Design,Synthesis And Cardiovascular Activlty Studies Of Novel Factor Xa Inhibitors And Ligustrazine Derivatives

There are two main parts in this thesis:the first part is the design, synthesis and anti-coagulation activity research of novel factor Xa inhibitors; the second part is the design, synthesis and anti-platelet activity and anti-oxidation activity research of novel ligustrazine derivatives.Cardiovascular disease (CVDs) is a class of diseases caused by dysfunction of circulatory system, which seriously threaten people’s health and life. CVDs is the leading cause of death world wide, especially in Asian regions. In China, nearly3million people die of CVDs annually, taking up about41%of the total death toll. Therefore it is an urgent task for researchers to develop novel and efficient anti-CVDs agents.Thrombosis plays a critical role in the pathogenesis of some cardiovascular diseases such as acute coronary syndromes. Until relatively recently, pharmacologic prophylaxis and treatment of venous thromboembolism were based on two types of anticoagulants, heparins (unfractionated heparin, low-molecular-weight heparins and fondaparinux) and vitamin K antagonists (e.g. warfarin). Although these agents considerably reduce thromboembolic events, they have several limitations, such as inconvenient for long-term use, slow onset of action, a narrow therapeutic window and their multiple drug and food interactions. To overcome these shortcomings, the research and development of new anticoagulants with high selectivity, better anticoagulative activity and low toxicity is needed.Part1.Design, synthesis and anti-coagulation activity research of novel factor Xa inhibitorsFXa is a trypsin-like serine protease that sits at the junction of both the extrinsic and the intrinsic pathways. The FXa is therefore an attractive and specific druggable target for new anticoagulant agents.Up to now, three oral direct FXa inhibitors, rivaroxaban, apixaban and edoxaban, have been approved for anti-thrombosis therapy for their high efficiency and high selectivity. However, in addition to a great success in the clinical application, rivaroxaban and other drugs have been found to have some side effects, such as causing anemia or bleeding. Therefore, research and development for new FXa inhibitors with higher efficacy and lowertoxicity is an important direction of the current anti-thrombotic drug development.There have been numerous studies on FXa’s structure and its interaction with FXa inhibitors. Based on these studies, we designed two series of new FXa inhibitors by changing P4area and linker region of leading compounds. In order to verify the reasonableness of the design, we carried out the docking analysis of the designed compounds, using compouter-aided drug design software sybyl7.3. The docking results showed that the design of the target compounds is theoretically rational. The synthesized compounds were tested for their in vitro anti-coagulation activities and FXa inhibitory activities. Results showed that some of the compounds exhibited good activities. Among which, H17displayed higher FXa inhibitory activity (IC50=1.9μM) and better anti-coagulant activity (PTCT2=1.0μM) than that of the positive control drug rivaroxaban (IC50=3.3μM, PTCT2=1.3μM).In summary, we designed and synthesize two series of FXa inhibitors with potent anticoagulation activities and FXa inhibitory activity, which are worth further investigation and development.Part2. Design, synthesis and biological evaluation of novel Ligustrazine derivatives Ligustrazine is an active component of Chinese traditional medicine Chuanxiong. Currently ligutrazine is an important drug for the treatment of ischemic cardiovascular disease in China. It is reported that ligustrazine exihibited various cardiovascular activities, such as free radical-scavenging, endothelium-protection and antiplatelet. However, ligustrazine was found dissatisfactory to be applicated in clinical practice because of its moderate activity, rapid metabolism and short half life time. Thus, it is meaningful to develop new generation of the ligustrazine drugs for treatment of CVDs from molecular modification.During our structure modification studies of ligustrazine, we found that some ligustrazine metabolites possessed improved cardiovascular activities than ligustrazine. The main metabolite of ligustrazine,3,5,6-trimethylpyrazine-2-carboxylic acid, displayed moderate activity on lowering the level of serum cholesterol and low density lipoprotein in vivo, and potent anticoagulation activity. Cinnamic acid and some of its derivatives have been reported to display anti-oxidative property. It is believed that cinnamic acids are potential scavengers of free radicals (such as O2·) and other oxidative species. Moreover, ozagrel, a derivative of cinnamic acid, is reported as a highly selective thromboxane (TXA2) synthase inhibitor, which has the ability to preclude the TXA2induced platelet aggregation.Inspired by the structural characteristics and the drug-like properties of cinnamic acid and in continuation of our work, we conjugated the Ligustrazinyloxy group with cinnamic acid group to obtain a novel series of trimethylpyrazine-2-carbonyloxy-cinnamic acids, in the hope of getting novel ligustrazine derivatives with high anti-oxidative activity or anti-platelet aggregation activity.This series of ligstrazine derivatives were evaluated for their anti-platelet aggregation activity and endothelial cells-protection activity.Anti-platelet aggregation assay:The inhibition of platelet aggregation of the compounds was tested using microplate-reader-nephelometry. The positive control drugs are ozagrel and clopidogrel. Among all the synthesized compounds, F’1, F2, F3, F5, F’7and F’9were the most potent platelet aggregation inhibitors with IC50values24.4,26.4,9.6,41.8,28.2and37.5μM, respectively, much more active than that of ozagrel (IC50=144.1μM), but slightly lower than that of clopidogrel (IC50=7.6μM). Protecting vascular endothelial cells against hyperoxic acute injury assay:These ligustrazine derivatives have been tested for protecting vascular endothelial cells against hyperoxic acute injury. The viability of injured endothelial cells is assessed by methylthiazolyltetrazolium (MTT) assay. Among all the ligustrazine derivatives, F2, F5, F’5, F’9, F10, F’10displayed remarkable protection activity (EC50=24.0,8.8,29.9,21.4,2.2,1.7μM, respectively). Particularly, the compound F’10presented almost40times higher potency than lipoic acid.In summary, a series of novel ligustrazine derivatives were designed, synthesized and biologically evaluated for their inhibitory activities against the platelet aggregation, and their protective effect on the damaged vacular endothelial cells. The results showed that some compounds exhibited high activities in one or both of the assays, demonstrating the potential values in further development of cardiovascular agents.