Synthesis, characterization, and antioxidant activity of Zn2+ and Cu2+ coordinated polyhydroxychalcone complexes

Article abstract of DOI:10.1007/s00706-016-1822-7

Four new metal complexes [Cu(ISO)₂], [Cu(BUT)₂] and [Zn(ISO)₂], [Zn(BUT)₂] of the polyhydroxychalcones (isoliquiritigenin and butein) are synthesized, structurally characterized and their antioxidant activity is investigated. The formation of the complexes [Cu(ISO)₂] and [Zn(ISO)₂] is followed by Job’s plot using NMR titration. The resulting compounds are characterized by mass spectrometry, IR spectroscopy, and elemental analysis. Studies on the radical scavenging activity are performed using DPPH as substrate. The results showed that the antioxidant activities of isoliquiritigenin and butein are enhanced after binding to copper or zinc. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-016-1822-7

Monatsh Chem
DOI 10.1007/s00706-016-1822-7
ORIGINAL PAPER
Synthesis, characterization, and antioxidant activity of Zn2+
and Cu2+ coordinated polyhydroxychalcone complexes
Chiara Sulpizio1 Simon T. R. Mu¨ller1 Qi Zhang1 Lothar Brecker2 Annette Rompel1
•
•
•
•
Received: 24 May 2016 / Accepted: 11 July 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Four new metal complexes [Cu(ISO)2],
[Cu(BUT)2] and [Zn(ISO)2], [Zn(BUT)2] of the polyhy-
droxychalcones (isoliquiritigenin and butein) are
synthesized, structurally characterized and their antioxidant
activity is investigated. The formation of the complexes
[Cu(ISO)2] and [Zn(ISO)2] is followed by Job’s plot using
NMR titration. The resulting compounds are characterized
by mass spectrometry, IR spectroscopy, and elemental
analysis. Studies on the radical scavenging activity are
performed using DPPH as substrate. The results showed
that the antioxidant activities of isoliquiritigenin and butein
are enhanced after binding to copper or zinc.
Keywords Bioinorganic chemistry Á
Carbonyl compounds Á Metal complex Á
NMR spectroscopy Á Structure activity relationship
Introduction
Oxidation processes are essential for life, nevertheless they
can have a toxic potential as they generate free radicals
which can cause degradation in food, chemicals, and in
biological systems [1–4]. Hence, antioxidants play a cen-
tral role in human health as they scavenge potentially
harmful free radicals such as reactive oxygen species
(ROS). Chalcones, being a subgroup of the flavonoid
family, have received considerable attention as radical
scavengers and show an impressive range of biological
activities such as antibacterial [5, 6], antiviral [7–9], and
anti-inflammatory properties [10, 11]. Polyhydroxychal-
cones can be considered the most active chalcones due to
their ability to form stabilized phenoxy radicals [11–14].
Hwang et al. [15] have shown that the position of the
hydroxyl groups on arene ring B (Fig. 1) has a major
impact on radical scavenging properties of polyhydroxy-
chalcones. While meta-substitution on ring B has no
beneficial effect, ortho- and para-substitutions increase the
antioxidant activity of chalcones significantly, presumably
due to the ability to form quinones as oxidation products.
Recently, it has been shown that the antioxidant activity
of polyphenols, structurally similar to polyhydroxychal-
cones, can be significantly enhanced by metal ion
complexation [16–19]. In contrast to the interest that metal
complexes of polyphenolic substances have received, little
is known about the synthesis and activity of polyhydroxy-
chalcone metal complexes, and how metal complexation
would impact the antioxidant activity of these powerful
Graphical abstract
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-016-1822-7) contains supplementary
material, which is available to authorized users.
& Annette Rompel
annette.rompel@univie.ac.at
1
¨
Institut fu¨r Biophysikalische Chemie, Fakultat fur Chemie,
Universitat Wien, Althanstraße 14, 1090 Vienna, Austria
¨
¨
2
¨
Institut fu¨r Organische Chemie, Fakultat fur Chemie,
Universitat Wien, Wahringer Straße 38, 1090 Vienna, Austria
¨
¨
¨
123

C. Sulpizio et al.
complex formation between polyhydroxychalcones and
metal ions show various affects on the electronic distri-
bution in the molecule, resulting in possible changes of
biological properties such as radical scavenging ability.
Copper(II) and zinc(II) are our metals of interest, since
they are endogenous metals and they have shown promising
features as metallodrug candidates [24, 25]. In fact Cu(II) and
Zn(II) complexes of the flavonoids quercetin and rutin have
been shown to greatly increase the antioxidant activity of this
class of compounds [26, 27]. In many complexes Zn(II)
prefers a tetrahedral coordination sphere [28] while the Cu(II)
cation prefers a square planar coordination. Based on the
polyhydroxychalcones scaffold other coordination geometries
such as distorted octahedral geometry have been reported for
copper [29]. In this communication we disclose synthesis,
characterization as well as studies on antioxidant activities of
four polyhydroxychalcone metal complexes.
Fig. 1 Proposed structure of a general M(II)–chalcone complex
secondary plant metabolites. Although in literature three
metal complexes of 2’-hydroxychalcones with Zn, Cd, and
Hg are reported [20], no complexes of polyhydroxychal-
cone have been synthesized, isolated, and characterized
until now. Thus in particular the possible structure of metal
ion coordination needs to be investigated. There are several
options how metals can coordinate polyhydroxychalcones
(see Fig. 1) [21, 22]. Both, the carbonyl moiety and the 20-
hydroxyl group of the 20-hydroxychalcones have been
suggested to coordinate a metal(II) ion [23]. Different
Results and discussion
Synthesis of polyhydroxychalcone and their metal
complexes
The synthesis of the polyhydroxychalcones was performed
via a well-documented three-step procedure in basic
Scheme 1
O
R=-THP 5a
R=-THP 7a
R=-MOM 7b
R=-TBDMS 7c
HO
R=-MOM 5b
R=-TBDMS 5c
3
O
OH
O
OH
O
RO
HO
OH
RO
OR
5
H+
9 ISO
7
OH
O
OH
O
-
OH
-
HO
RO
OH
2
1
OH
2´
O
OH
O
β
6
1´
1
R=-THP 2a
5
O
3´
4´
α
R=-MOM 2b
RO
RO
R=-TBDMS 2c
6´
2
4
HO
OH
RO
OR
H+
3
5´
OH
OR
10 BUT
8
6
O
HO
HO
R=-THP 6a
R=-THP 8a
R=-MOM 8b
R=-TBDMS 8c
R=-MOM 6b
R=-TBDMS 6c
4
reagents and conditions: ; 2a (3,4-dihydro-α-pyran (DHP), pyridinium-para-toluensulfonate (PPTS), CH2Cl2, r.t., 24h); 2b (Br-MOM, K2CO3, Acetone, rfx-50°C, 1-4h ); 2c (Cl-
TBDMS, imidazole, N2 inert atmosphere, THF, 0°C, 8h); 5a (DHP, PPTS, CH2Cl2, r.t., 24h); 5b (Br-MOM, K2CO3, Acetone, rfx-50°C, 1-4h ); 5c (Cl-TBDMS, imidazole, N2 inert
atmosphere, THF, 0°C, 8h); 6a (DHP, PPTS, CH2Cl2, r.t., 24h); 6b (Br-MOM, K2CO3, Acetone, rfx-50°C, 1-4h ); 6c (Cl-TBDMS, imidazole, N2 inert atmosphere, THF, 0°C, 8h)
7a,8a (MeOH, Ba(OH)2* 8H2O, rfx-35°C, 24 h); 7b,8b (MeOH, KOH, r.t., 24-78 h.); 7c,8c (LiHMDS, THF, rfx- 50°-70°C, 2h-8h); deprotection for 7a,8a (MeOH, PPTS, rfx-50 °C,
1h); deprotection for 7b,8b (MeOH, HCl, rfx-50 °C, 45 min.)
123

Synthesis, characterization, and antioxidant activity of Zn2? and Cu2? coordinated…
condition according to [30] (see Scheme 1). First com-
pound 1 was protected with one of the three protecting
groups, methoxymethyl (MOM), tetrahydropyranyl (THP),
and tert-butyldimethylsilyl-ether (t-BDMS) to get 2. Pro-
tection proceeded in all cases in moderate to good yields.
Subsequently, a base-catalyzed aldol condensation reaction
(Claisen–Schmidt condensation) with a protected ben-
zaldehyde 5 or 6 was performed to obtain the protected
chalcones 7 and 8. Finally, deprotection was performed to
give the desired polyhydroxychalcones isoliquiritigenin (9,
ISO) and butein (10, BUT).
accordance with the work published by Lee et al. [35], who
obtained the final hydroxychalcone in an overall yield of
10 % when they protected the 4-hydroxy group with
methoxymethyl prior to the Claisen–Schmidt condensation.
It should be noted that the use of a Sephadex LH-20 for
liquid column chromatography to purify the final polyhy-
droxychalcone increased the yield significantly compared
to a silica gel-based liquid chromatography.
The two complexes [Cu(ISO)2] and [Cu(BUT)2] were
prepared by mixing copper(II) acetate with (E)-1-(2,4-di-
hydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one (9,
ISO) and (E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxy-
phenyl)-2-propen-1-one (10, BUT), respectively, in a 1:2
(M, M = metal: L, L = ligand) molar ratio. The reactions
were carried out in methanol as solvent.
In order to optimize the synthesis of the polyhydroxy-
chalcones, different protecting groups have been applied in
the course of the butein synthesis (Scheme 1). MOM as a
protection group presents several disadvantages as it is highly
carcinogenic [31], and the purification of the protected inter-
mediate with liquid column chromatography is challenging.
Nevertheless, the protection reaction of the hydroxyl group
with MOM leads to a yield of 67 % for benzaldehyde and
64 % for acetophenone. Moreover, the removal of the MOM
group requires drastic acidic conditions [32], which often lead
to polymerization and oxidation of the desired product. t-
BDMS worked better during the protection step providing
similar and higher yields (64 % for the benzaldehyde and
83 % for the acetophenone). The coupling was performed
using LiHMDS [33], but difficulties with the purification
made isolation of the pure product problematic. Although
THP is a little less efficient in protecting benzaldehyde (60 %
yield) it protects the acetophenone most efficiently (89 %),
and has been advantageous in all other aspects as it is easy to
insert and remove. Furthermore, it is stable under the alkaline
coupling conditions [30]. As a consequence THP was chosen
as the protection group for the synthesis of both polyhydrox-
ychalcones ISO and BUT.
The complexes [Zn(ISO)2] and [Zn(BUT)2] were pre-
pared by mixing zinc(II) chloride with (E)-1-(2,4-
dihydroxyphenyl)-3-(4-hydroxyphenyl)-prop-2-en-1-one
(9, ISO) and (E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxy-
phenyl)prop-2-en-1-one (10, BUT) in a 1:2 (M:L) molar
ratio. The reactions were carried out in acetonitrile. Four
new Cu(II) and Zn(II) complexes of polyhydroxychalcones
termed [Cu(ISO)2], [Cu(BUT)2], [Zn(ISO)2], and
[Zn(BUT)2] have been obtained with yields of about 50 %.
Analysis of the polyhydroxychalcone metal
complexes
The polyhydroxychalcones remain structurally unchanged
in the metal complexes, as exemplarily shown by NMR
data of compound 9, ISO. In particular, the metal com-
plexes exist in the E-form, which was confirmed by the
characteristic coupling constant between the a and b pro-
tons of the double bond [see 1H NMR of (9, ISO),
J = 15.4 Hz, Fig. 3, ratio 10:0]. Despite several attempts
we were not able to obtain single crystals of [Cu(ISO)2],
[Zn(ISO)2], [Cu(BUT)2], and [Zn(BUT)2].
Independently of the used protection group, all the
condensation reactions worked with moderate yields of 45
and 52 %, respectively (see Table 1). For some substrates
MOM-ether worked better during the deprotection step
than the THP-ether [34]. Nevertheless, this strongly
depended on the substrate as for some THP-ether provided
better results. The overall yields for the three steps were
15 % for the MOM protecting group and 11 % for the THP
protecting group (see Table 1). These overall yields are in
Stoichiometry of the metal complexes using Job’s
plot
Studies to explore the stoichiometry between the polyhy-
droxychalcones and Zn2? and Cu2? were performed using
Table 1 Comparison of protecting groups for synthesis of butein
Protecting
group, yield/%
Protection
benzaldehyde,
yield/%
Protection
acetophenone,
yield/%
Coupling reaction,
yield/%
Deprotection,
yield/%
Overall,
yield/%
MOM
THP
67
60
64
64
89
83
45
52
55
15
25
11
t-BDMS
\15
n.d.
n.d.
n.d. not determined
123

C. Sulpizio et al.
Job’s plot on the basis of 1H NMR measurements [36]. The
product concentration of the metal ion and the shift dif-
ference of some indicative protons was plotted against the
molar fraction (see Fig. 2). Chemical shift changes of H-50
of 9, ISO (6.42 ppm) were measured as a function of molar
fraction. The rather large deviation in the region of the low
isoliquiritigenin (9, ISO) concentration is caused by quite
moderate S/N ratio and in particular by reasonable line
broadening. The latter is likely caused by a higher amount
of compound 9 being involved in complex formation with
Cu(II) bearing unpaired electrons. The maximum at a mole
fraction of ca. 0.66 is, however, even detectable in this case
and indicates a 1:2 stoichiometry of the investigated
complex (Fig. 2). Similar measurements with Zn(II) pro-
vide comparable results with an even better S/N ratio and
less line broadening. Hence, isoliquiritigenin (9, ISO)
coordinates to Zn2? and Cu2? in a ratio of 2:1 ISO:metal.
The predominately shifted signals are attributed to the
0
hydrogen of the double bond (Ha, Hb) and aromatic H (H3 ,
0
0
H5 , and H6 ) on ring A (see Figs. 1 and 3), which confirm
the keto function and the hydroxyl group at position C-20 to
be very likely involved in the coordination process. The
NMR complexation studies give further insights on the side
where the complexation occurs by showing a more distinct
line broadening of signals from protons influenced by the
Fig. 2 Job’s plot determined from the chemical shift changes of H-50
of isoliquiritigenin (9, ISO) with Cu(OAc)2 in methanol-d4. Summed
concentration of isoliquiritigenin (9, ISO) and Cu(OAc)2 is 13 mM
Fig. 3 1H NMR titration of the
Job’s plot of isoliquiritigenin (9,
ISO) and Zn2? with increasing
amount of Zn2? from the
bottom to the top (methanol-d4).
Colored indications highlight
additional small signals
0
attributed to H6 , Hb, and Ha
(color figure online)
123

Synthesis, characterization, and antioxidant activity of Zn2? and Cu2? coordinated…
0
0
Mass spectrometry
metal ions (Fig. 3). These are in particular H5 and H6 , as
the hydroxyl group C-20 ring A is involved in binding.
Addition of Zn(OAc)2 to the free ligand leads to the
formation of some new small peaks which are shifted
Electrospray ionization mass spectrometry (ESI-MS) is
performed on the Cu2? and Zn2? complexes of butein (10,
BUT) and isoliquiritigenin (9, ISO), respectively. The ESI
spectra show a typical isotope splitting matching the
masses of ligand to metal complexes with a stoichiometry
of 2:1, confirming their existence.
1
into a more shielded area in the H NMR (Fig. 3), likely
indicating a side reaction induced by the complexation.
In all attempts to record a Job’s plot with butein (10,
BUT) the resulting complexes were not completely soluble
and a pale yellow precipitate was formed after certain time.
Hence no 1H NMR spectra for the complex between butein
and zinc as well as between butein and copper can be
presented. Additionally 13C NMR studies have been carried
out to investigate possible coordination modes but the
analysis did not show any significant shift originating from
metal complexation. These observations are in accordance
with previous published data where the NMR spectra of the
ligand in the presence of the metal do not show any dif-
ferences from the pure ligand spectrum [37]. A possible
explanation is that the butein–metal complex is removed
from the solution due to precipitation.
For [Cu(ISO)2] these peaks can be found at m/
z = 573.0612 for 63Cu and at 575.0604 for 65Cu (see
Fig. 4). The peak corresponding to the free ligand was
observed at m/z = 255.0664 in negative mode and also the
adduct of two isoliquiritigenin molecules [(2 ISO-H?)] is
present at m/z = 511.1391. In the case of butein the peaks
of the copper adduct are present at 603.0362 for 63Cu and
605.0404 for 65Cu (see Fig. 4). These masses are attributed
to the formula [Cu(C15H10O5)2]2? indicating that the
butein coordinates the copper ions in the oxidized form
thereby explaining the loss of two Hs and we assumed that
the catechol functionality on the B ring is oxidized to the o-
Fig. 4 Electrospray ionization mass spectra of Cu2?/polyhydroxy-
chalcones 1:2 system for [Cu(ISO)2] (a) in negative mode,
[Cu(BUT)2] (b) in positive mode. The spectra a and b show the
typical isotopic spectral pattern of the experimentally observed
copper complexes Cu/L 1:2 in comparison with the corresponding
calculated one (blue). The spectra c and d show the typical isotopic
spectral pattern of the experimentally observed zinc complexes Zn/L
1:2 in comparison with the corresponding calculated one (blue) (color
figure online)
123

C. Sulpizio et al.
chinone (catecholase activity, E.C. 1.10.3.1). Oxidation of
flavonols may occur after copper complexation [38, 39].
The presence of the two peaks at m/z = 271.0599
(C10H15O5?H?) and 293.0422 (C10H15O5?Na?) results
from the oxidized butein.
diphenyl-2-picrylhydrazyl (DPPH) radical assay test,
which is one of the most common methods used to deter-
mine radical scavenging ability. The DPPH test was chosen
as it is a non-enzymatic method widely used to provide
basic information on the capability of compounds in
scavenging free radicals. It has already been proven to be a
reliable method for flavonoids and other organic com-
pounds. The test is performed in methanol because it is
well known that DPPH is not soluble in water [45–47]. ESI,
NMR, and IR spectroscopic investigations on the com-
plexes point towards metal coordination at the keto enol
moiety of the polyhydroxychalcones. Thus, the hydroxy
groups are able to participate in the free radical scavenging
activity. The activity of the metal complexes of isoliquir-
itigenin (9, ISO) with copper and zinc and of butein (10,
BUT) with copper have been measured and compared with
the one of the free ligands. Catechol has been used as a
positive control and studies were performed on the system
catechol-Cu2? and catechol-Zn2? as the free radical scav-
enging activity of phenols is not always enhanced upon
metal binding [48]. Blanks of the sole metals Cu2? and
Zn2? have also been carried out, too. The results are
summarized in Table 2.
For [Zn(ISO)2] the peaks confirming a stoichiometry of
two ligands per one zinc ion are found at m/z = 575.0676
(63Zn2?) and 577.0656 for 65Zn2?, while for [Zn(BUT)2]
these peaks are observed at m/z = 607.0576 for 63Zn2? and
609.0564 for 65Zn2?. The mass spectra did not show higher
coordinated metal complexes of stoichiometry 3:1 or 4:1
(ligand to metal). There is no peak detectable, indicating a
1:1 stoichiometry of ligand to metal.
IR spectroscopy
Infrared spectra of the Cu2? and Zn2? compounds were
recorded and compared to the free ligands butein (10,
BUT) and isoliquiritigenin (9, ISO). The intensity of the
OH-vibration at 3300 cm-1 is reduced and broadened for
both copper complexes, indicating that one or more
hydroxyl groups could be interacting with the metal center
[29]. Interestingly, the effect is more pronounced in the
case of butein possessing the catechol type functionality on
ring B. There is a shift of the carbonyl stretch from 1640 to
1610 cm-1 for both ligands in the presence of copper,
meaning that the bond strength of the C=O bond is
weakened. This is a strong indication for a participation of
the carbonyl moiety in the complexation process [40].
Additionally, a new band is observed at 500 cm-1 in the
spectra of the two metal complexes. In both cases these
bands are attributed to the m (Cu–O). This result is in
accordance with the disclosure of McWhinnie [41], who
has previously assigned the copper oxygen vibration at
500 cm-1 for related hydroxy-bridged copper compounds.
The IR spectra of the two polyhydroxychalcones with
Zn2? are quite complex as they in general exhibit a large
number of bands of varying intensities. The IR spectra of
isoliquiritigenin with Zn2? do not show any significant
shift compared to the free ligand, while the butein–Zn2?
complex shows a shift of the C=O stretching mode from
1630 to 1610 cm-1, indicating coordination of carbonyl
oxygen to metal ion [42–44]. In combination with the
Cu2? and Zn2? ions coordinated to the keto enol func-
tionality of isoliquiritigenin (9, ISO) alter the antioxidant
activity of the chalcone. In fact at the final molar ratio of
1.5 (0.8 mM of ligand and 0.4 mM of metal) the metal
complexes [Cu(ISO)2] and [Zn(ISO)2] show 72 and 88 %
inhibition of the DPPH radical, whereas the sole ligand
shows 10 % inhibition. A similar behavior is observed for
Table 2 The percentage of inhibition of the free DPPH radical in the
presence of different Me-polyhydroxychalcone systems at 1.5 molar
ratio (mol Me-polyhydroxychalcone/mol DPPH) for the studies with
isoliquiritigenin (9, ISO) and the Me-isoliquiritigenin system and 0.75
molar ratio for the butein (10, BUT) and the Cu-butein system
Compound
DPPH radical scavenging
activity/% inhibition
ISO
10
72
88
19
66
–
ISO-Cu2?
ISO-Zn2?
BUT
BUT-Zn2?
BUT-Cu2?
Catechol
1
results obtained from our H NMR titration studies, the IR
data support the formation of the complex on the 20-hy-
94
94
Catechol-
Zn2?
droxy/carbonyl side.
Cu2?
Zn2?
11
11
Free radical scavenging ability of metal chalcone
complexes
A solution of the Me-polyhydroxychalcone system at a stock con-
centration of 1.2 mM (0.8 mM of ligand and 0.4 mM of metal) has
been used. Positive controls with Cu2?, Zn2?, catechol, and catechol-
Zn2? have been performed at 1.5 molar ratio
The antioxidant activity of the metal polyhydroxychal-
cones complexes has been evaluated applying a 1,1-
123

Synthesis, characterization, and antioxidant activity of Zn2? and Cu2? coordinated…
butein (10, BUT) when complexating with Cu2? (Table 2).
The scavenging effect of the [Cu(ISO)2] was higher than
that one of the sole ligand ISO. For [Cu(BUT)2] the free
radical scavenging activity could only be observed up to a
0.75 molar ratio with 66 % inhibition in the presence of
copper while the crude butein (10, BUT) shows only 19 %
inhibition. In the preparation of the system butein/zinc a
red precipitate was immediately formed, therefore it was
not possible to perform the measurements. Pekal et al. [49]
provided the antioxidant properties of a Cu2?/quercetin
complex and Bratu et al. [26] on a Zn2?/rutin complex
using the DPPH method. Both studies conclude that the
radical scavenging activity of the metal flavonoid-com-
plexes is higher than the one of the ligands alone. The
results obtained support the mechanism suggested by
Souza et al. [21]. The higher antioxidant activity of the
complexes is due to the acquisition of additional superox-
ide dismutating centers, which causes an increase of the
molecule’s ability to stabilize unpaired electrons and
therefore to scavenge free radicals.
are the most stable compounds, as after 8 h only 20 and
30 % degraded vs. 40 and 45 % of [Zn(ISO)2] and
[Cu(ISO)2], respectively. This data points towards a rela-
tionship between the aromatic substituents and the kinetics
profiles. In particular, the presence of an ortho/para substi-
tution on the B ring seems to be fundamental in order to
achieve stability. This is probably due to the possibility to
form an intramolecular hydrogen bond, while the hydroxyl
group on the A ring looks to be of minor importance.
Therefore, the substitution pattern on the aromatic rings
seems to be a key factor in slowing the degradation process
of this class of metal complexes. At physiological pH, they
have a kinetic stability comparable with other metal com-
plexes of isoflavonoids [49].
Conclusion
In this work, two polyhydroxychalcones isoliquiritigenin
(ISO) and butein (BUT) were synthesized by Claisen
condensation and characterized applying different spec-
troscopic and spectrometric techniques. The two chalcones
have been reacted with Cu2? and Zn2? ions leading to
the coordination compounds: [Cu(ISO)2], [Zn(ISO)2],
[Cu(BUT)2], and [Zn(BUT)2].
Solubility and kinetic stability of [Cu(ISO)2],
[Zn(ISO)2], [Cu(BUT)2], and [Zn(BUT)2]
The synthesized complexes [Cu(ISO)2], [Zn(ISO)2],
[Cu(BUT)2], and [Zn(BUT)2] are insoluble in water only.
Solubility is achieved in the presence of organic solvents.
In fact the four complexes are soluble in methanol and
dimethyl sulfoxide. 35 % of water is the maximum of
water that is tolerated in the presence of dimethyl sulfoxide
and methanol until a precipitate is formed.
The Job’s plot for the system isoliquiritigenin/metal(II)
(metal = Cu2?, Zn2?) showed that isoliquiritigenin (9,
ISO) forms a complex in solution with stoichiometry two
to one, with the metal coordinating the keto function and
the hydroxyl group at position C-20. The IR and the mass
spectra confirmed that both isoliquiritigenin and butein
form stable complexes with copper and zinc. The metals
coordinate through the carbonyl function and the hydroxyl
function in ortho-position on ring A, again indicating that
zinc and copper coordinate the polyhydroxychalcones with
a ligand/metal stoichiometry of 2:1.
Several methods for a spectrophotometric determination
of the stability of metal complexes of flavonoids and related
structures have been described in the literature. We chose
the method described by Ferrari et al. [50] because the
reproducibility and accuracy are satisfactory, and it has
already been used to assess the kinetic stability of a similar
class of natural compounds such as curcumin derivatives.
We tested the stability of the complexes under physiological
conditions. Figures S1 and S2, reported in the Supplemen-
tary Material, show the decomposition kinetics profile of the
four complexes and their linear fittings. Table S1 in the
supporting information summarizes the hyperbolic function
parameters (a and b) together with their reciprocal values
and statistical parameters R2. The reciprocal of a (1/a) is the
percentage of residual compounds that remains intact under
physiological conditions. The value (-1/b) is related to the
degradation rate: the higher the value the faster the degra-
dation process. [Zn(ISO)2] is characterized by the lowest
values of 1/a and -1/b. In fact it decomposes up to *40 %
in 16 h, and a similar value of *45 % is observed for
[Cu(ISO)2]. [Cu(BUT)2] and [Zn(BUT)2] posses the highest
values for 1/a which are 0.64 and 0.74, respectively. They
Finally, the antioxidant activity of the metal polyhy-
droxychalcones was determined with the DPPH test. The
investigated complexes [Cu(ISO)2], [Zn(ISO)2], and [Zn
(BUT)2] possess higher antioxidant activity than the free
ligands isoliquiritigenin and butein.
Experimental
Chemicals were purchased from Sigma Aldrich, they were
of reagent grade and used without any further purification.
All NMR spectra were recorded in the NMR Core Facility
of the University of Vienna with a Bruker Avance III
500 MHz NMR spectrometer at 500.32 (1H), 125.81 (13C),
in CDCl3 or methanol-d4, at ambient temperature. The
1
splitting of proton resonances in the H NMR spectra are
defined as s = singlet, d = doublet, dd = doublet of
123

C. Sulpizio et al.
1.58–1.52 (m, 1H, H-10b) ppm; 13C NMR (125 MHz,
CDCl3): d = 202.6 (C=O), 164.8 (C-2), 163.1 (C-4), 132.3
(C-6), 114.5 (C-1), 108.5 (C-5), 103.4 (C-3), 96.1 (C-7),
63.0 (C-11), 30.6 (C-8), 30.0 (C-a), 25.4 (C-10), 19.7 (C-9)
ppm; HRMS (ESI-MS): m/z = 259.0931 ([M?Na]?),
237.1452 ([M?H]?, calcd. for C13H16O4 237.1121).
doublets, ddd = doublet of doublets of doublets, t = tri-
plet, and m = multiplet. Numbering of carbon atoms and
proton refers to Fig. 1 and Scheme 1. Elemental analyses
were carried out in the Mikroanalytisches Laboratorium of
the University of Vienna using an ‘‘EA 1108 CHNS-O’’
elemental analyzer by Carlo Erba Instruments. Electro-
spray ionization mass spectra were recorded in the
Massenspektrometriezentrum of the University of Vienna
with a Bruker Esquire 3000 with an orthogonal ESI source
with MeOH/ACN as solvent, the molecular mass was
determined in the positive as well as in the negative mode.
The spectrophotometer Shimadzu UV 1800 has been used
to recorder the UV–Vis spectra. The infrared spectra were
recorded with an infrared spectrometer Bruker Tensor 27
FTIR equipped with a globar 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).
4-(Tetrahydro-2H-pyran-2-yloxy)benzaldehyde
(5a, C12H14O3)
4-Hydroxybenzaldehyde (6.60 mmol) and pyridinium
para-toluenesulfonate (0.16 mmol) were dissolved in
30 cm3 of dichloromethane and the solution was stirred
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
column chromatography (hexane/ethyl acetate 7:3) to
obtain pure 5a. 1H NMR (500 MHz, CDCl3): d = 9.97
(s, 1H, C=O), 7.87–7.81 (dd, J = 8.9, 2.0 Hz, 2H, H-2,
H-6), 7.20–7.13 (dd, J = 8.9, 2.0 Hz, 2H, H-3, H-5), 5.55
(t, J = 3.1 Hz, 1H, H-7), 3.93–3.82 (m, 1H, H-11a), 3.64
(m, 1H, H-11b), 2.08–1.96 (m, 1H, H-8a), 1.93–1.82 (m,
2H, H-8b, H-9a), 1.80–1.60 (m, 2H, H-9b, 10a), 1.58–1.50
(m, 1H, H-10b) 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).
Synthesis of the polyhydroxychalcones
(E)-1-(2,4-Dihydroxyphenyl)-3-(4-hydroxyphenyl)-2-pro-
pen-1-one and (E)-1-(2,4-dihydroxyphenyl)-3-(3,4-
dihydroxyphenyl)-2-propen-1-one were synthesized per-
forming a Claisen–Schmidt condensation between the
benzaldehyde and acetophenone derivatives according to
the previously published procedure [30]. The 3,4-dihydro-
a-pyran is used to protect the hydroxyl group present on
the aromatic ring before performing the aldol reaction in
basic condition. The yields of each reaction are summa-
rized in Table 1. The synthesized compounds were
3,4-Bis(tetrahydro-2H-pyran-2-yloxy)benzaldehyde
(6a, C17H22O5)
3,4-Dihydroxybenzaldehyde (6.60 mmol) and pyridinium
para-toluenesulfonate (0.16 mmol) were dissolved in
30 cm3 of dichloromethane and the solution was stirred
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
obtain 6a as a mixture of stereoisomers. 1H NMR
(500 MHz, CDCl3): d = 9.87 (s, 1H, CHO), 7.50–7.49
(d, J = 8.0 Hz, 1H, H-6), 7.25 (s, 1H, H-2), 7.07–7.05 (d,
J = 8.2 Hz, 1H, H-5), 5.55 (t, J = 2.7 Hz, 2H, H-7, H-70),
3.93–3.82 (m, 2H, H-11a, H-110a), 3.64 (m, 2H, H-11b,
H-11b), 2.08–1.96 (m, 2H, H-8a, H-8a0), 1.93–1.82 (m, 4H,
H-8b, H-8b0, H-9a, H-9a0), 1.80–1.60 (m, 4H, H-9b, H-9b0,
H-10a, H-10a0), 1.58–1.50 (m, 2H, H-10b, 10b0) 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
1
characterized by H NMR, mass spectrometry, and IR.
2-Hydroxy-4-(tetrahydro-2H-pyran-2-yloxy)acetophenone
(2a, C13H16O4)
2,4-Hydroxyacetophenone (1, 6.60 mmol) and pyridinium
para-toluenesulfonate (0.16 mmol) were dissolved in
30 cm3 of dichloromethane and the solution was stirred
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
column chromatography (hexane/ethyl acetate 9:2) to
1
obtain pure 2a. H NMR (500 MHz, CDCl3): d = 12.62
(s, 1H, Ar–OH), 7.65 (d, J = 8.8 Hz, 1H, H-6), 6.63 (d,
J = 2.5 Hz, 1H, H-3), 6.57 (dd, J = 8.8, 2.5 Hz, 1H, H-5),
5.49 (t, J = 3.1 Hz, 1H, H-7), 3.85 (ddd, J = 11.3, 9.9,
3.1 Hz, 1H, H-11a), 3.66–3.61 (m, 1H, H-11b), 2.58 (s, 3H,
H-a), 2.04–1.95 (m, 1H, H-8a), 1.90–1.85 (m, 1H, H-8b),
1.75–1.65 (m, 2H, H-9a, H-9b), 1.65–1.59 (m, 1H, H-10a),
123

Synthesis, characterization, and antioxidant activity of Zn2? and Cu2? coordinated…
([M?Na]?), 307.1534 ([M?H]?, calcd. for C17H22O5
unpurified reaction mixture. HRMS (ESI-MS): m/z =
547.2291 ([M?Na]?), 525.2472 ([M?H]?).
307.1461).
(E)-1-[2-Hydroxy-4-(tetrahydro-2H-pyran-2-yloxy)-
(E)-1-(2,4-Dihydroxyphenyl)-3-(4-hydroxyphenyl)-prop-2-
en-1-one (9, ISO, C15H12O4)
7a (1.26 mmol) was dissolved in 50 cm3 of methanol.
phenyl]-3-[4-(tetrahydro-2H-pyran-2-yloxy)-
phenyl]prop-2-en-1-one (7a, C25H28O6)
Compound 2a (4.24 mmol) and 8.48 mmol of 5a 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 HCl (0.1 M), and extracted with ethyl
acetate. The organic layer was separated, dried over
Na2SO4, and then concentrated under vacuum. The reac-
tion mixture was purified by flash column chromatography
(hexane/ethyl acetate 7:3) to obtain pure 7a. 1H NMR
(500 MHz, methanol-d4): d = 8.07 (d, J = 8.8 Hz, 1H,
H-60), 7.85 (d, J = 15.4 Hz, 1H, H-b), 7.73 (d, J = 8.8 Hz,
2H, H-2, H-6), 7.70 (d, J = 15.4 Hz, 1H, H-a), 7.14 (d,
J = 8.8 Hz, 2H, H-3, H5), 6.67 (dd, J = 8.8, 2.5 Hz, 1H,
H-50), 6.62 (d, J = 2.5 Hz, 1H, H-30), 5.58 (t, J = 3.1 Hz,
1H, H-7), 5.53 (t, J = 3.1 Hz, 1H, H-70), 3.92–3.80 (m,
2H, H-11a, H-11a0), 3.69–3.59 (m, 2H, H-11b, H-11b0),
2.07–1.96 (m, 2H, H-8a, H-8a0), 1.93–1.77 (m, 4H, H-8b,
H-80b, H-9a, H9a0), 1.76–1.55 (m, 4H, H-9b, H-9b0, H-10a,
H-10a0), 1.66–1.54 (m, 2H, H-10b, H-10b0) ppm; 13C NMR
(125 MHz, methanol-d4): d = 192.0 (C=O), 164.7 (C-40),
163.5 (C-20), 159.3 (C-4), 144.3 (C-b), 132.9 (C-60), 132.2
(C-2, C-6), 128.2 (C-1), 118.1 (C-a), 116.7 (C-5), 116.5
(C-3), 114.4 (C-10), 108.5 (C-50), 104.0 (C-30), 96.1 (C-70),
94.7 (C-7), 62.9 (C-110), 62.1 (C-11), 30.6 (C-80), 29.9 (C-
8), 25.4 (C-100), 24.9 (C-10), 19.7 (C-90), 18.5 (C-9) ppm;
HRMS (ESI-MS): m/z = 447.1771 ([M?Na]?), 425.1956
([M?H]?, calcd. for C25H28O6 425.1880).
Subsequently
pyridinium
para-toluenesulfonate
(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 purified by flash column chromatogra-
phy (dichloromethane/methanol 9.5:0.5). 1H NMR
(500 MHz, methanol-d4): d = 7.98 (d, J = 9.1 Hz, 1H,
H-60), 7.80 (d, J = 15.4 Hz, 1H, H-b), 7.62 (d, J = 8.9 Hz,
2H, H-2, H-6), 7.60 (d, J = 15.4 Hz, 1H, H-a), 6.87 (d,
J = 8.9 Hz, 2H, H-3, H-5), 6.42 (dd, J = 8.8, 2.5 Hz, 1H,
H-50), 6.29 (d, J = 2.5 Hz, 1H, H-30) ppm; 13C NMR
(125 MHz, methanol-d4): d = 193.7 (C=O), 167.6 (C-40),
166.5 (C-20), 161.7 (C-4), 145.8 (C-b), 133.5 (C-60), 131.9
(C-2, C-6), 128.0 (C-1), 118.5 (C-a), 117.0 (C-3, C-5),
114.8 (C-10), 109.2 (C-50), 103.9 (C-30) ppm; FT-IR (KBr):
vꢀ = (C–OH) 3300, (C=O) 1640, (C=C) 1600, 1587,
1520 cm-1
;
HRMS
(ESI-MS):
m/z = 279.0627
([M?Na]?), 257.0807 ([M?H]?, calcd. for C15H12O4
257.0730).
(E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dihydroxy-
phenyl)prop-2-en-1-one (10, BUT, C15H12O5)
8a (1.26 mmol) was dissolved in 50 cm3 of methanol.
Subsequently
pyridinium
para-toluenesulfonate
(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 purified by column flash chromatogra-
phy (Sephadex LH-20, dichloromethane/methanol 9.5:0.5).
1H NMR (500 MHz, methanol-d4): d = 7.95 (d,
J = 9.2 Hz, 1H, H-60), 7.77 (d, J = 15.4 Hz, 1H, H-b),
7.53 (d, J = 15.4, 1H, H-a), 7.19 (d, J = 2.2 Hz, 1H, H-2),
7.12 (dd, J = 8.0, 2.2 Hz, 1H, H-6), 6.84 (d, J = 8.0 Hz,
1H, H-5), 6.42 (dd, J = 8.8, 2.5 Hz, 1H, H-50), 6.29 (d,
J = 2.5 Hz, 1H, H-30) ppm; 13C NMR (125 MHz,
methanol-d4): d = 191.9 (C=O), 166.0 (C-40), 164.4 (C-
20), 148.4 (C-5), 145.3 (C-4), 144.5 (C-b), 131.8 (C-60),
126.9 (C-1), 122.1 (C-6), 116.8 (C-a), 115.1 (C-5), 114.2
(C-2), 113.1 (C-10), 107.7 (C-50), 102.4 (C-30) ppm; FT-IR
(KBr): vꢀ = (C–OH) 3300, (C=O) 1640, (C=C) 1600, 1587,
(E)-3-[3,4-Bis(tetrahydro-2H-pyran-2-yloxy)phenyl]-1-[2-
hydroxy-4-(tetrahydro-2H-pyran-2-yloxy)phenyl]prop-2-
en-1-one (8a, C30H36O8)
Compound 2a (4.24 mmol) and 8.48 mmol of 6a 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 it was concentrated in vacuum, quenched with HCl
(0.1 M), and extracted with ethyl acetate. The organic layer
was separated, dried over Na2SO4, and then concentrated
under vacuum. The residue yielded the crude chalcone 8a
as a yellow powder which was a mixture of stereoisomers
and was used for the next step without any further
purification. NMR data have not been recorded from the
1520 cm-1
;
HRMS
(ESI-MS):
m/z = 293.1756
([M-2H??Na?]), 271.0617 ([M-H]?, calcd. for
C15H11O5 271.0611).
Bis[(E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-
2-en-1-one]copper ([Cu(ISO)2], C30H22CuO8)
ISO (0.78 mmol) was dissolved in 15 cm3 of methanol
under reflux. Subsequently sodium methoxide (0.8 mmol)
123

C. Sulpizio et al.
was added to the reaction mixture. After a few minutes a
solution of 15 cm3 methanol containing copper acetate
monohydrate (0.35 mmol) was added dropwise and the
color of the reaction mixture changed from orange to red-
brown. The reaction mixture was stirred over night under
reflux at 35 °C, then raised to room temperature and a
brown precipitate was formed, which was filtered, washed
with diethyl ether, and analyzed via mass spectrometry and
IR spectroscopy. Yield: 52 %; FT-IR (KBr): vꢀ = (C–OH)
3300, (C=O) 1610, (C=C) 1600, 1587, 1520, (Cu–O)
analyzed via mass spectrometry and IR spectroscopy.
Yield: 50 %; FT–IR (KBr): vꢀ = (C–OH) 3300, (C=O)
1610, (C=C) 1600, 1587, 1520 (Cu–O) 500 cm-1; HRMS
(ESI-MS): m/z = 607.0576 ([2 BUT ? 63Zn2? ? H]?,
calcd. for [C30H22ZnO8 ? H]? 607.0577), 609.0564 [2
ISO ? 65Cu2? ? H].
Spectroscopic data: Job’s plot
For the Job’s plot (method of continuous variations) stock
solutions of the polyhydroxychalcones 9 and 10 and Cu(OAc)2,
Zn(OAc)2 were prepared in methanol-d4. For each polyhy-
droxychalcone/divalent cations system, eleven 5 mm high-
precision NMR sample tubes were filled with 0.6 cm3 of the
compounds in the ratios [cation]/[polyhydroxychal-
cone] = 0:10, 1:9 up to 9:1, and 10:0, respectively. The total
concentration was kept constantly at 13 mM (600 mm3).
500 cm-1
;
HRMS (ESI-MS): m/z = 573.0612 [2
ISO ? 63Cu2? ? e-], 575.0604 ([2 ISO ? 65Cu2? ? e-],
calcd. for C30H22CuO8 573.0605).
Bis[(E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)-
prop-2-en-1-one]copper
([Cu(BUT)2], C30H20CuO10)
BUT (0.36 mmol) was dissolved in 5 cm3 of methanol at
reflux. Subsequently a solution containing 35 mg of copper
acetate monohydrate (0.17 mmol) was added dropwise.
The solution was stirred under reflux for 1 h, then a red/
brown precipitate was formed, which was filtered, washed
with diethyl ether, and analyzed via mass spectrometry and
IR spectroscopy. Yield: 50 %; FT–IR (KBr): vꢀ = (C–OH)
3250, (C=O) 1610, (C=C) 1600, 1570, 1490, (Cu–O)
500 cm-1; HRMS (ESI-MS): m/z = 603.0362 ([2 BUT–
4H ? 63Cu2?], calcd. for C30H20CuO10 603.034),
605.0404 [2 BUT–4H ? 65Cu2?].
Antioxidant activity (DPPH radical scavenging method)
The antioxidant activity test is conducted according to the
methoddescribed byFerrari et al. [48]. The test is based onthe
usage of 2,2-diphenyl-1-picrylhydrazyl, which is a nitrogen-
centered stable radical that gives a specific absorption with a
maximum at 517 nm. The free radical species DPPH reacts
withtheantioxidantcompoundandisconsequentlyreducedto
the non-radical species which producesa changeinabsorption
[51]. The resulting decrease in absorbance was used to
quantify the antioxidant activity. 1 cm3 of 6 9 10-5 mM
DPPHradical solutionispreparedinmethanolandmixedwith
a variable amount (10, 25, 50, 75 mm3, etc.) of a methanolic
solution containing the system metal-chalcone 1.2 mM.
Immediately the absorbance of the mixture is measured every
secondupto30 minat517 nm. Forthebaselinecontrol 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. [48]:
Bis[(E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-
2-en-1-one]zinc ([Zn(ISO)2], C30H22ZnO8)
ISO (0.195 mmol) was dissolved in 5 cm3 of acetonitrile.
After a few minutes a solution of 2.5 cm3 acetonitrile
containing zinc chloride (0.09 mmol) was added. The
reaction mixture was stirred at room temperature for 24 h.
The solvent was removed under reduced pressure at room
temperature to half of the initial volume. After a few hours an
orange precipitate was formed, which was filtered, washed
with diethyl ether, dried in air, and analyzed via mass
spectrometry and IR spectroscopy. Yield: 53 %; FT–IR
(KBr): vꢀ = (C–OH) 3300, (C=O) 1610, (C=C) 1600, 1587,
1520, 500 (Cu–O) cm-1; HRMS (ESI-MS): m/z = 575.0676
([2 ISO ? 63Zn2? ? H]?, calcd. for [C30H22ZnO8 ? H]?
575.0679), 577.0656 ([2 ISO ?65Zn2? ? H])?.
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 determinations were
performed in triplicate and the values of absorbance were
corrected considering the factor of dilution.
Solubility and kinetic stability
Bis[(E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxy-
phenyl)prop-2-en-1-one]zinc
To test the solubility of [Cu(ISO)2], [Zn(ISO)2],
[Cu(BUT)2], and [Zn(BUT)2] in water, we used the method
of the shaken-flask. 100 mm3 stock solution of each
compound in methanol (52 mM) were mixed in a vessel
with regular addition of 10 mm3 of water and then shaken.
The procedure was repeated until the formation of precip-
itate was observed.
([Zn(BUT)2], C30H22ZnO8)
BUT (0.2 mmol) was dissolved in 5 cm3 of acetonitrile.
After a few minutes a solution of 2.5 cm3 acetonitrile
containing zinc chloride (0.09 mmol) was added. The
reaction mixture was stirred at room temperature for 24 h.
After few hours a yellow precipitate was formed, which
was filtered, washed with diethyl ether, dried in air, and
The chemical stability of the four complexes was
investigated at 37 °C in the darkness applying UV–Vis
123

Synthesis, characterization, and antioxidant activity of Zn2? and Cu2? coordinated…
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spectroscopy by measuring the change in absorbance in the
200–600 nm range over an overall period of 16 h. Then
50 lM solutions of the complexes were prepared in DMSO
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t
¼ at þ b;
ð1Þ
f%
where f% is the fraction of residual compound at time
t (min) expressed as a percentage referred to starting con-
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Acknowledgments Open access funding provided by University of
Vienna. The authors acknowledge Ao. Univ.-Prof. Mag. Dr. Markus
Galanski from the Institut fu¨r Anorganische Chemie of the University
of Vienna for NMR measurement. Ing. Peter Unteregger is
acknowledged for support with the ESI-MS measurements at the
Massenspektrometriezentrum of the University Vienna. We thank
also Ao. Univ.-Prof. Eugen Libowitzky for access to ATR-IR mea-
surements. The authors wish to thank Dr. Federica Cappa for valuable
discussion regarding the organic synthesis.
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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
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