1 Introduction

Isatin and its derivatives have demonstrated diverse pharmacological properties1 such as antibacterial,2 anti-cancer,3 anti-tubercular,4 anti-HIV and antimalarial.5 Furthermore, isatin compounds are also widely applied in organic synthesis as a synthetic intermediate to obtain a diversity of target molecules.6 For example, the presence of two carbonyl groups allows the preparation of isatin derivatives through nucleophilic addition reactions under appropriate conditions.

Similarly, thiosemicarbazones and thiosemicarbazones complexes have also shown several applications.7,8,9,10 For instance, they have been employed for the treatment of innumerous diseases, including cancer,11,12,13 tropical infectious diseases, such as Chagas disease, human African trypanosomiasis, leishmaniasis, and malaria,14 as antifungal in food,15 as antiparasitic agent for veterinary use,16 among others applications.

In particular, the combination of isatin and thiosemicarbazones bioactive moieties at the same molecule has attracted interest to researchers since it allows the development of new candidate drugs. Generally, the isatin-thiosemicarbazone derivatives are efficiently synthesized via condensation reaction of isatins with thiosemicarbazones, which provide the respective products with unique applications including chemical, biological and pharmacological properties.17,18,19 In this regard, Hall et al.20 studied the properties of a series of isatin-β-thiosemicarbazones and it was notably found that some compounds selectively kill cells that express P-glycoprotein (P-gp, MDR1). Thiosemicarbazones derived from isatin have also demonstrated application in the treatment of smallpox,22 and derivatives from isatin-β-thiosemicarbazones show anti-HIV activity and inhibitor of Encephalitis Japanese among other viruses.23,24 In particular, our group has employed isatin-3-(N4-benzylthiosemicarbazone) as an antioxidant,25 and also for reactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) after contamination with organophosphates (OPs), which are widely used as pesticides and are inhibitors of these compounds in human body.26

Moreover, these kinds of isatin derivatives have also been studied as ligands in coordination compounds, which also have demonstrated pharmacological properties. In this context, Khan et al.27 have demonstrated the cytotoxic effect of isatin-N1-methyl-thiosemicarbazone and isatin-N1-ethyl-thiosemicarbazone copper complexes against L123 (human lung cells) and HepG2 cell (Hepatocellular carcinoma cells). Nonetheless, there is still a deficiency regarding the electrochemistry evaluation of isatin-thiosemicarbazone compounds.

On the other hand, the knowledge of electrochemical properties of biologically active molecules has been considered especially important to understand the reactivity of each reaction system through electron transfer reactions.32 In this context, these reactions might be correlated with the mechanisms of action of target molecules containing pharmacological activity,9 which provide useful information on the mechanism of the compounds with living cells. For example, Moreno and co-workers33 studied the anti-tuberculosis activity of a series of quinoxaline-2-carboxamide 1,4-di-N-oxides and concluded that the insertion of an electron-withdrawing group on the quinoxaline ring results in a less negative reduction potential and makes the bio-reduction more facile. The major mechanism of anticancer activity of thiosemicarbazone copper complexes is suggested to involve the thiol-mediated reduction of copper(II) to copper(I), and generation of reactive oxygen species (ROS) by copper(I) complex, which is re-oxidized to copper(II).11,12,13 The efficiency of each copper complex depends on its redox potential.

Notably, the electrochemical properties of isatin have been particularly studied by Oliveira et al.28 Similarly, there are studies of electrochemical properties of thiosemicarbazones derivatives such as di-2-thienyl ketone thiosemicarbazone,29 di-2-pyridyl ketone thiosemicarbazone30 and phenanthrenequinone thiosemicarbazone.31 The electrochemical characterization of target molecules containing the nitro group is also important due to the versatility of this class of nitrogen derivatives. Furthermore, nitro-group-containing drugs have been efficiently used as antineoplastic, antibiotic, antiparasitic agents, as well as insecticides and herbicides. Notably, this substituent might interact with biological species present in living systems by electron transfer involving transformations. Moreover, nitro groups can undergo reduction, which allows them to serve as prodrugs due to their bioactivation by enzymatic reduction, generating reactive species and ultimately inducing biological effects.34 Therefore, the study of electrochemical properties of isatin-thiosemicarbazones containing nitro groups in their structure is the great interest to researchers.

Despite the well-recognized applications of isatin-3-thiosemicarbazones to our knowledge is still highly desirable a study concerning reactivity and mechanisms of electrochemical transformations of this class of compounds. Thus, in this work, we have described the electrochemical characterization of a series of four isatin-thiosemicarbazone derivatives. These four molecules, which contain an analogous structure corresponding to the isatin-3-thiosemicarbazone (ITSC), are illustrated in Scheme 1.

Scheme 1
scheme 1

Chemical structure of the isatin-thiosemicarbazone derivatives: isatin-3-thiosemicarbazone (ITSC), isatin-3-(N4-benzylthiosemicarbazone) (ITSC-Ph), 1-(5-nitro-2-oxoindolin-3-ylidene)thiosemicarbazide (NO2-ITSC) and 1-(5-nitro-2-oxoindolin-3-ylidene)-4-phenylthiosemicarbazide (NO2-ITSC-Ph).

Full size image

2 Experimental

2.1 Reagents and method

Chemicals and analytical grade reagents (95.0–99.9% purity) obtained from commercial suppliers (Sigma Aldrich/Merck) were used as received. Elemental analysis (C, H, N) were analyzed on a Perkin–Elmer 2400 instrument. Melting points were recorded on a 430D Fisatom melting point instrument. The Fourier transform infrared spectra were recorded in the range 4000–400 cm−1 by diffuse reflectance on a Shimadzu-IR Prestige-2121 FT-IR spectrometer.

2.2 Synthesis of the isatin-thiosemicarbazone derivatives

The synthesis of the desired isatin-thiosemicarbazone was adapted from a procedure previously reported by Campaigne and Archer.35 The hydrochloric acid (3 drops) catalyzed reactions of the equal molar amounts of appropriate (un)substituted isatin (5,2 mmol) and thiosemicarbazide (5,2 mmol) moieties, in ethanol (60 mL) were refluxed for 6 h. After cooling, the obtained solid was filtered, washed with cold water and dried under vacuum to obtain the pure product as a solid. The formation of the desired products was confirmed by the appearance of the ν(C=N) band in the infrared spectra in the region of 1492-1664 cm−1, and the disappearance of the ν(C=O) band, at 1751 cm−1, due to the ketonic β-carbonyl group of the isatin.43

ITSC was synthesized by the reaction of isatin and thiosemicarbazide, as described elsewhere.20 Yield: 77%. Yellow solid. M.p.: 235 °C. Anal. Calcd. for C9H8N4OS (%): C, 49.08%; H, 3.66%; N, 25.44%. Found: C, 45.69%; H, 3.65; N, 23,54%. FT-IR (cm−1): 3422-3144 ν(N-H), 1682 ν(C=O), 1612 ν(C=N), 740 ν(C=S).

ITSC-Ph was synthesized by the reaction of isatin and 4-phenylthiosemicarbazide. The synthesis and crystal structure are described in a previous paper of our group.36 Yield: 80%. Orange solid. M.p.: 237 °C. Anal. Calcd. for C15H12N4OS (%): C, 60.80%; H, 4.08%; N, 18.91%. Found: C, 60.79%; H, 3.98%; N,18.62%. FT-IR (cm−1): 3298-3178 ν(N-H), 1693 ν(C=O), 1593 ν(C=N), 744 ν(C=S).

For the synthesis of NO2-ITSC, 5-nitroisatin and thiosemicarbazide were used. Our group had also described the synthesis and crystal structure.37 Yield: 91%. Yellow solid. M.p.: 265-270 °C dec.. Anal. Calcd. for C9H7N5O3S (%): C, 40.75; H, 2.68; N, 26.40. Found: C, 40.8; H, 2.15; N, 25.19. FT-IR (cm−1): 3196, 3275 ν(N-H), 1697 ν(C=O), 1620 ν(C=N), 1138 ν(C=S), 1338 ν(NO2) and 1516 νas(NO2).

NO2-ITSC-Ph was synthesized by the reaction of 5-nitroisatin and 4-phenylthiosemicarbazide, as described elsewhere.20 Yield: 94%. Orange solid. M.p.: 245-251 °C dec. Anal. Calcd. for C15H11N5O3S (%): C, 52.78%; H, 3.25%; N, 20.52%. Found: C,52.56%; H, 3.15%; N, 20.52%. FT-IR (cm−1): 3304, 3169 ν(N-H), 1693 ν(C=O), 1535 ν(C=N), 1153 ν(C=S), 1483, 1464 νas(NO2), 1352 ν(NO2).

2.3 Voltammetric parameters and electrochemical cells

Electrochemical measurements were carried out using an Autolab potentiostat/galvanostat (PGSTAT302). It was equipped with a three-electrode cell, with platinum wire as a counter electrode and 2.00 mm diameter glassy carbon as a working electrode (GCE). Calomelan reference electrode Hg/Hg2Cl2 in saturated KCl was used for all electrochemical experiments. Measurements were performed in 5 × 10−3 mol L−1 solutions of the isatin-thiosemicarbazone derivatives, in dimethylsulfoxide (DMSO) containing tetrabutylammonium hexafluorophosphate (TBAPF6) 0.1 mol L−1 as supporting electrolyte. Deaeration of solutions was accomplished by passing a stream of nitrogen through the solution for 15 min. prior to the measurement and then maintaining a blanket atmosphere of nitrogen over the solution during the measurement. The GCE was polished using alumina powder (0.05 µm) before every electrochemical assay. Ferrocene was used as an internal reference, and the redox potentials presented in this work are related to the standard ferrocene/ferrocenium redox couple (Fc/Fc+).

3 Results and Discussion

3.1 Isatin-thiosemicarbazone derivatives oxidation

Figure 1 represents the positively initiated scan cyclic voltammograms of the four isatin-thiosemicarbazones derivatives. It can be observed that the four molecules presented irreversible oxidation processes (wave 1). The oxidation potentials are shown in Table 1. The ligands ITSC, ITSC-Ph and NO2-ITSC-Ph showed one well-defined oxidation wave at 0.97, 0.88 and 0.98 V, respectively. The ligand NO2-ITSC showed two not well-defined oxidation waves at 0.92 and 1.03V.

Figure 1
figure 1

(a) Cyclic voltammograms of ITSC, (b) ITSC-Ph, (c) NO2-ITSC and (d) NO2-ITSC-Ph at 100 mV s−1 scan rate.

Full size image
Table 1 Oxidation potentials of the isatin-thiosemicarbazone derivatives.
Full size table

Voltammograms at different scan rates of this oxidation process were obtained for ITSC. It was observed linear dependence of peak current on the square root of the scan rate (I × v1/2) indicating the diffusion-controlled nature of the process.

Bakir and co-workers29 studied the oxidation of di-2-thienyl ketone thiosemicarbazone in DMSO and proposed an oxidation mechanism based on the removal of two electrons from the C=N double bond, and addition of OH groups from the residual water present in the solvent to both atoms, with subsequent cleavage of carbon-nitrogen bond. A similar reaction mechanism was proposed for the isatin-thiosemicarbazone derivatives, as represented in Scheme 2. Nonetheless, the products obtained in the present electrochemical reaction were isatin (6), thiourea (5) and nitroxyl.

Scheme 2
scheme 2

Mechanism proposed for the isatin-thiosemicarbazone derivatives oxidation, and reduction of the oxidatively generated isatin.

Full size image

On the reverse scan, two oxidatively generated reduction processes were clearly observed, which are indicated as waves 2 and 3 in Figure 1. Furthermore, the potentials of these processes are shown in Table 2. These waves are not observed in the voltammograms with reversal at less positive switching potentials (Figure 1), confirming to be due to the reduction of oxidatively generated products. Therefore, these waves are most likely attributed to the reduction of isatin which was generated in the decomposition of the isatin-thiosemicarbazone derivatives after their oxidation (Scheme 2). In addition, isatin presents two successive reduction processes, due to the reduction of the two ketone groups to form hydroxyl species. Yeagley and co-workers38 found the first reduction process at − 1.34 V for unsubstituted isatin and at − 1.07 V for isatin substituted with the nitro group. The nitro-substituted isatins reduce easier than unsubstituted analogues due to the electron-withdrawing character of the nitro groups. However, in our work, we have observed that wave 2 appears at −1.20 V in the ITSC and ITSC-Ph voltammograms, and at −0.96 and −0.93 V in the NO2-ITSC and NO2-ITSC-Ph voltammograms. The more positive values of wave 2 in the NO2-ITSC and NO2-ITSC-Ph voltammograms are most probably due to the nitro group which was attached at 5-position of isatin moiety. Moreover, these obtained results are in accordance with the proposed reaction pathway illustrated in Scheme 2.

Table 2 Reduction potentials of the oxidatively generated products.
Full size table

3.2 Isatin-thiosemicarbazone derivatives reduction

To understand better the reduction processes of the target isatin-thiosemicarbazone derivatives, voltammograms at negative potential ranges with different switching potentials were obtained (Figure 2).

Figure 2
figure 2

Cyclic voltammograms of ITSC, ITSC-Ph, NO2-ITSC and NO2-ITSC-Ph, at 100 mV s−1 scan rate.

Full size image

In the cyclic voltammograms of ITSC and ITSC-Ph (Figure 2, AI and BI), three principal waves of reduction indicated as 4, 5 and 6, and a shoulder indicated as 7, were observed. The potentials are represented in Table 3.

Table 3 Reduction potentials of the isatin-thiosemicarbazone derivatives.
Full size table

Voltammograms at different scan rates of these reduction processes were obtained for the ITSC molecule. The relationship of the peak currents as a function of the scan rates were analyzed for the processes observed at peaks 4, 5 and 6 (Figure 2). For the three waves, it was observed linear dependence of peak current on the square root of the scan rate (I × v1/2) indicating the diffusion-controlled nature of the processes.

A three steps reduction process is in accord with the reduction of similar compounds which were previously described in the literature. Thus, supported by the literature and based on our experimental results a reaction mechanism was conveniently proposed for ITSC and ITSC-Ph (Scheme 3). This mechanism is similar to that proposed by Bakir and co-workers29 for di-2-thienyl ketone thiosemicarbazone, by Bakir and Brown30 for di-2-pyridyl ketone thiosemicarbazone, by Reddy and co-workers39 for aryl hydrazones containing 1,3,4-oxadiazole moiety and pyrazoline-3-one moiety and by Afrasiabi and co-workers31 for phenanthrenequinone thiosemicarbazone. It is also similar for the reduction of the intermediate formed by the reduction of 3-diazooxindol, as proposed by Cardinali and co-workers.40

Scheme 3
scheme 3

Mechanism proposed for ITSC (1a) and ITSC-Ph (1b) reduction.

Full size image

The first reduction process (wave 4) appears at − 1.62 and − 1.57 V for the ITSC and ITSC-Ph, respectively. When the cathodic scan is reversed after this first reduction process (Figure 2, AIII and BIII), the reductively generated product is oxidized at 0.05 and 0.10 V (wave 11) for ITSC (1a) and ITSC-Ph (1b), respectively (Table 4). Wave 11 may be attributed to the oxidation of thiourea moiety (5) into corresponding formamidine disulfide. The electrochemical behavior of thiourea in acetonitrile was previously studied by Mouanga and Berçot.41 Based on this study, wave 7 most likely is attributed to the reduction of thiourea into respective sulfide, cyanide and ammonium ions. This proposition is supported by the previous contribution of Bakir and co-workers29 where the authors also observed reduction and oxidation of thiourea after reduction of di-2-thienyl ketone thiosemicarbazone, with the generation of less intensity waves when using glassy carbon electrode instead of the platinum electrode.

Table 4 Oxidation potential of the reductively generated products.
Full size table

The reduction of the intermediate 10 (wave 5) is observed at − 2.19 and − 2.06 V for ITSC and ITSC-Ph, respectively. When the cathodic scan is switched after wave 5 (Figure 2, AII and BII) the reductively generated compound 12 (3-iminoindolin-2-one) is oxidized at − 1.45 and − 1.39 V (wave 12) (Table 4), for ITSC and ITSC-Ph, respectively. The last reduction process (wave 6) for the generation of 3-aminoindolin-2-one 13 was observed at − 2.54 and − 2.58 V for the ITSC and ITSC-Ph, respectively.

In the cyclic voltammograms of NO2-ITSC and NO2-ITSC-Ph (Figure 2, CI and DI) waves 4 and 5 are also present but wave 6 is not observed, and three other waves (8, 9 and 10) are generated. Based on these experimental observations and in accordance with previous reports,28,42 a plausible reaction mechanism is illustrated in Scheme 4. The waves 4 and 5 are most likely attributed to similar processes as compared to ITSC and ITSC-Ph. However, the other three reduction waves (8, 9 and 10) may be attributed to reductions of the nitro groups (Scheme 4).

Scheme 4
scheme 4

Mechanism proposed for NO2-ITSC (1c) and NO2-ITSC-Ph (1d) reduction.

Full size image

It was found that the first reduction process of NO2-ITSC and NO2-ITSC-Ph (wave 4) appears at − 1.40 V for both molecules (Table 3). In these two compounds with a nitro group attached at the isatin phenyl ring, the potential values are more positive when compared with their analogues without substitution. Furthermore, these two nitro-substituted molecules are more easily reduced, indicating that this group plays an important role in this transformation withdrawing electronic density from the C=N-NH fragment.

The next reduction process is represented by wave 5, observed at −1.75 and −1.66 V for NO2-ITSC and NO2-ITSC-Ph, respectively. This process in these two nitro-substituted molecules show less negative potentials values when compared with ITSC and ITSC-Ph, confirming the electron-withdrawing character of the nitro groups. No oxidation of the reductively generated product 12 was observed in the voltammograms with switching potentials after wave 5 (Figure 2, CIII and DIII).

In fact, the next reduction is a reversible process proposed to be due to the reduction of –NO2 group to –NO2H,42 generating the intermediate 14 represented in Scheme 4. In the cyclic voltammograms CII and DII illustrated in Figure 2 it was observed the cathodic waves 8 at − 1.90 and − 1.92 V for NO2-ITSC and NO2-ITSC-Ph, respectively. The corresponding anodic waves 14 are at − 1.86 and − 1.80 V, respectively, for NO2-ITSC and NO2-ITSC-Ph (Table 4).

The –NO2H group is proposed to reduce to –N(OH)2 at − 2.00 and − 2.03 V (wave 9) in the voltammograms of NO2-ITSC and NO2-ITSC-Ph, respectively, generating the compound 15 (Scheme 4). Subsequently, this compound loses an equivalent of water, affording the product 16, which is oxidized at − 1.30 and − 1.22 V (wave 13), respectively, for NO2-ITSC and NO2-ITSC-Ph.

Finally, the –NO group is reduced to –NHOH, as indicated by wave 10, at − 2.76 and − 2.80 V, respectively for NO2-ITSC and NO2-ITSC-Ph (Table 3). Thus, thiourea moieties 5 and 5-hydroxyamino-3-iminoindolin-2-one 17 are conveniently proposed to be the final products of the reduction process of NO2-ITSC and NO2-ITSC-Ph.

Additionally, in the reverse scan, it is possible to observe oxidation of the final product 5-hydroxyamino-3-iminoindolin-2-one 17 in the range from − 0.20 to − 0.09 V (wave 15), which is in accordance with Andres and co-workers,42 who observed the oxidation of Ph-NHOH at about − 0.1 V.

On the other hand, it should be noted, also in accordance with Andres and co-workers,42 who studied the reduction of Ph-NO to Ph-NHOH, that side reactions as well as parallel products could also be formed in the reduction of –NO to –NHOH. They observed that some protonation of the PhNO−· occurs leading to dimerization and formation of PhN=N(O)Ph. In the sequence, PhN=N(O)Ph is reduced to PhN=N(O)Ph-, which can also undergo further chemically irreversible reduction to the azo, PhN=NPh, and then the hydrazo, PhNHNHPh, products. Considering this as a model, we can also propose that in the reduction of R-NO to R-NHOH (where R represents the ITSC or ITSC-Ph moieties) (wave 10), dimerization occurs leading to the formation of the azoxy (RN=N(O)R), azo (RN=NR) and the hydrazo (RNHNHR) products. In the study of Andres and co-workers,42 oxidation of PhN=N(O)Ph was observed at − 1.92 V, which indicates that wave 18, in the range from − 1.58 to − 1.56 V, is possibly due to RN=N(O)R oxidation.

Finally, the last two waves observed in the anodic scan waves 16 and 17, maybe related to oxidation of the azo (RN=NR) and the hydrazo (RNHNHR) reductively generated products. Although considering that different mechanisms, involving one or two steps, and different oxidation potentials may be observed for azo44 and hydrazo45 compounds, depending strongly on the identity of the organic groups attached to both nitrogen atoms, more studies should be necessary for a precise attribution of these two waves.

4 Conclusions

In summary, the electrochemical characterization of four isatin-thiosemicarbazone derivatives was performed, including the study of the electrochemical behavior of the group nitro. Upon oxidation, it was proposed to break the C=N bond and generate isatin and thiourea moieties for all evaluated molecules. Regarding the reduction, three processes were observed for the isatin-thiosemicarbazone derivatives, which were not substituted with the group nitro. Thus, the cleavage of the N-N bond as well as the generation of 3-aminoindolin-2-one and thiourea moieties was proposed. Furthermore, it was notably observed the reduction of the nitro groups from nitro-substituted isatin-thiosemicarbazone derivatives, resulting in 5-hydroxyamino-3-iminoindolin-2-one and thiourea moieties as final products. Studies regarding the preparation of different isatin-thiosemicarbazone derivatives and coordination compounds along with their structural characterization and electrochemical evaluation are still under investigation in our laboratory.