General
All chemical reagents and organic solvents were purchased from FUJIFILM Wako Pure Chemicals (Osaka, Japan), Tokyo Chemical Industries (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), and BLD Pharmatech (Shanghai, China), and were used without further purification. Proton nuclear magnetic resonance (1H-NMR) and carbon 13 nuclear magnetic resonance (13C-NMR) spectra were recorded on ECS-400 (JEOL, Tokyo, Japan), ECA-500 (JEOL), and ECZ-600R (JEOL) spectrometers. The chemical shifts of the 1H-NMR and 13C-NMR spectra were reported as δ values (ppm) relative to tetramethyl silane (0 ppm) and relative to CDCl3 (77.0 ppm) or dimethyl sulfoxide (DMSO)-d6 (39.6 ppm). The splitting patterns were reported as s (singlet); d (doublet); t (triplet); q (quartet); m (multiplet); and br (broad). The coupling constants (J values) are given in hertz (Hz). The electrospray ionization–mass spectrometry (ESI–MS) spectra were recorded on a Q-Exactive Plus spectrometer (Thermo Scientific, Waltham, MA, USA). High-resolution fast atom bombardment mass spectra (HRMS) were acquired using an NMS-SX102 102A spectrometer (JEOL). Column chromatography was performed using Wako-Gel C-200 (100–200 mesh). The purities of the synthesized compounds for biological testing were > 98% as determined by analytical high-performance liquid chromatography (HPLC). Unless otherwise stated, radioactivity was measured using an IGC-3R Curiemeter (Hitachi Aloka Medical, Tokyo, Japan). HPLC was performed using a JASCO HPLC system (JASCO, Tokyo, Japan): effluent radioactivity was monitored using a NaI (Tl) scintillation detector system.
Chemical synthesis
6-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-amino benzo[d]thiazole (8)
A mixture comprising 2-amino-6-bromobenzothiazole (7; 2.29 g, 10.0 mmol), bis(pinacolato)diboron (2.79 g, 11.0 mmol), K2CO3 (2.94 g, 30 mmol), and Pd(dppf)2 dichloromethane adduct (0.8 g, 1.0 mmol) in 1,4-dioxane (40 mL) was refluxed under argon for 10 h. The reaction mixture was filtered through a celite bed and washed with AcOEt. After solvent removal, the residue was purified by column chromatography (silica gel, CH2Cl2/AcOEt = 100/0 to 80/20) to give 8 as a white powder (2.06 g) with a melting point (mpt) of 218–219 °C (recrystallized form n-hexane). 1H-NMR (400 MHz, CDCl3) δ 8.06 (1H, s), 7.75 (1H, dd, J = 0.9, 9.0 Hz), 7.52 (1H, d, J = 8.1 Hz), 5.59 (2H, br), 1.35 (12H, s). 13C-NMR (151 MHz, CDCl3) δ 167.31, 154.47, 132.43, 131.24, 127.70, 118.51, 83.78, 31.57, 24.86. MS (ESI) [M + H]+ calculated for (C19H17BN2O2S) requires m/z, 277.1177; found m/z, 277.1170.
2-Acetylamino 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)benzo[d]thiazole (9a)
Acetylchloride (0.4 mL, 6.0 mml) in CH2Cl2 (2.0 mL) was added to a mixture comprising 8 (1.10 g, 4.0 mmol), Et3N (1.39 mL, 10.0 mmol), and N,N-dimethylaminopyridine (DMAP) (15 mg, 0.12 mmol) in CH2Cl2 (8 mL), which was stirred at room temperature overnight. The reaction was quenched by brine and extracted with CH2Cl2. The organic layer was washed with brine, dried over sodium sulfate, and evaporated. The residue was purified by column chromatography (silica gel, n-hexane/AcOEt = 90/10 to 80/20) to give 9a as a white powder (0.96 g) with a mpt of 235–236 °C (decomp). 1H-NMR (400 MHz, DMSO-d6) δ: 12.45 (1H, brs), 8.27 (1H, s), 7.72 (2H, s), 2.21 (3H, s), 1.31 (12H, s). 13C-NMR (125.7 MHz, CDCl3): δ 168.8, 150.0, 149.9, 132.5, 131.4, 128.6, 125.8, 119.6, 83.0, 24.9, 23.5. MS (FAB) [M + H]+ m/z, 319.
tert-Butyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]thiazol-2-yl-carbamate (9b)
Di-tert-butyl dicarbonate (1.64 g, 7.5 mmol) was added to a mixture comprising 7 (2.0 g. 7.24 mmol), Et3N (1.39 mL, 10.0 mmol), and DMAP (30 mg, 0.25 mmol) in CH2Cl2 (25 mL), which was stirred at room temperature overnight. The reaction mixture was quenched with brine and extracted with CH2Cl2. The organic layer was washed with brine, dried over sodium sulfate, and evaporated. The residue was purified by column chromatography (silica gel, n-hexane/AcOEt = 90/10 to 80/20) to give 9b as a white powder (2.17 g) with a mpt > 350 °C (decomp.) 1H-NMR (400 MHz, CDCl3): δ 11.77 (1H, br), 8.26 (1H, s), 7.91 (1H, d, J = 8.1 Hz), 7.82 (1H, dd, J = 0.9, 8.1 Hz), 1.60 (9H, s), 1.38 (12H, s). 13C-NMR (151 MHz, CDCl3): δ 163.02, 152.84, 150.97, 131.86, 131.08, 128.00, 120.08, 83.94, 83.39, 28.39, 24.86. HRMS (ESI) [M + H]+ calculated for (C18H26BN2O4S) requires m/z, 377.1701; found m/z, 377.1695.
Cyclohexyl 4-nitrophenyl carbonate (11)
A mixture comprising 4-nitrophenyl chloroformate (10; 6.05 g, 30.0 mmol), cyclohexanol (3.30 g, 33.0 mmol), and DMAP (0.36 g, 3.0 mmol) in tetrahydrofuran (THF) (15 mL) was stirred at 0 °C for 3 h and then at room temperature for 3 h. The reaction mixture was diluted with n-hexane and purified by column chromatography (silica gel, n-hexane/AcOEt = 95/5) to give 11 as a white powder (3.83 g) with a mpt of 63–64 °C. 1H-NMR (400 MHz, CDCl3): δ 8.28 (2H, d, J = 9.2 Hz), 7.39 (2H, d, J = 9.2 Hz), 4.73–4.80 (1H, m), 1.99–2.05 (2H, m), 1.79–1.84 (2H, m), 1.55–1.64 (4H, m), 1.26–1.47 (2H, m). 13C-NMR (151 MHz, CDCl3): δ 155.66, 151.83, 145.20, 125.23, 121.78, 78.71, 31.33, 25.06, 23.50. MS (FAB) [M + H]+ m/z, 266.
Cyclohexyl (5-bromo-2-methylpyridine-3-yl) carbamate (12)
A solution of potassium bis(trimethylsilyl)amide (5.6 mmol) in toluene (0.5 M, 11.2 mL) was added slowly to the mixture of 2-methyl-3-amino-5-bromopyridine (0.5 g, 2.67 mmol) in dry THF (5 mL) at 0 °C. After stirring for 15 min at 0 °C, a solution of 11 (0.85 g, 3.2 mmol) in THF (5 mL) was added slowly to the mixture described above. The reaction mixture was stirred for 30 min at 0 °C. The mixture was quenched with brine and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and then evaporated. The residue was purified by column chromatography on silica gel (n-hexane/AcOEt = 90/10 to 80/20) to give 12 as a pale-yellow powder (0.63 g) with a mpt of 95–96 °C. 1H-NMR (400 MHz, CDCl3): δ 8.48 (1H, br), 8.26 (1H, d, J = 2.0 Hz), 6.43 (1H, s), 4.76 (1H, m), 2.46 (3H, s), 1.74–1.97 (4H, m), 1.22–1.60 (6H, m). 13C-NMR (151 MHz, CDCl3): δ 152.82, 144.25, 133.67, 129.02, 118.30, 115.63, 74.74, 31.82, 25.24, 23.78, 20.26. MS (FAB) [M + H]+ m/z, 313.
Cyclohexyl 5-(2-acetylamino-benzo[d]thiazol-6-yl)-2-methylpyridin-3-ylcarbamate (PK68, 5)
A mixture comprising 12 (478 mg, 1.5 mmol), 9a (376 mg, 1.0 mmol), K2CO3 (520 mg, 3.75 mmol), and Pd(Ph3P)4 (173 mg, 0.15 mmol) in 1,4-dioxane/H2O (15 mL/3 mL) was refluxed under argon for 12 h. The reaction mixture was filtered through a celite bed and washed with AcOEt. The crude product was extracted with AcOEt, and the organic layer was washed with brine and dried over MgSO4, then evaporated. The residue was purified by column chromatography on silica gel (CH2Cl2/CH3OH = 100/0 to 90/10) to give PK68 (5) as a pale-yellow powder (440 mg) with a mpt >350 °C (decomp). 1H-NMR (400 MHz, DMSO-d6): δ 9.14 (1H, s), 8.59 (1H, d, J = 2.0 Hz), 8.30 (1H, d, J = 1.6 Hz), 8.10 (1H, d, J = 1.8 Hz), 7.81 (1H, d, J = 8.5 Hz), 7.71 (1H, dd, J = 1.9, 8.4 Hz), 4.62–4.68 (1H, m), 3.45 (1H, br), 2.46 (3H, s), 2.21 (3H, s), 1.91–1.94 (2H, m), 1.71–1.74 (2H, m), 1.21–1.52 (6H, m). 13C-NMR (125.7 MHz, DMSO-d6): δ 169.5, 158.6, 154.0, 150.3, 148.4, 142.6, 133.3, 132.9, 132.6, 132.2, 129.0, 120.9, 119.7, 72.9, 31.6, 24.9, 23.5, 22.8, 20.7. HRMS (FAB) [M + H]+ calculated for (C22H25N4O3S) requires m/z, 425.1647; found m/z, 425.1652.
Cyclohexyl 5-(2-tert-butoxycarbonyl-aminobenzo[d]thiazol-6-yl)-2-methylpyridin-3-ylcarbamate (13)
A mixture comprising 12 (313 mg, 1.0 mmol), 9b (376 mg, 1.0 mmol), K2CO3 (210 mg, 1.5 mmol), and Pd(Ph3P)4 (116 mg, 0.1 mmol) in 1,4-dioxane/H2O (10 mL/2 mL) was refluxed under argon for 12 h. The reaction mixture was filtered through a celite bed and washed with AcOEt. The crude product was extracted with AcOEt and the organic layer was washed with brine, dried over MgSO4, and then evaporated. The residue was purified by column chromatography on silica gel (CH2Cl2/CH3OH = 100/0 to 90/10) to give 13 as a pale-yellow powder (206 mg) with a mpt >350 °C (decomp). 1H-NMR (400 MHz, DMSO-d6): δ 11.87 (1H, br), 9.14 (1H, s), 8.59 (1H, d, J = 2.0 Hz), 8.28 (1H, d, J = 1.8 Hz), 8.10 (1H, s), 7.76 (1H, d, J = 8.3 Hz), 7.69 (1H, d, J = 10.1 Hz), 4.62–4.68 (1H, m), 2.46 (3H, s), 1.91–1.99 (2H, m), 1.72–1.74 (2H, m), 1.53 (9H, s), 1.29–1.45 (6H, m). 13C-NMR (151 MHz, DMSO-d6) δ: 160.2, 153.6, 150.2, 149.2, 142.6, 133.3, 132.9, 132.6, 131.8, 131.5, 128.8, 124.8, 120.6, 119.5, 81.8, 72.6, 31.6, 27.9, 24.9, 23.4, 20.7. HRMS (ESI) [M + H]+ calculated for (C25H31N4O4S) requires m/z, 483.2066; found m/z, 483.2055.
Cyclohexyl-5-(2-aminobenzo[d]thiazol-6-yl)-2-methylpyridin-3-ylcarbamate (14)
A mixture comprising 13 (100 mg, 0.21 mmol in CH3OH (10 mL) and 2 mol/L HCl (5 mL) was stirred at 60 °C overnight. Et3N (1.5 mL) was added to the reaction mixture and evaporated. The crude product was washed with water and purified by column chromatography (silica gel CH2Cl2/CH3OH = 100/0 to 97/3) to give 14 as a white powder (44.3 mg) with a mpt >350 °C (decomp). 1H-NMR (400 MHz, DMSO-d6): δ 9.10 (1H, s), 8.53 (1H, d, J = 1.8 Hz), 8.02 (2H, d, J = 11.9 Hz), 7.61 (2H, br), 7.51 (1H, dd, J = 1.5, 8.2 Hz), 7.41 (1H, d, J = 8.3 Hz), 4.61–4.67 (1H, m), 2.44 (3H, s), 1.90–1.93 (2H, m), 1.72–1.77 (2H, m), 1.16–1.48 (6H, m). 13C-NMR (151 MHz, DMSO-d6): δ 167.14, 153.93, 152.78, 149.68, 142.26, 133.51, 132.77, 132.09, 129.46, 124.16, 118.92, 118.01, 72.78, 40.03, 24.88, 23.42, 20.64. HRMS (ESI) [M + H]+ calculated for C20H22N4O2S) requires m/z, 383.1542; found m/z, 383.1531.