Synthesis And Antitumor Activity Of Novel Quinoline EGFR/HER2 Dual Target Inhibitors

As one of the most recognized major diseases in the world,cancer seriously endangers human physical and mental health and affects people’s quality of life.Studies have shown that in normal cells,the expression of EGFR and HER2 is appropriate,which can maintain the normal operation of the body and stable downstream signal transduction.Once EGFR and HER2 mutation or overexpression occurs,it will over-activate related downstream signaling pathways,which will trigger the occurrence and development of tumors.Therefore,EGFR and HER2 have become two attractive targets for the development of new anti-tumor drugs.In this paper,we designed and synthesized 12 new quinoline EGFR/HER2 dual target inhibitors,numbered 6a,6b,6c,6d,6e,6f,6g,6h,6i,6j,6k,6l.Then use EGFR overexpressing A431 cells,HER2 overexpressing SK-BR-3 cells,and SW620cells with less EGFR/HER2 expression as cell models,using Lapatinib and Neratinib preliminary screening of antitumor activity in vitro as a positive control drug.In addition,the inhibitory activity of 12 compounds on EGFR/HER2 kinase were tested,and compounds with better activity were initially screened for further in vitro antitumor activity testing.Experimental methods:The MTT method detects the growth inhibition of the target compounds on the cells,the enzyme-linked immunosorbent assay（ELISA）method detects the compounds’inhibitory activity on EGFR/HER2 kinase.Then observe the changes in the growth state of the cells after the compound acts on it under the microscope,wound healing assay detects the migration ability of the cells after the compound is applied.Flow cytometry to detect the effects of the compound on cell apoptosis and cycle.Western blot method to detect the expressions of p-ERK1/2,p-AKT and p-EGFR protein in A431 cells,Western blot method to detect the expressions of p-ERK1/2,p-AKT and p-HER2 protein in SK-BR-3 cells.Experimental results:MTT results showed that the 11 compounds had concentration-dependent inhibitory effects on A431 and SK-BR-3 cells except for 6l.Among them,compounds 6a,6d,6g and 6h had obvious inhibitory effects on the proliferation of A431 and SK-BR-3 cells.In particular,compound 6d had IC₅₀values of 1.89μmol/L and 1.93μmol/L on A431 cells and SK-BR-3 cells,respectively.There is no significant difference in the inhibitory effect of the positive drug Lapatinib,but compared with the positive drug Neratinib,the effect of compound 6d is better.The results of in vitro kinase inhibition experiments showed that compounds6a,6b,6d,6e and 6j had inhibitory effects on EGFR and HER2 kinases.Among them,compound 6d had the best inhibitory effect on the two kinases.Observation of cell morphology under the microscope and wound healing assay showed that with the increasing concentration of compound 6d,compared with the control group,the cell morphology changed significantly,and the cell migration ability was significantly reduced.The results showed that compound 6d could inhibit cell growth and affect cell state.The results of flow cytometry showed that after compound 6d acted on A431 and SK-BR-3 cells respectively,with the increase of the 6d concentration,the apoptotic rate of the two cell lines gradually increased,and the proportion of S-phase cells also gradually increased.It is revealed that compound 6d could induce cell apoptosis and block cell division in S phase,and presented a certain dose-dependent.Western blot experiment found that compound 6d could inhibit the expressions of p-ERK1/2,p-AKT and p-EGFR protein in A431 cells.Similarly,compound 6d could also inhibit the expressions of p-ERK1/2,p-AKT and p-HER2 protein in SK-BR-3cells,thereby inhibiting the excessive activation of downstream signal transduction and inhibiting tumor growth.Conclusions:Except for 6l,the remaining 11 new quinoline EGFR/HER2 dual target inhibitors have a certain inhibitory effect on A431 and SK-BR-3 cells.Among them,compound 6d has the best inhibitory effect on tumor cells and EGFR/HER2kinase.Compound 6d can inhibit cell migration,induce apoptosis,affect cell cycle distribution,and can inhibit the expression of p-ERK1/2,p-AKT,p-EGFR and p-HER2 proteins in cells.