Design,Synthesis And Bioactivity Of Small Molecular EGFR/c-Met Dual Targeting Inhibitors With 6-substituted Phenylamine Purine Structure

Epidermal growth factor receptor（EGFR）is a transmembrane receptor that combines with its ligands and produces dimerization.Subsequently,through tyrosine kinase activity,it activates receptor autophosphorylation,triggers a series of intracellular pathways such as PI3K/AKT pathway,RAS/RAF/MAPK pathway and JAK/STAT pathway,and then activates the proliferation and metastasis of cancer cells,eventually leading to tumor formation.After about 20 years of drug research,three generations of EGFR-TKIs have achieved good results.Clinical studies have shown that all generations of EGFR-TKIs develop resistance problems after a period of use.Among the various mechanisms leading to resistance to EGFR-TKIs,amplification of c-Met accounts for approximately 5-20%of acquired resistance.C-Met is a hepatocyte growth factor receptor（HGFR）.The amplification of c-Met can activate the MAPK-ERK1/2T pathway and the ERB3-PI3K-Akt pathway,and initiate a series of cellular processes to bypass the phosphorylation process blocked by EGFR inhibitors,leading to the resistance problem of EGFR-TKIs.At present,for the acquired resistance caused by c-Met amplification,the combination of EGFR-TKIs and c-Met inhibitors is mostly adopted in clinical treatment,but the interaction of the two drugs often leads to toxicity and side effects.Research on double-targeted inhibitors of EGFR/c-Met is still in its infancy and no candidate drug has been identified for clinical study.Purine is a kind of parent nucleus with low toxicity and extensive pharmacological activity.By comparing the structural fragments of EGFR-TKIs and c-Met inhibitors,we designed and synthesized a series of small molecule EGFR\c-Met double targeting inhibitors with substituted aniline structure,which both EGFR-TKIs and c-Met inhibitors had,at the position 6 of purine and the"five-atom connection"part of the c-Met small molecule inhibitor according to the combination principle.Firstly,1,1,3,3-tetramethyl propane is taken as an initial raw material to synthesize a side chain part of a’5-atom connector’through nucleophilic substitution reaction,intramolecular cyclization reaction,acylation reaction and hydrolysis reaction,then that side chain and a substitute aniline group are connected through acylation reaction to synthesize a key intermediate,purine parent nucleus is modify through acylation reaction,and finally the parent nucleus part and the intermediate part are subjected to substitution reaction to synthesize a final compound.The structure of the target compounds were confirmed by MS and ~1H NMR.All of them were new structures which were not reported in the literature after being retrieved by Scifinder.Using Gefitinib and Carboplatin as positive control drugs,respectively,the cell proliferation inhibitory activity of the target compounds were tested by MTT test.The results showed that compounds A-2,A-6 and B-3 exerted good anti-proliferation activity on A549 cells(IC₅₀ values were 28.65μM,6.31μM and 12.34μM,respectively).Compounds A-5,A-6,B-5,B-6 and B-7 exerted good proliferation inhibitory activity on A431 cells(IC₅₀ values were 28.4μM,9.94μM,26.32μM,18.99μM,29.29μM,respectively)Compound A-6 exhibited potent antiproliferative effects on both cell lines,with IC₅₀ values of 9.94μM and 6.31μM,respectively.Subsequently,further bioactivity evaluations of the preferred compound A-6 were performed:cell cloning experiments showed that compound A-6 had a good anti cell invasion effect.After being administered and cultured in A549 cells and A431 cells for 14 days,the clone formation rate in group A-6 was significantly lower than that in the blank control group,and the clone formation rate was decreased with the increase of concentration.These results indicated that compound A-6 had certain anti invasion ability to A549 cells and A431cells in a concentration-dependent manner.The results of AO/EB double staining showed that compound A-6 could induce the appearance of apoptotic bodies,and the induction ability was enhanced with the increase of concentration.These results indicated that compound A-6 had the ability to induce apoptosis of A549 cells and A431cells in a certain concentration-dependent manner.It was verified that compound A-6had certain inhibitory activity on both EGFR and c-Met.In summary,in this study,27 purine compounds were designed and synthesized using EGFR-TKIs and c-Met inhibitors as template compounds.The in vitro anti-proliferation experiment showed that compound A-6 had dual targeted inhibitory activity of EGFR/c-Met,which paved the way for the subsequent research.