Discovery of a vorapaxar analog with increased aqueous solubility

Article abstract of DOI:10.1016/j.bmcl.2010.09.009

An analog of the thrombin receptor antagonist vorapaxar (SCH 530348) with increased aqueous solubility, compound 9c (SCH 602539), was discovered through incorporation of polar substituents on the pyridine ring of the himbacine-derived lead series. This analog retained the excellent potency, pharmacokinetic and safety properties of vorapaxar while increasing the aqueous solubility by 20-fold. Also presented are in vivo evaluations of this compound in a cynomolgus monkey platelet aggregation assay and in a Folts model of thrombosis in anesthetized monkeys.

Full text of DOI:10.1016/j.bmcl.2010.09.009

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6676–6679
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a vorapaxar analog with increased aqueous solubility
⇑
Yan Xia , Samuel Chackalamannil, William J. Greenlee, Yuguang Wang , Zhiyong Hu, Yuriko Root,
Jesse Wong, Jianshe Kong, Ho-Sam Ahn, George Boykow, Yunsheng Hsieh, Stan Kurowski, Madhu Chintala
Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An analog of the thrombin receptor antagonist vorapaxar (SCH 530348) with increased aqueous solubil-
ity, compound 9c (SCH 602539), was discovered through incorporation of polar substituents on the pyr-
idine ring of the himbacine-derived lead series. This analog retained the excellent potency,
pharmacokinetic and safety properties of vorapaxar while increasing the aqueous solubility by 20-fold.
Also presented are in vivo evaluations of this compound in a cynomolgus monkey platelet aggregation
assay and in a Folts model of thrombosis in anesthetized monkeys.
Received 10 August 2010
Accepted 1 September 2010
Available online 15 September 2010
Keywords:
Thrombin receptor antagonist
PAR-1 antagonist
Solubility
Ó 2010 Elsevier Ltd. All rights reserved.
Vorapaxar analog
SCH 530348 analog
SCH 602539
Atherothrombosis is triggered by the rupture of an atheroscle-
rotic plaque in the coronary artery and it is the major cause of cardio-
vascular death.1 Plaque rupture leads to a spectrum of clinical
conditions, collectively known as acute coronary syndrome (ACS),
that range from unstable angina to acute myocardial infarction.2 In
addition to surgical interventions such as stent implantation, the
pharmacological treatment of ACS includes administration of
antiplatelet agents such as aspirin (inhibitor of thromboxane A2 bio-
synthesis) and clopidogrel (ADP antagonist). However, these agents
suffer from several limitations such as modest efficacy and bleeding
side effect, leaving room for a more potent oral antiplatelet agent
with an improved safety margin. In our efforts to identify such an
agent, we have reported the discovery of thrombin receptor antago-
nist vorapaxar (SCH 530348, 4, Fig. 1), based on the molecular tem-
plate of the natural product himbacine (1).3 Vorapaxar is currently
undergoing Phase-III clinical trials for ACS and secondary prevention
of cardiovascular events.4 In a Phase-II clinical trial, vorapaxar met
the primary endpoint of absence of thrombolysis in myocardial
infarction (TIMI) major plus minor bleeding and showed a numerical
reduction in outcome end points such as myocardial infarction (MI)
and major adverse cardiac events (MACE).5,6
that binds intramolecularly to the proximally located part of the
receptor, causing intracellular signaling events. Since thrombin
receptor activation represents the most potent platelet activation
mechanism, a thrombin receptor antagonist was expected to pro-
duce potent antiplatelet effects. Additionally, since the procoagu-
lant fibrin-generating activity of thrombin is unaffected and
platelet activation needed for normal hemostasis by other platelet
receptor agonists such as collagen are left intact, such an agent was
expected to provide improved safety margin compared to currently
available antiplatelet agents.9
Vorpaxar is a potent antagonist of PAR-1.3 In a PAR-1 binding
assay, vorapaxar showed a Ki of 8.5 nM and demonstrated a potent
oral antiplatelet effect in a cynomolgus monkey model of ex vivo
platelet aggregation (100% inhibition of platelet aggregation for
24 h after oral administration at 0.1 mg/kg).
Since vorapaxar has limited aqueous solubility (vide infra) making
intravenous formulations challenging, we undertook an effort to
identify analogs with greater aqueous solubility while retaining its
excellentspectrumofactivities. In thisLetterwedescribethesuccess-
ful outcome of these efforts that led to the discovery of compound 9c,
which is equipotent to vorapaxar at the PAR-1 receptor while being
20-foldmoresoluble in water. Also reported herein are invivostudies
carried out on 9c including an oral platelet aggregation inhibition as-
say ina cynomolgusmonkey model andanintravenous study ina Fol-
ts model of thrombosis in anesthetized monkeys.
In an effort to preserve the excellent potency and selectivity of
the lead series, we evaluated polar heteroaryl groups at the C-50
position of the pyridine moiety. Based on our previous SAR
studies,3,10 we knew that this part of the molecule was more toler-
ant to changes (Fig. 1). Scheme 1 illustrates the synthesis of target
The thrombin receptor, also known as protease activated recep-
tor-1 (PAR-1), is the most potent cell surface inducer of platelet
activation.7 Mechanistically, thrombin activates PAR-1 by proteo-
lytic cleavage of the extracellular loop of the G-protein coupled
PAR.8 The newly unveiled amino terminus acts as a tethered ligand
⇑
Corresponding author. Tel.: +1 908 740 3510; fax: +1 908 740 7164.
E-mail address: yan.xia@merck.com (Y. Xia).
Present address: ChemPartner Co., Ltd, Shanghai, China.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.009

Y. Xia et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6676–6679
6677
O
H
H
H
H
O
H
H
H
NHCO2Et
O
H
H
H
H
O
NHCO2Et
O
O
Me
H
Me
Me
Me
Me
N
N
N
5
5
9
4
1
, (+)-Himbacine
R
F
Figure 1.
compounds 9a–k. Utilizing our previously reported procedures,11
a
Table 2 summarizes the solubility and pharmacokinetic prop-
erties of the more potent compounds in this series. The solubility
assay was done using a high-throughput kinetic solubility test.15
The pharmacokinetic assay was performed in rat in a high-
throughput format as reported.16 As shown in Table 2, polar sub-
stitutions on the phenyl ring (9a and 9b) increased the solubility
only slightly compared to compound 4. Changing the phenyl
group to heteroaryl groups had a more pronounced effect on
solubility with the 2-pyridyl (9c) having the highest solubility.
Compound 9c also gave high plasma levels in the rat pharmaco-
kinetics model as shown by the high AUC and Cmax values. Com-
pound 9c was further evaluated in a 24 h equilibrium solubility
assay and it was found to be 20 times more soluble than com-
Wadsworth–Emmons–Horner reaction between the aldehyde 511
and the phosphonate 612 followed by hydrolysis provided the ke-
tone 7. Ketone 7 was then converted to the intermediate 8 in a sim-
ilar sequence as we previously described (reductive amination,
isolation of the major
a
isomer, and carbamate formation).3 The
intermediate 8 was then converted to target compounds 9a–k
through palladium-catalyzed coupling reactions of the bromopyri-
dine 8 with appropriate boronic acids, organotin reagents, or
organozinc reagents under the Suzuki, Stille, or Negishi protocols
as shown in Scheme 1.
Thrombin receptor (PAR1) binding studies on compounds 9a–
k were carried out as previously reported using human platelet
membranes as the PAR-1 source and tritiated high affinity throm-
bin receptor activating peptide (haTRAP) as the radioligand.13
PAR-1 Ki values for compounds 9a–k are shown in Table 1. The
2-cyanophenyl (9a) and the 3-methoxyphenyl (9b) substitutions
are well tolerated giving similar Ki’s as compound 4. Of the pyri-
dyl substituted compounds (9c–e), only the 2-pyridyl (9c) is po-
tent. The 3- and 4-pyridyl derivatives are roughly 10-fold less
potent (see 9d and 9e). The 5-methyl-2-pyridyl group (9f) was
tolerated but the 4-methyl-2-pyridyl group (9g) was not. A 4-
methoxy substitution on the 3-pyridyl improved the potency
three fold (9i vs 9d). Other heteroaryl groups such as pyrimidine
(9h), furan (9j), and thiazole (9k) gave moderate to poor potency.
pound 4 (67 lM vs 2.8 lM, Table 2).
Compound 9c was selected for further pharmacological evalua-
tions as shown in Table 3. It inhibited TRAP-induced platelet aggre-
gation in washed human platelets with an IC50 of 0.18 lM. In the
ex vivo platelet aggregation assay in cynomolgus monkeys after
oral administration,17 compound 9c showed complete inhibition
of platelet aggregation at 0.3 mpk for 24 h. Even at 0.1 mg/kg this
compound completely inhibited agonist-induced platelet aggrega-
tion up to 6 h with ca. 70% inhibition occurring at 24 h (Fig. 2). It is
worth noting that, being a pyridine derivative, compound 9c did
not show untoward activities in our enzyme (p450) inhibition or
enzyme induction (PXR)18 assays. It has good permeability in the
O
H
H
H
H
O
O
H
H
H
O
1. W-E-H
2. HCl
(ref. 9)
P(O)(OEt)2
N
O
+
O
O
Me
H
Me
CHO
Br
N
7
5
6
Br
O
O
Suzuki
H
H
H
H
H
H
H
H
9a-b, 9d, 9h-j
NHCO2Et
NHCO2Et
RB(OH)2
O
O
Stille
Me
Me
9c, 9e, 9k
9f-g
RSn(n-Bu)3
N
N
Negishi
RZnBr
8
R
Br
Scheme 1.

6678
Y. Xia et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6676–6679
Table 1
Table 2
Compound number
Kinetic solubility (
lM)
Rat AUC, Cmax (ng h/mL,
ng/mL)
O
H
H
H
H
NHCO2Et
<5a
12
<5
3064, 974
5218, 1571
319, 368
10, 096, 3069
NA
O
4
9a
9b
9c
9f
9i
9j
Me
75a
12
12
12
N
NA
NA
a
Equilibrium solubility: 4, 2.8
lM; 9c, 67 lM; NA, not available.
Ar
Compound number
Ar
Ki (nM) SEM
Table 3
4
8
1
Assays
9c
F
Platelet aggregation in
washed human platelets
p450 Inhibition
IC50 = 0.18 lM
CYP3A4, CYP2D6, CYP2C9 & CYP2C19 IC50
>30 M (co-incubation & pre-incubation)
0.13 @ 10
283 (nm/s)
1 mpk, po, methylcellulose:
:
NC
9a
9b
9c
9d
15
12
19
7
5
1
l
PXR
lM
Caco-2 permeability
Monkey pharmacokinetics
AUC0–24h = 2520 ng h/mL, Cmax = 236 ng/mL,
Tmax = 0.5 h
0.1 mpk, po, methylcellulose:
AUC0–24h = 50 ng h/mL; Cmax = 16 ng/mL;
Tmax = 1 h
MeO
N
N
30
25
20
15
10
5
240 47
250 19
0.1 mpk
0.3 mpk
9e
9f
N
N
42
4
Me
0
N
9g
9h
9i
144 70
337 55
49 18
0
6
12
18
24
Time (hr)
Me
Figure 2. Inhibition of ex vivo haTRAP-induced platelet aggregation by compound
9c in cynomolgus monkeys (n = 3) after oral administration at 0.3 and 0.1 mg/kg.
N
N
Compound 9c was evaluated alone and in combination with the
ADP antagonist cangrelor in a Folts model of thrombosis in anes-
thetized monkeys.19 Increasing doses of 9c, cangrelor or both were
administered intravenously to the same animal in a sequential
manner and the ability to inhibit cyclic flow reductions (CFR’s)
was monitored. Compound 9c inhibited thrombosis in the Folts
model in monkeys in a dose-dependent manner. The efficacy of
9c was additive to that of cangrelor.
In summary, we have discovered a potent thrombin receptor
antagonist 9c with 20-fold increased aqueous solubility compared
to vorapaxar through incorporation of polar substituents on the
pyridine ring of the lead series. Compound 9c showed PAR-1 affin-
ity and antiplatelet effect in vivo in a cynomolgus monkey model
comparable to those of vorapaxar. It also showed excellent
pharmacokinetic properties and no untoward activities such as
enzyme inhibition or enzyme induction. The increased aqueous
N
OMe
9j
61 22
75 28
O
S
9k
N
Caco-2 assay and good oral exposures in monkey pharmacokinetic
assays.

Y. Xia et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6676–6679
6679
9. For recent reviews on thrombin receptor (PAR-1) antagonists as antithrombotic
agents, see: (a) Chackalamannil, S. J. Med. Chem. 2006, 49, 5389; (b)
Chackalamannil, S.; Xia, Y. Expert Opin. Ther. Pat. 2006, 16, 493; (c)
Scarborough, R. M.; Pandey, A.; Zhang, X. Annu. Rep. Med. Chem. 2005, 40, 85;
(d) Maryanoff, B. E.; Zhang, H.-C.; Andrade-Gordon, P.; Derian, C. K. Curr. Med.
Chem.: Cardiovasc. Hematol. Agents 2003, 1, 13.
10. Xia, Y.; Chackalamannil, S.; Clasby, M.; Doller, D.; Eagen, K.; Greenlee, W. J.;
Tsai, H.; Agans-Fantuzzi, J.; Ahn, Ho-Sam; Boykow, G. C.; Hsieh, Y.; Lunn, C. A.;
Chintala, M. Bioorg. Med. Chem. Lett. 2007, 17, 4509.
11. Clasby, M. C.; Chackalamannil, S.; Czarniecki, M.; Doller, D.; Eagen, K.;
Greenlee, W. J.; Kao, G.; Lin, Y.; Tsai, H.; Xia, Y.; Ahn, H.-S.; Agans-Fantuzzi,
J.; Boykow, G.; Chintala, M.; Foster, C.; Smith-Thoran, A.; Alton, K.; Bryant, M.;
Hsieh, Y.; Lau, J.; Palamanda, J. J. Med. Chem. 2007, 50, 129.
12. Chelliah, M. V.; Chackalamannil, S.; Xia, Y.; Eagen, K.; Clasby, M. C.; Gao, X.;
Greenlee, W.; Ahn, H.-S.; Agans-Fantuzzi, J.; Boykow, G.; Hsieh, Y.; Bryant, M.;
Palamanda, J.; Chan, T.-M.; Hesk, D.; Chintala, M. J. Med. Chem. 2007, 50, 5147.
13. Ahn, H.-S.; Foster, C.; Boykow, G.; Arik, L.; Smith-Torhan, A.; Hesk, D.;
Chatterjee, M. Mol. Pharmacol. 1997, 51, 350. A modification of the assay was
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duplicate. Compounds of high interest (IC50 <100 nM) were assayed multiple
times (n P5, SD 20%)..
solubility of compound 9c facilitated its evaluation in a Folts model
of thrombosis via intravenous administration. In this study com-
pound 9c showed dose-dependent antithrombotic efficacy and this
efficacy was additive when co-administered with the ADP antago-
nist cangrelor.
Acknowledgments
We thank Dr. Charles McNemar and Dr. Prudence Bradley’s
groups at Merck Research Laboratories for solubility measure-
ments. We also acknowledge the support and encouragement of
Dr. Catherine Strader and Dr. Michael Graziano.
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