Evaluation of the antioxidant activity of synthetic diarylamines in the benzo[b]thiophene series

Abstract

The damaging consequences of free radicals that result from different kinds of oxidative stress have been implicated in a variety of human disorders, from cardiac ischemia to those affecting the central nervous system. So, there is an increasing interest in the development of antioxidants that can protect cells beyond their natural defences. In this study we investigated the antioxidant properties of two new diarylamines, with pharmacological potential. Both diarylamines have a benzo[b]thiophene nucleus, which is an indole analogous, one bearing a p-methoxyanilino group on the benzene ring (MJQ1) and the other an aminopyridine group and an ester group on the thiophene ring (MJQ2). The approaches used to assess the antioxidant potential of these molecules involved studies at mitochondrial level and studies with whole cells, namely the fibroblast cell line L929 and the neurological model PC12 cell line. The effects of the compounds tested on ADP/Fe2+ induced lipid peroxidation (a model of severe oxidative stress), in isolated rat liver mitochondria, were examined by oxygen consumption and quantification of thiobarbituric acid reactive substances. The results obtained showed that MJQ1 and MJQ2 have a maximal antioxidant activity at the concentrations of 50 nM and 60 μM, respectively (under the same conditions, the IC50 value for Trolox C is 18.8 μM, a well known antioxidant analog of alpha-tocopherol). At twice these concentrations both compounds were effective in the prevention of transmembranar electric potential collapse induced by ADP/Fe2+ on respiring mitochondria, evaluated with the TPP+ electrode. These drugs stabilizing action on mitochondrial inner membrane will preserve the capability of mitochondria to proceed energy linked processes such as oxidative phosphorylation in noxious conditions such as those associated with oxidative stress. We also demonstrated that until the concentration showing maximal antioxidant effects none of the compounds showed to be toxic as evaluated by the fluctuations on membrane potential associated with respiration and phosphorylation cycle; moreover, the respiratory control and the ADP/oxygen ratio were not depressed by the compounds. The diarylamines were screened for toxicity with whole cells (L929 cell line), using the MTT reducing test as indicator of cell viability. None of them revealed cytotoxicity for the concentrations at which they showed maximal antioxidant effect on mitochondria (50nM and 60 μM). At the same concentrations the compounds also revealed a decrease of lipid peroxidation, induced by the pair ascorbate/Fe2+ in the PC12 cell model, to the basal levels. The protector effect of these diarylamines is not due to the prevention of Fenton reaction by iron chelating because there is no difference between the compounds absorption spectra in the presence and absence of iron. In conclusion, this study demonstrated that these new diarilamines have antioxidative effects for nontoxic concentrations, at mitochondria and whole cell levels, which makes them molecules with therapeutic interest.