Discovery of nor-seco himbacine analogs as thrombin receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.01.138

Discovery of a novel nor-seco himbacine analog as potent thrombin receptor (PAR-1) antagonist is described. Despite low plasma level, these new analogs showed excellent ex vivo efficacy in the monkey platelet aggregation assay. A potent hydroxy metabolite generated in vivo was identified as the agent responsible for the ex vivo efficacy. Following this discovery, the metabolite series was optimized to obtain analogs that showed very good ex vivo efficacy along with excellent pharmacokinetic profile in c. monkey.

Full text of DOI:10.1016/j.bmcl.2012.01.138

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2544–2549
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of nor-seco himbacine analogs as thrombin receptor antagonists
Mariappan V. Chelliah a, , Samuel Chackalamannil a, Yan Xia a, Keith Eagen a, William J. Greenlee a,
⇑
Ho-Sam Ahn b, Jacqueline Agans-Fantuzzi b, George Boykow b, Yunsheng Hsieh c, Matthew Bryant c,
Tze-Ming Chan d, Madhu Chintala b
a Central Nervous System and Cardiovascular Chemical Research, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Cardiovascular and Metabolic Disease Research, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
c Drug Metabolism and Pharmacokinetics Research, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
d Department of Analytical Spectroscopy, 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:
Discovery of a novel nor-seco himbacine analog as potent thrombin receptor (PAR-1) antagonist is
described. Despite low plasma level, these new analogs showed excellent ex vivo efficacy in the monkey
platelet aggregation assay. A potent hydroxy metabolite generated in vivo was identified as the agent
responsible for the ex vivo efficacy. Following this discovery, the metabolite series was optimized to
obtain analogs that showed very good ex vivo efficacy along with excellent pharmacokinetic profile in
c. monkey.
Received 14 November 2011
Accepted 31 January 2012
Available online 16 February 2012
Keywords:
Vorapaxar
PAR-1
Ó 2012 Elsevier Ltd. All rights reserved.
Thrombin receptor antagonist
SCH 530348
Platelet aggregation
Himbacine
Antiplatelet agents play an essential role in the treatment of
atherothrombosis.1,2 Upon rupture of an atherosclerotic plaque,
the platelet activation mechanism and coagulation mechanism
synergize leading to the formation of a thrombus at the site of in-
jury which often occludes the coronary artery. The obstruction of
blood flow and the resulting myocardial underperfusion can lead
to a spectrum of clinical conditions known as acute coronary syn-
drome (ACS) that include Q-wave myocardial infarction, non-Q-
wave myocardial infarction and unstable angina.3 Aggregated
platelets contribute significantly to the formation of arterial
thrombi and as such and antiplatelet agents such as aspirin and
clopidogrel comprise the current standard of care for ACS. How-
ever, there remains an unmet clinical need for antiplatelet drugs
with greater potency and improved safety margin with regard to
hemorrhagic side effects, which are complicating factors for the
current antiplatelet treatments.4
in template bleeding times.13 In a Phase-II clinical study in patients
who underwent non-emergent PCI, SCH 530348 met the primary
end point of absence of significant TIMI major plus minor bleeding
when compared with placebo.14 For the secondary outcome end-
point, SCH 530348 was associated with a non-statistically signifi-
cant numerical reduction in periprocedural myocardial infarction.
Two additional Phase-II studies conducted in Japanese patients
(one in patients with acute coronary syndromes and the other in
patients with ischemic stroke) have confirmed the lack of signifi-
cant TIMI bleedings associated with the drug.13 Additionally, the
study performed in Japanese patients with acute coronary syn-
dromes demonstrated a significant reduction in periprocedural
myocardial infarctions. Complete results of two large scale clinical
studies on vorapaxar are anticipated in the near future.15,16
The tricyclic ring skeleton of vorapaxar is derived from the nat-
ural product himbacine, although it has an absolute chirality oppo-
site to that of the natural product. In an extensive SAR study of the
tricyclic himbacine-based PAR-1 antagonists, we have made sev-
eral modifications to the right-hand side ring (C-ring) by introduc-
ing substituents, and by replacing the C-ring with aryl and
heterocyclic rings.17–20 As reported previously, the tetrahydrothio-
pyranyl analog 2 (Fig. 1), which has the C7 carbon of the tricyclic
skeleton replaced with a sulfur atom, has PAR-1 affinity compara-
ble to the prototypical PAR-antagonist 1.19 Despite the fact that the
utility of compound 2 was limited due to its potential in vivo
metabolism to the corresponding less active sulfone, it lent itself
As part of our efforts to address this need, we have reported the
discovery of vorapaxar5 (SCH 530348) which is a potent antagonist
of the platelet surface thrombin receptor, also known as protease
activated receptor-1 (PAR-1).6–12 In preclinical non-human pri-
mate models of ex vivo platelet aggregation and arterial thrombo-
sis, SCH 530348 showed potent antiplatelet effects without change
⇑
Corresponding author. Tel.: +1 732 594 7261.
E-mail address: mariappan.chelliah@merck.com (M.V. Chelliah).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.138

M. V. Chelliah et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2544–2549
2545
reaction, followed by saponification to provide the dienoic acid 10.
The acid was converted to its acid chloride and coupled with the
previously reported alcohol 11 to provide the ester 12.17 Hydroge-
nation of the alkyne with Lindlar catalyst gave the cis-olefin 13.
Intramolecular Diels–Alder cyclization of 13 at 185 °C, followed
by in situ treatment with DBU to effect C7 epimerization, gave a
mixture of exo and endo products (ꢀ1:1) in 66% yield. The two iso-
mers were separated by silica gel column chromatography and the
exo isomer was used for the subsequent transformations. Debenzy-
lation of 14 over Pd-C/H2 followed by double bond reduction over
PtO2/H2 gave the carboxylic acid 16 which was converted to the
aldehyde 17 via its acid chloride. Coupling of several substituted
pyridyl phosphonates 18 with the aldehyde 17 gave the substi-
tuted nor-seco analogs represented by structure 19. Alternatively,
aldehyde 17 was coupled with phosphonate 20 to give the bro-
mo-substituted pyridyl analog 21 which could be further elabo-
rated to the desired target 19 by Suzuki coupling as shown.
The in vitro binding assays were carried out as described before
on PAR-1 receptors isolated from human platelets and [3H]haTRAP
as the radioligand.21 Table 1 presents the in vitro PAR-1 binding
affinity for the nor-seco analogs. Similar to the tricyclic himbacine
series, both the ortho and meta substituted phenyl derivatives
showed excellent binding affinity (compound 19a–h). Specific
requirements for disubstitution of the phenyl ring was also noted.
The 2,3 dichloro substituted analog (19i) was very potent
(IC50 = 13 nM), whereas the corresponding 2,5-dichloro analog
19j (IC50 = 513 nM), showed considerably weaker affinity. Selected
analogs were evaluated in a rat pharmacokinetic assay at an oral
dose of 10 mg/kg and the plasma levels were analyzed up to 6 h.
As shown in Table 1, the rat plasma levels of the parent were uni-
formly low for these compounds.
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
S
O
O
O A
B
C
N
N
N
2
3
1
CF3
CF3
CF3
Thr IC50 = 12 nM
Thr IC50 = 22 nM
Thr IC50 = 225 nM
Figure 1.
to the discovery of a new C-ring-opened chemical series which we
now wish to report. Raney nickel desulfuration of 2 resulted in the
anticipated sulfur extrusion with concomitant reduction of the
double bond to give 3. This new bicyclic analog showed an encour-
aging, albeit modest PAR-1 binding (IC50 = 225 nM). Since we knew
from the previous SAR studies in the original tricyclic series that
the trans-ethylene-linked pyridine was essential for optimal PAR-
1 affinity, we synthesized the trans-ethenyl analog 8 as shown in
Scheme 1.
The synthesis started with the previously reported aldehyde19
which was reduced to the corresponding alcohol and subsequently
reduced under Raney nickel conditions to provide the desulfurated,
nor-seco himbacine intermediate 5. The alcohol was oxidized to
the corresponding aldehyde which was subjected to Horner–
Wadsworth–Emmons reaction with phosphonate 7 to give the tar-
get compound 8. In line with our expectation, this novel nor-seco
himbacine analog 8 showed PAR-1 affinity comparable to the par-
ent tricyclic analog 1 in the radioligand binding assay. We further
set out to explore the SAR of the nor-seco himbacine series of PAR-
1 antagonists in detail.
A number of PAR-1 antagonists from this series were also tested
in a cynomolgus monkey ex vivo platelet aggregation inhibition as-
say. In this assay, the drug was dosed orally in HPbCD and blood
samples were drawn at various time points. A 1 lM solution of ha-
TRAP was added to the whole blood to initiate platelet aggregation,
which was quantified using an aggregometer. This assay provided a
direct measurement of the antiaggregatory response of the drug
under conditions of PAR-1 activation by an exogenously added
agonist.22 Compound 19b showed about 45% inhibition of platelet
aggregation at 3 mg/kg oral dose at the 24-h time point whereas at
the same dose analog 19c completely inhibited haTRAP-induced
platelet aggregation at 24 h. Upon further dosing down 19c
showed robust platelet aggregation inhibition even at 1 mg/kg
with 69% inhibition of aggregation at 24 h. Compound 19i also
showed excellent inhibition of haTRAP induced platelet aggrega-
tion (95% at 24 h) at a 3 mg/kg dose.
Analysis of the monkey plasma levels from efficacy studies indi-
cated relatively low plasma concentrations for the parent drugs
and high plasma levels of a (M+16) metabolite. For example 19c
showed an AUC(0–24 h) = 451 ng h/mL (Cmax = 62 ng/mL). How-
ever, M+16 metabolite concentration was three times higher
(AUC(0–24 h) = 1400 ng h/mL, Cmax = 98 ng/mL). In a similar fash-
ion, 19i also showed considerable amount of circulating hydroxy
metabolite (parent AUC(0–24 h) = 573 ng h/mL; (M+16) metabolite
AUC(0–24 h) = 813 ng h/mL). Since these compounds showed high
efficacy despite the low plasma level of the parent, we suspected
that the circulating hydroxy metabolite might likely be contribut-
ing to the observed efficacy. This prompted our effort to identify
and characterize the hydroxy metabolite generated in vivo.
Analysis of the rat plasma samples from PK studies using LC-
ESI/MS/MS on the Micromass Q–T of mass spectrometer indicated
the presence of a major M+16 metabolite arising from the metab-
olism of the six-membered carbocyclic ring. In an effort to further
confirm this characterization, 19c was incubated with rat liver
microsomes and the major hydroxy metabolite isolated. Proton
A modified synthetic approach that facilitated the SAR study in
this series is shown in Scheme 2. Commercially available (E)-2-
methyl-2-pentenal was subjected to Horner–Wadsworth–Emmons
O
O
O
H
H
H
H
H
H
H
H
a, b
c
S
O
H
O
H
O
H
H
H
O
H
OH
H
O
4
6
5
O
H
H
H
d
O
H
P(O)(OEt)2
N
H
N
CF3
7
8
CF3
Thr IC50 = 24 nM
Scheme 1. Reagent and condition: (a) NaBH4, MeOH; (b) Ra-Ni, MeOH-THF, heated
at reflux; (c) TPAP, NMO, 4 Å molecular sieves; (d) 7, BuLi, THF then 6.

2546
M. V. Chelliah et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2544–2549
O
O
c, d
OH
a, b
O
O
HO
CO2Bn
9
10
CO2Bn
O
12
11
O
O
H
H
H
H
g
f
e
O
O
H
+
O
H
H
H
CO2Bn
CO2Bn
exo
CO2Bn
endo
13
O
O
H
H
H
O
H
H
H
h, i
c, j
O
H
O
H
+
O
H
H
H
H
H
CO2Bn
endo
15
CO2Bn
CO2H
exo
14
16
O
exo:endo~1:1
H
H
H
O
k
H
H
H
O
H
O
(O)(OEt)2
H
N
H
H
O
18
Ar
N
17
P(O)(OEt)2
19
Ar
l
N
20
Br
O
H
H
m
O
H
H
H
N
21
Br
Scheme 2. Reagents and conditions: (a) (EtO)2P(O)CH2CO2Et, NaH, THF; (b) KOH, THF-MeOH-H2O; (c) (COCl)2, cat. DMF; (d) 11, Et3N, DMAP; (e) H2, Lindlar catalyst,
quinoline; (f) toluene, 185 °C, in a sealed tube; (g) DBU, rt; (h) 14, H2, Pd-C; (i) H2, PtO2; (j) Bu3SnH, cat. Pd(Ph3P)4; (k) 18, LHMDS then Ti(OiPr)4 and 17; (l) 20, LHMDS then
Ti(OiPr)4 and 17; (m) ArB(OH)2, Pd(Ph3P)4, K2CO3, toluene–ethanol–water, 100 °C in a sealed tube.
NMR study indicated that this metabolite corresponded to the C7a
hydroxylated derivative 22 (Fig. 2). In order to further corroborate
the identity of this oxidative metabolite, we synthesized an
authentic sample by treating 19c with LHMDS followed by oxida-
tion with (1S)-(+)-(10-camphorsulfonyl)oxaziridine to give 22. This
synthetic sample of 22 was identical to the sample isolated from
liver microsomal incubation of 19c.
In the rat pharmacokinetic studies, hydroxyl derivative 22
showed a fivefold improvement in parent AUC under similar dos-
ing conditions. In the c. monkey ex vivo efficacy assay, the 7a-hy-
droxy metabolite 22 showed 100% inhibition of platelet
aggregation at 24 h at a 3 mg/kg oral dose and at a lower dose of
1 mg/kg, complete inhibition was maintained up to 6 h. Analysis
of the c. monkey plasma samples showed excellent plasma concen-
tration of parent 22. The monkey AUC of 22 at an oral dose of 1 mg/
kg was 4280 ng h/mL which was more than nine times the AUC of
19c following a 3 mg/kg oral dose.
low plasma levels of the parents that we observed for the original
series in the rat PK studies could be explained by this facile meta-
bolic conversion. Since the hydroxylated metabolite 22 showed
improved pharmacokinetic profile while retaining the potency of
its parent 19c, we decided to further evaluate the C7a-OH analogs
of other nor-seco derivatives. The synthesis of these compounds
is described in Scheme 3. The intermediate 21 was readily oxidized
at it C7a position via the lactone enolate to give 23 which was con-
verted to the target analogs 24a–l. Representative examples of
PAR-1 binding and ex vivo studies of 7a-hydroxy nor-seco deri-
vates are presented in Table 2. The phenyl analog 24a showed
excellent binding affinity. As before, substitutions at both the ortho
and meta positions of the phenyl group were tolerated very well,
the corresponding para substituted analogs were either inactive
(24e) or substantially less potent (24h). We also evaluated several
heteroaryl analogs that replaced the phenyl substituent on the pyr-
idine moiety. Substitution with 3-thiophene (24j) and 3-furan
(24k) moieties resulted in analogs with excellent potencies com-
pared with the corresponding pyridyl analog 24i. Non-aromatic
Thus the original nor-seco derivatives seemed to be liable to
fast in vivo oxidation to the C7 hydroxy derivatives. The relatively

M. V. Chelliah et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2544–2549
2547
Table 1
Binding, PK and ex-vivo data for compounds 19a–k
O
H
H
H
H
O
H
N
Ar
Compound #
Ar
PAR-1 IC50 (nM)
Rat AUCa
C. monkey ex vivo platelet aggregation inhibitionb
19a
19b
19c
19d
19e
19f
19g
19h
19i
Phenyl
8.8
22
15
25
28
20
45
21
13
513
672
852
230
(2-Fluoro)-phenyl
(3-Fluoro)-phenyl
(2-Chloro)-phenyl
(3-Chloro)-phenyl
(2-Methyl)-phenyl
(3-Methyl)-phenyl
(3-Cyano)-phenyl
(2,3-Dichloro)-phenyl
(2,5-Dichloro)-phenyl
45% (3 mpk/24 h)
100% (3 mpk/24 h)
1146
95% (3 mpk/24 h)
19j
a
AUC from 0 to 6 h in ng h/mL and at 10 mg/kg oral dose (0.4% methylcellulose).
Reduction in haTRAP induced platelet aggregation in c. monkey following oral dose (20% PEG-HPBCD).
b
O
H
H
H
O
OH
7a
H
H
CYP P450
7a
O
H
O
H
H
H
1) LHMDS, THF 0°C
N
N
2) oxaziridine, -78°C to rt
19c
22
F
F
PAR-1 IC50 = 15 nM
PAR-1 IC50 = 27 nM
Ex-vivo: 1 mpk, 24 hrs
Ex-vivo: 1 mpk, 24 hrs
RR AUC0-6h = 230 ng.hr/ml (10 mg/kg)
Monkey AUC0-24hr (3 mg/kg, po) =
451 ng.hr/ml (parent); 1400 ng.hr/ml (M+16)
RR AUC0-6h = 1136 ng.hr/ml (10 mg/kg)
Monkey AUC0-24hr (1 mg/kg, po) = 4280 ng.hr/ml
Figure 2.
heterocycles were also tolerated in the place of phenyl group (e.g.,
24l).
O
H
H
H
H
O
O
OH
H
H
OH
H
H
Several of the hydroxylated nor-seco analogs were evaluated in
the rat pharmacokinetic assay. In general, the rat pharmacokinetic
profile was greatly improved from the C7a-H parent series (Tables 1
and 2). In the ex vivo platelet aggregation inhibition assay in cyno-
molgus monkey, several of these compounds showed impressive
oral antiplatelet effect that lasted, in many cases, for 24 h. For
example 24a completely inhibited the platelet aggregation at
3 mg/kg dose (Fig. 3) for 24 h. At a lower dose of 1 mg/kg, it inhib-
ited ꢀ65% of the platelet aggregation at the 24 h time point, and
even at 0.3 mg/kg dose it showed partial inhibition at earlier time
points. The (3-cyano)-phenyl analog 24d showed only moderate
rat plasma level, however the monkey efficacy was comparable
to 24a. Despite good rat plasma level, the (3-methoxy)phenyl
O
H
O
H
O
H
b or c
H
H
a
N
N
N
21
23
24a-l
Br
Br
R
Scheme 3. Reagent and condition: (a) LHMDS then (1S)-(+)-(10-camphorsulfo-
nyl)oxaziridine; (b) ArB(OH)2, cat. Pd(Ph3P)4, K2CO3, toluene–ethanol–water, 100 °C
in a sealed tube; for compounds 24a–k; (c) pyrrolidine, cat. Pd(OAc)2, 2-(dicyclo-
hexylphosphino)biphenyl, K3PO4, toluene, 100 °C; for compound 24l.

2548
M. V. Chelliah et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2544–2549
Table 2
Binding, PK and ex-vivo data for compounds 24a–l
O
OH
H
H
O
H
H
N
Ar
Compound #
Ar
PAR-1 IC50 (nM)
Rat AUCa
Ex vivob
Monkey AUCc
24a
24b
24c
24d
24e
24f
24g
24h
24i
Phenyl
6.4
33
35
3297
3159
203
1084
—
168
2441
—
1895
397
148
486
65% (1 mpk/24 h)
55% (3 mpk/6 h)
100% (3 mpk/6 h)
60% (1 mpk/24 h)
—
3825 (1 mg/kg)
2720 (3 mg/kg)
2770 (3 mg/kg)
6284 (1 mg/kg)
—
—
1210 (1 mg/kg)
—
—
4740 (3 mg/kg)
4110 (3 mg/kg)
—
(2-Methyl)-phenyl
(3-Methyl)-phenyl
(3-Cyano)-phenyl
(4-Cyano)-phenyl
(2-Methoxy)-phenyl
(3-Methoxy)-phenyl
(4-Methoxy)-phenyl
3-Pyridyl
20
Inactive
8.1
18
451
49
4.6
8.6
64
—
Inactive (1 mpk)
—
45% (3 mpk/24 h)
90% (3 mpk/24 h)
35% (3 mpk/24 h)
100% (3 mpk/6 h)
24j
24k
24l
3-Thiophene
3-Furan
1-Pyrrolidine
a
b
c
AUC from 0 to 6 h in ng h/mL and at 10 mg/kg oral dose (0.4% methylcellulose).
Reduction in haTRAP induced platelet aggregation in c. monkey following oral dose (20% PEG-HPBCD) of the hydrochloride salt.
AUC from 0 to 24 h in ng h/mL (data obtained from the ex-vivo plasma sample).
h/mL. The oral Cmax was 397 ng/mL with a Tmax of 1.0 h. Also,
the half-life was 10.7 h. The compound showed a low volume of
distribution (Vd(ss) = 1.5 (L/kg)) and showed
a clearance of
Platelet Aggregation Inhibition by 24a (20%HPBCD)
3.3 mL/min/kg. Since, many of earlier analogs showed extremely
slow clearance,18,19 we decided to evaluate the clearance of [3H]24a
in c. monkey. Following a 1 mg/kg iv dose of 3H–24a in the
c. monkey, total excreted radioactivity in urine and bile was quan-
tified. It was found that the entire radioactivity was accounted for
by 6 days indicating a very ideal clearance profile for this analog.
In summary, we have discovered a novel series of thrombin
receptor antagonists by truncation of the tricyclic ring system of
the prototypical compounds and by introduction of a C7a-hydroxyl
group, guided by the major active in vivo metabolite. Further opti-
mization of the aryl substitution resulted in the discovery of sev-
eral potent analogs with single-digit nanomolar PAR-1 affinity
and excellent oral efficacy in an ex vivo cynomolgus monkey plate-
let aggregation inhibition model. The benchmark nor-seco analog
24a showed complete ablation of agonist-induced ex vivo platelet
aggregation following 1 mg/kg oral dose and a robust pharmacoki-
netic profile in rat and monkey models.
35
30
25
20
15
10
5
3mpk
1mpk
0.3mpk
0
0
1
2
3
4
5
6
24
Hours
Figure 3. Ex vivo platelet aggregation inhibition in cynomolgus monkey following a
single oral dose (3 mg/kg, 1 mg/kg and 0.3 mg/kg in 20% PEG-HPBCD) 24a.
Acknowledgment
The authors like to thank Drs. Birendra Pramanik and Pradip
Das for mass spectral data, Dr. Mohindar Puar for NMR data and
Dr. David Hesk for radio labeling studies.
analog 24g did not show any efficacy at a 1 mg/kg dose. The 3-pyr-
idyl analog 24i showed good rat plasma level and efficacy up to
24 h at a 3 mg/kg dose. Though the 3-thiophene and 3-furan ana-
logs showed very poor rat plasma levels, analog 24j inhibited
90% of platelet aggregation (3 mpk/24 h) and analog 24k inhibited
about 35% of platelet aggregation (3 mpk/24 h). The 1-pyrrolidine
analog 24l showed low rat plasma level, however it maintained
good efficacy up to 6 h at a 3 mg/kg dose.
Due to the excellent ex vivo efficacy and rat plasma level exhib-
ited by compound 24a, it was subjected to a full monkey pharma-
cokinetic assay. Cynomolgus monkeys were dosed at 1 mg/kg oral
dose in 0.4%MC and 1 mg/kg iv dose in 20% HPBCD. Compound 24a
showed an oral bioavailability of 78% with AUC(0–24 h) = 3980 ng
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