Research paper
Synthesis and biological evaluation of novel FK228 analogues as potential isoform selective HDAC inhibitors

https://doi.org/10.1016/j.ejmech.2016.05.031Get rights and content

Highlights

  • Eight new FK228 analogues were synthesized.

  • These analogues were evaluated against HDACs and 39 human cancer cell lines.

  • Some of these analogues exhibited very high HDAC1 (class I) isoform selectivity.

  • Potent and highly isoform-selective HDAC1 inhibitors were identified.

  • Novel aspects of SAR were revealed.

Abstract

Novel C4- and C7-modified FK228 analogues were efficiently synthesized in a highly convergent and unified manner. This synthesis features the amide condensation of glycine-d-cysteine-containing segments with d-valine-containing segments for the direct assembly of the corresponding seco-acids, which are key precursors of macrolactones. The HDAC inhibition assay and cell-growth inhibition analysis of the synthesized analogues revealed novel aspects of their structure-activity relationship. This study demonstrated that simple modification at the C4 and C7 side chains in FK228 is effective for improving both HDAC inhibitory activity and isoform selectivity; moreover, potent and highly isoform-selective class I HDAC1 inhibitors were identified.

Introduction

The reversible acetylation and deacetylation of histones plays an important role for regulating gene expression by altering chromatin architecture [1]. Histone acetyltransferases (HATs) catalyse the transfer of acetyl groups from acetyl-CoA to the ε-amino groups on histone lysine residues, which relaxes the chromatin structure and epigenetically promotes gene transcription. Conversely, histone deacetylases (HDACs) catalyse the removal of acetyl groups from the N-acetyl lysine residues on histones, which contracts the chromatin structure and epigenetically suppresses gene transcription. The inhibition of HDAC enzymatic activity has been demonstrated to affect transcriptional events that are involved in growth arrest, differentiation, proliferation, cell cycle regulation, protein turnover and/or apoptosis in transformed tumour cell cultures. Consequently, HDAC-inhibiting compounds are considered to be potential drugs for targeted cancer therapy [2].

There are 18 human HDAC isoforms, which are grouped into four major classes: class I (HDACs 1, 2, 3 and 8), class II (class IIa: HDACs 4, 5, 7 and 9; class IIb: HDACs 6 and 10), and class IV (HDAC 11) are Zn2+-dependent metallohydrolases, whereas class III HDACs (SirTs 1–7) are NAD+-dependent sirtuins [3]. Inhibiting class I HDACs is considered to be an effective mechanism for anticancer agents, whereas inhibiting class II HDACs may cause undesirable side effects such as serious cardiac hypertrophy [4]; therefore, in cancer chemotherapy, the potent and selective inhibition of class I HDACs is highly desirable.

In 2009, the U.S. Food and Drug Administration (FDA) approved the bicyclic depsipeptide HDAC inhibitor FK228 (romidepsin, 1, Fig. 1) for treating cutaneous T-cell lymphoma and other peripheral T-cell lymphomas [5], [5](a), [5](b); however, FK228 is known to be associated with an unresolved cardiotoxicity issue [5](b), [6], which highlights the requirement for further identifying and developing new HDAC inhibitors with high efficacy and low toxicity. FK228 was first discovered by researchers from Fujisawa Pharmaceutical Co. Ltd. (now Astellas Pharma Inc.) in 1994, who isolated it from a culture broth of Chromobacterium violaceum (No. 968) [7]. Yoshida et al. proposed a molecular mechanism by which FK228 inhibits HDAC [8]. In that mechanism, FK228, which itself serves as a stable prodrug, is activated by reductive cleavage of the disulfide bond after incorporation into the cells, and the released sulfhydryl group in the butenyl side chain interacts with the zinc cation located at the binding pocket of the HDACs, resulting in a potent inhibitory effect. The structural features of FK228 are a 16-membered bicyclic depsipeptide comprising dehydrobutyrine (Dhb), l-valine, d-cysteine, d-valine, (3S,4E)-3-hydroxy-7-mercapto-4-heptenoic acid (Hmh) and a characteristic disulfide bond linkage. Since the discovery of FK228, several structurally similar bicyclic depsipeptide HDAC inhibitors have been isolated from bacterial fermentations, including spiruchostatins A–D (25) [9], FR901375 (6) [10], burkholdacs A (7) and B (8) [11] and thailandepsins A–F (712) [12] (thailandepsins A and C are identical to burkholdacs B and A, respectively). The potentially therapeutic biological properties and unique structural features of 112 have made them attractive targets for total synthesis, and several such syntheses of these natural products have been published in the literature [13], [14], [15], [16], [17], [18], [19].

During our research on the synthesis and biological evaluation of bicyclic depsipeptide HDAC inhibitors such as 15, 7 and 8 [13](a), [14](c), [15](b), [16], [18](a), we became interested in synthesizing FK228 analogues and evaluating their isoform selectivity in order to identify the potent and class I-selective HDAC inhibitors. There are several studies in the literature on synthesizing FK228 analogues [20]. Note that the synthesis and evaluation of FK228 analogues, which was reported by Ganesan et al., revealed novel aspects of their structure–activity relationship (SAR) [20c]. From this SAR study, three primary critical features that were related to the HDAC inhibitory activity of 1 were identified; i) the 16-membered macrocyclic scaffold is indispensable, ii) the unsaturated Dhb moiety can be replaced with other amino acids and iii) the C4 isopropyl group in the l-valine moiety is not always necessary. Although these results are very useful for designing novel FK228 analogues, no information on the effects of the stereochemistry in the amino acid components was reported.

Based on the results presented by Ganesan et al., we designed eight novel C4- and C7-modified FK228 analogues, which were denoted as FK-A1 to FK-A8 (13ah, Fig. 2). Note that the l-valine and Dhb moieties of 1 were replaced by simple amino acids such as glycine, d-/l-alanine, d-/l-phenylalanine and d-/l-leucine. We envisaged that 13ah could be readily synthesized via the total synthesis we had previously developed for bicyclic depsipeptide natural products.

In this study, we describe the synthesis of FK228 analogues 13ah using a synthetic strategy that was developed previously in our laboratory. Then, to investigate the effect of the C7 stereochemistry and amino acid substituent groups, these synthesized analogues were subjected to an HDAC inhibition assay and antiproliferative analysis.

Section snippets

General synthetic strategy for the novel FK228 analogues FK-A1 to FK-A8 (13ah)

The selection of the method that was used for constructing the 16-membered macrocyclic ring is crucial to successfully achieve the total synthesis of this class of natural products. In our previously reported total syntheses of FK228 (1) [13a], spiruchostatins A–D (25) [14](c), [15](b), [16] and burkholdacs A (7) and B (8) [18a], we employed two different reliable methods such as Shiina’s macrocyclization [21] and Mitsunobu macrocyclization [22]. For synthesizing 25, 7 and 8, we employed

Conclusion

We synthesized eight new FK228 analogues, i.e. FK-A1 to FK-A8 (13af), in a highly convergent and unified manner. The present preliminary biological evaluation revealed novel aspects of the SAR for this class of depsipeptide HDAC inhibitor. In particular, replacement of the C4 isopropyl and C7 ethylidene substituents of FK228 by a hydrogen atom and a d-amino acid side chain, respectively (13c, 13e and 13g), was quite effective for improving HDAC1 inhibitory activity and isoform selectivity.

Chemistry

All reactions involving air- and moisture-sensitive reagents were carried out using oven dried glassware and standard syringe-septum cap techniques. Routine monitorings of reaction were carried out using glass-supported Merck silica gel 60 F254 TLC plates. Flash column chromatography was performed on Kanto Chemical Silica Gel 60N (spherical, neutral 40–50 nm) with the solvents indicated.

All solvents and reagents were used as supplied with following exceptions. Tetrahydrofuran (THF) was freshly

Acknowledgements

We are grateful to the Screening Committee of New Anticancer Agents, which is supported by a Grant-in-Aid for Scientific Research onInnovative Area ‘Scientific Support Programs for Cancer Research (No. 221S0001, 2010–2014)’ from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), for assistance with the biological evaluation of compounds 1 and 13ah. This study was also supported by a Grant-in-Aid for the Strategic Research Foundation Program at Private Universities

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