Preparation Of Rotundic Acid, Design,Synthesis And Antitumor Study Of Rotundic Acid Derivatives

As one of the most serious diseases, cancer has become the main cause of death inhumans followed by cardiovascular disease. Recently, cancer appears a younger trend,and its incidence has been increasing year by year. It is of great urgence andsignificance to find antitumor drugs with high efficiency and low toxicity because mostof the currently available drugs had the disadvantages of low affection and highlytoxicity.Rotundic acid （RA,3β,19α,23-trihydroxy-urs-12-en-28-oic acid）, a pentacyclictriterpene acid, is the major component isolated from the dry bark of Ilex rotundaThumb. RA also can be isolated from Mussaenda Pubescens, Guettarda platypoda,Olea europaea, Planchonella duclitan, Nauclea officinalis. Although RA could beobtained from the above plant resources, there was little report on it. The activity reportsare limited to antitumor and lowering blood. It has been broadly proved in mostantitumor activity studies that pentacyclic triterpene acid might present specialanti-tumor activity. We particularly concerned about the anti-tumor activity ofpentacyclic triterpene acid. According to the literatures, RA certainly has anti-tumoractivity. However, its anti-tumor activity still need to be improved compared toanticancer drugs applied in clinic. To improve the anti-tumor activity and find leadingcompounds with more effective antitumor activity, the synthesis of RA derivatives werecarried out in the thesis.Firstly, we studied the preparation process and quality control methods of RA. Theinfluence factors of RA preparation process were determined by single factorexperiment and orthogonal test, and the optimum process parameters were determined.Extraction process parameters: extracted three times with80%ethanol,2h per time.Purification process parameters are as follows: add3fold water （on the basis of herb）, adjust pH to6-7with dilute hydrochloric acid, filtered, to obtain total saponins.Hydrolysis process parameters:50-fold amount of the1mol L^-1alkali solution of30%ethanol was used to be hydrolyzed for4h. Pure rotundic acid was obtained by repeatedsilica gel chromatography. This progress is a method of low energy consumption, mildcondition and high efficiency. The yield was more than5%. Quality control methods ofRA were established to control the quality of RA. The method was proved to fit qualitycontrol of RA by the experiment of specificity, linearity durability, precision, stability,repeatability, accuracy and so on. The purity of RA was≥98%.According to the bioisosteres and other principles of medicinal chemistry,the sixtyRA derivatives were designed and synthesized, among which58ones are newcompounds. Their structures were identified by MP, IR, MS,1H-NMR and13C-NMR.Some compounds were identified by HMBC, HMQC and COSY when necessary.The antitumor activities in vitro were evaluated on A-375, SPC-A1, Hela, HepG2and NCI-H446cancer cells by MTT assay. The50%inhibitory concentration （IC50）indicated that most of the RA derivatives had stronger antitumor activity than RA,among which more than40derivatives showed significant differences compared withRA.（P<0.05）. Seventeen RA derivatives including N4, N5, N6, N7possessed low IC50value（IC50<10μM）. Among which compounds N4and N7had better antitumor effectsand the inhibitory activity are1.7～23times and1.6～10.3times stronger than RA, andwere expected to be potential antitumor drugs.On the basis of better activity of compound N4, protein expression related withapoptosis on HeLa cells treated with compound N4were assessed by western blot. Theresults indicated that N4could decrease the protein expression of BCL-2, and increase aproapoptotic protein BAX level, the ratio of BAX to BCL-2was remarkable increasedin a dose dependent manner. Then cytochrome C released from mitochondrial tocytoplasm, which would trigger the cleave of executor Caspase, Caspase-3. The cleavedCaspase-3will finally induce the DNA fragmentation in cancer cell. Compound N4induces apoptosis through the mitochondrial pathway, it might be one of the antitumormechanism.3D-QSAR of RA derivative was investigated by CoMFA according to the Hela biological screening. The Cross-validation coefficient q²was0.571, Best principalcomponent was10, regression coefficients was0.980, SD was0.069, and F（10,40）was200.759. The data indicated that distribution of electrostatic field and steric field hadbetter correlation with antitumor activity. the QSAR was obtained through the analysisof electrostatic field and steric field. In conclusion, the QSAR provided the theoreticalbasis and foundation for the design of antitumor drug and structural modification of RA.In a word, preparation process, quality control methods, structure modification ofRA, antitumor actibity, mocular mechanism, and structure-activity relationship of RAderivatives were investigated systematically. The results obtained in the present thesiswill base the foundation for further study of RA.