Nitro Radical Research Articles

The effect of 5-substitution on the electrochemical behavior and antitubercular activity of PA-824

Nitroimidazole PA-824 is part of an exciting new class of compounds currently undergoing clinical evaluation as novel TB therapeutics. The recently elucidated mechanism of action of PA-824 involves reduction of the nitroimidazole ring and subsequent nitric oxide release. The importance of this compound and its unique activity prompted us to explore how substitution of the nitroimidazole ring would affect electrochemical reduction and antitubercular activity. We prepared analogs of PA-824 with bromo, chloro, cyano, and amino substituents in the 5-position of the aromatic ring. We found that substitution of the imidazole ring greatly influences reduction and the stability of the corresponding nitro radical anion. Further, the antitubercular activities of the bromo and chloro analogs may indicate that an alternate nitroreductase pathway within Mycobacterium tuberculosis exists.

Reduction of 1-(2-Chloroethyl)-2-nitrobenzene and 1-(2-Bromoethyl)-2-nitrobenzene at Carbon Cathodes: Electrosynthetic Routes to 1-Nitro-2-vinylbenzene and 1H-Indole

Studies of the electrochemical reductions of 1-(2-chloroethyl)-2-nitrobenzene (1) and 1-(2-bromoethyl)-2-nitrobenzene (2) at carbon cathodes in dimethylformamide (DMF) containing tetramethylammonium tetrafluoroborate have been undertaken. Cyclic voltammograms for 1 and 2 exhibit three irreversible cathodic peaks, the first of which is attributed to one-electron reduction of the nitro group, and the resulting nitro radical–anion immediately reacts as a base with the adjacent alkyl halide moiety via an E2 elimination to produce 1-nitro-2-vinylbenzene. At a potential corresponding to the first cathodic peak, bulk electrolyses of 1 or 2 in the absence of a proton donor afford 1-nitro-2-vinylbenzene as principal product, along with -indole and a dimeric species. However, when 1 or 2 is electrolyzed in the presence of either phenol or 2,4-pentanedione as a proton source, the only significant product is -indole. A mechanistic picture for the reductions of 1 and 2 is proposed, and a separate examination of the electrochemical behavior of 1-nitro-2-vinylbenzene has been included as part of this work.

Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.

Regioselective 3-Nitration of Flavones: A New Synthesis of 3-Nitro- and 3-Aminoflavones

A new, general, and regioselective method for the 3-nitrationof flavones have been developed. The nitration reaction is solventdependent, proceeds via a nitro radical pathway, and the corresponding3-nitroflavones have been obtained in moderate to very good yields(up to 81%). The reduction of 3-nitroflavones allowed thepreparation of the corresponding 3-aminoflavones in very good yields(up to 96%).

Mitochondrial Superoxide Dismutase and Glutathione Peroxidase in Idiosyncratic Drug-Induced Liver Injury

Drug-induced liver injury (DILI) susceptibility has a potential genetic basis. We have evaluated possible associations between the risk of developing DILI and common genetic variants of the manganese superoxide dismutase (SOD2 Val16Ala) and glutathione peroxidase (GPX1 Pro200Leu) genes, which are involved in mitochondrial oxidative stress management. Genomic DNA from 185 DILI patients assessed by the Council for International Organizations of Medical Science scale and 270 sex- and age-matched controls were analyzed. The SOD2 and GPX1 genotyping was performed using polymerase chain reaction restriction fragment length polymorphism and TaqMan probed quantitative polymerase chain reaction, respectively. The statistical power to detect the effect of variant alleles with the observed odds ratio (OR) was 98.2% and 99.7% for bilateral association of SOD2 and GPX1, respectively. The SOD2 Ala/Ala genotype was associated with cholestatic/mixed damage (OR = 2.3; 95% confidence interval [CI] = 1.4-3.8; corrected P [Pc] = 0.0058), whereas the GPX1 Leu/Leu genotype was associated with cholestatic injury (OR = 5.1; 95%CI = 1.6-16.0; Pc = 0.0112). The presence of two or more combined risk alleles (SOD2 Ala and GPX1 Leu) was more frequent in DILI patients (OR = 2.1; 95%CI = 1.4-3.0; Pc = 0.0006). Patients with cholestatic/mixed injury induced by mitochondria hazardous drugs were more prone to have the SOD2 Ala/Ala genotype (OR = 3.6; 95%CI = 1.4-9.3; Pc = 0.02). This genotype was also more frequent in cholestatic/mixed DILI induced by pharmaceuticals producing quinone-like or epoxide metabolites (OR = 3.0; 95%CI = 1.7-5.5; Pc = 0.0008) and S-oxides, diazines, nitroanion radicals, or iminium ions (OR = 16.0; 95%CI = 1.8-146.1; Pc = 0.009). Patients homozygous for the SOD2 Ala allele and the GPX1 Leu allele are at higher risk of developing cholestatic DILI. SOD2 Ala homozygotes may be more prone to suffer DILI from drugs that are mitochondria hazardous or produce reactive intermediates.

Voltammetric reduction of 4-nitroimidazole derivatives: Influence of the N-1 substitution in protic and aprotic media

The voltammetric reduction of 1-methyl- and 1-H- 4-nitroimidazole derivatives was studied in different protic and aprotic media to investigate the influence of the N-1 substitution in the mechanism of reduction, the susceptibility of the nitro group to reduction, and the stability of the nitro radical anion. The elucidation of their voltammetric behavior was carried out using differential pulse polarography and cyclic voltammetry with two different mixed media (Britton-Robinson/ethanol: 70/30 and DMF/citrate: 60/40) and an aprotic media (DMF) at the mercury electrode. In addition, we used UV–vis spectroscopy for the study of their chemistry in solution and quantum-chemical calculations to evaluate LUMO energies, HOMO and LUMO energy gaps, dipole moments and electron affinity, using water and DMF as solvents. The mechanism of reduction was strongly dependent on both the substitution at the N-1 position and the nature of the media. In all media, the methyl-substituted derivative (M-4-NImOH) was always more easily reduced than the demethylated species (H-4-NImOH). On the other hand, the nitro radical anion from M-4-NImOH was more stable than the nitro radical anion from H-4-NImOH.

Mode of action of Nifurtimox and N-oxide-containing heterocycles against Trypanosoma cruzi: Is oxidative stress involved?

Chagas disease is caused by the trypanosomatid parasite Trypanosoma cruzi and threatens millions of lives in South America. As other neglected diseases there is almost no research and development effort by the pharmaceutical industry and the treatment relies on two drugs, Nifurtimox and Benznidazole, discovered empirically more than three decades ago. Nifurtimox, a nitrofurane derivative, is believed to exert its biological activity through the bioreduction of the nitro-group to a nitro-anion radical which undergoes redox-cycling with molecular oxygen. This hypothesis is generally accepted, although arguments against it have been presented. In the present work we studied the ability of Nifurtimox and five N-oxide-containing heterocycles to induce oxidative stress in T. cruzi. N-Oxide-containing heterocycles represent a promising group of new trypanosomicidal agents and their mode of action is not completely elucidated. The results here obtained argue against the oxidative stress hypothesis almost for all the studied compounds, including Nifurtimox. A significant reduction in the level of parasitic low-molecular-weight thiols was observed after Nifurtimox treatment; however, it was not linked to the production of reactive oxidant species. Besides, redox-cycling is only observed at high Nifurtimox concentrations (>400 μM), two orders of magnitude higher than the concentration required for anti-proliferative activity (5 μM). Our results indicate that an increase in oxidative stress is not the main mechanism of action of Nifurtimox. Among the studied N-oxide-containing heterocycles, benzofuroxan derivatives strongly inhibited parasite dehydrogenase activity and affected mitochondrial membrane potential. The indazole derivative raised intracellular oxidants production, but it was the least effective as anti- T. cruzi.

Peroxynitrite and nitrosoperoxycarbonate, a tightly connected oxidizing-nitrating couple in the reactive nitrogen-oxygen species family: new perspectives for protection from radical-promoted injury by flavonoids

Peroxynitrite is the product of the reaction of nitric oxide with superoxide radical and is implicated in the pathogenesis of a wide variety of human diseases, being responsible for in-vivo oxidation/nitration events. Nitrosoperoxycarbonate anion, formed by the interaction of peroxynitrite with CO(2)/bicarbonate at physiological concentrations, provides a new interpretation of oxidative/nitrative processes formerly attributed to peroxynitrite. The aim of this review is to summarize the chemistry and biology of peroxynitrite and radical species related to nitrosoperoxycarbonate anion, as well as the information available regarding the molecular mechanisms that determine and regulate radical-promoted injury by the two tightly connected species at physiological concentrations. Interception of carbonate and nitro radicals produced by interaction of peroxynitrite with CO(2)/bicarbonate, as in-vivo prevention of pathological events, creates new perspectives for the evaluation of safe scavengers of oxidative/nitrative stress at the physiological level. In this respect, natural products such as flavonoids hold a preeminent position among the vast array of compounds endowed with such properties.

Electrochemical study, on mercury, of a Meta-nitroarylamine derivative of nor-β-lapachone, an antitumor and trypanocidal compound

The electrochemistry of 2,2-dimethyl-(3H)-3-(N-3'-nitrophenylamino)naphtho[1,2- b]furan-4,5-dione ([Q]-PhNO2), on mercury was investigated. The first peak is consistent with a quasi-reversible one-electron reduction of the ortho-quinone, forming [Q•-]-PhNO2, while the second one, bielectronic, corresponds to the simultaneous reduction of the latter radical to a dianion and the nitro group to a nitro radical anion. The second order rate constant, kdisp, for the decay of [Q•-]-PhNO2 is 15.188 x 103 ± 827 mol1 L s1 and the t1/2 equals 0.06 s. E17Ic values for [Q]-PhNO2 and its precursor, nor-β-lapachone, are similar. The ease of semiquinone generation and its stability are parameters statistically relevant in the correlation biochemical/theoretical aspects.

ESR and electrochemical study of 1,2-disubstituted 5-nitroindazolin-3-ones and 2-substituted 3-alkoxy-5-nitro-2H-indazoles: Reactivity and free radical production capacity in the presence of biological systems

Two families of 5-nitroindazole derivatives were electrochemically studied in an aprotic solvent using cyclic voltammetry (CV) technique. The produced nitro-anion radical species were characterized using electron spin resonance spectroscopy (ESR). Also, we examined the interaction between the radical species generated from nitroindazole derivatives and glutathione (GSH). Moreover, the capacity of intraparasite and intramammals-free radical production, through ESR spectroscopy, was performed.

Voltammetric Determination of Nitronaphthalenes at a Silver Solid Amalgam Electrode

Electrodes based on amalgam materials were re-introduced in electroanalytical chemistry in the year 2000, partially as reaction to unsubstantiated public fears of liquid mercury. In this publication, the voltammetric behavior of 1-nitronaphthalene and 2-nitronaphthalene was investigated at a mercury meniscus-modified silver solid amalgam electrode. The reduction mechanism in mixed neutral buffer-methanol medium includes the four-electron reduction to hydroxylaminoderivative followed by a two-electron reduction to the amine in acidic medium, similarly to mercury electrodes. In alkaline media, both compounds show the splitting of the main four-electron reduction peak typical for mercury electrodes in two new ones, the first one corresponding to a one electron reduction of the nitroderivative to the nitro radical anion, which was confirmed by microcoulometry. Using optimized conditions (differential pulse voltammetry, Britton-Robinson buffer pH 7.0 – methanol (9:1) medium) the calibration dependences are linear in the range of 2·10−7 (4·10−7) to 1·10−4 mol L−1 for 1-nitronaphthalene (2-nitronaphthalene). After preconcentration of the analytes from drinking and river water samples using solid phase extraction the limit of determination was lowered to ∼3·10−8 mol L−1.

Electrochemical Reduction Using Glassy Carbon Electrode in Aqueous Medium of a Potential Anti-Chagas Drug: NFOH

Nitrofurazone (NF) presents activity against Chagas’ disease, yet it has a high toxicity. Its analog, hydroxymethylnitrofurazone (NFOH), is more potent against Trypanosoma cruzi and much less toxic than the parent drug, NF. The electrochemical reduction of NFOH in an aqueous medium using a glassy carbon electrode (GCE) is presented. By cyclic voltammetry in anacidic medium, one irreversible reduction peak related to hydroxylamine derivative formation was registered, being linearly pH dependent. However, from , a reversible reduction peak at a more positive potential appears and corresponds to the formation of a nitro radical anion. The radical-anion kinetic stability was evaluated by the current ratio of the redox couple. The nitro radical anion decays with a second-order rate constant of 6.07, 2.06, and corresponding to pH 8.29, 9.29, and 10.2, respectively, with a corresponding half-time life of 0.33, 0.97, and 1.4 s for each pH value. By polishing the GCE surface with diamond powder and comparing with the GCE surface polished with alumina, it is shown that the presence of alumina affects the lifetime of the nitro radical anion.

Substituent Effect of 4-Nitroimidazole Derivatives: Acidic Hydrogen as Modulator of the Nitro Radical Kinetic Stability

The electrochemical reduction of 1-methyl-4-nitro-2-carboxyimidazole in a nonaqueous medium produced two reduction signals, which were influenced by the dissociation of the compound. The first reduction signal was caused by the reduction of the acidic species, and the second signal was a quasi-reversible couple caused by the reduction of the conjugate base to the radical dianion. According to the theory developed by Nicholson for the study of the electrode reaction kinetics, we have obtained the heterogeneous rate constant, , for the reduction of the conjugate base to the radical dianion. The voltammetric behavior of and its comparison with the behavior of another related compound such as 1-methyl-4-nitro-2-hydroxymethylimidazole is a good example to show the applicability of the general theory about the mechanism of self-protonation reactions in organic electrochemical processes to the specific case of nitroimidazole derivatives. According to the presented results, the presence of acidic hydrogen as a substituent in the nitroimidazole moiety would permit the modulation of the nitro radical anion stability, thus affecting its potential biological activity.

SOLVENT EFFECT ON ELECTROPHILIC AND RADICAL SUBSTITUTION OF TOLUENE MONONITRATION REACTIONS

Two kinds of nitrating reagents, a nitronium cation [Formula: see text] and a nitro radical (· NO2), were used in the gaseous phase toluene mononitration reaction. The closed shell calculation for electrophilic substitution and open shell calculation for radical substitution were both performed in solventless, H2O -solvated, and CH3OH -solvated molecular reaction systems. In the series of electrophilic toluene nitration reactions, both ortho-nitro toluene (o-MNT) and para-nitro toluene (p-MNT) are more abundant products than meta-nitro toluene (m-MNT), no matter what solvent is used in the reaction system. The reaction energy barrier for obtaining each kind of mononitro toluene follows a stepwise decreasing trend when the reaction is carried out in the solventless, H2O -solvated, and CH3OH -solvated systems. In all radical toluene nitration reactions, solventless or solvated, m-MNT is the most abundant product. The energy barrier data also show that the nitration reaction is more feasible in a solvated than in a solventless system. H2O has a more obvious solvent effect than CH3OH in the · NO2radical substitution reaction, and the H2O -solvated system provides a lower activation energy reaction path.

Cyanometallate incorporated supramolecular networks based on a nitroalkyl-substituted Cu IIN 4 precursor: Synthesis, crystal structure, thermal and electrochemical studies

Two new copper(II) complexes, viz. [Cu(nelin)(H 2O) 2] 2[Fe(CN) 6] · 6H 2O ( 2) and [{Cu(nelin)} 2Ni(CN) 4](ClO 4) 2 · 2H 2O ( 3), have been synthesized using [Cu(nelin)](ClO 4) 2 ( 1) (nelin = 1,9-diamino-5-methyl-5-nitro-3,7-diazanonane) as a nitroalkyl-substituted Cu IIN 4 precursor, and their structures and supramolecular networks have been fully explored using the single crystal X-ray diffraction technique. The H-bonded 1D chains of 2 run along the a-axis, being generated from supramolecular synthons using cations and anions, and are further propagated into a 3D array to form irregular honeycomb-like channels which are divided into two halves, with each half accomodating a helical water chain running in opposite directions to each other. In complex 3, the trinuclear units are arranged in successive rows in a herringbone fashion and bifurcated hydrogen bonding through the uncoordinated terminals of the [Ni(CN) 4] 2− units give rise to a supramolecular (4,4) network. A comparison of the PXRD pattern of complex 2 and its dehydrated form indicate marked changes in the diffraction pattern with the development of a quasi glassy nature in the dehydrated form. The electrochemical properties of 1, 2 and 3 have been investigated in comparative ways using the cyclic voltammetric technique in aqueous and MeCN solutions with Ag/AgCl as a reference electrode. Electrochemical reduction generates the one-electron reduced nitro-radical anion. In water–alcohol glass at 77 K complex 2 exhibits a typical four-line hyperfine EPR spectra with g || = 2.11, g ⊥ = 2.02, A || = 150 Oe and A ⊥ = 5 Oe at ν = 9.45 MHz.

Spectral and electrochemical studies of ruthenium(II) complexes with N4-methyl-4-nitrobenzaldehyde and N4-methyl-4-nitrobenzophenone thiosemicarbazone: Potential anti-trypanosomal agents

N 4-Methyl-4-nitrobenzaldehyde thiosemicarbazone (H4NO 2Fo4M), N 4-methyl-4-nitrobenzophenone thiosemicarbazone (H4NO 2Bz4M) and their ruthenium(II) complexes [Ru(4NO 2Fo4M) 2(PPh 3) 2] ( 1), [Ru(4NO 2Bz4M) 2(PPh 3) 2] ( 2), [Ru(4NO 2Fo4M) 2(dppb)] ( 3) and [Ru(4NO 2Bz4M) 2(dppb)] ( 4) (dppb = 1,4-bis(diphenylphospine)butane) were obtained and characterized. The crystal structure of H4NO 2Fo4M has been determined. Electrochemical studies have shown that the nitro anion radical, one of the proposed intermediates in the mechanism of action of nitro-containing anti-trypanosomal drugs, is formed at approximately −1.00 V in the free thiosemicarbazones as well as in their corresponding ruthenium(II) complexes, suggesting their potential to act as antitrypanosomal drugs. The natural fluorescence of H4NO 2Fo4M, H4NO 2Bz4M and complexes ( 1)–( 4) provides a way to identify and to monitor their concentration in biological systems.

Study of 5-nitroindazoles' anti- Trypanosoma cruzi mode of action: Electrochemical behaviour and ESR spectroscopic studies

New indazole derivatives have been developed to know about structural requirements for adequate anti- Trypanosoma cruzi activity. In relation to position 1 of indazole ring, we have observed that a butylaminopentyl substituent ( 14) affords good activity, but N-oxidation of ω-tertiary amino moiety yields completely inactive compounds ( 17, 18); the substituent at position 3 of indazole ring affects drastically the in vitro activity, 3-OH derivative 13 being completely inactive. On the other hand, since compound 22, denitro-analogue of active compound 4, does not show activity, the 5-nitro substituent of indazole ring seems to be essential. Intramolecular cyclization of side chain at position 1 also affords inactive compounds ( 19, 20). The electrochemical studies showed that the trypanocidal 5-nitroindazole derivatives yielded nitro-anion radical via one-electron process at physiological pH. This electrochemical behaviour occurs in the parasite according to ESR experiment with the T. cruzi microsomal fraction showing that 5-nitroindazole derivatives suffer bio-reduction without reactive oxygen species generation.

Theoretical Study of the Decomposition Reactions in Substituted Nitrobenzenes

The influence of substituent nature and position on the unimolecular decomposition of nitroaromatic compounds was investigated using the density functional theory at a PBE0/6-31+G(d,p) level. As the starting point, the two main reaction paths for the decomposition of nitrobenzene were analyzed: the direct carbon nitrogen dissociation (C6H5 + NO2) and a two step mechanism leading to the formation of phenoxyl and nitro radicals (C6H5O + NO). The dissociation energy of the former reaction was calculated to be 7.5 kcal/mol lower than the activation energy of the second reaction. Then the Gibbs free energies were computed for 15 nitrobenzene derivatives characterized by different substituents (nitro, methyl, amino, carboxylic acid, and hydroxyl) in the ortho, meta, and para positions. In meta position, no significant changes appeared in the reaction energy profiles whereas ortho and para substitutions led to significant deviations in energies on the decomposition mechanisms due to the resonance effect of the nitro group without changing the competition between these mechanisms. In the case of para and meta substitutions, the carbon-nitro bond dissociation energy has been directly related to the Hammett constant as an indicator of the electron donor-acceptor effect of substituents.

Electrochemical Study of Methyl 2-[p-Nitrophenyl(hydroxy)methyl]acrylate: An Anticancer Drug and Its Reactivity Toward GSH and Oxygen

Electrochemical experiments with methyl 2-[-nitrophenyl(hydroxy)methyl]acrylate were performed in protic (, , pH 6.9; , or , pH 9.4 and , , pH 9.6) and aprotic media. The primary reduction behavior in aprotic medium was typical of nitroaromatics along with an additional wave related to the reduction of the acrylate function. Kinetic analysis carried out in aprotic and aqueous basic media pointed out to the high stability of the electrogenerated nitro radical anion, especially in . Reduced (GSH) and oxidized (GSSG) gluthatione in phosphate buffer influenced the reduction behavior of , due mainly to protonation effects. Direct reduction of , in the presence of GSH, led to a transient nitroso-GS adduct. In the presence of GSSG, hydrogen-bonding-associated GSSG-hydroxylamine was the main product. Electrochemical studies of , in the presence of oxygen, showed no chemical reactivity between and . These electrochemical results help in the understanding of the anticancer activity of that can be considered a bioreductive agent with a glutathione depleting function.

Comparative spectroscopic and electrochemical study of nitroindazoles: 3-Alcoxy, 3-hydroxy and 3-oxo derivatives

Cyclic voltammetry and electron spin resonance techniques were used in the investigation of novel 3-alkoxy- and 3-hydroxy-1-[ω-(dialkylamino)alkyl]-5-nitroindazole derivatives. A self-protonation process involving the protonation of the nitro group was observed. The reactivity of the nitro-anion radical for these derivatives with glutathione, a biological relevant thiol, was also studied by cyclic voltammetry. These studies demonstrated that glutathione could react with radical species from 5-nitroindazole system. Also we demonstrated that nitro-anion radicals show three different patterns of delocalization where the indazole 1-lateral chain does not have major influence.