Chemistry methods
All solvents and reagents obtained from commercial sources were used without further purification, unless otherwise noted. Compounds 6a and 6f were purchased from Oakwood Chemical (West Columbia, SC) and purified prior to use. All reactions were carried out under an argon atmosphere unless otherwise noted. All final molecules were >95 % pure as judged by high-performance liquid chromatography (HLPC). HPLC analyses were performed on an Agilent 1220 Infinity system with an Agilent column (Poroshell 120 EC-C18, 4.6 × 150 mm, gradient of 0.1 % trifluoroacetic acid/acetonitrile). 1H and 13C NMR analyses were performed on a Varian Mercury 300 MHz spectrophotometer at 300 and 75 MHz, respectively. Chemical shifts are given in ppm in reference to tetramethylsilane (TMS) as an internal standard. Multiplicities are given as s (singlet), d (doublet), t (triplet), m (multiplet), and br s (broad signal). Low-resolution mass spectral data were obtained on an Agilent 1260 Infinity single quadrupole LCMS system. Melting points were taken on a Mel-Temp apparatus and are uncorrected. Thin layer chromatography (TLC) was performed on silica gel 60 F254-coated glass plates purchased from EMD Millipore, and visualized with UV light and/or basic KMnO4.
General procedure for the synthesis of dithiolethiones from β-keto esters, exemplified by 5-methyl-3H-1,2-dithiole-3-thione, 5a [41]
To a suspension of elemental sulfur (123 mg, 3.85 mmol), phosphorus pentoxide (1.03 g, 2.31 mmol), hexamethyldisiloxane (2.76 mL, 11.6 mmol), in toluene (10 mL) was added β-oxo ester 2a (500 mg, 3.85 mmol). The mixture was heated under reflux conditions until complete as judged by TLC (generally between 1 and 3 h), at which time the reaction mixture was cooled to 0 °C. Saturated aqueous K2CO3 was added (5 mL) to destroy any unreacted phosphorus pentoxide. The crude product was then extracted with ethyl acetate (10 mL × 3), dried (Na2SO4), filtered, concentrated, and purified by column chromatography (hexanes/ethyl acetate, 4:1) to give a low-melting red solid (521 mg, 91 %). R
f
= 0.65 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 2.52 (d, J = 0.99 Hz, 3 H), 7.00–7.07 (m, 1 H). 13C NMR (75 MHz, CDCl3) δ: 18.43, 139.41, 172.22, 216.66. Calc. 148, found 149 [M+H]+.
4-(4-Nitrophenyl)-3H-1,2-dithiole-3-thione, 4a [42]
Prepared from 1a [43]. Red solid (92 %). Mp 152–154 °C. R
f
= 0.37 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 7.89 (d, J = 8.73 Hz, 2 H), 8.30 (ds, J = 8.90 Hz, 2 H), 9.34 (s, 1 H). 13C NMR (75 MHz, CDCl3): δ = 128.67, 135.59, 145.44, 151.15, 152.50, 166.47, 218.57. Calc. 255, found 256 [M+H]+.
4-Ethyl-3H-1,2-dithiole-3-thione, 4b [44]
Prepared from 1b [45]. Yellow oil (81 %). R
f
= 0.46 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 1.15 (t, J = 7.43 Hz, 3 H), 2.48–2.73 (m, 2 H), 8.86 (t, J = 0.82 Hz, 1 H). 13C NMR (75 MHz, CDCl3): δ 13.03, 23.52, 150.79, 155.45, 215.12. Calc. 162, found 163 [M+H]+.
Ethyl 3-thioxo-3H-1,2-dithiole-4-carboxylate, 4c [46]
Prepared from diethyl 2-(ethoxymethylene)malonate, 1c. Red solid (47 %). Mp 61–62 °C. R
f
= 0.48 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 1.37 (t, J = 7.07 Hz, 3 H), 4.35 (q, J = 7.19 Hz, 2 H), 9.18 (s, 1 H). 13C NMR (75 MHz, CDCl3): δ 14.35, 62.12, 138.30, 160.81, 165.22, 211.31. Calc. 207, found 208 [M+H]+.
5-(4-Fluorophenyl)-3H-1,2-dithiole-3-thione, 5b [47]
Red solid (74 %). Mp 98–100 °C. R
f
= 0.84 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 7.12–7.26 (m, 2 H) 7.39 (s, 1 H) 7.59–7.72 (m, 2 H). 13C NMR (75 MHz, CDCl3): δ 116.97/117.26 (CF, d, J = 22 Hz), 129.19, 129.31, 136.13, 163.45/166.83 (CF, d, J = 254 Hz), 171.62, 215.66. Calc. 228, found 229 [M+H]+.
5-(Pyridin-4-yl)-3H-1,2-dithiole-3-thione, 5c [48]
Red solid (34 %). Mp decomposed. R
f
= 0.09 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 7.50 (s, 1 H) 7.52–7.59 (m, 2 H) 8.81 (d, J = 5.93 Hz, 2 H). 13C NMR (75 MHz, CDCl3): δ 121.02, 121.9, 145.67, 150.01, 175.25, 214.27. Calc. 211, found 212 [M+H]+.
5-(Furan-2-yl)-3H-1,2-dithiole-3-thione, 5d [49]
Red solid (63 %). Mp 97–100 °C. R
f
= 0.71 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 6.61 (dd, J = 3.53, 1.72 Hz, 1 H), 6.95–7.02 (m, 1 H), 7.38 (s, 1 H), 7.64 (dd, J = 1.81, 0.54 Hz, 1 H). 13C NMR (75 MHz, CDCl3): δ 113.53, 113.59, 133.27, 146.60, 146.71, 160.27, 214.50. Calc. 200, found 201 [M+H]+.
Ethyl 5-methyl-3-thioxo-3H-1,2-dithiole-4-carboxylate, 6b [50]
Red solid (78 %). Mp 64–66 °C. R
f
= 0.84 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 1.37 (t, J = 7.16 Hz, 3 H), 2.57 (s, 3 H), 4.39 (q, J = 7.07 Hz, 2 H). 13C NMR (75 MHz, CDCl3): δ 14.35, 19.11, 62.50, 140.80, 163.28, 174.05, 211.82. Calc. 220, found 221 [M+H]+.
4-Chloro-5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione, 6g [51]
Prepared from 3g [52]. Yellow solid (91 %). Mp 125–127 °C. R
f
= 0.63 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 3.90 (s, 3 H), 7.07 (d, J = 9.06 Hz, 2 H), 7.67 (d, J = 9.06 Hz, 2 H). 13C NMR (75 MHz, CDCl3): δ 55.57, 114.78, 124.12, 130.39, 123.43, 162.45, 165.62, 206.59. Calc. 274, found 275 [M+H]+.
4-Chloro-5-phenyl-3H-1,2-dithiole-3-thione, 6h [51]
Prepared from 3h [53]. Yellow solid (87 %). Mp 105–107 °C. R
f
= 0.74 (2 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 7.49–7.73 (m, 5 H). 13C NMR (75 MHz, CDCl3): δ 127.07, 128.88, 129.49, 129.79, 131.91, 165.63, 206.88. Calc. 244, found 245 [M+H]+.
4-Chloro-5-ethyl-3H-1,2-dithiole-3-thione, 6i [54]
Prepared from 3i [55]. Yellow solid (59 %). Mp 83–84 °C. R
f
= 0.71 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 1.40 (t, J = 7.52 Hz, 3 H), 2.98 (q, J = 7.61 Hz, 2 H). 13C NMR (75 MHz, CDCl3): δ 12.80, 27.99, 158.84, 171.46, 206.64. Calc. 196, found 197 [M+H]+.
Ethyl 5-acetamido-3-thioxo-3H-1,2-dithiole-4-carboxylate, 6c [56]
Compound 6a (100 mg, 0.452 mmol) was refluxed in acetic anhydride (5 mL) for 30 min. The solution was then cooled, concentrated to dryness, and the crude material purified by column chromatography (hexanes/ethyl acetate, 3:1) to give 6c as a red solid (104 mg, 88 %). Mp 156–157 °C. R
f
= 0.39 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 1.43 (t, J = 7.16 Hz, 3 H), 2.40 (s, 3 H), 4.42 (q, J = 7.13 Hz, 2 H), 12.72 (br s, 1 H). 13C NMR (75 MHz, CDCl3): δ 14.15, 23.97, 62.68, 118.75, 166.36, 170.63, 174.56, 208.25. Calc. 263, found 264 [M+H]+.
General procedure for the syntheses of dithiolethiones from nitriles, exemplified by 5-amino-4-(4-chlorophenyl)-3H-1,2-dithiole-3-thione, 6d
To an ice-cooled suspension of NaH (263 mg, 6.58 mmol), carbon disulfide (220 μL, 3.62 mmol), and elemental sulfur (116 mg, 3.62 mmol) in DMF (5 mL) was added 3d (500 mg, 3.29 mmol) in DMF (1 mL). The mixture was allowed to stir at 0 °C for 30 min, at which time saturated Na2CO3 (10 mL) was added. The mixture was then extracted with ethyl acetate (10 mL × 3), washed with water (10 mL × 3), dried (Na2SO4), filtered, concentrated, and purified by column chromatography (hexanes/ethyl acetate 4:1) to yield 6d as a red solid (838 mg, 95 %). Mp 106–107 °C. R
f
= 0.29 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 6.35 (br s, 2 H), 7.29 (d, J = 8.70 Hz, 2 H), 7.48 (d, J = 8.70 Hz, 1 H). 13C NMR (75 MHz, CDCl3): δ 130.00, 132.22, 132.27, 134.85, 151.04, 175.69, 234.84. Calc. 259, found 260 [M+H]+.
5-Amino-4-(phenylsulfonyl)-3H-1,2-dithiole-3-thione, 6e [30]
Red solid (69 %). Mp decomposed. R
f
= 0.13 (20 % EtOAc/Hex). 1H NMR (300 MHz, CDCl3): δ 7.50–7.78 (m, 3 H), 7.91–8.05 (m, 2 H), 9.01 (bs 1 H), 10.09 (bs, 1 H). 13C NMR (75 MHz, CDCl3): δ 117.75, 127.39, 128.77, 133.74, 140.45, 180.23, 203.60. Calc. 289, found 290 [M+H]+.
Biological methods
Cell culture conditions
The SH-SY5Y human neuroblastoma cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in DMEM:F-12 media (1:1) supplemented with FBS (10 %) and 100 U/mL penicillin and 100 μg/mL streptomycin in 150 cm2 culture flasks in a humidified atmosphere of 5 % CO2. The media was replaced every 3–4 days, and cells were subcultured once a confluence of 70–80 % was reached. All test compounds were dissolved in DMSO and diluted in media (final DMSO concentration of 0.1 % v/v).
Measurement of intracellular GSH levels
SH-SY5Y cells were seeded in white 96-well plates and allowed to adhere overnight. Media was removed and replaced with media containing either test compounds (100 μM) or DMSO (0.1 %) for 24 h. Total glutathione levels (GSH + GSSG) were then measured using GSH/GSSG Glo© assay from Promega (Madison, WI). GSH levels were expressed as a percentage of control.
Neuroprotection assay
SH-SY5Y cells were seeded in white 96 well plates and allowed to attach overnight. Media was removed and replaced with media containing either test compounds (100 μM) or DMSO for 24 h. Next, 6-OHDA (Aldrich) in media (final concentration of 40 μM) of media was added and the cells were co-treated for 24 h. Cellular viability was assessed using the CellTiter Glo© assay from Promega (Madison, WI). Viability was expressed as a percentage of control.
Statistical analyses
One-way analysis of variance (ANOVA) was used to test for significant differences using GraphPad Prism software (La Jolla, CA). P values less than 0.05 were considered to be statistically significant. Results are expressed as mean ± SEM.