Properly substituted cyclic bis-(2-bromobenzylidene) compounds behaved as dual p300/CARM1 inhibitors and induced apoptosis in cancer cells

Article abstract of DOI:10.3390/molecules25143122

Bis-(3-bromo-4-hydroxy)benzylidene cyclic compounds have been reported by us as epigenetic multiple ligands, but different substitutions at the two wings provided analogues with selective inhibition. Since the 1-benzyl-3,5-bis((E)-3-bromobenzylidene)piper

Full text of DOI:10.3390/molecules25143122

molecules
Article
Properly Substituted Cyclic Bis-(2-bromobenzylidene)
Compounds Behaved as Dual p300/CARM1
Inhibitors and Induced Apoptosis in Cancer Cells
Rossella Fioravanti 1,†, Stefano Tomassi 2,†, Elisabetta Di Bello 1, Annalisa Romanelli 1,
Andrea Maria Plateroti 3, Rosaria Benedetti 4 , Mariarosaria Conte 4, Ettore Novellino 2,
Lucia Altucci 4 , Sergio Valente 1,
*
and Antonello Mai 1,
*
1
Dipartimento di Chimica e Tecnologie del Farmaco, ‘Sapienza’ Università di Roma, 00185 Roma, Italy;
rossella.fioravanti@uniroma1.it (R.F.); elisabetta.dibello@uniroma1.it (E.D.B.);
annalisa.romanelli@uniroma1.it (A.R.)
2
3
4
Dipartimento di Farmacia, Università di Napoli ‘Federico II’, 80131 Napoli, Italy;
stefano.tomassi@unina.it (S.T.); ettore.novellino@unina.it (E.N.)
Dipartimento di Neuroscienze, Salute Mentale e Organi di Senso–Nesmos, ‘Sapienza’ Università di Roma,
00185 Roma, Italy; andrea.plateroti@gmail.com
Dipartimento di Medicina di Precisione, Università degli Studi della Campania Luigi Vanvitelli,
80138 Napoli, Italy; rosaria.benedetti@unicampania.it (R.B.); mariarosaria.conte@unicampania.it (M.C.);
lucia.altucci@unicampania.it (L.A.)
*
†
Correspondence: sergio.valente@uniroma1.it (S.V.); antonello.mai@uniroma1.it (A.M.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Maria Novella Romanelli and Silvia Dei
Received: 5 June 2020; Accepted: 6 July 2020; Published: 8 July 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Bis-(3-bromo-4-hydroxy)benzylidene cyclic compounds have been reported by us
as epigenetic multiple ligands, but different substitutions at the two wings provided analogues with
selective inhibition. Since the 1-benzyl-3,5-bis((E)-3-bromobenzylidene)piperidin-4-one
dual p300/EZH2 inhibition joined to cancer-selective cell death in a panel of tumor cells and in in vivo
xenograft models, we prepared a series of bis((E)-2-bromobenzylidene) cyclic compounds 4a to
test in biochemical (p300, PCAF, SIRT1/2, EZH2, and CARM1) and cellular (NB4, U937, MCF-7,
SH-SY5Y) assays. The majority of 4a exhibited potent dual p300 and CARM1 inhibition, sometimes
3 displayed
–
n
–
n
reaching the submicromolar level, and induction of apoptosis mainly in the tested leukemia cell
lines. The most effective compounds in both enzyme and cellular assays carried a 4-piperidone
moiety and a methyl (4d), benzyl (4e), or acyl (4k–m) substituent at N1 position. Elongation of
the benzyl portion to 2-phenylethyl (4f) and 3-phenylpropyl (4g) decreased the potency of compounds
at both the enzymatic and cellular levels, but the activity was promptly restored by introduction
of a ketone group into the phenylalkyl substituent (4h–j). Western blot analyses performed in NB4
and MCF-7 cells on selected compounds confirmed their inhibition of p300 and CARM1 through
decrease of the levels of acetyl-H3 and acetyl-H4, marks for p300 inhibition, and of H3R17me2,
mark for CARM1 inhibition.
Keywords: epigenetics; histone acetylation; histone methylation; drug discovery; multi-target agents
1. Introduction
Polypharmacology is a new emerging approach for chemotherapy, particularly useful when
the single-targeted therapy tailored for a specific disease remains without or loses its effect [1]. This can
happen because multifactorial diseases, such as neurodegenerative disorders or cancer, typically
feature multiple dysfunctions of several biological pathways, which can elicit resistance towards a
Molecules 2020, 25, 3122; doi:10.3390/molecules25143122
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single target modulator through their robustness and redundancy [2]. Multi-targeting compounds
differ from promiscuous drugs because the first are directed towards different targets and pathways
all related to the same pathology, whereas the latter possess a broad spectrum of biological activities
directed against targets unrelated to the same disease, and are joined to a plethora of adverse reactions
due to the presence of off-target effects [3]. Particularly in cancer, the contribution of genetic, epigenetic,
and metabolic dysfunctions may sustain the insurgence and the development of the disease, thus its
treatment could likely require complex therapeutic approaches, hijacking different signal pathways
and cross-talks, all related to epigenetic and not epigenetic phenomena [4].
Transcriptional dysregulations and epigenetic dysfunctions are common and recurring themes
in cancer. Alterations in histone acetylation and/or methylation or in DNA methylation modify
the transcription regulation leading to aberrant events characterized by either gene silencing or
activation [5–9]. Then, such dysfunctions can activate selected cellular programs for cell cycle arrest
and block of differentiation and/or apoptosis allowing cancer initiation and development, and can be
managed by using orthosteric and allosteric inhibitors, peptidomimetics, and degraders [10].
In 2008, pursuing our studies on design, synthesis, and biological evaluation of small molecule
modulators of epigenetic targets as anticancer agents, we prepared a large series of compounds with
general formula 1 (Figure 1). These structures hold two bromo- or dibromo-hydroxyphenyl portions
separated by spacers with different length and size, based on the similarity with some dyes previously
reported as histone methyltransferase inhibitors (HMTs) in a pioneering study [11]. We tested these
compounds against HMTs (belonging to both PRMT (protein arginine methyltransferase) and KMT
(histone lysine methyltransferase) families) and, due to their strict similarities with some described
histone acetyltransferase (HAT) or sirtuin modulators, against p300 HAT and SIRT enzymes [12,13].
Among such compounds, those carrying two 3,5-dibromo-4-hydroxyphenyl/benzylidene moieties
linked through a penta-1,4-dien-3-one, (hetero)cycloalkanone, or 1,1-(1,3-phenylene)diprop-2-en-1-one
spacer behaved as epigenetic multiple ligands (epi-MLs), capable to inhibit at the same time all the tested
enzymes [13]. When tested on the U937 human acute monocytic leukemia (AML) cell line, some epi-MLs
induced high apoptosis levels and/or massive, dose-dependent cytodifferentiation, whereas the related
single-target inhibitors were ineffective or showed a weak effect [13]. A combination of distinct
substitution patterns on the two phenyl/benzylidene wings together with an appropriate spacer
provided compounds with a better spectrum of activities [12–16]. The insertion of the 3-bromo-
4-hydroxybenzylidene portions at the 3,5 positions of the 1-benzylpiperidin-4-one yielded
2
(Figure 1), inactive against PRMT1, SET7, and p300 HAT, and exhibiting selective inhibition
of CARM1 [17] and EZH2 (unpublished results), a KMT highly involved in cancer diseases.
The change of 3-bromo-4-hydroxy- into 3-bromo-4-methoxyphenyl/benzylidene group applied to
the penta-1,4-dien-3-one or cyclohexan-1-one scaffold led to loss of inhibition towards PRMT1
and CARM1, and gain of activity against SETD8 and, to a lesser extent, EZH2 in a panel of five tested
KMTs (SET7, SETD8, G9a, SET7/9, and EZH2) [14
from furnished MC2884 ( , Figure 1), a dual p300/EZH2 inhibitor able to induce cancer-selective cell
death in both solid and blood cancers in vitro, ex vivo, and in vivo xenograft models [21,22].
The considerable anticancer activity observed with prompted us to explore the biochemical
,18–20]. The removal of the 4-hydroxy substituents
2
3
3
and biological effects of the shift of the bromine substituents from the 3- to the 2-benzylidene positions,
using the cyclohexanone, tetrahydro-4H-pyran-4-one or piperidin-4-one moiety as connection units
(compounds 4a
benzyl, 2-phenethyl, 3-phenylpropyl, 2-oxo-2-phenylethyl, 3-oxo-3-phenylpropyl, 4-oxo-4-phenylbutyl,
benzoyl, 2-phenylacetyl, 3-phenylpropanoyl, or 3-phenylacryloyl substituent. Compounds 4a have
–n, Figure 1). At the piperidone N1 position, besides hydrogen we inserted a methyl,
–
n
been evaluated in p300, PCAF, SIRT1/2, EZH2 and CARM1 enzyme assays, and in NB4 human acute
promyelocytic leukemia (APL), U937 AML, MCF-7 breast cancer and SH-SY5Y neuroblastoma cells,
to determine their effects in cell cycle phases and apoptosis (sub-G1 peaks) induction. Western blot
analyses of the levels of acetyl-H3, acetyl-H4, acetyl-H3K9/14, and acetyl-α-tubulin, marks of p300
activity in cells, as well as of those of H3K27me3 (trimethylated histone 3 lysine 27) and H3R17me2

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(dimethylated histone 3 arginine 17), histone marks of cellular activity of EZH2, and CARM1,
respectively, have been performed for selected derivatives in NB4 and MCF-7 cells to correlate
the observed biological effects with the modulation of the cited epigenetic targets.
Figure 1. Variously substituted phenyl/benzylidene groups connected by a penta-1,4-dien-3-one or
a (hetero)cycloalkanone led to compounds 1–4 displaying different selectivity for several epi-targets.
Compounds 4a–n are described in the present paper.
2. Results and Discussion
2.1. Chemistry
The known 2,6-bis((E)-2-bromobenzylidene)cyclohexan-1-one 4a
[
23], 3,5-bis((E)-2-bromobenzylidene)
25], and 3,5-bis((E)-
25]werepreparedthroughaldolcondensationbetween
2-bromobenzaldehyde and the appropriate 6-membered cyclic ketones by replacing sodium hydroxide,
used as the basic catalyst in the previous reports [23 25], with barium hydroxide and methanol for 2 h
at room temperature. Starting from 4c, compounds 4e were obtained by alkylation with suitable alkyl
chlorides heating at 60 ◦C for 2 h in the presence of K2CO3 in dry acetonitrile (MeCN) (4e
), or by one-pot
reductive amination with 4-oxo-4-phenylbutanal and sodium triacetoxyborohydride [NaBH(AcO3)3] in dry
dichloromethane (DCM) (4j) for 2 h at room temperature (Scheme 1). Compounds 4k were prepared by
tetrahydro-4H-pyran-4-one 4b
[24], 3,5-bis((E)-2-bromobenzylidene)piperidin-4-one 4c
[
2-bromobenzylidene)-1-methylpiperidin-4-one4d
[
24
,
–
–j
–i
–n
acylating the piperidin-4-one N1 nitrogen with different acyl chlorides, characterized by increasing distance
between the carbonyl group and the phenyl ring, in the presence of triethylamine (TEA) in dry DCM for 1 h
at room temperature (Scheme 1).

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Scheme 1. Synthesis of bis(2-bromobenzylidene) cyclic derivatives 4e–n.
2.2. p300, PCAF, SIRT1/2, EZH2, and CARM1 Enzyme Assays
The newly synthesized compounds 4a
–n
were tested to determine their effects against acetylation
and methylation epigenetic targets. First, 4a–n were screened in vitro against the two HAT enzymes p300
and PCAF. The IC50 values, when possible, were determined in a 10-dose mode, in triplicate, with 2-fold
serial dilution starting from 200
M solutions. In these assays, histone H3 and [acetyl-3H]-acetyl-CoA
were used as substrate and co-substrate, respectively, and C-646 (for p300) and anacardic acid (for PCAF)
were added as reference compounds. To determine the activity against SIRT1 and -2, 4a were tested
starting from 200 M solutions using a modified Fluor de Lys fluorescence-based assay with amino
µ
–n
µ
acids 379–382 (Arg-His-Lys-Lys(Ac)) of human p53 conjugated with aminomethylcoumarin as substrate
and NAD+ as co-substrate. The SIRT1 inhibitor selisistat was used as internal reference compound.
Furthermore, 4a–n were tested against a human five-membered Polycomb repressive complex 2 (PRC2)
containing EZH2, embryonic ectoderm development (EED), suppressor of zeste 12 (SUZ12), RbAp48,
and adipocyte enhancer-binding protein (AEBP2), to assess their capability to inhibit the catalytic unit
EZH2. According to a 2-fold serial dilution pattern, 4a
–
n
were tested in a 10-dose IC50 evaluation
3
in duplicate starting from 400
µM as initial concentration. The core histone and H-SAM were
used as substrate and co-substrate, respectively, whereas S-adenosyl-l-homocysteine (SAH) was used
as reference compound. Finally, 4a–n were screened against CARM1 in agreement to the experimental
protocol already reported above for p300 and PCAF, but using histone H3 and 3H-SAM as substrate
and co-substrate, respectively, and SAH as the internal reference.
When tested toward histone (de)acetylation enzymes, the majority of 4a
consistently inhibit the HAT p300, with some compounds displaying a submicromolar activity.
Differently, 4a were completely ineffective against PCAF as well as SIRT1/2 (Table 1). In particular,
4a 4b 4d, and 4e, bearing the cyclohexanone, the tetrahydro-4H-4-pyranone and the N-methyl-
and N-benzyl-4-piperidone spacer, exhibited single-digit micromolar IC50 values against p300 activity.
The elongation of the benzyl substituent at the N1 4-piperidone position (see compounds 4f and 4g
significantly decreased the acetyltransferase inhibition, that was promptly restored when a carbonyl
function was added to the N1-alkylphenyl chain (4h , stabilized on single-digit micromolar values).
The replacement of the alkylphenyl group at the N1 4-piperidone position with an acyl moiety,
such as in 4k , gave a gain of potency against p300 in particular with 4l and 4m (IC50 values
in the submicromolar range).
–n demonstrated to
–n
,
,
)
–
j
–
n

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Table 1. Biochemical data for 4a–n tested against p300, PCAF, SIRT1, SIRT2, PRC2/EZH2, and CARM1.
IC50, µM
SIRT2
Compound
p300
PCAF
SIRT1
PRC2/EZH2
CARM1
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
1.38 ± 0.24
2.56 ± 0.23
23.2 ± 0.51
2.66 ± 0.15
2.19 ± 0.77
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
117 ± 6
16.8 ± 3.4
23.3 ± 0.5
44.8 ± 6.0
3.24 ± 1.22
6.50 ± 0.86
18.5 ± 0.9
12.6 ± 2.6
5.52 ± 0.78
6.96 ± 0.90
4.80 ± 1.72
1.33 ± 0.14
0.43 ± 0.12
0.79 ± 0.06
7.14 ± 2.54
>200
>200
46.7 ± 1.1
44.2 ± 0.2
>200
30.17
±
2.71
40.7 ± 2.39
1.03 ± 0.22
1.57 ± 0.72
2.13 ± 1.35
2.09 ± 0.79
0.45 ± 0.03
0.46 ± 0.10
1.23 ± 0.29
0.12 ± 0.01
>200
46.0 ± 8.1
60.6 ± 10.5
68.5 ± 6.2
40.7 ± 15.3
15.2 ± 2.2
11.3 ± 0.08
13.9 ± 1.8
4n
C-646
anacardic acid
selisistat
SAH
40.0 ± 10.9
0.16 ± 0.01
48.5 ± 1.9
33.8 ± 1.4
0.30 ± 0.07
Towards PRC2/EZH2, 4a–n generally displayed low, if any, inhibition (Table 1). Only N1-substituted
4-piperidone compounds exhibited some activity, with the N1-acyl derivatives 4l and 4m being the most
effective. The N-methyl and N-benzyl analogues 4d and 4e showed low EZH2 inhibition, which
was lost with 4f and 4g (carrying longer substituents at N1) and partially restored by the introduction
of the ketone function in the alkylaryl chain.
Against CARM1, the N1-substituted 4-piperidone compounds displayed the highest activity,
reaching in some cases IC50 values at low micromolar/submicromolar levels (Table 1). The insertion of
the methyl or benzyl group at N1 furnished quite potent compounds (4d and 4e), while 2-phenylethyl
(4f) and 3-phenylpropyl (4g) substitution were less effective. Single-digit micromolar IC50 values were
shown by compounds bearing a carbonyl group (ketone or amide function) in the N1-side chain, while
4l and 4m were the most potent (IC50 values at submicromolar level).
2.3. Biological Evaluation in Cancer Cells
Compounds 4a–n were tested at 5 µM for 30 h in NB4 PML and U937 AML cells among
hematological cancers, and in MCF-7 breast cancer and SH-SY5Y neuroblastoma cells among solid
cancers, to determine their effects in cell cycle and apoptosis induction. In NB4 cells, the majority of
compounds (4a–c, 4g–k) gave a block in G2/M phase, while in U937 cells only 4f, 4l, and 4m displayed
the same effect, and 4g and 4n arrested the cell cycle at the G1/S phase (Figure 2A,B). In MCF-7 cells,
most compounds exhibited a profound alteration of the cell cycle with a block in the G1/S phase
and the almost total absence of cells either in the G2/M phase (4a
phase (4f 4k 4l, and 4n) (Figure 2C). SH-SY5Y cells treated with 4a
of the cell cycle, with 4h 4j 4l 4m and 4d 4f 4k displaying an arrest in the G1/S phase or in the G2/M
phase, respectively (Figure 2D).
,
4c
,
4d
,
4e
, 4j, 4m, and 4n) or in the S
–i
,
,
–n showed moderate alteration
,
,
,
,
,

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Figure 2. Cont.

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Figure 2. Effects of 4a–n on cell cycle phases in NB4 (A), U937 (B), MCF-7 (C), and SH-SY5Y (D) cells
treated at 5 µM for 30 h.
In the same cell lines, the induction of apoptosis has been evaluated through the percentage of
the sub-G1 peaks. In NB4 cells, 4l and 4m displayed the highest induction (% sub-G1 peaks = 67
and 56, respectively) (Figure 3A), demonstrating the crucial role of the insertion of the phenylacetyl
(4l) and 3-phenylpropionyl (4m) moiety at the N1 4-piperidone position to elicit this cellular effect.
It is noteworthy that 4l and 4m are also the most potent among the described dual p300/CARM1
inhibitors, reaching IC50 values at submicromolar level in both the enzymatic assays (Table 1).
In the same cell line, 4d
4l, and 4m exhibited a percentage of apoptosis over 30% (Figure 3B), the others being less effective.
In the tested solid tumor cell lines, MCF-7 and SH-SY5Y, the compounds were generally less potent
in inducing apoptosis respect to blood cancers: In MCF-7 cells, only the N1-acyl derivatives 4k
, 4e, 4h, and 4k showed from 30 to 40% of sub-G1 peaks. In U937 cells, 4e, 4k,
4
–
m
exceeded 20% of sub-G1 peaks (Figure 3C), and in SH-SY5Y cells 4m was the most potent with a
percentage of apoptosis induction of 17% (Figure 3D).
The most potent derivatives as apoptosis inducers, 4l and 4m, were tested in mesenchymal
progenitor MePR2B cells, an immortalized non-cancer cell system [26
toxicity. After treatment at 5 M for 30 h, 4l and 4m reduced the percentage of cell viability to 61.2
and 85.5 ± 3.7, respectively, in comparison with the control (100%).
Western blot analyses were carried out on NB4 and MCF-7 cells treated with a subset of compounds
M, 30 h), to confirm that the observed effects are due to modulation of p300, CARM1, and eventually
,
27], to determine their differential
µ
±
0.8
4
(5 µ
EZH2 activities. Suberoylanilide hydroxamic acid (SAHA), a FDA-approved HDAC inhibitor [28],
and GSK-126 [29], a well-known EZH2 inhibitor, were added as internal controls. In NB4 cells,
the levels of acetyl-H3 (acH3) and acetyl-H4 (acH4) as well as those of acetyl-H3K9/14 (AcH3K9/14, see
Supplementary material) and of the non-histone substrate acetyl-α-tubulin (Ac-Tubulin) were detected
as an index of p300 inhibition (Figure 4). In the same cell line, the levels of H3K27me3, histone mark of
the EZH2 catalytic activity (Figure 4), as well as the level of the EZH2 expression (see Supplementary
material) have been determined. Last, the amount of H3R17me2, the functional read-out of CARM1
inhibition, was determined (Figure 4).

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Figure 3. Cont.

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Figure 3. Induction of apoptosis by 4a
treated at 5 µM for 30 h.
–n in NB4 (A), U937 (B), MCF-7 (C), and SH-SY5Y (D) cells
Figure 4. Western blot analyses of the levels of acetyl-H3, acetyl-H4, acetyl-
α
-tubulin, H3K27me3,
and H3R17me2 in NB4 cells treated with 4d–n at 5 µM for 30 h. H3, H4, and GAPDH were used for
equal loading.
Consistently with their IC50 values for p300 inhibition in enzyme assay (Table 1), the majority of
the tested compounds showed a marked decrease of the levels of acetyl-H3 in NB4 cells with the only
exception of the N1-phenylethyl- and -3-phenylpropyl-substituted 4f and 4g, which were less effective.
A similar scenario was observed with the acetyl-H4, acetyl-H3K9/14, and acetyl-α-tubulin levels, which

Molecules 2020, 25, 3122
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decreased with almost all the tested compounds, thus confirming their p300 inhibition in this cell
context (Figure 4 and Figure S1 in Supplementary material).
Against H3K27me3, the mark of the catalytic activity of PRC2/EZH2, compounds 4d–j showed
no effects, according to their weak, if any, inhibition in enzyme assay (Table 1). Only compounds 4h
and 4m displayed a reduction of the H3K27me3 levels (Figure 4). In addition, all the tested compounds
reduced the level of the EZH2 expression in NB4 cells, confirming the behavior previously observed
with the close analogue 3 in the same cell line [21] (Figure S1 in Supplementary material).
Regarding CARM1 inhibition, almost all the tested compounds decreased the levels of
the H3R17me2 histone mark, with 4h, 4l, and 4m, the last two potent at submicromolar level
in Table 1, being the most effective (Figure 4).
In MCF-7 cells, the levels of acetyl-H3, acetyl-H4, H3K27me3, and H3R17me2 were detected by
western blot analyses as functional tests for p300 (acH3 and acH4), EZH2 (H3K27me3) and CARM1
(H3R17me2) inhibition in cells, respectively (Figure 5).
Figure 5. Western blot analyses of the levels of acetyl-H3, acetyl-H4, H3K27me3, and H3R17me2
in MCF-7 cells treated with 4d–n at 5 µM for 30 h. H3, H4, and GAPDH were used for equal loading.
In acetyl-H3 assay all the compounds led to a consistent reduction of the levels of the acetylation
mark, except for 4f and 4g (Figure 5). In acetyl-H4 and acetyl-
the tested compounds (4e 4i 4j 4l 4n for acetyl-H4, and 4i and 4k for acetyl-
to give a decrease of the acetylation levels. About EZH2, few compounds (4e
α
-tubulin assays, a limited number of
-tubulin) were able
4h 4j 4m, and 4n)
,
,
,
,
α
,
,
,
displayed a reduction of the H3K27me3 levels (Figure 5). Towards CARM1, 4i and 4m were the most
effective in reducing the H3R17me2 levels (Figure 5).
3. Materials and Methods
3.1. Chemistry
Melting points were determined on a Buchi 530 melting point apparatus (Buchi Italia, Cornaredo,
1
Italy). H-NMR and 13C-NMR spectra were recorded at 400 MHz using a Bruker AC 400 spectrometer

Molecules 2020, 25, 3122
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(Bruker Italia, Milano, Italy); chemical shifts are reported in
δ
(ppm) units relative to the internal
reference tetramethylsilane (Me4Si). Mass spectra were recorded on an API-TOF Mariner by Perspective
Biosystem (Stratford, TX, USA), samples were injected by a Harvard pump using a flow rate of
5–10 µL/min, infused in the Electrospray system. All compounds were routinely checked by thin layer
chromatography (TLC). TLC was performed on aluminum-backed silica gel plates (Merck DC, Alufolien
Kieselgel 60 F254) with spots visualized by UV light (254 and 365 nm) or using a KMnO4 alkaline
solution as staining reagent. All solvents were reagent grade and, when necessary, were purified
and dried by standard methods. Concentration of solutions after reactions and extractions involved
the use of a rotary evaporator operating at reduced pressure of ~20 Torr. Organic solutions were
dried over anhydrous sodium sulfate (Na2SO4). Analytical results are within 0.40% of the theoretical
values. All chemicals were purchased from Sigma Aldrich s.r.l. (Milan, Italy) or from TCI Europe
N.V. (Zwijndrecht, Belgium), and were of the highest purity. As a rule, samples prepared for physical
and biological studies were dried in high vacuum over P2O5 for 20 h at temperatures ranging from 25
to 40 ◦C, depending on the sample melting point.
3.1.1. General Procedure for the Synthesis of the N-Alkylaryl 3,5-Bis((E)-2-bromobenzylidene)
piperid-4-ones (4e–i). Example: 1-benzyl-3,5-bis((E)-2-bromobenzylidene)piperidin-4-one (4e)
3,5-Bis((E)-2-bromobenzyliden)piperidin-4-one 4c
bromide (1.38 mmol, 164
[25] (0.46 mmol, 200 g, 1 eq.) and benzyl
L, 3 eq.) were added to a suspension of anhydrous K2CO3 (0.69 mmol,
954 mg, 1.5 eq.) in acetonitrile (10 mL), and the resulting suspension was stirred at 60 ◦C. After 2 h
µ
the solvent was evaporated, water (50 mL) was added and the aqueous solution was extracted with
dichloromethane (3
×
30 mL). The collected organic phases were washed with a saturated solution
of NaCl (30 mL) and then dried with anhydrous Na2SO4, filtered and evaporated under vacuum to
afford a crude that was purified on silica gel (AcOEt/n-hexane 1:3) to obtain the desired compound
1
(170 mg, 67%) as a pale yellow solid. M.p.: 144–146 ◦C (toluene). Yield: 82%. H-NMR (DMSO-d6)
δ
ppm: 7.75–7.73 (m, 4H), 7.40 (t, 2H, J = 7.2 Hz), 7.34–7.30 (m, 4H), 7.17 (bs, 5H), 3.71 (bs, 4H), 3.60 (s,
2H). 13C-NMR (DMSO-d6)
δ
ppm: 161.4, 137.9 (2C), 135.3 (2C), 135.1 (2C), 134.7, 133.4 (2C), 131.4 (2C),
131.2 (2C), 129.2 (2C), 128.5 (2C), 128.2, 127.6 (2C), 124.9 (2C), 59.9, 53.3 (2C). MS (ESI-MS): Calculated:
524.00 for C26H21Br2NO [M + H]+, Found: 524.02. Calculated for M.W. 523.27, %: C 59.68; H 4.05;
N 2.68; Br 30.54. Found, %: C 59.44; H 3.96; N 2.85; Br 30.68.
3,5-Bis((E)-2-bromobenzylidene)-1-phenethylpiperidin-4-one (4f). M.p.: 105–107 ◦C (cyclohexane).
1
Yield: 65%. H-NMR (CDCl3)
δ ppm: 7.98 (bs, 2H), 7.69 (d, 2H, J = 7.2 Hz), 7.36 (t, 2H, J = 7.2 Hz),
7.28–7.27 (m, 1H), 7.25–7.20 (m, 5H), 7.16 (d, 1H, J = 6.8 Hz), 7.09 (d, 2H, J = 7.6 Hz), 3.76 (bs, 4H),
2.75–2.73 (m, 2H), 2.70–2.65 (m, 2H). 13C-NMR (CDCl3)
δ
ppm: 182.6, 146.4 (2C), 140.0 (2C), 136.9,
135.2 (2C), 133.4 (2C), 129.9 (2C), 129.3 (2C), 128.9 (2C), 128.5 (2C), 127.0 (2C), 126.0, 125.1 (2C), 60.2,
52.4 (2C), 32.8. MS (ESI-MS): Calculated: 538.02 for C27H24Br2NO [M + H]+, Found: 538.03. Calculated
for M.W. 537.30, %: C 60.36; H 4.31; N 2.61; Br 29.74. Found, %: C 60.59; H 4.39; N 2.42; Br 29.51.
3,5-Bis((E)-2-bromobenzylidene)-1-(3-phenylpropyl)piperidin-4-one (4g). M.p.: 93–95 ◦C
(cyclohexane). Yield: 67%. 1H-NMR (CDCl3)
δ ppm: 7.97 (bs, 2H), 7.68 (d, 2H, J = 8.0 Hz),
7.36 (t, 2H, J = 7.2 Hz), 7.29–7.26 (m, 2H), 7.25–7.20 (m, 4H), 7.16 (d, 1H, J = 6.8 Hz), 7.06 (d, 2H,
J = 7.2 Hz), 3.68 (bs, 4H), 2.56 (t, 2H, J = 7.6 Hz), 2.49 (t, 2H, J = 7.6 Hz), 1.65 (q, 2H, J = 7.5 Hz).
13C-NMR (CDCl3)
δ ppm: 182.5, 146.7 (2C), 140.3 (2C), 137.4, 135.1 (2C), 133.9 (2C), 129.5 (2C), 129.4 (2C),
128.9 (2C), 128.6 (2C), 127.7 (2C), 126.4, 125.0 (2C), 58.2, 53.6 (2C), 32.0, 29.8. MS (ESI-MS): Calculated:
552.04 for C28H26Br2NO [M + H]+, Found: 552.07. Calculated for M.W. 551.32, %: C 61.00; H 4.57;
N 2.54; Br 28.99. Found, %: C 60.85; H 4.49; N 2.62; Br 29.11.
3,5-Bis((E)-2-bromobenzylidene)-1-(2-oxo-2-phenylethyl)piperidin-4-one (4h). M.p.: 109–111 ◦C
(cyclohexane). Yield: 78%. 1H-NMR (CDCl3)
δ
ppm: 7.99 (bs, 2H), 7.88 (d, 2H, J = 7.2 Hz), 7.65
(d, 2H, J = 7.6 Hz), 7.57 (t, 1H, J = 7.6 Hz), 7.44 (t, 2H, J = 7.6 Hz), 7.34 (t, 2H, J = 7.6 Hz), 7.24–7.21 (m, 4H),
4.01 (s, 2H), 3.98 (bs, 4H). 13C-NMR (DMSO-d6)
ppm: 197.3, 186.6, 136.0 (2C), 135.2 (2C), 134.7,
134.7 (2C), 133.8, 133.4 (2C), 131.4 (2C), 131.3 (2C), 129.1 (2C), 128.3 (2C), 128.3 (2C), 125.0 (2C), 62.2,
δ

Molecules 2020, 25, 3122
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53.9 (2C). MS (ESI-MS): Calculated: 551.99 for C27H21Br2NO2 [M + H]+, Found: 552.00. Calculated for
M.W. 551.28, %: C 58.83; H 3.84; N 2.54; Br 28.99. Found, %: C 58.99; H 3.91; N 2.32; Br 28.79.
3,5-Bis((E)-2-bromobenzylidene)-1-(3-oxo-3-phenylpropyl)piperidin-4-one (4i). M.p.: 95–97 ◦C
(cyclohexane). Yield: 69%. 7.89 (d, 2H, J = 7.6 Hz), 7.75 (bs, 2H), 7.56–7.51 (m, 4H), 7.44 (t, 2H,
J = 7.6 Hz), 7.32–7.31 (m, 3H), 7.29 (s, 2H), 3.88 (s, 4H), 3.13 (t, 2H, J = 6.0 Hz), 2.60 (t, 2H, J = 6.4 Hz).
13C-NMR (CDCl3)
δ ppm: 196.4, 182.1, 144.8 (2C), 140.5 (2C), 137.2, 135.5 (2C), 134.0, 132.3 (2C),
130.5 (2C), 129.3 (2C), 128.5 (2C), 127.3 (2C), 127.0 (2C), 125.0 (2C), 59.3, 52.4 (2C), 40.7. MS (ESI-MS):
Calculated: 566.01 for C28H23Br2NO2 [M + H]+, Found: 566.02. Calculated for M.W. 565.31, %: C 59.49;
H 4.10; N 2.48; Br 28.27. Found, %: C 59.27; H 3.96; N 2.61; Br 28.45.
3.1.2. Synthesis of 3,5-Bis((E)-2-bromobenzylidene)-1-(4-oxo-4-phenylbutyl)piperidin-4-one (4j)
To a stirring solution of the 3,5-bis((E)-2-bromobenzylidene)piperidin-4-one (4c) [25] (0.46 mmol,
200 mg) and 4-oxo-4-phenylbutanal (0.46 mmol, 75 mg) in anhydrous dichloromethane (10 mL),
sodium triacetoxyborohydride (0.55 mmol, 117 mg, 1.2 eq.) was added portion wise and the resulting
suspension was stirred at room temperature. After 2 h the reaction was quenched with H2O (30 mL)
and extracted with dichloromethane (4
with a NaCl saturated aqueous solution (30 mL) and then dried with anhydrous Na2SO4, filtered
×
20 mL). The collected organic phases were then washed
and evaporated under vacuum to afford a crude that was purified on silica gel (AcOEt/n-hexane 1:3)
◦
to obtain the desired compound (197 mg, 74%). M.p.: 84–86 C (cyclohexane). 1H-NMR (CDCl3)
δ
ppm: 7.90 (d, 2H, J = 7.6 Hz), 7.72 bs, 2H), 7.54–7.52 (m, 5H), 7.42 (t, 2H, J = 8 Hz), 7.33–7.32 (m, 4H),
3.80 (s, 4H), 2.99 (t, 2H, J = 7.2 Hz), 2.67 (t, 2H, J = 6.4 Hz), 1.94–1.90 (q, 2H, J = 7.2 Hz). 13C-NMR (CDCl3)
ppm: 196.2, 182.3, 144.2 (2C), 141.0 (2C), 137.3, 135.5 (2C), 134.4, 132.1 (2C), 129.5 (2C), 128.3 (2C),
δ
128.1 (2C), 127.7 (2C), 127.5 (2C), 125.1 (2C), 57.3, 52.5 (2C), 37.7, 25.0. MS (ESI-MS): Calculated: 580.02
for C29H25Br2NO2 [M + H]+, Found: 580.01. Calculated for M.W. 579.33, %: C 60.12; H 4.35; N 2.42;
Br 27.58. Found, %: C 60.32; H 4.45; N 2.29; Br 27.34.
3.1.3. General Procedure for the Synthesis of N-Acyl-3,5-Bis((E)-2-bromobenzylidene)piperidin-4-ones
(4k–n). Example: 3,5-Bis((E)-2-bromobenzylidene)-1-cinnamoylpiperidin-4-one (4n)
Cinnamoyl chloride (0.67 mmol, 100
(0.45 mmol, 195 mg) and Et3N (0.76 mmol, 110
The resulting mixture is then allowed to stir at room temperature. After 1 h the reaction was quenched
with water (50 mL) and extracted with dichloromethane (3 30 mL). The collected organic layers were
washed with 2N HCl (3 30 mL) and then with a saturated solution of NaCl (30 mL). The organic
µ
L) was slowly added at 0 ◦C to a stirring solution of 4c
L, 1.13 eq.) in anhydrous dichloromethane (5 mL).
[25]
µ
×
×
phase was dried with anhydrous Na2SO4, filtered and evaporated under vacuum to afford a crude that
was then purified on silica gel (AcOEt/n-hexane 1:3) to afford the desired product (152 mg, 60%). M.p.:
1
98–100 ◦C (cyclohexane). H-NMR (CDCl3)
δ
ppm: 7.95 (bs, 2H), 7.71 (d, 2H, J = 8 Hz), 7.54 (d, 2H,
J = 15.6 Hz), 7.44–7.40 (m, 3H), 7.39–7.31 (m, 5H), 7.20 (d, 2H, J = 6.8 Hz), 6.38 (d, 1H, J = 15.6 Hz),
4.80–4.78 (m, 4H). 13C-NMR (CDCl3)
δ
ppm: 185.6, 165.3, 145.5 (2C), 143.5, 141.9 (2C), 137.0, 135.1 (2C),
133.6 (2C), 129.2 (2C), 128.7 (2C), 128.3, 127.9 (2C), 127.6 (2C), 126.0 (2C), 125.6 (2C), 117.1, 48.6 (2C). MS
(ESI-MS): Calculated: 563.99 for C28H21Br2NO2 [M + H]+, Found: 564.01. Calculated for M.W. 563.29;
%: C 59.70; H 3.76; N 2.49; Br 28.37. Found, %: C 59.49; H 3.65; N 2.70; Br 28.54.
1-Benzoyl-3,5-bis((E)-2-bromobenzylidene)piperidin-4-one (4k). M.p.: 124–126 ◦C (cyclohexane/toluene).
1
Yield: 76%. H-NMR (CDCl3)
δ
ppm: 8.01 (s, 2H), 7.69 (d, 2H, J = 8.8 Hz), 7.23–7.21 (3H, m), 7.14–7.11 (8H, m),
ppm: 182.5, 169.1, 144.8 (2C), 139.8 (2C), 136.1, 133.8 (2C), 132.1 (2C),
4.75–4.45 (m, 4H). 13C-NMR (CDCl3)
δ
128.9, 128.5 (2C), 128.3 (2C), 127.8 (2C), 126.1 (2C), 125.8 (2C), 124.7 (2C), 50.1 (2C). MS (ESI-MS): Calculated:
537.98 for C26H19Br2NO2 [M + H]+, Found: 538.00. Calculated for M.W. 537.25, %: C 58.13; H 3.56; N 2.61;
Br 29.75. Found, %: C 58.32; H 3.68; N 2.49; Br 29.67.
3,5-Bis((E)-2-bromobenzylidene)-1-(2-phenylacetyl)piperidin-4-one (4l). M.p.: 126–128 ◦C
1
(cyclohexane/benzene). Yield: 67%. H-NMR (CDCl3)
δ ppm: 7.95 (d, 2H, J = 18.8 Hz), 7.76 (d, 1H,
J = 6.8 Hz), 7.68 (d, 1H, J = 7.6 Hz), 7.45–7.36 (m, 4H), 7.26–7.24 (m, 1H), 7.20–7.18 (m, 3H), 7.08 (d, 1H,

Molecules 2020, 25, 3122
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J = 6.8 Hz), 6.84–6.82 (m, 2H), 4.77 (bs, 2H), 4.45 (bs, 2H), 3.50 (s, 2H). 13C-NMR (DMSO-d6)
δ ppm:
186.1, 169.8, 135.2 (2C), 134.4, 134.0, 133.6 (4C), 131.8 (2C), 131.4 (2C), 128.8 (5C), 128.4 (2C), 127.0 (2C),
125.5 (1C), 47.8, 44.5, 42.8. MS (ESI-MS): Calculated: 551.99 for C27H21Br2NO2 [M + H]+, Found: 552.00.
Calculated for M.W. 551.28, %: C 58.83; H 3.84; N 2.54; Br 28.99. Found, %: C 59.04; H 3.97; N 2.35;
Br 28.75.
◦
3,5-Bis((E)-2-bromobenzylidene)-1-(3-phenylpropanoyl)piperidin-4-one (4m). M.p.: 95–97 C
(cyclohexane). Yield: 69%. H-NMR (CDCl3)
1
δ ppm: 7.94 (d, 2H, J = 18.8 Hz), 7.68 (d, 2H, J = 8.0 Hz),
7.45–7.39 (m, 3H), 7.25–7.21 (m, 4H), 7.19–7.14 (m, 2H), 6.98 (d, 2H, J = 7.2 Hz), 4.76 (bs, 2H), 4.41 (bs,
2H), 2.83 (t, 2H, J = 8.0 Hz), 2.39 (t, 2H, J = 8.0 Hz). 13C-NMR (CDCl3)
δ
ppm: 182.3, 168.4, 144.3 (2C),
142.1, 140.1 (2C), 134.1 (2C), 133.0 (2C), 128.6 (2C), 128.3 (2C), 127.3, 128.0 (2C), 126.6 (2C), 125.0 (2C),
124.7 (2C), 48.5 (2C), 35.7, 32.9. MS (ESI-MS): Calculated: 566.01 for C28H23Br2NO2 [M + H]+, Found:
566.01. Calculated for M.W. 565.31, %: C 59.49; H 4.10; N 2.48; Br 28.27. Found, %: C 59.29; H 3.97;
N 2.72; Br 28.45.
3.2. Biochemistry
3.2.1. HAT Assays
The effect of tested derivatives on the catalytic activity of p300 and PCAF was determined
in a HotSpot HAT activity assay by Reaction Biology Corporation (Malvern, PA, USA) according to
the company’s standard operating procedure. In brief, the recombinant catalytic domains of PCAF
(aa 492–658, 250 nM) or p300 (aa 1284–1673, 30 nM) were incubated with histone H3 as substrate
(5 µ µM) as an acetyl donor in reaction buffer (50 mM
M) and [Acetyl-3H]-Acetyl Coenzyme A (3
Tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1% DMSO) for 1 h at 30 ◦C
in the presence or absence of a dose titration of the compounds. The reaction mixture was delivered to
filter-paper for detection. IC50 values were analyzed using Excel and GraphPad Prism 6.0 software
(GraphPad Software Inc., San Diego, CA, USA). The experiments for p300 were performed in triplicate.
3.2.2. SIRT1/2 Assay
The SIRT inhibition assay was performed using human recombinant SIRT1 and SIRT2 produced
in E. coli. Compounds were tested using a modified Fluor de Lys fluorescence-based assay kit based
on the method described in the BioMol product sheet (AK-555, AK-556, Milan, Italy). The assay
procedure has two steps: in the first part the SIRT1/2 substrate, an acetylated Lys side chain comprising
amino acids 379-382 (Arg-His-Lys-Lys(Ac)) (BioMol KI177, Milan, Italy) of human p53 conjugated
◦
with aminomethylcoumarin is deacetylated during incubation at 37 C for 1 h by SIRT1 or SIRT2
in the presence of NAD+ and the tested compounds. The second stage is initiated by the addition of
the Developer II (KI176), including nicotinamide (NAM), nicotinamide that stops the SIRT1/2 activity,
and the fluorescent signal is produced. The fluorescence was measured on a fluorometric reader
(Infinite 200 TECAN, Männedorf, Switzerland) with excitation set at 360 nm and emission detection
set at 460 nm.
3.2.3. EZH2/PRC2 Assay
The EZH2 substrate (0.05 mg/mL core histone) was added in the freshly prepared reaction buffer
(50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1% DMSO). The PRC2
complex (complex of human EZH2, human EED, human SUZ12, human AEBP2, and human RbAp48)
was delivered into the substrate solution and the mixture was mixed gently. Afterwards, the tested
compounds dissolved in DMSO were delivered into the enzyme/substrate reaction mixture by using
Acoustic Technology (Echo 550, LabCyte Inc. Sunnyvale, CA, USA) in nanoliter range, and 3H-SAM
was added into the reaction mixture to initiate the reaction. The reaction mixture was incubated for 1 h
at 30 ◦C and then it was delivered to filter-paper for detection. The data were analyzed using Excel
and GraphPad Prism software for IC50 curve fits. The experiments were performed in duplicate.

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3.2.4. CARM1 Assay
Histone H3 (5
µM) was added in freshly prepared reaction buffer (50 mM Tris-HCl (pH 8.5),
5 mM MgCl2, 50 mM NaCl, 0.01% Brij35, 1 mM DTT, 1% DMSO). CARM1 was delivered into
the substrate solution and the mixture was mixed gently. Afterwards, the tested compounds dissolved
in DMSO were delivered into the enzyme/substrate reaction mixture by using Acoustic Technology
(Echo 550, LabCyte Inc. Sunnyvale, CA) in nanoliter range, and 1 µ
M 3H-SAM was also added into
the reaction mixture to initiate the reaction. The reaction mixture was incubated for 1 h at 30 ◦C and then
it was delivered to filter-paper for detection. The data were analyzed using Excel and GraphPad Prism
software for IC50 curve fits. The experiments were performed in triplicate.
3.3. Cellular Studies
3.3.1. Cell Cultures
NB4 tumor cell line was purchased by DSMZ, Braunschweig Germany, and U937, NB4, MCF7,
and SH-SY5Y tumor cell lines by American Type Culture Collection (ATCC, Milan, Italy). Cell lines have
been tested and authen◦ticated following manufacturer’s instruction. All cell lines were maintained
in an incubator at 37 C and 5% CO2. The human leukemia cells were grown in RPMI-1640
(Sigma-Aldrich, Milan, Italy) while human breast cancer and neuroblastoma cells in Dulbecco’s
Modified Eagle Medium (DMEM) (Sigma-Aldrich) culture media, 1% l-glutamine (EuroClone, Milan,
Italy), 10% heat-inactivated Fetal Bovine Serum (FBS) (GIBCO, Monza, Italy) and antibiotics. MePR2B
cells were grown as previously reported [26].
3.3.2. Cell Proliferation, Cell Cycle, and Cell Death Analyses
For colorimetric exclusion, the cells (2
After stimulations at different times and concentrations, cells were diluted in the ratio 1:1 in Trypan
×
105 cells/mL) were plated in multiwells in triplicate.
Blue (Sigma) and counted with an optical microscope. For cell cycle analyses, cells were plated
(2
×
105 cells/mL) and were harvested, centrifuged at 1200 rpm for 5 min and resuspended in 500
µ
L of
a hypotonic solution containing 1
×
PBS, Sodium Citrate 0.1%, 0.1% NP-40, RNAase A and 50 mg/mL
Propidium Iodide (PI). After 30 min at room temperature (RT) in the dark, samples were acquired
by FACS-Calibur (BD Bioscences, San Jose, CA, USA) using CellQuest software (BD Biosciences).
The percentage in different phases of the cell cycle was determined by ModFit LT V3 software (Verity).
All experiments were performed in triplicate. Cell death was measured as percentage of cells in sub-G1
phase as in [30].
3.3.3. Western Blot Analyses
The procedures were performed as described in [30
Ac-H3 (Millipore, Milan, Italy); Ac-H4 (Millipore, Milan, Italy); Ac-H3K9-14 (Diagenode, Ougr
Belgium); Ac- -tubulin (Sigma-Aldrich), H3K27me3 (Diagenode); H3R17me2 (Diagenode); EZH2
,
31]. Primary antibodies used were:
ée,
α
(Abcam, Cambridge, UK). H3 (Sigma-Aldrich), H4 (Abcam), GAPDH (Sigma) and ERKs (Santa
Cruz, Biotechnology, TX, USA) were used to normalize the total protein extracts (see also
Supplementary Material).
4. Conclusions
Curcuminoids are a wide family of variegated compounds bearing a plethora of therapeutic
activities, including anti-inflammatory, antidiabetic, neuroprotective, and anticancer activities [24,25,32–38].
Some related compounds reported by us as epi-MLs exerted biochemical inhibition of a number
of epigenetic targets (PRMTs, KMTs, p300, SIRT1/2), joined to potent induction of apoptosis and/or
cytodifferentiation in U937 AML cells [13]. The changes at decoration of the two phenyl rings as well
as the use of different spacers connecting them were able to focus the activity of such compounds

Molecules 2020, 25, 3122
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against specific epigenetics targets, leading to more selective derivatives [12,14,17]. In particular,
the 1-benzyl-3,5-bis((E)-3-bromobenzylidene)piperidin-4-one behaved as dual p300/EZH2 inhibitor
3
and displayed cancer-selective growth arrest and cell death in hematological malignancies as well as solid
tumors, in in vitro, ex vivo, and in vivo models [21,22].
Starting from these data, we decided to explore the effect of the shift of the bromine substitution
at the two phenyl ring from the 3 to the 2 position, using as connection unit cyclohexanone,
tetrahydro-4H-pyran-4-one or piperidin-4-one moiety (compounds 4a–n). Such compounds were
evaluated in p300, PCAF, SIRT1/2, EZH2 and CARM1 enzyme assays, and in NB4 APL, U937 AML,
MCF-7 breast cancer and SH-SY5Y neuroblastoma cells, to determine their effects in cell cycle
phases and apoptosis induction when used at 5
µM for 30 h. Generally, in biochemical assays
4a exhibited high inhibitory activities against p300 and CARM1, weak activity (if any) against
–
n
PRC2/EZH2, whereas were totally inactive against PCAF and SIRT1/2. In particular, compounds
carrying the cyclohexanone (4a), tetrahydro-4H-pyran-4-one (4b), and piperidin-4-one substituted at
N1 with a methyl, benzyl, oxophenylalkyl and acyl moieties (4d
with 4l and 4m reaching submicromolar IC50 values for both p300 and CARM1 enzymes. In cellular
assays, the majority of 4a gave an alteration of the cell cycle with pro-apoptotic effect in the tested
–n) displayed the highest inhibition,
–n
cell lines, with the leukemia cells being generally more sensitive than the solid cancer cells. In NB4
cells, 4l and 4m induced the strongest apoptosis (near 70%). In U937 cells, in addition to 4l and 4m
also the N1 benzyl (4e) and benzoyl (4k) analogues exhibited near or over 40% apoptosis induction.
In MCF-7 and in SH-SY5Y cells the effect is lower: the N1 2-oxo-2-phenylethyl 4h and the N1 acyl
derivatives 4k–m were the most potent but the induction of apoptosis was around 25% (MCF-7)
or less than 20% (SH-SY5Y) at the tested conditions. Despite some apparent discrepancies, due to
the differences between the in vitro enzymatic assays and the more complex cellular system, Western
blot analysis of selected derivatives in NB4 and MCF-7 cells, performed to detect the levels of
acetyl-H3/acetyl-H4/acetyl-α-tubulin, H3K27me3 and H3R17me2 as histone marks for cellular activity
of p300, EZH2, and CARM1, respectively, supports the role of the recruitment and inhibition of
these epigenetic targets, mainly p300 and CARM1, in the mechanism of anticancer activities of
the studied compounds.
Supplementary Materials: The following are available online. Figure S1: Western blot analyses of the levels of
acetyl-H3K9/14 and EZH2 in NB4 cells treated with 4d
for 4d–n.
–n at 5 µ
M for 30 h. 1H and representative 13C spectra
Author Contributions: Conceptualization, A.M., S.V., L.A.; Data curation, S.V., R.F., S.T., E.D.B., A.R.;
Formal analysis, S.V., A.M., R.F., S.T., R.B., M.C., A.M.P., A.R.; Investigation, S.V., R.F., S.T., E.D.B., R.B.,
M.C., A.M.P., A.R.; Methodology, A.M., S.V., L.A., R.F., S.T., R.B., M.C.; Resources, A.M., L.A.; Supervision, A.M.,
S.V., E.N., L.A.; Validation, A.M., S.V., R.F., S.T., E.N., L.A.; Visualization, A.M., S.V., R.F., S.T., A.R.; Writing–original
draft, R.F., S.T., A.M.; Writing–review and editing, A.M., S.V., R.F., S.T., L.A. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Associazione Italiana per la Ricerca sul Cancro (AIRC IG19162 to
AM and AIRC IG17217 to LA), the Italian Ministry for University and Research (PRIN2015-20152TE5PK, to AM
and LA), the Italian Ministry of Health (PE-2013-02355271, to AM), the project “Epigenetic Hallmarks of Multiple
Sclerosis” (acronym Epi-MS) (id:415, Merit Ranking Area ERC LS) in VALERE 2019 Program (to RB); Blueprint
282510 (to AM and LA); EPICHEMBIO CM1406 (to AM and LA); VALERE: Vanvitelli per la Ricerca; Campania
Regional Government Technology Platform Lotta alle Patologie Oncologiche: iCURE (to LA); Campania Regional
Government FASE2: IDEAL (to LA); MIUR, Proof of Concept POC01_00043 (to LA); POR Campania FSE 2014-2020
ASSE III (to LA). S.T. acknowledges MIUR, PON R&I 2014-2020-AIM (Attraction and International Mobility),
project AIM1873131 - 2, linea 1. The APC was funded by MDPI.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 3122
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Sample Availability: Samples of the compounds 4a–n are available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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