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Background: Despite the promising preclinical antitumor activity of MET-targeting therapies, most clinical trials have failed. We introduced a new concept of quantitation of stroma-induced hepatocyte growth factor (HGF) to assess the actual MET signalling activity in gastric cancer (GC)., Methods: We treated serially diluted HGF and conditioned media (CM) from cancer-associated fibroblasts (CAFs) on low MET-expressing cancer cells and investigated the phenotypical and signalling changes. Stromal proportion and MET expression in GC samples were assessed, and gene set enrichment analysis (GSEA) from the public database was performed. The antitumor effect of anti-MET treatment was examined, especially when cancer cells were activated in a ligand-dependent manner., Results: Relatively high doses of HGF or high-concentrated CM fully activated MET signalling cascades and promoted cell proliferation/invasion. High stromal proportion denoted worse patient survival in MET-positive GCs than in MET-negative ones. GSEA showed that the gene sets regarding proliferation, migration, and CAF as well as MET pathway signature were enriched in simultaneously MET- and HGF-positive samples. Sufficient ligand-dependent MET signalling activation increased the sensitivity to crizotinib., Conclusions: We conclude that patients whose tumours have a high stromal proportion and at least low MET expression may benefit more from MET-targeted therapies.

Despite the huge success of tyrosine kinase inhibitors as anticancer agents, severe side effects are a major problem. In order to overcome this drawback, the first hypoxia-activatable 2-nitroimidazole-based prodrugs of the clinically approved ALK and c-MET inhibitor crizotinib were developed. The 2-aminopyridine functionality of crizotinib (essential for target kinase binding) was considered as ideal position for prodrug derivatization. Consequently, two different prodrugs were synthesized with the nitroimidazole unit attached to crizotinib either via carbamoylation (A) or alkylation (B) of the 2-aminopyridine moiety. The successful prodrug design could be proven by docking studies and a dramatically reduced ALK and c-MET kinase-inhibitory potential. Furthermore, the prodrugs showed high stability in serum and release of crizotinib in an enzymatic nitroreductase-based cleavage assay was observed for prodrug A. The in vitro activity of both prodrugs was investigated against ALK- and c-MET-dependent or -overexpressing cells, revealing a distinct hypoxia-dependent activation for prodrug A. Finally, inhibition of c-MET phosphorylation and cell proliferation could also be proven in vivo. In summary of the theoretical, chemical and biological studies, prodrug derivatization of the 2-aminopyridine position can be considered as a promising strategy to reduce the side effects and improve the anticancer activity of crizotinib., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To more accurately and rapidly achieve quantitative detection of clinical crizotinib samples, stable isotope labeled crizotinib was required as an internal standard. We have developed a method to prepare racemic [D 9 ] crizotinib using a base-catalyzed H/D exchange of both nitroso compound 2 and the acetophenone compound 6 with D 2 O and NaBD 4 reduction of 7 as the key steps to introduce the 9 deuterium atoms. Starting with 4-hydroxypiperidine, 14-step synthesis furnished the desired racemic [D 9 ] crizotinib 18. The deuterium-labeled compound 18 with the chemical purity of 99.62% was applicable for use as internal standards in the drug clinical study., (© 2018 John Wiley & Sons, Ltd.)

c-Met kinase has been considered as an attractive target for developing antitumor agents. The strong interactions between Tyr1230 and the inhibitors emphasized its importance for ligand binding. The clinically related Tyr1230 mutations have made negative impacts on current c-Met kinase inhibitors, especially the exquisitely selective ones, like PF-04217903, while the multi-targeted inhibitors, like Crizotinib, were not affected so much. In this study, the protein-ligand interactions between c-Met kinase domain (wild, Y1230C and Y1230H) and these inhibitors were compared. The binding site was expanded and the post-mutated regions became solvent accessible. The heavy dependency of PF-04217903 on the interactions with Tyr1230 resulted in the steep decrease of its potency against the Y1230 mutants. It was found that the ligand entrance region contributed consistently to the binding of Crizotinib, but not PF-04217903. Additional groups substituted in the ligand entrance region with stable interactions should be beneficial for improving the inhibitory activity of PF-04217903 against the Y1230 mutants. These findings will facilitate the discovery of potent inhibitors against Y1230 mutated c-Met kinase.