Starting from the N-hydroxy-3-(4-(2-phenylbutanoyl)amino)phenyl)acrylamide 5b previously described by us as HDAC inhibitor, we prepared four aza-analogues of 5b (6-8, 9b) as regioisomers containing the pyridine nucleus. A preliminary screening against mHDAC1 highlighted the N-hydroxy-5-(2-(2-phenylbutanoyl)amino)pyridyl)acrylamide 9b as the most potent inhibitor. Thus, we further developed both pyridylacrylic- and nicotinic-based hydroxamates (9a, 9c-f, and 11a-f) and 2′-aminoanilides (10a-f and 12a-f), related to 9b, to be tested against HDACs. Among them, the nicotinic hydroxamate 11d displayed subnanomolar potency (IC50: 0.5 nM) and selectivity up to 34000-fold over HDAC4 and from 100- to 1300-fold over all the other tested HDAC isoforms. The 2′-aminoanilides were class I-selective HDAC inhibitors, generally more potent against HDAC3, with the nicotinic anilide 12d being the most effective (IC50HDAC3 = 0.113 μM). When tested in U937 leukemia cells, the hydroxamates 9e, 11c, and 11d blocked over 80% cells in G2/M phase, whereas the anilides did not alter the cell cycle progress. In the same cell line, the hydroxamate 11c and the anilide 10b induced about 30% apoptosis, and the anilide 12c displayed about 40% cytodifferentiation. Finally, the most potent compounds in leukemia cells 9b, 11c, 10b, 10e, and 12c were also tested in K562, HCT116, and A549 cancer cells, displaying antiproliferative IC50 values at single-digit to sub-micromolar level.
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