PKM2-IN-1 (compound 3k) exhibits inhibitory activity against PKM2 with an IC50 of 2.95 μM, while the IC50 for PKM1 is 4-5 times higher.

PKM2:2.95 μM

Results show that most of the tested compounds exhibit some degree of PKM2 inhibition and some compounds, such as PKM2-IN-1 (compound 3k) and 6d, display more potent activity than the positive control shikonin. The representative compounds PKM2-IN-1, 6d display dose-dependent inhibition of PKM2 with less inhibition of PKM1 and PKL like shikonin. Among all tested compounds, the most potent compounds are 3a, PKM2-IN-1 and 3r, which exhibit IC50 values against HCT116 and Hela cells ranging from 0.39 to 0.41 μM, 0.18 to 0.29 μM and 0.18 to 0.38 μM, respectively.

Cell lines (HCT116, Hela, H1299, BEAS-2B) are cultured in RPMI 1640 containing 9% fetal bovine serum (FBS) at 37°C in 5% CO2. Cell viability is detected with the MTS assay according to the manufacturer's instructions. Briefly, 5000 cells in per well are plated in 96-well plates. After incubated for 12 h, the cells are treated with different concentration of tested compound (including PKM2-IN-1) or DMSO (as negative control) for 48 h. Then 20 μL MTS is added in per well and incubated at 37°C for 3 h. The absorbance of each well is determined by a microplate reader at a 490 nm wavelength. The IC50 values are calculated using Prism Graphpad software of the triplicate experiment.