Antioxidant capacity of two novel bioactive Fe(III)-cyclophane complexes

Article abstract of DOI:10.3390/molecules18021762

The cyclophanes 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7, 10, 24,27,30,33-octaaza-17,40-dioxa[10.1.10.1]paracyclophane (PO) and 2,9,25,32-tetraoxo- 4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33- octaaza[10.1.10.1]paracyclophane

Full text of DOI:10.3390/molecules18021762

Molecules 2013, 18, 1762-1774; doi:10.3390/molecules18021762
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antioxidant Capacity of Two Novel Bioactive Fe(III)-Cyclophane
Complexes
1
1,2
2
2
Alex J. Salazar-Medina , Rocío Sugich-Miranda , Eli Teran-Cabanillas , Jesús Hernández ,
2
3
1,2,
Gustavo A. González-Aguilar , Enrique Rudiño-Piñera , Rogerio R. Sotelo-Mundo * and
1
,
Enrique F. Velázquez-Contreras *
1
Departamento de Investigación en Polímeros y Materiales (DIPM), Universidad de Sonora,
Calle Rosales y Blvd, Luis Encinas s/n, Col. Centro, PO Box 130. Hermosillo Sonora 83000, Mexico
2
Centro de Investigación en Alimentación y Desarrollo (CIAD), A.C. Carretera a Ejido La Victoria
Km 0.6, PO Box 1735, Hermosillo Sonora 83304, Mexico
3
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología,
Universidad Nacional Autónoma de México (IBT-UNAM) Av. Universidad #2001,
Col. Chamilpa, Cuernavaca, Morelos 62250, Mexico
*
Authors to whom correspondence should be addressed; E-Mails: rrs@ciad.mx (R.R.S.-M.);
evlzqz@guaymas.uson.mx (E.F.V.-C.); Tel./Fax: +52-662-259-2161 (E.F.V.-C.).
Received: 13 November 2012; in revised form: 6 January 2013 / Accepted: 7 January 2013 /
Published: 29 January 2013
Abstract: The cyclophanes 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,
2
4
4,27,30,33-octaaza-17,40-dioxa[10.1.10.1]paracyclophane (PO) and 2,9,25,32-tetraoxo-
,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza[10.1.10.1]paracyclophane
(
2 2
PC) were coordinated with iron to form cationic binuclear Fe(III) Fe PO and Fe PC
complexes, respectively. Their antioxidant capacity, superoxide dismutase and peroxidase
mimetic activity, as well as their toxicity toward peripheral blood mononuclear cells
(
PBMCs) were evaluated. Both Fe PO and Fe PC are interesting biomimetics with antioxidant
2 2
capacity similar to that of ascorbic acid that prevent mortality in cultured PBMCs, with the
potential to have bioactive and protective functions in disease animal models.
Keywords: cyclophane; macrocycle; iron; biomimetic; antioxidant

Molecules 2013, 18
. Introduction
Reactive oxygen species (ROS) are toxic molecules implicated in several human pathological
1763
1
processes including tissue injury, inflammation, ageing, cancer, cardiovascular, pulmonary and
neurodegenerative diseases [1]. Aside from many enzymatic and chemical antioxidants, several
synthetic metal complexes have shown activity as biomimetic antioxidants [2]. Transition metal
complexes [e.g., complexes of Mn(II), Mn(III), Cu(II) and Fe(III)] have important antioxidant
properties [1], due to their ability to change their oxidation state and their chemical interactions with a
wide number of negatively charged ions, radicals and molecules. These characteristics have prompted
the design and development of metal-based drugs with promising pharmacological application which
might offer unique therapeutic opportunities [3].
The first line of defense against ROS in cellular systems is superoxide dismutase (SOD), that
•−
catalyzes the conversion of O2 to H O and O [4]. Some macrocyclic complexes have been proposed
2
2
2
as SOD mimetics, mainly because of their high thermal stability and flexible ligand arrangement that
•
−
allows redox processes based on their metal centers [5]. The action of SOD produces H O by the O
2
2
2
•−
dismutation, a much less reactive molecule compared to O
2 2 2
, but still toxic to the cell. H O in the
presence of transition metals, it is rapidly converted via the Fenton reaction to the hydroxyl radical
•
•
(
OH). The OH is widely accepted to be the most damaging and cytotoxic ROS produced by cells.
Two enzymes participate in the removal of H from the cellular environment, peroxidases (POx)
and catalases (CAT), converting the H into water molecules and diatomic oxygen [6]. For these
2
O
2
2
O
2
reasons we examined the use of Fe(III) ions coordinated to cyclophane ligands generating a new
binuclear Fe(III)-cyclophane group of complexes, which could represent a new class of antioxidants.
The study of these molecules will provide a deeper insight into the understanding of complexes that
could mimic some enzymes related to ROS contention mechanisms.
2
. Results and Discussion
2
.1. Synthesis of the PO and PC Iron Complexes
The purified chelating cyclophanes 2,9,25,32-tetraoxo-4,7,27,30-tetrakis (carboxymethyl)-1,4,7,10,
2
4,27,30,33-octaaza-17,40-dioxa[10.1.10.1] paracyclophane (PO) and 2,9,25,32-tetraoxo-4,7,27,30-
tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza[10.1.10.1] paracyclophane (PC), were synthesized
as previously reported [7]. Both molecules were derived from ethylenediaminetetraacetic acid (EDTA)
and provided with amino, amide and carboxymethyl pendant donor groups. Their purity was confirmed
by melting/decomposition point (MDP) and NMR studies (see Experimental section below). Reactions
between FeCl
3
.6H
2
O (at pH ~ 3) and PO and PC (both at pH ≤ 7), gave binuclear Fe(III) complexes
PO and Fe PC, respectively (Figure 1). We propose that PO and PC metal
2
+
[Fe
2
L] , labeled Fe
2
2
chelates have a structure in which each Fe atom is coordinated by two carboxylate oxygens, two amine
nitrogens and two amide oxygen atoms, with a feasible water molecule axial coordinated in aqueous
solutions, on the basis of the well-known ferric EDTA complexes [8] and the coordination mode
found in the X-ray crystal structure of a related cyclophane (3,10,21,28-tetraoxo-5,8,23,26-
2
tetrakis(carboxymethyl)-2,5,8,11,20,23,26,29-octaaza[12.12] paracyclophane) bound to zinc [9]. Fe PO

Molecules 2013, 18
1764
was soluble in aqueous solutions in a pH range from 4.2 to 10.0, while Fe PC was soluble in aqueous
2
solutions in a pH range from 5.2 to 10.0.
2 2
Figure 1. Chemical structures proposed of Fe PO and Fe PC cyclophane iron (III) complexes.
2.2. Mass Spectrometry
To confirm the formation of the metal complexes, the mass spectra of methanol-water solutions of
3
+ 4− 2+
Fe
2 2 2 2
PO and Fe PC were obtained. Fe PO showed a major peak assigned to [Fe L ] specie at
m/z = 510 with an abundance of 100%. Another peak that supports the formation of the binuclear Fe(III)
3
+ 4− 2−
complex correspond to [Fe
2
L Cl ] at m/z = 1,092 with an abundance of 6%. For Fe
2
PC a major
3
+ 4− 2+
peak assigned to [Fe
ion peaks corresponding to mononuclear species [Fe L ] were not detected for Fe
2
L ] specie was observed at m/z = 508 with an abundance of 100%. Molecular
3
+ −4 −
2
2
PO or Fe PC.
2.3. Electron Paramagnetic Resonance (EPR)
The X-band EPR spectra observed for Fe PO and Fe PC in 60% aqueous methanol solutions at 6 K
2
2
are shown in Figure 2. The spectra exhibit an intense peak at g ~ 4.3 in the region of 1,500 G, typical
of high-spin (S = 5/2) iron(III) complexes with low symmetry, as well as commonly found in a variety
of solid state materials, chelates and metallo proteins [10,11].
2 2
Figure 2. g values of the X-band EPR spectra for Fe PO and Fe PC. Experimental
conditions: methanol/water (6:4) matrix, temperature 6 K, microwave frequency 9.338 GHz,
modulation 1 G.

Molecules 2013, 18
.4. Infrared Spectroscopy
The coordination mode for Fe PO and Fe PC complexes was analyzed by IR spectroscopy by
1765
2
2
2
comparison of absorption bands of the non-coordinated ligand vs. the metal complex (Figure 3). The
−
1
−1
−1
stretching carbonyl bands gave shifts from 1,684 cm (PO) to 1,620 cm (Fe PO) and 1,674 cm
2
−
1
−1
(
2
PC) to 1,624 cm (Fe PC). We also observed a broad OH band at the 3,200–3,600 cm region. The
changes in the position of the absorption bands of Fe-bound and unbound cyclophanes provide us an
indicative of the formation of the metal complex and its preferred coordination sites in the ligand
structures, as well as the observed for Fe(III)-EDTA complexes [8].
Figure 3. IR spectra for KBr pellets of (a) PO and Fe PO (solid and dotted line respectively);
2
and (b) PC and Fe PC (solid and dotted line respectively).
2
2
.5. Electronic Absorption Spectra
The electronic spectra for Fe
2
PO solution were pH dependent, as shown in Figure 4(a). In a pH
−
1
−1
range of 6.0 to 10.0 the spectra show two bands with a λmax of 350 nm (εpH7.0 = 11,830 M cm ) and
−
1
−1
4
75 nm (εpH7.0 = 4,680 M cm ), assigned to a ligand-to-metal charge transferences (LMCT) [12].
With an increase of the pH, the molar absorptivity also increases, and the λmax of each band was
slightly displaced to a major wavelengths. Above pH ~ 10, the molar absorptivity of the LMCT bands
decreases due to the precipitation of the complex in the working solution. The data obtained in the
®
UV-Vis experiment were analyzed through the program Excel Worksheets for Spectrometry [13] and
we determined the presence of different protonation state species of the Fe
where the non-deprotonated species Fe L(H O) reaches its maximum concentration at pH 6.0 and then
start to decrease. Over this pH, the Fe PO complex solution becomes into a dark red colored solution
and the mono deprotonated species Fe L(H O)(OH) and bi deprotonated species Fe L(OH) appears, to
2
PO complex [Figure 5(a)],
2
2
2
2
2
2
2
2
finally shift into a yellow colored suspension (precipitate complex) at pH 11.0. These spectrophotometric
observations are consistent with those corresponding to the complex Fe(III)-EDTA [14].

Molecules 2013, 18
Figure 4. Solution electronic spectra of (a) Fe PO and (b) Fe PC at different pH values.
1766
2
2
White and black circles spectra corresponded to unbound cyclophane and Fe(III) respectively,
both at pH ~ 7, as a controls.
2
The electronic spectra of Fe PC also present two LMCT bands, the first one at 307 nm
−
1
−1
−1
−1
(
ε
pH7.0 = 8,710 M cm ), and the second band at 334 nm (εpH7.0 = 6,270 M cm ). In this case, the
intensity of the two bands decreases with the augmentation of the pH [Figure 4(b)]. The same method
of analysis was used to establish the presence of different protonation state species of Fe PO, and it
was utilized with the UV-Vis data spectra of Fe PO complex [Figure 5(b)]. The non-deprotonated
species Fe L(H O) moves its maximum concentration percentage from pH 6.0 for Fe PO to
pH 6.5 for Fe PC and then starts to decrease. Over pH ≈ 8.0 the deprotonated species appears,
Fe L(H O)(OH) and Fe L(OH) , and the complex solution becomes a dark red colored solution, to
finally shift into a yellow colored suspension due to the formation of the precipitate complex over pH
2
2
2
2
2
2
2
2
2
2
2
1
1.0, as well as that observed to Fe
As a consequence of the pH dependence mentioned above, a mixture of the species Fe
L(H O)(OH) of Fe PO as well as Fe PC, was present in each experiment described below (Figure 6).
2
PO.
2
2 2
L(H O) and
Fe
2
2
2
2

Molecules 2013, 18
Figure 5. Correlation between the species distribution and the absorption spectra of Fe PO
1767
2
2
at top and Fe PC at bottom. (% = Percentage of species distribution in solution).
2 2
Figure 6. Changes in protonation state of the aqueous solutions of Fe PO and Fe PC
complexes in dependence of pH.
The differences between the substituent groups X = -O- for Fe
Figure 1), provide the Fe(III) complexes with distinct properties, even with the no participation of the
substituent groups in the coordination sphere with iron. X = -O- allows a major flexibility to Fe PO
2 2 2
PO and X = -CH - for Fe PC
(
2
against the Fe
2 2
PC, because the more rigidity property of X = -CH - and this characteristic is
responsible of the differences in the UV-Vis spectra behavior between Fe PO and Fe PC. Even
2
2

Molecules 2013, 18
1768
though the two iron atoms coordinate to the same groups in PO and PC ligands, a higher tension was
expected to Fe PC complex to maintain the coordination. In addition, Fe PO has shown major
2
2
2
solubility in aqueous solution, letting us to study it at lower pHs than Fe PC do. In the species
distribution diagram we can see the non-protonated and protonates species of Fe PO and Fe PC
2
2
appear at different pH values, behavior attributable to the structural rigidity in each metal complex.
2.6. Antioxidant Capacity Assay
The characterization of antioxidant capacity is of great importance because the multiple
biological and medical applications of such synthetic molecules. The Fe PO had more antioxidant
2
−1
−1
capacity (0.0690 g eq Trolox mol ) than Fe
2
PC (0.0443 g eq Trolox mol ), both within the range
of the antioxidant capacity presented by ascorbic acid at the same molar concentration
−1
(
0.0755 g eq Trolox mol ) (Figure 7). The behavior of the Fe(III) complexes was similar to the
observed for the Cu(II) complexes reported by Sugich-Miranda and co-workers [5]. The difference
between Fe PO and Fe PC antioxidant capacity could be due to differences in conformational
2
2
•+
2
flexibility of Fe PC, resulting in a less favorable interaction with the ABTS radicals in solution
3
causing a lower antioxidant capacity. The antioxidant capacity of iron (FeCl ) was undetectable in the
TEAC assay.
2 2
Figure 7. Antioxidant capacity of Fe PO and Fe PC complexes by the TEAC assay.
Ascorbic acid and a solution of Fe(III) were used as a controls. Bars represent the standard
error of the mean (±SEM) of triplicate experiments. Ferric chloride had no detectable
antioxidant capacity and was not plotted.
2
.7. Superoxide Dismutase-Like Activity
The SOD-like activity was measured by inhibition of the oxidation of nitrobluetetrazolium (NBT)
•
−
induced by the presence of the O
2
radical generated by the xanthine and xanthine oxidase enzymatic
•−
system. We tested bovine SOD as the main scavenger of O
2
radical, producing 89.40% of NBT
oxidation inhibition with a 60 µM concentration of the enzyme. Under the same conditions the NBT
oxidation inhibition of Fe PO was 39.53% and for Fe PC was 41.97% (Figure 8). The values obtained
2
2
of both metal complexes, reaches over a 44% of NBT oxidation inhibition generated by bovine SOD.

Molecules 2013, 18
Figure 8. SOD-like activities of Fe PO and Fe PC complexes as inhibitors of the NBT
1769
2
2
oxidation. Bars represent the standard error of the mean (±SEM) of triplicate values.
2
.8. Peroxidase-Like Activity
In order to investigate the bioactive capacity as a POx mimetic, the POx-like activity of Fe
2
PO and
®
Fe
2
PC complexes was determined using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit.
®
−1
−1
The results observed were 0.13 and 0.29 nm of Amplex Red oxidized min mg complex for Fe
and Fe PC, respectively. Both Fe PO and Fe PC complexes kept the POx-like activity for about 48 h,
as observed with a continuous oxidation of the Amplex Red reagent in presence of H
This prolonged capacity of Fe PO and Fe PC to act as a POx mimetic towards H is desirable in a
2
PO
2
2
2
®
2
2
O (Figure 9).
2
2
2 2
O
chemical with pharmacological properties. For example, the HRPO, as the main peroxidase tested in
this assay, was only active in vitro for 30 min, compared to the 48 h of the cyclophane biomimetic
complexes. Likewise, for a hemoglobin recombinant protein of the plant Arabidopsis thaliana, another
class of POx mimetic, a rapid inactivation was observed after 30 s, although the great initial specific
activity [15]. Therefore, the study of the possible mechanisms of these mimetic chemicals will give
useful knowledge for the medical and pharmacological area.
Figure 9. Peroxidase-like activity of ● FeCl
3 ▲ ▼ 2 2
, ■ PO, PC, Fe PO and ♦ Fe PC. Bars
represent the standard error of the mean (±SEM) of triplicate values.
2
.9. Cellular Assay
To evaluate the bio-protective effect of Fe(III)-cyclophane complexes, different concentrations of
Fe PO and Fe PC (30, 60 and 120 µM) were incubated with PBMCs in the presence of xanthine plus
2
2

Molecules 2013, 18
1770
xanthine oxidase to generate reduced species of oxygen and induce cell damage. The percentage of
mortality was evaluated by trypan blue dye exclusion assay. Figure 10(a) shows that Fe PO reduced
2
mortality percentage in all the concentrations probed from 10.6% in control untreated cells to 5.06%
with the highest concentration (120 µM) tested, after 24 h of culture. A similar trend was for Fe PC
2
[
Figure 10(b)], reducing the mortality from 10.7% in control untreated cells to 4.70% with the highest
concentration tested, after 24 h of culture. We previously evaluated the toxicity after 24 h culture of
the Fe(III)-cyclophane complexes and the highest percentages of mortality for Fe PO and Fe PC was
2
2
0.9% and 0.5%, respectively. These results are similar to those previously reported by our group [5],
where we found no toxic but a protective effect of the Cu PO and Cu PC in PBMCs by reducing
2
2
mortality in 6% compared to control untreated cells. These results indicate that both cyclophane-Fe(III)
complexes were not cytotoxic during the first 24 h of culture, even when high doses were used.
Therefore, these molecules could be considered as potential candidates for further antioxidant studies
using animal models and cancer cell lines in order to probe its possible beneficial effects. Over 24 h of
culture the cell mortality increases because lacking of nutrients in the culture medium.
Figure 10. Cellular protection of (a) Fe PO and (b) Fe PC in PBMCs with induced
2
2
damage by ROS. The line marked as blank corresponds to PBMCs in RPMI medium with
no treatment. Data comes from a representative experiment. Points represent the standard
error of the mean (±SEM) of triplicate values.

Molecules 2013, 18
1771
3. Experimental
3
.1. Syntheses of Cyclophanes and Iron Complexes
PO was synthesized by adding bis(4-aminophenyl) ether (Aldrich, 1.5 g) in dimethylformamide
(
DMF, 70 mL) dropwise to ethylenediamine tetraacetic (EDTA) dianhydride (Aldrich, 2.0 g) in DMF
200 mL) under a nitrogen atmosphere. The light yellow solution obtained was concentrated and
(
mixed with water, and a pale brown solid was obtained. This crude cyclophane was converted to the
lithium salt, and recrystallized twice from hot water. Acidification (pH ~ 3) of the purified lithium salt
gave PO in the acid form as a colorless solid. Yield: 15%. MDP: 262.0 °C. The absence of impurities
1
such as oligomers was confirmed by H-NMR (D O/Na CO , pD = 9.5, 400 MHz, DSS): δ = 2.86
2
2
3
H
(8H, s, Hb), 3.29 (8H, s, Ha), 3.43 (8H, s, Hc), 6.85 (8H, d, He), 7.18 (8H, d, Hd).
PC was synthesized by essentially the same method as for PO by the use of the diamine bis
(
4-aminophenyl)methane (Aldrich). The crude product was converted to the lithium salt, which was
recrystallized from 50% ethanol. When an aqueous solution of the purified lithium salt was acidified
with diluted HCl to pH ~ 3, PC in the acid form was obtained as a colorless solid. The product was
1
washed with water. Yield: 12%. MDP: 256.0 °C. H-NMR (D
2
O, pD = 10.4; 400 MHz; DSS): δ = 2.81
H
(
8H, s, Hb); 3.28 (8H, s, Ha), 3.38 (8H, s, Hc), 3.78 (4H, s, Hf), 6.98 (8H, d, He), 7.12 (8H, d, Hd).
To obtain the iron (III) complexes, each host molecule (either PO or PC) was dissolved in water
and solubilized by adding a minimum amount of solid Na CO . This solution was added to an aqueous
solution containing a slight excess of FeCl ·6H O, the binuclear iron complex precipitated in a
quantitative yield. The melting decomposition point (MDP) was obtained in a Mel-Temp II equipment
Laboratory Devices). The electrospray ionization (ESI) mass spectra were obtained by the use of a
JEOL HX 110A spectrometer for sample solutions of methanol-water mixture.
2
3
3
2
(
2
+
3+ 4− 2+
Fe
Fe
2
2
PO. MDP: 301.7 °C. MS (ESI): m/z (for (C44
H
44Fe
2 8 2
N O14) ) = 510 (100) ([Fe L ] );
2
+
3+ 4− 2+
PC. MDP: 283.8 °C. MS (ESI): m/z (for (C46
H
48Fe
2 8 2
N O12) ) = 508 (100) ([Fe L ] ).
3
.2. Spectroscopic Measurements
The EPR spectra of the binuclear Fe(III) complexes were obtained on an X-band continuous wave
Elexsys E500 (Bruker) EPR spectrometer, for 60% aqueous methanol solutions. The final concentration
of each complex was approximately 1 mM. Infrared spectra were recorded on a Perkin Elmer
−1
Spectrum GX FT-IR system, in the range 4,000–400 cm . The samples were prepared as KBr pellets.
The solution electronic spectra were obtained by the use of an Agilent 8453A UV-VIS diode array
spectrometer. The pH of the samples solutions was adjusted by adding the necessary amount of NaOH
solution. The species distributions were determined using the data obtained in the UV-Vis analysis
®
varying the pH, with the program Excel Worksheets for Spectrometry [13].
3.3. Antioxidant Capacity Assay
The Trolox equivalent antioxidant capacity (TEAC) assay is based on determination of Trolox-
equivalents, which is a water-soluble analog of vitamin E. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-
carboxylic acid) was purchased from Aldrich (Milwaukee, WI, USA). ABTS (2,2-azino-bis(3-
2

Molecules 2013, 18
1772
ethylbenzthiazoline-6-sulfonic acid), potassium persulfate (K S O ) and ascorbic acid (vitamin C)
2
2
8
were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were analytical
grade or better. Fe PO and Fe PC complexes were evaluated at 30 and 60 µM concentrations using a
modified TEAC method to determine their antioxidant activity. The TEAC method measures the
2
2
•
+
antioxidant capacity to scavenging the blue-green ABTS radical cation versus the scavenging
•+
capacity of Trolox. The radical cation ABTS was obtained by mixing 5 mL of 7 mM ABTS solution
•
+
and 88 µL of a 140 mM K S O solution. One milliliter of the ABTS was dissolved in 88 mL of PBS.
2
2
8
•+
In a quartz cell, the reaction contained 245 µL of an ABTS and 5 µL of each test sample or dilutions
of the Trolox standards. The absorbance was read within 80 min after the initial mixing. Calculations
were made correlating the scavenging capacity of Trolox versus each sample. The antioxidant capacity
was expressed as grams equivalent Trolox per mol of test sample.
3
.4. Superoxide Dismutase-Like Activity
SOD activity was determined according to Beyer and Fridovich [16] with slight modifications as
follows: the stock solution consisted of 2 mM xanthine (50 µL), 0.3% Triton X-100 solution (50 µL),
.2 mM nitrobluetetrazolium (NBT, 50 µL), 0.1 mM phosphate buffer solution (770 µL, pH 7.8) and
1
−1
xanthine oxidase enzyme (600 mU mL , 20 µL) to start the reaction; 1 mM (60 µL) of each sample,
Fe PO or Fe PC (for a final concentration of 60 µM) were added to the quartz cell by triplicate. Then,
2
2
the quartz cell was mounted into a UV-visible spectrophotometer (Varian), and the absorbance was
measured (at 540 nm for 5 min) as absorbance increment due to NBT formation per unit of time.
Assays were performed at room temperature (25 °C). We reported the inhibition percentage of
tetrazolium formazan formation, under the conditions describe above.
3
.5. Peroxidase-Like Activity
POx activity was determined using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit.
®
The assay consisted in placing into individual wells of a microplate 50 µL of working solution
®
prepared as follows: 10 mM Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) reagent stock
2 2 2
solution (50 µL), 20 mM H O solution (500 µL) and 1X Reaction Buffer (4.45 mL) and Fe PO or
Fe PC (50 µL) were added with a final concentration of 0.1 mM. The POx activity was determined
2
®
spectrophotometrically monitoring the oxidation of the Amplex Red reagent to Resourfin in the
presence of H
2
O
2
and a POx or POx mimetic, using a microplate reader at 550 nm after 0.5, 12, 24 and
4
8 h of incubation.
3
.6. Cellular Assay
PBMCs were isolated from heparinized blood obtained from human healthy donors by a density
5
gradient centrifugation using Ficoll-Hypaque. Isolated PBMCs (5 × 10 cells per well) were resuspended
in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum, 50 mM 2-mercapoethanol,
−
1
1
00 U mL amphotericin band seeded onto 96-well tissue culture plates for stimulation with
−1
phytohemagglutinin (PHA, 5 µg mL ) in the presence of enzymatic system, xanthine plus xanthine
oxidase (XO), as an enzymatic generator of reactive oxygen species, i.e., superoxide, hydrogen

Molecules 2013, 18
1773
−1
peroxide and hydroxyl radicals [16] (8 mM of xanthine and 23 mU mL XO), and a different
concentrations of Fe PO and Fe PC (0, 30, 60 and 120 µM). Cells were incubated 6, 18 and 24 h at
. Cell viability was evaluated by trypan blue dye exclusion assay.
2
2
37 °C in 5% CO
2
4
. Conclusions
Two novel Fe(III)-cyclophanes have shown bioactive properties as antioxidants, reducing the
•+
synthetic ABTS radical, within the range observed for ascorbic acid, which was used as the reference
antioxidant. Reported results for similar Cu(II) complexes [5] demonstrated the biomimetic potential
of these metallic cyclophane derivatives [7]. Aside of their antioxidant capacity comparable to
phenolic acids, flavonoids and vitamins C and E [17] is the lack of need for negative interactions with
cells. For that matter, a bioassay using PBMC cells injured with ROS was done to test their antioxidant
2 2
capacity in vivo. In fact, both Fe PO and Fe PC were able to scavenge the superoxide produced by
xanthine and xanthine oxidase, leading to an increased cellular survival.
The differences in biomimetic activity between Fe PO and Fe PC could be due to structural
2
2
differences between the cyclophanes. The action of iron-cyclophanes against the superoxide radical
was similar in the SOD assay, while when a stable radical (ABTS) was used to test antioxidant
2 2
capacity, Fe PO was better than Fe PC. It is known that PC is more rigid compared to PO, and since
ABTS is a large molecule with a mass over 500 Da, we postulate that steric effects arise at the interaction
with the synthetic antioxidant. Considering that these molecules could remove superoxide radical and
led to molecular water that not affect cells, they appear to be very interesting molecules for further
studies to evaluate their antioxidant and bioactive properties in animal models and cancer cell lines.
Acknowledgments
Alex Salazar-Medina and Eli Teran-Cabanillas are thankful for a Ph.D. scholarship from
CONACYT. This project was partially funded by grant IPYM11/I157 from División de Ingeniería,
Universidad de Sonora. R. Sotelo-Mundo is on sabbatical leave at Universidad de Sonora, Departamento
de Investigación en Polimeros y Materiales, Universidad de Sonora.
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Sample Availability: Samples of the compounds PO, PC, Fe PO and Fe PC are available from
the authors.
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