Cyclic disulfides as functional mimics of the histone deacetylase inhibitor FK-228

Article abstract of DOI:10.1016/j.tetlet.2007.04.123

FK-228 is a potent histone deacetylase (HDAC) inhibitor with tremendous therapeutic potential against a wide array of human cancers. We describe the development of analogs that share FK-228's novel mechanism of activation and HDAC inhibition.

Full text of DOI:10.1016/j.tetlet.2007.04.123

Tetrahedron Letters 48 (2007) 4579–4583
Cyclic disulfides as functional mimics of the histone
deacetylase inhibitor FK-228
a
a
b
´
Jared R. Mays, Jose A. Restituyo, Rebeccah J. Katzenberger,
David A. Wassarmanb and Scott R. Rajskia,*
aSchool of Pharmacy, University of Wisconsin, Madison, WI 53705, United States
bDepartment of Pharmacology, University of Wisconsin, Madison, WI 53706, United States
Received 14 March 2007; revised 19 April 2007; accepted 25 April 2007
Available online 29 April 2007
Abstract—FK-228 is a potent histone deacetylase (HDAC) inhibitor with tremendous therapeutic potential against a wide array of
human cancers. We describe the development of analogs that share FK-228’s novel mechanism of activation and HDAC inhibition.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Histone deacetylases (HDACs) convert neutral acetyl
lysine side chains to their positively charged ammonium
salts1 and play a key role in the regulation of gene
expression.2–8 Histone deacetylation correlates most
often with transcriptional repression; there are now
numerous examples of human cancers believed to arise
as a result of accidental transcriptional repression.9 It
is therefore no surprise that HDAC inhibitors are highly
attractive anticancer agents. Among the most promising
Figure 1. Mechanism of FK-228 activation en route to HDAC
inhibition.
of these is the cyclic disulfide natural product FK-228
(1) which displays impressive anticancer and antiangio-
genic activities by virtue of its capacity for HDAC inhi-
bition.10–13 FK-228 is currently in Phase II clinical trials
for T cell lymphomas and other phase II projections
involve non-Hodgkin’s lymphoma, acute myelogenous
leukemia, and pancreatic cancer.14 Phase I clinical trials
for thyroid and other advanced malignancies, combina-
tion therapy for lung cancer, and for leukemias are also
underway. Alarmingly, the advancement of 1 through
clinical trials has been hindered as a result of shortages
of the natural product and its high degree of synthetic
complexity.13
adheres to a tripartite structure characteristic of most
effective HDAC inhibitors, as it contains an HDAC rec-
ognition or affinity element attached to an active site
binding/inactivating group via a linker devoid of elabo-
rate functionality.16 The increased levels of disulfide
reductants (i.e., glutathione, thioredoxin (Trx) and thio-
redoxin reductase (TrxR)) found in many cancer cells
likely render such cells particularly susceptible to the ac-
tions of 1 and provide added enthusiasm for the pursuit
of functional mimics of 1.17 Although advances have
been made toward acyclic thiol and masked thiol-based
HDAC inhibitors, the reductive mechanism displayed
by 1 has not yet been mimicked by non-peptidic, more
easily diversified scaffolds.18 We report here, the synthe-
sis and biological study of small, readily-diversifiable
disulfide motifs that share FK-228’s novel mechanism
of activation and HDAC inhibition.
Importantly, HDAC inhibition by 1 is a consequence of
disulfide reduction to 2. The thiol-capped butene moiety
likely binds an active site zinc (Fig. 1).15 Dithiol 2
*
Corresponding author. Tel.: +1 608 262 7582; fax: +1 608 262
5345; e-mail: srrajski@pharmacy.wisc.edu
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.04.123

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2. Results and discussion
ker lengths between the presumed Zn2+-binding thiol
and the remainder of these simple agents. We envisioned
a retrosynthesis wherein 3–5 would result from cycliza-
tion of di-S-trityl 6 using well-established thiol deprotec-
tion/oxidation conditions (Fig. 2). Compounds like 6
would be generated from simple peptide coupling of
the suitably functionalized acids 7 and S-trityl cysteine
methyl ester 8.
Our attention focused on cyclic disulfides 3–5, bearing 4,
5, and 6 methylene unit-containing linkers between the
proposed Zn2+-binding thiol and the rest of the ‘cap’
linked molecular framework (Fig. 2). Based upon the
elegant structure:activity studies of Yoshida and co-
workers, we expected that different linker lengths would
correlate to altered efficiencies of HDAC inhibition.19
For the purposes of preliminary biological studies here,
we have relegated efforts to the assessment of in vitro
activities. We envisioned 3–5 to be important intermedi-
ates en route to an array of more elaborate cyclic disul-
fides. Methyl esters 3–5 would provide a handle with
which to install lipophilic cap groups via simple
amide-forming reactions with various primary and sec-
ondary amines. Our specific interest in lipophilic cap
groups was motivated in large part by the vast experi-
ence of other groups.8,16 In particular, crystal structures
of histone deacetylase homolog protein (HDAC-like)
bound to known HDAC inhibitors Trichostatin A
(TSA) and SAHA highlight the importance of inhibitor
interactions with a number of phenylalanine residues
lining the enzyme channel leading to a deeply buried
active site.20 Moreover, amides derived from 3–5 would
permit delineation of the impact of slightly different lin-
A representative synthesis of disulfides 3–5 is shown in
Scheme 1 using compound 4. Commercially available
bromoacids of type 9 were readily converted to their
S-trityl carboxylic acids of type 10; peptide coupling of
10 and S-trityl cysteine methyl ester 8 using 1,10-carbon-
yldiimidizole (CDI) afforded di-S-trityl compounds such
as 11.21 Cyclization of 11 to 4 was accomplished using
I2/MeOH, as previously reported in the total synthesis
of FK-228 and related disulfides.13,22 The purity of 3–
1
5 was assessed to be >96% by H NMR. Saponification
of 4 with LiOH provided acid 12 which, followed by
peptide coupling to various amines, could produce an
assortment of derivatives bearing different cap groups
ancillary to the cyclic disulfide scaffold (13). Interest-
ingly, it was found that, while core structures 3 and 4
could be readily hydrolyzed and further manipulated,
the larger heterocycle 5 was not amenable to saponifica-
tion. Attempts to produce amides from this substance
were often complicated by significant decomposition.
As such, only a limited number of amide-diversified ana-
logs of 5 were successfully constructed.
We hypothesized that 3–5 would be cell permeable.
Thus, 3–5 were tested for their ability to induce histone
hyperacetylation in Drosophila S2 cells. Drosophila, with
its small well-defined genome and amenability to gene
ablation provides an excellent eukaryotic model.23 Dro-
sophila S2 cells were subjected to varying concentrations
of methyl esters 3–5 for 48 h at ambient temperature,
after which time nuclei were isolated and histones ana-
lyzed. Histone H4 lysine-acetylation status was assessed
using Western blots utilizing anti-(Ac)4-H4 antibody.
Much to our delight, H4 hyperacetylation was observed
in cells subjected to methyl esters 3–5. Hyperacetylation
was observed at disulfide concentrations as low as
20 lM (Fig. 3) and was particularly striking in cells sub-
Figure 2. Envisioned disulfide core structures and relevant retro-
synthesis.
Scheme 1. Synthesis of cyclic disulfides.

J. R. Mays et al. / Tetrahedron Letters 48 (2007) 4579–4583
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Figure 3. Cyclic disulfides induce histone H4 hyperacetylation in cells.
jected to disulfide 4 containing the 5 methylene-unit lin-
ker. The concomitant use of anti-H2B antibody revealed
that alterations in (Ac)4-H4 signal result from altered
acetylation status and not variation in absolute protein
loadings. Differences in H4 signal are clearly small mole-
cule-dependent and not a result of uneven protein load-
ing. These results confirmed that simple cyclic disulfides
devoid of the relatively large and lipophilic cap groups
characteristic of both natural and synthetic effective
HDAC inhibitors can induce significant histone hyper-
acetylation in cells. Additionally, this suggested that
diversified analogs like 13 may serve as good leads for
new types of HDAC inhibitors inspired by FK-228.
methyl esters 3–5 and amide-diversified analogs 14–36
(Fig. 4) were assayed for HDAC inhibition using the
Fluor-de-Lys fluorescence-based assay with HeLa cell
extracts.24 To emulate the reducing environment prom-
inent within many tumor cells because of high glutathi-
one, Trx and TrxR levels, Fluor-de-Lys assays were
performed in the presence and absence of the reducing
agent dithiothreitol (DTT).17,25 Due to the limited avail-
ability of FK-228, we selected the highly potent, com-
mercially-available and well-studied HDAC inhibitors
Scriptaid and Trichostatin A (TSA) as positive controls.
The results of this assay are depicted in Figure 4.
Most members of the library displayed a clear depen-
dence upon DTT to effect HDAC inhibition. For
instance, disulfide 22 bearing the 2,3-dimethoxybenzyl
amine cap E is devoid of activity in the absence of
DTT. However, in the presence of DTT, 22 (20 lM)
exhibits substantial HDAC inhibition. There also ap-
The intracellular reduction of 1 to its corresponding
dithiol 2 plays a crucial role in the agent’s HDAC inhi-
bition profile. We hypothesized that structurally simpli-
fied disulfides might capitalize on this activating event en
route to HDAC inhibition. To test this hypothesis,
Figure 4. Dependence of redox-triggered alterations in HDAC activity on linker length and cap group.26 The large standard deviations of these
preliminary data have prompted us to assess HDAC inhibition using alternative methods. The results of these studies will be reported shortly.

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peared to be a relationship between the number of meth-
ylene units and observed HDAC inhibition. This trend is
exemplified through comparison of 23 and 24. Although
both compounds contain the same recognition/affinity
group, 2,3-dimethoxybenzylamine, significantly greater
HDAC inhibition is observed for 24 than 23. A similar
trend is observed for many other members of the tested
cyclic disulfide panel. The cap group identity also
appeared to play a role in the moderate HDAC inhibition
activities displayed by members of the library. Even
subtle changes in cap group structure led to significantly
altered bioactivity. For example, 34 and 35 differ only in
their bromide substitution pattern. Yet, these com-
pounds differ significantly in their HDAC inhibition
profiles. That such alterations in activity are observed
with even subtle structural alteration from one com-
pound to the next is supported by the well-established
impact that both linker and cap identities have upon
HDAC inhibitor effectiveness.8,19 Overall, these data
provide preliminary evidence that small cyclic disulfides
may serve as more-easily constructed and readily-diver-
sifiable mimics of 1.
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In conclusion, we have shown that simple functional
mimics of 1 can be readily generated. Core methyl ester
structures 3–5 induced H4 hyperacetylation in Drosoph-
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nuclear lysates, thus supporting early assertions of cell
permeability by these synthetic agents. Methyl esters
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We thank the UW Advanced Opportunity Fellowship
(AOF) program, the NIH sponsored Chemistry and
Biology Interface (CBI) training grant for predoctoral
support of J.A.R. and J.R.M. The generous contribu-
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Innovation Award), the American Cancer Society,
UW Graduate School and UW School of Pharmacy
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Supplementary data
Experimental procedures and characterization for all
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