Synthesis And Antitumor Activities Of Novel Coumarin-piperazine Derivatives

Since the success of taxol and their related derivatives as anticancer agents,developing other novel natural compounds and their derivatives with better efficiency and low toxicity are regard as lead compounds to appropriate structural transformation and modification,this is an important way to anticancer drugs developed in recent years.Coumarin derivatives have traditionally been good source of new medicinal leads,and they play an important role in drug discovery,especially in the area of cancer pharmacology.7-hydroxy-4-methyl coumarin are natural coumarin,clinically there hydroxymethyl coumarin oral tablets for the treatment of cholecystitis,cholelithiasis and biliary tract infections and other diseases,has anti-bacterial,anti-leukemia,anti-oxidation,anti-HIV and anti-tumor and other broad biological activity.However,the presence of water-soluble of natural coumarins poor,weak anti-tumor activity and bioavailability and low defects.Piperazine and its derivatives are a class of compounds having diverse biological activities,especially having anti-tumor activity and can be used as a tumor-associated protease inhibitor.Based on the concept of hybrid molecules with a dual mode of action,twenty new coumarin piperazine conjugates were designed and synthesized in our study.All compounds were characterized by means of 1H NMR,13C NMR and MS.The anti-proliferative activities were determined by the standard method(MTT assay)against nine human tumor cell lines(T24,NCI-H460,HepG2,A549,MGC-803,Hct-116,Spca-2,7404 and Hela tumor cell lines).The activities of derivatives against HUVEC cells were also examined to test the toxicity of these compounds against human normal cells.The resulted showed that most of derivatives had significant antitumor activities and the IC50 of all compounds was lower than that of coumarin.Compounds 41 were selected as a representative to investigate their mechanism of antitumor against NCI-H460 cell line.Fluorescence staining assays(AO/EB and Hoechst 33258 staining)and flow cytometric analysis(Annexin V-FITC/propidium iodide test)showed that compounds 41 induced apoptosis.In order to further investigate their mechanism of antitumor,RT-PCR demonstrated that these coumarin piperazine derivatives increased gene expressions levels of TP53,cytochrome c and AIF,activated caspase-3,-8 and-9.Moreover,these compounds increased BAX and BAD-X gene expressions levels and decreased Bcl-2 and Bcl-xL levels.Further analysis indicated that these derivatives increased the intracellular level of ROS and Ca2+ production.And flow cytometry test results also show that compounds 41 involved G2 phase cell cycle arrest on NCI-H460 cell line.The levels of caspase-3,caspase-8,cytochrome c,PARP,p53,NF-kb,cyclinB1and p21 of compound 41 were measured by Western blot.Our results suggest that 41 increased the expression levels of caspase-3,cytochrome c,cleavg-PARP,p53 and p21,and decreased the expression levels of pro-PARP,pro-caspase 8 and Bcl-2.From the result of western blot,it can be suggested compound 41 could induce NCI-H460 cells apoptosis and arrest cell cycle in G2 phase,which may promote the apoptosis induced of 41.These data suggested that the compound 41 may induce apoptosis through extrinsic and intrinsic apoptotic signaling pathways.In summary,the hybrid of coumarin and piperazine could improve the anticancer activities of leader compound.Therefore,these compounds may be considered as the agents with high potential anti-cancer activity and good candidates for more advanced screening.