Design,Synthesis,and Biological Evaluation Of Betti Base Derivatives As EGFR Allosteric Inhibitors

The mortality rate of lung cancer ranks first among cancers,with non-small cell lung cancer accounting for 80%-85%.EGFR is an important drivers for the occurrence and development of non-small cell lung cancer.In order to target the treatment of NSCLC,some EGFR tyrosine kinase inhibitors have been developed.Both the first generation Gefitinib and Erlotinib have quinazoline rings,which are effective for exon 19 deletion and exon 21 mutation,but also effective for wild-type,with poor selectivity,and the problem of drug resistance quickly emerges.The effectiveness of the second generation of Afatinib is better than that of the first generation,but it still cannot completely solve the T790M drug resistance mutation and is prone to serious adverse reactions.The third generation Osimertinib and Olmutinib are effective for T790M mutation,exon 19 and 21 mutation,and have weak effects and good selectivity for wild type.However,with the application of the third generation of inhibitors,drug resistance still occurs,the most important of which is the C797S mutation.For such mutations,market supply is again black.Since 2016,some EGFR allosteric inhibitors have been reported.They binds to the allosteric site,which is different from the ATP competitive EGFR-TKIs binding site,wherein the representative ones are EAI045,JBJ-04-125-02,JBJ-09-063,etc.Due to the poor effect of single use,the need to combine monoclonal antibodies to take effect,and the inability to resolve some drug resistance mutations,there have still not obtained a listed EGFR allosteric inhibitor.In order to obtain a novel EGFR allosteric inhibitor,the hit compound la was obtained through virtual screening,and designed and synthesized 28 Betti base derivatives by analyzing the binding mode between la and EGFRT790M protein.ADP-GloTM kinase assay results showed that both 2b and 4a had inhibitory activity against the egFRL858R/T790M kinase and the EGFRL858R/T790M/C797S kinase.2b inhibits EGFRL858R/T790M kinase and EGFRL858R/T790M/C797S kinase with IC50 of 0.702 nM and 6.517 nM respectively.2b inhibits EGFRL858R/T790M kinase and EGFRL858R/T790M/C797S kinase with IC50 of 0.669 nM and 3.002 nM respectively.The results of the cell proliferation inhibition assay and ADP-GloTM kinase assay showed that 2b was the most active(IC50=0.40±0.01μM)against H1975 cells(EGFRL858R/T790M),which was better than the positive EAI045(IC40>100 μM and JBJ-04-125-02(IC50=8.59±0.32μM),but was equally effective against A549 cells(EGFRWT)(IC50=5.31±2.12 μM)with poor selectivity.4a showed better inhibitory activity against H1975 cells(IC50=1.16±0.14 μM)than the positive;against Ba/F3-EGFRL858R/T790M/C797S cells(IC50=3.12±0.05 μM),which equivalent to the positive EAI045(IC50=2.07±0.04 μM)and JBJ-04-125-02(IC50=0.21±0.01 μM);for A549 cells with IC50>100 μM and good selectivity.The results of ATP competition experiments showed that the IC50 of 2b for EGFRL858R/T790M kinase did not change significantly at different ATP concentrations(1 μM,10 μM,100 μM,1 mM),indicating that 2b acted at the allosteric site;the results of the combination of 2b/4a and Osimertinib showed that 2b/4a inhibited the proliferation of H1975 cells in the presence of Osimertinib,indicating that 2b and 4a acted at the allosteric site;the results of Western Blot showed that 2b and 4a could effectively inhibit EGFR phosphorylation,with dose dependent manner.Cell cycle and apoptosis assays showed that compounds 2b and 4a could block the cell cycle and induce cell apoptosis.In conclusion,2b and 4a are effective EGFR allosteric inhibitors,and it is hoped that subsequent structural modification and optimization will provide theoretical and practical basis for the clinical development of EGFR inhibitors to overcome C797S mutation resistance.