Synthesis Of Ursodeoxycholic Acid(UDCA)Derivatives And Study Its Preliminary Mechanism Of Anti-hepatoma Activity

Liver cancer is a type of malignancy that forms on the liver.The incidence and mortality of liver cancer in China accounted for about 50%of the world.The severe situation has brought a heavy burden on our society and medical care.Ursodeoxycholic acid(UDCA)is the main drug for the treatment of chronic hepatitis,hyperlipidemia,constitutional cholangitis,constitutional cirrhosis,and induced cholangitis.In our previous research,U12,a UDCA derivative,showed significant anti-hepatocarcinoma activity in vivo and vitro.However,the low polarity,hemolysis and large doses limited the druggability of U12.Thus,in order to find more active UDCA derivatives with low toxicity,UDCA was choosed as the raw material to further investigate the structural modification and optimization of U12 and discuss the structure-activity relationship.In this project,25 UDCA derivatives of A and B series were synthesized by substitution reaction,esterification reaction,and amidation reaction.The A series contains halogen-substituted derivatives,and the B series contains 24 different amidations.The derivatives were screened for cytotoxic activities towords hepatoma cell lines SMMC-7721 and HepG2.As a result,one of the derivative Ua2 showed better anti-hepatocarcinoma activity than U12.MTT results suggested that Ua2,Uc1,Uc2,and Ue1 could effectively inhibit the growth of SMMC-7721 and HepG2 at a concentration of 25 ?M.The inhibition rate was 4-8 times higher than U12.Among them,Ua2 showed less cytotoxicity toward normal hepatocytes(HL-7702)than compounds Uc1 Uc2,and Ue1.The preliminary study of structure-activity relationship showed that the derivatives with halogen substituted at the C-23 didn't significantly increase the anti-hepatoma activity and showed obvious cyctoxicities towords normal hepatocytes.Besides,the anti-hepatoma activity of 7-hydroxy acetylation derivatives were enhanced.A piperazine ring group substituted at 24-position enhanced the anti-hepatoma activity,but also showed obvious killing activity towords normal hepatocytes;when a benzene group linked to the piperazine ring,the cytotoxic effects of hepatoma and normal cells was decreased;with the growth of carbon chain between piperazine and benzene ring,the cytotoxicity of hepatoma and normal cells was decreased.Therefore,24-position piperazine ring is an important active group and the length of the carbon chain between the ring and the piperazine ring is also an important factor for anti-hepatoma activity.Mechanism studies showed that Ua2 could induce apoptosis of HepG-2 cell by activating the caspase signaling pathway.Western blot analyses indicate that Ua2 could induce PARP cleavage on HepG2 cells in a time and concentration dependent manner.In addition,flow cytometry Annexin V-FITC/PI double staining method further confirmed that Ua2 has an apoptosis-inducing effect.Besides,Caspase enzyme activity assay results showed that treatment of 25 ?M Ua2 for 6 h could activate caspase signaling pathway.In summary,Ua2 could induce apoptosis through activating caspase signaling pathway,and its anti-hepatoma effect is stronger than U12.Therefore,Ua2 could be considered as a promising candidate for liver cancer treatment.