Table : Activity against cancer cell lines Cal27 and HepG2, and cytotoxicity against MRC-5. Caspase 3/7 mediated apoptosis in Cal27 cells. The compounds demonstrating the most potent anticancer activity in the MTT assays, 9b, 9d, and 13f, were further evaluated using a caspase 3/7 activation assay in Cal27 cells. Caspase activation was measured through fluorescence image analysis and vorinostat and quisinostat were included as reference HDAC inhibitors and cisplatin as a positive control (Figure ). Additionally, compound 18b was tested to determine whether it can induce apoptosis by activating caspase 3/7 or if it lacks this effect and the accompanying cytotoxicity. After 48 hours of treatment with the respective compounds at both the IC50 and three times the IC50 concentrations determined from the MTT assay, almost all tested compounds induced caspase 3/7 activation (Figure ). The exception was 13f at 1 µM, here statistically significant caspase activation compared to the vehicle could not be observed. Nevertheless, at 3 µM a highly significant increase of caspase activation could be measured. Furthermore, 18b did not show a significant increase in caspase 3/7 activation even at 25 µM, which corroborates the previously observed low cytotoxicity in the MTT assays. Compound 9d exhibited the most pronounced effect, with a 37% increase in caspase activation at 1 µM and a 75% increase at 3 µM. Hyperacetylation of α-tubulin and histone H3. To determine cellular target engagement and downstream effects of HDAC inhibition we assessed the acetylation levels of α-tubulin, a substrate of HDAC6, and histone H3, a substrate of class I HDACs. Cal27 cells were treated with compounds 9b, 9d, 13f, 18b, and vorinostat and quisinostat as controls for 24 h at their Cal27 MTT IC50 concentrations, before assessing the acetylation levels by western blot (Figure ). All compounds showed hyperacetylation of α-tubulin. 9b, 9d, 13f, 18b, and quisinostat showed a weaker band than vorinostat, which is in good accordance with the isoform selectivity profile of these compounds, as 9b, 9d, 13f, 18b and quisinostat are preferential HDAC1 inhibitors while vorinostat shows stronger inhibition of HDAC6 than of HDAC1. Elevation of acetyl-histone H3 levels were also detected by all of the tested compounds. 9b and 9d stood out with the strongest band, even more pronounced than those of the control compounds quisinostat and vorinostat. Further in vitro characterization of 9b, 9d, and 13f. Since 9b, 9d, and 13f showed the most potent anticancer activities in the cell viability and caspase 3/7 activation assays, these compounds were further characterized through various in vitro experiments. First, the isoform selectivity profile against additional HDAC isoforms was determined. For this purpose, the compounds were tested for inhibition of the remaining class I enzymes HDAC2, 3, and 8 and against the class IIa enzyme HDAC4. The results are depicted in Table . All compounds showed low nanomolar activity against HDAC2 and 3. These results suggest a clear preference of the compounds for class I HDACs. The most potent HDAC2 and 3