Design, Synthesis And In Vitro Anti-tumor Activity Evaluation Of Podophyllotoxin Derivatives

Background:Malignant tumors are one of the major diseases that currently pose a serious threat to human life and health.Among them,lung cancer still ranks first with high morbidity rate(11.6%)and high mortality rate(18.4%).Finding effective ways to overcome tumors is still a difficult problem to be solved.Podophyllotoxin,a major antimitotic lignin,had become the focus of scientific interest for its pronounced biological activity such as antineoplastic,anthelminthic and antiviral.However,its shortcomings of the side effects and poor water solubility made its further development and utilization limited.Etoposide and teniposide,which had been obtained through extensive structural modification,had been successfully used in the clinical treatment of various cancers,but they still had obvious problems of gastrointestinal reactions,myelosuppression,multidrug resistance and poor specific selectivity to tumor tissues.According to our previous researches and a large number of literature reports,it was found that the introduction of amino acids and ligustrazine which was the major effective ingredients of Ligusticum chuanxiong Hort,could significantly enhance antitumor activity and the selectivity of the compound.It provides an important direction for the development of strong selectivity,high efficiency and low toxicity antitumor drugs.Research purpose:According to the principle of combination of drug design,different amino acids and ligustrazine were introduced into podophyllotoxin.Combined in vitro activity evaluation method,the paper was aimed to obtain podophyllotoxin derivatives with strong selectivity,high efficiency and low toxicity.Its preliminary antitumor mechanism and the pharmacological evaluation were researched,which provided reference for the further development and application of podophyllotoxin.Research methods:In this paper,a series of podophyllotoxin amino acid ligustrazine derivatives were obtained by esterification and amidation reaction.The structures were determined by 1H-NMR,13C-NMR and HRMS,and their physical parameters were characterized by polarimeter and melting point apparatus;MTT assay was used to evaluate the activity of podophyllotoxin derivatives on human lung cancer cells A549,human breast cancer cells MCF-7,human liver cancer cells HepG2 and toxicity to human normal liver cellsL-02 and screened out the dominant derivative with strong selectivity,high efficiency and low toxicity;The preliminary antitumor mechanism of the dominant derivative was investigated by cell staining observation and flow cytometric analysis;and the stability of pharmacological of the dominant compound was evaluated by HPLC,which laid the foundation for the discovery and development of podophyllotoxin antitumor derivatives.Research results:In this paper,twenty-three podophyllotoxin derivatives had been designed and synthesized.The structures and physical parameters of the derivatives had been confirmed.Through acticity evaluation in vitro,it was found that the antitumor activity of all the podophyllotoxin derivatives was significantly stronger than that of the positive drug etoposide,and most of them were stronger than doxorubicin.Among them,compound P-02 showed strong inhibitory effect on A549 cells(IC50= 9.5±0.03 nM)and exhibited strong cytotoxicity selectivity and the selectivity index between A549 cells and L-02 cells was 16.9;DAPI staining and flow cytometry analysis indicated that compound P-02 could fragment the nucleus of A549 cells and L-02 cells,promote intracellular Ca2+ concentration,induce mitochondrial transmembrane potential depolarization and induce cells early apoptosis.All of the effect of this compound on A549 cells was stronger than that of L-02 cells.In addition,the compound prevented the S phase and G0/G1 phase of A549 cells and L-02 cells,respectively;The stability evaluation in vitro showed that the residual rate of compound P-02 in simulated gastric fluid and rat plasma for 4 hours was 50.844%and 44.273%,respectively,which was indicated that the compound P-02 had certain stability in simulated gastric fluid and rat plasma.And the stability of compound P-02 in simulated gastric fluid was better than in rat plasma.Conclusion:After introducing amino acids and ligustrazine into podophyllotoxin,the activity and toxicity of most of the compounds were obviously improved.Among them,compound P-02 showed strong inhibitory effect on A549 cells and exhibited strong cytotoxicity selectivity,and the stability in vitro was good,which achieved the research purpose of this paper and provided some reference values for the development and utilization of podophyllotoxin.