CHMFL-BMX-078 is a highly potent and selective type II irreversible inhibitor of BMX kinase, with an IC50 of 11 nM.

BMX kinase:11 nM.

CHMFL-BMX-078 displays a high selectivity profile against the 468 kinases/mutants in the KINOMEscan evaluation and achieves at least 40-fold selectivity over BTK kinase (IC50=437 nM). For inactive state of BMX kinase, CHMFL-BMX-078 displays a binding Kd of 81 nM, while for the active state of BMX kinase, it exhibits a binding Kd of 10200 nM. CHMFL-BMX-078 exhibits antiproliferative effects against BaF3-TEL-BMX cells (GI50=0.016 μM) and selectivity over parental BaF3 cells. CHMFL-BMX-078 is about 80-fold more potent against BMX wt (EC50=5.8 nM) than C496S mutant (EC50=459 nM) for the inhibition of BMX total tyrosine phosphorylation.

CHMFL-BMX-078 also displays an acceptable Cmax (13565.23 ng/mL) and AUC0-t (1386.41 ng/mL h) in iv injection. However, it is not absorbed by oral administration, indicating that this compound could be administrated through i.v. or i.p. injection when used as a research tool.

The kinase reaction system contains BMX or BTK, 1 μL of serially diluted CHMFL-BMX-078, and substrate Poly peptidewith 100 μM ATP. The reaction in each tube is started immediately by adding ATP and kept going for an hour under 37 °C. After the tube cooled for 5 min at room temperature, 5 μL solvent reactions are carried out in a 384-well plate. Then 5 μL of ADP-Glo reagent is added into each well to stop the reaction and consume the remaining ATP within 40 min. At the end, 10 μL of kinase detection reagent is added into the well and incubated for 30 min to produce a luminescence signal. Luminescence signal is measured with an automated plate reader.

CHMFL-BMX-078 is dissolved in 55% saline containing 5% DMSO and 40% PEG400 by vortex. The final concentration of the stock solution is 1 mg/mL for administration.Rat: Six 8-week-old male Sprague?Dawley rats are fasted overnight before starting drug treatment via intravenous and oral administration. Animal blood collection time points are as follows. For groups 1, 3, and 5 (intravenous): 1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h before and after administration is selected. For group 2, 4, and 6 (oral): 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h before and after dosing. The plasma is collected for analysis.