Synthesis and evaluation of new heteroaryl nitrones with spin trap properties.

A new series of heteroaryl nitrones were synthesized and evaluated as free radical traps due to the results showed in our previous report. The physicochemical characterization of these new nitrones by electron spin resonance (ESR) demonstrated their high capability to trap and stabilize different atom centered free radicals generated by the Fenton reaction. Additionally, we intensely studied them in terms of their physicochemical properties. Kinetic studies, including the use of a method based on competition and the hydroxyl adduct decay, gave the corresponding rate constants and half-lives at the physiological pH of these newly synthesized nitrones. New nitrones derived from quinoxaline 1,4-dioxide heterocycles were more suitable than DMPO to trap hydroxyl free radicals with a half-life longer than two hours. We explain some of the results using computational chemistry through density functional theory (DFT).

Supramolecular hydrogels of ß-cyclodextrin linked to calcium homopoly-l-guluronate for release of coumarins with trypanocidal activity.

Association constants and thermodynamicsparameters on inclusion of four 3-amido coumarins that present trypanocidal activity, into 6-amino-ß-cyclodextrin (1:1 stoichimetry) were determined. In addition, pure homopolymeric-α-l-guluronate fraction prepared by partial hydrolysis of sodium alginate from Sub-Antarctic Kelp Durvillaea antarctica was conjugated with 6-amino-ß-cyclodextrin (64% yield). To glycoconjugates, 3-amido coumarins were incorporated (73% of encapsulation) and supramolecular hydrogels were prepared by gelation with Ca2+ ions. The trypanocidal activity of the inclusion complexes increased by 10%. Likewise, an increase in diffusion in artificial membrane was observed (13%). It was found that the inclusion complexes increased the variation of the mitochondrial potential of T. cruzi (17%). The lowest release of substituted amidocoumarins (ACS) from supramolecular hydrogels occurred at pH 1.2 whereas the maximum release (34%) was observed at pH 8.0. Encapsulation of lipophilic bioactive compounds in supramolecular hydrogels allows the generation of release systems sensitive to pH with potential application in biomedicine.

Chemical structure and biological properties of sulfated fucan from the sequential extraction of subAntarctic Lessonia sp (Phaeophyceae).

This work is related to the structural characterization of the sulfated polysaccharide from Lessonia sp and the study of its antioxidant and antiparasitic properties. Sequential extraction afforded D-mannitol as the only low MW sugar alcohol. Extraction with 2% CaCl2 afforded in 3.0% yield, a sulfated fucan (SF). Its major fraction (48.5% yield), isolated by ion-exchange chromatography corresponds to a linear polymer of α-l-fucopyranosil residues linked 1→3, sulfated at the O-4 and partially at O-2 positions. By alkaline extraction, sodium alginate (10.3% yield) was obtained. The antioxidant capacity of SF by ESR showed high elimination index (IC50, mg/mL) of hydroxyl (0.27), alkoxy (10.05), and peroxyl (82.88) radicals in relation to commercial mannitol. SF showed activity against the epimastigote form of Trypanosoma cruzi parasite (250 µg/mL) and low cytotoxicity in murine cells (367 µg/mL). The elimination capacity of radicals in aqueous medium of SF would allow its potential biomedical application.

New insights into the antioxidant activity of hydroxycinnamic and hydroxybenzoic systems: spectroscopic, electrochemistry, and cellular studies.

A series hydroxycinnamic and gallic acids and their derivatives were studied with the aim of evaluating their in vitro antioxidant properties both in homogeneous and in cellular systems. It was concluded from the oxygen radical absorbance capacity-fluorescein (ORAC-FL), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and cyclic voltammetry data that some compounds exhibit remarkable antioxidant properties. In general, in homogeneous media (DPPH assay), galloyl-based cinnamic and benzoic systems (compounds 7-11) were the most active, exhibiting the lowest oxidation potentials in both dimethyl sulfoxide (DMSO) and phosphate buffer. Yet, p-coumaric acid and its derivatives (compounds 1-3) disclosed the highest scavenging activity toward peroxyl radicals (ORAC-FL assay). Interesting structure-property- activity relationships between ORAC-FL, or DPPH radical, and redox potentials have been attained, showing that the latter parameter can be a valuable antioxidant measure. It was evidenced that redox potentials are related to the structural features of cinnamic and benzoic systems and that their activities are also dependent on the radical generated in the assay. Electron spin resonance data of the phenoxyl radicals generated both in DMSO and phosphate buffer support the assumption that radical stability is related to the type of phenolic system. Galloyl-based cinnamic and benzoic ester-type systems (compounds 9 and 11) were the most active and effective compounds in cell-based assays (51.13 ± 1.27% and 54.90 ± 3.65%, respectively). In cellular systems, hydroxycinnamic and hydroxybenzoic systems operate based on their intrinsic antioxidant outline and lipophilic properties, so the balance between these two properties is considered of the utmost importance to ensure their performance in the prevention or minimization of the effects due to free radical overproduction.

Electron spin resonance as a powerful tool for studying antioxidants and radicals.

The study of antioxidants and radicals has always been a complex task due to the special characteristics of these species such as reactions at low concentrations and short half-lives. Current techniques do not always produce good results and in some cases they can only be applied in chemical models. From this point of view, the development of electron spin resonance (ESR) has allowed the study of the antioxidant capacity of a wide variety of compounds and the detection of radicals in the reactions in which they are involved. The DPPH technique allows only the study of antioxidants in pure chemical models. The ORAC-ESR assay, based on the spin trapping technique, emerges as an interesting tool for identifying and quantifying the antioxidant capacity of different samples. Furthermore, the spin trapping technique allows us to characterize radicals in in vivo/ex vivo models. The present review discusses the current available techniques associated with ESR for the study of antioxidants and radical species.

ESR, electrochemical and cyclodextrin-inclusion studies of triazolopyridyl pyridyl ketones and dipyridyl ketones derivatives.

The electron spin resonance (ESR) spectra of free radicals obtained by electrolytic reduction of triazolopyridyl pyridyl ketones and dipyridyl ketones derivatives were measured in dimethylsulfoxide (DMSO). The hyperfine patterns indicate that the spin density delocalization is dependent of the rings presented in the molecule. The electrochemistry of these compounds was characterized using cyclic voltammetry, in DMSO as solvent. When one carbonyl is present in the molecule one step in the reduction mechanism was observed while two carbonyl are present two steps were detected. The first wave was assigned to the generation of the correspondent free radical species, and the second wave was assigned to the dianion derivatives. The phase-solubility measurements indicated an interaction between molecules selected and cyclodextrins in water. These inclusion complexes are 1:1 with betaCD, and HP-betaCD. The values of Ks showed a different kind of complexes depending on which rings are included. AM1 and DFT calculations were performed to obtain the optimized geometries, theoretical hyperfine constants, and spin distributions, respectively. The theoretical results are in complete agreement with the experimental ones.

Phenazine 5,10-dioxide derivatives as hypoxic selective cytotoxins: Part II. Structure-activity relationship studies.

The synthesis and evaluation as hypoxic selective cytotoxins of new derivatives of 2-amino or 2-hydroxyphenazine 5,10-dioxide are described. The compounds were developed as structural analogs of other bioreductive compounds and its in vitro cytotoxicities on V79 cells under hypoxic and aerobic conditions were determined. To gain insight into its mechanism of action electrochemical behavior, interaction with DNA experiments and QSAR studies were performed.

Applications of electron spin resonance and spin trapping in tropical parasitic diseases.

Free radicals may be reaction intermediates in biological systems in more situations than are presently recognized. However, progress in detecting such species by Electron Spin Resonance (ESR) has been relatively slow. ESR is a very sensitive technique for free radical detection and characterization. It can be used to investigate very low concentrations of radicals provided that they are stable enough for their presence to be detected. For unstable radicals special techniques have to be employed. One of these methods is called Spin Trapping. Parasitic diseases in tropical and subtropical areas constitute a major health and economic problem. The range of antiparasitic drugs varies widely in structural complexity and action at the subcellular and molecular levels. However, a number of these drugs are thought to exert their action by generating free radicals. Most of the free radical producing drugs used against parasites are: quinones, naphtoquinones, quinone-imines, aminoquinolines, N-oxides and nitroheterocyclic compounds. This review summarizes some of the more relevant achievements of ESR and Spin Trapping applications in parasitic diseases studies. The use of ESR spectroscopy to obtain relevant information about free radical characterization and the analysis of the mechanisms of action of drugs involved in several parasitic diseases is also presented.

Electrochemical and ESR study of 5-nitrofuryl-containing thiosemicarbazones antiprotozoal drugs.

Cyclic voltammetry and electron spin resonance (ESR) techniques were used in the investigation of several potential antiprotozoal thiosemicarbazones nitrofurane derivatives. A self-protonation process involving the protonation of the nitro group due to the presence of an acidic proton in the thiosemicarbazone moiety was observed in the first step of a CEE(rev) reduction mechanism of these derivatives. ESR spectra of the free radicals obtained by electrolytic reduction were characterized and analyzed. AM1 methodology was used to obtain the optimized geometries and UB3LYP calculations were performed to obtain the theoretical hyperfine coupling constants. The theoretical study exhibited an unusual assignment of the spin densities showing a free radical centered in the thiosemicarbazone moiety rather than the nitro which are in agreement with the experimental hyperfine pattern.

ESR and electrochemical studies of 2-acylpyridines and 6,6'-diacyl-2,2'-bipyridines.

The ESR spectra of radicals obtained by electrolytic reduction of 2-acylpyridines and 6,6'-diacyl-2,2'-bipyridines were measured in dimethylsulfoxide (DMSO) and analyzed by quantum chemical calculations. The electrochemistry of these compounds was characterized using cyclic voltammetry, in DMSO solvent. The results showed a two step reduction mechanism, first wave was assigned to the generation of the correspondent free radical species, and the second wave was assigned to the dianion derivatives. AM1 and DFT calculations were performed to obtain the optimized geometries, theoretical hyperfine constants, and spin distributions, respectively. The theoretical results are in complete agreement with the experimental ones.

Reactivity of 1,4-dihydropyridines toward SIN-1-derived peroxynitrite.

PURPOSE: To study the reactivity of C4-substituted 1,4-dihydropyridines (1,4-DHP), with either secondary or tertiary nitrogen in the dihydropyridine ring, toward SIN-1-derived peroxynitrite in aqueous media at pH 7.4. METHODS: Reactivity was followed by changes in the absorptivity of the UV-Vis bands corresponding to 1,4-DHP. Gas Chromatography/ Mass Spectrometer (GC-MS) and Electron Paramagnetic Resonance (EPR) spin trap techniques were used to characterize the final product and the intermediates of the reaction, respectively. RESULTS: 1,4-DHPs significantly reacted toward peroxynitrite at varied rates, according to the calculated kinetic rate constants. By EPR spectroscopy, a carbon-centered radical from the 1,4-DHP was intercepted with N-tert-butylamine-alpha-phenylnitrone (PBN), as the intermediate for the reaction with peroxynitrite. Likewise, the oxidized derivative (i.e., the pyridine) was identified as the final product of the reaction by GC-MS. By using the technique of deuterium kinetic isotope effect, the participation of the hydrogen of the 1-position on the 1,4-DHP ring was shown not to be the rate-limiting step of the reaction. CONCLUSIONS: The direct participation of the 1,4-DHP derivatives in the quenching of SIN-1-derived peroxynitrite has been demonstrated. Kinetic rate constant of tested 1,4-DHP toward peroxynitrite showed a direct relationship with the oxidation peak potential values; that is, compounds reacting faster were more easily oxidized.

Electrochemical and microsomal production of free radicals from 1,2,5-oxadiazole N-oxide as potential antiprotozoal drugs.

The electron spin resonance (ESR) spectra of free radicals obtained by electrolytic or microsomal reduction of several potential antiprotozoal 1,2,5-oxadiazoles were characterized and analyzed. Ab initio molecular orbital calculations were performed to obtain the optimized geometries and the theoretical hyperfine constant was carried out using ZINDO semiempirical methodology. Density functional theory was used to rationalize the reduction potentials of these compounds.

1,2,5-Oxadiazole N-oxide derivatives as potential anti-cancer agents: synthesis and biological evaluation. Part IV.

Several new 1,2,5-oxadiazole N-oxide derivatives and some deoxygenated analogues were synthesized to be tested as potential selective hypoxic cell cytotoxins. Compounds prepared were designed in order to gain insight into the mechanism of action of this kind of cytotoxin. Compounds were tested in oxia and hypoxia and they proved to be non-selective. 3-Cyano-N(2)-oxide-4-phenyl-1,2,5-oxadiazole showed the best cytotoxic activity in oxia. The cytotoxicity observed for these derivatives could be explained in terms of the electronic characteristics of the 1,2,5-oxadiazole substituents. Electrochemical and ESR studies were performed on the more cytotoxic derivative.

Electron spin resonance studies and theoretical quantum calculations of free radicals generated from anthracenetrione by electrochemical and microsomal reduction.

The ESR spectra of radicals obtained by electrolytic reduction of 4,4-dimethylanthracene-1,9,10 (4H)-trione (1) and the regioisomeric quinones 8-acetyloxymethyl-4,4,5-trimethyl- (2), and 5-acetyloxy-methyl-4,4,8-trimethyl-(4H)-1,9,10-anthracenetrione (3) were measured in DMSO and analyzed by quantum chemical calculations. The electrochemistry of these compounds was characterized using cyclic voltammetry, in DMSO and DMF solvents and compared with nifurtimox. The quinones were also reduced by microsomal NADPH-cytochrome P-450 reductase and the corresponding radicals species were also detected by ESR spectroscopy. AMI, INDO, and ADF calculations were performed to obtain the optimized geometries, theoretical hyperfine constants, and spin distributions, respectively. Density functional theory was used to rationalize the reduction potential of these compounds.

Possible role of salsolinol quinone methide in the decrease of RCSN-3 cell survival.

The endogenous dopamine-derived neurotoxin salsolinol was found to decrease survival in the dopaminergic neuronal cell line RCSN-3, derived from adult rat substantia nigra in a concentration-dependent manner (208 microM salsolinol induced a 50% survival decrease). Incubation of RCSN-3 cells with 100 micro;M dicoumarol and salsolinol significantly decreased cell survival by 2.5-fold (P < 0.001), contrasting with a negligible effect on RCHT cells, which exhibited nearly a 5-fold lower nomifensine-insensitive dopamine uptake. The levels of catalase and glutathione peroxidase mRNA were decreased when RCSN-3 cells were treated with 100 microM salsolinol alone or in the presence of 100 microM dicoumarol. In vitro oxidation of salsolinol to o-quinone catalyzed by lactoperoxidase gave the quinone methide and 1,2-dihydro-1-methyl-6,7-isoquinoline diol as final products of salsolinol oxidation as determined by NMR analysis. Evidence of the formation of salsolinol o-semiquinone radical has been provided by ESR studies during one-electron oxidation of salsolinol catalyzed by lactoperoxidase.

Electrochemical, UV--visible and EPR studies on nitrofurantoin: nitro radical anion generation and its interaction with glutathione.

This paper deals with the reactivity of the nitro radical anion electrochemically generated from nitrofurantoin with glutathione. Cyclic voltammetry (CV) and controlled potential electrolysis were used to generate the nitro radical anion in situ and in bulk solution, respectively and cyclic voltammetry, UV--Visible and EPR spectroscopy were used to characterize the electrochemically formed radical and to study its interaction with GSH. By cyclic voltammetry on a hanging mercury drop electrode, the formation of the nitro radical anion was possible in mixed media (0.015M aqueous citrate/DMF, 40/60, pH 9) and in aprotic media. A second order decay of the radicals was determined, with a k2 value of 201 and 111 M(-1) s(-1), respectively. Controlled potential electrolysis generated the radical and its detection by cyclic voltammetry, UV--Visible and EPR spectroscopy was possible. When glutathione (GSH) was added to the solution, an unambiguous decay in the signals corresponding to a nitro radical anion were observed and using a spin trapping technique, a thiyl radical was detected. Electrochemical and spectroscopic data indicated that it is possible to generate the nitro radical anion from nitrofurantoin in solution and that GSH scavenged this reactive species, in contrast with other authors, which previously reported no interaction between them.

Synthesis and antitrypanosomal evaluation of E-isomers of 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives. structure-activity relationships.

Several novel semicarbazone derivatives were prepared from 5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde and semicarbazides bearing a spermidine-mimetic moiety. All derivatives presented the E-configuration, as determined by NMR-NOE experiments. These compounds were tested in vitro as potential antitrypanosomal agents, and some of them, together with the parent compounds, 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives, were also evaluated in vivo using infected mice. Structure-activity relationship studies were carried out using voltammetric response and lipophilic-hydrophilic balance as parameters. Two of the compounds (1 and 3) displayed the highest in vivo activity. A correlation was found between lipophilic-hydrophilic properties and trypanocidal activity, high R(M) values being associated with low in vivo effects.

Singlet oxygen-dependent hydroxyl radical formation during uroporphyrin-mediated photosensitization in the presence of NADPH.

The conversion of singlet oxygen ((1)O2) to hydroxyl radical (*OH) during photosensitization of uroporphyrin (UP) in the presence of NADPH was examined by a spin-trapping technique with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Significant electron spin resonance (ESR) signals of DMPO-OH adduct were observed during irradiation of the UP-NADPH system with visible light. Scavengers of *OH reduced the signal intensity to 3-30% of control, indicating that more than 70% of DMPO-OH results from freely diffusing *OH. The ESR signal was almost completely lost when quenchers of (1)O2 were added, and was enhanced when the amount of deutrated solvent was increased. The appearance of (1)O2, as determined by the oxidation of 2,2,6,6-tetramethyl-4-piperidone (TEMPD), was delayed with an increase in the concentration of NADPH, whereas the production of *OH was upregulated. These observations indicate that conversion of (1)O2 to *OH occurs quickly in the presence of NADPH. Hydrogen peroxide (H2O2) was produced (1)O2-dependently during irradiation of UP in the presence of NADPH. However, neither catalase nor desferrioxamine decreased the DMPO-OH signal, and addition of H2O2 did not increase the signal. SOD increased the signal only slightly. These results suggest that the production of *OH from (1)O2 involves neither superoxide anion radical nor H2O2.

1,2,5-Oxadiazole N-oxide derivatives and related compounds as potential antitrypanosomal drugs: structure-activity relationships.

The syntheses of a new series of derivatives of 1,2,5-oxadiazole N-oxide, benzo[1,2-c]1,2,5-oxadiazole N-oxide, and quinoxaline di-N-oxide are described. In vitro antitrypanosomal activity of these compounds was tested against epimastigote forms of Trypanosoma cruzi. For the most effective drugs, derivatives IIIe and IIIf, the 50% inhibitory dose (ID50) was determined as well as their cytotoxicity against mammalian fibroblasts. Electrochemical studies and ESR spectroscopy show that the highest activities observed are associated with the facile monoelectronation of the N-oxide moiety. Lipophilic-hydrophilic balance of the compounds could also play an important role in their effectiveness as antichagasic drugs.

Scavenging of the one-electron reduction product from nisoldipine with relevant thiols: electrochemical and EPR spectroscopic evidences.

PURPOSE: To determine the formation of the one-electron reduction product from nisoldipine and its reactivity with relevant thiols in mixed medium. METHODS: Cyclic voltammetry (CV) and electron paramagnetic resonance (EPR) techniques were used to determine the one-electron reduction product corresponding to the nitro radical anion. CV was employed to assess both the rate constants corresponding to the decay of the radicals and its interaction with relevant thiols. RESULTS: The nisoldipine radical anion follows second order kinetics, with an association rate constant of 283+/-16 l mol(-1) sec(-1). Nitro radical anion from nisoldipine significantly reacted with thiol compounds. This reactivity was significantly higher than the natural decay of the radical in mixed medium. EPR spectra recorded in situ using DMF/ 0.1 N NaOH (pH 13) confirmed the formation of the nitro radical anion, giving a well-resolved spectra in 35 lines using 0.1 G modulation. CONCLUSIONS: Electrochemical and EPR data indicated that all the tested thiols scavenged the nitro radical anion from nisoldipine. The following tentative order of reactivity towards the thiols can be proposed: cysteamine approximately glutathione > N-acetylcysteine > captopril > penicillamine.