Thioester derivatives of the natural product psammaplin A as potent histone deacetylase inhibitors

Article abstract of DOI:10.3762/bjoc.9.11

There has been significant interest in the bioactivity of the natural product psammaplin A, most recently as a potent and isoform selective HDAC inhibitor. Here we report our preliminary studies on thioester HDAC inhibitors derived from the active monomeric (thiol) form of psammaplin A, as a means to improve compound delivery into cells. We have discovered that such compounds exhibit both potent cytotoxicity and enzymatic inhibitory activity against recombinant HDAC1. The latter effect is surprising since previous SAR suggested that modification of the thiol functionality should detrimentally affect HDAC potency. We therefore also report our preliminary studies on the mechanism of action of this observed effect.

Full text of DOI:10.3762/bjoc.9.11

Thioester derivatives of the natural product
psammaplin A as potent histone
deacetylase inhibitors
Matthias G. J. Baud1, Thomas Leiser2, Vanessa Petrucci3,
Mekala Gunaratnam3, Stephen Neidle3, Franz-Josef Meyer-Almes2
and Matthew J. Fuchter*1
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 81–88.
1Department of Chemistry, Imperial College London, London SW7
2AZ, United Kingdom, 2Department of Chemical Engineering and
Biotechnology, University of Applied Sciences, Schnittspahnstraβe
12, 64287 Darmstadt, Germany and 3Cancer Research UK
Biomolecular Structure Group, UCL School of Pharmacy, 29-39
Brunswick Square, London WC1N 1AX, United Kingdom
doi:10.3762/bjoc.9.11
Received: 29 September 2012
Accepted: 11 December 2012
Published: 15 January 2013
This article is part of the Thematic Series "Synthetic probes for the study
of biological function".
Email:
Matthew J. Fuchter* - m.fuchter@imperial.ac.uk
Guest Editor: J. Aubé
* Corresponding author
© 2013 Baud et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
epigenetics; histone deacetylase; natural product; prodrug;
psammaplin A; thioester
Abstract
There has been significant interest in the bioactivity of the natural product psammaplin A, most recently as a potent and isoform
selective HDAC inhibitor. Here we report our preliminary studies on thioester HDAC inhibitors derived from the active monomeric
(thiol) form of psammaplin A, as a means to improve compound delivery into cells. We have discovered that such compounds ex-
hibit both potent cytotoxicity and enzymatic inhibitory activity against recombinant HDAC1. The latter effect is surprising since
previous SAR suggested that modification of the thiol functionality should detrimentally affect HDAC potency. We therefore also
report our preliminary studies on the mechanism of action of this observed effect.
Introduction
Chromatin is a macromolecular complex consisting of DNA, [1,2]. In recent years, it has become increasingly apparent that
histone and nonhistone proteins. The epigenetic control of chro- misregulation of epigenetic pathways contributes to oncogen-
matin organization plays a major role in the regulation of gene esis [3,4] and small molecule inhibitors of these pathways have
expression, and consequently cell differentiation, proliferation emerged as highly attractive targets for anticancer therapies
and survival. Such control is mediated by a myriad of remodel- [5,6]. Inhibitors of epigenetic pathways should not only be
ling proteins, able to bind to, and covalently modify, chromatin useful as anticancer drugs, but also as molecular probes to study
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Beilstein J. Org. Chem. 2013, 9, 81–88.
the causative relationships between specific epigenetic modifi-
cations, their biological outcomes, and how their misregulation
is involved in diseases such as cancer [1,2].
The dynamic post-translational acetylation/deacetylation of
histone proteins is one of the most commonly studied epige-
netic events, and occurs at specific lysine residues on the
N-terminal histone tails, which project out from the nucleo-
some (the fundamental repeating unit of chromatin). Acetyla-
tion/deacetylation of such lysine residues is achieved by the
action of histone acetyltransferases (HATs) and histone
deacetylases (HDACs), respectively. Histone deacetylation by
HDACs causes transcriptional repression through both chro-
matin condensation and chromatin signalling. To date, 18
human genes encoding proven or putative HDACs have been
identified [7]. HDACs fall into two categories: the zinc-depen-
dent enzymes (class I, II and IV) and the NAD+-dependent
Figure 1: FDA approved HDAC inhibitors for the treatment of CTCL.
enzymes (class III, also called sirtuins) [5]. Class I HDACs further discovery of structurally novel HDAC inhibitors that, in
(HDAC1, 2, 3, 8) are mostly present in the nucleus, whereas particular, exhibit improved isoform selectivity.
class II HDACs are tissue specific and shuttle between the cyto-
plasm and the nucleus [8,9]. Class II can be further subdivided Among the myriad of previously reported HDAC inhibitors,
into class IIa (HDAC4, 5, 7, 9) and class IIb (HDAC6, 10). psammaplin A [15-18] (3, X = OH, Scheme 1, left) displays an
HDAC11 constitutes its own class IV. Despite their name, intriguing structure. It is a symmetric, dimeric hydroxyiminoty-
several HDACs are able to deacetylate a number of nonhistone rosine-based natural product, characterised in 1987, and repre-
protein substrates [10,11]. Sirtuins are structurally and mecha- sents the first example of a disulfide and oxime containing
nistically distinct enzymes.
metabolite isolated from a marine sponge. Since its initial report
by Crews and co-workers as a potent HDAC inhibitor [16],
To date, only two compounds that inhibit HDACs have been psammaplin A has provided inspiration for the development of
FDA approved: suberoylanilide hydroxamic acid (SAHA, 1, new HDAC inhibitors with novel structures [19]. Recently, we
trade name Zolinza by Merck & Co.) and romidepsin 2 (trade [20] and others [21] reported an in-depth structure–activity rela-
name Istodax by Celgene) for the treatment of cutaneous T-cell tionship of this natural product against its HDAC targets.
lymphoma (CTCL, Figure 1) [12-14]. The success of these Dissection of its activity against a panel of HDACs allowed us
compounds in the clinic has led to a significant interest in the to highlight structural features responsible for its high inhibitory
Scheme 1: SAR of psammaplin A against zinc-dependant HDACs. Adapted from Baud et al. [20].
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Beilstein J. Org. Chem. 2013, 9, 81–88.
potency and selectivity. In particular, we unambiguously higher than the potencies of their corresponding dimeric disul-
demonstrated that, similarly to the natural product and clini- fide analogues, and this was thought to reflect the rate of
cally approved romidepsin, psammaplin A is a prodrug, thioester hydrolysis versus the rate of disulfide reduction. We
requiring reduction of its disulfide functionality to the corres- therefore commenced a study to prepare thioester derivatives of
ponding thiol monomer 4 (X = OH), in order to potently inhibit our psammaplin A analogues to investigate whether this would
HDACs (Scheme 1, right). The resulting thiol moiety acts as a be an effective strategy to optimise these potent and selective
zinc binding group within the active site of the HDAC protein. HDAC inhibitors.
Furthermore, we demonstrated the importance of the oxime unit
Results and Discussion
of psammaplin A and related analogues for high potency and
Synthesis of acetate-protected thiol
analogues of psammaplin A
selectivity against recombinant HDAC1 (rHDAC1) in vitro
(Scheme 1). More recently, we disclosed highly potent hetero-
cyclic N-thioethylamide-based HDAC inhibitors based on the
psammaplin A pharmacophore and rationalised the results using
computational modelling [22].
We designed and synthesised several acetate-protected
psammaplin A analogues. The structures of our thioester-based
probes are shown in Scheme 2. We recently demonstrated [20]
that variation of the aromatic substitution pattern had only a
marginal influence on the HDAC inhibitory potency of
psammaplin A analogues in vitro. Therefore, the native phenol
was replaced by its methylated homologue for ease of synthesis,
notably to avoid side reactions during the carbodiimide-medi-
ated coupling step, whereby we had previously found the
phenol to act as a competitive nucleophile. Since we had previ-
ously demonstrated the importance of the oxime (Scheme 1) for
HDAC potency and selectivity, we prepared probes with these
different functionalities in order to allow comparison of our
data to our previously generated in vitro and in cell SAR data
[20].
While prereduced psammaplin A and thiol-containing
analogues displayed nanomolar to subnanomolar potencies in
vitro, they only displayed modest potencies in cell-based assays
against A549 (human lung carcinoma), MCF7 (human breast
carcinoma) and WI38 (normal human lung fibroblast) cell lines.
We attributed this to the low permeability and/or stability of the
free thiol in cells. While the use of nonreduced disulfide func-
tionality (e.g., present in the parental psammaplin A (3),
X = OH) is one strategy to “protect” the free thiol and allow for
its effective dosing into cells, this prodrug strategy is reliant on
intracellular reduction of the disulfide to the active thiol form.
As such, cellular potency would be expected to correlate signifi-
cantly with the cellular levels of reductants such as glutathione Condensation between acid 5 and O-methylhydroxylamine, fol-
[23]. An alternative prodrug approach to “protect” the thiol lowed by EDC coupling with S-2-aminoethyl ethanethioate 6
active form of psammaplin A analogues would be to form the [26] afforded thioacetate analogue 7 (Scheme 2). Condensation
corresponding thioester; the active thiol being generated in cells of acid 5 with hydroxylamine, followed by coupling with 6
after cleavage of the acyl group by nonselective esterases. In using DCC and N-hydroxyphthalimide [27] as coupling
support of this approach, Miyata and co-workers synthesised a reagents afforded thioacetate 8. The latter conditions were also
number of SAHA-derived thioesters during their research of applied to 5 and afforded thioacetate analogue 9. The isolated
nonhydroxamate inhibitors of HDACs, which exhibited yields for products 8 and 9 were unoptimised, and our previous
moderate to high potency in enzymatic and cell-based assays work suggests that these could be improved with further refine-
[24,25]. Interestingly, the potencies of their compounds were ment of the reaction conditions [28].
Scheme 2: Synthesis of 7–9. Conditions: (i) HCl·H2NOMe, pyridine, rt, 12 h; (ii) EDC, NHS, dioxane, rt, 3 h; (iii) 6, Et3N, dioxane/MeOH, rt;
(iv) HCl·H2NOH, pyridine, rt, 12 h; (v) DCC, NOHP, 6, Et3N, dioxane, rt, 24 h.
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Beilstein J. Org. Chem. 2013, 9, 81–88.
Thioesters 7–9 were assayed against A549, MCF7 and WI38 were found to be moderately active to completely inactive
cell lines, in addition to recombinant human rHDAC1 (class I) against rHDAC6 in each case, highlighting the important
and recombinant human HDAC6 (rHDAC6, class II) as previ- isoform selectivity of these compounds.
ously reported [20]. Psammaplin A (3), prereduced psammaplin
A thiol (4), and SAHA (1) were included as control compounds. The mechanistic origin for the high potency of the supposed
The results are shown in Table 1. IC506/1 is defined by the ratio prodrug thioesters in cell-free assays was unclear: Such com-
IC50HDAC6/IC50HDAC1 and was used as an indicator of isoform pounds were designed to be cleaved to give the active (thiol)
selectivity in vitro.
inhibitor in cells, and presumably exhibit a decreased potency
against the target HDAC, prior to cleavage of the acetyl group.
The synthesised thioesters displayed modest but significant As previously mentioned, Miyata and co-workers synthesised a
cytotoxic activity in our cell-based assays (Table 1, columns number of SAHA-based thioesters, which exhibited moderate to
1–3). Compound 8, which contains an oxime moiety, was the high potency in both enzymatic and cell based assays. The
most potent compound in each case, followed by methyloxime enzymatic assays they employed, however, used cell extracts as
7, and finally ketone 9. These findings parallel our previous the source of HDACs, potentially containing esterases, which
SAR data for the corresponding thiols [20], and reflect the may have cleaved the thioester during the assay. On the
previously established potency ranking for the AB system: contrary, we observed the same pattern with purified rHDAC1
NOH > NOMe > O. The most sensitive cell line to treatment and purified rHDAC6, which obviously contains no such poten-
was the MCF7 breast cancer cell line, with the most potent tial for esterase-driven hydrolysis. Recently, Williams reported
thioester 8 displaying an IC50 of 3.2 μM. Such sensitivity corre- the high potency of the natural product largazole, bearing an
lates with previous SAR data [20]. While being moderately octanoyl-protected thiol, against HDACs [29]. While the depro-
potent against MCF7 cells (21 μM), 9 was inactive against tected analogue displayed nanomolar to subnanomolar potency
A549 and WI38 cells at the highest concentration tested in vitro against purified HDACs, the native thioester was still
(50 μM). Similar to our previously reported SAR and mecha- highly potent. They hypothesized that the octanoyl group was
nistic studies, a correlation between HDAC inhibition and cyto- cleaved in situ to liberate the active thiol; however, no rigorous
toxicity is clearly observable for these psammaplin A prodrugs. studies were reported to confirm this.
Notably, in enzyme assays, the potencies of our acetate- We therefore undertook preliminary studies to attempt to shed
protected analogues approached those of their corresponding light on the reasons for the high potency of our thioesters in the
thiols (Table 1, see notea), while being approximately 10-fold enzymatic assays. We first envisaged that our assay buffer
more potent than the parental disulfide series (Table 1, see could potentially be responsible for thioester hydrolysis. We
notea). For example, 8 was found to be highly potent against prepared and used synthetic probe 10 [30] (Scheme 3) in order
rHDAC1, displaying an IC50 of 5 nM. With the exception of 9, to test this hypothesis. Coumarin-based probe 10 is known to
acetyl-protected compounds were found to be 2.5 (8) to 8 times react extremely fast with thiols through a Michael addition reac-
(7) less potent than the corresponding free thiol analogues tion. While the fluorescence of 10 is efficiently quenched by the
against rHDAC1. Curiously, acetyl-protected compounds 7–9 intramolecular double bond, upon reaction with thiols, a highly
Table 1: Biological data.
A549
IC50 (μM)
MCF7
IC50 (μM)
WI38
IC50 (μM)
rHDAC1
IC50 (μM)
rHDAC6
IC50 (μM)
Compound
IC506/1
SAHA (1)
PSA (3)
n.d.
7.5
2.5
n.d.
1.3
2.4
n.d.
3.4
3.4
0.030
0.045
0.001
0.21
2.8
7
62
PSA-SH (4)
0.36
360
8.3
(4.1/0.16)
3.2
(0.63/0.61)
5.2
(2.2/1.1)
0.005
(0.043/0.002)
23
(2.0/3.2)
4560
(46/1772)
8a
7a
9a
44
(46/5.1)
12
(13/12)
17
(n.d./10)
0.12
(0.22/0.015)
>50
(>50/2.7)
>417
(>230/180)
>50
(10/11)
21
(3.9/3.4)
>50
(4.1/14)
0.48
(9.0/1.1)
9.5
(8/0.25)
>20
(0.9/0.22)
aIn brackets are data for the corresponding disulfides and thiols respectively, obtained from Baud et al [20].
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Beilstein J. Org. Chem. 2013, 9, 81–88.
Scheme 3: Top: Generation of the fluorescent adduct 11 after reaction of probe 10 with thiols. Bottom left: Fluorescence intensity at 465 nm (Y-axis,
A.U.) as a function of the number of equivalents of 2-mercaptoethanol added (X-axis). Bottom right: Fluorescence intensity at 465 nm (Y-axis, A.U.)
as a function of the incubation time (X-axis, minutes) of probe 10 with thioester 8.
fluorescent conjugate 11 is produced. This system has been rHDAC1 was measured (at the IC50 concentration of thioester)
found to be particularly efficient to quantify thiol concentration as a function of the initial incubation time (1–60 minutes) of
in biological systems [30].
rHDAC1 with thioesters 7 and 9 (Figure 2). No variation of
enzymatic inhibition could be observed in each case, excluding
Control experiments were performed using 2-mercaptoethanol rHDAC1 as the source of hydrolysis. Finally, we assessed
as the thiol source [30]. Fluorescence spectra of 10 (10−6 M 10 whether the use of thioester inhibitors could influence the assay
in buffer FB-188 [31]) were recorded after incubation with 0, readout, when using our coupled HDAC assay. Trypsin-depen-
0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 equiv 2-mercaptoethanol dant generation of the fluorescence intensity (2nd step of our
(Scheme 3, bottom left). As expected, a linear relationship coupled assay) was discounted as a source of false-positive
between fluorescence intensity and the concentration of thiol activity, since trypsin was used in a large excess (430 μM)
could be observed between 0 and 1.0 equiv, confirming the compared to the thioesters (low micromolar to nanomolar).
sensitivity and reliability of this system at low concentration. Direct compound inhibition of trypsin would therefore not be
When our HDACi thioester 8 was incubated with probe 10 significant in the overall readout on stoichiometry grounds.
(Scheme 3, bottom right) under the buffer conditions, however,
no significant variation of the fluorescence intensity was Conclusion
observed. The standard incubation time of thioesters in our In conclusion, we have demonstrated that highly potent and
HDAC inhibitor assay is 20–30 minutes; however, no evidence selective HDAC inhibitors can be discovered by preparing
of thiol formation was observed after up to 100 minutes of incu- thioester derivatives of the natural product psammaplin A and
bation in the assay buffer. This data therefore does not support close analogues. Such thioesters display significant cytotoxicity
the hydrolysis of our thioester inhibitors in the assay buffer. against several cancer cell lines. While initially envisaged as a
Two explanations could be envisaged to explain this result: (1) prodrug approach, we found these thioesters to retain highly
The thioester is stable to the assay buffer and therefore does not potent enzymatic activity using purified HDAC enzymes. Our
contribute to the in situ generation of the free thiol. (2) The preliminary results in the investigation of the origin of this
quantity of free thiol generated in situ in this experiment was effect have discounted hydrolysis of the thioester under the
too low to be quantified.
buffered conditions of the assay and direct cleavage of the
acetyl group by the deacetylase enzyme. It therefore remains
An alternative hypothesis for the high potency of the thioester highly plausible that the thioacetate group can function as a
inhibitors is that the HDAC enzyme cleaves the acetyl group potent zinc-binding group in its own right. While this hypoth-
directly, utilizing its intrinsic deacetylase activity. Inhibition of esis requires further validation, it opens up exciting new possi-
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Beilstein J. Org. Chem. 2013, 9, 81–88.
Figure 2: rHDAC1 was incubated with a predetermined IC50 concentration of 7 (left) and 9 (right) for 1–60 minutes, and the remaining rHDAC1
activity (Y-axis, %) was recorded and plotted as a function of the incubation time (X-axis, minutes).
(E)-(S)-2-(3-(3-Bromo-4-methoxyphenyl)-2-
bilities to prepare HDAC inhibitors bearing diverse thioester
(hydroxyimino)propanamido)ethyl
ethanethioate (8)
zinc-binding groups. Variation of the thioester functionality to
protect its lability in cells and potentially orient suitable func-
tionality into the internal cavity of HDACs [32] remain exciting
avenues for future research.
Experimental
(E)-(S)-2-(3-(3-Bromo-4-methoxyphenyl)-2-
(methoxyimino)propanamido)ethyl
ethanethioate (7)
To a solution of crude acid 5 (1 equiv) in freshly distilled pyri-
dine (2 mL/mmol), under argon, was added hydroxylamine
hydrochloride (1.5 equiv). The resulting mixture was stirred
overnight at rt. Pyridine was then removed in vacuo, and the
residue was dissolved in 1 N HCl (5.5 mL/mmol) and extracted
three times with ethyl acetate (5.5 mL/mmol). The combined
organic layers were dried (Na2SO4), filtered and concentrated to
afford the relatively pure oxime intermediate, used for the next
Compound 7 (216 mg, 30% from acid 5) was prepared step without further purification. To a solution of this crude
according to previously reported procedures [28] and obtained oxime derivative in dioxane (10 mL/mmol) under argon was
as a yellowish oil after purification by flash column chromatog- added DCC (1 equiv) and N-hydroxyphthalimide (1 equiv).
raphy (AcOEt/CH2Cl2, 2:98). Rf 0.35 (AcOEt/CH2Cl2 2:98); After 2 hours of stirring at rt, 2-acetylsulfanylethylammonium
IR: 1674, 1520, 1495, 1044 cm−1; 1H NMR (400 MHz, CDCl3) chloride (6, 1 equiv) and triethylamine (2.1 equiv) were added,
δ 2.33 (s, 3H, CH3C), 3.04 (t, J = 6.6 Hz, 2H, CH2S), 3.48 (m, and the resulting mixture was stirred overnight at rt. On the
2H, CH2N), 3.81 (s, 2H, 4-CH2), 3.84 (s, 3H, CH3O-1), 4.01 (s, following day, the solvent was evaporated, and the product
3H, CH3O-N), 6.78 (d, J = 8.4 Hz, 1H, 2-H), 6.97 (br t, 1H, (50 mg, 19% from acid 5) was obtained as a white powder after
NH), 7.21 (dd, J = 8.4, 2.1 Hz, 1H, 3-H), 7.46 (d, J = 2.1 Hz, purification by flash column chromatography (AcOEt/CH2Cl2
1H, 5-H); 13C NMR (100 MHz, CDCl3) δ 28.4, 28.6, 30.6, 2:8) and trituration in warm AcOEt. Mp 167–169 °C; Rf 0.6
39.1, 56.2, 63.1, 111.4, 111.8, 129.4, 129.8, 133.9, 151.4, 154.4, (AcOEt/CH2Cl2, 2:8); 1H NMR (400 MHz, CD3OD) δ 2.29 (s,
162.6, 195.6; HRMS (ESI+) m/z: [M + H]+ calcd for 3H, CH3C), 3.01 (t, J = 6.6 Hz, 2H, CH2S), 3.41 (m, 2H,
C15H20BrN2O4S, 403.0322; found, 403.0336; Anal. calcd for CH2N), 3.82 (s, 2H, 4-CH2), 3.83 (s, 3H, CH3O), 6.91 (d, J =
C15H19BrN2O4S: C, 44.67; H, 4.75; N, 6.95; found: C, 44.74; 8.5 Hz, 1H, 2-H), 7.22 (dd, J = 8.5, 2.1 Hz, 1H, 3-H), 7.44 (d, J
H, 4.78; N, 6.89.
= 2.1 Hz, 1H, 5-H); 13C NMR (100 MHz, CDCl3) δ 28.7, 29.4,
86

Beilstein J. Org. Chem. 2013, 9, 81–88.
30.4, 39.9, 56.7, 112.1, 113.1, 130.5, 131.8, 134.7, 152.9, 155.9, The reaction mixture was incubated at 30 °C in the dark and
165.9, 197.1; HRMS (ESI+) m/z: [M + H]+ calcd for stopped after 60 min by the addition of a mixture of 70 µM
C14H18BrN2O4S, 389.0165; found, 389.0164; Anal calcd for trypsin and 200 nM SAHA. The fluorescence of AMC served as
C14H17BrN2O4S: C, 43.20; H, 4.40; N, 7.20; found: C, 43.29; an indirect measure of HDAC enzyme activity. The kinetics of
H, 4.32; N, 7.32.
AMC release was measured on a PolarStar fluorescence plate
reader (BMG) with an excitation wavelength of 340 nm and an
emission wavelength of 460 nm. Complete cleavage of deacety-
lated Boc-Lys-AMC by trypsin was achieved after about
10–15 min. The fluorescence intensity of the plateau was aver-
aged over at least 5 min and normalized with respect to the
percentage of enzyme activity. Finally, the normalized fluores-
cence intensities were plotted versus the concentration of test
compounds and fitted to a four-parameter logistic model to
calculate the IC50 values.
(S)-2-(3-(3-Bromo-4-methoxyphenyl)-2-
oxopropanamido)ethyl ethanethioate (9)
To a solution of acid 5 under argon in dioxane (5.5 mL/mmol)
was added DCC (1 equiv), N-hydroxyphthalimide (1 equiv). Acknowledgements
After 2 hours of stirring at rt, 2-acetylsulfanylethylammonium This work was supported by the Association for International
chloride (6, 1 equiv) and triethylamine (2.1 equiv) were added Cancer Research (AICR) (08-0407).
and the resulting mixture was stirred overnight at rt. The day
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