Design,Synthesis And Biological Evaluation Of Novel Thieno[3,2-d]pyrimidines As EGFR^L858R/T790M Mutant Inhibitors

Background and Objective:Lung cancer is an extremely serious threat to human health which has the highest mortality rates among cancers.Non-small-cell lung cancer（NSCLC）accounts for approximately 85%of lung cancers.Epidermal growth factor receptor（EGFR）overexpression and mutations are closely linked to the tumorigenesis of NSCLC.The EGFR tyrosine kinase inhibitors（EGFR-TKIs）are the first-line drugs for clinical targeted therapy of NSCLC.The third-generation of EGFR-TKIs in treating EGFR^L858R/T790M drug-resistant mutant NSCLC has been a research hotspot.Olmutinib,as a representative one,was restricted by the Korean FDA shortly after its launch due to severe skin ulceration observed in patients taking the drug.Therefore,continuing to explore new high-selectivity and low-toxicity EGFR^L858R/T790M inhibitors remains a top priority for NSCLC targeted therapy.The current study was designed to modify olmutinib’s structure and to obtain novel EGFR^L858R/T790M targeted inhibitors with high selectivity and low toxicity.Methods:Based on literature researches,this subject retained the thieno[3,2-d]pyrimidine skeleton of olmutinib and cycled up the phenylacrylamide open-chain structure into quinolin-2（1H）-ones according to the design concept of conformational restriction and skeleton transition.Then the 2-position of the thienopyrimidine skeleton was substituted with various amines to obtain the targeted compounds.All compounds were evaluated for EGFR kinase inhibition in vitro via Mobility shift assay.Further Molecular docking was used to reveal the structural basis of selective inhibition of 6l and 6o.The Cellular thermal shift assay was used to detect the binding ability of 6l and 6o to EGFR proteins.All compounds were evaluated for growth inhibitory effects on several cancer cell lines carrying EGFR mutation or overexpression by MTT assay and calculated for Drug-likeness by Molsoft.We selected compound 6o based on these biological evaluation and used NSCLC cell lines:H1975(EGFR^L858R/T790M)and A549(EGFR^WT)for follow-up comparative study.The Clone formation assay,Wound healing assay and Western Blot were carried out to explore the effects of 6o on proliferation,migration ability and phosphorylation of key proteins of EGFR pathway in two cells.Results:1.Eighteen novel thieno[3,2-d]pyrimidine derivatives bearing quinolin-2（1H）-ones were designed and synthesized.2.At the kinase molecular level,half of compounds had good targeted inhibition on EGFR^L858R/T790M(0.11≤IC₅₀<0.33μM),but no significant inhibition on EGFR^WT(IC₅₀>10μM).Among these compounds,selectivity of 61 and 6o for EGFR^L858R/T790M858R/T790M were 113-and 61-fold of that for EGFR^WT,respectively,which were significantly higher than that of olmutinib（43-fold）.3.The introduction of the quinolinone and the hydrophobic amines enhanced the hydrophobic interaction of 6l and 6o with ATP binding region in EGFR^L858R/T790M,thus contributing to improve selectivity and reduce toxicity.4.The binding ability of compounds 6l and 6o to the EGFR^L858R/T790M protein was stronger than that of the EGFR^WT at the cellular level.5.At the cellular level,most of compounds showed good cancer cells proliferation inhibitory activity and certain safety,and compound 6o had the best selective inhibition effect on H1975 cells(IC₅₀=3.20±0.58μM).6.Compound 6o can obviously inhibit the clone formation,wound healing ability and phosphorylation of key proteins of EGFR pathway（p-EGFR,p-ERK）in H1975compared with A549 cells.Conclusion:18 novel thieno[3,2-d]pyrimidine containing 2（1H）-quinolinone were designed and synthesized.Most of the compounds showed good targeting inhibitory activity to EGFR^L858R/T790M kinase and cell proliferation inhibition activity.Especially,6o showed good targeting inhibition on proliferation,migration and EGFR pathway of H1975 cells expressing EGFR^L858R/T790M.The above indicated that appropriate non-covalent binding mode and different hydrophobic aniline side chains were effective methods for designing EGFR^L858R/T790M targeting inhibitors.We expected compound 6o can provide new ideas for future structure optimization and development of targeting anticancer drugs for EGFR mutant NSCLC.