Synthesis, in vitro biological evaluation, and oxidative transformation of new flavonol derivatives: The possible role of the phenyl-N,N-dimethylamino group

Article abstract of DOI:10.3390/molecules23123161

Six new flavonols (6a–f) were synthesized with Claisen–Schmidt and Suzuki reactions and they were fully characterized by spectroscopic methods. In order to evaluate their antioxidant activities, their oxygen radical absorption capacity and ferric reducing antioxidant power were measured, along with their free radical scavenging activity against 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 2,2-diphenyl-1-picrylhydrazylradicals. In addition, their cytotoxicity on H9c2 cardiomyoblast cells was also assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Compounds bearing the phenyl-N,N-dimethylamino group (6a, 6c, and 6e) exhibited promising antioxidant potency and did not have any cytotoxic effect. After a consideration of these data, the oxidative transformation of the 6c compound was investigated in vitro with a chemical Fenton reaction and the identification of the formed oxidation products was performed by mass spectrometry. Two potential metabolites were detected. Based on these results, compound 6c can be a model compound for future developments. Overall, this work has proved the involvement of the phenyl-N,N-dimethylamino group in the antioxidant activity of flavonols.

Full text of DOI:10.3390/molecules23123161

molecules
Article
Synthesis, In Vitro Biological Evaluation, and
Oxidative Transformation of New Flavonol
Derivatives: The Possible Role of the
Phenyl-N,N-Dimethylamino Group
Peter Szabados-Furjesi 1, David Pajtas 2, Aliz Barta 2, Evelin Csepanyi 1, Attila Kiss-Szikszai 3
,
Arpad Tosaki 4 and Istvan Bak 1,
*
1
Department of Bioanalytical Chemistry, Faculty of Pharmacy, University of Debrecen, Debrecen H-4032,
Hungary; szabados-furjesi.peter@pharm.unideb.hu (P.S.-F.); csepanyi.evelin@pharm.unideb.hu (E.C.)
Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen,
Debrecen H-4032, Hungary; pajtas.david@med.unideb.hu (D.P.); barta.aliz@med.unideb.hu (A.B.)
Department of Organic Chemistry, Faculty of Science and Technology, University of Debrecen,
Debrecen H-4032, Hungary; kiss.attila@science.unideb.hu
2
3
4
Department of Pharmacology, Faculty of Pharmacy, University of Debrecen, Debrecen H-4032, Hungary;
tosaki.arpad@pharm.unideb.hu
*
Correspondence: bak.istvan@pharm.unideb.hu; Tel./Fax: +36-52-255-586
Academic Editor: Luciano Saso
Received: 13 November 2018; Accepted: 29 November 2018; Published: 30 November 2018
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Six new flavonols (6a–f) were synthesized with Claisen–Schmidt and Suzuki reactions and they
were fully characterized by spectroscopic methods. In order to evaluate their antioxidant activities, their
oxygen radical absorption capacity and ferric reducing antioxidant power were measured, along with
their free radical scavenging activity against 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
and 2,2-diphenyl-1-picrylhydrazylradicals. In addition, their cytotoxicity on H9c2 cardiomyoblast
cells was also assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
Compounds bearing the phenyl-N,N-dimethylamino group (6a, 6c, and 6e) exhibited promising
antioxidant potency and did not have any cytotoxic effect. After a consideration of these data, the oxidative
transformation of the 6c compound was investigated in vitro with a chemical Fenton reaction and the
identification of the formed oxidation products was performed by mass spectrometry. Two potential
metabolites were detected. Based on these results, compound 6c can be a model compound for future
developments. Overall, this work has proved the involvement of the phenyl-N,N-dimethylamino group
in the antioxidant activity of flavonols.
Keywords: antioxidant; flavonoid; flavonol; cytotoxicity; oxidative metabolism
1. Introduction
During oxidative/nitrosative stress, there is a lack of balance between the endogenous antioxidant
system and the prooxidants, which could result in the excess formation of the latter. Reactive oxygen
(ROS) and nitrogen species (RNS) can irreversibly damage lipids, proteins, nucleic acids, and other
biomacromolecules contributing to the development and progression of several diseases, for instance,
attention deficit hyperactivity disorder [
1], cancer [2], Parkinson’s disease [
3], Alzheimer’s disease [4],
atherosclerosis [ ], heart failure [ ], and myocardial infarction [7
5
6
]. In order to inhibit lipid peroxidation
and protect the cell membranes, proteins, and nucleic acids from the ROS- and RNS-caused damages,
the balance must be restored. This can be achieved by providing exogenous antioxidants to act
as an active contributors of the endogenous antioxidant defense system. The most frequently
Molecules 2018, 23, 3161; doi:10.3390/molecules23123161
www.mdpi.com/journal/molecules

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used exogenous antioxidants are ascorbic acid, vitamin E, carotenoids, and different polyphenolic
compounds, including flavonoids. These exogenous antioxidants are mostly produced by plants,
and possess promising biological effects [
past decades; they possess a broad range of beneficial biological effects, such as anti-inflammatory,
antiviral, antibacterial, and antiallergic activities [11 12]. In further studies, flavonoids were proved to
be powerful antioxidants [13 14]. Based upon the structural complexity, flavonoids can be divided into
six groups: anthocyanins, flavan-3-ols, flavanones, flavones, isoflavones, and flavonols, which are a
3-hydroxyflavone backboned subclass of flavonoids with a wide range of biological activity [15 17].
4,8–10]. The flavonoids have been studied thoroughly in the
,
,
–
Furthermore, the authors of the present study have previously investigated the antioxidant effects of
different flavonoid-type chromone derivatives [18] and found that the 40-N,N-dimethylamino-flavon
was the most active tested compound. Hence, the amino group promotes free-radical scavenger
activity [19]; i.e., the introduction of differentially N-substituted amino groups into the molecule
could modify the antioxidant activity of the different flavonoids [20
O-methylation of flavonoids could result in higher metabolic stability, increased bioavailability,
and better tissue distribution [23 25]; however, the methylation of hydroxyl groups could decrease
–22]. On the other hand,
–
the antioxidant activity [26]. Based on these results, the aim of the present work was to evaluate
the antioxidant potential and cytotoxic activity of six, newly synthetized O-methylated flavonol
derivatives (Table 1) and investigate the effect of phenyl-N,N-dimethylamino and methoxy groups on
the antioxidant potency.
Table 1. The structures of the investigated flavonol derivatives.
ID
R
R’
Position of R‘-Ph
6a
6b
6c
6d
6e
6f
H
H
H
H
OMe
OMe
3-N(CH3)2
4-CH2(O)CH3
3-N(CH3)2
4-CH2(O)CH3
3-N(CH3)2
6
6
7
7
7
7
4-CH2(O)CH3
The tested compounds were synthetized by Claisen-Schmidt condensation followed by a ring closure.
The side chains were built in with a Suzuki reaction. The radical scavenging activity was determined by
0
2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
radical scavenging assays. Moreover, the oxygen radical absorption capacity (ORAC) and ferric reducing
antioxidant power (FRAP) of the compounds were also measured. In further experiments, the cytotoxicity
of the compounds was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay on H9c2 cardyomyoblastoma cell cultures. In addition, we investigated the oxidative transformation
of the molecules by the chemical Fenton reaction as a biomimetic model system of biotransformation.
2. Results and Discussion
2.1. Chemistry
Scheme 1 illustrates the preparation of the flavonol backbone (4a
compounds of interest (6a–f).
–c) used for the synthesis of the

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Scheme 1. Synthesis of 6a-f. Reagents and reaction condition: (i) 1a
50% aq. NaOH (4 equiv.), MeOH, room temperature, 24 h; (ii) 3a
H2O2 (14.1 equiv.), EtOH, room temperature, 2 h; (iii) 4a 5a
,
–
b
, benzaldehyde 2a
,b (1.05 equiv.),
c, 8% aq. NaOH (3.1 equiv.), 30%
–
c,
,
b
(2 equiv.), KF (3 equiv.), Pd(OAc)2
◦
(5 mol%), XPhos (10 mol%), toluene/t-BuOH (6:1), argon atmosphere, 100 C, 4 h.
As a first step, the Claisen-Schmidt condensation of the commercially available bromoacetophenones
(
1a
,
b
) and benzaldehydes (2a
,
b
) in MeOH provided the corresponding 3a chalcones (88–97%) using
–
c
4 equiv. NaOH as a base. The cyclization of these chalcones (3a–c) into the 4a–c key intermediates (57–76%)
was performed using 14.1 equiv. 30% H2O2 in EtOH. Then, the Suzuki coupling of bromoflavonol
derivatives (4a ) with the appropriate boronic acid (5a ) resulted in the desired 6a flavonols (63–74%)
–c
,b
–f
using potassium-fluoride, Pd(OAc)2 as a palladium source, and XPhos as a ligand in toluene/t-BuOH (6:1)
under argon atmosphere.
2.2. Antioxidant Activity
To determine the antioxidant properties of the target compounds, four assays were performed.
During the ABTS assay, the ABTS radical cation scavenging ability was evaluated. The half-maximal
scavenging rate (IC50) was calculated for each compound from the inhibition percentage at 120 min in
the concentration range of 10–200 µM (Figure 1).

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50
45
40
35
30
25
20
15
10
5
0
6a
6b
6c
6d
6e
6f
Quercetin
+
Figure 1. The IC50 ABTS scavenging rate of the tested flavonol derivatives and quercetin standard.
The inhibition percentage was determined at 120 min in the concentration range of 10–200 µM.
The results show that compound 6c demonstrated the highest scavenging potency by having
the lowest IC50 value, followed by 6e and 6a, in the concentration range of 10–200
phenyl-N,N-dimethylamino group, these compounds (6a 6c, and 6e) are better electron donors than
compounds with methoxymethyl groups (6b 6d, and 6f) because of the electronegativity difference.
µM. By having the
,
,
During the single electron-transfer process, the ABTS radical cation abstracts an electron and the
flavonol may act as a donor and, thus, regenerates the original form and causes a detectable change
in the absorbance at 737 nm. The greater the change in the absorbance, the higher the scavenging
ability of the investigated compound. The principle of the DPPH assay is similar to that of the ABTS
assay; i.e., DPPH is a stable radical and its solution is deep violet, since decolorization due to the
reduction of the radical by the antioxidant can be detected at 515 nm by a spectrophotometer. Figure 2
depicts the calculated IC50 values for each compound from the inhibition percentage at 90 min in the
concentration range of 10–200 µM.
120
100
80
60
40
20
0
6a
6b
6c
6d
6e
6f
Quercetin
Figure 2. The IC50 DPPH scavenging rate of the tested flavonol derivatives and quercetin standard.
The inhibition percentage was determined at 90 min in the concentration range of 10–200 µM.
It can be seen that compound 6c was the most potent scavenger against the DPPH radical with
the lowest IC50 value, followed by 6a, 6d, and 6b at 200 µM concentrations These derivatives (6a, 6b,
6c, and 6d) have two methoxy groups on the B ring. In a previous report, Kim B. T. et al. reported the
effect of the substitution pattern of two hydroxyl groups on the B ring in the case of chalcones; the
ortho- and para- substitution showed much stronger antioxidant potency than meta-substitution, due to
the efficiency of the ortho- and para-dihydroxylated benzene ring system to delocalize electrons [27].
The steric hindrance is higher in the case of the three methoxy groups on the B ring, which perturbs
planarity, causing the lower antioxidant potency of these derivatives (6f and 6e) as the hydrogen
abstraction is easier in planar geometrical configuration [28]. Moreover, in both cases, 6c was the
most potent compound, which bears the phenyl-N,N-dimethylamino group. These observations are
in good correlation with Culhaoglu et al., who investigated the antioxidant activity of dialkylamino

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substituted 3-OH-flavon derivatives; the authors found that 40-N,N-dimethylamino-3-OH-flavon
showed significant scavenging of ABTS and DPPH radicals, and the potency was comparable to that
of quercetin [20]. Furthermore, earlier, we observed that 40-N,N-dimethylamino-flavon was also a
good radical scavenger [18]. Thus, the presence of the phenyl-N,N-dimethylamino-group could have a
crucial role in the radical scavenger activity of the molecules. Figure 3 shows the results of the FRAP
assay of the investigated compounds expressed in µM ferrous equivalents.
500
450
400
350
300
250
200
150
100
50
0
6a
6b
6c
6d
6e
6f
Quercetin
200 µM
100 µM
50 µM
20 µM
10 µM
Figure 3. FRAP values of the investigated compounds. The fresh FRAP working solution consisted
◦
of acetate buffer, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), and FeCl3, and was incubated at 37 C for
15 min before each experiment. After the activated FRAP reagent was added to the solution of the
tested compounds and the quercetin standard, the plate was incubated for 30 min. The absorbance was
measured at 593 nm. The FRAP values are expressed as µM Ferrous equivalents calculated based on
iron(II) sulfate heptahydrate calibration standard solutions. The experiments were run in duplicate
and repeated three times.
The results show that compounds 6c
derivatives at all the tested concentrations, while compounds 6d
lower ferrous equivalents. It has to be mentioned that the FRAP value of each compound (6a
,
6a, and 6e had the highest FRAP values among the
6f, and 6b exhibited significantly
) is
,
–
f
significantly decreased compared to the standard quercetin. However, this result is not surprising,
since it had been shown that methoxylated flavonoids are weaker antioxidants than the unmethylated
ones [26,29]. In their study, Deng et al. found that quercetin had a 650-times higher FRAP value then
the methylated form. In our experiments, we did not observe such a big difference, which could have
originated from the presence of the free OH group at position 3 and the phenyl-N,N-dimethylamino
group at ring A. Furthermore, since 6c, 6e, and 6a showed a significantly higher FRAP value than 6b,
6d, and 6f suggest that the presence of the phenyl-N,N-dimethylamino group is crucial, because the
latter three compounds do not bear this group, but they also have the free OH group at position 3.
Figure 4 depicts the results of the ORAC assay.

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50
45
40
35
30
25
20
15
10
5
0
6a
6b
6c
6d
6e
6f
Quercetin
2 µM
10 µM
Figure 4. ORAC values of the compounds of interest expressed in the net area under the curve
(AUC). To the mixture of tested compounds and fluorescein, the 2,20-azobis(2-amidinopropane)
dihydrochloride (AAPH) solution was added rapidly. The fluorescence was monitored at 485 nm
excitation and 520 nm emission wavelength for 2 h in every 2 min. The assay was performed in duplicate
and repeated three times. The netAUC was calculated by subtracting the blankAUC from sampleAUC.
Similarly to the findings of Deng et al., the ORAC values did not change as much as the
FRAP values [29]. The results show that compounds 6e
Additionally, in the case of 6e and 6a, the results were comparable to quercetin at a 10
,
6a, and 6b had the highest ORAC value.
M concentration.
µ
Surprisingly, compound 6c exhibited only the fourth highest capacity for the absorption of the oxygen
radical at each concentration.
2.3. Cytotoxic Effect
Figure 5 presents the results of the MTT assay; the cells were treated with 20 µM flavonol solutions
for 12 h. Compounds with the phenyl-N,N-dimethylamino functional group (6a, 6c and 6e) did not
exhibit cytotoxic activity; their cell viability percentage was comparable to the untreated control value
and the quercetin standard.
140
120
100
80
60
40
20
0
6a
6b
6c
6d
6e
6f
Control Quercetin
Figure 5. The effect of the tested compounds and the quercetin standard in 20 µM concentrations on
the viability of H9c2 cells evaluated by the MTT assay. The cardiomyoblastoma cells were treated for
12 h and following the addition of the MTT solution, the plates were incubated for 4 h. Absorbance was
measured at 540 and 630 nm. The measurements were carried out in triplicate and repeated two times.
The results are consistent with the findings of Luo W. et al., who found that 4-dimethylamine
flavonoid derivatives are protective against oxidative stress-induced cell death in PC12 neurons [30].
Moreover, treatment with compounds 6b, 6d, and 6f significantly decreased the viability of cells, which

Molecules 2018, 23, 3161
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was the most dominant in the case of 6d, which reduced the cell viability to 46% compared to the
control. These results correlate well with the findings of other research groups who studied the effect
of the number and position of methoxy groups on the flavonoid backbone on antioxidant and cytotoxic
potency [24,29,31]. The presence of methoxy groups in these flavonols and other flavonoids could
be the reason of their cytotoxicity depending on their number and/or positions; however, the exact
structure-activity relationship is not completely understood.
2.4. Oxidative Transformation
Based on the beneficial characteristics of 6c demonstrated by the antioxidant and cytotoxicity
assays, this compound was selected to determine the possible oxidative transformation pathways
and identify its potential metabolites. The chemical Fenton reaction was used as a model of phase I
biotransformation, since it is suitable for modeling phase I metabolic processes, such as N-dealkylation,
O-dealkylation, S-oxidation, benzylic hydroxylation, and aromatic hydroxylation, as it has been
shown previously [32,33]. Scheme 2 shows the oxidation routes and the potential metabolites of
compound 6c generated with the chemical Fenton system and detected with electrospray ionization
mass spectrometry (ESI-MS, spectra not shown); an aromatic hydroxylation on the B ring resulted in
the product 6c+OH (m/z 434.2) and O-demethylation gave the most abundant 6cO-CH3 product (m/z
404.1) based on the peak intensity.
Scheme 2. The oxidative transformation routes of compound 6c based on the information obtained
with the chemical Fenton system.
These results were also confirmed by LC-MS analysis. The place of the aromatic hydroxylation and
O-dealkylation was deduced from the characteristic retro Diels-Alder rearrangement fragmentation
pattern of the 3-hydroxyflavone backbone; however, due to the limitations of the detection method
used, the exact positions are yet to be determined. The combination of electrochemical oxidation and
the porphyrin system could yield additional oxidation products, such as secondary amine 6cN-CH3
after N-dealkylation, which would have the same m/z as 6cO-CH3. The 6cN-CH3 could undergo
another N-dealkylation, which results in the 6cN-2CH3 primary amine.

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3. Materials and Methods
3.1. Chemistry
3.1.1. General Information
Thin-layer chromatography with aluminium backed TLC plates of silica gel 60 F254
(0.2 mm, Merck, Darmstadt, Germany) under UV light was used to monitor the reactions.
Column chromatography was performed on silica gel (60, 70–230 mesh, Merck, Darmstadt,
Germany). Melting points were determined by Büchi B-540 apparatus (Büchi Labortechnik AG, Flawil,
Switzerland). The purity of the compounds was evaluated using an LTQ XL linear ion trap mass
spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with positive ESI. 1H-NMR and 13C-NMR
1
spectra were acquired on a Bruker AM 360 (360.13 MHz for H, 90.03 MHz for 13C) spectrometer
(Bruker, Billerica, MA, USA). Chemical shifts (
δ
) are given from internal CHCl3 (
δ = 7.26 ppm) or TMS
(δ
= 0.00 ppm) signals for 1H-NMR and CHCl3 (
δ
= 77.00 ppm) or DMSO (
δ
= 39.52 ppm) for 13C-NMR
and reported in parts per million (ppm, d). Coupling constants (J) were measured and reported in
hertz (Hz). Spectra can be found in Supplementary Materials. Elemental analyses (C, H, N) were
performed using the Elementar Vario MicroCube instrument (Elementar Analysensysteme GmbH,
Langenselbold, Germany). Infrared spectra were obtained in KBr discs using a JASCO FT-IR 4100A
Fourier-transform infrared spectrometer (Jasco Inc, Easton, MD, USA).
3.1.2. General Procedure for the Synthesis of 3a–c
To the stirred solution of the acetophenone (1a
,b, 1.29 g, 6 mmol) in MeOH (10 mL) 50% aq.
NaOH (1.26 mL, 24 mmol) was added. Benzaldehyde (2a
,
b, 6.3 mmol) suspension in MeOH (5 mL)
was added to the solution of the acetophenone and the mixture was stirred for 1 h, and then it was
allowed to stand at room temperature for one day. HCl solution (10%, w/v) was added to reach pH 1;
the precipitate was filtered off and washed with water (3 × 30 mL) to give 3a–c (88–97%).
1
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3a). H NMR (300 MHz, 298 K,
0
CDCl3):
J = 7.35 Hz, 1H, 6-H), 7.37-7.41 (d, J = 15.25 Hz, 1H,
15.34 Hz, 1H, β-H), 8.00–8.00 (d, J = 1.62 Hz, 60-H), 12.89 (s, 1H, OH).
δ
(ppm) = 3.95–3.98 (m, 6H, OMe), 6.89–6.93 (m, 2H, 5-H, 3 -H), 7.17 (s, 1H, 2-H), 7.27–7.29 (d,
α
-H), 7.53–7.56 (dd, 1H, 40-H), 7.87–7.92 (d, J =
1
(E)-1-(4-Bromo-2-hydroxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3b). H NMR (360 MHz, 298 K,
CDCl3): (ppm) = 3.94–3.96 (m, 6H, OMe), 6.89–6.91 (d, J = 8.36 Hz, 1H, 5-H), 7.03–7.06 (dd, 1H, 6-H),
δ
0
0
7.14–7.19 (m, 2H, 2-H, 3 -H), 7.23–7.26 (dd, 1H, 5 -H), 7.38–7.42 (d, J = 15.51 Hz, 1H, α-H), 7.73–7.76 (d,
J = 8.36 Hz, 1H, 60-H), 7.85-7.89 (d, J = 15.63 Hz, 1H, β-H), 13.09 (s, 1H, OH).
(E)-1-(4-Bromo-2-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3c). 1H NMR (400 MHz,
298 K, CDCl3):
δ (ppm) = 3.92–3.93 (m, 9H, OMe), 6.87 (s, 2H, 2-H, 6-H), 7.05–7.07 (d, J = 7.70 Hz,
1H, 50-H), 7.20 (s, 1H, 30-H), 7.41–7.45 (d, J = 15.40 Hz, 1H, -H), 7.74–7.77 (d, J = 7.70 Hz, 1H, 60-H),
α
7.82–7.86 (d, J = 15.40 Hz, 1H, β-H), 12.99 (s, 1H, OH).
3.1.3. General Procedure for the Synthesis of 4a–c
To the water-bath cooled suspension of chalcone (3a–c, 2.7 mmol) in EtOH (15 mL), 8% aq.
NaOH (3.9 mL, 8.41 mmol) was added to result in a solution. To the mixture, 30% H2O2 (3.9 mL,
38.2 mmol) was added dropwise, and then it was stirred at room temperature for 2 h. The reaction
mixture was poured into ice-water mixture (250 mL) and 10% HCl solution was added to reach pH 1.
The precipitate was allowed to sedimentate for one day, then filtered off and washed with cc. NaHCO3
solution (2 × 50 mL) and water (4 × 50 mL) to give 4a–c (57–76%).
6-Bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one (4a). 1H NMR (360 MHz, 298 K,
DMSO-D6):
δ
(ppm) = 3.85 (s, 6H, OMe), 7.14–7.16 (d, J = 7.85 Hz, 1H, 50-H), 7.80–7.82 (m, 3H,

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20-H, 60-H), 7.87–7.90 (d, J = 8.16 Hz, 1H, 8-H), 7.93–7.96 (dd, 7-H), 8.16–8.16 (d, J = 1.92 Hz, 1H, 5-H),
9.72 (s, 1H, OH).
7-Bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one (4b).1H NMR (400 MHz, 298 K, DMSO-D6):
0 0
(ppm) = 3.85–3.86 (m, 6H, OMe), 7.14–7.16 (d, J = 8.02 Hz, 1H, 5 -H), 7.61–7.64 (dd, 1H, 6 -H) 7.81–7.81
δ
(d, J = 1.67 Hz, 1H, 20-H), 7.90–7.93 (dd, 1H, 6-H), 8.00–8.02 (d, J = 8.55 Hz, 5-H), 8.20–8.20 (d, J =
1.44 Hz, 1H, 8-H), 9.63 (s, 1H, OH).
7-Bromo-3-hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (4c). 1H NMR (400 MHz, 298 K,
DMSO-D6):
δ
(ppm) = 3.75 (s, 3H, 40-MeO), 3.86 (s, 6H, 30-MeO, 50-MeO), 7.56 (s, 2H, 20-H, 60-H),
7.59–7.62 (d, J = 8.46 Hz, 1H, 6-H), 7.98–8.00 (d, J = 8.46 Hz, 5-H), 8.21 (s, 1H, 8-H), 9.74 (s, 1H, OH).
3.1.4. General Procedure for the Synthesis of 6a–f
Into a pressure tube, under argon, the mixture of 3-hydroxyflavone (4a–c, 0.265 mmol), KF
(46.3 mg, 0.795 mmol), Pd(OAc)2 (3 mg, 0.0133 mmol) and XPhos (12.6 mg, 0.0265 mmol), and boronic
acid (5a,b, 0.53 mmol) in toluene/t-BuOH (6:1, 3.5 mL) were added. The mixture was stirred and
heated in a 100 ◦C oil bath for 4 h. The solvent was removed under reduced pressure, and the residue
was purified by absorptive filtration using toluene/EtOAc (2:1) as the eluent. The crude product was
washed with i-Pr2O and filtered to give pure products 6a–f (63–74%).
2-(3,4-Dimethoxyphenyl)-6-[3-(dimethylamino)phenyl]-3-hydroxy-4H-chromen-4-one (6a). Yellow solid;
1
yield 69.1 mg (63%). Mp. 187.7–188.8 ◦C. Rf: 0.35 (toluene/EtOAc, 2:1). H NMR (360 MHz, 298 K,
CDCl3):
δ (ppm) = 3.02 (s, 6H, N(Me)2), 3.95–3.99 (m, 6H, OMe), 6.75–6.77 (d, J = 7.35 Hz, 1H, 6”-H),
6.97–6.99 (m, 3H, 8-H, 50-H, 2”-H), 7.13–7.17 (m, 1H, 20-H), 7.30–7.34 (m, 1H, 5”-H), 7.60 (s, 1H, OH),
7.85–7.90 (m, 3H, 7-H, 6 -H, 4”-H), 8.42 (s, 1H, 5-H). 13C NMR (91 MHz, 298 K, CDCl3):
δ (ppm) = 40.8
0
(C-N(Me)2), 56.0, 56.1 (C-30-MeO, 40-MeO), 110.8 (C-20), 111.1 (C-50), 111.3 (C-2”), 112.2 (C-4”), 115.7
(C-6”), 118.5 (C-8), 120.8 (C-10), 121.6 (C-60), 123.1 (C-5), 123.8 (C-4a), 129.7 (C-5”), 132.8 (C-7), 137.9
0
0
(C-3), 138.7 (C-6), 140.3 (C-1”), 145.2 (C-4 ), 148.9 (C-3 ), 150.8(C-3”), 151.1 (C-2), 154.6 (C-8a),173.3 (C-4).
LC-MS: m/z = 418.42 [M + H]+. IR (KBr, cm−1):
= 3292, 2934, 2839, 1605, 1561, 1516, 1489, 1463.71,
ν
1430, 1381, 1338, 1271, 1247, 1216, 1195, 1149, 1118, 1042, 1024, 991, 964, 933, 901, 869, 814, 770, 724, 694,
665, 634. Anal. Calcd for C25H23NO5: C, 71.93; H, 5.55; Found: C 71.79; H 5.52.
2-(3,4-Dimethoxyphenyl)-3-hydroxy-6-[4-(methoxymethyl)phenyl]-4H-chromen-4-one (6b). Yellow solid;
1
yield 80.3 mg (72%). Mp. 192.8–194.3 ◦C. Rf: 0.30 (toluene/EtOAc, 2:1). H NMR (360 MHz, 298 K,
CDCl3):
δ
(ppm) = 3.43 (s, 3H, Bn-MeO), 3.96–4.00 (m, 6H, 30-MeO, 40-MeO), 4.52 (s, 2H, Bn-H2),
7.00–7.02 (d, J = 7.37 Hz, 1H, 50-H), 7.08 (s, 1H, 20-H), 7.43–7.45 (d, J = 7.55 Hz, 2H, 2”-H, 6”-H),
7.62–7.69 (m, 3H, OH, 3”-H, 5”-H), 7.86–7.93 (m, 3H, 7-H, 8-H, 6”-H), 8.43 (s, 1H. 5-H). 13C NMR
(91 MHz, 298 K, CDCl3):
δ
(ppm) = 56.1, 56.1 (C-30-MeO, 40-MeO), 58.4 (C-Bn-MeO), 74.4 (C-Bn-CH2),
110.9 (C-20), 111.1 (C-50), 118.8 (C-8), 120.9 (C-10), 121.6 (C-60), 123.1 (C-5), 123.8 (C-4a), 127.3 (C-2”,
C-6”), 128.4 (C-3”, C-5”), 132.4 (C-7), 137.4 (C-4”),138.0 (C-3), 138.1 (C-6), 138.7 (C-1”), 145.2 (C-40),
149.0 (C-30), 150.9 (C-2), 154.7 (C-8a), 173.2 (C-4). LC-MS: m/z = 419.33 [M + H]+. IR (KBr, cm−1):
ν =
3265, 3030, 2993, 2939, 2833, 2737, 2603, 2039, 1922, 1843, 1801, 1714, 1601, 1561, 1516, 1483, 1459, 1411,
1388, 1336, 1298, 1269, 1218, 1197, 1174, 1148, 1112, 1025, 967, 944, 932, 904, 875, 857, 815, 806, 784, 770,
727, 699, 661, 629. Anal. Calcd for C25H22O6: C, 71.76; H, 5.30; Found: C 71.95; H 5.29.
2-(3,4-Dimethoxyphenyl)-7-[3-(dimethylamino)phenyl]-3-hydroxy-4H-chromen-4-one (6c). Pale-brown solid; yield
1
79.0 mg (71%). Mp. 191.6moved outside the brackets192.7 ◦C. Rf: 0.39 (toluene/EtOAc, 2:1). H NMR
(400 MHz, 298 K, CDCl3):
1H, 6”-H), 6.97–7.02 (m, 3H, 8-H, 50-H, 2”-H), 7.10 (s, 1H, 20-H), 7.33–7.37 (m, 1H, 5”-H), 7.63–7.65 (d, J =
7.84 Hz, 1H, 6-H), 7.75 (s, 1H, OH), 7.87–7.91 (m, 2H, 60-H, 4”-H), 8.24–8.26 (d, J = 7.83 Hz, 1H, 5-H). 13
δ (ppm) = 3.04 (s, 6H, N(Me)2), 3.96–3.99 (m, 6H, OMe), 6.79–6.81 (d, J = 7.84 Hz,
C
NMR (101 MHz, 298 K, CDCl3):
δ
(ppm) = 40.8 (C-N(Me)2), 56.1, 56.2 (C-30-MeO, 40-MeO), 110.9 (C-20),
111.0 (C-50), 111.4 (C-2”), 112.9 (C-4”), 115.9 (C-6”), 116.2 (C-8), 119.4 (C-10), 121.5 (C-60), 123.9 (C-4a), 124.1
(C-6), 125.7 (C-5), 129.8 (C-5”), 138.0 (C-3), 140.3 (C-1”), 145.3 (C-40), 147.8 (C-7), 149.0 (C-30), 150.8 (C-3”),

Molecules 2018, 23, 3161
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151.1 (C-2), 155.6 (C-8a), 173.0 (C-4). LC-MS: m/z = 418.42 [M + H]+. IR (KBr, cm−1):
ν
= 3228, 3028, 2992,
2919, 2847, 2800, 2592, 2034, 1940, 1885, 1713, 1617, 1576, 1555, 1514, 1489, 1453, 1428, 1397, 1346, 1265,
1236, 1213, 1171, 1147, 1111, 1039, 1024, 994, 969, 921, 883, 861, 847, 835, 821, 771, 717, 699, 648, 639. Anal.
Calcd for C25H23NO5: C, 71.93; H, 5.55; Found: C 71.97; H 5.52.
2-(3,4-Dimethoxyphenyl)-3-hydroxy-7-[4-(methoxymethyl)phenyl]-4H-chromen-4-one (6d). Yellow solid;
1
yield 79.3 mg (72%). Mp. 212.1–214.2 ◦C. Rf: 0.31 (toluene/EtOAc, 2:1). H NMR (400 MHz, 298 K,
CDCl3):
δ
(ppm) = 3.44 (s, 3H, Bn-MeO), 3.96–4.00 (m, 6H, 30-MeO, 40-MeO), 4.53 (s, 2H, Bn-H2),
6.99–7.01 (d, J = 7.46 Hz, 1H, 50-H), 7.10 (s, 1H, 20-H), 7.46–7.48 (m, 2H, 2”-H, 6”-H), 7.62–7.69 (m, 3H,
6-H, 3”-H, 5”-H), 7.75 (s, 1H, OH), 7.85–7.91 (m, 2H, 8-H, 60-H), 8.25–8.27 (d, J = 7.46 Hz, 1H, 5-H).
13C NMR (101 MHz, 298 K, CDCl3):
δ
(ppm) = 56.1, 56.1 (C-30-MeO, 40-MeO), 58.4 (C-Bn-MeO), 74.3
(C-Bn-CH2), 110.8 (C-20), 111.0 (C-50), 116.0 (C-8), 119.5 (C-10), 121.6 (C-60), 123.7 (C-6), 123.8 (C-4a),
125.9 (C-5), 127.5 (C-2”, C-6”), 128.4 (C-3”, C-5”), 138.0 (C-3), 138.5 (C-4”), 139.1 (C-1”), 145.3 (C-40),
146.3 (C-7), 149.0 (C-30), 150.8 (C-2), 155.6 (C-8a), 172.9 (C-4). LC-MS: m/z = 419.42 [M + H]+. IR (KBr,
cm−1):
ν = 3234, 3022, 2977, 2920, 2839, 2597, 2030, 1923, 1848, 1797, 1714, 1611, 1574, 1555, 1515, 1488,
1467, 1450, 1417, 1397, 1354, 1333, 1271, 1243, 1212, 1173, 1147, 1103, 1039, 1020, 914, 858, 843, 818, 807,
774, 706, 635. Anal. Calcd for C25H22O6: C, 71.76; H, 5.30; Found: C 71.70; H 5.32.
7-[3-(Dimethylamino)phenyl]-3-hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (6e). Pale-brown
1
solid; yield 88.2 mg (74%). Mp. 179.5–181.8 ◦C. Rf: 0.47 (toluene/EtOAc, 2:1). H NMR (360 MHz,
298 K, CDCl3):
δ
(ppm) = 3.04 (s, 6H, N(Me)2), 3.95 (s, 3H, 40-MeO), 3.97 (s, 6H, 30-MeO, 50-MeO),
6.80–6.82 (d, J = 7.17 Hz, 1H, 6”-H), 6.97–7.03 (m, 2H, 8-H, 2”-H), 7.15–7.17 (d, J = 7.17 Hz, 1H, 4”-H),
7.34–7.37 (m, 1H, 5”-H), 7.56 (s, 2H, 20-H, 60-H), 7.64–7.66 (d, J = 7.96 Hz, 1H, 6-H), 7.76 (s, 1H, OH),
8.25–8.27 (d, J = 8.22 Hz, 1H, 5-H). 13C NMR (91 MHz, 298 K, CDCl3):
δ (ppm) = 40.8 (C-N(Me)2), 56.4
(C-30-MeO, 50-MeO), 61.1 (C-40), 105.6 (C-20 C-60), 111.5 (C-2”), 113.0 (C-4”), 115.9 (C-6”), 116.3 (C-8),
119.3 (C-10), 126.4 (C-4a), 124.3 (C-6), 125.7 (C-5), 129.9 (C-5”), 138.4 (C-3), 140.2 (C-1”), 144.8 (C-40),
0
0
148.1 (C-7), 151.1 (C-2), 151.2 (C-3”), 153.3 (C-3 , C-5 ), 155.7 (C-8a), 173.2 (C-4). LC-MS: m/z = 448.33 [M
+ H]+. IR (KBr, cm−1):
= 3247, 3079, 2999, 2968, 2938, 2838, 1603, 1579, 1556, 1504, 1453, 1427, 1393,
ν
1353, 1283, 1263, 1242, 1209, 1172, 1127, 1057, 1027, 1011, 994, 928, 841, 831, 821, 773, 734, 714, 697, 652.
Anal. Calcd for C26H25NO6: C, 69.79; H, 5.63; Found: C 69.86; H 5.59.
3-Hydroxy-7-[4-(methoxymethyl)phenyl]-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (6f). Brown solid;
1
yield 82.0 mg (69%). Mp. 208.1–209.2 ◦C. Rf: 0.48 (toluene/EtOAc, 2:1). H NMR (400 MHz, 298 K,
CDCl3):
2H, Bn-H2), 7.15 (s, 1H, 8-H), 7.48 (s, 2H, 20-H, 60-H), 7.56 (s, 2H, 2”-H, 6”-H), 7.63–7.69 (m, 3H, 6-H,
δ
(ppm) = 3.45 (s, 3H, Bn-MeO), 3.95 (s, 3H, 40-MeO), 3.98 (s, 6H, 30-MeO, 50-MeO), 4.54 (s,
3”-H, 5”-H), 7.77 (s, 1H, OH), 8.26–8.28 (d, J = 7.47 Hz, 1H, 5-H). 13C NMR (101 MHz, 298 K, CDCl3):
δ
(ppm) = 56.4 (C-30-MeO, 50-MeO), 58.4 (C-Bn-MeO), 61.1 (C-40), 74.3 (C-Bn-CH2), 105.5 (C-20), 116.1
(C-8), 119.4 (C-10), 123.9 (C-6), 126.4 (C-4a), 126.0 (C-5), 127.5 (C-2”, C-6”), 128.4 (C-3”, C-5”), 138.4
(C-3), 138.5 (C-4”), 139.2 (C-1”), 140.1 (C-2), 144.8 (C-40), 146.6 (C-7), 153.3 (C-30, C-50), 155.7 (C-8a),
173.1 (C-4). LC-MS: m/z = 449.33 [M + H]+. IR (KBr, cm−1):
ν = 3278, 3010, 2983, 2969, 2939, 2899, 2837,
2154, 2064, 2010, 1928, 1602, 1583, 1574, 1551, 1508, 1491, 1451, 1409, 1394, 1379, 1338, 1296, 1244, 1211,
1194, 1172, 1130, 1114, 1056, 1026, 1011, 970, 936, 930, 913, 887, 844, 824, 779, 765, 712, 671, 624. Anal.
Calcd for C26H24O7: C, 69.63; H, 5.39; Found: C 69.57; H 5.43.
3.2. Antioxidant Activity and Cytotoxicity
3.2.1. Chemicals
Water (ultra-pure) was prepared with the SolPure 78 water purification system from
POL-LAB (Bielsko-Biała, Poland). Ethylenediaminetetraacetic acid disodium salt dihydrate
(EDTA-Na2), L(+)-ascorbic acid, ethanol (96%), acetic acid, and formic acid were obtained
from Scharlab Magyarország Kft.
(Debrecen, Hungary).
DMSO, methanol, hydrogen
peroxide solution, 2,2-diphenyl-1-picrylhydrazyl (DPPH), glacial acetic acid, sodium acetate, HCl,

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2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), FeCl3, iron(II) sulfate heptahydrate, fluorescein, 2,20-azobis
(2-amidinopropane) dihydrochloride (AAPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), potassium hydrogen phosphate, monopotassium phosphate, and K2S2O8 were
obtained from Sigma-Aldrich Kft. (Budapest, Hungary). Acetonitrile (ACN) was ordered from
Merck (Darmstadt, Germany). 2,20-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium
salt (ABTS salt) was purchased from Fluorochem Ltd. (Hadfield, United Kingdom).
3.2.2. ABTS Assay
To measure the ABTS radical scavenging activity, the work of Sugahara et al. and Re et al. was
followed closely, applying few alterations [34
K2S2O8 in water was kept in the dark for 16 h to form ABTS•+. The working solution was prepared
by adding 150 L of this working solution
L of ABTS•+ solution to 2.9 mL MeOH. Aliquots of 180
were pipetted into wells containing 20 L of test samples in DMSO with different concentrations (10,
20, 50, 100, and 200 M). Absorbance was measured at 737 nm after 10 s mixing using a Multiskan
,35]. A mixture containing 7 mM ABTS and 2.45 mM
µ
µ
µ
µ
GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 0, 1, 2, 3, 4, 5,
10, 15, 30, 45, 60, 75, 90, 105, and 120 min. Quercetin was used as the standard. The IC50 values were
calculated based on the inhibition percentage measured at 120 min. The experiments were carried out
in duplicate and repeated three times.
3.2.3. DPPH Assay
The method used for the DPPH radical scavenging assay was adopted from Clarke et al., with
modifications [36]. A total of 180
µL freshly made 0.2 mM DPPH solution in methanol was added to
20 L of the investigated compounds in various concentrations (10, 20, 50, 100, and 200
µ
µM) in DMSO.
After a gentle, 10-s mixing period, the plate was kept in the dark for 90 min. and the absorbance
was read at 515 nm using a Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific,
Waltham, MA, USA) at 0, 1, 2, 3, 4, 5, 10, 15, 30, 45, 60, 75, and 90 min. Blanks (20 µL DMSO with 180 µL
MeOH) and quercetin standards in the same concentration as the tested compounds were measured
simultaneously. The IC50 values were calculated based on the inhibition percentage measured at
90 min. The measurements were performed in duplicate and repeated three times.
3.2.4. FRAP Assay
The ferric reducing antioxidant power of the compounds was tested with a modified method
of Benzie and Strain [37]. The FRAP reagent consisted of 10 mL acetate buffer (300 mM, pH 3.6),
1 mL TPTZ solution (10 mM) in 40 mM HCl, and 1 mL FeCl3 (10 mM). The mixture was incubated
at 37 ◦C for 15 min and used on the same day. The reaction mixture was prepared by 20
µL of the
investigated compound (10, 20, 50, 100, and 200 µM) and 180 µL activated FRAP reagent in a 96-well
plate. Following a 30 min incubation period, the absorbance was measured at 593 nm by a Multiskan
GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Iron (II) sulfate
heptahydrate was used as the standard in different concentrations (0.5, 1, 5, 10, 50, 100, and 500
µM).
The FRAP values are expressed as
repeated three times.
µM ferrous equivalents. The experiments were run in duplicate and
3.2.5. ORAC Assay
The oxygen radical absorbance capacity was evaluated using the ORAC assay. The test was
carried out in a black 96-well plate with a black bottom. The stock solutions of 50 nM fluorescein and
180 mM AAPH in phosphate buffer (75 mM, pH 7.00) were prepared freshly and used on the same day.
To each well, 20
of blank samples, 20
20
monitored at 485 nm excitation and 520 nm emission wavelength for 2 h every 2 min by the FLUOstar
µ
L of compound in ACN (2 and 10
µ
M) was added to 160
µ
L fluores◦cein. In the case
µL of ACN was added. The plate was incubated for 15 min at 37 C, and then the
µL AAPH solution was rapidly added to each well to initiate the reaction. The fluorescence was

Molecules 2018, 23, 3161
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OPTIMA (BMG Labtech, Ortenberg, Germany) plate reader. The assay was performed in duplicate
and repeated three times. The area under the curve (AUC) was calculated as follows:
AUC = 0.5 + (A1/A0) + (A2/A0) + (A3/A0) + · · · + (An/A0),
(1)
where A0 is the fluorescence reading at 0 min and An is the fluorescence reading at n min. The reported
ORAC values were calculated by subtracting the blank AUC from the sample AUC and expressed
as AUCnet
.
AUCnet = AUCsample − AUCblank
(2)
3.2.6. MTT Assay
The MTT assay was used to determine the effect of the investigated compounds on the cell
viability. H9c2 cells (ATCC, CRL-1446, LGC Standards GmbH, Wesel, Germany) were seeded into
96-well plates at a density of 6000 cells/well in 200
Thermo Fischer Scientific with 10% FBS and supplemented with penicillin and streptomycin) and
cultured overnight. The flavonols were added to the cells in 20 M concentration. After 12 h, 20
of MTT solution (5 mg/ L in PBS) was added to each well, and the plates were incubated for 4 h.
Afterwards, the media was carefully removed and 100 L DMSO was added to each well to dissolve
µL media (DMEM, high glucose, pyruvate from
µ
µL
µ
µ
the formazan crystals. Absorbance was measured at 540 and 630 nm using a Synergy HT plate reader
(BioTek, Winooski, VT, USA). The tests were carried out in triplicate and repeated two times.
3.3. Oxidative Transformation
Chemical Fenton Reaction
The Fenton reaction of the compounds was carried out based on a method reported previously by
Csepanyi et al. [18]. The reaction mixture consisted of 400
ACN, 50 L 20 mM FeCl3, 50 L 20 mM EDTA-Na2, and 500
(50:50) and 2 L 30% hydrogen peroxide. The reaction mixture was stirred at room temperature at
µL 2.5 mM of the investigated compound in
µ
µ
µL 10 mM ascorbic acid in ACN/H2O
µ
700 rpm. Samples were drawn at 2, 24, 72, and 144 h and were diluted with ACN/H2O (50:50) and
analyzed immediately by an API 2000 Triple Quadrupole mass spectrometer (Applied Biosystems,
Waltham, MA, USA) equipped with a syringe pump with the following parameters: 20 µL/min flow
rate, 10 PSI curtain gas, 20 V declustering potential, 4000 V ion spray voltage, 400 V focusing potential,
and 200 ◦C ion source temperature. The positive ion electrospray mass spectra were recorded in the
range of m/z 100–500 using Analyst 1.5.1. Software (AB SCIEX, Concord, ON, Canada). In additional
experiments, the reaction mixture was separated and analyzed with an LTQ XL linear ion trap mass
spectrometer coupled with the Accela LC system (Thermo Fisher Scientific, Waltham, MA, USA).
The HPLC separation was performed using a Kinetex XB-C18 2.6
µm column, 0.1% formic acid in
water, and ACN with 0.1% formic acid with gradient elution, and the flow rate was set to 300
µL/min.
The method parameters for mass spectrometry were the followings: 35 a.u. sheath gas flow rate, 5000 V
spray voltage, 300 ◦C capillary temperature, 31 V capillary voltage, 150 V tube lens voltage, and 34 V
skimmer voltage.
4. Conclusions
In conclusion, six new flavonol derivatives were designed and synthetized, and their biological
activity was evaluated. The results of four antioxidant assays showed that three compounds (6a
and 6e) were potent antioxidants. Each of these molecules bear with a phenyl-N,N-dimethylamino
group on ring A. Furthermore, compounds 6a 6c, and 6e did not show any cytotoxic activity on
H9c2 cells during the MTT assay, when 20 M solutions were tested. According to the experimental
, 6c,
,
µ
data, the position of the phenyl-N,N-dimethylamino group did not significantly affect the antioxidant
potential. However, because compound 6c showed improved antioxidant activity among compounds

Molecules 2018, 23, 3161
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with a phenyl-N,N-dimethylamino substituent, this molecule was selected for further studies, in which
the oxidative biotransformation of the molecule was modeled by a biomimetic system; the chemical
Fenton reaction. As a result of these experiments, two potential metabolites were detected, which
were the products of O-demethylation and aromatic hydroxylation of compound 6c. These results
identify compound 6c as a potential subject of additional investigations and a prototype of promising
flavonol-type antioxidants in further developments. Furthermore, to the authors’ best knowledge,
this is the first time that the dimethylamino group has been proven to be involved in the antioxidant
activity in flavonols.
Supplementary Materials: The supplementary materials are available online.
Author Contributions: Conceptualization, I.B.; Data curation, P.S.-F.; Funding acquisition, A.T. and I.B.;
Investigation, P.S.-F., D.P., A.B., and A.K.-S.; Methodology, A.K.-S.; Project administration, I.B.; Resources, I.B.;
Supervision, I.B.; Visualization, P.S.-F., D.P., and A.B.; Writing – original draft, P.S.-F.; Writing—review & editing,
P.S.-F., E.C., A.K.-S., and I.B.
Funding: This study was supported by the GINOP-2.3.2-15-2016-00043 project and co-financed by the European
Union and the European Regional Development Fund. The project was subsidized by the European Union and
co-financed by the European Social Fund.
Acknowledgments: The work of Peter Szabados-Furjesi and David Pajtas was supported by the
EFOP-3.6.1-16-2016-00022 “Debrecen Venture Catapult Program”. Evelin Csepanyi assisted in fundamental
research in the frame of TÁMOP-4.2.4. A/2-11-1-2012-0001. National Excellence Program-elaborating and
operating an inland student and researcher personal support system was realized with personal support.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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