Fluorescent analogs of peptoid-based HDAC inhibitors: Synthesis, biological activity and cellular uptake kinetics

Article abstract of DOI:10.1016/j.bmc.2019.07.055

Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.

Full text of DOI:10.1016/j.bmc.2019.07.055

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorescent analogs of peptoid-based HDAC inhibitors: Synthesis, biological
activity and cellular uptake kinetics
Rick Raudszusa,b, Robert Nowotnyc, Christoph G.W. Gertzend,e, Andrea Schölera,
Andor Krizsanf,g, Ines Gockelc, Hermann Kalwab, Holger Gohlked,e, René Thiemec,
⁎
Finn K. Hansena,
a Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Medical Faculty, Leipzig University, Brüderstraße 34, 04103 Leipzig, Germany
b Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, Leipzig University, Härtelstraße 16-18, 04107 Leipzig, Germany
c Department of Visceral, Transplant, Thoracic and Vascular Surgery, University Hospital of Leipzig, Liebigstraße 20, 04103 Leipzig, Germany
d Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
e John von Neumann Institute for Computing (NIC), Jülich Supercomputing Centre (JSC) and Institute for Complex Systems – Structural Biochemistry (ICS-6),
Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany
f Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany
g Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered
as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC)
inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a
dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model.
Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by
the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were
tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to
rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in
the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-
(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory
capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that
compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all
compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative
properties.
Cancer
Histone deacetylase
HDAC inhibitor
Peptoid
Fluorescent probes
1. Introduction
remodeling, several non-histone proteins are also modulated by dif-
ferent HDAC isoforms.6 Comparing the 11 zinc-dependent isoforms in
Histone deacetylases (HDACs) catalyze the removal of acetyl groups
of lysine side chains of histones and thereby play an important role in
the regulation of transcription and protein expression.1,2 A dysregu-
lated HDAC activity has been associated with cancer development and
HDACs are often overexpressed in cancer cells.3,4 To date, 18 different
HDAC isoforms were identified in the human genome and divided into
four classes (I-IV). Only class I (HDACs 1, 2, 3 and 8), class IIa (HDACs
4, 5, 7, and 9) class IIb (HDACs 6 and 10), and class IV (HDAC 11)
isoforms contain zinc-dependent catalytic domains, whereas class III
isoforms (also called sirtuins) are NAD+-dependent enzymes.5 Al-
though many effects of HDACs 1–3 are related to chromatin
their localization, structure and substrates, HDAC6 is structurally and
functionally unique. HDAC6 is the only isoform mainly located in the
cytoplasm, is the only HDAC with two independent functional catalytic
domains, and it is primarily acting via the deacetylation of non-histone
substrates such as α-tubulin, cortactin or Hsp90.2,7,8
HDACs are considered emerging epigenetic drug targets and four
HDAC inhibitors (HDACi) have been approved by the U. S. Food and
Drug Administration (FDA) to treat T-cell lymphoma or multiple mye-
loma (see Fig. 1).5 Furthermore, the class I selective aminoanilide-based
HDACi tucidinostat (chidamide) was approved for the treatment of
relapsed or refractory peripheral T-cell lymphoma (PTCL) in China.5,9
⁎ Corresponding author.
E-mail address: finn.hansen@uni-leipzig.de (F.K. Hansen).
https://doi.org/10.1016/j.bmc.2019.07.055
Received 26 March 2019; Received in revised form 23 July 2019; Accepted 31 July 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:RickRaudszus,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.07.055

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Fig. 1. Approved histone deacetylase inhibitors.
The molecular structure of HDACi can be described by a simple cap-
linker-chelator pharmacophore model.5 The chelator or zinc-binding
group (ZBG) chelates the catalytic zinc ion in the active site, the linker
occupies the narrow hydrophobic tunnel inside the enzyme and the cap
groups interacts with the surface area.10 First-generation HDACi are
usually non-selective pan-inhibitors, which possess several side effects
such as fatigue, diarrhea, weight loss, bone marrow depression, and
cardiac arrhythmias.1 Therefore, current drug discovery efforts focus on
the development of class- or even isoform-preferential HDACi. The in-
creasing number of available X-ray co-crystal structures has fueled the
development of selective compounds and several class- and isoform-
preferential HDACi are currently investigated in clinical trials.11 How-
ever, there is still a lack of knowledge about the cellular penetration
and subcellular location of different types of HDACi. Fluorescent tag-
ging for cellular imaging and cell-based microscopy studies allow in-
vestigating subcellular localization and dynamics of drug targets.12
Furthermore, fluorescent tagging represents a powerful alternative to
radio-labelling to study cellular uptake kinetics. In this work, we de-
scribe the rational design, synthesis, biological activity and cellular
uptake kinetics of a small series of novel fluorescent HDACi.
class I selective HDACi tucidinostat, entinostat and mocetinostat are all
utilizing a benzyl linker in combination with the aminoanilide ZBG.
Thus, compound 3 was designed as a potential class I selective HDACi
featuring a benzyl group as linker and an aminoanilide ZBG.
The target compounds 1–3 were synthesized as summarized in
Schemes 1–3. In the first step benzylamine 4 was acylated with bro-
moacetyl bromide 5 to provide the bromoacetyl amide 6. Next, methyl
4-(aminomethyl)benzoate hydrochloride was reacted with 6 using
triethylamine as base to afford the secondary amine 7. Subsequently,
the fluorescent tag was introduced by the treatment of 7 with dansyl
chloride. The synthesis of the target compound 1 was finally accom-
plished by the hydroxylaminolysis of the methyl ester derivative 8
(Scheme 1). The target compound 2 was synthesized using a similar
synthetic pathway (Scheme 2). Briefly, the alkylation of methyl 6-
aminohexanoate hydrochloride with 6 in the presence of triethylamine
in DCM provided the alkyl-substituted secondary amine 9. The in-
troduction of the fluorophore followed by hydroxylaminolysis yielded
the target compound 2. The aminoanilide-based HDACi 3 was prepared
as illustrated in Scheme 3. The saponification of the key intermediate 8
afforded carboxylic acid 11. Next, 11 was coupled with mono-Boc-
protected phenylenediamine 12 using HATU as coupling agent to fur-
nish the Boc-protected compound 13. Finally, the treatment of 13 with
trifluoroacetic acid in dichloromethane provided the desired aminoa-
nilide-based HDACi 3.
2. Results
2.1. Design and synthesis of target compounds
Peptoids (or N-substituted glycines) are peptidomimetic analogues
of naturally occurring peptides. We have recently reported two types of
peptoid-based HDACi that are easily accessible by a multi-component
approach.13–16 Depending on the linker type and the nature of the cap
group, the isoform preference can be altered from HDAC6 preferential
to HDAC1-3 preferential.13–16 The aim of this work was to retain the N-
substituted glycine (peptoid) scaffold and to introduce a fluorescent tag
at the capping group, as this group is most solvent-exposed.
2.2. Inhibition of HDAC1, 2, 3 & 6, cytotoxicity and hyperacetylation of
histone H3 in esophageal adenocarcinoma cells OE33 & OE19
All synthesized fluorescent tagged compounds (1–3) were screened
in a biochemical assay for their inhibition of selected HDAC isoforms
using ZMAL (Z-Lys(Ac)-AMC) as substrate and compared to the in-
hibition capacity of vorinostat. HDAC1-3 were chosen as
a re-
presentative class I isoforms. HDAC6 was selected because it is the main
target of the peptoid-based HDACi of type I. The results are presented in
Table 1. Compound 1 bearing a benzyl linker showed potent activity
against HDAC6 (IC50: 0.132 µM) and reduced activity against HDAC1
(IC50: 0.915 µM), HDAC2 (IC50: 3.50 µM) and HDAC3 (IC50: 1.02 µM).
Compound 2 containing an alkyl linker displayed unselective HDAC
inhibition with IC50 values in the double-digit nanomolar range
Accordingly, the HDACi pharmacophore model tolerates a variety of
cap groups and this region has been used previously for labelling12,17,18
and hybridization19–21 approaches. The dansyl group is a widely used
fluorophore and is structurally related to typical HDACi caps. For in-
stance, both naphthyl and 4-dimethylaminophenyl residues were tol-
erated in our peptoid-based HDACi of type I and II.13,15,16 Therefore,
the dansyl moiety was chosen as fluorescent tag in this study. Our de-
sign strategy included the variation of the linker and ZBG (Fig. 2).
Compound 1 containing a benzyl linker was designed based on our
HDAC6 preferential inhibitors of type I, whereas compound 2 is derived
from our class I preferential peptoids of type II. The aminoanilide group
(as realized in e.g. tucidinostat) is an alternative ZBG with class I se-
lectivity and slow on, slow off binding kinetics.22 The well-established
(HDAC1 IC50
: 0.034 µM; HDAC2 IC50: 0.081 µM; HDAC3 IC50:
0.087 µM; HDAC6 IC50: 0.046 µM). As expected, the aminoanilide 3 was
inactive against HDAC6 (IC50: > 10 µM) and showed submicromolar
inhibition of HDAC1 (IC50: 0.676 µM) and HDAC2 (IC50: 0.668 µM).
Subsequently, compounds 1–3 and vorinostat were used in pro-
liferation assays in the esophageal adenocarcinoma cell lines OE33 and
OE19 for 72 h (Table 1). The esophageal adenocarcinoma still has a
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Fig. 2. Design of fluorescent analogs of peptoid-based HDAC inhibitors.
poor prognosis and only 50% of treated patients respond to che-
motherapy. Therefore, novel concepts and treatment regimens, like
HDACi, are needed to increase susceptibility to common chemother-
apeutics and to improve patient outcome.23 Compared with vorinostat
(GI50 OE33: 1.12 µM; GI50 OE19: 1.42 µM) compounds 1 and 3 showed
relatively high GI50 values and failed to inhibit the proliferation suffi-
ciently in esophageal adenocarcinoma cells. However, HDACi 2 had an
increased anti-proliferative activity with sub-micromolar GI50 values of
0.776 µM (OE33) and 0.925 µM (OE19). To confirm the HDAC in-
hibitory activity in vitro, histone H3 (Lys9) acetylation was analyzed by
western blot analysis after HDACi treatment for 48 h (1: 6.5 µM; 2:
2.25 µM; 3: 0.6 µM). All compounds induced histone H3 hyperacetyla-
tion in comparison to untreated controls in OE33 and OE19 cells,
clearly demonstrating the inhibitory capacity of HDACi 1–3 in an in
vitro cellular model. Representative western blots for OE33 and OE19
are shown in Fig. S1 (Supporting Information).
X-ray crystal structures of HDAC1 and HDAC6 as representatives for
HDAC classes I and IIb, respectively. We identified binding poses of all
compounds interacting with the zinc ions via their ZBG except for
compound 3 in HDAC6 (Table 2), likely due to its spacious ZBG. This is
in accordance with compound
3 being inactive towards HDAC6
(Table 1). For the remaining ligand-receptor combinations, the re-
spective IC50 values roughly correlate with the size of the largest pose
cluster, a proxy for how favorable the docking is.24 As such, compound
2, which exhibits the lowest IC50 towards all tested HDAC isoforms,
always contains > 50% of all docking poses in that cluster, although it
does not always show the lowest docking energy (Table 2). The binding
poses of the three compounds in HDAC1 exhibit a similar orientation,
with the benzyl moiety interacting with H28 and F150, while the dansyl
moiety interacts with Y204 and L271 (Fig. 3A-C). As to differences
among the HDACi, compound 2 binds to the zinc ion with optimal
geometry (distance ≈ 2.1 Å), and compound 1 cannot form π-stacking
interactions with F205. Compound 3, in turn, forms π-stacking inter-
actions with F205 but shows an increased distance to the zinc ion by
1.4 Å compared to compound 2. This could explain why compound 2
exhibits the lowest IC50 towards HDAC1. In HDAC6 compound 2 forms
equally favorable contacts as in HDAC1, while the distance and binding
geometry of the ZBG of compound 1 is more favorable in HDAC6
(Fig. 3C, D). Hence, our docking reproduces the selectivity profile of
compounds 1–3.
Taken together, the benzyl-based HDACi 1 revealed HDAC6 pre-
ferential inhibition, whereas compound 2 with an alkyl linker showed
potent and unselective inhibition of HDAC1-3 and HDAC6 with IC50
values in the double-digit nanomolar concentration range. The ami-
noanilide 3 was identified as a moderate and preferential inhibitor of
HDAC1-3. In agreement with the results from the biochemical assays,
compound 2 displayed the highest anti-proliferative activity of all
compounds and exceeded activity of the reference HDACi vorinostat.
2.3. Molecular modelling and docking studies
2.4. Photophysical data
In order to find a structural explanation for the selectivity profiles of
In order to characterize the photophysical properties of the syn-
thesized compounds, the absorption and emission spectra as well as the
the HDACi, compounds 1–3 were docked into the binding pockets of the
Scheme 1. Synthesis of compound 1.
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Scheme 2. Synthesis of compound 2.
Stokes shift of the three fluorescent-tagged compounds (1–3) were de-
termined in three different polar solvents (Table S1, Supporting
Information). Water as solvent generated lower Stokes shifts
(138–166 nm) because of the higher wavelengths of the absorption
maxima and the lower wavelengths of the emission maxima.
The opposite was observed for methanol and DMSO, which resulted
in higher Stokes shifts (186–202 nm).
the fluorescence signal after HDACi treatment for 48 h (Fig. S2,
Supporting Information).
3. Conclusion
In summary, we have synthesized three novel HDACi with fluor-
escent properties and studied their biological activity and cellular up-
take kinetics. The widely used dansyl group was chosen as the fluor-
escent tag and was introduced in the cap region of the HDACi
pharmacophore model. The target compounds were synthesized using a
straightforward submonomer protocol followed by the introduction of a
hydroxamic acid or 2-aminoanilide function as ZBG. All three com-
pounds were screened for their inhibition of recombinant HDAC1-3 and
HDAC6. As expected, compound 1 with a benzyl linker displayed pre-
ferential inhibition of HDAC6, whereas the alkyl-based HDACi 2
showed potent and unselective inhibition of all tested isoforms with
IC50 values in the double-digit nanomolar concentration range. The
aminoanilide-based HDACi 3 revealed moderate and preferential in-
hibition of HDAC1-3. Molecular modelling and docking studies pro-
vided structural explanations for the observed differences in the HDAC
isoform profiles of 1–3. Notably, compound 2 showed the highest anti-
proliferative activity in proliferation assays with esophageal adeno-
carcinoma cells (OE33 andOE19) and exceeded the activity of the FDA-
approved drug vorinostat. It can be assumed that this effect is related to
its potent and unselective HDAC inhibitory activity. In addition, our
results from a fluorescence microscopy study demonstrated differences
in the cellular uptake of compounds 1–3. The rapid and increased
cellular uptake of HDACi 2 might therefore contribute to its high anti-
proliferative properties.
2.5. Fluorescence microscopy und cellular uptake kinetics
Fluorescent tagging represents a simple and efficient method to
study cell penetration of small molecules. Therefore, we utilized the
fluorescent properties of our compounds 1–3 to monitor their cellular
uptake by fluorescence microscopy. OE33 (Fig. 4A) and OE19 (Fig. 4B)
cells were treated with 1–3 (1.25 µM) for 30 min. We were able to
monitor an uptake kinetic for all three inhibitors. After 30 min the
absolute fluorescence intensity was higher for all three compounds in
the OE19 cells than the OE33 cells. Interestingly, inhibitor 2 displayed
in both cell lines the fastest uptake kinetic and reached the highest
absolute fluorescence intensity. Representative images are shown after
HDACi treatment for 15 min (Fig. 4C). HDACi 1 and 3 showed com-
parable uptake kinetics but displayed slower uptake ratios than 2.
Notably, HDACi 2 showed the strongest anti-proliferative activity of all
three compounds and exceeded the activity of the reference compound
vorinostat (Table 1). Previously, it has been hypothesized that it might
be beneficial to enhance the cytotoxicity of HDACi by the simultaneous
inhibition of class I and IIb isoforms.25–27 Thus, the high activity of
HDACi 2 in the proliferation assays is most probably related to its un-
selective inhibition of class I HDAC isoforms and HDAC6 (Table 1).
However, the results from our fluorescence microscopy study indicate
that the rapid and more complete cellular uptake of HDACi 2 might
contribute to the increased anti-proliferative activity of this compound
compared to its analogs 1 and 3.
Taken together, our novel fluorescent HDACi 1–3 are promising
starting points to study the uptake kinetics of HDACi into cancer cells
and to investigate the subcellular location of histone deacetylases.
Finally, in order to investigate possible differences in the cellular
retention of the fluorescent probes 1–3, we performed confocal mi-
croscopy studies and observed predominantly cytoplasmic retention of
all three compounds (Fig. 4D). This is in accordance with previously
published results.17,18 Additionally, we noticed a similar distribution of
4. Experimental data
4.1. Chemistry
All reagents and solvents were commercially available and used
Scheme 3. Synthesis of compound 3.
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Table 1
Inhibition of HDAC1, 2, 3 and 6 and inhibition of proliferation in the esophageal adenocarcinoma cells OE33 and OE19.
Compound
HDAC1
HDAC2
HDAC3
HDAC6
OE33
OE19
IC50 [µM]
IC50 [µM]
IC50 [µM]
IC50 [µM]
GI50 [µM]
GI50 [µM]
1
0.915
0.034
0.676
0.089
0.006
0.001
0.130
0.001
3.50
0.33
1.02
0.13
0.132
0.046
> 10
0.038
0.003
0.004
6.23
0.776
4.59
1.12
0.91
0.195
0.16
0.35
12.9
0.925
9.10
1.42
1.2
0.037
0.72
0.10
2
0.081
0.668
0.183
0.003
0.021
0.007
0.087
1.13
0.008
0.006
0.004
3
Vorinostat
0.105
0.001
(pumps), Gynkotek UVD 340U (UV-detector) using a Macherey-Nagel
EC 250/4 NUCLEODUR 100-5 C18ec column. UV absorption was de-
tected at 254 nm with a linear gradient of 10% B to 95% B in 7 min
followed by isocratic elution at 95% B for 10 min using HPLC-grade
water +0.1% TFA (solvent A) and HPLC-grade acetonitrile +0.1% TFA
(solvent B) for elution at a flow rate of 1 mL/min. The purity of all final
compounds was 95% or higher.
Table 2
Results of the docking of compounds 1–3 into the X-ray crystal structures of
HDAC1 and HDAC6.
Compound
HDAC11
HDAC61
1
2
3
−11.61/20
−15.22/55
−17.77/29
−12.93/20
−16.04/53
n/a2
1
2
Docking energy in kcal mol−1/percent of poses in largest cluster.
No poses complexing the zinc ion were found.
4.1.1. N-Benzyl-2-bromoacetamide (6)
Benzylamine 4 (1.1 mL, 10 mmol, 1.0 eq) and DIPEA (1.7 mL,
10 mmol, 1.0 eq) were dissolved in dichloromethane (10 mL).
Bromoacetyl bromide 5 (0.87 mL, 10 mmol, 1.0 eq) was added dropwise
at 0 °C and the reaction was stirred for 16 h at room temperature.
Afterwards the solvent was removed under reduced pressure. The re-
maining residue was suspended in 5% aqueous hydrochloric acid
(5 mL) and extracted with dichloromethane (3 × 15 mL). The combined
organic layers were dried over sodium sulfate and concentrated under
reduced pressure to provide the bromoacetyl amide 6 as a yellow oil
(2.27 g, 99%). 1H NMR (400 MHz, CDCl3) δ 7.41–7.31 (m, 5H), 6.79 (s,
1H), 4.51 (d, J = 5.7 Hz, 2H), 3.96 (s, 2H) ppm. 13C NMR (100 MHz,
CDCl3) δ 165.3, 137.2, 128.8, 127.8, 127.7, 44.2, 29.1 ppm. HRMS
(ESI) m/z calculated for (M + Na)+ 249.984, found 249.9786.
without further purification. The high-resolution mass spectra were
measured by the Leipzig University Mass Spectrometry Service using a
Bruker Daltonics ESQUIRE 3000Plus (ESI). The nuclear magnetic re-
sonance spectrometry was performed using Varian Mercury-300BB
(300 MHz) or Varian Mercury-400BB (400 MHz) spectrometers.
Chemical shifts (δ) are given in ppm relative to the residual signals of
the respective solvents. 1H NMR signals marked with an asterisk (*)
correspond to peaks assigned to the major rotamer conformation.
Coupling constants (J) are reported in hertz (Hz). Following types of
signals were qualified: singlet (s), doublet (d), double doublet (dd),
triplet (t), quartet (q) and multiplet (m). See Supporting Information for
spectroscopic characterization. A Barnstead Electrothermal 9100 ap-
paratus was used for the determination of melting points. Thin layer
chromatography was carried out using Machery-Nagel precoated alu-
minum foil sheets. Vorinostat was synthesized according to the litera-
ture.28 Analytical HPLC analysis were carried out on a HPLC system
equipped with a Gynkotek Gina 50 (autosampler), Dionex P680 HPLC
4.1.2. Methyl 4-({[2-(benzylamino)-2-oxoethyl]amino}methyl)-benzoate
(7)
Methyl 4-(aminomethyl)benzoate hydrochloride (2.0 g, 10 mmol,
1.0 eq) and triethylamine (2.8 mL, 20 mmol, 2.0 eq) were dissolved in
dichloromethane (10 mL). After adding 6 (2.3 g, 10 mmol, 1.0 eq), the
Fig. 3. Predicted binding poses of compounds 1 (A, D, green), 2 (B, E, gray), and 3 (C, salmon) in the X-ray crystal structures of HDAC1 (A-C, blue) and HDAC6 (D, E,
orange). All three compounds complex the zinc ion (sphere) with their ZBGs.
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Fig. 4. Cellular uptake of compounds 1–3. A) OE33 and B) OE19 cells were treated with 1.25 µM of compound 1 (red), 2 (green) or 3 (blue) and the increase of the
fluorescence intensity were measured (Eex = 370 nm and Eem = 530 nm) every 10 sec for 30 min. C) Representative pictures were shown for compound 1–3 and a
control after 15 min. D) Confocal microscopy studies of OE33 and OE19 (50 µm scale) after 30 min incubation with compounds 1–3 (1.25 µM).
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reaction was stirred for 16 h at room temperature after which the sol-
vent was removed under reduced pressure. The remaining residue was
suspended in water and the aqueous solution was extracted with di-
chlormethane (3 × 15 mL). The combined organic layers were washed
with sodium bicarbonate solution (3 × 10 mL) and brine (1 × 10 mL)
dried over sodium sulfate and concentrated under reduced pressure.
The crude product was purified by column chromatography (petroleum
ether/ethyl acetate 75:25 to 0:100) to yield 7 as a yellow oil (2.2 g,
71%). 1H NMR (400 MHz, CDCl3) δ 8.00–7.95 (2·d, J = 8.2 Hz, 2H),
7.46–7.29 (m, 7H), 6.95 (s, 1H), 5.32 (s, 1H), 4.49*/4.44 (2·d, J = 5.8/
5.9 Hz, 2H), 3.94 (s, 3H), 3.85*/3.81 (2·s, 2H), 3.39*/3.29 (2·s, 2H)
ppm. 13C NMR (100 MHz, CDCl3) δ 171.0, 166.8, 144.4, 138.1, 129.9,
129.3, 128.7, 127.9, 127.7, 127.5, 58.3, 53.6, 52.0, 43.0 ppm. HRMS
(ESI) m/z calculated for (M + H)+ 313.1547, found 313.1504.
1H), 5.33 (s, 1H), 4.35 (d, J = 5.7 Hz, 2H), 3.90 (s, 2H), 3.69 (s, 3H);
3.34 (t, J = 8.0 Hz, 2H), 2.97 (s, 6H), 2.19 (t, J = 7.4 Hz, 2H),
1.54–1.46 (m, 4H), 1.24–1.14 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3)
δ 173.7, 168.3, 137.5, 133.1, 131.1, 130.4, 130.0, 129.9, 129.8, 128.7,
128.6, 127.6, 127.5, 123.3, 118.6, 115.4, 51.5, 51.4, 49.7, 45.4, 43.4,
33.6, 27.7, 26.1, 24.2 ppm. HRMS (ESI) m/z calculated for (M−H)−
524.2225, found 524.2385.
4.1.6. 4-[({N-[2-(Benzylamino)-2-oxoethyl]-5-(dimethylamino)-
naphthalene}-1-sulfonamido)methyl]-N-hydroxybenzamide (1)
Sodium hydroxide (88 mg, 2.2 mmol, 10 eq) and hydroxylamine
50% aqueous (0.40 mL, 6.6 mmol, 30 eq) were dissolved in methanol
(3 mL) and dichloromethane (1 mL). Then, ester 8 (0.12 g, 0.22 mmol,
1.0 eq) was added and the reaction was stirred at 0 °C for 2 h. The re-
action was monitored by thin layer chromatography. Upon completion,
the solvents were removed under reduced pressure. The solid residue
was suspended in water (1 mL) and the pH was adjusted to 7–8 using
10% aqueous hydrochloric acid. The precipitate was isolated by filtra-
tion and washed with cold water and cold diethyl ether to yield hy-
droxamic acid 1 as a yellow solid (0.11 g, 93%). Mp 127 °C; 1H NMR
(300 MHz, DMSO‑d6) δ 9.03 (s, 1H), 8.47 (d, J = 8.5 Hz, 1H), 8.34–8.26
(m, 2H), 8.22 (d, J = 8.6 Hz, 1H), 7.63–7.53 (m, 4H), 7.32–7.16 (m,
5H), 7.15–7.07 (m, 4H), 4.61 (s, 2H), 4.13 (d, J = 5.8 Hz, 2H), 3.93 (s,
2H), 2.82 (s, 6H) ppm. 13C NMR (75 MHz, DMSO‑d6) δ 208.5, 167.5,
163.9, 151.8, 139.3, 139.0, 135.8, 132.7, 130.3, 129.8, 129.6, 129.3,
128.6, 128.5, 127.7, 127.6, 127.2, 124.0, 119.3, 115.6, 51.3, 48.4,
45.5, 42.4 ppm. HRMS (ESI) m/z calculated for (M−H)− 545.1864,
found 545.2056.
4.1.3. Methyl 6-{[2-(benzylamino)-2-oxoethyl]amino}hexanoate (9)
Methyl 6-aminohexanoate hydrochloride (1.5 g, 8.0 mmol, 1.0 eq)
and triethylamine (2.2 mL, 16 mmol, 2.0 eq) were dissolved in di-
chloromethane (10 mL). Next, 6 (1.8 g, 8.0 mmol, 1.0 eq) was added
and the mixture was stirred for 16 h at room temperature. The solvent
was removed under reduced pressure and the crude product was pur-
ified by column chromatography (DCM/MeOH 100:0 to 90:10) to yield
9 as a yellow oil (0.75 g, 32%): 1H NMR (400 MHz, CDCl3) δ 7.65 (s,
1H), 7.38–7.29 (m, 5H), 7.27 (s, 1H), 4.49 (d, J = 5.9 Hz, 2H), 3.67 (s,
3H), 3.35 (s, 2H), 2.62 (m, 2H), 2.29 (m, 2H), 1.60 (m, 2H), 1.48 (m,
2H), 1.34 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ 173.9, 171.3,
138.4, 128.6, 127.6, 127.3, 52.4, 51.5, 49.8, 42.9, 33.8, 29.5, 26.5,
24.5 ppm. HRMS (ESI) m/z calculated for (M + H)+ 293.1860, found
293.1720.
4.1.7. 6-({N-[2-(Benzylamino)-2-oxoethyl]-5-(dimethyl-amino)-
4.1.4. Methyl 4-[({N-[2-(benzylamino)-2-oxoethyl]-5-(dimethyl-amino)
naphthalene}-1-sulfonamido)-N-hydroxyhexanamide (2)
naphthalene}-1-sulfonamido)-methyl]benzoate (8)
Sodium hydroxide (0.19 g, 4.8 mmol, 10 eq) and hydroxylamine
50% aqueous (0.88 mL, 14 mmol, 30 eq) were dissolved in methanol
(3 mL) and dichloromethane (1 mL). Then, ester 10 (0.25 g, 0.48 mmol,
1.0 eq) was added and the reaction was stirred at 0 °C for 2 h. The re-
action was monitored by thin layer chromatography. Upon completion,
the solvents were removed under reduced pressure. The solid residue
was suspended in water (1 mL) and the pH was adjusted to 7–8 using
10% aqueous hydrochloric acid. The precipitate was isolated by filtra-
tion and washed with cold water and cold diethyl ether to yield hy-
droxamic acid 2 as yellow powder (0.21 g, 85%). Mp 84 °C; 1H NMR
(400 MHz, DMSO‑d6) δ 9.42 (s, 1H), 8.55 (s, 1H), 8.44 (d, J = 8.8 Hz,
1H), 8.21–8.16 (m, 2H), 7.61–7.54 (m, 2H), 7.32–7.14 (m, 7H), 4.22 (s,
2H), 4.02 (s, 2H), 3.28–3.24 (m, 2H), 2.80 (s, 6H), 1.74 (t, J = 7.4 Hz,
2H), 1.42–1.34 (m, 2H), 1.32–1.24 (m, 2H), 1.04–0.96 (m, 2H) ppm.
13C NMR (100 MHz, DMSO‑d6) δ 168.8, 168.1, 151.7, 139.5, 136.0,
130.0, 129.8, 129.6, 128.7, 128.6, 128.4, 127.6, 127.2, 124.0, 119.4,
115.5, 49.1, 48.4, 45.5, 42.5, 32.8, 27.3, 26.1, 25.3 ppm. HRMS (ESI)
m/z calculated for (M−H)− 525.2177, found 525.2264.
Compound
7 (0.31 g, 1.0 mmol, 1.0 eq) was dissolved in di-
chloromethane (15 mL) and the reaction was cooled down to 0 °C be-
fore first pyridine (89 µL, 1.1 mmol, 1.1 eq) and second dansyl chloride
(0.30 g, 1.1 mmol, 1.1 eq) were added consecutively. The reaction was
stirred at room temperature for 48 h. Afterwards the solvent was re-
moved under reduced pressure and the residue was suspended in water.
The aqueous solution was extracted with dichloromethane (3 × 15 mL),
washed with saturated sodium bicarbonate solution (10 mL), 2% aqu-
eous hydrochloric acid (10 mL) and brine (10 mL). The organic phase
was dried over sodium sulfate and the solvent was removed under re-
duced pressure. Finally, the residue was purified by column chroma-
tography (petroleum ether/ethyl acetate 75:25 to 0:100) which yielded
the ester 8 as a yellow solid (0.28 g, 52%). Mp 89–90 °C; 1H NMR
(400 MHz, CDCl3) δ 8.62 (d, J = 8.5 Hz, 1H), 8.33–8.27 (m, 2H), 7.88
(d, J = 8.0 Hz, 2H), 7.55 (q, J = 8.3 Hz, 2H), 7.33–7.30 (m, 1H),
7.27–7.18 (m, 4H), 7.05–7.00 (m, 2H), 6.49–6.41 (m, 1H), 5.32 (s, 1H),
4.58 (s, 2H), 4.12 (d, J = 5.7 Hz, 2H), 3.94 (s, 3H), 3.89 (s, 2H), 2.93 (s,
6H) ppm. 13C NMR (100 MHz, CDCl3) δ 167.4, 166.4, 152.1, 139.6,
137.2, 132.9, 131.4, 130.7, 130.1, 130.1, 129.8, 128.9, 128.8, 128.6,
127.5, 127.4, 126.4, 123.2, 118.4, 115.4, 52.8, 52.1, 50.8, 45.3,
43.3 ppm. HRMS (ESI) m/z calculated for (M + H)+ 546.1984, found
546.1946.
4.1.8. 4-({N-[2-(Benzylamino)-2-oxoethyl]-5-(dimethylamino-
naphthalene)-1-sulfonamido}methyl)benzoic acid (11)
To a solution of sodium hydroxide (62 mg, 1.5 mmol, 4.0 eq) in
water (0.5 mL), methanol (0.5 mL) and tetrahydrofuran (4 mL) was
added ester 8 (0.21 g, 0.39 mmol, 1.0 eq) and the reaction was stirred at
room temperature for 24 h. The solvents were removed under reduced
pressure and the solid residue was dissolved in a sodium bicarbonate
solution (15 mL). After extraction of the aqueous solution with ethyl
acetate (3 × 15 mL) the pH of the aqueous phase was adjusted to 5
using 10% aqueous hydrochloric acid. The aqueous phase was extracted
with ethyl acetate (3 × 15 mL) and the combined organic layers were
washed with brine and dried over sodium sulfate. Removing the solvent
under reduced pressure provided the carboxylic acid 11 as a yellow
solid (0.19 g, 94%). Mp 130–131 °C; 1H NMR (400 MHz, DMSO‑d6) δ
12.94 (s, 1H), 8.48 (d, J = 8.5 Hz, 1H), 8.35–8.32 (m, 2H), 8.24 (d,
J = 8.6 Hz, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.60 (t, J = 8.0 Hz, 2H),
4.1.5. Methyl 6-({N-[2-(benzylamino)-2-oxoethyl]-5-(dimethyl-amino)-
naphthalene}-1-sulfonamido)hexanoate (10)
Compound
9 (0.75 g, 2.6 mmol, 1.0 eq) was dissolved in di-
chloromethane (20 mL) and the mixture was cooled down to 0 °C before
first pyridine (0.23 mL, 2.8 mmol, 1.1 eq) and second dansyl chloride
(0.76 g, 2.8 mmol, 1.1 eq) were added consecutively. The reaction was
stirred at room temperature for 48 h. The solvent was removed under
reduced pressure and the residue was purified as described in 4.1.4 to
yield the ester 10 as a yellow oil (0.96 g, 71%). 1H NMR (400 MHz,
CDCl3) δ 8.70 (s, 1H), 8.34 (s, 1H), 8.22 (d, J = 7.2 Hz, 1H), 7.63–7.52
(m, 2H) 7.36–7.30 (m, 3H), 7.18 (d, J = 8.0 Hz, 2H), 6.88–6.83 (m,
7

R. Raudszus, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
7.30–7.26 (m, 3H), 7.24–7.20 (m, 3H), 7.14–7.11 (m, 2H), 4.67 (s, 2H),
4.16 (d, J = 5.8 Hz, 2H), 3.99 (s, 2H), 2.84 (s, 6H) ppm. 13C NMR
(100 MHz, DMSO‑d6) δ 167.5, 167.4, 151.8, 142.2, 139.2, 135.8, 130.4,
130.3, 129.8, 129.7, 129.5, 129.3, 128.7, 128.6, 128.5, 127.6, 127.2,
124.0, 119.3, 115.6, 51.4, 48.7, 45.5, 42.4 ppm. HRMS (ESI) m/z cal-
culated for (M−H)− 530.1755, found 530.1885.
trypsin buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl) were added,
followed by further incubation at 37 °C for 30 min. Fluorescence was
measured with an excitation wavelength of 355 nm and an emission
wavelength of 460 nm using a Fluoroskan Ascent microplate reader
(Thermo Scientific). All compounds were evaluated in duplicate in at
least two independent experiments.
4.1.9. tert-Butyl (2-{4-[({N-[2-(benzylamino)-2-oxoethyl]-5-(di-methylamino)
naphthalene}-1-sulfonamido)methyl]benzamido}-phenyl)carbamate (13)
The carboxylic acid 11 (0.35 g, 0.65 mmol, 1.0 eq), tert-butyl(2-
aminophenyl) carbamate 12 (0.14 g, 0.65 mmol, 1.0 eq) and HATU
(0.25 g, 0.65 mmol, 1.0 eq) were dissolved in N,N-dimethylformamide
(5 mL) and DIPEA (0.11 mL, 0.65 mmol, 1.0 eq) was added. The reac-
tion was stirred at room temperature for 24 h after which the solvent
was removed under reduced pressure. The residue was dissolved in
dichloromethane (20 mL). After washing with 1% aqueous hydrochloric
acid (3 × 15 mL), saturated aqueous sodium bicarbonate solution
(3 × 15 mL) and brine (15 mL), the organic phase was dried over so-
dium sulfate. The solvent was removed under reduced pressure to yield
compound 13 as a brown solid (0.43 mg, 92%). Mp 93 °C; 1H NMR
(300 MHz, CDCl3) δ 9.16 (s, 1H), 8.63 (d, J = 8.5 Hz, 1H), 8.32 (d,
J = 8.7 Hz, 1H), 8.27 (dd, J = 7.4 Hz, 1.2 Hz, 1H), 7.78 (d, J = 8.0 Hz,
3H), 7.53 (q, J = 8.2 Hz, 2H), 7.32–7.27 (m, 2H), 7.25–7.13 (m, 7H),
7.01 (d, J = 6.0 Hz, 2H), 6.89 (s, 1H), 6,47 (t, J = 5.9 Hz, 1H), 4.57 (s,
2H), 4.12 (d, J = 5.6 Hz, 2H), 3.87 (s, 2H), 2.92 (s, 6H), 1.50 (s, 9H)
ppm. 13C NMR (100 MHz, CDCl3) δ 167.4, 164.9, 154.6, 138.5, 137.3,
134.1, 133.2, 131.3, 130.7, 130.6, 130.0, 129.8, 129.1, 128.8, 128.6,
127.8, 127.5, 126.0, 125.9, 125.7, 124.5, 123.4, 115.6, 81.4, 52.6,
50.6, 45.4, 43.3, 38.6, 28.3, 22.6 ppm. HRMS (ESI) m/z calculated for
(M−H)− 720.2861, found 720.2957.
4.3. Photophysical data
For the absorption and emission spectra the compounds 1–3 were
dissolved in water, methanol and DMSO each to get 1 M stock solutions.
These stocks were diluted 1:10 three times and 200 µL of each dilution
step plus blank were pipetted in a black 96-well plate. The absorption
spectra (250 nm to 750 nm) and the emission spectra (400 nm to
650 nm) were measured in 2 nm steps using a SpectraMax Gemini EM
Microplate Reader. The emission was stimulated by a wavelength near
the absorption maximum.
4.4. Cell culture
The esophageal adenocarcinoma cells OE33 (ECACC-96070808)
and OE19 (ECACC-96071721) were purchased from Sigma-Aldrich
(Taufkirchen, Germany) and grown as described previously.31 For
proliferation assays 3500 cells were seeded to 96-well plates and grown
for 24 h before treated with either vorinostat or HDACi 1–3 for 72 h.
Cells were incubated with PrestoBlue Cell Viability Reagent (Thermo-
Fisher, Darmstadt, Germany) accordingly to the manufacturer's pro-
tocol.
Western blot analysis were porfomed as previousely described.32
Briefly, 500,000 OE33 and 750,000 OE19 cells were seeded to 6-well
plates and treated by HDACi 1–3 for 48 h. Cells were harvested, lysed
and histones were isolated.33 5 µg of nuclear extract were separated on
15% sodium dodecyl sulphate (SDS)-polyacrylamide gels and blotted.
Afterwards the membrane was blocked by 5% low fat milk/TBST for
1 h, incubated with a specific antibody against acetyl-histone H3 (Lys9)
(#9649, CellSignaling, Danvers, USA) and histone H3 (#4499, Cell-
Signalling, Danvers, USA) over night at 4 °C. A peroxidase coupled goat-
anti-rabbit antibody (111-035-045, Jackson Immuno Research, Suffolk,
UK) was used for the detection (1 h at room temperature) and visulized
by ECL chemiluminiszence (Millipore, Billerica, USA).
4.1.10. N-(2-Aminophenyl)-4-[({N-[2-(benzylamino)-2-oxoethyl]-5-
(dimethylamino)naphthalene}-1-sulfonamido)methyl]benz-amide (3)
Compound 13 (0.36 g, 0.50 mmol, 1.0 eq) was dissolved in di-
chloromethane (2.5 mL). Subsequently, trifluoroacetic acid (1.0 mL,
13 mmol, 26 eq) was added dropwise and the reaction was stirred at
room temperature for 3 h. To stop the reaction, the pH was adjusted to
9 using sodium carbonate solution. Then, water (5 mL) was added to
dilute the aqueous phase. Extraction with dichloromethane
(3 × 15 mL), drying over sodium sulfate and removing the solvent
under reduced pressure provided the aminoanilide 3 as a brown solid
(0.27 g, 87%). Mp 116–117 °C; 1H NMR (400 MHz, CDCl3) δ 8.61 (d,
J = 8.5 Hz, 1H), 8.34–8.28 (m, 2H), 7.76 (d, J = 7.9 Hz, 2H), 7.55 (q,
J = 7.6 Hz, 3H), 7.40–7.30 (m, 4H), 7.27–7.18 (m, 4H), 7.14–7.08 (m,
2H), 7.04 (d, J = 6.0 Hz, 2H), 6.97–6.89 (m, 2H), 6.58–6.53 (m, 1H),
4.59 (s, 2H), 4.13 (d, J = 5.8 Hz, 2H), 3.86 (s, 2H), 2.93 (s, 6H) ppm.
13C NMR (100 MHz, CDCl3) δ 167.5, 165.2, 152.2, 140.4, 138.6, 137.3,
134.0, 133.1, 131.4,130.7, 130.0, 129.8, 129.3, 128.9, 128.6, 127.8,
127.6, 127.5, 127.3, 125.2, 124.5, 123.3, 119.8, 118.5, 118.4, 115.5,
52.5, 50.4, 45.4, 43.3 ppm. HRMS (ESI) m/z calculated for (M + H)+
622.2488, found 622.2624.
For the HDACi uptake kinetics, 500,000 cells were seeded to glass
cover slips in 6-well plates and grown for 48 h. The analyses were done
by HDACi treatment for 1 min and afterwards the fluorescence intensity
was recorded by Eex = 370 nm and Eem = 530 nm every 10 sec for
30 min using a Till Photomics Polychrome IV and a Zeiss Axiovert 100
equipped with a 40× Fluar 1.30.
For confocal microscopy studies, cells were treated as mentioned
before. Additionally, cells were transfected with cDNA coding for his-
tone H2b fuzed to the red fluorescent protein (H2B-RFP) with Fugene
(Promega) according to the manufacturers protocol with a DNA to re-
agent ratio 1:1. Imaging experiments were performed with a Leica SP8
microscop using the lasX software package in combination with a 63x
magnification lens and a 405 nm laser for excitation.
4.2. HDAC inhibition assays
The in vitro inhibitory activity of compounds 1–3 and vorinostat
against human HDAC1, HDAC2, HDAC3/NcoR2 and HDAC6 were
measured using a previously published protocol.29 OptiPlate-96 black
microplates (Perkin Elmer) were used with an assay volume of 50 µL.
5.0 µL test compound or control, diluted in assay buffer (50 mM Tris-
HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 0.1 mg/mL
BSA), were incubated with 35 µL of the fluorogenic substrate ZMAL (Z-
Lys(Ac)-AMC)30 (21.43 µM in assay buffer) and 10 µL of human re-
combinant HDAC1 (BPS Bioscience, Catalog# 50051), HDAC2 (BPS
Bioscience, Catalog# 50052), HDAC3/NcoR2 (BPS Bioscience, Cat-
alog# 50003) or HDAC6 (BPS Bioscience, Catalog# 50006) at 37 °C.
After an incubation time of 90 min, 50 µL of 0.4 mg/mL trypsin in
4.5. Molecular docking
For the molecular docking, compounds 1–3 were drawn and con-
verted into a 3D structure with Maestro,34 and energy-minimized with
Moloc.35,36 The HDACi were then docked into crystal structures of
HDAC1 (PDB ID: 4BKX37) and HDAC6 (PDB ID: 5EDU7) utilizing Au-
toDock338,39 as a docking engine and the DrugScore201840 distance-
dependent pair-potentials as an objective function. In the docking, de-
fault parameters were used, with the exception of the clustering RMSD
cutoff, which was set to 2.0 Å, to consider the flexibly connected sa-
turated and unsaturated carbon cycles, as done previously.13,16,25
Docking solutions with more than 20% of all configurations in the
8

R. Raudszus, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
largest cluster were considered sufficiently converged.13,16,25 The con-
figuration in the largest cluster with the lowest docking energy and
with a distance < 3 Å between the hydroxamic acid oxygen and the
zinc ion in the binding pocket was used for further evaluation.
anticancer activity of a focused histone deacetylase (HDAC) inhibitor library with
peptoid-based cap groups. J Med Chem. 2017;60:5493–5506.
17. Kong Y, Jung M, Wang K, et al. Histone deacetylase cytoplasmic trapping by a novel
fluorescent HDAC inhibitor. Mol Cancer Ther. 2011;10:1591–1599.
18. Fleming CL, Ashton TD, Nowell C, et al. A fluorescent histone deacetylase (HDAC)
inhibitor for cellular imaging. Chem Commun. 2015;51:7827–7830.
19. Ganesan A. Multitarget drugs: an epigenetic epiphany. ChemMedChem.
2016:1227–1241.
Acknowledgements
20. de Lera AR, Ganesan A. Epigenetic polypharmacology: from combination therapy to
multitargeted drugs. Clin Epigenetics. 2016;8:105.
We thank Prof. Dr. Ralf Hoffmann (Leipzig University) for providing
us access to the SpectraMax Gemini EM Microplate Reader. We are
grateful to the John von Neumann Institute for Computing (NIC) and
the Jülich Supercomputing Centre for computing time on the super-
computer JURECA (NIC project HKF7 to H.G.). The authors would like
to thank Franziska Rolfs for her excellent technical support.
21. Bhatia S, Krieger V, Groll M, et al. Discovery of the first-in-class dual histone dea-
cetylase-proteasome inhibitor. J Med Chem. 2018;61:10299–10309.
22. Chou CJ, Herman D, Gottesfeld JM. Pimelic diphenylamide 106 is a slow, tight-
binding inhibitor of class I histone deacetylases. J Biol Chem.
2008;283:35402–35409.
23. Al-Batran SE, Homann N, Pauligk C, et al. Perioperative chemotherapy with fluor-
ouracil plus leucovorin, oxaliplatin, and docetaxel versus fluorouracil or capecitabine
plus cisplatin and epirubicin for locally advanced, resectable gastric or gastro-oeso-
phageal junction adenocarcinoma (FLOT4): a randomised, phase 2/3 trial. Lancet.
2019;393:1948–1957.
Appendix A. Supplementary data
24. Cosconati S, Forli S, Perryman AL, Harris R, Goodsell DS, Olson AJ. Virtual screening
with AutoDock: theory and practice. Expert Opin Drug Dis. 2010;5:597–607.
25. Kalin JH, Bergman JA. Development and therapeutic implications of selective his-
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hibitors increase cisplatin potency in chemoresistant cancer cell lines. J Med Chem.
2017;60:5334–5348.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.07.055.
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