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A nitro radical anion formation from nifedipine: an electrochemical approach
inwtoctio~ F I.SEVIER S('IENTII'I( P[ BIISHFRK IR[ I.ANI)
Chemico-Biological Interactions 89 (1993) 197-205
A nitro radical anion formation from nifedipine:
an electrochemical approach J.A. Squella *a, C. Solabarrieta a, L.J. Nufiez-Vergara b aLaboratorio de Electroquimica, hLaboratorio de Farmacologia, Facultad de Ciencias Quimicas y Farmac~uticas, Universidad de Chile, Casilla 233 Santiago 1 Chile
(Received 29 March 1993; revision received 14 June 1993; accepted 15 June 1993)
Abstract The cyclic voltammetric behaviour of nifedipine was studied. The addition of three aprotic solvents to nifedipine in an aqueous citrate buffer system was examined. Qualitatively they result in separation of the initial irreversible 4 electron reduction into two stages, the NO2/RNO2" - and RNO 2" -, 4H+/RNHOH, H20 couples, respectively. Particular attention was directed to the l-electron RNO:,/RNO 2" - couple as measured by the cyciic voltammetric mode in mixed media. Analysis of the cyclic voitammetric response as a function of scan rate and non-aqueous solvent content yields information on the stability of the radical anion. The chemical forward reaction of the radical anion follows a second order kinetics with a stability constant of 1.1 × 10 -3 1 mol -I s -l and a half-life time of 0.09 s for 1 mM of nifedipine in aqueous citrate buffer, pH 7.4/DMF; 50:50. Key words: Nitro radical anion; Nifedipine; Cyclic voltammetry
1. Introduction Several studies [ ! - 4 ] have suggested that the r a d i c a l - a n i o n s (one-electron reduction products, R N O 2" -) o f n i t r o a r o m a t i c a n d nitroheterocyclic c o m p o u n d s (RNO2) are obligate intermediates in the enzymatic reduction o f these c o m p o u n d s to h y d r o x y l a m i n e s o r amines a c c o r d i n g to the following equation: ArNO2 + ' N i t r o r e d u c t a s e s '
-
ArNO2" -
" Corresponding author. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0009-2797(93)03217-1
(1)
198
J.A. Squella et al. ~Chem.-Biol. Interact. 89 (1993) 197-205
The use of nitroheterocyclic drugs as antibacterial, antiprotozoal and anticancer agents is well established and newer derivatives have attracted much attention in the treatment of hypoxic tumours. The cytotoxic activity of such drugs depends upon reduction of the nitro group, usually at low redox potentials which are normally unattainable in well-oxygenated cells. The relative reduction rates in hypoxia or anoxia and under toxic conditions is the basis of their selective toxicity and therapeutic differential. However, there are at present many other nitrocompounds extensively used in medicine, but where the pharmacological activity is not directly related to the reduction of the nitro group. 4-Nitroaryl- 1, 4 dihydropyridines extensively used for its cardioactivity belong to the above mentioned compounds. Very soon after the discovery of the cardiovascular properties of i,4-dihydropyridines, these substances were found to be inhibitors of the entry of Ca 2÷ into the cells of cardiac and vascular muscle through the voltage-dependent calcium channels [5]. To this day, the 1,4dihydropyridines are still the most potent group of calcium channel blockers or 'calcium antagonists'. Some of the most widely used members of the above family are: nifedipine, nitrendipine, nimodipine, nisoldipine and nicardipine. Nifedipine, the first member of 1,4-dihydropyridine family was successfully introduced to the pharmaceutical market at the beginning of 1975 for the treatment of coronary diseases
I51. Although these dihydropyridines are nitroaromatic compounds, its pharmacological activity is related to the dihydropyridine ring [5]. However, there is no chemical basis to discard a metabolic route with formation of nitro radical anions. Obviously, the formation of these free radicals from the nitroaromatic dihydropyridines would produce undesirable side-effects to the pharmacological ones. The combination of identification and measurement of steady-state concentrations of radicals by electron spin resonance (E.S.R.) and spectrophotometric monitoring of reaction kinetics following radical generation by pulse radiolysis [6] has provided considerable insight into the most important reactions and properties of nitro radical anions. On the other hand, electrochemical techniques are valuable tools for studying the kinetics and mechanistic aspects of ion radical reactions [7-9], one major advantage is that the ion radicals are generated and the kinetics of their follow-up reactions can be monitored during the same experiment. Electrochemical techniques, specifically cyclic voltammetry, have been used in studies related to nitro radical anion formation from nitroheterocyclic compounds of biological interest [10-15]. Recently, a cyclic voltammetric study of the electrochemically generated nitro radical anion from nitrendipine and nimodipine has been reported [16,17]. An 'in vitro' nitro radical anion formation from nifedipine is presented in order to emphasize the unreported nitroaromatic character of some 1,4-dihydropyridine calcium antagonists. Besides, a method capable of studying further reactions of the free radical is shown.
2. Experimental procedures 2.1. Drugs and reagents
Nifedipine (NFD) was supplied
by Bayer Lab. Santiago, Chile (100%
J.A. Squella et al. ~Chem.-Biol. Interact. 89 (1993) 197-205
199
chromatographically pure, 99.8% activity). Dimethylformamide (DMF), acetonitrile (ACN) and dioxane (DX) were of spectroscopic grade.
2.2. Buffer systems 1.5 mM Trisodium citrate buffer containing various proportions of either ethanol or aprotic solvent (DMF, ACN, DX) was used as the electrochemical solvent (expressed as % v/v of the ethanol or aprotic solvent content). The ionic strength was kept constant at 0.3 M with KC. 2.3. Drug solutions Stock solutions of N F D either in ethanol or DMF, ACN and DX were prepared and protected from daylight to avoid photodecomposition [18]. The routine drug concentration was maintained in 1.0 mM at all non-aqueous solvent percent. 2.4. Cyclic voltammetry Electrochemical measurements were carried out with an lnelecsa assembly similar to one described in a previous paper [16]. 2.5. Electrodes A Metrohm hanging mercury drop electrode with a drop surface of 1.39 mm 2 as working electrode and a platinum wire as the counter electrode were used. All potentials were measured against a saturated calomel electrode (SCE). All cyclic voltammograms were carried out at a constant temperature of 25°C and the solutions were purged with pure nitrogen for ten minutes before the voltammetric runs. The return-to-forward peak current ratio, ip,a/ip,c, for the reversible first electron transfer (RNO2/RNO2" - couple) was measured, varying the scan rate (v) from 0.1 up to 150 Vs -t. The experimental current ratios were calculated according to Nicholson's procedure [19].
3. Results and discussion
The cyclic voltammetric behaviour of nifedipine indicates that the mechanism depends markedly on the solvent system and the supporting electrolyte used. Fig. la shows a cyclic voltammogram of 1 mM nifedipine solution at pH 7.4 in a citrate buffer with ethanol (60/40). A irreversible cathodic peak I with a peak potential (Ep) o f - 0 . 9 7 V corresponds to the four-electron reduction of the nitro group of nifedipine according to equation 1. An anodic peak (II) also appears with an Ep of -0.4 V that corresponds to the oxidation of the hydroxylamine derivative formed in the cathodic sweep which is represented by equation 2: 1
RNO 2 + 4e- + 4H +
- R N H O H + H20
(1)
11
RNHOH
:
-- R N O + 2 e - + 2 H Ill
÷
(2)
200
J.d. Squella et al. / Chem.-Biol. Interact. 89 (1993) 197-205
I
[
V
2o uA
f
II
I
III
II
295
12.50
- E
r1~v
Fig. I. Cyclic voltammogram of I mM nifedipine at pH 7.4 in citrate buffer:ethanol (60:40), (a) first sweep and (b) second sweep.
Fig. lb shows a cathodic peak (III) at -0.33 V, in the second sweep, corresponding to the reduction of the nitroso derivative according to equation 2. The above results are consistent with several earlier works from which a similar scheme was established for nitrobenzene and related aromatic nitro compounds [20]. This shows that from the electrochemistry point of view, nifedipine can be considered an aromatic nitro compound. Consequently, the above character permits to postulate a reduction pathway as a possible metabolic route for nifedipine. On the other hand, the addition of an aprotic solvent (dimethylformamide, acetonitrile and dioxane) to an aqueous citrate buffered nifedipine solution at pH 7.4 permits to establish the presence of other different reduction processes. The voltammogram in Fig. 2 allows us to distinguish a new couple (IV-V) with a cathodic peak potential (Ep.¢) of -0.92 V and an anodic peak potential (Ep.a) of -0.86 V. The AEp value of 60 mV implies that a monoelectronic reduction of the nitro group
201
J.A. Squella et al. / Chem.-Biol. Interact. 89 (1993) 197-205
lo uA
50
1750
-E
mV
Fig. 2. Cyclic voltammogram of nifedipine in a mixed media (aqueous citrate buffer:DMF) 50:50.
is involved in this couple in order to produce the nitro radical anion. An irreversible couple appears at more cathodic potentials due to the three-electron reduction of the nitro radical anion. Consequently,the observed voltammogram in mixed media can be described by the following equations: v
RNO 2 + e -
-- -- RNO2"-
(3)
IV I
R N O 2 " - + 3e- + 4H ÷
• RNHOH + 2H20
(4)
In order to study in isolation the RNO2/RNO2"-, the switching potential (Ev) was chosen at positive potentials relative to the second irreversible reduction peak. The tendency of an electrochemically generated species to undergo chemical following reactions is reflected by the ip.a/ip,c ratio [21]. Thus this ratio equals to unity in absence of 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 RNO 2" - species by changing electrochemical and chemical conditions, and then by measuring the ip,a/ ip.¢ values of the nitro~anion radical nitro couple. The first chemical condition under study was the dependence of the aprotic solvent content on the current ratio. The results show that while the aprotic solvent percentage was increased, the ip.a/ip,c ratio increased as well. This was true up to a certain value (depending on solvent nature) above which
J.A. Squella et al. / Chem.-Biol. Interact. 89 (1993) 197-205
202
no further changes in the ratio were observed. These results illustrate the extended lifetime of the R N O 2 " - species, i.e., the lack of protons in the media favours the stability of the free radical by hindering the later protonation. This procedure permits to examine the influence of pH on the radical lifetimes. These studies have shown that the stability of RNO2" - is greatly extended under alkaline conditions, i.e., there is an increase in the ip.a/ip,c ratio when adding of NaOH to the electrolytic medium. However, determining radical stability in mixed solvents as a function of pH is of limited value due to the non-reliability of pH measurements under these conditions [22]. On the other hand, we also studied the stability of the RNO2" - species by changing the electrochemical conditions, e.g., the scan rate, and keeping the chemical conditions of the solutions constant. The results show that as the scan rate increased, the ip.a/ip,c increased towards unity, a typical behaviour for an irreversible chemical reaction following a charge-transfer step, i.e., the EC process [23] In order to check the order of the following chemical reaction, we studied the dependence between the ip.a/ip.c ratio and nifedipine concentration. It must be stressed that a diagnostic criterion suitable for discriminating EC processes involving chemical reactions with order n ~ 1 from those with n = 1 is given by the dependence of ip,a/ip,c ratio with the concentration of the reactant (RNO2), which is observed only in the first case [23]. Our results clearly reveal a dependence between the ip.a/ip.¢ ratio and nifedipine concentration, suggesting a second order reaction for the chemical step according to the following mechanism: RNO 2 + e -
:
:
RNO2"-
2 RNO 2
~ Products
(5) (6)
The theory of cyclic voltammetry for a second order reaction electrochemically initiated was exhaustively studied by Olmstead et al. [23]. According to this theory, we have obtained linear relationships between the kinetic parameters o~ and r. This is conclusive of a second order chemical reaction for the nitro radical anion decomposition. On the basis of the results obtained by the reduction schemes of nitro groups [20], presumably the second order chemical reaction is the dismutation of the nitro radical anion. Furthermore, the theory developed by Olmstead et al. [24] permits us to calculate the rate constant k2 of the second order chemical reactions, using a single voltammogram. The procedure is as follows: (a) the ip.a/ip,c ratio from a single voltammogram is measured. (b) The above ratio is interpolated in the working curve described [24] for this type of process, obtaining the values of k 2, Co and r. As Co and r are known, k2 is attained. Table I exhibits the rate constant k2 and the halflife times for the second order reaction of the radical anion of nifedipine in three different mixed media. The half-life time is concentration-dependent for a second order chemical kinetics thus, in order to calculate this value we have used both, a concentration value of 1 mM (voltammetric concentration of nifedipine) and another concentration value of 0.057 tzM (similar to the in vivo concentrations of nifedipine)
203
J.A. Squella et aL ~Chem.-Biol. Interact. 89 (1993) 197-205
Table 1 k2 Rate constants and half-life times for the second order chemical reaction of the nitro radical anion formed from nifedipine Solventa
k2 x 10-3 (1 mol -t s -j)
tl;2 (s)b
tl;2 (s)c x 10-3
DMF ACN DX
I1 3.7 2.8
0.09 0.27 0.34
1.58 4.73 6.34
aThe solvent cOntains aqueous citrate buffer, pH 7.4/non-aqueous solvent, 50:50. t'l mM Nifedipine radical anion. c0.057 v.M Nifedipine radical anion.
[25]. Our results show that in vivo concentrations o f nifedipine produce a more stable nitro radical anion (hi2 = 1580 s) and consequently, the feasibility o f the appearance o f toxic effects with chronic treatment with this type o f drugs seems to be a matter o f high interest. The reproducibility o f the method was tried for the ip.a /ip. c ratio and log k2 measuring ten independent runs of the same drug solutions under the same experimental conditions (50% D M F , pH 7.4 and 1 V/s sweep rate). An average variation coefficient o f 3.6% and 2.2% was obtained for both current ratio and log k2, respectively. Our study demonstrates that nifedipine electrochemical behaviour is o f a nitroaryl nature and, consequently, the one-electron reduction with formation o f a free radical derivative is the first step in its reduction pathway. Furthermore, we have compared some nitroaryl 1,4 dihydropyridines whose ability to form n i t r o radical anions have been demonstrated only in vitro [16,17] with other known, biologically active nitroaromatics (Table 2). According to these results, we can conclude that there are no significant differences between 1,4 dihydropyridines and the other nitroaromatics, particularly tinidazole, metronidazole and nitrobencene. This implies that the energy requirements for reduction are relatively similar. On the other hand, the feasibility o f further in vivo reactions (with 02, D N A , GSH, etc.) is highly probable
Table 2 Peak potentials for the one-electron reduction of different nitroaromatic compounds Nitrocompound
Ep.c (V vs. SCE)
Tinidazole Metronidazole Furazolidone Nitrobenzene Nifedipine Nitrendipine Nimodipine
-0.80 -0.84 -0.58 -0.85 -0.92 -0.83 -0.82
Values are obtained by cyclic voltammetry in aqueous citrate buffer (pH 7.4)/DMF: 50:50.
204
J.A. Squella et al. ~Chem.-Biol. Interact. 89 (1993) 197-205
because of the high stability of the nitro radical anion. Therefore, the availability of a method capable of generating the radical anion and studying its reactions in the same experiment, is a very important tool for this research area. 4. Acknowledgements This research was supported by grants 91/881 and Q-3118/9223 from FONDECYT and DTI Universidad de Chile, respectively. The authors also express their thanks to Prof. C. Telha for his help in revising this manuscript. 5. References 1 P. Wardman, Some reactions and properties of nitro radical anions important in biology and medicine, Environ. Health Perspect., 64 (1985) 309-320. 2 R. Docampo, S. Moreno, A. Stoppani, W. Leon, F. Cruz, F. Villalta and R. Mu~iz, Mechanism of nifurtimox toxicity in different forms of Trypanosoma cruzi, Biochem. Pharmacol., 30 (1981) 1947-1951. 3 S. Moreno, R.P. Mason, R. Muffiz, F. Cruz and R. Docampo, Generation of free radicals from metronidazole and other nitroimidazoles by Tritichomonas foetus, J, Biol. Chem., 258 (1983) 4051-4054. 4 R. Mason and J. Holtzman, ESR spectra of free radicals formed from nitroaromatic drugs by microsomal nitroreductase, Pharmacologist, 16 (1974) 277. 5 S. Goldmann and J. Stoltefuss, 1,4-Dihydropyridines: effects of chirality and conformation on the calcium antagonist and calcium agonist activities, Angew. Chem. Int. Ed. Engl., 30 (1991) 1559-1578. 6 P. Wardman, Application of pulse radiolysis methods to study the reactions and structure of biomolecules, Rep. Prog. Phys., 41 (1978) 259-302. 7 C. Corvaja, G. Farnia and E. Vianello, Kinetics of decay of nitrophenol radical anions and reduction mechanism of nitrophenols in aqueous alkaline media, Electrochim. Acta II (1966) 919-929. 8 L.H Piette, P. Ludwig and R.N. Adams, EPR and electrochemistry, Studies of electrochemically generated radical ions in aqueous solution, Anal. Chem., 34 (1962) 8. 9 W.H. Smyth and A.J. Bard, Electrochemical reactions of organic compounds in liquid ammonia, 1I, Nitrobenzene and nitrosobenzene, J. Am. Chem. Soc., 97 (1975) 5203-5210. 10 J.H~ Tocher and D.i. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, I The influence of solvent, Free Rad. Res. Commun., 4(5) (1988) 269-276. I 1 J.H. Tocher and D.I. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, II! Nitroso derivative formation, Free Rad. Res. Commun., 5(6) (1989) 327- 332. 12 J.H. Tocher and D.I. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, IV Lifetime of the metronidazole radical anion, Free Rad. Res. Commun., 6(I) (1989) 39-45. 13 J.H. Tocher and D.I. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, V Measurements and comparison of nitroradical lifetimes, Int. J. Radiat. Biol., 57(I) (1990) 45-53. 14 T. Symonds, J.H. Tocher, D. Tother and D. Edwards Electrochemical characteristics of nitroheterocyclic compounds of biological interest, VIi Effect of electrode material, Free. Rad. Res. Commun., 14(1) (1991) 33-40. 15 J. Tocher and D.I. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, VIii Stability of nitro radical anions from cyclic voltammetric studies, Free Rad. Res. Commun., 16(1) (1992) 19-25. 16 J.A. Squella, J. Mosre, M. Blazquez and L.J. Nunez-Vergara, Cyclic voltammetric study of the nitro radical anion from nitrendipine generated electrochemically, J. Electroanal. Chem., 319(I) (1991) 177-184.
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17 18 19 20 21 22 23 24 25
205
L.J. Nunez-Vergara, S. Bollo, A. Alvarez, J.A. Squella and M. Biazquez, Nitro radical anion formation from nimodipine, J. Electroanal. Chem., 345(I,2) (1993) 121-134. J.A. Squella, E. Barnafi, S. Perna and L.J. Nunez-Vergara, Nifedipine: differential pulse polarography and photodecomposition, Talanta, 36(3) (1989) 363-366. R.S. Nicholson, A simple procedure to calculate the current ratio, Anal. Chem., 36 (1964) 1406. B. Kastening, Free radicals in organic polarography, Nitro and nitroso compounds, in: P. Zuman and L. Meites (Eds.), Progress in Polarography, Vol. 3, Chap. 4, 1972, pp. 261. R.S. Nicholson and I. Shain, Theory of stationary electrode polarography, single scan and cyclic methods applied to reversible, irreversible and kinetic systems, Anal. Chem., 36 (1964) 706. A. Albert and E.P. Sorgeant, Ionization contents of acids and bases, Methuen, London, 1962. G. Bontempelli, F. Magno, G. Mazzochin and R. Seeger, Linear sweep and cyclic voltammetry, Annail di Chimica, 79 (1989) 138. M. Olmstead, R. Hamilton and R.S. Nicholson, Theory of cyclic voltammetry for a dimerization reaction initiated electrochemically, Anal. Chem., 41 (1969) 260. L.Z. Benet and R.L. Williams, Dise~o y optimizaci6n de los regimenes de dosificaci6n, datos far-, macocin6ticos, in: A. Goodman, T.W. Rail, A. Nies and P. Taylor (Eds.), Las bases farmacol6gicas de la terapdutica, Editorial Panamericana, 1990, pp. 1642.
Chemico-Biological Interactions 89 (1993) 197-205
A nitro radical anion formation from nifedipine:
an electrochemical approach J.A. Squella *a, C. Solabarrieta a, L.J. Nufiez-Vergara b aLaboratorio de Electroquimica, hLaboratorio de Farmacologia, Facultad de Ciencias Quimicas y Farmac~uticas, Universidad de Chile, Casilla 233 Santiago 1 Chile
(Received 29 March 1993; revision received 14 June 1993; accepted 15 June 1993)
Abstract The cyclic voltammetric behaviour of nifedipine was studied. The addition of three aprotic solvents to nifedipine in an aqueous citrate buffer system was examined. Qualitatively they result in separation of the initial irreversible 4 electron reduction into two stages, the NO2/RNO2" - and RNO 2" -, 4H+/RNHOH, H20 couples, respectively. Particular attention was directed to the l-electron RNO:,/RNO 2" - couple as measured by the cyciic voltammetric mode in mixed media. Analysis of the cyclic voitammetric response as a function of scan rate and non-aqueous solvent content yields information on the stability of the radical anion. The chemical forward reaction of the radical anion follows a second order kinetics with a stability constant of 1.1 × 10 -3 1 mol -I s -l and a half-life time of 0.09 s for 1 mM of nifedipine in aqueous citrate buffer, pH 7.4/DMF; 50:50. Key words: Nitro radical anion; Nifedipine; Cyclic voltammetry
1. Introduction Several studies [ ! - 4 ] have suggested that the r a d i c a l - a n i o n s (one-electron reduction products, R N O 2" -) o f n i t r o a r o m a t i c a n d nitroheterocyclic c o m p o u n d s (RNO2) are obligate intermediates in the enzymatic reduction o f these c o m p o u n d s to h y d r o x y l a m i n e s o r amines a c c o r d i n g to the following equation: ArNO2 + ' N i t r o r e d u c t a s e s '
-
ArNO2" -
" Corresponding author. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0009-2797(93)03217-1
(1)
198
J.A. Squella et al. ~Chem.-Biol. Interact. 89 (1993) 197-205
The use of nitroheterocyclic drugs as antibacterial, antiprotozoal and anticancer agents is well established and newer derivatives have attracted much attention in the treatment of hypoxic tumours. The cytotoxic activity of such drugs depends upon reduction of the nitro group, usually at low redox potentials which are normally unattainable in well-oxygenated cells. The relative reduction rates in hypoxia or anoxia and under toxic conditions is the basis of their selective toxicity and therapeutic differential. However, there are at present many other nitrocompounds extensively used in medicine, but where the pharmacological activity is not directly related to the reduction of the nitro group. 4-Nitroaryl- 1, 4 dihydropyridines extensively used for its cardioactivity belong to the above mentioned compounds. Very soon after the discovery of the cardiovascular properties of i,4-dihydropyridines, these substances were found to be inhibitors of the entry of Ca 2÷ into the cells of cardiac and vascular muscle through the voltage-dependent calcium channels [5]. To this day, the 1,4dihydropyridines are still the most potent group of calcium channel blockers or 'calcium antagonists'. Some of the most widely used members of the above family are: nifedipine, nitrendipine, nimodipine, nisoldipine and nicardipine. Nifedipine, the first member of 1,4-dihydropyridine family was successfully introduced to the pharmaceutical market at the beginning of 1975 for the treatment of coronary diseases
I51. Although these dihydropyridines are nitroaromatic compounds, its pharmacological activity is related to the dihydropyridine ring [5]. However, there is no chemical basis to discard a metabolic route with formation of nitro radical anions. Obviously, the formation of these free radicals from the nitroaromatic dihydropyridines would produce undesirable side-effects to the pharmacological ones. The combination of identification and measurement of steady-state concentrations of radicals by electron spin resonance (E.S.R.) and spectrophotometric monitoring of reaction kinetics following radical generation by pulse radiolysis [6] has provided considerable insight into the most important reactions and properties of nitro radical anions. On the other hand, electrochemical techniques are valuable tools for studying the kinetics and mechanistic aspects of ion radical reactions [7-9], one major advantage is that the ion radicals are generated and the kinetics of their follow-up reactions can be monitored during the same experiment. Electrochemical techniques, specifically cyclic voltammetry, have been used in studies related to nitro radical anion formation from nitroheterocyclic compounds of biological interest [10-15]. Recently, a cyclic voltammetric study of the electrochemically generated nitro radical anion from nitrendipine and nimodipine has been reported [16,17]. An 'in vitro' nitro radical anion formation from nifedipine is presented in order to emphasize the unreported nitroaromatic character of some 1,4-dihydropyridine calcium antagonists. Besides, a method capable of studying further reactions of the free radical is shown.
2. Experimental procedures 2.1. Drugs and reagents
Nifedipine (NFD) was supplied
by Bayer Lab. Santiago, Chile (100%
J.A. Squella et al. ~Chem.-Biol. Interact. 89 (1993) 197-205
199
chromatographically pure, 99.8% activity). Dimethylformamide (DMF), acetonitrile (ACN) and dioxane (DX) were of spectroscopic grade.
2.2. Buffer systems 1.5 mM Trisodium citrate buffer containing various proportions of either ethanol or aprotic solvent (DMF, ACN, DX) was used as the electrochemical solvent (expressed as % v/v of the ethanol or aprotic solvent content). The ionic strength was kept constant at 0.3 M with KC. 2.3. Drug solutions Stock solutions of N F D either in ethanol or DMF, ACN and DX were prepared and protected from daylight to avoid photodecomposition [18]. The routine drug concentration was maintained in 1.0 mM at all non-aqueous solvent percent. 2.4. Cyclic voltammetry Electrochemical measurements were carried out with an lnelecsa assembly similar to one described in a previous paper [16]. 2.5. Electrodes A Metrohm hanging mercury drop electrode with a drop surface of 1.39 mm 2 as working electrode and a platinum wire as the counter electrode were used. All potentials were measured against a saturated calomel electrode (SCE). All cyclic voltammograms were carried out at a constant temperature of 25°C and the solutions were purged with pure nitrogen for ten minutes before the voltammetric runs. The return-to-forward peak current ratio, ip,a/ip,c, for the reversible first electron transfer (RNO2/RNO2" - couple) was measured, varying the scan rate (v) from 0.1 up to 150 Vs -t. The experimental current ratios were calculated according to Nicholson's procedure [19].
3. Results and discussion
The cyclic voltammetric behaviour of nifedipine indicates that the mechanism depends markedly on the solvent system and the supporting electrolyte used. Fig. la shows a cyclic voltammogram of 1 mM nifedipine solution at pH 7.4 in a citrate buffer with ethanol (60/40). A irreversible cathodic peak I with a peak potential (Ep) o f - 0 . 9 7 V corresponds to the four-electron reduction of the nitro group of nifedipine according to equation 1. An anodic peak (II) also appears with an Ep of -0.4 V that corresponds to the oxidation of the hydroxylamine derivative formed in the cathodic sweep which is represented by equation 2: 1
RNO 2 + 4e- + 4H +
- R N H O H + H20
(1)
11
RNHOH
:
-- R N O + 2 e - + 2 H Ill
÷
(2)
200
J.d. Squella et al. / Chem.-Biol. Interact. 89 (1993) 197-205
I
[
V
2o uA
f
II
I
III
II
295
12.50
- E
r1~v
Fig. I. Cyclic voltammogram of I mM nifedipine at pH 7.4 in citrate buffer:ethanol (60:40), (a) first sweep and (b) second sweep.
Fig. lb shows a cathodic peak (III) at -0.33 V, in the second sweep, corresponding to the reduction of the nitroso derivative according to equation 2. The above results are consistent with several earlier works from which a similar scheme was established for nitrobenzene and related aromatic nitro compounds [20]. This shows that from the electrochemistry point of view, nifedipine can be considered an aromatic nitro compound. Consequently, the above character permits to postulate a reduction pathway as a possible metabolic route for nifedipine. On the other hand, the addition of an aprotic solvent (dimethylformamide, acetonitrile and dioxane) to an aqueous citrate buffered nifedipine solution at pH 7.4 permits to establish the presence of other different reduction processes. The voltammogram in Fig. 2 allows us to distinguish a new couple (IV-V) with a cathodic peak potential (Ep.¢) of -0.92 V and an anodic peak potential (Ep.a) of -0.86 V. The AEp value of 60 mV implies that a monoelectronic reduction of the nitro group
201
J.A. Squella et al. / Chem.-Biol. Interact. 89 (1993) 197-205
lo uA
50
1750
-E
mV
Fig. 2. Cyclic voltammogram of nifedipine in a mixed media (aqueous citrate buffer:DMF) 50:50.
is involved in this couple in order to produce the nitro radical anion. An irreversible couple appears at more cathodic potentials due to the three-electron reduction of the nitro radical anion. Consequently,the observed voltammogram in mixed media can be described by the following equations: v
RNO 2 + e -
-- -- RNO2"-
(3)
IV I
R N O 2 " - + 3e- + 4H ÷
• RNHOH + 2H20
(4)
In order to study in isolation the RNO2/RNO2"-, the switching potential (Ev) was chosen at positive potentials relative to the second irreversible reduction peak. The tendency of an electrochemically generated species to undergo chemical following reactions is reflected by the ip.a/ip,c ratio [21]. Thus this ratio equals to unity in absence of 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 RNO 2" - species by changing electrochemical and chemical conditions, and then by measuring the ip,a/ ip.¢ values of the nitro~anion radical nitro couple. The first chemical condition under study was the dependence of the aprotic solvent content on the current ratio. The results show that while the aprotic solvent percentage was increased, the ip.a/ip,c ratio increased as well. This was true up to a certain value (depending on solvent nature) above which
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no further changes in the ratio were observed. These results illustrate the extended lifetime of the R N O 2 " - species, i.e., the lack of protons in the media favours the stability of the free radical by hindering the later protonation. This procedure permits to examine the influence of pH on the radical lifetimes. These studies have shown that the stability of RNO2" - is greatly extended under alkaline conditions, i.e., there is an increase in the ip.a/ip,c ratio when adding of NaOH to the electrolytic medium. However, determining radical stability in mixed solvents as a function of pH is of limited value due to the non-reliability of pH measurements under these conditions [22]. On the other hand, we also studied the stability of the RNO2" - species by changing the electrochemical conditions, e.g., the scan rate, and keeping the chemical conditions of the solutions constant. The results show that as the scan rate increased, the ip.a/ip,c increased towards unity, a typical behaviour for an irreversible chemical reaction following a charge-transfer step, i.e., the EC process [23] In order to check the order of the following chemical reaction, we studied the dependence between the ip.a/ip.c ratio and nifedipine concentration. It must be stressed that a diagnostic criterion suitable for discriminating EC processes involving chemical reactions with order n ~ 1 from those with n = 1 is given by the dependence of ip,a/ip,c ratio with the concentration of the reactant (RNO2), which is observed only in the first case [23]. Our results clearly reveal a dependence between the ip.a/ip.¢ ratio and nifedipine concentration, suggesting a second order reaction for the chemical step according to the following mechanism: RNO 2 + e -
:
:
RNO2"-
2 RNO 2
~ Products
(5) (6)
The theory of cyclic voltammetry for a second order reaction electrochemically initiated was exhaustively studied by Olmstead et al. [23]. According to this theory, we have obtained linear relationships between the kinetic parameters o~ and r. This is conclusive of a second order chemical reaction for the nitro radical anion decomposition. On the basis of the results obtained by the reduction schemes of nitro groups [20], presumably the second order chemical reaction is the dismutation of the nitro radical anion. Furthermore, the theory developed by Olmstead et al. [24] permits us to calculate the rate constant k2 of the second order chemical reactions, using a single voltammogram. The procedure is as follows: (a) the ip.a/ip,c ratio from a single voltammogram is measured. (b) The above ratio is interpolated in the working curve described [24] for this type of process, obtaining the values of k 2, Co and r. As Co and r are known, k2 is attained. Table I exhibits the rate constant k2 and the halflife times for the second order reaction of the radical anion of nifedipine in three different mixed media. The half-life time is concentration-dependent for a second order chemical kinetics thus, in order to calculate this value we have used both, a concentration value of 1 mM (voltammetric concentration of nifedipine) and another concentration value of 0.057 tzM (similar to the in vivo concentrations of nifedipine)
203
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Table 1 k2 Rate constants and half-life times for the second order chemical reaction of the nitro radical anion formed from nifedipine Solventa
k2 x 10-3 (1 mol -t s -j)
tl;2 (s)b
tl;2 (s)c x 10-3
DMF ACN DX
I1 3.7 2.8
0.09 0.27 0.34
1.58 4.73 6.34
aThe solvent cOntains aqueous citrate buffer, pH 7.4/non-aqueous solvent, 50:50. t'l mM Nifedipine radical anion. c0.057 v.M Nifedipine radical anion.
[25]. Our results show that in vivo concentrations o f nifedipine produce a more stable nitro radical anion (hi2 = 1580 s) and consequently, the feasibility o f the appearance o f toxic effects with chronic treatment with this type o f drugs seems to be a matter o f high interest. The reproducibility o f the method was tried for the ip.a /ip. c ratio and log k2 measuring ten independent runs of the same drug solutions under the same experimental conditions (50% D M F , pH 7.4 and 1 V/s sweep rate). An average variation coefficient o f 3.6% and 2.2% was obtained for both current ratio and log k2, respectively. Our study demonstrates that nifedipine electrochemical behaviour is o f a nitroaryl nature and, consequently, the one-electron reduction with formation o f a free radical derivative is the first step in its reduction pathway. Furthermore, we have compared some nitroaryl 1,4 dihydropyridines whose ability to form n i t r o radical anions have been demonstrated only in vitro [16,17] with other known, biologically active nitroaromatics (Table 2). According to these results, we can conclude that there are no significant differences between 1,4 dihydropyridines and the other nitroaromatics, particularly tinidazole, metronidazole and nitrobencene. This implies that the energy requirements for reduction are relatively similar. On the other hand, the feasibility o f further in vivo reactions (with 02, D N A , GSH, etc.) is highly probable
Table 2 Peak potentials for the one-electron reduction of different nitroaromatic compounds Nitrocompound
Ep.c (V vs. SCE)
Tinidazole Metronidazole Furazolidone Nitrobenzene Nifedipine Nitrendipine Nimodipine
-0.80 -0.84 -0.58 -0.85 -0.92 -0.83 -0.82
Values are obtained by cyclic voltammetry in aqueous citrate buffer (pH 7.4)/DMF: 50:50.
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because of the high stability of the nitro radical anion. Therefore, the availability of a method capable of generating the radical anion and studying its reactions in the same experiment, is a very important tool for this research area. 4. Acknowledgements This research was supported by grants 91/881 and Q-3118/9223 from FONDECYT and DTI Universidad de Chile, respectively. The authors also express their thanks to Prof. C. Telha for his help in revising this manuscript. 5. References 1 P. Wardman, Some reactions and properties of nitro radical anions important in biology and medicine, Environ. Health Perspect., 64 (1985) 309-320. 2 R. Docampo, S. Moreno, A. Stoppani, W. Leon, F. Cruz, F. Villalta and R. Mu~iz, Mechanism of nifurtimox toxicity in different forms of Trypanosoma cruzi, Biochem. Pharmacol., 30 (1981) 1947-1951. 3 S. Moreno, R.P. Mason, R. Muffiz, F. Cruz and R. Docampo, Generation of free radicals from metronidazole and other nitroimidazoles by Tritichomonas foetus, J, Biol. Chem., 258 (1983) 4051-4054. 4 R. Mason and J. Holtzman, ESR spectra of free radicals formed from nitroaromatic drugs by microsomal nitroreductase, Pharmacologist, 16 (1974) 277. 5 S. Goldmann and J. Stoltefuss, 1,4-Dihydropyridines: effects of chirality and conformation on the calcium antagonist and calcium agonist activities, Angew. Chem. Int. Ed. Engl., 30 (1991) 1559-1578. 6 P. Wardman, Application of pulse radiolysis methods to study the reactions and structure of biomolecules, Rep. Prog. Phys., 41 (1978) 259-302. 7 C. Corvaja, G. Farnia and E. Vianello, Kinetics of decay of nitrophenol radical anions and reduction mechanism of nitrophenols in aqueous alkaline media, Electrochim. Acta II (1966) 919-929. 8 L.H Piette, P. Ludwig and R.N. Adams, EPR and electrochemistry, Studies of electrochemically generated radical ions in aqueous solution, Anal. Chem., 34 (1962) 8. 9 W.H. Smyth and A.J. Bard, Electrochemical reactions of organic compounds in liquid ammonia, 1I, Nitrobenzene and nitrosobenzene, J. Am. Chem. Soc., 97 (1975) 5203-5210. 10 J.H~ Tocher and D.i. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, I The influence of solvent, Free Rad. Res. Commun., 4(5) (1988) 269-276. I 1 J.H. Tocher and D.I. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, II! Nitroso derivative formation, Free Rad. Res. Commun., 5(6) (1989) 327- 332. 12 J.H. Tocher and D.I. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, IV Lifetime of the metronidazole radical anion, Free Rad. Res. Commun., 6(I) (1989) 39-45. 13 J.H. Tocher and D.I. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, V Measurements and comparison of nitroradical lifetimes, Int. J. Radiat. Biol., 57(I) (1990) 45-53. 14 T. Symonds, J.H. Tocher, D. Tother and D. Edwards Electrochemical characteristics of nitroheterocyclic compounds of biological interest, VIi Effect of electrode material, Free. Rad. Res. Commun., 14(1) (1991) 33-40. 15 J. Tocher and D.I. Edwards, Electrochemical characteristics of nitro-heterocyclic compounds of biological interest, VIii Stability of nitro radical anions from cyclic voltammetric studies, Free Rad. Res. Commun., 16(1) (1992) 19-25. 16 J.A. Squella, J. Mosre, M. Blazquez and L.J. Nunez-Vergara, Cyclic voltammetric study of the nitro radical anion from nitrendipine generated electrochemically, J. Electroanal. Chem., 319(I) (1991) 177-184.
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