Journal of Inorganic Biochemistry 114 (2012) 38–46

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Journal of Inorganic Biochemistry

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Hydroxy(thio)pyrone and hydroxy(thio)pyridinone iron chelators: Physico-chemical

properties and anti-oxidant activity
Sílvia Chaves a, Sónia Canário a, Marta P. Carrasco a, Lurdes Mira b, M. Amélia Santos a,⁎
a
Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais 1, 1049‐001 Lisboa, Portugal
b
Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749‐016 Lisboa, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: The O,S-donor analogues of maltol and deferiprone (DMHP), respectively, thiomaltol and DMHTP, have been in-
Received 2 December 2011 vestigated in solution for their iron-complexation ability, as well as their electrochemical behaviors, in the pres-
Received in revised form 26 April 2012 ence and absence of iron, aimed at the rationalization of their anti-oxidant activity, particularly, as hydroxyl
Accepted 26 April 2012
radical scavengers and inhibitors of lipid peroxidation. The results were compared with those of the O,O-donor
Available online 4 May 2012
compounds and revealed that all the compounds are good iron chelators (pFe= 14.1–20.2), but the O,S-donor
Keywords:
ligands being somewhat weaker than the corresponding oxo-analogues. Also all the ligands appear to be able
O,S-ligands to prevent the redox cycling of iron, a relevant anti-oxidant activity, which seems to be primary due to their
Hydroxy(thio)pyridinones high capacity to form iron complexes which are not effective in promoting free radical reactions. This is a signif-
Hydroxy(thio)pyrones icant feature for the development of leading analogues as drug candidates with co-adjuvant roles in oxidative-
Iron chelators stress dependent pathologies.
Antioxidants © 2012 Elsevier Inc. All rights reserved.
Oxidative stress

1. Introduction
Chronic iron overload and related diseases, requiring frequent blood
transfusions, have been managed by chelation therapy protocols, main-
ly using the bacterial siderophore deferrioxamine, a compound that
needs to be injected intravenously, and/or orally active compounds
such as deferiprone (DMHP). An important goal of the research on the
field of iron chelators has been focused on the design of selective
drugs, which can be orally administered, forming inert iron complexes
with low toxicity [1]. The redox cycling of iron constitutes a critical as-
pect of its toxicity. In fact, iron has a major role in the production of sev-
eral oxygen free radicals in living organisms, by promoting the
generation of the very reactive hydroxyl radical (HO•) through the
Fenton and Haber–Weiss reactions [2,3]. In the Fenton reaction, the
hydroxyl radical is produced from hydrogen peroxide (H2O2):
nþ − · ðnþ1Þþ
H2 O2 þ M →HO þ HO þ M
:
In the metal-catalyzed Haber–Weiss reaction, the superoxide radical
(O2•−) reduces the metal ion, which then reacts with hydrogen peroxide
by Fenton chemistry to yield the hydroxyl radical:
·− ðnþ1Þþ nþ
O2 þ M →O2 þ M
⁎ Corresponding author. Tel.: + 351 218419273; fax: + 351 218464455.
E-mail address: masantos@ist.utl.pt (M.A. Santos).
nþ − · ðnþ1Þþ
H2 O2 þ M →HO þ HO þ M
In addition, through a Fenton type reaction, preformed lipid hydro-
peroxides (ROOH), resulting from oxidation of unsaturated fatty acids,
are decomposed to form the alkoxyl radicals (RO•), strong oxidants
that can propagate the reaction chain of lipid peroxidation [4]:
nþ · · ðnþ1Þþ
ROOH þ M →RO þ HO þ M
Free radicals are continuously being formed in small amounts by
normal metabolic processes and many of them serve useful physiolog-
ical functions [5,6]. Nevertheless, when overproduced, they can damage
biomolecules and be implicated in the aetiology of several diseases
(atherosclerosis, diabetes, ischemia-reperfusion, chronic inflammation,
cancer) and ageing [7,8]. The antioxidants, even if present at much
lower concentrations than the “oxidizable substrates”, can significantly
delay or prevent oxidative processes [9]. Their properties can be due to
their ability to scavenge free radicals and other reactive species that can
damage biomolecules, or even to synergistic effects with other drugs
[10]. Another anti-oxidant mechanism may result from metal chelation
and concomitant hampering of the participation of these metal ions in
free radical generating reactions, such as those above described [2–4].
Therefore, the development of low-molecular-weight iron chelators,
with anti-oxidant activity may lead to potential drugs with co-adjuvant
roles in oxidative-stress dependent pathologies. In fact, by reducing the
level of plasma labile iron, the iron chelator can also simultaneously
suppress redox cycling, avoiding oxidative stress and preventing iron

0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2012.04.019
 S. Chaves et al. / Journal of Inorganic Biochemistry 114 (2012) 38–46 39

overload‐related neurologic disorders such as Alzheimer disease, Table 1

Parkinson disease, Friedreich's ataxia and multiple sclerosis [11]. Protonation constantsa (log Ki), partition coefficientsb (log P) and redox potentialsc
(Ep) of maltol, thiomaltol, DMHP and DMHTP, together with some literature data.
Hydroxypyrones and hydroxypyridinones are well known for their
synthetic versatility and high affinity for a range of metal ions, thus ren- Ligand Maltol Thiomaltol DMHP DMHTP
dering them as excellent choices for the formulation of therapeutic log K1 8.54 (1) 8.09 (1) 9.77 (1) 9.45 (1)
metal sequestering drugs and diagnostic metallopharmaceuticals [12]. 8.67 (3)d,e 8.27 (1)d 9.82 (1)d 9.70 (1)d
Following our interest on the development of O,O-donor ligands of 8.513f 8.16 (2)h 9.76j 9.47h
8.7g 8.12 (2)i 9.749 (4)k 9.44 (1)i
hydroxypyridinone type and their O,S-analogues [12–14], we report
9.64l
herein the results of studies performed with two O,S-donor analogues, log K2 – – 3.64 (1) 0.95 (3)m
thiomaltol and DMHTP (see Scheme 1), alone or in the presence of 3.74 (2)d
iron. For comparison, their corresponding O,O-analogues, maltol (3- 3.62j
hydroxy-2-methyl-4-pyrone) and DMHP (3-hydroxy-1,2-dimethyl-4- 3.655 (7)k
3.56l
pyridinone), are also studied under the same experimental conditions.
log P − 0.04 0.54 − 0.82n − 0.59
In particular, solution physico-chemical properties are evaluated for − 1.03o
this set of compounds (acid‐base behavior, lipo-hydrophilic character, Ep (V) 1.304 1.113 0.913 0.752
redox potentials) and their iron complexes, namely the thermodynamic a
I = 0.1 M KCl, 5% DMSO/H2O, T = 25.0 ± 0.1 °C.
stability constants and the redox properties. Finally, their antioxidant b
1-octanol/water at pH 7.4.
c
activity is assessed and discussed on the basis of their capacity for hy- I = 0.1 M KNO3, 20% DMSO/H2O by CV (pH 5, scan rate 100 mV/s).
d
droxyl radical scavenging, metal chelation and lipid peroxidation Ref. [13] (I = 0.1 M KCl, 15% DMSO/H2O, T = 25.0 ± 0.1 °C).
e
Ref. [15] (I = 0.16 M NaCl, T = 37 °C, in water).
inhibition. f
Ref. [16].
g
Ref. [17].
h
2. Results and discussion Ref. [18] (I = 0.1 M KCl, T = 25.0 °C, in water).
i
Ref. [19] (I = 0.16 M NaCl, T = 25.0 °C, in water).
j
Ref. [20] (I = 0.1 M KCl, T = 25.0 °C, in water).
2.1. Physico-chemical characterization of the compounds k
Ref. [21] (I = 0.2 M KCl, T = 25.0 °C, in water).
l
Ref. [22].
The ligands were firstly characterized in terms of their most relevant m
Ref. [13] (determined by 1H NMR titration).
n
physico-chemical properties, namely the stepwise protonation con- Ref. [23].
o
Ref. [24].
stants (log Ki) in 5% (v/v) DMSO/H2O medium, the partition coefficients
(log P) between 1-octanol and a TRIS-buffered aqueous solution at pH
7.4 and also the oxidation potentials (EP) in 20% (v/v) DMSO/H2O, at
pH 5. The set of values obtained herein, together with others reported
in the literature for different experimental conditions, are summarized
in Table 1 [13,15–24]. The option for a mixed DMSO/H2O media was stabilization of the conjugate bases of the pyrones, due to the lower
due to solubility limitations of the thio-derivatives in water, namely electronegativity and higher polarizability of the sulphur atom with
for the CV working conditions (Section 2.1.3). subsequently larger delocalization of the negative charge. Other
A detailed analysis of the obtained values is presented below. examples of this observation are pyromeconic acid (7.69 [26]) and
thiopyromeconic acid (7.29 [18]) or ethylmaltol (8.72 [15]) and
2.1.1. Acid‐base properties ethylthiomaltol (8.16 [19]). The second conclusion results from the
For all the ligands investigated, the stepwise protonation constants fact that the ring O-atom is more electronegative than the ring
were obtained from potentiometric titrations and curve fitting analysis N-atom, thus contributing to a further stabilization of the pyrone
with the HYPERQUAD program [25], providing one or two constants for conjugate base.
the hydroxy(thio)pyrones and the hydroxy(thio)pyridinones, respec-
tively. The first protonation constants are attributed to the hydroxyl 2.1.2. Lipo-hydrophilic character
group of those compounds, while the second one is attributed to the The lipophilicity of the compounds was estimated through the cal-
N-pyridinyl group. Analysis of Table 1 evidences that the log Ki values culation of their partition coefficients (log P) between 1-octanol and a
obtained herein are in good agreement with those found in the literature TRIS-buffered aqueous phase at pH 7.4. Table 1 shows that thiomaltol
for different experimental conditions. From these results, two conclu- and DMHTP present a higher lipophilic character than the respective
sions can be drawn: the oxo-compounds (maltol, DMHP) present higher oxo-derivatives (maltol and DMHP). Since, at the physiological pH, all
log Ki values than the corresponding thio-derivatives (thiomaltol, the compounds have the neutral species HL (83–99% formation) as
DMHTP); the hydroxyl groups of the pyrones (maltol, thiomaltol) are the predominant form, a moderate lipophilic nature would be expected
more acidic than those of the corresponding pyridinones (DMHP, for them. Nevertheless, partition coefficients are slightly different
DMHTP). The first conclusion may be explained by the additional (−0.82 b log P b 0.54), because the molecular charge is not the only
relevant factor for the lipo-hydrophilic character, but solute–solvent
interactions can also be determinant features. In fact, the herein calcu-
lated log P values are in accordance with the fact that both thio-
compounds are less soluble in water than the oxo-analogues, probably
due to a lesser degree of H-bond formation as well as to the lesser
strength of the [O–H…S] hydrogen bonds, as compared with that of
the [O–H…O] analogues.

2.1.3. Redox behavior of the compounds

Scheme 1. Structural formulae of the studied compounds.
In order to determine the redox potential of the compounds, their
electrochemical behavior was analyzed by cyclic voltammetry (CV) at
a glassy carbon electrode, in a 20% (v/v) DMSO in 0.1 M KNO3 aqueous
solution. The option for the glassy carbon electrode was required to
circumvent problems due to adsorption of the thio-derivatives by the
40 S. Chaves et al. / Journal of Inorganic Biochemistry 114 (2012) 38–46

mercury dropping electrode and also to limitations of the working
potential window in the case of the oxo-compounds. Thus, the glassy
carbon electrode appeared adequate to make studies both with the
oxo- and the thio-compounds. The use of DMSO in electrochemistry is
also well established [27].
Some preliminary CV studies of the compounds performed at pH 3, 5
and 7 showed that the oxidation peak potential for each ligand
remained constant in that pH range. Therefore, a final option for
working at a fixed pH 5 was taken to guarantee the presence of 100%
HL species for all the compounds. Furthermore, under our experimental
conditions (pH 5), no splitting in two of the oxidation peaks of DMHP or
DMHTP was observed, as found by El-Jammal and Templeton [28], due
to the existence of two structurally distinct electroactive forms related
by a slow tautomeric equilibrium between phenolic and zwitterionic
phenolate forms.
In fact, for scan rates between 100 and 800 mV/s, the compounds
exhibited single irreversible anodic waves (see Fig. 1) with peak poten-
tials (vs Ag/AgCl electrode) at 1.304 V (maltol), 1.113 V (thiomaltol),
0.913 V (DMHP) and 0.752 V (DMHTP). The plot of the experimental
anodic peak currents (iP) versus the square root of the scan rate (v1/2)
obtained from the cyclic voltammograms of the four ligands showed
linear relationships, indicating diffusion controlled waves for irrevers-
ible oxidation of these compounds in the range of pH studied and for
scan rates higher than 200 mV/s. Moreover, in the case of DMHP, the
iP/v 1/2 versus v1/2 graph presents a negative slope which points towards
a process involving a CE (chemical reaction followed by an electron
transfer process). The oxidation process involves two electrons in the
case of the pyrones and one electron for the pyridinones. It was found
that the redox potentials of the oxo-compounds (maltol, DMHP) are
higher than those of the corresponding thio-compounds (thiomaltol,
DMHTP) and so the oxo-compounds appear as worse reductants than
the thio-analogues.
a)
4 × 10-4
3 × 10-4
2 × 10-4
1 × 10-4
2.2. Iron complexes
2.2.1. Iron‐chelating capacity
The iron-chelating ability of the compounds was evaluated, in
order to obtain the respective complex formation model and species
distribution curves. The global stability constants obtained for the iron
complexes of the four compounds are presented in Table 2, as well as
some literature values previously obtained for the O,O-analogues.
They were determined from the fitting analysis of measured pH-
dependent UV/visible (UV/Vis) spectra (PSEQUAD program [30]),
since at the beginning of the potentiometric titration the extent of
complexation was too high to allow the use of this method. To deter-
mine the value of log βFeL, spectrophotometric titrations at 1:3 (Fe/L)
stoichiometry and pH b 2 were performed, using a batch procedure in
which the exact amount of acid to be added was calculated for the
total volume of solution. The subsequent log βFeL2 and log βFeL3 global
constants were calculated from fitting analysis of the 1:3 Fe/L spec-
trophotometric titration data at pH > 2, while fixing the previously
determined log βFeL value; in the case of the Fe(III)/thiomaltol system,
a 1:10 stoichiometric ratio was used because under 1:3 conditions pre-
cipitation occurred at pH ca 3.3.
Analysis of Table 2 indicates good iron-chelating capacities for all the
compounds, although the commercially available DMHP presents the
highest chelating capacity (pFe= 20.2); also all the iron–ligand systems
include three species (FeL, FeL2 and FeL3) in the complexation model
and the values herein calculated for the O,O-analogues are in good
agreement with those obtained by other authors [22,29]. All the com-
pounds present a bidentate coordination to iron, via the phenol and
(thio)keto groups, but the O,O‐ligands prove to be better Fe(III) chela-
tors than the corresponding O,S-analogues, in agreement with the
Pearson´s principle of hard soft [Lewis] acid base (HSAB) [31]. Analysis
of spectral titration data (Fig. 2) evidences two absorbance maxima at
410 and 468 nm, corresponding to the formation of the FeL3 complex
(L ≡ maltol), while the isosbestic point at 506 nm gives support to the
existence of an equilibrium between the FeL2 and FeL3 complexes.
Moreover, analysis of the data shown in Fig. 2 reveals a maximum
absorption at 530 nm corresponding to the bischelate (FeL2) charge-
transfer band (LMCT), while the spectrum obtained at the lowest
pH (0.8) can be fitted with formation of ca 40% FeL and 8% FeL2.
Analysis of Fig. 3, in comparison with Fig. 2, evidences the relatively
weaker Fe–ligand interaction of the FeL3 complex in the case of the
Fe(III)/thiomaltol system as compared with that of Fe(III)/maltol,
namely based on the red shift of absorption maximum wavelength
(500/615 vs 410/468 nm). Data analysis of the electronic spectra
obtained for Fe(III)/DMHTP also indicates the existence of three

Table 2
Global stability constantsa (log βMLi), pFeb and redox potentialsc (EP) for the Fe(III)
complexes of maltol, thiomaltol, DMHP and DMHTP.

b) Ligand Maltol Thiomaltol DMHP DMHTP

1 × 10-4 log βFeL 10.91 (1) 10.56 (1) 14.82 (1) 11.82 (2)
11.2d 15.14e
14.56f
log βFeL2 21.26 (4) 18.00 (2) 27.02 (1) 22.39 (1)
21.9d 26.68e
26.75f
5 × 10-5 log βFeL3 29.81 (6) 23.14 (5) 36.81 (1) 31.19 (1)
28.2d 35.92e
36.4f
pFe 16.9 14.1 20.2 15.6
EP (V) − 0.588 − 0.459 − 0.933 − 0.703
a
I = 0.1 M KCl, 5% DMSO/H2O, T = 25.0 ± 0.1 °C.
b
pH = 7.4, CL/CFe = 10, CFe = 10− 6 M.
c
I = 0.1 M KNO3, 20% DMSO/H2O by CV (CL/CFe = 5, CFe = 200 μM, pH = 7, scan rate
100 mV/s).
d
Ref. [29].
e
Fig. 1. Typical cyclic voltammograms for oxidation of: a) maltol and thiomaltol, b) DMHP Ref. [20] (I = 0.1 M KCl, T = 25.0 °C, in water).
f
and DMHTP (CL = 6.25 × 10− 3 M, pH 5, scan rate 100 mV/s). Ref. [22].
 S. Chaves et al. / Journal of Inorganic Biochemistry 114 (2012) 38–46
a)
0.8
0.7
0.6
0.5
A 0.4
0.3
0.2
0.1
0
350 400 450 500 550 600 650
λ (nm)
b)
FeL3
FeL2
FeL
Fe
Fig. 2. a) Electronic spectra of the 1:3 Fe(III)/maltol system, pH 0.8–6.2; b) species distri-
bution curves with molar extinction coefficients at the maximum absorption wavelengths
(CL/CFe = 3, CFe = 2 × 10− 4 M).
iron complexes, although only one broad band with wavelength
maximum at 620 nm was observed, apparently corresponding to
the FeL2 and FeL3 complexes (see Fig. 4).
In the solid state, the octahedral Fe(III)–(thiomaltol)3 complex
was found to have a twist angle of 48.2 o [32], that is slightly lower
than the reported value for Fe(III)-(maltol)3 [33], thus suggesting for
thiomaltol a substantial trans influence and a concomitant facial
complex geometry, which should be enthalpically favourable. Appar-
ently, this is in opposition to the relative magnitude order of the
corresponding Fe(III)-complex stabilities found for these species in
aqueous solution.
2.2.2. Redox behavior of the iron complexes
The potential toxicity of the Fe(III)-chelates associated to the
Fenton reaction depends on different factors, such as the accessibility
of the iron centre to reductants, the kinetics of complex dissociation
and the potentials involved in the redox cycling. Accordingly, the
redox potential of the iron complexes is expected to be quite depen-
dent on the type of chelator. Thus, the series of iron complexes with
the ligands under study was generated in situ and their redox proper-
ties were assessed by CV at neutral pH. These electrochemical studies
were performed under micromolar ligand concentrations and using a
mixed solvent (20% (v/v) DMSO/0.1 M KNO3 aqueous solution) to guar-
antee the absence of precipitates; excess of ligand (1:5 metal/ligand
ratio, CFe = 200 μM–1 mM) was used, to assure full metal coordination
and decrease the complex vulnerability to ligand exchange (hydrolysis)
[34]. The Fe(III) complex formation was confirmed by the solution
colour formation, namely orange (maltol, DMHP), brownish orange
(thiomaltol) or violet (DMHTP). The option for KNO3 as the supporting
electrolyte, instead of KCl, was based on the fact that the nitrate (log
KFeL = −0.22) is a weaker ligand for the hard Fe(III) than the chloride
(log KFeL = 0.63) [35]. This fact avoids the competition of the anion of
41
a)
700
b) FeL3
FeL FeL2
Fe
Fig. 3. a) Electronic spectra of the 1:10 Fe(III)/thiomaltol system, pH 2.5–8.3; b) species
distribution curves with molar extinction coefficients at the maximum absorption
wavelengths (CL/CFe = 10, CFe = 9 × 10− 5 M).
the electrolyte salt with the chelators for the metal coordination, name-
ly under the conditions of low concentration used in these electrochem-
ical studies.
Table 2 contains the redox potentials (EP) for the four iron com-
plexes obtained at a scan rate of 100 mV/s and CFe = 200 μM. Analy-
sis of this table shows that the reduction of the complexes with the
O,S-donor ligands occurs at more positive potentials than the re-
spective O,O-analogues, which may be ascribed to the higher stabi-
lization of reduced Fe(II) complexes with the softer O,S-ligands.
Analysis of the voltammograms obtained for the iron–maltol and
–thiomaltol complexes, at scan rates between 100 and 800 mV/s,
shows the existence of single quasi-reversible peaks (Fig. 5a)) at pH 7,
with peak potentials of −0.588 V and −0.459 V (vs Ag/AgCl electrode),
respectively. Regarding the voltammograms for the iron complexes
(presumably, FeL3) with the corresponding pyridinone analogues
(DMHP and DMHTP (Fig. 5b), it was observed the existence of one re-
duction peak and two reoxidation peaks [DMHP (−0.521, −0,832 V);
DMHTP (−0.580, −0.640 V)], which may be due to some stabilization
of the nascent oxidized species as FeL2 and FeL3 [36]. In fact, the pres-
ence of ligand excess (1:5, Fe:L ratio) can be a guarantee of a significant
amount of reoxidation involving the tris-complex (FeL3) formation, es-
pecially at lower scan rates, but the split in two peaks, which is even
more evident at higher scan rates, must be related with the slow attain-
ment of the equilibrium concentration of the zwitterionic tautomer
thus compromising the stabilization of the nascent oxidized species
exclusively as FeL3 [36].
In order that the iron complexes can participate in redox cycling,
the oxidized complex must be reduced by endogenous species such
as ascorbate, which means that iron complexes with redox potentials
42 S. Chaves et al. / Journal of Inorganic Biochemistry 114 (2012) 38–46

a) a)
-6.0 × 10-6

-4.0 × 10-6

i(A)
-2.0 × 10-6

400 450 500 550 600 650 700

0
b)
2.0 × 10-6
FeL3
FeL2
E(V)
FeL

b)
-1.2 × 10-5

Fe

-8 × 10-6

i(A) -4 × 10-6

Fig. 4. a) Electronic spectra of the 1:3 Fe(III)/DMHTP system, pH 0.8–6.8; b) species dis-
tribution curves with molar extinction coefficients at the maximum absorption wave-
lengths (CL/CFe = 3, CFe = 2 × 10− 4 M).
0

lower (more negative) than + 0.386 V (determined herein for the

ascorbyl/monohydroascorbate couple, pH 7) cannot catalyze the fol- 4 × 10-6
lowing reaction,

3þ − 2þ þ ·− E(V)
Fe L þ HAsc →Fe L þ H þ Asc
Fig. 5. Cyclic voltammograms of a) Fe(III)/Maltol and Fe(III)/Thiomaltol (CFe = 200 μM)
thus rendering impossible the Fenton reaction. The redox potentials and b) Fe(III)/DMHP and Fe(III)/DMHTP (CFe = 400 μM) systems in 20% DMSO aqueous
obtained for the complexes at pH 7 and 1:5 metal/ligand ratio, but solution at a three-electrode cell (glassy carbon work, platinum auxiliary and Ag/AgCl
at different Fe(III) concentrations are represented in Fig. 6. Analysis reference electrodes) (CL/CFe = 5, pH 7, I = 0.1 M KNO3, scan rate 100 mV/s).
of this figure shows the increase (become more positive) of the
redox potentials with decreasing iron concentration. Thus, under presented in Fig. 6, it is possible to conclude that these chelators must
physiological conditions, involving μM concentrations of iron and prevent the redox cycling of iron if they are present at concentrations
chelator, slightly higher electrode potentials should be expected. sufficient to fully complex Fe(III). Obviously, this conclusion is of rele-
Identical trend was observed for the redox potentials of other cou- vance for the potential medicinal applications of these compounds.
ples, which also increased under physiological conditions (for [O2] =
10− 5 M and [O2•−] = 10− 10 M, the redox potential has not the standard
value of −0.35 V but +0.136 V [37]). On the other hand, R.C. Hider et al.
[34] studied hydroxyl radical production from the Fe(III)/DMHP system
and evidenced its dependence on the molar ratio of iron to DMHP;
specifically, the proportion of the Fe(DMHP)2 species decreased with
increasing excess of free ligand and, under biological conditions, any
Fe(DMHP)2 complex formed intracellularly was rapidly reduced by
ascorbate.
Although the herein described voltammetric studies have not been
performed under the physiological concentrations, the redox potential
of the ascorbyl/monohydroascorbate couple at such diluted conditions
should be expected to be higher (more positive) than the herein deter-
mined value (+0.386 V); recently, the electrode potential for this
system, under specific physiological conditions (10 − 6 and 10− 3 M,
respectively, in a cell) was reported +105 mV vs normal hydrogen
electrode (NHE) (ca −94 mV vs Ag/AgCl [38]). Anyway, based on the Fig. 6. Redox potentials of the compounds as a function of iron concentration at pH 7 and
previously reported redox behavior for the DMHP/Fe couple [34] and Fe/L 1:5 (scan rate 100 mV/s) in 20% DMSO aqueous solution at a three-electrode cell
the trend of redox potential found for the series of ligand/Fe couples (glassy carbon work, platinum auxiliary and Ag/AgCl reference electrodes).
 S. Chaves et al. / Journal of Inorganic Biochemistry 114 (2012) 38–46 43

2.3. Anti-oxidant properties

2.3.1. Hydroxyl radical scavenging activity

Hydroxyl radicals, generated in “free” solution by a mixture of
ascorbic acid, H2O2 and Fe(III)–EDTA complex (through Fenton chem-
istry), can attack deoxyribose (DR) to form products that, upon
heating with thiobarbituric acid (TBA) at low pH, yield a pink chromo-
gen. When hydroxyl radical “scavengers” are added, they compete with
DR for the radical species and diminish the chromogen formation. Thus,
the rate constant for reaction of the compounds with OH• can be de-
duced from the inhibition of colour formation. When pulse radiolysis fa-
cilities are not available, the deoxyribose method appears as a simple
“test-tube” assay for determination of approximate rate constants for
reactions of compounds with hydroxyl radicals [39]. From the slopes
of competition plots obtained in the presence of EDTA (see Experimen-
tal), second-order rate constants were calculated (Table 3).
Based on the values obtained for the four ligands, and taking into
account that most compounds react with OH • with rate constants of
10 9–10 10 M − 1 s − 1, we can infer that the present compounds are
moderately good scavengers of OH•. In addition, the studied ligands also
Fig. 7. Inhibition of deoxyribose degradation by DMHP, maltol, DMHTP, and thiomaltol.
inhibited deoxyribose degradation in reaction mixtures without EDTA. Each assay contained, in a final volume of 1.0 mL, 20 mM KH2PO4/K2HPO4 buffer pH
For the lowest concentration tested, a powerful inhibitory effect was ob- 7.4, 2.8 mM deoxyribose, iron, ligand, 0.2 mM H2O2 and 0.1 mM ascorbic acid. Iron
served, suggesting that they have a very high degree of metal binding ca- was added to the reaction mixtures either as 0.1 mM Fe(III)–0.1 mM EDTA (▬) or as
0.1 mM FeCl3 (•••••). Results are presented as means ± SD of four (maltol) and two
pability. This effect is shown in Fig. 7 for the DMHP, as example. In these
(other ligands) independent experiments performed in triplicate.
assays without EDTA, the substances that inhibit deoxyribose degrada-
tion are those that bind iron ions strongly enough to remove them from
deoxyribose and form complexes less reactive in generating OH• [40]. OH• generated in free solution, whereas for higher ligand concentra-
Fig. 7 shows the profiles of the percentage of inhibition of deoxyri- tions the inhibition is due to iron chelation by ligands forming metal
bose degradation by the ligands in reactions mixtures containing complexes in a less redox‐active form compared with EDTA–metal
EDTA, in which both the effects of OH• scavenging and iron chelation complex.
by the ligands are expected to be involved.
Analysis of this figure shows that, for each of these ligands, the 2.3.2. Inhibition of lipid peroxidation
percentage of inhibition efficiency increases with the ligand concen- Lipid peroxidation is a free radical chain reaction leading to forma-
tration. However, the O,O-donor ligands are more effective than the tion of multiple molecules of lipid radicals and peroxides. The oxidation
O,S- analogues, DMHP presenting the lowest IC50 value while both of liposomes was induced by the Fe(III)–ascorbate system and was used
thiomaltol and DMHTP present similarly higher IC50 values (Table 3). as a model for studying the efficacy of the compounds to protect poly-
The fact that the effectiveness of O,O-donors is more significant for unsaturated fatty acids against oxidation. Protection by compounds is
higher ligand concentrations, suggests that the inhibition of deoxyri- achieved by radical scavenging and/or iron chelation and it can also be
bose degradation must be predominantly due to iron chelation by the influenced by their lipophilicity.
ligands competing with EDTA at higher ligand concentrations, where The inhibitory activity of the compounds on the oxidation of lipo-
this competition with EDTA is favoured. In fact, this competition effect somes is illustrated in Fig. 8. It is observed that, at low concentrations,
can be seen from the calculated concentration ratios ([FeEDTA]/[FeL3]) the lipid peroxidation is more effectively inhibited by maltol and
contained in Table 4. DMHTP than by thiomaltol and DMHP, respectively. However, under
In addition, the higher Fe(III) chelating efficacy (higher pFe values) higher ligand concentrations, the inhibition increases considerably for
of the O,O-donor ligands, as compared with O,S-analogues, is accompa- thiomaltol and DMHP whereas it only slightly increases for maltol and
nied by an increased difficulty of the reduction of the corresponding DMHTP. These different behaviors can be explained on the basis of the
iron chelates (EredFeL3 −0.6 and −0.9 V, see Table 2); however, in any redox potentials of ligands and of respective iron complexes as well as
case, these chelates are much more difficult to reduce than the on lipophilic character of ligands.
FeEDTA
Fe(III)–EDTA chelate (Ered −0.1 V) [42] and so less OH• is produced Since the oxidation potentials of maltol (1.304 V) and thiomaltol
and a lower deoxyribose degradation results. (1.113 V) are particularly high, they are weak reductants and conse-
Therefore, the above results give support to the conclusion that, in quently the inhibition of lipid peroxidation should occur through a
reaction mixtures containing EDTA, the inhibition of deoxyribose degra- mechanism independent of free radical scavenging, eventually involv-
dation, for lower ligand concentrations, results from the scavenging of ing iron chelation. In fact, as thiomaltol is a weaker iron chelator than

Table 4
Table 3 Ratio of Fe(III)–EDTA to Fe(III)–L3 chelate concentrationsa for CL 0.6 and 3 mM, at the
Second-order rate constants (K)* for the reaction of the compounds with HO• obtained experimental conditions of deoxyribose degradation method (pH= 7.4, CFe = CEDTA =
by the deoxyribose method. 0.1 mM).

Compound K (M− 1 s− 1) IC50 (mM) System [FeEDTA]/[FeL3] [FeEDTA]/[FeL3]

(CL = 0.6 mM) (CL = 3 mM)
Maltol 7.27 × 107 0.81 ± 0.06
Thiomaltol 4.31 × 107 1.02 ± 0.15 Fe/maltol 5.2 0.3
DMHP 1.56 × 108 0.63 ± 0.09 Fe/thiomaltol 2904 250
DMHTP 1.76 × 107 0.99 ± 0.21 Fe/DMHP 0.3 0.002
Fe/DMHTP 22 1.8
The rate constants were calculated from the slopes of the straight lines obtained in the
presence of EDTA (Kligand = slope × KDR × [DR] × Aº, where KDR = 3.1 × 109 M− 1 s− 1, a
Calculations performed using the protonation and stability constants contained in
[DR] = 2.8 mM and Aº is the absorbance in the absence of ligand) [40]. Table 1, Table 2 and literature values [41] for the Fe/EDTA system.
44 S. Chaves et al. / Journal of Inorganic Biochemistry 114 (2012) 38–46
Fig. 8. Effect of a) maltol, thiomaltol and b) DMHP and DMHTP on the peroxidation of
rat liver liposomes. Each assay contained, in a final volume of 1.0 mL, 50 mM Tris–HCl
pH 7.4 buffer including 100 mM NaCl, liposomes containing 2.5 μmol of lipid phosphorus
per ml, 50 μM Fe(III) and different concentrations of ligands. After mixing, peroxidation
was initiated by the addition of 50 μM ascorbate. The reaction mixtures were incubated
at 37 °C for 60 min and the lipid peroxidation extent was evaluated by TBA method.
Results are presented as means ± SD of at least two independent experiments performed
in triplicate.
maltol, the presence of thiomaltol results in a higher concentration of
free iron than in the presence of the oxo-compound. Therefore, more
radicals should exist and less lipid peroxidation inhibition occurs for
low concentrations of thiomaltol. Nevertheless, thiomaltol has also a
higher lipophilic character than the other three compounds, which
may account for a better insertion of the iron chelates in the membrane
for high thiomaltol concentrations. This means that the iron chelate of
thiomaltol becomes less accessible to ascorbic acid and so more difficult
to be reduced with consequent better inhibition of lipid peroxidation.
On the other hand, DMHP and DMHTP have some reductant charac-
ter and thus, for lower ligand concentrations, the mechanism of
lipid peroxidation inhibition may be predominantly related to radical
scavenging. Since the redox potential of DMHTP is lower (0.752 V)
than that of DMHP (0.913 V), DMHTP is a better reductant and thus a
better inhibitor of lipid peroxidation than DMHP. Moreover, DMHTP is
relatively more lipophilic than DMHP, thus a better protector of the
membranes against oxidation. However, for high ligand concentrations
(>160 μM), the lipid peroxidation inhibitory mechanism seems to be
more dependent on the chelation effect than on free radical scavenging
activity. In fact, the O,O-donor ligands are harder bases than the
O,S- analogues and so they preferentially chelate Fe(III) over Fe(II).
Accordingly, for high ligand concentrations, DMHP is a better inhibitor
of lipid peroxidation than DMHTP, besides the iron-DMHP chelate is
less easily reduced than the corresponding DMHTP chelate, in agree-
ment with the redox potentials presented in Table 2.
3. Conclusions
Since the toxicity caused by excessive amounts of iron in iron-
overload diseases is mediated by the catalytic generation of free radi-
cals, one of the goals of iron chelation therapy development is that the
therapeutic agents should reduce the levels of plasma labile iron but
do not participate in in vivo redox cycling. Following this aim, four com-
pounds (two O,S‐donor ligands and the corresponding O,O‐analogues)
were studied in terms of their iron‐chelating capacities and hydroxyl
radical scavenging activity. All the compounds were moderately
good scavengers of OH• and efficient iron chelators, but the O,O-
analogues presenting somewhat stronger chelating efficacy than the
corresponding O,S-derivatives. In addition, all the ligands are able to
prevent the redox cycling of iron and therefore the inhibition of deoxy-
ribose degradation seems to be predominantly due to iron chelation by
the ligands competing with EDTA. This is an important issue, consider-
ing the potential therapeutic use of the compounds. In fact, it seems
quite likely that, in vivo, an antioxidant, which can prevent damage
caused by OH•, acts not by scavenging it, but by iron chelation and
preventing OH• formation. Finally, iron is a known catalyst of
lipid peroxidation and this role requires “redox active” iron. Our
lipoperoxidation studies support the assumption that the activity of
these ligands on the lipid peroxidation inhibition results predominantly
from iron chelation. These iron complexes can then prevent the reduc-
tion of Fe(III) to Fe(II) by ascorbate (according to the electrochemical
studies) and concomitantly inhibit the lipid peroxidation. The studies
performed herein encourage further research on non-toxic iron chela-
tors as anti-oxidant drugs.
4. Experimental
4.1. General information
Electronic spectra for the spectrophotometric titrations were
recorded with a Hitachi U 2000 spectrophotometer, using 1 cm path
length cells, at 25.0 ± 0.1 °C. Cyclic voltammetry was performed at
room temperature using an AUTOLAB potentiostat and also a Metrohm
744 pH-meter with a Metrohm combined electrode 6.0228.000 PT1000
for the respective pH determinations. In the study of the antioxidant
properties of the compounds, the absorbance values were recorded
with a Unicam UV2 spectrophotometer.
4.2. Materials
All the reagents were of analytical reagent grade and used as
supplied. Maltol (3-hydroxy-2-methyl-4-pyrone) and DMHP (3-
hydroxy-2-methyl-4-pyridinone) were from Aldrich; thiomaltol
and DMHTP were synthesized in our laboratory according to already
published procedures [13]. For the deoxyribose degradation assay
and lipoperoxidation inhibition studies, both trichloroacetic and
2-thiobarbituric acids were obtained from Merck; deoxyribose
was from Lancaster and L-ascorbic acid was purchased from Sigma.
In the complexation studies, the aqueous Fe(III) stock solution
(1.77 × 10 − 2 M) was obtained from Merck and standardized by
atomic absorption. For the electrochemical measurements, the sol-
vent used was DMSO from Aldrich and the supporting electrolyte
KNO3 was obtained from Merck.
Adult male Sprague–Dawley rats (3–4 months old) were obtained
from the Instituto de Investigação Científica Bento da Rocha Cabral
 S. Chaves et al. / Journal of Inorganic Biochemistry 114 (2012) 38–46
(Lisboa, Portugal). Rats were maintained, two per cage, in a room at
22 °C under 12 h dark/light cycling and ad libitum access to water
and regular chow.
4.3. Potentiometric and spectroscopic studies
4.3.1. Titrant solution
The titrant (0.1 M KOH) was prepared from a carbonate-free com-
mercial concentrate (Titrisol), standardized by potentiometric titration
with potassium hydrogen phtalate and discarded when the percentage
of carbonate (Gran's method [43]) was about 0.5% of the total amount of
base.
4.3.2. Measurements
Potentiometric titrations of the ligands (CL = 2.0–3.0 × 10− 3 M) and
spectrophotometric titrations of their iron complexes (350–700 nm) in
5% (v/v) DMSO/H2O solution were performed at ionic strength (I) 0.1 M
KCl and the working temperature was maintained at 25.0± 0.1 °C. To
improve the low water solubility of the thio-derivatives, studies were
performed in a 3% (v/v) DMSO/water medium. For the complexometric
studies, the total volume was 20 mL, the ligand concentration was
about 6.0× 10− 4 M and the Fe(III) concentration was 2.0 × 10− 4 M,
using iron in (1:3) stoichiometric relation. All Fe(III)–L systems were
studied for pH below 2 (0.8–2) using a batch titration (7 points), in
which the amount of acid to be added (from standard 0.1 or 1 M HCl
solutions) was calculated for the total volume solution used. In the
case of the Fe(III)/thiomaltol system, a 1:10 stoichiometry was used to
avoid early precipitate formation. Under the experimental conditions
used, the value determined for pKw was 13.85.
4.3.3. Calculation of equilibrium constants
The stepwise protonation constants, Ki = [HiL] / [Hi − 1L][H], and
the overall iron-complex stability constants, βFemHhLl = [FemHhLl] /
[Fe] m[H] h[L] l, were calculated by fitting analysis of the potentiometric
data of the ligand and of the spectroscopic data of the ligand in the
presence of Fe(III), respectively, with the HYPERQUAD 2003 [25]
and the PSEQUAD programs [30]. The Fe(III) hydrolytic species [44]
were included in the equilibrium model and the species distribution
curves were plotted with the HYSS program [25].
4.4. Partition coefficients
Partition coefficients of the ligands between 1-octanol and Tris buff-
er 0.01 M pH 7.4, based on the concentration ratio of each compound
between the two phases, were determined at room temperature using
the “shakeflask” method [45]. An aliquot of the ligand solution in Tris
buffer (saturated with 1-octanol) was added to a mixture of the Tris
buffer and 1-octanol (saturated with Tris buffer pH 7.4) in equal
volumes. The aqueous phase was separated from the two-phase system
and its absorbance value recorded using the benzenoid bands of the
ligands.
4.5. Cyclic voltammetry
Voltammetric measurements were performed in 20% (v/v) anhy-
drous DMSO and 0.1 M KNO3 using a three-electrode cell consisting of
a glassy carbon working, a platinum auxiliary and Ag/AgCl reference
electrodes. The glassy carbon electrode surface was hand polished
with 0.05 μm alumina to a mirror like finish [46]. In a typical experi-
ment, the cell volume was 12 mL and for the determination of the
redox potential of the compounds the concentration of ligand was
6.25 × 10− 3 M and the pH ca 5; for the determination of the redox
potential of the iron complexes, the ligand and Fe(III) concentrations
were about 1–5 × 10− 3 M and 0.2–1 × 10− 3 M, respectively, and pH 7.
All the solutions were degassed with type U nitrogen previously bub-
bled in anhydrous DMSO. Sweep rates were varied between 100 and
45
800 mV/s, in order to check the reversibility of the redox couple and
the number of electrons involved in the reaction. Data are reported at
a scan rate of 100 mV/s and room temperature (≈25 °C). The redox po-
tential for the 1 × 10− 3 M ascorbate/dehydroascorbate system was
measured under identical conditions and used as a reference measure-
ment (EP = 0.386 V, 100 mV/s); the Fe(III)/DMHP 1:5 system was used
as an internal standard of potential.
4.6. Deoxyribose degradation assays
The HO • scavenging activity of the ligands was determined by the
DR degradation assay, as described by Halliwell et al. [39], with slight
modifications [47]. Hydroxyl radicals were generated in free solution
by a mixture of ascorbic acid, H2O2 and Fe(III)–EDTA complex. Each
assay contained, in a final volume of 1.0 mL, 20 mM KH2PO4/K2HPO4
buffer pH 7.4, 2.8 mM deoxyribose, 0.1 mM FeCl3, 0.1 mM EDTA, ligand
in a range of concentrations (0.6–3.0 mM), 0.2 mM H2O2 and 0.1 mM
ascorbic acid. Solutions of FeCl3–EDTA, H2O2, ascorbic acid and TBA,
were made up immediately before use and the ascorbic acid was
added to start the reaction. After incubation at 37 °C for 1 h, the color
was developed by adding 1 mL of 2.8% (w/v) trichloroacetic acid and
1 mL of 1% (w/v) TBA in 0.05 M NaOH. The reaction mixtures were
then heated in a boiling water bath for 15 min. After cooling, the absor-
bance of the resulting solutions was measured spectrophotometrically at
532 nm. The rate constants for the reaction of the ligands with HO • were
calculated from the slopes of the straight lines obtained representing 1/A
against [ligand]. The slope was Kligand/KDR [DR] A°, where the absorbance
in the presence of ligand was A =KDR [DR] [HO•] and the absorbance in
the absence of ligand was Aº =Kligand [ligand] [HO•] + KDR [DR] [HO•].
The DR concentration was 2.8 mM and the rate constant for the reaction
of deoxyribose with HO• was KDR = 3.1× 109 M− 1 s− 1.
For all concentrations of ligands, controls in which deoxyribose was
omitted from the reaction mixture were performed. DMHP and DMHTP
did not release TBA-reactive material when attacked by HO•. However,
maltol and thiomaltol released TBA-reactive products when attacked
by HO•, and the absorbance values obtained for these controls were sub-
tracted from the values of complete assays plus the ligands. The control
TBA values ranged between 13 and 19% of the absorbance measured for
the complete system without ligand.
Data were expressed as mean ± S.E. determined from triplicate
analysis. The ligands concentrations which caused 50% of inhibition
of the system (IC50) were determined using Origin 7 ® software.
4.7. Effects of ligands on oxidation of liposomes
Liposomes were prepared from rat hepatic microsomes isolated
from male Sprague–Dawley rats by differential centrifugation. Total
microsomal lipid was extracted from freshly prepared microsomes by
the method of Folch et al. [48]. Lipid content was measured as the
amount of total lipid phosphorus by the method of McClare et al. [49]
The solvents were purged with nitrogen and all operations were
performed under nitrogen at 4 °C. The extracted lipid was stored
at –20 °C under nitrogen in chloroform. Aqueous suspensions of
microsomal lipid were prepared by transferring an aliquot of the stock
lipid solution to a round bottom flask and the chloroform was removed
under a stream of nitrogen. Then 50 mM Tris–HCl buffer pH 7.4 was
added to the thin lipid film and the flask was capped under nitrogen.
The final concentration of lipid in the suspension was 5 μmol of lipid
phosphorus per ml.
The oxidation of liposomes was induced by the Fe(III)-ascorbate sys-
tem and the lipid peroxidation extent was evaluated by measuring the
formation of malondialdehyde (MDA) through the 2-thiobarbituric
acid (TBA) method. TBA method is a non-specific assay for MDA, never-
theless “free” MDA is produced during the peroxidation of liposomes
prepared from rat hepatic microsomes [7]. In addition, as it was
shown by some of us, a very good correlation exists between the
46 S. Chaves et al. / Journal of Inorganic Biochemistry 114 (2012) 38–46
concentration of MDA measured as a TBA‐reactive substance and the
concentration of “free” MDA, determined by HPLC during peroxidation
of microsomal liposomes [50]. The assays were performed as follows
[51]: reaction mixtures were made to a final volume of 1.0 mL with
50 mM Tris–HCl pH 7.4 buffer including 100 mM NaCl, liposomes con-
taining 2.5 μmol of lipid phosphorus per ml, 50 μM Fe(III) and different
concentrations of ligands. After mixing, peroxidation was initiated by
addition of 50 μM ascorbate. The reaction mixtures were incubated at
37 °C for 60 min. A control assay was performed in the absence of
ligand. After the incubation period, 1 mL of 20% (w/v) trichloroacetic
acid and 1 mL of 0.67% (w/v) TBA were added to each assay. The
mixtures were heated in a boiling water bath for 15 min. After cooling,
3 mL of 1-butanol was added and the solution was mixed for 1 min to
extract the chromophore. After 10 min, phases were separated and
the absorbance of the clear upper butanol phase was read at 532 nm
against appropriate blanks. Results obtained in the presence of the
compound were expressed in terms of percentage of lipoperoxidation
inhibition in comparison with a control assay.
Acknowledgments
The authors thank the Portuguese Foundation for Science and
Technology (FCT) for financial support, specifically for projects PCDT/
QUI/56985/04 and PEst-OE/QUI/UI0100/2011.
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