Design,Synthesis And Biological Evaluation Of Quinazoline EGFR Allosteric Inhibitors

Lung cancer is currently one of the diseases with the highest mortality rate in the world,and non-small cell lung cancer（NSCLC）patients account for approximately 85%of lung cancer patients.Among the current molecular targeted therapies,epidermal growth factor receptor（EGFR）has been widely studied as an important oncogenic gene in NSCLC.Up to now,EGFR inhibitors have been developed for four generations,and these drugs can competitively bind to the ATP binding pocket of EGFR,thereby inhibiting the excessive activation of tyrosine kinase.However,with the continuous application of drugs in clinical practice,patients often experience varying degrees of resistance,so there is an urgent need to find new methods to overcome resistance.The discovery of EGFR allosteric pockets has helped researchers find new binding sites,located on the back of the ATP pocket.Inhibitors do not need to compete with ATP,but instead bind to the allosteric pockets to stabilize the EGFR protein in an inactive conformation,unable to exert catalytic effects and effectively inhibit the proliferation of lung cancer cells.This is a brand new strategy for overcoming the drug resistance issues caused by existing EGFR inhibitors.In this paper,allosteric inhibitor BDTX-189 and non allosteric inhibitor Poziotinib were used as lead compound to analyze the structural characteristics of lead compound.Computer aided drug design technology was used to analyze the binding mode of compounds and EGFR.With quinazoline as the parent core,21 compounds with novel structures were designed and synthesized.Among them,Class I compounds try to integrate the covalent warheads at position6 and 7 of the two lead compounds with hydrophilic groups at position 7 from the perspective of simplifying the structure according to the interaction mode of the lead compound at position6 and 7 with the solvent accessible region.At the same time,based on the principle of bioelectronic isotopes,the effects of covalent warhead types and hydrophobic groups such as pyrazine ring and pyridine ring on the activity of compounds were explored.Based on the structure-activity relationship of class I compounds,class II compounds further modified the piperidine ring in compounds with better activity,and explored the binding mode of different substituted piperidine rings and solvent accessible regions.Class III compounds are mainly based on BDTX-189,exploring the effects of rigid hydrophobic groups such as naphthalene ring and indole ring,as well as hydrophilic groups such as piperidine,on the activity of the compounds.The structure of the synthesized compound was confirmed by HRMS,¹H NMR,and ¹³C NMR.This paper first tested the EGFR（L858R+T790M）inhibitory activity of the synthesized compound at 500 n M.The kinase test results showed that the inhibition rates of three compounds I-1～I-3 in Class I compounds were 94.3%±0.4%,94.6%±0.2%,and 94.4%±0.04%,respectively,which were equivalent to the activity of positive compounds BDTX-189（inhibition rate:95.0%±0.3%）and Poziotinib（inhibition rate:95.7%±1.5%）.Class II compounds modify the piperidine ring on the basis of Class I,but unfortunately,their activity has decreased.The test results of Class III compounds showed that the combination of naphthalene ring as the hydrophobic group and morpholine ring as the hydrophilic group resulted in the most active compound III-5 of this series,with an inhibition rate of 90.6%±1.6%.In order to verify whether the obtained compounds are allosteric inhibitors,this paper conducted ATP concentration dependent inhibition activity tests for I-1～I-3,III-3,and III-5based on kinase screening results,with ATP concentrations set to,respectively 2μM、20μM and 200μM.All 5 compounds were not affected by ATP concentration and showed similar inhibitory activity.Subsequently,this paper measured the kinase IC₅₀ and cell IC₅₀ of the selected compounds,and the results showed that compounds I-2(kinase IC₅₀=0.52 n M)and III-5(kinase IC₅₀=1.28 n M)exhibited similar kinase inhibitory activity to the positive compounds BDTX-189(kinase IC₅₀=0.89 n M).I-2 and III-5 exhibited good anti cell proliferation activity.In this paper,the structural design,synthesis,mechanism verification and biological activity evaluation of quinazoline derivatives were carried out systematically,and I-2,III-5 and other compounds with further research value were found,which provided theoretical reference and molecular entity for the related research of EGFR allosteric inhibitors.