The scavenging of DPPH, galvinoxyl and ABTS radicals by imine analogs of resveratrol

Article abstract of DOI:10.3390/molecules21010127

Resveratrol (3,5,41-trihydroxystilbene) is a phytoalexin produced by plants. Resveratrol is known for its anti-cancer, antiviral and antioxidant properties. We prepared imine analogs of resveratrol ((hydroxyphenyliminomethyl)phenols) and tested their anti

Full text of DOI:10.3390/molecules21010127

molecules
Communication
The Scavenging of DPPH, Galvinoxyl and ABTS
Radicals by Imine Analogs of Resveratrol
1
Peter Kotora 1,*, František Šeršenˇ , Juraj Filo 1, Dušan Loos 1, Juraj Gregánˇ 2,3,* and
4
Fridrich Gregánˇ
Received: 14 December 2015 ; Accepted: 14 January 2016 ; Published: 21 January 2016
Academic Editor: Derek J. McPhee
1
Institute of Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina,
842 15 Bratislava, Slovakia; f.sersen@gmail.com (F.Š.); filo@fns.uniba.sk (J.F.); dusan.looos@chello.sk (D.L.)
Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina,
2
842 15 Bratislava, Slovakia
3
Department of Chromosome Biology, MFPL, University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria
Department of Chemistry, Faculty of Natural Sciences, Matej Bell University, Tajovského 40,
4
974 01 Banská Bystrica, Slovakia; Fridrich.Gregan@umb.sk
*
Correspondence: kotora@fns.uniba.sk (P.K.); juraj.gregan@univie.ac.at (J.G.); Tel.: +421-2-60296-412 (P.K.);
+421-2-60296-158 (J.G.)
Abstract: Resveratrol (3,5,41-trihydroxystilbene) is a phytoalexin produced by plants. Resveratrol is
known for its anti-cancer, antiviral and antioxidant properties. We prepared imine analogs of
resveratrol ((hydroxyphenyliminomethyl)phenols) and tested their antioxidant activity. All prepared
resveratrol analogs were able to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH), galvinoxyl radical
1
(GOR) and 2,2 -azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) radicals. The antioxidant
activity efficiency correlated with the number and position of hydroxyl groups. The most effective
antioxidants were resveratrol analogs containing three hydroxyl groups in the benzylidene part
of their molecules. These results provide new insights into the relationship between the chemical
structure and biological activity of resveratrol analogs.
Keywords: resveratrol analogs; iminophenols; antioxidant activity
1. Introduction
The structure of (hydroxyphenyliminomethyl)phenols (Figure 1) is similar to the structure of
resveratrol (3,5,41-trihydroxystilbene), which exhibits various biological activities. There are two
geometric isomers of resveratrol—the trans configuration undergoes isomerisation to the cis form
following exposure to ultraviolet light. Resveratrol is a phytoalexin produced by several plants that
was initially identified in the flowering plant Veratrum grandiflorum. Relatively high concentrations
of resveratrol are also found in grape skins and seeds, as well as red wine. Resveratrol has gained
widespread attention due to its ability to extend the lifespan of various organisms, to protect against
age-related diseases such as cancer and a broad range of biological activities including antiviral and
antioxidant properties [
wine drinking (“French paradox”, the observation of reduced incidence of cardiovascular disease in
regions of France where red wine and fats are consumed in large quantities) are due to resveratrol [ ].
Molecular mechanisms underlying the health-enhancing effects of resveratrol are not well understood
but some of them may be due to its antioxidant properties [ ]. Although basal level of oxidative stress
is important for normal cell function, severe oxidative stress often leads to oxidative damage, cell
death and various diseases [ ]. Resveratrol supplementation decreases the oxidative stress caused by
1–4]. However, it is not clear whether the health benefits associated with
5
6
7
Molecules 2016, 21, 127; doi:10.3390/molecules21010127
www.mdpi.com/journal/molecules

Molecules 2016, 21, 127
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consumption of a high calorie meal [
8]. In addition, resveratrol is a potent activator of SIRT1, member
of sirtuin family of protein deacetylases implicated in counteracting various age-related diseases [9].
8
OH
R
5
B
R
A
B
4
3
HO
7
R
R
R
N
A
6
R
1
R
2
OH
R
resveratrol
iminophenols
Figure 1. Structure of resveratrol and studied Hydroxyphenyliminomethyl)phenols.
Previous studies showed that modification of resveratrol could enhance its biological
activities [10–12]. It is therefore important to synthesize resveratrol analogs and study their
physico-chemical and biological properties. Imines are often used as ligands for the complexation
of metals; they are used for the preparation of liquid crystals and are also used in analytical and
pharmaceutical chemistry [13]. The traditional method of the imine preparation is based on heating
aldehydes with amines in an organic solvent (benzene or toluene) [13–15]. Synthesis in which the
organic solvent is replaced with water [16] or other recycle medium such as polypropylene glycol [17
has been described as “green” alternative to these preparations.
]
The goal of this work was to prepare various imine analogs of resveratrol (some of which have
not been described in the literature yet) and to test their antioxidant activity. We analyzed antioxidant
properties of twenty-one (hydroxyphenyliminomethyl)phenols (Figure 1) where Ri = OH or H.
The studied (hydroxyphenyliminomethyl)phenols consist of two parts; one is aminophenol (AP)
and the other is phenolic part from hydroxybenzaldehyde (HBA). Some of these compounds are
produced as secondary metabolites in plants. 4-HBA and 3,4-dihydroxy-benzaldehyde (DHBA) are
present in Origanum vulgare L. [18], oil palm biomass [19], Potentilla fulgens [20], sugarcane molasses [21
and vanilla pods [22]. 4-HBA and 4-AP are present in Dilobeia thouarsii [23]. Many HBAs are known
for their flavoring and antimicrobial properties [24 25]. Antioxidant properties of some HBAs (2-HBA,
3-HBA, 3,4-DHBA) were studied by the Tsimidou group [26 27]. Some of imine resveratrol anologs
were studied as multifunctional drugs in the treatment of Alzheimer’s dissease [28] and they also
exhibit antioxidant activity [28 29]. Some hydroxybenzene diimine derivatives possess important
]
,
,
,
biological activities such as antioxidant [30], antiinflammatory and anti-analgesics [31].
2. Results and Discussion
2.1. Chemistry
The starting compounds for preparation of (hydroxyphenyliminomethyl)phenols were mono
aminophenols (APs) and mono-, di- and trihydroxybenzaldehydes (HBAs). Twenty-one (hydroxy
phenyliminomethyl)phenols (compounds 1–21, for full names see Section 3.2. Synthesis) were prepared
by condensation reactions where the first step was addition of amino group on carbonyl group followed
by elimination of water according to protocols of Tanaka and Shiraishi [16] and Lu et al. [29] (Scheme 1).
Briefly, equimolar amounts of˝appropriate AP and HBA were stirred in a small amount of distilled
water at room temperature (25 C) for 2 h. Obtained products were filtered, washed several times with
˝
distilled water and dried at 45 C in vacuum. Yields of (hydroxyphenyliminomethyl)phenols ranged
between 26% and 95%. The compounds were characterized by melting point, elemental analysis,
1H- and 13C-NMR, IR and MS spectra.

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8
R
5
2
5
2
R
R
B
4
8
4
3
7
R
CHO
R
R
water, rt, 2 h
R
N
+
6
+ H2O
B
A
A
R
7
1
3
1
R
R
R
R
R
H N
2
6
R
R
R
Scheme 1. Preparation of iminophenol analogs of resveratrol.
2.2. Antioxidant Activity
All prepared (hydroxyphenyliminomethyl)phenols
picrylhydrazyl (DPPH), galvinoxyl and 2,2 -azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS)
radicals (Table 1). This table shows that some of the prepared (hydroxyphenyliminomethyl)phenols
1–21 were able to scavenge 2,2-diphenyl-1-
1
were more effective scavengers of DPPH (compounds:
8, 10, 12, 14, 17–21) or galvinoxyl (GOR)
(compounds 13 16 17 21) as compared to resveratrol (Table 1). On the other hand all prepared
2,
9,
,
,
–
(hydroxyphenyliminomethyl)phenols exhibited lower efficiency of ABTS scavenging as resveratrol,
their SC50 values ranged from 1.98 to 18.16 µmol/dm3.
Table 1. SC50 and proton affinity (PA) + electron transfer enthalpy (ETE) values of studied (hydroxy
phenyliminomethyl)phenols.
DPPH SC50/r2
GOR SC50/r2
ABTS SC50/r2
PA + ETE in
Methanol (kJ/mol) Water (kJ/mol)
PA + ETE in
Compound
(µmol/dm3)
(µmol/dm3)
(µmol/dm3)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
27.90/0.951
38.26/0.963
560/0.893
967/0.976
383/0.936
184/0.985
48.27/0.998
3075/0.809
415/0.995
393/0.903
251/0.999
11.64/0.969
8.50/0.809
8.50/0.928
3.54/0.993
6.74/0.869
3.86/0.994
6.57/954
14.39/0.972
18.16/0.922
6.4/0.988
3.05/0.847
3.31/0.968
6.40/0.977
5.83/0.988
3.74/0.918
2.53/0.962
2.01/0.989
2.83/0.956
2.46/0.977
2.92/0.9997
1.98/0.994
1.43/0.959
546.8
550.9
547.7
565.4
547.1
550.1
547.4
543.1
520.8
539.6
554.6
541.8
542.7
547.5
540.3
554.9
537.4
525.8
531.1
548.4
528.5
548.6
562.2
566.0
562.2
580.7
562.1
564.4
562.8
551.9
537.4
554.7
569.9
554.0
557.6
573.9
555.4
570.2
552.7
540.7
546.5
563.1
544.0
563.5
88.64/0.974
53.98/0.937
21.00/0.997
43.00/0.991
19.00/0.994
83.00/0.947
24.00/0.970
18.00/0.988
12.52/0.964
42.00/0.986
149/0.979
22.05/0.988
12.60/0.996
9.05/0.988
18.05/0.988
8.77/0.943
26.37/0.849
184/0.979
127/0.971
39.96/0.945
2300/0.987
73.78/0.998
456/0.995
23.34/0.997
102/0.865
55.43/0.727
173/0.988
17
18
19
20
21
25.24/0.989
27.67/0.993
72.10/0.958
23.92/0.985
15.39/0.994
72.66/0.910
Resveratrol
r2 is an average square deviation.
Antioxidant activities of compounds
SC50 values reported by Lu et al. [29] and Li et al. [28] are similar to those presented in our current
study. However, our melting points NMR spectra of compounds and do not correspond with those
reported by Li et al. [28]. The chemical shift for the compound published by Li et al. [30] for OH
(in meta position of ring A) = 14.21 is too high and = 9.63 for compound is too small. This raises the
1, 2, 3, 4, 5, 7, 9 and 16 have already been studied [28,29].
2
9
2
δ
δ
9
possibility that compounds 2 and 9 prepared by Li et al. [28] are not identical to the denoted structures.
We found that the efficiency of the radical scavenging correlated with the position and number
of hydroxyl groups in prepared compounds. Regarding the position of the hydroxyl group in
hydroxyphenylimino parts of prepared compounds, most (hydroxyphenyliminomethyl)phenols
with the OH group in ortho position of the ring A were the most potent scavengers of DPPH and
galvinoxyl radicals. (Hydroxyphenyliminomethyl)phenols with three OH groups in the ring A were

Molecules 2016, 21, 127
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the most potent scavengers of ABTS radicals. (Hydroxyphenyliminomethyl)phenols 2, 4, 6, 7, 11, 16
which contained one or two OH groups in positions 3 and 4 of the ring A exhibited relatively low
ability to scavenge DPPH or GOR radicals (their values of SC50 ranged from 38 to 3075
mol/dm3).
However, compounds and 5, which have OH group in the ortho position of the benzene ring A,
µ
3
exhibited low antioxidant activity. On the other hand, compunds 17 and 21, which do not have OH
groups in the ortho position of the phenyl ring A, exhibited high antioxidant activity.
Based on the 1H-NMR spectra analyses, we assumed that proton could be most easily dissociation
from the OH group in the ortho position of the ring A. (Hydroxyphenyliminomethyl)phenols with
R1 = OH could form intramolecular hydrogen bond with the nitrogen of imine group. Due to this
interaction acidity of hydrogen at this position increased, which was confirmed by the high chemical
shift (δ = 12.25 to 14.19 ppm). On the other hand, the chemical shift of the OH protons bound in the
meta or para position were <8.10 ppm.
It is generally assumed that the mechanism of radical scavenging by phenolic compounds is
associated with the ability to release hydrogen atom, proton or electron from the antioxidant molecule.
We speculated that releasing of the proton from OH group could be the main mechanism respossible
for the antioxidant activity of studied (Hydroxyphenyliminomethyl)phenols. Consistent with this
assumption were the SC50 values of studied compounds with higher
δ which exhibited higher
antioxidant activity as compared to other (Hydroxyphenyliminomethyl)phenols.
To find a possible correlation between the antioxidant activity of (hydroxyphenyliminome
thyl)phenols and a position of the hydroxyl groups, we used the method PM6 (see the Experimental
Section) to calculate the energy associated with the release of hydrogen (bond dissociation enthalpy,
BDE); the proton relaxation (PDE); release of electron (IP ionization potential) and the energy
associated with combined transfer of the electron and proton (ETE and PA, respectively) for the
studied (Hydroxyphenyliminomethyl)phenols. The values of these energies in methanol or in water
are presented in Tables S1–S8.
The antioxidant mechanism associated with the release of a hydrogen atom can be described by
the Equation (1). From the point of view of thermodynamics the strength of the antioxidant effect in
the model describes BDE (bond dissociation enthalpy, Equation (2)):
‚
‚
‚
ArOH ` R Ñ ArO ` R
(1)
(2)
‚
‚
BDE “ HpArO q ` HpH q ´ HpAr ´ OHq
The other antioxidant mechanism of (hydroxyphenyliminomethyl)phenols may be associated
with combined transfer of electrons and protons to radical (R‚). In the first step, the electron is
transferred to the radical (Equation (3)). The possibility of such transition is determined by the size of
ionization potential of IP, which describes the Equation (4):
‚
‚`
´
ArOH ` R Ñ Ar ´ OH ` R
(3)
‚`
´
IP “ HpAr ´ OH q ` Hpe q ´ HpAr ´ OHq
(4)
In the second step, a proton is transferred to the anion radical R´ (Equation (5)). The possibility
of such a transfer is characterized by the value of PDE (proton dissotiation enthalpy, Equation (6)):
‚`
´
‚
Ar ´ OH ` R Ñ ArO ` RH
(5)
(6)
‚
‚`
PDE “ HpArO q ` HpH+q ´ HpAr ´ OH
q
Another possible mechanism of the antioxidant effect of (hydroxyphenyliminomethyl)phenols is
the gradual loss of a proton followed by electron transfer (Equations (7) and (8)):
´
ArOH Ñ ArO ` H+
(7)

Molecules 2016, 21, 127
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´
‚
‚
´
ArO ` R Ñ ArO ` R
(8)
(9)
´
R
` H+ Ñ RH
The reaction enthalpy of the first step (Equation (7)) corresponds to the proton affinity PA
(Equation (10)) whereas the reaction enthalpy of the second step (Equation (8)) describes enthalpy of
electron transfer (ETE, Equation (11)):
´
PA “ HpArO q ` HpH+q ´ HpAr ´ OHq
(10)
(11)
‚
´
´
ETE “ HpArO q ` Hpe q ´ HpArO q
Comparison of enthalpies required for the above mentioned antioxidant reactions suggests that
the preferred mechanism seems to be the one described by Equations (7)–(11), those characterized by
the values of PA + ETE. Based on the above mentioned ideas, we speculated that the lower PA + ETE
values of studied (hydroxyphenyliminomethyl)phenols may imply their higher antioxidant activity,
i.e., lower value of SC50. Figure 2 shows the dependence of SC50 scavenging DPPH radicals on the sum
of PA + ETE enthalpy. As can be seen from these figures, there is no obvious relationship between these
parameters. This discrepancy could be caused by forming of (hydroxyphenyliminomethyl)phenols
self-aggregates which could reduce the antioxidant activity of original compounds.
1000
DPPH
GOR
3000
2500
2000
1500
1000
500
800
600
400
200
0
0
520
540
560
520
540
560
PA + ETE [kJ/mol]
PA + ETE [kJ/mol]
20
ABTS
15
10
5
0
540
550
560
570
580
PA + ETE [kJ/mol]
Figure 2. Dependence of SC50 of DPPH, GOR and ABTS scavenging by studied (Hydroxypheny
liminomethyl)phenols on PA + ETE entalpies. Dashed lines denote the SC50 values of resveratrol.

Molecules 2016, 21, 127
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To test this assumption we performed the following experiment. According to the work of
Cigánˇ et al. [32], creation of self-aggregates can be determined from concentration-dependence of
absorption or fluorescence spectra. The formation of self-aggregates should be manifested by a shift
of emission maxima or by a shape change in absorption/fluorescence spectra with an increase of
concentration of the studied compound. We observed that the corresponding changes in absorption
spectra were very small. Also in fluorescence emission spectra, relatively small batochromic shifts
were observed with increasing concentrations. Compound
5 with lower antioxidant activity exhibited
approximately 11 nm, whereas compound 14 with higher antioxidant activity showed only 4 nm
(data not shown). The formation of self-aggregates was apparent from the fluorescence excitation
spectra. We observed changes in shapes of these spectra with the increasing concentrations of
(Hydroxyphenyliminomethyl)phenols (Figure 3). These results suggest that one possible explanation
for the lower antioxidant activity of some of the studied (Hydroxyphenyliminomethyl)phenols is
formation of self-aggregates. A possible structure of aggregates for compound
Figure S1.
3
is presented in
3
3
3
5 mg/dm
5 mg/dm
A
0.9 mg/dm
C
B
1.0
0.8
0.6
0.4
0.2
0.0
3
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
3
3
50 mg/dm
= 500 nm
50 mg/dm
= 530 nm
9 mg/dm
λ
em
λ
λ
= 500 nm
em
em
300
350
400
450
300
350
400
450
300
350
400
450
500
λ
[nm]
λ
[nm]
λ
[nm]
exc
exc
exc
Figure 3. Fluorescence excitation spectra of studied (Hydroxyphenyliminomethyl)phenols:
and 15 (C
5
(
A
); 14 (B)
) in methanol at concentration of 0.9 mg/dm3. The excitation and emission slits were 2 resp.
10 nm.
Numerous studies showed that various modifications of resveratrol molecule enhance its
biological activities including the antioxidant properties [10 12 33]. It is therefore important to
–
,
synthesize new resveratrol analogs and study their physico-chemical and biological properties.
We aimed to identify novel analogues of resveratrol that could potently scavenge radicals and serve
as leads for the development of future drugs. To find out more about potential pharmacological and
nutritional relevance of (Hydroxyphenyliminomethyl)phenols synthesized in our current study, it will
be important to test in vivo activities of these compounds. However, given their unstability in water,
additional chemical modifications may be required to achieve high in vivo activity. Our current study
brings new insights into the relationship between the chemical structure and antioxidant properties of
resveratrol analogs.
3. Experimental Section
3.1. General Information
Methyl alcohol p.a. was purchased from Centralchem (Bratislava, Slovakia). 2-Aminophenol,
3-amino-phenol, 4-aminophenol, 2-hydroxybenzaldehyde, 3-hydroxybenzaldehyde, 4-hydroxy-
benzaldehyde, 2,3-dihydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde,
3,4-dihydroxybenzaldehyde, 3,5-dihydroxybenzaldehyde, 2,3,4-trihydroxybenzaldehyde, 2,4,6-trihyd
roxybenzaldehyde, 3,4,5-trihydroxybenzaldehyde, 2,2-diphenyl-1-picrylhydrazyl, were purchased
1
from Alfa Aesar (Heysham, UK). 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt (ABTS) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Molecules 2016, 21, 127
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Absorption spectra were recorded by a Genesis 6 spectrophotometer (Thermo–Scientific, Waltham,
MA, USA). Fluorescence emission spectra were recorded by a FSP 920 spectrofluorimeter (Edinburgh
Instruments, Livingston, UK) and 1H- (300 MHz) and 13C-NMR (75 MHz) spectra were registered on a
VNMRS 300 spectrometer (Varian, Salt Lake City, UT, USA) in DMSO-d6 with tetramethylsilane as
internal standard. Melting points were determined on a Kofler hot plate apparatus and are uncorrected.
Elemental analyses were obtained on an Elemental Analyzer CHNS-OEA 1108 (Carlo Erba, Wigan,
UK). FTIR spectra (in solid phase) were recorded on a Nicolet 6700 spectrometer (Thermo–Scientific
(Nicolet), Waltham, MA, USA) using the ATR technique.
MS spectra were recorded by a LC-MS spectrometer consisting of an Agilent 1200 HPLC (Walbron,
Germany), with a MSD 6110 MS detector (Agilent Technologies, Santa Clara, CA, USA).
3.2. Synthesis
General Procedure for the Synthesis of (Hydroxyphenyliminomethyl)phenols
Twenty-one (hydroxyphenyliminomethyl)phenols were prepared following the protocol
described by Tanaka and Shiraishi [16]. Briefly, equimolar amounts of mono-, di- and tri- hydroxy
benzaldehydes (HBA) and 2-, 3- or 4-aminophenols (AP) were stirred (2 h) in distilled water at room
temperature (25 ˝C). Obtained products were filtered, washed by water and dried at 45 ˝C. Yields of
(Hydroxyphenyliminomethyl)phenols ranged between 36%–95%. The prepared compounds were
characterized by melting point, elemental analysis, 1H- and 13C-NMR, IR and MS spectra.
3-{[(2-Hydroxyphenyl)imino]methyl}phenol (
(213.24) C, 73.22; H, 5.20; N, 6.57. Found: C, 73.43; H, 5.04; N, 6.74%. IR: 1627.4 (v/C=N). 1H-NMR
(DMSO-d6) : 13.77 (s, 1H), 9.71 (s, 1H), 8.96 (s, 1H), 7.61 (dd, J = 7.9, 1.3 Hz, 1H), 7.46–7.27 (m, 2H),
1
): Yield 77%; m.p. 182–184 ˝C. Anal. Calc. for C13H11NO2
δ
7.12 (ddd, 1H), 7.03–6.80 (m, 4H). 13C-NMR (DMSO-d6)
δ: 162.10, 161.16, 151.56, 135.36, 133.26, 132.74,
128.49, 120.03, 120.00, 119.93, 119.16, 117.12, 116.95; positive LC-MS m/z: 214.1 [M + H]+ calc. for
C13H12NO2+, 214.09, found 214.1
2-{[(3-Hydroxyphenyl)imino]methyl}phenol (
(213.24) C, 73.22; H, 5.20; N, 6.57. Found: C, 73.47; H, 5.09; N, 6.50%. IR: 1619.9 (v/C=N).1H-NMR
(DMSO-d6) : 9.64 (s, 1H), 8.97 (s, 1H), 8.58 (s, 1H), 7.41 (d, J = 7.8 Hz, 2H), 7.30 (dd, J = 7.7 Hz, 1H), 7.14
(d, J = 7.7 Hz, 1H), 7.07 (dd, J = 7.0 Hz, 1H), 7.00–6.73 (m, 3H).13C-NMR (DMSO-d6)
: 160.04, 158.02,
2
): Yield 71%; m.p. 116–118 ˝C. Anal. Calc. for C13H11NO2
δ
δ
151.35, 138.64, 138.16, 130.09, 127.58, 120.77, 119.89, 119.81, 118.91, 116.40, 115.05; positive LC-MS m/z:
214.1 [M + H]+ calc. for C13H12NO2+, 214.09, found 214.1.
2-{[(3-Hydroxyphenyl)imino]methyl}phenol (
(213.24) C, 73.22; H, 5.20; N, 6.57. Found: C, 73.10; H, 5.13; N, 6.51%. IR: 1602.8 (v/C=N). 1H-NMR
3
): Yield 69%; m.p. 119–120 ˝C. Anal. Calc. for C13H11NO2
(DMSO-d6)
δ
: 13.12 (s, 1H), 9.64 (s, 1H), 8.90 (s, 1H), 7.65 (dd, J = 7.6, 1.5 Hz, 1H), 7.41 (ddd, J = 8.2
,
1.6 Hz, 1H), 7.25 (dd, J = 7.9 Hz, 1H), 6.97 (dd, J = 8.3 Hz, 2H), 6.83 (d, J = 7.8 Hz, 1H), 6.77 (dd, J = 2.0 Hz
,
1H), 6.73 (dd, J = 8.0, 1.7 Hz, 1H). 13C-NMR (DMSO-d6)
δ
: 163.64, 160.74, 158.75, 149.70, 133.65, 133.02,
130.62, 119.66, 119.52, 116.99, 114.51, 112.49, 108.56; positive LC-MS m/z: 214.1 [M + H]+ calc. for
C13H12NO2+, 214.09, found 214.1.
3-{[(4-Hydroxyphenyl)imino]methyl}phenol (
(213.24) C, 73.22; H, 5.20; N, 6.57. Found: C, 73.38; H, 5.23; N, 6.62%. IR: 1625.5 (v/C=N). 1H-NMR
(DMSO-d6) : 10.09 (s, 1H), 9.44 (s, 1H), 8.39 (s, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.15 (t, J = 7.9 Hz, 1H), 6.87
4
): Yield 78%; m.p. 210–212 ˝C. Anal. Calc. for C13H11NO2
δ
(d, J = 8.4 Hz, 2H), 6.60 (dd, 3H). 13C-NMR (DMSO-d6)
δ: 161.00, 160.01, 158.49, 153.82, 131.07, 130.22,
127.94, 116.07, 112.86, 112.02, 108.19; positive LC-MS m/z: 214.1 [M + H]+ calc. for C13H12NO2+, 214.09,
found 214.1.
2-{[(4-Hydroxyphenyl)imino]methyl}phenol (
(213.24) C, 73.22; H, 5.20; N, 6.57. Found: C, 73.40; H, 4.98; N, 6.49%. IR: 1614.3 (v/C=N). 1H-NMR
(DMSO-d6) : 13.42 (s, 1H), 9.67 (s, 1H), 8.90 (s, 1H), 7.59 (dd, J = 7.6, 1.4 Hz, 1H), 7.47–7.21 (m, 3H),
5
): Yield 38%; m.p. 138–139 ˝C. Anal. Calc. for C13H11NO2
δ

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7.08–6.89 (m, 2H), 6.86 (dd, J = 5.9 Hz, 2H). 13C-NMR (DMSO-d6)
δ
: 160.61, 160.58, 157.40, 139.61,
132.93, 132.63, 123.08, 119.86, 119.39, 116.89, 116.39; positive LC-MS m/z: 214.1 [M + H]+ calc. for
C13H12NO2+, 214.09, found 214.1.
3-{[(4-Hydroxyphenyl)imino]methyl}phenol (
(213.24) C, 73.22; H, 5.20; N, 6.57. Found: C, 73.04; H, 5.25; N, 6.41%. IR: 1622.4. (v/C=N),1H-NMR
(DMSO-d6) : 9.62 (s, 1H), 9.48 (s, 1H), 8.51 (s, 1H), 7.41–7.23 (m, 3H), 7.18 (d, J = 8.7 Hz, 2H), 6.99–6.84
6
): Yield 84%; m.p. 172–173 ˝C. Anal. Calc. for C13H11NO2
δ
(m, 1H), 6.80 (d, J = 8.7 Hz, 2H). 13C-NMR (DMSO-d6)
δ: 157.67, 157.18, 156.27, 142.59, 137.86, 129.78,
122.51, 119.99, 118.19, 115.74, 113.89; positive LC-MS m/z: 214.1 [M + H]+ calc. for C13H12NO2+, 214.09,
found 214.1.
4-{[(4-Hydroxyphenyl)imino]methyl}phenol (
(213.24) C, 73.22; H, 5.20; N, 6.57. Found: C, 73.48; H, 5.02; N, 6.33%. IR: 1639.6 (v/C=N). 1H-NMR
(DMSO-d6) : 10.00 (s, 1H), 9.37 (s, 1H), 8.44 (s, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.11 (d, J = 8.7 Hz, 2H),
7
): Yield 82%; m.p. 183–185 ˝C. Anal. Calc. for C13H11NO2
δ
6.86 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 8.7 Hz, 2H). 13C-NMR (DMSO-d6)
δ: 160.10, 156.89, 155.68, 143.21,
130.18, 127.91, 122.15, 115.64, 115.58; positive LC-MS m/z: 214.1 [M + H]+ calc. for C13H12NO2+, 214.09,
found 214.1.
3-{[(2-Hydroxyphenyl)imino]methyl}benzene-1,2-diol (
C13H11NO3 (229.24) C, 68.11; H, 4.84; N, 6.11. Found: C, 68.29; H, 5.06; N, 6.14 %. IR: 1627.7 (v/C=N).
1H-NMR (DMSO-d6)
: 14.19 (s, 1H), 9.83 (s, 1H), 9.02 (s, 1H), 8.94 (s, 1H), 7.41 (dd, J = 7.8, 1.5 Hz, 1H),
7.13 (ddd, J = 7.8, 1.5 Hz, 1H), 7.04 (dd, J = 7.8, 1.5 Hz, 1H), 6.98 (dd, J = 8.1, 1.2 Hz, 1H), 6.94–6.85
8
): Yield 75%; m.p. 168–169 ˝C. Anal. Calc. for
δ
(
m, 2H), 6.72 (dd, J = 7.8 Hz, 1H). 13C-NMR (DMSO-d6)
δ: 161.63, 152.02, 151.29, 146.49, 134.38, 128.40,
122.89, 120.06, 119.60, 119.41, 118.47, 118.30, 116.91; positive LC-MS m/z: 230.1 [M + H]+ calc. for
C13H12NO3+, 230.08, found 230.1.
˝
9): Yield 77%; m.p. 152–155 C Anal. Calc. for
4-{[(2-Hydroxyphenyl)imino]methyl}benzene-1,3-diol (
C13H11NO3 (229.24) C, 68.11; H, 4.84; N, 6.11. Found: C, 68.29; H, 5.06; N, 6.14%. IR: 1627.7 (v/C=N),
1H-NMR (DMSO-d6)
: 14.20 (s, 1H), 10.13 (s, 1H), 9.62 (s, 1H), 8.76 (s, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.28
(dd, J = 7.9, 1.5 Hz, 1H), 7.10–7.01 (m, 1H), 6.91 (dd, J = 8.0, 1.3 Hz, 1H), 6.83 (td, J = 7.8, 1.4 Hz, 1H),
6.32 (dd, J = 8.5, 2.3 Hz, 1H), 6.21 (d, J = 2.3 Hz, 1H). 13C-NMR (DMSO-d6)
: 165.40, 162.90, 160.66,
δ
δ
150.90, 134.91, 134.62, 127.52, 120.03, 119.47, 116.75, 112.66, 107.94, 103.09; positive LC-MS m/z: 230.0
[M + H]+ calc. for C13H12NO3+, 230.08, found 230.0.
2-{[(2-Hydroxyphenyl)imino]methyl}benzene-1,4-diol (10): Yield 71%; m.p. 135–137 ˝C. Anal. Calc. for
C13H11NO3 (229.24) C, 68.11; H, 4.84; N, 6.11. Found: C, 67.92; H, 4.72; N, 5.88 %. IR: 1638.5 (v/C=N),
1H-NMR (DMSO-d6)
δ: 12.86 (s, 1H), 9.63 (s, 1H), 9.03 (s, 1H), 8.83 (s, 1H), 7.32 (dd, J = 7.9, 1.5 Hz,
1H), 7.16–7.05 (m, 1H), 7.03–6.72 (m, 5H). 13C-NMR (DMSO-d6)
δ: 162.02, 153.79, 151.48, 149.80, 135.91,
128.28, 121.09, 120.05, 120.01, 119.90, 117.56, 117.31, 116.90; positive LC-MS m/z: 230.1 [M + H]+ calc.
for C13H12NO3+, 230.08, found 230.1.
5-{[(2-Hydroxyphenyl)imino]methyl}benzene-1,3-diol (11): Yield 26%; m.p. 174–175 ˝C. Anal. Calc. for
C13H11NO3 (229.24) C, 68.11; H, 4.84; N, 6.11. Found: C, 68.32; H, 4.69; N, 5.86%. IR: 1628.1 (v/C=N).
1H-NMR (DMSO-d6):
δ
= 9.45 (s, 2H), 8.98 (s, 1H), 8.44 (s, 1H), 7.10 (dd, J = 7.8, 1.5 Hz, 1H), 7.05 (ddd,
J = 7.8, 7.7, 1.5, 1H), 6.87 (dd, J = 8.0, 1.3 Hz, 1H), 6.85 (d, J = 2.2 Hz, 2H), 6.81 (ddd, J = 7.7, 7.4, 1.3 Hz,
1H), 6.36 (dd, J = 2.2 Hz, 1H). 13C-NMR (DMSO-d6):
δ = 160.40, 158.97, 151.18, 138.87, 138.64, 127.40,
119.98, 119.88, 116.35, 107.30, 105.99; positive LC-MS m/z: 230.1 [M + H]+ calc. for C13H12NO3+, 230.08,
found 230.1.
3-{[(3-Hydroxyphenyl)imino]methyl}benzene-1,2-diol (12): Yield 79%; m.p. 184–185 ˝C. Anal. Calc. for
C13H11NO3 (229.24) C, 68.11; H, 4.84; N, 6.11. Found: C, 68.32; H, 4.65; N, 5.87%. IR: 1632.9 (v/C=N),
1H-NMR (DMSO-d6)
δ
: 13.25 (s, 1H), 9.64 (s, 1H), 9.18 (s, 1H), 8.86 (s, 1H), 7.25 (dd, J = 7.9 Hz, 1H),
7.09 (dd, J = 7.8, 1.4 Hz, 1H), 6.94 (dd, J = 7.8, 1.4 Hz, 1H), 6.88–6.67 (m, 4H). 13C-NMR (DMSO-d6)

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δ
: 163.57, 158.34, 149.48, 149.01, 145.62, 130.24, 122.81, 119.28, 118.91, 118.72, 114.07, 111.97, 108.13;
positive LC-MS m/z: 230.1 [M + H]+ calc. for C13H12NO3+, 230.08, found 230.1.
2-{[(3-Hydroxyphenyl)imino]methyl}benzene-1,4-diol (13): Yield 89%; m.p. 175–177 ˝C. Anal. Calc. for
C13H11NO3 (229.24) C, 68.11; H, 4.84; N, 6.11. Found: 68.30; H, 4.73; N, 5.91%. IR: 1634.1 (v/C=N),
1H-NMR (DMSO-d6)
δ: 12.27 (s, 1H), 9.62 (s, 1H), 9.08 (s, 1H), 8.78 (s, 1H), 7.23 (dd, J = 7.9 Hz, 1H),
7.03 (d, J = 2.9 Hz, 1H), 6.91–6.64 (m, 5H). 13C-NMR (DMSO-d6)
δ: 163.29, 158.70, 153.50, 150.11, 150.02,
130.56, 121.47, 119.65, 117.55, 117.38, 114.32, 112.46, 108.53; positive LC-MS m/z: 230.1 [M + H]+ calc.
for C13H12NO3+, 230.08, found 230.1.
4-{[(4-Hydroxyphenyl)imino]methyl}benzene-1,3-diol (14): Yield 55%; m.p. 130–132 ˝C. Anal. Calc. for
C13H11NO3 (229.24) C, 68.11; H, 4.84; N, 6.11. Found: 68.27; H, 5.06; N, 5.90%. IR: 1617.4 (v/C=N).
1H-NMR (DMSO-d6)
δ
: 13.80 (s, 1H), 10.13 (s, 1H), 9.55 (s, 1H), 8.72 (s, 1H), 7.37 (d, J = 8.5 Hz, 1H),
7.29–7.16 (m, 2H), 6.86–6.76 (m, 2H), 6.38 (dd, J = 8.4, 2.3 Hz, 1H), 6.27 (d, J = 2.3 Hz, 1H). 13C-NMR
(DMSO-d6) δ: 163.19, 162.23, 160.18, 156.67, 139.86, 134.33, 122.57, 116.31, 112.59, 107.98, 102.79; positive
LC-MS m/z: 230.1 [M + H]+ calc. for C13H12NO3+, 230.08, found 230.1.
2-{[(4-Hydroxyphenyl)imino]methyl}benzene-1,4-diol (15): Yield 92%; m.p. 196–197 ˝C. Anal. Calc. for
C13H11NO3 (229.24) C, 68.11; H, 4.84; N, 6.11. Found: 67.92; H, 4.97; N, 6.26%. IR: 1624.5 (v/C=N).
1H-NMR (DMSO-d6)
δ: 12.58 (s, 1H), 9.64 (s, 1H), 9.04 (s, 1H), 8.79 (s, 1H), 7.29 (d, J = 8.7 Hz, 2H), 6.98
(d, J = 2.7 Hz, 1H), 6.93–6.52 (m, 4H). 13C-NMR (DMSO-d6)
δ
: 160.39, 157.26, 153.35, 149.94, 139.99,
123.05, 120.70, 119.82, 117.40, 117.27, 116.35; positive LC-MS m/z: 230.1 [M + H]+ calc. for C13H12NO3
,
+
230.08, found 230.1.
5-{[(4-Hydroxyphenyl)imino]methyl}benzene-1,3-diol (16): Yield 92%; m.p. 118–120 ˝C. Anal. Calc. for
C13H11NO3 (229.24) C, 68.11; H, 4.84; N, 6.11. Found: 67.92; H, 4.97; N, 6.26%. IR: 1620.5 (v/C=N).
1H-NMR (DMSO-d6)
δ: 9.44 (s, 3H), 8.38 (s, 1H), 7.15 (d, J = 8.7 Hz, 2H), 6.87–6.69 (m, 4H), 6.32
(
dd, J = 2.1 Hz, 1H). 13C-NMR (DMSO-d6)
δ: 159.05, 157.85, 156.61, 143.02, 138.78, 122.90, 116.12,
106.80, 105.66; positive LC-MS m/z: 230.1 [M + H]+ calc. for C13H12NO3+, 230.08, found 230.1.
4-{[(3-Hydroxyphenyl)imino]methyl}benzene-1,2,3-triol (17): Yield 89%; m.p. 254–256 ˝C. Anal. Calc. for
C13H11NO4 (245.24) C, 63.67; H, 4.52; N, 5.71. Found: 63.96; H, 4.48; N, 5.50%. IR: 1606.2 (v/C=N).
1H-NMR (DMSO-d6)
δ
: 13.70 (s, 1H), 9.63 (d, 2H), 8.72 (s, 1H), 8.43 (s, 1H), 7.22 (dd, J = 7.9 Hz, 1H),
6.94 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 8.9 Hz, 1H), 6.76–6.62 (m, 2H), 6.40 (d, J = 8.5 Hz, 1H). 13C-NMR
(DMSO-d6) δ: 163.25, 158.96, 152.61, 150.89, 149.38, 133.05, 130.83, 124.74, 114.04, 112.82, 112.23, 108.41;
positive LC-MS m/z: 246.1 [M + H]+, 268.0 [M + Na]+. Calc. for C13H12NO3+, 246.08, found 246.1.
2-{[(3-Hydroxyphenyl)imino]methyl}benzene-1,3,5-triol (18): Yield 94%; m.p. 257–259 ˝C. Anal. Calc. for
C13H11NO4 (245.24) C, 63.67; H, 4.52; N, 5.71. Found: 63.87; H, 4.39; N, 5.78%. IR: 1626.1 (v/C=N).
1H-NMR (DMSO-d6)
δ: 12.25 (s, 2H), 10.07 (s, 1H), 9.56 (s, 1H), 8.87 (s, 1H), 7.20 (dd, J = 11.1, 5.5 Hz,
1H), 6.68 (dd, J = 18.1, 7.3 Hz, 3H), 5.81 (s, 2H). 13C-NMR (DMSO-d6)
δ: 163.91, 162.62, 158.33, 157.10,
149.27, 130.28, 113.11, 111.57, 107.33, 101.46, 94.10; positive LC-MS m/z: 246.1 [M + H]+, calc. for
C13H12NO3+, 246.08, found 246.1.
5-{[(3-Hydroxyphenyl)imino]methyl}benzene-1,2,3-triol (19): Yield 47%; m.p. 283–285 ˝C. Anal. Calc. for
C13H11NO4 (245.24) C, 63.67; H, 4.52; N, 5.71. Found: 63.77; H, 4.39; N, 5.78%. IR: 1644.2 (v/C=N).
1H-NMR (DMSO-d6)
δ
: 9.42 (s, 1H), 9.17 (s, 2H), 8.76 (s, 1H), 8.22 (s, 1H), 7.14 (dd, J = 7.9 Hz, 1H), 6.87
: 192.00, 160.66, 158.66, 153.92, 150.43, 146.78, 146.68,
(s, 2H), 6.70–6.48 (m, 3H)m. 13C-NMR (DMSO-d6)
δ
140.86, 137.71, 130.43, 130.07, 128.10, 127.40, 112.97, 112.21, 109.41, 108.56, 108.35, 106.07, 103.94, 101.60;
positive LC-MS m/z: 246.1 [M + H]+, 268.1 [M + Na]+. Calc. for C13H12NO3+, 246.08, found 246.1.
2-{[(4-Hydroxyphenyl)imino]methyl}benzene-1,3,5-triol (20): Yield 37%; m.p. 173–175 ˝C. Anal. Calc. for
C13H11NO4 (245.24) C, 63.67; H, 4.52; N, 5.71. Found: C, 63.76; H, 4.69; N, 5.88%. IR: 1616.0 (v/C=N).
1H-NMR (DMSO-d6)
δ: 12.27 (s, 2H), 9.94 (s, 1H), 9.48 (s, 1H), 8.85 (s, 1H), 7.14 (d, J = 8.7 Hz, 2H), 6.79

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(d, J = 8.7 Hz, 2H), 5.79 (s, 2H). 13C-NMR (DMSO-d6)
δ
: 163.53, 156.34, 155.39, 140.10, 122.21, 116.39,
101.85, 94.44; positive LC-MS m/z: 246.1 [M + H]+, calc. for C13H12NO3+, 246.08, found 246.1.
5-{[(4-Hydroxyphenyl)imino]methyl}benzene-1,2,3-triol (21): Yield 89%; m.p. 228–229 ˝C. Anal. Calc. for
C13H11NO4 (245.24) C, 63.67; H, 4.52; N, 5.71. Found: C, 63.80; H, 4.41; N, 5.87%. IR: 1640.0 (v/C=N).
1H-NMR (DMSO-d6)
δ: 9.35 (s, 1H), 9.11 (s, 2H), 8.59 (d, 1H), 8.26 (s, 1H), 7.23–7.00 (m, 2H), 6.85
(
d, J = 2.8 Hz, 2H), 6.81–6.67 (m, 2H). 13C-NMR (DMSO-d6)
δ: 168.82, 148.74, 145.74, 140.95, 136.83,
124.97, 116.02, 115.85, 107.57; positive LC-MS m/z: 246.1 [M + H]+, 268.0 [M + Na]+. Calc. for
C13H12NO3+, 246.08, found 246.1.
3.3. DPPH Assay
Scavenging of DPPH radicals by prepared (hydroxyphenyliminomethyl)phenols was performed
according to our previous work Šeršenˇ et al. [34]. Briefly, various amounts of tested (Hydroxy
phenyliminomethyl)phenols were added into methanol solution of DPPH and the final DPPH
concentration was kept constant (c = 10´4 mol dm´3). 20 min after the addition of the tested substance,
¨
absorbance was measured at 517 nm. The antioxidant activity was evaluated using the values SC50,
i.e., concentration of the studied compound, which causes a 50% decrease in absorbance at 517 nm as
compared to the control sample. Methanol was used as a blank.
3.4. Scavenging of Galvinoxyl Radicals
Various amounts of (hydroxyphenyliminomethyl)phenols were added into methanol solution of
galvinoxyl radical such that the final galvinoxyl concentration was c = 10´4 mol dm´3. 20 min after the
¨
addition of (hydroxyphenyliminomethyl)phenols, absorbance was measured at 862 nm. SC50 values
were calculated from the absorbance vs. the concentration dependence. Methanol was used as a blank.
3.5. ABTS Assay
ABTS cation radicals were prepared according to Kurin et al. [35] with minor adaptations.
Briefly 7 mM aqueous solution of ABTS was mixed in equimolar ratio with 2.45 mM K2S2O8, and let
24 h to stand in the dark at room temperature. After 24 h 1.1 mL of concentrated solution of the radical
was diluted with 50 mL of phosphate buffer (solution A: 900 mg of Na2HPO4 in 500 mL H2O, the
solution B: 340 mg of KH2HPO4 in 500 mL H2O, we combined 9 parts of solution A and 1 part of
solution B, the pH we set to 7.4). To a solution of 1.8 mL of ABTS we added 0, 25, 50, 75, 100, 125, 150,
175 or 200 mL of the test substance and completed with 200, 175, 150, 125, 100, 75, 50, 25 or 0 mL of
ethanol (to a final volume of 2 mL. The absorbance at 734 nm of prepared solutions was measured
after 6 min. We calculated the appropriate values of the SC50 from the linear part of the absorbance vs.
the concentration of (hydroxyphenyliminomethyl)phenols. The water was used as blank.
3.6. Molecular Calculations
The prepared (hydroxyphenyliminomethyl)phenols, their anions and radicals were studied
using the quantum chemical method PM6 [36], which is part of the program MOPAC2012 [37].
Optimal structures of compunds were calculated (keyword PRECISE). The effect of solvents on
the above mentioned compounds were studied by COSMO-method [38], which is also part of the
MOPAC2012 [39]. Ionization potentials and heats of formations used for the calculation of PDE, BDE,
PA and ETE according to the Equations (2), (6), (10) and (11) were obtained using the method PM6.
4. Conclusions
In this work we prepared twenty-one (hydroxyphenyliminomethyl)phenols, which were
synthesized by condensation reactions of the corresponding aminophenols and hydroxybenzaldehydes.
The chemical structures of these compounds were confirmed by 1H-NMR, IR spectroscopy,
elemental analysis and GCMS. All prepared (hydroxyphenyliminomethyl)phenols were able to

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scavenge DPPH, GOR and ABTS radicals. Some of the (hydroxyphenyliminomethyl)phenols
were more potent scavengers of DPPH (compounds: 10 12 14 11 27) and GOR (compounds:
12 15 17 21) radicals as compared to resveratrol. The efficiency of antioxidant activity
8,
,
–
,
–
2
,
9
,
,
,
–
correlated with the number and position of hydroxyl groups. The most efficient antioxidants were
(hydroxyphenyliminomethyl)phenols containing three hydroxyl groups in the benzylidene part
of molecules.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/1/127/s1.
Acknowledgments: This work was supported by the Slovak Grant Agency VEGA 1/0612/11 and 1/0196/14,
projects ITMS 26240220071, APVV-0334-12, APVV-0061-11, PCIG11-GA-2012-322300 and The Austrian Science
Fund (FWF) grants P23609 and P21437. We are greatful to Julia Greganova for her help.
Author Contributions: F.G., F.Š., P.K. and J.F. performed experiments. D.L. performed calculations. F.Š., F.G. and
J.G. analysed results and wrote the manuscript. All authors contributed to the paper and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
1
ABTS
AP
2,2 -azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid
aminophenol
BDE
DPPH
ETE
GOR
HBA
IP
bond dissociation enthalpy
2,2-diphenyl-1-picrylhydrazyl
electron transfer enthalpy
galvinoxyl radical
hydroxybenzaldehyde
ionization potential
infrared
IR
MS
NMR
PA
mass spectrometry
nuclear magnetic resonance
proton affinity
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