Histone Deacetylase 2 (HDAC2) Inhibitors Containing Boron

Article abstract of DOI:10.1002/cbic.202000131

Histone deacetylase enzymes (HDACs) are responsible for the global silencing of tumour-suppressor genes. Treatment with a histone deacetylase inhibitor (HDACi) can reverse this process and restore normal cell function. Herein, we report a small series of boron-based (boronic acid, boronate ester and closo-1,2-carborane) HDAC2 inhibitors with IC₅₀ values in the nanomolar range. The boronate ester 4 b was the most potent compound assessed in this study (IC₅₀=40.6±1.5 nM), followed closely by the 1,2-closo-carborane (IC₅₀=42.9±1.5 nM). Compound 4 b exceeds the potency of the related gold-standard HDAC pan-inhibitor vorinostat (1) toward this particular HDAC isoform.

Full text of DOI:10.1002/cbic.202000131

Combining Chemistry and Biology
Accepted Article
Title: Histone deacetylase 2 (HDAC2) inhibitors containing boron
Authors: Poya Kavianpour, Madeleine Gemmell, Jan Kahlert, and
Louis Rendina
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000131
Link to VoR: https://doi.org/10.1002/cbic.202000131
01/2020

10.1002/cbic.202000131
ChemBioChem
FULL PAPER
Histone deacetylase 2 (HDAC2) inhibitors containing boron
Poya Kavianpour[a], Madeleine C. M. Gemmell[a], Jan U. Kahlert[a] and Louis M. Rendina* [a],[b]
[a]
Mr. P. Kavianpour, Ms. M.C.M. Gemmell, Dr. J.U. Kahlert and Prof. L.M. Rendina
School of Chemistry
The University of Sydney
The University of Sydney, F11, Eastern Ave, Sydney NSW 2006, Australia
E-mail: lou.rendina@sydney.edu.au
[b]
Prof. L.M. Rendina
The University of Sydney Nano Institute
Camperdown NSW 2050, Sydney NSW 2006, Australia
Abstract: Histone deacetylase enzymes (HDACs) are known to be
responsible for the global silencing of the tumour suppressor genes.
Treatment with histone deacetylase inhibitors (HDACi) can reverse
this process and restore normal cell function. Herein we report a small
series of boron-based (boronic acid, boronate ester and closo-1,2-
carborane) HDAC2 inhibitors with IC50 values in the nM range. The
boronate ester 4b was the most potent compound assessed in this
study (IC50 = 40.6 ± 1.5 nM), followed closely by the 1,2-closo-
carborane derivative (IC50 = 42.9 ± 1.5 nM). Compound 4b exceeded
the potency of the gold-standard HDAC pan-inhibitor vorinostat 1
toward this particular HDAC isoform.
deacetylation of histones triggers characteristic elements of
cancer growth and progression: cell proliferation, inflammation,
[11, 15-16]
and tumorigenesis.
The acetylation of lysine residues of
histone tails is commonly associated with transcriptionally active
chromatin.[16] In the inactive phase, the negatively-charged DNA
phosphodiester backbone is bounded tightly to the positively-
[3, 12]
charged histone proteins.
The net positive charge of the
histone proteins is extinguished by acetylating the ԑ-amino group
of they lysine residue through the covalent attachment of an acetyl
[7, 17]
group from acetyl-CoA in the presence of HATs.
Consequently, the nucleosome structure is relaxed at the
modified site, exposing the DNA to transcriptional proteins.
Deacetylation dynamically counters the effect of acetylation, thus
restoring the positive charge of the lysine sidechain. [4, 11, 15] When
histones are deacetylated, the DNA is packed, and the chromatin
architecture cannot be accessed by the cell’s transcriptional
machinery. The widespread silencing of tumour genes triggered
by hyper-deacetylation has been established as a biomarker of
cancer.[6, 18-19] With an appropriate small molecule inhibitor, this
process can potentially be reversed.
Introduction
The rapidly growing field of epigenetics has shown great promise
in the development of first-in-class anti-cancer drugs. The ability
to regulate genes without altering their chromosomal DNA
sequence is an attractive alternative to currently used methods
due to its early biomarker detectability.[1] It is known that the
human genome experiences constant dynamic modifications that
are tightly regulated by two opposing factors, a set of genes
contributing to a malignant phenotype and inherent tumour
suppressor genes that have the ability to maintain homeostasis.[1-
2] Disruption of the genes essential for normal cellular growth can
result in dysfunctional cell behavior, including the expression of
oncogenes affiliated with cancer.[3]
The first FDA-approved HDAC inhibitor (HDACi), vorinostat (1), is
currently used for the treatment of cutaneous T-cell lymphoma
(CTCL). [20-24] Vorinostat is known as a ‘pan-HDAC inhibitor’ as it
possesses an ability to interact with several isoforms of HDAC
metalloenzymes at the Zn2+ site by coordination involving its
25-26]
hydroxamic group.[20,
Though a potent chemotherapeutic
agent, vorinostat lacks selectivity for any specific HDAC
isoform(s). Class I HDAC inhibitors that have shown potent
binding to the Zn2+ co-factor in HDACs possess ‘hard’ Lewis
bases, e.g. hydroxyl, carbonyl, sulfonyl and carboxylic acids, that
are ideal for ‘hard’ Lewis acid target engagement of the Zn2+
ion.[26-28] In recent years, boron drug discovery is making great
inroads in medicinal chemistry with the approval of three
boronated drugs (bortezomib, crisaborole, and tavaborole) for
clinical use. However, examples of boronated HDACi’s are rare.
Indeed, such studies have solely focused on the use of boronic
acids to coordinate to the Zn2+ ion in Class I, II and IV HDACs but
with only limited success.[29-31] It has been shown that, to date,
hydroxamic acid-based drugs such as 1 are still amongst the most
potent inhibitors of Class I HDACs.[20, 26, 32-33]
Global mutations at the genomic level have long been associated
4-5]
as hallmarks of cancer.[1,
In cancer, the regulatory tumour
mechanism that control normal cell growth and homeostasis are
turned off leading to tumorigenesis.[6-7] Recent studies have
shown the epigenetic state of a cell is malleable and prone to
evolve in response to its environment.[1, 3, 8-10] These responses
have been correlated to a histone remodeling mechanism,
involving the covalent re-organization of histone proteins as a
critical component of its biomarker for early detection. The highly
dynamic acetylation status of the amino acid residues in histone
proteins is controlled by two families of enzymes, histone
acetyltransferases (HATs) and histone deacetylase (HDACs),
6, 11]
which can be modulated in an opposing manner.[3,
Of
increasing interest are the histone deacetylase enzymes
(HDACs) which are known to be responsible for the global
8,
silencing of the tumour suppressor genes.[4,
10-14]The
1
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10.1002/cbic.202000131
ChemBioChem
FULL PAPER
Scheme 1. Synthesis of boronated derivatives of 1.
To the best of our knowledge, modification of the phenyl capping
group in 1 with boron moieties has not been reported previously.
There is compelling evidence that modification of the capping
group in 1 can potentially direct exquisite HDAC selectivity.[2, 30, 33]
To date, the X-ray crystal structures of all human HDAC Class I
have been reported.[34-38] With the potential use of isoform-
selective HDACi’s, Class I HDACs appear to be the most
abundant and overexpressed isoforms in various cancers thereby
rationalizing the requirement for novel Class I inhibitors that might
lead to improved cancer therapeutics. Indeed, the targeting of
overexpressed proteins in tumor cells with boron moieties has
been the subject of intense interest in recent years.[39-41] Of
notable interest is recent discovery of carborane-based epidermal
and reproducibility of this particular synthetic method over other
known routes.
Commercially-available octanedioic acid was readily converted
into the anhydride oxonane-2,9-dione (2) in quantitative yield by
means of condensation reaction involving acetic anhydride. By
using similar conditions to those reported in the preparation of 1,
the commercially-available 4-aminophenylboronic acid pinacol
ester and (3-aminophenyl) boronic acid was stirred with 2 in THF
solution for 1 h (Scheme 1). The success of this reaction was
found to be dependent on the ability of the poorly-nucleophilic
amine attacking the electrophilic carbonyl atom of the anhydride.
Short reaction times coupled with use of dry solvent successfully
afforded 3b (85%) and 3d (80%), respectively, in excellent yields.
42]
growth factor receptor (EGFR) inhibitors by Couto et al.[39,
These authors determined that the incorporation of closo-1,7-
carborane cages into an existing EGFR inhibitor led to a
significant (ca. 10-fold) increase in inhibition of this receptor when
compared to the parent (organic) inhibitor.[40, 42]
1-(p-Nitrophenyl)-closo-1,2-carborane
was
prepared
as
described previously by Hawthorne et al.[44] The reduction of the
nitro group was achieved by means of a hydrogenation reaction
employing a Pd/C catalyst in MeOH, and the resulting anilino-
closo-1,2-carborane precursor was purified by means of column
chromatography in high yield (85%). The preparation of 3c was
successfully achieved by reacting the anilino-closo-1,2-carborane
with 2. Longer reaction times than those used to prepare the other
boronated derivatives of 3 allowed for high conversion to 3c (80%)
with no further purification required due to its crystallisation from
aqueous solution.
X-ray crystallographic analyses of Class I HDACs has shown that
the capping group of HDACi is exposed to solvent and interacts
with amino acid residues located near the entrance of the active
catalytic channel.[33, 36] Given the capping region of the substrate
channel is the most variable amongst the HDAC family, potential
modifications at this site could generate inhibitors with excellent
isoform selectivity. Of direct interest in this work are the boron
clusters such as the closo-carboranes which have shown high
protein receptor affinity and inhibition of enzymes by binding to
hydrophobic cavities at or near the receptor site.[27, 33, 43] Herein,
we report the synthesis and a preliminary in vitro inhibition study
of selected group of boronated HDAC2 inhibitors in which the
nature of the boron moiety was varied from boronic acid to
boronate ester to 1,2-closo-carborane. Such modifications will
necessarily change the nature of the hydrophobicity/hydrophilicity
and steric bulk of the capping group in this class of HDACi’s, and
these effects upon target engagement will be explored in this
study.
The conversion of the carboxylic acid groups of 3b and 3d to the
hydroxamic acid derivatives required first, the formation of ethyl
carbonic anhydride intermediate by ethyl chloroformate in the
presence of triethylamine. Second, the addition of the freshly
prepared hydroxylamine resulted in the formation of the desired
target compounds 4b and 4d in 65% and 70% yields, respectively.
Purification of crude 4b proved to be non-trivial, however, owing
to its highly hygroscopic nature.
In the final conversion of 3c to the desired 4c, the potential risk of
cage deboronation of the closo-1,2-carborane in the presence of
hydroxylamine was considered. To minimize the attack of the
nucleophilic base at the boron cluster, a short reaction time (10
min) was used. 11B{1H} NMR spectroscopy revealed that a minor
amount of deboronation of the carborane cage did indeed occur
despite the short reaction time, with the nido-carborane peaks
observed at the expected upfield shifts (δ = -21.06 to -37.12).
Given the hydrophilic nature of the nido-7,8-carborane anion by-
product over the hydrophobic closo-carborane, purification was
performed by means of an aqueous workup to yield the desired
Results and Discussion
There are several established synthetic routes for the synthesis
of the archetypal HDAC inhibitor 1. The widely used methodology
described by Mai et al.[23] was employed in order to validate new
synthetic routes for the synthesis of 4b-d owing to the high-yield
2
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10.1002/cbic.202000131
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of Y198 and HDAC8 with a noticeable loop L1, formed by the
insertion of seven amino acid residues (located at positions 30-
36).[51] The presence of these secondary features allows for a
more facile exploitation and selective binding of an inhibitor due
to their absence from other Class I HDACs. Unsurprisingly,
HDACi research over the past decade has focused on the design
of HDAC3 and HDAC8 inhibitors due to their significant structural
differences.[31, 52-55]
closo-1,2-carborane 4c in good yield (75%). The product was
confirmed by means of ESI-MS with a molecular ion peak
observed at m/z 431.3079 for [M + Na]+ (calculated: m/z
431.3079).
Table 1. IC50 values for the assessed compounds 1 and 4b-d.
Compound
IC50 ± SEMa (nM)
1
44.3 ±1.6
40.6 ±1.5
42.9 ±1.5
61.2 ±1.7
To date, integration of boron moieties into HDAC inhibitors such
as 1 has only had limited success, with their in vitro potency being
surpassed by more traditional organic fragments. Previous work
in this field has focussed on the re-design the skeletal structure of
1 by altering either (a) its linker or (b) Zn2+-binding domain to
improve the drug’s metal-complexing capability. For instance,
Suzuki and co-workers prepared a library of boronic acid
structures by altering the linker component of the skeletal
structure.[30] Here, the researchers replaced the hydroxamic acid
moiety with boronic acid group. Their design logic was consistent
with the mechanism of action of inhibitors such as 1 where the
hydroxamic acid moiety interacted with the Zn2+ ion which is
located near the ‘foot’ pocket of the enzyme active site. It was
thought that the increased electrophilicity of the carbonyl oxygen
in the substrate would result from coordination to the Lewis acidic
Zn2+ ion. The coordination of the metal ion made the carbonyl
group prone to a nucleophilic attack by a water molecule activated
by the nearby His140 and His141 residues. This particular
interaction results in a tetrahedral transition state which is
stabilised by the Zn-O interaction and additional hydrogen
bonding interactions with Tyr303 and His140. The proton transfer
from His141 to the N-atom of the intermediate triggers scission of
the C-N bond to afford the acetate and lysine products. By
replacement of the hydroxamic acid by boronic acids, Suzuki et
al. proposed the vacant p-orbital of the boron atom would interact
with the water molecule at the active site to afford a stable
tetrahedral boronate complex. This complex would then bind the
Zn2+ ion and form two hydrogen bonds to Tyr303 and His140
(HDAC1 numbering), thus leading to HDAC inhibition. Despite
their efforts, the authors failed to obtain a boronic acid inhibitor
which was at least comparable to 1 in terms of potency.
4b
4c
4d
aSEM = standard error of the mean (n = 3).
The ability of compounds 1 and 4b-d to inhibit HDAC2 activity was
assessed by means of an in vitro HDAC2 assay. Notably, all
compounds assessed in this work were found to exhibit potent
HDAC2 inhibition at the nM level (Table 1). Compound 4b
(pinacol ester) was found to be the most potent inhibitor of HDAC2
followed by 4c (closo-1,2-carborane), 1 and 4d (boronic acid). It
is clear that the electrophilic boronate ester group enhanced
HDAC2 inhibition compared to 1 but, notably, the bulkier and
more hydrophobic closo-1,2- carboranyl group also led to a potent
derivative 4c even though it does not possess any electrophilic
boron atoms. These results indicate that hydrophobicity of the
capping group might be an important parameter governing the
nature of the capping group. Indeed, the hydrophilic boronic acid
4d was the least potent inhibitor prepared in this study. The Hill
coefficient was determined for 4b-d and, in each case, it was
equal to 1, consistent with independent (non-cooperative) binding
of the boronated inhibitors to HDAC2. Motifs such as cyclic
peptides and macrocyclic capping groups that can potentiate
greater interactions with amino-acid residues at the rim of the
channel than the traditional phenyl cap in 1 have been explored
for their ability to confer HDAC selectivity.[45-49] The boronated
compounds prepared in this work add to this group of motifs and
demonstrate that the nature of the capping group motif does not
significantly alter the nature of target engagement but instead
increased hydrophobicity and/or steric bulk appears to confer
selectivity for Class I HDACs, in this case HDAC2.
Another past study of relevance to this work was a computational
study undertaken by Bakri et al. where they developed an in-silico
query using various integrated software packages.[31] In this
study, multiple sequence alignment was performed against HDAC
class II human enzymes, resulting in an idealised molecular
docking simulation. In conjunction with Lipinski’s ‘rule of five’, and
Egan’s and Veber’s rules, the paper reported four new boronated
ligands that met these parameters with good predicted oral
bioavailability. Further analysis of the ligand’s health impact
including ADME (absorption, distribution, metabolism, excretion)
and toxicology were estimated using the Benigni-Bossa rules.
These four ligands also were found to have more negative
ΔGbinding values than that of 1. It was concluded by these authors
that the use of boron derivatives of 1 have promising potential as
HDAC II enzyme candidates but no such candidates have been
prepared to date.
Current HDACis are mostly pan-inhibitors with a number of side-
effects including fatigue, dehydration and thrombocytopenia.
Even though the therapeutic advantages of isoform-selective
HDACis are yet to be proven clinically proven, it is possible that
higher selectivity for one particular HDAC isoform may result in a
better therapeutic index with fewer adverse side-effects. By
studying the currently-available X-ray crystal structures of Class I
HDAC enzymes, an elucidation of both mechanism and relative
orientation of the inhibitor at the active site can be determined. X-
ray diffraction data have shown that HDAC1 and HDAC2 possess
a single residue difference at S262 and A264, respectively.[50-51]
The ability to design a small molecule inhibitor that can
successfully distinguish between these two isoforms would allow
one to determine whether exploiting the small difference between
the HDAC1 and HDAC2 isoforms can lead to a higher isoform
selectivity and potency. HDAC3 and HDAC8 have a more
pronounced difference in their protein structure, with HDAC3
having a single insertion of F199 residue altering the orientation
3
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10.1002/cbic.202000131
ChemBioChem
FULL PAPER
fluorescence intensity value at the specified compound concentration. A
positive control (Trichostatin A) was used.
Conclusion
We have successfully prepared a small library of boronated
compounds that possess the ability to alter the Zn2+-binding
domain of 1. Our lead inhibitor 4b shows a higher HDAC2 potency
over the gold standard HDAC drug 1. One unique feature of Class
I HDAC enzymes such as HDAC2 over the other enzyme classes
is the presence of a 14 Å internal cavity, located at the internal
bottom channel adjacent to the Zn2+ binding site.[33, 35, 56] It is clear
that HDACis incorporating hydroxamates are the most potent
enzyme inhibitors to date, however, X-ray diffraction studies show
only a minimal set of non-covalent interactions between the
hydroxamic acid and adjacent cavity [57]. The exploitation of this
cavity is one intriguing way of enhancing enhance isoform
selectivity. Research in the field suggests that 2-aminoanilide is
Compounds 1, 2 and 3a were prepared by the methods of Mai et al.[23] and
were isolated without further purification. Compound 3a was isolated as
two polymorphic white solids (2.41 g, 63%) and (0.50 g, 13%),
respectively.
8-Oxo-8-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)amino)octanoic acid (3b): Compound 3b was prepared by
adapting a procedure described by Mai et al. Compound 2 (1.41 g, 9.1
mmol) and 4-aminophenylboronic acid pinacol ester were stirred in THF
for 1 h at RT. The resulting mixture was filtered off to give 3b as an off-
white powder (2.5 g, 85% yield). 1H NMR (300 MHz d6-DMSO, δ): 1.28 (s,
12H), 1.34 (m, 4H), 1.56 (m, 4H), 2.18 (m, 2H), 2.30 (m, 2H), 6.52 (d, 2H,
3JHH = 8.46 Hz, Ph), 7.31 (d, 2H, 3JHH = 8.40 Hz, Ph), 9.84 (s, 1H, NH) and
11.95 (s, 1H, COOH). ESI-MS (m/z): 398 (100%, [M + Na]+).
an excellent fragment with a higher selectivity for HDACs 1-3
[52,
when compared to related hydroxamates
58-59]. An X-ray
8-(1-(p-aminophenyl)-closo-1,2-carborane)-8-oxooctanoic acid (3c):
Compound 3c was prepared by adapting a procedure described by Mai et
al. Compound 1 (0.9 g, 1.2 mmol) 1-(p-aminophenyl)-closo-1,2-carborane
were stirred THF for 1.5 h at RT. The resulting mixture was filtered off to
give 3c as a yellow powder (2.5 g, 85%). ATR-FTIR (cm-1): 3292 (br, OH),
2935 (CH), 1656 (C=O), 1599 (C=O). 1H NMR (400 MHz, d6-DMSO, δ):
1.22-3.40 (br, BH), 1.36 (br, 4H), 1.61 (br, 4H), 2.22 (t, 2H), 2.29 (t, 2H),
3.91 (s, 2H). 4.41 (br, CH), 4.9 (br, NH), 7.58 (d, 2H, 3JHH = 7.77 Hz, Ph),
crystallographic study of HDAC2 with an 2-aminoanilide inhibitor
has confirmed an anilide interaction with the ‘foot’ pocket adjacent
to the catalytic region. By expanding on the substituted 5-position
of the ring, compounds containing the anilide moiety have shown
up to a 31-fold higher selectivity for HDAC1 over HDAC2, with
potencies in the nM range [60]. Of greater interest is the inclusion
of bulky, branched capping groups allowing for an additional 10-
3
7.71 (d, 2H, JHH = 7.71 Hz, Ph), 8.57 (br, COOH). 11B{1H} (128 MHz,
fold increase in potency[61]
. The inclusion of 2-aminoanilide into
DMSO-d6, δ): -12.4, -11.0, -8.7, -4.5, -2.4. ESI-MS (m/z): 414.20 (61%,
[M+Na]+), 415.21 (100%, [M+Na]+), 416.20 (80%, [M+Na]+), 417.21 (43%,
[M+Na]+).
our boronated structures has the potential to give low nM HDAC2
inhibitory activity, and we are currently studying the effects of
enhancing the bulk of the boronic ester at the capping position in
conjunction with the incorporation of a substituted 2-aminoanilide
ring. The results of this work will be reported in due course.
8-((3-Boronophenyl)amino)-8-oxooctanoic acid (3d): Compound 3d
was prepared by adapting a procedure described by Mai et al. Compound
1 (1.51 g, 9.1 mmol) and (3-aminophenyl)boronic acid (2.0 g, 9.1 mmol)
were stirred in THF for 1.5 h at RT. The resulting mixture was filtered off
to give 3d as an off-white powder (2.5 g, 85%). ATR-FTIR (cm-1): 3292 (br,
OH), 2935 (CH), 1656 (C=O), 1599 (C=O). 1H NMR (300 MHz, d6-DMSO,
δ): 1.33 (m, 4H), 1.49 (m, 4H), 1.60 (m, 4H), 2.18 (m, 2H), 2.29 (m, 2H),
7.45 (d, 2H, 3JHH = 7.46 Hz, Ph), 7.71 (d, 2H, 3JHH = 7.71 Hz, Ph), 9.76 (s,
1H, NH) and 11.94 (s, 1H, COOH). ESI-MS (m/z): 316 (100%, [M + Na]+).
Experimental Section
Material and methods: NMR spectra were recorded on a Bruker Avance
III 300 or 400 spectrometers in DMSO-d6 unless otherwise noted (1H at
400.13 MHz and 11B at 128.37 MHz). All chemical shifts (δ) are given in
ppm relative to TMS = 0, using trace isotopic protons in deuterated
solvents as the internal reference, relative integral, multiplicity (s= singlet,
br = broad singlet, d = doublet, dd= doublet of doublets, dt = double of
triplets, t = triplet, m = multiplet), and coupling constants (J, Hz). Low
resolution mass spectrometry was performed using ThermoQuest
Finnigan LCQ-Deca ion trap mass spectrometer with electrospray
ionization in positive (+ESI) or negative (-ESI) mode. Column
chromatography was carried out over Scharlau 60 40-60 µm silica. Thin
layer chromatography (TLC) was carried out on Merck Kiesel gel 60 F254
aluminum back plates. Visualization of carborane-containing compounds
on TLC plates was achieved using an acidified PdCl2 (1%) stain and heat.
1,2-closo-carborane was purchased from Katchem (Czech Republic).
Compounds 4b - 4d were prepared by adapting a procedure described
Mai et al.[23] At 0 oC, solutions of 3b - d were treated with excess amounts
of triethylamine and ethyl chloroformate, followed by the addition of freshly-
prepared hydroxylamine (4.3 mmol).
N1-Hydroxy-N8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pheny-
l)octanediamide (4b): Brown oil (312 mg, 71%). 1H NMR (400 MHz, d6-
DMSO, δ): 1.28 (s, 12H), 1.34 (m, 4H), 1.56 (m, 4H), 2.18 (m, 2H, H21,22),
2.30 (m, 2H), 6.52 (d, 2H, 3JHH = 8.46 Hz, Ph), 7.31 (d, 2H, 3JHH = 8.40 Hz,
Ph), 9.84 (s, 1H, NH) and 10.95 (br, 1H, OH). 11B{1H} (128 MHz, DMSO-
d6, δ): 30.9. HRMS (+ESI): Calc. for C20H31BN2O5: 413.22182, found
413.22183 ([M+Na]+).
The fluorogenic assays was performed using the HDAC2 assay kit (Green)
purchased from BPS Bioscience (San Diego, CA). A 50 µL buffer mixture
containing the human recombinant HDAC2 enzyme isoform, test
compound (dissolved in <1% DMSO), and the corresponding HDAC2
substrate was added into a 96-well assay plate. The plate was incubated
at 37 oC for 30 min, followed by the addition of 50 µL of HDAC developer
reagent and incubated at room temperature for an additional 20 min.
Fluorescence intensity of the assay plates was measured on a BMG
Labtech 96-microplate reader using an excitation wavelength of 355 nm
and detection wavelength of 460 nm for all compounds. The fluorescence
intensity data was analyzed using Prism (GraphPad Software, San Diego,
CA). The DMSO controls (Ft) and enzyme-free controls (Fb) were defined
as 100% and 0% HDAC2 activity, respectively. The percent (%) activity of
each compound was calculated as (F-Fb)/(Ft-Fb), where F represents the
N1(1-(p-aminophenyl)-closo-1,2-carboranyl)-N8-hydroxyoctanediam-
ide (4c): Yellow powder (391 mg, 75%). 1H NMR (400 MHz, DMSO-d6, δ):
1.22-3.40 (br, BH), 1.36 (br, 4H), 1.61 (br, 4H), 2.22 (t, 2H), 2.29 (t, 2H),
3.91 (s, 2H). 4.41 (br, CH), 4.9 (br, NH), 8.57 (br, OH). 11B{1H} (128 MHz,
d6-DMSO, δ): -12.8, -10.8, -8.9, -4.3, -2.0. HRMS (+ESI): 427.30229 (8%,
C10H3010B711B3N2NaO3 [M+Na]+, gives 427.30778), 428.29866 (38%,
C10H3010B611B4N2NaO3 [M+Na]+, gives 428.29883), 429.29503 (74%,
C10H3010B511B5N2NaO3 [M+Na]+, gives 429.29851), 430.30140 (100%,
C10H3010B411B6N2NaO3 [M+Na]+, gives 430.30756), 431.30790 (89%,
C10H10B311B7N2NaO3 [M+Na]+, gives 431.30791), 432.30414 (41%,
C10H10B211B8N2NaO3 [M+Na]+, gives 432.30485), 433.31096 (10%,
C10H10B111B9N2NaO3 [M+Na]+, gives 433.31021).
4
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(3-(8-(Hydroxyamino)-8-oxooctanamido)phenyl)boronic acid (4d):
White powder (416 mg, 81%). 1H NMR (400 MHz, d6-DMSO, δ): 1.33 (m,
4H), 1.49 (m, 4H), 1.60 (m, 4H), 2.18 (m, 2H), 2.29 (m, 2H), 7.43 (m, 1H,
Ph), 7.50 (m, 3H, Ph), 8.98 (s, 1H, NH) and 10.94 (br, 1H, COOH). 11B{1H}
(128 MHz, DMSO-d6, δ): 29.0. HRMS (+ESI): Calc. for C14H21BN2O5:
309.1532, found 331.14357 ([M+Na]+).
Acknowledgements
We thank Dr Ian Luck for assistance with the NMR spectra, and
Dr Nick Proschogo for assistance with the MS. We also thank the
National Breast Cancer Foundation of Australia for funding.
Keywords: epigenetics • cancer • boron • HDAC inhibitor •
Vorinostat
5
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Entry for the Table of Contents
O
H
N
OH
R
N
H
O
HO
R =
C
BH
B
B
O
O
H
OH
Insert text for Table of Contents here. We report the first boron-based histone deacetylase inhibitors of HDAC2, showcasing boron’s
untapped potential in the design of new classes of epigenetic enzyme inhibitors.
Institute and/or researcher Twitter usernames: @GroupRendina
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