The second modification involved the replacement of the middle phenyl ring of entinostat with a pyrazine ring, in addition to the replacement of the pyridine ring (capping group) with the bulkier 3-indolyl scaffold as in the case of compound IV, which resulted in higher inhibitory activity against HDAC 1-3 compared to the reference inhibitors (Table ). The preliminary testing of the developed prodrugs showed that they exhibited very weak HDAC inhibitory activity compared to the corresponding parent inhibitors. The prodrugs and their parent inhibitors were then tested in a cell viability assay via a tetrazolium salt-based assay on wild-type THP1 acute myeloid leukemia (AML) cells and THP1 cells that had been lentivirally transfected with the E. coli nitroreductase NfsB. We had used this system successfully before to activate bioreductive prodrugs of inhibitors of the lysine-specific demethylase 1 (LSD1) . We pre-screened all compounds at 1, 10, and 50 µM concentrations in the wild-type and transfected cell lines (% inhibition against NTR-THP1 at 10 µM is presented in Table ). When sub-µM inhibition of HDACs in vitro and more than 50% inhibition of cell viability were achieved by the prodrugs in the NTR-THP1 cells, a GI50 value was determined. For inhibitors II and IV, the more potent prodrugs 5 and 6, respectively, were selected.