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Cathodic reduction of 2-nitronaphthothiophen-4,9-quinone: evidence of catalysis by proton donors and its simulation
Journal of Electroanalytical Chemistry 462 (1999) 195 – 201
Cathodic reduction of 2-nitronaphthothiophen-4,9-quinone: evidence of catalysis by proton donors and its simulation Fabiane C. de Abreu a,b, Josealdo Tonholo a, Ota´vio L. Bottecchia c, Carlos L. Zani d, Marı´lia O.F. Goulart a,* b
a Departamento de Quı´mica, CCEN, Uni6ersidade Federal de Alagoas, Maceio´, 57.072.970 Alagoas, Brazil Departamento de Quı´mica Fundamental, CCEN, Uni6ersidade Federal de Pernambuco, Recife, Pernambuco, Brazil c Departamento de Quı´mica, Uni6ersidade Federal de Uberlaˆndia, Minas Gerais, Brazil d Centro de Pesquisas Rene´ Rachou, Fundac¸a˜o Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil
Received 23 June 1998; received in revised form 1 October 1998
Abstract 2-Nitronaphtho[2,3-b]thiophen-4,9-quinone (1) is biologically active. The reducible groups have conjugate interaction. Electrochemical experiments (cyclic voltammetry and electrolysis) were performed in order to verify possible intramolecular electron transfer or secondary redox systems and to gain insight into the redox behaviour to help in the understanding of its trypanocidal mechanism of action. Cyclic voltammograms of 1 at the Hg electrode, in DMF+ TBAP or DMF+ TEAP showed the presence of at least three waves, the two first related to quinone reduction and the third one relative to a catalytic process. After cathodic reduction, at potentials close to the third electron uptake, protons from adventitious water or ammonium quaternary salts can be reduced. Hydrogen formation, with the regeneration of the quinone dianion could be the cause of its catalytic nature. This effect is more pronounced with TEAP. Macroscale electrolyses reinforce the findings. This reaction can be hampered by addition of electrophiles to the medium. Simulated curves fit the experimental ones well. The fourth wave, present at fast scan rates, where the catalysis is not effective, is related to further reduction of the nitro radical anion to the hydroxylamino derivative. At the time scale of cyclic voltammetry, no intramolecular electron transfer was observed. The biological activity of 1 is, possibly, related to the very electrophilic quinone group, generating reactive radical oxygen species through redox cycling. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Catalysis; Reduction; Nitrothiophenequinone; Cyclic voltammetry; Simulation; Electrolysis
1. Introduction Chagas’ disease or American trypanosomiasis is a protozoan infection caused by the flagellate Trypanosoma cruzi [1]. It is a real and important medical and social problem in Latin America. Human malaria, caused mainly by Plasmodium falciparum, is one of the most important parasitic infections on the planet [2]. Trypanosomes lack the enzymes catalase and glutathione peroxidase and are substantially more sensitive * Corresponding [email protected].
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to oxidative stress than their biological hosts [2]. This is consistent with the use of redox cyclers and oxygen radical producers as drugs, mainly nitroheterocycles and some quinones, which effectively catalyse biological electron transfer. Their major reaction is reduction to stable anion radicals, which can transfer free electrons to acceptor molecules or radicals, under back formation of the parent compounds. The acceptor may be another electrogenerated species, or molecular oxygen, which is reduced to the superoxide anion, leading under special conditions to oxidative stress. Radical oxygenated species play a dual role in malaria. Besides their protective effect against the parasites, it might contribute to the
0022-0728/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved. PII: S 0 0 2 2 - 0 7 2 8 ( 9 8 ) 0 0 4 1 1 - 2
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pathology observed in severe and cerebral malaria. Another promising approach in malaria chemotherapy is the use of iron chelators [3]. 2-Nitronaphtho[2,3-b]thiophen-4,9-quinone (1), which carries two pharmacophoric groups exhibits satisfactory anti-plasmodial and trypanocidal activity [4,5]. Compound 1 was also tested [4] against the enzyme trypanothione reductase [6], an important target for drug development in Chagas’ disease [7], with 42% inhibition at 100 mmol l − 1 [4]. The knowledge of electrochemical properties is essential for a better understanding of the biological activity and properties of free radical species emerging in the course of conversions of redox cyclers [8]. Comparison of trypanocidal activities with the first reduction potential of several quinones suggested a contribution of the ease of reduction to the biological activity [5].
The present communication deals with the cathodic reduction of the biologically active 1, which contains two different electrochemically reducible sites and presents a conjugate interaction. Electroreduction of nitro derivatives of quinones had been described [9 – 14], but without considering any relationship with pharmacological activities and any evidence of mechanistic complications. Since a potential intramolecular redox system can occur here, a question arises as to whether the application of external voltage can induce intramolecular electron transfer in such molecules or is responsible for the occurrence of secondary redox systems [15,16]. Also, as the electrophilic character of quinones has been proved to be relevant for anti-protozoan activity [5] and considering that the presence of nitro substituents certainly makes the electron capture by the quinone moiety easier, the paper intends to consider the possible role of redox behaviour into the mechanism of biological activity of 1.
2. Experimental
2.1. Samples Compound 1 and naphtho[2,3-b]thiophen-4,9quinone (2) were prepared according to the literature [17,4]. 2-Nitrothiophene (Aldrich) (3) was purified through flash chromatography, eluted by dichloromethane+ hexane (1:1). Cyclic voltammetry (CV) was carried out in anhydrous N,N%-dimethylform-
amide+ n-Bu4NClO4 (TBAP) or Et4NClO4 (TEAP), both 0.1 mol l − 1. TBAP and TEAP were prepared as described [18] and recrystallized from ethyl acetate+ water and ethyl acetate, respectively. They were dried in a vacuum oven at 70°C. N,N%-dimethylformamide was distilled at reduced pressure after treatment with anhydrous cupric sulphate. Just before the experiment, it was passed through an activated (400°C for 24 h) neutral Al2O3 (Aldrich) column.
2.2. Apparatus and procedure CV was performed using a computer assisted PAR model 273A potentiostat/galvanostat coupled with an SMDE 303A/EG & G PAR hanging mercury electrode (area 0.0097 cm2) used as the working electrode. A Pt wire was used as the counter-electrode and a homebuilt Ag AgCl, 0.1 mol l − 1 NaCl reference electrode, isolated from the solution by a Vycor® rod. The scan rate was varied from 0.020 to 20 V s − 1. All the electrochemical parameters, except under specially mentioned conditions, were obtained at n=0.100 V s − 1. The concentration of 1 used for studies of dependence on concentration was in the range of 0.11–5.04 mmol l − 1. The range of temperature was from 1 to 35°C. In all the experiments, the cell was covered with aluminium foil to suppress possible photoreactions. 1 H-NMR analyses (200 MHz) were performed on a Bruker apparatus. A Perkin–Elmer model 1600 FT spectrometer was used for IR spectra. All the solutions were degassed with a fast stream of Ar or N2 ultra purum grade.
2.3. Electrolyses and product analyses Macro-scale controlled potential electrolyses of 1 (0.07 mmol) were performed in a divided cell, in DMF+ TBAP, using a Hg pool as the working electrode (38.5 cm2), a platinum grid as auxiliary and Ag AgCl 0.1 M Cl − as reference electrodes. The chosen potentials were near the third (− 2.00 V) and fourth (− 2.50 V) waves. In the latter case, a small amount of water was added. The current was integrated electronically, with the passage of 12 and 13 mol electrons mol − 1, respectively. Work up with addition of water and CHCl3 extraction furnished, after solvent evaporation and filtration of TBAP in a short silica gel column eluted by ethyl acetate+hexane (1:1), a crude complex mixture, from whence the hydroxylamino derivative of 1 was isolated in 24 and 17%, respectively. Additional electrolysis of 1 (0.18 mmol) using DMF+ TEAP, with Eap = − 1.70 V, was performed. After 9.7 mol electrons mol − 1, the reaction was interrupted without reaching the residual current. Partial recovery of 1 was obtained.
F.C. de Abreu et al. / Journal of Electroanalytical Chemistry 462 (1999) 195–201
2.4. Simulation studies The program used was codified in Fortran and run in an IBM9672 mainframe under the CMS operational system. 3. Results and discussion CVs of 1, at all scan rates (n), showed the process to be a multistep one, with the presence of four waves and occurrence of some unexpected phenomena (Fig. 1A– red C). Peaks 1 and 2 (E red p1 = − 0.400 V, E p2 = −0.926 V) are related to reversible diffusion controlled processes, as can be concluded from the linear dependence of peak current, I red p , and sweep rate and usual reversibility red red independent of diagnostic criteria (I ox p /I p #1, E p red red log n, E p − E p/2 = 59 mV). These peaks were attributed to the successive uptake of electrons by the quinone moiety, generating the semiquinone and the hydroquinone dianion. At n= 0.100 V s − 1, the third red (E red p3 = −1.816 V) and fourth (E p4 = −2.488 V) waves have no related anodic waves and undergo significant changes with scan rate. At very slow n, the third wave shape is very sharp and intense. With the increase of n, its current height decreases sharply, with 1/2 evidence of reversibility. The value of I red increases p /n markedly on decreasing the sweep rate and its peak height becomes less pronounced, until it gets close to the first two waves, typical of a catalytic mechanism (Fig. 1C). The slight cathodic displacement of this wave with increasing n may be attributed to a coupled fast chemical reaction, following a reversible heterogeneous process, once it is lower than expected for an irreversible electronic transfer. Another parameter in acred red cordance with E red p3 reversibility is the value, E p − E p/2, red which is very close to the ones obtained for E p1 and E red p2 (Fig. 1C). The catalytic mechanism is probably related to the reduction of the nitro group to its anion radical, whose stability is lower than the stability of the electrogenerated semiquinone forms (waves 1 and 2) which are totally reversible in all sweep rates. In relation to the third wave, based on the ratio of the kinetic peak current represented by I red p3 and on the diffusion-controlled peak current, represented by I red p1 as a function of log(1/n), three regions can be observed: a pure diffusion-controlled region (PD, n\ 5 V s − 1), an intermediate one, with mixed control (I, 1B nB5 Vs − 1) and a kinetically-dominant region (KD, nB 0.5 V s-1) (Fig. 2) [19]. These regions will be always considered. E red p4 shifts negatively with increasing scan rate (Fig. 1A), characteristic of an irreversible wave. This shift is also due to an ohmic drop effect when higher currents flow. At high scan rates (n\ 20 V s − 1), it is followed by an additional anodic wave (E ox p4 = −0.454 V) at potenox tials between E ox and E (Fig. 1B). p1 p2
197
The electrochemical data from 1 were compared with the those from 2 [18] and 3, hitherto not reported in the literature, for n= 0.100 V s − 1, in the PD region. Considering the three first E red values for 1 and comparing p them with E red of the model systems (3, E red p p1 = −1.040 red V; 2, E red p1 = − 0.795 V and E p2 = − 1.474 V), the expected and important contribution of the nitro group, which facilitates reduction, becomes evident. In contrast, the nitro group is reduced to its anion-radical,
Fig. 1. Effect of scan rates on CV of 1; DMF + TBAP, Hg, c1 = 2.98 mmol l − 1. (A) Electrochemical potential domain, 0 to −3.0 V; (B) n = 20 V s − 1; (C) El (inversion potential) 70 mV after E red p3 .
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red red Fig. 2. Curve (I red p3 /I p1 ) vs. log 1/n; I p1 considered as a pure diffusion-controlled current.
at more negative potentials. The quinone group, after the two-electron uptake, acts as an electrodonating substituent to the nitrothiophene portion of the molecule. In this region, the fourth wave corresponds to further reduction of the nitro group to hydroxylamino derivative [11] with protons furnished by the supporting electrolyte. Its corresponding anodic wave (Fig. 1B) is due to the oxidation of the hydroxylamino group to the nitroso derivative [20]. The electrochemical behaviour of 1, with emphasis on the third wave, was studied as a function of concentration and temperature, the nature of the supporting electrolyte, as well as with addition of proton sources and electrophiles. Results will always be referred to the first wave which, due to its reversible and hardly altered nature, will be considered as an internal standard.
3.1. The effect of concentration The three previously mentioned regions (Fig. 2) were used in the concentration dependence analysis of I red p1 , red red I red and I . I was not analysed. It is useful to report p2 p3 p4 red that I red p1 and I p2 variations with the concentration of 1 were always linear, independent of n, unlike that for red I red p3 . In the pure diffusion-controlled (PD) region, I p3 is proportional to the concentration of 1. In the other two regions, there is a great increase in I red p3 with the concentration of 1, especially at low scan rate. This result implies participation of a higher order chemical reaction following the heterogeneous transfer.
Fig. 3. Effect of temperature on CVs of 1; DMF+ TBAP, c1 = 2.82 mmol l − 1. T (---)= 308 K, ( — )=298 K, (·-·-·-)= 274 K.
is 10% °C − 1, for T\ 10°C, that confirms the kinetic nature of I red p3 (Fig. 3).
3.3. The effect of the nature of the supporting electrolyte The effects of the counterions on the peak potentials of the first, second and third charge transfers in DMF can be observed in Fig. 4. One observes for TEAP in comparison with TBAP, at n =0.100 V s − 1, the absence of the fourth reduction wave (even at very fast n), red negative shifts for E red p1 (− 0.457 V) and E p2 (−0.968 red red V) and a positive shift for E p3 (− 1.763 V). I red p3 /I p1 is higher than in DMF+TBAP. The role of quaternary ammonium salts, as a proton source is well known. TEAP is more susceptible than TBAP to Hofmann elimination due to steric reasons
3.2. The effect of the temperature The values of E red were not greatly affected by the p temperature. In relation to peak current, I red p1 acted as an internal standard and did not change. In the kinetically-dominant region (KD, Fig. 2), at 0.100 V s − 1, I red p3 was reduced to 28% of its original height, with a temperature decrease of 34.5°C. There is a further decrease as n decreases. The current/temperature slope
Fig. 4. Effect of supporting electrolytes on CVs of 1; n = 0.100 V s − 1, ( —) DMF+0.1 mol l − 1 TBAP, c1 =2.98 mmol l − 1. (---) DMF + 0.1 mol l − 1 TEAP, c1 =2.77 mmol l − 1. T =298 K.
F.C. de Abreu et al. / Journal of Electroanalytical Chemistry 462 (1999) 195–201
Fig. 5. Effects of different amounts of water added on purpose on the CV of 1, DMF + 0.1 mol l − 1 TBAP, c1 = 2.66 mmol l − 1, n = 0.100 V s − 1. El 70 mV after E red p3 .
[21]. It is very probable that the highly basic electrogenerated intermediate can react with the supporting electrolyte, reducing the proton. In this situation, the proton would be the partner in the homogeneous electron transfer, suggesting an EEEC% electrodic mechanism, with the indirect reduction of protons. This type of chemical catalysis was observed for aromatic nitrogen heterocycles [22]. To investigate the role of protons in the mechanism, different proton sources were added on purpose.
3.4. The effect of a proton source The effects of proton donors of different acidities were analysed, mainly in the pure diffusion-controlled region (n=0.100 V s − 1) (Fig. 5). Addition of water (cH2O = 0.111 and 1.089 M) leads to significant positive shifts of the third and fourth waves (34 and 52 mV, respectively) together with a great increase of the curred rent ratio (68 and 133%) of I red p3 . I p4 decreases concomitantly, until final collapse of both waves into one. The first two wave changes were compatible with the rered ported behaviour of quinones [DE red p2 (70 mV)\DE p1 (40 mV)]. At low water concentration, no change of E red p1 was found (Fig. 5). The addition of phenol at lower concentrations did not change the features of the waves. At phenol concentrations greater than twice the concentration of 1, a new wave (E red p = − 1.459 V), intermediate between red E red and E , appeared. A slight increase of I red p2 p3 p3 was observed. The first wave remained unaltered during the whole procedure. Greater amounts of phenol caused the increase of the intermediate wave and the latter two waves coalesced into one (Fig. 6). Preprotonation effects can be considered since a dianionic species is generated at the second wave. The intermediate reduction peak, in the presence of phenol could be attributed to this effect. The combined data corroborated the possibility of chemical catalysis due to proton reduction.
199
Fig. 6. Effects of different amounts of phenol added on purpose on the CV of 1, DMF+0.1 mol l − 1 TBAP, c1 =2.24 mmol l − 1, n =0.100 V s − 1.
3.5. The effect of added electrophile Acetic anhydride (Ac2O) was added during CV studies of 1 and its effects analysed at low scan rates (0.035 V s − 1). Small amounts of Ac2O (1/6 in relation to 1) promoted a slight positive shift of E red p2 , a decrease of I ox p2 and the complete disappearance of the catalytic effect. A reversible one-electron wave (E red p3 = −1.654 V) appeared, followed by an irreversible one, three times bigger (E red p4 = − 2.000 V), probably related to further reduction of the nitro group. A new wave was red observed in between E red p2 and E p3 ’ and could be related to adsorption (Fig. 7). Increased amounts of Ac2O led to very complex voltammograms and were not explored further.
3.6. Electrolyses Results from electrolyses, related mainly to charge consumption are indicative of catalysis. The low yield of the hydroxylamino derivative 4 is expected, due to the main reaction mechanism, C%, as well as due to the instability of the derivative [23].
Fig. 7. Effects of acetic anhydride (c = 0.5 mmol l − 1) on the CV of 1, DMF+ 0.1 mol l − 1 TBAP, c1 =3 mmol l − 1, n =0.035 V s − 1.
F.C. de Abreu et al. / Journal of Electroanalytical Chemistry 462 (1999) 195–201
200
3.7. Simulation studies A catalytic current is frequently observed with N-heterocyclic compounds, thiols, proteins, and similar compounds. The following mechanism generally applies: B + HA X BH + + A − BH + +e − X BH 2BH 2B+ H2 where HA is a proton donor. The presence of B, the organic catalyst, reduces the activation energy for reduction of hydrogen ions, which depends on the concentration of hydrogen ions and on the kinetics of the above first two reactions. The organic catalyst B is electrochemically reduced and catalyses hydrogen ion reduction as well [24]. In order to explain the catalytic effect, the third wave analysed out assuming a feedback of Ox3 occurring through an irreversible homogeneous chemical reaction with a Bro¨nsted acid (namely, tetrabutylamonium perchlorate used as supporting electrolyte or even residual water), via Hofmann elimination, with reduction of the abstracted proton [step (4)]: Ox1 + e − X Ox2
(1)
Ox2 + e − X Ox3
(2)
Ox3 + e
−
X Ox4
Ox4 + HB Ox3 +1/2H2 +B
(3) −
(4)
The expected voltammograms for the above mechanism were simulated by solving the differential equations, which describe them: ([Ox1] ( [Ox1] =D (t (x 2 2
([Ox2] ( 2[Ox2] =D (t (x 2 ([Ox3] ( 2[Ox3] =D + k[HB][Ox4] (t (x 2 ( 2[Ox4] ([Ox4] =D − k[HB][Ox4] (t (x 2 ([HB] ( 2[HB] =D (x 2 (t The brackets indicate molar concentration. The concentrations are functions of time, t, and the co-ordinate, x, referred to the surface electrode. The required parameters are standard potentials and the heterogeneous rate constants for steps (1), (2) and (3), E°1, ks1, E°2, ks2, E°3, ks3, respectively, the chemical rate constant of step (4), k, and the diffusion coefficients, all assumed to be given by D. The initial conditions are t =0, x ]0: [Ox1] =[Ox1]0 = c*, [Ox2]= 0, [Ox3]= 0, [Ox4]= 0,
Fig. 8. Experimental vs. simulated CVs of 1, DMF+0.1 mol l − 1 TBAP, n =0.200 V s − 1.
[HB]= [HB]0; the boundary condition is: t\ 0, x=0: #[HB]/#x = 0. The Runge–Kutta method of second-order was used [25] and the concentration profiles were calculated. The current density was calculated according to the method of Polcyn and Shain [26] extended to three charge transfers, and taking into account the one electron transfer for steps (1) to (3): I/(FA)=
([Ox1] (x
+ + +
x=0
([Ox1] (x
x=0
([Ox1] (x
([Ox2] + (x x=0
([Ox3] (x
+
([Ox2] (x
x=0
x=0
x=0
Simulation of the above mechanism showed that the peak height of the third wave depends on the rate of step (4). The greater the rate constant, the greater is the peak height of the third wave. This effect is more pronounced as the proton donor concentration (HB) increases. Further, the third peak narrows as the rate constant increases. The narrowing is of little importance for lower values of the proton donor concentrations. This information was used as a basis for varying the several parameters necessary for simulation and further visual fitting of theoretical voltammograms to the experimental ones. Using E°1 = − 0.370 V; E°2 = − 0.890 V; E°3 = − 1.700 V; k/(nc*)=1000 (n= sweep rate), and heterogeneous rate constants high enough to guarantee a reversible behaviour of the charge transfers, a very good fit was obtained (Fig. 8).
4. Conclusions The cathodic reduction of 1, in the kinetically-dominant region (KD), occurs through an EEEC% mecha-
F.C. de Abreu et al. / Journal of Electroanalytical Chemistry 462 (1999) 195–201
nism, where the catalytic current is due to reduction of protons from adventitious water or ammonium quaternary salts. In the pure diffusion-controlled region (PD), further reduction of the nitro group to the hydroxylamino derivative (fourth wave) is observed, and the corresponding anodic wave due to oxidation of hydroxylamino group to the nitroso derivative. In the time scale of CV, no intramolecular electron transfer was observed. Substituent effects, mainly resonance effects are operative in the process of electron uptake. The presence of the conjugate nitro group facilitates reduction of the quinone moiety. In contrast, the nitro group is reduced to its anion-radical, at more negative potentials. The quinone group, after the two-electron uptake, acts as an electron-donating substituent to the nitrothiophene portion of the molecule. Although inconclusive toward biological activity due to the lower solubility of 1 in water or even in mixed solvents and the few tests performed, some features might be suggested. The group responsible for the activity should be the quinone, presenting, in the present case, a very electrophilic nature. Generation of reactive oxygenated radicals, emerging from the redox cycle, in the presence of O2, is probably the reason for trypanocidal activity of 1. Trypanocidal activity is definitely not related to the reduction of nitro group, that occurs at high negative potential values. Anti-plasmodial activity can have the same cause or could be explained by iron complexation. These results are encouraging and further experiments must be performed. Further structural modifications will be necessary to improve the solubility and in vivo activity of 1. Additional assays, concerning anti-plasmodial and trypanocidal activities are in progress.
Acknowledgements The authors acknowledge financial support by CNPq, RHAE, FAPEAL, CAPES and PADCT. The authors wish to thank Dr Marcelo Navarro for helpful discussions.
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Cathodic reduction of 2-nitronaphthothiophen-4,9-quinone: evidence of catalysis by proton donors and its simulation Fabiane C. de Abreu a,b, Josealdo Tonholo a, Ota´vio L. Bottecchia c, Carlos L. Zani d, Marı´lia O.F. Goulart a,* b
a Departamento de Quı´mica, CCEN, Uni6ersidade Federal de Alagoas, Maceio´, 57.072.970 Alagoas, Brazil Departamento de Quı´mica Fundamental, CCEN, Uni6ersidade Federal de Pernambuco, Recife, Pernambuco, Brazil c Departamento de Quı´mica, Uni6ersidade Federal de Uberlaˆndia, Minas Gerais, Brazil d Centro de Pesquisas Rene´ Rachou, Fundac¸a˜o Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil
Received 23 June 1998; received in revised form 1 October 1998
Abstract 2-Nitronaphtho[2,3-b]thiophen-4,9-quinone (1) is biologically active. The reducible groups have conjugate interaction. Electrochemical experiments (cyclic voltammetry and electrolysis) were performed in order to verify possible intramolecular electron transfer or secondary redox systems and to gain insight into the redox behaviour to help in the understanding of its trypanocidal mechanism of action. Cyclic voltammograms of 1 at the Hg electrode, in DMF+ TBAP or DMF+ TEAP showed the presence of at least three waves, the two first related to quinone reduction and the third one relative to a catalytic process. After cathodic reduction, at potentials close to the third electron uptake, protons from adventitious water or ammonium quaternary salts can be reduced. Hydrogen formation, with the regeneration of the quinone dianion could be the cause of its catalytic nature. This effect is more pronounced with TEAP. Macroscale electrolyses reinforce the findings. This reaction can be hampered by addition of electrophiles to the medium. Simulated curves fit the experimental ones well. The fourth wave, present at fast scan rates, where the catalysis is not effective, is related to further reduction of the nitro radical anion to the hydroxylamino derivative. At the time scale of cyclic voltammetry, no intramolecular electron transfer was observed. The biological activity of 1 is, possibly, related to the very electrophilic quinone group, generating reactive radical oxygen species through redox cycling. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Catalysis; Reduction; Nitrothiophenequinone; Cyclic voltammetry; Simulation; Electrolysis
1. Introduction Chagas’ disease or American trypanosomiasis is a protozoan infection caused by the flagellate Trypanosoma cruzi [1]. It is a real and important medical and social problem in Latin America. Human malaria, caused mainly by Plasmodium falciparum, is one of the most important parasitic infections on the planet [2]. Trypanosomes lack the enzymes catalase and glutathione peroxidase and are substantially more sensitive * Corresponding [email protected].
author.
Fax:
+ 55-82-2141615;
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to oxidative stress than their biological hosts [2]. This is consistent with the use of redox cyclers and oxygen radical producers as drugs, mainly nitroheterocycles and some quinones, which effectively catalyse biological electron transfer. Their major reaction is reduction to stable anion radicals, which can transfer free electrons to acceptor molecules or radicals, under back formation of the parent compounds. The acceptor may be another electrogenerated species, or molecular oxygen, which is reduced to the superoxide anion, leading under special conditions to oxidative stress. Radical oxygenated species play a dual role in malaria. Besides their protective effect against the parasites, it might contribute to the
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pathology observed in severe and cerebral malaria. Another promising approach in malaria chemotherapy is the use of iron chelators [3]. 2-Nitronaphtho[2,3-b]thiophen-4,9-quinone (1), which carries two pharmacophoric groups exhibits satisfactory anti-plasmodial and trypanocidal activity [4,5]. Compound 1 was also tested [4] against the enzyme trypanothione reductase [6], an important target for drug development in Chagas’ disease [7], with 42% inhibition at 100 mmol l − 1 [4]. The knowledge of electrochemical properties is essential for a better understanding of the biological activity and properties of free radical species emerging in the course of conversions of redox cyclers [8]. Comparison of trypanocidal activities with the first reduction potential of several quinones suggested a contribution of the ease of reduction to the biological activity [5].
The present communication deals with the cathodic reduction of the biologically active 1, which contains two different electrochemically reducible sites and presents a conjugate interaction. Electroreduction of nitro derivatives of quinones had been described [9 – 14], but without considering any relationship with pharmacological activities and any evidence of mechanistic complications. Since a potential intramolecular redox system can occur here, a question arises as to whether the application of external voltage can induce intramolecular electron transfer in such molecules or is responsible for the occurrence of secondary redox systems [15,16]. Also, as the electrophilic character of quinones has been proved to be relevant for anti-protozoan activity [5] and considering that the presence of nitro substituents certainly makes the electron capture by the quinone moiety easier, the paper intends to consider the possible role of redox behaviour into the mechanism of biological activity of 1.
2. Experimental
2.1. Samples Compound 1 and naphtho[2,3-b]thiophen-4,9quinone (2) were prepared according to the literature [17,4]. 2-Nitrothiophene (Aldrich) (3) was purified through flash chromatography, eluted by dichloromethane+ hexane (1:1). Cyclic voltammetry (CV) was carried out in anhydrous N,N%-dimethylform-
amide+ n-Bu4NClO4 (TBAP) or Et4NClO4 (TEAP), both 0.1 mol l − 1. TBAP and TEAP were prepared as described [18] and recrystallized from ethyl acetate+ water and ethyl acetate, respectively. They were dried in a vacuum oven at 70°C. N,N%-dimethylformamide was distilled at reduced pressure after treatment with anhydrous cupric sulphate. Just before the experiment, it was passed through an activated (400°C for 24 h) neutral Al2O3 (Aldrich) column.
2.2. Apparatus and procedure CV was performed using a computer assisted PAR model 273A potentiostat/galvanostat coupled with an SMDE 303A/EG & G PAR hanging mercury electrode (area 0.0097 cm2) used as the working electrode. A Pt wire was used as the counter-electrode and a homebuilt Ag AgCl, 0.1 mol l − 1 NaCl reference electrode, isolated from the solution by a Vycor® rod. The scan rate was varied from 0.020 to 20 V s − 1. All the electrochemical parameters, except under specially mentioned conditions, were obtained at n=0.100 V s − 1. The concentration of 1 used for studies of dependence on concentration was in the range of 0.11–5.04 mmol l − 1. The range of temperature was from 1 to 35°C. In all the experiments, the cell was covered with aluminium foil to suppress possible photoreactions. 1 H-NMR analyses (200 MHz) were performed on a Bruker apparatus. A Perkin–Elmer model 1600 FT spectrometer was used for IR spectra. All the solutions were degassed with a fast stream of Ar or N2 ultra purum grade.
2.3. Electrolyses and product analyses Macro-scale controlled potential electrolyses of 1 (0.07 mmol) were performed in a divided cell, in DMF+ TBAP, using a Hg pool as the working electrode (38.5 cm2), a platinum grid as auxiliary and Ag AgCl 0.1 M Cl − as reference electrodes. The chosen potentials were near the third (− 2.00 V) and fourth (− 2.50 V) waves. In the latter case, a small amount of water was added. The current was integrated electronically, with the passage of 12 and 13 mol electrons mol − 1, respectively. Work up with addition of water and CHCl3 extraction furnished, after solvent evaporation and filtration of TBAP in a short silica gel column eluted by ethyl acetate+hexane (1:1), a crude complex mixture, from whence the hydroxylamino derivative of 1 was isolated in 24 and 17%, respectively. Additional electrolysis of 1 (0.18 mmol) using DMF+ TEAP, with Eap = − 1.70 V, was performed. After 9.7 mol electrons mol − 1, the reaction was interrupted without reaching the residual current. Partial recovery of 1 was obtained.
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2.4. Simulation studies The program used was codified in Fortran and run in an IBM9672 mainframe under the CMS operational system. 3. Results and discussion CVs of 1, at all scan rates (n), showed the process to be a multistep one, with the presence of four waves and occurrence of some unexpected phenomena (Fig. 1A– red C). Peaks 1 and 2 (E red p1 = − 0.400 V, E p2 = −0.926 V) are related to reversible diffusion controlled processes, as can be concluded from the linear dependence of peak current, I red p , and sweep rate and usual reversibility red red independent of diagnostic criteria (I ox p /I p #1, E p red red log n, E p − E p/2 = 59 mV). These peaks were attributed to the successive uptake of electrons by the quinone moiety, generating the semiquinone and the hydroquinone dianion. At n= 0.100 V s − 1, the third red (E red p3 = −1.816 V) and fourth (E p4 = −2.488 V) waves have no related anodic waves and undergo significant changes with scan rate. At very slow n, the third wave shape is very sharp and intense. With the increase of n, its current height decreases sharply, with 1/2 evidence of reversibility. The value of I red increases p /n markedly on decreasing the sweep rate and its peak height becomes less pronounced, until it gets close to the first two waves, typical of a catalytic mechanism (Fig. 1C). The slight cathodic displacement of this wave with increasing n may be attributed to a coupled fast chemical reaction, following a reversible heterogeneous process, once it is lower than expected for an irreversible electronic transfer. Another parameter in acred red cordance with E red p3 reversibility is the value, E p − E p/2, red which is very close to the ones obtained for E p1 and E red p2 (Fig. 1C). The catalytic mechanism is probably related to the reduction of the nitro group to its anion radical, whose stability is lower than the stability of the electrogenerated semiquinone forms (waves 1 and 2) which are totally reversible in all sweep rates. In relation to the third wave, based on the ratio of the kinetic peak current represented by I red p3 and on the diffusion-controlled peak current, represented by I red p1 as a function of log(1/n), three regions can be observed: a pure diffusion-controlled region (PD, n\ 5 V s − 1), an intermediate one, with mixed control (I, 1B nB5 Vs − 1) and a kinetically-dominant region (KD, nB 0.5 V s-1) (Fig. 2) [19]. These regions will be always considered. E red p4 shifts negatively with increasing scan rate (Fig. 1A), characteristic of an irreversible wave. This shift is also due to an ohmic drop effect when higher currents flow. At high scan rates (n\ 20 V s − 1), it is followed by an additional anodic wave (E ox p4 = −0.454 V) at potenox tials between E ox and E (Fig. 1B). p1 p2
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The electrochemical data from 1 were compared with the those from 2 [18] and 3, hitherto not reported in the literature, for n= 0.100 V s − 1, in the PD region. Considering the three first E red values for 1 and comparing p them with E red of the model systems (3, E red p p1 = −1.040 red V; 2, E red p1 = − 0.795 V and E p2 = − 1.474 V), the expected and important contribution of the nitro group, which facilitates reduction, becomes evident. In contrast, the nitro group is reduced to its anion-radical,
Fig. 1. Effect of scan rates on CV of 1; DMF + TBAP, Hg, c1 = 2.98 mmol l − 1. (A) Electrochemical potential domain, 0 to −3.0 V; (B) n = 20 V s − 1; (C) El (inversion potential) 70 mV after E red p3 .
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red red Fig. 2. Curve (I red p3 /I p1 ) vs. log 1/n; I p1 considered as a pure diffusion-controlled current.
at more negative potentials. The quinone group, after the two-electron uptake, acts as an electrodonating substituent to the nitrothiophene portion of the molecule. In this region, the fourth wave corresponds to further reduction of the nitro group to hydroxylamino derivative [11] with protons furnished by the supporting electrolyte. Its corresponding anodic wave (Fig. 1B) is due to the oxidation of the hydroxylamino group to the nitroso derivative [20]. The electrochemical behaviour of 1, with emphasis on the third wave, was studied as a function of concentration and temperature, the nature of the supporting electrolyte, as well as with addition of proton sources and electrophiles. Results will always be referred to the first wave which, due to its reversible and hardly altered nature, will be considered as an internal standard.
3.1. The effect of concentration The three previously mentioned regions (Fig. 2) were used in the concentration dependence analysis of I red p1 , red red I red and I . I was not analysed. It is useful to report p2 p3 p4 red that I red p1 and I p2 variations with the concentration of 1 were always linear, independent of n, unlike that for red I red p3 . In the pure diffusion-controlled (PD) region, I p3 is proportional to the concentration of 1. In the other two regions, there is a great increase in I red p3 with the concentration of 1, especially at low scan rate. This result implies participation of a higher order chemical reaction following the heterogeneous transfer.
Fig. 3. Effect of temperature on CVs of 1; DMF+ TBAP, c1 = 2.82 mmol l − 1. T (---)= 308 K, ( — )=298 K, (·-·-·-)= 274 K.
is 10% °C − 1, for T\ 10°C, that confirms the kinetic nature of I red p3 (Fig. 3).
3.3. The effect of the nature of the supporting electrolyte The effects of the counterions on the peak potentials of the first, second and third charge transfers in DMF can be observed in Fig. 4. One observes for TEAP in comparison with TBAP, at n =0.100 V s − 1, the absence of the fourth reduction wave (even at very fast n), red negative shifts for E red p1 (− 0.457 V) and E p2 (−0.968 red red V) and a positive shift for E p3 (− 1.763 V). I red p3 /I p1 is higher than in DMF+TBAP. The role of quaternary ammonium salts, as a proton source is well known. TEAP is more susceptible than TBAP to Hofmann elimination due to steric reasons
3.2. The effect of the temperature The values of E red were not greatly affected by the p temperature. In relation to peak current, I red p1 acted as an internal standard and did not change. In the kinetically-dominant region (KD, Fig. 2), at 0.100 V s − 1, I red p3 was reduced to 28% of its original height, with a temperature decrease of 34.5°C. There is a further decrease as n decreases. The current/temperature slope
Fig. 4. Effect of supporting electrolytes on CVs of 1; n = 0.100 V s − 1, ( —) DMF+0.1 mol l − 1 TBAP, c1 =2.98 mmol l − 1. (---) DMF + 0.1 mol l − 1 TEAP, c1 =2.77 mmol l − 1. T =298 K.
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Fig. 5. Effects of different amounts of water added on purpose on the CV of 1, DMF + 0.1 mol l − 1 TBAP, c1 = 2.66 mmol l − 1, n = 0.100 V s − 1. El 70 mV after E red p3 .
[21]. It is very probable that the highly basic electrogenerated intermediate can react with the supporting electrolyte, reducing the proton. In this situation, the proton would be the partner in the homogeneous electron transfer, suggesting an EEEC% electrodic mechanism, with the indirect reduction of protons. This type of chemical catalysis was observed for aromatic nitrogen heterocycles [22]. To investigate the role of protons in the mechanism, different proton sources were added on purpose.
3.4. The effect of a proton source The effects of proton donors of different acidities were analysed, mainly in the pure diffusion-controlled region (n=0.100 V s − 1) (Fig. 5). Addition of water (cH2O = 0.111 and 1.089 M) leads to significant positive shifts of the third and fourth waves (34 and 52 mV, respectively) together with a great increase of the curred rent ratio (68 and 133%) of I red p3 . I p4 decreases concomitantly, until final collapse of both waves into one. The first two wave changes were compatible with the rered ported behaviour of quinones [DE red p2 (70 mV)\DE p1 (40 mV)]. At low water concentration, no change of E red p1 was found (Fig. 5). The addition of phenol at lower concentrations did not change the features of the waves. At phenol concentrations greater than twice the concentration of 1, a new wave (E red p = − 1.459 V), intermediate between red E red and E , appeared. A slight increase of I red p2 p3 p3 was observed. The first wave remained unaltered during the whole procedure. Greater amounts of phenol caused the increase of the intermediate wave and the latter two waves coalesced into one (Fig. 6). Preprotonation effects can be considered since a dianionic species is generated at the second wave. The intermediate reduction peak, in the presence of phenol could be attributed to this effect. The combined data corroborated the possibility of chemical catalysis due to proton reduction.
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Fig. 6. Effects of different amounts of phenol added on purpose on the CV of 1, DMF+0.1 mol l − 1 TBAP, c1 =2.24 mmol l − 1, n =0.100 V s − 1.
3.5. The effect of added electrophile Acetic anhydride (Ac2O) was added during CV studies of 1 and its effects analysed at low scan rates (0.035 V s − 1). Small amounts of Ac2O (1/6 in relation to 1) promoted a slight positive shift of E red p2 , a decrease of I ox p2 and the complete disappearance of the catalytic effect. A reversible one-electron wave (E red p3 = −1.654 V) appeared, followed by an irreversible one, three times bigger (E red p4 = − 2.000 V), probably related to further reduction of the nitro group. A new wave was red observed in between E red p2 and E p3 ’ and could be related to adsorption (Fig. 7). Increased amounts of Ac2O led to very complex voltammograms and were not explored further.
3.6. Electrolyses Results from electrolyses, related mainly to charge consumption are indicative of catalysis. The low yield of the hydroxylamino derivative 4 is expected, due to the main reaction mechanism, C%, as well as due to the instability of the derivative [23].
Fig. 7. Effects of acetic anhydride (c = 0.5 mmol l − 1) on the CV of 1, DMF+ 0.1 mol l − 1 TBAP, c1 =3 mmol l − 1, n =0.035 V s − 1.
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3.7. Simulation studies A catalytic current is frequently observed with N-heterocyclic compounds, thiols, proteins, and similar compounds. The following mechanism generally applies: B + HA X BH + + A − BH + +e − X BH 2BH 2B+ H2 where HA is a proton donor. The presence of B, the organic catalyst, reduces the activation energy for reduction of hydrogen ions, which depends on the concentration of hydrogen ions and on the kinetics of the above first two reactions. The organic catalyst B is electrochemically reduced and catalyses hydrogen ion reduction as well [24]. In order to explain the catalytic effect, the third wave analysed out assuming a feedback of Ox3 occurring through an irreversible homogeneous chemical reaction with a Bro¨nsted acid (namely, tetrabutylamonium perchlorate used as supporting electrolyte or even residual water), via Hofmann elimination, with reduction of the abstracted proton [step (4)]: Ox1 + e − X Ox2
(1)
Ox2 + e − X Ox3
(2)
Ox3 + e
−
X Ox4
Ox4 + HB Ox3 +1/2H2 +B
(3) −
(4)
The expected voltammograms for the above mechanism were simulated by solving the differential equations, which describe them: ([Ox1] ( [Ox1] =D (t (x 2 2
([Ox2] ( 2[Ox2] =D (t (x 2 ([Ox3] ( 2[Ox3] =D + k[HB][Ox4] (t (x 2 ( 2[Ox4] ([Ox4] =D − k[HB][Ox4] (t (x 2 ([HB] ( 2[HB] =D (x 2 (t The brackets indicate molar concentration. The concentrations are functions of time, t, and the co-ordinate, x, referred to the surface electrode. The required parameters are standard potentials and the heterogeneous rate constants for steps (1), (2) and (3), E°1, ks1, E°2, ks2, E°3, ks3, respectively, the chemical rate constant of step (4), k, and the diffusion coefficients, all assumed to be given by D. The initial conditions are t =0, x ]0: [Ox1] =[Ox1]0 = c*, [Ox2]= 0, [Ox3]= 0, [Ox4]= 0,
Fig. 8. Experimental vs. simulated CVs of 1, DMF+0.1 mol l − 1 TBAP, n =0.200 V s − 1.
[HB]= [HB]0; the boundary condition is: t\ 0, x=0: #[HB]/#x = 0. The Runge–Kutta method of second-order was used [25] and the concentration profiles were calculated. The current density was calculated according to the method of Polcyn and Shain [26] extended to three charge transfers, and taking into account the one electron transfer for steps (1) to (3): I/(FA)=
([Ox1] (x
+ + +
x=0
([Ox1] (x
x=0
([Ox1] (x
([Ox2] + (x x=0
([Ox3] (x
+
([Ox2] (x
x=0
x=0
x=0
Simulation of the above mechanism showed that the peak height of the third wave depends on the rate of step (4). The greater the rate constant, the greater is the peak height of the third wave. This effect is more pronounced as the proton donor concentration (HB) increases. Further, the third peak narrows as the rate constant increases. The narrowing is of little importance for lower values of the proton donor concentrations. This information was used as a basis for varying the several parameters necessary for simulation and further visual fitting of theoretical voltammograms to the experimental ones. Using E°1 = − 0.370 V; E°2 = − 0.890 V; E°3 = − 1.700 V; k/(nc*)=1000 (n= sweep rate), and heterogeneous rate constants high enough to guarantee a reversible behaviour of the charge transfers, a very good fit was obtained (Fig. 8).
4. Conclusions The cathodic reduction of 1, in the kinetically-dominant region (KD), occurs through an EEEC% mecha-
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nism, where the catalytic current is due to reduction of protons from adventitious water or ammonium quaternary salts. In the pure diffusion-controlled region (PD), further reduction of the nitro group to the hydroxylamino derivative (fourth wave) is observed, and the corresponding anodic wave due to oxidation of hydroxylamino group to the nitroso derivative. In the time scale of CV, no intramolecular electron transfer was observed. Substituent effects, mainly resonance effects are operative in the process of electron uptake. The presence of the conjugate nitro group facilitates reduction of the quinone moiety. In contrast, the nitro group is reduced to its anion-radical, at more negative potentials. The quinone group, after the two-electron uptake, acts as an electron-donating substituent to the nitrothiophene portion of the molecule. Although inconclusive toward biological activity due to the lower solubility of 1 in water or even in mixed solvents and the few tests performed, some features might be suggested. The group responsible for the activity should be the quinone, presenting, in the present case, a very electrophilic nature. Generation of reactive oxygenated radicals, emerging from the redox cycle, in the presence of O2, is probably the reason for trypanocidal activity of 1. Trypanocidal activity is definitely not related to the reduction of nitro group, that occurs at high negative potential values. Anti-plasmodial activity can have the same cause or could be explained by iron complexation. These results are encouraging and further experiments must be performed. Further structural modifications will be necessary to improve the solubility and in vivo activity of 1. Additional assays, concerning anti-plasmodial and trypanocidal activities are in progress.
Acknowledgements The authors acknowledge financial support by CNPq, RHAE, FAPEAL, CAPES and PADCT. The authors wish to thank Dr Marcelo Navarro for helpful discussions.
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