CHEMBL5736793

Determination of the Effect of the Compounds of the Present Invention Against FGFR Kinase Activity: The following method was used to determine the inhibition degree of the kinase activity of recombinant human FGFR protein by the compounds of the present invention under in vitro conditions. Cisbio Company's HTRF® KinEASE-TK tyrosine kinase kit(Cat. No: 62TKOPEB) was used in the present method, the kit was used to reflect the inhibitory effect of the compounds on FGFR kinase activity by determination of the phosphorylation degree of FGFR protein-mediated biotinylated polypeptide substrates based on the principle of time-resolved fluorescence energy resonance transfer (TF-FRET). For detailed experimental procedures, refer to the kit instructions. Recombinant human FGFR protein was purchased from Carna bioscience (Japan, Cat. No: FGFR1 #08-133, FGFR2 #08-134, FGFR3 #08-135, and FGFR4 #08-136).The experimental procedure is briefly described as follows: the test compound was first dissolved in DMSO to prepare a stock solution, and then gradiently diluted with the buffer provided in the kit, and the final concentration of the test compound in the reaction system ranges from 10 μM to 0.1 nM. The concentration of ATP solution (Sangon Biotech (Shanghai) Co., Ltd., A600311) used in the test is the ATP Km concentration corresponding to each FGFR subtype measured in advance, and the ATP Km concentration corresponding to FGFR1˜4 is 100 μM, 40 μM, 40 μM and 120 μM respectively. The reaction was carried out in a 384-well microplate, the compound and a certain amount of FGFR protein was firstly added to the well, and incubated at room temperature for 5-30 minutes, then the ATP solution and the biotinylated polypeptide substrate solution were added to the reaction solution, and incubated for 50 minutes with shaking at room temperature. Subsequently, an anti-phosphotyrosine antibody coupled with a europium compound and streptavidin coupled with the modified allophycocyanin XL665 were added to the reaction, and incubation was continued for 1 hour at room temperature with shaking. After the incubation ended, the fluorescence intensity values of the respective wells at an excitation wavelength of 304 nm and emission wavelengths of 620 nM and 665 nM were measured in a TF-FRET mode on a microplate reader. The percentage inhibition of the compound at each concentration was calculated by comparison with the fluorescence intensity ratio of the control group (0.1% DMSO), and the nonlinear regression analysis was performed on the logarithm values of the concentrations of the compounds—inhibition rate by GraphPad Prism 5 software to obtain the IC50 value of compounds.