Production Process Development And Pharmacokinetics Study Of PAR-1 Inhibitor Andropaxar

Thrombotic diseases,such as acute coronary syndrome（ACS）,myocardial infarction and apoplexy have become a major threat to human life in modern society,causing more than 20 million deaths every year in the world.Antiplatelet drugs are most widely used in the prevention and treatment of thrombotic diseases.Antiplatelet drugs mainly contains anti arachidonic acid metabolic drugs,antiplatelet activation drugs and 5-HT receptor antagonist.Anti arachidonic acid metabolic drugs include cyclooxygenase（COX）inhibitors（such as Aspirin）,and TXA2 synthetase and receptor inhibitors（such as Ozagrel,Ridogrel and Terutroban）.Antiplatelet activation drugs include ADP receptor inhibitors（such as Clopidogrel,Prasugrel and Ticagrelor）,protease activated receptor antagonists（such as Vorapaxar）,and 5-HT receptor antagonists（such as Sarpogrelate,Citalopram）.Antiplatelet aggregation drugs include platelet membrane glycoprotein Ⅱb/Ⅲa receptor inhibitors（such as Abciximab,Tirofiban,and Integrilin）,phosphodiesterase（PDE）inhibitors（such as Dipyridamole and Cilostazol）.Other antiplatelet drugs,acting on other sites of platelet activation pathways,such as CD40 and PI3K,most of which are small molecule drugs and biological drugs,are under development.And they provide more options and probabilities for the treatment of thrombotic diseases.Inevitably,all antithrombotic drugs could increase the risk of bleeding.Therefore,inhibitory activity,prototype drugs,metabolites,half-life and other factors should be taken into comprehensive consideration when new antiplatelet drugs are developed.Meanwhile,the mechanism of platelet aggregation and related signaling pathways should be further studied.Development of drugs with dual or multiple targets at platelet aggregation had become one of the research directions of antiplatelet drugs.Andropaxar is developed under the financial assistance of the National Natural Science Foundation of China after several years of independent research.It shows significant inhibitory activity against PAR-1 with a completely new structure,clear target and mechanism,as well as less side-effects and high safety,which makes it an excellent candidate for antiplatelet drugs.It has completely independent intellectual property rights.Andropaxar industrialization research has great research significance and excellent development prospects.To realize the industrial production of Andropaxar,we have conducted in-depth research.Several problems in the reported synthesis route hinder the industrial production of Andropaxar.Firstly,the synthesis route remained to be further optimized.The synthesis of compound 6 can be simplified while the yield can be improved.The preparation of compound 10 from compound 8 can be achieved through one pot method.Secondly,experimental operations are tedious.Column chromatography is used in the separation and purification of each intermediate and final product which is not conducive to industrial production.Thirdly,reaction conditions are harsh.Compound 11 is prepared under the tempreture of-78℃ which is inappropriate for industrial production.The usage of O3 claims high requirements for the corrosion resistance of the equipment as well as the gas distribution performance.And it is not realistic to achieve desulfurization reduction with large amount of Raney nickel that is inflammable and explosive.Fourthly,some of the materials are unavailable,such as hypertoxic diethyl chlorophosphate.Fifthly,some of the agents used in the synthesis route are unreasonable.For example,aluminum oxide used in the preparation of compound 8 is not necessary.The overuse of pyridine solution in the preparation of compound 11 does not only increase the cost,and improves the difficulty of post-processing.Amberlyst15 used in the preparation of 13 is expensive and it has complex synthetic materials which is not beneficial for quality control of intermediate products and can be replaced with cheap chemicals.And DCM can be recycled.Thus it can be seen that there is still a long way to go to achieve the industrial production of Andropaxar.Therefore,we have conducted deep and systematic optimization of process parameters based on existing work.Through optimization,the synthesis route was shortened by one step,and some agents were replaced with other low cost agents,and reaction conditions were more mild.The optimized synthesis route is more appropriate for industrial production which has been realized at kg level.In addition,to expand the candidate library of PAR-1 inhibitors,Andropaxar analogues were synthesized,and their in vitro PAR-1 inhibitory activities and pharmacokinetics were studied.The specific research contents mainly include the following four parts:1.Process development of Andropaxar API.Based on reported synthetic route and relevant national technical guidelines for drug registration,compound 12 was selected as the starting material of Andropaxar,and the preparation processes of compound 12 and Andropaxar were studied respectively.Firstly,the preparation process of starting material compound 12 was improved and optimized.The preparation route that was suitable for industrial production was finally determined.Compound 12 was prepared from commercially available andrographolide over seven steps.At present,the industrial production of compound 12 had been realized.Secondly,the registered process of Andropaxar was improved and optimized.According to relevant national regulations for drug registration,the process was systematically studied,and the process parameters were optimized.Finally,a registered process suitable for industrial production was determined.At present,the pilot production of Andropaxar had been completed.2.Establishment of detection method for related substances and purity of Andropaxar.In order to ensure the quality of Andropaxar and its intermediate products,it is necessary to develop their detection methods of related substances.This chapter described the instruments and method used in the process of method establishment,which provided an efficient way to detect the related substances and purity of Andropaxar and its intermediate product.In the meanwhile,diastereomers of Andropaxar could also be detected using this method.3.Studies on Andropaxar analogues.Although Vorapaxar Sulfate has good anticoagulant activity,it still has some disadvantages,such as complex structure,long synthesis route,high preparation cost and obvious side effects.The preparation of two PAR-1 inhibitor analogues which had high similarity with Vorapaxar in their nuclear structure was expatiated in this part.The two analogues were synthesized through five and four steps respectively starting from sclareolide,a common marketed natural product.Both of them showed good in vitro anticoagulant activity with an IC50 of 0.256 nM and 23.83 nM,respectively.They can be used as candidate compounds for further research to prevent and/or treat thrombotic diseases.4.Pharmacokinetics study of Andropaxar and its preparations.The suitable preparation of Andropaxar was preliminarily screened,and self microemulsion was optimal according to the preparation process and the difficulty of industrialization.Preliminary pharmacokinetics study showed that the half life of Andropaxar prototype drug was about 1.5 h and the average residence time is about 3.5 h.In addition,the bioavailability of the prototype drug was 48.2%which was 24%higher than its salt.To sum up,the starting materials and registered process of Andropaxar that was suitable for industrialization were determined according to relevant national guidelines for drug registration.Pilot production of Andropaxar had been completed and quality research of Andropaxar and its intermediate products were carried out.Related substances and purity detection methods were established.Two Andropaxar analogues with PAR-1 inhibitory activity had been developed,and their IC50 values were at nanomolar level,which could be used as candidate compounds for further research and development.In addition,the pharmacokinetics study of Andropaxar and its preparation were conducted,and the pharmacokinetic parameters were statistically analyzed to provide the basis for further development and research of the preparation.