Later, the binding affinity of the compounds was determined with microscale thermophoresis (MST) measurements, using NT-647-labeled recombinant CTD of Hsp90α protein. A nonlinear regression curve was fitted with the K D formula, and, as expected, substances showing weak inhibition have high dissociation constants (6: 249 µM, 7a: 286 µM; Figure ). The lowest K D value was observed for 5b with 3.42 µM (Figure ). Next, the in vitro cytotoxicity assessment of compounds 5a-d, 6, 7a, and 7b revealed 5b as a promising candidate (with low IC 50 : 1.8 ± 0.3 µM) in a BCR-ABL1+ tested leukemia cell line K562 (Figure ).