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Boletín de la Sociedad Chilena de Química

versión impresa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.45 n.1 Concepción mar. 2000

http://dx.doi.org/10.4067/S0366-16442000000100013 

NITRORADICAL ANION FORMATION FROM NITROFURANTOIN IN CARBON ELECTRODES

M. MERINO1, J. CARBAJO2, L.J. NUÑEZ-VERGARA1 AND J.A. SQUELLA1*

1Laboratorio de Bioelectroquímica, Facultad de Ciencias Químicas y Farmacéuticas,
Santiago, Chile
2Departamento de Química Física y Química Orgánica, Universidad de Huelva, Huelva,
España
(Received: July 15, 1999 - Accepted: November 18, 1999)

SUMMARY

The electrochemical nitroreduction of nitrofurantoin has been studied on carbon paste and glassy carbon electrodes. We can observe a monoelectronic reversible couple ArNO2/ArNO2.- and an irreversible peak due to the further reduction of nitro radical to the hidroxilamine via three electrons.

According to the experimental results, the reduction process shows a typical behavior of an EC mechanism. The k2 obtained values showed that the nitroradical anion was better stabilized on carbon paste electrode.

KEY WORDS: Electroreduction, carbon paste electrode, glassy carbon electrode, nitrofurantoin.

RESUMEN

La formación electroquímica del nitro anión radical de nitrofurantoína ha sido estudiada sobre electrodos de carbono vítreo y pasta de carbono. Se encontró que sobre ambos tipos de electrodos, existe un proceso monoelectrónico reversible correspondiente a la cupla redox ArNO2/ArNO2.-, seguido de un pico irreversible correspodiente a la reducción vía tres electrones del anión radical a la correspondiente hidroxilamina. De acuerdo a los resultados obtenidos, el proceso de reducción ocurre a través de un mecanismo EC, donde los valores de k2 encontrados, indican que el anión radical nitro es mejor estabilizado sobre electrodos de pasta de carbono.

PALABRAS CLAVES: Electroreducción, electrodo de pasta de carbono, electrodo de carbon vítreo, nitrofurantoína.

INTRODUCTION

Nitrofurantoin N-(5-nitro-2-furfurylidine)-1-aminohydantoin) (Figure 1), is a synthetic nitrofurane widely used to treat urinary tract infections1). Although the molecular mechanism leading to nitrofurantoin-induced cell toxicity is still uncertain, the antimicrobial activities as well as other clinical toxicities of nitrofurantoin may be due to the reductive metabolic activation of the 5-nitro function to the anion radical, nitroso and hydroxilamine derivatives2).

FIG. 1. Chemical structure of nitrofurantion.

Studies "in vitro" of interaction between metabolites generated by the reduction of nitrofurantoin and DNA, bacterial and of mammals, have revealed that the damage is highest on the bacterial DNA than on mammals' DNA. This is explained partly, by the impediment that tries the complex structure of the mammals' DNA3).

Like adverse effect to the treatment with nitrofurantoin, it has been associated with pulmonary fibrosis, neuropathy, hepatitis and hemolytic anemia in humans and animals of experimentation4). Studies "in vitro" suggest that a redox cycling of nitrofurantoin is a critical event for their cytotoxic mechanims5).

In aerobic conditions, the chemical activation of nitrocompounds could take place via one electron reduction, of the group nitro RNO2 to their nitro radical anion RNO2.-. This reaction is catalyzed by the enzyme cytochrom P450-NADPH reductase. The radical is reoxidized quickly, regenerating the initial nitrocompound and forming the anion radical superoxide (O2.-). This is able to dismute spontaneously or through catalysis by the superoxide dismutase, forming hydrogen peroxide (H2O2). The latter could undergo a Haber-Weiss reaction and form hidroxyl radical (·OH) 6). All these oxygen-reactive species are able to produce important cellular changes, i.e., altering essential functions like unstabilization of membranes7).

In the last decades, extensive studies of the electrochemical properties of nitroheterocyclic compounds8-29) have been published. In this way, nitrofurantoin8,9,29) has received special attention and it has been used as a model for these studies. By analogy with other nitroheterocycles, it is assumed that reductive activation of the nitro group is a prerequisite for the biological action9).

In order to develop an electrochemical model for the bioreductive route of the nitroheterocyclic compounds, most of the studies have been done on a mercury working electrode. Symons and co.9) have determined that changes in the electrochemical solvent alter the redox mechanism of these compounds. In a purely aqueous media, reduction occurs in a single irreversible 4-electron reduction to yield the hidroxylamine derivative10). However, in an aprotic or mixed aqueous/aprotic medium, reduction takes place in two different steps. First a reversible 1-electron reduction, to give the nitro radical anion, followed by an irreversible 3-electron reduction, giving the hidroxylamine derivative9). As the amount of the aprotic solvent in the mixed medium is increased, the stability of the nitro radical anion also increases10,23). Moreover, the working electrode also plays a significant role in the reduction process, and does not merely acts as an electron source. Altering the electrode material might also influence the redox mechanism9).

In this work, we have studied the electrochemistry of nitrofurantoin in a mixed buffered aqueous/DMF medium using cyclic voltammetry. Also, a comparative study of the reduction mechanism of this drug on carbon paste and carbon vitreous is included. Further work will be focused on the study of the electrocatalytic effect of modifying the carbon paste with enzymes included in metabolic nitroreduction processes.

EXPERIMENTAL

Drug and reagents

Nitrofurantoin was supplied by Laboratorio Chile S.A. (Santiago, Chile). Dimethylformamide (DMF), spectroscopic grade, was purchased from Merck.

Buffer systems

Citrate buffer (1.5 mM) containing aprotic solvent (DMF 60%) at pHs between 9-12, was used as the electrochemical solvent. The ionic strength was kept constant at 0.3 M with KCl.

Apparatus and electrodes

A thermostatic cell with three electrodes was employed. A platinum wire and a saturated calomel electrode (SCE) were used as auxiliary and reference electrodes rerspectively. A carbon vitreous and carbon paste electrodes were used as working electrodes for voltammetric experiments.

Electrochemical measurements were carried out in a CV-50 W BAS potentiostat connected to pc GATEWAY 2000.

Procedure

All the electrochemical runs were carried out in nitrogen atmosphere. Scan rates were varied between 0.05 and 5 V/s. The potential sweep was varied between 0 and -0.75 V or between 0 and -1.5 V. Between each run, the working electrode was cleaned by an electrochemical pulse at 0 V for 5 s.

RESULTS AND DISCUSSION

Nitrofurantoin has been reducible in all type of electrode, i.e., mercury and solid electrodes. In the present work a comparative study of the reduction of nitrofurantoin on carbon paste and glassy carbon electrodes was carried out. In Figure 2, we can observe the cyclic voltammograms (first sweep) of nitrofurantoin solution in a mixed media containing 40:60 citrate buffer (pH 9) / DMF on both electrodes. We can observe a monoelectronic reversible couple ArNO2/ArNO2.- with a cathodic potential peak (Ep.c) of -0.65 and -0.69 V on carbon paste and glassy carbon, respectively. Furthermore, an irreversible peak due to the further reduction via three electrons at -1.1 and -0.95 V, respectively, was also observed. The main characteristics of these peaks registered at a scan rate of 1 V/s are shown in Table I.

In order to study in isolation the ArNO2/ArNO2.- couple, the switching potential (El) was chosen at positive potentials relative to the second irreversible reduction peak. The tendency of an electrochemically generated species to undergo chemical reaction is reflected by the ip.a/ip.c ratio8). Thus, this ratio is equal to unity in the absence of a coupled reaction but decreases if the reduction product reacts later, i.e., a decline in the return wave occurs. Therefore, the cyclic voltammetric experiment can be used to prove the stability of the ArNO2.- species by changing the electrochemical (sweep rate) and chemical (pH) conditions and then by measuring the ip.a/ip.c values of the nitro/nitro radical anion couple.

The first condition under study was the dependence of pH on the current ratio. These studies have shown that the stability of ArNO2.- is greatly extended under alkaline conditions. In Figure 3, the voltammograms of the isolated nitro radical anion at several pH values on both types of electrodes are shown. The main parameters obtained from these couples are summarized in Table I. From these results, we can observe an increase in the cathodic peak potential as pH is increased, showing an enlarged energy requirement for the reduction in alkaline media. On the other hand, from the increase in the current ratio when pH is increased, we can assume the stabilization of the free radical in alkaline media.

FIG. 2. Cyclic voltammogram of nitrofurantoin 10-2 M in mixed citrate buffer/DMF 60%, pH = 9. vb = 1V/s.
a) Carbon paste electrode and b) glassy carbon electrode.

TABLE I. Current ratio and cathodic peak potentials of electrochemical reduction of NFT.


Glassy Carbon
Carbon paste
                 
pH Ep.cI (mV) (DEp) Ep.cII (mV) (ip.a/ip.c) Ep.cI(mV) (DEp) Ep.cII (mV) (ip.a/ip.c)

9 -690 130 -1150 0.759 -650 220 -950 0.888
10 -700 130 -1190 0.801 -660 190 -960 0.992
11 -710 110 -1240 0.922 -670 220 -940 0.994
12 -720 110 -1250 0.924 -680 230 -950 0.996

Furthermore, we have also studied the stability of the nitro radical anion from nitrofurantoin by changing the electrochemical conditions. The results on both types of electrodes show that as the scan rate increased the current ratio, ip.a/ip.c, increased towards unity (Figures 4a and 4b), typical behavior of an irreversible chemical reaction following a charge transfer step (EC process)9), in accordance with the following mechanism:

In consequence, at low scan rates the species reduced in step 1 is consumed in step 2, and therefore, not all is oxidized in the scan of return, i.e., (ip.a/ip.c < 1).

The results have revealed a dependence between current ratio, ip.a/ip.c, and the concentration of nitrofurantoin, which suggests a reaction of order different from one. According to the procedure described by Olmstead et al.10) we have evaluated the plot omega versus tau (Figure 5a and 5b), concluding that the chemical step is of second order. Also, the peak current was found to increase linearly with the square root of the scan rate, indicating a diffusion controlled electrode process.

FIG. 3. Effect of the pH on the cyclic voltammogram of nitrofurantoin 10-2 M in mixed citrate buffer/DMF 60%. vb = 1 V/s. a) carbon paste electrode and b) glassy carbon electrode.

FIG. 4. Variation of current ratio with log v at different pH values. a) Carbon paste electrode and b) glassy carbon electrode.

FIG. 5. Plot w versus t at different pH values. a) Carbon paste electrode and b) glassy carbon electrode.

The theory developed by Olmstead et al.10) allows us to calculate the rate constant k2 of the second order chemical reactions, from a single voltmmogram. According to this procedure, we have obtained the k2 values at different pHs on a carbon paste and glassy carbon electrodes (Table II). From the above results, we can conclude that alkalinity stabilizes the nitroradical, probably because its protonation is more difficult. Further, the nitro radical anion, at all pH values, is better stabilized on carbon paste electrodes than glassy carbon electrodes.

TABLE II. Constant of decline of the radical nitro in the reduction of NFT


k2(L/mol . s) 
pH Glassy Carbon Carbon paste

9 176 ± 13 126 ± 11
10 144 ± 9 105 ± 8
11 95 ± 7 68 ± 6
12 70 ± 5 51 ± 4

CONCLUSIONS

The nitro radical anion from nitrofurantoin was generated in both carbon paste and glassy carbon electrodes. According to the k2 values obtained, the nitro radical anion was better stabilized on carbon paste. These results are very interesting because gave support to the possible modification of the carbon paste with enzymes, in order to study nitroreduction in the presence of biocatalysts.

ACKNOWLEDGEMENTS

The authors express their gratitude for financial support from FONDECYT through PROJECT OF POST DOCTORATE N 3970023.
_______________________________
*To whom correspondence should be addressed: P.O. Box 233 Santiago 1, Fax: 56-2-7378920,
e-mail: asquella@ll.ciq.uchile.cl

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