Design,synthesis And Antitumor Evaluation Of Novel Pyridazine And Triazine Derivatives As C-Met Kinase Inhibitors

Cancer is a major disease that threatens human life in today’s world.A lot of research shows that c-Met is over-activated,which may initiate the transformation of normal cells into tumor cells and further drive its subsequent events such as invasion,metastasis,and spread.Therefore,the inhibition of c-Met signaling has become an important strategy for cancer treatment,and the search for new and efficient c-Met kinase inhibitors has become a hot topic in the medical community.Based on the summary of TypeⅡc-Met kinase inhibitors reported in the literature.Using altiratinib and foretinib as the lead compounds,4-phenoxypyridine was retained.Using the classic principle of equal arrangement of electrons and skeletal transition,3-pyridazinone,4-pyridazinone,and 1,2,4-triazinone with rigid structures were introduced into the Linker part,and different substituents at the terminal benzene ring（R₁）were introduced,three types（23 compounds）of unreported c-Met kinase inhibitors were designed and synthesized.The structure of the target compounds were confirmed by IR,MS,¹H NMR and ¹³C NMR.MTS method of kinase test results showed that the target compound（GYL-1～GYL-23）have moderate to high inhibitory activity on c-Met kinase(IC₅₀=0.016～3.54μM).Representative compound GYL-8(IC₅₀=0.044μM),GYL-9(IC₅₀=0.057μM),GYL-15(IC₅₀=0.016μM),and GYL-16(IC₅₀=0.031μM).MTT colorimetry was used to test the antitumor activity of the target compound on A549cell line,HT-29 cell line and H460 cell line.The text results showed that the target compound has moderate to strong cytotoxicity to the tumor cells of the three cell lines tested.Among the compounds,the activity of GYL-9(HT-29:IC₅₀=0.63μM)and GYL-15(HT-29:IC₅₀=0.56μM;H460:IC₅₀=0.72μM)were better than the positive control drug Foretinib.The results of soft agar cloning experiments showed that A549and HT-29 cells were sensitive to the target compound GYL-15.The antitumor mechanism was studied with the preferred target compound GYL-15.AO/EB staining results showed that GYL-15 induced apoptosis in A549 cell line and HT-29 cell line in a concentration-dependent manner;flow cytometry analysis of Annexin V-FITC and PI dual-labeling detection also showed that GYL-15had the apoptosis-inducing effect of A549 cell line and HT-29 cell line in a concentration-dependent manner;the results of scratch experiments showed that the target compound GYL-15 could significantly inhibit the migration of A549 cells at both concentrations.Based on the results of MTS kinase detection and MTT cell detection,the structure-activity relationship of the target compounds were preliminarily summarized:（1）The target compounds whose Linker part were 1,2,4-triazinone showed good biological activity;（2）Terminal benzene compounds with a single electron-withdrawing group（F or Cl）on the ring are usually more active than compounds with a double-electron-withdrawing group or an electron-donating group;（3）The biological activity of compounds with a substituent at the 4-position of the benzene ring were superior to other locations.The docking results of GYL-15 with c-Met receptor protein（PDB ID code:3LQ8）showed that the N atom of pyridine ring,NH of amide fragment and O atom of carbonyl in GYL-15 formed three hydrogen bonds with three amino acid residues（Met1160,Asp1222 and Lys1110）of c-Met protein,respectively.Meanwhile,pyridyl ring、the phenyl ring at 4-position of pyridine and the phenyl ring at 1-position of triazine formed threeπ–πinteractions with Tyr1159、Phe1223 and Phe1134,respectively.Through the study of this subject,new ideas were provided for the design and synthesis of c-Met inhibitors.At the same time,it laid a foundation for further study of typeⅡc-met kinase inhibitors.