Design,Synthesis And Anticancer Activity Studies Of Afatinib Derivatives

It has been found that Epidermal Growth Factor Receptor?EGFR?signaling plays an crucial role in the development of tumor and regulates the important biological processes in the development of many tumors which including cell proliferation,differentiation,protein synthesis,Metastasis,inhibition of apoptosis promote angiogenesis and so on.Therefore,the inhibition of the signal pathway has become a hot spot for cancer prevention and treatment,and attracting the attention of many research institutions and researchers.This paper mainly introduces the research of EGFR inhibitors and the design,synthesis and anti-tumor activity of novel EGFR inhibitors.Basing on the structure-activity relationship of afatinib and its derivatives,it was discovered that the 4-anilinquinazoline structure which is the crucial activity site of alfatinib.And the structure of?,?-unsaturated amide can form an irreversible covalent binding mode with the amino acid residue Cys797.According to the to the latest literature,we retained the 4-aniline quinazoline moiety and the Michael receptor,the small molecule tetrahydrofuran were substituted with methyl group.What's more,inspired by LP-7,the nitrogen-containing side chains in lead compound were replaced by different substituted aryl moeity to examine its effect on the activity,and obtained compounds T-1^T-22.Compound T-11 showed excellent biological activity in vitro and evaluated for activity against EGFR kinase with IC₅₀0 of 3.6 nM.In our later research we found that compound T-11 showed unsatisfactory results in kinase and solubility.In order to solve the question we introduced the aryl semicarbazone part to hope provided more nitrogen atoms combined with the receptor.In addition,aryl semicarbazone scaffolds can provide more nitrogen to improve solubility.Therefore,compounds T-23^T-52 were designed and synthesized.The solubility of these compounds is generally not good except the compound T-37 and evaluated for activity against EGFR kinase with IC₅₀0 of 56 nM.Inspired by compound T-37,we found that improving the solubility of the compounds may be beneficial to the activity.In order to improve the solubility of the compounds,used of bioisosterism,compounds T-53^T-62 were synthesized by replacing the aryl moeity with the structure of furan,thiophene and pyrrole.Moreover,We discovered that the target compound T-56 which containing the pyrrole group exhibited potent antitumor cell activity in vitro.So we introduced the structure of the flexible chain and the aqueous solubility amine to the compound T-56 to improve the solubility of the compound,and yielding compounds T-63^T-86.Taking consideration of being a drug,we finally use small molecular amine to replace the pyrrole amine structure to design and synthesis of compounds T-87^T-100.The structures of target compounds were confirmed by 1H-NMR and MS spectra and some compounds also have ¹³C-NMR and HRMS spectra data.All afatinib derivatives?T-1^T-100?were tested three or four cell lines for their cytotoxic activities on A549,PC-3,MCF-7,Hela and HepG2 by the MTT method,using afatinib as positive controls.In order to determine their target,Some preferred compounds were evaluated for their activities against EGFR kinase by the HTRF kinase assay,using afatinib and Staurosporine as positive controls.The results of activity in vitro bearing cinnamamide moiety showed that most of the target compounds showed moderate to good cytotoxicity against three or four tumor cells lines.The compound T-11 showed the best activity against A549,PC-3,MCF-7 and Hela cancer cell lines,with the IC₅₀0 values of 0.07±0.02?M,7.67±0.97?M,4.65±0.90?M and 4.83±1.28?M,which were equal to more active than afatinib?0.05±0.01?M,4.1±2.47?M,5.83±1.89?M and 6.81±1.77?M?,respectively.The results of the activity against EGFR kinase indicated that cinnamamide compounds?T-5?T-11?were potent inhibitor of EGFR inhibitors and the most promising compound T-11 showed strong antitumor activities against EGFR kinase with the IC₅₀0 values of 3.6 nM was equal to the reference compound afatinib(IC₅₀0 1.6 nM).In addition,dose-dependent experimental data indicated that four tumor cells were dose-dependent for compounds T-5 and T-11.The cytotoxicity results showed that most of the aryl semicarbazone scaffolds compounds show lower activity on four tumor cells lines.The compound T-37showed the best activity against A549,HepG2,MCF-7 and PC-3 cancer cell lines,with IC₅₀0 values of 1.32±0.38?M,0.07±0.61?M,0.91±0.29?M and 4.89±0.69?M,which were equal to more active than afatinib?1.33±1.28?M,2.63±1.06?M?respectively.Compound T-37 also showed a strong inhibitory activity against EGFR kinase,with an IC₅₀0 of 56 nM.The results of flow cytometry method of operation showed that compound T-37 could block the proliferation of A549 tumor cells in G2/M and induce apoptosis of A549.The cytotoxicity results showed that the target compounds which containing pyrrole amines moiety had potent antitumor activity in vitro.Especially for tumor cells A549 and HepG2.Most of the compounds showed more activity than the control compound against the two tumor cells,and some even 20 times more activity than afatinib.Most of the elected compounds showed the best activity against EGFR kinase,which were equal to more active than afatinib.The most promising compound T-64 showed strong antitumor activities against EGFR kinase,which were 10-fold more active than afatinib.The cytotoxicity results showed that the target compound?T-87^T-100?exhibited excellent activity against the A549,HepG2,PC-3 and MCF-7 tumor cell lines.Most of the elected compounds showed the best activity against EGFR kinase,which were equal to more active than afatinib.The most promising compound T-88showed strong antitumor activities against EGFR kinase,which were 5-fold more active than afatinib.In addition,the results of flow cytometry method of operation showed that compound T-88 could block the proliferation of A549 tumor cells in G2/M and induce apoptosis of A549.According to the results of activities against tumor cell lines and EGFR kinase,we preliminary analyzed and discussed the structure-activity relationships?SARs?of target compounds.We found that methoxy substitution had a significant impact on the activity and methoxy substituted on C-4 or C-2,3,4 position was benefit for the activity.Structure-activity relationships and docking studies of pyrrole amines and small molecular amines semicarbazone indicated that the two moiety were benefit for the activity and aryl semicarbazone moiety was unfavorable for the activity.According the anti-tumor activities and molecular docking results of afatinib derivatives,we learn that quinazoline moiety play an important role in maintain the activities of these compounds.They can form hydrogen bonds with the amino acid residues of EGFR kinase,which makes them closely integrated with EGFR kinase.These results of structure-activity relationship could provide ideas and give us new research directions for the further study of EGFR inhibitors.