Cell experiment:

Methylene blue staining proliferation assay (MBS): The effect of compounds on cell proliferation is assessed using HuH-7 hepatocellular carcinoma cells are cultured in the vendor-recommended medium. Specifically, 5000 cells/well are seeded in 96-well tissue culture plates in a total media volume of 100 μL/well and increasing compound dilutions or DMSO are added 24 hours thereafter in triplicates. 72 hours after compound addition, cells are fixed by adding 25 and incubated for 10 minutes at room temperature. Cells are washed three times with H2O. Cells are washed 3 times with H2O, 200 mL/well and then lysed by adding 200 mL/well of 3% HCl for 30 minutes at room temperature with shaking. Optical density is measured at A650 nm. The concentration of compound providing 50% of proliferation inhibition with respect to DMSO-treated cells is determined (IC50) using XLFit software.

FGFR4-IN-1 is a potent inhibiotr of FGFR4 with IC50 of 0.7 nM.

FGFR4-IN-1 significantly inhibits the proliferation of HuH-7 hepatocellular carcinoma cells with IC50 of 7.8 nM[1].

References:
[1]. Ring-fused bicyclic pyridyl derivatives as fgfr4 inhibitors. WO 2015059668 A1