Design,Synthesis And In Vitro Evaluation Of Novel Ursolic Acid Derivatives As Potential Anticancer Agents

Ursolic acid(UA,3b-hydroxy-urs-12-en-28-oic acid),molecular formula for C30H48O3,a pentacyclic triterpene acid,exists abundantly in the plant kingdom.It is reported that ursolic acid was isolated from hundreds of plants,many of which is a traditional Chinese medicinal materials,such as herb plants and fruit trees.Ursolic acid is colorless crystals,m.p is 283?285 ?.Ursolic acid and its derivatives have been reported to have interesting bioactivity,including anti-HIV,antihepatodamage,antimalarial,antimicrobial,anti-inflammatory activity,antioxidant,anti-tumor and so on.Ursolic acid is widely used in fields of medicine function of health care,food,daily cosmetics and so on,and the high-efficiency antitumor activity of Ursolic acid has become a hot spot in research recently.In this paple study,the,natural products UA was chosen as the lead compound,a series of novel UA derivatives modified at the C-3 and the C-28 positions containing Piperazine-thiourea structure were designed and synthesized in an attempt to develop potent antitumor agents,their structures were confirmed by 1H NMR,13CNMR and high resolution mass spectrum(HRMS).The in vitro antitumor activities were evaluated against five cancer cell lines(MGC-803,HCT-116,T24,HepG2 and A549)and a normal cell(HL-772)by MTT assay.The result indicated that the synchronous introduction of piperazine-thiourea at C-28 and an acyl group at C-3 may improve the antitumor activity of UA,It also indicated that thehydrolyzing acyl group at C-3 may diminish the antitumor activity.So it is significant that an acetyl group was introduced at the 3-OH position.In addition,target compound 6r displayed the highest cytotoxicity against HepG2 cancer cells with IC50 values of 5.40 ± 0.79 ?M and showed lower cytotoxicity against HL-7702 cells with IC50 values of greater than 100?M,making them good candidates as antitumor drugs.The induction of apoptosis and affects on the cell cycle distribution of compound 6r were investigated by acridine orange/ethidium bromide staining,Hoechst 33258 staining,JC-1 mitochondrial membrane potential staining and flow cytometry,which revealed that the antitumor activity of 6r was achieved through the induction of cell apoptosis by G1 cell-cycle arrest.Western blot and qRT-PCR(quantitative real-time PCR)experiments demonstrated that compound 6r may induce apoptosis through both of intrinsic and extrinsic apoptosis pathway.The work of this master dissertation includes the following several aspects:1?3-acetyl ursolic acid was synthesized by the treatment of ursolic acid with acetic anhydride in dry pyridine in the presence of 4-dimethylaminopyridine(DMAP)3-acetyl ursolic acid was treated with oxalyl chloride to give the intermediate 28-acyl chloride,whichwas highly reactive and was then coupled with piperazine to produce compounds 4.Compound 4 was hydrolyzed in the presence of sodium hydroxide to afford 5.Isothiocyanates were obtained according to the method of the literature.UA derivatives(6a-6t and 7a-7r)were obtained by the condensation of isothiocyanates with 4(or 5)at room temperature.The structures of UA derivatives 6a-6t and 7a-7r were then confirmed by 1H NMR,13CNMR and high resolution mass spectrum(HRMS).2.The piperazine-thioureas UA derivatives were synthesized and their antitumor activities were evaluated against five cancer cell lines(MGC-803,HCT-116,T24,HepG2 and A549)and a normal cell HL-7720.Further investigation of the mechanism on the mode of action of representative compound 6r found that it can effectively induce cell apoptosis in HeG2 cells.3.In order to investigate whether the growth inhibitory activity of compound 6r was related to the induction of apoptosis,changes in the morphological character of HepG2 cells were determined using acridine orange(AO)/ethidium bromide(EB),Hoechst 33258 staining and JC-1 mitochondrial membrane potential staining under fluorescence microscopy.