Catalytic Antioxidant Activity of Bis-Aniline-Derived Diselenides as GPx Mimics

Herein, we describe a simple and efficient route to access aniline-derived diselenides and evaluate their antioxidant/GPx-mimetic properties. The diselenides were obtained in good yields via ipso-substitution/reduction from the readily available 2-nitroaromatic halides (Cl, Br, I). These diselenides present GPx-mimetic properties, showing better antioxidant activity than the standard GPx-mimetic compounds, ebselen and diphenyl diselenide. DFT analysis demonstrated that the electronic properties of the substituents determine the charge delocalization and the partial charge on selenium, which correlate with the catalytic performances. The amino group concurs in the stabilization of the selenolate intermediate through a hydrogen bond with the selenium.

Notable among the polyfunctionalized diselenides, the presence of an amino or carbonyl group in close proximity to the selenium moiety has some unique biological features due to non-bonding interactions [33][34][35]. For example, the bis-2-aniline diselenide is reported to be a good antioxidant, preventing the oxidative stress caused by peroxynitrite and hydroperoxides [36][37][38]. Moreover, the aniline-derived diselenides, mainly with an amino group in the ortho position, give these compounds two possible reactive centers, Se-Se bond cleavage and the unshared pair of electrons on the nitrogen. This makes this class of compounds extremely flexible in functional group interconversions, making it appropriate for several transformations, mainly in the formation of selenium-containing heterocycles, such as selenamides [39], benzoselenazines [40], benzoselenazoles [41] and triazole diselenides [42].
Recently, we reported a new robust methodology for the synthesis of o-aniline-derived diselenides from the reduction of o-nitrobenzene diselenides. As part of our wider research program aimed at efficient methodologies for the synthesis of organoselenium compounds and their biological evaluation [43][44][45][46][47][48][49][50], herein, we report the application of oaniline-derived diselenides as potential GPx mimics. For this purpose, different physicalchemical studies were performed to demonstrate their biological properties, i.e., kinetic profile. Furthermore, DFT studies were also carried out in order to study the electronic properties of the substituents for determining the charge delocalization on the selenium atom and its influence on catalytic performance.

Results and Discussion
The bis-o-nitrobenzene diselenides were initially prepared through the nucleophilic aromatic substitution of o-halonitrobenzenes with K2Se2 (generated in situ) from a modified simple methodology [51]. After this, we carried out the reduction of bis-o-nitrobenzene diselenides from a well-established procedure described in the literature [52] using low-cost iron sulfate heptahydrate (FeSO4.7H2O) to synthesize the aniline-derived diselenides (Scheme 1).  Among organoselenium compounds, diorganyl diselenides present important antioxidant and anticancer properties mainly because of the ability of these diselenides to act as mimetics of the enzyme glutathione peroxidase (GPx) [6,8,11]. This selenoenzyme possesses a residue of selenocysteine in its active site and is responsible for the reduction of peroxides to water in our organism, protecting it from oxidative stress and related diseases [32].
Notable among the polyfunctionalized diselenides, the presence of an amino or carbonyl group in close proximity to the selenium moiety has some unique biological features due to non-bonding interactions [33][34][35]. For example, the bis-2-aniline diselenide is reported to be a good antioxidant, preventing the oxidative stress caused by peroxynitrite and hydroperoxides [36][37][38]. Moreover, the aniline-derived diselenides, mainly with an amino group in the ortho position, give these compounds two possible reactive centers, Se-Se bond cleavage and the unshared pair of electrons on the nitrogen. This makes this class of compounds extremely flexible in functional group interconversions, making it appropriate for several transformations, mainly in the formation of selenium-containing heterocycles, such as selenamides [39], benzoselenazines [40], benzoselenazoles [41] and triazole diselenides [42].
Recently, we reported a new robust methodology for the synthesis of o-aniline-derived diselenides from the reduction of o-nitrobenzene diselenides. As part of our wider research program aimed at efficient methodologies for the synthesis of organoselenium compounds and their biological evaluation [43][44][45][46][47][48][49][50], herein, we report the application of o-anilinederived diselenides as potential GPx mimics. For this purpose, different physical-chemical studies were performed to demonstrate their biological properties, i.e., kinetic profile. Furthermore, DFT studies were also carried out in order to study the electronic properties of the substituents for determining the charge delocalization on the selenium atom and its influence on catalytic performance.

Results and Discussion
The bis-o-nitrobenzene diselenides were initially prepared through the nucleophilic aromatic substitution of o-halonitrobenzenes with K 2 Se 2 (generated in situ) from a modified simple methodology [51]. After this, we carried out the reduction of bis-o-nitrobenzene diselenides from a well-established procedure described in the literature [52] using lowcost iron sulfate heptahydrate (FeSO 4 .7H 2 O) to synthesize the aniline-derived diselenides (Scheme 1).  Among organoselenium compounds, diorganyl diselenides present important antioxidant and anticancer properties mainly because of the ability of these diselenides to act as mimetics of the enzyme glutathione peroxidase (GPx) [6,8,11]. This selenoenzyme possesses a residue of selenocysteine in its active site and is responsible for the reduction of peroxides to water in our organism, protecting it from oxidative stress and related diseases [32].
Notable among the polyfunctionalized diselenides, the presence of an amino or carbonyl group in close proximity to the selenium moiety has some unique biological features due to non-bonding interactions [33][34][35]. For example, the bis-2-aniline diselenide is reported to be a good antioxidant, preventing the oxidative stress caused by peroxynitrite and hydroperoxides [36][37][38]. Moreover, the aniline-derived diselenides, mainly with an amino group in the ortho position, give these compounds two possible reactive centers, Se-Se bond cleavage and the unshared pair of electrons on the nitrogen. This makes this class of compounds extremely flexible in functional group interconversions, making it appropriate for several transformations, mainly in the formation of selenium-containing heterocycles, such as selenamides [39], benzoselenazines [40], benzoselenazoles [41] and triazole diselenides [42].
Recently, we reported a new robust methodology for the synthesis of o-aniline-derived diselenides from the reduction of o-nitrobenzene diselenides. As part of our wider research program aimed at efficient methodologies for the synthesis of organoselenium compounds and their biological evaluation [43][44][45][46][47][48][49][50], herein, we report the application of oaniline-derived diselenides as potential GPx mimics. For this purpose, different physicalchemical studies were performed to demonstrate their biological properties, i.e., kinetic profile. Furthermore, DFT studies were also carried out in order to study the electronic properties of the substituents for determining the charge delocalization on the selenium atom and its influence on catalytic performance.

Results and Discussion
The bis-o-nitrobenzene diselenides were initially prepared through the nucleophilic aromatic substitution of o-halonitrobenzenes with K2Se2 (generated in situ) from a modified simple methodology [51]. After this, we carried out the reduction of bis-o-nitrobenzene diselenides from a well-established procedure described in the literature [52] using low-cost iron sulfate heptahydrate (FeSO4.7H2O) to synthesize the aniline-derived diselenides (Scheme 1). The aniline-derived diselenides 3a-e were evaluated with regard to GPx-like antioxidant activity. The catalytic parameters were obtained using the Tomoda [53] reaction model, where the synthesized diselenides were applied as catalysts in the formation of diphenyl disulfide (PhSSPh) through the reduction of hydrogen peroxide (H 2 O 2 ) in the presence of thiophenol (PhSH), which is accompanied by an increase in UV/vis absorbance in 305 nm ( Figure 2a). Then, the absorbance was plotted against diphenyl disulfide concentration to determine the molar absorptivity at 305 nm ( Figure 2b). The aniline-derived diselenides 3a-e were evaluated with regard to GPx-like antioxidant activity. The catalytic parameters were obtained using the Tomoda [53] reaction model, where the synthesized diselenides were applied as catalysts in the formation of diphenyl disulfide (PhSSPh) through the reduction of hydrogen peroxide (H2O2) in the presence of thiophenol (PhSH), which is accompanied by an increase in UV/vis absorbance in 305 nm ( Figure 2a). Then, the absorbance was plotted against diphenyl disulfide concentration to determine the molar absorptivity at 305 nm ( Figure 2b). The catalytic parameters were obtained by fitting the kinetic profiles, that is, initial rate versus initial PhSH concentration, as shown in Figure Table 1 shows the catalytic constant (kcat), the Michaelis-Menten constant (Km) and the catalytic efficiency (η where η = kcat/Km) for the reaction with the aniline-derived The catalytic parameters were obtained by fitting the kinetic profiles, that is, initial rate versus initial PhSH concentration, as shown in Figure  The aniline-derived diselenides 3a-e were evaluated with regard to GPx-like antioxidant activity. The catalytic parameters were obtained using the Tomoda [53] reaction model, where the synthesized diselenides were applied as catalysts in the formation of diphenyl disulfide (PhSSPh) through the reduction of hydrogen peroxide (H2O2) in the presence of thiophenol (PhSH), which is accompanied by an increase in UV/vis absorbance in 305 nm (Figure 2a). Then, the absorbance was plotted against diphenyl disulfide concentration to determine the molar absorptivity at 305 nm (Figure 2b). The catalytic parameters were obtained by fitting the kinetic profiles, that is, initial rate versus initial PhSH concentration, as shown in Figure Table 1 shows the catalytic constant (kcat), the Michaelis-Menten constant (Km) and the catalytic efficiency (η where η = kcat/Km) for the reaction with the aniline-derived  Table 1 shows the catalytic constant (k cat ), the Michaelis-Menten constant (K m ) and the catalytic efficiency (η where η = k cat /K m ) for the reaction with the aniline-derived diselenides 3a to 3e and, for comparison, the well-known catalysts ebselen [35] and diphenyl diselenide [53]. diselenides 3a to 3e and, for comparison, the well-known catalysts ebselen [35] and diphenyl diselenide [53].  diselenides 3a to 3e and, for comparison, the well-known catalysts ebselen [35] and diphenyl diselenide [53]. diselenides 3a to 3e and, for comparison, the well-known catalysts ebselen [35] and diphenyl diselenide [53]. The results show that the catalytic efficiency of the aniline-derived diselenides is structure-dependent, especially regarding the electronic character of the substituents at the para position related to selenium, with an increase in the catalytic efficiency with the electron-withdrawal capacity of the substituent (compound 3b), once cleavage of the Se-Se bond is facilitated. These results suggest that the mechanism of these catalyzed reactions involves the formation of a zwitterionic form of the selenolate intermediate with a negative density charge in the selenium atom (Scheme 2a), which is similar to the mechanism proposed by Tomoda et al. [53].
The structures 3a-3e have been optimized by Density Functional Theory (DFT) at the BP86-D3/def2-TZVP level of theory, using the zero-order regular approximation (ZORA) to take the relativistic effects into account. In all the optimized geometries (except 3b), an intramolecular hydrogen bond (HB) exists between the two amine moieties, with distances that range from 2.624 (3d) to 3.139 (3a) Å . In the case of 3b, the electron-withdraw- The results show that the catalytic efficiency of the aniline-derived diselenides is structure-dependent, especially regarding the electronic character of the substituents at the para position related to selenium, with an increase in the catalytic efficiency with the electron-withdrawal capacity of the substituent (compound 3b), once cleavage of the Se-Se bond is facilitated. These results suggest that the mechanism of these catalyzed reactions involves the formation of a zwitterionic form of the selenolate intermediate with a negative density charge in the selenium atom (Scheme 2a), which is similar to the mechanism proposed by Tomoda et al. [53].
The structures 3a-3e have been optimized by Density Functional Theory (DFT) at the BP86-D3/def2-TZVP level of theory, using the zero-order regular approximation (ZORA) to take the relativistic effects into account. In all the optimized geometries (except 3b), an intramolecular hydrogen bond (HB) exists between the two amine moieties, with distances that range from 2.624 (3d) to 3.139 (3a) Å . In the case of 3b, the electron-withdrawing -CF3 group likely makes the lone pair of the nitrogen less available for HBs. The electronic effect of the group in the para position influences all the atomic charges of the diselenide system. Indeed, the atomic charges have been computed through the Natural Population Analysis (see Computational Details) as implemented in NBO 6.0, and for the The results show that the catalytic efficiency of the aniline-derived diselenides is structure-dependent, especially regarding the electronic character of the substituents at the para position related to selenium, with an increase in the catalytic efficiency with the electron-withdrawal capacity of the substituent (compound 3b), once cleavage of the Se-Se bond is facilitated. These results suggest that the mechanism of these catalyzed reactions involves the formation of a zwitterionic form of the selenolate intermediate with a negative density charge in the selenium atom (Scheme 2a), which is similar to the mechanism proposed by Tomoda et al. [53]. Of the aniline-derived diselenides, the highest catalytic efficiency was observed for compound 3b (Table 1, entry 4), which was 5 and 2 times more active than the standards ebselen and diphenyl diselenide, respectively. It is worth noting that the diselenide 3b was more effective than ebselen, which is a pre-clinical drug candidate with pronounced biological activities, including, recently, the inhibition of protease M pro from COVID-19 (SARS-CoV-2) virus [29][30][31].
Due to the high antioxidant activity of the diselenide 3b as a mimetic of GPx, we decided to investigate the effectiveness of this methodology at the gram scale. Thus, we performed the reaction from 20.0 mmol (5.40 g) of the o-halonitrobenzene 1c to afford the desired nitro-diselenide 2b, followed by its reduction to obtain the bis-aniline-derived 3b without a significant decrease in the yields (Scheme 3), proving that this protocol could be used as a robust method in the larger-scale synthesis of this privileged structure.

GPx-Like Experimental Procedure
The kinetic profile of the oxidation reaction was conducted in a UV-vis Spectrophotometer, following the wavelength of diphenyl disulfide formation at 305 nm. Spectroscopic methanol was used as solvent in the oxidation reaction, and the final volume of cuvettes was kept at 2000 µ L. The H2O2 and catalyst concentration were fixed in 15 × 10 −3 mol.L −1 and 1 × 10 −5 mol.L −1 respectively, and the PhSH concentration was varied from 0.5 × 10 −3 to 15 × 10 −3 mol L −1 . The temperature was kept at 25 °C, and each experiment was run at least 2 times.

Michaelis-Menten Equation
The GPx-like kinetic profiles were treated using the Michaelis-Menten nonlinear Equation (1)  The structures 3a-e have been optimized by Density Functional Theory (DFT) at the BP86-D3/def2-TZVP level of theory, using the zero-order regular approximation (ZORA) to take the relativistic effects into account. In all the optimized geometries (except 3b), an intramolecular hydrogen bond (HB) exists between the two amine moieties, with distances that range from 2.624 (3d) to 3.139 (3a) Å. In the case of 3b, the electron-withdrawing -CF 3 group likely makes the lone pair of the nitrogen less available for HBs. The electronic effect of the group in the para position influences all the atomic charges of the diselenide system. Indeed, the atomic charges have been computed through the Natural Population Analysis (see Computational Details) as implemented in NBO 6.0, and for the selenium, it ranges from 0.063 to 0.123 e for 3c (the most electron-donating group) and 3b (the most electron-withdrawing one), respectively. The atomic charge on the selenium qualitatively correlates with η, according to which the best catalysts have a more positive charge on the selenium and a less negative charge on the nitrogen (Figure 4 and Supplementary Materials). In addition, a similar correlation can be observed between the atomic charge of the ammonium-selenolate and η: in this case, the best catalysts have a less negative charge on the selenium, leading to a larger degree of charge delocalization and, consequently, a more stable intermediate. This is in agreement with the mechanism proposed by Tomoda [53]. Furthermore, the hydrogen bonding between the ammonium protons and the selenolate moiety is quite strong and stabilizes the intermediate, having an orbital interaction of 8-9 kcal/mol depending on the substituent (Supplementary Materials), hence making the catalyst more active.
Tomoda et al. [53] also proposed that another reactive intermediate is formed in the initial step from the reaction of the diselenide with PhSH, that is, the selenyl sulfide (Scheme 2b). In the case of the aniline-derived diselenides, it seems that the formation of this intermediate is destabilized by the inductive electron donor capacity of the amine groups at the ortho position, reflected in their lower catalytic efficiency when compared with the diphenyl diselenide (Table 1, entries 3 and 2, respectively).
Of the aniline-derived diselenides, the highest catalytic efficiency was observed for compound 3b (Table 1, entry 4), which was 5 and 2 times more active than the standards ebselen and diphenyl diselenide, respectively. It is worth noting that the diselenide 3b was more effective than ebselen, which is a pre-clinical drug candidate with pronounced biological activities, including, recently, the inhibition of protease M pro from COVID-19 (SARS-CoV-2) virus [29][30][31].
Due to the high antioxidant activity of the diselenide 3b as a mimetic of GPx, we decided to investigate the effectiveness of this methodology at the gram scale. Thus, we performed the reaction from 20.0 mmol (5.40 g) of the o-halonitrobenzene 1c to afford the desired nitro-diselenide 2b, followed by its reduction to obtain the bis-aniline-derived 3b without a significant decrease in the yields (Scheme 3), proving that this protocol could be used as a robust method in the larger-scale synthesis of this privileged structure.
charge of the ammonium-selenolate and η: in this case, the best catalysts have a less negative charge on the selenium, leading to a larger degree of charge delocalization and, consequently, a more stable intermediate. This is in agreement with the mechanism proposed by Tomoda [53]. Furthermore, the hydrogen bonding between the ammonium protons and the selenolate moiety is quite strong and stabilizes the intermediate, having an orbital interaction of 8-9 kcal/mol depending on the substituent (Supplementary Materials), hence making the catalyst more active. Tomoda et al. [53] also proposed that another reactive intermediate is formed in the initial step from the reaction of the diselenide with PhSH, that is, the selenyl sulfide (Scheme 2b). In the case of the aniline-derived diselenides, it seems that the formation of this intermediate is destabilized by the inductive electron donor capacity of the amine Of the aniline-derived diselenides, the highest catalytic efficiency was observed for compound 3b (Table 1, entry 4), which was 5 and 2 times more active than the standards ebselen and diphenyl diselenide, respectively. It is worth noting that the diselenide 3b was more effective than ebselen, which is a pre-clinical drug candidate with pronounced biological activities, including, recently, the inhibition of protease M pro from COVID-19 (SARS-CoV-2) virus [29][30][31].
Due to the high antioxidant activity of the diselenide 3b as a mimetic of GPx, we decided to investigate the effectiveness of this methodology at the gram scale. Thus, we performed the reaction from 20.0 mmol (5.40 g) of the o-halonitrobenzene 1c to afford the desired nitro-diselenide 2b, followed by its reduction to obtain the bis-aniline-derived 3b without a significant decrease in the yields (Scheme 3), proving that this protocol could be used as a robust method in the larger-scale synthesis of this privileged structure.

GPx-Like Experimental Procedure
The kinetic profile of the oxidation reaction was conducted in a UV-vis Spectrophotometer, following the wavelength of diphenyl disulfide formation at 305 nm. Spectroscopic methanol was used as solvent in the oxidation reaction, and the final volume of cuvettes was kept at 2000 µ L. The H2O2 and catalyst concentration were fixed in 15 × 10 −3 mol.L −1 and 1 × 10 −5 mol.L −1 respectively, and the PhSH concentration was varied from 0.5 × 10 −3 to 15 × 10 −3 mol L −1 . The temperature was kept at 25 °C, and each experiment was run at least 2 times.

Michaelis-Menten Equation
The GPx-like kinetic profiles were treated using the Michaelis-Menten nonlinear Equation (1)

GPx-Like Experimental Procedure
The kinetic profile of the oxidation reaction was conducted in a UV-vis Spectrophotometer, following the wavelength of diphenyl disulfide formation at 305 nm. Spectroscopic methanol was used as solvent in the oxidation reaction, and the final volume of cuvettes was kept at 2000 µL. The H 2 O 2 and catalyst concentration were fixed in 15 × 10 −3 mol L −1 and 1 × 10 −5 mol L −1 respectively, and the PhSH concentration was varied from 0.5 × 10 −3 to 15 × 10 −3 mol L −1 . The temperature was kept at 25 • C, and each experiment was run at least 2 times.

Michaelis-Menten Equation
The GPx-like kinetic profiles were treated using the Michaelis-Menten nonlinear Equation (1): where the V 0 was the initial velocity and k cat and K m were the catalytic rate constant and Michaelis-Menten constant, respectively. The [cat] and [PhSH] represent the concentration of the catalyst and thiophenol, respectively.

Computational Details
All geometries were optimized with ORCA 4.1.0, [54] using the BP86 functional in conjunction with a triple-ζ quality basis set (ZORA-TZVP) and def2/J auxiliary basis. For heavy elements (such as selenium and bromine), relativistic effects have been accounted by using the Zeroth Order Regular Approximation (ZORA) scalar correction. The dispersion corrections were introduced using the Grimme D3-parametrized correction and the Becke−Johnson damping to the DFT energy [55]. All the diselenide structures were confirmed to be local energy minima (no imaginary frequencies). Selenolate species show an unavoidable imaginary frequency correlated with the rotation of the -NH 3 moiety. The atomic charges have been computed by the Natural Population Analysis (NPA) as implemented in NBO6 [56].

Conclusions
In conclusion, we have developed a short and robust synthetic route for the synthesis of nitro aryl and aniline-derived diselenides in good overall yields. The aniline-derived diselenides were evaluated as GPx mimetics and the diselenide 3b substituted with the CF 3 group showed the best results, being 5 and 2 times more effective as a GPx mimetic than the standard catalysts ebselen and diphenyl diselenide, respectively. Furthermore, DFT analysis was performed for all the diselenides, which demonstrated non-bonding interaction. This correlates with the GPx activities of these diselenides.

Supplementary Materials:
The following are available online, 1 H, and 13 C NMR spectra of the synthesized compounds (3a-e). Figure S1 Figure S3: HRMS spectrum of compound 2a. Figure S4  Spectrum of compound 3b. Figure S20: HRMS spectrum of compound 3b. Figure S21 Figure S26: HRMS spectrum of compound 3d. Figure S27 Figure S29: ESI-MS spectrum of compound 3e. Figure S30: 77 Se NMR (76 MHz, CDCl 3 ) Spectrum of compound 3b. Figure S31: Absorbance plotted against diphenyl disulfide concentration. The red line represents the linear fit. The coefficient of molar absorptivity in 305 nm was 1415 L mol −1 cm −1 (R 2 = 0.9996). Figure S32: Initial rate (V 0 ) plotted against substrate concentration. The initial rates were calculated from at least two experiments for each concentration of PhSH. The concentrations of ebselen and H 2 O 2 were fixed at 5 × 10 −5 and 15 × 10 −3 mol L −1 , respectively. The red line represents the Michaelis-Menten fit. Figure S33: Initial rate (V 0 ) plotted against substrate concentration. The initial rates were calculated from at least two experiments for each concentration of PhSH. The concentrations of diphenyl disulfide and H 2 O 2 were fixed at 5 × 10 −5 and 15 × 10 −3 mol L −1 , respectively. The red line represents the Michaelis-Menten fit. Figure S34: Initial rate (V 0 ) plotted against substrate concentration. The initial rates were calculated from at least two experiments for each concentration of PhSH. The concentrations of 3c and H 2 O 2 were fixed at 5 × 10 −5 and  Figure S35: Initial rate (V 0 ) plotted against substrate concentration. The initial rates were calculated from at least two experiments for each concentration of PhSH. The concentrations of 3a and H 2 O 2 were fixed at 5 × 10 −5 and 15 × 10 −3 mol L −1 , respectively. The red line represents the Michaelis-Menten fit. Figure S36: Initial rate (V 0 ) plotted against substrate concentration. The initial rates were calculated from at least two experiments for each concentration of PhSH. The concentrations of 3b and H 2 O 2 were fixed at 5 × 10 −5 and 15 × 10 −3 mol L −1 , respectively. The red line represents the Michaelis-Menten fit. Figure S37: Initial rate (V 0 ) plotted against substrate concentration. The initial rates were calculated from at least two experiments for each concentration of PhSH. The concentrations of 3d and H 2 O 2 were fixed at 5 × 10 −5 and 15 × 10 −3 mol L −1 , respectively. The red line represents the Michaelis-Menten fit. Figure S38: Initial rate (V 0 ) plotted against substrate concentration. The initial rates were calculated from at least two experiments for each concentration of PhSH. The concentrations of 3e and H 2 O 2 were fixed at 5 × 10 −5 and 15 × 10 −3 mol L −1 , respectively. The red line represents the Michaelis-Menten fit. Table S1: Atomic charges of nitrogen and selenium according to NPA. Table S2: Donor-acceptor second order perturbation analysis. Table S3: DFT-computed energies for optimized geometries (in kcal/mol).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.