Design,Synthesis And Anticancer Activity Studies Of AZD9291 Derivatives Overcoming T790M/L858R Drug Resistance Mutation

Lung cancer remains the highest morbidity and death rate disease in China,with720,000 deaths,accounting for 25%of the total cancer deaths.Non-small cell lung cancer（NSCLC）accounts for 80%of the total number of patients with lung cancer and has a high mortality.Studies have shown that epidermal growth factor receptor（EGFR）overexpression rate and mutation rate in NSCLC patients are as high as 75%.Therefore,inhibiting EGFR signaling pathways related to cell growth,proliferation and apoptosis can significantly inhibit the growth and proliferation of tumor cells.In recent years,small molecule anti-tumor inhibitors targeting EGFR have achieved remarkable therapeutic effects in clinical treatment.The first generation of EGFR inhibitors represented by gefitinib and erlotinib and the second generation of EGFR inhibitors represented by alfatinib have potent inhibitory effects on EGFRL858R and EGFRT790M mutations,respectively.AZD9291,as the first FDA approved third generation EGFR inhibitor,is obviously superior to the first two generations of EGFR inhibitors in terms of biological activity and drug resistance.Although there are many EGFR inhibitors have approved by FDA,they all have some shortcomings（low kinase selectivity,easy drug resistance,high toxic and side effects,etc.）.Consequently,the author took AZD9291 as the lead compound and used computer-aided drug design software to summarize and perfect the structure-activity relationship of AZD9291.According to the combination principle and bioisosterism principle,we first introduced small molecular alkyl substituents or halogen groups in the ortho position of acrylamide structure to change the Michael addition reaction activity of acrylamide and reduce toxic and side effects.Next,different groups（electron withdrawing group-CN,electron donating group-CH₃）were introduced at the C-5 and C-6 positions of the pyrimidine ring to change the hydrogen bonding forces between the pyrimidine ring and the hinge region residue Met793.Then,the electron-withdrawing group F atom was introduced into the indole ring to weaken the methylation capability of N-methylindole and the interaction force between the indole ring and surrounding amino acid residues.Finally,the aliphatic heterocycle structures were introduced at the C-5 and C-6 positions of the pyrimidine ring to fully expand the pyrimidine space structure and explored the effect of the space volume of the pyrimidine ring on the antiproliferative activity.Four series of pyrimidine amine derivatives containing acrylamide structure containing acrylamide structure were designed and synthesized.The structures of all the target compounds were confirmed by ¹H NMR,¹³C NMR and TOF-MS.The thesis tested their inhibitory activities against EGFR^T790M/L858R、EGFR^WTT kinase and nti-proliferative activities of H1975、A549、HepG-2 and Hela cells and used AZD9291 as positive control.The compounds with excellent kinase activity and cell activity in vitro were preferably analyzed by flow cytometry,apoptosis analysis and immunofluorescence staining.In vitro biological activity results showed that most of the target compounds have high inhibitory activity on EGFR^T790M/L858R790M/L858R mutant kinase and A549 cells.Compound ZBB-26 has high inhibitory activity against EGFR^T790M/L858R mutant kinase(with IC₅₀0 values of 8 nM),while low inhibitory activity against EGFR^WT kinase(with IC₅₀0 values of 8 nM).The selectivity against EGFR^T790M/L858R was 191.3-fold.The compounds ZBB-16,ZBB-17 and ZBB-26have higher inhibitory activity on H1975 cell（EGFRT⁷90M/L858R90M/L858R double mutant）,with IC₅₀ values of 1.107μM,0.823μM and 0.254μM,respectively.In addition,it is preferred that compound ZBB-26 significantly inhibited A549 cell proliferation at a concentration of 0.72μM and blocked cell cycle at G2/M phase.A549 cells also showed obvious concentration-dependent tolerance.Molecular docking results of compound ZBB-26 also showed that the compound can be well embedded into EGFR protein pocket and form bidentate hydrogen bond with hinge region residue Met793.The spatial orientation and action capability of the compound are similar to those of the lead compound.Compounds with pyrimidothipyran core skeleton not only retain the inhibitory activity on A549 cells,but also exhibit excellent anti-proliferation activity on Hela cells.Compound ZBB-68 showed potent dual inhibitory activity on A549 cells and Hela cells,with IC₅₀0 values of 0.057μM and 0.104μM,respectively,and its inhibitory ability was superior to that of lead compound AZD9291(IC₅₀ values to A549 cell and Hela cell were 0.073μM and 0.615μM,respectively).At the same time,computer molecular docking technology and quantitative structure-activity relationship analysis method were applied to carry out detailed structure-activity relationship analysis on pyrimidinothiazoles derivatives,and the 3D-QSAR model of EGFR inhibitors with cervical cancer inhibitory activity was established.The3D-QSAR results showed that the established CoMFA model（q2=0.765,R²=0.965）and CoMSIA model（q²=0.875,R²=0.956）have high practical value and prediction ability,providing sufficient theoretical basis for designing new EGFR^T790M790M inhibitor and lung/cervical cancer dual inhibitor.