Metalloenzymes superoxide dismutases (SODs) are key enzymes in the metabolism of oxygen free radicals whose primary activity is the dismutation of superoxide radical anion into hydrogen peroxide and molecular oxygen. The iron-dependent superoxide dismutases

enzymes in the metabolism of oxygen free radicals whose primary activity is the dismutation of superoxide radical anion into hydrogen peroxide and molecular oxygen. The iron-dependent superoxide dismutases (Fe-SODs), structurally distinct from the Cu/Zn family of SODs and, to a lesser extent, from the manganese family of SODs (MnSODs), constitute in a number of parasitic protozoan organismsthe primary enzymatic defence against damage caused by the superoxide anion radical O2· , and its derivatives such as peroxynitrite. As a consequence, it has been proposed that the development of specific inhibitors of parasitic iron superoxide dismutases, which are different from their host’s Cu/Zn or Mn-dependent enzymes, may lead to the perturbation of the antioxidant defence mechanism, and thus could contribute to the development of new drugs with anti-parasitic properties. The bis-catechol derivative N,N-bis(dihydroxybenzoyl)-1,6-diaminohexane 8 (Chart 1) has been shown to be a selective and irreversible inhibitor of the iron-SOD from Crithidia fascicula, a non-parasitic trypanosomatid related to trypanosomes. The authors showed that inhibition was caused by a mechanism other than chelation, and hypothesized that this bis-catechol was oxidized to a quinone before forming a covalent bond with the protein. In addition to any inhibitory effects, there is an interest in studying the reactivity of catechol derivatives towards biological molecules such as proteins, whose structural modifications are often associated in the development of many diseases and in aging. Indeed, a number of drugs can undergo oxido-reductive transformations in cells, such as O-demethylation reactions catalyzed by cytochrome P450-dependent mono-oxygenases, or oneor two-electron oxidation mediated by peroxidases and tyrosinases, leading to the formation of catechol and/or ortho-quinone moieties as major metabolites. In the present work, as part of a program to develop novel iron-superoxide dismutase inhibitors, we synthesised and evaluated a series of catechol derivatives. The influence of added SOD on the rate of auto-oxidation reaction of these compounds to their corresponding ortho-quinones was also investigated. Results Synthesis of Catechol Derivatives (Chart 1) Commercially available 2,3-dihydroxybenzoic acid 1 was used as starting material for the synthesis of all catechol derivatives 2—8. Catechol 2 was prepared from 1 and [(tert-butyloxycarbonyl)amino]ethylamine with 1,3-dicyclohexylcarbodiimide (DCC) as coupling reagent. Removal of the butyloxy carbonyl protecting group (Boc) from 2 under acidic conditions using trifluoroacetic acid gave hydrochloride compound 3. Compounds 4 and 6 were obtained from 2,3-dihydroxybenzoic acid using the DCC method in the presence of glycine ethyl ester or N-hydroxysuccinimide, respectively. Treatment of 4 in aqueous sodium hydroxide and subsequent acidification gave compound 5, and reaction of 6 with 1,2-diaminoethane and 1,6-diaminohexane yielded the bis-catechol derivatives 7 and 8, respectively. All compounds were then fully characterised by mass spectrometry, H-, C-NMR and IR spectroscopies. Oxidation Kinetic Measurements The oxidation of catechols to their corresponding ortho-quinone derivatives was determined by UV-visible spectroscopy by monitoring the increase in absorbance at l5280 nm. As shown in Fig. 1A, autoxidation of compound 5 at 400 mM occurred through a very slow process in a 50 mM Tris buffer (pH 8.2) at 25 °C, with a first order constant value of 0.079 h (t1/259 h). The addition of either Escherichia coli Fe-SOD or bovine erythrocytes Cu/Zn-SOD had no significant effect on the oxidation rate with half-life time values of about 11 and 10 h, respectively (Fig. 1A). A similar effect was observed on all other compounds (data not shown), thus providing proof that this class of enzyme neither catalyses nor inhibits the conversion of catechol compounds to their corresponding quinones. In these experiments, it should be noted that catechols (400 mM) were in large excess compared to enzyme amount (0.3 mM), explaining why an inhibition of auto-oxidation rate, which could be due to substrate consumption by enzyme through covalent bonding, was not observed. In comparison, when tyrosinase, known to catalyse the production of ortho-quinones from phenol and catechol compounds, was added, oxidation of compound 5 occurred much more rapidly (Fig. 1B). Inhibition Studies The activities of catechol-incubated 578 Chem. Pharm. Bull. 50(5) 578—582 (2002) Vol. 50, No. 5