Synthesis And Activity Evaluation Of The Analogue Of Thrombin Receptor Antagonist Vorapaxar

At present,more than 3 million people die of cerebrovascular diseases every year in China.Antithrombotic therapy is the main way to prevent cerebrovascular events.Thrombin receptor which was discovered in 1991 is a new target of antiplatelet aggregation.Vorapaxar is the only thrombin receptor antagonist approved by FDA and it was discovered by Merck for cardiovascular disease,it was used to prevent cerebrovascular diseases in patients with a history of heart attack.However Vorapaxar can significantly increase the risk of intracranial hemorrhage in patients with a history of stroke,and vorapaxar can not be used to the patients with the history of transient ischemic attack or stroke,which greatly limits the scope of application of vorapaxar.Therefore,in order to improve the side effects of intracranial hemorrhage and improve the the scope of application,many researchers study the pharmacological activity and structure-activity relationship of these vorapaxar analoguesto carry out structural modification and physical and chemical properties optimization.In this project,vorapaxar is used as the original drug.On the basis of the principle,avoiding the patent protection scope of vorapaxar,we intend to improve the shortcomings of vorapaxar by increasing the polarity of the compound and reducing the ability of the compound to enter the brain.Therefore,three series of vorapaxar derivatives were designed on the structure of the tricyclic mother nucleus of vorapaxar,and the inhibitory activity of these compounds on PAR-1 was evaluated by Ca²⁺transport inhibition fluorescence detection.By the way of the pharmacokinetic test to evaluate the pharmacokinetic behavior and the ability of drugs into the brain,we hope to find the candidate thrombin receptor antagonists with good anticoagulant activity in vitro and low ability to enter the brain.The main research contents and results are as follows:In the first part,the research progress of pharmacology of vorapaxar analogues in recent years is reviewed.The research status of thrombin receptor antagonists and the clinical characteristics of vorapaxar are briefly introduced.The purpose,content and significance of this project are described.In the second part,according to the patent protection scope and the structure-activity relationship of vorapaxar,9 analogues of vorapaxar were designed and synthesized.The synthesis routes were preliminarily explored.The key intermediates and target products were characterized by ESI-MS,NMR and other means.In the third part,the derivatives of vorapaxar were tested in a biological assay to determine their inhibition activities against PAR-1.The antagonist activity of compounds at the human PAR-1 receptor expressed in the transfected KNRK cells were evaluated by measuring their effects on agonist-induced intracellular Ca²⁺ion mobilization using a fluorimetric detection method.Cellular antagonist effects were calculated as a percentage of inhibition of the agonist response for each compound.The compounds A2,A4,B1,B2,B3and C1 showed similar inhibition activities with vorapaxar,whereas compounds A1,A3 and C2 displayed decreased activities.Among the active compounds,compound A4 with an amino acetonitrile substituent at the C7 position exhibited good PAR-1 inhibitory activity with an IC₅₀ value of 0.18μM,which was slightly better than vorapaxar(IC₅₀=0.25μM).Compounds A2,B3 and C1 showed a little diminished PAR-1 inhibitory activities,with IC₅₀values of 0.61,0.76 and 0.55μM.The substituents with moderate polarity are benefitial to keep the inhibitory activity on PAR-1 and may reduce the ability to enter the brain.The pyridine ring at C-4 was important（C1 and C2）,and minor changes may have major impact on the activity.This was because of the basicity and orientation of the lone pair of electrons on nitrogen in the 2-pyridyl group was important for binding to human PAR-1 receptor.In the fourth part,representative compounds were also evaluated for pharmacokinetic characteristics in rats to assess their bioavailabilities and concentrations in the brain.SD rats were administered at 5mg/kg oral dose or at 1mg/kg iv injection dose of vorapaxar,A2,A4,B1,B3 and C1.Compound A4 exhibited an excellent oral bioavailability of 98%and oral half-life of 6.4h.However,the bioavailability of A2,B3 and C1（23%,34%and 44%）were lower than vorapaxar（94%）.The brain/plasma concentration ratio at 8h after oral administration were measured which could reflect the ability of drug to reach the brain.The brain/plasma concentration ratios in rats for A4,B3 and C1 were significantly lower than that of vorapaxar,but the lower oral bioavailabilities of B3 and C1 may limit their potentials.Overall,compound A4 presented a favorable profile of good PAR-1 inhibition activity,high orall bioavailability and low brain/plasma ratio.At the same time,molecular docking test showed that the binding sites of compound A4 and thrombin protein were well matched,which was similar to the binding conformation of vorapaxa.It was verified from the target that A4 might be a new PAR-1 antagonist candidate for reducing intracranial hemorrhage.