As already mentioned above, the detailed understanding on the voltammetric reduction of nitroheterocyclic compounds is consolidate [7, 20, 21]. As the NTBS and NFBS designing was based on nitrofurazone as a prototype, its voltammetric behavior in aqueous media was taken as reference for present study, considering the drug determination in different matrixes by using modified electrodes [33–35], boron-doped diamond electrode [36], carbon fiber electrode [37], as well as the voltammetric study of nitro-anion radical kinetic stability by applying different working electrodes such as mercury [38], glassy carbon [22, 23], boron-doped diamond [39], DNA biosensor [40], carbon fiber [24] and carbon nanotubes [41]. Thus, from Fig. 2, the voltammetric registers for NTBS (Fig. 2A) and NFBS (Fig. 2B) in the forward scan at negative potential show two cathodic peaks, being Ecp1 -0.397 V and − 0.365 V and Ecp2 -0.861 V and − 0.835 V, respectively. These records correspond to the respective reduction processes of nitro group forming the hydroxylamine derivative followed by the amine, as depicted below:
R-NO2 + 4e + 4H+ → R-NHOH + H2O
R-NHOH + 2e + 2H+ → R-NH2 + H2O
In the reverse scan, the anodic peak Eap1 also is observed for both cases similarly to the previously registered for nitrofurazone [22, 23, 39], being 0.278 V for NTBS and 0.240 V for NFBS. This peak corresponds to the hydroxylamine oxidation to the nitroso derivative, which differently than nitroaromatic compounds such as chloramphenicol [22, 42–44], the nitroso-hydroxylamine couple was not detected at low scan rate values. The hydroxylamine oxidation occurs according to the following reaction:
R-NHOH → R-NO + 2e + 2H+
The voltammetric records in blue line in respective Figs. 2A and 2B refer to the results obtained for the analogues of NTBS and NFBS without the nitro group, resembling cyclic voltammograms of blank without any peak registered. This confirms that in the potential range studied, the nitro group is the electroactive moiety of the molecules studied. Additionally, it is evident that the hydroxylamine derivative formation is irreversible by absence of corresponding anodic record. The irreversibility of this voltammetric reduction was reinforced by the linear relationship between Ecp1 values as function of scan rate on logarithmic scale, showing that Ecp1 values were shifted to the more negative potential values by 41 mV and 30 mV per tenfold increase in scan rate for NTBS and NFBS, respectively. From this behavior and applying the Laviron equation [45, 46], the electron transfer coefficient values (αn) and the rate constant of the electrochemical reaction (ks) were calculated from the following equation:
$${\text{Ecp}}_{\text{1}}\text{ }\text{=}{\text{ }\text{E}}^{\text{0}}\text{+}\frac{\text{2.303RT}}{\text{αnF}}\text{log}\left(\frac{\text{RT}{\text{k}}_{\text{s}}}{\text{αnF}}\right)\text{-}\frac{\text{2.303RT}}{\text{αnF}}\text{log}\text{ }\text{}\text{,}$$
in which R is the universal gas constant (8.314 J/K mol), T is the temperature at Kelvin, F is the Faraday constant (96,485 C/mol) and Eº is the formal potential, which can be deduced with the correlation between Ecp1versus scan rate by the extrapolation on the ordinate when ν = 0. Thus, the Eº values obtained were − 0.346 V and − 0.337 V for NTBS and NFBS, respectively. In a complementary way, taking the calculated intercept and slope by the linear fit of the inset graphs in Figs. 2A and 2B, the obtained αn values were 1.42 and 1.54 and ks values were 0.843 s− 1 and 0.989 s− 1 for NTBS and NFBS, respectively.
Usually, the αn values between 1 and 2 indicate that there are two electrons involved in the rate-determining step of the electrode reaction [47]. In fact, the reduction potential can be reflecting the transfer of the first or second electron in the voltammetric reduction of nitroheterocyclic compounds [48,49], in which the formation of nitroso compound is the slowest step of the first reduction wave [22, 23, 48], according to the steps below at acidic medium:
R-NO2 + 2e + 2H+ → R-N(OH)2
R-N(OH)2 → R-NO + H2O
R-NO + 2e + 2H+ → R-NHOH + H2O
In sequence, considering the mass transport process as diffusional for irreversible systems of hydroxylamine derivative formation involving a total of 4 electrons, the Randles-Ševičk equation of multi-electron electrode processes of organic species [50, 51] was applied as prediction of this behavior for NTBS and NFBS, being the equation:
$${\text{Icp}}_{\text{1}}\text{=}\left(\text{2.99}\text{ }\text{× }{\text{10}}^{\text{5}}\right)\text{A}{\text{D}}^{\text{0.5}}\text{C}{\text{}}^{\text{0.5}}\text{n}{\text{(n}\text{' }\text{+}\text{ }\text{α}\text{)}}^{\text{0.5}}$$
,
in which Icp1 is the peak current (A), n is the total number of electrons, A is the area of the electrode (cm2), D is the diffusion coefficient of drug (cm2/s), C is the bulk concentration of the analyte (mol/cm− 3), ν is the scan rate (V/s), n' is the number of electrons transferred before the rate determining step and α rate determining step is the transfer coefficient. Consequently, it is possible to verify by Figs. 2C and 2D that the voltammetric reduction of drugs was diffusion-controlled (blue line) in accordance to the simulated model (red line) only at low scan rates, since from 100 mV/s significant linearity change is observed. The logarithmic relationship between Icp1 and ν is linear for all scan rate range studied for both drugs (insets Figs. 2C and 2D) and it is empirically known that an intermediate slope value between 0.5 (diffusional) and 1 (adsorptive) suggests a mixed diffusion-adsorption-controlled system [52]. Therefore, being the slopes of 0.65 for NTBS and 0.70 for NFBS confirm a mixed mass transfer process.
The voltammetric reductions of NTBS and NFBS suffer significant influence of the reaction medium, specially the pH in aqueous media. The Figs. 3A and 3B show the changes of profile voltammetric under different pH values, in which it is possible to observe clear changes in the intensity and position of the generated peak. Current values decrease with increasing pH, reflecting a drop in the protonation rate until reaching the lowest values recorded in an alkaline medium [49]. Moreover, at pH 9.0 it is observed a split of the main peak correspondent to the Ecp1 with a second peak register around -0.5 V having also a corresponding oxidation peak. This phenomenon can be better evaluated by Figs. 3C and 3D, where it is verified that Ecp1 values (blue line) were displaced toward a more negative potential region with the increase of pH, besides to the recording of another peak at less negative potential values (Ecp2, red line) from pH 9.0 and which remains constant in a more alkaline media, being the Ecp2 values of -0.589 V and − 0.555 V for NTBS e NFBS, respectively. A similar voltammetric behavior was observed for nitrofurazone and its other derivatives [22–24, 39–41] as also other nitroheterocyclic compounds, such as nitrofurans and nitroimidazoles [20,21,24,25,38]. Thus, to this reduction peak is attributed to the reversible one-electron reduction to the radical anion and the other peak is due to the further three-electron reduction of the radical anion to the hydroxylamine.
Figure 3 clearly shows that the peak potential values for NTBS and NFBS were pH dependent. In the acidic range, Ecp1 is linearly shifted with slope value of 44 mV/pH for both studied compounds, although it is observable that at pH 6.0 linearity is changed for NFBS while the same occurs for NTBS only at pH 8.0. This behavior shows that the decreased acidity jeopardizes the electrode reaction with the fast protonation step occurring before the charge transfer process. However, the slope value indicates that one proton is involved in the rate-determining step of the reaction which may correspond to a second slow protonation reaction of the nitro group reduced to the nitroso intermediate. Additionally, the behavior change occurred in the range 6 < pH < 8 might be associated to an intermediary situation, in which the nitro-anion radical was not sufficiently and kinetically stable to produce the reversible redox couple on the GCE surface. Identical results were recorded for nitrofurazone and other nitrofuran analogues [23,24,34,35,38,41,48,52] which have been exhaustively studied [20,23,49,53].
At pH 12, the first reduction step of NTBS and NFBS was registered in restricted potential range as shown in Fig. 4 (A and B), which corresponds to the \(\text{R-N}{\text{O}}_{2}/\text{R-N}{\text{O}}_{2}^{•-}\) couple with Ecp2 = -0.589 V and − 0.555 V and the respective reverse anodic peak with Eap2 = -0.504 V and − 0.468 V, respectively. The cyclic voltammograms simulated by Digisim software are also shown, following the procedure described above and by using the diffusion coefficients of both drugs with the involvement of one electron for the nitro-anion radical generation, promoting a good match between the experimental record and simulated voltammograms, which corroborates the same potential range (Ecp2) and similar peak heights (cathodic current peak, Icp2). Both reduction processes were diffusion governed following the mass transport model for a reversible system [47, 50], in which Icp2 values varied linearly with the square root of scan rate, being Icp2 = 0.313–6.74 ν1/2 (R2 = 0.993) for NTBS and Icp2 = 0.113–9.34 ν1/2 (R2 = 0.996) for NFBS.
The radical kinetic stability was evaluated through the current ratio Iap2/Icp2 analysis corresponding to the one-electron reversible couple referent to the nitro-anion radical generation at pH 12, in which the biggest distance between Ecp2 and Ecp1 was registered. Figure 4 (C and D) shows the scan rate effect on current ratio, indicating that the reversibility of the redox system referring to the \(\text{R-N}{\text{O}}_{2}/\text{R-N}{\text{O}}_{2}^{•-}\)couple is increased with Iap2/Icp2 ratio reaching unity for the highest scan rates, presenting a similar tendency for both studied drugs with the same result set experimental and simulated. Additionally, from the cyclic voltammograms simulation, the dependence of the current ratio on drug concentration (from 0.01 to 0.1 mmol/L) was predicted, indicating a decreasing linear relationship, being Ipa2/Ipc2 = 0.869 − 0.298 [NTBS] (R2 = 0.999) and Ipa2/Ipc2 = 0.876 − 0.275 [NFBS] (R2 = 0.997). Similarly, this behavior also was previously observed for nitrofurazone [23,24,38,39,41]. This set of results meets the necessary diagnostic criteria to classify this redox system as a coupled homogeneous electrode process, in which a reversible charge transfer step is followed by an irreversible second-order chemical reaction [20, 21, 31].
The consolidated model developed by Olmstead and co-authors [20, 31, 32, 54] was applied taking into account the nitro-anion radical generation in aqueous medium (protic medium), in which the presence of enough protons favors the nitro-anion radical decay, facilitating the radical protonation reaction, the nitro group regeneration and the nitroso derivative formation, as depicted below:
2\(\text{R-N}{\text{O}}_{2}^{•-}\) + 2H+ → R-NO2 + R-NO
This irreversible disproportionation reaction is considered as second-order, being the respective kinetic parameters calculated based on the previous known model [32]. Figure 5 shows the plots between ω and τ parameters, from which the rate constant values for the second-order reaction (k2) were calculated from the slope of the linear fit. These parameters were also calculated with the simulated cyclic voltammograms by Digisim software, as described above. For both studied compounds, NTBS and NFBS, the simulated slopes were similar to the slopes values experimentally obtained. Additionally, Table 1 presents calculated k2 and half-life time (t1/2) values for the studied compounds, considering the experimental and simulated values. It is possible to observe that for both compounds the recorded results present close magnitude orders, in which the simulation satisfactorily corroborated the experimental values, reinforcing the adequacy of the adopted kinetic model as already similarly described for nitrofurazone and other nitrofuran analogues [23,24,34,35,38,41,48,52]. Thus, it can be observed that the NFBS, furanic nucleus derivative, showed higher stability and lower decay for the nitro-anion radical generated under these experimental conditions described in relation to the NTBS, thiophenic nucleus derivative.
Table 1
Stability kinetic data of \(\text{R-N}{\text{O}}_{2}^{•-}\) at pH 12 and cell growth inhibition of epimastigotes forms of T. cruzi
Compound | Cyclic voltammetry | Digisim | inhibition % * |
k2 (L mol-1 s-1) | t1/2 (s) | k2 (L mol-1 s-1) | t1/2 (s) |
NTBS | 20,585 | 0.486 | 23,021 | 0.434 | 97.6 |
NFBS | 10,923 | 0.915 | 11,127 | 0.899 | 93.0 |
*[compound] = 100 µmol/L (GATTI, 2015).
Unfortunately, it was not possible to establish a direct correlation between the kinetic stability and the percentage of cell inhibition of the epismatigote form of T. cruzi. This could indicate that other associated physicochemical properties determined the trypanocidal action of these derivatives, although confirming that the compounds containing the nitro group have significant biological activity (~ 100% inhibition at 0.1 mmol/L).