Novel histone deacetylase inhibitors: Cyclic tetrapeptide with trifluoromethyl and pentafluoroethyl ketones

Article abstract of DOI:10.1016/j.bmcl.2004.08.016

Cyclic tetrapeptides containing trifluoromethyl and pentafluoroethyl ketone as zinc binding functional group were synthesized as potent HDAC inhibitors. Evaluation by human HDAC inhibition assay and p21 promoter assay showed that these inhibitors are promising anticancer agents.

Full text of DOI:10.1016/j.bmcl.2004.08.016

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5343–5346
Novel histone deacetylase inhibitors: cyclic tetrapeptide with
trifluoromethyl and pentafluoroethyl ketones
a
Binoy Jose,a Yusuke Oniki,b Tamaki Kato,b Norikazu Nishino,a,b, Yuko Sumida and
*
Minoru Yoshidaa,c
aCREST Research Project, Japan Science and Technology Agency, Saitama 332-0012, Japan
bGraduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan
cRIKEN, Saitama 351-0198, Japan
Received 21June 2004; revised 2 August 2004; accepted 7 August 2004
Available online 3 September 2004
Abstract—Cyclic tetrapeptides containing trifluoromethyl and pentafluoroethyl ketone as zinc binding functional group were syn-
thesized as potent HDAC inhibitors. Evaluation by human HDAC inhibition assay and p21promoter assay showed that these
inhibitors are promising anticancer agents.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Post-translational modification of N-terminal tails of
histone proteins by reversible acetylation with histone
acetyl transferase (HAT) and histone deacetylase
(HDAC) enzymes is involved in chromatin remodeling
and plays a crucial role in gene expression.1 The inap-
propriate recruitment of HDACs has been clearly linked
to carcinogenesis and the inhibition of HDACs results in
transcriptional reactivation, cell-cycle arrest and termi-
nal differentiation of transformed cells. HDAC inhibi-
tors have potential for the prevention and treatment of
cancer and emerged as an attractive target as anticancer
drugs.2
HDAC inhibitory activity.4 Other HDAC inhibitors
have thiol,5 epoxide,6 carboxylic acid,7 and amide8 as
zinc binding functionalities. Very recently, we reported
retrohydroxamates as potent HDAC inhibitors.9 The
catalytic mechanism for the deacetylation of acetylated
lysine and various reported ligands of cyclic tetrapeptide
inhibitors of HDAC are shown in Figure 1. Many of
inhibitors have potent antitumor effect in vivo in tumor
bearing animals. However, the potential for drug devel-
opment could be limited due to many reasons such as
low potency, lack of selectivity, cytotoxicity, low solubil-
ity in aqueous vehicle and low stability in cell culture.
Poor pharmacokinetic properties associated with the
hydroxamic acid group in matrix metalloproteinases
inhibitors10 prompted us to explore the possibility of
other zinc binding functional groups for cyclic
tetrapeptides.
A number of structurally diverse natural and synthetic
compounds have been so far reported exhibiting HDAC
inhibitory activity.2 These compounds consist of a
hydrophobic scaffold with a spacer, that is attached to
a functional group which can interact with zinc ion pre-
sent in the active site pocket. Compounds containing
hydroxamic acid as a functional group is reported as
the most potent inhibitor for HDACs, which include
the natural product trichostatin A.3 In our previous
work, we synthesized cyclic hydroxamic acid-containing
peptides (CHAPs) and found that they have very high
Trifluoromethyl ketones are potent inhibitors of aspar-
tyl11 and serine12 proteases, by formation of stabilized
hemiketals and hemithioketals at the active site. A re-
cent report on HDAC inhibitors showed that trifluoro-
methyl ketone attached to aromatic amides has
micromolar inhibitory activity for HDAC.13 We de-
signed and synthesized cyclic tetrapeptides containing
trifluoromethyl ketone as HDAC inhibitors. We specu-
late that the cyclic tetrapeptide trifluoromethyl ketone
and related compounds can give better inhibitory activ-
ity. The functional groups selected for this study is
Keywords: Histone deacetylase; Inhibitor; Cyclic tetrapeptide; Tri-
fluoromethyl ketone; Pentafluoroethyl ketone.
*
Corresponding author. Tel./fax: +8193695606;1e-mail:
life.kyutech.ac.jp
nishino@
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.016

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B. Jose et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5343–5346
Figure 1. Binding of substrate to zinc ion in HDAC1, reported ligands of cyclic tetrapeptide inhibitors and proposed functional groups. (a) Binding
with substrate, (b) ketoepoxide, (c) hydroxamic acid, (d) retrohydroxamic acid, (e) sulfhydryl, (f) trifluoromethyl ketone, (g) pentafluoroethyl ketone,
(h) trifluoromethyl ketone with a sulfur atom in the spacer.
shown in Figure 1. The hydrophobic scaffold for the
proposed inhibitor is the cyclic tetrapeptide portion of
CHAP31, which was found to have excellent antitumor
activity.4
ammonia to obtain the thiol derivative. The thiol deriv-
ative is reacted with 3-bromo-1,1,1-trifluoroacetone to
yield the proposed compound 3 (Scheme 2).16
HDAC inhibitory activity of these electrophilic ketones
depends on the degree of hydration of the ketone, which
can chelate with the zinc ion present in the deep pocket
of HDAC binding site. We verified the degree of hydra-
tion using HPLC and 19F NMR studies. HPLC of com-
pound 1 showed a single peak at 8.0min indicating that
the compound is completely hydrated under the HPLC
conditions.17 Compound 2 showed two peaks, one at
8.8min and the other at 10.5min in the ratio of 3:7 indi-
cating that the tendency to form hydrate in the case of 2
is less (Fig. 2). Compound 3 shows the similar behavior
as 1 with a single peak at 8.2min.
The cyclic tetrapeptide cyclo(-L-Asu(OBzl)-D-Tyr(Me)-
L-Ile-D-Pro-) was synthesized by the reported procedure,
where Asu is aminosuberic acid.4a The benzyl side chain
protection of the cyclic tetrapeptide was removed by
hydrolysis with lithium hydroxide and the resulted lith-
ium salt was then treated with trifluoroacetic anhydride
under modified Dakin–West conditions14 to give the de-
sired trifluoromethyl ketone 1 in 40% yield. Using pen-
tafluoroacetic anhydride, cyclic tetrapeptide containing
pentafluoroethyl ketone 2 was also synthesized (Scheme
1).
Next we synthesized a cyclic tetrapeptide containing
1,1,1-trifluoro(3-thio)acetone as the functional group.
For this, we used a cyclic tetrapeptide containing an L-
amino-6-bromohexanoic acid (L-Ab6).15 The cyclic
tetrapeptide, cyclo(-L-Ab6-D-Tyr(Me)-L-Ile-D-Pro-) was
synthesized by the reported procedure.5 This cyclic pep-
tide is treated with potassium thioacetate to yield thioac-
etate derivative, and the product was then treated with
19F NMR of 1 shows only one singlet at d À82.5 in
CDCl3. In methanol 1 shows a doublet at d 82.0 indicat-
ing hemiacetal formation. In a mixture (7:3) of methanol
and water, a new peak was observed at d À86.5 due to
the hydrate formation. The intensity ratio of 1:2 for
the peaks at d À82 and À86.5 indicate the strong ten-
dency of trifluoromethyl ketone to form hydrate. On
the other hand, under similar conditions, a lower rate
Scheme 1. Reagents and conditions: (a) LiOH, H2O, THF; (b) for 1, pyridine, trifluoroacetic anhydride for 2, pyridine, pentafluoropropionic
anhydride.
Scheme 2. Reagents and conditions: (a) AcSK, DMF; (b) NH3/MeOH; (c) BrCH2COCF3, DMF.

B. Jose et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5343–5346
5345
Figure 2. (a) HPLC chart for 1 and (b) 2. The conditions for HPLC
analysis is given in Ref. 17.
of hydration was observed from the 19F NMR of com-
pound 3. Similar differences in the degree of hydration
of trifluoromethyl ketones and pentafluoromethyl ke-
tones were observed by Peet and co-workers during their
study on human neutrophil elastase inhibitors.12
Figure 3. In vivo stability test of 1 and trichostatin A.
The compounds synthesized were tested for the HDAC
inhibitory activity using HDAC1, HDAC4, and
HDAC6 prepared from 293T cells.18 The results of
HDAC inhibitory activity and p21promoter assay of
the compounds are shown in Table 1. For comparison
the activity of trichostatin A is also shown. All three
compounds show HDAC inhibitory activity in nanomo-
lar concentrations. The activity of compound 3 is higher
than the other two compounds probably due to the pres-
ence of the C–S–C bond, that may provide a different
mode of ligation with Zn ions. More studies are needed
to find the binding nature of 3 within the enzyme pocket.
When comparing the activity between compounds 1 and
2, trifluoromethyl ketone is more active than pentafluoro-
ethyl ketone. This may be ascribed to the presence of the
bulky pentafluoroethyl group that causes a decrease in
activity. Since Frey and co-workers described on the
instability of trifluoromethyl ketone inhibitors in their
work, we carried out in vivo stability tests of inhibitor
1. The inhibitor was stable under the p21promoter as-
say conditions though not much better than trichostatin
A (Fig. 3).
Figure 4. Proposed mode of interaction of the inhibitor functional
groups with Zn in the active site of HDAC1.
In summary, in order to find novel non-hydroxamate
HDAC inhibitors, we designed and synthesized
CHAP31based electrophilic ketones. These inhibitors
have displayed potent HDAC inhibition activity in vivo
and in vitro. These results further confirm the hypothe-
sis that structural modification of the terminal residues
of HDAC inhibitors can lead to potent HDAC inhi-
bitors that may have potential as anticancer agents.
Further studies on compound 1 and 3 are under way.
Although the mechanism of inhibition on these inhibi-
tors is not completely clear, it seems that the hydration
of the ketone is very important for the inhibitory activ-
ity. Based on this observation, we propose the mode of
interaction of the functional groups of these inhibitors
with the zinc ion present in the active site of HDAC
as shown in Figure 4.
References and notes
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Table 1. HDAC inhibition and p21promoter assay data for the
compounds
Compound
IC50 (lM)
EC50
(lM)
HDAC1HDAC4 HDAC6 HDAC8
1
0.73
0.85
.440.611 0.37
3.09
0.78
0.23
2
NTa
0.19
5.70
0.18
31.10
6.64
0.77
3
0.047
0.022
TSAb
0.020
0.028
0.040
a Not tested.
b Trichostatin A.

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a
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