Design,Synthesis And Anti-tumor Activity Studies Of 2-aryl-4-arylmethylaminopyrimidines As Fourth-generation EGFR Inhibitors

Lung cancer provides the highest incidence rate and mortality rate in China,while non-small cell lung cancer（NSCLC）accounts for 80%of lung cancer mortality.Studies have shown that EGFR signaling pathway is closely related to tumor invasion and metastasis.Therefore,inhibition of EGFR signaling pathway could significantly inhibit the growth and proliferation of tumor cells.In recent years,small molecule inhibitors targeting EGFR have achieved significant effects in clinical treatment,but the inevitable emergence of drug resistance problems,resulting in the reduced efficacy of EGFR in patients with NSCLC.Therefore,it is urgent to develop novel EGFR inhibitors that could effectively inhibit EGFR^T790M/C797S mutation.In this paper,the structure-activity relationship of the EGFR inhibitor Angew2017-7634-1 reported in the literature was summarized based on computer-aided drug design.By meaning of structural assembly and bioelectronic isometric principles,three series of 62 2-aryl-4-arylmethylaminopyrimidine EGFR inhibitors were designed and synthesized.The main transformations were as follows:（1）According to the principle of bioelectronic isostery and other principles,the active structure of the lead compound Angew2017-7634-1 was retained,and the hydrogen bond donor/acceptor substituted at different positions were introduced into the 2-aryl group.The different hydrogen bond donors/acceptors were introduced at the 4-amino group,and the carbon chain length of heterocyclic and 4-amino group was increased to explore the factors affecting the formation of hydrogen bond interaction between the compound and protein amino acid SER797.The first series of2-aryl-4-aminoquinazoline derivatives ZZH-1～ZZH-38 were design and synthesized,which a total of 38 target compounds.（2）In order to improve the poor solubility of the first series of target compounds,a small molecule secondary amine structure was introduced into the4-arylmethylamino group on the basis of previous studies,hoping to enhance the solubility of the compounds,and then increase the inhibitory activity of the compounds.The second series of pyrimidine methylamine-substituted quinazoline derivatives ZZH-39～ZZH-46 were designed and synthesized,with a total of 8 target compounds.（3）Based on the results of the first and second series of anti-tumor activity and the previous research of the research group,using the principle of structural assembly,the pyranopyrimidine skeleton was substituted for the quinazoline structure,and the small molecule secondary amine structure was retained.The third series of2-aryl-4-aminothiopyranopyrimidine derivatives ZZH-47～ZZH-62 were designed and synthesized,with a total of 16 target compounds.With Angew2017-7634-1 as the lead compound,A549,H460,MCF-7,H1975and Ba/F3 cell as the test cell,the anti-tumor activity of 62 target compounds were tested in vitro by MTT method.The results showed that the most of the target compounds exhibited moderate to strong anti-tumor activity against the tested cell compared with the lead compound Angew2017-7634-1.Among them,the compounds ZZH-24,ZZH-41 and ZZH-61,as the most promising derivatives of each series,not only exhibited excellent anti-tumor activity against the tested cell lines,but also could effectively inhibit EGFR^{Del19/T790M/C797S} triple mutant Ba/F3 cell at a concentration of10μM,with inhibition rates of 91.0%,71.43%and 99.8%,respectively.According to the results of the anti-tumor activity,compounds with better anti-tumor activity were screened for EGFR^WT,EGFR^L858R/T790M and EGFR^{Del19/T790M/C797S} kinase inhibitory activity assays.The results showed that the inhibitory effects of most of the tested compounds against EGFR^WT and EGFR^L858R/T790M kinases greater than 10μM.Fortunately,the compounds ZZH-24,ZZH-41 and ZZH-61 displayed moderate to strong inhibitory activity against EGFR^L858R/T790M,with IC₅₀ values of 0.74,8.88 and0.33μM,respectively.Moreover,the promising compounds ZZH-24 and ZZH-61exhibited effective inhibitory activity against EGFR^{Del19/T790M/C797S} with IC₅₀ values of475.5 n M and 133.5 n M,respectively,and the inhibition rate of ZZH-61 against EGFR^{Del19/T790M/C797S} kinase reached 98.2%at 10μM concentration.The activity of preferred compounds ZZH-24,ZZH-41 and ZZH-61 were further studies,such as AO cell staining,Axinnex V-FITC cell apoptosis Flow cell cycle assays and Fluorescence quantitative PCR analysis,etc.The results showed that the compounds ZZH-24,ZZH-41 and ZZH-61 not only induced late apoptosis of A549 cell lines in a dose-dependent manner,but also blocked A549 cell cycle arrest in S-phase in a dose-dependent manner.Molecular docking study showed that compounds ZZH-24,ZZH-41 and ZZH-61 could bind tightly to EGFR protein,and form 4 hydrogen bonds with EGFR^T790M/C797S protein.The introduction of small secondary amine structure into the solvent region was helpful to enhance the affinity of compounds.Based on the in vitro anti-tumor activity of the compounds and the results of molecular docking,the structure-activity relationship of the compounds was summarized as follows:（1）The 2-aryl o-hydroxyl group could form hydrogen bond interactions with the amino acid residues MET793 and GLN791 in the EGFR^T790M/C797S protein,which is the key group to maintain the biological activity of the compounds.（2）The heteroatom（N/O）and NH in the heterocycle of the 4-amino side chain could form 2 hydrogen bonds with SER797,which was essential for maintaining the activity of EGFR^{Del19/T790M/C797S} mutant cell lines.（3）the introduction of fluorine atoms was not conducive to the anti-tumor activity of the compounds;（4）the introduction of the thiopyrimidine structure was conducive to enhancing the anti-tumor activity and kinase activity of the compound in vitro,but t its oxide loses the inhibitory activity of EGFR^L858R/T790M kinase.（5）The introduction of small molecule secondary amine structure could increase the solubility of the compounds,thereby increasing the activity of the compounds.In conclusion,in this paper,the three series of 62 novel2-aryl-4-arylmethylaminopyrimidine EGFR inhibitors were obtained by the modification of Angew2017-7634-1 as the lead compound,and their structure-activity relationship and mechanism of action have been preliminarily analyzed and discussed,which lays a foundation for further study of these inhibitors.