Exploring the 2′-hydroxy-chalcone framework for the development of dual antioxidant and soybean lipoxygenase inhibitory agents

Article abstract of DOI:10.3390/molecules26092777

2^′-hydroxy-chalcones are naturally occurring compounds with a wide array of bioactiv-ity. In an effort to delineate the structural features that favor antioxidant and lipoxygenase (LOX) inhibitory activity, the design, synthesis, and bioactivity profile of a series of 2^′-hydroxy-chalcones bearing diverse substituents on rings A and B, are presented. Among all the synthesized derivatives, chalcone 4b, bearing two hydroxyl substituents on ring B, was found to possess the best combined activity (82.4% DPPH radical scavenging ability, 82.3% inhibition of lipid peroxidation, and satisfac-tory LOX inhibition value (IC₅₀ = 70 μM). Chalcone 3c, possessing a methoxymethylene substituent on ring A, and three methoxy groups on ring B, exhibited the most promising LOX inhibitory activity (IC₅₀ = 45 μM). A combination of in silico techniques were utilized in an effort to explore the crucial binding characteristics of the most active compound 3c and its analogue 3b, to LOX. A common H-bond interaction pattern, orienting the hydroxyl and carbonyl groups of the aromatic ring A towards Asp768 and Asn128, respectively, was observed. Regarding the analogue 3c, the bulky (-OMOM) group does not seem to participate in a direct binding, but it induces an orientation capable to form H-bonds between the methoxy groups of the aromatic ring B with Trp130 and Gly247.

Full text of DOI:10.3390/molecules26092777

molecules
Article
0
Exploring the 2 -Hydroxy-Chalcone Framework for the
Development of Dual Antioxidant and Soybean Lipoxygenase
Inhibitory Agents
1
1
1
1
Ioanna Kostopoulou , Andromachi Tzani , Nestor-Ioannis Polyzos , Maria-Anna Karadendrou ,
Eftichia Kritsi 2 , Eleni Pontiki , Thalia Liargkova , Dimitra Hadjipavlou-Litina
,3
4
4
4
,
Panagiotis Zoumpoulakis 2
,3
and Anastasia Detsi *
1,
1
Laboratory of Organic Chemistry, Department of Chemical Sciences, School of Chemical Engineering,
National Technical University of Athens, Heroon Polytechniou 9, Zografou Campus, 15780 Athens, Greece;
ioanna.th.kostopoulou@gmail.com (I.K.); andromachi.tzani@gmail.com (A.T.);
nestor_polyzos@hotmail.com (N.-I.P.); markarad96@gmail.com (M.-A.K.)
Institute of Chemical Biology, National Hellenic Research Foundation, 48, Vas. Constantinou Avenue,
2
1
1635 Athens, Greece; ekritsi@eie.gr (E.K.); pzoump@uniwa.gr (P.Z.)
3
4
Department of Food Science and Technology, University of West Attica, Ag. Spyridonos, 12243 Egaleo, Greece
Laboratory of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences, Aristotle University
of Thessaloniki, 54124 Thessaloniki, Greece; epontiki@pharm.auth.gr (E.P.); thalialiargkova@yahoo.gr (T.L.);
hadjipav@pharm.auth.gr (D.H.-L.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
*
Correspondence: adetsi@chemeng.ntua.gr; Tel.: +30-210-7724126
ꢀ
ꢁꢂꢃꢄꢅꢆ
0
Citation: Kostopoulou, I.; Tzani, A.;
Polyzos, N.-I.; Karadendrou, M.-A.;
Kritsi, E.; Pontiki, E.; Liargkova, T.;
Hadjipavlou-Litina, D.;
Abstract: 2 -hydroxy-chalcones are naturally occurring compounds with a wide array of bioactiv-
ity. In an effort to delineate the structural features that favor antioxidant and lipoxygenase (LOX)
inhibitory activity, the design, synthesis, and bioactivity profile of a series of 2 -hydroxy-chalcones
bearing diverse substituents on rings A and B, are presented. Among all the synthesized derivatives,
chalcone 4b, bearing two hydroxyl substituents on ring B, was found to possess the best combined
activity (82.4% DPPH radical scavenging ability, 82.3% inhibition of lipid peroxidation, and satisfac-
0
Zoumpoulakis, P.; Detsi, A. Exploring
0
the 2 -Hydroxy-Chalcone Framework
for the Development of Dual
Antioxidant and Soybean
tory LOX inhibition value (IC50 = 70
on ring A, and three methoxy groups on ring B, exhibited the most promising LOX inhibitory activity
IC50 = 45 M). A combination of in silico techniques were utilized in an effort to explore the crucial
µM). Chalcone 3c, possessing a methoxymethylene substituent
Lipoxygenase Inhibitory Agents.
Molecules 2021, 26, 2777. https://
doi.org/10.3390/molecules26092777
(
µ
binding characteristics of the most active compound 3c and its analogue 3b, to LOX. A common
H-bond interaction pattern, orienting the hydroxyl and carbonyl groups of the aromatic ring A
towards Asp768 and Asn128, respectively, was observed. Regarding the analogue 3c, the bulky
Academic Editor: Luisella Verotta
(
-OMOM) group does not seem to participate in a direct binding, but it induces an orientation capable
Received: 24 January 2021
Accepted: 30 April 2021
Published: 8 May 2021
to form H-bonds between the methoxy groups of the aromatic ring B with Trp130 and Gly247.
Keywords: chalcones; aurones; butein; sulfuretin; antioxidant activity; lipoxygenase inhibition;
molecular docking
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Chalcones (1,3-diaryl-2-propen-1-ones) (Figure 1) constitute a large and important
group of bioactive compounds belonging to the flavonoid family. They possess an impor-
tant role in medicinal chemistry since they are privileged scaffolds used as lead compounds
for the discovery of new drugs. Thus, the isolation from natural sources (plants, flowers,
fruits, vegetables etc.), as well as the organic synthesis of chalcones have been intensified
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
during the last decades due to their wide spectrum of bioactivity. The presence of a
unsaturated carbonyl system in chalcone derivatives enable them to exhibit a wide range
of biological activities such as antioxidant, anti-inflammatory, [ ], anticancer [ ], antibacte-
rial [ ], antimalarial [ ], tyrosinase inhibitory activity [ ], anti-HIV [ ], anti-leishmanial [ ],
while they are also potential neuroprotective agents [ ]. The chalcone derivatives which
α,β-
1
2
3
4
5
6
7
8,9
4
.0/).
Molecules 2021, 26, 2777. https://doi.org/10.3390/molecules26092777
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2777
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possess hydroxyl and methoxy substitution on aromatic rings A and B have demonstrated
potential anti-leishmanial and anti-trypanosomal activities by acting on a number of molec-
ular targets. Moreover, free hydroxyl groups are essential to increase their activity (such as
antioxidant and antimicrobial) while methoxy groups decrease it. Regarding antitumor
activity, it is possibly positively affected in the presence of methoxy substitutions on the
aromatic ring [10].
Figure 1. General structure and numbering scheme of the chalcone and aurone framework.
Another flavonoid subclass, aurones ((Z)-2-benzylidenebenzofuran-3-(2H)-ones)
(
Figure 1) are oxidative cyclization products of chalcones that rarely appear in nature
and are less studied than chalcones. So far aurones have been exploited via isolation from
flowering plants [11 12], invasive weeds [13], mosses [14], and marine brown algae [15], via
organic synthesis [16 17] and biosynthesis from chalcone precursors by the key enzyme au-
,
,
reusidin synthase [18]. Sulfuretin [19] and aureusidin [20] consist representative naturally
occurring aurones, bearing different hydroxylation patterns, while some methoxy-aurones
also occur in nature [21,22]. Aurones constitute promising bioactive compounds exhibiting
among others, antioxidant [17], antifungal [23], anticancer [24], antimicrobial [25], anti-
leishmanial [16], antimalarial [26], anti-inflammatory activity [27], and consist potential
effective agents for the treatment of Alzheimer’s disease [28]. Consequently, during the
last decades, there is an increase in publications concerning their isolation, synthesis, and
bioactivity evaluation.
In this work, among the synthesized compounds the synthesis of two naturally
occurring chalcones and aurones, such as butein (4a) and sulfuretin (7a), is also presented.
0
0
Butein (2 ,4 ,3,4-tetrahydroxy-chalcone) occurs in many unrelated genera including
Butea Dahlia, Coreopsis, and Searsia and has been isolated in good yields [29]. Chemically,
butein has been synthesized through various organic synthetic methods including mi-
crowave irradiation in the presence of PdCl (PPh ) , ammonium formate in THF [30], aldol
2
3 2
condensation catalyzed by the novel acid catalyst thionyl chloride (SOCl )/ethyl alcohol
2
(EtOH) [31], Claisen–Schmidt condensation between protected 2,4-dihydroxyacetophenone
and 3,4-dihydroxybenzaldehyde in the presence of basic catalyst, and subsequent removal
of the protecting groups [30,32].
0
0
Among naturally occurring aurones, sulfuretin (6,3 ,4 -trihydroxyaurone) is known to
have anti-inflammatory effects and has been isolated among others from the heartwood of
Rhus verniciflua, Bidens, Cosmos, and Dahlia flowers [33,34]. Furthermore, the total synthesis
of sulfuretin has been exploited by scientists starting by the chemical synthesis of a 3-
coumaranone, obtained by gold(I)-catalyzed oxidative cyclization of an o-ethynylanisole
and its condensation with 3,4-dimethoxybenzaldehyde in basic conditions, followed by
demethylation with BBr [35]. To our knowledge, the synthesis of sulfuretin has not
3
been previously achieved using the oxidative cyclization of the OMOM protected butein
precursor.
Natural and synthetic chalcones have shown to possess strong radical scavenging
0
0
potential, while some of them, such as 2,3,4,6-tetrahydroxychalcone and butein (2 ,4 ,3,4-
tetrahydroxy-chalcone), exhibit more potent radical-scavenging activity than reference
antioxidants such as vitamin C or α-tocopherol [36,37]. It is considered that hydroxyl

Molecules 2021, 26, 2777
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groups consist key moieties which can enhance the antioxidant activity of chalcones [23,38].
Aurones have been less studied than chalcones concerning their antioxidant and anti-
inflammatory activity whereas their ability to inhibit soybean lipoxygenase (LOX) and
histone deacetylase (HDAC) have been recently reported [27,39–41].
The combined anti-inflammatory and antioxidant activity of novel drugs could be an
asset for the treatment of several diseases and several natural and synthetic antioxidants
have been shown to possess potent anti-inflammatory properties [42,43]. In this framework,
and as a continuation of our studies towards the synthesis and biological evaluation of
novel bioactive compounds [16
,
17
,
44
,
45], we present herein the synthesis and structure
0
identification of synthetic and naturally occurring 2 -hydroxy-chalcones and selected au-
0
rones. Among the 2 -hydroxy-chalcones series (3a
–3l, 4b, and 5) and aurones (6a–6b and
7a–
7b), the chalcones 3c
,
3h
,
4b
,
5
and the aurone 6a are presented, to our knowledge, for
the first time in the literature. The novel chalcones were prepared via Claisen–Schmidt
condensation in alkaline media (Method A: 20% w/v KOH, EtOH, Method B: 60% w/w
NaH, DMF). Chalcones 3f
condensation reaction between appropriately substituted 2 -hydroxy-acetophenones and
benzaldehydes carried out using aqueous KOH in ethanol, as previously reported in the
–
3g and 3i
–
3l, were prepared through the Claisen–Schmidt
0
literature [41
,46–49]. Aurones 6b and 7b were synthesized via oxidative cyclization of
the corresponding chalcones using mercury (II) acetate in pyridine followed by depro-
tection of the methoxylated aurone using BBr in CH Cl , respectively. The synthesis of
3
2
2
aurones 6b and 7b has been previously presented [50,51] via Knoevenagel condensation of
benzofuranone with the appropriate benzaldehyde using polyphosphoric acid (PPA).
The synthesized compounds were evaluated in vitro for their antioxidant activity
as well as their ability to inhibit soybean LOX. Structure/activity relationship and in
silico studies were undertaken, attempting to discover the structural characteristics of
compounds 3b and 3c that contribute to their LOX inhibitory activity.
2. Results and Dicussion
2.1. Chemistry
First, 2-Hydroxy-4-(methoxymethoxy)-acetophenone 1a and 3,4-bis(methoxymethoxy)-
benzaldehyde 2a were synthesized from the corresponding hydroxy-compounds using
0
MOM-Cl and K CO in acetone. Further, 2 -hydroxy-acetophenones 1b
–1e and benzalde-
2
3
hydes 2b–2h were commercially available.
Chalcones 3a 3l were obtained in 22–85% yields via Claisen–Schmidt condensation of
-hydroxy-acetophenones 1a
basic conditions (Scheme 1).
–
2
–1e with appropriately substituted benzaldehydes 2a–2h in
0
Scheme 1. Synthetic procedure of 2 -hydroxychalcones 3a–3l.

Molecules 2021, 26, 2777
4 of 24
The hydroxylated chalcones 4a and 4b were prepared by deprotection of the corre-
sponding chalcones 3a and 3h, respectively, after removal of the methoxymethyl-protecting
group, using 10% aqueous HCl in methanol. Chalcone
5 was synthesized by Fischer
esterification of the corresponding chalcone 3k in methanol, using H SO as a catalyst
2
4
(
Scheme 2).
0
Scheme 2. Synthetic procedure of 2 -hydroxychalcones 4a, 4b, and 5.
Furthermore, aurones 6a and 6b were obtained in moderate yields (60 and 74% re-
spectively) via the oxidative cyclization of the corresponding chalcones 3a and 3b, using
mercury(II) acetate in pyridine as has been described in detail in one of our previous
publications [17]. Aurone 7a was prepared by deprotection of the corresponding aurone
6a
, by removing the methoxymethyl-protecting group using 10% aqueous HCl in MeOH,
whereas aurone 7b was prepared by deprotection of the methoxylated aurone 6b using
BBr in CH Cl as the deprotecting agent (Scheme 3).
3
2
2
Scheme 3. Synthetic procedure of aurones 6a, 6b, sulfuretin (7a) and 7b.

Molecules 2021, 26, 2777
5 of 24
1
The structural characterization of the synthesized compounds was confirmed by H-
and 13C-NMR, MS/ESI (+/
−
), and HRMS spectroscopy (for those compounds that have
not been reported in the literature).
1
0
The H-NMR spectra of 2 -hydroxy-chalcones are characterized by a signal at a low
0
field (
≈
13 ppm) attributed to the proton of the 2 -OH group which is deshielded as a
result of taking part in a strong intramolecular hydrogen bond with the neighboring
carbonyl group, and the pair of AB doublets with characteristic J values of 15.0–17.0 Hz,
1
confirming the E-geometry of the double bond. In the H-NMR spectra of chalcones
possessing methoxy group on position 2 (chalcones 3b
shift of the AB doublets is observed for the vinyl protons which appear at 8.01–8.21 ppm
for Hb) and 7.54–7.92 ppm (for Ha) as compared to the same signals in the spectra of the
chalcones (3a 3d 3f 3i 3k 3l 4a 4b) without substitution at position 2 (7.68–8.05 ppm
, 3c, 3e), a significant downfield
(
–
,
–
,
,
,
,
for Hb and 7.45–7.87 ppm for Ha, respectively). This can be attributed to the ‘ortho’
1
effect of the methoxy group. In the H-NMR spectra of the synthesized aurones, the most
characteristic peak is the singlet owed to the exocyclic vinylic proton at
δ 6.78–7.50 ppm for
0
the substituted on ring A. The presence of a substituent on position 2 in aurones 6b and 7b
causes a downfield shift of the vinylic proton signal at 7.78 ppm and 7.74 ppm, respectively.
This difference in chemical shifts is also attributed to the ‘ortho’ effect [17].
2.2. In Silico Determination of Lipophilicity as ClogP
Lipophilicity is an important physicochemical property related to the biological ac-
tivity and ADME properties. We theoretically calculated the lipophilicity values of all
compounds as ClogP using the CLOGP Program of Biobyte Corp. [52].
2.3. Bioactivity Assessment
In this work, we evaluated in vitro the bioactivity of a series of chalcones and aurones
that were expected to offer protection against inflammation and free radical attack.
Free radicals, which are generated during the biochemical function of all aerobic
organisms, are very reactive species that can attack and damage biological targets such
as lipids and DNA. Normally, the endogenous antioxidant mechanisms of the organisms
(redox enzymes and small molecules such as vitamins) scavenge the free radicals, however
these mechanisms are not effective enough when excessive free radical production occurs.
In this case, exogenous antioxidants have to be used in order to prevent damage caused by
oxidative stress. Antioxidants are defined as substances that even at low concentration,
significantly delay or prevent oxidation of easily oxidizable substrates [53,54].
There is an increased interest of using antioxidants for medicinal purposes in the
recent years, as it is widely accepted that free radicals play a crucial role in many patho-
logical conditions such as inflammation, cancer, and neurodegenerative disorders [55–58].
Moreover, several nonsteroidal anti-inflammatory agents have been reported to act ei-
ther as inhibitors of free radical production or as radical scavengers [59]. Consequently,
compounds possessing antioxidant properties could be expected to offer protection in
inflammation and to lead to potentially effective drugs.
In order to estimate the antioxidant activity of a compound, different experimental
approaches are used. In this way, factors such as solubility or steric hindrance, which may
be of overriding importance in one environment but not in another, can be varied and the
antioxidant ability of a compound in a variety of milieus may be evaluated. Most of them
require a spectrophotometric measurement and a certain reaction time in order to obtain
reproducible results [60].
In this context, the in vitro antioxidant capacity of the synthesized chalcones and
aurones (Table 1) was monitored in terms of: (a) the interaction with the stable free radical
2
,2-Diphenyl-1-picrylhydrazyl (DPPH); (b) the anti-lipid peroxidation activity; (c) the
hydroxyl radical scavenging ability; and (d) the 2,2-azino-bis(3-ethylbenzothiazoline-6-
•
+
sulfonic acid (ABTS) radical cation (ABTS ) reduction-decolorization ability.

Molecules 2021, 26, 2777
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Table 1. Substituents of all the synthesized chalcone and aurone derivatives.
Generic Chalcone Structure
Compound
R1
H
H
H
H
H
H
Br
H
Br
H
H
H
H
H
H
R2
R3
H
R4
H
R5
OMOM
H
R6
OMOM
OCH3
OCH3
OCH3
H
R7
3
a
OMOM
H
OCH3
OCH3
H
3
b
H
OMOM
H
H
OCH3
OCH3
H
3
c
H
H
3
d
Cl
Cl
Br
Br
Br
Br
H
OCH3
OCH3
OCH3
OCH3
OMOM
OMOM
H
3
e
H
OCH3
H
H
3
f
H
OCH3
OCH3
OMOM
OMOM
COOH
COOH
H
H
3
g
H
H
H
3
h
H
H
H
3
i
j
H
H
H
3
H
H
H
3
k
H
Cl
H
H
H
H
3
l
H
H
H
H
4
a
OH
H
H
H
OH
OH
H
4
b
Br
H
H
OH
OH
H
5
H
H
H
COOCH3
H
Generic Aurone Structure
Compound
R1
H
H
H
H
R2
H
H
H
H
R3
OMOM
H
R4
H
R5
OMOM
H
R6
OMOM
OCH3
OH
R7
H
6
a
6
b
OCH3
H
OCH3
H
7
a
OH
OH
7
b
H
OH
H
OH
OH
The first assay refers to the interaction of the compounds with the stable free radical
DPPH and it is a fast, simple, cost-effective, and widely used method, where the DPPH rad-
ical reacts directly with the antioxidant and is decolorized. This interaction indicates their
radical scavenging ability in an iron-free system. The scavenging effect of the synthesized
analogues on the DPPH radical was evaluated according to the methods of Hadjipavlou
et al. [61].
The second technique is based on the ability of the compounds to inhibit lipid peroxi-
0
dation of linoleic acid induced by 2,2 -Azobis(2-amidinopropane) dihydrochloride (AAPH)

Molecules 2021, 26, 2777
7 of 24
radical (Table 2). AAPH-induced linoleic acid oxidation has been developed as a quick and
reliable method for measuring the antioxidant activity. The thermal free radical producer
(
AAPH) generates free radicals in the solution which cause the oxidation of linoleic acid,
and the method is a measure of how effective antioxidants protect against lipid peroxida-
tion in vitro. Oxidation of linoleic acid by AAPH is followed by UV spectrophotometry in
a highly diluted sample [61].
Table 2. In vitro antioxidant evaluation of the synthesized compounds, through the DPPH scavenging ability and the
inhibition of linoleic acid assays, as well as antioxidant activity of selected analogues via their ability to scavenge hydroxyl
radicals and the ABTS radical cation decolorization assay.
Interaction with the Free Radical DPPH (%)
Inhibition of Lipid Peroxidation
·
+·
%
100 µM
HO (%)
1
ABTS
Compound
of Linoleic Acid Induced by
AAPH Radical (%) 100 µM
1
2
00 µM
100 µM
60 min
00 µM
0 min
18.9
no
3a
21.6
no
11.4
67.3
39.4
no
40
no
*
93
4
3b
3c
no
2.3
no
*
3d
no
*
*
3
e
no
no
20.0
no
*
*
3
f
no
no
*
*
3
g
no
no
31.0
65.6
93.0
no
*
*
3h
5.3
6.0
no
*
*
3
i
no
*
*
3
j
no
no
*
*
3k
no
no
no
*
*
3
l
no
no
87.6
24.4
82.3
100
18.5
29.4
53.2
13.4
*
*
*
4a
95.7
82.4
no
95.3
82.0
no
68
100
*
86
no
*
4b
5
6a
no
no
43
no
no
77
*
86
64
71
no
*
6b
no
no
7a
89.0
100
87.0
93.0
91.2
100
93.0
93.6
7b
NDGA
Trolox
92.0
82
93
no: no action under the experimental conditions; *: not tested.
•
Among the reactive oxygen species (ROS), the hydroxyl ( OH) free radical is possibly
the most toxic, reacting with several biological important molecules such as DNA, lipids,
or carbohydrates. Polyunsaturated fatty acids are particularly vulnerable by free radicals.
Iron released from damaged cells is more likely to be readily available to catalyze the
•
generation of OH radicals. Thus, we tried to test the ability of our compounds to scavenge
hydroxyl radicals determining the competition of selected chalcones and aurones with
3+
DMSO for hydroxyl radicals. Hydroxyl radicals were generated using the Fe /ascorbic
acid system and expressed as a percentage inhibition of formaldehyde production in the
presence of each compound at 100 µM.
The ABTS radical cation decolorization assay was used as another antioxidant activity
estimation protocol (Table 2). Oxidation of ABTS with potassium persulfate generates
directly the radical with no involvement of an intermediate radical, which is then reduced
upon adding electron-donating antioxidants.
Furthermore, as an indication of their anti-inflammatory activity, all the molecules
were tested as inhibitors of soybean lipoxygenase (LOX). The enzyme lipoxygenase cat-

Molecules 2021, 26, 2777
8 of 24
alyzes the first two steps in the metabolism of arachidonic acid, which is cleaved from
membrane phospholipids to leukotrienes (LTB4). LTB4 generation is considered to be
important in the pathogenesis of neutrophil-mediated inflammatory diseases. For this
study, the UV absorbance-based soybean LOX assay was used, which is a plant enzyme
with satisfactory homology with the human 5-LOX [61–66].
2.3.1. Antioxidant Activity
The results of the DPPH scavenging ability of all the synthesized compounds (Table 2
)
revealed that, for the active analogues, the result was not time dependent (from 20 to
0 min). In the DPPH assay, the basic chemical reaction involved is the reduction of
6
the DPPH radical by a single electron transfer (ET) from the antioxidant. Particularly
effective antioxidants via this mechanism are the phenoxide anions derived from phenolic
compounds, like catechol and derivatives, such as Nordihydroguaiaretic acid (NDGA).
Chalcone analogues 3a and 3f
seem either to be totally inactive (3f
weak activity (3a 3h); whereas their hydroxylated analogues 4a and 4b exhibit significant
–3i, which possess -OCH or -OMOM substituents at ring B,
3
,
3g, 3i) in scavenging the DPPH radical, or to possess
,
interaction with the radical showing DPPH radical scavenging ability (95.7% and 82.4%,
respectively).
The evaluation of the antioxidant activity of aurone derivatives via the DPPH method
shows that aurones 6a and 6b exhibit no interaction with the radical, whereas their hydrox-
ylated analogues, sulfuretin (7a) and aurone 7b, are potent DPPH scavengers with 89%
and 100% inhibition, respectively. NDGA was used as a reference compound (Table 2).
0
All the synthesized chalcones possess a phenolic hydroxyl group at position 2 of the
aromatic ring A. However, this OH cannot effectively interact with the DPPH radical as it
is involved in a strong hydrogen bond with the adjacent carbonyl oxygen. The antioxidant
0
activity of 2 -hydroxy-chalcones arises mainly from the other substituents on rings A and
B, as was also observed in our previous work regarding quinolinone-chalcone hybrids [67].
Furthermore, it seems that the presence of bromo-substituent in chalcone derivatives,
in combination with the presence of hydroxyl groups, enhances the antioxidant activity of
the compounds. The chalcones 4a and 4b exhibit high antioxidant activity regarding the
DPPH assay (95.7% and 82.0%, respectively), while the 4b derivative, which possesses one
0
bromo-substituent at position 5 of the aromatic ring A, shows significantly higher lipid
peroxidation inhibitory activity (82.3%), than the 4a analogue, which is almost inactive
(
3
24.4%). The insertion of the Br substituent renders the chalcone 4b more lipophilic (ClogP
.14) than chalcone 4a (ClogP 1.65) and this can possibly explain the enhanced lipid
peroxidation inhibitory activity of 4b. Aurones 6a and 6b did not show any DPPH radical
scavenging ability, exactly as their chalcone precursors 3a and 3b.
Sulfuretin (7a) showed potent DPPH scavenging activity (89.0/91.2%) while the 2,4,5-
trihydroxy aurone 7b was one of the most active compounds in this assay, showing 100%
inhibition.
Lipophilicity as ClogP values does not seem to play any significant role in the DPPH
scavenging ability of the compounds (Table 5).
As far as the lipid peroxidation inhibitory ability of the tested chalcones is concerned,
it seems that the presence of bromo-substituents enhances anti-lipid peroxidation. Chal-
cone 3h, which bears two -OMOM substituents at positions 3 and 4 of ring B and 3,4-
0
dihydroxychalcone derivative 4b, which both possess a bromo-substituent at position 5 ,
exhibit satisfactory inhibitory activity of 65.6% and 82.3%, respectively. The introduction
0
of one more bromo-substituent at position 3 , leads to compound 3i, which is strong in-
hibitor of lipid peroxidation (93%). However, chalcones 3f and 3g, which also possess
bromo-substituents, are inactive against lipid peroxidation. This is maybe attributed to the
existence of two methoxy groups at the aromatic ring B, which seem to lead to inactive
inhibitors, as it also happens in the case of chalcones 3d and 3e. Furthermore, according to
Table 2, it seems that the presence of a chloro-substituent at the aromatic ring A of chalcone
moiety leads to inactive molecules. Among all the evaluated chalcones, compound 5 with a

Molecules 2021, 26, 2777
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methoxy-carbonyl substituent at aromatic ring B showed the best inhibitory activity (100%).
The studied aurones exhibited low activity overall, with the exception of aurone 7b which
showed a moderate lipid peroxidation inhibition of 53.2%. With some exceptions anti-lipid
peroxidation goes in parallel to lipophilicity values.
The % values for DPPH scavenging activity and AAPH anti-lipid peroxidation activity
in relation to time, for the most potent compounds are given in Tables 3 and 4. Both
activities seem to be constantly high during the 10 min of observation and measurement.
These results verify the strong antioxidant capacity of the tested compounds, which is
exhibited immediately after their addition to the assay medium (t = 0 min) and rapidly
reaches a plateau at t = 9 min.
Table 3. In vitro % values for DPPH scavenging activity of the most potent compounds in relation
to time.
Interaction with the Free Radical DPPH (%)
Compound
0
min
3 min
95.4
100
6 min
94.9
100
9 min
95.4
100
4
a
95.4
100
4
b
7
a
92.3
98.2
94.4
99.3
94.9
99.5
94.9
99.8
7
b
Table 4. In vitro % values for AAPH anti-lipid peroxidation activity of the most potent compounds
in relation to time.
Inhibition of Lipid Peroxidation of Linoleic Acid Induced by AAPH
Radical (%)
Compound
0
min
3 min
98.6
93.2
82.6
96.9
6 min
96.9
95.7
82.5
92.5
9 min
99.2
94.3
81.5
98.9
3
3
i
l
95.6
87.6
81.8
95.4
4
b
5
Representative compounds were selected for the competition with DMSO for hydroxyl
radicals assay (Table 2). The best activity was shown by chalcone 4b whereas aurone 7b
,
0
0
0
with a 2 ,4 ,5 -trihydroxy-substitution pattern on ring B, follows. The presence of a hydroxyl
substituent at ring A of the aurone scaffold seems to affect this type of activity: the natural
product sulfuretin (7a), with a 5-OH at ring A and a catecholic moiety at ring B was inactive
in this assay. Chalcone 4a, the precursor of sulfuretin (7a) shows 68% competition with
DMSO for hydroxyl radicals. The highly oxygenated aurone 6a presents moderate activity,
equal to its precursor, chalcone 3a (43 and 40%, respectively). Trolox was used as a reference
compound (Table 2).
In the ABTS radical cation (ABTS +) decolorization assay (Table 2), the tested chalcones
•
showed better activity than the corresponding aurones (compare 3a with 6a and 4a with
0 0 0
7a), with the exception of the 2 ,4 ,5 -trimethoxy chalcone 3b and chalcone 4b, which were
both inactive, whereas aurone 6b showed 64% activity. Trolox was used as a reference
compound (Table 2).
2.3.2. Soybean LOX Inhibitory Activity
In the present series of compounds, chalcones were generally found to possess signifi-
cantly higher LOX inhibitory activity than aurones (Table 5). This is in accordance with our
previous study [17].

Molecules 2021, 26, 2777
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Table 5. In vitro determination of soybean inhibition; theoretically calculated lipophilicity as
ClogP values.
Inhibition of Soybean
ClogP $
Compound
Lipoxygenase 100 µM (%) or IC50 µM
(40.2% at 100 µM) #
IC50 = 75 µM
3
a
2.30
3.48
3.08
4.49
4.69
4.64
5.33
3.86
4.54
3.77
4.00
3.42
1.65
3.14
3.39
1.72
2.96
1.97
1.31
3.92
3
b
3
c
IC50 = 45 µM
(32.0% at 100 µM) #
3
d
3
e
IC50 = 100 µM
(28.0% at 100 µM) #
IC50 = 68.5 µM
3
f
3
g
3
h
IC50 = 55.0 µM
3
i
j
IC50 = 67.5 µM
3
IC50 = 100 µM
3
k
IC50 = 67.5 µM
3
l
IC50 = 100 µM
(39.3% at 100 µM) #
4
a
4
b
IC50 = 70.0 µM
5
IC50 = 100 µM
(24.4% at 100 µM) #
(13.0% at 100 µM) #
(24.5% at 100 µM) #
no
6
a
6
b
7
a
7
b
NDGA
IC50 = 0.45 µM
No: no activity under the reported experimental conditions; #: These compounds were less potent therefore
$
no IC50 values were determined for them; : theoretically calculated ClogP values using the C-QSAR Program,
Biobyte [36].
Among all the tested chalcones, the most potent lipoxygenase inhibitor is chalcone
0
3c
(IC50 = 45
µ
M) which possesses one -OMOM group at position 4 of ring A and three
methoxy groups at positions 2, 4, and 5 at ring B. The -OMOM groups seems to play
0
an important role in the LOX inhibitory activity of this series of 2 -hydroxy-chalcones:
Chalcone 3b, which possesses the same methoxy substitution pattern as chalcone 3c but
lacks the -OMOM group at ring A, shows lower activity (IC = 75
µ
M) than 3c. Chalcone
50
0
3a, with a 4 -OMOM and 3,4-di-OMOM substitution is a poor LOX inhibitor (40.2%). The
presence of two -OMOM substituents at positions 3 and 4 of ring B favors LOX inhibitory
activity in the case of chalcones 3h and 3i, which also bear bromine substituents at ring
0
A. In fact, chalcone 3h with a 5 -Br substitution pattern is the second most active LOX
0
inhibitor of the series (IC50 = 55
µ
M) whereas 3i, with an additional Br atom at position 3 ,
shows IC50 = 67.5 µM. NDGA was used as a reference compound (Table 5).
The -OMOM group seems also to be a structural feature that affects activity depending
0
0
on the substituents at ring A: Chalcone 3i, with 3 , 5 -dibromo pattern at ring A and 3,4-
di-OMOM at ring B, is almost equipotent with its 3,4-dimethoxy analogue 3g (IC50 = 67.5
and 68.5
µ
M, respectively). However, chalcone 3h, one of the most active of the series
0
with one bromine substituent at position 5 , is transformed to an inactive compound when
the -OMOM groups are replaced by -OCH (chalcone 3f, 38% inhibition). On the other
3
hand, removal of the -MOM protecting group results to the active 3,4-dihydroxy chalcone
analogue 4b (IC50 = 70 µM).

Molecules 2021, 26, 2777
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Chalcone 3d, the chlorine-containing analogue of 3f, is also inactive but a change of
the position of the methoxy groups at ring B results to a significant improvement in activity:
chalcone 3e with a 2,3-dimethoxy substitution pattern shows IC50 = 100 µM.
The transformation of chalcone 3b to the analogous aurone 6b diminishes the activity,
showing only 13.0% LOX inhibition. The same decrease in inhibition is observed in the
transformation of 4a to 7a and of 3a to 6a.
As a general remark, lower inhibition results are observed by the compounds that
possess lower lipophilicity values.
2.4. In Silico Studies
2.4.1. Molecular Docking Studies
The lack of a co-crystallized ligand in the crystal structure of soybean lipoxygenase
isoform 1 (LOX-1) (PDB ID: 3PZW) imposes the identification of potential allosteric binding
sites apart from the iron-binding site and the substrate-binding cavity. To this extent, three
putative binding sites were identified using SiteMap’s approach [68
is located and mapped at the interface of the amino terminal -barrel (PLAT domain) and
of the -helical domain (houses the catalytic iron) while Sites 2 and 3 belong to the -helical
domain (Figure S1, Supplementary Materials) [71]. Evaluation of Site Score, indicate Site 1
site score 1.113) as a putative allosteric site for further docking studies. Interestingly, Site 1
–70]. In particular, Site
1
β
α
α
(
has been previously identified and utilized for docking studies on LOX-1, as presented in
recent publications [67,72].
Molecular docking was utilized to study the binding mode of the most promising syn-
thesized chalcone 3c and its analogue 3b, which does not possess an -OMOM substituent,
in an effort to provide a structure-activity explanation on soybean LOX-1 inhibitory activity.
Additionally, for comparison reasons, the binding mode of the standard LOX inhibitor
NDGA (IC50 = 0.45
compounds interact with the soybean LOX-1 through allosteric interactions.
Especially, the analysis of the docked poses of the examined compounds 3b IC50 = 75 µM
and 3c (IC50 = 45 M) illustrated a common H-bond interaction pattern as depicted in
µM) was determined. In general, the results suggested that the studied
(
)
µ
Figure 2, orienting the hydroxyl (-OH) and carbonyl (-CO) groups of their aromatic ring
A towards Asp768 and Asn128, respectively. The binding motif of NDGA includes the
formation of H-bonds between the hydroxyl groups (-OH) of the aromatic ring A with
Asp768 and Asn128, highlighting their crucial role in the binding and potentially in the
inhibitory activity. Additionally, the hydroxyl groups (-OH) of the aromatic ring B of
NDGA create a strong hydrogen bond network, including Cys127, Arg182, Arg141, and
Ile142, which is not presented in the examined 3b and 3c. However, in the case of 3c
,
although the bulky (-OMOM) group seems not to participate in a direct binding, it induces
an orientation capable to form H-bonds between the methoxy (-OCH ) groups in positions
3
4
and 5 of the aromatic ring B with Trp130 and Gly247. In the case of 3b only, the methoxy
(
-OCH ) group in position 3 forms a H-bond with Phe144, probably inducing the reduction
3
of the inhibitory activity (Figure 2).
2.4.2. Molecular Dynamics Simulations Studies
In a step further, in an effort to evaluate (a) the stability of the tested complexes, (b) the
0
binding mode, and (c) the influence of -OMOM substitution in position 4 of compound
3c, short length (10 ns) molecular dynamics simulations were implemented in the docking
poses of compounds 3b and 3c. LOX-1 interactions as derived from the MD simulations
with the testing compounds are depicted in Figure 3. The RMSD results indicated that
the structural model backbone of LOX-1, as well as compounds 3b and 3c, remain stable
during the simulation (Figure S2, Supplementary Materials).

Molecules 2021, 26, 2777
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Figure 2. 2D and 3D representative binding poses of (
A) nordihydroguaiaretic acid (NDGA), (B) Com-
pound 3b, and ( ) Compound 3c are depicted. Hydrogen bonds are illustrated with pink (2D)
C
and yellow (3D) color lines. Figure made with MAESTRO (Schrödinger Release 2020-4: Maestro,
Schrödinger, LLC, New York, NY, USA, 2020).
Figure 3. Schematic illustration of LOX-1 interactions throughout the simulation time (10 ns) with (
and ( ) Compound 3c. Interactions that occur more than 20% of the simulation time are presented. Hydrogen bonds are
illustrated with pink color lines. Figure made with Desmond software [66].
A) Compound 3b
B
Both compounds exhibit the interactions presented in docking studies (Figure S3,
Supplementary Materials). Specifically, the MD results indicate that both 3b and 3c preserve
the direct H-bonds of their hydroxyl and carbonyl groups with the side chains of Asp768

Molecules 2021, 26, 2777
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and Asn128 although not in high percentage (Figure 3). In addition, their methoxy (-OCH3)
groups in position 2 interact with the backbone (in the case of 3b) and the side chain (in the
case of 3c) of Asn128, respectively. Moreover, a weaker water-bridged H-bond is formed
between the hydroxyl group of compound 3b and the backbone of Cys127, while the same
interaction is shown in the case of compound 3c with the side chain of Glu244. Regarding
compound 3c, its carbonyl and methoxy (-OCH ) group in position 2 interact through
3
H-bonds with the side chains of Asn769 and Arg533, respectively. In contrast, the described
interactions are not present for compound 3b, offering a putative explanation of the higher
inhibitory activity of compound 3c.
Regarding the interactions of methoxy (-OCH ) groups at positions 4 and 5 of 3b and
3
3c, a different profile is observed in agreement with the docking results. Especially, for
compound 3b, the methoxy (-OCH ) groups share H-bonds with Phe144 backbone while
3
in the case of compound 3c, H-bonds are formed with Gly246 and Trp130.
With regard to the -OMOM chain of compounds 3c, the MD simulations indicates the
formation of a hydrogen bond with Tyr525.
Overall, the binding of 3b and 3c is reinforced by additional hydrophobic interactions.
In detail, compound 3b interacts with Val126, Val520, and Trp772 while compound 3c
develops interactions with Leu246 and Phe108 (Figure S3, Supplementary Materials).
3. Materials and Methods
NMR spectroscopy: Synthesized compounds were structurally elucidated using Var-
ian Gemini 300 MHz (Palo Alto, CA, USA) at the School of Chemical Engineering, NTUA
and Varian 600 MHz (Palo Alto, CA, USA) at the Institute of Chemical Biology, NHRF,
NMR spectrometers using DMSO-d , CDCl 99.9 atom % D and CD OD-d as solvents.
6
3
3
4
1
H-NMR spectra were recorded at 298 K under the following parameters. Spectral width
SW) = 2–14 ppm, number of scans (ns) = 32. Coupling constants (J) are expressed in hertz
Hz). Chemical shifts ( ) of NMR are reported in parts per million (ppm) units relative to
(
(
−
δ
the solvent.
Melting points were determined on a Gallenkamp MFB-595 melting point apparatus
London, UK) and are uncorrected. ESI-MS spectra were recorded on a Varian 500 MS IT
(
Mass Spectrometer (Palo Alto, CA, USA). For the in vitro tests, a Lambda 20 (Perkin–Elmer,
Norwalk, CT, USA) UV–Vis double beam spectrophotometer was used.
3
.1. Synthesis and General Procedures
-hydroxy-4-(methoxymethoxy)-acetophenone (1a) [17]: To a mixture of 2,4-dihydroxy-acetophenone
531.1 mg, 3.5 mmol) and anhydrous K CO (1.9 g, 10.0 mmol) in dry acetone (39 mL)
2
(
2
3
was added dropwise chloromethyl methylether (CH OCH Cl) (0.4 mL, 5.3 mmol). The
3
2
mixture was refluxed for 24 h, cooled to room temperature, filtered, washed with acetone,
and the solvent was evaporated. The resulting orange oil was purified using flash column
chromatography (petroleum ether/ethyl acetate 9:1) to give (1a) as a low melting yellow
1
solid. Yield: 548 mg (80%); H-NMR (300 MHz, CDCl ):
δ (ppm) 12.58 (s, 1H, -OH), 7.65 (d,
3
J = 8.8 Hz, 1H, H-6), 6.60 (d, J = 2.4 Hz, 1H, H-3), 6.56 (dd, J = 8.8, 2.4 Hz, 1H, H-5), 5.22 (s,
H, 2 × -OCH -), 3.50 (s, 3H, 3 × -OCH ), 2.59 (s, 3H, 3 × -CH ).
2
2
3
3
Note: Chloromethyl methylether is a highly toxic compound and should not be
ingested or inhaled. All contact with this material should be avoided. It is a flammable
material, corrosive to eyes and skin on contact. It is a possible carcinogen. It is lachrymator;
Heat and sources of ignition should be avoided.
3
,4-bis(methoxymethoxy)-benzaldehyde (2a) [17]: Prepared following the procedure described
for the synthesis of acetophenone 1a, compound 2a was prepared from 3,4-dihydroxy-
benzaldehyde (500 mg, 3.6 mmol). After evaporation of the solvent, the oily resulting
product was purified using flash column chromatography (petroleum ether/ethyl acetate
8
:2 (2 × 100 mL) and 7:3 (2 × 100 mL) with dry packing to give benzaldehyde 2a as a low
◦
1
melting white solid. Yield: 503.6 mg (62%). mp 61.2–62 C; H-NMR (300 MHz, CDCl ):
3
δ
(ppm) 9.82 (s, 1H, HC = O), 7.64 (d, J = 1.9 Hz, 1H, H-3), 7.47 (dd, J = 8.3, 1.9 Hz, 1H,

Molecules 2021, 26, 2777
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H-2), 7.23 (d, J = 1.0 Hz, 1H, H-6), 5.30 (s, 2H, -OCH -), 5.27 (s, 2H, -OCH -), 3.51 (s, 3H,
2
2
-
COCH ), 3.51 (s, 3H, -COCH ).
3 3
3.1.1. General Procedure for the Synthesis of Chalcones 3a–3l
(1) Method A
To a stirred solution of the appropriate 2-hydroxy-acetophenone (1 eq) and a sub-
stituted benzaldehyde (1 eq) in ethanol is added KOH (20% w/v aqueous solution). The
mixture is refluxed for 4 h or stirred in room temperature for 24 h. The completion of the
◦
reaction is monitored by TLC. At the end of the reaction, the mixture is cooled to 0 C
(ice-water bath) and acidified with HCL (10% v/v aqueous solution). The yellow precipitate
formed is filtered and washed with 10% aqueous HCl solution.
(2) Method B
To a stirred solution of the appropriate 2-hydroxy-acetophenone (1 eq) and a substi-
tuted benzaldehyde (1 eq) in dry dimethylformamide (DMF) is added NaH (60% w/w)
and the mixture is stirred at room temperature for 24 h. The completion of the reaction is
◦
monitored by TLC. At the end of the reaction, the mixture is cooled to 0 C (ice-water bath)
and acidified with HCl (10% v/v aqueous solution). An orange-yellow oil is formed and the
mixture is extracted with diethyl ether; the extracts are dried with Na SO and the solvent
2
4
is evaporated under reduced pressure to give the chalcone as an orange-yellow solid.
Note: NaH dispersed in oil was used for the reaction. Special attention must be paid
when using NaH, as it can ignite in air, especially upon contact with water to release
hydrogen, which is also flammable.
0
0
2
-hydroxy-4 ,3,4 -methoxymethoxy-chalcone (3a): Prepared according to the general procedure
method B) starting from 2-hydroxy-4-(methoxymethoxy)-acetophenone (1a) (418 mg,
.1 mmol) and 3,4-bis(methoxymethoxy)benzaldehyde (2a) (504 mg, 2.1 mmol), dissolved
(
2
in 4.5 mL dry DMF and NaH (60% w/w, 255.43 mg). After the work-up procedure, the
product was obtained upon recrystallization from hexane/ethyl acetate as an orange-yellow
powder. The orange-yellow solid was recrystallized from hexane/ethyl acetate. Yield: 342
◦
◦
1
mg (40%). m.p. 98–100 C (ref. [73] m.p.107.8 C); H-NMR (300 MHz, CDCl ):
1
δ (ppm)
3
0
3.30 (s, 1H, OH), 7.84 (d, J = 8.7 Hz, 1H, H-6 ), 7.81 (d, J = 15.3 Hz, 1H, Hb), 7.43 (m, 2H,
H-5, Ha), 7.29 (m, 1H, H-2), 7.19 (d, J = 8.1 Hz, 1H, H-6), 6.63 (d, J = 2.1 Hz, 1H, H-3 ), 6.59
dd, J = 8.7, 1.8 Hz, 1H, H-5 ), 5.96 (br, 4H, 2
0
0
(
3
×
-OCH O-), 5.22 (s, 2H, -OCH O-), 3.56 (s,
2 2
1
3
H, -CH ), 3.53 (s, 3H, -CH ), 3.50 (s, 3H, -CH ); C-NMR (150 MHz, CDCl3): δ (ppm)
3 3 3
1
1
91.95, 166.13, 163.51, 149.62, 147.42, 144.35, 131.28, 129.19, 124.11, 118.73, 116.18, 116.10,
14.95, 108.116, 103.93, 95.54, 95.10, 94.01, 56.37, 56.35, 56.33.
0
2
-hydroxy-2,4,5-trimethoxy-chalcone (3b): Prepared according to the general procedure
(method A) starting from 2-hydroxy-acetophenone (1b) (500 mg, 3.7 mmol) and 2,4,5-
trimethoxy-benzaldehyde (2b) (721 mg, 3.7 mmol), dissolved in 6 mL ethanol and 3.1 mL
of 20% w/v aqueous solution KOH. The mixture was refluxed for 4 h. After the work-up
procedure, the product was obtained upon recrystallization from hexane/ethyl acetate
◦
as an orange-yellow powder. Yield: 546.3 mg (47%). m.p. 129–130 C; (ref. [74] m.p.
◦
1
1
1
7
7
24–125 C); H-NMR (300 MHz, CDCl3):
δ
(ppm) 13.04 (s, 1H, -OH), 8.22 (d, J = 15.3 Hz,
H, Hb), 7.93 (dd, J = 8.1, 1.5 Hz, 1H, H-6 ), 7.62 (d, J = 15.3 Hz, 1H, Ha), 7.48 (ddd, J = 8.5,
.2, 1.6 Hz, 1H, H-4 ), 7.13 (s, 1H, H-6), 7.02 (dd, J = 8.4, 0.9 Hz, 1H, H-3 ), 6.94 (ddd, J= 8.5,
.2, 1.2 Hz, 1H, H-5 ), 6.54 (s, 1H, H-3), 3.98 (s, 3H, -OCH ), 3.96 (s, 3H, -OCH ), 3.94 (s, 3H,
0
0
0
0
3
3
-
OCH3).
0
0
2
-hydroxy-4 -methoxymethoxy-2,4,5-trimethoxy-chalcone (3c): Prepared according to the gen-
eral procedure (method B) starting from 2-hydroxy-4-(methoxymethoxy)-acetophenone (1a
60 mg, 0.3 mmol) and 2,4,5-trimethoxy-benzaldehyde (2b) (60 mg, 0.3 mmol), dissolved in
.8 mL dry DMF and NaH (60% w/w, 36.67 mg). After the work-up procedure, the residue
was purified using flash column chromatography (petroleum ether/ethyl acetate 7.5:2.5)
)
(
0
◦
1
to give 3c as an orange solid. Yield: 145 mg (39%). mp 86.2–86.8 C; H-NMR (600 MHz,

Molecules 2021, 26, 2777
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CDCl ):
δ
(ppm) 13.51 (s, 1H, -OH), 8.17 (d, J = 15.6 Hz, 1H, Hb), 7.85 (d, J = 8.7 Hz, 1H,
3
0
0
H-6 ), 7.54 (d, J = 15.6 Hz, 1H, Ha), 7.11 (s, 1H, H- 6), 6.63 (d, J= 1.8 Hz, 1H, H-3 ), 6.58 (dd,
J = 8.4, 1.8 Hz, 1H, H-5 ), 6.52 (s, 1H, H-3), 5.22 (s, 2H, -OCH O-), 3.95 (s, 3H, -OCH ), 3.92
0
2
3
13
(
br, 6H, 2
×
-OCH ), 3.49 (s, 3H, -OCH OCH ); C-NMR (150 MHz, DMSO-d6): δ (ppm)
3 2 3
1
1
92.63, 166.22, 163.41, 155.08, 152.93, 143.45, 140.21, 131.34, 118.24, 115.53, 115.33, 112.02,
08.07, 104.09, 96.97, 94.17, 56.77, 56.52, 56.50, 56.23; HR-MS m/z (neg): 373.1291 C H O
2
0
22
4
(
calcd. 374.1366).
0
0
5
-chloro-3,4-dimethoxy-2 -hydroxy-chalcone (3d): Prepared according to the general proce-
dure (method A) starting from 5-chloro-2-hydroxyacetophenone (1c) (500 mg, 2.9 mmol)
and 3,4-dimethoxy-benzaldehyde (2c) (486.9 mg, 2.9 mmol), dissolved in 7.2 mL ethanol
and 2.5 mL of 20% w/v aqueous solution KOH. The mixture was stirred at room temperature
for 24 h. After the work-up procedure, the product was obtained upon recrystallization
◦
from methanol/dichloromethane as a yellow powder. Yield: 630 mg (72%); m.p. 170–172 C
◦
1
(
ref. [73] m.p. 167–168 C]; H-NMR (600 MHz, DMSO-d ):
δ
(ppm) 12.56 (s, 1H, -OH),
.25 (d, J = 2.6 Hz, 1H, H-6 ), 7.87 (d, J = 15.0 Hz, 1H, Hb), 7.81 (d, J = 15.6 Hz, 1H, Ha), 7.57
6
0
8
(
3
1
m, 2H, Ar), 7.47 (dd, J = 7.8, 1.2 Hz, 1H, Ar), 7.04 (m, 2H, Ar), 3.86 (s, 3H, -OCH ), 3.83 (s,
3
H, -OCH3); 13C-NMR (150 MHz, DMSO-d6):
35.45, 129.38, 127.09, 124.43, 122.70, 122.14, 119.71, 119.11, 111.61, 111.15, 55.83, 55.60.
δ (ppm) 192.45, 160.08, 151.76, 148.94, 146.17,
0
0
5
-chloro-2,3-dimethoxy-2 -hydroxy-chalcone (3e) [31]: Prepared according to the general pro-
cedure (method A) starting from 5-chloro-2-hydroxy-acetophenone (1c) (341 mg, 2.0 mmol)
and 2,3-dimethoxy-benzaldehyde (2d) (330 mg, 2.0 mmol), dissolved in 5 mL ethanol and
1.6 mL of 20% w/v aqueous solution of KOH. The mixture was stirred at room temperature
for 24 h. After the work-up procedure, the product was obtained upon recrystallization
◦
from methanol/dichloromethane as a yellow powder. Yield: 390 mg (50%). mp 133–134 C;
1
0
H-NMR (600 MHz, DMSO-d6):
δ
(ppm) 12.18 (s, 1H, -OH), 8.18 (d, J= 2.4 Hz, 1H, H-6 ),
8
7
.06 (d, J = 15.6 Hz, 1H, Hb), 7.75 (d, J = 15.6 Hz, 1H, Ha), 7.66 (dd, J = 7.2, 1.8 Hz, 1H, H-6),
0
0
.57 (dd, J = 9.0, 2.4 Hz, 1H, H-4 ), 7.17 (m, 2H, H-4, H-5), 7.04 (d, J = 9.0 Hz, 1H, H-3 ), 3.85
13
(s, 3H, -OCH ), 3.80 (s, 3H, -OCH ); C-NMR (75 MHz, DMSO-d6): δ (ppm) 192.30, 162.50,
3 3
1
1
55.80, 148.30, 138.60, 135.10, 129.70, 127.80, 125.50, 124.10, 123.60, 122.90, 115.60, 112.40,
12.16, 60.80, 55.58.
0
0
5
-bromo-3,4-dimethoxy-2 -hydroxy-chalcone (3f) [41]: Prepared according to the general pro-
cedure (method A) starting from 5-bromo-2-hydroxy-acetophenone (1d) (500 mg, 2.0 mmol)
and 3,4-dimethoxy-benzaldehyde (2c) (330 mg, 2.0 mmol), dissolved in 5 mL ethanol and
1.6 mL of 20% w/v aqueous solution of KOH. The mixture was stirred at room temperature
for 24 h. After the work-up procedure, the product was obtained upon recrystallization
◦
from methanol/dichloromethane as a yellow powder. Yield: 300 mg (50%). mp 178–179 C;
1
0
H-NMR (600 MHz, DMSO-d6):
δ
(ppm) 12.56 (s, 1H, -OH), 8.33 (s, 1H, H-6 ), 7.86 (d,
J = 15.6 Hz, 1H, Hb), 7.80 (d, J = 15.0 Hz, 1H, Ha), 7.68 (dd, J = 9.0, 2.4 Hz, 1H, H-4 ), 7.55
0
(
6
d, J= 1.8 Hz, 1H, H-2), 7.47 (dd, J = 8.4, 1.8 Hz, 1H, H-6), 7.04 (d, J = 8.4 Hz, 1H, H-5),
0
.97 (d, J = 8.4 Hz, 1H, H-3 ), 3.86 (s, 3H, -OCH ), 3.83 (s, 3H, -OCH ); C-NMR (75 MHz,
3 3
13
DMSO-d6):
δ (ppm) 192.90, 160.50, 152.10, 149.60, 146.30, 138.40, 132.30, 127.10, 124.50,
123.00, 119.30, 112.02, 111.58, 111.30, 110.20, 55.80, 55.50.
0
0
0
3
,5 -dibromo-3,4-dimetoxy-2 -hydroxy-chalcone (3g) [41]: Prepared according to the general
procedure (method A) starting from 3,5-dibromo-2-hydroxy-acetophenone (1e) (500 mg,
.7 mmol) and 3,4-dimethoxy-benzaldehyde (2c) (282.5 mg, 1.7 mmol), dissolved in 5 mL
1
ethanol and 1.4 mL of 20% w/v aqueous solution of KOH. The mixture was stirred at
room temperature for 24 h. After the work-up procedure, the product was obtained upon
recrystallization from methanol/dichloromethane as a yellow powder. Yield: 470 mg
◦
1
(
(
63%). mp 165–166 C; H-NMR (600 MHz, DMSO-d ):
δ
(ppm) 13.75 (s, 1H, -OH), 8.55
6
0
0
d, J= 2.4 Hz, 1H, H-6 ), 8.11 (d, J= 2.4 Hz, 1H, H-4 ), 7.95 (d, J = 15.6 Hz, 1H, Hb), 7.91 (d,
J = 15.6 Hz, 1H, Ha), 7.60 (d, J= 1.8 Hz, 1H, H-2), 7.53 (dd, J = 8.4, 1.8 Hz, Hz, 1H, H-6),
13
7
.06 (d, J = 8.4 Hz, 1H, H-5), 3.87 (s, 3H, -OCH ), 3.84 (s, 3H, -OCH ); C-NMR (75 MHz,
3 3

Molecules 2021, 26, 2777
16 of 24
DMSO-d6):
δ
(ppm) 195.30, 161.60, 155.20, 152.00, 150.80, 143.50, 135.10, 130.20, 128.20,
121.00, 119.20, 115.50, 114.70, 114.50, 113.00, 58.80, 58.60.
0
0
5
-bromo-3,4-dimethoxymethoxy-2 -hydroxy-chalcone (3h): Prepared according to the gen-
eral procedure (method A) starting from 5-bromo-2-hydroxy-acetophenone (1d) (430 mg,
2
5
.0 mmol) and 3,4-dimethoxymethoxy-benzaldehyde (2a) (500 mg, 2.0 mmol), dissolved in
mL ethanol and 1.6 mL of 20% w/v aqueous solution of KOH. The mixture was stirred at
room temperature for 24 h. After the work-up procedure, the product was purified using
flash column chromatography (petroleum ether/ethyl acetate 8:2) to give 3h as a yellow
◦
1
solid. Yield: 38 mg (22%). mp 105–106 C; H-NMR (600 MHz, DMSO-d ):
δ (ppm) 12.37 (s,
6
0
1
H, -OH), 8.28 (d, J = 2.4 Hz, 1H, H-6 ), 7.83 (d, J= 15.0 Hz, 1H, Hb), 7.75 (d, J= 15.6 Hz, 1H,
Ha), 7.68 (dd, J = 9.0, 2.4 Hz, 1H, H-4 ), 7.66 (d, J = 1.8 Hz, 1H, H-2), 7.55 (dd, J = 8.4, 1.2 Hz,
H, H-6), 7.18 (d, J = 8.4 Hz, 1H, H-3 ), 6.98 (d, J = 8.4 z, 1H, H-5), 5.29 (s, 4H, 2
0
0
1
×
-OCH -),
2
1
3
3
.44 (s, 3H, -OCH ), 3.41 (s, 3H, -OCH ); C-NMR (150z MHz, DMSO-d6):
δ (ppm) 192.25,
3
3
1
1
4
60.19, 149.87, 146.65, 145.39, 138.05, 132.34, 128.46, 124.96, 123.24, 120.48, 120.06, 117.53,
16.28, 110.28, 94.92, 94.42, 55.94, 55.91; HR-MS m/z (neg): 421.0288 C H BrO (calcd.
19
19
6
22.0365).
0
0
0
3
,5 -bromo-3,4-dimethoxymethoxy-2 -hydroxy-chalcone (3i): Prepared according to the general
procedure (method A) starting from 3,5-dibromo-2-hydroxy-acetophenone (1e) (588 mg,
.0 mmol) and 3,4-dimethoxymethoxy-benzaldehyde (2a) (500 mg, 2.0 mmol), dissolved
2
in 5 mL ethanol and KOH 1.6 mL of 20% w/v aqueous solution of KOH. The mixture
was stirred at room temperature for 24 h. After the work-up procedure the product was
◦
◦
obtained as a yellow powder. Yield: 38 g (22%). mp 134–135 C (ref. [46] m.p. 129–131 C);
1
0
H-NMR (600 MHz, CDCl3):
δ
(ppm) 13.63 (s, 1H, -OH), 7.98 (d, J = 2.4 Hz, 1H, H-6 ), 7.92
d, J = 15.0 Hz, 1H, Hb), 7.87 (d, J = 2.4 Hz, 1H, H-4 ), 7.48 (d, J = 1.8 Hz, 1H, H-2), 7.39 (d,
0
(
J = 15.0 Hz, 1H, Ha), 7.33 (dd, J = 8.4, 1.2 Hz, 1H, H-6), 7.22 (d, J = 8.4 Hz, 1H, H-5), 5.31 (br,
H, 2 × -O-CH -O-), 3.56 (s, 3H, -OCH ), 3.53 (s, 3H, -OCH ).
4
2
3
3
0
4
-carboxy-2 -hydroxy-chalcone (3j) [41]: Prepared according to the general procedure (method
A) starting from 2-hydroxy-acetophenone (1b) (1 g, 7.3 mmol) and 4-carboxy-benzaldehyde
2g) (1.102 g, 7.3 mmol), dissolved in 18 mL ethanol and 6 mL of 20% w/v aqueous solution of
(
KOH. The mixture was stirred at room temperature for 24 h. After the work-up procedure,
the product was obtained upon recrystallization from methanol/dichloromethane as a
◦
1
yellow powder. Yield: 813 mg (63%). mp 253–255 C; H-NMR (600 MHz, DMSO-d ):
δ
6
0
(ppm) 13.13 (s, 1H, -COOH), 12.31 (s, 1H, -OH), 8.22 (d, J = 8.2 Hz, 1H, H-6 ), 8.10 (d,
J = 15.6 Hz, 1H, Hb), 7.99 (m, 4H, H-2, H-3, H-5, H-6), 7.84 (d, J = 15.6 Hz, 1H, Ha), 7.56 (t,
0
0
0
13
J = 7.8 Hz, 1H, H-5 ), 6.99 (m, 2H, H-3 , H-4 ); C-NMR (150 MHz, DMSO-d6):
δ
(ppm)
1
92.51, 166.33, 161.30, 142.98, 138.51, 136.47, 132.41, 131.08, 129.86, 129.26, 124.44, 121.19,
119.5, 118.05.
0
0
4
-carboxy-5 -chloro-2 -hydroxy-chalcone (3k) [48]: Prepared according to the general proce-
dure (method A) starting from 5-chloro-2-hydroxy-acetophenone (1c) (500 mg, 2.9 mmol)
and 4-carboxy-benzaldehyde (2g) (440 mg, 2.9 mmol), dissolved in 7.2 mL ethanol and
2
.5 mL of 20% w/v aqueous solution of KOH. The mixture was stirred at room temperature
for 24 h. After the work-up procedure, the product was obtained upon recrystallization
◦
from methanol/dichloromethane as a yellow powder. Yield: 710 mg (85%). mp 284–286 C;
1
H-NMR (600 MHz, DMSO-d ):
δ (ppm) 13.15 (s, 1H, -COOH), 12.15 (s, 1H, -OH), 8.23 (d,
J = 3.0 Hz, 1H, H-6 ), 8.08 (d, J = 15.6 Hz, 1H, Hb), 8.01 (m, 4H, H-2, H-3, H-5, H-6), 7.84 (d,
J = 15.6 Hz, Ha), 7.58 (dd, J = 8.4, 2.4 Hz, 1H, H-4 ), 7.04 (d, J = 9.0 Hz, 1H, H-3 ); C-NMR
150 MHz, DMSO-d6): (ppm) 192.64, 167.22, 160.16, 143.91, 138.88, 136.05, 132.83, 130.18,
30.07, 129.67, 124.91, 123.43, 123.11, 120.22.
6
0
0
0
13
(
δ
1
0
2
-hydroxy-chalcone (3l): Prepared according to the general procedure (method A) starting
from 2-hydroxy-acetophenone (1b) (500 mg, 3.7 mmol) and benzaldehyde (2h) (392.6 mg,
.7 mmol), dissolved in 6 mL ethanol and 3.1 mL of 20% w/v aqueous solution KOH. The
mixture was stirred at room temperature for 24 h. After the work-up procedure, the product
3

Molecules 2021, 26, 2777
17 of 24
was obtained upon recrystallization from methanol/dichloromethane as a yellow powder.
◦
◦
1
Yield: 697 mg (84%); m.p. 85–86 C (ref. [49] m.p. 82–85 C); H-NMR (600 MHz, CDCl ):
3
δ
(ppm) 12.83 (s, 1H, -OH), 7.93 (m, 2H, aromatic H), 7.67 (m, 4H, aromatic H and Hb),
7
.51 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H, aromatic H), 7.45 (m, 3H, aromatic H and Ha), 7.04 (d,
J = 8.4 Hz, 1H, aromatic H), 6.96 (t, J = 7.2 Hz, 1H aromatic H).
3.1.2. General Procedure for the Synthesis of Chalcones 4a and 4b
Chalcones 4a and 4b were produced by removing the MOM protecting group. The
corresponding methoxymethoxy-chalcone wasdissolved in methanol and 10% aqueous
◦
HCl was added dropwise. The mixture was stirred at 65 C for 15 min. The reaction was
monitored by TLC. After completion of the reaction, the mixture was diluted with water
and extracted with ethyl acetate. The organic extracts were dried with Na SO and the
2
4
solvent was evaporated under reduced pressure to give the chalcone as a solid product.
0
0
2
,3,4,4 -tetrahydroxy-chalcone (Butein) (4a): Prepared according to the general procedure,
chalcone (3a) (47.5 mg, 0.1 mmol) was dissolved in 2.2 mL methanol followed by dropwise
addition HCl (10% aqueous solution, 1 mL). The mixture was refluxed for 15 min, until a
clear red solution was obtained. After the work-up procedure, a red solid was acquired.
◦
1
Yield: 22.4 mg (70%). m.p. > 180 C; H-NMR (300 MHz, DMSO-d ):
δ
(ppm) 13.53 (s, 1H,
6
0
0
OH-2 ), 10.60 (s, 1H, OH-4 ), 9.71 (s, 1H, OH-4), 9.06 (s, 1H, OH-3), 8.11 (d, J = 8.9 Hz, 1H,
H-6 ), 7.63 (m, 2H, Ha, Hb), 7.25 (d, J = 1.8 Hz, 1H, H-3 ), 7.18 (dd, J = 8.1, 1.8 Hz, 1H, H-5 ),
.79 (d, J = 8.1 Hz, 1H, H-5), 6.38 (dd, J = 8.7, 2.1 Hz, 1H, H-6), 6.25 (d, J = 2.4 Hz, 1H, H-2);
MS (ESI) (positive): m/z = 273.16 (50%) [M + 1] , 206.61 (77%) [M
ESI) (negative): m/z = 271.2 (100%) [M − 1] , 542.79 (92%) [2 × (M − 1)] .
0
0
0
6
+
+
−
4
×
-OH + 2] ; MS
+
+
(
0
0
5
-bromo-2 ,3,4-trihydroxy-chalcone (4b): Prepared according to the general procedure, chal-
cone (3h) (200 mg, 0.5 mmol) was dissolved in 9.8 mL methanol followed by dropwise
addition of HCL (10% aqueous solution, 4 mL). The mixture was refluxed for 15 min.
After the work-up procedure, the product was obtained upon recrystallization from
◦
1
methanol/dichloromethane. Yield: 500 mg (50%). mp 198–199 C; H-NMR (600 MHz,
DMSO-d6): (ppm) 12.59 (s, 1H, -OH), 9.88 (brs, 1H, Ar-OH), 9.14 (brs, 1H, Ar-OH), 8.34
d, J= 2.4 Hz, 1H, H-6 ), 7.71 (m, 2H, Ha, Hb), 7.66 (dd, J = 8.4, 2.4 Hz, 1H, H-4 ), 7.34 (d,
δ
0
0
(
0
J= 1.2 Hz, 1H, H-2), 7.24 (dd, J = 8.4, 1.8 Hz, 1H, H-6), 6.96 (d, J = 9.0 Hz, 1H, H-3 ), 6.83
d, J = 7.8 Hz, 1H, H-5); 13C-NMR (150 MHz, DMSO-d6):
(ppm) 192.16, 160.46, 149.47,
46.70, 145.68, 137.98, 132.36, 126.07, 123.18, 122.98, 120.04, 118.04, 116.01, 115.75, 110.25;
HR-MS m/z (neg): 332.9763 C H BrO (calcd. 333.9841).
(
1
δ
15
11
4
0
3
.1.3. Synthetic Procedure of 4-Methoxycarbonyl-2 -Hydroxy-Chalcone (5)
Chalcone (3j) (500 mg, 1.9 mmol) was added to methanol (50 mL), followed by
◦
dropwise addition of H SO (5 mL). The mixture was stirred at 70 C for 24 h. The reaction
2
4
was monitored by TLC. After completion of the reaction, the solvent was evaporated
◦
under reduced pressure and the residue was cooled at
−
19 C. The precipitate formed
◦
1
was filtered and washed with methanol. Yield: 317 mg (60%). mp: 128–131 C; H-NMR
0
(600 MHz, DMSO-d6):
δ
(ppm) 12.31 (s, 1H, -OH), 8.24 (dd, J = 8.4, 1.2 Hz, 1H, H-6 ), 8.14 (d,
J = 15.6 Hz, 1H, Hb), 8.04 (m, 4H, H-2,H-3,H-5,H-6), 7.86 (d, J = 15.6 Hz, 1H, Ha), 7.58 (ddd,
0
0
0
13
J = 8.4, 7.8, 1.8 Hz, 1H, H-4 ), 7.02 (m, 2H, H-3 , H-5 ), 3.88 (s, 3H, -COOCH ); C-NMR
3
(150 MHz, DMSO-d6):
δ
(ppm) 193.3, 165.7, 161.7, 142.8, 139.0, 136.5, 131.0, 129.6, 129.2,
1
24.6, 121.0, 119.2, 117.7, 109.6, 52.3; HR-MS m/z (neg): 281.0809 C H O (calcd. 282.0892).
17
14
4
3.1.4. General Procedure for the Synthesis of Aurones 6a and 6b
To a solution of mercuric acetate (1.25 eq) in pyridine was added the desirable chalcone
◦
(
1 eq) at room temperature and the mixture was stirred at 110 C for 1 h. The reaction
was monitored by TLC. The cooled reaction mixture was poured into ice cold water and
acidified with HCl (10% aqueous solution). The precipitated solid was extracted with

Molecules 2021, 26, 2777
18 of 24
dichloromethane, the extracts were dried (Na SO ) and the solvent was evaporated to give
2
4
a solid which was further purified by recrystallization from methanol/dichloromethane.
Note: Pyridine is a harmful reactant and should be handled with care as it can be
harmful in case of inhalation, ingestion, or when in contact with the skin. Pyridine is
also known to reduce male fertility. Pyridine has a very noticeable and unpleasant smell,
therefore, good ventilation is essential, when used. To neutralize pyridine addition of a
◦
weak acid, such as 10% aqueous HCl, at 0 C is suggested.
Mercuric acetate is a harmful reagent. It is fatal if swallowed, in contact with skin or if
inhaled. It may cause damage to organs through prolonged or repeated exposure. It is very
toxic to aquatic life with long lasting effects. Protective gloves/protective clothing should
be worn throughout the period of handling this reagent.
0
0
3
,4 ,6-tri(methoxymethoxy)-aurone (6a): Prepared according to the general procedure starting
from chalcone (3a) (140.4 mg, 0.3 mmol), dissolved in 3.5 mL pyridine. After the work-up
procedure, the product was obtained upon recrystallization from methanol/dichloromethane
◦
1
as a yellow solid. Yield: 84 mg (60%). mp 140.2–141.4 C; H-NMR (300 MHz, CDCl ):
3
δ
(ppm) 7.71 (m, 2H, H-4, H-10), 7.55 (d, J = 8.4 Hz, 1H, H-5), 7.22 (d, J= 8.7 Hz, 1H, H-
0
0
0
5
), 6.95 (s, 1H, H-7), 6.86 (d, J = 8.4 Hz, 1H, H-6 ), 6.78 (s, 1H, H-2 ), 5.31 (s, 6H, 3
×
13
-OCH O-), 3.57 (s, 3H, -CH OCH ), 3.53 (br, 6H, 2
×
-CH OCH ); C-NMR (150 MHz,
2
2
3
2 3
CDCl3):
δ (ppm) 183.16, 168.09, 164.82, 149.03, 147.29, 147.22, 126.98, 126.84, 125.93, 119.99,
1
16.47, 116.04, 113.27, 112.24, 99.48, 95.90, 95.29, 94.59, 56.58, 56.54, 56.48; HR-MS m/z (pos):
4
03.1352 C H O (calcd. 402.1315).
21
22
8
0
0 0
2
,4 ,5 -trimethoxy-aurone (6b): Prepared according to the general procedure starting from
chalcone (3b) (150 mg, 0.4 mmol) and mercury acetate (184.25 mg, 0.6 mmol) dissolved
in 4.8 mL pyridine. After the work-up procedure, the product was obtained upon recrys-
tallization from methanol/dichloromethane as a yellow solid. Yield: 112.7 mg (74%). mp
◦ ◦
81.2–183.4 C (ref. [50] m.p. 191–193 C); H-NMR (300 MHz, DMSO-d ): δ (ppm) 7.78 (s,
6
1
1
1
7
H, H-10), 7.75 (m, 2H, H-4, H-5) 7.56 (d, J = 8.4 Hz, 1H, H-7), 7.28 (t, J = 7.5 Hz, 1H, H-6),
.17 (s, 1H, H- 6 ), 6.77 (s, 1H, H-3 ), 3.91 (br, 6H, 2 × -OCH ), 3.82 (s, 3H, -OCH ).
0
0
3
3
0
0
3
.1.5. Synthetic Procedure of 3 ,4 ,6-Trihydroxy-Aurone (Sulfuretin) (7a)
Aurone (7a) was prepared via deprotection reaction of aurone (6a). Aurone (6a
48.2 mg, 0.1 mmol) was added to 2.25 mL methanol followed by dropwise addition of
)
(
HCl (10% aqueous solution, 1 mL). The mixture was refluxed for 15 min, until a clear
orange solution was obtained. The reaction was monitored by TLC. After completion of the
reaction, the mixture was diluted with water and extracted with ethyl acetate. The organic
extracts were dried with Na SO and the solvent was evaporated under reduced pressure
2
4
◦
1
to give the product as an orange solid. Yield: 29.6 mg (92%). mp > 250 C; H-NMR
0
0
(
300 MHz, DMSO-d6):
δ
(ppm) 11.04 (s, 1H, OH-3 ), 9.60 (s, 1H, OH-4 ), 9.23 (s, 1H, OH-6),
7
6
.57 (d, J = 8.1 Hz, 1H, H-4), 7.42 (d, J = 1.8 Hz, 1H, H-7), 7.22 (dd, J = 8.4, 1.8 Hz, 1H, H-5),
0
0
.81 (d, J = 8.4 Hz, 1H, H-5 ), 6.72 (d, J = 1.5 Hz, 1H, H-2 ), 6.68 (dd, J = 8.4, 1.8 Hz, 1H,
0
13
H-6 ), 6.61 (s, 1H, H-10); C-NMR (150 MHz, CD OD-d ):
δ
(ppm) 184.47, 169.77, 168.24,
3
4
1
49.37, 147.69, 146.70, 126.83, 126.40, 125.53, 118.94, 116.66, 114.90, 114.70, 114.03; MS (ESI)
+
+
(positive): m/z = 271.14 (67%) [M + 1] , 562.75 (42%) [2
×
M + Na] ; MS (ESI) (negative):
+
+
m/z = 269.23 (33%) [M − 1] , 538.86 (100%) [2 × (M − 1)] .
0
0
0
3
.1.6. Synthetic Procedure of 2 ,4 ,5 -Trihydroxy-Aurone (7b)
Aurone (7b) was prepared via deprotection reaction of aurone (6b). Aurone (6b
)
(
(
50 mg, 0.2 mmol) was added to 2 mL dry dichloromethane followed by addition of BBr
2.4 mL, 2.4 mmol). The mixture was stirred for 2 h at 0 C, until a clear orange solution
3
◦
was obtained. The reaction was monitored by TLC. After completion of the reaction,
the resulting mixture was poured into ice-cold water and left to stand for 25 min. The
residue was extracted with ethyl acetate, the organic extracts were dried with Na SO ,
2
4
and the solvent was evaporated under reduced pressure to give the product as a red solid.

Molecules 2021, 26, 2777
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◦
1
Yield: 26.8 mg (62%). mp > 250 C; H-NMR (300 MHz, CD OD-d ):
δ
(ppm) 7.74 (s, 1H,
3
4
0
H-10), 7.70 (m, 2H, H-4, H-5), 7.44 (s, 1H, H-6 ), 7.37 (d, J = 8.4 Hz, 1H, H-7), 7.247. (t,
J = 7.2 Hz, 1H, H-6), 6.38 (s, 1H, H-3 ); MS (ESI): m/z = 271.2 (55%) [M + 1] , 255.3 (100%)
0
+
[
M − 1 × OH + 1]+.
3.2. Biological In Vitro Assays
Each in vitro experiment was performed at least in triplicate and the standard devia-
tion of absorbance was less than 10% of the mean. For the in vitro assays, a stock solution
1% DMSO in the appropriate buffer with the tested compound diluted under sonification)
was prepared from which several dilutions were made with the appropriate buffer.
(
3
2
.2.1. Determination of the Reducing Activity of the Stable Radical
,2-Diphenyl-1-Picrylhydrazyl (DPPH)
To an ethanolic solution of DPPH (100
µM) in absolute ethanol, an equal volume
of the compounds dissolved in DMSO was added (100
µM). The mixture was shaken
vigorously and allowed to stand for 20 or 60 min; absorbance at 517 nm was determined
spectrophotometrically, and the percentage of activity was calculated. All tests were
undertaken on three replicates, and the results presented in Table 2 were averaged and
compared with the appropriate standard nordihydroguaiaretic acid (NDGA) [61].
3.2.2. Inhibition of Linoleic Acid Lipid Peroxidation
Production of conjugated diene hydroperoxide by oxidation of sodium linoleate in an
0
aqueous solution is monitored at 234 nm. 2,2 -Azobis(2-amidinopropane) dihydrochloride
(AAPH) was used as a free radical initiator. Ten microliters of the 16 mM linoleic acid
sodium salt solution was added to the UV cuvette containing 0.93 mL of 0.05 M phosphate
◦
◦
buffer, pH 7.4 prethermostated at 37 C. The oxidation reaction was initiated at 37 C under
air by the addition of 50 L of 40 mM AAPH solution. Oxidation was carried out in the
presence of the synthesized compounds (10 L, from a stock solution of 10 mM in DMSO).
µ
µ
In the assay without antioxidant, lipid oxidation was measured in the presence of the same
◦
level of DMSO. The rate of oxidation at 37 C was monitored by recording the increase in
absorption at 234 nm caused by conjugated diene hydroperoxides and compared with the
appropriate standard trolox [61].
3
.2.3. Competition of the Tested Compounds with DMSO for Hydroxyl Radicals
The hydroxyl radicals generated by the Fe3+ / ascorbic acid system, were detected
by the determination of formaldehyde produced from the oxidation of DMSO (Table 2).
A negative control using the same % DMSO was used. Trolox was used as reference
compound [75].
+
3
.2.4. ABTS —Decolorization Assay for Antioxidant Activity
+
·
In order to produce the ABTS radical cation (ABTS ), ABTS stock solution in water
7 mM) was mixed with potassium persulfate (2.45 mM) and left in the dark at room
(
temperature for 12–16 h before use. Our published experimental technique was used (1).
The results are recorded after 1 min of the mixing solutions at 734 nm. The results were
compared to the appropriate standard inhibitor Trolox [75].
3.2.5. Soybean LOX Inhibition Study In Vitro
The tested compounds dissolved in DMSO were incubated at room temperature with
−
f
sodium linoleate (0.1 mL) and 0.2 mL of enzyme solution (1/9
×
10 w/v in saline) in Tris
buffer pH 9. The conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm
was recorded and compared with the appropriate standard inhibitor NDGA [61].

Molecules 2021, 26, 2777
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3.3. In Silico Studies
3.3.1. Molecular Docking Studies on Soybean Lipoxygenase Seed Isoform 1 (LOX-1)
The synthesized compounds 3b and 3c were primarily sketched in 2D format and pre-
pared at the optimum pH = 7.0
Subsequently, molecular docking simulations were carried out applying the Schrödinger
induced fit docking (IFD) protocol [76 77]. The referred protocol, based on glide and prime
±
0.5, using LigPrep program of Schrödinger platform. [76]
,
modules, offers an accurate prediction of ligand favorable binding modes with concomitant
structural changes in the receptor (simultaneous flexibility of the side chains, backbone, or
both). The crystal structure of soybean LOX-1 (PDB ID: 3PZW) [63] complexed with ferrous
iron (Fe2 —inactive form) was subjected to protein preparation [78
+
,
79]. Particularly, all wa-
ter molecules were removed, missing residues and hydrogens were added, and restrained
energy minimization was followed using OPLS3 force field. Identification of potential al-
losteric binding sites apart from the iron-binding site and the substrate-binding cavity was
performed using SiteMap calculations [68–70]. The maximum number of docking poses
was set equal to 10, they were visually inspected and their binding modes were analyzed.
3.3.2. Molecular Dynamics Simulations
Molecular dynamics simulations (MD) of the tested ligand-enzyme complexes were
performed using the Desmond software of the Schrödinger platform, release 2020-3 [80].
For the examined systems setup, TIP4P was defined as the solvent model and the examined
3
complexes were inserted in an orthorhombic box with volume 850,054 Å . Additionally,
+
OPLS2003 force field was utilized and the systems were neutralized by adding 7 Na ions.
Subsequently, modeled systems were relaxed and then subjected to 10 ns MD simulations
in the NPT ensemble class. During the simulation, the temperature was maintained at
300 K using the Nose–Hoover chain thermostat method with a 1.0 ps relaxation time and
the pressure was maintained at 1 bar using Martyna–Tobias–Klein barostat method. The
root mean square deviation (RMSDs) was calculated for both the docked compounds and
Ca atoms of the enzyme, and also interactions and contacts (H-bonds, hydrophobic, ionic,
water bridges) of the examined compounds with the LOX-1 residues were monitored
throughout the simulation.
4
. Conclusions
In conclusion, this project reports the synthesis of sixteen 2 -hydroxy chalcone deriva-
tives and four aurone analogues, from which, to our knowledge, compounds 3c 3h 4b
, and 6a are presented in the literature for the first time. The synthesized chalcones
0
,
,
,
5
and aurones were evaluated for their antioxidant activity using different in vitro tech-
niques, while they were also tested as inhibitors of soybean LOX, as an indication of
their anti-inflammatory effect. The bioassays’ results revealed that compound 4b is the
most promising antioxidant agent (82.4% for DPPH scavenging ability and 82.3% in lipid
peroxidation inhibition and 100% scavenging of hydroxyl radicals), while analogue 3c
is the most potent LOX inhibitor (IC50 = 45 µM). Chalcones 3c and 3b, which share the
common structural feature of a 2,4,5-trimethoxy substitution at ring B, were further stud-
ied to explore their mechanism of action on the soybean LOX-1 enzyme using molecular
docking studies and molecular dynamics simulations. The results indicate that these com-
pounds share common binding characteristics and interact with LOX-1 enzyme through
allosteric interactions.
Supplementary Materials: The following are available online: Figure S1: Predicted binding sites of
Soybean Lipoxygenase seed isoform 1 (LOX-1), Figure S2: RMSD calculations for (A) all C
a
enzyme
atoms (blue) in LOX-1-Compound 3b complex and for all atoms of compound 3b (red), (B) all C
a
enzyme atoms (blue) in LOX-1-Compound 3c complex and for all atoms of compound 3c (red), Figure
S3: Categorization of (A) LOX-1-Compound 3b and (B) LOX-1-Compound 3c interactions, during
1
the molecular dynamics simulation (10 ns). The H-NMR spectra of all the synthesized compounds
13
and C-NMR spectra of the novel synthesized compounds.

Molecules 2021, 26, 2777
21 of 24
Author Contributions: Conceptualization, A.D.; Data curation, I.K., A.T., N.-I.P., M.-A.K., E.K.,
E.P. and T.L.; Methodology, I.K., A.T., N.-I.P., M.-A.K., E.P. and T.L.; Project administration, A.D.;
Resources, P.Z. and A.D.; Software, E.K. and P.Z.; Supervision, D.H.-L., P.Z. and A.D.; Writing—
original draft, I.K., A.T., N.-I.P., M.-A.K., E.K., E.P., T.L., D.H.-L. and P.Z.; Writing—review & editing,
I.K., A.T., N.-I.P., M.-A.K., E.K., E.P., T.L., D.H.-L., P.Z. and A.D. All authors have read and agreed to
the published version of the manuscript.
Funding: I.K. would like to thank the Eugenides Foundation for financial support (scholarship for
postgraduate studies).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: D. Hadjipavlou-Litina would like to be thankful to Biobyte and A. Leo for
providing free access to Biobyte platform.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
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