Inhibitors of histone deacetylases (HDACs) are a new class of anticancer agents that affect gene regulation. We had previously reported the first simple synthetic HDAC inhibitors with in vitro activity at submicromolar concentrations. Here, we present structure-activity data on modifications of a phenylalanine-containing lead compound including amino acid amides as well as variations of the amino acid part. The compounds were tested for inhibition of maize HD-2, rat liver HDAC, and for the induction of terminal cell differentiation and inhibition of proliferation in Friend leukemic cells. In the amide series, in vitro inhibition was potentiated up to 15-fold, but the potential to induce cell differentiation decreased. Interestingly, an HDAC class selectivity was indicated among some of these amides. In the amino acid methyl ester series, a biphenylalanine derivative was identified as a good enzyme inhibitor, which blocks proliferation in the submicromolar range and is also a potent inducer of terminal cell differentiation.

a Histone deacetylase (HDAC) inhibitors are a novel class of promising anti-cancer agents. Little information is available on the capacity of structurally different HDAC inhibitors to induce terminal cell differentiation in different cell types in relation to enzyme inhibition and subtype selectivity. Consequently, the aim of this study was to provide a comprehensive comparison of these effects. New biarylalanine inhibitors of HDAC were synthesized and compared to a series of standard inhibitors from different laboratories. Chromatographically purified rat liver and immunoprecipitated FLAG-tagged recombinant human HDACs were used as sources of HDAC activity. Enzyme inhibition was studied using a fluorescent substrate and its conversion was monitored by high-performance liquid chromatography. The ability to induce cell differentiation was compared in murine (Friend DS-19) and human (K562) erythroleukemic cell lines, and was quantified by benzidine staining. Inhibition of cell proliferation was evaluated by cell counting. All HDAC inhibitors were identified as potent inhibitors of erythroleukemic cell proliferation. However, we observed a complex pattern of differentiation induction: structurally similar inhibitors resulted in disparate activity profiles, whereas similar profiles were detected within distinct structural classes. Among the newly synthesized biarylalanine compounds, a 3 0 -methoxy derivative was identified as a very effective inducer of terminal cell differentiation. We conclude that investigation of subtype selectivity of selected HDAC inhibitors does not provide a clear link between selectivity and the observed cellular activity profile. The predictive value of in vitro HDAC inhibition assays for identifying anti-proliferative compounds has been emphasized.