Synthesis and biological evaluation of a novel photo-activated histone deacetylase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2020.127291

Hydroxamic acid-based histone deacetylase inhibitors (HDACi) are a class of epigenetic agents with potentially broad therapeutic application to several disease states including post angioplasty mediated neointimal hyperplasia (NIH). Precise spatiotemporal

Full text of DOI:10.1016/j.bmcl.2020.127291

Bioorganic & Medicinal Chemistry Letters 30 (2020) 127291
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a novel photo-activated histone
deacetylase inhibitor
a
b
c
c
a
Gopal R. Sama , Hongbin Liu , Simon Mountford , Phillip Thompson , Andrea Robinson ,
b,⁎
Anthony E. Dear
a
b
c
Department of Chemistry, Monash University, Melbourne, Victoria, Australia
Department of Medicine, Eastern Health Clinical School, Monash University, Melbourne, Victoria, Australia
Monash Institute of Pharmaceutical Sciences, Melbourne, Victoria, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hydroxamic acid-based histone deacetylase inhibitors (HDACi) are a class of epigenetic agents with potentially
broad therapeutic application to several disease states including post angioplasty mediated neointimal hyper-
plasia (NIH). Precise spatiotemporal control over the release of HDACi at the target blood vessel site is required
for the safe and successful therapeutic use of HDACi in the setting of drug eluting balloon catheter (DEBc)
angioplasty treatment of NIH. We aimed to develop and characterise a novel photoactive HDACi, as a potential
coating agent for DEBc.
Metacept-3
Caged metacept-3
HDACi
DEBc
Photolytic release of HDACi
Metacept-3 1 was caged with a photo-labile protecting group (PPG) to synthesise a novel UV365nm active
HDACi, caged metacept-3 15. Conversion of caged metacept-3 15 to active/native metacept-3 1 by UV365nm
was achievable in significant quantities and at UV365nm power levels in the milliwatt (mW) range.
In vitro evaluation of the biological activity of pre and post UV365nm activation of caged metacept-3 15
identified significant HDACi activity in samples exposed to short duration, mW range UV365nm. Toxicity studies
performed in human umbilical vein endothelial cells (HUVEC’s) identified significantly reduced toxicity of caged
metacept-3 15 pre UV365nm exposure compared with native metacept-3 1 and paclitaxel (PTX).
Taken together these findings identify a novel photo-activated HDACi, caged metacept-3 15, with pharma-
cokinetic activation characteristics and biological properties which may make it suitable for evaluation as a
novel coating for targeted DEBc angioplasty interventions.
7
Inhibition of histone deacetylase (HDAC) activity leads to the ac-
cumulation of acetylated histones and other cellular proteins with re-
sultant modulation of gene expression and associated cellular differ-
entiation, growth arrest, apoptosis together with additional cellular
current PTX coatings, demonstrated potentially promising therapeutic
8
6
results and whilst metacept-3 1 has exhibited low systemic toxicity
local vascular endothelial cell toxicity remained problematic particu-
larly at higher concentrations associated with DEBc-mediated drug
1
–4
8
processes. As such, HDACi have emerged as promising candidates for
a number of diverse disease states including cancer and inflammatory
conditions including cardiovascular, gastrointestinal and neurological
delivery.
To circumvent potential local and deployment-mediated systemic
toxicity in the setting of metacept-3 1 coated DEBc angioplasty design
of an inert, photo-inducible formulation of metacept-3 1 able to be
rapidly released upon light exposure once in contact with the vessel
intima was investigated. Metacept-3 1 was caged with a 2-nitrobenzyl
based photolabile protecting group enabling UV365nm photo-activa-
tion. Quantification of UV365nm-mediated conversion of caged meta-
cept-3 15 to native metacept-3 1 was determined together with biolo-
gical activity of post UV365nm activated caged metacept-3 15 and
cellular toxicity.
3
,4
disorders.
The HDACi metacept-3 1, a derivative of the hydroxamic acid
5
HDACi oxamflatin, has recently been evaluated in vitro and in vivo
demonstrating attenuation of vascular smooth muscle cell migration,
proliferation, and inhibition of neointimal hyperplasia (NIH) in a
6
murine model. Exploration in an ovine model as to the potential
therapeutic efficacy of metacept-3 1 as an alternative DEBc coating for
the treatment of NIH, prompted by safety concerns in relation to
⁎
Corresponding author at: Department of Medicine, Eastern Health Clinical School, Monash University, Level 2, 5 Arnold Street Box Hill 3128, Melbourne,
Victoria, Australia.
E-mail address: Anthony.dear@monash.edu (A.E. Dear).
https://doi.org/10.1016/j.bmcl.2020.127291
Received 2 April 2020; Received in revised form 21 May 2020; Accepted 26 May 2020
Available online 30 May 2020
0960-894X/ © 2020 Elsevier Ltd. All rights reserved.

G.R. Sama, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127291
Scheme 1. Synthesis of hydroxylamine 8 fragment. Reagents and reaction conditions: a) K
2
CO
3
, ethyl-4-bromobutyrate, DMF, 50 °C, 15 h; b) acetic anhydride, HNO
3
,
0
°C, 3 h; c) NaBH
4
, MeOH, rt, 3 h; d) SOCl
2
, DCM, rt, 3 h; e) N-Hydroxyphthalimide, TEA, DMF, 60 °C, 12 h; f) NH
2
NH ·H O, EtOH, reflux, 2 h.
2
2
The caging of metacept-3 1 was achieved using a convergent syn-
10 was reduced to the amine 11 in the presence of freshly added 10%
Pd(0)/C catalyst under a hydrogen atmosphere. The amine 11 was then
treated with mesyl chloride to give sulfonamide ester 12. Finally, sa-
ponification of the ester in the presence of aq. NaOH produced the
thetic strategy. Starting from commercially available acetovanillone 2,
hydroxylamine fragment 8 was synthesized over six steps in 35%
overall yield (Scheme 1). The ketoester 3 was synthesized by activation
5
of acetovanillone 2 with K
2
CO
3
, followed by the addition of ethyl-4-
target sulfonamide carboxylic acid 13 in good yield.
bromobutyrate. The ketoester 3 was subjected to an aromatic sub-
The two fragments, o-nitrohydroxylamine 8 and biphenyl car-
boxylic acid 13, were coupled in the presence of PyBOP and NMM in
stitution reaction in the presence of a nitration mixture consisting of
1
0
HNO
3
and acetic anhydride at 0 °C to yield compound 4. Reduction of
DMF to furnish the caged metacept-3 ester 14 (Scheme 3). Finally, the
target caged metacept-3 15 was obtained by hydrolysis of the ester with
the ketone to the secondary alcohol 5 was achieved with NaBH in
4
1
0
MeOH. Following reduction, o-nitrobenzyl chloride 6 was synthesized
in the presence of thionyl chloride and later subjected to a nucleophilic
substitution reaction with N-hydroxyphthalimide to furnish the imide-
protected hydroxylamine 7. Finally, deprotection of 7 with hydrazine
Novozyme-435 in phosphate buffer and dioxane (1:5 mixture).
Human umbilical vein endothelial cells (HUVECs) ((Lonza (CC-
2519, pooled donor), Basel, Switzerland)) were maintained in Media-
199 (Sigma, USA) supplemented with penicillin/streptomycin, 20%
foetal calf serum (FCS), 20 µg/mL endothelial cell growth factor
hydrate in EtOH gave the first fragment, hydroxylamine 8, as a yellow
oil in accordance with previously published methods9
Supporting information).
–12
(see
(Sigma) and 20 µg/mL heparin and kept in a 5% CO incubator at 37 °C.
2
HL60 cells were cultured in RPMI-1640 (Gibco; Thermo Fisher
Scientific, Inc., Waltham, MA, USA) containing 10% heat-inactivated
The second fragment, metacept-3 carboxylic acid 13, was synthe-
5
sised according to a previous literature report (Scheme 2). Biphenyl
fetal calf serum (Thermo Fisher Scientific, Inc.) and kept in a 5% CO
incubator at 37 °C.
2
nitro compound 10 was synthesized using a Suzuki coupling of 3-ni-
trophenyl boronic acid 9 and 4-bromomethyl ester in the presence of
The agents (caged metacept-3 15, native metacept-3 1 and PTX)
Na
2
CO
3
and 10% Pd(0)/C catalyst. Subsequently, the crude compound
were dissolved in 100% DMSO and added to plates at a final
Scheme 2. Synthesis of metacept-3 carboxylic acid 13. Reagents and reaction conditions: a) Ethyl 4-bromobenzoate, Na
2
CO
3
, 10% Pd/C, EtOH, reflux, 24 h; b) H ,
2
1
0% Pd/C, EtOAc, 48 h; c) MeSO
2
Cl, pyridine, rt, 12 h; d) 1 M NaOH (aq), MeOH, rt, 3 h.
2

G.R. Sama, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127291
Scheme 3. Synthesis of the metacept-3 photolabile linker 15. Reagents and reaction conditions: a) PyBOP, NMM, DMF, rt, 15 h; b) Phosphate buffer, dioxane,
Novozyme-435, 30 °C, 5 days.
Scheme 4. Photolysis of caged metacept-3 15 at 365 nm UV light.
concentration of 1.0 mmol/L for 24 h.
Protein (25 μg per lane) was loaded onto a 15% precast SDS poly-
acrylamide electrophoresis gel (BioRad). After electrophoresis (100 V,
90 min), separated proteins were transferred (15 mA, 60 min) to a
polyvinylidene difluoride membrane (BioRad). Nonspecific binding
sites were blocked with 5% non-fat milk (GE Healthcare, USA) for
120 min at room temperature, and blots were then incubated with
antibodies against Histone H3 Acetyl (Santa Cruz Biotechnology Inc,
USA) overnight at 4 °C. Anti-rabbit horseradish-peroxidase-conjugated
IgG (1:1000; DakoCytomation) was used to detect the binding of its
correspondent antibody. Blots were also re-probed for β-actin (Cell
Signalling) to ensure equal protein loading. Protein expression was
detected with ECL Advanced Western Blotting Detection Kit (GE
Healthcare) and quantified using Quantity One (v.4.6.7) analysis soft-
ware (BioRad).
Cell viability was assessed using 0.4% trypan blue staining im-
mediately after culture. Black staining cells were considered as non-
viable cells and unstained bright cells as viable. Cell viability at 24hrs
(
%)=(the total number of viable cells at 24 h-the total number of viable
cells at the beginning of the experiment/the total number of viable cells
at the beginning of the experiment). All experiments were repeated a
minimum of 3 times with averages displayed graphically.
HL60 cells treated with 10.0 µM caged metacept-3 15 (with and
without UV treatment), eluted from the surface of a coated DEBc, and
metacept-3 1 for 24 h were washed with phosphate-buffered saline
(
PBS). Protein extracts were obtained from HL60 cells using extraction
buffer with protease inhibitors (Roche) and PhosSTOP (Roche).
Histones were isolated by acid extraction. Briefly, cells were lysed in
ice-cold lysis buffer (10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl
2
,
Results of in vitro studies were expressed as means ± standard
error of the mean (SEM), and analyzed using GraphPad Prism 5 soft-
ware (GraphPad Software, Inc., La Jolla, CA, USA), using unpaired t-
tests for two-group comparisons and one-way analysis of variance and
Dunnett's post hoc (ANOVA) for three or more group comparisons. P-
value of < 0.05 was considered to be statistically significant.
1
0
mmol/L KCl, 0.5 mmol/L DTT, and 1.5 mmol/L phe-
nylmethylsulfonyl fluoride), with protease inhibitors (ABCAM) and
5
mol/L H
2
4
SO was added. After incubation on ice for 1 h the sus-
pension was centrifuged and the supernatant was harvested, mixed
with acetone at a ratio of 9:1, and incubated at −20 °C overnight. After
centrifugation, the pellet was washed with 70% ethanol, air dried, and
Caged metacept-3 analogue 15 was subjected to photolysis at
UV365 nm using a UVP® CL-1000® Ultraviolet Crosslinker (5.0 cm
distance from light source to sample) to study the photolytic release of
the acid-soluble histone fraction dissolved in H O. Protein concentra-
2
tions were determined using Bradford protein assay (BioRad).
3

G.R. Sama, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127291
Table 1
Caged metacept-3 analogue 15 photolysis results.
Time interval
Caged metacept-3 15
Metacept-3 1
%
%
Initial
100
53.3
18
0
3
4
6
7
9
0 min
5 min
0 min
5 min
0 min
46.7
82
8.4
2.4
0.7
91.6
97.6
99.3
Fig. 1. Effect of caged metacept-3 15 pre-UV activation, metacept-3 1 and PTX
on HUVEC toxicity (*p < 0.05 vs 1.0 mmol/L metacept-3 1 and 1.0 mmol/L
PTX). Control = Untreated cells.
the target hydroxamic acid (metacept-3, 1) in a polar solvent (Scheme
4
). Initially, a 4.26 mM solution of caged metacept-3 analogue 15 was
prepared in a MeCN and water (1:3) solvent system and irradiated with
an 8-Watt UV365nm light. Subsequent HPLC analysis of the photolytic
reaction aliquots collected at 30, 45, 60, 75, and 90 min after UV ex-
posure revealed formation of the required hydroxamic acid 1 as the
major product (Scheme 4, Table 1). The analysis showed that 47% of
angioplasty balloon at a dose of 3.0ug/mm2 using a pre-programmed
SonoTek Exactacoat Ultrasonic Spray Coating System with an AccuMist
8
ultrasonic nozzle as previously described and exposed to UV365nm
light (65mW) for 4 min. Western blot experiments using a HL-60 cell
line were performed to evaluate compound release and subsequent
HDAC inhibitory behaviour as measured by level of histone H3 acet-
ylation (blue arrowhead). HDACi activity of caged metacept-3 analogue
the available metacept-3
1
(m/z 307.0751, calculated for
+
(
C
14
H
15
N
2
O
4
S) ), peak at ~6.6 min) was released after 30 min of ex-
posure to UV light and the remaining 53% of the caged metacept-3 15
was unchanged.
1
5 (10.0 µM) eluted from coated DEBs without UV exposure (caged
For every 15 min, half of the starting material 15 was cleaved and
after 90 min, 99% of the caged metacept-3 analogue 15 had undergone
photolysis. Significantly, negligible amount of amide formation (i.e.
formation of 16, peak at ~8.3 min) was observed (for chromatograms
see supporting information).
metacept-3 15 without UV) demonstrated no significant increase in
histone H3 acetylation compared to control (untreated cells). Caged
metacept-3 15 (10.0 µM) eluted from coated DEBs with UV exposure
(
Caged metacept-3 15 + UV) demonstrated increased histone H3
acetylation (blue arrowhead) similar to levels identified with metacept-
1. This experiment provides support for the release of metacept-3 1
Subsequently, the caged metacept-3 15 photolysis experiment was
repeated using a 85 µM concentration in a DMSO, EtOH and water
3
after photolysis in its bioactive form and preservation of its biological
activity as a histone deacetylase inhibitor (HDACi) of similar potency to
metacept-3 1 (Fig. 2). Β-Actin levels indicate equal protein loading. A
replicate western blot is available in Supporting information Fig. 3.
Photo-activation of small molecules with potentially significant
pharmacological and therapeutic activity may aid in provision of tar-
geting treatment strategies particularly in the setting of novel DEBc
coatings. In order to investigate the potential therapeutic utility of
photo-activation of the HDACi metacept-3 1 a caged metacept-3 ana-
logue 15 was synthesised using a convergent synthetic approach. Two
fragments were synthesised, O-substituted hydroxylamine intermediate
(
4:4:92 v/v/v) solvent mixture. Aliquots of samples were withdrawn at
2
1
, 4 and 6-minute time intervals after exposure at 5.0 cm to UV365nm,
00mW (OmniCure S2000 Spot UV Curing System). In contrast to the
earlier experiment, an increase in the hydroxamic acid compound
(
metacept-3 1, peak at 6.8 min) was observed and the photolysis was
faster. After 6 min of irradiation, only 8.2% of the caged metacept-3 15
standard (Peak at 14.6 min) remained unchanged. In addition, an in-
crease in the concentration of carboxamide 16 (peak at 8.2 min) was
observed and may reflect the effects of using a different solvent to
undertake these studies (see Supporting information for chromato-
grams).
8
1
from acetovanillone 2, and metacept-3 carboxylic acid intermediate
3 from 3-nitrophenyl boronic acid 9. These two intermediates were
On decreasing the concentration of the caged metacept-3 standard
1
5 from 4.26 mM to 85 µM, approximately 62% of metacept-3 1 was
then coupled to give caged metacept-3 ester 14 and enzymatic hydro-
lysis of ester group furnished the target caged metacept-3 15. The caged
metacept-3 analogue 15, and all intermediates leading to its synthesis,
were fully characterised. Photolysis HPLC studies confirmed the for-
mation of metacept-3 1 after UV 365 nm light-induced cleavage from
the linker. Significant reduction in the toxicity in HUVEC cell lines was
observed for the caged metacept-3 analogue 15 compared to metacept-
observed after only 2 min of UV exposure with 91% of the caged me-
tacept-3 undergoing photolysis after 6 min of exposure to the 365 nm
UV light (Table 2).
Human umbilical vein endothelial cells (HUVECs) were treated with
1
.0 mmol/L solution of metacept-3 1, 1.0 mmol/L solution of caged
metacept-3 analogue 15 and 1.0 mmol/L solution of PTX. A significant
reduction in cellular toxicity was observed after 24 h of treatment with
caged metacept-3 analogue 15 when compared to native metacept-3 1
and PTX (Fig. 1). We have previously investigated MCT-3-mediated
3
1 and PTX. Metacept-3 1 formed after photolysis of the caged meta-
cept-3 analogue 15 was found to induce acetylated histone H3 activity
in vitro, with levels comparable to native metacept-3 1, thereby con-
firming its HDACi activity and therapeutic potential.
8
concentration dependent effects on HUVEC cell viability and specifi-
cally investigated the highest (1.0 mmol/L) concentration of all drugs
assayed in order to demonstrate the maximum concentration dependent
differences in cell viability attributable to each compound.
Previous studies have identified UV and visible light-mediated
13–15
synthesis and activation of HDACi.
To our knowledge this is the
first study investigating photo-activation of an hydroxamate HDACi,
using the convergent synthetic strategy previously outlined, for the
specific purposes of DEBc-mediated vascular targeting. Given the rela-
tively short clinical time frames associated with DEB intervention
(< 3–4 min) identification of a low toxicity, time efficient, photo-ac-
tivatable agent such as caged metacept-3 analogue 15 may afford an
improvement to current DEB coatings which have been associated with
Caged metacept-3 15 was coated to the surface of an inflated
Table 2
Caged metacept-3 15 photolysis results in DMSO/EtOH/H2O.
Time interval
Caged metacept-3 15
Metacept-3 1
Carboxamide 16
7
,16
2
4
6
Minutes
Minutes
Minutes
28.9
11.1
8.2
61.6
79
9.3
both local and systemic toxicity issues.
Future studies will in-
9.8
vestigate the viability of using the caged metacept-3 analogue 15 as an
alternative DEBc coating in in vitro and in vivo models.
81.4
10.2
4

G.R. Sama, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127291
Fig. 2. Effect of caged metacept-3 15 with and without UV treatment on histone H3 expression in HL-60 cells.
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Gopal Reddy Sama is grateful to Monash University for the award of
Monash Graduate Scholarship (MGS), Monash International
Postgraduate Research Scholarship (MIPRS) and the School of
Chemistry for Dean's Postgraduate Research Scholarship (DPRS) and
Dean's International Postgraduate Research Scholarship (DIPRS).
Professor Helmut Thissen and Mr Paul Pasic, CSIRO Manufacturing,
Melbourne, Victoria, Australia for assistance with ultrasonic coating.
This work was supported, in part, by the Eastern Health Foundation
Research Grants Program.
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