Synthesis And Antitumor Activity Of Tetrandrine Derivatives

Objective:The discovery of valuable antitumor drugs from plants has become a research hotspot.Tetrandrine is the major alkaloid component in the root of Stephania tetrandra S.Moore and has a wide range of pharmacological effects.Our previous study has shown that substitution at the C-5 position of tetrandrine could increase its antitumor activity.On this basis,tetrandrine as a lead compound was modified selectively in this paper.The in vitro antitumor activity was screened in order to synthesize novel derivatives with better biological activity and provide more sufficient data for the study of structure-activity relationships.The preliminary study on the anti-tumor mechanism of the preferred compound will contribute to the future design,development and application of new derivatives of tetrandrine.Methods:1.Tetrandrine was first derivatized at C-5 position by bromination under low temperatures and acidity.Catalyzed by Pd?PPh₃?₄,the resulting 5-bromotetrandrine was used as a common intermediate for the synthesis of new tetrandrine derivatives via the Suzuki-Miyaura cross-coupling reaction,then,16 new derivatives were obtained by the reaction.The structures of the compounds were confirmed by melting point determination,ESI-MS,¹H NMR,and ¹³C NMR.2.Against HepG2?human hepatoma cell?and A549?human non-small lung cancer cell?,16 compounds,as well as tetrandrine?as a positive control drug?,were initially tested for anti-tumor in vitro at a drug concentration of 10?M by MTT assay.Then the compounds with inhibition rate higher than 50%were further screened to optimize the compound with better activity.The effect of the preferred compound on the survival of A549 cell was determined.3.The effects of compound 12 on the apoptosis rate and cell cycle of A549 cells were analyzed by flow cytometry;JC-1 staining was used to detect the change of mitochondrial transmembrane potential;the cleavage of apoptosis-associated protein,caspase-3,PARP,the changes of Bax,and Bcl-2 were detected by Western blot.Results:1.According to the structure identification and corresponding spectral analysis,16compounds synthesized in this paper were consistent with the structure of the target compounds,and have not been reported in related literature.2.In vitro antitumor activities by MTT method showed that:For HepG2 cells,the preliminary screening results showed that the inhibitory rates of 5,7,8,10,11 and 12were more than 50%.Among them,the antitumor activity of compound 8 and 12 was better than that of tetrandrine.For A549 cells,the results of the preliminary screening showed that the inhibitory rates of all the compounds except the compounds 10,13,and16 were higher than 50%and the IC₅₀0 values of all the compounds were less than that of tetrandrine.Compound 12 exhibited the best anti-proliferative activity against HepG2and A549 cells with IC₅₀0 values of 4.58±0.51?M and 3.04±0.82?M,respectively.3.Effect of A549 cell viability:The preferred compounds inhibited the survival of tumor cells in a time-concentration response relationship.4.The results indicated that compound 12 could promote the apoptosis of A549 cells in a dose-dependent manner.At the same time,it caused cell cycle arrest at S-phase and reduced mitochondrial membrane potential.Western blot results showed that the treatment of compound 12 with A549 cells for 72 h enhanced the expression of Bax protein and inhibited the expression of Bcl-2 protein.Compared to the control group,the activation of caspase-3 in cells and the cleaved-PARP?89KD?were both significantly increased.Conclusion:In this paper,16 new derivatives of tetrandrine were obtained through screening conditions and none of them have been reported in related literatures.Anti-tumor activity screening results showed that compounds exhibited generally low effect on HepG2,but had higher selectivity to A549.Compound 12 had higher inhibitory activity on both tumor cells.Structure-activity relationship analysis:Compared with tetrandrine,the addition of methoxy groups in the newly added group could improve its anti-tumor activity.When a pyridine group was introduced,the parent structure had a higher antitumor activity in the para position of the N than in the ortho position of the N.Tetrandrine derivatives could induce apoptosis in tumor cells through cell cycle arrest,changing the proportion of apoptosis-related proteins and affecting mitochondrial function.This study provided an effective method for the synthesis and antitumor activity of tetrandrine derivatization in vitro.