Design,Synthesis And Biological Activity Studies Of Novel Factor Xa Inhibitors

Cardiovascular and Cerebrovascular diseases are a class of diseases caused by disorder of circulatory system,which seriously threaten people's health and life in the world wide,especially in Asian regions.Thromboembolic disease,such as myocardial infarction,stroke,deep venous thrombosis,has become one of diseases with high morbidity and mortality worldwide.Therefore,thrombosis plays a key role in the pathogenesis of cardiovascular and Cerebrovascular diseases.So far,there are two types of anticoagulants used for pharmacologic prophylaxis and treatment of venous thromboembolism: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.FXa is at the intersection of both the extrinsic and the intrinsic pathways.If we can inhibit Xa factor,the both coagulation routes can be blocked at the same time,The FXa therefore has become an attractive and specific druggable target for new anticoagulant agents.Up to now,three are several marketed oral direct FXa inhibitors,such as rivaroxaban,apixaban and edoxaban.Although the novel FXa inhibitors overcome the shortcomings of traditional anticoagulants,these drugs still have some defects,such as the narrow use of drug indications,damage to liver and kidney function,as well as the lack of effective antidote for the occurrence of bleeding.Because of the efficacy and safety of the inhibitor itself,there is an urgent need to develop a more safe and effective new FXa inhibitor for clinical use.Based on these studies of FXa's structure and its interaction with FXa inhibitors,we found that there are two important binding pockets in Xa factor target:specific binding pocket S1 and large hydrophobic pocket S4,with 80 °angle between the two pockets;therefore,molecule of rivaroxaban can also be broken up into three parts:P1 area,P4 area and the Linker area.The specific binding pocket S1 is a smaller hydrophobic pocket that can accommodate 5-chlorothiophene the P1 region of rivaroxaban while the 5-chlorothiophene forms a Cl-? force with Tyr228 at the bottom of the S1 pocket;the big hydrophobic pocket S4 formed ?-? stacking effect with the benzene ring of P4 region of rivaroxaban;rivaroxaban's Linker region formed two hydrogen bonds with Gly219 of FXa.According to the combination mode of the selected lead compound and FXa,the P4 region and the Linker region of the lead compound were modified by changing the volume and hydrophobicity of the P4 region and increasing the force of the Linker and FXa.We designed three series of new FXa inhibitors:pyrrolidone-,thiadiazine-,and indole-based FXa inhibitors,for obtaining novel skeletal Xa factor inhibitors with high activity.In order to verify the rationality of design ideas,we analyzed the three kinds of compounds designed by using compouter-aided drug design software sybyl X 1.3.The docking results showed that the structural modification of the target compound was reasonable in theory.Three series of FXa inhibitors were synthesized by directional reaction,with condensation reaction and acylation reaction.The structures of the designed inhibitors were confirmed by spectroscopy.The synthesized FXa inhibitors were tested for their in vitro anti-coagulation activities.The PT of platelet-poor plasma were tested by using the STAGO prothrombin time kit and the prothrombin time analyzer,and the "PTCT2,clotting time doubling concentration for prothrombin time" was calculated by the tested results,which evaluated the anticoagulant activity of the compounds.Some of these compounds showed good anticoagulant activity,such as compounds H2,H3,H15,PTCT2 values were 23.4,33.7,45.9 ?M,respectively,which were lower than the positive control drug rivaroxaban(PTCT2 =1.8 ?M).Next,the compound with good anticoagulant activity were tested for their FXa inhibitory activities.The chromogenic substrate S-2222 can be decomposed into polypeptides and p-nitroaniline by the catalyze of FXa,and the latter has a UV absorption peak at 405 nm.The results showed that some compounds exhibited good activity,such as compounds H2,H14,H15,with IC50 values = 0.82,0.55 and 0.52?M,respectively,which were lower than the positive control drug rivaroxaban(IC50 ?5 nM).In conclusion,three series of novel skeleton of the FXa inhibitors were designed,synthesized and evaluated for their--based on the target drug design and molecular simulation.Although the activity of the target compounds were not improved signficantly,compared with the lead compound rivaroxaban,The study of SAR has a guiding significance for us to further design and synthesis higher active FXa inhibitors.