Histone deacetylase inhibitors: Synthesis of cyclic tetrapeptides and their triazole analogs

Article abstract of DOI:10.1016/j.tetlet.2010.06.050

Synthesis of nine macrocyclic peptide HDAC inhibitors and three triazole derivatives is described. HDAC inhibitory activity of these compounds against HeLa cell lysate is evaluated. The biological data demonstrate that incorporation of a triazole unit improves the HDAC inhibitory activity.

Full text of DOI:10.1016/j.tetlet.2010.06.050

Tetrahedron Letters 51 (2010) 4357–4360
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Histone deacetylase inhibitors: synthesis of cyclic tetrapeptides and their
triazole analogs
*
Erinprit K. Singh, Lidia A. Nazarova, Stephanie A. Lapera, Leslie D. Alexander, Shelli R. McAlpine
Department of Chemistry and Biochemistry, 5500 Campanile Dr., San Diego State University, San Diego, CA 92182-1030, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of nine macrocyclic peptide HDAC inhibitors and three triazole derivatives is described. HDAC
inhibitory activity of these compounds against HeLa cell lysate is evaluated. The biological data demon-
strate that incorporation of a triazole unit improves the HDAC inhibitory activity.
Published by Elsevier Ltd.
Received 9 May 2010
Revised 4 June 2010
Accepted 11 June 2010
Available online 16 June 2010
During the cell cycle, post-translational modifications to the
e
-
are 11 metal-dependent HDACs currently known, and it is unclear
which isoforms are responsible for silencing tumor suppressor
genes. Given the uncertainty of the role played by the individual
HDACs, chemotherapy has focused on inhibitors that target endog-
enous HDACs, which are multiple isoforms.
amino-terminal tails of histone proteins are made by a number of
different enzymes, including histone acetyl transferases (HATs)
and histone deacetylases (HDACs).1 Histone tails contain ꢀ40 lysine
residues, which are acetylated by HATs. Acetylation induces a con-
formational change within chromatin, allowing the transcriptional
machinery access to DNA thus promoting gene expression.1,2
HDACs repress the gene expression by deacetylating the lysine tails,
allowing the positively charged lysines to bind tightly to the nega-
tively charged DNA and denying the transcriptional machinery
access to genes, thereby repressing gene expression. Thus, these
post-translational modifications play a key role in directing gene
expression, and can create a phenotype that is unrelated to changes
in DNA.3 Inappropriate up-regulation of HDACs’ silences specific
tumor suppressor genes, which are responsible for cell prolifera-
tion, differentiation, and apoptosis.4,5 Molecules that interfere with
HDAC activity have shown a great promise as anticancer agents as
they inhibit this silencing process and allow tumor suppressor
genes to be transcribed and control the cell’s growth.6–8 With a
number of HDAC inhibitors in clinical trials and suberoylanilide
hydroxamic acid (SAHA, ZolinzaÒ) recently approved by the FDA
for the treatment of cutaneous T cell lymphoma (CTCL), HDAC inhi-
bition proves to be a worthy strategy for cancer therapy.9
HDAC inhibitors consist of three components: (1) the active site
metal-binding unit, (2) surface recognition domain, and (3) a linker
that connects the two domains.10 They operate by binding the sur-
face recognition domain located at the rim of the HDAC pocket and
placing the metal-binding unit within the pocket (Fig. 1).11 HDAC
inhibitors can be divided into five structural categories: short chain
fatty acids, hydroxamic acids, electrophilic ketones, benzamides,
and cyclic peptides.12 These five structural categories are known
to inhibit the three classes of metal-dependent HDACs.13 There
Romidepsin (IstodaxÒ), a cyclic peptide, has recently been ap-
proved as an HDAC inhibitor against CTCL, indicating that cyclic
peptides are pharmacophores of interest in this field.14 In this Let-
ter, we describe the synthesis of twelve HDAC inhibitors. Nine of
these tetrapeptides were synthesized and modifications were fo-
cused on the surface recognition domain and incorporating diverse
metal-binding units: acetyl lysines, trifluoroacetyl lysines, and
guanidine moieties. The poor solubility of these macrocycles, cou-
pled with the fact that rigid structures bind more effectively to pro-
tein targets than their structurally similar flexible counterparts, led
us to incorporate triazole units within the surface recognition do-
main in three molecules.15 We anticipated that the presence of a
triazole will rigidify the macrocyclic structure, restricting bond
rotations. This may lead to a more potent inhibitor if the metal-
binding unit is placed in the correct orientation within the HDAC
pocket. In the triazole molecules, only 2 of the 3 metal-binding
units were incorporated into their design: acetyl lysines and triflu-
oroacetyl lysines as, during the course of this work, we found that
the guanidines were poor inhibitors.
The design of compounds 2–9 was based on our lead compound
1 (Fig. 1).16 Compound 2 was generated by modifying 1, replacing
the acetyl lysine metal-binding unit with a guanidine unit. Revers-
ing the stereochemistry from
L to D of the homoarginine in 2 pro-
duced 3, while incorporation of arginine and a six-membered
ring at positions 3 and 4, respectively, gave 4. Integrating a six-
membered ring or a triazole at position 4 into our lead structure
gave compounds 5 and 6, respectively. Finally, substitution of the
acetyl moiety with a trifluoroacetyl moiety as the metal-binding
unit and subsequent inclusion of a six-membered ring or triazole
unit at position 4 produced compounds 7, 8, and 9, respectively.
* Corresponding author.
E-mail address: mcalpine@chemistry.sdsu.edu (S.R. McAlpine).
0040-4039/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2010.06.050

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E. K. Singh et al. / Tetrahedron Letters 51 (2010) 4357–4360
Figure 1. Peptide HDAC inhibitors based on lead compound 1.
A second series of compounds were synthesized, where these
were based on apicidin, a cyclic tetrapeptide HDAC inhibitor with
low nanomolar protozoan histone deacetylase inhibition.17 Four
compounds were made (Fig. 2), where all the four employed a free
tryptophan as opposed to the methoxy-protected indole present in
Apicidin. Both compounds 10 and 11 contain an acetyl lysine as the
metal-binding unit, and 10 includes a proline unit at position 4
whereas 11 contains a triazole unit. Compounds 12 and 13
replaced the six-membered ring at position 4 of Apicidin with a
D
-proline and incorporated a trifluoroacetyl moiety as the metal-
binding unit. Derivative 12 maintained the -stereochemistry of
the tryptophan, while 13 reversed it to a -tryptophan.
L
D
Syntheses of our peptide HDAC inhibitors were completed via a
convergent solution-phase route (Scheme 1), which allowed for an
easy substitution of amino acids at each position.16 TBTU and DI-
PEA were used to form the dipeptide fragments 1–2 and 3–4,
where acid-protected residues 1(a–d) and N-Boc-protected residue
2(a–c) furnished dipeptide MeO-1-2-Boc, and acid-protected resi-
dues 3(a–d) and N-Boc-protected residues 4(a,b) were coupled to
give dipeptides MeO-3-4-Boc (84–98% yield) (Scheme 1). Dipep-
tide acids of MeO-1-2-Boc were deprotected with lithium hydrox-
ide (52–80% yield), whereupon they were coupled to free amine
dipeptides MeO-3-4 to give linear tetrapeptides (41–90% yield
depending on the substrate). The linear tetrapeptide was acid
and amine deprotected using standard conditions, whereupon it
was cyclized by dissolving it in a 1:1 ratio of CH2Cl2 and CH3CN
(0.007–0.1 M). Addition of DIPEA (8–10 equiv) and three coupling
agents (HATU, DEPBT, and TBTU 2 equiv total) to the reaction
Figure 2. Peptide HDAC inhibitors based on apicidin.

E. K. Singh et al. / Tetrahedron Letters 51 (2010) 4357–4360
4359
Scheme 1. Cyclic peptide and triazole analog synthesis. Reagents and conditions: (a) TBTU, HATU, and/or DEPBT (1.2 equiv total), DIPEA (3–6 equiv), CH2Cl2 (0.1 M), yields
41–98%; (b) LiOH (4–8 equiv), CH3OH (0.1 M), yields 52–80%; (c) TFA (20–25%), anisole (2 equiv), CH2Cl2 (0.1 M); (d) TBTU, HATU, and DEPBT (2.0 equiv total), DIPEA (8–
10 equiv), CH2Cl2/CH3CN (1:1, 0.1–0.007 M), yields 3–45%; (e) K2CO3 (3 equiv) p-TsN3 (3 equiv), dimethyl(2-oxypropyl)phosphonate (3 equiv), CH3CN/CH3OH (1:1, 0.25 M),
yields 42–73%; (f) CuSO4 (1.5 mM), sodium ascorbate (45 mM), CH3OH/H2O (1:1, 0.005 M), yields 3–9%.
raphy, followed by HPLC furnished final products, which were con-
firmed via LC/MS and 1H NMR (yields ranged from 3% to 45%
depending on substrate). For compounds 2–4 deprotection of
amines on residues 3(a–b) was accomplished via hydrogenolysis
following cyclization. Compounds were dissolved in EtOH (0.1 M)
and treated with H2 gas in the presence of pure Pd/C. The reaction
mixtures were stirred for 3–5 h under H2, followed by filtration
with Celite, to yield final derivatives.
Synthesis of triazole containing HDAC inhibitors followed a lin-
ear approach (Scheme 1) whereby each residue was sequentially
added onto the molecule in a solution-phase. An amino aldehyde
residue 1(a–b) was converted to its corresponding alkyne with
the Bestmann–Ohira reagent (42–73% yield).18 The alkyne was
amine deprotected with TFA and subsequently coupled to residue
2(a–b) to give 1-2-Boc (92–98% yield). Treatment of 1-2-Boc with
TFA afforded free amine 1–2. This intermediate was coupled to res-
Figure 3. HDAC inhibition assays, where each column represents percent of HDAC
activity from the average of three independent trials at 200 lM of apicidin and
idue 3(d–e) to furnish 1-2-3-Boc (74–92% yield), which was amine
deprotected to furnish 1-2-3. The linear precursor was obtained
upon coupling 1-2-3 with residue 4 (66–93% yield).19 To generate
our final triazole containing macrocycles, we employed a Cu(I)-cat-
alyzed azide-alkyne cycloaddition as reported by Sharpless and co-
workers.20 Sodium ascorbate and copper sulfate were dissolved in
water and added to reaction flask with 10% of the solvent mixture
compounds 1–13, respectively. HDAC inhibition activity was relative to the DMSO
control.
produced a clear solution. The reactions were complete in approx-
imately 2–4 h. Work-up with methylene chloride and ammonium
chloride, concentration in vacuo, purification via flash chromatog-

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E. K. Singh et al. / Tetrahedron Letters 51 (2010) 4357–4360
1:1 ratio of CH3OH and H2O (0.005 M). The linear precursor was
dissolved in the remaining solvent mixture and added dropwise
to the reaction flask overnight. Upon completion of the reaction,
CH3OH was removed under reduced pressure and the product
was extracted with CH2Cl2, concentrated in vacuo, and purified
via flash chromatography, followed by HPLC to furnish final prod-
ucts. Final compounds were confirmed via LCMS and 1H NMR
(yield ranged from 3% to 9% yield).
Acknowledgments
We thank the Frasch foundation (658-HF07) for support of
E.K.S., L.A.N., S.A.L., and L.D.A. We thank NIH (1U54CA132379-
01A1) for support of L.D.A. and S.R.M. and (1R01CA137873) for
support of E.K.S., L.D.A., and S.R.M. We also thank NIH MIRT pro-
gram for support of E.K.S. and L.D.A.
Upon completion of the synthesis, we tested our molecules in
HDAC inhibition assays. The twelve compounds were assayed at
Supplementary data
200 lM concentration against endogenous HDACs from HeLa cell
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.06.050.
lysates using a fluorogenic substrate, as previously described.16
Apicidin (Fig. 2) was used as a positive control (2% 0.3% deacetyl-
ase activity at 1 lM) and DMSO was used as a negative control (set
to 100% deacetylase activity) (Fig. 3). Compounds 6 and 9 were the
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Although our HDAC Inhibition data show that our compounds
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activity. Second, changes from 4 to 5 atoms on the linker do not af-
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