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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.55 n.2 Concepción jun. 2010 

J. Chil. Chem. Soc, 55, N° 2 (2010), págs.: 244-249





1 Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Olivos 1007, Independencia, Santiago, Chile (
2 Instituto de Química Medica, Consejo Superior de Investigaciones Científicas, Madrid, España


Cyclic Voltammetry (CV) and electrón spin resonance (ESR) techniques were used in the investigation of several potentially antiprotozoal nitro derivatives of dihydroquinoxaline and indazole. A self-protonation process involving protonation of the nitro group due to the presence of an acidic protón in the structure of the nitro compound was observed in the first step of an ECErev reduction mechanism. ESR spectra of the free radicals obtained by electrochemical reduction in situ were characterized and analyzed. The ESR spectra showed three different hyperfine patterns for both families of compounds. In order to evaluate the free radical scavenger properties of these nitrocompounds we carried out ORAC-Fl (Oxygen Radical Antioxidant Capacity) assay. These derivatives showed a low antioxidant capacity.

Keywords: ESR; Chagas' disease; ORAC; Cyclic Voltammetry, Nitro compounds.


The trypanosomiases form is a widespread group of parasitic diseases that affect over several hundreds of millions of people, especially inthe tropical and subtropical countries '. The parasite Trypanosoma cruzi is the causative agent of Chagas' disease, an endemic infection that affects human populations mainly in Central and South America, where 18 million people are infected, and causes approximately 400,000 deaths per year2. Nifurtimox (Nfx) and Benznidazole (Bnz), the drugs used in the treatment of this pathology, are effective only during the acute phase of the disease. Moreover, these compounds cause serious side effects, such as peripheral neuropathy, anorexia, vomiting, allergy, and cardiac and renal toxicities3.

Experiments carried out on the drugs used in Chagas' treatment suggest that intracellular reduction followed by redox cycling yielding reactive oxygen species (ROS) may be their major mode of action against T. cruzi. These ROS can cause cellular damage directly by reacting with various biological macromolecules or indirectly by generation of the highly reactive hydroxyl radical via iron-mediated Haber-Weiss and Fenton reactions4.

On the other hand, an important number of pharmacologically active compounds including Nfx and Bnz have a nitro aromatic group in their molecular structures 5,6,7,8,9,10. Due to the ability of these drugs to be reduced at the nitro group, they are metabolized to the corresponding amines via the formation of nitroso and hydroxylamine derivatives.

Nevertheless, it is known that the biological activity of nitro compounds is not due to the final producís of reduction but to the formation of intermediates, possibly free radicals11.

Olea-Azar C. et al. studied12 the electrochemistry of 3-alkoxy and 3-hydroxy-5-nitroindazole derivatives by Cyclic Voltammetry, in DMSO as solvent. They found that the reduction mechanism depends on the acidic moieties in their structures. A self-protonation process involving protonation of the nitro group was observed. Some derivatives presented a one-electron reversible transfer corresponding to the generation of the nitro anion radical by an Erev mechanism. Others presented a reduction mechanism involving a self-protonation process and the generation of the nitro anion radical from uncharged species by an Erev and ECErev mechanism.

A series of indazole V-oxide derivatives were synthesized by Gerpe A. et al13 and their antichagasic properties were studied. These authors found that 3-cyano-2-(4-iodophenyl)-2H-indazole V-oxide exhibited an interesting activity against the two T. cruzi strains and the two life-stages evaluated, and also showed leishmanicidal activity in the three parasite strains evaluated.

Rodríguez J. et al14 studied two new groups of 5-nitroindazole derivatives using electrochemical and spectroscopic techniques. They observed a self-protonation process involving protonation of the nitro group. The reactivity of the nitro-anion radical of these derivatives with glutathione, a biologically relevant thiol, was also studied by cyclic voltammetry.

These studies demonstrated that glutathione could react with radical species containing the 5-nitroindazole system. It was also demonstrated that these nitro-anion radicals show three different patterns of delocalization in which the indazole side-chain at NI does not have a major influence.

Another important group of nitro compounds of biological interest are the nitrofuran derivatives that have been studied in terms of their mechanisms of action and their ability to act as alternative chemotherapeutic agents for Chagas' disease15,16,17,18,19.

Recently, we studied the effect of labile hydrogen present in the different structures of Ru and Pd complexes with nitrofuran derivates as ligands, in order to verify the ORAC-FI values, and we related these results to the biological activity of these compounds.20,21.

In accordance with the references discussed above, we set out to study two new families of heterocyclic derivatives of nitroquinoxaline (NQ) and 5-nitroimidazole (NI) with the aim of evaluating their physical-chemical properties in order to characterize them as new potential antichagasic drugs.

The study of these two families (Tables 1 and 2) is mainly based on the scaffold and the functional groups of these molecules. These compounds have a nitro group (RNO2) that could be reduced to generate the respective anion radical (R-NO2- ) and, in some cases, an amide (H-N-CO), amine (R-NH2) or other group bearing a labile hydrogen (thiosemicarbazones and semicarbazones) could easily give up a hydrogen atom 12,13,14,20,21,22,23. This ability could quench the free radical generated and thus modify their antiprotozoal capacity.

The electrochemical behavior of these derivatives was studied in DMSO using cyclic voltammetry, and the radical species were characterized using electrón spin resonance.

In discussing the electrochemical assays, these will be separated into two sections; one for molecules without labile hydrogen and another for those that possess labile hydrogen in their structure.



The NQ and NI (Tables 1 and 2) were synthesized according to reported methods14,24,25a,b.

Cyclic voltammetry

Dimethylsulfoxide (DMSO) was obtained from Aldrich. Tetrabutylammonium perchlorate (TBAP), used as a support electrolyte, was obtained from Fluka. CV was carried out using a Metrohm 693 VA instrument with a 694 VA Stand convertor and a 693 VA Processor, in DMSO (ca. 1.0 x 10-3 mol/L), under a nitrogen atmosphere at room temperature, with TBAP (ca. 0.1 mol/L), using a three-electrode cell. A hanging mercury drop electrode was used as the working electrode, a platinum wire as the auxiliary electrode, and saturated calomel as the reference electrode.

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. The anion radicals were generated by electrolytic reduction in situ under the same conditions of temperature, atmosphere and concentrations stated for the CV experiment. The ESR spectra were simulated using the program WINEPR Simfonia version 1.25.

Oxygen Radical Absorbance Capacity Assay (ORAC-Fluorescein)


2,2'-Azo-bis(2-amidinopropane) dihydrochloride (AAPH) was used as the peroxyl radical source. Fluorescein (FL) and Trolox (6-hydroxy-2,5,8-tetramethylchroman-2-carboxylic acid), were purchased from Sigma-Aldrich.

Solutions and ORAC determination.

The methodology proposed by Ou et al. 26 was modified for this study. The reaction involving AAPH, fluorescein (FL) and antioxidant (NI and NQ) was carried out in 3000 uL quartz cells. Constant quantities of AAPH and FL were added to the cell while the volume of phosphate buffer (75 niM, pH 7.4) was decreased to compénsate the volume of the compounds studied. For all measurements 240 mL 12 mM AAPH, and 215 mL of 70 nM FL, were added21.

The five nitro compounds studied (NQ-1,4,5,6, NI-1) were dissolved in DMSO to obtain a concentration of 10-4M. Increasing amounts ranging from 15 to 60 mL were added, with final concentrations between 0.5 and 2.0 mM.

The standard reference compound Trolox used to build the calibration curve was dissolved in phosphate buffer (75 mM) in a range of concentrations from 0.5 to 2.0 mM.

A blank without antioxidant was prepared under the same conditions as the samples, and the measurements were carried out at 50°C

A luminescence spectrometer (Perkin-Elmer model LS 50B, Boston, MA, USA) connected to a thermostated bath (Haake Fisons model DC1-B3, Karlsruhe, Germany) was used in this analysis. To detect the fluorescence, the excitation wavelength was adjusted to 490 nm and emission was measured at 515 nm.


Cyclic voltammetry

Molecules without labile hydrogen (NQ-2,3 and NI-2,3,4).

In order to achieve the best experimental conditions that ensure nitro-anion radical stability, DMSO was used as an aprotic medium and TBAP was used as the support electrolyte. Under these conditions, all compounds displayed similar electrochemical behaviour. Figure 1 shows the cyclic voltammogram of NQ-3, with only one well-defined couple with a peak EcII/Ealionear -1.26 V for NQ. These derivatives reveal a quasireversible monoelectronic transfer corresponding to the generation of the nitro anion radical (RNO2·- ).

The characterization of the voltammetric wave took place using parameters DE and Ipa/Ipc. The Ipa/Ipc values were obtained from Nicholson and Shain's equation27.

The electrochemical parameters obtained for these compounds showed that when the sean rate was increased (from 0.25 to 2 V/s) the Ipa/Ipc values approached unity. The parameter DE did not reach 0.06 V for either NI or NQ derivatives, indicating that this is a quasireversible process.

For each of the compounds studied, Ipc vs. v½ curve is linear, indicating that the electrón transfers are diffusion-controlled and are not due to the species adsorbed on the surface of the electrode28.

The reduction mechanism proposed for these compounds is shown in scheme 1.

Moolecules with labile hydrogen (NQ-1,4,5,6 and NI-1)

Figure 2 shows the cyclic voltammogram of NQ-5. The electrochemical behavior is similar for these five derivatives. The voltammograms obtained for each compound showed two reduction waves, one cathodic peak EIc/EIa (c.a. -1.16 V) corresponding to nitro anion radical RNO·- and asecond wave at higher cathodic potential (EIIc/EIIa, c.a. -1.38 V), v/s SCE, corresponding to the reduction of the anion -ORNO2 generated through a self-protonation reaction as a chemical complementary equilibrium (Cl).

This process corresponds to an acid-base equilibrium in the aprotic medium, a typical self-protonation phenomenon displayed by nitro-compounds with acidic moieties in their structures 29,30,31.

It is possible that the higher negative potential (wave EIIc/EIIa) of the nitro derivatives corresponds to a diminished capacity to accept electrons due to its negative charge.

In order to obtain suitable conditions to observe the nitro anion radical of these derivatives and verify the self-protonation mechanism proposed, we carried out experiments in the presence of increasing amounts of NaOH (0.1 M).

Figure 3 show the voltammogram obtained for NQ-5 in the presence of different amounts of alkali. The electroreduction wave Ele gradually disappears with increasing NaOH concentration on going from 0 to 1 mM (Fig. 3).

The calculated Ipa/Ipc ratio using the Nicholson and Shain equation 25,26 increases to about 1.0 with the addition of NaOH for peak EIIc/EIIa. These results confirm the ECErev mechanism proposed for these derivatives given by the increment in the Ipa/Ipc ratio toward the reversibility of its final peak. Figure 2 shows voltammograms characteristic of these derivatives.

This study confirms the presence of an acidic protón in the chemical structures of NI and NQ derivatives. The proposed mechanism forthe reduction of these molecules is presented in Scheme 2.

NI derivatives presented a similar mechanism to that suggested in Scheme 2 for NQ derivatives.

In addition to the two electrochemical reduction waves (E1 and E2). an electrochemical oxidation peak (EaO) with a potential close to -0.5V is observed. This wave corresponds to the electrochemical oxidation of a hydroxylamine (RNHOH) to nitroso compound as described as a general mechanism of reduction of aromatic nitro compounds32.The hydroxylamine is generated as a reduction product of the nitro compound studied as shown in Scheme 3.

With this feature it is possible to attribute the EaO signal to an irreversible oxidation process of the hydroxylamine to the corresponding nitro compound as shown in scheme 4.

The results obtained by cyclic voltammetry for all the compounds studied are summarized in Table 3.

Oxygen Radical Absorbance Capacity Assay (ORAC-Fluorescein)

In order to study the hydrogen donating ability of the nitro compounds and its possible relationship to trypanosomicidal activity, the free-radical scavenger capacity of NI-1 and NQ-1,4,5 and 6 was further evaluated using the ORAC assay. The ORACFL assay expresses antioxidant activity relative to a standard (Trolox) while measuring the oxidation of the fluorescent substrate by peroxy radicals generated during the reaction. This method follows a hydrogen atom transfer pathway, where the antioxidant and a peroxyl radical interact with each other thus breaking the reaction of oxidation chain.

To compare the antioxidant capacity of these molecules we used Trolox (range of concentration from 0.5 mM to 2.0 mM) as a reference antioxidant (figure 4).

Figure 5 shows the decay characteristics in the ORACFl test for nitro compounds NI and NQ.

The decay curve shown in Figure 5 is similar for the five nitro compounds studied. Here, it is possible to see a slight increase in the área under the curve from the blank with increasing concentrations of NI and NQ, which translates into a low antioxidant capacity of the compounds, to slightly retarded oxidation of fluorescein.

ORACFL values of the nitro compounds studied were close to the reference value of Trolox.The compounds studied here have low antioxidant capacity. This feature indicates that the labile hydrogen present in the structure of the compound should not interfere with their ability to generate free radicals.

The results obtained by the ORACFL assay for NI and NQ derivatives are summarized in Table 4.

Electron Spin Resonance

In order to evaluate the generation of nitro anion radical species proposed in the electrochemical mechanism, ESR experiments for selected nitrocompounds were done. NI and NQ free radicals were prepared in situ by electrochemical reduction in DMSO applying the potential corresponding to peak Ecl or EcII obtained from the CV experiments. All the studied structures produced stable paramagnetic intermediates in the first reduction step. The interpretation of the ESR by means of a simulation process confirmed the stabilities of these radical species due to delocalized unpaired electrons. The simulated spectrum of the NQ-2 radical (Fig. 6) shows two triplets, one for the nitro group nitrogen and the other for nitrogen atom (NI) of the quinoxaline group, and four doublets for the ring H-1, H-2, H-3 and H-4.

The spectrum was simulated using the following parameters: line width = 0.30 G, Lorentzian/Gaussian ratio = 1 and hyperfine constants are included in Table 5.

Figure 7 displays a simulated spectrum for NI-1 in terms of three triplets corresponding to the nitrogen atoms of the nitro group and N1 and N2 of the indazole system and three doublets assigned to nuclei H-1, H-2 and H-3 of the ring.

The NQ-6 free radical (Figure 8) displays a simulated spectrum of two triplets, one for the nitro group nitrogen and the other for nitrogen atom (NI) of the quinoxaline system, and three doublets due to H-1, H-2, H-3 and H-4 of the ring.

The three simulated spectra indicated in this report represent the hyperfine pattern for the ten nitro compounds studied.


Our CV results show that NI or NQ derivatives are electrochemically reduced via formation of a nitro anion radical (RNO2·- ). The reduction mechanism depends on the acidic moieties in their structures. A self-protonation process involving protonation of the nitro group was also observed. The reduction mechanism proposed for derivatives with labile hydrogen is an ECErev one corresponding to the generation of the nitro anion radical from the uncharged species, followed by a self-protonation process from a hydroxyl moiety and the generation of a nitro anion radical from the negatively charged species.

On the other hand, derivatives without labile hydrogen displayed a reduction mechanism via formation of the nitro anion radical (RNO2·- ).

The ORACFL assay showed that all compounds studied have low antioxidant capacities, indicating that the labile hydrogen present in the structure of the compounds should not interfere with their ability to generate RNO2·- free radicals.

Stable free radicals were generated using electrochemical reduction at potentials corresponding to the Ele and Elle wave and were characterized by ESR spectroscopy.

The NI and NQ compounds studied showed three different spectral patterns depending on their structural characteristics. These results indicated that the side chain does not have a major influence on the electrón delocalization.

The ESR spectral pattern was similar for NQ derivatives. In the case of NI compounds, the different ESR pattern with respect to the other NQ derivatives could be explained in terms of the molecular structure due to the fact that they show a structure which facilitates delocalization of the unpaired electrón in the heteroeyelie system.


This research was supported by FONDECYT grant 1071068 and a CONICYT scholarship.



1., acessed in November 05, 2007.        [ Links ]

2.  J.D. Maya, S. Bollo, L.J. Núñez, J.A. Squella, A. Morello, Biochem. Pharmacol. 65, 999, (2003).        [ Links ]

3.  J.D. Maya, B.K. Cassels, P. Iturriaga-Vásquez, J. Ferreira, M. Faúndez, N. Galanti, A. Ferreira, A. Morello, Comp. Biochem. Physiol. A Mol. Integr. Physiol.,146, 601, (2007).        [ Links ]

4.  H. Cerecetto, B. Mester, S. Onetto, G. Seaone, M. González, F. Zinola, Bioorg. Med. Chem., 47, 1207, (1992).        [ Links ]

5.  F. C. de Abreu, J. Tonholo, O. Bottecchia; C. Zani, L. Goulart, M.OF.Goulart, J. Electroanal. Chem., 462, 195, (1999).        [ Links ]

6.  Q. Li, X. Zhao, J. M. S. Gee, S. J.; Kurth, M. J. Seiber, J. N. Hammock, J. Agric. Food Chem.,39, 1685, (1991).        [ Links ]

7.  V. Chopineaux-Courtois; C. Reymond, F.; Bouchard, G.; Carrupt, P. A.; Testa, B.; H. Girault,. J. Am. Chem. Soc., 121, 1743, (1999).        [ Links ]

8.  H. Nivinskas; R. L. Koder; I. Anusevieius; J. Sarlauskas, N. Miller, Acta Biochim. Pol., 47, 941, (2000).        [ Links ]

9.  Z. She, M. Gao, Ch. Jin, Y. Chen, L. Yu, J. Process Biochem., 40, 3017, (2005).        [ Links ]

10.  B.  Blanchard-Fillion, D. Prou, M. Polydoro, D. Spielberg, E. Tsika, Z. Wang, S. L. Hazen, M. Koval, S. Przedborski, H. Ischiropoulos,. Jour. Neurosci., 26, 6124, (2006).        [ Links ]

11.  Y. Oh. Zkan, K. Sentujrk, Jour. Pharm. Biomed. Anal, 17, 299, (1998).        [ Links ]

12.  C. Olea-Azar, H. Cerecetto, A. Gerpe, M. González, V. Aran, C. Rigol, L. Opazo, Spectrochimica Acta, Part A, 63, 36 (2005).        [ Links ]

13.   A. Gerpe, G. Aguirre, H. Cerecetto, M. González, C. Olea-Azar, Bioorg. Med. Chem., 14, 3467, (2006).        [ Links ]

14.   J. Rodríguez, A. Gerpe, G. Aguirre, O.E. Piro, C. Olea-Azar, M. González, H. Cerecetto, Spectrochimica Acta Part A, 70, 519, (2008).        [ Links ]

15.   J. A. Bautista-Martínez, I. González, M. Aguilar-Martínez, Electrochim. Acta, 49, 3403,(2004).        [ Links ]

16.   M. Bonta, G. Chauviere, J. Perie, L. Nuñez-Vergara, J.A. Squella, Electrochim. Acta, 47, 4045, (2002).        [ Links ]

17.   R. Sokolová, M. Hromadová, J. Fiedler, L. Pospísil, S. Giannarelli, M. Valásek, J. Electroanal. Chem., 622, 211 (2008).        [ Links ]

18.   J. Giglio, A. Rey, H. Cerecetto, I. Pirmettis, M. Papadopoulos, E. León, A. Monge, A. López de Ceráin, A. Azqueta, M. González, M. Fernández, A. Paolino, A. León, Eur. J. Med. Chem., 41, 1144, (2006).        [ Links ]

19.   J. Arguello, L. J. Núñez-Vergara, S. Bollo, J. A. Squella, ,Bioelectrochemistry, 69, 104, (2006).        [ Links ]

20.   C. Olea-Azar, E. Norambuena, D. Gambino, L. Otero, Europ. Jour. of Med. Chem., 44, 4937,(2009).        [ Links ]

21.   L. Otero, Ch. Folch, G. Barriga, C. Rigol, L. Opazo, M. Vieites , D. Gambinoa, H. Cerecetto , E. Norambuena, C. Olea-Azar, Spectrochimica Acta Part A, 70, 519, (2008).        [ Links ]

22.   J.A. Bautista-Martínez , I. González, M. Aguilar-Martínez, Electrochim. Acta, 49, 3403, (2004).        [ Links ]

23. N A. Hamdy, A. M. Gamal-Eldeen, Eur. J. Med. Chem., 44, 4547, (2009).        [ Links ]

24 V.J. Aran, C. Ochoa, L. Boiani, P. Buccino, H. Cerecetto, A. Gerpe, M. González, D. Montero, J.J. Nogal, A.Gómez-Barrio, A. Azqueta, A. López de Cerain, O.E. Piro, E.E. Castellano, Bioorg. Med. Chem., 13, 3197, (2005).        [ Links ]

25.   (a) S. de Castro et al., J. Chem. Soc, Perkin Trans, 1, 790, (2002), (b) S. Ancizu, E. Moreno, E. Torres, A. Burguete, S. Pérez-Silanes, D. Benítez, R. Villar, B. Solano, A. Marín, Aldana I, H. Cerecetto, M. González, A. Monge, Molecules, 14, 2256, (2009).        [ Links ]         [ Links ]

26.   Ou, B.; Hampsch-Woodill, M.; Prior, R. L, J. Agric. Food Chem., 49, 4619, (2001).        [ Links ]

27.   R.S Nicholson. and I. Shain, Anal. Chem., 36, 706, (1964).        [ Links ]

28.   R.S. Nicholson., Anal. Chem.,38, 1406, (1966).        [ Links ]

29.   J. Bautista, I. González , M. Aguilar, Electrochim. Acta, 48, 4239, (2003).        [ Links ]

30.   J. Carbajo, S. Bollo, L.J. Nuñez-Vergara, A. Campero, J.A. Squella, Electroanal. Chem., 53, 187, (2002).        [ Links ]

31.   G. Kokkinidis, A. Kelaidopoulou, J. Electroanal. Chem., 414,197, (1996).        [ Links ]

32.   C. Rigol, C. Olea Azar, F. Mendizabal, L. Otero, D. Gambino, M. González and H. Cerecetto, spectrochimica Acta part A, 61, 2933, (2005).        [ Links ]


(Received: September 14, 2009 - Accepted: April 29, 2010)

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