Synthesis And Antitumor Activity Evaluating In Vitro Of Betulinic Acid Derivatives

purpose:Cancer is the disease with the highest morbidity and mortality in the world and it is a serious threat to human life and health.More than 10 million people worldwide suffer from cancer every year.Cancer is hyperproliferative due to the loss of normal cells in the body,which caused by a variety of causes.Currently,the main clinical treatment for cancer is chemotherapy and radiotherapy.Platinum,alkylating agents and anti-metabolite chemicals can also cause serious and irreversible damage to the normal cells of the body while treating cancer,which seriously affects the therapeutic effect of cancer.Therefore,it is imperative to develop new effective and selective anti-tumor drugs for the treatment of clinical cancer patients.Betulinic acid(BA)is a pentacyclic triterpenic acid found in various plants in nature.BA has a wide range of biological activities,such as anti-inflammatory,anti-malarial,anti-HIV,etc.In recent years,a large number of experimental studies have shown that betulinic acid also has good pharmacological activity in anti-tumor.However,due to its poor water solubility and its poor selectivity in the treatment of cancer,therapeutic effect of the BA is not particularly satisfactory.Based on these studies,we modified the structure of betulinic acid to obtain betulinic acid derivatives,which is expected to improve its water solubility and selectivity and reduce toxic and side effects,providing experimental basis for clinical development of new anti-tumor drugs.In this study,betulinic acid derivatives zx-1 and zx-2 were designed and synthesized,and then we detected the anti-tumor activity of the two compounds in vitro and preliminarily explore their anti-tumor mechanism.Methods:1.Design and synthesis of betulinic acid derivatives zx-1 and zx-22.MTT assay was used to detect the inhibitory effects of the two compounds on various cancer cells,and the control group and the blank group were set.3.The effects of the two compounds on the growth status of non-small cell lung cancer A549 cells were observed and photographed under an inverted microscope.4.The cell scratch test was performed to detect the effects of the two compounds on the migration of A549 cells.Samples were taken and photographed under microscope.5.Annexin V-FITC/PI double staining method was used to detect the effect of two compounds on the apoptosis rate of A549 cells,and the apoptosis rate was detected by flow cytometry.6.PI single staining method was used to detect the effect of two compounds on the cell cycle distribution of A549 cells,and the cell cycle distribution was detected by flow cytometry.7.Western blot was used to detect the expression of P53,Bax,Bcl-2 and Caspase-3 proteins in A549 cells treated with compound zx-2.Results:1.We synthesized and obtained two betulinic acid derivatives.2.The results of MTT assay demonstrated that two compounds with different concentrations significantly inhibited the proliferation of non-small cell lung cancer(A549)cells,gastric adenocarcinoma(SGC-7901)cells,liver cancer(LUC-7721)cells,and breast cancer(MCF-7)cells.3.According to the cell morphology observation under the microscope,it was found that the morphology of the cells changed significantly after the two compounds acted on A549 cells for 24 h including the cell volume becomes smaller and rounder,the edge is blurred,and the cytoplasm shrinks.4.The results of cell scratches test showed that the area of scratches of A549 cells treated with the two compounds was significantly increased compared with the control group in a concentration-dependent manner.5.The results of flow cytometry showed that the apoptosis rate of A549 cells increased significantly after treated with the two compounds for 48 h,and the apoptosis rate of the A549 cells increases as the concentration increases.The results also showed that the effect of compound zx-2 on the apoptosis of A549 cells was more significant than that of zx-1 at the same concentration.6.It is known from the test results of flow cytometry that the two compounds affected the cell cycle distribution of A549 cells.The cell growth cycle of A549 cells could be blocked in G0/G1 phase after the effect of zx-1,while the cell growth cycle of A549 could be blocked in S phase after the effect of zx-2.7.The results of western blot showed that zx-2 can decrease the expression of P53 and Bcl-2 proteins and promote the expression of caspase-3 and Bax proteins.Conclusions:The betulinic acid derivatives zx-1 and zx-2 can inhibit the growth and proliferation of various tumor cells,it also can induce apoptosis and necrosis of non-small cell lung cancer A549 cells,regulate the cell cycle,inhibits cell migration,decrease the expression of P53 and Bcl-2 proteins and promote the expression of caspase-3 and Bax proteins.These may be related to the mechanism by which two betulinic acid derivatives exert anti-tumor effects.