Depsipeptide HDAC inhibitors

Total Synthesis of Depsipeptide HDAC Inhibitors - Routes to the fi-Hydroxy Acid Fragment... 

Despite the overall similarities between the effects of hydroxamic acid and depsipeptide HDAC inhibitors on cancer cells, we have identified some important class-specific effects (in addition to the selective induction of a-tubulin acetylation). Importantly, the kinetics of inhibition of cellular HDACs by these inhibitors varies widely. While hydroxamic acids induce rapid histone acetylation in intact cells, the onset of action of the depsipeptide inhibitors is much slower (Fig. 12-10). Also, following removal of compound by extensive washing, histone acetylation is rapidly lost in hydroxamic acid treated cells, but is maintained for protracted periods in cells treated with depsipeptide inhibitors. 

Depsipeptide HDAC inhibitor FTC133 and 236, follicular cancer cell lines X X ... 

Figure 4.16 Examples of the FK228 family of depsipeptide HDAC inhibitors. Enzyme inhihition values are given for the free thiol.

There have been many total syntheses of the depsipeptide HDAC inhibitors by academic groups. In the first, by Simon, macrolactonization of the linear seco-hydroxy acid 41 (Scheme 4.3) to the FK228 precursor 43 proved to be challenging under the usual conditions of carboxylic acid activation due to competing elimination of the allylic alcohol. Instead, the cyclization was accomplished by alcohol inversion under Mitsunobu conditions using large... 

The total syntheses and SAR studies, notably by Ganesan with FK228 and Williams with largazole, have identified the key features within depsipeptide HDAC inhibitors that are required for HDAC inhibition (Figure 4.17). The macrocyclic backbone is essential as linear uncyclized compounds are inactive. Within the macrocycle, the zinc binding thiol warhead needs to be of D-stereochemistry and the epimer is virtually inactive, while the other amino acid residues present can be varied without loss in activity. The... 

One of the first HDAC inhibitors to be identified and characterized was sodium butyrate, where it was found to alter the histone acetylation state (Riggs et al, 1977), and further determined to inhibit HDAC activity both in vitro and in vivo (Candido et al, 1978). Almost a decade later trichostatin A (TSA), a fungistatic antibiotic, was found to induce murine erythroleukemia cell differentiation (Yoshida et al, 1987). To date, a wide range of molecules have been described that inhibit the activity of Class I and Class II HDAC enzymes, and with a few exceptions, can be divided into structural classes including (1) small-molecule hydroxamates, such as TSA, suberoylanilide hydroxamic acid (SAHA), scriptaid and oxamflatin (2) short-chain fatty-acids, such as sodium butyrate, sodium phenylbutyrate and valproic acid (VPA) (3) cyclic tetrapeptides, such as apicidin, trapoxin and the depsipeptide FK-228 and (4) benzamides, such as MS-275 and Cl-994 (for reviews see Remiszewski et al, 2002 Miller et al, 2003). Some of these molecules are represented in Fig. 4. 

Konstantinopoulos PA, Vandoros GP, Papavassihou AG (2006) FK228 (depsipeptide) A HDAC inhibitor with pleiotropic antitumor activities. Cancer Chemother Pharmacol 58 711-715. 

The marine sponge-derived HDAC inhibitors can be classified as cyclic peptides or bicyclic depsipeptides. 

This HDAC inhibitor (Fig. 15) was isolated from a cyanobacterium Symplaca sp. collected from the Florida Keys. This depsipeptide was described by Leusch and coworkers in lOOSP It has been shown to be potent at low nanomolar ranges against mammary cancer cell lines MDA-MB-231, HT29 colon cancer cells, IMR-32 neuroblastoma cells and U20S fibroblastic osteosarcoma cells. ... 

To achieve selectivity in a classical metal-binding HDAC inhibitor, the cap needs to contain functionality for additional interactions with the rim . Of the inhibitors described above, the cyclic tetrapeptides have this potential due to their large macrocyclic scaffold, but have yet to result in clinical candidates. Structurally, the most complex HDAC inhibitors are the depsipeptide natural products exemplified by FK228. These compounds, which are treated separately in the next section, have even more elaborate caps , and are the best-documented example of selective HDAC inhibitors. 

The depsipeptide FK228 (originally called FR901,228) was isolated [28] by Fujisawa Pharmaceuticals from an extract of the bacteria Chromobacterium violaceum No. 968 on the basis of an assay for phenotypic reversal of ras-transformed tumor cells. The compound was shown to be active in a tumor xenograft animal model, and to have effects [29] similar to the known HDAC inhibitors, trichostatin A and trapoxin. Superficially, FK228 (Fig. 12-7) does not... 

Another patent by Fujisawa disclosed [31] the structure of FR-901375 from an extract of Pseudomonas chloroaphis No. 2522. While it is a likely H DAC inhibitor, no data have been reported in this regard and the decision seems to have been made to promote FK228 instead as the clinical candidate. In 2001, additional depsipeptide natural products, the spiruchostatins, were reported [32] by Shin-ya s group at the University of Tokyo and Yamanouchi Pharmaceuticals. These compounds were isolated from an extract of Pseudomonas sp. Q71576, on the basis of the ability to increase expression of luciferase driven by the plasminogen activator inhibitor (PAI-1) promoter. Given the structural similarity to FK228, the spiruchostatins were likely to be HDAC inhibitors and this was confirmed in a later patent [33] and in our biochemical studies (see below) with the natural product prepared by total synthesis. 

Compared to other classes of HDAC inhibitors, the depsipeptides exhibit two impressive features. Firstly, they are highly potent with IC50S in the low nanomolar range. Secondly, they are significantly more active against class I HDACs compared to class II HDACs. Fortuitously, it is the former that are more heavily implicated in cancer and cardiac hypertrophy. On the other hand, the depsipeptides are structurally the most complicated class of HDAC inhibitors. Their elaborate framework has apparently deterred the pharmaceutical industry from the preparation of unnatural analogs and the iterative improvement of their properties. The Fujisawa and Yamanouchi patents only cover the natural products and so far only academic groups have described the total synthesis of depsipeptides. 

We have also compared the activities of depsipeptide and hydroxamic acid HDAC inhibitors on cellular responses of malignant and normal cells. When tested at equipotent concentrations, these inhibitors have remarkably similar effects, inducing essentially identical levels of histone acetylation and p21clP1/wafl protein expression. However, only SAHA and TSA induced robust a-tubulin acetylation. This is consistent with previous findings [42] that... 

PDB code 1T69) (b) outline catalytic mechanism of hydrolytic lysine deacetylation by Class I, II and IV HDACs (c) representative HDAC inhibitor chemotypes, including two clinically approved inhibitors, the hydroxamic acid SAHA/Vorinostat, and the depsipeptide FK228/ Romidepsin. 

After the cyclic tetrapeptides, the bicyclic depsipeptides were the second class of macrocyclic natural product HDAC inhibitor to be discovered. The first and foremost example is FK228 (36, Figure 4.14), discovered by high-throughput screening at Fujisawa using a phenotypic assay for compounds with the ability to revert the morphology of ras-transformed cancer cells and... 

After 5 years in the FDA s fast track development program, Romidepsin (Fig. 18) was approved by the FDA for refractory cutaneous T-cell lymphoma on November 6,2009. In the literature, romidepsin has also been called depsipeptide, FK228, FR901228, and NSC-630176. It was isolated from bacterial fermentation extracts from Chromobacterium violaceum and is a potent inhibitor of HDAC. In some human cancer cell lines, romidepsin inhibits HDACs at levels ten times that of TSA. 

Table 12-2 Inhibition values of depsipeptides against HDAC and HDAC6. For comparison, the values with the nonselective inhibitor trichostatin A are shown...

These findings clearly demonstrate that the relative selectivity of depsipeptide inhibitors for class I enzymes does not limit their anticancer activity, at least in vitro, and confirm that inhibition of HDAC6 is not required for the effects [46] of HD AC inhibitors on cell-cycle arrest and gene expression. Such observations are consistent with the predominant expression of class I HDACs in malignant cells, and the correlation between expression of these enzymes and outcome in malignancies. 

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