2-Trifluoroacetylthiophenes, a novel series of potent and selective class II histone deacetylase inhibitors Philip Jones, * Matthew J. Bottomley, Andrea Carfı ´, Ottavia Cecchetti, Federica Ferrigno, Paola Lo Surdo, Jesus M. Ontoria, Michael Rowley, Rita Scarpelli, Carsten Schultz-Fademrecht and Christian Steinku ¨ hler IRBM/Merck Research Laboratories, Via Pontina km 30,600, 00040 Pomezia, Italy Received 18 November 2007; revised 7 February 2008; accepted 9 February 2008 Available online 14 February 2008 Abstract—The identification of class II HDAC inhibitors has been hampered by lack of efficient enzyme assays, in the preceding paper two assays have been developed to improve the efficiency of these enzymes: mutating an active site histidine to tyrosine, or by the use of a trifluoroacetamide lysine substrate, allowing screening to identify class II HDAC inhibitors. Herein, 2-trifluoro- acetylthiophenes have been demonstrated to inhibit class II HDACs, resulting in the development of a series of 5-(trifluoroace- tyl)thiophene-2-carboxamides as novel, potent and selective class II HDAC inhibitors. X-ray crystal structures of the HDAC 4 catalytic domain with a bound inhibitor demonstrate these compounds are active site inhibitors and bind in their hydrated form. Ó 2008 Elsevier Ltd. All rights reserved. The role of two counteracting enzyme families, the his- tone acetyl transferases (HATs) and the histone deacet- ylases (HDACs) in controlling the post-transcriptional acetylation status of lysine residues on histone tails, and a number of other proteins, is well-documented. 1,2 This equilibrium is a crucial determinant of chromatin structure, and hence gene transcription. The HDAC enzyme family has been implicated in a diverse array of processes, including: neoplasias, skeletal and muscle formation, cardiac hypertrophy, T-cell differentiation and neuronal survival. 2,3 However, the role of each of the individual HDAC isoforms or indeed classes in each of these biological processes is still being established. 3,4 Consequently, there is an urgent need for selective small molecule HDAC inhibitors (HDACi), both as research tools and ultimately as therapeutic agents. The HDAC enzyme family can be divided into two clas- ses: class I (HDACs 1, 2, 3 + 8), and class II, which can be further subdivided into class IIa (HDACs 4, 5, 7 + 9) and class IIb (HDACs 6 + 10). The class II HDACs differ from their class I counterparts in the fact that in addition to their catalytic domain, they contain a long N-terminal regulatory domain, 3–5 which has been impli- cated in gene regulation through protein–protein inter- actions. 3,5,6 The sub-division of the class II HDACs is the result of the presence of a second known or putative catalytic domain within class IIb HDACs. Aside from HDAC6 which has been demonstrated to function as a tubulin deacetylase, 7 there has been an extensive debate in the literature as to whether other class II HDACs, in particular class IIa enzymes, possess any intrinsic deace- tylase activity. 5,6 In the preceding paper we have shown that pure class IIa HDACs do possess weak but measur- able deacetylase activity, 8,9 and this activity can be enhanced and measured either by an H-Y mutation in the active site to give a ‘gain of function’ (GOF) enzyme, or by use of the wild-type (WT) enzyme with an ‘unnat- ural’ trifluoroacetamide lysine substrate. With this tool- box a program was initiated to identify selective class II HDAC inhibitors, targeting in particular HDAC4. A focused screen of both in-house and commercial derivatives containing known zinc binding groups was initially carried out on both HDAC4 GOF 10 and 4WT. 11 Specific interest was given to trifluoromethyl ke- tone derivatives given that trifluoroacetamide lysine was a substrate for HDAC4WT, and that alkyl trifluoro- methyl ketones had already been shown to inhibit both HDAC4 assays in the tens of nanomolar range. 9 These 0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.02.026 Keywords: Histone deacetylase; HDAC; Trifluoroacetyl ketones; Hydrated ketones. * Corresponding author. Tel.: +39 0691093559; fax: +39 0691093654; e-mail: philip_jones@merck.com Available online at www.sciencedirect.com Bioorganic & Medicinal Chemistry Letters 18 (2008) 3456–3461