Protective effect of piceatannol and bioactive stilbene derivatives against hypoxia-induced toxicity in H9c2 cardiomyocytes and structural elucidation as 5-LOX inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.07.033

Stilbenes with well-known antioxidant and antiradical properties are beneficial in different pathologies including cardiovascular diseases. The present research was performed to investigate the potential protective effect of resveratrol (1) and piceatannol (2), against hypoxia-induced oxidative stress in the H9c2 cardiomyoblast cell line, and the underlying mechanisms. Compounds 1 and 2 significantly inhibited the release of peroxynitrite and thiobarbituric acid levels at na no- or submicromolar concentrations, and this effect was more evident in piceatannol-treated cells, that significantly increased MnSOD protein level in a concentration dependent manner. Furthermore, since piceatannol, which is far less abundant in natural sources, displayed a higher bioactivity than the parent compound, we hereby report on a very fast synthesis and detailed structure-based design of a focused stilbene library. Finally, taking into account that hypoxia-induced ROS accumulation also increases expression and activity of 5-lipoxygenase (5-LOX) with production of leukotrienes, we have disclosed structural key factors crucial for 5-LOX activity. Among the synthesized analogues (3–7), compound 7 was the most effective in improving cardiomyocytes viability and in 5-LOX inhibition. In conclusion, modeling and experimental studies provided the basis for further optimization of stilbene analogues as multi-target inhibitors of the inflammatory and oxidative pathway.

Full text of DOI:10.1016/j.ejmech.2019.07.033

European Journal of Medicinal Chemistry 180 (2019) 637e647
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Protective effect of piceatannol and bioactive stilbene derivatives
against hypoxia-induced toxicity in H9c2 cardiomyocytes and
structural elucidation as 5-LOX inhibitors
,
,
,
Mariarosaria Boccellino c 1, Maria Donniacuo d 1, Ferdinando Bruno d 1, Barbara Rinaldi d,
Lucio Quagliuolo c, Marika Ambruosi d e, Simona Pace e, Mario De Rosa d,
,
Abdurrahman Olgaç f, Erden Banoglu f, Nicola Alessio c, Antonio Massa g, Haroon Kahn h,
Oliver Werz e, Antonio Fiorentino i, Rosanna Filosa a
, b, *
a Consorzio Sannio Tech-AMP Biotec, Appia Str. 7, Apollosa, BN, 82030, Italy
b Institute of Food Sciences, National Research Council, Roma Str. 64, Avellino, 83100, Italy
c Department of Biochemistry, Department of Precision Medicine, University of Campania “L. Vanvitelli”, Via L. De Crecchio 7, Naples, 80138, Italy
d Department of Experimental Medicine, University of Campania, “L. Vanvitelli”, Via L. De Crecchio 7, Naples, 80138, Italy
e Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University of Jena, Philosophenweg 14, 07749, Jena,
Germany
f Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Yenimahalle, Ankara, 06330, Turkey
g
ꢀ
Dipartimento di Chimica e Biologia, Universita di Salerno, Via Giovanni Paolo II, 132, 84084, Fisciano, SA, Italy
h Department of Pharmacy, University of Peshawar, Peshawar, 25120, Pakistan
i
ꢀ
Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche -DiSTABiF, Universita Degli Studi Della Campania “Luigi Vanvitelli”, Via
Vivaldi 43, I-81100, Caserta, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Stilbenes with well-known antioxidant and antiradical properties are beneficial in different pathologies
including cardiovascular diseases. The present research was performed to investigate the potential
protective effect of resveratrol (1) and piceatannol (2), against hypoxia-induced oxidative stress in the
H9c2 cardiomyoblast cell line, and the underlying mechanisms. Compounds 1 and 2 significantly
inhibited the release of peroxynitrite and thiobarbituric acid levels at na no- or submicromolar con-
centrations, and this effect was more evident in piceatannol-treated cells, that significantly increased
MnSOD protein level in a concentration dependent manner. Furthermore, since piceatannol, which is far
less abundant in natural sources, displayed a higher bioactivity than the parent compound, we hereby
report on a very fast synthesis and detailed structure-based design of a focused stilbene library. Finally,
taking into account that hypoxia-induced ROS accumulation also increases expression and activity of 5-
lipoxygenase (5-LOX) with production of leukotrienes, we have disclosed structural key factors crucial for
5-LOX activity. Among the synthesized analogues ( 3e7), compound 7 was the most effective in
improving cardiomyocytes viability and in 5-LOX inhibition. In conclusion, modeling and experimental
studies provided the basis for further optimization of stilbene analogues as multi-target inhibitors of the
inflammatory and oxidative pathway.
Received 6 April 2019
Received in revised form
7 July 2019
Accepted 9 July 2019
Available online 10 July 2019
Keywords:
Resveratrol (RS)
Piceatannol (PC)
Pterostilbene (PT) analogues
Cardiomyocytes
ROS
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
Hypoxia-induced oxidative stress and cardiomyocyte apoptosis
are considered as essential processes in the progression of heart
failure. Hypoxia, caused by an imbalance in the demand and supply
of oxygen, often occurs in physiologic conditions such as high
altitude and in pathological conditions including ischemia and
cardiovascular disorders in which the heart is very sensitive due to
* Corresponding author. Consorzio Sannio Tech-AMP Biotec, Appia Str. 7, Apol-
losa, BN, 82030, Italy.
E-mail address: rosanna.filosa@sanniotech.com (R. Filosa).
1
Authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2019.07.033
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

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M. Boccellino et al. / European Journal of Medicinal Chemistry 180 (2019) 637e647
its high oxygen consumption [1,2]. Sufficient supply of oxygen is
essential for the proper functioning of cardiomyocytes [3]. Many
studies indicated that hypoxia has adverse effects on myocardial
cells [4e6], but the mechanism involved in the occurrence of
hypoxia injury is far from being fully understood. Cell hypoxia is
associated with the generation of reactive oxygen species (ROS).
Increased amounts of ROS, generated via NADPH oxidase, mito-
chondrial electron transport, xanthine oxidase, lipoxygenases, and
nitric oxide synthase (NOS) can lead to apoptosis, resulting in lethal
cell injuries [7].
Thus, by focusing on the scavenging of ROS as a promising
therapeutic target for hypoxia injury, antioxidant compounds have
been of great interest as potential therapeutic compounds for
hypoxia-induced oxidative stress. In this regard, nature has always
been a fascinating source of interest to continually discover new
substances [8]. Many antioxidant compounds were isolated from
plant extracts, including polyphenolic derivatives, which were
studied to clarify their mechanism of action [9]. On that basis,
research efforts on the human health effects of natural polyphenols
has grown in the last 50 years [10]. Studies have brought to light the
strong contribution of polyphenols in prevention and treatment of
cardiovascular pathologies, cancer, osteoporosis, diabetes mellitus
and neurodegenerative disorders such as Parkinson's and Alz-
heimer's disease [11e15].
Although polyphenols are mainly of natural origin, it is possible
to develop synthetic substances mimicking natural counterparts
since they can be relatively inexpensive, easily accessible and with
highly reproducible biological properties, including antioxidant
activity [16]. Note that biological activity of polyphenols is deter-
mined by several factors; the most important is related to direct
interaction with receptors and enzymes involved in signal trans-
duction that can modify the redox status of cells and promote a
series of redox-dependent reactions in addition to the possibility of
express the well-known antioxidant and free radicals scavenging
activity, which protect cells against oxidative damage [17,18]. The
main antioxidants are characterized by the presence of phenolic
groups in their chemical structure, particularly, catechol, pyrogallol
or hydroquinone groups [19].
Studies have also suggested that some of the activities of
resveratrol are, in fact, related to its biotransformation into picea-
tannol by the enzyme CYP1B1 of the cytochrome P450 family
[44,45]. Among its many activities, piceatannol, the “less-known
congener” of the famous resveratrol, can help preventing the
development and progression of cardiovascular diseases (CVDs)
through multiple mechanisms such as inhibition of low-density
lipoprotein cholesterol (LDL-c) oxidation, inhibition of platelet ag-
gregation, mediation of cardiac cell function, and reduction of
myocardial tissue damage during ischemic events by anti-
inflammatory activity [46].
During pathologic states such as ischemia or congestive heart
failure, eicosanoids contribute to multiple maladaptive changes
including inflammation, alterations of cellular growth programs,
and activation of multiple transcriptional events leading to the
deleterious sequel of these pathologic states. Myocardial phos-
pholipids serve as primary reservoirs of arachidonic acid (AA),
which is liberated through the rate-determining hydrolytic action
of cardiac phospholipases A2 (PLA2b). AA released by PLA2-cata-
lysed hydrolysis of phospholipids serves as a precursor for eicosa-
noids generated by cyclooxygenase (COX), lipoxygenase (LOX), and
cytochrome P450 (CYP) pathways [47]. The metabolism of AA in the
heart is primarily determined by three cell types (i.e. myocardial,
endothelial, and vascular smooth muscle), which rely on complex
intercellular communication through paracrine signalling to coor-
dinate blood flow, contractile state, and haemodynamic function
[48].
Inflammatory cells such as neutrophils or monocytes can
penetrate acutely into the myocardium following ischemic damage
and produce a wide variety of biologically active oxidized eicosa-
noids. For example, the LOX
products
5-and 12-
hydroxyeicosatetraenoic acid (5-HETE and 12-HETE) levels were
found to dramatically increase in cultured canine myocytes
following 45 min of hypoxia and 5 h re-oxygenation [49]. Similarly,
ischemic rabbit myocardium produced greater amounts of leuko-
triene B4, 5-HETE, and 12-HETE than non-ischemic controls, with
the latter being the major product [50].
The roles of LOXs and their products in myocardial function
remain relatively unexplored, nowadays inhibition of 5-LOX is
known for the treatment of asthma [51], but initial investigations
have revealed an up-regulation of LOX enzymes and/or LOX prod-
ucts following myocardial ischemia/infarction which may
contribute to cardiac hypertrophy, myocyte apoptosis, and fibrosis.
Therefore, the purpose of the current study was to investigate
the effects of resveratrol and piceatannol on H9c2 cardiomyocytes
subjected to hypoxia-induced oxidative stress. To this end, we have
investigated the oxidative balance between ROS release and anti-
oxidant defence, moreover, taking into account that Hypoxia-
induced ROS accumulation also increases expression and activity
of 5-LOX in pulmonary artery endothelial cells with production of
leukotrienes and induction of cell proliferation [49] we have eval-
uated also their potential as direct inhibitors of 5-LOX.
Our groups have long been involved in a variety of drug dis-
covery projects involving painstaking molecular-level syntheses
and modifications as the basic components of research and devel-
opment, using lead structures that may still have suboptimal
structural features requiring modification to fit better to their
respective target(s) [52e58].
To this end, considering piceatannol our lead structure and
further extend our verification of the inhibitory effect, we synthe-
size novel stilbene analogues (Table 1) and evaluated their 5-LOX
inhibitory activity and protective effects on cardiomyocytes
viability vs the natural lead.
Hydroquinones and catechols are very easily converted to qui-
nones by oxidation catalysed by virtually any oxidative enzyme or
metal ions, in this context the biological activities of this class of
compounds can be correlated with the known properties of the
parent compound(s) [20e25].
Recent studies focused their attention on stilbenoids that are
poly-hydroxy natural products with resveratrol representing one of
the highest exponents of the class [26]. Trans-resveratrol (trans-
30,4,50-trihydroxystilbene), is a natural phytoalexin identified for
the first time in the wine grape Vitis vinifera in 1976, and today has
been found in more than 70 plant species. It is often related to so-
called “French paradox”, the phenomenon whereby in France,
despite the high consumption of saturated fatty acids rich food,
drinking of red wine reduces the incidence of mortality for car-
diovascular diseases [27e30]. The biological properties of resvera-
trol are described in detail in several reviews [31e37]. Its beneficial
properties continuously inspired researchers to search new and
more effective analogues [38e41].
Piceatannol (trans-2,30,4’,5-tetrahydroxystilbene, also known as
Astringine), a hydroxy analogue of resveratrol, is known as the least
abundant in nature. It has been identified only in some species:
Melaleuca leucadendron (White tea tree), Cassia garretiana (Asian
legume), Rheum undulatum (Corean rhubarb) and in cell culture
suspension of Vitis vinifera. [42] Having a catechol group, piceatannol
might have more biological activities than the analogous structures.
It is a potent antioxidant and an anti-leukemic substance, shows
anti-allergic effects and induces cellular apoptosis [43].

M. Boccellino et al. / European Journal of Medicinal Chemistry 180 (2019) 637e647
639
Table 1
Structures of resveratrol, piceatannol and stilbene structural analogues 1-7.
ꢁ
ꢁ
Compd.
Pos.3 (¼ R1)
Pos.4 (¼ R2)
Pos.5 (¼ R3)
Pos.3(¼ R4)
Pos.4(¼ R5)
1 (Resveratrol)
2 (Piceatannol)
eOH
eOH
eH
eH
eH
eH
eH
eH
eH
eOH
eOH
eH
eOH
eH
eOH
eOH
eOH
eOCH3
eOCH3
eOH
3
4
5
6
7
eOCH3
eOCH3
eOCH3
eOCH3
eOCH3
eOCH3
eOCH3
eOCH3
eOCH3
eOCH3
eH
eOH
eOCH3
eOH
eOH
2. Results and discussion
2.2. Evaluation of anti-oxidant activity against hypoxia-induced
oxidative stress in H9c2 cells
2.1. Chemistry
Hypoxia-induced oxidative stress and the resulting cell
apoptosis are central in the pathogenesis of heart failure [13e15].
H9c2 cells are rat embryonic myoblast cells and retain several
characters similar to cardiac cells and have been extensively used as
in vitro models to study cardiac ischemia/hypoxia [16e18]. In this
study, we observed that H9c2 cells were highly sensitive to
hypoxia-induced injury in terms of loss of cell viability. In fact, 48 h
of hypoxia reduced H9c2 viability by 30% compared to normoxic
control cells. Eighteen hours pretreatment and co-treatment with
different concentrations of resveratrol (compound 1), ranging from
Methoxylated (3e7) piceatannol and resveratrol structural an-
alogues were synthesized using standard chemical methodologies,
under microwave irradiation by Suzuki-coupling conditions.
Essentially, 1 mmol of the corresponding brominated compound
were added to
a
solution of 1.2 mmol of trans-2-(3,5-
dimethoxyphenyl)-vinylboronic acid pinacol ester (obtained from
a commercial supplier and was of analytical grade) in EtOH in
presence of catalytic amount of [1,10 bis(diphenylphosphino)
ferrocene] dichloropalladium (II) and K2CO3. The reaction mixture
was stirred and irradiated by microwave at 110 ꢀC for 30 min to
yield the desired methoxystilbenes. Finally, demethylation of the
phenolic ethers (6) was successfully established with boron tri-
bromide at room temperature to obtain compound 2 (Scheme 1).
Those procedures afforded all desired compounds in a good yield.
80 nM to 7
Indeed, 20
m
M, significantly recovered cell viability up to 95%.
m
M compound 1 was cytotoxic in both treatment con-
ditions (~80% cell death) (Fig. 1A). In the same way, 18 h pretreat-
ment and co-treatment with different concentrations of
piceatannol (compound 2), ranging from 80 nM to 7
mM, signifi-
cantly recovered cell viability (Fig. 1B). A concentration of 20
mM
compound 2 in both treatment conditions drastically reduced cell
viability (~80%) (Fig. 1B). On the basis of these results, we chose to
perform the next experiments with a pre-treatment protocol to
mimic a condition of prevention care in hypoxia-induced disease,
such as myocardial infarction.
Hypoxia is best defined as an imbalance between supply and
demand for oxygen. In particular, under normal cellular conditions
there is a constant balancing act between the production of ROS
and their degradation by antioxidant systems including enzymes
such as superoxide dismutase, glutathione peroxidase, and cata-
lase. Hearts of mice overexpressing the antioxidant enzyme su-
peroxide dismutase present reduced infarct size associated with
increased superoxide radical removal after an ischemia-reperfusion
event compared with non-transgenic controls [59].
To evaluate the anti-oxidant activity of resveratrol and picea-
tannol in our experimental hypoxic system we measured the
release of peroxynitrite and the levels of lipid peroxidation in car-
diomyocytes exposed to 48 h hypoxia compared to normoxic cells.
Forty-eight hours of hypoxia induced significant release of perox-
ynitrite compared to normoxic control cells. 80 nM and 2 mM pre-
treatment with resveratrol (1) or piceatannol (2) significantly
reduced NO release. This effect was more evident after a pre-
treatment with 80 nM PC (Fig. 2).
The levels of lipid peroxidation were increased in car-
diomyocytes exposed to 48 h hypoxia compared to normoxic cells.
Pre-treatment with 80 nM and 2 mM compound 1 or compound 2
Scheme 1. General synthetic route for synthesis of the resveratrol (1) and piceatannol
(2) analogues 3e7. Reagents and conditions: (a) dppf (0.005 mmol), K2CO3 (2 mmol),
EtOH, 110 ꢀC, 300, MW; (b) BBr3, CH2Cl2, rt.

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M. Boccellino et al. / European Journal of Medicinal Chemistry 180 (2019) 637e647
Fig. 1. Effects of resveratrol (1) and piceatannol (2) in pre-treatment (18 h) and co-treatment on the cell viability of H9c2 cells exposed to hypoxia for 48 h *p < 0.05 vs CTRL;
ꢀp < 0.05 vs 20
M 1 or 2 pre-treatment.
m
Fig. 2. Changes of released peroxynitrite in H9c2 cells under hypoxia treated with
ꢀ
resveratrol (1) or picetannol (2) *p
1e80 nM.
<
0.05 vs HYPOXIA
p < 0.05 vs compound
Fig. 3. Levels of thiobarbituric acid reactive substances (TBARS) in H9c2 cells under
hypoxia exposed to aꢀpre-treatment (18 h) with resveratrol (1) or picetannol (2).
*p < 0.05 vs HYPOXIA p < 0.05 vs 1e80 nM #p < 0.05 vs 1e2 mM.
significantly and in a concentration-dependent manner reduced
this effect (Fig. 3).
Several studies have demonstrated that the induction of MnSOD
expression is related to the mechanism underlying the tolerance to
hypoxia [60,61]. Forty-eight hours of hypoxia significantly induced
a decrease of MnSOD protein expression and 18 h pre-treatment
with
2 mM resveratrol (1) or piceatannol (2) significantly

M. Boccellino et al. / European Journal of Medicinal Chemistry 180 (2019) 637e647
641
Fig. 4. Western blot analysis of MnSOD expression in H9c2 cells under hypoxia
exposed to a pre-treatment (18 h) with resveratrol (1) or picetannol (2) at two con-
centrations (80 nM, 2 microM). *p < 0.05 vs HYPOXIA #p < 0.05 vs resveratrol 2
microM.
Fig. 6. The graph show the percentage of the DCF-DA positive cells after pre-treatment
(18 h) with 2
m
M of compounds 2, 3 and 8 on H9c2 cells and then grown in normoxia
ꢀ
and hypoxia condition.*p < 0.05 vs CTRL Normoxia p < 0.05 vs CTRL Hypoxia.
(MTT) assay [62]. As shown in Fig. 5, the preliminary tests of ana-
logues 3e7 at 80 nM revealed a recovery of cell viability of approx.
25e35% for all tested compounds.
2.4. Evaluation of endogenous ROS after hypoxia-induced oxidative
stress in H9c2 cells of synthetic analogues
Taking into account the interesting protective effect of picea-
tannol (2) we evaluated its ability to prevent reactive oxygen spe-
cies (ROS) generation in cardiomyocytes exposed to hypoxia, in
comparison with pterostilbene (3) and Zileuton (8). As shown in
Fig. 6, a reduction of endogenous ROS formation, assessed using
20,7’ edichlorofluorescin diacetate (DCF-DA) assay, was revealed
only for piceatannol (2) 38% vs 45% detected in hypoxia-induced
cells.
2.5. Evaluation of effect on apoptosis after hypoxia-induced
oxidative stress in H9c2 cells of synthetic analogues
Finally piceatannol (2) pterostilbene (3) and Zileuton (8) were
tested with annexin V FITC to measure early apoptosis occurring
during cytotoxicity in cardiomyocytes exposed to hypoxia. As
shown in Fig. 7, the preliminary evaluation of analogues 2, 3 and
8 at 2 mM revealed a reduction of early apoptosis only for derivate 3
(25% vs 60% detected in hypoxia-induced cells).
2.6. Evaluation of 5-LOX activity and structure-activity
relationships
Analysis of test compounds as 5-LOX inhibitors was routinely
carried out in two different test systems, a cell-free assay using
isolated human recombinant 5-LOX and a cell-based assay using
human neutrophils. The cell-free assay allows identifying com-
pounds that directly interfere with 5-LOX catalytic activity,
whereas the cell-based test system considers cellular regulatory
aspects of 5-LOX product synthesis, and as such offers several
points of attack of a given compound (e.g., inhibition of 5-LOX-
activating protein (FLAP), interference with 5-LOX-activating lipid
hydroperoxides, protein kinases or Ca2þ mobilization, and 5-LOX
translocation/membrane association). The reference 5-LOX inhibi-
tor N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea (zileuton) was
used as the reference drug.
Fig. 5. Effects of a pre-treatment (18 h) with compounds 2e7 on the cell viability of
H9c2 cells at 80 nM (A) and 2 mM (B) exposed to hypoxia. *p < 0.05 vs HYPOXIA
p < 0.05 vs compound 2.
ꢀ
increased MnSOD protein levels compared to the concentration of
80 nM. This effect was more evident with piceatannol (2) treatment
(Fig. 4).
2.3. Evaluation of protective activity against hypoxia-induced
oxidative stress in H9c2 cells of synthetic analogues
All synthetic derivatives were tested in cardiomyocytes exposed
to 48 h hypoxia (Fig. 5). The protective effect of piceatannol ana-
logues against hypoxia-induced cell injury was assessed using 3-
As shown in Table 2, all tested compounds caused significant
inhibition of 5-LOX activity in both cell-based and cell-free assay,
with IC50 values in the low micromolar range. Of note, the
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide

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M. Boccellino et al. / European Journal of Medicinal Chemistry 180 (2019) 637e647
Fig. 7. The graph show the percentage of the Annexin-V positive cells after pre-treatment (18 h) with 2
hypoxia condition. *p < 0.05 vs CTRL Normoxia p < 0.05 vs CTRL Hypoxia.
mM of compounds 2, 3 and 8 on H9c2 cells and then grown in normoxia and
ꢀ
Table 2
Inhibition of 5-LOX activity of compounds 1e7 in a cell-based assay (intact PMNL) and in a cell-free assay.
Compound
Inhibition of 5-LOX activity (IC50 values [mM])
Intact
Cell-free
1
4.9 0.6
63.4 2.1%a
2
0.24 0.09
1.1 0.27
3
4
0.53 0.12
0.76 0.19
3.91 0.65
3.62 0.36
5
5.48 1.99
5.49 0.22
6
7
0.07 0.01
0.05 0.01
0.78 0.22
0.32 0.03
8
Zileuton
1.1 0.40*
0.56 0.1*
Data are given as mean S.E.M., n ¼ 3e4. * [58].
a
Remaining activity at 10 mM (%).
pterostilbene derivative 4 was more potent than resveratrol in both
cell-free and cell-based experiments. Moreover, 30-hydroxypter-
ostilbene 7 (trans-3,5-dimethoxy-30,40-dihydroxystilbene), one of
metabolites of pterostilbene and 30-methoxypterostilbene (trans-
3,5-dimethoxy-30,40-methoxystilbene) 6 were revealed as the most
It is interesting to note that when the 40-OH group is converted
into a methoxy group, the 5-LOX-inhibitory activity of the com-
pounds radically decreased (7 vs 5) but this variation was not so
evident with pterostilbene scaffold derivatives (3 vs 4) Moreover,
the introduction of 3,5-dimethoxy motif at the A-phenyl ring of
resveratrol, improved the 5-LOX inhibitory activity in both cell free
and cell based evaluations (7 vs 2, 3 vs 1). This is in agreement with
potent direct 5-LOX inhibitors with IC50 values of 0.32 and 0.78
mM
in cell-free, and 50 and 70 nM in cell based assays, respectively.

M. Boccellino et al. / European Journal of Medicinal Chemistry 180 (2019) 637e647
643
the recent findings, where pterostilbene, exhibited promising
chemotherapeutic properties, may be due to the higher lip-
ophilicity of pterostilbene scaffold, which may result in better
permeability.
were carried out in 10 mL glass tubes, sealed with aluminium/
Teflon crimp tops, which can be exposed to 300 ꢀC and 30 bar in-
ternal pressure. After the irradiation period, the reaction vessel was
cooled rapidly (60e120s) to ambient temperature by gas jet cool-
ing. Flash chromatography was performed on Carlo Erba silica gel
60 (230e400 mesh; Carlo Erba, Milan, Italy). TLC was carried out
using plates coated with silica gel 60F 254 nm purchased from
Merck (Darmstadt, Germany). 1H and 13C NMR spectra were
registered on a Brucker AC 300. Chemical shifts are reported in
ppm. The abbreviations used are follows s, singlet; d, doublet; dd
double doublet; bs, broad signal. MS spectrometry analysis ESI-MS
was carried out on a Finnigan LCQDeca ion trap instrument.
2.7. Docking studies with 5-LO
All of the seven molecules were docked into the crystal structure
of human 5-LOX (PDB code: 3O8Y) [63]. After docking experiments,
the best and the most energetically favorable pose of each ligand
was selected. The protein-ligand complexes were analyzed to
identify the most conserved interactions. The most active mole-
cules compound 6 and compound 7 in the series (IC50 ¼ 0.07 and
0.05
mM, respectively) are able to make strong interactions through
4.1.1. General procedure for synthesis of compounds 3,4,5,6,7
The synthesis of piceatannol structural analogues was carried
out under microwave irradiation by Suzuky-coupling conditions.
To a solution of trans-2-(3,5-dimethoxyphenyl)-vinylboronic
acid pinacol ester (1.2 mmol) in EtOH abs. (2,5 mL), [1,10 bis(di-
phenylphosphino)ferrocene]dichloropalladium (II) (0.005 mmol),
K2CO3 anhydrous (2 mmol) and the corresponding brominated
compound (1 mmol) were added. The reaction mixture was stirred
and irradiated by microwave at 110 ꢀC for 30 min. Then the reaction
was filtered and concentrated under reduced pressure. The residue
was purified by flash chromatography on silica gel using hexane/
ethyl acetate (from 95:5 to 7:3) as eluent to give the corresponding
derivatives.
hydrogen bonding with the side chains of both His372 and His367
as well as filling the hydrophobic region with 3,5-dimethoxy
groups (Fig. 8F and G). Compounds 2, 3 and 4 with IC50 values in
the range of 0.24e0.76 mM are also positioned in the active site to
establish similar H-bonding interactions with these His residues
through their p-hydroxy or p-methoxy groups (Fig. 8B and C and D).
The docking poses of the least potent compound 1 and compound 5
indicates that these compounds are not oriented appropriately to
form these conserved H-bond interactions, which is in good
agreement with the observed activity loss (IC50 ¼ 4.9 and 5.48
mM,
respectively).
3. Conclusion
4.1.2. 4-(3,5-dimethoxystyryl)phenol (3)
Research and development of stilbene-based medicinal chem-
istry have become rapidly evolving with increasingly active com-
pounds covering almost the whole range of therapeutic fields.
The results of the present work indicated that piceatannol (2)
exerts a profound effect against hypoxia-induced injury for H9c2
cardiomyocytes through suppression of oxidative stress, while,
with a direct evaluation of early apoptotic cells, pterostilbene (3)
demonstrate suppressive effects on apoptosis. Moreover, picea-
tannol and synthetic pterostilbene derivatives showed significant
activity in inhibiting 5-LOX in both cell-based and cell-free assay,
with IC50 values in the low micromolar range. Additionally, the
results indicate that minimal differences in the structure of poly-
methoxystilbenes can substantially affect both the inhibitory 5-LOX
activity and the protection capability over cardiomyocyte injury. In
this context, we highlight fast chemical methodologies for the
synthesis of stilbene derivatives and outline the successful design
of novel stilbene based hybrids as antiinflammatory and antioxi-
dant compounds, that could be of interest in the development of
targeted pharmaceutical approaches for limiting ischemic damage.
This information may be useful in further design of stilbene-based
molecules as new leads for the development of novel agents with
clinical potential or as effective chemical probes. The results pre-
sented in this research cannot be generalized to animal models, but
our new lead stilbene derivatives appear to be highly suitable for a
posterior evaluation to dissect biological processes in the progres-
sion of myocardial tissue damage during ischemia. To this end,
further studies are in progress to confirm the possibility of using
these compounds in human therapies.
Yield: 43%. Rf: 0.4 in 8:2 hexane/ethyl acetate 1H NMR (CDCl3,
300 MHz):
d
: 7.42 (d, 2H); 6.99 (dd, 2H); 6.85 (d, 2H); 6.68 (s, 2H);
6.42 (t, 1H); 3.86 (s, 6H). 13C NMR (CDCl3, 300 MHz):
d
55.4, 99.7,
104.4, 115.7, 126.6, 128.0, 128.7, 130.1, 139.7, 155.4, 160.9 ESI(m/z):
256.3 [Mþ þ 1].
4.1.3. 1-(3,5-dimethoxystyryl)-4-methoxybenzene (4)
Yield: 46%. Rf: 0.4 in 9:1 hexane/ethyl acetate 1H NMR (CDCl3,
300 MHz):
d
: 7.47 (d, 2H); 6.99 (dd, 2H); 6.91 (d, 2H); 6.68 (s, 2H);
6.40 (t, 1H); 3.86 (s, 9H). 13C NMR (CDCl3, 300 MHz):
d
55.4, 99.6,
104.3, 114.1, 126.6, 127.8, 128.7, 129.9, 139.7, 159.4, 160.9 ESI(m/z):
271.6 [Mþ þ 1].
4.1.4. 5-(3,5-dimethoxystyryl)-2-methoxyphenol (5)
Yield: 44%. Rf: 0.6 in 6:4 hexane/ethyl acetate 1H NMR (CDCl3,
300 MHz):
d
: 7.42 (d, 2H); 6.99 (dd, 2H); 6.85 (d, 2H); 6.68 (s, 2H);
6.42 (t, 1H); 3.86 (s, 6H). 13C NMR (CDCl3, 300 MHz):
d
55.4, 56.0,
60.4, 99.7, 104.4, 110.6, 111.9, 119.4, 127.1, 128.8, 130.9, 139.6, 145.8,
146.5, 160.9 ESI(m/z): 309.3 [Mþ þ 23].
4.1.5. 4-(3,5-dimethoxystyryl)-2-methoxyphenol (6)
Yield: 42%. Rf: 0.7 in 7:3 hexane/ethyl acetate 1H NMR (CD3OD,
300 MHz):
d: 7.12 (s, 1H); 6.98 (d, 2H); 6.97 (dd, 2H); 6.79 (d, 2H);
6.36 (t, 1H); 3.89 (s, 3H); 3.79 (s, 3H). 13C NMR (CD3OD, 300 MHz):
d
55.4, 56.0, 60.4, 99.7, 104.4, 110.6, 111.9, 119.4, 127.1, 128.8, 130.9,
139.6, 145.8, 146.5, 160.9 ESI(m/z): 309.3 [Mþ þ 23].
4.1.6. 4-(3,5-dimethoxystyryl)benzene-1,2-diol (7)
Yield: 43%. Rf: 0.6 in 6:4 hexane/ethyl acetate 1H NMR (CD3OD,
4. Experimental section
300 MHz):
6,31 (d,2H); 6.56 (d,1H); 6.63 (s, 1H); 6, 76 (d, 1H); 6,88 (d, 2H). 13
NMR (CD3OD, 300 MHz): 55.9, 56.0, 101.4, 107.2, 114.1, 118.8, 121.7,
d: 3.74 (s, 3H); 3.77 (s, 3H); 5,21 (s,-OH); 6.16 (s, 1H);
C
4.1. Chemistry
d
128.8, 128.9, 131.0, 136.6, 147.1, 147.3, 161.3, 161.9 ESI(m/z): 272.3
All reagents were analytical grade and purchased from
SigmaeAldrich (Milano, Italy). All microwave irradiation experi-
ments were carried out in a Biotage® Initiator þ Microwave syn-
thesizer (Biotage, Sweden AB, Uppsala, Sweden). The reactions
[Mþ þ 23].
4.1.7. General procedure for synthesis of compounds 2
To a solution of 6 (1 mmol) in CH2Cl2 (4 mL), was added

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M. Boccellino et al. / European Journal of Medicinal Chemistry 180 (2019) 637e647
Fig. 8. A, B) Binding mode analysis of Resveratrol (1), Piceatannol (2) and Pterostilbene (3) depicted with interacting 5-LO (PDB code 3O8Y) residues. (D, E, F, G) Binding mode
analysis of synthetic analogues 4e7. Main interactions are represented schematically with VMD 1.9.3. [74]

M. Boccellino et al. / European Journal of Medicinal Chemistry 180 (2019) 637e647
645
dropwise 1 mL of BBr3 (solution 1 M in CH2Cl2) at temperature of
0 ꢀC. The reaction mixture was stirred at room temperature over
night and after was quenched by addition of 4 mL of water at 0 ꢀC.
The resulting mixture was stirred for 30 min at 0 ꢀC. The aqueous
layer was extracted with ethyl acetate and the organic extracts
were combined, washed with brine, dried over Na2SO4 and
concentrated by rotary evaporation. The crude product was purified
by flash chromatography on silica gel using hexane/ethyl acetate
(from 7:3 to 6:4) as eluent to obtain 62% of compound 2.1H NMR
data are in agreement with those reported in literature [35e37].
of each point were used to measure nitrite levels. NO is rapidly
converted into stable end products nitrite and nitrate. Nitrite was
measured by the Griess reaction as reported in literature. Briefly,
100 mL of supernatant was mixed with an equal volume of Griess
reagent (0.5% sulfanilamide, 2.5% H3PO4, and 0.05% naphthyl-
ethylene diamine in H2O) and incubated for 10 min at room tem-
perature. Absorbance was assayed at 550 nm and compared with a
standard curve obtained using sodium nitrite.
4.2.6. TBARS levels
104 cells were seeded in 6-well plates and after 24 h incubation
at 37 ꢀC were washed once and stimulated with resveratrol and
4.2. Biology
piceatannol (80 nM and 2
hypoxic chamber.
m
M) at 37 ꢀC 18 h and then were put in
4.2.1. Cell culture
H9c2 rat cardiomyoblast cell line was purchased from the
American Type Culture Collection (ATCC) and cultured in mixed
growth medium (Dulbecco's modified Eagle's medium (DMEM)
(Hyclone)) supplemented with 10% heat-inactivated FBS (Hyclone).
Cells were kept in an incubator in an atmosphere of 5% CO2 and 95%
air at 37 ꢀC and passaged at 1:3 ratio when they reached 80%
confluence.
At the end of the hypoxic stress cells were washed two times
with cold PBS before lysis using 4 vol of modified RIPA buffer
(50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.25% Na deoxycholate,
2% Triton-X100, 1 mM PMSF, 2 mM leupeptin). The protein samples
were incubated with 0.5 ml of 20% acetic acid (pH 3.5) and 0.5 ml of
0.78% aqueous thiobarbituric acid solution. The mixture was heated
to 95 ꢀC for 45 min and centrifuged at 1600ꢁg for 5 min. To quantify
the amount of TBARS in the supernatant fractions, spectropho-
tometry was performed at an absorbance of 532 nm [27] and data
were expressed as TBARS/serum protein in mM/mg. Data are pre-
sented as the average of triplicate measurements from duplicate
experiments.
4.2.2. Hypoxic stress
For hypoxic stress 104 cells were seeded in 96-well plates and
after 24 h incubation at 37 ꢀC were washed once and stimulated
with resveratrol and piceatannol at various concentrations (from
80 nM to 20 m
M) at 37 ꢀC both 18 h before the hypoxic stress that
just before. After incubation, the cells were put in a humidified
chamber sealed (Billups-Rothenburg, Del Mar, California) supplied
with 5% carbon monoxide and 95% nitrogen for 48 h. Finally, MTT
assay to test cell viability was performed. Using the same time
points of treatment, before and just before the hypoxic stress, H9c2
cells were also treated with synthetic piceatannol derivatives (de-
4.2.7. Western blot analysis
106 H9c2 cells were lysed by incubation on ice for 30 min with
RIPA lysis buffer and 10 mL/mL leupeptin, 5 mL/mL aprotinin, 1 mmol/
L pepstatin, and 10 mmol/L DTT were added before use. After
centrifugation, the supernatant was collected, and the protein
concentration was measured using a commercial kit (Bio-Rad
Laboratories, Milan, Italy) according to the manufacturer's in-
rivatives 3, 4, 5, 6 and 7 at 80 nM and 20 mM).
structions. 80 mg of protein samples were run on 12% SDSePAGE
4.2.3. Cell proliferation assay
gels and then transferred to polyvinylidene difluoride (PVDF)
membranes (Bio-Rad Laboratories, Milan, Italy). Membranes were
blocked for 1 h at room temperature with 5% milk in tris buffer
saline (TBS) with 0.1% Tween 20 (T-TBS), followed by incubation at
4 ꢀC overnight with primary antibodies against manganese super-
oxide dismutase (MnSOD) (Merck Millipore, Germany) Membranes
were then washed three times with 0.1% T-TBS solution, and
incubated for 1 h at room temperature with a secondary antibody
goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP respectively,
according to the primary antibodies data sheet (Santa Cruz
Biotechnology, Milan, Italy). GAPDH antibody (Santa Cruz
Biotechnology, Milan, Italy) was used as an internal standard. The
immunoreactive bands were visualized using an enhanced chem-
iluminescence system (SuperSignal West Femto Maximum Sensi-
tivity Substrate, Pierce, Rockford, USA). The protein bands were
scanned and quantitated with Gel Doc-2000 (Bio-Rad, Milan, Italy).
Cell proliferation was determined by 3-(4,5-Dimethyl-thiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Van Meerloo J).
H9c2 cells (3 ꢁ 104 cells/mL) were seeded into the wells of 96-well
culture plates as described above. MTT solution at 10% was added to
each well and incubated for about 2 h. Then, excess medium was
removed and 100 mL of a solution of 1 N hydrochloric acid at 10% in
isopropanol to dissolve the formazan crystals. The mixture was
shaken for about 20 min and the optical density in each well was
measured using
a microplate spectrophotometer (Microplate
Reader Model 550, BIO-RAD, California, USA) at 570 nm. Triplicate
experiments were performed for treatment with each concentra-
tion. The percentage cell viability (%) was calculated by comparison
with a sample's corresponding control.
4.2.4. DCF-DA assay
The generation of reactive oxygen species (ROS) was monitored
by the conversion of fluorogenic 20,70-dichlorodihydrofluorescein
4.2.8. Apoptosis assay
diacetate
(DCFH-DA)
to
highly
fluorescent
dichloro-
Apoptotic cells were detected using fluorescein-conjugated
annexin V with a Nexin® kit on a Guava® easyCyte™ (Millipore
Sigma, Burlington, MA, USA) flow cytometer following the manu-
facturer's instructions. Samples were acquired on a Guava® easy-
Cyte™ flow cytometer (Millipore, Sigma) and analysed using
easyCyte™ software.
fluoresceindiacetate within cells by ROS.
The cells cultures were incubated for 30 min at RT with 2 mM
DCFH-DA in PBS. Cells were washed with PBS acquired on a Guava®
easyCyte™ flow cytometer (Millipore, Sigma) and analysed using
easyCyte™ software.
4.2.5. Griess test
4.2.9. Evaluation of 5-LOX activity
104 cells were seeded in 6-well plates and after 24 h incubation
at 37 ꢀC were washed once and stimulated with resveratrol and
4.2.9.1. Determination of 5-LOX product formation in cell-based as-
says. Polymorphonuclear leukocytes (PMNL) were isolated from
human blood of adult healthy volunteers, with consent, obtained
from the Institute of Transfusion Medicine, University Hospital
piceatannol (80 nM and 2
hypoxic chamber. At the end of the hypoxic stress the supernatants
m
M) at 37 ꢀC 18 h and then were put in

646
M. Boccellino et al. / European Journal of Medicinal Chemistry 180 (2019) 637e647
Jena, as described [64]. PMNL (5 ꢁ 106 in 1 ml PBS plus 1 mg/ml
glucose and 1 mM CaCl2) were preincubated with the test com-
pounds (15 min, 37 ꢀC) and 5-LOX product formation was started by
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addition of 2.5 m
M A23187 [65]. After 10 min at 37 ꢀC, the reaction
was stopped with 1 ml of methanol. Formed 5-LOX metabolites
were extracted and analyzed by HPLC as described [66]. 5-LOX
product formation is expressed as ng of 5-LOX products per
106 cells, which includes LTB4 and its all-trans isomers, 5(S),12(S)-
di-hydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid (5(S),12(S)-
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E. coli, and determination of5-LOX activity in cell-free systems.
E.coli BL21 was transformed with pT3-5LO plasmid, human re-
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described [67], and immediately used for 5-LOX activity assays. 5-
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LOX (0.5
mg) was diluted with PBS/EDTA and pre-incubated with
test compounds. After 15 min at 4 ꢀC, samples were pre-warmed for
30 s at 37 ꢀC, and 2 mM CaCl2 plus 20
mM arachidonic acid were
added to start 5-LOX product formation. After 10 min at 37 ꢀC
formed metabolites were analyzed by HPLC as described for intact
cells above.
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€
Wizard [70] protocol of the Schrodinger Suite 2017-1for force field
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protonation states and atom types of the molecule with OPLS2005
forcefield [72]. The grid generation and docking runs were done in
single precision mode (GlideScore SP) by Glide 7.4. [73] The best
ranking pose was visualized with VMD 1.9.3. [74].
Acknowledgements
This work was supported by the following grants PON 01 01226
“Dal nutraceutico al farmaco per strategie integrate- Nutrafast, and
POR Campania FSE 2007-2013, Assi IV e V Progetto “Strategie Ter-
apeutiche Innovative” (STRAIN).
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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