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Inorganic Chemistry Communications 2 (1999) 358–360

Interpretation of the sod-like activity of a series of copper(II) complexeswith thiosemicarbazones

Alicia Dıaz, Roberto Cao *, Alex Fragoso, Ileana Sanchez´ ´Laboratory of Bioinorganic Chemistry, Faculty of Chemistry, Havana University, Havana 10400, Cuba

Received 11 March 1999

Abstract

In the present paper we report the SOD-like activity of a series of copper(II) complexes with acetaldehyde, acetophenone, 2-oxo-glutaricacid, methyl-furfural, piruvic acid, salicylaldehyde, 2-acetylpyridine thiosemicarbazones and ribose bis(thiosemicarbazone). The complexeswere studied by EPR spectroscopy. A linear correlation between fsg≤/A≤ and the SOD-like activity was observed. The higher f valuescorrespond to copper(II) complexes with enhanced tetrahedral distortion. q 1999 Elsevier Science S.A. All rights reserved.

Keywords: Copper(II) complexes; Thiosemicarbazone; SOD-like activity; EPR

1. Introduction

Biological properties of thiosemicarbazones have beenstudied since 1956, when Brockman et al. [1] reported theantitumoral properties of 2-formylpyridine thiosemicarba-zone. A series of a-(N)-heterocyclic carboxaldehyde thio-semicarbazones was later studied [2] for their anticanceractivity and, since then, this and other biological propertiesof thiosemicarbazones have been reported [3]. Earlier thisdecade, reports of some copper(II) thiosemicarbazone com-plexes able to dismutate the superoxide radical anion beganto appear [4–6].

The intracellular enzyme superoxide dismutase(EC.1.15.1.1) contains copper(II) at the active center andzinc(II) bound to it through an imidazolate bridge. Thisenzyme, represented as Cu,ZnSOD, dismutates the super-oxide radical according to the following reaction

xy q2O q2H ™O qH O2 2 2 2

The high catalytic activity of Cu,Zn-SOD is diffusion con-trolled and pH independent [7,8]. A key to this behavior isthe imidazolate bridge formed between Cu(II) and Zn(II)by His-61. Reduction of Cu(II), at the active site, in the cyclicreaction has been shown to occur with the uptake of oneproton per subunit [9]. This species should protonate thebridging imidazolate (His-61), once the Cu–Im bond is bro-ken, as follows

* Corresponding author. Tel.: q53-7-792 145; Fax: q53-7-333502/335774; E-mail: cao@fq.oc.uh.cu

xy q(His) Cu(II)-Im-Zn(II)(His) AspqO qH3 2 2

™(His) Cu(I)qImH-Zn(II)(His) AspqO3 2 2

The deprotonation of coordinated ImH takes place whenCu(I) is reoxidized and then hydroperoxide anion is formed

xy(His) Cu(I)qImH-Zn(II)(His) AspqO3 2 2

y™(His) Cu(II)-Im-Zn(II)(His) AspqHO3 2 2

In this manner, the imidazolate bridge plays an importantrole in supplying protons to effect the overall reaction.

A protonated residue Arg-141, held about 6 A from theactive site plays the important role of a proton source as wellas meeting other important requirements for functional activ-ity. Its positive charge electrostatically attracts the substratetoward the active site, and once O2

xy is coordinated toCu(II),it stabilizes it through H-bond formation. In this bindingmotif, Cu(II) acquires a distorted square pyramidal structure.If the enzyme is not interacting with the substrate, a watermolecule can occupy the nascent copper(II) superoxidecoor-dination site [8].

Other features that play important roles in Cu,Zn-SODactivity and are related to the redox process of copper includethe geometric distortion of the Cu(II) coordination sphereand/or ligand flexibility and ‘softness’ of the donor atoms.These features have already been pointed out by Addison andco-workers [10–12] as influencing the redox properties ofcopper complexes. For example, a twist of 608 in the chelatingring of pyrrole-2-carboxaldehyde has been observed to raise

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Wednesday Jul 21 11:42 AM StyleTag -- Journal: INOCHE (Inorganic Chemistry Communications) Article: 231

Fig. 1. Geometric rearrangements in Cu,ZnSOD during superoxide radicaldismutation.

Table 1EPR parameters and SOD-like activities of the copper(II) thiosemicarba-zones complexes

Ligand pIC50 A≤

a Aoa g≤ go f (cm) b

AlTSC c 6.7 139 51 2.360 2.173 170ATSC 5.0 144 67 2.147 2.070 149MFuTSC 6.0 146 65 2.151 2.075 147PyTSC 5.2 158 78 2.180 2.106 138PTSC c 4.6 169 72 2.191 2.090 130GTSC 4.8 173 70 2.184 2.092 126SaTSC 4.3 193 74 2.190 2.106 113RibTSC c 4.0 188 82 2.125 2.063 113

a 104 (cmy1).b fsg≤/A≤.c Ref. [23].

the reduction potential of the Cu(II) Schiff base complex byapproximately 200 mV. For this series of complexes, anincrease in g≤ accompanies the increase in E1/2 [13].

When Cu(II), with a distorted square planar geometry inCu,Zn-SOD, is reduced to Cu(I), rearrangement to a tetra-hedral geometry takes place [14]. This is schematically rep-resented in Fig. 1. Therefore, a more favorable redox processshould be accommodated by copper(II) mimetic complexesfeaturing highly distorted square planar geometries and/orflexible ligands. Although a correlation between the distor-tion of the square planar geometry of Cu(II) in different type2 copper enzymes and their reduction potentials has beenobserved [13], this feature has not been sufficiently consid-ered when designing Cu(II) complexes as SOD mimetics.

The goal of the present paper is to obtain a correlationbetween the geometric distortion of square planar copper(II)complexes and their SOD-like activity. For this study, a seriesof copper(II) thiosemicarbazone complexes was selectedbecause, for these compounds, the other features affectingSOD-like properties are greatly attenuated.

2. Experimental

Acetaldehyde (AlTSC), acetophenone (ATSC), 2-oxo-glutaric acid (GTSC), methyl-furfural (MFuTSC), piruvicacid (PTSC), salicylaldehyde (SaTSC) and 2-acetylpyri-dine (PyTSC) thiosemicarbazones were prepared by the con-densation reaction between the corresponding aldehyde orketone and thiosemicarbazide. The synthesis of ribosebis(thiosemicarbazone) is reported in Ref. [15]. The syn-thesis of the copper complexes is described in Refs.[6,15,16].

2.1. EPR studies

The EPR spectra were obtained with Bruker 200 D SRCand Bruker ESP 300 X-band spectrometers, in 50% DMSOat room temperature and 130 K. The frequencies were meas-ured with XL Microwave model 3120 and Hewlett-Packard5352B frequency counters, respectively.

2.2. SOD-like activity

The method used to study the SOD-like activity is similarto that described by Fridovich and co-workers [17,18], withthe same specifications as reported elsewhere [19], usingnitroblue tetrazolium chloride (NBT) as indicator, and thexanthine–xanthine oxidase system as the superoxide radicalgenerator (in a 10 mM pH 7.8 phosphate buffer solution).NBT reduction by O2

xy was spectrophotometrically moni-tored at 560 nm. The IC50 values were determined by regres-sion analysis and interpolation of the % inhibition versusassay concentration curve for at least five experimental pointsfor each system, with inhibition values ranging from 10 to70%. In all cases a linearity greater than 0.960 was achieved.

3. Results and discussion

In the present paper we selected a series of aliphatic andaromatic thiosemicarbazone copper(II) complexes with sim-ilar electronic properties. Ligands with NS (AlTSC, ATSC,MFuTSC), ONS (GTSC, PTSC, SaTSC), NNS (PyTSC)and NSSN (RibTSC) donor sets coordinated to copper(II)were selected.

The SOD-like activity is expressed in terms of IC50 values(in mM), which corresponds to the concentration necessaryto dismutate 50% of superoxide radical generated by thexanthine–xanthine oxidase system.

In order to quantify the degree of distortion of the cop-per(II) complexes, we selected the f factor (g≤/A≤) obtainedfrom the EPR spectra of frozen solutions. Although the ffactor, which is considered an empirical index of tetrahedraldistortion [10], has been related to redox properties of cop-per(II) complexes [10,12], it has only been qualitativelyconsidered in a few reports on SOD mimetics [20–22].

A tendency for g≤ to increase as A≤ decreases was observedfrom the EPR spectra of all the complexes. Ao and go vary ina similar way, as do A≤ and g≤ (Table 1). The f factor assumesvalues smaller than 135 cm for square planar copper(II)complexes, and increases with increasing tetrahedral distor-tion. The f value of Cu,ZnSOD is equal to 160 cm, indicatinga tetrahedral distortion from square planar geometry, as evi-denced by crystal structure determinations [14]. This con-siderable distortion of the geometry of copper(II) inCu,ZnSOD is one of the features that enhances the catalytic

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Fig. 2. Correlation between the pIC50 values and the empirical ratio fsg≤/A≤.

activity of this enzyme. Therefore, it ought to be expectedthat copper(II) mimetic complexes exhibiting appreciablesquare planar distortion should also present high SOD-likeactivity. That is the interpretation we give to the correlationfound between SOD-like activity and the values of f factorrepresented in Fig. 2.

Consider ribose thiosemicarbazonate copper(II), whichexhibits the smallest SOD-like activity of all the complexesstudied by us. The NSSN ligand of RibTSC is tetradentate,exhibiting the most rigid and least distorted square planarenvironment about copper(II). On the contrary, the com-plexes with NS coordinated thiosemicarbazones present thehighest SOD-like activity. Tetrahedral distortion in thesecomplexes is largest since these ligands are the most flexible,especially AlTSC which is aliphatic. Note that the obtainedcorrelation holds whether the ligand is aromatic or not, sug-gesting that the electronic factors of the ligand do not play animportant role in the observed correlation.

To the best of our knowledge, this is the first paper report-ing a good correlation between f factor and SOD-like activityof copper(II) complexes. We are currently analyzing similartrends in other Cu(II) complexes with cyclodextrinderivatives.

Acknowledgements

The authors are very grateful to Prof. R. Basosi (Universitadi Siena, Italy) and Prof. T. Rojo (Universidad del Paıs´Vasco, Spain) for the utilization of their EPR spectrometers.

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