Discovery of potent orally active thrombin receptor (protease activated receptor 1) antagonists as novel antithrombotic agents

Article abstract of DOI:10.1021/jm0502236

Structurally novel thrombin receptor (protease activated receptor 1, PAR-1) antagonists based on the natural product himbacine are described. The prototypical PAR-1 antagonist 55 showed a K_i of 2.7 nM in the binding assay, making it the most po

Full text of DOI:10.1021/jm0502236

5884
J. Med. Chem. 2005, 48, 5884-5887
Letters
agent is likely to have less bleeding liability than the
currently existing antithrombotic drugs. A PAR-1 an-
tagonist may also inhibit thrombin mediated proinflam-
matory and proliferative processes in vascular endo-
thelial cells and smooth muscle cells, offering additional
cardiovascular utility in the treatment of atherosclerosis
and restenosis.14,15
The cellular activation of thrombin is mediated by a
unique tethered ligand mechanism in which thrombin
selectively cleaves the extracellular domain of the
receptor at Arg41-Ser42. The newly unmasked amino
terminus binds intramolecularly to the proximal recep-
tor eliciting transmembrane signaling.16-19 There were
several reports that strongly suggest the potential
utility of a PAR-1 antagonist for the treatment of
thrombotic disorders.20-26 These studies, while provid-
ing encouraging proof-of-concept for the antithrombotic
effect of PAR-1 antagonism, employed modestly potent
PAR-1 antagonists that required iv or subcutaneous
administration of the drug.
We report the discovery of high affinity, orally active,
low molecular weight non-peptide PAR-1 antagonists
based on the natural product himbacine (1).27,28 One of
the synthetic analogues of himbacine, (()-12, was
identified in high-throughput screening as a lead for the
PAR-1 antagonist program. The synthesis outlined in
Scheme 1 employs a highly diastereoselective intra-
molecular Diels-Alder (IMDA) reaction to generate the
tricyclic intermediate 9 in a protocol that was adapted
from the total synthesis of himbacine.27 The synthesis,
which was originally carried out in the racemic form,
commences with commercially available 3-butyn-2-ol,
which was O-protected to give 2 and subsequently
elaborated to the pentynoic acid benzyl ester 3 as
described. Esterification of 3 with dienoic acid 4 and
subsequent Lindlar reduction of the triple bond gave
the IMDA precursor 6. Thermal cyclization of 6 gave
65% of the required tricyclic carboxylic ester 8 after a
brief in situ treatment with DBU to epimerize the
stereogenic center R to the lactone carbonyl group.
Catalytic hydrogenation of 8 over platinum oxide ef-
fected diastereospecific reduction of the double bond as
well as O-debenzylation to produce the tricyclic car-
boxylic acid 9. The acid chloride generated from 9 was
reduced to the aldehyde 10 by treatment with tributyl-
tin hydride in the presence of palladium.29 Horner-
Wadsworth-Emmons reaction of aldehyde 10 with the
appropriate phosphonate 11 gave the vinylpyridine
derivatives in excellent yields. Halogen or O-triflate
substituted pyridine (e.g., 13) could be further elabo-
rated via palladium catalyzed coupling reactions to
alkyl, aryl, amino, and other substituted pyridine
derivatives.
Discovery of Potent Orally Active
Thrombin Receptor (Protease Activated
Receptor 1) Antagonists as Novel
Antithrombotic Agents
Samuel Chackalamannil,* Yan Xia,
William J. Greenlee, Martin Clasby, Dar`ıo Doller,
Hsingan Tsai, Theodros Asberom,
Michael Czarniecki, Ho-Sam Ahn, George Boykow,
Carolyn Foster, Jacqueline Agans-Fantuzzi,
Matthew Bryant, Janice Lau, and Madhu Chintala
Schering-Plough Research Institute, 2015 Galloping Hill
Road, Kenilworth, New Jersey 07033
Received March 9, 2005
Abstract: Structurally novel thrombin receptor (protease
activated receptor 1, PAR-1) antagonists based on the natural
product himbacine are described. The prototypical PAR-1
antagonist 55 showed a Ki of 2.7 nM in the binding assay,
making it the most potent PAR-1 antagonist reported. 55 was
highly active in several functional assays, showed excellent
oral bioavailability in rat and monkey models, and showed
complete inhibition of agonist-induced ex vivo platelet ag-
gregation in cynomolgus monkeys after oral administration.
Currently available antithrombotic therapy suffers
from low efficacy and side effects mainly associated with
bleeding.1 Additionally, most of the marketed anti-
thrombotic agents are not orally bioavailable.2 Several
efforts to achieve an orally active antithrombotic agent
based on GpIIb/IIIa antagonism failed in clinical trials.3
Therefore, there exists an unmet clinical need for a
potent, orally active antithrombotic agent with a better
safety margin. Inhibition of thrombin mediated platelet
activation represents a unique opportunity to discover
novel antithrombotic agents with potent antiplatelet
effects.
In addition to its fibrin generating properties, throm-
bin activates a number of cell types via proteolytic
activation of specific G-protein-coupled cell surface
receptors known as protease activated receptors
(PARs).4-7 Four PARs are known, among which the
prototypical PAR-1 receptor, also known as thrombin
receptor, is widely distributed on human and monkey
platelets, endothelial cells, and smooth muscle cells.
Thrombin is the most potent activator of platelets, and
thrombin mediated platelet activation plays a critical
role in the pathophysiology of thrombosis.8-10 Activated
platelets bind to fibrinogen, causing platelets to ag-
gregate at the site of cardiovascular injury to form a
thrombus that is further stabilized by a thrombin-
generated fibrin network.11-13 Since PAR-1 antagonists
are expected to be specific for the cellular activation of
thrombin and do not inhibit fibrin generation, such an
In vitro binding studies were carried out using puri-
fied human platelet membrane as PAR-1 source and
tritiated high-affinity thrombin receptor activating
peptide, alanine-p-fluorophenylalanine-arganine-cyclo-
* To whom correspondence should be addressed. Phone: 908-740-
3474. Fax: 908-740-7164. E-mail: samuel.chackalamannil@spcorp.com.
10.1021/jm0502236 CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/24/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5885
Scheme 1a
Scheme 2
groups at C-6 (19-21, 25) also had comparable activity,
whereas branched chain substituents (23, 24) and polar
substituents (26, 27) were less active. Pyridine substi-
tuted at the 6-position with an n-hexyl group (22) was
far less active, and the 6-phenyl derivative 31 was
totally inactive.
In an effort to further characterize the PAR-1 antago-
nist profile of the himbacine series, 12 and 18 were
resolved using chiral HPLC (Scheme 2). The (+)-
enantiomer was found to be approximately 10 times
more active than the (-)-enantiomer in each case. The
absolute stereochemistry of the more active series was
established using an enantiospecific synthesis starting
from (R)-3-butyn-2-ol. Subsequent resolution of a num-
ber of active compounds confirmed that the ent-himba-
cine absolute stereochemistry is favored for PAR-1
antagonism.
In the radioligand binding assay, compound (+)-18a
showed a Ki of 12 nM against PAR-1. In the haTRAP
induced human platelet aggregation assay,31 this com-
pound showed dose-dependent inhibition of aggregation
with an IC50 of 70 nM. This compound was further
evaluated in an ex vivo23 platelet aggregation assay in
conscious cynomolgus monkeys after iv infusion (10 mg/
kg, 30 min). Nearly complete inhibition of platelet
aggregation induced by exogenously added peptide
agonist (haTRAP) to the plasma drawn from the drug
treated group was noted for 2 h. However, this com-
pound showed poor oral bioavailability in rat (f < 3%)
perhaps due to rapid metabolism (iv T1/2 < 1 h).
Having achieved excellent PAR-1 affinity and ex vivo
efficacy, we further turned our attention to generating
compounds with good oral bioavailability. Although C-5
alkyl substitution gave weaker PAR-1 affinity than the
corresponding C-6 alkyl substitution (17 vs 12), the C-5
methoxy substituted derivative 33 showed modest ac-
tivity and the corresponding O-benzyl analogue 34
showed excellent PAR-1 inhibition. However, 34 showed
no oral bioavailability presumably because of rapid
metabolism.
C-5 aryl substituted derivatives were examined, and
their data are presented in Table 2. The C-5 phenyl
substituted derivative 35, which had an IC50 of 27 nM,
was the first compound in this series to show a promis-
ing pharmacokinetics profile. In preliminary rat phar-
macokinetics studies, which measured plasma levels of
the parent compound for 4 h after oral administration
of the drug in 20% hydroxypropyl-â-cyclodextrin (HP-
a Reagents and conditions: (a) n-BuLi/THF, BnOCOCl, -78 °C;
(b) DOWEX/CH3OH; (c) 4, 4-pyrrolidinylpyridine, DCC/CH2Cl2; (d)
H2, Lindlar/THF; (e) o-xylene, 185 °C; (f) DBU; (g) H2, PtO2/
CH3OH; (h) (i) SOCl2/toluene, 80 °C, (ii) Bu3SnH, (Ph3P)4Pd; (i)
11, n-BuLi(hex)/THF, 0 °C; (j) (i) PdCl2(dppf), KOAc, diboron
pinnacol ester/dioxane 80 °C, (ii) Ar-Br, PdCl2 (dppf), K3PO4, 80
°C.
Table 1. SAR of Non-Aryl Substituted Pyridine Derivatives
(Compound Type A)
IC50
IC50
compd
(()-12 6-CH3
(()-14
(()-15 3-CH3
(()-16 4-CH3
(()-17 5-CH3
(()-18 6-Et
(()-19 6-vinyl
(()-20 6-n-Pr
(()-21 6-n-Bu
R
(nM)a
compd
R
(nM)a
300
(()-25 6-cy-Pr
(()-26 6-NHCH3
(()-27 6-CH2OH
210
1250
1500
H
4000
>5000
2100
1100
85
(()-28 6-CH2OCH3 850
(()-29 6-CH2Ph
(()-30 6-OCH3
(()-31 6-Ph
900
inactive
inactive
3681
325
150
250
(()-32 5-Bn
143
(()-33 5-OCH3
(()-34 5-OBn
(+)-35 5-Ph
(()-22 6-n-hex 3500
19
27
(()-23 6-i-Pr
(()-24 6-i-Bu
725
550
a PAR-1 binding assay ligand: [3H]haTRAP, 10 nM (Kd ) 15
nM).30
hexylalanine-homoarganine-[3H]phenylalanine amide
([3H]haTRAP, Kd ) 15 nM), as ligand as reported
previously.30 This assay, which was carried out in 96-
well plates, tolerates long incubation time and up to 5%
of DMSO concentration, which was important for solu-
bilizing compounds of low aqueous solubility.
Initial SAR studies were directed at optimizing the
substitution pattern on the pyridine ring. An examina-
tion of Table 1 indicates that the presence of an alkyl
group at the C-6 position of the pyridine ring (12, 18,
20-24) is preferred over alkyl groups at other positions.
The monosubstituted pyridine derivative 14 and the
C-3, C-4, and C-5 substituted derivatives (15-17) were
less active. An ethyl substituent at the 6-position (18)
gave the best PAR-1 affinity. Other small hydrophobic
BCD), modest plasma levels were detected (AUC0-4h
917 nM‚h; Cmax ) 453 nM). The limited plasma levels
were presumed to be due to the facile metabolism of the
)

5886 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Letters
Table 2. SAR of 5-Aryl Substituted Pyridine Derivatives
(Compound Type B)
IC50
rat PKb
AUC, Cmax
compd
Ar
(nM)a
(+)-35
(()-36
(()-37
(()-38
(()-39
(()-40
(+)-41
(+)-42
(+)-43
(+)-44
(+)-45
(+)-46
(+)-47
(+)-48
(+)-49
(+)-50
(+)-51
(+)-52
(+)-53
(+)-54
(+)-55
Ph
27
325
204
467
1000
>300
14
46
44
11
22
26
35
10
25
25
13
19
28
100
11
917, 453
ND
ND
ND
ND
(p-CH3)-phenyl
(p-OCH3)-phenyl
(p-F)-phenyl
(p-Cl)-phenyl
(p-CF3)-phenyl
(o-CH3)-phenyl
(o-CF3)-phenyl
(o-CO2Et)-phenyl
(o-OCH3)-phenyl
(o-F)-phenyl
ND
2805, 1222
2350, 1004
ND
413, 419
2467, 1077
4285, 1785
3553, 1516
4112, 1457
ND
Figure 1. Inhibition of ex vivo haTRAP-induced platelet
aggregation by 55 in cynomolgus monkeys (n ) 3) after oral
administration at 1, 3, and 10 mg/kg.
(o-Cl)-phenyl
(m-F)-phenyl
(m-Cl)-phenyl
(m-Br)-phenyl
(m-CN)-phenyl
(m-CH3)-phenyl
(m-iPr)-phenyl
(m-OCH3)-phenyl
(m-SO2NH2)-phenyl
(m-CF3)-phenyl
ND
oral dose was 1.16 µM, and the half-life was 3.2 h
following intravenous administration. The oral bioavail-
ability of 55 in cynomolgus monkey was 50%, and the
half-life was 12.4 h after intravenous administration.
The effect of 55 on haTRAP induced ex vivo platelet
aggregation in whole blood in conscious, fasted cyno-
molgus monkeys was determined (Figure 1). After oral
administration of the drug, blood samples were collected
at various intervals and aggregation response to 1 µM
haTRAP was performed in a whole blood aggregometer.
The compound showed dose-dependent inhibition of
platelet aggregation up to the time point measured.
While the aggregation response to 1 mg/kg showed some
recovery at the 6 h time point, aggregation inhibition
observed for the 3 and 10 mg/kg groups was sustained
throughout the experiment.
Compound 55 did not affect clotting parameters (PT,
prothrombin time; APTT, activated partial thrombo-
plastin time), confirming that its mechanism of action
is not by active site inhibition of thrombin or other
coagulation proteases. The compound was selective
against a number of GPCRs, ion channels, and receptors
that it was tested against at the CEREP laboratories
and was inactive in the PAR-2 and PAR-4 functional
assays.
331, 380
258, 289
12, 20
ND
6116, 2300
a PAR-1 binding assay ligand: [3H]haTRAP, 10 nM (Kd ) 15
nM).30 b Compounds were dosed in 20% HPBCD. AUC measure-
ments are given in nM‚h and Cmax in nM.
unsubstituted phenyl ring. In an effort to further
optimize the binding and pharmacokinetics properties
of 35, substitutions on the phenyl ring were examined.
As the data in Table 2 indicate, para substitution of the
phenyl ring of 35 resulted in weaker PAR-1 binding
(36-40). However, several ortho and meta substituted
phenyl compounds showed excellent PAR-1 affinity.
More importantly, several of these compounds showed
improved pharmacokinetics parameters in the rat phar-
macokinetics screening model. Compounds with halo-
gens at the ortho and meta positions in general showed
excellent PAR-1 binding and good plasma levels in rats
after oral dosing (e.g., 45-48). The m-(trifluoromethyl)-
phenyl derivative 55 had the best overall profile in this
group with an IC50 of 11 nM against PAR-1, an AUC of
6116 nM‚h, and Cmax of 2300 nM in the rat pharmaco-
kinetics model.
In the radioligand binding assay, 55 showed a Ki of
2.7 nM against PAR-1, making it the most potent PAR-1
antagonist reported to date. Scatchard plots of satura-
tion binding in the presence and absence of 55 were
consistent with a competitive binding profile to the PAR-
1. The effect of 55 on aggregation responses to thrombin
(1 U/mL), haTRAP (1 µM), ADP (20 µM), and collagen
(5 µg/mL) in human platelet rich plasma (PRP)21 was
evaluated in a comparative experiment. Compound 55
showed robust inhibition of thrombin-induced platelet
aggregation with an IC50 of 44 nM and haTRAP induced
platelet aggregation with an IC50 of 24 nM, whereas it
showed no inhibition of ADP and collagen induced
aggregation of platelets. In the thrombin induced cal-
cium transient assay32 in human coronary artery smooth
muscle cells, this compound showed a Kd of 2.6 nM. The
compound inhibited thrombin-induced thymidine incor-
poration32 in human coronary artery smooth muscle
cells with a Ki of 13.0 nM. Detailed pharmacokinetics
parameters of 55 were determined in rat and cynomol-
gus monkey models. In rat, 55 showed an oral bioavail-
ability of 30% at a dose of 10 mg/kg. The Cmax following
In summary, we have reported here the first instance
of orally active PAR-1 antagonists that are also highly
potent. The prototypical PAR-1 antagonist 55 (SCH
205831) with a Ki of 2.7 nM was more potent than any
PAR-1 antagonists reported to date in receptor binding
and several functional assays. The unique properties of
this series are further underscored by the excellent oral
bioavailability of 55 in rat and monkey models, excellent
potency against thrombin and haTRAP induced human
platelet aggregation, and the sustained and complete
ex vivo platelet aggregation inhibition in cynomolgus
monkey model after oral administration. We believe that
the high potency of 55 as an antiplatelet agent is due
to its tight binding to the thrombin receptor where it
effectively competes against the tethered ligand that
enjoys considerable entropic advantage over an incom-
ing antagonist. Further mechanistic and in vivo studies
of these compounds will be published.
Acknowledgment. The authors thank Drs. Cathe-
rine Strader, John Piwinski, Michael Graziano, Ashit
Ganguly, John Hunter, Ronald White, and Ted Sybertz

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