Carbonyl- and sulfur-containing analogs of suberoylanilide hydroxamic acid: Potent inhibition of histone deacetylases

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Abstract

Suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylase, is used in clinical trials for a variety of advanced cancers. Twelve new analogs of SAHA were synthesized and tested as in vitro inhibitors of isolated histone deacetylases (HDACS) and in vivo inhibitors of interferon regulated transcriptional responses (a marker for HDAC activity). The analogs containing an α-mercaptoketone or an α-thioacetoxyketone were more potent than SAHA in both assays.

Introduction

Epigenetic regulation of gene expression is mediated by several mechanisms, including DNA methylation, ATP-dependent chromatin remodeling, and post-translational modifications of histones, such as methylation, phosphorylation, ubiquitinylation, or acetylation. Dynamic acetylation of ε-amino groups of lysine residues on core histone tails is regulated by two opposing enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC).1 Transcriptional regulation is controlled by HAT enzymes, which add acetyl groups to lysine residues of histones and non-histone proteins, and HDACs, which remove these modifications, thereby mediating chromatin remodeling and gene expression.2 Pharmacological HDAC inhibitors comprise a novel class of cancer chemotherapeutics in clinical development that target HDAC enzymatic activity thereby inducing hyperacetylation of HDAC substrates, which results in altered chromatin structure and gene expression, and has been shown to induce growth arrest, cell differentiation, and apoptosis of tumor cells.3 Furthermore, they suppress cell proliferation in a variety of transformed cells in culture and in tumor, bearing animals, and have shown great promise as new anticancer drugs.4 Several structurally diverse HDAC inhibitors now in clinical trials have demonstrated encouraging antitumor activity in a variety of cancer types, enhancing the rationale for continued development of new HDAC inhibitors.5

Suberoylanilide hydroxamic acid (SAHA) (Fig. 1) is one of the early HDAC inhibitors,6 which inhibits cell growth, induces terminal differentiation in tumor cells,7 prevents the formation of malignant tumors in mice,8 and is currently in phase III clinical trials.9 Crystal structures of a hyperthermophilic bacterium HDAC10 and of human HDAC811 with SAHA bound show that the hydroxamic acid moiety coordinates to the active-site zinc ion. Hydroxamates, however, have been found to exhibit unfavorable pharmacokinetic behavior resulting from glucuronidation and sulfation,12 and from metabolic hydrolysis,13 all of which result in short in vivo half-lives of the hydroxamic acid group. Consequently, new potent inhibitors have been sought. Non-hydroxamate HDAC inhibitors discovered to date, however, have reduced potency compared to hydroxamates and have not shown any metabolic advantage.14

Although hydroxamates bind to zinc ions, they are not necessarily the optimal coordinators. Sulfur ligands are well known to bind tightly to zinc-containing enzymes.15 This was clearly demonstrated by the invention of captopril, a thiol-containing inhibitor of the zinc-dependent enzyme angiotensin-converting enzyme (ACE).16

We have designed a series of SAHA analogs as potential inhibitors of HDACs, in which the hydroxamic acid was replaced by sulfur-containing moieties, which may have enhanced binding affinities for zinc. The optimal ligand from this series can be utilized as the preferred ligand for other HDAC inhibitor compounds.

Section snippets

Chemistry

The hydroxamate group of SAHA was separated into two moieties: A and B, as shown in Figure 1, and a 12-membered library of SAHA analogs was designed (Fig. 2). The type of carbonyl moiety (A) was compared (Fig. 2, 1 vs 2, 3, 6,and 11). To compare the effectiveness of a sulfur atom versus an oxygen atom, compounds in which a sulfur or oxygen at the same position were synthesized (Fig. 2, 1 vs 2; 3 vs 4 and 5; 6 vs 7; 12 vs 13). The group at B was investigated as well (Fig. 2, 4 vs 9; 7 vs 8 and 10

Conclusions

An α-mercaptoketone and α-thioacetoxylketone are improved zinc-binding ligands relative to hydroxamate in this study and should be valuable groups to attach to molecules that target not only histone deacetylase, but other zinc-dependent enzymes.

Experimental

Conventional organic solvents were purchased from Fisher. All of the reagents were purchased from Aldrich Chemical Co. and were used without further purification unless stated otherwise. Methylene chloride was distilled under N2 from calcium hydride. Flash chromatography was performed with Merck silica gel (230–400 mesh). TLC plates (silica gel 60-F254) were purchased from VWR Scientific. All 1H NMR spectra were recorded on Varian Gemini 300 MHz, Mercury 400, or Inova 500 spectrometers (75, 100,

Acknowledgments

The authors are grateful to the National Institutes of Health for financial support of this research (Grant R41 CA116971 to R.B.S. and R01 GM063872 to C.M.H.).

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