Discovery and synthesis of a novel series of quinoline-based thrombin receptor (PAR-1) antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.12.042

The design, synthesis, and SAR studies of a structurally novel series of highly potent thrombin receptor (PAR-1) antagonists are described. Compound 30 is a highly potent thrombin receptor antagonist (IC₅₀ = 6.3 nM), a related compound 36 showing efficacy in a monkey ex vivo study.

Full text of DOI:10.1016/j.bmcl.2005.12.042

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1544–1548
Discovery and synthesis of a novel series of
quinoline-based thrombin receptor (PAR-1) antagonists
Martin C. Clasby,a,* Samuel Chackalamannil,a Michael Czarniecki,a Dario Doller,a
Keith Eagen,a William J. Greenlee,a Yan Lin,a Hsingan Tsai,a Yan Xia,a Ho-Sam Ahn,b
Jacqueline Agans-Fantuzzi,b George Boykow,b Madhu Chintala,b Carolyn Foster,b
Matthew Bryantc and Janice Lauc
aCentral Nervous System and Cardiovascular Chemical Research, Schering-Plough Research Institute,
2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
bCardiovascular and Metabolic Disease Research, Schering-Plough Research Institute,
2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
cDrug Metabolism and Pharmacokinetics Research, Schering-Plough Research Institute,
2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Received 13 October 2005; revised 5 December 2005; accepted 9 December 2005
Available online 27 December 2005
Abstract—The design, synthesis, and SAR studies of a structurally novel series of highly potent thrombin receptor (PAR-1) antag-
onists are described. Compound 30 is a highly potent thrombin receptor antagonist (IC50 = 6.3 nM), a related compound 36 show-
ing efficacy in a monkey ex vivo study.
Ó 2005 Elsevier Ltd. All rights reserved.
Thrombin is a multi-functional protease involved in
hemostasis and wound healing.1 It plays a key role in
the coagulation cascade by converting fibrinogen to fi-
brin which is crosslinked to form a clot.2 In addition,
thrombin activates various cell types including platelets,
leukocytes, endothelial cells, and vascular smooth mus-
cle cells. The cellular activity of thrombin is mediated
via proteolytic activation of specific cell surface recep-
tors known as protease-activated receptors (PARs).3–7
Four PAR subtypes are known: PAR-1, PAR-3, and
PAR-4 are activated by thrombin, while PAR-2 is acti-
vated by trypsin and tryptase. PAR-1 is the most impor-
tant of these receptors and the mechanism by which
thrombin activates PARs such as PAR-1 is unique.
Thrombin binds to the receptor through its exo-site
anion binding region, and it cleaves the extracellular do-
main at Arg41-Ser42 to reveal an amino terminus which
binds intramolecularly to the receptor.8–11 Thrombin
receptor-activating peptides (TRAPs),12 designed to
mimic the amino terminus of the activated receptor,
were shown to elicit a functional agonist response. Mu-
tant receptors that do not contain cleavable functional-
ity in the extracellular domain are unresponsive to
thrombin but are activated by TRAPs.10
Inhibition of platelet activation by a PAR-1 antagonist
is hypothesized to have significant utility in the treat-
ment of thrombotic disorders such as platelet-driven
arterial thrombosis. Additionally, a PAR-1 antagonist
may be effective in treating vascular disorders such as
atherosclerosis and restenosis by inhibiting proinflam-
matory and proliferative processes that are mediated
by PAR-1 activation in endothelial and vascular smooth
muscle cells. Because a thrombin receptor antagonist
would only inhibit the cellular effects of thrombin and
not its enzymatic role in fibrin generation, this mecha-
nism of action would have a reduced bleeding liability
relative to current antithrombotic agents.13,14
Recently, we have reported a structurally novel class of
potent, orally active PAR-1 antagonists15 (shown in
Fig. 1). This study showed that an alkyl substitution
was optimal at the 6-position, but not at the 5-position,
whereas an aryl substitution was optimal at the 5-posi-
Keywords: Thrombin; Receptor; Antagonist; PAR-1; Quinoline.
*
Corresponding author. Tel.: +1 908 740 2244; fax: +1 908 740
7152; e-mail: martin.clasby@spcorp.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.042

M. C. Clasby et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1544–1548
1545
(R)-3-butyn-2-ol, respectively, in three steps.15 Propar-
gylic alcohol 3 was coupled with acid 416 and the prod-
uct hydrogenated using Lindlar’s catalyst to give triene
5, which underwent an intramolecular Diels–Alder reac-
tion at 185 °C to furnish 6. After in situ treatment of 6
with DBU, tricyclic ester 7 was isolated in 66% yield
for the 3 steps. The tricyclic ester 7 was converted to
the acid 8 via hydrogenation which, in turn, was con-
verted to the aldehyde 9 using tributyltin hydride-medi-
ated reduction of its acid chloride derivative.17
Phosphonates were synthesized via treatment of the lith-
ium anion of a substituted 2-methylquinoline with dieth-
ylchlorophosphate. Finally, the targets 12–24 were
synthesized via Horner–Wadsworth–Emmons reaction
of the aldehyde 9 with the appropriate phosphonate.
The N-methyl derivative 25 and the N-oxide 26 were
synthesized by treatment of 12 and 14 with methyl
iodide and m-CPBA, respectively.
O
H
H
H
H
O
H
H
H
H
O
O
Me
Me
N
1
2
2
N
6
Et
5
CF3
PAR-1 IC50 = 11 nM
PAR-1 IC50 = 20 nM
Figure 1. Thrombin receptor antagonists.
tion and not tolerated at the 6-position. In order to ex-
plore the possibility of a synergistic effect between these
positions, and to further refine this lead, we synthesized
a series of quinoline derivatives (Scheme 1).
Compounds 12–26 were screened in the in vitro binding
assay using purified human platelet membranes as a
PAR-1 source and tritiated high affinity thrombin recep-
tor activating peptide, alanine-p-fluorophenylalanine-
arganine-cyclohexylalanine-homoarganine-[3H]phenyl-
alanine amide, ([3H]haTRAP) as the ligand as previous-
ly reported.15,18 Representative SAR is shown in Table
1. These initial results showed that substitution at the
6-position of the quinoline ring with an electron-donat-
ing group gave the most potent compounds. The
6-methoxy-substituted compound 14 gave comparable
binding data (IC50 = 15 nM) to lead structures 1 and 2
thus supporting our initial hypothesis. Substitution,
however, at other positions or quaternization of
the pyridyl nitrogen led to substantially less active
compounds. Although not shown in the table, removal
of the olefin linker via hydrogenation led to at least a
10-fold loss in activity.
Propargylic alcohol 3 was readily available in both race-
mic and enantiopure forms from ( )-3-butyn-2-ol or
O
O
H
HO2C
HO
a,b
c
O
O
4
H
H
O
Me
BnO
Me
BnO
Me
BnO
5
6
O
O
3
O
O
H
O
H
H
H
H
H
d
e
f,g
O
O
O
H
O
H
H
O
H
H
H
O
Me
BnO
Me
Me
7
8
9
HO
O
H
H
Me
P(O)(OEt)2
O
Encouraged by these results, we then set about further
exploring the SAR of the 6-position of the quinoline
moiety. A representative synthesis of various analog types
is outlined in Schemes 2 and 3. Phenol 27 was protected
as its TIPS ether and converted to the phosphonate 29
h
N
H
H
i
Me
N
R
R
N
6
10
11
12-24
4
8
R
Table 1. SAR of quinoline derivatives of 1
k
Compound
R group
PAR-1 IC50 (nM)a
O
H
H
H
H
j
( )12
( )13
( )14
( )15
( )16
( )17
( )18
( )19
( )20
( )21
( )22
(+)23
(+)24
( )25
( )26
—
6-Me
60
100
O
H
H
H
O
6-OMe
6-Cl
6-F
15
155
O
Me
H
Me
730
150
O
N
7-Me
8-OMe
8-Me
Me
700
N
R
390
4-OMe
4-OBn
6-OMe, 5-Cl
6-OCF3
5,6-Dimethoxy
—
Inactiveb
Inactiveb
800
R
6
25
26
Scheme 1. Synthesis of thrombin receptor antagonists. Reagents and
conditions: (a) DCC, 4-pyrrolidinylpyridine, CH2Cl2, 63%; (b) Lindlar
Catalyst, TEA, H2, THF; (c) xylenes, 185 °C; (d) DBU, THF, 66% 3
steps; (e) PtO2, H2, MeOH, 75%; (f) (COCl)2, DMF (cat), CH2Cl2; (g)
Bu3SnH, Pd(Ph3P)4, PhMe, 0 °C to rt, 88% 2 steps; (h) t-BuLi,
(EtO)2POCl, THF, À78 °C to rt, 68–96%; (i) n-BuLi (0 °C), then 9 0 °C
to rt, THF, 34–94%; (j) MeI, CH2Cl2, 99%; (k) mCPBA, CH2Cl2.
Inactiveb
50
Inactiveb
620
6-OMe
a PAR-1 binding assay ligand: [3H]haTRAP, 10 nM (Kd = 15 nM).15,18
b Inactive at a concentration of 1000 lM.

1546
M. C. Clasby et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1544–1548
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
O
P(O)(OEt)2
N
O
O
Me
Me
Me
N
N
a
c, d
b
e
f
9
N
N
N
29
OTIPS
28
27
OH
OTIPS
32-34
30
31
Ar
OTf
OH
O
j
NHBoc
g
i
Br
NR1R2
HO
R
R
44
37
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
H
Me
Me
Me
Me
Me
h
k
N
O
N
N
O
N
N
O
35
O
O
O
36
45
38-43
46
NH2
NHBoc
NR1R2
OEt
OH
O
O
R
R
Scheme 2. Reagents and conditions: (a) TIPSCl, imidazole, DMF, 88%; (b) LDA, (EtO)2POCl, THF, À78 °C to rt, 82%; (c) n-BuLi (0 °C), then 9
0 °C to rt, THF; (d) TBAF, THF, 0 °C, 65% 2 steps; (e) Tf2O, TEA, CH2Cl2, 99%; (f) Pd(Ph3P)4 (5%), K2CO3, ArB(OH)2, PhMe/EtOH/H2O (4:2:1);
(g) ethyl bromoacetate, NaH, DMF; (h) LiOH, MeOH/H2O (3:1); (i) R = H, NaH, DMF; R = Me, NaH, 1,4-dioxane, 100 °C; (j) Ph3P, DEAD,
THF; (k) TFA, CH2Cl2.
O
H
H
H
H
O
H
H
H
H
O
H
H
H
H
O
P(O)(OEt)2
N
O
N
O
N
Me
Me
Me
N
N
c
a
b
e
d
47
N
NH2
9
N
N
N
Si Si
Si Si
HSi HSi
49
Si Si
NH
50
R
52
NH2
48
53-57
O
51
Scheme 3. Reagents and conditions: (a) (Ph3P)3RhCl, 120 °C, 41%; (b) LDA, (EtO)2POCl, THF, À78 °C to rt, 44%; (c) n-BuLi (0 °C), then 9 0 °C to
rt, THF, 77%; (d) 1 N HCl, THF/MeOH (1:1), 80%; (e) RCOCl, TEA, CH2Cl2, 0 °C.
in the usual manner. Horner–Wadsworth–Emmons reac-
tion of 29 with the aldehyde 9 followed by fluoride-medi-
ated deprotection gave the key intermediate 30. The 6-
aryl-substituted analogs 32–34 were synthesized via Suzu-
ki reaction of the triflate 31. The O-linked analogs 36, 38–
43, 45, and 46 were synthesized via alkylation or Mitsun-
obu chemistry followed by derivatization when appropri-
ate. The N-linked derivatives 53–57 provided more of a
challenge. Finding an appropriate protecting group for
the aminoquinoline 47 proved to be a problem since the
group would have to survive strongly basic reaction con-
ditions. Additionally, protecting group removal via cata-
lytic hydrogenation was not feasible because we required
an olefin linker in the final targets for activity.
removal under mildly acidic conditions. The key inter-
mediate 52 was converted to amides 53–57 via treatment
with various acid chlorides in the presence of
triethylamine.
The PAR-1 binding data for this series of compounds are
shown in Table 2. The most potent compound was the
phenol 30 with a PAR-1 IC50 of 6.3 nM. Other com-
pounds of note were 36, 40, 52, and amide 55 all with
IC50 in the 30–50 nM range. We observed that increasing
steric bulk within a subseries generally led to a loss in
activity; for example, 40 and 41 in the hydroxyacetamide
subseries and 55, 57, and 56 in the quinolinylalkylamide
subseries. For proof-of-principle studies we used an
ex vivo model of platelet aggregation in cynomolgus
monkeys. After administration of the drug, blood sam-
ples were collected at various intervals and aggregation
The BSB (benzostabase) group19 was eventually chosen
because it possessed the desired stability and ease of

M. C. Clasby et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1544–1548
1547
Table 2. SAR of quinoline 6-position
Table 2 (continued)
Compound
Z
PAR-1 IC50 (nM)a
O
H
H
H
N
O
Me
(+)55
28
2151
219
H
H
Me
O
Me
Me
H
N
(+)56
(+)57
( )58
(+)59
N
Z
Me
O
O
H
N
Compound
Z
PAR-1 IC50 (nM)a
6.3
O
Me
125
(+)30
OH
OBn
1250
(+)32
(+)33
(+)34
(+)36
(+)38
(+)39
300
50
CF3
a PAR-1 binding assay ligand: [3H]haTRAP, 10 nM (Kd = 15 nM).15,18
b Inactive at a concentration of 1000 lM.
S
response to 1 lM of haTRAP was measured in a whole
blood aggregometer. When tested in our ex vivo model
of platelet aggregation, compound 36 showed almost
complete inhibition 2 h after IV dosing in cynomolgus
monkeys. It was not active when dosed orally due to
poor oral bioavailability.20 Surprisingly, compounds 14
and 30 did not show substantial activity in the ex vivo
model of platelet aggregation, which is presumably due
to rapid conjugation and clearance.
S
300
29
O
O
OH
O
O
1000
800
O
NH2
NH2
In conclusion, we have discovered a series of potent
PAR-1 receptor antagonists. The most potent com-
pound up to this point is 30 with an IC50 of 6.3 nM.
A closely related compound, 36, was shown to have
activity in an ex vivo platelet aggregation model. Efforts
toward producing orally active antagonist in this series
will be reported in due course.
O
Me
O
Me
(+)40
(+)41
(+)42
25
500
500
O
N
H
O
O
N
H
Me
O
Me
Acknowledgments
The authors thank Drs. Catherine Strader, John Piwin-
ski, Ashit Ganguly, and Ted Sybertz for helpful discus-
sions, and Drs. Birendra Pramanik and Pradip Das for
mass spectral data.
O
N
O
O
(+)43
( )45
( )46
(+)52
(+)53
(+)54
290
275
O
O
N
H
References and notes
NHBoc
NH2
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O
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aggregation.