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. 2013 Dec 18;5(2):183–187. doi: 10.1021/ml400452v

Himbacine-Derived Thrombin Receptor Antagonists: C7-Aminomethyl and C9a-Hydroxy Analogues of Vorapaxar

Mariappan V Chelliah 1,*, Samuel Chackalamannil 1, Yan Xia 1, William J Greenlee 1, Ho-Sam Ahn 1, Stan Kurowski 1, George Boykow 1, Yunsheng Hsieh 1, Madhu Chintala 1
PMCID: PMC4027727  PMID: 24900795

Abstract

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We have synthesized several C7-aminomethyl analogues of vorapaxar that are potent PAR-1 antagonists. Many of these analogues showed excellent in vitro binding affinity and pharmacokinetics profile in rats. Compound 6a from this series showed excellent PAR-1 activity (Ki = 5 nM). We have also synthesized a C9a-hydroxy analogue of vorapaxar, which showed very good PAR-1 affinity (Ki = 19.5 nM) along with excellent rat pharmacokinetic profile and ex vivo efficacy in the cynomolgus monkey.

Keywords: Himbacine, vorapaxar, PAR-1 antagonist, thrombin receptor, platelet aggregation

Cardiovascular disease is a major cause of death in both developed and developing countries. A large number of these deaths is associated with platelet mediated atherothrombotic events.1,2 When an atherosclerotic rupture occurs in the vascular endothelium, it leads to the adhesion of platelets to the site of injury, which subsequently form a platelet rich thrombus. Though the formation of thrombus plays a vital role in hemostasis, the formation of thrombi often occludes the coronary artery leading to acute coronary syndrome such as unstable angina and myocardial infarction. Thus, antiplatelet agents play a vital role in treating patients prone to atherothrombotic events. Aspirin and clopidogrel are two widely used antiplatelet agents for people afflicted by acute coronary syndrome. Aspirin works by the antagonism of thromboxane A2 (TXA2) mediated platelet activation, whereas clopidogrel works by the antagonism of the predominant ADP receptor P2Y12. Though platelet activation is initiated by several factors, thrombin is the most potent activator of platelets. This activation is mediated by thrombin action on two GPCR receptors located on the surface of the platelets called thrombin receptors (also known as protease activated receptors; PAR-1 and PAR-4).3−9 Among these two receptors, PAR-1 plays a major role in primate platelet activation. Agents that antagonize the activation of this PAR-1 receptor can be expected to be potent antiplatelet agents.

We have published a series of himbacine-derived antiplatelet agents that are potent antagonists of PAR-1 receptor.10−16 Our systematic optimization of the himbacine based hit led to the discovery of vorapaxar (SCH530348), a potent PAR-1 antagonist.14 Vorapaxar showed excellent ex vivo platelet aggregation inhibition in a preclinical monkey efficacy model.17 In the phase-II clinical study, it met the primary end point of absence of TIMI major and minor bleeding.18 In the phase-III clinical trial for the secondary prevention of atherothrombotic events, there was a significant reduction in overall ischemic events with vorapaxar when added to background antiplatelet therapy.19,20 Thus, treatment of atherothrombotic events with antagonists of the PAR-1 receptor has been validated by the results of the phase-III clinical trial of vorapaxar.

In our lead optimization of the himbacine based thrombin receptor antagonists, we replaced the C7 carbon of the tricyclic himbacine scaffold with nitrogen, which resulted in the discovery of 1 (Figure 1).12 This heterohimbacine analogue 1 maintained the excellent antithrombotic activity while solving the critical enzyme induction and clearance issues encountered in the corresponding carbocyclic series.12 When compound 1 was dosed in cynomolgus monkey, with 20% PEG-HPBCD as the dosing vehicle, it showed excellent ex vivo efficacy in the platelet aggregation inhibition assay. Unfortunately, this compound showed poor aqueous solubility (<2 μM) and the ex vivo efficacy was markedly reduced when the dosing vehicle was changed from 20% PEG-HPBCD to 0.4% methylcellulose. In the subsequent SAR study of this series, nitrogen atom of the C-ring was moved exocyclic to the ring to give the C7-amino substituted tricyclic himbacine analogues, which led to the discovery of vorapaxar.14 In this letter, we disclose the variation of this C-ring exocyclic amine to give the homologated analogues 6a–j where a methylene linker is introduced between the amino group and the C-ring along with the carboxamide analogues 9a–h. We also synthesized the C9a-hydroxy analogue 11 of vorapaxar. This analogue was prepared in reflection of a recent disclosure of novel nor seco himbacine analogues as potent PAR-1 antagonists.16 In the nor seco series, we observed a dramatic improvement in the rat plasma level by the introduction of a hydroxy functionality at the carbon alpha to the lactone carbonyl group. We wanted to see whether a similar improvement in pharmacokinetic profile will result for vorapaxar by the introduction of a similar C9a-hydroxy group. Herein we describe the synthesis and the PAR-1 activity results of these analogues.

Figure 1.

Figure 1

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Himbacine-derived thrombin receptor antagonists.

The synthesis of the C7-aminomethyl analogues 6a–g (Scheme 1) starts with the known ketone 2,14 which was subjected to a Wittig reaction followed by the hydrolysis of the resulting vinyl ether to give the aldehyde 3. Reduction of this aldehyde gave the alcohol 4, which was mesylated, and the mesylate was subsequently displaced with sodium azide to give the azide intermediate, which was reduced to the amine 5 using trimethyl phosphine. This amine was subsequently treated with chloroformates, acid chlorides, sulfonyl chlorides, and isocyanates to give the corresponding carbamates, sulfonamides, and ureas 6a–j. The preparation carboxamide analogues 9a–h started with the ketone 7,11 which was converted to the carboxylic acid 8. Coupling of this acid with amines under standard condition gave the amide analogues 9a–h.

Scheme 1. Synthesis of C7-Aminomethyl Carbamates and Carboxamides.

Scheme 1

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Reagents and conditions: (a) Ph3P+CH2OCH3 Cl–, tBuOK, THF; (b) HCl in dioxane/water, rt; (c) sodium borohydride, THF–MeOH; (d) methanesulfonyl chloride, Et3N; (e) sodium azide, DMSO, 65 °C; (f) Me3P, H2O; (g) ROCOCl, RCOCl, RSO2Cl, RNCO; (h) TFA–DCM (for compounds 6e–g only); (i) NaClO2, H2O2, NaH2PO4; (j) RNH2, HATU, Et3N.

Synthesis of the C9a-hydroxy analogue of vorapaxar 11 is described in Scheme 2. The amino functionality of the carbamate group was protected with di-tert-butyldicarbonate, and the resultant product was treated with lithium bis(trimethylsilyl)amide to generate the C9a-enolate. This enolate was stirred under an oxygen atmosphere to introduce the C9a-hydroxy functionality. Deprotection of the tert-butyl carbamate group under acidic conditions gave the target 11.

Scheme 2. Synthesis of C9a-Hydroxy Analogue of Vorapaxar.

Scheme 2

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Reagents and conditions: (a) (Boc)2O, Et3N, DMAP, CH3CN, 60 °C; (b) LHMDS, THF then oxygen; (c) TFA–DCM, 0 °C to rt.

The above synthesized analogues were evaluated in the in vitro binding assay using PAR-1 receptors isolated from human platelets and [3H]haTRAP as the radioligand.22 Table 1 presents the PAR-1 binding affinity for the C7-aminomethyl derivatives, and Table 2 presents the affinity for the carboxamide analogues. Binding affinity for compound 11 is presented in Scheme 2. Both the C7-hydroxymethyl analogue 4 and the unsubstituted aminomethyl analogue 5 showed good binding affinity. The methyl carbamate 6a (Ki = 5 nM) showed excellent binding affinity, while the ethylcarbamate 6b (Ki = 14 nM) was 3-fold less active. Both the acetamide 6c and the propionamide 6d analogues showed very good affinity. Amides 6e–g containing amino groups indicate that polar groups are tolerated at this position. The amide derivative of piperidine-4-carboxylic acid 6g showed very good affinity, though the corresponding analogues of alanine (6e) and proline (6f) analogues showed 2-fold less affinity. The methane sulfonamide (6h), ethane sulfonamide (6i), and N-ethyl urea (6j) analogues showed good affinity.

Table 1. Binding and PK Data for Compounds 4, 5, and 6a–j.

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a

n = 2 or more.

a

AUC from 0 to 6 h in ng·h/mL and at 10 mg/kg oral dose (0.4% methylcellulose).

Table 2. Binding Data for Compounds 9a–h.

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a

n = 2 or more.

The carboxamide analogues 9a–c containing N-methyl, N-ethyl, and N-isopropyl amides showed good affinity. However, introduction of polar group reduces the affinity as indicated by the amino ethanol amide analogue 9d. Amides of 4-aminopyridine and 3-aminopyridines 9e–f showed considerable reduction in affinity. Both analogues of 3-hydroxy pyrrolidine (9g–h) showed reduced affinity indicating that polar groups at this position reduces the affinity. Also, the C9a-hydroxy analogue of vorapaxar, 11, showed good affinity (Scheme 2) indicating that the introduction of the hydroxyl group at the C9a position is tolerated in this series

Selected analogues were evaluated in the rat pharmacokinetic assay at an oral dose of 10 mg/kg, and the plasma levels were analyzed up to 6 h. Several of these analogues showed good plasma levels. While the ethyl carbamate analogue 6b showed good rat plasma level (AUC = 1213 ng·h/mL), the corresponding methyl carbamate analogues 6a showed about 4-fold higher plasma level (AUC = 4863 ng·h/mL). Compared with the acetamide 6c, the propionamide analogue 6d showed 2-fold increase in plasma level (AUC = 7728 ng·h/mL). Analogue 6g showed very poor plasma level (AUC = 5 ng·h/mL) indicating that the polar amino group severely affects the absorption. While methane sulfonamide 6h showed excellent plasma level, the N-ethyl urea analogue 6j showed a low level of plasma. Rat plasma level for the C9a-hydroxy analogue 11 was excellent (AUC = 7431 ng·h/mL) as indicated in Scheme 2. Compared with vorapaxar (AUC = 3064 ng·h/mL), analogue 11 showed a 2-fold increase in rat plasma level. This observation is in parallel to the nor seco himbacine analogues where introduction of the hydroxy group at alpha to the carbonyl group showed increased plasma level.16

We evaluated compounds 11 and 6a in the ex vivo platelet aggregation inhibition assay in cynomolgus monkey (Figure 2). The compound was given as an oral dose and blood was drawn at various points of time. The exogenous agonist peptide, haTRAP, was added as a 1 M solution to the blood sample, and the extent of aggregation induced by this agonist was quantified using an aggregometer as described before.23 At a dose of 1 mg/kg, 11 completely inhibited platelet aggregation, and the inhibition was maintained at this level for up to 24 h. This clearly indicated that the introduction of the C9a-hydroxy group, while improving the rat plasma level, also maintained excellent ex vivo efficacy profile. In marked contrast, compound 6a showed only transient efficacy despite showing excellent binding affinity and rat plasma levels.

Figure 2.

Figure 2

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Ex vivo platelet aggregation inhibition in cynomolgus monkey following a single oral dose (1 mg/kg in 20% PEG-HPBCD) for 11 and 6a.

In summary, we have synthesized several C7-aminomethyl and carboxamide analogues of vorapaxar. The aminomethyl analogues in general showed excellent binding affinity while maintaining excellent rat plasma levels for many analogues. Introduction of a hydroxy group at the C9a position of vorapaxar improved rat plasma level while maintaining complete inhibition of platelet aggregation in cynomolgus monkey.

Acknowledgments

We acknowledge the support of Drs. John Piwinski, Catherine Strader, Birendra Pramanik, Pradip Das, Tze-Ming Chan, Jesse Wong, Jianshe Kong, and Richard Morrison.

Glossary

Abbreviations

PAR-1

protease activated receptor-1

TXA2

thromboxane A2

PEG-HPBCD

poly(ethylene glycol)–hydroxypropyl-beta-cyclodextrin

haTRAP

high affinity thrombin receptor-activating peptide

ADP

adenosine diphosphate receptor

GPCR

G protein coupled receptor

PK

pharmacokinetics

DMAP

4-dimethylaminopyridine

LHMDS

lithium bis(trimethylsilyl) amide

DCM

dichloromethane

HATU

1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

Supporting Information Available

Experimental details for the preparation of compounds 6a, 9a, and 11 is provided. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Present Address

† S.C.: Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854–8020, United States.

Author Present Address

‡ Y.X.: School of Environmental and Municipal Engineering, Qingdao Technological University, 11 Fushun Road, Qingdao, Shandong 266033, China.

Author Present Address

§ W.J.G.: MedChem Discovery Consulting, LLC, 115 Herrick Avenue, Teaneck, New Jersey 07666, United States.

The authors declare no competing financial interest.

Supplementary Material

ml400452v_si_001.pdf (199KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml400452v_si_001.pdf (199KB, pdf)

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