Design,Synthesis And Anti-Tumor Activity Studies Of Afatinib Derivatives

Malignant tumor is serious threat to human health,and the number of people killed due to cancer is increasing every year.Therefore,it has become a research hotspot to conquer and cure cancer in many countries.In recent years,with the further understanding of the biological characteristics of tumors,several new anti-tumor targets have been discovered.Epidermal Growth Factor Receptor?EGFR?signaling pathway plays an important role in tumorigenesis.Including the apoptosis,proliferation,differentiation,migration and cell cycle of the cell,and closely related to the formation and deterioration of the tumor.Therefore,inhibition of EGFR signaling pathway has become a hot spot for cancer prevention and treatment.This paper mainly introduced the research status of related EGFR inhibitors,and design,synthesis and anti-tumor activity of novel EGFR inhibitors.Using afatinib as a lead compound,combined with the structure-activity relationship study and the reported results of small molecule inhibitors of the EGFR signaling pathway,the structural modification of the lead compound afatinib was introduced.The structure was optimized and modified mainly at the C-6 and C-7positions of quinazoline.In order to screen out compounds with significant antiproliferation activity,eleven series of afatinib derivatives containing indoles structures were designed and synthesized.In order to investigate the effect of N-H bonds at the substituents of C-4 position of quinazoline to the antiproliferative activity,we introduce to the structure of2,3-dihydro-indole and 1,2,3,4-tetrahydroquinoline,two series of afatinib derivatives?YQ-1^YQ-21?containing small molecule amide structures were obtained.Among them,compound YQ-20 showed excellent biological activity in vitro,the IC₅₀0 values was 33 nM against EGFR kinase.However,the experiments revealed that YQ-20 had solubility problems in vitro.Therefore,we have introduced a small molecule secondary amine structure based on this type of compounds.By introducing a water-soluble group,the solubility can be improved,and two series of afatinib derivatives?YQ-22^YQ-51?containing a 2-secondary amine acetamide structure were designed and synthesized.Through studies of antiproliferative activity,compound YQ-48 showsd excellent antiproliferative activity in vitro,but the IC₅₀values was 154 nM against EGFR kinase,which did not reach the intended purpose of the original design of the compound.After consulting the literature and research group's previous studies,it was found that the acrylamide structure can act as an electrophilic warhead and form a covalent bond with the cysteine residue?Cys797?in EGFR,making the combination of the compound and the ATP binding site more stabler.Therefore,the acrylamide structure was introduced and two series of compounds?YQ-52^YQ-81?were synthesized.However,from the activity data of these compounds,it has been found that the water solubility of the compounds is not good,which may affect the effect of the compounds in vitro.In order to increase water solubility,inspired by the afatinib lead compound,the structure of acrylamide was retained,and small molecule secondary amine groups were introduced to increase water solubility.Two series of compounds?YQ-82^YQ-113?were synthesized.According to the results of activity tests,most compounds showed excellent antiproliferative activity,and compounds YQ-84,YQ-88 and YQ-93showed excellent activity of against EGFR kinase,and their IC₅₀0 values was 3 nM,4nM and 5 nM against EGFR kinase,respectively.In order to investigate the effect of the formation of cyclic structures on the quinazoline C-6 and C-7 positions,the oxazole ring structure was introduced and fourteen compounds?YQ-114^YQ-127?were synthesized.Since the compounds containing oxazole ring showed excellent activity of against EGFR kinase,according to the principle of bioisosterism,the imidazole ring structure was introduced,and ten compounds?YQ-128^YQ-137?were synthesized.In order to increase the water solubility,the structure of small molecule secondary amine was introduced at the end of the flexible chain,and thirteen compounds?YQ-138^YQ-150?were synthesized.The structures of all the target compounds were characterized by ¹H-NMR and MS spectra.Some of the compounds were confirmed by ¹³C-NMR.Using MTT colorimetric assay,A549,PC-3,MCF-7,and HepG2 were used as test cell lines,afatinib was a positive control,and one hundred and fifty target compounds were tested for anti-tumor activity.In order to investigate its targets,LANCE^?Ultra enzyme activity assay was used,and afatinib was used as a positive control.Several excellent activity target compounds were tested on against EGFR kinase activities.The activity of the target compounds containing the small molecule amide structure showed that most compounds showed moderate inhibitory activity against four tumor cells.Among them,when the substituent at the C-7 position of quinazoline was ethoxy,the activity was significantly decreased.When the C-7 position of quinazoline was methoxy or tetrahydrofuran,the cell activity was better than other substituents.The kinase activity test results showed that the optimal compounds had excellent anti-tumor activity against EGFR kinases on YQ-11 and YQ-20,and the IC₅₀0 reached the nM level.In addition,the dose-dependent experimental data indicate that compound YQ-20 was dose-dependent on four tumor cells.The activity of the target compounds containing 2-secondarylamine acetamide structure showed that most of the compounds showed moderate to excellent activity on the tumor cells?A549,HepG2,MCF-7 and PC-3?.Overall,the compounds were substituted with diethylamine slightly increased antiproliferative activity against A549,HepG-2,MCF-7 and PC-3 cell lines relative to substituted with morpholine.In general,the compounds were substituted with?S?-3-hydroxytetrahydrofuran at the C-7 position of the quinazoline facilitate proliferative activity.The activity of the target compounds containing an acrylamide structure revealed that most of the compounds exhibited excellent antiproliferative activity against different test cancer cells.Overall,the Compounds?YQ-82^YQ-113?was more effective than compounds?YQ-52^YQ-81?for the three types of cancer cells.The results showed that the introduction of a small molecule secondary amine compounds was beneficial to antiproliferative activity.We hypothesized that small-molecule secondary amines may found for increase the water-solubility and contribute to high binding affinity.Among them,compound YQ-93 showed the best antiproliferative activity against the three human cancer cell lines.The IC₅₀0 values of compound YQ-93 were 1.09?M,1.34?M and 1.23?M against A549,MCF-7 and PC-3,respectively,and reached the same level as lead compounds afatinib?0.71?M,0.93?M and 2.51?M?.Further EGFR kinase activity studies revealed that the EGFR kinase activity of compounds YQ-84,YQ-88 and YQ-93 reached the same level as the lead compound afatinib.In addition,flow cytometry results and AO staining experiments showed that compound YQ-93 can induce A549 apoptosis.The activity of the target compounds containing oxazole and imidazole structure showed that the test compounds had higher inhibitory activity against A549 than MCF-7 and PC-3.The results showed that these compounds have certain selectivity for A549 cell lines.More importantly,from the data of antiproliferative activity,it can be seen that the compounds?YQ-138^YQ-150?has more pronounced effect than the compounds?YQ-114^YQ-137?on the three cancer cell lines.According to the test results of the antiproliferation activity and enzyme activity,we conducted a preliminary analysis and discussion on the structure-activity relationship of the target compounds.In addition,the docking results showed that the structure of quinazoline plays an important role in maintaining the activity of the compounds,which was due to the fact that the structure of quinazoline can form hydrogen bonds with the amino acid residues on the crystals of the EGFR kinase protein,allowing the compound to bind tightly to the protein crystals.The analysis of the structure-activity relationship provides optimization and transformation ideas for the study of EGFR inhibitors,pointing the way for later research.