Synthesis And Preliminary Pharmacological Activity Of Stilbene-Pyrimidine Derivatives As EGFR Inhibitors,and Related Study Of Drug Liposomes For The Optimized Compound

Objective: A series of diphenylpyrimidine derivatives bearing a 4-(styryl)aniline functionality were synthesized and biologically evaluated as potential epidermal growth factor receptor(EGFR)inhibitors for intervention of non-small-cell lung cancer(NSCLC)in order to identify promising molecules with better antiproliferative activity,lower toxicity and higher anti-resistance.Methods: Led by the third generation EGFR inhibitor Rociletinib,a series of novel stilbene-stubstituted pyrimidine derivatives were designed and synthesized according to the molecular hybrid strategy.All these newly obtained compounds were characterized by 1H NMR,13 C NMR,HRMS and MS.Their inhibitory activity against EGFRWT(wild-type EGFR)and EGFRT790M/L858R(Thr790→Met790,Leu858→Arg858)kinase was determined by fluorescence kinase assay in vitro.MTT colorimetric assay was used to determine the cell viability after the new compounds of different concentrations acting on the selected cell lines for 72 hours.DAPI staining and MTT assay were used to determine the antitumor activity of the optimized inhibitor SAR-007 and Gefitinib under the same conditions.The effect of compound SAR-007 on the apoptosis in lung cancer cell line H1975 was investigated by flow cytometry.Compound SAR-007 was prepared as pharmaceutical liposomes by thin film dispersion.The effect of drug liposomes prepared by SAR-007,blank liposomes and SAR-007 on the survival rate of non-small cell lung cancer cell line H1975(harboring EGFRL858R/T790 M mutation)was determined by MTT assay.Result: A total of 12 potent EGFR inhibitors featuring a stilbene-pyrimidine template were synthesized and,and their molecular structure were determined by NMR and HRMS technology.Kinase test results showed that their IC50 values ranged from6.8 nMol/L to 29.4 nMol/L,which were 40-176 times higher than Gefitinib.Compound SAR-007 showed a strong inhibitory effect on EGFRT790M/L858 R kinase(IC50: 11.0nMol/L),and higher selectivity(SI = 49,SI = EGFRWT: EGFRT790M/L858R).The results of MTT assay showed that most of the new compounds had strong antiproliferative activity against A431 cells(harboring EGFRoverexpression)and A549 cells(harboring EGFRK-ras mutation),which were 2-35 times higher than that of Gefitinib.In addition,the toxicity of these compounds against the normal lung epithelial HBE cells was lower than that of Gefitinib.In particular,compound SAR-006(IC50: 2.318 μMol/L)and compound SAR-007(1C50: 2.91 μMol/L)also showed good inhibitory activity against gefitinib-resistant lung cancer cells H1975,with an IC50 value of higher than 20 μMol/L.The results of DAPI staining showed that the inhibitory activity of compound SAR-007 against H1975 cells was remarkable stronger than that of the listed drugs.The drug concentration-cell survival rate line chart indicated that the strong killing effect acted on H1975 by compound SAR-007 was more remarkable than that of Gefitinib.Apoptosis test results showed that the inhibitory effects of molecule SAR-007 were clearly increased in a concentration dependent manner,with an apoptosis rate ranging from40.4 to 79.8%.The drug liposomes prepared by the film dispersion method appeared uniform size distribution and good appearance(Z-Average: 197.7nm,PdI: 0.354).The results of MTT assay of the drug liposomes showed that the inhibitory activity of SAR-007 against H1975 cells was slightly improved.Conclusion: In this work,a total of 12 novel stilbene pyrimidine derivatives were discovered as potent EGFR inhibitors,which showed good inhibition activityagainst EGFRWTand EGFRT790 M kinases.We also found that compound SAR-007 exhibited more inhibitory activity,higher selectivity and less cytotoxicity in restraining the EGFRT790 Mmutant.Compound SAR-007 could effectively inhibit the acquired resistance of lung cancer cells by inteacting with the mutant EGFRT790 M kinase.The liposome experiments provided a valuable clue for improving their anti-NSCLC activity.Overall,all these results are of great value for the development of stilbene-substituted pyrimidines as effective EGFRT790 M mutant inhibitors.