Design,Synthesis And Anti-Triple Negative Breast Cancer Activity Evaluation Of Veratramine Derivatives As Novel AP-1 Inhibitors

Triple negative breast cancer（TNBC）is a disease that seriously threatens women’s health.Currently,chemotherapy is the main treatment method,with large toxic and side effects and drug resistance of patients.It is urgent to develop effective targeted therapy clinically.The mechanism research showed that the transcription factor AP-1 is highly expressed in triple negative breast cancer cells.It is of great significance to develop inhibitors targeting AP-1.In our previous studies,it was found that Veratramine and its analogues can specifically recognize and bind to the AP-1 target gene sequence,thus inhibiting the expression of various downstream proteins related to tumor formation,invasion and metastasis,and further playing an anti-tumor role.In this project,Veratramine was identified as the lead compound and its structure was modified.Through analyzing the binding characteristics of Veratramine and AP-1targeted DNA sequence,the structural modification of Veratramine is mainly designed from four aspects:（1）Connecting nitrogen atoms of the E ring to side chains of different lengths and types;（2）Oxidizing the hydroxyl group at position 3 into carbonyl group accompanied by double bond shift,and constructing the carbonyl group into a group with hydrogen bond donor or acceptor through nucleophilic addition reaction;（3）Using acetylation conditions to protect the hydroxyl group at position 3,oxidizing the hydroxyl group at position 23 to construct a key intermediate,and synthesizing derivatives such as fluorine substitution;（4）Converting double bonds into epoxy bridges or ortho-dihydroxy groups.A total of 50 derivatives were synthesized,and all intermediates and target compounds were identified by HR-MS,¹H-NMR and ¹³C-NMR.Secondly,this subject verified the preliminary mechanism of the synthesized derivatives and studied the proliferation inhibitory activity of triple negative breast cancer cells in vitro.HEK293/AP-1 luciferase reporter gene system was established using HEK293 as model cell.The inhibitory effects of derivatives on AP-1 signaling pathway were measured at 30μM and 10μM respectively.A total of 13 compounds were screened out to have stronger inhibitory effects on AP-1 signaling pathway than the lead compound Veratramine.In addition,considering that NF-κB signaling pathway plays an important role in the regulation of normal physiological functions,the HEK293/NF-κB luciferase reporter gene system was constructed to test the inhibitory effect of target compounds on NF-κB signaling pathway.Based on the test results of Veratramine derivatives in the above two reporter gene systems,a total of 8 derivatives VT-18,VT-19,VT-22,VT-23,VT-27,VT-38,VT-39,VT-42 had no obvious inhibitory effect on NF-κB signaling pathway while maintaining strong AP-1 signaling pathway inhibitory activity.Therefore,this subject tested the proliferation inhibitory activity of 8 preferred Veratramine derivatives on triple negative breast cancer cells and the results showed that the derivatives VT-38 and VT-39 showed better in vitro proliferation inhibitory activity than the lead compound(IC₅₀=10.37μM),with IC₅₀ values of 3.87 and 4.22μM respectively.Further mechanism research showed that the preferred derivatives effectively inhibited the formation of AP-1/DNA complex by binding to the target DNA sequence,thereby inhibiting the expression of MMP-2 and MMP-9 in the downstream signaling pathways,and exerting anti-invasion and metastasis effects of triple negative breast cancer.Finally,molecular docking technology showed that the hydrogen bond between the preferred compounds and DNA base is similar to the binding mode between the lead compound Veratramine and DNA base,and the hydroxyl group at position 23 is an important pharmacodynamic group.It is suggested that the 23-hydroxyl group should be retained in its original form in the next structural modification,To sum up,the active natural product Veratramine was taken as the lead compound in this project,and a series of Veratramine derivatives with strong selective inhibitory activity on AP-1 signaling pathway have been obtained through structural optimization and biological activity test research.Among them,two derivatives showed strong cell proliferation inhibitory activity on triple negative breast cancer cells in vitro test,which is worthy of further research.The novel AP-1 signaling pathway inhibitors in this subject provide new lead compounds for the research and development of new anti-triple negative breast cancer drugs,and also provide new ideas for the design of anti-tumor drugs.