Seleninic Acid Potassium Salts As Water-Soluble Biocatalysts with Enhanced Bioavailability

materials

Article Seleninic Acid Potassium Salts as Water-Soluble Biocatalysts with Enhanced Bioavailability

Magdalena Obieziurska 1, Agata J. Pacuła 1, Anna Laskowska 1, Angelika Długosz-Pokorska 2, 2 1, , Anna Janecka and Jacek Scianowski´ * † 1 Department of Organic Chemistry, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Street, 87-100 Torun, Poland; [email protected] (M.O.); [email protected] (A.J.P.); [email protected] (A.L.) 2 Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland; [email protected] (A.D.-P.); [email protected] (A.J.) * Correspondence: [email protected] Dedicated to Professor Grzegorz Mlosto´non the occasion of his 70th birthday. † Received: 20 December 2019; Accepted: 31 January 2020; Published: 2 February 2020

Abstract: Organoselenium compounds are well-known glutathione peroxidase (GPx) mimetics that possess antioxidants/prooxidant properties and are able to modulate the concentration of reactive oxygen species (ROS), preventing oxidative stress in normal cells or inducing ROS formation in cancer cells leading to apoptosis. The purpose of this study was the synthesis of potent GPx mimics with antioxidant and anticancer activity along with improved bioavailability, as a result of good solubility in protic solvents. As a result of our research, glutathione peroxidase (GPx) mimetics in the form of water-soluble benzeneseleninic acid salts were obtained. The procedure was based on the synthesis of 2-(N-alkylcarboxyamido)benzeneselenenic acids, through the oxidation of benzisoselenazol-3(2H)-ones or analogous arenediselenides with an amido group, which were further converted to corresponding potassium salts by the treatment with potassium tert-butanolate. All derivatives were tested as potential antioxidants and anticancer agents. The areneseleninic acid salts were significantly better peroxide scavengers than analogous acids and the well-known organoselenium antioxidant ebselen. The highest activity was observed for the 2-(N-ethylcarboxyamido)benzeneselenenic acid potassium salt. The strongest cytotoxic effect against breast cancer (MCF-7) and human promyelocytic leukemia (HL-60) cell lines was found for 2-(N-cyclohexylcarboxyamido)benzeneselenenic acid potassium salt and the 2-(N-ethylcarboxyamido)benzeneselenenic acid, respectively. The structure–activity correlations, including the differences in reactivity of benzeneseleninic acids and corresponding salts were evaluated.

Keywords: areneseleninic acids; areneseleninic acid salts; amphiphilic compounds; antioxidant activity; antiproliferative activity

1. Introduction Designing catalysts inspired by enzymes is one of the crucial tactics aimed at inﬂuencing cell physiology or reversing a pathological state that triggers a disease to develop. The catalytic systems protecting cells from reactive oxygen/nitrogen species (ROS/RNS) and further oxidative damage, involving selenoenzymes from the glutathione peroxidase (GPx) family, can operate due to the presence of the reactive selenol moiety incorporated in the structure of the amino acid selenocysteine [1–3]. In the GPx-catalytic cycle, the initial redox-active -SeH, plays the crucial role at the enzyme’s active site and initiates the cycle through the rapid reaction with H2O2. As it has been presented by Mugesh et al., the regulation of ROS concentrations depends on the levels of peroxide and the thiol co-factor, mostly

Materials 2020, 13, 661; doi:10.3390/ma13030661 www.mdpi.com/journal/materials Materials 2020, 13, 661 2 of 12 Materials 2019, 12, x FOR PEER REVIEW 2 of 12 the glutathione (GSH) [4]. Under physiological conditions, the active selenol 1 is ﬁrst oxidized to the co-factor, mostly the glutathione (GSH) [4]. Under physiological conditions, the active selenol 1 is selenenic acid 2 and next regenerated in the presence of two GHS molecules. In the state of oxidative first oxidized to the selenenic acid 2 and next regenerated in the presence of two GHS molecules. In stress, when the H2O2 level is higher, with a lower amount of thiol, the cycle is extended and further the state of oxidative stress, when the H2O2 level is higher, with a lower amount of thiol, the cycle is oxidation of the seleninic acid 3 takes place (Scheme1). extended and further oxidation of the seleninic acid 3 takes place (Scheme 1).

Scheme 1. GPx activity cycle at physiological conditions and elevated ROS levels. Scheme 1. GPx activity cycle at physiological conditions and elevated ROS levels. Since it was discovered that small organoselenium compounds can mimic the activity of glutathione Since it was discovered that small organoselenium compounds can mimic the activity of peroxidase and reduce peroxides in a similar manner to the above-presented cycle, a lot has been glutathione peroxidase and reduce peroxides in a similar manner to the above-presented cycle, a lot accomplished in the synthesis of redox-active Se-catalysts [5]. Several compounds have been identified has been accomplished in the synthesis of redox-active Se-catalysts [5]. Several compounds have been and selected as biologically potent, however, low selectivity, leading to higher toxicity, along with identified and selected as biologically potent, however, low selectivity, leading to higher toxicity, solubility problems are still limiting the applicability. When designing new GPx mimics in the form of along with solubility problems are still limiting the applicability. When designing new GPx mimics modified “selenocysteine-like” catalysts, the direct approach to synthetize compounds bearing a free in the form of modified “selenocysteine-like” catalysts, the direct approach to synthetize compounds selenol moiety is problematic due to the high instability of the -SeH group and rapid formation of the bearing a free selenol moiety is problematic due to the high instability of the -SeH group and rapid corresponding diselenide. Thus, an efficient organoselenium antioxidant should possess a masked formation of the corresponding diselenide. Thus, an efficient organoselenium antioxidant should Se-moiety that could be easily unmasked and transformed to a reactive selenol at the specific place of possess a masked Se-moiety that could be easily unmasked and transformed to a reactive selenol at action. This can be accomplished by synthetizing stable compounds that correspond structurally to a the specific place of action. This can be accomplished by synthetizing stable compounds that certain isoform of the GPx selenocysteinyl redox center. Herein, we present a series of new peroxide correspond structurally to a certain isoform of the GPx selenocysteinyl redox center. Herein, we scavengers in the form of seleninic acids 4 and corresponding seleninic salts 5. Both of these Se(IV) present a series of new peroxide scavengers in the form of seleninic acids 4 and corresponding compound series 4 and 5 can be qualified as the derivatives of the primal GPx in the form of seleninic seleninic salts 5. Both of these Se(IV) compound series 4 and 5 can be qualified as the derivatives of acid 3. The structure of the designed molecules is composed of two important features: the hydrophilic the primal GPx in the form of seleninic acid 3. The structure of the designed molecules is composed -SeOOH/SeOOK group and the hydrophobic N-alkylbenzamide moiety that connects together exhibit of two important features: the hydrophilic -SeOOH/SeOOK group and the hydrophobic N- amphiphilic properties. As the structure of derivative 4 corresponds to the specific benzseleninic acid alkylbenzamide moiety that connects together exhibit amphiphilic properties. As the structure of intermediated formed in the GPx catalytic cycle of the known drug candidate ebselen, the mechanism derivative 4 corresponds to the specific benzseleninic acid intermediated formed in the GPx catalytic of action should include the “ebselen-like” catalytic pathway A. However, we assume that in the case cycle of the known drug candidate ebselen, the mechanism of action should include the “ebselen- of analogue salt 5, the formation of the Se-N bond will not be achieved, which can accelerate the speed like” catalytic pathway A. However, we assume that in the case of analogue salt 5, the formation of of peroxide reduction (pathway B, Scheme2). the Se-N bond will not be achieved, which can accelerate the speed of peroxide reduction (pathway Amphiphilic molecules, that can easily penetrate lipid bilayer membranes, may create a delivery B, Scheme 2). system with an incorporated pharmacophore. The Se(IV) molecules can act as “the hidden selenol”, as postulated by Rocha [1], be transported to the specific place of action as stable and soluble RSeOOH/RSeOOK and, at the final stage, be transformed into the bioactive RSeH. Till now, seleninic acids have found several application routes, including their utilization as ligands [6–10] and reagents in oxidation [11–14], dehydrogenation [15] and oxidative deoximation reactions [16]. However, their applicability in medicinal chemistry seems to be still an unexplored field with promising perspectives. Only few examples of such areneseleninic acids have been published by Mugesh et al. [4,17–19], but analyzed only as intermediates of corresponding benzisoselenazolones, with no activity evaluation. Similar amphiphilic seleninic acids with p-amido function have been

Materials 2020, 13, 661 3 of 12 presented by Jacobs et al. [20]. The studies revealed that the compounds can easily penetrate cell membranes and possess signiﬁcantly better antibacterial activity than phenyl seleninic acid and simple surfactants. Nevertheless, the “ebselen-like” catalysts have never been synthetized in the form of seleninic acid salts. The purpose of this study was the synthesis of GPx mimics in the form of - + + RSeOO M . The presence of the -SeOO−M moiety can improve the solubility in water and change the physicochemical properties of the molecules which, in turn, may strongly inﬂuence the metabolism of theMaterials presented 2019, 12 potential, x FOR PEER drug REVIEW candidates. 3 of 12

Scheme 2. Origin of the structure of the designed antioxidants 4 and 5. Scheme 2. Origin of the structure of the designed antioxidants 4 and 5. 2. Materials and Methods Amphiphilic molecules, that can easily penetrate lipid bilayer membranes, may create a delivery 2.1.system General with an incorporated pharmacophore. The Se(IV) molecules can act as “the hidden selenol”, as postulated by Rocha [1], be transported to the specific place of action as stable and soluble Melting points were measured with a Büchi Tottoli SPM-20 heating unit (Büchi Labortechnik AG, RSeOOH/RSeOOK and, at the final stage, be transformed into the bioactive RSeH. Flawil, Switzerland) and were uncorrected. Nuclear magnetic resonance (NMR) spectra were recorded Till now, seleninic acids have found several application routes, including their utilization as on Bruker Avance III/400 or Bruker Avance III/700 (Karlsruhe, Germany) for 1H and 176.1 MHz or ligands [6–10] and reagents in oxidation [11–14], dehydrogenation [15] and oxidative deoximation 100.6 MHz for 13C. Chemical shifts were recorded relative to SiMe (δ0.00) or solvent resonance (CDCl reactions [16]. However, their applicability in medicinal chemistry4 seems to be still an unexplored3 δ7.26, CD OD δ3.31). Multiplicities were given as: s (singlet), d (doublet), dd (double doublet), ddd field with3 promising perspectives. Only few examples of such areneseleninic acids have been (double double doublet), t (triplet), dt (double triplet), and m (multiplet). 77Se NMR spectra were published by Mugesh et al. [4,17–19], but analyzed only as intermediates of corresponding recorded on Bruker Avance III/ 400 or Bruker Avance III/ 700 with diphenyl diselenide as an external benzisoselenazolones, with no activity evaluation. Similar amphiphilic seleninic acids with p-amido standard. NMR spectra were carried out using ACD/NMR Processor Academic Edition. All original function have been presented by Jacobs et al. [20]. The studies revealed that the compounds can easily NMR spectra are presented in Supplementary Materials. Infrared spectra (IR) were measured on penetrate cell membranes and possess significantly better antibacterial activity than phenyl seleninic Alpha FT-IR spectrometer from Bruker (Karlsruhe, Germany). Elemental analyses were performed acid and simple surfactants. Nevertheless, the “ebselen-like” catalysts have never been synthetized on a Vario MACRO CHN analyzer. Commercially available solvents dimethylformamide (DMF), in the form of seleninic acid salts. The purpose of this study was the synthesis of GPx mimics in the dichloromethane (DCM), and MeOH (Aldrich, St. Louis, MO, USA) and chemicals were used without form of RSeOO-M+. The presence of the -SeOO−M+ moiety can improve the solubility in water and further puriﬁcation. Column chromatography was performed using Merck 40-63D 60Å silica gel change the physicochemical properties of the molecules which, in turn, may strongly influence the (Merck, Darmstadt, Germany). metabolism of the presented potential drug candidates. 2.2. Procedures and Analysis Data 2. Materials and Methods 2.2.1. Synthesis of 2-(N-alkylcarboxyamido)benzeneselenenic acids 10–15 2.1. General Method A: To a solution of N-alkylbenzisoselenazol-3(2H)-one (1.00 mmol) in methanol (5 mL), 30% hydrogenMelting points peroxide were (5.00 measured mmol) was with added a Büchi and Tottoli the mixture SPM-20 was heating stirred atunit 50 ◦(BüchiC for 1 Labortechnik h. Methanol AG, Flawil, Switzerland) and were uncorrected. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance III/400 or Bruker Avance III/700 (Karlsruhe, Germany) for 1H and 176.1 MHz or 100.6 MHz for 13C. Chemical shifts were recorded relative to SiMe4 (δ0.00) or solvent resonance (CDCl3 δ7.26, CD3OD δ3.31). Multiplicities were given as: s (singlet), d (doublet), dd (double doublet), ddd (double double doublet), t (triplet), dt (double triplet), and m (multiplet). 77Se NMR spectra were recorded on Bruker Avance III/ 400 or Bruker Avance III/ 700 with diphenyl diselenide as an external standard. NMR spectra were carried out using ACD/NMR Processor Academic Edition. All original NMR spectra are presented in Supplementary Materials. Infrared spectra (IR) were measured on Alpha FT-IR spectrometer from Bruker (Karlsruhe, Germany). Elemental analyses were performed on a Vario MACRO CHN analyzer. Commercially available solvents dimethylformamide (DMF), dichloromethane (DCM), and MeOH (Aldrich, St. Louis, MO,

Materials 2020, 13, 661 4 of 12 was evaporated, the obtained residue was dissolved in DCM, followed by addition of manganese oxide and anhydrous magnesium sulfate. The mixture was dried for 24 h, ﬁltered and evaporated. Method B: To a solution of diselenide (1.00 mmol) in methanol (5 mL), 30% hydrogen peroxide (5.00 mmol) was added and the mixture was stirred at 50 ◦C for 1 h. Methanol was evaporated, the obtained residue was dissolved in DCM, followed by addition of manganese oxide and anhydrous magnesium sulfate. Mixture was dried for 24 h, ﬁltered and evaporated. 2-(N-ethylcarboxyamido)benzeneselenenic acid 10 1 Yield: 90%, 84%; mp 135–139 ◦C; H NMR (700 MHz, DMSO) δ = 1.25 (t, J = 7.7 Hz, 3H, CH3), 3.63–3.68 (m, 1H, N-CH2), 3.75–3.81 (m, 1H, N-CH2), 7.75 (dt, J1 = 0.7, J2 = 7.7 Hz, 1H, 1Har), 7.82 (dt, 13 J1 = 1.4, J2 = 7.7 Hz, 1H, 1Har), 7.85 (d, J = 7.7 Hz, 1H, 1Har), 8.16 (d, J = 7.7 Hz, 1H, 1Har); C NMR (100.6 MHz, DMSO) δ = 15.83 (CH3), 37.00 (CH2), 127.25 (CHar), 127.82 (CHar), 131.36 (Car), 132.70 77 (CHar), 134.18 (CHar), 147.64 (Car), 168.31 (C=O); Se NMR (76 MHz, DMSO) δ = 1123.91 ppm; IR: 3242, 3074, 2973, 2928, 2872, 2363, 1703, 1686, 1619, 1587, 1567, 1549, 1445, 1380, 1360, 1313, 1291, 1271, 1 1246, 1145, 1115, 1090, 1056, 1024, 1007 cm− ; Elemental Anal. Calcd for C9H11NO3Se (260.90): C, 41.55; H, 4.26; N, 5.28 Found: C, 41.23; H, 4.18; N, 5.17. 2-(N-propylcarboxyamido)benzeneselenenic acid 11 1 Yield: 65%, 84%; mp 138–142 ◦C; H NMR (700 MHz, DMSO) δ = 0.91 (t, J = 7.7 Hz, 3H, CH3), 1.63–1.71 (m, 2H, CH2), 3.56–3.61 (m, 1H, N-CH2), 3.66-3.70 (m, 1H, N-CH2), 7.60 (dt, J1 = 0.7, J2 = 7.0 Hz, 1H, 1Har), 7.82 (dt, J1 = 1.4, J2 = 7.7 Hz, 1H, 1Har), 7.85 (d, J = 7.7 Hz, 1H, 1Har), 8.17 (d, 13 J = 7.7 Hz, 1H, 1Har); C NMR (100.6 MHz, DMSO) δ = 11.84 (CH3), 23.37 (CH2), 43.66 (CH2), 127.31 77 (CHar), 127.82 (CHar), 131.24 (Car), 132.71 (CHar), 134.20 (CHar), 147.67 (Car), 168.59 (C=O); Se NMR (76 MHz, DMSO) δ = 1126.91 ppm; IR: 3243, 3085, 2953, 2927, 2868, 1667, 1627, 1584, 1456, 1444, 1336, 1 1291, 1246, 1159, 1113, 1098, 1038, 1021 cm− ; Elemental Anal. Calcd for C10H13NO3Se (289.02): C, 45.84; H, 4.78; N, 5.11 Found: C, 43.53; H, 4.69; N, 5.27. 2-(N-butylcarboxyamido)benzeneselenenic acid 12 1 Yield: 60%, 47%; mp 121–125 ◦C; H NMR (700 MHz, DMSO) δ = 0.89 (t, J = 7.7 Hz, 3H, CH3), 1.34 (m, 2H, CH2), 1.58–1.67 (m, 2H, CH2), 3.58–3.63 (m, 1H, N-CH2), 3.70–3.74 (m, 1H, N-CH2), 7.42 (t, J = 7.0 Hz, 1H, 1Har), 7.82 (t, J = 7.7 Hz, 1H, 1Har), 7.84 (d, J = 7.7 Hz, 1H, 1Har), 8.16 (d, J = 7.7 Hz, 1H, 13 1Har); C NMR (100.6 MHz, DMSO) δ = 14.06 (CH3), 20.06 (CH2), 32.11 (CH2), 41.67 (CH2), 127.33 77 (CHar), 127.78 (CHar), 131.20 (Car), 132.75 (CHar), 134.23 (CHar), 147.59 (Car), 168.57 (C=O); Se NMR (76 MHz, DMSO) δ = 1126.33 ppm; IR: 3325, 3082, 2929,2870, 2851, 1668, 1586, 1586, 1446, 1444, 1357, 1 1324, 1299, 1248, 1231, 1182, 1150, 1112, 1065, 1022, 1007 cm− ; Elemental Anal. Calcd for C11H15NO3Se (289.02): C, 45.84; H, 5.25; N, 4.86 Found: C, 45.96; H, 5.34; N, 4.99. 2-(N-hexylcarboxyamido)benzeneselenenic acid 13 1 Yield: 33%, 36%; mp 84–87 ◦C; H NMR (700 MHz, DMSO) δ = 0.85 (s, 3H, CH3), 1.32–1.36 (m, 6H, 3 CH2), 1.63–1.65 (m, 2H, CH2), 3.57–3.60 (m, 1H, N-CH2), 3.65–3.72 (m, 1H, N-CH2), 7.76 (t, × 13 J = 7.7 Hz, 1H, 1Har), 7.81-7.85 (m, 2H, 2Har), 8.16 (d, J = 7.7 Hz, 1H, 1Har); C NMR (100.6 MHz, DMSO) δ = 14.36 (CH3), 22.45 (CH2), 26.48 (CH2), 29.97 (CH2), 31.35 (CH2), 41.93 (CH2), 127.33 (CHar), 77 127.82 (CHar), 131.21 (Car), 132.76 (CHar), 134.22 (CHar), 147.82 (Car), 168.58 (C=O); Se NMR (76 MHz, DMSO) δ = 1122.09 ppm; IR: 3243, 3076, 3058, 0958, 2926, 2856, 1682, 1622, 1585, 1568, 1552, 1460, 1 1445, 1376, 1329, 1298, 1250, 1215, 1108, 1039, 1026, 1010 cm− ; Elemental Anal. Calcd for C13H19NO3Se (317.05): C, 49.37; H, 6.06; N, 4.43 Found: C, 49.56; H, 6.14; N, 4.59. 2-(N-(3-methyl)butylcarboxyamido)benzeneselenenic acid 14 Yield: 52%, 72%; mp 119–123 C; 1H NMR (700 MHz, DMSO) δ = 0.89–0.92 (m, 6H, 2 CH ), ◦ × 3 1.53–1.57 (m, 2H, CH2), 1.62–1.66 (m, 1H, CH), 3.61–3.65 (m, 1H, N-CH2), 3.72–3.77 (m, 1H, N-CH2), 7.76 (t, J = 7.0 Hz, 1H, 1Har), 7.82 (dt, J1 = 0.7, J2 = 7.0 Hz, 1H, 1Har), 7.85 (d, J = 7.0 Hz, 1H, 1Har), 8.16 13 (d, J = 7.7 Hz, 1H, 1Har); C NMR (100.6 MHz, DMSO) δ = 22.78 (CH3), 22.77 (CH3), 25.75 (CH), 38.92 (CH2), 40.30 (CH2), 127.32 (CHar), 127.78 (CHar), 131.21 (Car), 132.75 (CHar), 134.22 (CHar), 147.57 (Car), 168.52 (C=O); 77Se NMR (76 MHz, DMSO) δ = 1126.14 ppm; IR: 3317, 3060, 2956, 2895, 2868, 1666, 1585, Materials 2020, 13, 661 5 of 12

1 1459, 1445, 1385, 1366, 1328, 1301, 1266, 1248, 1170, 1146, 1128, 1113, 1047, 1032, 1011 cm− ; Elemental Anal. Calcd for C12H17NO3Se (303.04): C, 47.69; H, 5.67; N, 4.63 Found: C, 47.87; H, 5.58; N, 4.79. 2-(N-cyclohexylcarboxyamido)benzeneselenenic acid 15 1 Yield: 47%, 66%; mp 108–110 ◦C; H NMR (700 MHz, DMSO) δ = 1.15–1.22 (m, 1H), 1.28–1.40 (m, 2H, CH2), 1.56–1.70 (m, 3H), 1.75–1.83 (m, 2H, CH2), 2.01–2.05 (m, 2H, CH2), 4.07–4.12 (m, 1H, N-CH), 7.75 (dt, J1 = 0.7, J2 = 7.7 Hz, 1H, 1Har), 7.82 (dt, J1 = 1.4,J2 = 7.7 Hz, 1H, 1Har), 7.85 (dd, J1 = 1.4, 13 J2 = 7.0 Hz, 1H, 1Har), 8.13 (d, J = 7.7 Hz, 1H, 1Har); C NMR (100.6 MHz, DMSO) δ = 25.44 (CH2), 25.79 (CH2), 25.91 (CH2), 33.71 (CH2), 33.91 (CH2), 54.38 (CH), 127.26 (CHar), 127.55 (CHar), 131.76 77 (Car), 132.71 (CHar), 134.16 (CHar), 147.42 (Car), 167.92 (C=O); Se NMR (76 MHz, DMSO) δ = 1113.88 1 ppm; IR: 3234, 2927, 2852, 1621, 1588, 1544, 1448, 1329, 1295, 1243, 1147, 1075 cm− ; Elemental Anal. Calcd for C13H17NO3Se (315.04): C, 49.69; H, 5.45; N, 4.46 Found: C, 49.40; H, 5.53; N, 4.32.

2.2.2. Synthesis of 2-(N-alkylcarboxyamido)benzeneselenenic acid potassium salts 16–21 To a solution of benzeneselenic acid (1.00 mmol) in absolute ethanol (5 mL), potassium tert-butanolate (1.00 mmol) in absolute ethanol (2 mL) was added portionswise and stirred at room temperature for 1 h. The solution was evaporated and the residue was washed with diethyl ether (3 2 mL). All derivatives were obtained as yellow oil. × 2-(N-ethylcarboxyamido)benzeneselenenic acid potassium salt 16 1 Yield: 98%; H NMR (700 MHz, D2O) δ = 1.12 (t, J = 7.7 Hz, 3H, CH3), 3.30–3.33 (m, 2H, N-CH2), 7.50 (dt, J1 = 1.4, J2 = 7.7 Hz, 1H, 1Har), 7.61 (dd, J1 = 0.7, J2 = 7.7 Hz, 1H, 1Har), 7.65 (dt, J1 = 1.4, 13 J2 = 7.7 Hz, 1H, 1Har), 7.99 (dd, J1 = 0.7, J2 = 7.7 Hz, 1H, 1Har); C NMR (100.6 MHz, D2O) δ = 13.53 (CH3), 35.13 (CH2), 124.47 (CHar), 127.36 (CHar), 130.96 (CHar), 132.34 (CHar), 133.14 (Car), 150.98 (Car), 77 169.27 (C=O); Se NMR (76 MHz, D2O) δ = 1142.60 ppm; IR: 3192, 3052, 2971, 2932, 1628, 1587, 1543, 1 1444, 1376, 1359, 1314, 1146, 1051 cm− ; Elemental Anal. Calcd for C9H10KNO3Se (298.85): C, 36.25; H, 3.38; N, 4.70 Found: C, 36.58; H, 3.29; N, 4.85. 2-(N-propylcarboxyamido)benzeneselenenic acid potassium salt 17 1 Yield: 97%; H NMR (700 MHz, D2O) δ = 0.87 (t, J=7.0 Hz, 3H, CH3), 1.53–1.57 (m, 2H, CH2), 3.28 (t, J = 7.0 Hz, 2H, N-CH2), 7.52 (dt, J1 = 1.4, J2 = 7.7 Hz, 1H, 1Har), 7.63 (dd, J1 = 1.4, J2 = 7.7 Hz, 1H, 13 1Har), 7.67 (dt, J1 = 1.4, J2 = 7.7 Hz, 1H, 1Har), 8.00 (dd, J1 = 1.4, J2 = 7.7 Hz, 1H, 1Har); C NMR (100.6 MHz, D2O) δ = 10.73 (CH3), 21.89 (CH2), 41.83 (CH2), 124.47 (CHar), 127.35 (CHar), 130.94 (CHar), 77 132.32 (CHar), 133.23 (Car), 150.94 (Car), 169.53 (C=O); Se NMR (76 MHz, D2O) δ = 1142.45 ppm; 1 IR: 3196, 3050, 2952, 2867, 1630, 1590, 1560, 1458, 1443, 1374, 1358, 1339, 1320, 1286, 1244, 1148 cm− ; Elemental Anal. Calcd for C10H12KNO3Se (312.96): C, 38.46; H, 3.87; N, 4.49 Found: C, 38.72; H, 3.93; N, 4.61. 2-(N-butylcarboxyamido)benzeneselenenic acid potassium salt 18 1 Yield: 96%; H NMR (700 MHz, D2O) δ = 0.83 (t, J=7.7 Hz, 3H, CH3), 1.28–1.33 (m, 2H, CH2), 1.49–1.55 (m, 2H, CH2), 3.31 (t, J = 7.7 Hz, 2H, N-CH2), 7.51 (t, J = 7.7 Hz, 1H, 1Har), 7.61 (d, J = 7.7 Hz, 13 1H, 1Har), 7.66 (dt, J1 = 0.7, J2 = 7.0 Hz, 1H, 1Har), 8.00 (d, J = 7.7 Hz, 1H, 1Har); C NMR (100.6 MHz, D2O) δ = 13.02 (CH3), 19.54 (CH2), 30.49 (CH2), 39.78 (CH2), 124.45 (CHar), 127.33 (CHar), 130.93 (CHar), 77 132.29 (CHar), 133.24 (Car), 150.93 (Car), 169.46 (C=O); Se NMR (76 MHz, D2O) δ = 1142.95 ppm; IR: 1 3216, 3061, 2957, 2930, 2870, 1628, 1588, 1547, 1460, 1440, 1318, 1258, 1148, 1125 cm− ; Elemental Anal. Calcd for C10H12KNO3Se (326.98): C, 40.49; H, 4.32; N, 4.29 Found: C, 40.23; H, 4.41; N, 4.44. 2-(N-hexylcarboxyamido)benzeneselenenic acid potassium salt 19 Yield: 89%; 1H NMR (700 MHz, D O) δ = 0.76 (t, J = 7.0 Hz, 3H, CH ), 1.20–1.22 (m, 4H, 2 CH ), 2 3 × 2 1.51–1.55 (m, 2H, CH2), 3.30 (t, J = 7.0 Hz, 2H, N-CH2), 7.50 (dt, J1 = 0.7, J2=7.7 Hz, 1H, 1Har), 7.59 (dd, J1 = 1.4, J2 = 7.0 Hz, 1H, 1Har), 7.65 (dt, J1 = 1.4, J2 = 7.0 Hz, 1H, 1Har), 7.99 (d, J = 7.7 Hz, 1H, 13 1Har); C NMR (100.6 MHz, D2O) δ=13.33 (CH3), 21.95 (CH2), 25.85 (CH2), 28.27 (CH2), 30.73 (CH2), 40.06 (CH2), 124.48 (CHar), 127.30 (CHar), 130.95 (CHar), 132.31 (CHar), 133.25 (Car), 150.98 (Car), 169.40 77 (C=O); Se NMR (76 MHz, D2O) δ = 1142.17 ppm; IR: 3218, 3058, 2955, 2926, 2856, 1626, 1588, 1543, Materials 2020, 13, 661 6 of 12

1 1459, 1436, 1400, 1376, 1314, 1252, 1120, 1012 cm− ; Elemental Anal. Calcd for C13H18KNO3Se (355.01): C, 44.06; H, 5.12; N, 3.95 Found: C, 44.38; H, 5.03; N, 4.19. 2-(N-(3-methyl)butylcarboxyamido)benzeneselenenic acid potassium salt 20 Yield: 91%; 1H NMR (700 MHz, D O) δ = 0.85 (d, J = 7.0 Hz, 6H, 2 CH ), 1.43–1.46 (m, 2H, CH ), 2 × 3 2 1.58–1.62 (m, 1H, CH), 3.34 (t, J = 7.0 Hz, 2H, N-CH2), 7.51 (dt, J1 = 0.7, J2 = 7.7 Hz, 1H, 1Har), 7.60 (dd, J1 = 1.4, J2 = 7.7 Hz, 1H, 1Har), 7.66 (dt, J1 = 1.4, J2 = 7.7 Hz, 1H, 1Har), 8.00 (d, J = 7.0 Hz, 1H, 13 1Har); C NMR (100.6 MHz, D2O) δ = 21.71 (2 CH3), 25.28 (CH), 37.24 (CH2), 38.43 (CH2), 124.49 × 77 (CHar), 127.34 (CHar), 130.95 (CHar), 132.32 (CHar), 133.28 (Car), 150.98 (Car), 169.40 (C=O); Se NMR (76 MHz, D2O) δ = 1142.29 ppm; IR: 3189, 3057, 2954, 2929, 2869, 1627, 1588, 1545, 1461, 1365, 1315, 1 1260, 1227 cm− ; Elemental Anal. Calcd for C12H16KNO3Se (340.99): C, 42.35; H, 4.74; N, 4.12 Found: C, 42.13; H, 4.68; N, 4.27. 2-(N-cyclohexylcarboxyamido)benzeneselenenic acid potassium salt 21 Yield: 89%; 1H NMR (700 MHz, D O) δ = 1.08 (m, 1H, CH), 1.22-1.31 (m, 4H, 2 CH ), 1.53–1.54 2 × 2 (m, 1H, CH), 1.64–1.68 (m, 2H, CH2), 1.85–1.86 (m, 2H, CH2), 3.70–3.73 (m, 1H, CH), 7.49 (dt, J1 = 0.7, J2 = 7.7 Hz, 1H, 1Har), 7.58 (dd, J1 = 0.7, J2=7.0 Hz, 1H, 1Har), 7.65 (dt, J1 = 0.7, J2 = 7.7 Hz, 1H, 1Har), 13 7.98 (d, J = 7.7 Hz, 1H, 1Har); C NMR (176.08 MHz, D O) δ = 24.16 (2 CH ), 24.56 (CH ), 31.54 (2 2 × 2 2 × CH2), 49.40 (CH), 123.95 (CHar), 126.97 (CHar), 130.46 (CHar), 131.79 (CHar), 133.03 (Car), 150.35 (Car), 77 168.16 (C=O); Se NMR (76 MHz, D2O) δ = 1142.44 ppm; IR: 3183, 3057, 2927, 2853, 1628, 1587, 1540, 1 1449, 1369, 1336, 1300, 1257, 1072, 1053 cm− ; Elemental Anal. Calcd for C13H16KNO3Se (340.99): C, 44.32; H, 4.58; N, 3.98 Found: C, 44.55; H, 4.64; N, 3.82.

2.3. Antioxidant Activity Assay Compounds 10–21 (0.1, 0,01 or 0,0075equiv.) and dithiothreitol DTTred (0.15 mmol) were dissolved 1 in 1.1 mL of CD3OD or D2O. Next, 30% H2O2 (0.15 mmol) was added and H NMR spectra were directly measured in speciﬁc time intervals. Following the changes in the integration on the 1H NMR spectra, the decay of the substrate was evaluated. 2.4. 3-(4,5-Dimethyldiazol-2-yl)-2,5 Diphenyl Tetrazolium Bromide (MTT) Viability Assay The MTT test was conducted by Mosmann methodology [21].

3. Results and Discussion The ﬁrst step of this study involved the synthesis of N-alkyl benzeneselenenic acids with o-amido function. The compounds were obtained by two diﬀerent methods based on the oxidation of N-alkylbenzisoselenazol-3(2H)-ones 8 - method A, or corresponding diselenides 9 - method B. Derivatives 8 and 9 were synthetized according to our previously published procedures [22–25]. The overall yields of both methods A and B were comparable. Next, benzeneseleninic acids 10-15 were transformed to the corresponding benzeneseleninic salts 16–21. The reaction was conducted using potassium tert-butanolate in anhydrous ethanol (Scheme3). The structures of both, benzeneseleninic acids and their potassium salts were fully characterized by 1H, 13C and 77Se NMR. The spectra were obtained in deuterated dimethyl sulfoxide (for compounds 10–15) and water (16–21). According to the best of our knowledge, NMR spectra of benzeneseleninic acids salts have never been published before. We observed that the stability of derivatives 10–15 in solution depends on the used solvent. Compound 15, dissolved in chloroform, was partially decomposed in 24 h and fully converted into the corresponding benzisoselenazolone in 7 days (Figure1). Materials 2019, 12, x FOR PEER REVIEW 7 of 12

Materials 2020, 13, 661 7 of 12 Materials 2019, 12, x FOR PEER REVIEW 7 of 12

Scheme 3. Sythesis of benzeneseleninic acids 10–15 and corresponding potassium salts 16–21.

The structures of both, benzeneseleninic acids and their potassium salts were fully characterized by 1H, 13C and 77Se NMR. The spectra were obtained in deuterated dimethyl sulfoxide (for compounds 10–15) and water (16–21). According to the best of our knowledge, NMR spectra of benzeneseleninic acids salts have never been published before. We observed that the stability of derivatives 10–15 in solution depends on the used solvent. CompoundSchemeScheme 15, dissolved 3.3. SythesisSythesis ofinof benzeneseleninicbenzeneseleninchloroform, wasic acidsacids partiall 10–1510–15y anddecomposedand correspondingcorrespondi inng 24 potassiumpotassium h and fully saltssalts converted 16–21.16–21. into the corresponding benzisoselenazolone in 7 days (Figure 1). The structures of both, benzeneseleninic acids and their potassium salts were fully characterized by 1H, 13C and 77Se NMR. The spectra were obtained in deuterated dimethyl sulfoxide (for compounds 10–15) and water (16–21). According to the best of our knowledge, NMR spectra of benzeneseleninic acids salts have never been published before. We observed that the stability of derivatives 10–15 in solution depends on the used solvent. Compound 15, dissolved in chloroform, was partially decomposed in 24 h and fully converted into the corresponding benzisoselenazolone in 7 days (Figure 1).

Figure 1. Decomposition of benzeneseleninic acid 15 to the corresponding benzisoselenazolone. Figure 1. Decomposition of benzeneseleninic acid 15 to the corresponding benzisoselenazolone. On the contrary, in DMSO, the decomposition was significantly slower and the sample could be storedOn the for contrary, one week in withDMSO, no the side-product decomposition formation. was significantly As it has been slower presented and the insample Scheme could2, the be transformationstored for one ofweek benzeneseleninic with no side-product acid 5 to ebselen formatio 7 takesn. As place it has through been thepresented formation in ofScheme the selenenic 2, the acidtransformation 6. This could of indicatebenzeneseleninic that the rate acid of the5 to reduction ebselen 7 of takes R-SeOOH place to through R-SeOH the can formation be influenced of the by theselenenic type of acid a solvent. 6. This could indicate that the rate of the reduction of R-SeOOH to R-SeOH can be influencedAll derivatives by the type were of a tested solvent. as antioxidants using the NMR activity assay proposed by Iwaoka and co-workersAllFigure derivatives 1. Decomposition [26]. were The tested efficiency of benzeneseleninicas antioxidants of the peroxide acidusing scavenging15 theto the NMR corresponding potentialactivity assay wasbenzisoselenazolone. measuredproposed byby theIwaoka rate red ox ofand dithiothreitol co-workers [26]. oxidation The efficiency (DTT ofto the DTT peroxide) in the scavenging presence potential of the selenocatalyst was measured and by equimolarthe rate of amountOn of the H 2contrary,O2. Compounds in DMSO, 10–15 the decomposition were applied in was 0.1 molarsignificantly equivalent slower and and dissolved the sample in deuterated could be methanol.stored for Resultsone week are presentedwith no side-product in Table1. formation. As it has been presented in Scheme 2, the transformation of benzeneseleninic acid 5 to ebselen 7 takes place through the formation of the selenenic acid 6. This could indicate that the rate of the reduction of R-SeOOH to R-SeOH can be influenced by the type of a solvent. All derivatives were tested as antioxidants using the NMR activity assay proposed by Iwaoka and co-workers [26]. The efficiency of the peroxide scavenging potential was measured by the rate of

Materials 2019, 12, x FOR PEER REVIEW 8 of 12

dithiothreitol oxidation (DTTred to DTTox) in the presence of the selenocatalyst and equimolar amount Materials 2020, 13, 661 8 of 12 of H2O2. Compounds 10–15 were applied in 0.1 molar equivalent and dissolved in deuterated methanol. Results are presented in Table 1.

Table 1.1. Results of the antioxidantantioxidant activity measurement. Catalyst (0.1 equiv.) Remaining DTTred (%) Catalyst (0.1 equiv.) Remaining DTTred (%) 33 min min 5 min5 min 15 min 30 min15 min 60 min 30 min 60 min Ebselen, 7 84 75 64 58 52 Ebselen,Benzseleninic 7 acids 84 75 64 58 52 10 89 86 78 67 51 Benzseleninic acids 11 81 75 70 63 51 10 12 8589 8286 75 6578 47 67 51 13 89 85 69 46 19 11 81 75 70 63 51 14 76 56 38 24 12 12 15 8585 6482 37 1875 2 65 47 Benzseleninic acid salts 0 0 0 0 0 13 16–21 89 85 69 46 19 14 76 56 38 24 12 All benzeneseleninic15 acids85 10–15 were64 more reactive37 than18 ebselen 2 (N-phenylbenzisoselenazol-3(2H)-one) 7. The activity was higher for compounds 14 and 15, possessing moreBenzseleninic bulky substituents. acid salts The 16–21 best result was0 obtained for N-cyclohexylbenzisoselenazol-3(2H)-one0 0 0 15,0 for which only 2% of the substrate was observed after 60 minutes of reaction time. When the -SeOOH All benzeneseleninic acids 10–15 were more reactive than ebselen (N-phenylbenzisoselenazol- group was exchanged for -SeOOK, the activity increased drastically. The reaction was completed in 3 3(2H)-one) 7. The activity was higher for compounds 14 and 15, possessing more bulky substituents. minutes. Consequently, all benzeneseleninic acid salts 16–21 were evaluated by the same procedure The best result was obtained for N-cyclohexylbenzisoselenazol-3(2H)-one 15, for which only 2% of but using 0.01 equivalent of the Se-catalyst (Table2). the substrate was observed after 60 minutes of reaction time. When the -SeOOH group was exchanged forTable -SeOOK, 2. Results the activity of the antioxidant increased activity drastically. measurement The reaction – 0.01 eq.was of completed Se-catalyst. in 3 minutes. Consequently, all benzeneseleninic acid salts 16–21 were evaluated by the same procedure but using Catalyst Mann-Whitney 0.01 equivalent of the Se-catalyst (TableRemaining 2). DTT (%) t-Test (0.01 equiv.) Test Table 2. Results3 min of the 5 antioxidant min 15 minactivity 30 me minasurement 60 min – 0.01p-value eq. of Se-catalyst.p-value Ebselen, 7 97 96 95 94 92 <0.001 <0.015 Catalyst16 24 11Remaining 0 DTT (%) 0 0 <0.005 t-test< 0.015 Mann-Whitney test 17 76 45 21 14 11 <0.005 <0.015 (0.01 equiv.) 18 71 63 26 17 14 <0.01 <0.015 19 3 min 81 5 63min 1517 min 0 30 min 0 60 min<0.001 p-value<0.015 p-value 20 59 16 0 0 0 <0.005 <0.015 Ebselen, 721 97 78 1896 395 094 0 92< 0.001 <0.001< 0.015 <0.015 16 24 11 0 0 0 <0.005 <0.015 In these conditions, almost no conversion was observed for ebselen. The N-ethyl derivative 16 17 76 45 21 14 11 <0.005 <0.015 was the most active one. Other results corresponded to the ones obtained for areneseleninic acids, with higher18 antioxidant 71 potential observed63 for26 more sterically17 hindered14 compounds<0.01 20 and 21. <0.015 The differences19 between antioxidant81 properties63 was17 initially evaluated0 by t-test.0 Due<0.001 to various variance<0.015 of tested groups, nonparametric Mann-Whitney test was additionally performed to confirm it. Both tests 20 59 16 0 0 0 <0.005 <0.015 showed that the antioxidant properties of areneseleninic acid salts are significantly better than the well-known21 organoselenium78 antioxidant18 ebselen.3 0 0 <0.001 <0.015 As salts 16–21 are soluble in water, they were additionally tested using the procedure proposed by SantiIn these et. al. conditions, [27], in which almost deuterated no conversion water was was used observed as a solvent. for ebselen. The reaction The N-ethyl was very derivative fast; thus, 16 allwas compounds the most active were usedone. inOther only results 0.0075 molarcorresponded equivalents. to the The ones amount obtained of asubstrate for areneseleninic was measured acids, e for each catalyst after 2 min of reaction time (Table3). MaterialsMaterialsMaterialsMaterials 20192019 2019 2019,, 1212,, ,12, x12x, , FOR FORx,x x FORFOR FOR PEERPEER PEERPEER PEER REVIEWREVIEW REVIEWREVIEW REVIEW 99 ofof9 9 of 1212 of 12 12

withwithwithwith higherhigher higher higher antioxidantantioxidant antioxidant antioxidant potentialpotential potential potential observedobserved observed observed forfor for for momo mo morerere stericallyresterically sterically sterically hinderedhindered hindered hindered compoundscompounds compounds compounds 2020 20 20andand and and 21.21. 21. 21. TheThe The The differencesdifferencesdifferencesdifferences betweenbetween between between antioxidantantioxidant antioxidant antioxidant propertiesproperties properties properties waswas was was initiainitia initia initiallyllyllylly lly evaluatedevaluated evaluatedevaluated evaluated byby byby byt-test.t-test. t-test.t-test. t-test. DueDue DueDue Due toto toto varioustovarious variousvarious various variancevariance variancevariance variance ofofof testedoftested tested tested groups,groups, groups, groups, nonparametricnonparametric nonparametric nonparametric MaMa Ma Mann-Whitneynn-Whitneynn-Whitneynn-Whitney testtest test test waswas was was additionallyadditionally additionally additionally performedperformed performed performed toto to confirmtoconfirm confirm confirm it.it. it. Bothit.Both Both Both teststeststeststests showedshowed showed showed thatthat that that thethe the the antioxidantantioxidant antioxidant antioxidant propertiesproperties properties properties ofof of arenofaren aren areneseleniniceseleniniceseleniniceseleninic acidacid acid acid saltssalts salts salts areare are are significantlysignificantly significantly significantly betterbetter better better thanthan than than thethethethe well-knownwell-known well-known well-known organoseleniorganoseleni organoseleni organoseleniumumumum antioxidantantioxidant antioxidant antioxidant ebselen.ebselen. ebselen. ebselen. AsAsAs As saltssalts salts salts 16–2116–21 16–21 16–21 areare are are solublesoluble soluble soluble inin in water,water,in water, water, theythey they they werewere were were additionallyadditionally additionally additionally testedtested tested tested usingusing using using thethe the the procedureprocedure procedure procedure proposedproposed proposed proposed bybyby bySantiSanti Santi Santi et.et. et. et.al.al. al. al.[27],[27], [27], [27], inin in whichwhichin which which deuterateddeuterated deuterated deuterated waterwater water water waswas was was usus us ededused edasas as aasa solvent.asolvent. a solvent. solvent. TheThe The The reactionreaction reaction reaction waswas was was veryvery very very fast;fast; fast; fast; thus,thus, thus, thus, allallall allcompoundscompounds compounds compounds werewere were were usedused used used inin in onlyonlyin only only 0.00750.0075 0.0075 0.0075 molarmolar molar molar equivalents.equivalents. equivalents. equivalents. TheThe The The amouamou amou amountntnt ofntof of aaof substrateasubstrate a substrate substrate waswas was was measuredmeasured measured measured ee forefore for for eacheach each each catalystcatalyst catalyst catalyst afterafter after after 22 minutes2minutes 2 minutes minutes ofof of reactionofreaction reaction reaction timetime time time (Table(Table (Table (Table 3).3). 3). 3).

Materials 2020, 13, 661 9 of 12 TableTableTableTable 3.3. 3.Results Results3. Results Results ofof of thetheof the the antioxidantantioxidant antioxidant antioxidant activiactivi activi activityty tyty measurementmeasurementty measurementmeasurement measurement performedperformed performedperformed performed inin inin DDin 22DDO OD22 O –2–O 0.00750.0075– – 0.0075 0.0075 eq.eq. eq. eq. ofof of Se-Se-of Se- Se- catalyst.catalyst.catalyst.catalyst.

Table 3. Results of the antioxidant activity measurement performed in D2O – 0.0075 eq. of Se-catalyst. 161616 16 171717 17 181818 18 191919 19 202020 20 212121 21 PhSeOOKPhSeOOKPhSeOOKPhSeOOK NoNoNoNo catalystcatalyst catalyst catalyst Catalyst.Catalyst.Catalyst.Catalyst. Catalyst. No 16 17 18 19 20 21 PhSeOOK (0.0075 equiv.) Catalyst (0.0075(0.0075(0.0075(0.0075(0.0075 equiv.)equiv.) equiv.)equiv.) equiv.) DTTred (%) 19 47 15 46 79 26 49 93 DTTDTTDTTDTTredredred red (%)(%)red (%)(%) (%) 191919 19 474747 47 151515 15 464646 46 797979 79 262626 26 49 49 49 49 93 93 93 93

TheTheTheTheThe bestbest best best activity activity activity activity waswas was was was observedobserved observed observed observed forfor forfor for N-butylN-butyl N-butylN-butyl N-butyl 1818 18 and18and and and N-ethylN-ethyl N-ethyl N-ethylN-ethyl derivativederivative derivative derivative 16,16, 16, 16, 16,asas as inasin as in thethein in the the test thetest test test testperformedperformed performed performed performed red inininin methanolin methanolmethanolin methanolmethanol methanol (Table(Table (Table(Table (Table2 2).2).). 2).2). 2).TheThe TheThe The amount amount amountamount amount ofof of ofof DTTDTTof DTT DTTDTT DTTredredredred waswasred waswaswas was 15%15% 15%15% 15% andand andand and 19%19% 19%19% 19% 19% respectively,respectively, respectively,respectively, respectively, respectively, whereaswhereas whereaswhereas whereas whereas whenwhen whenwhen when when nono nono no catalystcatalystno catalystcatalyst catalyst catalyst waswaswaswaswas present presentpresent present present stillstill still still 93% 93% 93% 93% ofof of substrateofsubstrate substrate substrate remainedremained remained remained remained inin inin theinthe thethe the rere re reactionaction actionreactionaction mixture.mixture. mixture. mixture.mixture. TheThe The The raterate rate rate rate ofof of theofthe of the the theprocessprocess process process process waswas was was was alsoalso also also also signiﬁcantlysignificantlysignificantlysignificantlysignificantly improved improved improved improved whenwhen when when when N-cyclohexylbenzeneselN-cyclohexylbenzenesel N-cyclohexylbenzeneseleninic N-cyclohexylbenzenesel N-cyclohexylbenzeneseleniniceniniceniniceninic acidacid acid acid saltsalt salt salt 2121 21 21was 21was was was was applied.applied. applied. applied. applied. TheThe The The Thetesttest test test testwaswas was was was alsoalsoalsoalsoalso performed performedperformed performed performed using using using using benzeneseleninicbenzeneseleninic benzeneseleninic benzeneseleninic acidacid acidacid acid potassiumpotassium potassiumpotassium potassium saltsalt salt saltsalt PhSeOOK.PhSeOOK. PhSeOOK. PhSeOOK.PhSeOOK. TheThe The The The obtainedobtained obtained obtained obtained resultresult result result result indicatesindicates indicates indicates indicates thatthatthatthatthat thethethe the the presencepresencepresence presence presence ofof of anofan an anelectronelectron electron electron electron withdrawingwithdrawing withdrawing withdrawing withdrawing grgr groupgr oupoupgroupoup suchsuch such suchsuch asas as asasthethe the thethe o-amidoo-amido o-amido o-amido functionfunction function function function cancan can can canimproveimprove improve improve improve thethe the the the antioxidantantioxidantantioxidantantioxidantantioxidant properties.properties. properties. properties. Finally,Finally,Finally,Finally,Finally, thethe the the cytotoxic cytotoxic cytotoxic cytotoxic activityactivity activity activity activity ofof ofof acidsofacids acidsacids acids 10–1510–15 10–1510–15 10–15 andand and and saltssalts salts saltssalts 16–2116–21 16–21 16–2116–21 againstagainst against against breastbreast breast breast breast cancercancer cancer cancer cancer MCF-7MCF-7 MCF-7 MCF-7 MCF-7 andand and and and humanhumanhumanhumanhuman promyelocytic promyelocyticpromyelocytic promyelocytic promyelocytic leukemialeukemialeukemia leukemia leukemia HL-60HL-60HL-60 HL-60 HL-60 cellcell cell cell lineslineslines lines lines waswaswas was was assessedassessedassessed assessed assessed usingusingusing using using thethethe the the cellcellcell cell cell viabilityviabilityviability viability viability assayassayassay assay assay (MTT)(MTT)(MTT) (MTT) (MTT) [21 ]. As[21].[21].[21]. a[21]. reference AsAs As As aa referenceareference a reference reference compound, compound,compound, compound, compound, wehave wewe we we havehave usedhave have usedused carboplatin.used used caca carboplatin. rboplatin.carboplatin.rboplatin. The TheThe resultsThe The resultsresults results results are areare shownare are shownshown shown shown in inin Table in TableinTable Table Table4. 4.4. 4. 4.

TableTableTable 4. 4. Cytotoxic4. Cytotoxic Cytotoxic activity activity activity of of acidsof acids acids 1–14 1–14 1–14 and and and corresponding corresponding corresponding salts salts salts 16–20. 16–20. 16–20. TableTableTable 4. 4. 4.CytotoxicCytotoxic Cytotoxic activity activityactivity of ofofacids acidsacids 1–14 1–14 and andand corresponding correspondingcorresponding corresponding salts saltssalts salts 16–20. 16–20.16–20. 16–20. StructureStructureStructureStructure MCF-7MCF-7MCF-7MCF-7 HL-60HL-60HL-60HL-60 StructureStructureStructureStructure MCF-7MCF-7MCF-7MCF-7 HL-60HL-60HL-60HL-60 Structure MCF-7MCF-7 HL-60HL-60 Structure MCF-7MCF-7 HL-60HL-60 Structure Structure ICICICIC505050IC (µM)5050(µM)(50 µ(µM)(µM) (µM)M) IC ICIC5050ICIC (µM)(µM)50505050 (µM)(µM) (µM)(µ M) ICICIC5050IC (µM)(µM)5050(µ50 (µM)(µM) M)(µM) ICICIC5050IC (µM)(µM)5050(µ50 (µM)(µM) M)(µM)

40.1 1.2 11.7 1.0 31.5 1.2 56.8 4.4 40.140.140.140.1 ±±± 1.21.2± ± 1.2 1.2 11.7 11.7 11.7 11.7 ±± 1.01.0± ± 1.0 1.0 31.531.531.531.5 ±± 1.21.2± ± 1.2 1.2 56.8 56.8 56.8 56.8 ±± 4.44.4± ± 4.4 4.4 101010 10 161616 16

71.9 4.1 23.5 1.1 20.6 2.5 63.1 0.5 71.971.971.971.9 ±±± 4.14.1± ± 4.1 4.1 23.5 23.5 23.5 23.5 ±± 1.11.1± ± 1.1 1.1 20.620.620.620.6 ±± 2.52.5± ± 2.5 2.5 63.1 63.1 63.1 63.1 ±± 0.50.5± ± 0.5 0.5 111111 11 171717 17

42.5 0.8 62.1 8.0 101.4 2.3 92.6 4.7 42.542.542.542.5 ±±± 0.80.8± ± 0.8 0.8 62.1 62.1 62.1 62.1 ±± 8.08.0± ± 8.0 8.0 101.4101.4101.4101.4 ±± 2.32.3± ± 2.3 2.3 92.6 92.6 92.6 92.6 ±± 4.74.7± ± 4.7 4.7 MaterialsMaterialsMaterialsMaterials 20192019 2019 2019,, 1212,, , 12 x12x FOR,FOR ,x x FOR FOR PEERPEER PEER PEER REVIEWREVIEW REVIEW REVIEW 1010 of10of10 1212of of 12 12 1818 18 121212 12 1818

65.465.465.465.4 ±± ±± 65.4 0.9 113 9.1 45 0.2 200 14.3 65.465.465.465.4 ±±± 0.90.9± ± 0.9 0.9 113 113113113 113 113 113 ± ±±± 9.1 9.1±9.19.1 ± ±9.1± 9.1 9.1 4545454545 ± 45±±±45 0.2 0.2±0.20.2 ± 0.2± 0.2 0.2 200 200 200 200 200 200 200± ±±± 14.3 14.3±14.314.3 ± 14.3± 14.3 14.3 0.90.90.9 0.9 1313131313 13 13 13 1919191919 1919

40.840.840.840.8 ±± ±± 40.8 4.5 51.5 4.5 26.5 3.5 72.5 4.3 ± 51.551.551.551.5 ±± 4.54.5 ± ±± 4.5 4.5 26.526.526.526.5 ± ±± 3.53.5 ±± 3.5 3.5 72.5 72.5 72.5 72.5± ±± 4.34.3 ±± 4.3 4.3 4.54.54.5 4.5 1414 1414 2020 2020

45.945.945.945.9 ±± ±± 45.9 5.3 70.8 1.3 16.6 1.1 42.1 3.1 ± 70.870.870.870.8 ±± 1.31.3 ± ±± 1.3 1.3 16.616.616.616.6 ± ±± 1.11.1 ±± 1.1 1.1 42.1 42.1 42.1 42.1± ±± 3.13.1 ±± 3.1 3.1 5.35.35.3 5.3 1515 1515 2121 2121 Carboplatin 3.80 0.45 2.9 0.1 ± ± CarboplatinCarboplatinCarboplatinCarboplatin 3.803.803.803.80 ±± ±± 2.92.92.9 2.9±± 0.10.1 ±± 0.1 0.1 0.450.450.450.45

TheTheTheThe highesthighest highesthighest cytotoxiccytotoxic cytotoxiccytotoxic activityactivity activityactivity againstagainst againstagainst MCF-7MCF-7 MCF-7MCF-7 cellcell cellcellss waswasss waswas foundfound foundfound forfor for for N-cyclohexylN-cyclohexyl N-cyclohexylN-cyclohexyl potassiumpotassium potassiumpotassium saltsalt saltsalt 2121 21 21 withwithwithwith ICIC 50IC 50IC 16.616.65050 16.6 16.6 ±± 1.11.1 ± ± 1.1 1.1 µM.µM. µM. µM. ForFor For For mostmost most most compounds,compounds, compounds, compounds, withwith with with thethe the the exex ceptionex ceptionexceptionception ofof ofacid acidof acid acid 12,12, 12, 12, thethe the the conversionconversion conversion conversion ofof of-SeOOH -SeOOHof -SeOOH -SeOOH toto toto-SeOOK-SeOOK -SeOOK-SeOOK improvedimproved improvedimproved cytotoxicity.cytotoxicity. cytotoxicity.cytotoxicity. InIn InInHL-60HL-60 HL-60HL-60 cellcell cellcell lineline lineline thethe thethe lowestlowest lowestlowest ICIC 50IC50IC valuevalue5050 valuevalue ofof ofof11.711.7 11.711.7 ±± 1.01.0±± 1.0 1.0 µMµM µMµM waswas waswas observedobservedobservedobserved forfor for for N-ethylN-ethyl N-ethylN-ethyl benzeneseleninicbenzeneseleninic benzeneseleninicbenzeneseleninic acidacid acidacid 10.10. 10. 10. InIn Ingeneral,Ingeneral, general,general, withwith withwith oneone oneone exceptionexception exceptionexception (derivative(derivative (derivative(derivative 21),21), 21),21), acidsacids acidsacids withwithwithwith -SeOOH-SeOOH -SeOOH-SeOOH groupgroup groupgroup werewere werewere moremore moremore cytotoxiccytotoxic cytotoxiccytotoxic thanthan thanthan thth ee thth correspondingcorrespondingee correspondingcorresponding salts,salts, salts,salts, inin incontrarycontraryin contrarycontrary toto tothetheto thethe ICIC 50IC50IC valuesvalues5050 valuesvalues obtainedobtainedobtainedobtained forfor for for MCF-7MCF-7 MCF-7MCF-7 cellcell cellcell line.line. line.line.

4.4. Conclusions4.Conclusions4. ConclusionsConclusions ThisThisThisThis articlearticle articlearticle presentspresents presentspresents thethe thethe synthesissynthesis synthesissynthesis ofof ofofthethe thethe firsfirs firsfirstt water-solublewater-solublett water-solublewater-soluble organoseleniumorganoselenium organoseleniumorganoselenium antioxidants.antioxidants. antioxidants.antioxidants. AA AA seriesseriesseriesseries ofof ofofN-alkylcarboxyamidobenzeneseleninicN-alkylcarboxyamidobenzeneseleninic N-alkylcarboxyamidobenzeneseleninicN-alkylcarboxyamidobenzeneseleninic acidsacids acidsacids andand andand N-alkylcarboxyamidobenzeneseleninicN-alkylcarboxyamidobenzeneseleninic N-alkylcarboxyamidobenzeneseleninicN-alkylcarboxyamidobenzeneseleninic acidacidacidacid potassiumpotassium potassiumpotassium saltssalts saltssalts werewere werewere obtainedobtained obtainedobtained andand andand evaluatedevaluated evaluatedevaluated asas aspotentialaspotential potentialpotential antioxidantsantioxidants antioxidantsantioxidants andand andand anticanceranticancer anticanceranticancer agents.agents. agents.agents. AllAllAllAll benzeneseleninicbenzeneseleninic benzeneseleninicbenzeneseleninic acidacid acidacid saltssalts saltssalts exhibitedexhibited exhibitedexhibited signifsignif signifsignificanticanticanticant peroxideperoxide peroxideperoxide scavengingscavenging scavengingscavenging properties,properties, properties,properties, bothboth bothboth inin inin methanolmethanolmethanolmethanol andand andand water.water. water.water. TheThe TheThe bestbest bestbest obtainedobtained obtainedobtained antioxidanantioxidan antioxidanantioxidant,t, 2-(N-ethylcarboxyamido)benzeneselenenic2-(N-ethylcarboxyamido)benzeneselenenict,t, 2-(N-ethylcarboxyamido)benzeneselenenic2-(N-ethylcarboxyamido)benzeneselenenic acidacid acidacid potassiumpotassiumpotassiumpotassium salt,salt, salt,salt, usedused usedused inin inonlyinonly onlyonly 0.010.01 0.010.01 equivalent,equivalent, equivalent,equivalent, significantlysignificantly significantlysignificantly increasedincreased increasedincreased thethe thethe raterate raterate ofof ofHofH 2 2HOOH222O Oreduction,reduction,22 reduction,reduction, inin inin comparisoncomparisoncomparisoncomparison toto totothethe thethe correspondingcorresponding correspondingcorresponding acid,acid, acid,acid, andand andand thethe thethe well-knownwell-known well-knownwell-known antioxidants,antioxidants, antioxidants,antioxidants, e.g.,e.g., e.g.,e.g., ebselenebselen ebselenebselen andand andand thethe thethe phenylseleninicphenylseleninicphenylseleninicphenylseleninic acidacid acidacid potassiumpotassium potassiumpotassium saltsalt saltsalt PhSeOOK.PhSeOOK. PhSeOOK.PhSeOOK. ItIt indicatesItindicatesIt indicatesindicates thethe thethe necessitynecessity necessitynecessity ofof ofaddingofadding addingadding thethe thethe N-alkyl-o-N-alkyl-o- N-alkyl-o-N-alkyl-o- amidoamidoamidoamido functionfunction functionfunction toto totothethe thethe structurestructure structurestructure ofof ofofthethe thethe designeddesigned designeddesigned catalysts.catalysts. catalysts.catalysts. TheThe TheThe compoundscompounds compoundscompounds cancan cancan bebe be beappliedapplied appliedapplied inin inin submicromolarsubmicromolarsubmicromolarsubmicromolar amountamount amountamount whatwhat whatwhat cancan cancan decreasedecrease decreasedecrease toxictoxic toxictoxic sisi dede siside deeffects.effects. effects.effects. TheThe TheThe highesthighest highesthighest cytotoxiccytotoxic cytotoxiccytotoxic activityactivity activityactivity waswas waswas observedobservedobservedobserved forfor for for 2-(N-cyclohexyl-carboxyamido)benzenese2-(N-cyclohexyl-carboxyamido)benzenese 2-(N-cyclohexyl-carboxyamido)benzenese2-(N-cyclohexyl-carboxyamido)benzeneseleneniclenenicleneniclenenic acidacid acidacid potassiumpotassium potassiumpotassium saltsalt saltsalt 2121 21 in21in inMCF-7MCF-7in MCF-7MCF-7 cellscells cellscells (IC(IC(IC(IC5050 16.616.65050 16.6 16.6 ±± 1.11.1 ± ± 1.1 1.1 µM)µM) µM) µM) andand and and forfor for for 2-(N-ethylcarboxyamido)benzeneselenenic2-(N-ethylcarboxyamido)benzeneselenenic 2-(N-ethylcarboxyamido)benzeneselenenic 2-(N-ethylcarboxyamido)benzeneselenenic acidacid acid acid 1515 15 in15in HL-60inHL-60in HL-60 HL-60 cellscells cells cells (IC(IC (IC (IC5050 11.711.75050 11.7 11.7 ±± 1.01.0±± 1.0 1.0 µM).µM). µM).µM). Generally,Generally, Generally,Generally, thethe thethe compoundscompounds compoundscompounds withwith withwith -SeOOK-SeOOK -SeOOK-SeOOK moietymoiety moietymoiety werewere werewere moremore moremore cytotoxiccytotoxic cytotoxiccytotoxic forfor for for breastbreast breastbreast cancercancer cancercancer cells,cells,cells,cells, whereaswhereas whereaswhereas compoundscompounds compoundscompounds withwith withwith -SeOOH-SeOOH -SeOOH-SeOOH groupgroup groupgroup werewere werewere moremore moremore potentpotent potentpotent againstagainst againstagainst leukemialeukemia leukemialeukemia cells.cells. cells.cells.

SupplementarySupplementarySupplementarySupplementary Materials:Materials: Materials: Materials: TheThe The The followingfollowing following following areare are are availableavailable available available onlineonline online online atat www.mdpi.com/xxx/s1,www.mdpi.com/xxx/s1,at at www.mdpi.com/xxx/s1, www.mdpi.com/xxx/s1, FigureFigure Figure Figure S1.S1. S1. S1.(a)(a) (a) 1(a)1HH 1 NMR,1NMR,HH NMR, NMR, (b)(b)(b) (b)1313CC 13 13 NMR,NMR,CC NMR,NMR, andand andand (c)(c) (c) 77(c)77SeSe 7777 SeNMRNMRSe NMRNMR spectraspectra spectraspectra ofof 2-(N-ethylcarboxyamido)bof2-(N-ethylcarboxyamido)bof 2-(N-ethylcarboxyamido)b2-(N-ethylcarboxyamido)benzeneselenenicenzeneselenenicenzeneselenenicenzeneselenenic acidacid acidacid 10.10. 10. 10.FigureFigure FigureFigure S2.S2. S2. S2.(a)(a) (a) (a)11HH 1 1HH NMR,NMR,NMR,NMR, (b)(b) (b) (b)1313CC 13 13 NMR,NMR,CC NMR,NMR, andand andand (c)(c) (c) 77(c)77SeSe 7777 SeNMRNMRSe NMRNMR spectraspectra spectraspectra ofof 2-(N-propylcarboxyamidoof 2-(N-propylcarboxyamidoof 2-(N-propylcarboxyamido2-(N-propylcarboxyamido)benzeneselenenic)benzeneselenenic)benzeneselenenic)benzeneselenenic acidacid acidacid 11.11. 11. 11.FigureFigure FigureFigure S3.S3. S3. S3. (a)(a) (a) 1(a)1HH 1 NMR,1NMR,HH NMR, NMR, (b)(b) (b) 13(b)13CC 13 13 NMR,NMR,CC NMR, NMR, andand and and (c)(c) (c) 77(c)77SeSe 77 77 SeNMRNMRSe NMR NMR spectraspectra spectra spectra ofof 2-(N-butylcarboxyamiof2-(N-butylcarboxyami of 2-(N-butylcarboxyami 2-(N-butylcarboxyamido)benzeneselenenicdo)benzeneselenenicdo)benzeneselenenicdo)benzeneselenenic acidacid acid acid 12.12. 12. 12.FigureFigure Figure Figure S4.S4.S4. S4.(a)(a) (a) (a)11HH 1 1NMR,HNMR,H NMR,NMR, (b)(b) (b) (b)1313CC 13 13 NMR,NMR,CC NMR,NMR, andand andand (c)(c) (c) 77(c)77SeSe 7777 SeNMRNMRSe NMRNMR spectraspectra spectraspectra ofof 2-(N-hexylcarboof2-(N-hexylcarboof 2-(N-hexylcarbo2-(N-hexylcarboxyamido)benzeneselenenicxyamido)benzeneselenenicxyamido)benzeneselenenicxyamido)benzeneselenenic acidacid acidacid 13.13. 13. 13. FigureFigureFigureFigure S5.S5.S5. S5. (a)(a) (a) (a) 11HH 1 1HH NMR,NMR,NMR,NMR, (b)(b)(b) (b) 1313CC13 13 CC NMR,NMR,NMR,NMR, andandandand (c)(c) (c) (c) 7777SeSe7777 SeSe NMRNMRNMRNMR spectraspectraspectraspectra ofof ofof 2-(N-(3-2-(N-(3-2-(N-(3-2-(N-(3- methyl)butylcarboxyamido)benzenesemethyl)butylcarboxyamido)benzenesemethyl)butylcarboxyamido)benzenesemethyl)butylcarboxyamido)benzeneseleneniclenenicleneniclenenic acidacid acidacid 14.14. 14. 14.FigureFigure FigureFigure S6.S6. S6. S6.(a)(a) (a) (a)11HH 1 1NMR,HNMR,H NMR,NMR, (b)(b) (b) (b)1313CC 13 13 NMR,NMR,CC NMR,NMR, andand andand (c)(c) (c) (c)7777SeSe 7777 SeNMRSeNMR NMRNMR spectraspectraspectraspectra ofof 2-(N-cyclohexylcarboxyamido)ben of2-(N-cyclohexylcarboxyamido)benof 2-(N-cyclohexylcarboxyamido)ben2-(N-cyclohexylcarboxyamido)benzeneseleneniczeneseleneniczeneseleneniczeneselenenic acidacid acidacid 15.15. 15. 15.FigureFigure FigureFigure S7.S7. S7. S7.(a)(a) (a) 1(a)1HH 1 NMR,1NMR,HH NMR,NMR, (b)(b) (b) (b)1313CC 13 13 NMR,NMR,CC NMR,NMR, andand andand (c)(c) (c) (c) 7777SeSe7777 SeNMRNMRSe NMRNMR spectraspectra spectraspectra ofof 2-(N-ethylcarboxyamido)benzeneseof2-(N-ethylcarboxyamido)benzeneseof 2-(N-ethylcarboxyamido)benzenese2-(N-ethylcarboxyamido)benzeneseleneniclenenicleneniclenenic acidacid acidacid potassiumpotassium potassiumpotassium saltsalt saltsalt 16.16. 16. 16.FigureFigure FigureFigure S8.S8. S8. S8.(a)(a) (a) (a)11HH 1 NMR,1HNMR,H NMR,NMR, (b)(b)(b) (b)1313CC 13 13 NMR,NMR,CC NMR,NMR, andand andand (c)(c) (c) 77(c)77SeSe 7777 SeNMRSeNMR NMRNMR spectraspectra spectraspectra ofof of2-(N-propylcarboxyamido)benzeneselenenic2-(N-propylcarboxyamido)benzeneselenenicof 2-(N-propylcarboxyamido)benzeneselenenic2-(N-propylcarboxyamido)benzeneselenenic acidacid acidacid potassiumpotassium potassiumpotassium saltsalt saltsalt 17.17. 17. 17. FigureFigureFigureFigure S9.S9. S9. S9.(a)(a) (a) (a)11HH 1 1NMR,HNMR,H NMR,NMR, (b)(b) (b) (b)1313CC 13 13 NMR,NMR,CC NMR,NMR, andand andand (c)(c) (c) (c)7777SeSe 7777 SeNMRSeNMR NMRNMR spectraspectra spectraspectra ofof of2-(N-butylcarboxyamido)benzeneselenenic2-(N-butylcarboxyamido)benzeneselenenicof 2-(N-butylcarboxyamido)benzeneselenenic2-(N-butylcarboxyamido)benzeneselenenic

Materials 2020, 13, 661 10 of 12

The highest cytotoxic activity against MCF-7 cells was found for N-cyclohexyl potassium salt 21 with IC 16.6 1.1 µM. For most compounds, with the exception of acid 12, the conversion of 50 ± -SeOOH to -SeOOK improved cytotoxicity. In HL-60 cell line the lowest IC value of 11.7 1.0 µM 50 ± was observed for N-ethyl benzeneseleninic acid 10. In general, with one exception (derivative 21), acids with -SeOOH group were more cytotoxic than the corresponding salts, in contrary to the IC50 values obtained for MCF-7 cell line.

4. Conclusions This article presents the synthesis of the first water-soluble organoselenium antioxidants. A series of N-alkylcarboxyamidobenzeneseleninic acids and N-alkylcarboxyamidobenzeneseleninic acid potassium salts were obtained and evaluated as potential antioxidants and anticancer agents. All benzeneseleninic acid salts exhibited significant peroxide scavenging properties, both in methanol and water. The best obtained antioxidant, 2-(N-ethylcarboxyamido)benzeneselenenic acid potassium salt, used in only 0.01 equivalent, significantly increased the rate of H2O2 reduction, in comparison to the corresponding acid, and the well-known antioxidants, e.g., ebselen and the phenylseleninic acid potassium salt PhSeOOK. It indicates the necessity of adding the N-alkyl-o-amido function to the structure of the designed catalysts. The compounds can be applied in submicromolar amount what can decrease toxic side effects. The highest cytotoxic activity was observed for 2-(N-cyclohexyl-carboxyamido)benzeneselenenic acid potassium salt 21 in MCF-7 cells (IC 16.6 1.1 µM) and for 2-(N-ethylcarboxyamido)benzeneselenenic acid 15 in HL-60 cells 50 ± (IC 11.7 1.0 µM). Generally, the compounds with -SeOOK moiety were more cytotoxic for breast 50 ± cancer cells, whereas compounds with -SeOOH group were more potent against leukemia cells.

Supplementary Materials: The following are available online at http://www.mdpi.com/1996-1944/13/3/661/s1, Figure S1. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-ethylcarboxyamido)benzeneselenenic acid 10. Figure S2. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-propylcarboxyamido)benzeneselenenic acid 11. Figure S3. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-butylcarboxyamido)benzeneselenenic acid 12. Figure S4. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-hexylcarboxyamido)benzeneselenenic acid 13. Figure S5. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-(3-methyl)butylcarboxyamido)benzeneselenenic acid 14. Figure S6. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-cyclohexylcarboxyamido)benzeneselenenic acid 15. Figure S7. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-ethylcarboxyamido)benzeneselenenic acid potassium salt 16. Figure S8. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-propylcarboxyamido)benzeneselenenic acid potassium salt 17. Figure S9. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-butylcarboxyamido)benzeneselenenic acid potassium salt 18. Figure S10. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 22-(N-hexylcarboxyamido)benzeneselenenic acid potassium salt 19. Figure S11. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-(3-methyl)butylcarboxyamido)benzeneselenenic acid potassium salt 20. Figure S12. (a) 1H NMR, (b) 13C NMR, and (c) 77Se NMR spectra of 2-(N-cyclohexylcarboxyamido)benzeneselenenic acid potassium salt 21. Author Contributions: Conceptualization, J.S.;´ Data curation, M.O., A.L. and A.J.P.; Formal analysis, M.O., A.L., A.D.-P. and A.J.P.; Investigation, M.O., A.L., A.J.P., A.D.-P. and A.J.; Writing—original draft, A.J.P. and J.S.; Writing—review & editing, A.J. and J.S.´ All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Science Centre, Poland, grant number 2019/03/X/ST4/00121. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Barbosa, N.V.; Nogueira, C.W.; Nogara, P.A.; de Bem, A.F.; Aschnerc, M.; Rocha, J.B.T. Organoselenium compounds as mimics of selenoproteins and thiol modiﬁer agents. Metallomics 2017, 9, 1703–1734. [CrossRef] [PubMed] 2. Orian, L.; Toppo, S. Organochalcogen peroxidase mimetics as potential drugs: a long story of a promise still unfulﬁlled. Free Rad. Biol. Med. 2014, 66, 65–74. Materials 2020, 13, 661 11 of 12

3. Weekley, C.M.; Harris, H.H. Which form is that? The importance of selenium speciation and metabolism in the prevention and treatment of disease. Chem. Soc. Rev. 2013, 42, 8870–8894. [CrossRef][PubMed] 4. Ungati, H.; Govindaraj, V.; Narayanan, M.; Mugesh, G. Probing the formation of a Seleninic Acid in Living Cells by a Fluorescence Switching of a Glutathione Peroxidase Mimetic. Angew. Chem. Int. Ed. 2019, 58, 8156–8160. [CrossRef] 5. Pacuła, A.J.; Mangiavacchi, F.; Sancineto, L.; Lenardao, E.J.; Scianowski,´ J.; Santi, C. An Update on “Selenium Containing Compounds from Poison to Drug Candidates: A Review on the GPx-like Activity”. Curr. Chem. Biol. 2015, 9, 97–112. [CrossRef] 6. Candrini, G.; Malavasi, W.; Preti, C.; Tosi, G.; Zannini, P. Substituted benzeneseleninic acids as bidentate ligands. Synthesis and spectroscopic studies of manganese(H) and iron complexes. Spetrochim. Acta. 1983, 39, 635–639. [CrossRef] 7. Graziosi, G.; Preti, C.; Tosi, G.; Zannini, P. Osmium (111) Halide Complexes with para- and meta Substituted Benzeneseleninato Ligands. Aust. J. Chem. 1985, 38, 1675–1684. [CrossRef] 8. Yao, N.-T.; Zhang, R.-F.; Zhang, S.-L.; Li, Q.-L.; Ma, C.-L. A novel octa-nuclear 32-membered zirconocene macrocycle based on the aromatic selenite. Dalton Trans. 2017, 46, 524–528. [CrossRef] 9. Ugandhar, U.; Baskar, V. Monoorganoantimony(V) Phosphonates and PhosphoSelininates. Dalton Trans. 2016, 45, 6269–6274. [CrossRef] 10. Yan, M.; Zhang, R.-F.; Zhang, S.-L.; Ru, J.; Du, J.-Y.; Ma, C.-L. Novel cobalt (II) coordination complexes based on 3,4,5-trifluorobenzeneseleninic acid and different N-donor ligands. Polyhedron 2018, 146, 172–179. [CrossRef] 11. Syper, L.; Młochowski, J. Benzeneperoxyseleninic Acids - Synthesis and Properties. Tetrahedron 1987, 43, 207–213. [CrossRef] 12. Hori, T.; Sharpless, K.B. Synthetic Applications of Arylselenenic and Arylseleninic Acids. Conversion of Olefins to Allylic Alcohols and Epoxides. J. Org. Chem. 1978, 43, 1689–1694. [CrossRef] 13. Taylor, T.R.; Flood, L.A. Polystyrene-Bound Phenylseleninic Acid: Catalytic Oxidations of Olefins, Ketones, and Aromatic Systems. J. Org. Chem. 1983, 48, 5160–5164. [CrossRef] 14. Back, G.T.; Collins, S.; Kerr, R.G. Oxidation of Hydrazines with Benzeneseleninic Acid and Anhydride. J. Org. Chem. 1981, 46, 1564–1570. [CrossRef] 15. Teskey, Ch.J.; Adler, P.; Gonzalves, C.; Maulide, N. Chemoselective α,β-Dehydrogenation of Saturated Amides. Angew.Chem. Int. Ed. 2019, 58, 447–451. [CrossRef] 16. Jing, X.; Yuan, D.; Yua, L. Green and Practical Oxidative Deoximation of Oximes to Ketones or Aldehydes with Hydrogen Peroxide/Air by Organoselenium Catalysis. Adv. Synth. Catal. 2017, 359, 1194–1201. [CrossRef] 17. Sarma, B.K.; Mugesh, G. Antioxidant Activity of the Anti-Inflmmatory Compound Ebselen: A Reversible Cyclization Pathway via Selenenic and Seleninic Acid Intermediates. Chem. Eur. J. 2008, 14, 10603–10614. [CrossRef] 18. Bhabak, K.P.; Mugesh, G. Amide-Based Glutathione Peroxidase Mimics: Effect of Secondary and Tertiary Amide Substituents on Antioxidant Activity. Chem. Asian J. 2009, 4, 974–983. [CrossRef] 19. Bhabak, K.P.; Vernekar, A.A.; Jakka, S.R.; Roy, G.; Mugesh, G. Mechanistic investigations on the efficient catalytic decomposition of peroxynitrite by ebselen analogues. Org. Biomol. Chem. 2011, 9, 5193–5200. [CrossRef] 20. Du, P.; Viswanathan, U.M.; Xu, Z.; Ebrahimnejad, H.; Hanf, B.; Burkholz, T.; Schneider, M.; Bernhardt, I.; Kirsch, G.; Jacob, C. Synthesis of amphiphilic seleninic acid derivatives with considerable activity against cellular membranes and certain pathogenic microbes. J. Haz. Mat. 2014, 269, 74–82. [CrossRef] 21. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef] 22. Pacuła, A.J.; Scianowski,´ J.; Aleksandrzak, K.B. Highly efficient synthesis and antioxidant capacity of N-substituted benzisoselenazol-3(2H)-ones. RSC Advances 2014, 4, 48959–48962. [CrossRef] 23. Pacuła, A.J.; Kaczor, K.B.; Wojtowicz, A.; Antosiewicz, J.; Janecka, A.; Długosz, A.; Janecki, T.; ´Scianowski, J. New glutathione peroxidase mimetics—Insights into antioxidant and cytotoxic activity. Bioorg. Med. Chem. 2017, 25, 126–131. [CrossRef][PubMed] 24. Pacuła, A.J.; Kaczor, K.B.; Antosiewicz, J.; Janecka, A.; Długosz, A.; Janecki, T.; Wojtczak, A.; Scianowski,´ J. New Chiral Ebselen Analogues with Antioxidant and Cytotoxic Potential. Molecules 2017, 22, 492–506. [CrossRef][PubMed] Materials 2020, 13, 661 12 of 12

25. Pacuła, A.J.; Obieziurska, M.; Scianowski,´ J.; Kaczor, K.B.; Antosiewicz, J. Synthesis of biologically active diaryl diselenides under water control. Arkivoc 2018, iii, 144–155. 26. Kumakura, F.; Mishra, B.; Priyadarsini, K.I.; Iwaoka, M. A Water-Soluble Cyclic Selenide with Enhanced Glutathione Peroxidase-Like Catalytic Activities. Eur. J. Org. Chem. 2010, 3, 440–444. [CrossRef] 27. Tidei, C.; Piroddi, M.; Galli, F.; Santi, C. Oxidation of thiols promoted by PhSeZnCl. Tetrahedron Lett. 2012, 53, 232–234. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).