| The c-Met RTK family is the only known high-affinity receptor for hepatocyte growth factor (HGF), also known as scatter factor (SF), which is produced by stromal and mesenchymal cells and acts primarily on c-Met-expressing epithelial cells in an endocrine and/or paracrine fashion. c-Met and HGF are widely expressed in a variety of tissues, and their expression is normally confined to cells of epithelial and mesenchymal origin, c-Met is expressed by most carcinomas and its elevated expression relative to normal tissue has been detected in a number of cancers including lung, breast, colorectal, prostate, pancreatic, head and neck, gastric, hepatocellular, ovarian, renal, glioma, melanoma, and a number of sarcomas. c-Met expression has been reported to be up-regulated in tumors by a number of epigenetic mechanisms including tumor secreted growth factors, tumor hypoxia, and activation of other oncogenes.In addition, c-Met gene amplification and subsequent overexpression has been reported in gastric cancer, colorectal cancer, and glioma. Activation of the HGF/c-Met signaling pathway has been shown to lead to a wide array of cellular responses including proliferation, survival, angiogenesis, wound healing, tissue regeneration, scattering, motility, invasion and branching morphogenesis. Since c-Met is at the crossing of many roads leading to tumorigenesis and metastasis, targeting this receptor could be a relatively simple way to interfere with many pathways simultaneously. HGF/c-Met signaling pathway has come into the spotlight as a promising therapeutic target for inhibiting tumor growth and has become one of the leading molecular targets in cancer. Currently various strategies are currently in development to disrupt the HGF-Met signal transduction pathway, in which small molecular inhibitors have been a particularly active field.1-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-b] pyrazine of Pfizer’s PF-04217903,3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine of Janssen’s JNJ-33377605and3H-[1,2,4]triazolo[4,3-b]pyridazin of SGX’s SGX-523are potent and selective c-Met inhibitors. Based on the crystal structure of the c-Met kinase "DFG-in" conformation, type I kinase inhibitors pharmacophore, structure-activity relationships of triazoles type I c-Met inhibitors and experience of optimization and modification on these compounds, this study designed a series of novel compounds using PF-04217903, JNJ-33377605, SGX-523and our previous patented compounds spiro[indoline-3,4’-piperidine]-2-ones and2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indoles as leading compouds by means of biological isostere principle, combination principles and molecular docking, then synthesized these compounds through directional synthesis and other method of chemical synthesis. We investigated their biological activities on cellular level to obtain more active compounds, analyzed and discussed binding mode of compounds and c-Met kinase through molecular docking experiments and discussed preliminary structure-activity relationship to lay the foundation for developing c-Met inhibitors with clinical application value.The main contents are summarized as follows:1. we designed and synthesized structurally relevant novel compounds by replacing1H-[1,2,3]triazolo[4,5-b]pyrazine and3H-[1,2,4]triazolo[4,3-b]pyridazin of leading compounds with3H-[1,2,3]triazolo[4,5-d]pyrimidine,7H-purine and indazole moiety:(1)3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidines WXY001, WXY002, WXY003, WXY004, WXY005were designed by introducing1-methyl-1H-pyrazole,1-(2-tetrahydro-2H-pyran-2-yloxyethyl)-7H-pyrazole,1-(2-hydroxyethyl)-1H-pyrazole,1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]-1 H-pyrazole and1-((1R,4R)-4-hydroxy-cyclohexyl)-1H-pyrazole to the5-position of3H-[1,2,3]triazolo[4,5-d]pyrimidine. The target compounds were prepared by the reaction of1-position substituted4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazoles with5-chloro-3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine via Suzuki coupling reaction.(2)7-(quinolin-6-ylmethyl)-purines WXY006, WXY007, WXY008, WXY009, WXY010were designed by introducing1-methyl-1H-pyrazole,1-(2-tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazole,1-(2-hydroxyethyl)-1H-pyrazole,1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]-1H-pyrazole and1-((1R,4R)-4-hy droxy-cyclohexyl)-1H-pyrazole to the2-position of purines, These compounds were synthesized using2-chloro-N4-(quinolin-6-ylmethyl)-4,5-pyrimidinediamino as material via cyclization reaction and Suzuki coupling reaction.(3)1(2)-(quinolin-6-ylmethyl)-1H-indazoles WXY011, WXY012, WXY013, WXY014, WXY015,WXY016,WXY017,WXY018, WXY019, WXY020can be obtained by introducing1-methyl-1H-pyrazole,1-(2-tetrahydro-2H-pyran-2-yloxyethy1)-1H-pyrazole,1-(2-hydroxyethyl)-1H-pyrazole,1-[(1R,4R)-4-(tert-butyldimethylsilyloxy) cyclohexyl]-1H-pyrazole and1-((1R,4R)-4-hydroxy-cyclohexyl)-1H-pyrazole to the6-position of indazoles, The4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole groups reacted with6-bromo-1H-indazole to obtain6-(1-methyl-pyrazol-3-yl)-1H-indazole,6-[1-(2-tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazol-4-yl]-1H-indazole, and6-{1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclo hexyl]-1H-pyrazol-4-yl}-1H-indazole respectively via Suzuki coupling reaction, then reacted with commercially available6-chloromethylquinoline to give1-(quinolin-6-ylmethyl)-1H-indazoles their isomers2-(quinolin-6-ylmethyl)-1H-indazoles as a result of two isomeric forms of indazole. All the products were characterized by1H-NMR and MS, and were evaluated in TR-FRET-based assay. The results showed that the3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo [4,5-d]pyrimidine and7-(quinolin-6-ylmethyl)-purines exhibited lower inhibitory effect while1(2)-(quinolin-6-ylmethy1)-1H-indazoles exhibited significant inhibitory effect on c-Met with IC50<0.9μM in in TR-FRET-based assay except WXY016and WXY019with IC50>10μM.2.Keeping1H-[1,2,3]triazolo[4,5-b] pyrazine or3H-[1,2,4]triazolo[4,3-b]pyridazine, methylenelink, quinoline of the lead compounds unchanged, the substituents of the pyrazine/pyridazine were replaced by the structure2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole to obtain novel2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles containing1H-[1,2,3]triazolo[4,5-b] pyrazine and3H-[1,2,4]triazolo[4,3-b]pyridazine WXY021, WXY022, WXY023, WXY024, WXY025, WXY026, WXY027, WXY028, these compounds were prepared as followed:The reaction of tert-butyl8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol e-2-carboxylate with6-bromo-l-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-b]pyrazine and5-chloro-3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine, respectively, gave WXY021and WXY022via Suzuki coupling reaction. Then, WXY021and WXY022were transformed to WXY023, WXY027and WXY024, WXY028by formylation and carbanoylation, and obtained WXY025, WXY026concurrently. All the products were characterized by1H-NMR and MS, and were evaluated in TR-FRET-based assay. The results showed that these compounds exhibited potent inhibition on c-Met, in which WXY022and WXY023had stronger with IC50values of0.0145μM and0.0995μM in TR-FRET-based assay than others with IC50<1μM.3.Keeping1H-[1,2,3]triazolo[4,5-b]pyrazine,3H-[1,2,4]triazolo[4,3-b]pyridazine,3H-[1,2,3]triazolo[4,5-d]pyrimidine,7H-purine, methylenelink and quinoline unchanged, the substituents of the pyrazine, pyridazine and7H-purine were replaced by the structure spiro[indoline-3,4’-piperidine]-2-one of our previous patented compounds to obtain novel spiro[indoline-3,4’-piperidine]-2-ones WXY029, WXY030, WXY031, WXY032, WXY033, WXY034, WXY035and WXY028, which were prepared by reaction of the pinacol borane esters of5and6position of spiro[indoline-3,4’-piperidine]-2-one with bromine substituted1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazine, 3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine,3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine and7-(quinolin-6-ylmethyl)-purine via Suzuki coupling. All the products were characterized by1H-NMR and MS, and were evaluated in TR-FRET-based assay. The results showed that these compounds exhibited potent inhibition on c-Met, in which WXY029, WXY030, WXY031, WXY032had stronger than WXY033, WXY034, WXY035,WXY036, and WXY029、WXY030stronger than WXY031, WXY032, WXY030had stronger with IC50values of0.0147μM in TR-FRET-based assay than WXY029with IC50values of0.227μM and WXY032(IC50=0.22μM) stronger than WXY021(IC50=0.28μM). These results suggested that spiro [indoline-3,4’-piperidine]-2-ones containing1-(quinolin-6-ylmethy1)-1H-[1,2,3] triazolo[4,5-b]pyrazine and3-(quinolin-6-ylmethyl)-3H-[1,2,4]triazolo[4,3-b]pyridazine were more effect on c-Met than others, of which1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo [4,5-b]pyrazine was stronger. Moreover,6position substituted spiro [indoline-3,4’-piperidine]-2-ones presented more effective activity than5position substituted compounds.4.For the strong inhibition on c-Met kinase, the compounds1(2)-(quinolin-6-ylmethyl)-1H-indazoles,2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles and spiro [indoline-3,4’-piperidine]-2-ones were evaluated in cell-based assay. The results showed that some target compounds exhibited significant inhibitory effect on c-Met with IC5o<10μM in cell-based assay.5. Molecular docking showed that all the compounds assumed a’U-shaped’binding mode, which is the typical character of type I c-Met inhibitors. The quinoline moiety engages the MET hinge region with a single canonical hydrogen bond between the quinoline nitrogen and backbone carbonyl of Met1178and a nonclassic C-H-O=C hydrogen bond between quinoline C8and the carbonyl oxygen of Pro1176, which was highly characteristic of all compounds bound to ATP binding site in kinase domains. Fused-rings1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazine,3-(quinolin-6 ylmethyl-3H-[1.2,4]triazolo[4,3-b]pyridazine,3-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine,7-(quinolin-6-ylmethyl)-purine and indazoles were swiched in between Tyr1230and Met1211through a π-π stacking interaction. The methylenelink could better match the sub hydrophobic pocket composed of Leul157, Arg1086and Tyr1230, or Va11109, Lys1110, Ala1226and Va11092and enhanced the π-π interaction, of which triazolopyridine was the best because triazolopyridazine, as an electron deficient group, was sandwiched in between Tyr1230and Metl211through a π-π stacking interaction. In addition, the hydrogen bond was important to the binding affinity and two nitrogen atoms of the triazolopyridine formed two hydrogen bonds with the back bone NH of Asp1222so it had higher affinity and lower IC50value.The spirocyclic pyridine ring also pointed out towards the solvent surface after "U-shape" folding, however, the solvent surface was far away from the hinge region. The hydrophobic interactions from Met1211and Tyr1230would contribute to the selectivity since the two amino acid were conserved among only three(c-Met, Axl, Mer) of the491kinases. The methylene connecting quinoline and triazolopyridazine was folded up to fit the subhydrophobic pocket composed by Leu1157, Arg1086and Tyr1230. The adjacent hydrophilic ring,5or6substituent of spiro [indoline-3,4’-piperidine]-2-ones, is extended to the binding pocket. Comparing to WXY030, WXY029could not match to the active site because of the tension from spire. It leaded to the conformation changes and larger distance between N from N-CH3-pyridine and the residue Asn1167, and could not generate hydrogen bond (shown in Fig.5). Therefore, WXY029had the lower affinity and the lower activity. Similar reasons explained why the analogues WXY032, WXY034, WXY036had more powerful activity than the analogues WXY031, WXY033, WXY035.In conclusion, we designed118new compounds,36of which were synthesized. Meanwhile,17key intermediates were also synthesized. All the target compounds were prepared via Suzuki coupling reaction. This coupling reaction with pinacol borane esters gave good yields under basic conditions using DMF/H2O as a solvent and Pd (PPh3)4as a catalyst. Indazoles,2.3.4.5-tetrahydro-1H-pyrido[4,3-b]indoles and spiro[indoline-3,4’- piperidine]-2-ones had the best inhibitory effect, in which IC50values of WXY030, WXY022and WXY023reached lOnM in TR-FRET-based assay and IC50values of some compounds reached1OμM in cell-based assay. Molecular docking experiments verified and explained the results.The study lay the foundation for developing c-Met inhibitors with clinical application value. |
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