Metalofármacos como doadores de óxido nítrico (NO•) e/ou nitroxil (HNO): desenvolvimento de novos agentes e investigação das atividades anticâncer, anti-hipertensivas e antituberculose

Abstract

Cancer, cardiovascular diseases and tuberculosis (TB) are major therapeutic challenges, for which various drug strategies have been developed, including the use of inorganic agents such as metallo-drugs and NO (nitric oxide)/HNO (nitroxyl). Within this prospect, the thesis objective addresses the design, synthesis, physical-chemical studies and biological evaluation of metal-based drugs capable of releasing NO and/or HNO with pharmacological effects. Two types of organic platforms are thus envisaged as ligands in FeII/III complexes devised as potential sources of NO/HNO: the spectator cyclam ligand (1,4,8,11-tetraazacyclotetradecane) and three reactive azine hydroxamic acid ligands (ArC(O)NHOH). The first chapter deals with the chemical reactivity of a trans-[Fe(cyclam)(NO)Cl]Cl2 complex, which is shown to decompose by releasing NO• under physiological pH and temperature conditions, the process being significantly accelerated upon light irradiation at 365 nm. In contrast, in vitro experiments in the presence of glutathione revealed that the same complex produces HNO. Angiogenesis studies showed that the complex is able to significantly inhibit the hypoxia-inducible factor (HIF-1α) induced by hypoxia or addition of a NO• donor (Spermine NONOate) in breast cancer cells: this result is consistent with a release of HNO by the nitrosyl complex. On the other hand, vasodilation assays using precontracted rat aortic rings revealed that the complex exhibits a relaxation IC50 of 910 nM, vs 24 nM for the reference drug, nitroprusside. The next chapters deal with the oxidation-promoted release of HNO from aromatic hydroxamic acids. Chapter 2 focuses on the oxidation mechanism of isonicotinoic, nicotinoic and pyrazinoic hydroxamic acids, mediated by potassium ferricyanide (K3[FeIII(CN)6]) in physiological pH conditions. It is shown that oxidative activation of aryl hydroxamic acids, mediated by FeIII, involves the production of the N,O-di(di)azinoylhydroxylamine intermediate with concomitant release of HNO, instead of the putative acyl nitroso species. The free hydroxamic acids were also evaluated for different biological activities and used as controls to be compared with iron complexes described in chapter 3. These pentacyanoferrate(II) complexes, Na3FeII(CN)5(ArCONHOH), initially devised as potential hybrid of isoniazid (INH, first-line anti-TB prodrug, for which the isonicotinoyl radical is the active metabolite) and delamanid (third-line anti-TB, for which HNO is the active metabolite), were synthesized and characterized by spectroscopic techniques, cyclic voltammetry, and DFT calculations. Using EPR and 1H NMR spectroscopy, oxidation of these FeII complexes with H2O2 was shown to result in the release of HNO and corresponding azinoic acids, through a likely intramolecular electron transfer. In contrast to what was observed from the INH complex (Na3[FeII(CN)5(INH)]), no azinoyl radical species was evidenced to be produced from the corresponding hydroxamic acid complexes. Instead, the corresponding carboxylic acids were found to be formed, in particular pyrazinoic acid which is the active metabolite of pyrazinamide, another antitubercular pro-drug: this result would be relevant for the treatment of pyrazinamide-resistant tuberculosis, as the activation could thus occur without assistance of the pyrazinamidase enzyme of Mycobacterium tuberculosis (Mtb). The free hydroxamic acids and corresponding complexes did not show any antibiotic action against an actively growing/non-resistant Mtb strain. However, the complexes showed a strong dose-dependent and reversible vasodilation activity, close to that of nitroprusside (64-250 nM vs 13 nM). The free hydroxamic acids were found ca. 19- to 76-fold less active than the corresponding complexes. On the basis of in vivo experiments, Na4[FeII(CN)5(PyCONHO-)] also showed a promising cardiovascular activity profile for the development of a new antihypertensive agent.