Synthesis And Evaluations Of Novel Ligustrazine Derivatives As Anticancer Agents

Background:Breast cancer is the most common malignancy in women worldwide and the leading cause of death in women.According to literature reports,the overexpression of Stat3(signal transduction and transcriptional activator 3)is closely related to the proliferation,metastasis and drug resistance of breast cancer.Ligustrazine is the main active component of the traditional Chinese medicine Ligusticum chuanxiong,which can inhibit the proliferation of breast cancer,induce apoptosis,inhibit migration,and has a variety of pharmacological activities such as antioxidant,anti-inflammatory,antithrombosis,analgesia,etc.Ligustrazine inhibits the activity of tumor cells by blocking the cell cycle of tumor cells,inducing apoptosis and autophagy of tumor cells,inhibiting angiogenesis,and inhibiting adhesion and invasion of tumor cells.Ligustrazine also has natural,safe and low-toxicity biological properties.Purpose:Through computer aided drug design,ligustrazine based small molecule inhibitor ligustrazine Stattic derivatives were synthesized to evaluate the proliferation inhibitory activity of different tumor cells,and the mechanism of inhibiting the proliferation and apoptosis of tumor cells was discussed.At the same time,to explore the mechanism and target verification of ligustrazine-Stattic derivatives on tumor cell migration.In vivo tumor model was established to evaluate the activity and safety of ligustrazine-Stattic derivatives in mice.Methods:1.Computer aided drug design and synthesis of ligustrazine-Stattic derivatives;2.The activity of ligustrazine-Stattic derivatives against different tumor cells was evaluated by MTT assay;3.To evaluate the effects of ligustrazine-Stattic derivatives on the proliferation of breast cancer cells by MTT and clone assay;4.The effects of ligustrazine-Stattic derivatives on apoptosis,mitochondrial membrane potential,reactive oxygen species content and cell cycle were evaluated by flow cytometry.5.WB(Western Blot)was used to verify the changes of apoptotic proteins and cyclins induced by ligustrazine-Stattic derivatives.6.WB method was used to evaluate the effects of ligustrazine-Stattic derivatives on cell target protein;6.The effects of ligustrazine-Stattic derivatives on cell migration were evaluated by scratch and WB assay.7.The subcutaneous tumor model of mouse breast cancer was established to evaluate the antitumor activity of ligustrazine-Stattic derivatives in vivo,and the safety of ligustrazine-Stattic derivatives was evaluated by weighing,blood biochemical analysis and HE.Results:1.Synthesis of ligustrazine-Stattic derivatives(Statmp-151 and Statmp-156);2.MTT assay confirmed that ligustrazine-Stattic derivatives had more significant activity in breast cancer,and the activity was much higher than ligustrazine monomer.3.MTT and clone experiments confirmed that ligustrazine-stattic derivatives can significantly inhibit the proliferation of breast cancer cells,and the activity of Statmp-151 is better than that of Statmp-156.4.Statmp-151 can induce apoptosis of breast cancer cells,meanwhile,the expression of Bcl-2 protein is decreased,the expression of Bax is not significantly changed,and the expression of Cleave Caspase 3 is significantly increased.Statmp-151 induced S-phase arrest of MDA-MB-231 cells,and the expression levels of CDK2 and CDK6 were decreased,while the expression levels of CDK4 were not significantly changed.The content of intracellular ROS decreased,and the mitochondrial membrane potential changed.5.WB assay confirmed that Statmp-151 could significantly inhibit the phosphorylation of Stat3.6.By comparing the migration of different cells at the same site for 0 h and 24 h,the results confirmed that Statmp-151 could significantly inhibit the migration of cells,and the expression levels of migration-related proteins MMP2 and MMP9 were decreased.7.In vivo tumor volume and tumor weight analysis of mice showed that Statmp-151 inhibited tumor cell growth.The body weight,blood biochemistry,tissue weight and HE staining of mice showed that Statmp-151 was a novel and relatively safe derivative of ligustrazine with no obvious toxicity.Conclusion:In summary,Statmp-151/156,a novel ligustrazine derivative designed and synthesized based on the structure of Stat3 protein,has been proved by pharmacological experiments to significantly inhibit the proliferation and migration of tumors in a timedose-dependent manner with good targeting.In vivo experiments confirmed that the new ligustrazine derivative Statmp-151 can significantly inhibit the growth of tumor,and the new ligustrazine derivative Statmp-151 is relatively safe and non-toxic.Ligustrazine and Stat3 small molecule inhibitor synthesis of new small molecule will be a new direction and field of cancer prevention and treatment.