Study On The Design, Synthesis And Antitumor Activity Of A Novel EGFR / VEGFR Double Target Inhibitor

Objective:In recent years, a multi-targeted tyrosine kinase inhibitor has become the hot spot of anti-tumor drugs. The purpose of this project is to get the target compounds by the method of computer aided drug design, chemical synthesis, and biological studies in molecular level, cellular level and animal models, in order to get a dual targeted anti-tumor drug candidate on inhibiting the activity of EGFR and VEGFR-2.Method:Through the study on the relationship between structure activity on4-anilino quinazoline derivatives as tyrosine kinase inhibitors, analysis of favorable groups or side chain structure of quinazoline parent nucleus can be connected, and protein crystal binding mode of the positive compounds vandetanib and EGFR, VEGFR. Using the method of computer aided drug design,4series of novel4-aniline quinazoline compounds were designed and synthesized by our laboratory. Homogeneous time-resolved fluorescence method (HTRF) was used to evaluate the compounds’ inhibitory activity against EGFR and VEGFR-2in vitro. The activity study of cells (A431, A549, and MDA-MB-231) proliferation of the compounds was carried out by MTS method. To evaluate the affect of the migration ability by cell scratch damage model in evaluation of compound in tumor cells (A431) and normal cell (HUVEC). Cell apoptosis were measured by flow cytometry. Nude mice xenograft model of A431cells was used to evaluate the anti-tumor activity in vivo. The Influence was detected by Western blottingting to assess a compound on the expression of EGFR and VEGFR-2in tumor tissues.Results:By design and synthesis we got totally4series of12new compounds of4-anilino quinazoline inhibitor. In HTRF method, TYIG4-TYIG12showed better activity from screening, which also had varying degrees of inhibition on cells (A431, A549, and MDA-MB-231) in MTS method. Among these eight compounds, TYIG10showed more obvious selectivity of inhibitory effects of cell proliferation. Cell scratch damage test showed strong migration inhibitory ability on compounds TYIG10in1μmol·L-1and3μmol·L-1on A431and HUVEC cells. Flow cytometry detected that compound TYIG10can induce apoptosis in A431cells at1μmol·L-1,3μmol·L-1,10mol·L-1, the apoptosis rate were18.7%,29.1%and35.4%, which could well restrain the tumor growth with dose-dependent tumor growth rate of44.0%,56.7%and69.6%on doses of25mg/kg,50mg/kg and75mg/kg in vivo. Western blottingting results showed that TYIG10had the ability to inhibit the expression of A431in tumor tissues of VEGFR-2, and also verified its anti VEGFR-2mechanism. Conclusion:TYIG10on inhibiting the activity of EGFR and VEGFR-2has well anti-tumor activity in vitro and in vivo, and needs more tests to further verify whether it can become a novel targeting tyrosine kinase inhibitor.