Himbacine-derived thrombin receptor antagonists: C7-Spirocyclic analogues of vorapaxar

Article abstract of DOI:10.1021/ml500008w

We have synthesized several C₇-spirocyclic analogues of vorapaxar and evaluated their in vitro activities against PAR-1 receptor. Some of these analogues showed activities and rat plasma levels comparable to vorapaxar. Compound 5c from this series showed excellent PAR-1 activity (K _i = 5.1 nM). We also present a model of these spirocyclic compounds docked to the PAR-1 receptor based on the X-ray crystal structure of vorapaxar bound to PAR-1 receptor. This model explains some of the structure-activity relationships in this series.

Full text of DOI:10.1021/ml500008w

Letter
pubs.acs.org/acsmedchemlett
Himbacine-Derived Thrombin Receptor Antagonists: C7‑Spirocyclic
Analogues of Vorapaxar
Mariappan V. Chelliah,* Keith Eagen, Zhuyan Guo, Samuel Chackalamannil,† Yan Xia,‡ Hsingan Tsai,
William J. Greenlee,§ Ho-Sam Ahn, Stan Kurowski, George Boykow, Yunsheng Hsieh,
and Madhu Chintala
Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033-1300, United States
S
* Supporting Information
ABSTRACT: We have synthesized several C7-spirocyclic
analogues of vorapaxar and evaluated their in vitro activities
against PAR-1 receptor. Some of these analogues showed
activities and rat plasma levels comparable to vorapaxar.
Compound 5c from this series showed excellent PAR-1
activity (Ki = 5.1 nM). We also present a model of these
spirocyclic compounds docked to the PAR-1 receptor based
on the X-ray crystal structure of vorapaxar bound to PAR-1
receptor. This model explains some of the structure−activity
relationships in this series.
KEYWORDS: PAR-1 antagonist, vorapaxar, thrombin receptor, himbacine
latelets play a central role in balancing the interplay
Optimization of our himbacine based lead led to the
Pbetween blood clotting and bleeding. Any atherosclerotic discovery of vorapaxar, a first-in-class antiplatelet agent that
works by antagonism of the PAR-1 receptor.8 In a recent Ph-III
plaque rupture in the artery will activate the aggregation of
platelets, which is a major component of blood clot.1 When a
clinical trial, vorapaxar was evaluated against the secondary
prevention of atherothrombotic events.18,19 Vorapaxar showed
clot occludes a small vessel, it can prevent the blood flowing to
a significant reduction of death from cardiovascular events.
Thus, the proof of concept of treating platelet aggregation by
the antagonism of PAR-1 receptor has been clinically validated.
Since the C7-carbamate side-chain of vorapaxar is a site of
metabolism,20 as part of our structure−activity studies on
vorapaxar analogues we were interested in analogues where the
carbamate is cyclized in a spirocyclic fashion to give an
analogue such as 5b (Figure 1). We believed that this might
address the carbamate metabolism observed in vorapaxar. In
this letter we disclose the synthesis, PAR-1 activity, and a
docking model of these novel spirocyclic analogues of
vorapaxar.
the heart and cause heart attack, which is a major cause of death
in developed countries. Drugs that inhibit platelet aggregation
can be used to treat patients with prior cardiovascular events
and prevent future heart attacks. Aspirin and thienopyridines
are two commonly prescribed classes of antiplatelet agents.
Aspirin works by blocking the activation of platelets by
thromboxane, whereas thienopyridines work by irreversibly
inhibiting the ADP receptor P2Y12.2,3 Despite the use of these
drugs, heart attack is still a major cause of mortality. There is an
unmet clinical need for novel drugs that work by an entirely
different mechanism compared with aspirin and thienopyr-
idines.
Synthesis of the spirocyclic oxazolidinone analogues 5a−d
(Scheme 1) starts with the known ketone 1,5 which was
converted to the olefine 8 using standard Wittig olefination
condition. Hydroboration of this alkene using 9-BBN gave
compound 3 as the major isomer, which was converted to the
carbamate 4 using trichloroacetyl isocyanate. Spirocyclization of
carbamate 4 to give the oxazolidinone 5a was achieved using
rhodium mediated C−H insertion as described by Dubois.21
The 3-fluorophenyl analogue 5b was synthesized by ozonolysis
of 5a giving aldehyde 6, which was coupled with the known
We have published on the activity of several series of
antiplatelet agents, derived from the natural product himbacine,
that work by a novel mechanism.4−10 These agents are
antagonists of the PAR-1 thrombin receptor (also called
protease activated receptor, PAR), a G-protein coupled
receptor, located on the platelet surface. Though two types of
PAR receptors are present in human (PAR-1 and PAR-4),
PAR-1 dominates the thrombin mediated platelet activa-
tion.11−17 Thrombin cleaves the extra-cellular domain of
these receptors exposing a tethered ligand, which activates
these receptors leading to the aggregation of platelets, which
ultimately lead to the formation of platelet rich thrombi. These
thrombi can often occlude the coronary arteries leading to heart
attack.
Received: January 10, 2014
Accepted: March 11, 2014
Published: March 11, 2014
© 2014 American Chemical Society
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pyridyl intermediate 9 from aldehyde 6 and phosphonate 8.
This enabled direct Suzuki type cross-coupling reaction on 9 to
afford the desired targets 5c−d.
The synthesis of the isomeric oxazolidinone spirocyclic
analogues, where the C7-amino group takes the β-orientation,
represented by structures 12a−f, is presented in Scheme 2. The
a
Scheme 2. Synthesis of Spirocyclic Analogues 12a−f
Figure 1. Spirocyclic analogue of vorapaxar.
a
Scheme 1. Synthesis of Spirocyclic Analogues 5a−d
a
Reagents and conditions: (a) trichloroacetyl isocyanate then K2CO3;
(b) Rh2(OAc)4, MgO, PhI(OAc)2; (c) acetyl chloride, triethyl amine,
and DMAP; (d) ozone, −78 °C then dimethyl sulfide, rt; (e) 8,
LHMDS, Ti(OiPr)4 then 13; (f) K2CO3, MeOH; (g) ArB(OH)2,
Pd(Ph3P)4, K2CO3 (for compounds 12c−e), and 2-pyridylzinc
chloride, Pd(Ph3P)4, THF (for compound 12f).
alcoholic functionality of 10a was converted to carboxamide
11a using trichloroacetyl isocyanate, which gave the desired
spirocyclic analogue 12a upon rhodium facilitated C−H
insertion.21 Analogue 12b was prepared in a similar fashion
starting from the alcohol 10b. To prepare analogues where the
phenyl substitution is varied, intermediate 15 was prepared.
Preparation of 15 starts with 10a, where the alcoholic
functionality was protected as the acetate and the double
bond was cleaved by ozonolysis to give the aldehyde 13.
Coupling of this aldehyde with the phosphonate 8 gave 14,
which was converted to the carbamate 15. Spirocyclization
followed by cross-coupling of the aryl bromide with
appropriately substituted aryl boronic acids or aryl zinc reagent
gave the target analogues 12c−f.
We also synthesized isomeric oxazolidinone analogues where
the oxygen atom of the oxazolidinone ring is directly attached
to the C7-position of the carbocyclic ring as represented by the
structures 20a−b (Scheme 3). Reaction of 1 with dimethylsul-
fonium methylide gave, after chromatographic separation,
epoxides 17a and 17b. Both epoxides were ring opened with
sodium azide to give the azides 18a−b, which on reduction
a
Reagents and conditions: (a) Ph3P+CH3 Br−, phenyl lithium, THF;
(b) 9-BBN then NaBO3·4H2O; (c) trichloroacetyl isocyanate then
K2CO3; (d) Rh2(OAc)4, MgO, PhI(OAc)2; (e) ozone, −78 °C then
dimethyl sulfide, rt; (f) 7, LHMDS, Ti(OiPr)4 then 6 ; (g) 8, LHMDS,
Ti(OiPr)4 then 6; (h) 2-cyano-phenyl boronic acid neopentylglycol
ester or 3-cyano-phenyl boronic acid, Pd(Ph3P)4, K2CO3.
phosphonate 7. To synthesize analogues where the phenyl
substitution is varied further, we prepared bromo-substituted
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ACS Medicinal Chemistry Letters
Letter
a
shows the best activity. This trend continues in the isomeric
oxazolidinone analogues 12a−f where the 2-cyanophenyl
analogue 12c (Ki = 8.9 nM) shows excellent activity. Unlike
the vorapaxar analogues, the spirocyclic series appears to be
sensitive to the effect of phenyl substitution as represented by
the marked reduction in potency for the 3-cyanophenyl
analogue 5d (Ki = 267 nM) and pyridyl analogue 12f (Ki =
232 nM). Among the two spirocyclic analogues 20a and 20b
where the oxygen of the oxazolidinone ring is directly attached
to the C7-carbon, the β-isomer 20a showed better activity than
the corresponding α-isomer 20b. Representative analogues
were also evaluated in rat pharmacokinetic studies. Analogues
5a, 5b, and 20a were dosed orally at 10 mg/kg, and they
showed good plasma levels as indicated by their AUCs up to
the 6 h time point that was evaluated.
Scheme 3. Synthesis of Spirocyclic Analogues 20a−b
To gain insight into protein ligand interactions and
understand the SAR, models of compounds complexed with
PAR1 were built by docking using the crystal structure of
vorapaxar bound to PAR-1.23 As in vorapaxar, the tricyclic
region of the compounds discussed here binds to the
extracellular loop region, with the spirocycle pointing toward
an opening to the solvent, while the biaryl region binds to a
hydrophobic pocket toward the trans-membrane region. As a
representative example, we have provided a model of
compound 5c bound to the PAR-1 receptor (Figure 2). On
a
Reagents and conditions: (a) NaH, trimethylsulfonium iodide,
DMSO; (b) NaN3, NH4Cl, DMF, 60 °C; (c) Me3P, ethyl acetate−
H2O; (d) triphosgene, DIPEA.
with trimethyl phosphine followed by treatment with
triphosgene gave the oxazolidinone analogues 20a−b.
In vitro activities for the target compounds were determined
using [3H]haTRAP as the radioligand as described before.22
PAR-1 receptors isolated from human platelets were used for
this study. The inhibition constants (Ki) for the spirocyclic
analogues are presented in Table 1. Among the spirocyclic
analogues 5a−d, the 2-cyanophenyl analogue 5c (Ki = 5.1 nM)
Table 1. Inhibition Constant and Pharmacokinetic Data for
Compounds 5a−d, 12a−f, and 20a−b
a
b
compd
Ar
Ki (nM) SEM
21
rat AUC
5a
3-CF3−Ph
3-F−Ph
2-CN−Ph
3-CN−Ph
3-CF3−Ph
3-F−Ph
2-CN−Ph
3-CN−Ph
2-F−Ph
2-pyridyl
3-CF3−Ph
3-CF3−Ph
9
4920
3245
Figure 2. Model of compound 5c bound to the PAR-1 receptor. The
inhibitor is represented as stick (green carbon) and the protein as
cartoon. The binding site pocket and select key residues are also
shown.
5b
15.5 1.5
5.1 0.5
267 19.5
26.5 11.5
28 13
5c
5d
12a
12b
12c
12d
12e
12f
20a
20b
the basis of the model, we attempt to rationalize the observed
SAR. In general, the SAR in the 5a−d and 12a−f series follows
a similar trend. For example, within each series, substitutions at
the 2-position with a nitrile have the most potency enhance-
ment (5c and 12c). This can be explained by the binding of the
biaryl region of these compounds to a subpocket involving
residues Pro236, Leu237, Leu262, Leu263, and Lys240 near the
bottom of the inhibitor binding site. Substituent 2-CN−Ph in
5c and 12c would fit nicely to the pocket. However, 2-F−Ph
8.9 3.1
43 10
25
1
232 21
31.5 4.5
392 78
3202
a
b
n = 2 or more. AUC from 0 to 6 h in ng·h/mL and at 10 mg/kg oral
dose (0.4% methyl cellulose).
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(12e) is slightly short and does not interact efficiently with the
protein. The increase in Ki of 2-pyridyl (12f) may be partly
attributed to the desolvation penalty of burying a polar atom in
the hydrophobic environment.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the preparation of all the compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(M.V.C.) E-mail: mchelliah@aol.com.
Present Addresses
†(S.C.) Ernest Mario School of Pharmacy, Rutgers, The State
University of New Jersey, Piscataway, New Jersey 08854−8020,
United States.
‡(Y.X.) School of Environmental and Municipal Engineering,
Qingdao Technological University, 11 Fushun Road, Qingdao,
Shandong 266033, China.
§(W.J.G.) MedChem Discovery Consulting, LLC, 115 Herrick
Avenue, Teaneck, New Jersey 07666, United States.
Notes
The authors declare no competing financial interest.
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We acknowledge the support of Drs. John Piwinski, Catherine
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ABBREVIATIONS
■
PAR-1, protease activated receptor-1; haTRAP, high affinity
thrombin receptor-activating peptide; ADP, adenosine diphos-
phate receptor; 9-BBN, 9-borabicyclo[3.3.1]nonane; DMAP,
dimethylaminopyridine; LHMDS, lithium bis(trimethylsilyl)
amide; THF, tetrahydrofuran; DMSO, dimethyl sulfoxide;
DIPEA, N,N-diisopropylethylamine; PK, pharmacokinetics;
AUC, area under the curve; SAR, structure−activity relation-
ship
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