The biological effects of peroxynitrite have been recently considered to be largely dependent on its reaction with carbon dioxide, which is present in high concentrations in intra- and extracellular compartments. Peroxynitrite anion (ONOO⁻) reacts rapidly with carbon dioxide, forming an adduct, nitrosoperoxocarboxylate (ONOOCO₂ ⁻), whose decomposition has been proposed to produce reactive intermediates such as the carbonate radical (CO·̄₃). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6–9. The radical was unambiguously identified by its EPR parameters (g = 2.0113; line width = 5.5 G) and by experiments with bicarbonate labeled with ¹³C. In this case, the singlet EPR signal obtained with ¹²C bicarbonate splits into the expected doublet because of ¹³C (a(¹³C)= 11.7 G). The singlet spectrum of the unlabeled radical was invariant between pH 6 and 9, confirming that in this pH range the detected radical is the carbonate radical anion (CO·̄₃). Importantly, in addition to contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct EPR spectroscopy.

In this review we compare and discuss results of compounds already reported as anticancer agents based on isatin-derivatives, metalated as well as non-metallated. Isatin compounds can be obtained from plants, marine animals, and is also found in human fluids as a metabolite of amino acids. Its derivatives include imines, hydrazones, thiosemicarbazones, among others, already focused on numerous anticancer studies. Some of them have entered in pre-clinical and clinical tests as antiangiogenic compounds or inhibitors of crucial proteins. As free ligands or coordinated to metal ions, such isatin derivatives showed promising antiproliferative properties against different cancer cells, targeting different biomolecules or organelles. Binding to metal ions usually improves its biological properties, indicating a modulation by the metal and by the ligand in a synergistic process. They also reveal diverse mechanisms of action, being able of binding DNA, generating reactive species that cause oxidative damage, and inhibiting selected proteins. Strategies used to improve the efficiency and selectivity of these compounds comprise structural modification of the ligands, metalation with different ions, syntheses of mononuclear and dinuclear species, and use of inserted or anchored compounds in selected drug delivery systems.

We characterized the pro-apoptotic activity of two new synthesized isatin-Schiff base copper(II) complexes, obtained from isatin and 1,3-diaminopropane or 2-(2-aminoethyl)pyridine: (Cu(isapn)) and (Cu(isaepy)(2)), respectively. We demonstrated that these compounds trigger apoptosis via the mitochondrial pathway. The early induction of the p53/p21 system indicates a role for p53 in cell death, however, experiments carried out with small interfering RNA against p53, or with cells lacking p53, support that a p53-independent mechanism can also occur. The extent of apoptosis mirrors the kinetics of intracellular copper uptake. Particularly, Cu(isaepy)(2) enters the cells more efficiently and specifically damages nuclei and mitochondria, as evidenced by atomic absorption analysis of copper content and by the extent of nuclear and mitochondrial integrity. Conversely, Cu(isapn), although less permeable, induces a wide-spread oxidative stress, as demonstrated by analyses of reactive oxygen species concentration, and oxidation of proteins and lipids. The increase of the antioxidant defense, through the overexpression of Cu,Zn-SOD, partially counteracts cell death; whereas retinoic acid-mediated differentiation completely rescues cells from apoptosis induced by both compounds. The activation of JNK- and Akt-mediated phosphorylative pathways has been found to be not functional for apoptosis induction. On the contrary, apoptosis significantly decreased when the analogous zinc complex was used or when Cu(isaepy)(2) was incubated in the presence of a copper chelator. Altogether, our data provide evidence for a dual role of these copper(II) complexes: they are able to vehicle copper into the cell, thus producing reactive oxygen species, and could behave as delocalized lipophilic cation-like molecules, thus specifically targeting organelles.