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Journal of Inorganic Biochemistry 101 (2007) 849–858

InorganicBiochemistry

Macrocyclic copper(II) complexes: Superoxide scavengingactivity, structural studies and cytotoxicity evaluation

Ana S. Fernandes a,b, Jorge Gaspar b, M. Fatima Cabral a, Catia Caneiras a, Rita Guedes a,Jose Rueff b, Matilde Castro a, Judite Costa a, Nuno G. Oliveira a,b,*

a CECF, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugalb CIGMH, Department of Genetics, Faculty of Medical Sciences, New University of Lisbon, Rua da Junqueira 96, 1349-008 Lisboa, Portugal

Received 31 July 2006; received in revised form 15 January 2007; accepted 29 January 2007Available online 11 February 2007

Abstract

Synthetic superoxide dismutase mimetics have emerged as a potential novel class of drugs for the treatment of oxidative stress relateddiseases. Among these agents, metal complexes with macrocyclic ligands constitute an important group. In this work we synthesized fivemacrocyclic copper(II) complexes and evaluated their ability to scavenge the superoxide anions generated by the xanthine–xanthine oxi-dase system. Two different endpoints were used, the nitro blue tetrazolium (NBT) reduction assay (colorimetric method) and the dihy-droethidium (DHE) oxidation assay (fluorimetric method). IC50 values in the low micromolar range were found in four out of fivemacrocyclic complexes studied, demonstrating their effective ability to scavenge the superoxide anion. The IC50 values obtained withthe NBT assay for the macrocyclic copper(II) complexes, were consistently higher, approximately threefold, than those obtained withthe DHE assay. Spectroscopic and electrochemical studies were performed in order to correlate the structural features of the complexeswith their superoxide scavenger activity. Cytotoxicity assays were also performed using the MTT method in V79 mammalian cells and wefound that the complexes, in the range of concentrations tested in the superoxide scavenging assays were not considerably toxic. In sum-mary, some of the presented macrocyclic copper(II) complexes, specially those with a high stability constant and low IC50, appear to bepromising superoxide scavenger agents, and should be considered for further biological assays.� 2007 Elsevier Inc. All rights reserved.

Keywords: Superoxide scavenging activity; Macrocycles; Copper(II) complexes; Superoxide dismutase; Antioxidant

1. Introduction

Reactive oxygen species (ROS) are implicated in severalhuman pathological processes including tissue injury,inflammation, ageing, cancer, cardiovascular, pulmonaryand neurodegenerative diseases [1]. Superoxide anion(O��2 ) may cause several harmful effects, leading to tissueinjury and inflammation [2,3]. By catalyzing the conversionof O��2 to H2O2 and O2, superoxide dismutases (SOD) rep-resent the first line of defence against O��2 . Preclinical stud-

0162-0134/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2007.01.013

* Corresponding author. Address: CECF, Faculty of Pharmacy, Uni-versity of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. Fax:+351 217 946 470.

E-mail address: ngoliveira@ff.ul.pt (N.G. Oliveira).

ies have revealed that SOD enzymes play a protective effectin animal models of several diseases [3]. MnSOD hasshown to suppress cancer phenotypes in a large numberof cancer models [4]. However, the therapeutic use of thenative SOD has several limitations related with low cellpermeability and short half-life. In addition, bovine CuZn-SOD was tested in clinical trials but immunological prob-lems lead to its withdrawal from the market [5]. Toovercome this problem, synthetic SOD mimetic com-pounds have emerged as a potential novel class of drugs.

Transition metal complexes [e.g. complexes of Mn(II),Mn(III), Cu(II) and Fe(III)] have notably shown impor-tant antioxidant properties, namely SOD mimetic activity[6,7]. The metal containing SOD mimetic agents moreextensively studied are manganese(III) metalloporphyrins,

N

O

N

N NN

R

R R

R

CO2H

L1 - R = H

L2 - R =

L5

CO2H

L3 - R = H

L4 - R =

N

O

R R

O

O

N

NNH H

H

Scheme 1.

850 A.S. Fernandes et al. / Journal of Inorganic Biochemistry 101 (2007) 849–858

manganese(III) salen complexes and manganese(II) macro-cyclic complexes [1].

Copper is an essential element involved in several biolog-ical functions, acting as a catalytic component of manymetalloenzymes, including SOD. Therefore, copper(II)complexes can enclose a SOD mimetic activity hinderingincreased levels of reactive oxygen species. Biological effectsof the SOD mimics are related to their structures. High sta-bility constants are required to avoid the dissociation of thecomplex in vivo. This could be achieved by using macrocy-clic ligands [6]. The macrocyclic nature of the ligand seemsimportant for the SOD mimetic activity of the correspond-ing complexes as well as for their stability in the presence ofproteins, even if the metal ion does not lie inside the cavity[8]. Several macrocyclic copper complexes have beenreported to scavenge the superoxide anion [7–11].

Previous studies have demonstrated that chemical mod-ifications in the ring size, donor atoms and substituents onthe macrocycles, may have profound effects both on thestability and the SOD-like activities of the respective com-plexes [10,11]. A copper(II) complex which possesses SODmimetic activity should have a flexible arrangement of theligands around the copper(II) ion in order to allow an easyreduction to copper(I). In addition, a copper(II) SODmimetic complex should enclose a certain stability, avoid-ing thus dissociation in the acid region and should possessan accessible site in order to easily bind the O��2 radical and,hence to give a quick reduction to copper(I). Finally, anequatorial field of medium strength is required becausestrong ones do not favour the attack of O��2 to the accessi-ble apical sites [12].

Although many copper(II) complexes have been studied,most of them are not thermodynamically stable or highlyactive at the range of physiological pH [13]. For this reason,the identification of new complexes with high stability andactivity, together with low toxicity, is still a challenging issuein bioinorganic chemistry. Another important feature is thesolubility of the compounds. Some of the SOD mimetics thathave been developed, despite of their effective catalytic activ-ity, are not water soluble. It is estimated that �40% of thecompounds that enter the development phase fail to reachthe market, mainly due to poor biopharmaceutical proper-ties, in which low aqueous solubility is included [14]. Hence,the water solubility presented by the complexes under studyis definitely an important favorable characteristic.

In the present work, we describe five low molecularweight copper(II) macrocyclic complexes, focusing on theirpossible application as superoxide scavenging agents. Theability of those copper(II) complexes to scavenge O��2 wasevaluated by two different methods: the nitro blue tetrazo-lium (NBT) and the dihydroethidium (DHE). Spectro-scopic studies were performed in order to correlate thestructural features of the complexes with their scavengingactivity. In addition, the cytotoxicity of these complexeswas assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in V79 Chi-nese hamster cells, a widely used and non-tumoral cell line.

2. Experimental

2.1. Chemicals

Phosphate saline buffer (PBS; 0.01 M, pH 7.4), caffeicacid, xanthine, xanthine oxidase (XO) (E.C.1.1.3.22), nitroblue tetrazolium (NBT), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] (MTT), trypsin, Ham’sF-10 medium, 30% hydrogen peroxide (w/w), newborn calfserum, penicillin–streptomycin solution and Cu,Zn-super-oxide dismutase from human erythrocytes (CuZnSOD;E.C.1.15.1.1) were obtained from Sigma-Aldrich. Dimeth-ylsulfoxide (DMSO) was obtained from Merck. Copper(II)nitrate-trihydrate, disodium ethylenediaminetetraacetate(Na2H2edta) and DHE were purchased from Fluka.

Stock solution of DHE (10 mM) was prepared inDMSO, aliquotized and stored at �18 �C. CuZnSODwas reconstituted in PBS. The concentration of the resul-tant solution (7.9 lM) was calculated taking into accounta molecular weight for the enzyme of 32,500 [15].

2.2. Synthetic procedures

2.2.1. Synthesis of the macrocycles

The macrocyclic ligands used for the preparation of thecopper(II) complexes studied in the present work areshown in Scheme 1 and were synthesized as previouslydescribed [16–18]. All the compounds were obtained ingood yield and were characterized by 1H and 13C NMRspectroscopy on a Bruker Avance 400 spectrometer. Thereferences used for the NMR measurements were 3-(tri-methylsilyl)propionic acid-d4 sodium salt for 1H NMRand 1,4-dioxan for 13C NMR.

L1 was synthesized by Richman and Atkins’ procedure[19], involving the condensation of the disodium salt of1,4,N,N 0-bis(p-toluenesulfonyl)-1,4-diaminoethane with 1-O,5-O-di(p-toluenesulfonyl)-3-oxapentane-1,5-diol, at 110 �Cin dry dimethylformamide. The protective groups of theditosylated cyclic amine were removed by a reductive cleav-age with a mixture of glacial acetic acid, 48% hydrobromicacid and phenol during 28 h under reflux [16]. 1H NMR(D2O): d 3.06 (t (triplet), 4 H), 3.34 (s (singlet), 4 H),3.75 (t, 4 H).

A.S. Fernandes et al. / Journal of Inorganic Biochemistry 101 (2007) 849–858 851

The macrocycle L2 was synthesized by condensation ofthe parent amine L1, with potassium bromoacetate in anaqueous basic solution [16]. 1H NMR (D2O): d 3.19 (t, 4H), 3.25 (s, 4 H), 3.68 (s, 4 H), 3.80 (t, 4 H). 13C NMR(D2O): d 52.16, 54.70, 57.27, 66.21, 171.53.

L3 was also prepared according to the Richman andAtkins’ method [19], involving the condensation of thedisodium salt of 3,6,N,N 0-tetra(p-toluenesulfonyl)-3,6-dia-zaoctane-1,8-diamine with 1-O,5-O-di(p-toluenesulfonyl)-3-oxapentane-1,5-diol, in dry dimethylformamide for 6 h.In a second step, the protective groups were removed bya reductive cleavage [17,20], as described above. 1HNMR (D2O): d 3.21 (s, 4 H), 3.29 (m (multiplet), 8 H),3.37 (t, 4 H), 3.73 (t, 4 H). 13C NMR (D2O): d 42.73,43.19, 43.87, 46.50, 65.67.

L4 was synthesized by condensation of the parent amineL3 with potassium bromoacetate in an aqueous alkalinesolution [18]. 1H NMR (D2O): d 3.42 (s, 4 H), 3.55 (t, 4H), 3.64 (t, 4 H), 3.77 (t, 4 H), 3.85 (s, 4 H), 4.09 (s, 4H), 4.15 (t, 4 H). 13C NMR (D2O): d 50.00, 51.72, 53.23,54.67 (d), 55.02, 64.89, 168.59, 171.79.

L5 was prepared by the same method of L3, using 1-O,8-O-di(p-toluenesulfonyl)-3,6-dioxaoctane-1,8-diol andthe disodium salt of 3,N,N 0-tri(p-toluenesulfonyl)-3-aza-pentane-1,5-diamine [17]. 1H NMR (D2O): d 3.52 (t, 4H), 3.69 (m, 8 H), 3.82 (s, 4 H), 3.94 (t, 4 H).13C NMR(D2O): d 42.73, 43.04, 45.90, 65.30, 69.86.

Aqueous macrocycles solutions were prepared at�2.5 · 10�3 M, and their concentrations were determinedby potentiometric titrations.

2.2.2. Synthesis of the macrocyclic copper(II) complexes

Copper(II) complexes were prepared by adding an aque-ous solution of the nitrate salt (4.0 lmol) previously stan-dardized by titration with Na2H2edta [21] to an aqueoussolution of the ligands (4.2 lmol). After stirring at roomtemperature, KOH solution was added until pH 7.4. Thesolvent was removed under reduced pressure and the resi-due was taken up in 2 mL of PBS. At micromolar concen-trations, water molecules can act as competing ligands forcopper(II), modifying the species distributions towards lesscoordinated species. To avoid this problem, when cop-per(II) complexes were prepared, a small excess of ligandwas used in order to increase the complexation of the cop-per(II) ions [22]. As shown by the distributions curves thecopper(II) complexes are completely formed at pH 7.4,except for L1 (see below). Additionally, the EPR resultsconfirmed the absence of aqueous copper(II) impurities.

The values of the stability constants of Cu(II) with theligands were previously determined from potentiometrictitrations, performed at 25.0 ± 0.1 �C and 0.10 M ionicstrength [16–18]. The constant values were calculated by fit-ting the potentiometric data obtained using the SUPER-QUAD [23] or HYPERQUAD programs [24].

Species distribution curves were calculated for the aque-ous solutions containing Cu(II) and each ligand (L1–L5) ata molar ratio of 1:1, using the Hyss program [25]. The spe-

cies concentrations in solution were determined at physio-logical pH. Simulations of the species distribution in thepresence of 0.01 M phosphate buffer were also performed.For these calculations, the stability constants for cop-per(II)-hydrogenophosphate [26] as well as the protonationconstants for phosphoric acid [27] were considered, in orderto calculate the percentage of each species present at pH 7.4.

2.3. Superoxide scavenging activity

Superoxide scavenging activity of the complexes wasstudied by using their ability to scavenge O��2 , generatedby the xanthine–xanthine oxidase (X–XO) system, troughtwo different endpoints: the reduction of NBT and the oxi-dation of DHE. Copper(II) and CuZnSOD from humanerythrocytes were used as controls. The evaluation of theinhibition of XO by the complexes was performed follow-ing the production of uric acid.

2.3.1. NBT assay

In this assay, while O��2 is generated, NBT is reduced,developing a blue formazan colour which is associated withan increase in the absorbance at 560 nm [28]. When a scav-enger compound is added, it competes with the NBT forthe oxidation of the generated superoxide anions. There-fore, there is a decrease in the rate of the NBT reduction,which leads to lower absorbance increases. The more effec-tive the compound, the lower the concentration whichinhibits the NBT reduction in 50% (IC50) [8].

The conditions of the NBT assay were adapted fromKovala-Demertzi et al. [28]. The reaction system (final vol-ume = 1 mL) contained 0.2 mM of xanthine, 0.6 mM ofNBT in phosphate buffer 0.1 M, pH 7.8. Each compoundwas added to the reaction mixture in different concentra-tions up to 40 lM. The reaction was started by the additionof XO (6 mU/mL), an activity which allowed to yield theabsorbance change between 0.030 and 0.040 per minute,at 560 nm, 25 �C. The extent of NBT reduction was fol-lowed spectrophotometrically, by measuring the increaseof the absorbance at 560 nm on a Hitachi U-2001 spectro-photometer, for 3 min. Each experiment was performed induplicate and each concentration generated a time-depen-dent curve. From its linear domain, we calculated the slope(Abs/min). The percentage of inhibition for each concen-tration was calculated as follows: [100 � (slope/slope con-trol) * 100]. The IC50 of each compound was defined asthe concentration which inhibited 50% of the NBT reduc-tion by O��2 produced in the X–XO system.

2.3.2. DHE assay

In the DHE assay, dihydroethidium is oxidized by O��2 ,giving a fluorescent compound. The fluorescence emissionis related to the amount of superoxide anion present inthe system [29]. This assay was performed in 96-well micro-plates. Each well (200 ll) contained 0.2 mM of xanthineand phosphate buffer 0.1 M, pH 7.8. The tested com-pounds, diluted in phosphate buffer pH 7.8, were added

852 A.S. Fernandes et al. / Journal of Inorganic Biochemistry 101 (2007) 849–858

to the reaction mixture (10 ll). Different concentrations upto 80 lM were tested for each compound (CuL1–CuL4). Inwhat concerns CuL5, since it revealed no activity using theNBT assay (see Section 3), we only tested the highest con-centration (80 lM).

DHE aliquots (10 mM, DMSO) were diluted 1:100 inphosphate buffer pH 7.8. DHE was added to each well ata final concentration of 10 lM. The reaction was startedby the addition of XO (5 mU/mL). The reaction wasperformed at 25 �C, and the extent of DHE oxidationwas followed by measuring the increase of the fluorescenceon a Zenyth 3100 microplate reader, for 60 min, usingkexcitation = 485 nm and kemission = 595 nm. Each experi-mental point was performed using eight replicates. Twoindependent experiments were performed for each concen-tration of the different complexes. Each concentrationgenerated a time course curve. From its linear domain,we calculated the slope. The percentage of inhibition foreach concentration was calculated as described above(Section 2.3.1). The IC50 of each compound was definedas the concentration which inhibited 50% of the DHE oxi-dation by the O��2 produced in the X–XO system.

2.3.3. Xanthine oxidase inhibition assay

We also evaluated if the generating system X–XO couldbe inhibited by the copper(II) complexes in study. This wasperformed by following at 293 nm, during 5 min, the uricacid produced after xanthine was oxidized by XO in aero-bic conditions concomitantly to the production of O��2 . Theassay was performed for each complex (80 lM) at the sameexperimental conditions described above, with the excep-tion of the NBT solution, which was replaced for equal vol-ume of phosphate buffer [30]. Caffeic acid (50 lM) wasused as positive control. Each experiment was performedin duplicate.

2.4. Structural studies

2.4.1. Spectroscopic studiesThe electronic spectra of the complexes were performed

using a UNICAM model UV-4 spectrophotometer. Thecomplexes were prepared in aqueous solutions at1.20 · 10�3 M (1:1 ratio) in 0.1 M in KNO3. For CuL2,CuL3, CuL4 and CuL5 the solutions’ pH was 7.38, 7.41,7.37, and 7.38, respectively.

EPR spectroscopy measurements of the copper(II) com-plexes were recorded with a Bruker EMX 300 spectrometerequipped with a continuous-flow cryostat for liquid nitro-gen, operating at X-band. The complexes were preparedat 1.25 · 10�3 M (1:1 ratio) in water (1.0 M in NaClO4).The spectra of CuL2, CuL3, CuL4 and CuL5 wererecorded at the pH values of 7.39, 7.35, 7.40 and 7.40,respectively.

2.4.2. ElectrochemistryA BAS CV-50 W Voltammetric Analyzer connected to a

BAS/Windows data acquisition software were used for the

electrochemical measurements. Cyclic voltammetry experi-ments were performed in a glass cell MF-1082 from BAS ina C-2 cell enclosed in a Faraday cage, at room temperatureunder nitrogen atmosphere.

The reference electrode was Ag/AgCl (MF-2079 fromBAS) and its potential was �44 mV relative to a saturatedcalomel electrode (SCE). The auxiliary electrode was a7.5 cm platinum wire (MW-1032 from BAS) with a gold-plated connector. The working electrode was a glassycarbon (MF-2012 from BAS). Between each cyclic voltam-metry scan the working electrode was electrocleaned bymulticycle scanning in the supporting electrolyte solution,polished on diamond 1 lm and on alumina 0.3 lm, accord-ing to standard procedures. The aqueous solutions of thecomplexes were prepared at 1.20 · 10�3 M (1:1 ratio) in0.1 M KNO3 (supporting electrolyte). The voltammogramsof CuL2, CuL3, CuL4 and CuL5 were performed at the pHvalues of 7.39, 7.35, 7.40 and 7.40, respectively. Cyclic vol-tammograms were recorded in the region from +1.0 to�1.0 V versus Ag/AgCl, varying the scan rate from 10 to100 mV s�1. The half wave potentials E1/2 were calculatedapproximately from (Epa + Epc)/2.

2.5. Cell survival evaluation

2.5.1. Cell cultureWild-type V79 Chinese Hamster Cells (MZ), kindly pro-

vided by Prof. H.R. Glatt (Potsdam, Germany), were rou-tinely maintained in 175 cm2 culture flasks (Sarstedt) usingHam’s F-10 Medium, supplemented with 10% newborncalf serum and 1% antibiotic solution (penicillin–strepto-mycin) as the cell culture medium. The cells were kept at37 �C, under an atmosphere containing 5% CO2.

2.5.2. MTT assay

The toxicity of macrocyclic copper(II) complexes wasevaluated using the MTT assay. In this method, MTT isconverted by mitochondrial enzymes of viable cells into aformazan which can be measured at 595 nm. The absor-bance is proportional to the number of viable cells [31].In our experimental protocol, approximately 5 · 103 cellswere cultured in 200 ll of culture medium per well in 96-well plates and incubated at 37 �C under a 5% CO2 atmo-sphere. The cells were grown for 24 h and then exposed todifferent concentrations of the macrocyclic copper(II) com-plexes (1, 10, 50 and 100 lM) dissolved in phosphate salinebuffer (pH 7.4), during a 24 h period. Hydrogen peroxide(10 mM) was used as a positive control. The cells werewashed with culture medium and MTT (dissolved in cul-ture medium) was added at a concentration of 0.5 mg/mlto each well [32]. The cells were grown for a further periodof 2.5 h and then carefully washed with PBS. At the end ofthe incubation period, the media was discarded and DMSO(200 ll) was added to each well to solubilize the formazancrystals. Absorbance was read in a Zenyth 3100 microplatereader at 595 nm. Two independent experiments were per-

Table 1Stability constants (log units) for copper(II) complexes

Equilibrium quotient L1a L2a L3b L4c L5b

[CuL]/[Cu][L] 10.80 13.37 20.34 19.23 15.72[CuHL]/[CuL][H] – – – 4.38 –[Cu2L]/[Cu][CuL] – – – 4.56 –[CuL2]/[CuL][L] 8.80 – – – –[CuL]/[CuLOH][H] – – 10.4 (8.1) 8.87

a I = 0.10 M KNO3; T = 25.0 �C [16].b I = 0.10 M KNO3; T = 25.0 �C [17].c I = 0.10 M N(CH3)4NO3; T = 25.0 �C [18].

Table 2Species distribution and pCu values calculated for an aqueous solutioncontaining Cu(II) (10 lM) and each ligand (10.5 lM) at a molar ratio of1:1 (charges on metal ions and complexes were omitted for simplicity)

Ligand Species (% relative to the total amount of Cu(II)) pCu

L1a 79.8% CuL + 12.4% CuL2 + 5.1% Cu+ 2.6% CuOH + 0.1% Cu(OH)2

6.29

L2b 100.0% CuL 8.90L3 99.9% CuL + 0.1% CuOH 15.39L4 0.1% CuHL + 83.3% CuL + 16.6% CuLOH 13.44L5 96.7% CuL + 3.3% CuLOH 11.22

a 12.8% CuL + 41.2% CuL2 + 27.2% CuHPO4 + 17.6% Cu(HPO4)2 +1.1% Cu(HPO4)3, in 0.01 M phosphate buffer, pH 7.4.

b 93.9% CuL + 3.6% CuHPO4 + 2.3% Cu(HPO4)2 + 0.2% Cu(HPO4)3,in 0.01 M phosphate buffer, pH 7.4.

A.S. Fernandes et al. / Journal of Inorganic Biochemistry 101 (2007) 849–858 853

formed and four replicate cultures were used for each com-plex concentration in each independent experiment.

3. Results and discussion

Macrocycles and their metal complexes have been sug-gested as promising agents for the diagnosis and treatmentof different diseases [33–35]. In addition, some macrocycliccomplexes have been suggested as a potential class of SODmimics, mainly because of their high thermodynamic sta-bility [6,8].

Most of the catalytic antioxidants have a redox-activemetal centre [1,6], but only a few metal ions have the abilityto catalyze the dismutation of O��2 to hydrogen peroxideand oxygen. It is well known that copper(II) aqueous ionis a very potent superoxide scavenger [6]. The low IC50 val-ues found using Cu(NO3)2 in this work are consistent withthe values found for other Cu(II) salts [8] and show the effi-cacy of Cu(II) in the disproportionation of O��2 .

Human serum albumin has a high-affinity site for cop-per(II) [36] precluding the use of free copper(II) as a ther-apeutic SOD mimetic. In fact, in blood plasma, albuminexists in very high concentration, binding to the non-ceru-loplasmin copper fraction and acting thus as a coppertransport protein [13,36]. Therefore, if free Cu(II) ions wereadministered, they would be immediately complexed byserum albumin, loosing their ability to dismutate the O��2[8]. Within mammalian cells, the binding of Cu(II) by cel-lular components also occurs, and copper chaperones, glu-tathione and metallothioneins, among other proteins, maybe involved [37]. Since the intracellular milieu has anextraordinary overcapacity for chelation of copper(II), freecopper availability is extremely restricted, even when cellsare exposed to an elevation of the medium’s copper con-centration [38].

In view of this, copper(II) must be enclosed in a stableligand, which protects it from being chelated by serumand cellular components. Additionally, this ligand mustallow copper(II) to switch its redox state and dismutatethe O��2 . It has been described that if the ligand is a macro-cycle, the metal complex may have higher biological stabil-ity [6,8]. In this study we present four macrocycliccopper(II) complexes possessing superoxide scavengingactivity with IC50 in the low lM range.

The knowledge of the stability constants values for thecopper(II) complexes is a very important issue for the pre-diction of their behaviour in vivo. High stability constantsare required to avoid dissociation of the complex inin vivo systems. The stability constants values of the macro-cyclic copper(II) complexes under study were determined inprevious works [16–18] and are shown in Table 1. All com-plexes, except CuL1, showed reasonably high values of sta-bility constants (log KML > 13). L3 and L4 have indeedshown the highest values (logKML = �20).

The species distribution diagrams and the pCu values(pCu = �log [Cu2+]) were obtained from the values of thestability constants of the macrocyclic copper(II) complexes,

using the Hyss program [25]. The concentration used inthese simulations was 10 lM of Cu(II) at a molar ratio1:1 with L1–L5. This value was chosen because it is withinthe range of IC50 values of the active superoxide scavengingcomplexes. The results obtained in these conditions and atphysiological pH (7.4) are shown in Table 2. In these sim-ulations, no free copper(II) ions are likely to be found insolution for the copper(II) macrocyclic complexes, exceptin the case of L1 (�5.1% of aqueous copper(II) ion). Thiscan be explained by the fact that under these conditions(molar ratio 1:1) the complexation of L1 with copper(II)is not fulfilled. EPR spectra confirmed the absence cop-per(II) impurities in the CuL2–CuL5 solutions.

The copper(II) complexes were initially redissolved inphosphate saline buffer and this solution was used in boththe biochemical and cytotoxicity assays. Taking this intoaccount and in order to give an even more accurate speci-ation in the used conditions, we considered the possiblecompetition between the phosphate species present in highlevels in the buffer and the copper(II) complexes understudy. Having this in mind, we recalculated the species con-centrations at pH 7.4. Using this approach, no changeswere found for the species in solution at pH 7.4 forCuL3, CuL4 and CuL5. In what concerns to CuL1 and,in a lesser extent to CuL2, we should consider that the pres-ence of high phosphate concentration derived in some dif-ferences in the species in solution, giving rise to theappearance of CuHPO4, Cu(HPO4)2 and Cu(HPO4)3 atpH 7.4 (Table 2 – footnote).

The SOD-like activity of the complexes was first deter-mined using NBT reduction assay. This reference method

Table 3Superoxide scavenging activity for the copper(II) compounds and nativeCuZnSOD

Compound IC50 (lM)

NBT assay DHE assay

CuL1 6.86 1.07CuL2 18.84 5.03CuL3 30.82 11.78CuL4 13.94 4.66CuL5 N.D. N.D.Cu(II) 0.28 0.11CuZnSOD 0.017 0.001

N.D. not determined.

854 A.S. Fernandes et al. / Journal of Inorganic Biochemistry 101 (2007) 849–858

is simple and convenient and has been widely used forsuperoxide quantification and as well as in SOD mimicsresearch [39]. Some authors have pointed out a few limita-tions for this method, especially the auto-oxidation of NBT[40] and the poor solubility of the formazan product gener-ated [39]. In addition, we used a different experimentalapproach, the DHE fluorimetric method, which has beendescribed by some authors as a more sensitive and specificassay for superoxide anion [40]. This method has beencommonly used in cell-based experiments to quantify O��2[29].

The superoxide scavenging effect of the macrocyclic cop-per(II) complexes determined by both the NBT and DHEmethods, are depicted in Fig. 1. The correspondent IC50

values determined for the complexes under study, as wellas for Cu(II), are presented in Table 3. A very good corre-lation between the NBT and DHE assays was found(r = 0.979). The IC50 values obtained for the macrocycliccopper(II) complexes using the NBT assay, were consis-tently higher, approximately threefold, than those obtainedwith the DHE assay (Table 3). The aforementioned differ-ences in the sensitiveness and specificity between bothmethods may somehow explain these results. However,other authors have pointed out that DHE could enhancethe rate of superoxide dismutation [41].

Since both NBT and DHE methods involve the activityof XO it is of utmost importance to ascertain that theresults obtained are in fact due to a SOD-like activityand not a consequence of the inhibition of XO. The mon-itoring of the production of uric acid revealed that theactive copper complexes (CuL1, CuL2, CuL3 and CuL4),at 80 lM, did not inhibit XO. Caffeic acid at the sameexperimental conditions inhibited 47.0% and 59.8% of uricacid production at 180 and 290 s, respectively. Someauthors have also pointed out that Cu(II) ion could inhibitXO at mM concentrations [42]. However, in our experi-mental conditions, using 80 lM of Cu(NO3)2, that inhibi-tion did not occur.

For both assays and for all the complexes studied, thenative human CuZnSOD was used as a positive control.As expected, a dose-dependent inhibition was found bothfor NBT reduction and DHE oxidation (data not shown).

a

-100

102030405060708090

100

0 10 20 30 40 50

[CuL] (μM)

Inhi

bitio

n (%

)

CuL1CuL2CuL3CuL4CuL5

Fig. 1. Effect of the macrocyclic copper(II) complexes CuL1–CuL5 on the ingenerated superoxide.

The IC50 values for the enzyme were determined in thesame experimental conditions used for the study of thecomplexes and revealed to be extremely low, in the nMrange (Table 3).

Four of the five macrocyclic copper(II) complexes stud-ied (CuL1, CuL2, CuL3 and CuL4) have an effective abilityto scavenge the O��2 with IC50 in the low micromolar range(Fig. 1, Table 3). The most active was CuL1 followed byCuL4, CuL2 and finally by CuL3. In what concerns toCuL5, no IC50 value was calculated because no superoxidescavenging activity was found using the NBT assay(Fig. 1a). Using the DHE method an inhibition of <40%was found at 80 lM, which may be considered as a lowsuperoxide scavenging activity (Fig. 1b).

The superoxide scavenging activities of the complexesunder study are in the same range of those reported forother metal complexes [43–45]. However, compounds withhigher activity (e.g. some Mn-salens and Mn-porphyrins)have also been described [43,46,47], despite some of themare not readily water soluble. Nevertheless, focusing onthe previously reported macrocyclic copper(II) complexes,which are more similar to the presented compounds, it isnoticeable that the complexes under study are generallymore active, having lower IC50 values [7,10].

In what concerns the four active macrocyclic copper(II)complexes and regardless the analytical method, we canobserve that CuL1 is the most effective compound forsuperoxide scavenging (Fig. 1, Table 3). However, as itwas aforementioned in this discussion, at physiological

0

20

40

60

80

100

0 15 30 45 60 75 90[CuL] (μM)

Inhi

bitio

n (%

)

CuL1 CuL2 CuL3 CuL4 CuL5

b

hibition of the NBT reduction (a) and DHE oxidation (b) by the X–XO

A.S. Fernandes et al. / Journal of Inorganic Biochemistry 101 (2007) 849–858 855

pH and in the molar ratio used, the complexation of L1with copper(II) is not complete (Table 2), a consequenceof the low stability constant of L1 for copper (logKML =10.8). Thus, we can not rule out that the presence of freecopper in the reaction system could contribute to the lowIC50 found for the complex of L1 (Tables 2 and 3). More-over, in the presence of phosphate buffer, the percentCu(II) complexed with L1 markedly decreased with a con-comitant increase of Cu(II) complexed with hydrogeno-phosphate (Table 2 – footnote). For CuL2 this problemis not so relevant, although we should not disregard thepresence of Cu(II) complexed with hydrogenophosphate,which may contribute in some extent to the dismutationof O��2 [48]. For the complexes CuL3, CuL4 and CuL5, thisquestion is not applicable.

In view of this, we attempted to find out a structural-O��2scavenging activity correlation for the complexes withhigher thermodynamically stability (CuL2–CuL5). For thispurpose, visible and EPR spectroscopic studies were per-formed. In addition, the electrochemical behaviour of thecomplexes was studied by cyclic voltammetry.

Spectroscopic visible data and EPR parametersfor Cu(II) complexes in water solution are collected inTable 4. The EPR spectra of the copper(II) complexesare shown in Fig. 2. The simulation of the spectra [49] indi-cates three different principal values of g, showing that theCu(II) ion in these complexes is in a rhombically distortedligand field. All the complex species show parameterswhich are characteristic of rhombic symmetry with elonga-tion of the axial bonds and a dx2�y2 ground state. Elongatedrhombic-octahedral, rhombic square-coplanar or distortedsquare pyramidal stereochemistries would be consistentwith these data, but trigonal–bipyramidal or tetragonalgeometries involving compression of axial bonds shouldbe excluded [18,50].

According to the ligand field theory [51–53], the gz valueincreases and the Az value decreases as the planar ligandfield becomes weaker or as the axial ligand field becomesstronger and this occurs with the simultaneous red-shiftof the d–d absorption bands in the electronic spectra. Thissequence, in principle, parallels the degree of distortionfrom square-planar to square pyramidal, C4v, and then tooctahedral (Oh) or tetragonal (D4h) geometries.

Table 4Spectroscopic X-band EPR data for the Cu(II) complexes of L2–L5 and CuZ

Complex Visible band kmax/nm (emolar/M cm�1) EPR para

Ai · 10�4

gx

CuL2 638 (100) 2.041CuL3 580 (141) 2.042CuL4-1 674 (86) 2.036CuL4-2 2.037CuL5 634 (111) 2.044CuZnSOD [58] 680 (150)

a Considering gz as gi and Az as Ai.

The EPR parameters for copper(II) complexes are deter-mined by the chemical composition and the physical con-straints on the atoms nearest to the metal ion. On thebasis of the Peisach and Blumberg approach [54], whichaccounts for the close relationship between gi and Ai forequatorially coordinated S = 1/2 paramagnetic metal com-plexes some important properties of the copper(II) com-plexes may be determined. In this case, the actualcomposition of the equatorial atom donor set may be eval-uated from the EPR data using the diagrams of gi and Aivalues compiled by the authors for a series of modelcompounds.

The spectrum of CuL2 (Fig. 2a) exhibits the presence ofone species. The gi value (considering gz as gi) and Ai value(considering Az as Ai) parameters are according with twonitrogen and two oxygen donor atoms coordinated to themetal center in the equatorial plane. These parametersand also the value of the maximum of the visible band(Table 4) suggest a square-pyramidal structure where theequatorial plane is determined by the two nitrogen andtwo oxygen atoms and one oxygen atom is in the apicalposition.

The data obtained for CuL3 spectrum, together with theposition of the d–d absorption band (Fig. 2b and Table 4)point out to a square-pyramidal structure where the equa-torial plane is determined by the four nitrogen atoms andone oxygen atom is in the apical position.

The spectrum of CuL4 is shown in Fig. 2c and exhibitsthe presence of two species (CuL4-1 and CuL4-2). TheEPR parameters and electronic spectrum determined inthis work (Table 4) are in agreement to those publishedbefore [18]. In CuL4-1, copper(II) is in a distorted octahe-dral environment having two nitrogen and two oxygenatoms coordinated directly to the metal ion. Two oxygenatoms accomplish the octahedral geometry. In what con-cerns to the CuL4-2 species the EPR parameters are com-pletely different, having lower gi and higher Ai values,indicating a stronger equatorial field. An octahedral dis-torted geometry is also proposed but having three nitrogenand one oxygen atoms in the equatorial plane and two oxy-gen atoms in axial positions. From these data it is not pos-sible to know if the oxygen atom of the macrocycle ring isinvolved in the metal ion coordination.

nSOD

meters

cm�1; gi/Ai · cm

gy gz Ax Ay Az gk=Aka

2.053 2.225 18.1 22.9 190.7 1172.046 2.192 30.0 27.9 199.4 1102.104 2.295 4.5 30.2 158.6 1452.076 2.239 1.7 13.4 176.2 1272.049 2.228 26.6 27.7 183.4 122– 2.271 – – 140 162

2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600

[G] [G]

[G]

a b

dc

[G]

2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600

2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600

Fig. 2. EPR X-band spectra of the Cu(II) complexes of L2–L5 in 1:1 ratio at the pH = 7.39 for CuL2 (a), pH = 7.35 for CuL3 (b), pH = 7.40 for CuL4 (c)and pH = 7.40 for CuL5 (d) in 1.0 M NaClO4. The spectra were recorded at 116 K, microwave power of 2.0 mW and modulation amplitude of 1.0 mT.The frequency (m) was of 9.41 GHz. The simulated spectra are below of the experimental ones (grey line).

856 A.S. Fernandes et al. / Journal of Inorganic Biochemistry 101 (2007) 849–858

Fig. 2d presents the spectrum of CuL5 and Table 4 liststhe spectral parameters obtained. The values of gi and Aiare consistent with two nitrogen and two oxygen donoratoms coordinated to the metal center in the equatorialplane. These parameters and also the value of the maxi-mum of the visible band (Table 4) indicate penta- or hexac-oordination environments for the Cu(II). Although we donot have the X-ray structure analysis of this copper(II)complex, we have found in the literature an X-ray structurefor a dimer of the same macrocycle with Ni2+. The crystalstructure of the compound consists of [NiL5Cl]+ and[NiL5(H2O)]2+ complex cations. The coordination envi-ronment for each metal is octahedral, determined by threenitrogen and two oxygen atoms of the ligand and com-pleted by secondary species from the medium (Cl� andwater, respectively) [55]. It is important to mention thatin this structure one nitrogen atom is in an axial position.Considering this X-ray structure and taking into accountour results data, we may propose that in the case ofCuL5, the same octahedral environment seems probable.

The SOD-like activity depends on structural features,namely on the conformation of the active site. Too stablesquare-planar geometries may not be favourable for theO��2 dismutation [10], while more distorted arrangementslead to higher SOD activity [56]. The quotient gi/Ai is an

empirical measure for the degree of tetrahedral distortion.This quotient ranges from �105 to 135 cm for square-pla-nar structures and increases markedly on the introductionof tetrahedral distortion [57]. In terms of the factor gi/Ai,CuL4-1 appears to have some tetrahedral distortion ofthe copper(II) arrangement, being the complex structurallymost similar to CuZnSOD [58] (Table 4).

The redox behaviour of CuL2–CuL5 in aqueous solu-tions was studied by cyclic voltammetry. All of the com-plexes exhibited irreversible waves, except CuL3. Thismay suggest that the SOD-like activity exhibited by thecomplexes CuL2 and CuL4 is due to a stequiometric scav-enging activity. The copper(II) complex of L3 displays asingle quasi-reversible one-electron (ipa/ipc = 1.3;Epa = �0.528 V; Epc = �0.617 V; DEp = 89 mV) reductionwave at �0.573 V versus Ag/AgCl, which is assigned toCu(II)/Cu(I). This may suggest a catalytic behaviour.However, the value of E1/2 for CuL3 is more negative thanthose reported for SOD mimetic compounds (from �0.240to �0.504 V). Also, the value of DEp is lower than thosereported for SOD mimetic compounds (from 114 to121 mV) [59], which might explain the mild activity exhib-ited by this complex on the biochemical assays.

While some of these compounds have shown to be effec-tive scavengers of the O��2 , they may also be toxic to mam-

0

20

40

60

80

100

120

CuL1 CuL2 CuL3 CuL4 CuL5 Cu(II)

% S

urvi

val

1μΜ 10 μΜ 50 μΜ 100 μΜ

Fig. 3. Cell survival of V79 Chinese hamster cells treated with different concentrations of the macrocyclic copper(II) complexes CuL1–CuL5 and Cu(II).The survival values are percentages relative to non-treated control cells. Each experimental point is an average value ± SD of two independentexperiments. Four replicate cultures were used for each complex concentration in each independent experiment.

A.S. Fernandes et al. / Journal of Inorganic Biochemistry 101 (2007) 849–858 857

malian cells. In fact, cytotoxicity is a common limitationfor the pharmaceutical use of new compounds. Therefore,we measured the cytotoxic effects of macrocyclic copper(II)complexes using the MTT test in V79 cells. Cell survivalwas evaluated using a standard 24 h incubation with thecopper(II) complexes. The concentrations evaluated werein the range of those tested in the superoxide scavengingassays. In general, the macrocyclic complexes showed onlya slight cytotoxic effect of approximately 5–15% and nodose–response effect was observed. Interestingly, CuL5,the complex that did not show a superoxide scavengingactivity, revealed the highest cytotoxic profile (Fig. 3).

Despite of the potential cytotoxicity of Cu(II), due to itsability to generate hydroxyl radical and to displace othermetal co-factors from metalloenzymes [37], in our experi-ments Cu(II) ions did not exhibit notable cytotoxic effects,in the range of concentrations studied (Fig. 3).

4. Conclusion

We have described four copper(II) complexes of macro-cyclic ligands which have the ability to effectively scavengethe O��2 . CuL1 is the most active one, but at physiologicalpH the complexation with copper(II) is not complete. Thisfact, along with the low stability constant value of CuL1and pCu, precludes its interest as a SOD mimic.

The non-scavenging activity of CuL5 may be justified bythe presence of an N atom of the macrocyclic backbone atthe axial position. This fact causes steric hindrance to theapproach of the O��2 .

CuL3 presents the lowest scavenging activity. The rea-son for this behavior can be related to the strong equatorialligand field which does not favour the attack of O��2 to theaccessible apical sites. Nevertheless, this complex also pre-sented low cytotoxicity in mammalian cells and high stabil-ity constant and pCu values.

CuL4 and CuL2 show the highest scavenging activitywhich can be correlated to the existence of an equatorialfield of medium strength for both complexes. Another rea-son may be due to the presence of N-methylcarboxylatesubstituents that provide a flexible arrangement of thedonor atoms around the copper(II) ion. EPR studiesrevealed the structural similarity of the copper(II) environ-ment in CuL4-1 with the Cu(II) centre of CuZnSOD.Despite of the irreversible behaviour shown by thiscomplex in the electrochemical studies, CuL4 gathers anumber of favourable characteristics. In fact, this com-plex has one of the highest stability constant and pCuvalues, being therefore less prone to undergo dissociationin vivo, an important requisite for a bioactive metal com-plex. In addition, CuL4 has a quite low IC50 and exhibitedlow cytotoxicity in the range of doses tested (up to100 lM).

This work underlines some important features for theresearch on macrocyclic copper(II) complexes as SODmimetics, and supports future evaluation of some of thesecompounds in cell-based and/or in animal experimentalprotocols.

5. Abbreviations

ROS reactive oxygen speciesSOD superoxide dismutaseNBT nitro blue tetrazoliumDHE dihydroethidiumMTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tet-

razolium bromidePBS phosphate saline bufferX–XO xanthine–xanthine oxidaseNMR nuclear magnetic resonanceEPR electronic paramagnetic resonance

858 A.S. Fernandes et al. / Journal of Inorganic Biochemistry 101 (2007) 849–858

Acknowledgements

The authors acknowledge the financial support fromFundacao para a Ciencia e Tecnologia (FCT) and POCTI,with co-participation of the European Community fundFEDER (Project POCTI/49114/QUI/2002). The authorsalso thank Rita Delgado and the Instituto de TecnologiaQuımica e Biologica, Universidade Nova de Lisboa, Portu-gal, for the spectroscopic and cyclic voltammetry experi-ments.

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