Highly potent and selective histone deacetylase 6 inhibitors designed based on a small-molecular substrate

Article abstract of DOI:10.1021/jm060554y

To find novel histone deacetylase 6 (HDAC6)-selective inhibitors and clarify the structural requirements for HDAC6-selective inhibition, we prepared thiolate analogues designed based on the structure of an HDAC6-selective substrate and evaluated the histo

Full text of DOI:10.1021/jm060554y

J. Med. Chem. 2006, 49, 4809-4812
4809
Chart 1. Examples of HDAC Inhibitors
Highly Potent and Selective Histone Deacetylase 6
Inhibitors Designed Based on a Small-Molecular
Substrate
Takayoshi Suzuki,*,† Akiyasu Kouketsu,† Yukihiro Itoh,†
Shinya Hisakawa,† Satoko Maeda,‡ Minoru Yoshida,‡,§
Hidehiko Nakagawa,† and Naoki Miyata*,†
Graduate School of Pharmaceutical Sciences,
Nagoya City UniVersity, 3-1 Tanabe-dori, Mizuho-ku, Nagoya,
Aichi 467-8603, Japan, RIKEN, Saitama 351-0198, Japan, and
CREST Research Project, Japan Science and Technology Agency,
Saitama 332-001, Japan
ReceiVed May 11, 2006
Abstract: To find novel histone deacetylase 6 (HDAC6)-selective
inhibitors and clarify the structural requirements for HDAC6-selective
inhibition, we prepared thiolate analogues designed based on the
structure of an HDAC6-selective substrate and evaluated the histone/
R-tubulin acetylation selectivity by Western blot analysis. Aliphatic
compounds 17b-20b selectively caused R-tubulin acetylation over
histone H4 acetylation. In enzyme assays using HDAC1, HDAC4, and
HDAC6, compounds 17a-19a exhibited HDAC6-selective inhibition
over HDAC1 and HDAC4.
Chart 2. Thiolate HDAC Inhibitors
Introduction
Histone deacetylasesa (HDACs) are responsible for the
deacetylation of the acetylated lysine residues in the N-terminal
region of the core histones and are involved in transcriptional
regulation, cell cycle progression, and developmental events.1
Thus far, eighteen HDAC family members have been identified,
and they are divided into two categories, i.e., zinc-dependent
enzymes (HDAC1-11) and NAD+-dependent enzymes (SIRT1-
7).1a,2 HDAC6, a zinc-dependent HDAC isoform, is unique in
that it is cytoplasmic and participates in the deacetylation of
nonhistone proteins, such as R-tubulin and HSP90, as well as
regulating important biological processes including microtubule
stability and function, and molecular chaperon activity.3 In
addition, it has recently been reported that inhibition of HDAC6
has an antitumor effect in multiple myeloma cells.4 Thus,
HDAC6-selective inhibitors are of interest not only as tools for
elucidating the more intricate biological functions of HDAC6,
but also as candidate antitumor agents.
A number of structurally diverse HDAC inhibitors have been
identified,5 including 1 (trichostatin A, TSA),6 2 (suberoylanilide
hydroxamic acid, SAHA),7 3 (CHAP31),8 4 (trapoxin B, TPX
B),6b,9 and 5 (MS-275)10 (Chart 1). Most hydroxamates, such
as 1 and 2, inhibit all of the HDAC isoforms, whereas most
non-hydroxamates, such as 4 and 5, do not inhibit HDAC6.3b,c,11
To date, the only known HDAC6-selective inhibitor is 6
(tubacin) (Chart 1), which was discovered using a combinatorial
approach.12 At present, there is little information about the
structural requirements for HDAC6-selective inhibition. There-
fore, there is a need to develop novel HDAC6-selective
inhibitors and then to study their structure-activity relationships.
We recently described a series of thiol-based analogues,
including 7 (NCH-26) and 8 (NCH-31) (Chart 2), which act as
novel non-hydroxamate HDAC inhibitors.13 Thiols are thought
to inhibit HDACs by coordinating the zinc ion which is required
for deacetylation of the acetylated lysine substrate. Further, the
S-isobutyryl prodrugs 9 (NCH-47) and 10 (NCH-51) (Chart 2),
which are thought to be hydrolyzed to the free thiols within
cells, showed potent cancer cell growth-inhibitory activities.14
Following these findings, we performed further investigation
of thiolate analogues, seeking to find HDAC6-selective inhibi-
tors. We describe here the HDAC6 selectivity of thiolates whose
designs were based on the structure of a small-molecular
HDAC6-selective substrate.
Results and Discussion
Since HDAC6 has been reported as an R-tubulin de-
acetylase,3b,3c inhibition of HDAC6 and that of other HDACs
can be assessed according to the accumulation of acetylated
R-tubulin and acetylated histones, respectively, using Western
blot analysis. We initially examined the histone/R-tubulin
acetylation selectivity of 2, 9, and 10 (Figure 1). As reported
previously,11b 2 caused the accumulation of both acetylated
histone H4 and acetylated R-tubulin. Like other non-
* To whom correspondence should be addressed. Tel and fax:
+81-52-836-3407; e-mail: suzuki@phar.nagoya-cu.ac.jp; miyata-n@
phar.nagoya-cu.ac.jp.
†
Graduate School of Pharmaceutical Sciences, Nagoya City University.
‡ RIKEN.
§ CREST Research Project.
a Abbreviations: HDAC, histone deacetylase; SIRT, sirtuin; AU, arbitary
unit.
10.1021/jm060554y CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/18/2006

4810 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Letters
Scheme 1a
Figure 1. Western blot detection of acetylated R-tubulin and histone
H4 levels in HCT116 cells after incubation with compounds 2, 9, and
10 for 8 h.
a Reagents: (a) (Boc)2O, Et3N, THF, H2O, rt, 96%; (b) ArNH2, POCl3,
pyridine, -15 °C, 10-48%; (c) RNH2, 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluroniumhexafluorophosphate, 1-hydroxybenzotriazole hydrate,
Et3N, DMF, rt, 35-63%; (d) thioisobutyric acid, Et3N, EtOH, rt, 19-58%.
Figure 2. Design of HDAC6-selective inhibitors.
Figure 4. Western blot detection of acetylated R-tubulin and histone
H4 levels in HCT116 cells after 8 h treatment with 12b-20b.
butyric acid under alkaline conditions to yield the desired
thioesters 12b-20b.
We initially evaluated compound 12b for the accumulation
of acetylated R-tubulin and histone H4 using Western blot
analysis (Figure 4). Although compound 12b produced an
increase in the accumulation of acetylated R-tubulin as compared
with 9 and 10 (Figure 1), the selectivity was insufficient. In an
attempt to improve the selectivity of the histone/R-tubulin
acetylation, we decided to carry out the structural conversion
of compound 12b. The coumarin structure derived from a
substrate for fluorescent enzyme assays was replaced with
various aromatic (13b-16b) or aliphatic (17b-20b) moieties
(Figure 3). Interestingly, although the aromatic compounds
13b-16b did not show high selectivity, the aliphatic compounds
17b-20b produced a dose-dependent increase of R-tubulin
acetylation without a major increase in acetylated histone H4
(Figure 4). These results indicated that the aliphatic compounds
17b-20b selectively inhibit HDAC6 in preference to other
HDACs in cells. To quantify the selectivity of cyclopentyl 17b,
one of the most active R-tubulin acetylating agents in this series,
acetylated R-tubulin and histone H4, were measured over a range
of concentrations (Figure 5). The estimated EC50 values of
cyclopentyl 17b for R-tubulin acetylation and histone H4
acetylation were 0.23 µM and >32 µM, respectively, and the
selectivity index (histone acetylation EC50/R-tubulin acetylation
EC50) was >140 which exceeded those of 2 (SI ) 2.0) and 6
(SI ) 75)11b (Figure 5 and Figure 1S of Supporting Information).
To confirm the HDAC6-selectivity of these aliphatic com-
pounds, we performed in vitro enzyme assays. For the enzyme
assays, we synthesized compounds 17a-19a, the corresponding
thiols of 17b-19b. Compounds 17a-19a were prepared by the
Figure 3. Structures of the thioesters 12b-20b.
hydroxamate HDAC inhibitors, compound 9 selectively caused
histone H4 hyperacetylation, which indicates that compound 7
selectively inhibits nuclear HDACs over cytoplasmic HDAC6.
However, unlike other non-hydroxamates, compound 10 in-
creased the acetylation state of both histone H4 and R-tubulin.
These results suggested that HDAC6-selective inhibitors might
be obtained by structural modification of thiolate HDAC
inhibitors.
In designing novel HDAC6-selective inhibitors, we focused
initially on a small-molecular HDAC6-selective substrate 1115
(Figure 2). Jung and co-workers found that compound 11 is
selectively deacetylated by HDAC6 in preference to HDAC1
and HDAC3. This indicated that the structure of N-Boc and
trifluoromethyl coumaryl amide of compound 11 is selectively
recognized by HDAC6, and so we considered that compound
12a, in which the acetamide of 11 is replaced by a thiol, might
behave as an HDAC6-selective inhibitor (Figure 2).
Since we used a cellular assay as the first screening,
compound 12b (Figure 3), the S-isobutyryl prodrug of com-
pound 12a, and its derivatives 13b-20b were initially prepared.
The route used for synthesis of compounds 12b-20b is shown
in Scheme 1. (S)-2-Amino-7-bromoheptanoic acid 2116 was
treated with (Boc)2O to give N-Boc compound 22. The
condensation of carboxylic acid 22 with an appropriate amine
afforded amides 23. Bromides 23 were treated with thioiso-

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4811
some sterically bulky alkyl groups can be placed and other
HDAC isoforms do not have such a pocket.
In conclusion, we have identified novel HDAC6-selective
inhibitors whose designs were based on the structure of the
HDAC6-selective substrate 11. As far as we could determine,
they are the first inhibitors that show significant HDAC6-
selective inhibition in both cellular and enzyme assays. We have
also established that the presence of a bulky alkyl group in these
compounds is important for HDAC6-selective inhibition. The
structures of the newly discovered HDAC6-selective inhibitors
17-20 are simpler than that of 6 and appear to be suitable as
lead structures for the further development of superior HDAC6-
selective inhibitors. These findings provide a basis for construct-
ing new tools for probing the biology of HDAC6 and for finding
new candidate antitumor agents with potentially fewer side
effects.
Figure 5. Quantification of acetylated R-tubulin and histone H4 levels
in HCT116 cells treated for 8 h with 17b. Compound 17b was insoluble
in 0.1% DMSO-McCoy5A culture medium at concentrations >300
µM.
Scheme 2a
Acknowledgment. This research was partly supported by
Grants-in Aid for Young Scientists (B) from the Ministry of
Education, Science, Culture, Sports, Science, Technology, Japan,
and grants from the Hori Information Science Promotion
Foundation, the Japan Securities Scholarship Foundation, the
Tokyo Biochemical Research Foundation, Takeda Science
Foundation, the TERUMO Lifescience Foundation and the
Program for the Promotion of Fundamental studies in Health
Science of the National Institute of Biomedical Innovation
(NIBIO), Japan. We thank the Screening Committee of New
Anticancer Agents, supported by a Grant-in-Aid for Scientific
Research on Priority Area “Cancer” from the Ministry of
Education, Culture, Sports, Science and Technology of Japan
for HDAC1 inhibition assay results.
a Reagents: (a) KSAc, EtOH, rt, 75-93%; (b) NaOH, H2O, EtOH, rt,
71-77%.
Supporting Information Available: Experimental procedures
including spectral data for compounds 12-20 and biological
methods (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
Table 1. In Vitro HDAC1-, HDAC4-, and HDAC6-Inhibitory Activities
of 17a-19aa
IC50 (nM)
selectivity
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