J. Med. Chem. 2008, 51, 6639–6641
6639
Efficient Solid-Phase Synthesis of FK228
Analogues as Potent Antitumoral Agents
Salvatore Di Maro,†,‡ Rey-Chen Pong,§ Jer-Tsong Hsieh,§
and Jung-Mo Ahn*,†
Department of Chemistry, UniVersity of Texas at Dallas,
Richardson, Texas 75080, and Department of Urology, UniVersity of
Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390
Figure 1. Structures of a novel FK228 analogue. (A) Analogue design
and (B) conformation (orange) overlaid on FK228 (green).
ReceiVed July 29, 2008
transfer reaction; however, no increase in overall yield was
achieved (13%).9
Abstract: Novel structural analogues of a HDAC inhibitor FK228 have
been synthesized by modifying the most synthetically challenging unit,
(3S,4E)-3-hydroxy-7-mercaptoheptenoic acid, with simple isosteric
substitutions. These changes did not alter the backbone structure from
FK228 but enabled facile and rapid synthesis by using readily available
starting materials and high-yielding reactions. FK228 analogues were
examined for their antitumoral activity on a variety of human cancer
cells and led to the identification of new potent compounds.
Despite its promising biological activity, these difficulties
appear to deter the synthesis of a series of FK228 analogues
that would facilitate structure-activity relationship studies and
reveal the requirements to achieve higher potency and specific-
ity. Thus, we report herein novel FK228 analogues that were
designed by employing simple isosteric substitutions, and its
efficient solid-phase synthesis to produce a large number of
FK228 analogues.
Histone deacetylases (HDACs) are involved in a post-
translational modification of histones by removing acetyl groups
at the ε-amino groups of histone tails, which results in changing
chromosomal DNA conformation and which then influences
gene expression, often referred as epigenetic gene regulation.1
Epigenetic gene regulation is known to play a critical role in
embryo development, cell proliferation, and differentiation. In
contrast, altered epigenetic regulation has been noticed in various
cancers. Thus, it is considered as an attractive target for the
treatment of cancers.2 Until now, a number of HDAC inhibitors
have been identified including TSA,a SAHA, and FK228.2
A natural product, FK228 (also known as FR901228) is a
highly potent HDAC inhibitor that is isolated from Chromo-
bacterium Violaceum (Figure 1A).3 Structurally unrelated to
other HDAC inhibitors, it is a unique bicyclic depsipeptide4
and a stable prodrug that becomes activated by the reduction
of its disulfide bond after uptake into cells.5 As Furumai et al.
have demonstrated, the released sulfhydryl group appears to be
important for the interaction with the zinc cation localized at
the active site of HDACs. In addition, it shows high selectivity
toward class I HDACs,6 which have been reported to be more
relevant for therapeutic intervention in oncology.7
To overcome the synthetic challenges identified from the
previous synthesis,8,9 (3S,4E)-3-hydroxy-7-mercapto-4-heptenoic
acid in FK228 was modified into a structure that can be easily
assembled using readily available starting materials, yet still
has a capability to retain the same conformation required for
the biological activity. First, the trans double bond in the
heptenoic acid was replaced by an isosteric amide bond. To
develop peptidomimetics, an amide bond in peptides has been
changed to a trans double bond because of its structural rigidity
and capability to present two alkyl chains on opposite sides.10
Second, the ester bond to form the depsipeptide was replaced
by another amide bond for facile ring closure that can provide
higher synthetic yield and increased in vivo stability. These two
simple modifications transformed the synthetically challenging
heptenoic acid into a structure that can be easily assembled with
an L-aspartic acid and a cysteamine (Figure 1A). In addition,
another unnatural amino acid in FK228, (Z)-dehydrobutyrine
(Dhb), was substituted with various L- and D-amino acids for
easy construction of FK228 analogues. All of these changes
allowed to achieve highly efficient synthesis (vide infra).
To ensure that the isosteric substitutions do not alter the
backbone structure from FK228, the novel FK228 analogue was
examined by molecular modeling. A Monte Carlo conforma-
tional search using MacroModel11 (version 9.1, Schro¨dinger)
and united atom AMBER force field showed an almost identical
structure compared to FK228 (rmsd ) 0.20 Å; Figure 1B).
For high synthetic efficiency, we have focused on developing
a solid-phase strategy, since this platform would ultimately allow
the production of a large number of FK228 analogues. As shown
in Scheme 1, a backbone amide linker (BAL)12 was coupled to
aminomethylpolystyrene resin, and a subsequent reductive
amination anchored a cysteamine on the resin. To the resulting
secondary amine (1), the first amino acid, Fmoc-L-Asp(OAl),
was coupled with HBTU13 for 12 h (80% yield). However, when
several coupling methods were screened, TFFH14 was found to
provide a higher yield (95%). Also, coupling the amino acid as
a symmetric anhydride by treating with DIC was effective (98%
yield). The aspartylcysteamine (2) was constructed by one
simple coupling reaction and is a surrogate for the challenging
Its first total synthesis was reported by Simon and co-workers
in 1996 and achieved by following a rather laborious synthetic
route giving a moderate yield (18% overall yield over 16 steps).8
Three principal challenges noticed are (1) the asymmetric
synthesis of the (3S,4E)-3-hydroxy-7-mercapto-4-heptenoic acid,
(2) the macrolactonization to form the 16-membered depsipep-
tide, and (3) the oxidation of thiols to create the 15-membered
ring. An improved synthesis was recently reported to prepare
the heptenoic acid using an asymmetric Noyori hydrogen
* To whom correspondence should be addressed. Phone: +1-972-883-
2917. Fax: +1-972-883-2925. E-mail: jungmo@utdallas.edu.
†
University of Texas at Dallas.
Present address: Department of Pharmacological and Toxicological
‡
Chemistry, University of Naples, “Federico II”, Naples 80100, Italy.
§
University of Texas Southwestern Medical Center at Dallas.
a Abbreviations: TSA, trichostatin A; SAHA, suberoylanilide hydroxamic
acid; BAL, 5-(4-formyl-3,5-dimethoxyphenoxy)butyric acid; HBTU, O-ben-
zotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate; TFFH, tet-
ramethylfluoroformamidinium hexafluorophosphate; DIC, diisopropyl car-
bodiimide; TFA, trifluoroacetic acid; L-1-Nal, L-1-naphthylalanine.
10.1021/jm800959f CCC: $40.75
2008 American Chemical Society
Published on Web 10/08/2008