Aspirin-inspired acetyl-donating HDACs inhibitors

Article abstract of DOI:10.1007/s12272-018-1045-z

Aspirin is one of the oldest drugs for the treatment of inflammation, fever, and pain. It is reported to covalently modify COX-2 enzyme by acetylating a serine amino acid residue. By virtue of aspirin’s acetylating potential, we for the first time developed novel acetyl-donating HDAC inhibitors. In this study, we report the design, synthesis, in silico docking study, and biological evaluation of acetyl-donating HDAC inhibitors. The exposure of MDA-MB-231 cells with compound 4c significantly promotes the acetylation of α-tubulin and histone H3, which are substrates of HDAC6 and HDAC1, respectively. In silico docking simulation also indicates that compound 4c tightly binds to the deep substrate-binding pocket of HDAC6 by coordinating the active zinc ion in a bidentate manner and forming hydrogen bond interactions with Ser531 and His573 amino acid residues. In particular, compound 4c (GI₅₀ = 147?μM) affords the significant enhancement of anti-proliferative effect on MDA-MB-231 cells, compared with its parent compound 2c (GI₅₀ > 1000?μM) and acetyl-donating group deficient compound 6 (GI₅₀ = 554?μM). Overall, compound 4c presents a novel strategy for developing acetyl-donating HDAC inhibitors.

Full text of DOI:10.1007/s12272-018-1045-z

Arch. Pharm. Res. (2018) 41:967–976
Online ISSN 1976-3786
Print ISSN 0253-6269
https://doi.org/10.1007/s12272-018-1045-z
RESEARCH ARTICLE
Aspirin-inspired acetyl-donating HDACs inhibitors
1
1
2
1
1
•
Jiah Lim
Byoungduck Park
•
Yoojin Song
1
•
Jung-Hee Jang
1
•
Chul-Ho Jeong
•
Sooyeun Lee
•
Young Ho Seo
Received: 13 March 2018 / Accepted: 4 June 2018 / Published online: 20 June 2018
Ó The Pharmaceutical Society of Korea 2018
Abstract Aspirin is one of the oldest drugs for the treat-
ment of inflammation, fever, and pain. It is reported to
covalently modify COX-2 enzyme by acetylating a serine
amino acid residue. By virtue of aspirin’s acetylating
potential, we for the first time developed novel acetyl-do-
nating HDAC inhibitors. In this study, we report the
design, synthesis, in silico docking study, and biological
evaluation of acetyl-donating HDAC inhibitors. The
exposure of MDA-MB-231 cells with compound 4c sig-
nificantly promotes the acetylation of a-tubulin and histone
H3, which are substrates of HDAC6 and HDAC1, respec-
tively. In silico docking simulation also indicates that
compound 4c tightly binds to the deep substrate-binding
pocket of HDAC6 by coordinating the active zinc ion in a
bidentate manner and forming hydrogen bond interactions
with Ser531 and His573 amino acid residues. In particular,
compound 4c (GI50 = 147 lM) affords the significant
enhancement of anti-proliferative effect on MDA-MB-231
cells, compared with its parent compound 2c (GI50-
Keywords Aspirin Á Histone deacetylases Á Cancers Á
Alzheimer’s disease Á Drug addiction
Introduction
Histone deacetylases (HDACs) have emerged as important
pharmaceutical targets for a wide range of diseases,
including cancer, Alzheimer’s disease, depression, and
drug addiction (Kazantsev and Thompson 2008; Cuadrado-
Tejedor et al. 2017; D’ydewalle et al. 2012; Golden et al.
2013; Omonijo et al. 2014; Chun 2018). The reversible
acetylation of lysine residues at the N-terminal tails of core
histones has a critical role in epigenetic regulation of gene
expression. This acetylation status is tightly regulated by
the balance of HDACs and histone acetyltransferases
(HATs) and the perturbation of this balance is closely
linked to the onset and progression of many diseases (Hong
et al. 1993; Ropero and Esteller 2007). Therefore, the
inhibition of HDACs helps in restoring the aberrant epi-
genetic changes associated with diverse disorders (Bolden
et al. 2006; Kazantsev and Thompson 2008). HDACs
catalyze the removal of e-N-acetyl groups from lysine
residues at the N-terminal tails of core histones, impeding
the accessibility of transcriptional factors to promoter
segments of DNA within the condensed chromatin (Lee
et al. 1993). HDACs substrates are not limited to histone
proteins. These enzymes also regulate the acetylation
levels of non-histone proteins such as a-tubulin, Hsp90,
p53, c-Myc, NF-jB, and E2P (Glozak et al. 2005). HDAC
inhibitors are commonly classified into four main classes
based on their chemical structure, hydroxamic acids, ben-
zamides, cyclic peptides, and short-chain fatty acids
(Nebbioso et al. 2012). To date, the U.S FDA has approved
four HDAC inhibitors as anticancer drugs and these include
[
1000 lM) and acetyl-donating group deficient com-
pound 6 (GI50 = 554 lM). Overall, compound 4c presents
a novel strategy for developing acetyl-donating HDAC
inhibitors.
Jiah Lim and Yoojin Song contributed equally to this work.
&
Young Ho Seo
seoyho@kmu.ac.kr
1
2
College of Pharmacy, Keimyung University, Daegu 42601,
Republic of Korea
Department of Pharmacology, School of Medicine,
Keimyung University, Daegu 42601, Republic of Korea
123

9
68
J. Lim et al.
SAHA (vorinostat) (Duvic et al. 2007), FK-228 (ro-
midepsin) (VanderMolen et al. 2011), PXD101 (belinostat)
Materials and methods
Chemistry
(
Lee et al. 2015), and LBH589 (panobinostat) (Raedler
016), while HBI-8000 (chidamide) (Lu et al. 2016) has
2
been approved by the Chinese FDA for the treatment of
cutaneous T cell lymphoma (Fig. 1). Although most
HDAC inhibitors have been primarily developed for the
hematological malignancies (Tambaro et al. 2010), an
increasing number of HDAC inhibitors are recently
investigated for the treatment of central nervous system
Unless otherwise noted, all reactions were performed under
argon atmosphere in oven-dried glassware. All purchased
reagents and solvents were used without further purifica-
tion. Thin layer chromatography (TLC) was carried out
using Merck silica gel 60 F254 plates. TLC plates were
visualized using a combination of UV, p-anisaldehyde,
ceric ammonium molybdate, ninhydrin, and potassium
permanganate staining. Column chromatography was
conducted under medium pressure on silica (Merck Silica
Gel 40–63 lm) or performed by MPLC (Biotage Isolera
One instrument) on a silica column (Biotage SNAP HP-Sil)
or C18 column (Biotage SNAP Ultra C18). NMR analyses
were carried out using a JNM-ECZ500R (500 MHz)
(
CNS) diseases such as Alzheimer’s disease, depression,
and drug addiction (Kazantsev and Thompson 2008).
Aspirin (acetylsalicylic acid) is one of the oldest and
most widely used drugs for the treatment of inflammation,
fever, and pain as well as for the prevention of cardio-
vascular diseases. Like many other nonsteroidal anti-in-
flammatory drugs (NSAIDs), its basic mechanism of action
as an anti-inflammatory drug is well documented to target
cyclooxygenase-2 (COX-2). More specifically, aspirin is
reported to covalently modify COX-2 through acetylation
of Ser530 near its active site, which prevents its native
substrate from properly binding to its active site (Lei et al.
1
13
manufactured by Jeol resonance. H and C NMR chem-
ical shifts are reported in parts per million (ppm). The
deuterium lock signal of the sample solvent was used as a
reference, and coupling constants (J) are given in hertz
(Hz). The splitting pattern abbreviations are as follows: s,
singlet; d, doublet; t, triplet; q, quartet; dd, doublet of
doublets; m, multiplet. The purity of all tested compounds
was confirmed to be higher than 95% by analytical HPLC
performed with a dual pump Shimadzu LC-6AD system
equipped with VP-ODS C18 column (4.6 mm 9 250 mm,
5 lm, Shimadzu).
2
015).
In recent years, epidemiological data and accumulating
scientific evidence have shown that aspirin can also reduce
overall risk of various types of cancers (Patrignani and
Patrono 2016). Although the precise mechanisms exerting
its anticancer effects are not yet clearly elucidated, newly
report suggests that aspirin’s ability to chemically acetylate
endogenous proteins has immense therapeutic significance
in cancers (Sonnemann et al. 2008). The unique acetylating
nature of aspirin intrigues us to investigate anticancer
effect of aspirin analogues and their therapeutic potential as
HDAC inhibitors.
3-Acetoxybenzoic acid (2b)
A mixture of 3-hydroxybenzoic acid (1.0 g, 138.12 mmol)
and acetic anhydride (5 mL) in pyridine (10 mL) was
stirred at 125 °C for 2 h, equipped with a refluxing con-
denser under argon. After cooling at room temperature, the
Fig. 1 Structures of FDA-approved HDAC inhibitors
1
23

Aspirin-inspired acetyl-donating HDACs inhibitors
969
1
reduced pressure to afford compound 3b in 68% yield. H
mixture was quenched with saturated aqueous NaHCO3,
acidified with 12 N HCl to pH 2. The resulting solid was
NMR (500 MHz, CDCl ) d 8.82 (s, 1H), 7.49 (d,
3
filtered, washed with H O (50 mL), and dried under high
2
1
vacuum to afford compound 2b in 94% yield. H NMR
J = 7.4 Hz, 1H), 7.43 (m, 3H), 7.41–7.36 (m, 4H), 7.22 (d,
13
J = 7.4 Hz, 1H), 5.01 (s, 2H), 2.29 (s, 3H). C NMR
(
500 MHz, CDCl ) d 7.99 (d, J = 8.0 Hz, 1H), 7.83 (s,
(125 MHz, CDCl ) d 169.4, 165.4, 150.7, 135.2, 133.5,
3
3
1
3
1
H), 7.50 (t, J = 8.0 Hz, 1H), 7.37–7.34 (m, 1H), 2.34 (s,
3
129.8, 129.4, 128.9, 128.7, 128.4, 127.9, 125.4, 124.5,
?
120.8, 78.4, 21.1. ESI MS (m/e) = 286.11 [M ? 1] .
1
H). C NMR (125 MHz, CDCl ) d 171.1, 169.3, 150.7,
3
30.8, 129.7, 127.7, 127.3, 123.5, 21.1. ESI MS (m/
-
e) = 179.03 [M-1] .
4-((Benzyloxy)carbamoyl)phenyl acetate (3c)
4
-(Hydroxycarbamoyl)phenyl acetate (2c)
A mixture of compound 2c (0.2 g, 1.11 mmol), o-benzyl-
hydroxylamine hydrochloride (0.19 g, 1.22 mmol), DIPEA
(0.39 mL) in DCM (25 mL) was stirred with EDC (0.43 g,
2.22 mmol) at room temperature for 4 h. The mixture was
diluted in DCM (100 mL). The organic layer was washed
with 1 N HCl (100 mL) twice and then with brine
(100 mL), dried over Na SO , concentrated under reduced
A mixture of 4-hydroxybenzoic acid (1.0 g, 138.12 mmol)
and acetic anhydride (5 mL) in pyridine (10 mL) was
stirred at 125 °C for 2 h, equipped with a refluxing con-
denser under argon. After cooling at room temperature, the
mixture was quenched with saturated aqueous NaHCO3,
acidified with 12 N HCl to pH 2. The resulting solid was
2
4
1
pressure to afford compound 3c in 60% yield. H NMR
(500 MHz, (CDCl ) d 7.69 (d, J = 8.6 Hz, 2H), 7.41–7.40
filtered, washed with H O (50 mL), and dried under high
2
1
vacuum to afford compound 2c in 66% yield. H NMR
3
(m, 2H), 7.37–7.33 (m, 3H), 7.08 (d, J = 8.6 Hz, 2H), 4.98
1
3
(
500 MHz, (CDCl ) d 8.14 (d, J = 8.6 Hz, 2H), 7.21 (d,
(s, 2H), 2.29 (s, 3H). C NMR (125 MHz, CDCl ) d 169.1,
3
3
1
J = 8.6 Hz, 2H), 2.34 (s, 3H). C NMR (125 MHz,
3
165.7, 153.5, 135.3, 129.6, 129.4, 128.9, 128.7, 128.4,
CDCl ) d 171.0, 169.0, 155.0, 131.9, 126.9, 121.9, 21.3.
122.0, 121.6, 78.4, 21.2. ESI MS (m/e) = 286.11
?
[M ? 1] .
3
-
ESI MS (m/e) = 179.03 [M-1] .
2
-((Benzyloxy)carbamoyl)phenyl acetate (3a)
3-(Hydroxycarbamoyl)phenyl acetate (4b)
A mixture of compound 2a (0.2 g, 1.11 mmol), o-benzyl-
hydroxylamine hydrochloride (0.19 g, 1.22 mmol), EDC
A mixture of compound 3b (0.203 g, 1.04 mmol) and Pd/C
(0.05 g) in MeOH (12.5 mL) was stirred at room temper-
ature for 2 h under hydrogen. The resulting mixture was
diluted in MeOH (50 mL), filtered, and concentrated under
reduced pressure, and purified by MPLC (Biotage SNAP
(
0.43 g, 2.22 mmol), and DIPEA (0.39 mL, 2.22 mmol) in
DCM (25 mL) was stirred at room temperature for 4 h. The
reaction mixture was diluted with DCM (100 mL) and
washed with 1 N HCl (100 mL) and then with brine
HP-Sil column) to afford compound 4b in 45% yield. R
f-
1
= 0.31 (7:3 ethyl acetate: hexane) H NMR (500 MHz,
(
100 mL), dried over Na SO , concentrated under reduced
2
4
pressure and purified by silica gel column chromatography
acetone-d6) d 7.71 (d, J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.50
1
3
to afford compound 3a in 71% yield. R = 0.32 (4:6 ethyl
f
(t, J = 7.7 Hz 1H), 7.30–7.28 (m, 1H), 2.28 (s, 3H).
C
1
acetate: hexane). H NMR (500 MHz, CDCl ) d 8.63 (s,
NMR (125 MHz, acetone-d ) d 169.6, 164.8, 151.9, 134.6,
3
6
1
H), 7.73 (d, J = 7.4 Hz, 1H), 7.50–7.44 (m, 3H), 7.39 (q,
130.3, 130.1, 125.8, 124.8, 121.5, 20.9. ESI MS (m/
-
e) = 194.08 [M-1] .
J = 7.3 Hz, 3H), 7.29 (t, J = 7.7 Hz, 1H), 7.09 (d,
1
J = 8.0 Hz, 1H), 5.03 (s, 2H), 2.10 (s, 3H). C-NMR
3
(
125 MHz, CDCl ) d 169.0, 164.1, 148.1, 135.4, 132.6,
4-(Hydroxycarbamoyl)phenyl acetate (4c)
3
1
2
30.0, 129.3, 128.9, 128.8, 126.4, 125.3, 123.3, 78.2,
?
0.94. ESI MS (m/e) = 286.11 [M ? 1] .
A mixture of compound 3c (0.203 g, 1.04 mmol) and Pd/C
(0.05 g) in MeOH (12.5 mL) was stirred at room temper-
3
-((Benzyloxy)carbamoyl)phenyl acetate (3b)
ature for 2 h under hydrogen. The resulting mixture was
dissolved in MeOH (50 mL), filtered, and concentrated
under reduced pressure, and purified by MPLC (Biotage
A mixture of compound 2b (0.2 g, 1.11 mmol), o-benzyl-
hydroxylamine hydrochloride (0.19 g, 1.22 mmol) with
DIPEA (0.39 mL) in DCM (25 mL) was stirred by EDC
SNAP HP-Sil column) to afford compound 4c in 39%
1
yield. R = 0.24 (7:3 ethyl acetate: hexane). H NMR
f
(
0.43 g, 2.22 mmol) at room temperature for 4 h. The
(500 MHz, acetone-d ) d 10.8 (s, 1H), 8.52 (s, 1H), 7.88 (d,
6
1
J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 2.28 (s, 3H). C
3
mixture was diluted in DCM (100 mL). The organic layer
was washed with 1 N HCl (100 mL) twice and then with
brine (100 mL), dried over Na SO , concentrated under
NMR (125 MHz, acetone-d ) d 168.6, 164.5, 153.5, 129.7,
6
-
128.4, 122.0, 20.2. ESI MS (m/e) = 194.08 [M-1] .
2
4
123

9
70
J. Lim et al.
4-Hydroxy-N-((tetrahydro-2H-pyran-2-
yl)oxy)benzamide (5)
27,000, and the number of evaluations to 2,500,000, while
the number of docking runs was set to 100 with a cutoff of
˚
1
A for the root-mean-square tolerance for the grouping of
A mixture of 4-hydroxybenzoic acid (1.0 g, 7.24 mmol), o-
each docking run. The docking model of HDAC6 with
compound 4c was depicted in Fig. 6 and rendering of the
picture was generated using PyMol (DeLanoScientific).
(
Tetrahydro-2H-pyran-2-yl)
.96 mmol), and EDC (2.78 g, 14.48 mmol) in DMF
25 mL) was stirred by at room temperature for 12 h. The
hydroxylamine
(0.93 g,
7
(
resulting mixture was dissolved in DCM (100 mL). The
organic layer was washed with 1 N HCl (100 mL) and then
washed with brine (100 mL), dried over Na SO , concen-
Biology
2
4
Dulbecco’s Modified Eagle’s medium (DMEM) with L-
glutamine was purchased from GenDEPOT (Barker, TX,
USA) and fatal bovine serum (FBS) and penicillin/strep-
tomycin were purchased Gibco BRL (Gaithersburg, MD,
USA). Antibodies specific for a-tubulin, Ac-a-tubulin,
histone H3, Ac-histone H3, b-actin and Ac-lysine were
purchased from Cell Signaling Technology (Boston, MA,
USA). Goat anti-rabbit IgG horseradish peroxidase conju-
gate was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Cell Titer 96 Aqueous One Solution cell
proliferation assay kit was purchased from Promega
trated under reduced pressure and purified by silica gel
column chromatography to afford compound 5 in 22%
1
yield. R = 0.53 (7:3 ethyl acetate: hexane). H NMR
f
(
500 MHz, DMSO-d ) d 11.4 (s, 1H), 10.1 (s, 1H), 7.63 (d,
6
J = 8.6 Hz, 2H), 6.80 (d, J = 9.2 Hz, 2H), 4.94 (s, 1H),
4
.06–4.02 (m, 1H), 3.51–3.48 (m, 1H), 1.70–1.53 (m, 6H).
1
3
C NMR (125 MHz, DMSO-d ) d 164.2, 160.2, 128.8,
6
1
22.8, 114.7, 100.8, 61.2, 27.7, 24.5, 18.1. ESI MS (m/
?
e) = 238.18 [M ? 1] .
TM
TM
N,4-dihydroxybenzamide (6)
(Madison, WI, USA). Amersham ECL select Western
blotting detection reagent was purchased GE Healthcare.
HDAC fluorogenic assay kits (HDAC1, HDAC3, HDAC6,
and HDAC7) were purchased BPS Bioscience (San Diego,
CA, USA).
A mixture of compound 5 (0.075 g, 0.49 mmol) in ace-
tonitrile (20 mL) and 3 N HCl (10 mL) was stirred at room
temperature for 4 h. The mixture was concentrated under
reduced pressure and purified by reverse-phase MPLC
1
SNAP Ultra C18) to afford compound 6 in 10% yield. H
(
Cell culture
NMR (500 MHz, DMSO-d ) d 7.77 (d, J = 8.6 Hz, 1H),
6
7
.60 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 6.79 (d,
3
MDA-MB-231 cells were grown in DMEM with L-glu-
tamine supplemented with streptomycin (500 mg/mL),
penicillin (100 units/mL), and 10% fatal bovine serum
1
J = 9.2 Hz, 1H). C NMR (125 MHz, DMSO-d ) d 167.2,
6
-
61.6, 131.5, 121.5, 115.1 ESI MS (m/e) = 152.04 [M-1] .
1
(FBS). Cells were grown to confluence in a humidified
Docking studies
atmosphere (37 °C, 5% CO2).
In silico docking of 4c with the 3D coordinates of the X-ray
crystal structures of HDAC6 (PDB code: 5EF7) was
accomplished using the AutoDock program downloaded
from the Molecular Graphics Laboratory of the Scripps
Research Institute. The AutoDock program was chosen
because it uses a genetic algorithm to generate the poses of
the ligand inside a known or predicted binding site utilizing
the Lamarckian version of the genetic algorithm where the
changes in conformations adopted by molecules after
in situ optimization are used as subsequent poses for the
offspring. In the docking experiments carried out, water
was removed from the 3D X-ray coordinates while Gas-
teiger charges were placed on the X-ray structures of
HDAC6 along with 4c using tools from the AutoDock
suite. A grid box centered on the substrate binding pocket
of HDAC6 enzyme with definitions of 60_60_60 points
Cell proliferation assay
Cell were seeded at 2000 cells per well in a clear 96-well
plate, the medium volume was brought to 100 mL, and the
cells were allowed to attach overnight. The next day,
varying concentrations of 2a–b, 4b–c, and 6 or 0.5%
DMSO vehicle control were added to the wells. The pro-
liferation of cells was determined using the Promega Cell
Titer 96 Aqueous One Solution cell proliferation assay.
After incubation with compounds, 20 mL of the assay
substrate solution was added to the wells, and the plate was
incubated at 37 °C for an additional 1 h. Absorbance at
Ò
490 nm was then read on FLUO star Omega (BMG
LABTECH), and values were expressed as percent of
absorbance from cells incubated in DMSO alone.
˚
and 0.375 A spacing was chosen for ligand docking
experiments. The docking parameters consisted of setting
the population size to 150, the number of generations to
1
23

Aspirin-inspired acetyl-donating HDACs inhibitors
971
Western blot
2c could promote the acetylation level of lysine e-amino
groups in MDA-MB-231 cells. Aspirin is well documented
to covalently modify COX-2 through acetylation of Ser530
near its active site and prevents its native substrate from
properly binding to its active site (Lei et al. 2015). How-
ever, there are only few studies reported about the acety-
lation capacity of aspirin on lysine residues (Hawkins et al.
1969). Therefore, we measured the acetylating capacity of
aspirin (2a), meta-analogue 2b, and para-analogue 2c on
lysine residues of whole proteins. MDA-MB-231 cells
were first treated with aspirin (2a), meta-analogue 2b, and
para-analogue 2c for 24 h and the acetylation level of
lysine residues was assessed by anti-Ac-lysine immunoblot
(Fig. 2). Interestingly, aspirin (2a) most efficiently pro-
moted the acetylation of lysine residues. Compound 2b and
2c were also capable of increasing the acetylation level of
lysine residues, compared with DMSO control. Encouraged
by the acetylating potential of compounds 2a–c, we set out
to develop a novel acetyl-donating HDAC inhibitors, uti-
lizing the scaffold of compounds 2a–c.
6
MDA-MB-231 cells (1 9 10 /dish) were seeded in 50 nm
culture dishes, and allowed to attach overnight. Cells were
incubated with compound 2a–c, or compound 4b–c for an
additional 24 h. For comparison, cells were also incubated
with 0.5% DMSO. Cells were harvested in ice-cold lysis
buffer (23 mM Tris–HCL pH 7.6, 139 mM NaCl, 1% NP-
4
0, 1% sodium deoxycholate, 0.1% SDS), and 30 lg of
lysate per lane was separated by SDS-PAGE and followed
by transferring to a PVDF membrane. The membrane was
blocked with 5% skim milk in TBS-T, and then incubated
with the corresponding antibody. After binding of an
appropriated secondary antibody coupled to horseradish
peroxidase, proteins were visualized by ECL chemilumi-
nescence according to the instructions of the manufacturer.
Results
Most HDAC inhibitors are structurally designed to
mimic its endogenous substrate, acetyl-lysine (Fig. 3)
Aspirin analogues 2b–c were synthesized following the
literature procedure with slight modifications (Scheme 1)
(
Bassett and Barnett 2014). The common feature of HDAC
(
Zhang et al. 2014; Hrast et al. 2014). Briefly, compounds
b–c were acetylated with acetic anhydride in pyridine to
afford 2b–c in 66–94% yield.
inhibitor pharmacophore, as exemplified by SAHA, is
characterized by three elements, a zinc-binding group
1
(
ZBG), a linker, and a cap group. Numerous HDAC inhi-
With compounds 2a–c in hand, we next investigated
whether aspirin (2a), meta-analogue 2b, and para-analogue
bitors have been developed to treat a variety of human
Scheme 1 Structure of aspirin and synthesis of aspirin analogue 2b–c, 4a–c, and 6. Reagents and conditions: (a) acetic anhydride, pyridine, rt,
2
4
h, 94% for 2b, 66% for 2c; (b) NH
5% for 4b, 39% for 4c; (d) NH OTHP, EDC, DIPEA, DMF, rt, 12 h, 59%; (e) 3 N HCl, MeCN, rt, 4 h, 10%
2 2
OBn, EDC, DIPEA, DCM, rt. 4 h, 71% for 3a, 68% for 3b, 60% for 3c; (c) H , 10% Pd/C, MeOH, rt, 2 h,
2
123

9
72
J. Lim et al.
aspirin, meta-analogue 2b, and para-analogue 2c, leading
to acetyl-donating HDAC inhibitors shown in Fig. 3.
The synthesis of acetyl-donating HDAC inhibitors 4a–
c is summarized in Scheme 1. We first carried out EDC-
mediated amide coupling of compound 2a–c with NH OBn
2
in the presence of DIPEA to provide benzyl-protected
compound 3a–c in 60–71% yield. The removal of benzyl-
protecting group under hydrogen atmosphere in the pres-
ence of 10% palladium catalyst successfully furnished
compound 4b–c in 39–45% yield. Unfortunately, the
attempt to cleave benzyl-protecting group from 3a under
the same reaction condition did not lead to a fruitful result.
It only provided a complex mixture, indicating compound
4a was not stable and decomposed upon the removal of
protecting group unlike other analogues (4b–c).
To clarify the mode of action of acetyl donating HDAC
inhibitor, we aimed at the synthesis of compound 6 that
lacks acetyl-donating group. The synthesis began with the
Fig. 2 Comparative acetylating capacity of aspirin (2a), meta-
analogue 2b, and para-analogue 2c on lysine residues. MDA-MB-
2
31 cells were treated with 5 mM of the indicated compound for 24 h
and the effect of compound 2a, 2b, and 2c on the acetylation status of
whole proteins was analyzed by western blot. DMSO (D) was used as
a negative control
coupling reaction of 1c with NH OTHP in the presence of
2
EDC and DIPEA in DMF, providing compound 5 in 59%
yield. Acidic cleavage of THP-protecting group from
compound 5 using 3 N HCl in acetonitrile successfully
furnished compound 6 in 10% yield.
diseases and the majority of these HDAC inhibitors
incorporate hydroxamic acid as a zinc-binding group,
including clinically approved SAHA, PXD101, and
LBH589 (Bassett and Barnett 2014). At the same time, the
phenyl linker of HDAC inhibitors has been widely used in
in the development of HDAC inhibitors due to its
hydrophobic nature, well fitting into the lipophilic channel
of HDAC substrate pocket. Accordingly, we designed new
HDAC inhibitors by installing a hydroxamic acid zinc-
binding group and a phenyl linker to the structures of
Upon completion of synthesis, we evaluated the anti-
proliferative activity of compound 2a–c, 4b–c, and 6
against MDA-MB-231 cell line, which is a highly aggres-
sive and invasive triple-negative breast cancer (TNBC) cell
line. It lacks estrogen receptor (ER), progesterone receptor
(PR), and human epidermal growth factor receptor (Her2).
As shown in Table 1, compound 2a (aspirin), 2b, and 2c
exhibited very poor anti-proliferative activities against
Fig. 3 Design of acetyl-
donating HDAC inhibitors.
ADG, acetyl-donating group;
ZBG, zinc-binding group
1
23

Aspirin-inspired acetyl-donating HDACs inhibitors
973
Table 1 Anti-proliferative
Compound
Structure
MDA-MB-231 (GI50; lM)
[ 1000
effect of compound 2a–c, 4b–c,
and 6 against breast cancer cell
line, MDA-MB-231
2
a (aspirin)
O
OH
OAc
2
b
c
720
O
O
AcO
OH
OH
2
[ 1000
AcO
AcO
4
b
c
169
147
O
O
OH
N
H
4
OH
N
H
AcO
6
554
MDA-MB-231 TNBC cell line, in that GI50 values of
compound 2a and 2c were higher than 1,000 lM and GI50
value of compound 2b was 720 lM. As expected, com-
pound 4b and 4c displayed a significantly improved anti-
cancer effect against MDA-MB-231 cell line with GI50
values of 169 lM and 147 lM, compared with their parent
compound 2b and 2c. Interestingly enough, compound 6
that lacks acetyl-donating group (ADG) demonstrated a
relatively poor anticancer effect (GI50 = 554 lM), com-
pared with compound 4c (GI50 = 147 lM), clearly indi-
cating that acetyl-donating group of compound 4c played
an important role in anticancer effect. Overall, the result
strongly validates molecular design of acetyl-donating
HDAC inhibitors in the discovery of novel HDAC
inhibitors.
Fig. 4 Time- and dose-dependent anti-proliferative effect of 4c on
MDA-MB-231 cells
To examine time- and dose-dependent effect of com-
pound 4c on the growth of MDA-MB-231 cells, we treated
MDA-MB-231 cells with compound 4c at various con-
centration (1, 10, 100, and 300 lM) for 1, 2, and 3 days
and measured the cell proliferation using MTS assay
exposure of cells with 300 lM of compound 4c displayed a
significant anticancer effect and impaired the growth of
MDA-MB-231 cells by 37, 21, and 22% for 1, 2, and
3 days, respectively.
We next assessed the comparative effect of aspirin, 4c,
and 4b on the acetylation status of a-tubulin and histone
H3, which were epigenetically removed by HDAC6 and
HDAC1 enzymes, respectively. As shown in Fig. 5, the
(
Fig. 4). The cell proliferation assay indicated that com-
pound 4c afforded relatively good cellular efficacy against
MDA-MB-231 cells in a dose-dependent manner. The
123

9
74
J. Lim et al.
Fig. 5 Comparative effect of aspirin, 4c, and 4b on the acetylation
status of a-tubulin and histone H3. MDA-MB-231 cells were treated
with 75 or 150 lM of aspirin, 4c, and 4b for 24 h and the expression
of the indicated proteins was determined by western blot. DMSO
(
D) was employed as a negative control
treatment of MDA-MB-231 cells with compound 4c sig-
nificantly promoted the acetylation of a-tubulin and histone
H3, while the reference drug, aspirin failed to induce the
acetylation of a-tubulin and histone H3. Compound 4b was
less effective on acetylating a-tubulin than compound 4c,
and incapable of acetylating histone H3. As expected, the
expression level of a-tubulin and histone H3 remain
unaffected by aspirin, 4c, and 4b. We next measured the
total acetylation status of whole proteins in MDA-MD-231
cells. Compound 4c acetylated e-amino group of lysine
residues more efficiently than the reference drug aspirin
and the other analogue 4b, evidenced by anti-Ac-lysine
immunoblot. Taken together, the result clearly demon-
strated that para-analogue 4c most significantly inhibited
HDAC1 and HDAC6 enzymes and promoted the acetyla-
tion of their substrates, histone H3 and a-tubulin,
respectively.
Fig. 6 Molecular docking model of 4c in the binding pocket of
HDAC6 (PDB code: 5EF7)
acetyl group formed a hydrogen bond interaction with
S531 residue. The phenyl ring of 4c projected into the
lipophilic channel of HDAC6, forming p–p interactions
with F583 and F643 residues. The hydroxamate OH and
To assess the precise binding pose of compound 4c to
HDAC6, we carried out in silico docking simulation of
compound 4c into the substrate binding pocket of HDAC6
C=O groups of compound 4c coordinated the active site
2?
Zn
ion in a bidentate manner. Additionally, the
hydroxamate OH group of 4c participated in a hydrogen
bond interaction with H573 residue.
(
PDB code: 5EF7). As shown in Fig. 6, the docking sim-
ulation indicated that compound 4c nicely occupied the
deep substrate binding pocket of HDAC6. Para-acetyl
group of compound 4c was located in the rim of the sub-
strate binding pocket and the carbonyl oxygen (C=O) of the
1
23

Aspirin-inspired acetyl-donating HDACs inhibitors
975
small-molecule that acts as a dual inhibitor of HDAC and PDE5
and that rescues hippocampal synaptic impairment in Alzhei-
mer’s disease mice. Neuropsychopharmacology 42:524–539
D’ydewalle C, Bogaert E, Van Den Bosch L (2012) HDAC6 at the
intersection of neuroprotection and neurodegeneration. Traffic
Discussion
With the aim of generating acetyl-donating HDAC inhi-
bitors, we designed, synthesized, and performed biological
evaluation of a series of aspirin derivatives. The results
revealed that aspirin (2a), together with its meta-analogue
1
3:771–779
Duvic M, Talpur R, Ni X, Zhang C, Hazarika P, Kelly C, Chiao JH,
Reilly JF, Ricker JL, Richon VM, Frankel SR (2007) Phase 2
trial of oral vorinostat (suberoylanilide hydroxamic acid, SAHA)
for refractory cutaneous T-cell lymphoma (CTCL). Blood
2
b and para-analogue 2c was able to promote the acety-
lation of lysine residues, establishing the proof of principle
for the strategy of developing acetyl-donating HDAC
inhibitors. By virtue of aspirin’s acetylating potential, we
developed novel acetyl-donating HDAC inhibitors by
modifying the scaffold of compound 2a–c. The biological
evaluation clearly indicated that compound 4c (GI50-
1
09:31–39
Glozak MA, Sengupta N, Zhang X, Seto E (2005) Acetylation and
deacetylation of non-histone proteins. Gene 363:15–23
Golden SA, Christoffel DJ, Heshmati M, Hodes GE, Magida J, Davis
K, Cahill ME, Dias C, Ribeiro E, Ables JL, Kennedy PJ, Robison
AJ, Gonzalez-Maeso J, Neve RL, Turecki G, Ghose S,
Tamminga CA, Russo SJ (2013) Epigenetic regulation of
RAC1 induces synaptic remodeling in stress disorders and
depression. Nat Med 19:337–344
= 147 lM) afforded the significant enhancement of anti-
cancer effect on TNBC MDA-MB-231 cells, compared
with its parent compound 2c (GI [ 1,000 lM) and ADG-
Hawkins D, Pinckard RN, Crawford IP, Farr RS (1969) Structural
changes in human serum albumin induced by ingestion of
acetylsalicylic acid. J Clin Invest 48:536–542
Hong L, Schroth GP, Matthews HR, Yau P, Bradbury EM (1993)
Studies of the DNA binding properties of histone H4 amino
terminus. Thermal denaturation studies reveal that acetylation
markedly reduces the binding constant of the H4 ‘‘tail’’ to DNA.
J Biol Chem 268:305–314
Hrast M, Anderluh M, Knez D, Randall CP, Barreteau H, O’neill AJ,
Blanot D, Gobec S (2014) Design, synthesis and evaluation of
second generation MurF inhibitors based on a cyanothiophene
scaffold. Eur J Med Chem 73:83–96
Kazantsev AG, Thompson LM (2008) Therapeutic application of
histone deacetylase inhibitors for central nervous system disor-
ders. Nat Rev Drug Discov 7:854–868
5
0
deficient analogue 6 (GI50 = 554 lM). The treatment of
MDA-MB-231 cells with compound 4c significantly pro-
moted the acetylation of a-tubulin and histone H3, which
are substrates of HDAC6 and HDAC1, respectively.
Moreover, the docking simulation demonstrated that
compound 4c tightly bound to the deep substrate-binding
2
?
pocket of HDAC6 by coordinating the active Zn ion in a
bidentate fashion and forming hydrogen bond interactions
with amino acid residues. Overall, this study for the first
time demonstrated the design, synthesis, and biological
evaluation of acetyl-donating HDAC inhibitors and pre-
sented the novel strategy for developing acetyl-donating
HDAC inhibitors.
Lee DY, Hayes JJ, Pruss D, Wolffe AP (1993) A positive role for
histone acetylation in transcription factor access to nucleosomal
DNA. Cell 72:73–84
Lee HZ, Kwitkowski VE, Del Valle PL, Ricci MS, Saber H,
Habtemariam BA, Bullock J, Bloomquist E, Li Shen Y, Chen
XH, Brown J, Mehrotra N, Dorff S, Charlab R, Kane RC,
Kaminskas E, Justice R, Farrell AT, Pazdur R (2015) FDA
approval: belinostat for the treatment of patients with relapsed or
refractory peripheral T-cell lymphoma. Clin Cancer Res
Acknowledgements This research was supported by Basic Science
Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education (NRF-
2
016R1A6A1A03011325 and 2016R1D1A1B01009559), and Korea
Institute of Planning and Evaluation for Technology in Food, Agri-
culture, Forestry and Fisheries (IPET) through Animal Disease
Management Technology Development Program, funded by the
Ministry of Agriculture, Food and Rural Affairs (MAFRA) (116102-
2
1:2666–2670
Lei J, Zhou Y, Xie D, Zhang Y (2015) Mechanistic insights into a
classic wonder drug–aspirin. J Am Chem Soc 137:70–73
Lu X, Ning Z, Li Z, Cao H, Wang X (2016) Development of
chidamide for peripheral T-cell lymphoma, the first orphan drug
approved in China. Intractable Rare Dis Res 5:185–191
0
3).
Compliance with ethical standards
Nebbioso A, Carafa V, Benedetti R, Altucci L (2012) Trials with
‘
Conflict of interest The authors declare no conflict of interest.
epigenetic’ drugs: an update. Mol Oncol 6:657–682
Omonijo O, Wongprayoon P, Ladenheim B, Mccoy MT, Govitrapong
P, Jayanthi S, Cadet JL (2014) Differential effects of binge
methamphetamine injections on the mRNA expression of histone
deacetylases (HDACs) in the rat striatum. Neurotoxicology
References
4
5:178–184
Patrignani P, Patrono C (2016) Aspirin and cancer. J Am Coll Cardiol
8:967–976
Bassett SA, Barnett MP (2014) The role of dietary histone deacety-
lases (HDACs) inhibitors in health and disease. Nutrients
6
6
:4273–4301
Raedler LA (2016) Farydak (Panobinostat): first HDAC inhibitor
approved for patients with relapsed multiple myeloma. Am
Health Drug Benefits 9:84–87
Bolden JE, Peart MJ, Johnstone RW (2006) Anticancer activities of
histone deacetylase inhibitors. Nat Rev Drug Discov 5:769–784
Chun P (2018) Therapeutic effects of histone deacetylase inhibitors
on kidney disease. Arch Pharm Res 41:162–183
Cuadrado-Tejedor M, Garcia-Barroso C, Sanchez-Arias JA, Rabal O,
Perez-Gonzalez M, Mederos S, Ugarte A, Franco R, Segura V,
Perea G, Oyarzabal J, Garcia-Osta A (2017) A first-in-class
Ropero S, Esteller M (2007) The role of histone deacetylases
(
HDACs) in human cancer. Mol Oncol 1:19–25
Sonnemann J, Huls I, Sigler M, Palani CD, Le Hong TT, Volker U,
Kroemer HK, Beck JF (2008) Histone deacetylase inhibitors and
123

9
76
J. Lim et al.
aspirin interact synergistically to induce cell death in ovarian
cancer cells. Oncol Rep 20:219–224
Tambaro FP, Dell’aversana C, Carafa V, Nebbioso A, Radic B,
Ferrara F, Altucci L (2010) Histone deacetylase inhibitors:
clinical implications for hematological malignancies. Clin Epi-
genetics 1:25–44
Vandermolen KM, Mcculloch W, Pearce CJ, Oberlies NH (2011)
Romidepsin (Istodax, NSC 630176, FR901228, FK228, dep-
sipeptide): a natural product recently approved for cutaneous
T-cell lymphoma. J Antibiot (Tokyo) 64:525–531
Zhang YJ, Shen LL, Cheon HG, Xu YN, Jeong JH (2014) Synthesis
and biological evaluation of glucagon-like peptide-1 receptor
agonists. Arch Pharm Res 37:588–599
1
23