Development of a Potent and Selective HDAC8 Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.6b00239

A novel, isoform-selective inhibitor of histone deacetylase 8 (HDAC8) has been discovered by the repurposing of a diverse compound collection. Medicinal chemistry optimization led to the identification of a highly potent (0.8 nM) and selective inhibitor of HDAC8.

Full text of DOI:10.1021/acsmedchemlett.6b00239

Letter
pubs.acs.org/acsmedchemlett
Development of a Potent and Selective HDAC8 Inhibitor
Oscar J. Ingham,† Ronald M. Paranal,‡ William B. Smith,‡ Randolph A. Escobar,† Han Yueh,†
Tracy Snyder,† John A. Porco, Jr.,† James E. Bradner,‡,§ and Aaron B. Beeler*,†
†Department of Chemistry and Center for Molecular Discovery (BU-CMD), Boston University, 590 Commonwealth Avenue,
Boston, Massachusetts 02215, United States
‡Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts 02215, United States
§Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: A novel, isoform-selective inhibitor of histone
deacetylase 8 (HDAC8) has been discovered by the
repurposing of a diverse compound collection. Medicinal
chemistry optimization led to the identification of a highly
potent (0.8 nM) and selective inhibitor of HDAC8.
KEYWORDS: Histone deacetylase, HDAC, histone deacetylase 8, HDAC8, triazole, epigenetic
Scheme 1. General Reactions for Preparation of Hydroxamic
Acids/Esters (A) and Representative Methyl Esters Utilized
in the Derivatization (B)
istone/protein deacetylase (HDAC) enzymes constitute
Ha family of proteins involved in epigenetic regulation of
gene expression by deacetylation of histones.1 Many HDACs
are also responsible for the deacetylation of non-histone cellular
proteins, a process that is critical for regulation function and
localization. There are 11 known “classical” zinc-dependent
HDAC isoforms, which are divided into three classes according
to their sequence homology and organization. In recent years,
HDAC enzymes have emerged as attractive targets for disease
biology2 including cancer,3 neurodegenerative diseases,4 auto-
immunity, and transplant rejection.5
It is well established that metal-chelating motifs bind tightly
to the Zn(II) ion in the active site of HDAC enzymes and can
lead to reversible inhibition of their biological function.6 HDAC
inhibitors (HDACi) have great potential as therapeutic agents,
with several advancing into clinical trials; and multiple pan-
active HDACi are FDA approved for T-cell lymphoma and
multiple myeloma.2 There is clear interest in continued
development of isoform-specific inhibitors as potential
therapeutic agents or as tools to further understand selectivity
and to discern the function of HDAC isoforms.
In an effort to discover new small molecules with HDAC
subtype selectivity,7 we looked to take advantage of an existing
small molecule library available at the Boston University Center
for Molecular Discovery (BU-CMD). We designed a study to
repurpose complex libraries and advanced synthetic inter-
mediates to focus them toward HDAC inhibition activity by
addition of strong Zn (II)-chelating moieties.8,9 Utilizing
established methodologies, we carried out direct, mild
conversion of esters to hydroxamic acids and methyl
hydroxymates (Scheme 1, A). In the presence of catalytic
cyanide and excess hydroxylamine, a set of methyl esters (1),
were converted to hydroxamic acids 2,10 and a similar
transformation was carried out with trimethyl aluminum and
methanolamine to afford methyl hydroxamates 3.11
A structurally diverse set of 134 esters (Scheme 1B) was
reacted in parallel on a one milligram scale. Products were
purified by mass-directed HPLC affording a total of 120
hydroxamic acids and methyl hydroxamates. Purified com-
pounds were assayed against HDAC isoforms 1−9 and a single
Received: June 17, 2016
Accepted: August 19, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00239
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ACS Medicinal Chemistry Letters
Letter
compound, triazole hydroxamic acid 4, showed good activity
(Figure 1). This compound was subsequently validated as a
potent inhibitor of HDAC812−16 (IC50 = 10 nM) with modest
inhibition of HDAC617 (3600 nM).
Scheme 2. General Synthetic Scheme for the Synthesis of
Triazole Analogues
enantiomer 14 and disubstituted triazole 15 were synthesized
and also found to be inactive (Figure 2).
With this preliminary information in hand, we synthesized an
array of analogs to determine more detailed SAR. Position R1
was varied utilizing alternative amino acids, and the R2 and R3
positions were modified using a series of terminal acetylenes.
We also synthesized saturated analogues to determine the
requirement of the alkyne. In total, 70 analogues were
synthesized and assayed for inhibition of HDAC8.
Figure 1. HDAC activity profile for triazole 4.
HDAC8 is classified as a class I HDAC but is unusual in
many respects. Unlike other isoforms, little is known about the
functions of HDAC8. Classical pan-active HDACi, such as
SAHA (vorinostat), bind to HDAC8 with substantially
diminished activity (IC50 = 2 μM), reflecting a unique binding
site of this isoform.18−21
Cellular functions of HDAC8 have only recently begun to be
identified.22 Deardorff and co-workers showed the correlation
of HDAC8 mutations to specific phenotypes in patients with
Cornelia de Lange syndrome and the apparent role of HDAC8
in deacetylation of SMC3, a critical protein in the cohesin
complex.23−25 Recently, Cristea and co-workers reported
interaction of HDAC8 with multiple cohesin proteins, SMC3,
SMC1a, STAG2, as well as an additional mitosis related protein
CROCC.26 These studies have begun to highlight the potential
role of HDAC8 in maintaining proper function of cohesin.
There is also evidence that HDAC8 modulates acetylation of
other proteins such as Oct3/4, Nanog, Cdh1, Rex1, p53, ERRα,
and CREB.27,28 A recent study by Lin and co-workers provides
evidence that HDAC8 may also have enzymatic function for
hydrolysis of larger fatty acids.29
The trisubstituted triazole scaffold was originally obtained via
an unusual copper-catalyzed tandem, [3 + 2]-cycloaddition-
coupling reaction between azide 5 and phenylacetylene.30 As
this approach was limited in yield and scope, a simple, high-
yielding synthetic route was developed to facilitate construction
of orthogonally substituted analogues (Scheme 2). Copper-
mediated Huisgen cycloaddition with phenylacetylene iodide 6
afforded iodotriazole 7.31 Sonogashira coupling with terminal
acetylenes afforded alkynyl triazoles 9, which were quantita-
tively converted to the desired hydroxamic acids 10.
In order to determine critical structure−activity relationships
(SAR), we first synthesized the corresponding carboxylic acid
11, methyl ester 12, and methyl hydroxamate 13 derivatives,
which were all found to be inactive against the entire profile of
HDACs, thereby establishing that the hydroxamic acid moiety
was necessary for HDAC8 inhibition. The corresponding (R)-
Across the total set of analogues, there was little effect on
isoform selectivity, but we observed significant impact on
HDAC8 inhibition potency.14 Small hydrophobic groups at the
R3 position, such as cyclopropane, were found to be beneficial
and afforded greater inhibition, highlighted by the 0.8 nM
activity of cyclopropyl congener 16 (OJI-1). Larger sub-
stituents were not tolerated as exemplified by the dramatic loss
of activity in p-ethylphenyl derivative 17 and biphenyl 18.
Introduction of alternative amino acids resulted in a loss of
activity compared to phenylalanine. For instance, indole
derivative 19 showed a 10-fold loss of potency and the valine
derived 20 was essentially inactive. Substitution at the 4-
position (R2) of the triazole was the most tolerant. Both
electron-donating (21) and electron-withdrawing (22) sub-
stituents were tolerated. Furthermore, heteroaromatic groups
such as thiophene (23) were well tolerated. However, there was
some loss of activity when larger hydrophobic substituents were
added such as t-butyl phenyl 24.
We also synthesized a number of analogues that evaluated
the oxidation level of the alkyne. Interestingly, reduction of the
alkyne resulted in loss of activity for all substrates except for the
n-pentyl and pentenyl derivatives 27 and 28, which were found
to be significantly less active.
Based on the available SAR, there appears to be five crucial
structural features necessary for selective HDAC8 inhibition
(Figure 3): (1) S-stereochemistry; (2) an aromatic moiety
adjacent to the hydroxamic acid; (3) small, aromatic ring fused
directly to the 4-position; (4) a small, hydrophobic acetylenic
functionality at the 5-position of the triazole; and (5) an alkyne
linker between the triazole and R3.
We also carried out evaluation of the most active analogue
(OJI-1) against a panel of HDACs (1−9) to verify selectivity
(Figure 4). As expected, there was very little activity against
other HDACs. Exceptions were HDAC6 and HDAC1, which
were mildly inhibited with IC50s of 1.2 and 4.3 μM, respectively.
OJI-1 is more potent and selective than what is reported for
PCI-34051 (HDAC8 IC50 = 10 nM, HDAC6 IC50 = 2.9 μM,
B
DOI: 10.1021/acsmedchemlett.6b00239
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ACS Medicinal Chemistry Letters
Letter
Figure 4. HDAC activity profile for triazole 16.
HDAC1 IC50 = 4 μM),15 which is a tool molecule most often
utilized to study the effects of HDAC8 inhibition.
In conclusion, by repurposing an existing collection of
structurally diverse molecules, we have discovered a selective
HDAC8 inhibitor. Medicinal chemistry efforts afforded robust
SAR resulting in one of the most potent and selective HDAC8
inhibitors reported. Ongoing efforts are focused on refining a
binding model through unambiguous crystallization studies and
utilizing OJI-1 to better understand the role of HDAC8 in
cellular function and its potential as a therapeutic target.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00239.
■
S
Experimental procedures, characterization data, and
HDAC inhibition data for all analogues (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: 617-358-3487. E-mail: beelera@bu.edu.
■
Funding
Financial support from Boston University and the Center for
Chemical Methodology and Library Development at Boston
University (GM-067041) are gratefully acknowledged. Work at
the BU-CMD is supported by R24-GM111625. NMR (CHE-
0619339) and MS (CHE- 0443618) facilities at Boston
University are supported by the NSF.
Figure 2. (A) Preliminary inactive analogues. (B) Representative R1−
R3 analogues and HDAC8 IC50. (C) Representative reduced
analogues.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Norman Lee (Boston University) for high-
resolution mass spectrometry data.
■
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Figure 3. SAR for HDAC8 Inhibition.
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