Skip to main content
NCBI home page
ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2014 Mar 11;5(5):561–565. doi: 10.1021/ml500008w

Himbacine-Derived Thrombin Receptor Antagonists: C7-Spirocyclic Analogues of Vorapaxar

Mariappan V Chelliah 1,*, Keith Eagen 1, Zhuyan Guo 1, Samuel Chackalamannil 1, Yan Xia 1, Hsingan Tsai 1, William J Greenlee 1, Ho-Sam Ahn 1, Stan Kurowski 1, George Boykow 1, Yunsheng Hsieh 1, Madhu Chintala 1
PMCID: PMC4027741  PMID: 24900880

Abstract

graphic file with name ml-2014-00008w_0008.jpg

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

Platelets play a central role in balancing the interplay between blood clotting and bleeding. Any atherosclerotic plaque rupture in the artery will activate the aggregation of platelets, which is a major component of blood clot.1 When a clot occludes a small vessel, it can prevent the blood flowing to 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 thienopyridines.

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 activation.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.

Optimization of our himbacine based lead led to the discovery of vorapaxar, a first-in-class antiplatelet agent that works by antagonism of the PAR-1 receptor.8 In a recent Ph-III clinical trial, vorapaxar was evaluated against the secondary prevention of atherothrombotic events.18,19 Vorapaxar showed 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.

Figure 1.

Figure 1

Open in a new tab

Spirocyclic analogue of vorapaxar.

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 phosphonate 7. To synthesize analogues where the phenyl substitution is varied further, we prepared bromo-substituted 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.

Scheme 1. Synthesis of Spirocyclic Analogues 5a–d.

Scheme 1

Open in a new tab

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.

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 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.

Scheme 2. Synthesis of Spirocyclic Analogues 12a–f.

Scheme 2

Open in a new tab

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).

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 dimethylsulfonium 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 with trimethyl phosphine followed by treatment with triphosgene gave the oxazolidinone analogues 20a–b.

Scheme 3. Synthesis of Spirocyclic Analogues 20a–b.

Scheme 3

Open in a new tab

Reagents and conditions: (a) NaH, trimethylsulfonium iodide, DMSO; (b) NaN3, NH4Cl, DMF, 60 °C; (c) Me3P, ethyl acetate–H2O; (d) triphosgene, DIPEA.

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) 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.

Table 1. Inhibition Constant and Pharmacokinetic Data for Compounds 5a–d, 12a–f, and 20a–b.

graphic file with name ml-2014-00008w_0007.jpg

compd Ar Ki (nM) ± SEMa rat AUCb
5a 3-CF3–Ph 21 ± 9 4920
5b 3-F–Ph 15.5 ± 1.5 3245
5c 2-CN–Ph 5.1 ± 0.5  
5d 3-CN–Ph 267 ± 19.5  
12a 3-CF3–Ph 26.5 ± 11.5  
12b 3-F–Ph 28 ± 13  
12c 2-CN–Ph 8.9 ± 3.1  
12d 3-CN–Ph 43 ± 10  
12e 2-F–Ph 25 ± 1  
12f 2-pyridyl 232 ± 21  
20a 3-CF3–Ph 31.5 ± 4.5 3202
20b 3-CF3–Ph 392 ± 78  
Open in a new tab
a

n = 2 or more.

b

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

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 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 enhancement (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 (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.

Figure 2.

Figure 2

Open in a new tab

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.

Although the substituent 3-CF3 is tolerated in 20a, the isomeric compound 20b shows ∼10-fold increase in Ki. We attribute this Ki shift to the difference in the stereo chemical orientation between these two isomers. From modeling, the NH in spirocycle 5a makes a hydrogen bond with the backbone C=O of the protein; in spirocycle 12a–f and 20a, this NH is replaced by a CH2 group, which is still tolerated (the NH in spirocycle 12a–f and O in 20a is close to His336). However, for 20b, the corresponding atom is an oxygen, which would cause electrostatic conflict with the C=O of the protein.

In summary, we have synthesized several C7-spirocyclic analogues of vorapaxar, evaluated their PAR-1 activities, and used the vorapaxar/PAR-1 crystal structure to build a docking model for these analogues. Compared with the vorapaxar series, the spirocyclic analogues appear to be more sensitive to phenyl substitution. The 2-cyanophenyl analogues 5c and 12c showed excellent in vitro activities, which could be explained by the excellent fit into a subpocket in the trans-membrane domain of the protein.

Acknowledgments

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

Glossary

ABBREVIATIONS

PAR-1

protease activated receptor-1

haTRAP

high affinity thrombin receptor-activating peptide

ADP

adenosine diphosphate 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 relationship

Supporting Information Available

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 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

ml500008w_si_001.pdf (492.8KB, pdf)

References

  1. David G.; Patrono C. Platelet Activation and Atherothrombosis. N. Engl. J. Med. 2007, 357, 2482–2494. [DOI] [PubMed] [Google Scholar]
  2. Miner J.; Hoffhines A. The Discovery of Aspirin’s Antithrombotic Effects. Tex. Heart Inst. J. 2007, 342179–186. [PMC free article] [PubMed] [Google Scholar]
  3. Testa L.; Biondi Zoccai G. G.; Valgimigli M.; Latini R. A.; Pizzocri S.; Lanotte S.; Laudisa M. L.; Brambilla N.; Ward M. R.; Figtree G. A.; Bedogni F.; Bhindi R. Current Concepts on Antiplatelet Therapy: Focus on the Novel Thienopyridine and Non-Thienopyridine Agents. Adv. Hematol. 2010, 595934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chackalamannil S.; Xia Y.; Greenlee W. J.; Clasby M.; Doller D.; Tsai H.; Asberom T.; Czarniecki M.; Ahn H.-S.; Boykow G.; Foster C.; Agans-Fantuzzi J.; Bryant M.; Lau J.; Chintala M. Discovery of Potent Orally Active Thrombin Receptor (Protease Activated Receptor 1) Antagonists as Novel Antithrombotic Agents. J. Med. Chem. 2005, 48, 5884–5887. [DOI] [PubMed] [Google Scholar]
  5. Clasby M. C.; Chackalamannil S.; Czarniecki M.; Doller D.; Eagen K.; Greenlee W.; Kao G.; Lin Y.; Tsai H.; Xia Y.; Ahn H.-S.; Agans-Fantuzzi J.; Boykow G.; Chintala M.; Foster C.; Smith-Torhan A.; Alton K.; Bryant M.; Hsieh Y.; Lau J.; Palamanda J. Metabolism-Based Identification of a Potent Thrombin Receptor Antagonist. J. Med. Chem. 2007, 50, 129–138. [DOI] [PubMed] [Google Scholar]
  6. Chelliah M. V.; Chackalamannil S.; Xia Y.; Eagen K.; Clasby M. C.; Gao X.; Greenlee W. J.; Ahn H.-S.; Agans-Fantuzzi J.; Boykow G.; Hsieh Y.; Bryant M.; Palamanda J.; Chan T.-M.; Hesk D.; Chintala M. Heterotricyclic Himbacine Analogues as Potent, Orally Active Thrombin Receptor (Protease Activated Receptor-1) Antagonists. J. Med. Chem. 2007, 50215147–5160. [DOI] [PubMed] [Google Scholar]
  7. Clasby M. C.; Chackalamannil S.; Czarniecki M.; Doller D.; Eagen K.; Greenlee W. J.; Lin Y.; Tagat J. R.; Tsai H.; Xia Y.; Ahn H.; Agans-Fantuzzi J.; Boykow G.; Chintala M.; Hsieh Y.; McPhail A. T. Himbacine Derived Thrombin Receptor Antagonists: Discovery of a New Tricyclic Core. Bioorg. Med. Chem. Lett. 2007, 17133647–3651. [DOI] [PubMed] [Google Scholar]
  8. Chackalamannil S.; Wang Y.; Greenlee W. J.; Hu Z.; Xia Y.; Ahn H.; Boykow G.; Hsieh Y.; Palamanda J.; Agans-Fantuzzi J.; Kurowski S.; Graziano M.; Chintala M. Discovery of a Novel, Orally Active Himbacine-Based Thrombin Receptor Antagonist (SCH 530348) with Potent Antiplatelet Activity. J. Med. Chem. 2008, 51113061–3064. [DOI] [PubMed] [Google Scholar]
  9. Xia Y.; Chackalamannil S.; Greenlee W. J.; Wang Y.; Hu Z.; Root Y.; Wong J.; Kong J.; Ahn H.; Boykow G.; Hsieh Y.; Kurowski S.; Chintala M. Discovery of a Vorapaxar Analog with Increased Aqueous Solubility. Bioorg. Med. Chem. Lett. 2010, 20226676–6679. [DOI] [PubMed] [Google Scholar]
  10. Chelliah M. V.; Chackalamannil S. L.; Xia Y.; Eagen K.; Greenlee W. J.; Ahn H.; Agans-Fantuzzi J.; Boykow G.; Hsieh Y.; Bryant M.; Chan T.; Chintala M. Discovery of Nor-Seco Himbacine Analogues as Thrombin Receptor Antagonists. Bioorg. Med. Chem. Lett. 2012, 2272544–2549. [DOI] [PubMed] [Google Scholar]
  11. Chackalamannil S. L.Antiplatelet Agents. In Burger’s Medicinal Chemistry, Drug Discovery, and Development, 7th ed.; Abraham D. J., Rotella D. P., Eds.; Wiley: New York, 2010; pp 409–476.
  12. Coughlin S. R.Protease-Activated Receptors in Handbook of Cell Signaling; Bradshaw R. A., Dennis E. A., Eds.; Elsevier: San Diego, CA, 2004; Vol. 1, pp 167–171. [Google Scholar]
  13. Coughlin S. R. Cold Spring Harbor Symposia on Quantitative Biology. Protease-Activated Receptors in the Cardiovascular System 2002, 67, 197–208. [DOI] [PubMed] [Google Scholar]
  14. Coughlin S. R. Protease-Activated Receptors in Vascular Biology. Thromb. Haemostasis 2001, 86, 298–307. [PubMed] [Google Scholar]
  15. Coughlin S. R. Protease-Activated Receptors in Hemostasis, Thrombosis and Vascular Biology. J. Thromb. Haemost. 2005, 3, 1800–1814. [DOI] [PubMed] [Google Scholar]
  16. Chackalamannil S. Thrombin Receptor (Protease Activated Receptor-1) Antagonists as Potent Antithrombotic Agents with Strong Antiplatelet Effects. J. Med. Chem. 2006, 49, 5389–5403. [DOI] [PubMed] [Google Scholar]
  17. Grand R. J.; Turnell A. S; Grabham P. W. Cellular Consequences of Thrombin-Receptor Activation. Biochem. J. 1996, 313, 353–368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tricoci P.; Huang Z.; Held C.; Moliterno D. J.; Armstrong P. W.; Van de Werf F.; White H. D.; Aylward P. E.; Wallentin L.; Chen E.; Lokhnygina Y.; Pei J.; Leonardi S.; Rorick T. L.; Kilian A. M.; Jennings L. H. K.; Ambrosio G.; Bode C.; Cequier A.; Cornel J. H.; Diaz R.; Erkan A.; Huber K.; Hudson M. P.; Jiang L.; Jukema J. W.; Lewis B. S.; Lincoff A. M.; Montalescot G.; Nicolau J. C.; Ogawa H.; Pfisterer M.; Prieto J. C.; Ruzyllo W.; Sinnaeve P. R.; Storey R. F.; Valgimigli M.; Whellan D. J.; Widimsky P.; Strony J.; Harrington R. A.; Mahaffey K. W.; Thrombin-Receptor Antagonist Vorapaxar in Acute Coronary Syndromes. N. Engl. J. Med. 2012, 3661520–33. [DOI] [PubMed] [Google Scholar]
  19. Morrow D. A.; Braunwald E.; Bonaca M. P.; Ameriso S. F.; Dalby A. J.; Fish M. P.; Fox K. A.; Lipka L. J.; Liu X.; Nicolau J. C.; Ophuis A. J.; Paolasso E.; Scirica B. M.; Spinar J.; Theroux P.; Wiviott S. D.; Strony J.; Murphy S. A.; Vorapaxar in the Secondary Prevention of Atherothrombotic Events. N. Engl. J. Med. 2012, 366151404–1413. [DOI] [PubMed] [Google Scholar]
  20. Ghosal A.; Lu X.; Penner N.; Gao L.; Ramanathan R.; Chowdhury S. K.; Kishnani N. S.; Alton K. B. Identification of Human Liver Cytochrome P450 Enzymes Involved in the Metabolism of SCH 530348 (Vorapaxar), a Potent Oral Thrombin Protease-Activated Receptor 1 Antagonist. Drug Metab. Dispos. 2011, 39, 30–38. [DOI] [PubMed] [Google Scholar]
  21. Espino C. G.; Du Bois J. A Rh-Catalyzed C–H Insertion Reaction for the Oxidative Conversion of Carbamates to Oxazolidinones. Angew. Chem., Int. Ed. 2001, 40, 598–600. [DOI] [PubMed] [Google Scholar]
  22. Ahn H.-S.; Foster C.; Boykow G.; Arik L.; Smith-Torhan A.; Hesk D.; Chatterjee M. Binding of a Thrombin Receptor Tethered Ligand Analogue to Human Platelet Thrombin Receptor. Mol. Pharmacol. 1997, 51, 350–356. [DOI] [PubMed] [Google Scholar]
  23. Zhang C.; Srinivasan Y.; Arlow D. H.; Fung J. J.; Palmer D.; Zheng Y.; Green H. F.; Pandey A.; Dror R. O.; Shaw D. E.; Weis W. I.; Coughlin S. R.; Kobilka B. K. High-Resolution C Structure of Human Protease-Activated Receptor. Nature 2012, 492, 387–392. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

ml500008w_si_001.pdf (492.8KB, pdf)

Articles from ACS Medicinal Chemistry Letters are provided here courtesy of American Chemical Society

RESOURCES