Himbacine derived thrombin receptor antagonists: Discovery of a new tricyclic core

Article abstract of DOI:10.1016/j.bmcl.2007.04.061

The synthesis and biological activity of a novel series of thrombin receptor antagonists is described. This series of compounds showed excellent in vitro and in vivo potency. The most potent compound 40 had an IC₅₀ of 7.6 nM and showed robust i

Full text of DOI:10.1016/j.bmcl.2007.04.061

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3647–3651
Himbacine derived thrombin receptor antagonists:
Discovery of a new tricyclic core
a
a
a
Martin C. Clasby,a, Samuel Chackalamannil, Michael Czarniecki, Darıo Doller,
*
´
Keith Eagen,a William J. Greenlee,a Yan Lin,a Jayaram R. Tagat,a Hsingan Tsai,a
Yan Xia,a Ho-Sam Ahn,a Jacqueline Agans-Fantuzzi,a George Boykow,a
Madhu Chintala,a Yunsheng Hsieha and Andrew T. McPhailb
aSchering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
bDuke University, Durham, NC 27708, USA
Received 8 March 2007; revised 6 April 2007; accepted 16 April 2007
Available online 25 April 2007
Abstract—The synthesis and biological activity of a novel series of thrombin receptor antagonists is described. This series of com-
pounds showed excellent in vitro and in vivo potency. The most potent compound 40 had an IC50 of 7.6 nM and showed robust
inhibition of platelet aggregation in a cynomolgus monkey model after oral administration.
Ó 2007 Elsevier Ltd. All rights reserved.
Thrombin, the main effector protease of the coagulation
cascade, converts soluble fibrinogen to fibrin and acti-
vates platelets which aggregate at the site of a vascular
injury. Fibrin monomers polymerize to an insoluble
fibrin meshwork that traps aggregated platelets and
other plasma particle leading to the formation of a
thrombus. The activation of platelets and other cell
types by thrombin is mediated via proteolytic activation
of specific cell surface receptors known as protease acti-
vated receptors (PARs).1–6 Four PARs are known
(PAR1-4). Among these, PAR-1, also known as throm-
bin receptor, is the most potent activator of human and
primate platelets. Thrombin binds to PAR-1 through its
exo-anion binding site. Cleavage of the extracellular
domain at Arg41-Ser42 reveals an amino terminus that
then binds intramolecularly to the receptor.7–9 This
intramolecular activation mechanism (also known as
the tethered ligand mechanism) makes it difficult to
identify a small molecule antagonist for the activated
thrombin receptor (PAR-1) due to entropic reasons.
useful in the treatment of disorders such as arterial
thrombosis, atherosclerosis and, restenosis. Such an
agent could have a significant advantage in safety with
regard to bleeding side effects over the current anti-
thrombotic therapies.10–13
We have recently reported the synthesis and biological
activity of a novel series of thrombin receptor (PAR-1)
antagonists (e.g., 1 and 2) based on the natural product
himbacine14–16 (Scheme 1). These compounds are potent
antagonists of PAR-1,17 and in the case of 2 show excel-
lent activity when dosed orally in an ex-vivo model of
O
O
O
H
H
H
H
H
H
H
H
H
H
OH
O
O
O
Me
Me
3
N
N
N
It has been hypothesized that by targeting only the cel-
lular action of thrombin, and not its role in the coagula-
tion cascade, a thrombin receptor antagonist would be
IC50 = 12000 nM
1
2
OH
CF3
IC50 = 16 nM
Keywords: Thrombin; PAR1; Himbacine; Antagonist.
*
IC50 = 6.3 nM
Corresponding author. Tel.: +1 908 740 2244; fax: +1 908 740
7152; e-mail: martin.clasby@spcorp.com
Scheme 1. Thrombin receptor antagonists.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.061

3648
M. C. Clasby et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3647–3651
platelet aggregation using high affinity thrombin recep-
tor-activating peptides (haTRAP) as the ligand.18,19
185–195 °C gave the tricyclic core 4c–7c along with aro-
matic byproducts 4d–7d. The core was then reduced to
give the tricyclic acids 4e–7e. The acids were then
reduced to aldehydes 4f–7f via reduction of the corre-
sponding acid chlorides.22 The aldehydes 4f–7f were
converted to the final targets 4g–7g and 4h–5h using a
Horner–Emmons reaction. Compounds 4g, 4h, 5g, and
5h were synthesized from enantiopure 4a and 5a, com-
pounds 6g and 7g were synthesized in racemic form.
When tested in the PAR-1 binding screen compound
4g was an immediate improvement over 3 with an
IC50 = 580 nM, its enantiomer 5g was much less potent,
a similar trend was observed for 4h (IC50 = 90 nM) and
its enantiomer 5h. Similarly to himbacine derived com-
pounds 1 and 2, it is the compounds derived from the
(R)-alcohol that were more potent. Our attempts to epi-
merize the aldehyde 4f to form compounds with the
identical relative stereochemistry to 1 and 2 were met
with failure. We were, however, pleased to see that the
racemic compounds 6g and 7g were very potent with
IC50 = 225 and 12.5 nM, respectively. Targets similarly
derived from aromatic byproducts 4d–7d were inactive.
Compound 7g was selected for further SAR study.
A
structurally related compound 3 was identified from
our compound collection to be a mild antagonist of
PAR-1 (IC50 = 12 lM). We were intrigued by the simi-
larities to our current leads and embarked on an optimi-
zation study for this series of compounds. In this
communication we describe the synthesis and biological
activity of compounds such as 3.
The general synthesis of analogs of 3 is described in
Scheme 2. The tricyclic portion of the targets was syn-
thesized using our reported literature procedure:20,21
condensation of cinnamic acid chloride with propargylic
alcohols 4a–7a followed by thermolysis in o-xylene at
O
O
Cl
(b)
(a)
O
R1
+
HO
R1
R2
CO2Bn
4a-7a
CO2Bn
4b-7b
R2
O
O
O
(d)
(e)
Our previous studies with compounds related to 1 and 2
containing the himbacine tricycle showed that 5-phenyl-
pyridines gave the greatest potency. This was also found
to be the case for this new tricyclic aryl himbacine series.
The best three examples, 9, 10, and 11, are shown in
Scheme 3. In general, the most potent compounds pos-
(c)
O
O
O
R1
R1
R1
R2
4e-7e
R2
4f-7f
R2
CO2H
CHO
CO2Bn
+
4c-7c
O
O
R1
O
O
O
H
H
H
H
H
H
R2
CO2Bn
4d-7d
O
O
O
O
O
P(O)(OEt)2
R1
R2
N
N
N
4) R1= H, R2 = Me
5) R1= Me, R2 = H
6) R1= R2 = Me
7) R1= R2 = H
N
(f)
N
8
4g-7g
4h-5h
OMe
CF3
9 IC50 = 10 nMa
F
Cl
10 IC50 = 7.5 nMa
11 IC50 = 14.5 nMa
OMe
O
H
O
H
O
H
O
O
O
H
H
H
H
H
(c)
(a)
(b)
O
O
O
O
O
O
H
HO
H
HN
H
HO
H
O
O
O
14
5g IC50 = 9170 nMa
HO
4h IC50 = 90 nMa
4g IC50 = 580 nMa
( ) 7e
12
H
Ph
O
H
+
+
O
H
O
H
O
O
H
H
(c)
O
O
O
O
O
H
H
H
H
H
( )7g IC50 = 12.5 nMa
HN
HO
( )6g IC50 = 225 nMa
5h IC50 = 3139 nMa
O
O
13
15
HO
H
a) PAR-1 binding assay ligand: [3H]haTRAP, 10 nM (Kd = 15 nM)17
Ph
a) PAR-1 binding assay ligand: [3H]haTRAP, 10 nM (Kd = 15 nM)17
Scheme 2. Synthesis of analogs of compound 3. Reagents and
conditions: (a) TEA, THF, 0 °C; (b) 185–195 °C, o-xylene; (c) PtO2,
EtOAC, H2; (d) (COCl)2, DMF, CH2Cl2; (e) Bu3SnH, Pd(Ph3P)4;
PhMe, 0 °C; (f) BuLi, THF, then 4f–7f.
Scheme 3. Reagents and condition: (a) (COCl)2, DMF, CH2Cl2; (b)
(S)-2-phenylglycinol, TEA, THF (75%, 2 steps); (c) 4 M HCl in
dioxane, reflux (99%).

M. C. Clasby et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3647–3651
3649
sess a chloro, fluoro, or trifluoromethyl group at the
meta-position of the phenyl ring of the 5-phenylpyri-
dine. It should also be noted that these compounds are
enantiopure. The resolution was achieved via chromato-
graphic separation of the diastereomeric amides derived
from the acid 7e and (S)-2-phenylglycinol (Scheme 3).
We were able to determine the absolute configuration
of 13 via X-ray crystallographic analysis23 and thus
the absolute configuration of 12. Acidic hydrolysis gen-
erated the enantiopure acids 14 and 15. Compounds de-
rived from 15 were found to be 10- to 15-fold less potent
than compounds derived from 14, the more potent enan-
tiomer typically showed a 2-fold improvement over race-
mic compound. This resolution was found to be general
for every tricycle of this kind studied, and allowed us to
generate compounds with enantiomeric excess greater
than 99% (determined by HPLC; CHIRALPAKÒ
ADÒ, CHIRALCELÒODÒ).
very promising result but we required a compound that
had at least 24 h duration at 3 mpk. In order to accom-
plish this goal we examined the effect of substitution on
both the tricyclic and 5-phenylpyridine parts of the
molecule.
One interesting aspect of this new series of compounds is
the relative ease by which the SAR of aromatic ring of
the tricycle can be studied; there are hundreds of com-
mercially available cinnamic acids. We have studied
the effect of substitution of various groups on the aro-
matic ring of the tricycle. Initial SAR showed that while
simple alkyl, alkoxy, carboxy, and hydroxyl led to com-
pounds with reduced activity (IC50 > 100 nM), simple
halogen substitution gave very potent compounds. The
synthesis used was identical to that described in Scheme
2 with notable exceptions shown in Scheme 4. The alde-
hyde 16 required for the synthesis of the 6,8-difluoro
compounds (24 and 28) was unstable. To circumvent
the use of this aldehyde we condensed the tert-butyl es-
ter 18 with the acid chloride 17 followed by acid medi-
ated decarboxylation to give 19. Palladium mediated
reduction of the derived enol-triflate 20 gave the desired
compounds 24 and 28. The 7-fluoro compounds (33 and
40) and the 5-fluoro compounds (35 and 42) were
For compounds such as 1 and 2 we have used an ex-vivo
model of platelet aggregation to determine efficacy in an
animal model. The drug is usually administered orally to
cynomolgus monkeys in 20% HPbCD at a dose of
3 mpk. Blood samples are then collected at various time
intervals (1, 2, 3, 4, 5, 6, and 24 h) from both the treat-
ment and control animals, and the aggregation response
to 1 lM of haTRAP is measured in a whole blood
aggregometer. This gives an aggregation versus time
graph. Compounds 9 and 10 were inactive in this assay
due to poor measured PK (AUC <300 ng h/ml). Only
compound 11 (AUC = 6300 ng h/ml) was shown to have
activity but with a limited duration (5 h). This was a
Table 1. Binding data of analogs of compound 7g
F
F
O
O
H
H
H
O
O
F
F
H
16
17
O
O
Cl
F
F
O
O
H
H
H
H
O
N
O
O
O
F
F
(a)
(b)
(c)
O
OTf
N
N
19
20
18
Ar
Ar
Ar
(d)
24,28
OTf
OTf
F
O
O
O
H
H
H
F
(e)
(e)
O
35,42
33,40
H
22
21
Scheme 4. Reagents and conditions: (a) BuLi, THF, then 17 (65%); (b)
TFA, CH2Cl2, 0 °C (76%); (c) Tf2O, TEA, CH2Cl2 (60%); (d)
Pd(OAc)2, HCO2H, Ph3P, DMF, 60 °C (59–76%); (e) Pd(dppf)Cl2,
HCO2H, Ph3P, DMF, 80 °C (20–74%).
aPAR-1 binding assay ligand: [3H]haTRAP, 10 nM (Kd = 15 nM).17

3650
O
M. C. Clasby et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3647–3651
44. Both of these compounds were about 3-fold less active
O
H
H
Me
H
than their unsubstituted precursors; this was found to be
typical for this type of substitution in this series of com-
pounds. Selected compounds were tested in our ex-vivo
platelet aggregation assay in cynomolgus monkeys; the
data are shown in Table 2. A majority of the compounds
tested showed activity at a dose of 3 mpk. Compound 40,
however, showed robust activity when dosed at 2 mpk for
24 h and intermittent activityat 1 mpk; this is the most po-
tent compound discovered to date in this series. Com-
pound 40 had excellent blood levels in both rats (Table
2) and cynomolgus monkeys (AUC0–24 h = 6200 ng h/ml
when dosed at 1 mpk). In addition compound 40 was
shown to have a slow disassociation rate14 from the recep-
tor with a measured half-life of 139 min. We believe that
because of the intramolecular nature of the PAR-1 mech-
anism this property will be important for any antagonist
to be effective in a clinical setting.12–14
O
O
F
F
(a)
N
N
IC50 = 30 nMa
43
OH
H
29
Cl
Cl
O
O
H
H
F
F
O
O
(b)
N
N
In summary, we have discovered a novel series of throm-
bin receptor antagonists based on the arylhimbacine ser-
ies. Compound 40 was shown to be the most potent with
activity in a monkey model of platelet aggregation at a
dose of 2 mpk. This compound had good blood levels
after oral dosing in both rats and monkeys. Compound
40 also has a slow disassociation rate from the receptor,
thus giving it an excellent chance to compete with the
intramolecular activation of PAR-1 initiated by throm-
bin. Results of additional studies will be reported in
due course.
IC50 = 26.5 nMa
44
40
F
F
a) PAR-1 binding assay ligand: [3H]haTRAP, 10 nM (Kd = 15 nM)17
Scheme 5. Reagents and conditions: (a) LDA, THF, MeI, À78 °C
(47%); (b) LHMDS, THF, À78 °C, (1S)-(10-camphorsulfonyl)oxazir-
idine (20%).
Table 2. In-vivo and ex-vivo data of selected compounds
Acknowledgments
The authors thank Drs. Catherine Strader, John Piwin-
ski, Michael Graziano, Ashit Ganguly, and Ronald
White for helpful discussions, Drs. Birendra Pramanik
and Pradip Das for mass spectral data and Drs. T.-M.
Chan and Mohindar Puar for NMR data.
References and notes
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given in Table 1. Compounds of high interest have been
highlighted; we found that fluoro substitution at either
the X2 or X3 positions led to the most potent com-
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followed by (1S)-(10-camphorsulfonyl)oxaziridine gave

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3651
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