Synthesis and histone deacetylase inhibitory activity of cyclic tetrapeptides containing a retrohydroxamate as zinc ligand

Article abstract of DOI:10.1016/j.bmcl.2004.03.018

Cyclic tetrapeptide retrohydroxamic acids were prepared as histone deacetylase (HDAC) inhibitors and evaluated the inhibitory activity and found that they have potential as anticancer drugs.

Full text of DOI:10.1016/j.bmcl.2004.03.018

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2427–2431
Synthesis and histone deacetylase inhibitory activity of cyclic
tetrapeptides containing a retrohydroxamate as zinc ligand
c
c
a
Norikazu Nishino,a,b, Daisuke Yoshikawa, Louis A. Watanabe, Tamaki Kato,
*
Binoy Jose,b Yasuhiko Komatsu,b Yuko Sumidab and Minoru Yoshidab,d
aGraduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan
bCREST Research Project, Japan Science and Technology Agency, Saitama 332-001, Japan
cFaculty of Engineering, Kyushu Institute of Technology, Kitakyushu 808-8550, Japan
dRIKEN, Saitama 351-0198, Japan
Received 13 February 2004; revised 6 March 2004; accepted 6 March 2004
Abstract—Cyclic tetrapeptide retrohydroxamic acids were prepared as histone deacetylase (HDAC) inhibitors and evaluated the
inhibitory activity and found that they have potential as anticancer drugs.
Ó 2004 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) are zinc hydrolases
responsible for the deacetylation of N-acetyl lysine res-
idues of histone and nonhistone protein substrates.1
Human HDACs are classified into two distinct classes,
the HDACs and sirtuins. The HDACs are divided into
two subclasses based on their similarity to yeast histone
deacetylases Rpd3 (Class I includes HDAC1, 2, 3, 8, and
11) and Hda1 (Class II includes HDAC4, 6, 7, 9, and
10).2 All of the HDACs have a highly conserved zinc
dependent catalytic domain. There is growing evidence
that the acetylation state of proteins and thus HDAC
enzyme family plays a crucial role in the modulation of a
number of biological processes, including transcription,3
microtubule structure, and function,4 and the cell cycle.5
Figure 1. HDAC inhibitors.
In the past few years, a number of HDAC inhibitors
such as trichostatin A (TSA),6 SAHA,7 and CHAPs8
(Fig. 1) have been reported as useful tools to study the
The essential feature of an HDAC inhibitor is the zinc
function of chromatin acetylation and deacetylation,
binding ligand with a spacer attached to a hydrophobic
and gene expression. Development of more inhibitors
scaffold. Most of the reported HDAC inhibitors have
for HDAC is necessary not only as probe for biological
hydroxamic acid as the zinc binding ligand. We have
functions of the different HDACs but also as thera-
reported the SAR study of a series of cyclic tetrapeptide
peutics such as inhibition of a specific HDAC that is
associated with a particular disease.9
hydroxamic acids (CHAPs) and found that CHAPs
having the cyclic tetrapeptide part of natural cyclic
peptide Cyl-1, as the best of the series with nanomolar
activity and therefore we retained the cyclic tetrapeptide
scaffold in this study.8 Recently, a group of researchers
from Merck reported the side chain modification of
Keywords: Histone deacetylase; Inhibitor; Retrohydroxamate; Cyclic
tetrapeptide.
apicidin for the synthesis of more active HDAC inhib-
itors by replacing the ethyl ketone of apicidin with other
zinc binding ligands.10 Other ligands such as, a-keto
* Corresponding author. Tel./fax: +81-936956061; e-mail: nishino@
life.kyutech.a.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.018

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N. Nishino et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2427–2431
amides11 and trifluoromethyl ketones12 were also
reported recently. We reported the synthesis and HDAC
activity of cyclic tetrapeptide containing sulfhydryl
group as zinc binding functionality.13 For the develop-
ment of novel HDAC inhibitors with improved biolog-
ical properties, we proposed to synthesize cyclic
tetrapeptides with other suitable zinc binding ligand.
Here we report the synthesis and HDAC inhibitory
activity of cyclic tetrapeptides with N-formyl hydroxyl-
amine (retrohydroxamate) and N-acetyl hydroxylamine
as zinc binding ligands.
O
N
R
Br
OBzl
OtBu
HN OBzl
c
a,b
( )n
( )n
( )n
Ac
OH
Ac
Ac
OtBu
N
H
N
N
H
H
O
O
O
4
5
6
O
O
N
R
R
N
OBzl
OBzl
For the synthesis of the cyclic tetrapeptide containing a
N-formyl hydroxylamine or N-acetyl hydroxylamine
functional groups at the side chain, we need an amino
acid containing retrohydroxamic acid at the x-position
of the side chain with a spacer of four to six methylene
units. For this we started with recently reported com-
pound, x-bromo-a-aminoalkanoic acid.14 Two routes
were selected for the synthesis of retrohydroxamates.
First route is, the reaction of protected a-amino-7-
bromoheptanoic acid 1 with N-formyl-O-benzylhydr-
oxylamine.15 This resulted in the formation of two
products as shown in Scheme 1. The required compound
2 was purified by silica gel column chromatography in
25% yield. In the second route, we started with N-acetyl-
DL-7-bromoheptanoic acid (4) to prepare the t-butyl
ester and reacted it with O-benzylhydroxylamine. The
product obtained was further treated with acetic anhy-
dride and subsequent trifluoroacetic acid treatment, and
enzymatic reaction yielded the desired product 7. It was
then protected with Boc group and 2-(trimethylsi-
lyl)ethanol to yield 2 (Scheme 2). The overall yield ob-
tained by both methods were almost same. After Boc
deprotection, the amino acid containing retrohydroxa-
f,g
d,e
( )n
( )n
Boc
OH
N
H
O
H2N
Tmse
O
O
2
7
R = H, CH3, n = 1, 2, 3
Scheme 2. Reagents and conditions: (a) Boc2O, DMAP, 100%; (b) O-
benzylhydroxyl amine, DIEA/MeOH, 40–50%; (c) Ac2O or HCOOH,
65–70%; (d) TFA, 100%; (e) acylase, 74–80%; (f) Boc2O, Et3N, 100%;
(g) TmseOH, DCC, DMAP, 90–100%.
mate 8 was coupled with Boc-D-Tyr(Me)-L-Ile-DL-Pip-
OH to yield the diastereomeric linear tetrapeptide 9.
After deprotection of the 2-(trimethylsilyl)ethanol group
and the Boc group, the tetrapeptide was cyclized using
HATU in DMF under high dilution condition to give
the cyclic peptide in 65% yield (Scheme 3). Cyclic tet-
rapeptides containing
D-Pip and L-Pip were separated
by silica gel column chromatography and characterized
by 1H NMR spectra. The benzyl group was deprotected
to yield the proposed cyclic tetrapeptide retrohydroxa-
mic acids 10a–l.16 Using the same strategy, we synthe-
sized 12 cyclic tetrapeptides containing a N-formyl
OBzl
O
R
OBzl
N
N
Scheme 3. Reagents and conditions: (a) TFA, 100%; (b) Boc-
D-
Br
O
Tyr(Me)- -Ile-X-Xaa-OH, HBTU, HOBtÆH2O, Et3N, 70–80%; (c)
L
R
TBAF/THF, 90–100%; (d) TFA, 100%; (e) HATU, DIEA, 50–60%; (f)
Pd/C, H2, 90–100%.
a
+
Boc
Boc
Boc
O
N
H
N
H
N
H
Tmse
O
O
hydroxylamine or N-acetyl hydroxylamine as the zinc
binding ligand as shown in Table 1.
Tmse
Tmse
O
O
O
2a R = H
3a R = H
1
2b R = CH3
3b R = CH3
These compounds were tested for the HDAC inhibitory
activity using HDAC prepared from mouse melanoma
B16/BL6 cells.17 The IC50 values of the compounds are
given in Table 1. Compounds 10e and 10f having five
Scheme 1. Reagents and conditions: (a) KI, K2CO3, dry acetone, N-
formyl-O-benzylhydroxylamine or N-acetyl-O-benzylhydroxylamine,
reflux, 36 h (25–35% of 2).

N. Nishino et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2427–2431
2429
Table 1. HDAC inhibitory data for retrohydroxamates and selected CHAPs
Compound Sequence Spacer and ligand
Configu-
ration
Chain length HDAC inhibitory
activity IC50 (lM)
O
O
10a
10b
10c
10d
cyclo(-
cyclo(-
cyclo(-
cyclo(-
L
L
L
L
-Lys(For,OH)-
-Lys(For,OH)-
D
D
-Tyr(Me)-
L
L
-Ile-
L
-Pip-)
LDLL
LDLD
LDLL
LDLD
4
4
4
4
1.392
N
H
H
OH
-Tyr(Me)-
-Ile-D
-Pip-)
0.957
88.5
N
OH
O
-Lys(Ac,OH)-
D
-Tyr(Me)-
L
-Ile-
L-Pip-)
N
CH3
OH
O
-Lys(Ac,OH)-
D
-Tyr(Me)-
L
-Ile-
D
-Pip-)
88.4
N
CH3
OH
O
O
10e
10f
cyclo(-
L
L
-Hly(For,OH)-
-Hly(For,OH)-
D
D
-Tyr(Me)-
L
-Ile-
L
-Pip-)
LDLL
LDLD
5
5
0.141
0.084
N
H
H
OH
cyclo(-
-Tyr(Me)-
L
-Ile-D
-Pip-)
N
OH
O
10g
10h
10i
10j
cyclo(-
cyclo(-
cyclo(-
cyclo(-
L
L
L
L
-Hly(Ac,OH)-
D
-Tyr(Me)-
L
-Ile-
L
-Pip-)
LDLL
LDLD
LDLL
LDLD
5
5
6
6
58.0
N
CH3
CH3
OH
O
-Hly(Ac,OH)-
D
-Tyr(Me)-
L
-Ile-D
-Pip-)
43.8
N
OH
O
-Aoc(For,OH)-
D
D
-Tyr(Me)-
L
-Ile-
L
-Pip-)
1.350
0.893
N
H
H
OH
O
-Aoc(For,OH)-
-Tyr(Me)-
L
-Ile-
D-Pip-)
N
OH
O
10k
10l
cyclo(-
L
L
-Hly(For,OH)-
-Hly(For,OH)-
D
-Tyr(Me)-
L
L
-Ile-
L
-Pro-)
LDLL
LDLD
5
5
NT
NT
N
H
OH
O
cyclo(-
D
-Tyr(Me)-
-Ile-D-Pro-)
N
H
OH
H
N
CHAP498b
CHAP508b
cyclo(-
L
L
-Asu(NHOH)-
D
-Tyr(Me)-
L
L
-Ile-
L
-Pip-)
LDLL
LDLD
5
5
0.00396
0.00481
OH
OH
O
H
N
cyclo(-
-Asu(NHOH)-
D
-Tyr(Me)-
-Ile-D-Pip-)
O
Hly is homolysine and Aoc is 2,8-diamino octanoic acid, NT ¼ not tested.
methylene group as spacer and retrohydroxamate as the
functional group showed high inhibitory activity. On the
other hand, compounds with the same spacer length and
same cyclic tetrapeptide scaffold, but containing an
acetyl group instead of the formyl group showed very
weak activity possibly due to the presence of the bulky
methyl group instead of the hydrogen atom. Com-
pounds with four methylene spacers and six methylene

2430
N. Nishino et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2427–2431
Table 2. HDAC inhibitory data for selected compounds
Compound
HDAC inhibitory activity IC50 (lM)
p21 Promoter assay
HDAC1
HDAC4
HDAC6
HDAC8
EC1000 (lM)
TSA
0.019
0.0044
0.0004
0.026
0.033
0.067
0.020
NT
0.028
0.110
0.013
0.782
0.029
0.178
0.04
NT
0.19
NT
CHAP30
CHAP31
10f
0.003
0.074
0.089
0.19
NT
0.022
NT
NT
0.046
0.034
0.059
10k
10l
0.61
NT ¼ not tested.
spacers showed weak activity in comparison with five
methylene spacer indicating that the spacer length is
optimum with five methylene units as in the case of
CHAPs. Compounds with LDLD combination showed
better activity than LDLL combination.
CHAPs. During the synthesis of these cyclic tetrapep-
tides containing retrohydroxamates, a report on straight
chain retrohydroxamates was published.21 Our cyclic
tetrapeptide retrohydroxamates are more than 10 times
potent than those reported retrohydroxamates indicat-
ing the importance of the cyclic tetrapeptide cap group.
The inhibitory activity toward the subclass of HDAC
for selected compounds is shown in Table 2.18 For
comparison, the inhibitory activities of TSA and corre-
sponding CHAPs, such as CHAP31 and CHAP 30 are
also shown. Retrohydroxamates inhibit HDACs in
almost equal extent as TSA. However, the inhibitory
activity of retrohydroxamates is lower than that of
CHAP31. In an early report on retrohydroxamate
inhibitor for thermolysin, the inhibitory activity was
also reduced about one tenth of the corresponding
hydroxamic acid.19 The HDAC inhibitory activity of
retrohydroxamates is almost similar for all the members
of the HDAC family. Proposed mode of interaction of
retrohydroxamates with zinc ion at the active site pocket
is shown in Figure 2.
We synthesized cyclic tetrapeptides containing a retro-
hydroxamic acid as suitable replacement of the
hydroxamic acid side chain of HDAC inhibitors. Some
of these compounds such as 10f and 10l showed inhib-
itory activity in nanomolar concentrations. In conclu-
sion, it has been shown that alternatives to the
hydroxamate zinc-binding group can lead to potent
inhibitors of HDAC and anticancer agents.
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