Identification of histone deacetylase inhibitors with (arylidene)aminoxy scaffold active in uveal melanoma cell lines

Article abstract of DOI:10.1080/14756366.2020.1835883

Uveal melanoma (UM) represents an aggressive type of cancer and currently, there is no effective treatment for this metastatic disease. In the last years, histone deacetylase inhibitors (HDACIs) have been studied as a possible therapeutic treatment for UM, alone or in association with other chemotherapeutic agents. Here we synthesised a series of new HDACIs based on the SAHA scaffold bearing an (arylidene)aminoxy moiety. Their HDAC inhibitory activity was evaluated on isolated human HDAC1, 3, 6, and 8 by fluorometric assay and their binding mode in the catalytic site of HDACs was studied by molecular docking. The most promising hit was the quinoline derivative VS13, a nanomolar inhibitor of HDAC6, which exhibited a good antiproliferative effect on UM cell lines at micromolar concentration and a capability to modify the mRNA levels of HDAC target genes similar to that of SAHA.

Full text of DOI:10.1080/14756366.2020.1835883

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Identification of histone deacetylase inhibitors
with (arylidene)aminoxy scaffold active in uveal
melanoma cell lines
Susanna Nencetti , Doretta Cuffaro , Elisa Nuti , Lidia Ciccone , Armando
Rossello , Marina Fabbi , Flavio Ballante , Gabriella Ortore , Grazia Carbotti ,
Francesco Campelli , Irene Banti , Rosaria Gangemi , Garland R. Marshall &
Elisabetta Orlandini
To cite this article: Susanna Nencetti , Doretta Cuffaro , Elisa Nuti , Lidia Ciccone , Armando
Rossello , Marina Fabbi , Flavio Ballante , Gabriella Ortore , Grazia Carbotti , Francesco
Campelli , Irene Banti , Rosaria Gangemi , Garland R. Marshall & Elisabetta Orlandini (2021)
Identification of histone deacetylase inhibitors with (arylidene)aminoxy scaffold active in uveal
melanoma cell lines, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 34-47, DOI:
10.1080/14756366.2020.1835883
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
020, VOL. 36, NO. 1, 34–47
2
https://doi.org/10.1080/14756366.2020.1835883
BRIEF REPORT
Identification of histone deacetylase inhibitors with (arylidene)aminoxy scaffold
active in uveal melanoma cell lines
a
a
a
a
a,b
c
Susanna Nencetti , Doretta Cuffaro , Elisa Nuti
, Lidia Ciccone
, Armando Rossello
, Marina Fabbi ,
d
a
c
c
a
c
ꢀ
Flavio Ballante , Gabriella Ortore , Grazia Carbotti , Francesco Campelli , Irene Banti , Rosaria Gangemi ,
Garland R. Marshall and Elisabetta Orlandini
d
b,e
a
b
c
Dipartimento di Farmacia, Universit ꢀa di Pisa, Pisa, Italy; Research Center “E. Piaggio”, Universit ꢀa di Pisa, Pisa, Italy; IRCCS Ospedale Policlinico
d
San Martino, Genova, Italy; Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO,
USA; Dipartimento di Scienze della Terra, Universit ꢀa di Pisa, Pisa, Italy
e
ABSTRACT
ARTICLE HISTORY
Uveal melanoma (UM) represents an aggressive type of cancer and currently, there is no effective treat-
ment for this metastatic disease. In the last years, histone deacetylase inhibitors (HDACIs) have been
studied as a possible therapeutic treatment for UM, alone or in association with other chemotherapeutic
agents. Here we synthesised a series of new HDACIs based on the SAHA scaffold bearing an (arylidene)a-
minoxy moiety. Their HDAC inhibitory activity was evaluated on isolated human HDAC1, 3, 6, and 8 by
fluorometric assay and their binding mode in the catalytic site of HDACs was studied by molecular dock-
ing. The most promising hit was the quinoline derivative VS13, a nanomolar inhibitor of HDAC6, which
exhibited a good antiproliferative effect on UM cell lines at micromolar concentration and a capability to
modify the mRNA levels of HDAC target genes similar to that of SAHA.
Received 29 July 2020
Revised 24 September 2020
Accepted 28 September 2020
KEYWORDS
Uveal melanoma; HDAC
inhibitors; HDAC6; SAHA;
(
arylidene)aminoxy-based
hydroxamates
7
1
. Introduction
tumour suppressor genes . The status of histone acetylation
depends on the balance between histone acetylation and deacety-
lation, induced by histone acetyltransferases (HATs), and HDACs,
respectively. Increasing evidence suggests that the alteration of
Uveal melanoma (UM) is a highly aggressive form of melanoma
and is the most common primary intraocular tumour in adults.
The traditional clinical treatment of patients with UM is the enu-
cleation of the affected eye or, to preserve the vision or the globe,
other clinical options are radiotherapy, photodynamic therapy,
8
HAT/HDAC activity is present in cancer . In mammalians, 18 differ-
ent HDACs have been identified and divided into four classes
(Class I, Class II, Class III, and Class IV) based on their sequence
homology to yeast proteins domain organisation and subcellular
localisation. Class I (subtypes 1,2,3,8), II (subtypes 4,5,6,7,9,10), and
IV (subtype 11) HDACs are zinc-dependent enzymes, while Class
III HDACs (SIRTs 1–7) require the cofactor NAD to express their
activity. Class I can generally be detected in the nucleus and is
ubiquitously expressed.
HDACs are overexpressed in a wide range of diseases including
tumours and consequently become attractive targets for the
1
and systemic chemotherapy . About 50% of the patients have
strong tendency to develop lethal metastasis, principally to the
liver (89%), that are usually identified 2–5 years after treatment of
the primary tumour and are poorly sensitive to chemotherapy.
Thus, the prognosis of these patients becomes poor with a
median survival of 2–18 months. UM can be grouped in two
classes on the basis of their gene expression profile: Class I
tumours with very low metastatic risk and Class II with a high risk
9
þ
2
of metastasis . In the last 10 years metastatic UM has been
1
0–12
development of drugs for cancer treatment
.
studied at the genetic and molecular levels to discover effective
target therapies. Epigenetic alterations, such as changes in DNA
methylation and histone acetylation status, are involved in tumour
HDAC inhibitors (HDACIs) interfere with the deacetylation pro-
cess mediated by HDACs with an increase of histone acetylation.
HDACIs induce cancer cell death through several pathways: apop-
3
progression .
In the past years, a group of enzymes involved in the epigen- tosis, differentiation, cell cycle arrest, and suppression of cell
etic regulation of gene expression, histone deacetylases (HDACs), migration. In UM HDACIs induce morphological differentiation and
5
,13,14
have generated increasing interest as potential therapeutic targets cell-cycle arrest and inhibit the growth of UM tumour in vivo
.
4–6
for UM . HDACs remove the acetyl groups from histone lysine
The first HDAC inhibitor approved by the FDA, in 2006, was
residues from diverse protein targets, resulting in a condensed Vorinostat (SAHA) for the treatment of cutaneous T-cell lymphoma
chromatin structure that downregulates gene expression, also of (CTCL) . To date other three HDAC inhibitors have been
1
5
CONTACT Susanna Nencetti
susanna.nencetti@unipi.it
Dipartimento di Farmacia, Universit ꢀa di Pisa, Via Bonanno 6, Pisa 56126, Italy; Marina Fabbi [Email
icon] marina.fabbi@hsanmartino.it [Postal address icon] IRCCS Ospedale Policlinico San Martino, Genova, Italy.
ꢀ
Present address: Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, Uppsala SE-751 24, Sweden
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
35
Figure 1. Representative compounds of the different structural classes of HDAC inhibitors. Chemical structures of clinically approved drugs are highlighted in box.
1
6
approved by FDA: Romidepsin (FK228)
(
in 2009, Belinostat
1
7
PXD101) in 2014 for the treatment of CTLC or Peripheral T-cell
lymphoma (PTCL), and Panobinostat (LBH589)
18
in 2015 for
Multiple myeloma and CTCL. Chidamide (HBI-8000) has been
approved by the Chinese FDA for relapsed or refractory PTCL in
1
9
2
015 (Figure 1). In vitro studies with some of these HDACIs have
shown a shift of the gene expression profile from Class II (high
metastatic risk) to a Class I (low metastatic risk) in UM cell cul-
5
tures . Moreover, it has been recognised that HDAC inhibitors
such as Vorinostat (SAHA) and Panobinostat (LBH589), may be
useful in adjuvant therapy in patients with high-risk Class II UM
Figure 2. Pharmacophore model of the newly synthesised compounds.
playing a role in preventing the progression of micrometa- mostly represented by a hydroxamic acid (Figure 2). Modifications
1
3
static disease .
Several structural classes of HDAC inhibitors, both natural and the ZBG, have become important strategies that have led to the
of these four structural parts such as the CAP group, the linker, or
2
1
synthetic, have been reported in the literature such as hydroxa- development of a large number of HDACIs .
mates, carboxylates, benzamides, and cyclic peptides (Figure 1). A
As part of our ongoing efforts in identifying new HDAC inhibi-
variety of derivatives of each class have been synthesised and tors potentially active in UM, we decided to develop a series of
2
0
characterised . All classes of HDAC inhibitors share a common hydroxamates based on SAHA scaffold. The choice of SAHA struc-
pharmacophore model, which consists of a CAP group, a connec- ture as a starting point of our study was based on two considera-
tion unit (CU), a linker and a zinc-binding group (ZBG) that is tions: SAHA is an anticancer drug in clinical use and, moreover, its

3
6
S. NENCETTI ET AL.
activity on UM has been already proved by Landreville et al. with aqueous hydroxylamine in basic conditions. The carboxylic
1
3
through an in vitro study on UM cell lines .
acids 3a–c (n ¼ 1) and 4a–d (n ¼ 2) were obtained by saponifica-
In this context, we envisioned developing new HDAC SAHA- tion with 2 N KOH in ethanol of the ethyl esters 6a–c and 7a–f.
based inhibitors of general structure A (Figure 2) in which the
amidic group of SAHA is replaced by a different connection unit
2.2. In vitro HDACs inhibition assays
(CU). In fact, in the synthesised compounds (1a–c, 2a–f, 3a–c and
4a–e, Scheme 1) the linker and the hydrophobic CAP group (R)
The hydroxamic acids 1a–c, 2a–f and carboxylic acids 3a–c, 4a––e
are connected via an oxime ether moiety (C ¼ N–O–) possessing a synthesised in this study were preliminarily tested on a HeLa
2
sp -hybridization, analogously to other CU used in many HDAC nuclear extract containing a mixture of HDACs, by using a com-
2
2
inhibitors .
mercially available HDAC assay kit (BIOMOL). Inhibitory data
Furthermore, since it is widely accepted that the CAP group expressed as IC50 values are reported in Table 1. SAHA and
plays an important role in the HDAC inhibition profile, we have Trichostatin A (TSA) were used as positive controls.
synthesised new derivatives in which the CAP group was changed
The results of this preliminary screening, reported in Table 1,
from a phenyl group of SAHA into a more bulky aromatic moiety. showed that compounds bearing a hydroxamic moiety as ZBG
We have also investigated the influence of the linker length on possess HDAC inhibitory activity while the carboxylic function as
HDAC inhibition activity synthesising compounds with two or four ZBG is detrimental to HDAC inhibition. The length of the linker
carbon atoms in the linear alkyl chain (Figure 2).
In the present paper, we report the synthesis and HDAC inhibi- phatic chain appears nearly optimal: IC50 values of compounds
tory activity of both hydroxamic compounds of type
with n ¼ 1 are in the micromolar range while the IC50 values of
influences the HDAC inhibitory activity and a 4-carbon linear ali-
A
(
R
2
¼NH–OH, 1a–c, 2a–f) and their corresponding carboxylic acid compounds with n ¼ 2 are in the nanomolar range. Among the
analogues (R ¼OH, 3a–c, 4a–e). The HDAC inhibitory activity of hydroxamic acids with n ¼ 2, those having bulkier CAP groups
2
the new compounds was first evaluated against HeLa cell nuclear exhibited superior activity than monoacrylic derivatives. The most
extract containing a mixture of Class I HDACs. Then, with the aim potent compounds, 2b (SN2), 2c (LD10), 2e (VS16), and 2f
to deeply investigate the biological properties of the four most (VS13), as shown in Table 1, resulted 3- to 5-folds more potent
promising hydroxamic derivatives, we performed in vitro assays on than SAHA and were selected for a deeper biological evaluation.
isolated Class I enzymes (HDACs 1,3,8) and Class II HDAC6.
The selected hydroxamates (SN2, VS16, LD10, and VS13) were
tested for their inhibitory activity towards four human isolated
HDACs: HDACs 1, 3, and 8 (Class I HDACs) and HDAC 6 (a Class II
HDAC) to investigate how the different CAP groups could influ-
2
3
Considering the expression of histone deacetylases in UM and
5
the potential therapeutic role of HDACIs in this tumour , the same
compounds were selected for further tests on UM cell lines.
Finally, docking studies were performed to investigate their bind-
ing mode.
2
4
ence the HDAC activity . In particular, HDAC6, which was not pre-
sent in the HeLa nuclear extract used for the preliminary tests, is
2
5–27
considered an important target in cancer therapy
. SAHA, enti-
nostat, and tubastatin A were used as positive controls. As sum-
marised in Table 2, all compounds (except LD10 at HDAC1) were
able to inhibit HDAC1, HDAC3, and HDAC6 in the nanomolar
range while they were less efficient against HDAC8, displaying
2
. Results and discussion
2.1. Chemistry
The synthesis of the target compounds is outlined in Scheme 1. IC50 values in the low micromolar range. Moreover, compounds
Ethyl esters 6a–c were obtained by Michael–type reaction of SN2, VS16, and LD10 resulted in more active on HDAC3 than on
oximes 5a–f with ethyl acrylate, while ethyl esters 7a–f were the other isoforms. For example, SN2 and VS16 had lower IC50
obtained by reaction of the opportune oximes 5a–f with the ethyl values (58 nM and 43 nM, respectively) for HDAC3 than those for
2 3
ester of 5-bromovaleric acid using Cs CO as base. The hydroxa- HDAC1 (698 nM and 593 nM, respectively), or for HDAC6 (316 nM
mic acids 1a–c (n ¼ 1) and 2a–f (n ¼ 2) were directly obtained and 406 nM, respectively). Dose-response curves are illustrated in
from the ethyl esters 6a–c and 7a–f, respectively, by treatment Supplementary Figure S1.
Scheme 1. Synthesis of the hydroxamic acids 1a-c, 2a-f and carboxylic acids 3a-c, 4a-e.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
37
Table 1. HDACs inhibitory activity of the newly synthesised compounds 1a–c, 2.3. Docking studies
a–f, 3a–c, 4a–e.
2
Compounds LD10, VS13, SN2, and VS16 were docked in HDAC1,
HDAC3, HDAC6, and HDAC8.
The superposition between the docked-pose of two investi-
gated compounds LD10 and VS13 and the PDB structures
2
8
(
human HDACs in complex with hydroxamate inhibitors ) showed
a quite different disposition of both ligands in the HDACs binding
site. Specifically, the CAP group of these inhibitors occupies a
region between loop3 and loop4 of HDACs, while that of the crys-
tallised hydroxamate inhibitors is allocated around loop1, loop2,
and loop4 (Figure 3).
In the HDAC-inhibitor crystal complexes, the region filled by
the CAP of our inhibitors is frequently (but not constantly) occu-
pied by water molecules which engaged hydrogen bonds with
the catalytic histidine. The different pose displayed by compounds
LD10 and VS13 could be related to the substitution of the typical
amidic connection unit, present in the HDAC inhibitors, with the
methyloximino moiety. This group seems to modify the flexibility
of the inhibitors and, consequently, the CAP fitting with the pro-
tein surface.
IC50 on Hela
a
Compd.
R
R
1
R
2
n
extract (mM)
b
1
3
a
a
H
3
3
CS
CS
H
H
NHOH
OH
1
1
NT
28
>100
2
4
a
a
H
H
H
NHOH
OH
2
2
2.880
>100
1
3
b
b
H
H
NHOH
OH
1
1
43.2
>100
The comparison of the four HDAC subtypes highlighted that
the core structure of the catalytic domain is well conserved
2
4
b (SN2)
b
H
H
NHOH
OH
2
2
0.056
>100
(Figure 4(a–d)). The only exception was the different orientation
displayed by Phe152, in HDAC8, with respect to Phe150 (HDAC1),
Phe144 (HDAC3), and Phe620 (HDAC6) (Figure 4(a–d), Figure 5).
Moreover, analysis of loops3 and 4 displayed a detectable
sequence and conformational variability among the four HDAC
isozymes (Figures 4 and 5).
1
3
c
c
H
H
NHOH
OH
1
1
3.346
>100
The major differences were found in HDAC8 where both loops
possess not conserved residues (Figure 5; alignment generated
2
9
2
4
c (LD10)
c
H
H
NHOH
OH
2
2
0.096
>100
using PRALINE ) which influence the binding site surface and the
backbone shape. Precisely, loop4 of HDAC8 opens the binding
cavity outside, with respect to the arrangement of the same loop
in HDAC1 and HDAC3 (see also the surface in Figure 4). On the
contrary, the arrangement of loop3 in HDAC8 (as in HDAC1)
shrinks the binding channel.
2
4
d
d
H
H
NHOH
OH
2
2
4.6
>100
GOLD calculation showed that all ligands have a zinc-coordi-
nated pose in the HDACs binding site.
2
4
e (VS16)
e
Me
Me
NHOH
OH
2
2
0.053
>100
Regarding the CAP group orientation, the differences between
loop3 and 4 regions among the HDAC subtypes can induce a dif-
ferent pose of the studied ligands. While in HDAC3 and 6 the CAP
group of LD10 and VS13 was oriented towards loop3, in HDAC8
it pointed towards loop4 (Figure 4). A similar trend to HDAC8 was
registered in HDAC1 for LD10 inhibitor.
2
f (VS13)
H
NHOH
2
0.054
In HDAC8, both ligands LD10 and VS13 coordinated the cata-
lytic zinc and established one polar interaction with the His143 of
the second binding shell (Figure 4(d)).
SAHA
TSA
a
0.280
0.037
In HDAC1 core, the hydroxamic moiety of both inhibitors
established two hydrogen bonds with His141 and Tyr303. In add-
ition, the aromatic portion of VS13 was orientated towards loop3
thanks to a weak polar interaction of the quinoline nitrogen with
the backbone (dH-N ¼ 2.8 Å) (Figure 4(a)).
Values are means of at least three experiments, SD values are <20% of the
b
mean; not tested.
In HDAC3, the LD10 and VS13 hydroxamate group was stabi-
lised through two hydrogen interactions with His135 and Tyr298.
Among the four selected compounds, the most active on all Both aromatic CAP groups pointed towards loop3, in particular,
the HDACs considered in this work was the quinoline derivative the quinoline nitrogen of VS13 engaged a strong hydrogen bond
VS13 that showed a stronger effect on HDAC6 than on the other with the loop3’s backbone (Figure 4(b)).
isoforms. In particular, VS13 was active at 10 nM on HDAC6, only
A similar ligands pose was calculated in HDAC6 where the
7
times less active than SAHA and ꢁ900 times more selective for hydroxamate group was further stabilised by the His610 of the
HDAC6 over HDAC8.
second catalytic shell (Figure 4(c)).
These compounds were chosen for further evaluation on UM
Therefore, docking results showed a progressive increment
cell lines and docking studies were carried out to explain their dif- from HDAC8 to HDAC1, HDAC3 and HDAC6 in the polar interac-
ferent selectivity profile. tions which strengthened the stabilisation of the inhibitors in the

3
8
S. NENCETTI ET AL.
Table 2. HDACs inhibitory selectivity profile of selected hydroxamic acids, SAHA, entinostat and tubastatin A.
a
IC50 (mM)
Compd.
Chemical structure
HDAC1
0.698
HDAC3
0.058
HDAC6
0.316
HDAC8
8.206
SN2
VS16
0.593
0.043
0.406
13.680
LD10
VS13
6.043
0.137
0.594
0.040
0.836
0.010
>30
9.29
SAHA
0.007
1.480
2.866
0.0014
0.790
0.766
0b.0014
0.495
>30
2.341
b
b
b
Entinostat
Tuastatin A
a
>30
0.015
Compounds were tested in duplicate in a 10-point dose curve with 3-fold serial dilution starting from 30 lM. In the case of SAHA a 15-point dose curve on HDACs
3
and 6 was performed.
b
34
See ref. .
Moreover, the computational analysis of compounds SN2 and
VS16 showed that both ligands are similarly located in the four
HDAC binding channel subtypes (Figure 6). The biphenyl CAP of
these ligands is longer than the one present in LD10 and VS13
and it linearly crossed the outer binding regions between loop3
and 4.
As previously mentioned, the loop3 of HDAC8 shrinked the
outer binding channel. This conformation pushed the CAP of SN2
and VS16 inhibitors deeply (more than 2 Å) towards the catalytic
core. This scrunched conformation caused a detrimental folding of
the spacer and allowed only one polar interaction with second
catalytic shell residues.
In the other HDAC subtypes, all SN2 and VS16 poses were
quite superposed. In the outer portion of the binding site, the
biphenyl CAP was located close to loop3 (in
a ranking
HDAC3 > HDAC1 > HDAC6) while, in the inner binding pocket, the
number of hydrogen bonds increased from one in HDAC8 to two
in HDAC6 and HDAC1, and finally three in HDAC3 (not shown for
clarity in Figure 6).
For all investigated ligands (LD10, VS13, SN2 and VS16) the
computational study showed tiny differences in the ligand-binding
poses, principally due to the sequence and shape variations of
Figure 3. Superposition of hydroxamate inhibitors co-crystallised in human
HDACs (light grey coloured) and docked-pose of inhibitors LD10 and VS13 (sal-
mon and green coloured, respectively). For clarity, inhibitor poses extracted from
different subtypes are shown in HDAC6 binding site.
binding site and promoted the zinc coordination. Analysing the loops3 and 4 among the four HDAC subtypes.
VS13 binding pose, it can be hypothesised that a nitrogen atom
However, the slight differences of the ligand-binding mode are
in the aromatic CAP ring allows better interaction with loop3 and, crucial to strengthen or decrease the inhibitor interactions and
3
0
therefore, enhances the inhibitory activity.
consequently binding affinity .

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
39
Figure 4. Docking of LD10 (salmon) and VS13 (green) in (a) HDAC1; (b) HDAC3; (c) HDAC6, and (d) HDAC8. Proper hydrogen bonds are highlighted in cyan.
2.4. Activity on human UM cell lines in vitro
Finally, we determined if the treatment of UM cells 92.1 and
Mel270 with our compounds could modify the mRNA levels of
HDAC target genes. We chose genes both down-regulated
The anti-proliferative effects of the most promising HDAC inhibi-
tors, VS16, VS13, SN2, and LD10 were evaluated on human UM
cell lines (92.1 and Mel270) and SAHA was used as a positive con-
trol. First, we assessed by MTT assay the cell viability after a 72 h
treatment at different concentrations. Figure 7 and Supplementary
Figure S2 show that compounds VS16, VS13, and SN2 have a
stronger effect on 92.1 and Mel270 cell viability than SAHA and
LD10. Similar results were found for ovarian carcinoma cell line
(
RAD54L, RAD51) and up-regulated (CLU, DHRS2, and CDKN1A) by
3
2,33
HDAC inhibitors in cancer cells
. As shown in Figure 9 and
Supplementary Figure S4, VS13 induced a pattern of gene modu-
lation, which overlapped that of SAHA. Of note, VS13 shares with
SAHA the high activity on HDAC6 (see Table 2), thus supporting
the hypothesis that these modulatory effects on genes could be
mainly mediated by HDAC6 inhibition on which the other com-
pounds have a lower effect.
A2780 and to
Figure S2).
a lesser extent for SKOV3 (Supplementary
We then investigated whether this anti-proliferative effect was
due to the blockade in the G0/G1 phase of the cell cycle, as
expected for SAHA . Flow cytometry analysis of cell cycle distri-
3. Conclusions
3
1
bution in 92.1 and Mel270 cells after treatment with the different UM represents an aggressive type of cancer and currently, there is
compounds or control DMSO, indeed showed that VS16, VS13, no effective treatment for metastatic UM. This cancer form is char-
SN2, and SAHA efficiently block the cell cycle at the G0/G1 phase acterised by an overexpression of HDACs and HDAC inhibitors
(
Figure 8 and Supplementary Figure S3) while LD10 behaved like have been shown to inhibit the growth of UM in vivo and in vitro.
the control DMSO. In this study, we synthesised a series of HDACIs based on SAHA

4
0
S. NENCETTI ET AL.
scaffold bearing an (arylidene)aminoxy moiety. All new com- VS13, bearing a quinoline as the CAP group, which showed a
pounds were tested first on HeLa nuclear extract to evaluate if nanomolar affinity for HDAC6 (IC50¼10 nM) and a 1000-fold select-
the chemical modification of the SAHA structure maintained the ivity over HDAC8. On these four hydroxamic acid derivatives dock-
HDAC inhibitory activity. Among all the synthesised hydroxamates ing studies were performed to explain their binding mode and
we tested the most active ones on isolated enzymes, HDAC1, 3, 6, the interaction inside the HDAC1, 3, 6, and 8 active sites. In par-
and 8. The best inhibitory results were obtained with compound ticular, the nitrogen of the quinoline ring was shown to play an
important role by forming a strong hydrogen bond with the back-
bone of loop3 present in HDAC6 catalytic site. The effect of HDAC
Figure 7. MTT. In vitro cytotoxicity at the 72 h time point of the different com-
pounds in human UM cell lines 92.1 and Mel270, as assessed by the MTT cell via-
Figure 5. Sequence alignment of loop3 and loop4 regions of HDAC1, 3, 6, and 8. bility assay. Data are expressed as percent of control with the DMSO solvent,
At bottom, superposition of aligned loop3 and loop4 crystal structures of the which was used at the same amount present in the highest compound concen-
four HDAC subtypes (HDAC1, HADC3, HDAC6, and HDAC8 are magenta, salmon, tration. Error bars represent SD of quadruplicates. One representative experiment
grey, and purple coloured, respectively).
is shown.
Figure 6. Docking poses of inhibitors SN2 and VS16. The colour code is protein dependent: the complex of both inhibitors in HDAC1, HADC3, HDAC6 and HDAC8 are
magenta, salmon, grey, and purple coloured, respectively. On the left are highlighted the hydrogen bonds engaged by both compounds with second shell catalytic res-
idues of HDAC3.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
41
Figure 8. Influence of the different compounds and DMSO solvent control on cell cycle distribution in UM 92.1 and Mel270 cells. The cells were treated with 10 mM
compound for 48h and cell cycle distribution was analysed by flow cytometry after fixation and staining with PI. The percentage of cells in each category is indicated.
One representative experiment is shown.
inhibition in living cells was evaluated by testing the most promis-
ing compounds on human UM cell lines, 92.1 and Mel270, in com-
parison with SAHA. VS13 showed a strong antiproliferative
activity by MTT assay and the ability to efficiently block the cell
cycle at the G0/G1 phase, similarly to SAHA. Finally, we deter-
mined if treatment with these compounds could modify the
mRNA levels of HDAC target genes in UM cells and VS13 induced
a pattern of gene modulation which overlapped that of SAHA.
Considering the particular activity of VS13 on HDAC6, these
results support the hypothesis that the modulatory effects on
genes could be mainly mediated by HDAC6 inhibition. However,
additional studies will be necessary to shed light on the mechan-
ism involved in the antitumor activity of HDACIs in this aggressive
type of cancer.
Figure 9. Modulation of RAD54L, RAD51 and CLU mRNA expression by the differ-
ent compounds in UM cells 92.1 and Mel270. Cells were treated for 48 h with
1
0 mM compound or the corresponding amount of DMSO. Data, normalised to
GAPDH housekeeping gene, are expressed as fold change relative to the DMSO
control. Error bars represent SD of triplicates. One representative experiment
is shown.
4
. Experimental section
4.1. Chemistry
4.2. General procedure for the synthesis of oximes 5a–c, e, f
Analytical grade reagents and solvents were purchased from
Sigma-Aldrich (St. Louis, MO), and were used as supplied. Melting
points were determined on a K o€ fler hot-stage apparatus and are
A solution of the appropriate commercially available aldehyde or
.
ketone (3.21 mmol) and NH
2
OH HCl (6 mmol) in a mixture of EtOH
ꢂ
9
8% (5.3 ml) and Pyridine (8.3 mmol) was stirred at 150 C for
1
13
uncorrected. H and C NMR spectra were recorded on a Bruker
Avance III HD 400 MHz spectrometer (F €a llander, Switzerland) and
on a Varian Gemini-200 MHz spectrometer. Chemical shifts (d) are
reported in parts per million and coupling constants (J) are
1
50 min. Then, the solvent was evaporated and the crude was
3
added with a saturated solution of NaHCO , filtered and washed
with water to obtain the oximes 5a–c,e,f.
13
reported in hertz (Hz). C NMR spectra were fully decoupled. The
following abbreviations were used to explain multiplicities: singlet
4.2.1. (E)-4-(methylthio)benzaldehyde oxime (5a)
The title compound was prepared from the appropriate aldehyde
(s), doublet (d), triplet (t), double doublet (dd), broad (br), and
following the general procedure to obtain 5a as a white solid
multiplet (m). Chromatographic separations were performed on
silica gel columns by flash column chromatography (Kieselgel 40,
1
(
(
64% yield). HNMR (CDCl
3
):8.03 (s, 1H); 7.52–7.48 and 7.25–7.21
m, 4H, J ¼ 8.42 Hz), 2.51 (s, 3H).
0
.040–0.063 mm, Merck). Reactions were monitored by thin-layer
chromatography (TLC) on silica gel plates containing a fluorescent
indicator (Merck Silica Gel 60 F254) and spots were detected
under UV light (254 nm) and hydroxamic acids were visualised
0
4
.2.2. (E)-1,1 -biphenyl-4-carbaldehyde oxime (5b)
The title compound was prepared from the appropriate aldehyde
following the general procedure to obtain 5b as a white solid
99% yield). H NMR (200 MHz, CDCl ) d: 8.19 (s, 1H); 7.88–7.83
and 7.55–7.47 (m, 8H);
3
with FeCl aqueous solution. Evaporation was carried in vacuo
(rotating evaporator). Sodium sulphate was always used as the
1
(
3
drying agent. Commercially available chemicals were purchased
from Sigma-Aldrich. The oxime 5d is commercially available and
was purchased from Sigma-Aldrich. Elemental analysis was used
to determine the purity of target compounds and was performed 4.2.3. (E)-2-naphthaldehyde oxime (5c)
by our analytical laboratory and agreed with the theoretical values The title compound was prepared from the appropriate aldehyde
to within ±0.4%.
following the general procedure to obtain 5c as a white solid

4
2
S. NENCETTI ET AL.
1
(64% yield). H NMR (200 MHz, CDCl
3
) d: 8.30 (s, 1H); 7.88–7.83 4.4. General procedure for the synthesis of ethyl
(E)–4–((arylideneamino)oxy)pentanoates (7a–f)
and 7.55–7.47 (m, 8H).
ꢂ
To a cooled (0 C) and stirred solution of the appropriate oxime
5
a–f (4.69 mmol) and ethyl 5-bromopentanoate (3.91 mmol) in
anhydrous DMF (25 ml), Cs CO (4.69 mmol) was added. The
The title compound was prepared from the appropriate ketone resulting mixture was stirred for 24 h at room temperature. Then,
following the general procedure to obtain 5e as a white solid water was added and the solution was extracted with Et O. The
organic phases were combined, dried with Na SO , filtered, and
0
4
.2.4. 1-([1,1 -Biphenyl]-4-yl)ethanone oxime(5e)
2
3
2
1
(
96% yield). H NMR (200 MHz, CDCl ) d: 7.74–7.59 (m, 6H);
2
4
3
evaporated under vacuum. The crude was purified affording exclu-
sively the E isomer.
7
.49–7.34 (m, 3H); 2,33 (s, 3H).
0
4
.2.5. 1-([1,1 -Biphenyl]-4-yl)ethanone oxime(5f)
4.4.1. Ethyl (E)-5-(((4-(methylthio)benzylidene)amino)oxy)penta-
noate (7a)
The title compound was prepared from the oxime 5a following
The title compound was prepared from the appropriate aldehyde
following the general procedure to obtain 5f as a white solid
1
(
90% yield). H NMR (200 MHz, CDCl
3
) d: 8.43 (s, 1H), 8.18–8.09 (m, the general procedure. The crude product was purified by flash
3
H), 7.97–7.93 (m, 1H), 7.84–7.70 (m, 1H), 7.60–7.52 (m,1H).
chromatography on silica gel (n-hexane/EtOAc 6:4) to give a yel-
1
low oil (48% yield). H NMR (200 MHz, CDCl
3
) d: 8.03 (s, 1H), 7.23
(m, 2H), 7.50 (m, 2H), 4.09–4.21 (m, 4H), 2.51 (s, 3H), 2.41–2.32 (m,
2
H), 1.77–1.80 (m, 4H),1.26 (t, J ¼ 7.14 Hz, 3H).
4.3. General procedure for the synthesis of ethyl
(
E)–3–((arylideneamino)oxy)propanoates 6a–c
0
To a solution of the appropriate oxime 5a–c (3.36 mmol) in MeCN 4.4.2. Ethyl (E)-5-((([1,1 -biphenyl]-4-ylmethylene)amino)oxy)penta-
(
1.5 ml), triphenylphosphine (0.67 mmol) and ethyl acrylate noate (7b)
(
3.36 mmol) were added. The obtained solution was stirred at The title compound was prepared from the oxime 5b following
ꢂ
6
5 C for 72 h. The reaction mixture was dried under reduced pres- the general procedure. The crude product was purified by flash
sure to give a crude oil that was purified by flash chromatography chromatography on silica gel (n-hexane/EtOAc 7:3) to give a yel-
1
on silica gel. The evaporation of the selected fractions gave a low oil (85% yield). H NMR (200 MHz, CDCl
3
) d: 8.01 (s, 1H),
7
.74–7.59(m, 6H), 7.48–7.32 (m, 3H) 4.25–4.17 (m, 2H), 4.21 (q,
pure yellow oil constituted exclusively by the E isomer.
J ¼ 7.14 Hz, 2H), 2.37–2.34 (m, 2H), 1.80–1.74 (m, 4H), 1.26 (t,
J ¼ 7.14 Hz, 3H).
4
.3.1. Ethyl (E)-3-(((4-(methylthio)benzylidene)amino)oxy)propa-
noate (6a)
4.4.3. Ethyl (E)-5-(((naphthalen-2-ylmethylene)amino)oxy)penta-
The title compound was prepared from the oxime 5a following noate (7c)
the general procedure. The crude product was purified by flash The title compound was prepared from the oxime 5c following
chromatography on silica gel (n-hexane/EtOAc 7:3) to give a yel- the general procedure. The crude product was purified by flash
1
low oil (30% yield). H NMR (200 MHz, CDCl
7
(
J ¼ 7.14 Hz, 3H).
3
) d: 8.03 (s, 1H), chromatography on silica gel (n-hexane/EtOAc 6:4) to give a yel-
1
.51–7.47 (m, 2H), 7.28–7.21 (m, 2H), 4.44 (t, J ¼ 6.41 Hz, 2H), 4.19 low oil (65% yield). H NMR (200 MHz, CDCl
) d: 8.22 (s, 1H),
3
q, J ¼ 7.14 Hz, 2H), 2.75 (t, J ¼ 6.41 Hz, 2H), 2.51 (s, 3H), 1.28 (t, 7.85–7.78 (m, 5H), 7.53–7.47 (m, 2H), 4.25–4.20 (m, 2H), 4.18–4.08
(q, J ¼ 7.14 Hz, 2H), 2.41–2.35 (m, 2H), 1.82–1.75 (m, 4H), 1.25 (t,
J ¼ 7.14 Hz, 3H).
0
4
.3.2. Ethyl (E)-3-((([1,1 -biphenyl]-4-ylmethylene)amino)oxy)propa-
4.4.4. Ethyl (E)-4-((benzylideneamino)oxypentanoate (7d)
noate (6b)
The title compound was prepared from the oxime 5d following
The title compound was prepared from the oxime 5b following the general procedure. The crude product was purified by flash
the general procedure. The crude product was purified by flash chromatography on silica gel (n-hexane/EtOAc 6:4) to give a yel-
1
chromatography on silica gel (n-hexane/EtOAc 7:3) to give a yel- low oil (80% yield). H NMR (200 MHz, CDCl ) 8.07 (s,1H),
3
1
low oil (35% yield). H NMR (200 MHz, CDCl
3
) d: 8.11 (s, 1H), 7.59–7.55(m, 2H), 7.37–7.35 (m, 3H), 4.21–4.14 (m, 2H), 4.12 (q,
7
.49–7.39 (m, 3H), 7.64–7.57 (m, 6H), 4.46 (t, J ¼ 6.41 Hz, 2H), 4.19 J ¼ 7.14 Hz, 2H), 2.40–2.33 (m, 2H), 1.79–1.72 (m, 4H), 1.25 (t,
(q, J ¼ 7.14 Hz, 2H), 2.76 (t, J ¼ 6.41 Hz, 2H), 1.27 (t, J ¼ 7.14 Hz, 3H).
J ¼ 7.14 Hz, 3H),
0
4
.4.5. Ethyl (E)-4-(((1-([1,1 -biphenyl]-4-yl)ethylidene)amino)oxy)bu-
4
.3.3. Ethyl (E)-3-(((naphthalen-2-ylmethylene)amino)oxy)propa-
tanoate (7e)
noate (6c)
The title compound was prepared from the oxime 5e following
the general procedure. The crude product was purified by flash
The title compound was prepared from the oxime 5b following
the general procedure. The crude product was purified by flash chromatography on silica gel (n-hexane/EtOAc 6:4) to give a yel-
1
chromatography on silica gel (n-hexane/EtOAc 9:1) to give a yel- low oil (66% yield). H NMR (200 MHz, CDCl ) d: 8.02 (s, 1H),
3
1
low oil (42% yield). H NMR (200 MHz, CDCl ) d: 8.21 (s, 1H), 7.74–7.59 (m, 6H), 7.48–7.32 (m, 3H), 4.25–4.20 (m, 2H), 4.21 (q,
3
7
.84–7.82 (m, 5H), 7.52–7.47 (m, 2H), 4.49 (t, J ¼ 6.41 Hz, 2H), 4.18 J ¼ 7.14 Hz, 2H), 2.41–2.30 (m, 2H), 2.26 (s, 3H), 1.94–1.78 (m, 4H),
(q, J ¼ 7.14 Hz, 2H), 2.77 (t, J ¼ 6.41 Hz, 2H), 1.27 (t, J ¼ 7.14 Hz, 3H), 1.26 (t, J ¼ 7.14 Hz, 3H).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
43
4
.4.6.
noate (7f)
The title compound was prepared from the oxime 5e following for C14
Ethyl
(E)-4-(((quinolin-3-ylmethylene)amino)oxy)buta-
d
6
) d: 167.1, 149.4, 134.0, 133.2, 130.1, 128.9, 128.8, 128.7, 128.2,
127.6, 127.3, 127.1, 70.3, 33.0, 14.9. Elemental analysis calcd. (%)
: C, 65.11; H, 5.46; N, 10.85; found: C, 65.19, H, 5.52,
H N O
14 2 3
the general procedure. The crude product was purified by flash N, 10.92.
chromatography on silica gel (n-hexane/EtOAc 7:3) to give a yel-
1
low oil (90% yield). H NMR (200 MHz, CDCl
3
) d: 8.30 (s, 1H),
4
.6. General procedure for the synthesis of
8
2
1
.14–7.94 (m,3H), 7.82–7.66 (m, 2H), 7.57–7.49 (m, 1H), 4.27 (m,
(E)–3–((arylideneamino)oxy)pentananamide (2a–f)
H), 4.12 (q, J ¼ 7.14 Hz, 2H), 2.40–2.32 (m, 2H), 1.80–1.72 (m, 4H),
.24 (t, J ¼ 7.14 Hz, 3H).
To a stirred solution of KOH (2.47 mmol) in MeOH (4 ml) was
added a 50% aqueous solution of NH OH (0.49 ml). This solution
2
was added dropwise in 30 min to a stirred and cooled solution
4
.5. General procedure for the synthesis of
ꢂ
(0 C) of the appropriate ethyl ester 7a2f (3.12 mmol) in
(
E)–N–hydroxy–3–((arylideneamino)oxy)propanamides (1a–c)
MeOH (2 ml).
After stirring at 0 C for 30 min. and at room temperature over-
ꢂ
To a stirred solution of KOH (1.32 mmol) in MeOH (2 ml) was
added a 50% aqueous solution of NH OH (0.089 ml). This solution night the mixture evaporated in vacuo. The residue was added
2
was added dropwise in 30 min to a stirred and cooled solution with water and the aqueous solution washed with Et O. Aqueous
2
ꢂ
(
0 C) of the appropriate ethyl ester 6a2c (0.087 mmol) in
phase was acidified with 10% HCl to pH ¼ 5 and then extracted
with EtOAc. Organic layers were collected, dried, and evaporated
in vacuo to give a crude solid.
MeOH (2 ml).
ꢂ
After stirring at 0 C for 30 min. and at room temperature over-
night the mixture was evaporated in vacuo. The residue was
added with water and the aqueous solution washed with Et O.
2
The aqueous phase was acidified with 10% HCl to pH ¼ 5 and 4.6.1. (E)-N-hydroxy-5-(((4-(methylthio)benzylidene)amino)oxy)pen-
then extracted with EtOAc. Organic layers were collected, dried,
and evaporated in vacuo to give a solid.
tanamide (2a)
The title compound was prepared from the ethyl ester 7a follow-
ing the general procedure. The crude product was purified by
flash chromatography on silica gel (n-hexane/EtOAc 3:7) to give a
4
.5.1.
(E)-N-hydroxy-3-(((4-(methylthio)benzylidene)amino)oxy)
1
yellow semisolid. (45% yield); H NMR (400 MHz, DMSO-d
6
): d
propanamide(1a)
The title compound was prepared from the ethyl ester 6a follow-
ing the general procedure. The crude product was purified by
flash chromatography on silica gel (n-hexane/EtOAc 3:7) to give a
semisolid (40% yield). H NMR (400 MHz, DMSO-d ): d 10.48 (bs,
1
7
0.48 (bs, 1H), 8.81 (bs, 1H), 8.18 (s, 1H), 7.55–7.53 (m, 2H),
.33–7.28 (m, 2H); 4.09 (t, J ¼ 6 Hz, 2H), 2.62 (t, J ¼ 6 Hz, 2H), 2.50
13
(s, 3H), 1.68–1.57 (m, 4H). C (100 MHz, DMSO-d ): d 167.1, 148.5,
6
1
41.0, 128.9, 128.4, 127.7, 126.2, 70.3, 32.9, 28.2, 21.6. Elemental
1
6
analysis calcd. (%) for C13 S: C, 55.30; H, 6.43; N, 9.92;
18 2 3
H N O
1
2
H), 8.81 (bs, 1H), 8.11 (s, 1H), 7.55–7.53 (m, 2H), 7.33–7.28 (m,
H); 4.35 (t, J ¼ 6 Hz, 2H), 2.50 (s, 3H), 2.41 (t, J ¼ 6 Hz, 2H).
found: C, 55.39, H, 6.52, N, 10.02.
1
3
C
(100 MHz, DMSO-d ): d 167.1, 148.5, 141.0, 128.9, 128.4, 127.7,
6
1
C
26.2, 70.3, 32.9, 14.8. Elemental analysis calcd. (%) for
0
4
.6.2. (E)-5-((([1,1 -biphenyl]-4-ylmethylene)amino)oxy)-N-hydroxy-
11
H
14 2
N O
3
S: C 51.95; H 5.55; N 11.02; found: C 52.02, H 5.61,
pentanamide (2b) (SN2)
N 11.25.
The title compound was prepared from the ethyl ester 7b follow-
ing the general procedure. The crude product was purified by
flash chromatography on silica gel (n-hexane/EtOAc 3:7) to give a
0
4
.5.2. (E)-3-((([1,1 -biphenyl]-4-ylmethylene)amino)oxy)-N-hydroxy-
ꢂ
1
yellow solid (35% yield). M.p. ¼160–162 C. HNMR (400 MHz,
propanamide (1b)
DMSO-d ): d 8.27 (s, 1H),7.73–7.69 (m, 6H, Ar), 7.50–7.46 (m, 2H,
6
The title compound was prepared from the ethyl ester 6b follow-
ing the general procedure. The crude product was purified by
flash chromatography on silica gel (n-hexane/EtOAc 3:7) to give a
Ar), 7.40–7.38 (m, 1H, Ar), 4.10 (m, 2H), 1.84 (t, J ¼ 7.1 Hz, 2H),
1
3
1
6
.64–1.47 (m, 4H). C NMR (100 MHz, DMSO-d ): d 175.5, 148.27,
ꢂ
1
141.7, 139.8, 131.8, 129.4, 128.3, 127.9, 127.5, 127.1, 74.5, 29.6,
3.5. Elemental analysis calcd. (%) for C H N O : C, 69.21; H,
18 20 2 3
.45; N, 8.97; found: C, 69.33, H, 6.52, N, 9.02.
yellow solid. (40% yield). M.p. ¼ 128–130 C. H NMR (400 MHz,
2
6
DMSO-d ) d: 10.47 (s, 1H), 8.79 (s, 1H), 8.26 (s, 1H),7.76–7.69 (m,
6
6
H, Ar), 7.57–7.46 (m, 2H, Ar), 7.41–7.38 (m, 1H, Ar), 4.32 (t,
13
J ¼ 6.4 Hz, 2H), 2.39 (t, J ¼ 6.4 Hz, 2H). C NMR (100 MHz, DMSO-
d ): d 167.0, 149.1, 141.9, 139.8, 131.9, 129.5, 128.3, 127.9, 127.5,
6
4
.6.3. (E)-N-hydroxy-5-(((naphthalen-2-ylmethylene)amino)oxy)pen-
tanamide (2c) (LD10)
The title compound was prepared from the ethyl ester 7c follow-
ing the general procedure. The crude product was purified by
1
6
27.1, 70.3, 33.0. Elemental analysis calcd. (%) for C H N O C,
16 16 2 3:
7.59; H, 5.67; N, 9.85; found: C, 69.99, H, 6.02, N, 10.02.
4
.5.3. (E)-N-hydroxy-3-(((naphthalen-2-ylmethylene)amino)oxy)pro- flash chromatography on silica gel (n-hexane/EtOAc 3:7) to give a
ꢂ
1
yellow solid. (35% yield). M.p. ¼ 229–231 C. HNMR (400 MHz,
panamide (1c)
DMSO-d ) d: 10.53 (bs, 1H), 8.81 (bs, 1H), 8.37 (s, 1H), 8.06 (s, 1H),
6
The title compound was prepared from the ethyl ester 6c follow-
ing the general procedure. The crude product was purified by 7.95–7.92 (m, 3H), 7.83–7.81 (m, 1H), 7.57–7.54 (m, 2H), 4.13 (t,
1
3
flash chromatography on silica gel (n-hexane/EtOAc 3:7) to give a J ¼ 6.8 Hz, 2H), 1.88 (t, J ¼ 7.2 Hz, 2H), 1.67–1.50 (m, 4H). C NMR
ꢂ
1
yellow solid. (47% yield). M.p. ¼ 153–155 C; HNMR (400 MHz, (100 MHz, DMSO-d
) d: 175.6, 148.9, 134.0, 133.3, 130.4, 128.9,
6
DMSO-d ) d: 10.48 (bs, 1H), 8.80 (s,1H), 8.35 (s, 1H), 8.07 (s, 1H), 128.7, 128.5, 128.2, 127.5, 127.2, 123.1, 74.5, 38.9, 29.6, 23.4.
6
7
18 2 3
.95–7.93 (m, 3H), 7.81 (m, 1H), 7.57–7.55 (m, 2H), 4.35 (t, Elemental analysis calcd. (%) for C16H N O : C, 67.12; H, 6.34; N,
13
J ¼ 6.24 Hz, 2H), 2.41 (t, J ¼ 6.24 Hz, 2H). C NMR (100 MHz, DMSO- 9.78; found: C, 67.33, H, 6.42, N, 9.82.

4
4
S. NENCETTI ET AL.
4
.6.4. (E)-5-((benzylideneamino)oxy)-N-hydroxypentanamide (2d)
3
14.7. Elemental analysis calcd. (%) for C11H13NO S: C, 55.21; H,
The title compound was prepared from the ethyl ester 7d follow- 5.48; N, 5.85; found: C, 55.23, H, 5.52, N, 5.89.
ing the general procedure. The crude product was purified by
flash chromatography on silica gel (n-hexane/EtOAc 3:7) to give a
0
ꢂ
1
4.7.2.
acid (3b)
(E)-3-((([1,1 -biphenyl]-4-ylmethylene)amino)oxy)propanoic
yellow solid. (35% yield). M.p. ¼ 134–136 C. HNMR (400 MHz,
DMSO-d ): d 10.51 (bs, 1H), 8.81 (bs, 1H), 8.22–8.21 (m, 1H, Ar),
6
The title compound was prepared from the ethyl ester 6b follow-
ing the general procedure white solid (48% yield). M.p. ¼
7
1
1
.61–7.59 (m, 2H, Ar), 7.41–7.39 (m, 2H, Ar), 4.11–4.05 (m, 2H),
1
3
.65–1.46 (m, 4H). C NMR (100 MHz, DMSO-d ): d 172.2, 148.8,
6
ꢂ
1
1
04–106 C. HNMR (400 MHz, DMSO-d ): d 8.28 (s, 1H), 7.74–7.69
6
32.6, 130.2, 129.2, 127.2, 73.7, 35.6, 28.7, 23.3. Elemental analysis
(
m, 6H, Ar), 7.50–7.46 (m, 2H, Ar), 7.41–7.38 (m, 1H, Ar), 4.32 (t,
16 2 3
calcd. (%) for C12H N O : C, 61.00; H, 6.83; N, 11.86; found: C,
1
3
J ¼ 6.4 Hz, 2H), 2.63 (t, J ¼ 7.1 Hz, 2H), C NMR (100 MHz, DMSO-
6
1.13, H, 6.92, N, 11.92.
d ): d 172.8, 149.1, 142.0, 139.8, 131.5, 129.5, 128.3, 127.9, 127.5,
6
1
27.13, 70.1, 34.7. Elemental analysis calcd. (%) for C H NO : C,
16 15 3
0
(E)-5-(((1-([1,1 -biphenyl]-4-yl)ethylidene)amino)oxy)-N- 71.36; H, 5.61; N, 5.20; found: C, 71.43, H, 5.62, N, 5.29.
4
.6.5.
hydroxypentanamide (2e) (VS16)
The title compound was prepared from the ethyl ester 7e follow-
4
.7.3.
(E)-3-(((naphthalen-2-ylmethylene)amino)oxy)propanoic
ing the general procedure. The crude product was purified by trit-
ꢂ
acid (3c)
uration with Et O to give a white solid (35% yield). M.p. ¼ 142 C;
2
1
The title compound was prepared from the ethyl ester 6c follow-
ing the general procedure white semisolid (56% yield). HNMR
HNMR (400 MHz, DMSO-d ) d: 10.72 (s, 1H), 9.39 (bs,
6
1
1
H),7.76–7.68 (m, 6H, Ar), 7.50–7.46 (m, 2H, Ar), 7.40–7.38 (m, 1H,
(400 MHz, DMSO-d
6
): d 12.48 (bs, 1H), 8.38 (s, 1H), 8.06 (s, 1H, Ar),
Ar), 4.13 (t, J ¼ 6.0 Hz, 2H), 3.40–3.28 (m, 2H), 2.12 (s, 3H), 1.90 (t,
1
3
7.96–7.92 (m, 3H, Ar), 7.81 (m, 1H, Ar), 7.57–7.55 (m, 2H, Ar), 4.35
J ¼ 7.1 Hz, 2H), 1.64–1.55 (m, 4H). C NMR (100 MHz, DMSO-d ): d
6
1
3
(
t, J ¼ 6.00 Hz, 2H), 2.66 (t, J ¼ 6.20 Hz, 2H). C NMR (100 MHz,
DMSO-d ): d 173.1, 149.5, 134.0, 133.2, 130.1, 128.9, 128.8, 128.7,
28.2, 127.6, 127.3, 123.1, 70.1, 35.3. Elemental analysis calcd. (%)
1
3
68.4, 153.7, 141.1, 139.9, 135.6, 129.4, 128.2, 127.1, 126.8, 73.9,
3.2, 29.1, 22.8, 12.7. Elemental analysis calcd. (%) for C19
6
22 2 3
H N O :
1
C, 69.92; H, 6.79; N, 8.58; found: C, 69.93, H, 6.82, N, 8.63.
3
for C14H13NO : C, 69.12; H, 5.39; N, 5.76; found: C, 69.13, H, 5.42,
N, 5.79.
4
.6.6. (E)-N-hydroxy-5-(((quinolin-3-ylmethylene)amino)oxy)penta-
namide (2f) (VS13)
The title compound was prepared from the ethyl ester 7f follow-
ing the general procedure. The crude product was purified by
4
.7.4.
(E)-5-(((4-(methylthio)benzylidene)amino)oxy)pentanoic
acid (4a)
The title compound was prepared from the ethyl ester 7a follow-
crystallisation with CHCl to give a white solid (42% yield). M.p. ¼
3
1
ing the general procedure white solid (76% yield). M.p. ¼
ꢂ
1
8
7
1
1
3
6
13 C; HNMR (400 MHz, DMSO-d ) d: 10.37 (bs, 1H), 8.68 (s,1H),
6
ꢂ
7
0–72 C; 1HNMR (400 MHz, DMSO-d
6
): d 12.10 (bs, 1H), 8.19 (s,
.39 (m, 1H) 8.31 (s, 1H), 8.02 (m, 2H), 7.93 (m, 1H), 7.80 (m, 1H),
1
H), 7.54 (m, 2H, Ar), 7.31 (m, 2H, Ar), 4.09 (t, J ¼ 6.00 Hz, 2H), 2.51
.65 (m, 1H), 4.22 (t, J ¼ 6.28 Hz, 2H), 2.01 (t, J ¼ 7.2 Hz, 2H),
13
1
3
(s, 3H), 2.26 (t, J ¼ 7.2 Hz, 2H), 1.68–1.63 (m, 4H).
C NMR
.71–1.61 (m, 4H). C NMR (100 MHz, DMSO-d ): d 169.3, 152.1,
6
(100 MHz, DMSO-d ): d 174.8, 148.5, 141.0, 128.9, 128.4, 127.6,
6
49.9, 147.8, 137.3, 130.6, 129.3, 128.5, 128.3, 127.8, 118.1, 74.5,
2.4, 28.6, 22.1. Elemental analysis calcd. (%) for C15
2.71; H, 5.96; N, 14.63; found: C, 62.73, H, 6.02, N, 14.69.
1
26.1, 73.6, 33.8, 28.6, 21.6, 14.7. Elemental analysis calcd. (%) for
17 3 3
H N O : C,
C
13
H17NO
3
S: C, 58.40; H, 6.41; N, 5.24; found: C, 58.55, H, 6.42,
N, 5.29.
4
.7. General procedure for the synthesis of
0
4
.7.5.
(E)-5-((([1,1 -biphenyl]-4-ylmethylene)amino)oxy)pentanoic
(
(
E)–3–((arylideneamino)oxy)propanoic acids (3a–c) and
E)–3–((arylideneamino)oxy)pentanoic acids (4a–e)
acid (4b)
The title compound was prepared from the ethyl ester 7b follow-
ing the general procedure white solid (64% yield). M.p. ¼
A solution of the appropriate ethyl ester 6a–c, 7a–d (1.35 mmol)
in anhydrous EtOH (0.21 ml) was added with a solution of 2 N
ꢂ
1
9
8–100 C; HNMR (400 MHz, DMSO-d ): d 12.02 (bs, 1H),8.28 (s,
6
KOH in anhydrous EtOH (4.90 ml). The resulting mixture was
1
H), 7.74–7.68 (m, 6H, Ar), 7.50–7.46 (m, 2H, Ar), 7.40–7.38 (m, 1H,
ꢂ
stirred at 40 C for 24 h. Ethanol was evaporated and the solid Ar), 4.13 (t, J ¼ 6.4 2H), 2.27 (t, J ¼ 7.2 Hz, 2H), 1.70–1.58 (m, 4H).
1
3
residue was dissolved in water and washed with Et
2
O. Aqueous
C NMR (100 MHz, DMSO-d ): d 174.8, 148.6, 141.8, 139.8, 131.6,
6
basic phase was then acidified with 1 N HCl until pH ¼ 5 was 129.5, 128.3, 127.8, 127.5, 127.1, 73.7, 33.8, 28.6, 21.6. Elemental
obtained, and the product was extracted with EtOAc. Organic analysis calcd. (%) for C H NO : C, 72.71; H, 6.44; N, 4.71; found:
1
8
19
3
layers were collected, dried, evaporated to dryness.
C, 72.73, H, 6.46, N, 4.79.
4
.7.1.
(E)-3-(((4-(methylthio)benzylidene)amino)oxy)propanoic 4.7.6.
acid (4c)
(E)-5-(((naphthalen-2-ylmethylene)amino)oxy)pentanoic
acid (3a)
The title compound was prepared from the ethyl ester 6a follow- The title compound was prepared from the ethyl ester 7c follow-
ing the general procedure white solid (45% yield). M.p. ¼ ing the general procedure. Orange solid (55% yield). M.p. ¼
ꢂ
1
ꢂ
1
7
1
(
6–78 C. HNMR (400 MHz, DMSO-d ): d 12.28 (bs, 1H), 8.17 (s, 68–70 C; HNMR (400 MHz, DMSO-d ) d: 12.01 (bs, 1H), 8.38 (s,
6
6
H), 7.53 (m, 2H, Ar), 7.28 (m, 2H, Ar), 4.28 (t, J ¼ 6.24 Hz, 2H), 2.60 1H), 8.06 (s, 1H, Ar), 7.96–7.92 (m, 3H, Ar), 7.81 (m, 1H, Ar),
13
t, J ¼ 6.24 Hz, 2H), 2.51 (s, 3H). C NMR (100 MHz, DMSO-d
6
): d 7.57–7.54 (m, 2H, Ar), 4.16 (t, J ¼ 6.4 Hz, 2H), 2.28 (t, J ¼ 7.2 Hz, 2H).
13
1
72.8, 149.0, 141.2, 130.1, 128.7, 127.7, 126.1, 125.3, 70.0, 34.7,
6
C NMR (100 MHz, DMSO-d ): d 174.8, 149.0, 134.0, 133.2, 130.2,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
45
1
28.9, 128.7, 128.6, 128.2, 127.5, 127.2, 123.1, 73.8, 33.9, 28.6, 21.6. duplicate and terminated by the addition of a stop buffer contain-
Elemental analysis calcd. (%) for C16
H
17NO
3
: C, 70.83; H, 6.32; N, ing 100 mM HEPES, 0.015% Brij-35, 10 mM EDTA, 0.1% CR-3, and
1.5 mM of the pan-HDAC inhibitor Panobinostat. A DMSO concen-
tration of 3.33% characterised both compounds’ titration and con-
trols wells. Buffers components, except for CR-3 (LabChip Coating
5
.16; found: C, 70.93, H, 6.36, N, 5.19.
4
.7.7. (E)-5-((benzylideneamino)oxy)pentanoic acid (4d)
Reagent 3, purchased from PerkinElmer, Product Number 760050)
The title compound was prepared from the ethyl ester 7d follow-
ing the general procedure. Colourless oil (47% yield). H NMR
400 MHz, DMSO-d ): d 12.03 (bs, 1H), 8.23 (s, 1H), 7.61–7.59 (m,
and
Technology)
Panobinostat
(LBH589,
purchased
from
ApexBio
1
3
5,36
were purchased from Sigma Aldrich. As standard
(
6
compounds (positive controls), three well-known HDAC inhibitors
3
7
38
39
2
1
1
H), 7.42–7.39 (m, 3H); 4.10 (t, J ¼ 6 Hz, 2H), 2.26 (t, J ¼ 6 Hz, 2H), were used: Entinostat (MS-275) , SAHA , and Tubastatin A (all
13
.68–1.55 (m, 4H). C (100 MHz, DMSO-d
6
): d 175.3, 149.4, 133.0, purchased from Selleck Chemicals). In the case of SAHA an add-
30.7, 129.7, 127.7, 74.1, 34.3, 29.2, 22.0. Elemental analysis calcd. itional 15-point dose curve screening was performed over HDACs
(
%) for C H NO : C, 65.14; H, 6.83; N, 6.33; found: C, 65.23, H, 3 and 6. Fluorescence intensity of electrophoretically separated
1
2
15
3
6
.86, N, 6.39.
substrate and the product was detected using the LabChip EZ
Reader and the inhibition percentage values were normalised
according to the associated 0% and 100% inhibition control wells.
The data were then analysed by non-linear regression using
0
(E)-5-(((1-([1,1 -biphenyl]-4-yl)ethylidene)amino)oxy)penta-
4
.7.8.
4
0
noic acid (4e)
GraphPad Prism 6.01 software to derive IC50 values.
The title compound was prepared from the ethyl ester 7e follow-
ing the general procedure. White solid (33% yield). M.p. ¼
ꢂ
1
1
37–139 C; HNMR (400 MHz, DMSO-d ) d: 7.78–7.67 (m, 6H), 4.10. Molecular modelling
6
7
.52–7.37 (m, 3H), 4.11 (t, 2H, J ¼ 6.0 Hz), 2.21 (s, 3H), 1.83 (t, 2H,
Crystal structures of human HDAC1, HDAC3, HDAC6, and HDAC8
1
3
J ¼ 7.3 Hz), 1.64–1.60 (m, 2H), 1.52–1.45 (m, 2H).
C
NMR
41
42
43
44
(codes: 5ICN , 4A69 , 5EDU , and 3F07 ) were downloaded
6
(100 MHz, DMSO-d ): d 175.5, 153.5, 141.0, 139.9, 135.7, 129.5,
from Protein Data Bank (PDB) and superposed for aligning their
1
28.2, 127.1, 126.8, 79.6, 74.4, 29.8, 23.6, 12.7. Elemental analysis
45
coordinates using Chimaera . Docking of LD10, VS13, VS16, and
calcd. (%) for C H NO : C, 73.29; H, 6.80; N, 4.50; found: C, 73.33,
H, 6.86, N, 4.59.
1
9
21
3
SN2 was carried out using the same procedure identified as the
best through preliminary cross- and re-docking studies, by means
4
6
of ASP fitness function of GOLD software . A scaffold match con-
straint on the hydroxamate position deduced from the crystallo-
graphic ligand structures, with a default strength of 5, was
4
.8. HDACs inhibition assays
HDAC inhibitory activity of newly synthesised compounds was applied. The region of interest for docking was defined in GOLD
evaluated using an HDAC Fluorimetric Assay/Drug Discovery Kit in such a manner that every protein contained all the residues
(AK-500, BIOMOL). Briefly, HeLa nuclear extract (0.5 ml/well) was within 10 Å from the superposed co-crystallised ligands. The “allow
added to each well already containing the assay buffer (control), early termination” command was deactivated. All ligands were
diluted trichostatin A (standard reference), or various concentra- submitted to 80 Genetic Algorithm runs, clustering the output ori-
ꢂ
tions of HDAC inhibitors and were incubated at 37 C with 25 mM entations using an RMSD cut-off of 1.5 Å. The metal coordination
TM
of Fluor de Lys Substrate. Reactions were stopped after 30 min of the zinc ion was set in an octahedral geometry, as in the crys-
TM
by adding Fluor de Lys
Developer. After the addition of this tallographic structures. The default GOLD parameters were used
developer, the plate was incubated at room temperature for for all other settings.
5 min, the fluorescence intensity of the wells was measured on a
The ligands were built using Maestro and then subjected,
fluorometric plate reader with excitation set at 360 nm and emis- after a minimisation step, to a conformational search of 1,000
sion detection set at 460 nm.
steps in a water environment (using the generalized-Born/surface-
4
7
1
4
8
area model) by means of Macromodel software. The algorithm
used was the Monte Carlo method with the MMFFs force field
and a distance-dependent dielectric constant of 1.0. Missing
hydrogens were added to the protein according to the predicted
protonation state at the physiological pH, 7.0. Asn and Gln resi-
dues were absent in the range of interest for the direct contacts
of docking; flip corrections were necessary only for the histidines
involved in the zinc coordination, which were protonated in such
a way to expose the lone pair towards the zinc atom.
4
.9. Microfluidic chip–based HDAC inhibition assay
Test compounds were screened against human histone deacety-
lases (HDACs) 1, 3, 6, and 8 using the Calliper off-chip mobility
shift assay technology. All HDACs were purchased from BPS
Bioscience and four selective substrates were used according to
the targeted isozyme. A (FAM)-labelled Substrate A peptide (pur-
chasable from PerkinElmer, Product Number CLS96000) was syn-
thesised in-house as previously reported and used for HDAC1
assays. The other three substrates were purchased from
PerkinElmer: two (FITC)-labelled peptides (p53 Acetylated Peptide
34
4.11. Cells and cell treatments
4
9
50
and Histone 4 Acetylated Peptide, Product Number 760512 and The human UM cell lines 92.1 and Mel270 (kindly provided to
60513 respectively) were used for HDAC 3 and 6 assays, respect- RG by M. J. Jager) and the ovarian cancer cell lines SKOV3 (ATTC),
ively; (FAM)-labelled peptide (Broad Substrate B, Product A2780 (ICLC) and A2774 (IST Genoa) were grown in RPMI 1640,
7
a
Number CLS960007) was employed as a substrate for HDAC8. Test with 2 mM L-glutamine, 10% heat-inactivated foetal calf serum,
ꢂ
and standard compounds (3-fold serial dilution starting at 30 mM, and 100 lg/ml penicillin-streptomycin (Lonza) at 37 C in a 5%
1
0 concentrations) were incubated with purified HDACs and 1 mM CO2 incubator. The cells were cultured for no longer than
of substrate for 60 min at room temperature in an assay buffer 3 months, when an aliquot of the original stock was thawed,
TM
composed of 25 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, which had been genotyped using the Cell ID System (Promega,
V
R
1
2
mM MgCl , and 0.01% BSA. Reactions were performed in G9500) and the GeneMapper software, version 4.0.

4
6
S. NENCETTI ET AL.
Table 3. QRT-PCR primers.
Disclosure statement
Gene
Forward primer
Reverse primer
No potential conflict of interest was reported by the author(s).
GAPDH
GAAGGTGAAGGTCGGAGT
GACAATGCAGAGAAGCTGG
CCCTTTCTTCCATCACCTCGCT
TCTCTGGCAGTGATGTCCTGGA
CATGGGTGGAATCATATTGGAA
GCAGGAAGACATCATCATCC
GCCTTAGAGCTGTAACCAGGAG
TAAAGGGCGGTGGCACTGTCTA
POLR2A
RAD54L
RAD51
Funding
CLU (TRPM2)
DHRS2 (Hep27) GGTGCTGTCATCCTGGTCTCTT
CDKN1A (P21)
TGCGGATGAAGGACCAGTGTGA TTTCCTGGTCAACCTCTCAGCG
CCAGCTCCAATGCCAGTGTTCT
AGGTGGACCTGGAGACTCTCAG TCCTCTTGGAGAAGATCAGCCG
This study was partially supported by the University of Pisa
[PRA_2017_51], by the Italian Ministry of University and Research
(MIUR) [FFABR-2017] (S.N.), by the Italian Ministry of Health
(5 ꢃ 1000 funds 2015, M.F.), and National Institutes of Health (NIH)
[5R01GM106974] (G.R.M.).
Treatments with the compounds were performed with slight
differences, according to the final use of the samples. For the cell
viability assay with MTT, cells were seeded in 96-well flat-bottom
3
plates in culture medium at 5 ꢃ 10 cells/well, whereas for cell
cycle analysis and QRT-PCR cells were seeded in 24-well plates at ORCID
4
5
ꢃ 10 cells/well. The day after, a culture medium containing the
Elisa Nuti
Lidia Ciccone
http://orcid.org/0000-0003-2669-5376
http://orcid.org/0000-0002-2762-1929
appropriate amount of compounds or their solvent control DMSO
was added, and treatments were carried out for the indi-
cated time.
Armando Rossello
http://orcid.org/0000-0002-6795-8091
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