HPLC/MS: = 590.2 [M+H]+ HRMS: 589.248129 (found); C29H39N3O8S, 589.24579 (calcd.). 2.67-2.69 (2H, m), 3.79 (3H, s, OMe), 5.93 (2H, br.s, NH2). 13C NMR (150 MHz, CDCl3): 22.8, 23.3, 24.5, 26.9, 50.6, 105.6, 117.7, 132.4, 161.8, 166.5. 2-(Boc-amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (3a) The mixture of 2a (10 mmol, 2.11 g), Boc2O (12.5 mmol, 2.56 g), DMAP (1 mmol, 122 mg) Rabbit Polyclonal to Collagen III in 25 mL of THF was stirring under reflux overnight. The additional 0.5 g of Boc2O was added, and stirring under reflux overnight. After cooling to RT, the combination was quenched by water, and extracted by DCM. The organic layer was dried, and concentrated. The residue was dissolved in 20 mL of ethanol, potassium hydroxide (2.24 g) and 20 mL of water was added. The combination was reflux for 5 hours. The reaction was quenched by water, the aqueous combination was washed by ether. The aqueous was the adjusted to pH = 6 with 1 M HCl aq. The product was collected by vacum filtration as yellow solids (1.42 g, 48%). HPLC/MS: = 296.0 [M-H]- HRMS: 297.102519 (found); C14H19NO4S, 297.1035 (calcd.). 1H NMR (600 MHz, CDCl3): 1.57 (9H, s), 1.79-1.82 (4H, m), 2.62-2.63 (2H, m), 2.82-2.83 (2H, m), 10.09 (1H, s). 13C JANEX-1 NMR (150 MHz, CDCl3): 22.7, 23.0, 24.2, 26.3, 28.2, 82.3, 109.2, 125.3, 131.7, 151.8, 170.7. Methyl 2-aminothiophene-3-carboxylate (2b) Triethylamine (50 mmol, 5.0 mL) was added dropwise to a mixture of 1,4-dithiane-2,5-diol (7.60 g, 50 mmol), methyl cyanoacetate (9.59 g, 100 mmol), and DMF (40 mL). The combination was stirred at 45 C for 30 min. After cooled to RT, the reaction combination was diluted with JANEX-1 aqueous acetic acid (0.4 M, 200 mL). The combination was extracted with ether (4 40 mL), and the combined organic layer was washed with water (2 40 mL). After dried over sodium sulfate, the organic layer was filtered through silica gel pad. After evaporation, 8.70 g of yellow solids were obtained (yield: 55%). HPLC/MS: = 158.2 [M+H]+ 1H NMR (600 MHz, CDCl3): 3.83 (3H, s), 5.94 (2H, br.s), 6.20 (1H, d, J = 5.4 Hz), 6.98 (1H, d, J = 6.0 Hz). 13C NMR (150 MHz, CDCl3): 51.0, 106.9, 107.0, 125.8, 162.7, 165.8. 2-(Boc-amino)-3-thiophenecarboxylic acid (3b) The mixture of 2b (20 mmol, 3.14 g), Boc2O (25 mmol, 5.15 g), DMAP (2 mmol, 244 mg) in 30 mL of THF was stirring under reflux overnight. After evaporation of the solvent, the combination was quenched by water, and extracted by DCM. The organic layer was dried, and concentrated. The residue was dissolved in 20 mL of ethanol, potassium hydroxide (4.5 g) JANEX-1 and 20 mL of water was added. The combination was reflux for 5 hours. The reaction was quenched by water, the aqueous combination was washed by ether. The aqueous was the adjusted to pH = 6 with 1 M HCl aq. The product was collected by vacum filtration and further purified by flash chromatography with ethyl acetate, 1.52 g of brown solids were obtained (yield: 31%). HPLC/MS: = 242.0 [M-H]- HRMS: 243.055945 (found); C10H13NO4S, 243.05653 (calcd.). 1H NMR (600 MHz, CDCl3): 1.58 (9H, s), 6.71 (1H, d, J = 5.4 Hz), 7.23 (1H, d, J = 6.0 Hz), 9.88 (1H, br.s). 13C NMR (150 MHz, = 234.3 [M+H]+ 1H NMR (600 MHz, CDCl3): 3.86 (3H, s), 6.02 (2H, br.s), 7.21-7.26 (2H, m), 7.33-7.46 (2H, m),7.45-7.46 (2H, m). 13C NMR (150 MHz, CDCl3): 51.1, 107.7, 124.7, 125.0, 126.6, 128.8, 133.9, 162.2, 165.8. 2-(Boc-amino)-5-phenylthiophene-3-carboxylic acid (3c) The mixture of 2c (10 mmol, 2.33 g), Boc2O (15 mmol, 3.10 g), DMAP (1 mmol, 122 mg) in 30 mL of THF was stirring under reflux overnight. After cooling to RT, the combination was quenched by water, and extracted by DCM. The organic layer was dried, and concentrated. The residue was dissolved in 20 mL of ethanol, potassium hydroxide (2.24 g) and 20 mL of water was added. The combination was reflux for 1 hour. The reaction was quenched by water, the aqueous combination was washed by ether. The aqueous was the adjusted to pH = 6 with 1 M HCl aq. The product was collected by vacum filtration.