Synthesis, structure, and antioxidant activity of methoxy- and hydroxyl-substituted 2'-aminochalcones

Article abstract of DOI:10.1007/s00706-016-1812-9

Abstract: Three 2'-aminochalcone derivatives (E)-1-(2-aminophenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one, (E)-1-(2-aminophenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one, and (E)-1-(2-amino-4,5-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one, have been synthesized, characterized, and tested in vitro in order to assess their antioxidant activity. All compounds were characterized on the basis of ¹H NMR, ¹³C NMR, ESI-mass spectrometry, FT-IR, UV/Vis, and elemental analysis. The X-ray crystal structures of (E)-1-(2-aminophenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one and (E)-1-(2-amino-4,5-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one were successfully determined showing a planar molecule geometry. Studies on the biological properties including test of free radical scavenging ability (DPPH test)?and superoxide dismutase mimetic activity were performed. The results indicate that the aminochalcone carrying two hydroxyl functionalities in adjacent meta and para position exhibits a stronger antioxidant activity than the other derivatives. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-016-1812-9

Monatsh Chem
DOI 10.1007/s00706-016-1812-9
ORIGINAL PAPER
Synthesis, structure, and antioxidant activity
of methoxy- and hydroxyl-substituted 2’-aminochalcones
Chiara Sulpizio1 Alexander Roller2 Gerald Giester3 Annette Rompel1
•
•
•
Received: 18 May 2016 / Accepted: 26 June 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Three 2’-aminochalcone derivatives (E)-1-(2-
aminophenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one, (E)-1-
(2-aminophenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one,
and (E)-1-(2-amino-4,5-dimethoxyphenyl)-3-(4-methoxy-
phenyl)prop-2-en-1-one, have been synthesized, charac-
terized, and tested in vitro in order to assess their
antioxidant activity. All compounds were characterized on
Graphical abstract
1
the basis of H NMR, 13C NMR, ESI-mass spectrometry,
FT-IR, UV/Vis, and elemental analysis. The X-ray crystal
structures
of
(E)-1-(2-aminophenyl)-3-(4-hydroxy-
and (E)-1-(2-amino-4,5-
phenyl)prop-2-en-1-one
dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one
were successfully determined showing a planar molecule
geometry. Studies on the biological properties including
test of free radical scavenging ability (DPPH test) and
superoxide dismutase mimetic activity were performed.
The results indicate that the aminochalcone carrying two
hydroxyl functionalities in adjacent meta and para position
exhibits a stronger antioxidant activity than the other
derivatives.
Keywords Antioxidant activity Á Claisen condensation Á
Structure activity relationship Á
X-ray structure determination
Introduction
Medicinal plants have always represented one of the most
excellent and ancient sources of pharmaceutical agents in the
history of medicinal chemistry. Among them chalcones are
natural colorful pigments present in several medicinal and
edible plants [1]. They act as intermediates in the flavonoid
biosynthesis serving different physiological functions such as
UV photoprotectors, insect repellents, and attractants of
pollinators [2, 3]. Besides these physiological aspects chal-
cones provide an attractive scaffold for medicinal chemistry,
as they possess a wide range of biological activities and are
compounds of high therapeutical interest [4].
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-016-1812-9) contains supplementary
material, which is available to authorized users.
& Annette Rompel
annette.rompel@univie.ac.at
1
¨
Fakultat fur Chemie, Institut fur Biophysikalische Chemie,
Universitat Wien, Althanstraße 14, 1090 Vienna, Austria
¨
¨
¨
In the past decades a large number of reports have been
published on the pharmacological effects of chalcones,
and it has repeatedly been claimed that this natural pro-
duct is efficient and safe for the prevention and treatment
of several diseases such as atherosclerosis and
2
3
¨
Fakultat fur Chemie, Institut fur Anorganische Chemie,
Universitat Wien, Wahringer Straße 42, 1090 Vienna, Austria
¨
¨
¨
¨
¨
Fakultat fur Geowissenschaften, Geographie und Astronomie,
Institut fu¨r Mineralogie und Kristallographie, Universitat
¨
¨
Wien, Althanstraße 14, 1090 Vienna, Austria
123

C. Sulpizio et al.
neurodegenerative disorders like Alzheimer disease, pre-
venting the low density lipoprotein (LDL) oxidation
[5, 6]. Chalcones have been investigated for their anti-
tumor [7], anti-inflammatory [8], antifungal and antibac-
terial properties [9] as well as for their radical scavenging
potential [10]. Structurally they possess a 1,3-diaryl-2-
propen-1-one skeleton (see Scheme 1). The presence of
the a,b-unsaturated keto functionality (2-propen-1-one
chain) seems to be responsible for the medicinal proper-
ties of the chalcones [11, 12].
In fact it has been proven in previous studies that the
electrophilicity of this a,b-unsaturated carbonyl moiety is
involved in the antioxidant [13, 14] activities of chalcones.
Additionally, although numerous antioxidant substances
belong to different chemical classes, they have a certain
electronic and steric characteristic in common: in particular
an important requisite would be a planar structure with two
hydrophobic moieties (aromatic groups) and an acidic
proton (the phenolic proton) [15]. These characteristics,
which are all present in chalcones, make them
123

Synthesis, structure, and antioxidant activity of methoxy- and hydroxyl-substituted 2-…
suitable candidates for the design of new drugs; moreover,
depending on the substitution pattern of the two rings they
can display different spectra of activity. In this work we
focused our attention on hydroxyl, methoxyl, and amino
substitution in order to observe how they impact the bio-
logical activities of chalcones.
Results and discussion
Synthesis of 2’-aminochalcone derivatives
All three final compounds (E)-1-(2-aminophenyl)-3-(4-
hydroxyphenyl)prop-2-en-1-one (5), (E)-1-(2-aminophe-
nyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one (5a), and
(E)-1-(2-amino-4-methoxyphenyl)-3-(3,4-dimethoxyphenyl)-
prop-2-en-1-one (6) were obtained with moderate-to-good
yields and purely in the E form confirmed by the means of
1H NMR and X-ray crystallography. The amino chalcones
5 and 5a were synthesized according to the most common
base-catalyzed Claisen Schmidt condensation using barium
hydroxide octahydrate [23]. 5 and 5a were obtained via a
three-step procedure (see Scheme 1). First the appropriate
benzaldehydes 1 and 1a were protected with tetrahydropy-
ranyl (THP) to obtain 2 and 2a. THP has been chosen as a
protecting group because it is easy to insert and remove and
it is also stable under normal alkaline coupling conditions
[24]. Protection proceeded in both cases in good yield (89 %
for 2 and 69 % for 2a). Subsequently a base-catalyzed aldol
condensation reaction between the aminoacetophenone with
protected benzaldehydes 2 and 2a was performed to furnish
the protected aminochalcones 4 and 4a species with a yield
of 39 % and 40 %, respectively. Compound 4a was imme-
diately used without any further purification and its identity
checked by ESI-mass spectrometry (see ‘‘Experimental’’
section compound 4a). Compound 4 instead was purified by
flash column chromatography. After purification 4 decom-
poses very fast turning from yellow to dark red, therefore
13C NMR measurements have not been recorded and 4 was
immediately used for the condensation step. Finally depro-
tection was performed to give the hydroxy-2’-
aminochalcones 5 and 5a with a yield of 25 % for 5 and
20 % for 5a. All the condensation reactions worked only
moderately, and purification with liquid column chro-
matography is challenging after condensation as well as
after the hydrolysis reaction. This may be due to the for-
mation of the cyclization side product between the amino
group on the A ring and the aromatic B ring, which is very
difficult to separate from the target compound by flash
column chromatography as the two molecules have the same
retention time. The cyclization of 2’-aminochalcones has
been reported before [25]. Moreover, the different attempts
pointed out that both time and temperature influence the
hydrolysis reaction.
Methoxylated and hydroxylated chalcones are also well
known for their powerful antioxidant activities. In fact it has
been reported in literature that natural chalcones carrying
methoxyl and hydroxyl substitutions such as butein,
isoliquiritigenin, cardamonin, flavokawain A and B, are able
to scavenge reactive oxygen species (ROS) [10]. Normally
living organisms are protected against highly reactive oxygen
species by an endogenous system of enzymes like superoxide
dismutase or other naturally occurring antioxidants widely
distributed in the biological system such as ascorbic acid or
vitamin E for instance [16]. In fact a high level of ROS can
attack essential biological molecules like lipids, proteins, and
DNA, and it has been already demonstrated that in case of
disease the production of ROS is increased [17]. As a con-
sequence, natural and synthetic molecules possessing
multifunctional antioxidant activities such as flavonoids are
of great interest and important in disease prevention and in
therapy [18]. Within this polyhedral class of compounds, 2’-
aminochalcone derivatives still represent a new domain of
investigation. Currently they have been investigated for their
antitumor activity [19, 20], and additionally they have shown
to be useful as photoprotectors in sunscreen formulations as
well as in textile polymers or fibers because of their high
UV–visible extinction coefficient [21, 22]. Apart from these
promising aspects, little is known about the reactivity/re-
sponse of aminochalcones towards reactive oxygen species.
Thus, an evaluation of the potential biological applications of
the novel amino chalcone-based compounds will give more
insights into their mode-of-action. Therefore, they could
represent in the future new templates for novel antioxidants.
In this work three new chalcones with methoxyl, hydro-
xyl, and amino substitutions on ring A as well as on ring B
were synthesized and characterized with the aim to investi-
gate their antioxidant properties and clarify their molecular
structure. The compounds are therefore characterized via 1H
NMR, 13C NMR, FT-IR, and UV–Vis and elemental analy-
sis. The crystal structures of the newly synthesized
molecules 1-(2-aminophenyl)-3-(4-hydroxyphenyl)prop-2-
en-1-one (5) and 1-(2-amino-4,5-dimethoxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (6) (see Scheme 1) were
solved by single-crystal X-ray diffraction in order to study
the conformation of the typical chalcones.
Synthesis of the 2’-aminochalcone 6 was carried out
between the 2-aminoacetophenone and the 4-methoxy-
123

C. Sulpizio et al.
benzaldehyde in one direct reaction leading to a yield of
40 % of the pure product.
Table 1 Crystal data and structure refinement for compounds 5 and 6
Identification code
5
6
Empirical formula
Mr/g cm-3
C15H13NO2
1.305
C18H19NO4
1.314
Spectroscopic and mass characterization
Space group
P21/c
Iba2
Characterization of the synthesized compounds was per-
formed by ESI-mass spectrometry, FT-IR spectroscopy,
Crystal system
Monoclinic
18.877(4)
16.644(3)
19.387(4)
90
Orthorhombic
13.1460(13)
29.598(3)
8.1392(6)
90
1
UV–Vis spectroscopy, H NMR, and 13C NMR. All tech-
˚
a/A
˚
b/A
niques confirm unambiguously the structure of the final
products 5, 5a, and 6. ESI-MS analysis shows the molec-
ular ion peak always with good intensity and in accordance
with the calculated empirical formula. The same is valid
for elemental analysis results. For compounds 5 and 6 the
typical strong NH2 asymmetric and symmetric vibrations
were visible at 3340/3042 cm-1 (5) and 3386/3267 cm-1
(6), respectively. For compound 5a the NH2 stretching
overlapped with the strong broad band of the two OH
groups [26]. All the absorptions arising from the aromatic
rings appear between 1400 and 1600 cm-1. The UV–Vis
absorption occurs in the range from 480 to 260 nm,
showing maxima of absorption at 240, 342, and 399 nm for
5, 364 and 487 nm for 5a, and 244, 323, and 412 nm for 6
[27]. These values are attributed to p–p* and n–p* tran-
sitions due to the excitation in the aromatic ring and the
C=O group [28].
˚
c/A
a/°
b/°
c/°
91.781(10)
90
90
90
3
˚
V/A
6088(2)
20
3166.9(5)
8
Z
l/mm-1
0.087
0.093
Reflns collected
Independent reflns
R(int)
GooF on F2
R1 [I [ 2r(I)]
wR2 (all data)
126774
15117
17995
2924
0.0638
1.025
0.0776
1.033
0.0579
0.1596
0.0403
0.0943
Finally, the characteristic high value of the coupling
constant of the double bond of the enone moiety (see also
1H NMR of and Ha and Hb, ‘‘Experimental’’ section)
indicates that the chalcones 5, 5a, and 6 were obtained in
the E form [29]. The Hb of the olefinic system is more
deshielded then Ha because of the p-bond delocalization,
and therefore they both appear in the aromatic region. This
result is in good agreement with the X-ray structure anal-
yses data obtained for 5 and 6.
bond in both compounds is in the E configuration and that
the OH group in 5 and the OCH3 group in 6 are located in
para position on ring B. The most interesting structural
feature is a plane molecular conformation: in fact the
hydrogen bonding interaction between the amino function
NH2 and the carbonylic O atom seems to play an important
role in defining the planar conformation of the molecule.
The oxygen atoms O(3A) in 5 and O(3) in 6 act as an
acceptor in a moderate intramolecular hydrogen bond
involved with the adjacent NA(1)–H2 and N(1)–H2 func-
tionalities, respectively (see Fig. S1 and S2). The same
O(3A) atom in 5 acts again as an acceptor in a likewise
moderate intermolecular hydrogen bond interaction with
the O(4)–H group. The N(1A)–O(3) and O(3)–O(4) dis-
X-ray crystallography
5 and 6 were crystallized at room temperature from a
mixture of dichloromethane/methanol 9:5 for 5 and hex-
ane/ethyl acetate 9:2 for 6. A summary of crystal
parameters and refinement details of 5 and 6 are given in
Table 1. Compounds 5 and 6 crystallize in the space groups
P21/c and Iba2, respectively. The asymmetric unit of
compound 5 contains five independent molecules, whereas
the asymmetric unit of 6 contains one molecule. All bond
distances and angles are comparable to previous reports
[30] and similar experimental value were previously found
for the 2’-aminochalcone (E)-1-(2-aminophenyl)-3-(pyr-
˚
tances are 2.621 and 2.640 A for compound 5 (see
Table 2). Similar distances are observed for compound 6
(Table 3). The intramolecular hydrogen bond seems to be
the most significant coadjuvant to the molecular packing
forces that support the planarity of the entire molecule,
although further contribution could originate also from
weak aromatic ring-stacking interactions between the
phenyl rings and the keto-enolic moiety.
idine-4-yl)prop-2-en-1-one
[31]
and
(E)-1-(4-
Antioxidant activity
aminophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one
[32]. The ORTEP diagrams of 5 and 6 prepared with
OLEX2 are shown in Fig. 1 along with the atom number-
ing scheme. The results confirm that the olefinic double
The antioxidant activity under in vitro conditions has
been explored through two different methods, namely
the superoxide dismutase (SOD) assay and the DPPH
123

Synthesis, structure, and antioxidant activity of methoxy- and hydroxyl-substituted 2-…
Fig. 1 ORTEP style drawings
based on X-ray structure
analysis of 5 (a) and 6 (b) with
atom numbering scheme.
Thermal parameters enclose
50 % probability
Table 2 Selected hydrogen
˚
˚
d(D-H)/A
˚
d(H-A)/A
˚
d(D-A)/A
D
H
A
D-H-A/°
bond distances/A and angles/°
for 5
O(4A)
N(1A)
a
H(4A)
O(3B)a
O(3A)
0.84
0.88
1.80
1.96
2.640(2)
2.621(3)
174.0
130.3
H(1AB)
?X, 1/2 - Y, -1/2 ? Z
Table 3 Selected hydrogen
˚
˚
d(D-H)/A
˚
d(H-A)/A
˚
d(D-A)/A
D
H
A
D-H-A/°
bond distances/A and angles/°
for 6
N(1)
N(1)
a
H(1A)
H(1B)
O(3)
O(3)a
0.88
0.88
1.93
2.03
2.584(4)
2.890(4)
130.4
166.8
?X,1-Y,-1/2?Z
radical scavenging method. The DPPH test relies on the
antioxidant ability to quench the radical by hydrogen
donation capacity, whereas the SOD-like assay is based
on the fact that antioxidants can suppress the formation
of reactive oxygen species by inhibiting the enzymes
involved in its generation and have superoxide scav-
enging activities themselves [33, 34]. Both methods have
been chosen because they have already been widely used
to determine the antioxidant activity of isoflavonoids
[35].
123

C. Sulpizio et al.
Free radical scavenging ability
antioxidant activity studies performed by Iqbal et al. [37]
on a series of 4-aminochalcones and sulfonamide-substi-
tuted chalcones, which also did not show a powerful
antioxidant activity. Previous studies performed on the
trimethoxy chalcone 1-(2,4-dimethoxyphenyl)-3-[3-(4-
methoxyphenyl)-1-(1-methylbuta-1,3-dienyl)-1H-pyrazol-
4-yl]propenone, suggested that the amino group is not
involved in the antioxidant mechanism [38]. The mea-
surements reported here evidence only a significant
inhibition of DPPH for 5a that exhibit two phenolic groups
in meta and para position on ring B, therefore this would
suggest that the mechanism of the H-transfer originate
mainly from the phenolic group rather than from the keto-
enolic moiety. The two adjacent hydroxyl groups seem to
be mainly responsible for the antioxidant activity of the
aminochalcone, supporting the mechanism proposed by
Hwang et al. [18].
The free radical scavenging ability of the synthesized
2’-aminochalcones was evaluated by the DPPH radical
assay. The results are summarized in Fig. 2. The
aminochalcone 5a shows an IC50 value (the antioxidant
concentration necessary to decrease the initial amount of
DPPH by 50 %) of 4.9 1 lM which is even better than
the one for catechol that in the same experimental condi-
tion showed an IC50 of 5.3 1 lM (see Table 4). The
results point out that the presence of the two hydroxyl
groups in ring B of 5a are important for the antioxidant
activity of the aminochalcones [10]. For the other two
compounds 5 and 6 the IC50 is not reached within the
concentrations applied. Our values are close to the ones
determined for other potent natural flavonoids [36]. Some
studies on antioxidant properties of chalcones highlighted a
mechanism involving H atom transfer from the phenolic
moiety of the ring B [18]. In contrast to these reports no
significant free radical scavenging ability is observed for
the aminochalcone 5. This result is comparable to previous
Superoxide scavenging ability
The superoxide radical anions are generated in vitro by a
xanthine/xanthine oxidase system causing the reduction of
the nitro blue tetrazolium (NBT2?) and consequently the
determination of the scavenging activity of the chalcones.
Applying this method O2.2 reduces the yellow colored
(NBT2?) to produce the blue formazan, which is measured
spectrophotometrically at 560 nm. Antioxidants are scav-
engers of oxygen free radicals and prevent the purple NBT
formation [39]. The compounds 5, 5a, and 6 were studied
as potential scavengers of oxygen free radicals and the
results are summarized in Fig. 3, which illustrates the
percentage of inhibition of NBT2? against the
Fig. 2 The percentage of inhibition of the free DPPH radical in the
presence of 5, 5a, and 6. The concentration of the different
compounds is expressed as lM
Table 4 IC50 value of the aminochalcones 5, 5a, and 6 for the
inhibition of xanthine oxidase and the inhibition of DPPH
Compound
SOD mimic activity
IC50/lM SDa
DPPH radical assay
IC50/lM SDa
5
38.6
37.1
–
1
1
–
5a
4.9
–
1
1
Fig. 3 Percentage of inhibition (% In) of superoxide radical formed
by xanthine/xanthine oxidase enzyme by different concentrations of
2’-aminochalcones assayed by NBT2? absorption at 560 nm in
presence of 5, 5a, and 6. The concentration of the different
compounds is expressed as lM
6
Caffeic acid
34.1
–
1
–
Catechol
5.3
a
Standard deviation
123

Synthesis, structure, and antioxidant activity of methoxy- and hydroxyl-substituted 2-…
concentration of the different synthesized 2’-aminochal-
cones 5, 5a, and 6. The IC50 value, that is the concentration
of 2’-aminochalcone necessary to reduce the absorbance of
the NBT2? to half of its initial value was calculated to
38.6 1 lM for 5 and 37.1 – 1 lM for 5a (see Table 4).
The IC50 value of the aminochalcones 5 and 5a is not so
much different to the ones previously found for other fla-
vonoids such as rutin (42.7 lM) and quercetin (42.3 lM)
[40]. For comparative purpose, we have also measured the
activity of caffeic acid which showed an IC50 of 34.1 lM
under the same experimental condition. The IC50 value is
not reached for 6 which showed a very low activity com-
parable to that one previously found for nobiletin and
tangeretin [39]. In previous studies it has been proposed
that the reaction takes place by donating the unpaired
electron from superoxide radical to the flavonoids [41].
According to this reaction mechanism the presence of at
least one hydroxyl group on the B ring seems to be
essential for the activity, and would explain the lack of
scavenging activity for compound 6. In fact the percentage
of inhibition of superoxide anions is not affected by
increasing the concentration of the aminochalcones 6.
Previous studies [42] on superoxide scavenging activity
performed on a series of 40-aminochalcones showed a dose-
dependent inhibition of superoxide radicals. In fact it has
been observed that the activity increased when the
aminochalcones dose level was higher. Interestingly the
same aminochalcones possess an electron-releasing group
at position 4 of the ring B, therefore this structural char-
acteristic seems to be essential for significant antioxidant
activity. Additionally it has been observed that the super-
oxide scavenging activity is sensitive to the planarity of the
molecule [39] present in 5 and 5a.
important prerequisite for the activity of the molecule. The
intramolecular hydrogen bond interaction with the adjacent
carbonylic oxygen functionality contributes to stabilize the
planar structure of the studied chalcones.
Experimental
Chemicals were purchased from Sigma Aldrich, they were
of reagent grade and used without any further purification
unless otherwise specified. All NMR spectra were recorded
¨
at the NMR core facility 1090 Wien, Wahringer Straße 38
of the University of Vienna with a Bruker Avance III
500 MHz NMR spectrometer at 500.32 MHz (1H),
125.81 MHz (13C), in CDCl3, methanol-d4, or acetone-d6
at ambient temperature. The splitting of proton resonances
in the 1H NMR spectra are defined as s = singlet,
d = doublet, dd = doublet of doublets, ddd = doublet of
doublets of doublets, t = triplet, and m = multiplet.
Numbering of carbon atoms and protons refers to
Scheme 1. Electrospray ionization mass spectra were
recorded at the Mass Spectrometry Center (MSC) of the
Chemical Faculty of the University of Vienna on a Bruker
Esquire 3000 with an orthogonal ESI source applying
MeOH/ACN as solvent. The molecular mass was deter-
mined in the positive mode. Elemental analyses were
carried out in the Mikroanalytisches Laboratorium of the
University of Vienna with a Perkin-Elmer 2400 CHN
elemental analyser. The spectrophotometer Shimadzu UV
1800 has been used to recorder the UV–Vis spectra.
Spectra were collected in methanol. The infrared spectra
were recorded with an infrared spectrometer Bruker Tensor
27 FTIR equipped with a global MIR light source, a KBr
beam splitter, and a DLaTGS detector. Sample and back-
ground spectra were averaged from 100 scans at 4 cm-1
resolution. Undiluted sample powder was pressed on the
diamond window of a Harrick MVP 2 diamond ATR
accessory. Background spectra were obtained from the
empty ATR unit. Data handling was performed with OPUS
5.5 software (Bruker Optik GmbH, 2005). Intensities of
reported IR bands are defined as br = broad, s = strong,
m = medium, and w = weak.
Conclusion
This study reports on the synthesis, characterization, and
evaluation of antioxidant activity of three different 2’-
aminochalcones. The antioxidant activity was evaluated
with two different methods: superoxide scavenging assay
and DPPH radical scavenging testing. Compound 5a hav-
ing two adjacent hydroxyl groups in the B ring exhibits
very high antioxidant activity compared to compounds 5
and 6 based on the DPPH test. The presence of the OH
groups in 5 (one in ring B) and 5a (two adjacent ones in
ring B) makes them good candidates for superoxide scav-
4-(Tetrahydro-2H-pyran-2-yloxy)benzaldehyde
(2, C12H14O3)
4-Hydroxybenzaldehyde (1, 6.60 mmol) and pyridinium
para-toluenesulfonate (0.16 mmol) were dissolved in
30 cm3 of dichloromethane and the solution was mixed
at room temperature. Subsequently, 3,4-dihydro-a-pyran
(19.73 mmol) was added dropwise. The reaction was
stirred for 24 h and the progress was monitored by TLC.
Then the mixture was washed with water, dried over
Na2SO4, concentrated under vacuum, and purified by flash
enging activity. Compound
6 having a methoxyl
substituent in ring B reveals to be less effective. Therefore,
this set of aminochalcones shows a clear correlation
between the number and the position of OH groups and
their activity. The planar structure evidenced by NMR
spectroscopy and X-ray structure analysis seems to be an
123

C. Sulpizio et al.
1
column chromatography (hexane/ethyl acetate 7:3) to
1
obtain pure 2. Yield: 89 %; H NMR (500 MHz, CDCl3):
obtain pure 4. Yield: 40 %; H NMR (500 MHz, CDCl3):
d = 1.50–1.58 (m, 1H, H-10b), 1.60–1.80 (m, 2H, H-9b,
10a), 1.82–1.93 (m, 2H, H-8b, H-9a), 1.96–2.08 (m, 1H,
H-8a), 3.64 (m, 1H, H-11b), 3.82–3.93 (m, 1H, H-11a),
5.55 (t, J = 3.1 Hz, 1H, H-7), 6.69–6.71 (m, 2H, H-50,
H-60), 6.3 (br. s, 2H, NH2), 7.08–7.10 (d, 2H, J = 8.8 Hz,
H-2, H-6), 7.50–7.53 (d, 1H, J = 15.4 Hz, H-a), 7.27–7.30
(t, 1H, J = 8.1 Hz, H-40), 7.57–7.59 (d, 2H, J = 8.8 Hz,
H-5, H-3), 7.71–7.74 (d, 1H, J = 15.4 Hz, H-b), 7.86–7.87
(d, 1H, J = 8.2 Hz, H-30) ppm; HRMS (ESI–MS): m/
z = 346.1426 ([M?Na]?), 324.1604 ([M?H]?, calcd. for
C20H21NO3 324.1594).
d = 1.50–1.58 (m, 1H, H-10b), 1.60–1.80 (m, 2H, H-9b,
10a), 1.82–1.93 (m, 2H, H-8b, H-9a), 1.96–2.08 (m, 1H,
H-8a), 3.64 (m, 1H, H-11b), 3.82–3.93 (m, 1H, H-11a),
5.55 (t, J = 3.1 Hz, 1H, H-7), 7.13–7.20 (dd, J = 8.9,
2.0 Hz, 2H, H-3, H-5), 7.81–7.87 (dd, J = 8.9, 2.0 Hz, 2H,
H-2, H-6), 9.97 (s, 1H, C=O) ppm; 13C NMR (125 MHz,
CDCl3): d = 191.1 (C=O), 161.7 (C-4), 132.4 (C-2,6),
131.9 (C-1), 115.9 (C-3,5), 96.1 (C-7), 63.0 (C-11), 30.0
(C-8), 25.3 (C-10), 19.6 (C-9) ppm; HRMS (ESI–MS): m/
z = 229.0829 ([M?Na]?, calcd. for C12H14O3Na
229.0835).
(E)-3-[3,4-Bis(tetrahydro-2H-pyran-2-yloxy)phenyl]-1-(2-
aminophenyl)prop-2-en-1-one (4a, C25H29NO5)
3,4-Bis(tetrahydro-2H-pyran-2-yloxy)benzaldehyde
(2a, C17H22O5)
2a (4.24 mmol) and 3 (8.48 mmol) were dissolved in
10 cm3 of methanol under reflux at 35 °C. Subsequently, a
solution of 20 cm3 of methanol containing barium hydrox-
ide octahydrate (16.96 mmol) was added dropwise to the
reaction mixture. The reaction mixture was stirred for 24 h
and the progress was monitored by TLC. Then the mixture
was concentrated under vacuum, quenched with 0.1 M HCl
and extracted with ethyl acetate. The organic layer was
separated, dried over Na2SO4 and then concentrated under
vacuum. The residue yielded the crude chalcone 4a as a
yellow powder and was used for the next step without any
further purification. Yield: 39 %; HRMS (ESI–MS): m/
z = 446.1955 ([M?Na]?), 424.2133 ([M?H]?, calcd. for
C25H29NO5 424.2118).
3,4-Dihydroxybenzaldehyde (1a, 6.60 mmol) and pyri-
dinium para-toluensulfonate (0.16 mmol) were dissolved
in 30 cm3 of dichloromethane and the solution was mixed
at room temperature. Subsequently, 3,4-dihydro-a-pyran
(39.46 mmol) was added dropwise. The reaction was
stirred for 24 h and the progress was monitored by TLC.
Then the mixture was washed with water, dried over
Na2SO4, concentrated under vacuum, and purified by flash
column chromatography (hexane/ethyl acetate 9:2) to
1
obtain 2a as a mixture of stereoisomers. Yield: 69 %; H
NMR (500 MHz, CDCl3): d = 1.58–1.50 (m, 2H, H-10b,
10b0), 1.60–1.80 (m, 4H, H-9b, H-9b0, H-10a, H-10a0), 1.
82–1.93 (m, 4H, H-8b, H-8b0, H-9a, H-9a0), 1.96–2.08 (m,
2H, H-8a, H-8a0) 3.64 (m, 2H, H-11b, H-11b), 3.82–3.93
(m, 2H, H-11a, H-110a), 5.55 (t, J = 2.7 Hz, 2H, H-7,
H-70), 7.05–7.07 (d, J = 8.2 Hz, 1H, H-5), 7.25 (s, 1H,
H-2), 7.49–7.50 (d, J = 8.0 Hz, 1H, H-6), 9.87 (s, 1H,
CHO) ppm; 13C NMR (125 MHz, CDCl3): d = 190.6
(C=O), 153.1 (C-4), 146.9 (C-3), 129.8 (C-1), 123.9 (C-6),
115.8 (C-5), 115.2 (C-2), 98.4 (C-7), 98.9 (C-70), 63.7 (C-
11), 63.3 (C-110), 30.1 (C-8), 30.1 (C-80), 25.4 (C-10), 25.2
(C-100) 19.4 (C-9), 18.8 (C-90) ppm; HRMS (ESI–MS): m/
z = 329.1356 ([M?Na]?), 307.1534 ([M?H]?, calcd. for
C17H22O5 307.1461).
(E)-1-(2-Aminophenyl)-3-(4-hydroxyphenyl)prop-2-en-1-
one (5, C15H13NO2)
4 (1.26 mmol) was dissolved in 50 cm3 of methanol.
Subsequently
pyridinium
para-toluensulfonate
(0.062 mmol) was added and the reaction was stirred
under reflux and the progress was monitored by TLC. Then
the reaction mixture was directly concentrated under
vacuum and then purified by flash column chromatography
(dichloromethane/methanol 9.5/0.5). Yield: 25 %; 1H
NMR (500 MHz, acetone-d6): d = 6.60–6.63 (t,
J = 6.62 Hz, 1H, H-40), 6.81–6.83 (d, 1H, J = 8.2 Hz,
H-60), 6.90–6.92 (d, 2H, J = 8.8 Hz, H-2, H-6), 7.04 (s,
2H, NH2), 7.24–7.27 (t, J = 8.3 Hz, 1H, H-50), 7.67–7.69
(d, 1H, J = 15.4 Hz, H-a), 7.67–7.69 (d, J = 8.5 Hz, 2H,
H-5, H-3), 7.72–7.75 (d, J = 15.4 Hz, 1H, C-b), 8.01–8.03
(d, J = 8.2 Hz, 1H, H-30), 8.83 (s, 1H, OH) ppm; 13C NMR
(126 MHz, acetone-d6): d = 190.8 (C=O), 159.5 (C-4),
152.0 (C-20), 133.7 (C-50), 130.9 (C-30), 130.1 (C-a), 127.1
(C-1), 126.8 (C-5, C-3), 120.02 (C-b), 117.6 (C-10), 115.7
(C-2, C-6), 115.3(C-60), 114.7 (C-40) ppm; FT-IR (KBr):
(E)-1-(2-Aminophenyl)-3-[4-(tetrahydro-2H-pyran-2-
yloxy)phenyl]prop-2-en-1-one (4, C20H21NO3)
2 (4.24 mmol) and 3 (8.48 mmol) were dissolved in
10 cm3 of methanol under reflux at 35 °C. Subsequently,
a solution of 20 cm3 of methanol containing barium
hydroxide octahydrate (16.96 mmol) was added dropwise
to the reaction mixture. The reaction mixture was stirred
for 24 h and the progress was monitored by TLC. Then the
mixture was concentrated under vacuum, quenched with
0.1 M HCl and extracted with ethyl acetate. The organic
layer was separated, dried over Na2SO4 and then concen-
trated under vacuum. The reaction mixture was purified by
flash column chromatography (hexane/ethyl acetate 7:3) to
ꢀ
m = 3340, 3042, 3000, 1640, 1600, 1587, 1520,
1326 cm-1
;
UV/Vis (methanol): kmax = 240, 342,
399 nm; HRMS (ESI–MS): m/z = 262.0832 ([M?Na]?),
240.1012 ([M?H]?, calcd. for C15H13NO2 240.1019).
123

Synthesis, structure, and antioxidant activity of methoxy- and hydroxyl-substituted 2-…
(E)-1-(2-Aminophenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-
1-one (5a, C15H13NO3)
4a (1.26 mmol) was dissolved in 50 cm3 of methanol.
m/z = 336.1206 ([M?Na]?), 314.1384 ([M?H]?, calcd.
for C18H19NO4 314.1386).
Subsequently,
pyridinium
para-toluensulfonate
Crystallographic structure determination
Single-crystal X-ray diffraction data were collected with
(0.062 mmol) was added and the reaction was stirred
under reflux at 50 °C and the progress was monitored by
TLC. Then the reaction mixture was directly concentrated
under vacuum and then purified by column flash chro-
matography (Sephadex LH-20, dichloromethane/methanol
Bruker diffractometers, equipped with
a multilayer
monochromator, INCOATEC microfocus sealed tube (k
´
˚
(MoKa) = 0.71073 A), and with a CMOS Photon Detector
1
9.5/0.5). Yield: 20 %; H NMR (500 MHz, acetone-d6):
at 100 K for 6, and with a APEXII CCD detector for 5 at
200 K, respectively. The data reductions were performed
using APEX2 (Bruker Analytical X-ray systems, Madison,
2004) software package. The structure solution was exe-
cuted using SHELXS [43] with the GUI OLEX2 [44]. The
structure refinements were realized with SHELXL [45]
embedded in GUI’s OLEX2 [44] and ShelXle [45]. The
crystallographic results were proofed with PLATON [46].
OLEX2 [44] calculated modified hkl-files to finalize the
refinement process. Both, the original and the modified hkl-
files, were uploaded to the CCDC database (http://www.
ccdc.cam.ac.uk/) with the accession number 1406883 for 5
and 1406882 for 6.
d = 6.62–6.64 (t, J = 8.2 Hz, H-40), 6.88–6.90 (d,
J = 8.2 Hz, 1H, H-6), 6.81–6.83 (d, 1H, J = 9.5 Hz, H-
60), 7.03 (s, 2H, NH2), 7.16–7.18 (dd, J = 10.4, 2.2 Hz,
1H, H-5), 7.24–7.27 (t, 1H, J = 8.4 Hz, H-50), 7.30 (s, 1H,
H-2), 7.61–7.58 (d, J = 15.4 Hz, 1H, C-b), 7.65–7.68 (d,
J = 15.4 Hz, 1H, C-a), 8.0–8.02 (d, J = 9 Hz, 1H, H-30),
8.06 (OH), 8.42 (OH) ppm; 13C NMR (126 MHz, acetone-
d6): d = 191.0 (C=O), 151.9 (C-20), 147.6 (C-3), 145.3(C-
4), 142.7 (C-b), 133.8 (C-50), 130.7 (C-30), 127.7 (C-10),
121.9 (C-5), 120.0 (C-a), 118.5 (C-1), 116.9 (C-60), 115.4
(C-40), 115.3 (C-6), 114.8 (C-2) ppm; FT-IR (KBr):
m = 3325, 1640, 1600, 1573, 1505, 1326 cm-1; UV/Vis
ꢀ
(methanol): kmax = 364, 487 nm; HRMS (ESI–MS):
m/z = 278.0780 ([M?Na] ?), 256.0961 ([M?H]?, calcd.
for C15H13NO3 256.0968).
Antioxidant activity (DPPH radical scavenging
method)
(E)-1-(2-Amino-4,5-dimethoxyphenyl)-3-
The antioxidant activity test is conducted according to the
method described by Ferrari et al. [47]. 1-x cm3 of
6 9 10-5 mM DPPH radical solution is prepared in
methanol and mixed with a variable amount x mm3 (x = 5,
10, 15, 20 mm3, etc.) of a methanolic solution containing
the 2’-aminochalcone (1.2 mM). The absorbance of the
mixture is measured immediately every second up to
30 min at 517 nm at 20 °C. For the baseline a control
sample of 1 cm3 of methanol was used. The percentage of
inhibition of the DPPH radical was calculated for each
sample referring to the following formula in accordance to
Ref. [47]:
(4-methoxyphenyl)prop-2-en-1-one (6, C18H19NO4)
1-(2-Amino-4,5-dimethoxyphenyl)ethanone (4.24 mmol)
and 4-methoxybenzaldehyde (4.24 mmol) were dissolved
in 10 cm3 of methanol at 35 °C under reflux. Subsequently,
a solution of 20 cm3 of methanol containing barium
hydroxide octahydrate (16.96 mmol) was added dropwise
to the reaction mixture. The reaction mixture was stirred
for 24 h and the progress was monitored by TLC. Then the
mixture was concentrated under vacuum, quenched with
0.1 M HCl and extracted with ethyl acetate. The organic
layer was separated, dried over Na2SO4 and then concen-
trated under vacuum. The reaction mixture was purified by
flash column chromatography (hexane/ethyl acetate 9:2) to
obtain pure 6. Yield: 40 %; 1H NMR (500 MHz, methanol-
d4): d = 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.86 (s,
3H, OCH3), 6.36 (s, 1H, H-60), 6.96–6.98 (d, J = 8.8 Hz,
2H, H-2, H-6), 7.39 (s, 1H, H-30), 7.56–7.60 (d, 1H,
J = 15.5 Hz, Ha), 7.60–7.63 (d, J = 15.5 Hz, 1H, H-b),
7.68–7.67 (d, J = 8.50 Hz, 2H, H-4, H-5) ppm; 13C NMR
(126 MHz, methanol-d4): d = 191.6 (C=O), 163.1 (C-40),
157.8 (C-50), 151.7 (C-4), 143.3 (C-b), 141.3 (C-10), 131.3
(C-5, C-3), 129.6 (C-20), 122.4 (C-a), 115.9 (C-30), 115.7
(C-2, C-6), 112.2 (C-1), 100.6 (C-60), 56.4 (C-OCH3), 56.2
A0 À At
%In ¼
 100;
A0
where A0 is the absorbance of the control (DPPH radical) at
time 0 and At is the absorbance of the mixture DPPH–
antioxidant at time t (30 min). All the determinations were
performed in triplicate and the values of absorbance were
corrected considering the factor of dilution. The IC50 value
was also calculated from the regression line.
Superoxide dismutase activity (SOD assay using
XO/NBT system)
ꢀ
(C-OCH3), 56.1(C-OCH3) ppm; FT-IR (KBr): m = 3386,
3267, 1630, 1563, 1501, 1401, 1342 cm-1; UV/Vis
The SOD-like activity is performed with the method
described by Ferrari et al. [48] with some modification.
(methanol): kmax = 244, 323, 412 nm; HRMS (ESI–MS):
123

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Acknowledgments Open access funding provided by University of
Vienna. The authors wish to kindly acknowledge Ao. Univ.-Prof.
Mag. Dr. Markus Galanski, NMR Centre, Faculty of Chemistry,
University of Vienna for NMR measurement and Ing. Peter
Unteregger for support with the ESI-MS measurements at the
Massenspektrometriezentrum of the University of Vienna. We thank
Amir Blazevic, MSc. and Dipl.-Ing. Matthias Pretzler for valuable
discussions about the data reported in the manuscript.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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