DOI: 10.1002/cmdc.201100531 Design, Synthesis, and Biological Activity of Hydroxamic Tertiary Amines as Histone Deacetylase Inhibitors Stefania Terracciano, Maria Giovanna Chini, Raffaele Riccio, Ines Bruno,* and Giuseppe Bifulco* [a] Introduction Histone deacetylases (HDACs) are a superfamily of proteins that catalyze one of the most important posttranslational modifications: the removal of acetyl groups from lysine resi- dues of nucleosomal cores. Eukaryotic HDACs are grouped into four classes: Classes I, II and IV (zinc-dependent proteins), and Class III, human sirtuin (SIRT; NAD + -dependent enzymes). [1] Class I HDACs include the nuclear isoforms 1, 2, 3, and 8. Mem- bers of Class II are subdivided into Class IIA (HDACs 4, 5, 7, and 9) and Class IIB (HDACs 6 and 10), and are localized in the nu- cleus and cytoplasm. Class IV includes only the nuclear isoform HDAC11. Under physiological conditions, HDACs [2] regulate a number of cellular functions essential for homeostasis, chroma- tin remodeling, transcriptional regulation, [3] intracellular trans- port, [4] metabolism, and cell proliferation. Histone acetylation status is also involved in the development of certain forms of malignancy, [5] controlling the abnormal activation of onco- genes and deactivating the transcription of tumor-suppressor genes. [6, 7] Therefore, over the past two decades HDAC inhibi- tors (HDACi) have been considered as promising anticancer agents. [8] They have been divided into four structural groups: [9] short-chain fatty acids, hydroxamic acids, benzamides, and cyclic tetrapeptides. Although a large number of HDACi have been obtained from both natural sources and through chemi- cal synthesis, and despite the fact that some are in clinical trials—such as valproic acid, [10] PXD101 (belinostat), [11] and MS- 275, [12] —there are only two HDAC inhibitors currently on the market: vorinostat (Zolinza) and the depsipeptide FK228 (romi- depsin, Istodax). [13] Both of these were approved by the US Food and Drug Administration (FDA) for the treatment of cuta- neous T-cell lymphoma (CTCL). [14] As part of our ongoing extensive investigations on the mechanism of HDAC inhibition by natural and synthetic prod- ucts, [15] and with the goal of developing new enzyme inhibitors as attractive drug candidates against tumor-related diseases, we investigated HDAC inhibition by a small set of hydroxamic tertiary amines. Based on our previous research, we decided to expand the chemical diversity of our collection of new mono- and bis-amides that show satisfactory levels of antiproliferative activity, thus emerging as effective HDAC inhibitors. [15e] Specifi- cally, we explored the efficiency of non-peptidic linear com- pounds equipped with a hydroxamic group as metal binder and two variously substituted arms as the cap group. Herein we report docking studies, synthesis, and biological evaluation of this new generation of hydroxamic tertiary amines. Results and Discussion Docking studies We designed a small set of hydroxamic tertiary amines, re- specting the well-established [16] structural features of a stan- dard HDAC inhibitor: 1) a hydrophobic region (cap group) in- volved in molecular recognition, 2) a Zn 2 + chelating element (metal binder), and 3) a five- to seven-atom spacer (linker) be- tween the cap group and the metal binder. We conjugated the same linker chain, presenting the hydroxamic group, with vari- ous aromatic moieties to optimize the size and physicochemi- cal properties of the cap groups (compounds 1–10, Figure 1). For the modeling studies we used the AutoDock 3.0.5 soft- ware package [17] and histone deacetylase-like protein (HDLP, PDB ID: 1C3R; [18] Figure 2) as the model enzyme, which has been used successfully in the interpretation of the inhibitory activities of several HDAC ligands. [15, 19] In particular, for the docking calculations we used the HDLP model previously opti- mized at the quantum mechanical level by our research Herein we describe the design, synthesis, and biological evalu- ation of new hydroxamic tertiary amines as histone deacety- lase (HDAC) inhibitors. These compounds have allowed us to clarify the influence of cap group dimension and hydrophobici- ty on HDAC inhibitory activity. This report also reveals the rec- ognition pattern between the linear compounds and the his- tone deacetylase-like protein (HDLP) model receptor, and dis- cusses the synthesis and in vitro evaluation of HDAC inhibitory activity in HeLa cell nuclear extracts. We obtained good quali- tative agreement between experimental results and theoretical predictions, confirming that appropriately substituted hydroxa- mic tertiary amines are potential active HDAC inhibitors. [a] Dr. S. Terracciano, Dr. M. G. Chini, Prof. R. Riccio, Prof. I. Bruno, Prof. G. Bifulco Dipartimento di Scienze Farmaceutiche e Biomediche Università degli Studi di Salerno Via Ponte Don Melillo, 84084 Fisciano (SA) (Italy) E-mail : brunoin@unisa.it bifulco@unisa.it 694  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2012, 7, 694 – 702 MED