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Journal of the Chilean Chemical Society

versão On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.48 n.4 Concepción dez. 2003 

J. Chil. Chem. Soc., 48, N 4 (2003) ISSN 0717-9324


Claudio Olea-Azar,1* Carolina Rigol,1 Lucía Opazo1, Antonio Morello,2 Juan Diego Maya,2 Yolanda Repetto2, Gabriela Aguirre3, Hugo Cerecetto3, Rossanna Di Maio3, Mercedes González3 and Williams Porcal3.

1. Department of Inorganic and Analytical Chemistry. Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Santiago , Chile
2. Department of Molecular and Clinical Pharmacology. Faculty of Medicine. University of Chile,
P.O. Box 70000, Santiago 7, Chile.
3. Department of Organic Chemistry, Faculty of Chemistry, University of the Republic, Montevideo, Uruguay

(Received: July 15, 2003 – Accepted: August 21, 2003)


The Electron Spin Resonance (ESR) spectra of radicals obtained from two new potential antitrypanosomal drugs by Trypanosoma cruzi reduction were analyzed. DMPO Spin Trapping was used to investigate the possible formation of free radicals in the trypanosome microsomal system. The Nitro 2 (4-(n-butyl)-1-(5-nitrofurfurylidene)semicarbazide) analogue of Nifurtimox showed better antiparasitic activity than N-oxide 1 (4-(n-butyl)-1-[(7-bromo-N1-oxidebenzo[1,2-c]1,2,5-oxadiazole-5-yl)methylidene]semicarbazide). Only Nitro 2 could produce oxygen redox cycling in T. cruzi epimastigotes. The ESR signal intensities were consistent with the trapping of hydroxyl radical. These results are in agreement with the biological observation that Nitro 2 showed antichagasic activity by an oxidative stress mechanism.

Keywords: Nitrofuran derivatives, N-Oxide derivatives, ROS scavenging, ESR spin trapping, T. cruzi, oxidative stress


Parasitic diseases in tropical and subtropical areas constitute a major health and economic problem. Chagas’ disease, produced by several strains of Trypanosoma cruzi, affects approximately 24 million people from Southern California to Argentina and Chile [1]. Nifurtimox and benznidazole are currently used to treat this disease [2]. A characteristic ESR signal corresponding to the nitro anion radical (R-NO2·) appears when nifurtimox is added to intact T. cruzi cells [3]. This and other experiments [4-6] suggest that intracellular reduction of nifurtimox followed by redox cycling, yielding O2-· and H2O2, may be the major mode of action against T. cruzi. However, the use of nifurtimox has the disadvantage of its side effects [7].

Nitro compounds, especially 5-nitrofuryl derivatives, have been documented to be of great value as antiparasitic drugs. Recently we have explored 5-nitro-2-furaldehyde derivatives to find new substances with fewer side effects than Nifurtimox [8-12]. We have also carried out three-dimensional quantitative structure-activity relationship (3-D QSAR) studies on the in vitro and in vivo antiparasitic activities against Trypanosoma cruzi to establish the mode of action for this kind of semicarbazone derivatives [13,14].

In general, the biological effects of nitroheterocyclic compounds, especially in T. cruzi, involve redox cycling of these compounds and oxygen radical production, two processes in which the nitroanion radicals play an essential role [15].

Previously, we reported studies on the antiprotozoal activities of 5-nitrofurfural and 5-nitrothiophene-2-carboxaldehyde derivatives, and we showed that these compounds generate nitro anion radicals, characterized by ESR spectroscopy [16-17].

We also reported studies on the 1,2,5-oxadiazole N-oxide family in order to determine their antitrypanosomal activities, tested in vitro against the epimastigote form of T. cruzi. Moreover, we have shown some ESR spectra that prove the facile electronation of the N-oxide moiety. Beside, these new structures were based on the conjunction of N-oxide systems and the semicarbazide moieties ("spermidine-mimetic") [18]. In addition, we recently reported the electrochemical studies and the evidence of microsomal production of free radicals for 1,2,5-Oxadiazole N-Oxide suggesting its potential antiprotozoal activity [19]. All N-oxide studied showed similar E1/2 to nifurtimox. Additionally, the side chains assessed in this work did not modify the E1/2, an aspect that might be important for the selectivity of these compounds towards trypanothione reductase. Stable free radicals generated using a microsomal system showed hyperfine coupling constants identical to those of the radicals obtained by electrochemical reduction. The ESR spectra also proved that the N-oxide group is protonated, as suggested by the reduction mechanism proposed from the cyclic voltammetric results.

In the present study, we report the ESR and spin trapping results of two new potential antitrypanosomal drugs: 4-(n-butyl)-1-[(7-bromo-N1-oxidebenzo[1,2-c]1,2,5-oxadiazole-5-yl)methylidene]semicarbazide (N-oxide1) and 4-(n-butyl)-1-(5-nitrofurfurylidene)semicarbazide (Nitro2) (Figure 1). Both molecules have the same side chain but with different groups generating free radical species such as the nitro and N-oxide groups. In this paper we have characterized the free radical species generated by T. cruzi reduction that correlate with the percentage of growth inhibition of T. cruzi epimastigotes in order to suggest a possible mechanism.

Fig.1.- Chemical structure of the potential antitrypanosomal drugs.


2.1. Samples.
The N-oxide 1 and Nitro 2 were synthesized according to methods described earlier [9, 20].

2.2. Reagents
Dimethylsulfoxide (DMSO) (spectroscopy grade), glutathione (GSH), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma Aldrich Co., St. Louis, MO. Tetrabutylammonium perchlorate (TBAP) used as supporting electrolyte was obtained from Fluka.

2.3. ESR Spectroscopy.
ESR spectra were recorded in the X band (9.85 GHz) using a Bruker ECS 106 spectrometer with a rectangular cavity and 50 kHz field modulation. The hyperfine splitting constants were estimated to be accurate within 0.05 G. ESR spectra of the anion radical drugs and the radical oxygen species were produced using a microsomal fraction (4 mg protein/mL) obtained from T. cruzi, in a reaction medium containing 1mM NADPH, 1mM EDTA and 100 mM DMPO, in 20mM phosphate buffer, pH 7.4. The ESR spectra were simulated using the program WINEPR Simphonia 1.25 version.

2.5. Parasites
Trypanosoma cruzi epimastigotes (Tulahuen strain), from our collection, were grown at 28 ºC in Diamond’s monophasic medium as reported earlier [21,22], with blood replaced by 4 m M hemin. Fetal calf serum was added to a final concentration of 4%. Parasites: 8 x 107 cells correspond to 1 mg protein or 12 mg of fresh weight.


3.1 ESR
We have obtained a well resolved ESR spectra for both anion radical derivatives when they are prepared in situ by electrochemical reductions in DMSO, applying a potential corresponding to the first wave as obtained from the cyclic voltammetric experiments (data not shown). However, when T. cruzi microsomes are incubated with both compounds, after a 10 min induction period to become the microsomes anaerobic, gave a not well-resolved ESR spectra (data not shown), probably attributable to both radical species.

3.2 Effect of Nitro 2 and N-Oxide 1 upon culture growth in Trypanosoma cruzi epimastigotes
Several drug concentrations were used in order to determine the respective IC50 Nitro 2 produced significant inhibition of epimastigote culture growth (IC50 of 7.4±0.5 m M). N-oxide 1 showed a much lower effect upon epimastigote growth (IC50 > 30 m M).

3.3 ESR spectra of DMPO-OH· and DMPO-N-Oxide adducts obtained with T. cruzi extracts
In order to analyse the antitrypanosomal mechanism of these drugs, we incubated both compounds with T. cruzi homogenates in the presence of NADPH and EDTA and DMPO (figures 2 and 3). A well-resolved ESR spectrum appeared when DMPO was added to the T. cruzi-Nitro 2 system. The ESR signal intensity was consistent with the trapping of the hydroxyl radical (DMPO-OH spin adduct aN=aH = 14.7 G) (Figure 2). These hyperfine constants are in agreement with the splitting constants of other DMPO-OH adducts by DMPO [23]. These results are in agreement with the above-mentioned activity for this compound. However, when the N-oxide 1 compound was incubated with T. cruzi in presence of DMPO (figure 3), six ESR line appeared. According with this hyperfine pattern and the hyperfine constants (aN = 15.6 G and aH = 21.6 G ) this spectrum was consistent with the trapping of the N-oxide radical (Figure 3), which can not produce radical oxygen species. These results agree with the low activity of these compounds. On the other hand, N-oxide-produced low growth inhibition of T. cruzi occurs at concentrations that do not stimulate hidroxyl radical generation. As seen in their structures, both molecules have the same side chain, which could indicate that the nitro group generated radical species more efficiently than N-oxide group.
Finally, for Nitro 2 the main toxic mechanism seems to be the production of oxidative stress because of the extensive redox cycling that Nitro 2 undergoes.

Fig. 2.- ESR spectra of DMPO-OH· adduct obtained with T. cruzi extracts with Nitro 2. The ESR spectra were observed 10 min after incubation at 37°C with T. cruzi microsomal fraction (4 mg protein/mL), NADPH (1mM), EDTA (1mM), in phophate buffer (20mM), pH 7,4 , DMPO (100mM), Nitro 2 (1mM in acetonitrile 10 v/v). Spectrometer conditions: microwave frequency 9.68 GHz microwave power 20 mW, modulation amplitude 0.4G, scan rate 0.83 G/s , time constant 0.25 s number scans: 10.

Fig. 3.- ESR spectra of DMPO-N-oxide adduct obtained with T. cruzi extracts with N-oxide 1. The ESR spectra were observed 10 min after incubation at 37°C with T. cruzi microsomal fraction (4 mg protein/mL), NADPH (1mM), EDTA (1mM), in phophate buffer (20mM), pH 7,4 , DMPO (100mM), N-oxide 1 (1mM in acetonitrile 10 v/v). Spectrometer conditions: microwave frequency 9.68 GHz microwave power 20 mW, modulation amplitude 0.4G, scan rate 0.83 G/s , time constant 0.25 s number scans: 10.


The ESR spectra of the anion radicals for N-oxide 1 and Nitro 2 generated by T. cruzi system showed low resolution. However, well resolved ESR spectra were obtained when DMPO was added to the system. For Nitro 2, the ESR signal intensity was consistent with the trapping of the hydroxyl radical, and for the N-oxide 1, its spectrum was consistent with the trapping of the N-oxide radical.

The biological studies and the ESR experiment with the T. cruzi system indicate that Nitro 2 and N-oxide 1 could have different mechanisms of toxicity. While Nitro 2 may act by production of oxidative stress throughout the increase in redox recycling of the molecule; N-oxide 1 seems to act through different inhibition mechanism.


This investigation was supported by FONDECYT – Chile grants No 1030949


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