1H, 13C and 77Se NMR spectra for all isolated compounds were recorded on 400 and 500 MHz (1H: 399.97 MHz; 13C: 100.6 MHz and 1H: 500 MHz; 13C: 125 MHz) and spectrometers, using the residual solvent peaks of CDCl3 (1H: δ 7.26; 13C: δ 77.2), as an indirect reference to TMS (δ = 0 ppm). 77Se NMR spectra were recorded on 400 MHz and 500 MHz (77Se: 76 MHz and 100 MHz) spectrometers with Ph2Se2 (δ = 460 ppm) as an indirect reference to Me2Se (δ = 0 ppm). Column chromatography was performed using silica gel (0.14 - 0.25 mm). Melting points are uncorrected for all solid compounds with digital melting point apparatus. The high resolution mass spectra (HRMS) were obtained using a time of flight (TOF) instrument equipped with electron ionization (EI) operating in the positive ion mode. Solvents were distilled first and then used for the column chromatography.
Preparation of Bis-[2-phenyl-1,2-benzisoselenazol-3-( 2H )-one-7-yl]diazene (8): To a brown suspension of in situ prepared Na2Se2 (12.46 mmol) in dry THF (20 mL) under an inert atmosphere was slowly added a solution of 2-bromo-3-nitro-N-phenylbenzamide (1 g, 3.113 mmol) in THF (10 mL) at room temperature. After heating at reflux for 5 h, the reaction was allowed to stir at room temperature. Water was added to quench the reaction, and the reaction mixture was further stirred at room temperature for additionally 30 min. Following extraction with CHCl3, the combined organic layers were dried over anhydrous Na2SO4. Removal of the solvent and purification of the residue by silica gel column chromatography, eluting first with hexane/ethyl acetate (1:1), then with CHCl3, and finally with CHCl3/MeOH (98:2), afforded compound 8 as dark black solid. Yield: (550 mg, 32%); mp > 300 ⁰C; 1H NMR (500 MHz, CDCl3): δ = 7.36 (d, J = 7.5 Hz, 2H), 7.46 (t, J = 7.5 Hz, 4H,), 7.62 (d, J = 7.5 Hz, 4H), 7.73 (t, J = 7.5 Hz, 2H), 8.32 (d, J = 7 Hz, 2H), 8.53 (d, J = 7.5 Hz, 2H). 13C NMR (125 MHz, CDCl3) : δ = 119.9, 125.1, 126.5, 128.1, 128.8, 129.4, 131.4, 131.9, 139.1, 144.2, 163.8 ppm. 77Se NMR (100 MHz, CDCl3): δ = 928 ppm.
All data correspond to literature.[27]
Typical Procedure for the Preparation of Diselenides:
Bis[3-Amino-N-phenylbenzamide-2-yl] Diselenide (9): This was prepared according to the procedure reported using azo-bis-ebselen 8 (500 mg, 0.870 mmol) and in situ prepared colourless solution of NaTePh prepared from Ph2Te2 (712 mg, 1.74 mmol) and NaBH4 (132 mg, 3.48 mmol) in ethanol (20 mL) at 0 oC. The mixture was then heated at 80 oC for 4 h and allowed to cool at room temperature. After adding water to the above mixture, the extraction was carried out with ethyl acetate and separated organic layers were dried over anhydrous MgSO4. Evaporation of the solvent and purification by column chromatography, using 80% ethyl acetate/n-pentane mixture as an eluent afforded the title compound as orange crystalline solid. Yield = (280 mg, 55%); mp 220-223 oC (lit. 222-224 oC). 1H NMR (500 MHz, CDCl3): δ = 4.46 (s, broad, 4H), 6.61 (d, J = 8 Hz, 2H), 6.72 (d, J = 7 Hz, 2H,), 7.01 (t, J = 7.5 Hz, 2H), 7.10 (t, J = 7 Hz, 3H), 7.23 (d, J = 8 Hz, 3H), 7.29 (d, J = 6 Hz, 6H), 8.40 (s, 2H). 13C NMR (125 MHz, CDCl3) : δ = 110.6, 115.8, 116.7, 120.7, 124.5, 128.6, 131.2, 137.5, 144.2, 149.6, 168.4 ppm. 77Se NMR (100 MHz, CDCl3): δ = 360 ppm. All data correspond to literature.[35-36]
Bis[3-Amino-N-(p-tolyl)benzamide-2-yl] Diselenide (19): Yield = (750 mg, 80%); mp 220-222 oC (lit. 219-221 oC). 1H NMR (500 MHz, CDCl3): δ = 2.29 (s, 6H), 4.43 (s, broad, 4H), 6.60 (d, J = 8 Hz, 2H), 6.68 (d, J = 7.5 Hz, 2H), 7.01 (m, 6H), 7.13 (d, J = 8 Hz, 6H), 8.22 (s, 2H). 13C NMR (125 MHz, CDCl3) : δ = 20.9, 110.7, 115.7, 116.7, 120.7, 129.1, 131.2, 134.1, 134.9, 144.4, 149.6, 168.2 ppm. 77Se NMR (100 MHz, CDCl3): δ = 360 ppm. All data correspond to literature.[27]
Reaction of compound 8 with H2O2 and PhSH: To the NMR tube containing compound 8 (50 mg, 0.087 mmol) in 1.0 mL of CDCl3 was added 8 equiv of PhSH (72 μL). The progress of the reaction was monitored by the 77Se NMR spectroscopy. Further, more total of 16 equiv of PhSH were added subsequently to the above mixture. A solution of 6 equiv of H2O2 (40 μL) was added to the above mixture. Then another more 6 equiv of H2O2 was added and the 77Se NMR spectrum recorded again. More 12 equiv of H2O2 was added subsequently to the NMR mixture. All the 77Se NMR spectra were recorded after every addition of the PhSH and H2O2 to the NMR tube. Water was added to the above mixture and the extraction was carried out with CHCl3. The separated organic layers were combined and dried over anhydrous MgSO4. Evaporation of the solvent and purification by column chromatography on silica gel, using 40% ethyl acetate/n-pentane mixture as an eluent afforded compounds 10, 12 and 7a.
3-Amino-N-phenyl-2-((phenylthio)selanyl)benzamide (10):
Yield: 18 mg (28%); mp 197-201 oC. 1H NMR (500 MHz, CDCl3): δ = 6.84 (t, J = 7 Hz, 1H), 7.10 – 7.21 (m, 4H), 7.34 (t, J = 7.5 Hz, 2H), 7.39 – 7.46 (m, 3H), 7.49 – 7.74 (m, 3H). 13C NMR (125 MHz, CDCl3) : δ = 116.6, 120.2, 120.6, 121.2, 124.6, 125.0, 127.3, 128.3, 128.9, 129.0, 129.2, 131.2, 132.4, 149.4, 167.4 ppm. 77Se NMR (76 MHz, CDCl3): δ = 452 ppm. HRMS (TOF MS ESI) m/z calcd for C19H16N2OSSe [M + H]+: 401.0227; found: 401.0226.
2-Phenyl-7-((phenylthio)amino)benzoselenazol-3(2H)-one (12):
Yield: 17 mg (25%); mp 211-215 oC. 1H NMR (500 MHz, CD3OD): δ = 6.51 (s, 1H), 6.65 (dd, J = 1, 7.5 Hz, 1H,), 6.67 (dd, J = 1, 8 Hz, 1H), 6.88 – 6.99 (m, 2H), 7.14 (t, J = 7.5 Hz, 1H), 7.21 (dd, J = 1, 8.5 Hz, 1H), 7.26 (dd, J = 0.5, 7.5 Hz, 3H), 7.38 (t, J = 7.5 Hz, 1H), 7.47 – 7.50 (m, 2H), 7.98 (t, J = 8 Hz, 1H). 13C NMR (125 MHz, CD3OD) : δ = 115.1, 115.4, 115.5, 120.9, 121.2, 124.1, 125.9, 127.1, 128.1, 128.4, 130.6, 137.8, 144.1, 150.7, 168.8 ppm. 77Se NMR (76 MHz, CDCl3): δ = 833 ppm. HRMS (TOF MS ESI) m/z calcd for C19H14N2OSSe [M + H]+: 399.0070; found: 399.067.
7-Amino-2-phenyl-1,2-benzisoselenazol-3(2H)-one (7a): Yield: 8 mg (16%); mp 180-183 oC (lit. 182-186 oC); 1H NMR (500 MHz, DMSO-d6): δ = 5.87 (s, 2H), 6.91 (d, J = 7.5 Hz, 1H,), 7.18 (d, J = 7.5 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.47 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 7.5 Hz, 2H). 13C NMR (125 MHz, DMSO-d6) : δ = 115.8, 116.1, 121.1, 125.0, 126.3, 127.8, 128.1, 129.3, 139.4, 144.4, 165.8 ppm. 77Se NMR (100 MHz, DMSO-d6): δ = 929 ppm. All data correspond to literature.[36]
Reaction of diselenide 19 with H2O2 and PhSH: To an NMR tube containing diselenide 19 (50 mg, 0.082 mmol) in 0.50 mL of DMSO-d6 was added 2 equiv of H2O2 (19 μL, 0.16 mmol) and mixed together. The oxidation reaction of was followed by the 77Se NMR spectroscopy. Thereafter, observing the signal corresponding to selenoxide35, an equimolar of PhSH (16 μL, 0.16 mmol) was added to the above mixture (data not shown here). Immediately, three signals were observed at δ 409, 842 and 929 ppm in the 77Se NMR spectrum (Figure S35 in the Supporting Information). The signal corresponding to diselenide 19 at δ 368 was fully disappeared. The mixture was extracted with ethyl acetate and purified by column chromatography. Purification was performed using 40% ethyl acetate/n-pentane as eluent produced products 20, 21, and 7b.
3-Amino-2-((phenylthio)selanyl)-N-(p-tolyl)benzamide (20): Yield: 18 mg (27%). mp 182-185 oC. 1H NMR (400 MHz, CDCl3): δ = 2.34 (s, 3H), 6.81 (dd, J = 4, 8.4 Hz, 2H,), 7.05 (s, 1H), 7.09 – 7.20 (m, 6H), 7.34 (d, J = 8 Hz, 2H), 7.43 ppm (d, J = 6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ = 21.1, 116.6, 116.7, 120.4, 127.6, 128.4, 129.1, 129.2, 129.5, 131.3, 132.6, 134.4, 135.2, 144.3, 149.5, 167.4 ppm. 77Se NMR (76 MHz, CDCl3): δ = 453 ppm. HRMS (TOF MS ESI) m/z calcd for C20H18N2OSSe [M + Na]+: 437.0198; found: 437.0191.
7-((Phenylthio)amino)-2-(p-tolyl)benzoselenazol-3(2H)-one (21): Yield: 20 mg (30%); mp 155-158 oC. 1H NMR (400 MHz, CDCl3): δ = 2.33 (s, 3H), 5.77 (s, 1H), 7.04 (d, J = 8 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.31 (m, 4H), 7.48 (dd, J = 1.2, 8 Hz, 2H), 7.72 ppm (d, J = 7.6 Hz 1H) (2 proton merge in the solvent). 13C NMR (100 MHz, CDCl3) : δ = 21.2, 117.6, 121.5, 124.5, 125.3, 127.7, 128.2, 129.5, 129.8, 130.0, 131.5, 136.2, 136.8, 140.7, 141.6 ppm (1 peak missing). 77Se NMR (76 MHz, CDCl3): δ = 856 ppm. HRMS (TOF MS ESI) m/z calcd for C20H16N2OSSe [M + H]+: 413.0223; found: 413.0228.
7-Amino-2-[(4-tolyl)-1,2-benzisoselenazol-3(2H)-one (7b): Yield: 5 mg (7%). mp 157-160 oC (lit. 159-162 oC). The 1H NMR spectrum corresponds to literature.[36]
Mechanistic study of compound 8 with H2O2 and PhSH: To the NMR tube containing compound 8 (50 mg, 0.082 mmol) in 1.0 mL of CDCl3 was added 8 equiv of PhSH (72 μL). The progress of the reaction was monitored by the 77Se NMR spectroscopy. Further, more total of 16 equiv of PhSH were added subsequently to the above mixture. The 77Se NMR spectra were recorded after each addition to the NMR tube. A solution of 6 equiv of H2O2 (40 μL) was added to the above mixture. The NMR spectra were recorded followed by 77Se NMR spectroscopy. Then another more 6 equiv of H2O2 was added and the 77Se NMR spectrum recorded again. More 12 equiv of H2O2 was added subsequently to the NMR mixture. All the 77Se NMR spectra were recorded after every addition of the PhSH and H2O2 to the NMR tube. See the Supporting Information for the NMR spectra of the mixture.
Mechanistic study of diselenide 9 with PhSH and H2O2: To the NMR tube containing diselenide 9 (30 mg, 0.05 mmol) in 0.5 mL of CDCl3 was added 1 equiv of PhSH (5 μL, 0.05 mmol). The progress of the reaction was followed by the 77Se NMR spectroscopy. The signal corresponding to selenenyl sulfide 10 was observed. Further addition of PhSH in excess (2-4 equiv), selenol peak was appeared in the spectrum. Thereafter, the signal corresponding to ebselenamine 7a was observed by adding 2 equiv of H2O2 (12 μL, 0.10 mmol) to the above mixture. Again, addition of extra PhSH (2 equiv) to the above mixture produced selenenyl sulfide 10 and diselenide 9. All the 77Se NMR spectra were recorded from time to time with subsequent addition of H2O2 and PhSH. See the Supporting Information for the 77Se NMR spectra.
Mechanistic Study of diselenide 9 with PhSSPh: The 1.5 equiv of PhSSPh (29 mg, 0.13) was added to the NMR tube containing diselenide 9 (50 mg, 0.086 mmol) in 0.5 mL of CDCl3. The 77Se NMR recorded next day. Only the diselenide 9 peak was appeared at 361 ppm. Further, 1.5 equiv of PhSSPh was added to the above mixture. The 77Se NMR spectra were recorded after each addition to the NMR tube. Now, added one more equiv (6 μL) of H2O2 in the same reaction mixture. The NMR spectra were recorded followed by 77Se NMR spectroscopy. Then another three more equiv of PhSSPh was added and the 77Se NMR spectrum recorded again. For the spectra related to kinetic experiment, please see the Supporting Information.
Mechanistic study of diselenide 19 with H2O2 and PhSH: To the NMR tube containing diselenide 16 (50 mg, 0.082 mmol) in 0.50 mL of DMSO-d6 was added 2 equiv of H2O2 (19 μL, 0.16 mmol) and mixed together. The oxidation reaction of was followed by the 77Se NMR spectroscopy. Thereafter, observing the signal corresponding to selenoxide[36], an equimolar of PhSH (16 μL, 0.16 mmol) was added to the above mixture. Immediately, three new signals corresponding to 20, 21 and 7b were observed in the 77Se NMR spectrum. See the Supporting Information for the NMR spectrum of the mixture.
HPLC Peroxidation Assay: The experimental setup for recording inhibition times (Tinh) and inhibited rates of peroxidation (Rinh) during azo-initiated peroxidation of linoleic acid using AMVN at 42 oC in a two-phase chlorobenzene/water system has been recently described with slight modifications.[45] Fresh linoleic acid was used as purchased. The values of Rinh and Tinh reported for reactions performed in the presence and absence of AscOH in Table 1 are means ± SD based on triplicates. As Rinh- and Tinh- values indicated slight variations depending on the amount of linoleic acid hydroperoxide which is always present in commercial samples as an impurity, and increases upon storage. In a fresh sample of linoleic acid was added small amounts of peroxidized linoleic acid from an older bottle until the concentration, as assessed by UV spectroscopy of conjugated diene formation at 234 nm was about 170-180 µM.
Coupled Reductase Assay: The GPx-like activities of compounds prepared were determined using UV-spectroscopy by following the reported protocol with slight modifications.[48] The test mixture contained GSH (2 mM), ethylene diamine tetra acetate (EDTA, 1 mM), GR (1.65 unit×mL-1), and NADPH (0.4 mM) in potassium phosphate buffer (100 mM), pH 7.5. Catalysts (80 µM) were added to the test mixture at 21 oC and the reaction was initiated by addition of H2O2 (1.6 mM). Initial reaction rates were based on the consumption of NADPH as assessed by UV-spectroscopy at 340 nm. The initial reduction rates were triplicated and calculated from the first 10 seconds of reaction by using 6.22 mM-1cm-1 as the extinction coefficient for NADPH. GPx-data reported in Table 1 are means ± SD.
Computational Details: Computational calculations for compounds 10,12 20, and 21 were executed by using the Gaussian 09 suite of quantum chemical programs.[49] The hybrid Becke 3-Lee-Yang-Parr (B3LYP) exchange correlation functional was implemented for density functional theory (DFT) calculations.[50] The geometry optimizations and frequencies were carried out at the B3LYP/6-311+G(d) basis sets. The quantifications of orbital interaction were done by natural bond analysis at B3LYP/6–311+G(d,p) level.[51] The 77Se NMR calculations were performed at B3LYP/6-311+G (d,p) level on B3LYP/6–311+G(d)-level-optimized geometries by using the gauge-including atomic orbital (GIAO) method (referenced with respect to the peak of Me2Se).[52]