Design,Synthesis And Anticancer Activity Studies Of Novel Pyrrolo [2,3-b] Pyridine Derivatives As Small Molecule C-Met Inhibitors

HGF/c-Met signal transduction pathway is highly expressed in various tumor tissues,and is closely related to tumor invasion and metastasis.Developing small molecule inhibitors targeting c-Met has become a potential strategy for cancer treatment.In this article,we mainly introduce the HGF/c-Met signaling pathway and its relationship with cancer,and review the research progress of small molecule c-Met inhibitors.In addition,the design,synthesis and antitumor activity in vitro of the novel small molecule c-Met inhibitor were studied on the basis of the preliminary work.According to their chemical structure and the binding mode with c-Met protein,small molecule c-Met inhibitors are divided into two types:Class I and Class II.Class?c-Met inhibitor has become the focus of research because of the better activity and the large modified steric of its structure.And the structure-activity relationship is summarized as follows:?1?The structure of Class II c-Met inhibitors can be divided into four fragments:mother nucleus,Linker 1,Linker 2 and hydrophobic fragments.?2?Linker 2 is a characteristic fragment of this class of inhibitors which can be a cyclic or chain structure,and follows the"5 atom"rule—there are five atoms,that is,six chemical bonds between the hydrophobic fragment and Linker 1.On this basis,taking Class?c-Met inhibitor Foretinib as the leading compound,the target compounds were designed,in which the quinoline structure was replaced by pyrrolo[2,3-b]pyridine as the mother nucleus,and the 4-phenoxy fragment of Linker 1 were preserved.?1?The Linker 2 section followed the"5 atom"rule and replaced the cyclopropyl malonamide structure with pyridine formamide fragment so that the"5atoms"fragment was designed as a cyclic structure,and substituted phenyl groups were used as hydrophobic fragments to design compounds W-1^W-15 bearing phenylpicolinamide structure.?2?In order to investigate the effect of"5 atom"fragment on the activity of the compounds,the target compound W-16^W-26 was designed by changing the position of the phenyl group on the pyridine ring and keeping it at a distance of 6 atoms.?3?According to the principle of skeleton transition and bioisosterism,the pyridine structure was replaced with pyrimidine structure,and 24 target compounds W-27^W-50 containing phenylpyrimidine-carboxamide structure were designed.?4?The N-acylhydrazone structure and the amide structure of Foretinib were introduced into the Linker 2 part by using the principle of flattening,and the target compounds W-51^W-77 containing aromatic hydrazone unit were designed.At the same time,the effects of the"5 atoms"fragment with chain structure on the activities of the compounds were also investigated.?5?The hydrophobic fragment of the compounds in?4?was modified by replacing the phenyl group with the pyrrole ring and introducing the small molecule amine on the N atom to increase the water solubility to design the target compounds W-78^W-91 bearing the heterocyclic hydrazone structure.The structures of all the target compounds were confirmed by¹H-NMR and MS spectra and some were confirmed by¹³C-NMR spectra.All of the 91synthesized target compounds were tested for their cytotoxic activities on A549,MCF-7 and PC-3 cell lines by the MTT method,using Foretinib as positive controls,and 41 aromatic hydrazone derivatives were also tested for cytotoxic activities on HepG2 cell lines.In addition,the preferred compounds W-4,W-8 and W-10^W-12 in phenylpicolinamide derivatives and compounds W-32,W-34,W-37 and W-44 in phenylpyrimidine-carboxamide derivatives were further tested on HepG2 and Hela cell lines.In order to determine their target,26 compounds of phenylpicolinamide derivatives and the preferred compounds W-32,W-34,W-37,W-44,W-68 and W-87 were evaluated for their activities against c-Met kinase by the Homogeneous Time Resolved Fluorescence?HTRF?assay,using c-Met inhibitor Foretinib as positive control.In order to investigate the target compounds can inhibit cell growth in a time and concentration dependent manner,compound W-68 was studied more deeply on A549 cell lines.Furthermore,the compound W-68 was subjected to acridine orange?AO?staining and Annexin V-FITC/PI flow cytometry to determine the target compounds can induce apoptosis.The results showed that most of the target compounds exhibited moderate to excellent cytotoxic activity against the tested cell lines.The most promising compounds W-10 exhibited the best activity in phenylpicolinamide derivatives with the IC₅₀0 values of 0.69?M against c-Met kinase,which were 0.62 to 19.5 times more active than Foretinib on the tested cell lines.The introduction of fluorine atoms on the part of Linker 1 led to the enhancement of the activities of the compounds.The activities of the compounds of which the phenyl group is substituted at the C-4position of the pyridine ring is better than that of the compounds at the C-5 position.The compounds W-37 of phenylpyrimidine-carboxamide derivatives showed the best activity with the IC₅₀0 values of 0.12?M against c-Met kinase,which were 1.60 to473.5 times more active than Foretinib on the tested cell lines.The introduction of fluorine atoms on Linker 1 had no significant effect on the activities of the compounds.In addition,the position of the amide on the pyrimidine ring affected the activities of the compounds.For example,the activity of pyrimidine-4-amide compounds is slightly superior to that of pyrimidine-2-formamide compounds.What's more,the inhibitory activity of the aromatic hydrazone series compounds W-68,W-72 and W-75 was superior to that of Foretinib,and W-68 showed excellent c-Met kinase activity with the IC₅₀0 values of 0.506?M.The introduction of the fluorine atom into the Linker 1 moiety had no significant effect on the activities of the compounds.The length of carbon chain between the N atom of the pyrrole ring and the small molecular amine had a certain influence on the activities of the compounds containing heterocyclic hydrazone.In general,the activities of the compounds containing the phenylhydrazone structure is superior to that of the heterocyclic hydrazone series.In addition,further studies of W-68 show that the compounds could inhibit the growth of A549 cells in a time and concentration-dependent manner and could induce apoptosis.According to the antitumor activity in vitro of the compounds,the structure-activity relationship was summarized.The"5 atom"structure between phenoxy and hydrophobic fragments plays an important role in the activity of the compounds.The introduction of fluorine atoms on the aminophenoxy groups enhances the activities of the phenylpicolinamide derivatives,while played no significant impact on activities of the phenylpyrimidine-carboxamide derivatives.In general,the activities of the phenylpyrimidine-carboxamide derivativeswere superior to that of phenylpicolinamide derivatives,and the activities of the benzene hydrazone derivatives were superior to that of the heterocyclic hydrazone series.In addition,the position of the substituent on the hydrophobic fragment plyed great impact on activity of the target compounds.The preliminary analysis and discussion on the structure-activity relationship of pyrrolo[2,3-b]pyridine compounds indicate the direction for later studies of this type of inhibitors.