthesis and biological evaluation of a series of new non-peptide PAR₁ mimetic receptor antagonists, based on conformational analysis of the S₄₂FLLR₄₆ tethered ligand (TL) sequence of PAR₁. These compounds incorporate the key pharmacophore groups in the TL sequence, guanidyl, amino and phenyl, which are essential for triggering receptor activity. Compounds 5 and 15 (50-100 μM) inhibited both TFLLR-amide (10 μM) and thrombin-mediated (0.5 and 1 U/ml; 5 and 10 μM) calcium signaling in a cultured human HEK cell assay.

Full text of DOI:10.1007/s00726-009-0306-z

Amino Acids (2010) 38:985–990
DOI 10.1007/s00726-009-0306-z
ORIGINAL ARTICLE
Design, synthesis and biological evaluation of non-peptide PAR1
thrombin receptor antagonists based on small bifunctional
templates: arginine and phenylalanine side chain groups are keys
for receptor activity
Maria-Eleni Androutsou Æ Mahmoud Saifeddine Æ
Morley D. Hollenberg Æ John Matsoukas Æ
George Agelis
Received: 20 February 2009 / Accepted: 9 May 2009 / Published online: 31 May 2009
Ó Springer-Verlag 2009
Abstract In the present study, we report the synthesis
and biological evaluation of a series of new non-peptide
PAR1 mimetic receptor antagonists, based on conforma-
tional analysis of the S42FLLR46 tethered ligand (TL)
sequence of PAR1. These compounds incorporate the key
pharmacophore groups in the TL sequence, guanidyl,
amino and phenyl, which are essential for triggering
receptor activity. Compounds 5 and 15 (50–100 lM)
inhibited both TFLLR-amide (10 lM) and thrombin-med-
iated (0.5 and 1 U/ml; 5 and 10 lM) calcium signaling in a
cultured human HEK cell assay.
2008; Nystedt et al. 1995; Kahn et al. 1998). The serine
proteinase, thrombin, cleaves and activates cellular PAR1
in many pathophysiological settings associated with
hemostasis, tissue injury, tumor invasion and the prolifer-
ation of vascular smooth muscle and tumor cells. Thrombin
cleaves the extracellular N-terminal peptide domain of
human PAR1 between Arg-41 and Ser-42 to expose a
truncated N terminus bearing the peptide activation motif
SFLLR (Matsoukas et al. 1996) that acts as a tethered
receptor-activating ligand (Hollenberg and Compton 2002;
Ramachandran and Hollenberg 2008). Synthetic peptides
containing this amino acid sequence have full PAR1 ago-
nist properties independent of thrombin activation. The
limited stability of peptides due to their peptidic nature
often restricts their medical application.
Keywords Thrombin Á PAR1 receptor Á SFLLR Á
Pharmacophore Á Mimetics
PAR1 peptide and non-peptide inhibitors have been
reported in the last decade, some of which are already in
clinical trials (Seiler and Bernatowicz 2003; Maryanoff
et al. 2003; Chackalamannil et al. 2003; Selnick et al.
2003). Therefore, in the present study, we have attempted
to synthesize a series of alternative new non-peptide PAR1
mimetics (5, 12, 15, 18), based on conformational analysis
of the SFLLR tethered ligand sequence of PAR1 using a
series of templates aimed at inhibiting the cellular actions
of thrombin (Alexopoulos et al. 2004). Structure–activity
relationships (SAR) and amino acid substitutions in com-
bination with NMR studies have determined the specific
role of each amino acid in the native SFLLR sequence
(Natarajan et al. 1995).
Introduction
Receptors that mediate thrombin action are attractive
drug discovery targets because of their involvement in
cardiovascular pathophysiology (dysregulation of platelet
aggregation and endothelial cell function) (Ogletree et al.
1994; Andrade-Gordon et al. 1999). The cellular actions of
thrombin are, in large part, caused by the activation of
proteinase-activated receptors (PARs) 1, 3 and 4 (Hollen-
berg and Compton 2002; Ramachandran and Hollenberg
M.-E. Androutsou Á J. Matsoukas Á G. Agelis (&)
Department of Chemistry, University of Patras,
26500 Patras, Greece
e-mail: aggelisgeorge@hotmail.com;
imats@chemistry.upatras.gr; minws13@hotmail.com
These synthetic compounds, which incorporate the
essential tethered ligand pharmacophore groups (Alexo-
poulos et al. 1999) guanidyl, amino and phenyl a sub-
stituents built onto four different small bifunctional
templates (piperidine, azetidine, cyclohexane and indole)
(Zhang et al. 2003), were subsequently tested for biological
M. Saifeddine Á M. D. Hollenberg Á J. Matsoukas
Department of Physiology and Pharmacology,
University of Calgary, Calgary T2N 4N1, Canada
123

986
M.-E. Androutsou et al.
activity in a PAR1-dependent calcium signaling assay (Ka-
wabata et al. 1999). Both compounds 5 and 15 (50–200 lM)
were able to block thrombin and TFLLR-NH2-triggered
elevations in intracellular calcium via PAR1. These active
peptide mimetic antagonists show promise for the develop-
ment of PAR1-targeted agents as alternatives to those cur-
rently being considered for use in cardiovascular and cancer
pathologies.
n-hexane (2 9 10 mL). A solution of Fmoc-Lys(Boc)-OH
(0.655 g, 1.4 mmol), HOBt (0.141 g, 1.05 mmol)/DIC
(0.12 mL, 0.77 mmol) in DMF was added to the pre-
swollen resin and the mixture was stirred at RT. After 3 h,
the resin was filtered and washed as previously described.
Cleavage from the resin using HFIP/DCM (3:7, 12 mL) for
2 h at RT, filtration, concentration of the solvent and pre-
cipitation by DEE, gave fully protected dipeptide as a
white solid. The resulting solid (0.65 mmol) was dissolved
in dry DCM, preactivated with HOBt (0.14 g, 1.04 mmol)/
DCC (0.145 g, 0.715 mmol) for 30 min at 0°C and a
solution of N-benzyl-5-amino indole (0.414 g, 0.65 mmol),
DIPEA (0.51 mL) in DCM was added. After 7.5 h at RT
the suspension was filtered off, the filtrate was diluted in
DCM, washed successively with NaHCO3 (1 9 5% w/v),
citric acid (3 9 10% w/v), H2O (93), dried over Na2SO4
and concentrated in vacuo. Precipitation by DEE afforded
crude 4 as a violet solid. Deprotection of Boc, Fmoc groups
occurred with TFA/DCM (3:7) in the presence of scav-
engers TMSBr/anisole/TES for 1.5 h and piperidine/DCM
(1:4) for 2 h. The solvents were removed and the crude 5
was isolated by trituration with DEE, purified by pre-
parative RP-HPLC and lyophilized to furnish final product
5 as a pure violet solid (0.25 g, 0.49 mmol, overall 70%).
A similar methodology was applied for the synthesized
compounds 11, 14, 17. All products were characterized by
1H NMR and ESI-MS.
Materials and methods
General remarks
All of the solvents and reagents were obtained commer-
cially and used as such, unless noted otherwise. TLC was
performed on silica gel 60 F254 plates (Merck, Germany).
HPLC was performed with Waters (Milford, MA, USA)
system 600 controller equipped with Millenium 2.1 oper-
ating system using a Waters 996 diode array UV/vis
detector. Purification of final products was performed with
Waters Preparative HPLC (Waters Prep LC Controller)
using SunFire Prep C18 column (50 9 100 mm) with
5 lm packing material. Separation was performed with
stepped linear gradient from 5 to 60% AcN over 45 min at
1
a flow rate of 12 mL/min. H NMR spectra were recorded
using Brucker 400 MHz spectometer. All chemical shifts
(d) were recorded as ppm and all samples were dissolved in
DMSO-d6. The coupling constants (J) were expressed in
Hz. ESI-MS was performed with an electrospray platform
(Waters) equipped with a Masslynx NT 2.3 operational
system. Synthetic PAR1-activating peptide, TFLLR-NH2
([95% pure by HPLC and mass spectral criteria) was
obtained from the University of Calgary Peptide Synthesis
Facility (peplab@ucalgary.ca). Human plasma thrombin
(approx. 3,200 U/mg) was from Calbiochem (San Diego,
CA, USA).
1
Data for compound 5: H NMR (400 MHz; DMSO-d6)
dppm = 7.48–7.12 (m, 13H, Ar), 6.58 (d, 1H, J = 3.2 Hz),
5.37 (s, 2H), 4.96 (m, 2H), 3.57 (m, 1H), 3.23–3.12 (m,
2H), 2.67 (m, 2H), 1.78 (m, 2H), 1.46 (m, 2H), 1.29 (m,
2H); ESI-MS, m/z, 516.50 (L ? 1), 518.45 (L ? 3),
519.46 (L ? 4).
General method for the guanylation of primary amine
derivatives 11, 14, 17
TFA salt 14 (0.65 mmol), 1H-pyrazole-1-carboxamide
hydrochloride (0.15 g, 1.04 mmol) and DIPEA (0.44 mL,
2.6 mmol) were dissolved in DMF sufficient to produce a
final concentration approximately 2 M. The reaction mix-
ture was stirred under nitrogen at RT while being moni-
tored by TLC Rf = 0.35 in n-BuOH/AcOH/H2O (4:1:1,
v/v). After 12 h DEE was added to precipitate the crude 15,
which was collected, washed with DEE and dried. Purifi-
cation by preparative RP-HPLC afforded pure 15
(0.53 mmol, 81%). Compounds 12, 18 were prepared by a
similar procedure.
Data for compound 15: 1H NMR (400 MHz; DMSO-d6)
dppm = 8.32, 8.10 and 7.92 (3 br s, 4H), 7.73–7.30 (m, 4H),
4.25–4.11 (m, 3H), 3.06 (q, 2H, J = 6.8 Hz), 2.41–2.20
(m, 2H), 1.99–1.29 (m, 14H); ESI-MS, m/z, 450.07 (L?),
451.39 (M ? 1), 452.46 (M ? 2).
General method for the synthesis of compound 5
2-Chlorotritylchloride resin (1 g, 1.6 mmol Cl-/g of resin)
was left to swell in dry DCM (8 mL) for 30 min, N-Fmoc-
4-fluoro-Phe-OH (0.338 g, 1 mmol), DIPEA (2.5 mmol)
were added and stirred at RT. After 1 h, the suspension was
filtered, washed successively with DCM/MeOH/DIPEA
(17:2:1, v/v) (2 9 10 mL 9 10 min), DMF (3 9 10 mL),
i-Pro (2 9 10 mL), n-hexane (2 9 10 mL) and dried in
vacuo for 24 h at RT. The loading of the amino acid/g of
substituted resin was 0.7 mmol/g, calculated by weight and
amino acid analysis. Removal of Fmoc group was achieved
by the repetitive treatment with piperidine/DMF (1:5,
10 mL) for 10 and 20 min and the resin was thoroughly
washed with DMF (3 9 10 mL), i-Pro (2 9 10 mL),
123

Design, synthesis and biological evaluation of non-peptide PAR1 thrombin receptor
987
Cl
C
Cl
Cl
H
C
H
C
H
H
C
H
N
b, c
a
P
O
NH Fmoc
P
O
C
O
N
C
O
Fmoc
O
CH2
CH2
3
1
2
NH
F
F
Boc
F
d, e
F
f, g
O
H
H
N
O
H
N
H2N
N
N
Fmoc
N
H
O
H
N
O
N
4
NH
Boc
5
NH2
Scheme 1 Reagents and conditions: a N-Fmoc-4-fluoro-Phe-OH,
DIPEA, DCM, 1 h; b piperidine/DMF (1:5), 30 min; c Fmoc-
Lys(Boc)-OH, HOBt/DIC, DMF, 3 h; d HFIP/DCM (3:7), 2 h;
e N-benzyl-5-amino indole, HOBt/DCC, DIPEA, DCM, 0–25°C, 8 h;
f TFA/DCM (3:7), TMSBr/anisole/TES, 1.5 h; g piperidine/DCM
(1:4), 2 h
1
Data for compound 12: H-NMR (400 MHz; CD3OD)
Finally, deprotection of Boc- and Fmoc-protecting groups
with TFA and piperidine solutions in DCM, respectively,
furnished the final product 5 in high yield (overall 70%
based on the calculated substitution of resin 2) (Scheme 1).
A similar methodology was applied for the synthesized
compounds 12, 15, 18 following the general principle of
solid phase organic synthesis-repeated cycles of coupling
and deprotection. In particular, 2-chlorotritylchloride resin
bound analogs 10, 13, 16 were synthesized using Fmoc
methodology. These compounds bear a variety of scaffolds
such as piperidine, azetidine and cyclohexane. Cleavage of
fully protected compounds from the resin followed by
selective deprotection of Boc group in acidic conditions
(TFA/DCM), in the presence of scavengers, afforded 11,
14, 17. Facile transformation of the amino to guanidino
group of 11, 14, 17 using 1H-pyrazole-1-carboxamide
hydrochloride (Bernatowicz et al. 1992) in DMF/DIPEA
and subsequent Fmoc deprotection of the isonipecotic
derivative led to 12, 15 and 18, respectively, in high yields
([80%) (Scheme 2). Purification of synthetic compounds
was performed by preparative RP-HPLC using C18 column
as stationary phase and UV peak detection. The purity of
the lyophilized target final products 5 and 12, 15, 18 was
determined by analytical RP-HPLC, followed by UV
detection at 254 nm. Separations were achieved with a
stepped linear gradient of acetonitrile (0.08% TFA) in
water (0.08% TFA) and identification by ESI-MS and
NMR.
dppm = 7.54–7.07 (m, 4H), 4.92–4.89 (m, 1H), 4.71–4.67
(t, 1H), 4.28–4.09 (t, 2H), 3.8 (s, 2H), 2.67 (t, 2H), 2.28 (m,
2H), 1.86 (m, 1H), 1.54 (m, 2H), 1.42 (m, 2H, H-6); ESI-
MS, m/z, 408.02 (L?), 409.03 (L ? 1).
Data for compound 18: 1H-NMR (CDCl3) dppm = 7.26–
6.98 (m, 4H), 4.41 (t, 1H), 3.91–3.88 (m, 2H,), 3.13–3.01
(m, 6H), 2.62 (t, 2H), 2.38–2.29 (m, 4H), 1.91 (m, 4H),
1.73–1.59 (m, 2H), 1.43 (m, 2H); ESI-MS, m/z, 465.46
(L ? 1), 466.38 (L ? 2).
Results and discussion
Synthesis of mimetics
The synthesis of compound 5 was accomplished using a
combination of solid and liquid phase organic synthesis.
Attachment of the first amino acid Fmoc-Phe(F)-OH to the
2-chlorotritylchloride resin (Barlos et al. 1989) was
achieved by a simple, fast and racemization-free reaction
using a solution of DIPEA in DCM at RT. Removal of
Fmoc protecting group using piperidine/DMF (1:4), fol-
lowed by the coupling of Fmoc-Lys(Boc)-OH in the
presence of activators HOBt/DIC, led to the fully protected
dipeptide 3. Cleavage of 3 from the resin with HFIP/DCM
(3:7) and coupling with N-benzyl-5-amino-indole, in the
presence of HOBt/DCC/DIPEA, afforded derivative 4.
123

988
M.-E. Androutsou et al.
Cl
Cl
Cl
C
H
C
H
N
H
C
a
b, c
O
C
O
R
O
NH Fmoc
P
P
O
7: R = Fmoc-azetidine
8: R = 1,4-dicarboxycyclohexane
9: R = Fmoc-isonipecotic acid
NH
NH
Boc
6
1
Boc
O
C
O
C
O
C
H
C
H
N
H
C
H
N
H
C
H
N
g, h
i
b, d
O
C
O
HO
C
HO
C
7
N
O
N
O
O
N
O
O
NH
NH
HN
NH2
10
12
11
F
F
F
NH2
TFA salt
Boc
O
O
O
C
O
C
O
O
H
C
H
N
H
N
H
H
H
N
H
H
N
H
g, h
b,e
i
HO
C
O
C
O
C
O
N
C
C
C
HO
C
O
C
N
C
8
NH
14
13
15
F
F
F
HN
NH
NH2
NH2
TFA salt
Boc
O
C
O
O
O
O
O
N C
Fmoc
NH
Fmoc
NH
H
C
H
N
H
H
N
H
C
H
N
i, j
g, h
b,f
HO
C
O
N C
NH2
O
C
C
N C
HO
C
O
C
C
9
O
HN
NH
NH
Boc
NH2
F
F
F
TFA salt
17
H2N
16
18
Scheme 2 Reagents and conditions: a Fmoc-Lys(Boc)-OH, DIPEA,
DCM, 1 h; b piperidine/DMF (1:5), 30 min; c N-Fmoc-azetidine or
1,4-dicarboxycyclohexane or Fmoc-isonipecotic acid, HOBt/DIC,
DMF, 3 h; d 4-fluoro-phenylacetic acid, HOBt/DIC, DMF, 2 h;
e 4-fluoro-benzylamine HOBt/DIC, DMF, 3 h; f N-Fmoc-4-fluoro-
Phe-OH, HOBt/DIC, DMF, 3 h; g HFIP/DCM (3:7), 2 h; h TFA/DCM
(1:1), TES, 1 h; i 1H-pyrazole-1-carboxamidine, DIPEA, DMF, 12 h;
j piperidine/DCM (1:4), 2 h
Effects of mimetics on PAR1-mediated calcium
signaling
calcium concentration that could be elevated further by
TFLLR-NH2 or thrombin action. In this setting, concen-
trations of compound 5 at 100 lM still completely blocked
the ability of either thrombin or TFLLR-NH2 to elevate
intracellular calcium further, via PAR1. At lower concen-
trations (5–10 lM), where compound 5 had little or no
effect on the baseline concentration of intracellular cal-
cium, it was still able to block thrombin-mediated eleva-
tions of intracellular calcium via PAR1. The mechanism
whereby these compound 5 stock solutions maintained in
the refrigerator caused an elevation of basal intracellular
calcium at concentrations greater than 25 lM was not
evaluated further, except to verify that the effect was still
present in PAR1–PAR2-desensitized HEK cells, indicating
an action independent of PARs. In contrast, compound 15
on its own had no effect on the resting level of intracellular
The ability of compound 5 to block PAR1 activation was
tested in the routine HEK cell calcium signaling assay in
which the concentration of calcium in the incubation buffer
is 5 mM (Kawabata et al. 1999). As shown in Fig. 1,
pretreatment of HEK cells with compound 5 was able to
block the calcium signal triggered either by thrombin or by
the PAR1-selective agonist, TFLLR-NH2. The data shown
in Fig. 1 were obtained from freshly prepared stock solu-
tions of compound 5 (7–9 mM) dissolved in DMSO. It is
important to point out for future work that when the stock
solution was allowed to stand for several days in the
refrigerator, compound 5 on its own (25–200 lM) caused a
PAR-independent elevation of the steady-state intracellular
123

Design, synthesis and biological evaluation of non-peptide PAR1 thrombin receptor
989
was lower than that of compound 5, because at 25 lM,
compound 15 only partially blocked the thrombin/PAR1-
mediated calcium response, whereas compound 5 blocked
the response to thrombin by abut 80% at this concentration
(Fig. 2). This action of compound 5 was PAR-selective,
because at a concentration of 25 lM under the same con-
ditions where thrombin action was blocked, the PAR1
antagonist did not affect the ability of trypsin to trigger a
calcium signal via PAR2 in the HEK cells (data not shown).
∆
∆
Thrombin
Compound 5
Compound 5
Thrombin
Structure and PAR1-targeted action of mimetics
TFLLR
TFLLR
Time (s)
Herein, the design of non-peptide mimetics that can
interact with and block PAR1 activation was based on the
current knowledge of the interactions of the tethered ligand
with the thrombin receptor. Given that the cyclic analog of
SFLLR (Adang et al. 1994; Moore et al. 1995) observed by
NMR is probably the bioactive species, along with the
observation that certain cyclic peptide derivatives are
biologically active, we were prompted to design molecules
that mimic the conformation adopted by SFLLR. This
cyclic conformation brings the Phenylalanine and Arginine
residues in close proximity. We therefore hypothesized that
replacement of the aliphatic residues of the active peptide
core with different scaffolds would yield novel PAR1-tar-
geted compounds.
Fig. 1 Inhibition of PAR1 calcium signaling by Compound 5 in HEK
cells. The figure shows the increase in HEK cell intracellular calcium
signaling (E530: upward deflection, arrow) caused by the activation of
PAR1 by either thrombin (1 U/ml, open triangle, upper tracings) or
the PAR1-activating peptide (TFLLR-amide, 10 lM, open square,
lower tracings) either without (left-hand tracings) or after (right-hand
tracings) pretreatment of the cells with compound 5 (100 lM, closed
circle). The directions of fluorescence (E530) and time (seconds) are
shown by the inset. The arrows indicate an upward deflection of 1 cm
and a time span of 2 min, respectively
In this report, according to this hypothesis and the
structure-activity profile of the native pentapeptide SFLLR,
different scaffolds (piperidine, azetidine, cyclohexane,
indole) were utilized providing accordingly either struc-
tural flexibility or rigidity. Furthermore, these templates
were selected due to the spatial requirements for displaying
the key substituents in a suitable conformation and offered
favorable synthetic considerations for their attachment.
Ultimately, the mimetics we have prepared could be of
value for the development of PAR1 antagonists in addition
to those that have been developed using a variety of other
approaches (Andrade-Gordon et al. 1999; Zhang et al.
2003; Clasby et al. 2006).
Fig. 2 Concentration–inhibition curve for blocking thrombin-medi-
ated PAR1 calcium signaling in HEK cells by compound 5. Cells were
pretreated with increasing concentrations of compound 5 and the
calcium signal caused by thrombin (0.5 U/ml) via PAR1 activation
was monitored. The inhibition of calcium signaling was expressed as
a percentage (% control) relative to the signal observed in cells that
had not been treated with compound 5. The error bars shown for the
data obtained at 25 lM compound 5 are representative of the
precision of the measurements. The IC50 for compound 5 was about
5 lM
Conclusions
This study describes the synthesis and biological evalua-
tion of thrombin receptor mimetics bearing the essential
phenylalanine/arginine pharmacophore groups of SFLLR
on four different templates (piperidine, azetidine, cyclo-
hexane and indole). Two of the templates yielded com-
pounds (5 and 15) that will be able to block thrombin
activation of PAR1 and that will be of value for further
optimization of non-peptide antagonists of PAR1. The
piperidine and azetidine scaffolds did not work as well as
cyclohexane and indole presumably because these
calcium. Notwithstanding, like compound 5, compound 15
(25–200 lM) was able to reduce PAR1-mediated eleva-
tions in calcium caused by either thrombin (0.5 U/ml;
5 nM) or TFLLR-NH2 (10 lM). The concentration–inhi-
bition curve for the ability of compound 5 to prevent
thrombin (0.5 U/ml)-triggered PAR1 calcium signaling is
shown in Fig. 2. The IC50 for this inhibitory action of
compound 5 was about 5 lM. In this regard, the potency of
compound 15 to block the activation of PAR1 by thrombin
123

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M.-E. Androutsou et al.
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scaffolds are rigid templates that do not allow a proper
orientation of the pharmacophore groups to interact with
the receptor. In particular, azetidine and piperidine are
small rigid moieties in contrast to the flexible cyclohexane
ring which can adopt both chair and boat conformations.
Indole is also a larger scaffold compared to azetidine and
piperidine which bear pharmacophores at the right distance
for triggering activity.
Acknowledgments This research project is co-financed by EU-
European Social Fund (75%) and the Greek Ministry of Develop-
ment-GSRT (25%) with ancillary support from the Canadian Insti-
tutes of Health Research (CIHR: MDH).
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