Mycophenolic acid is a natural compound, purchased from Chengdu Refines Biotechnology Co., Ltd., with a purity of 98%. The organic reagents required for the experiment are all analytically pure, and other chemicals are purchased from Aladdin reagent. The obtained products were monitored by thin-layer chromatography to check the progress of the reaction, and separation was carried out using a chromatography column. The melting point of the target product was measured in an open capillary (the temperature is not corrected); 1H-NMR and 13C-NMR used the chemical shift of TMS as the zero point, measured by AV-300 nuclear magnetic resonance instrument; The high resolution mass spectrum was measured by ESI mass spectrometer.
4.1 General procedure for the reaction of mycophenolic acid with different intermediates containing-amino
Mycophenolic acid (64 mg, 0.2 mmol), different amine compounds (0.24 mmol), carboxylic mixture 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (114.7 mg, 0.6 mmol) and catalyst Biphenyl-4-amidoxime (HOBt) (84.8 mg, 0.4 mmol) were taken in a 25 ml round bottom flask, and 5 ml dichloromethane as solvent, stirred at 0 ℃ for 4-6 h, progress of reaction was confirmed by TLC, the final products were purified using silica gel chromatography, and all the target compounds were obtained, which are white powder in appearance, and the yield is 48%-79%.
4.1.1 (E)-N-benzyl-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide (A1)
White powder; yield 52%; m.p. 96-98℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.67 (s, 1H), 7.35-7.30 (m, 2H), 7.27-7.20 (m, 3H), 5.77 (brs, 1H), 5.32 (t, J = 8.1 Hz, 1H), 5.19 (s, 2H), 4.41 (d, J = 5.7 Hz, 2H), 3.77 (s, 3H), 3.40 (d, J = 6.9 Hz, 2H), 2.36 (s, 4H), 2.15 (s, 3H), 1.84 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 172.91, 172.48, 163.63, 153.59, 144.04, 138.33, 134.60, 128.62 (2C), 127.58 (2C), 127.36, 123.06, 122.00, 116.76, 106.40, 70.03, 61.01, 43.48, 35.27, 35.09, 22.62, 16.12, 11.54. ESI-HRMS (m/z) calcd for C24H28NO5+ [M+H]+: 410.19620, found: 410.19528.
4.1.2 (E)-N-(4-fluorobenzyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide (A2)
White powder; yield 73%; m.p. 156-158℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.67 (s, 1H), 7.19 (dd, J = 8.4, 5.4 Hz, 2H), 6.98 (t, J = 8.7 Hz, 2H), 5.81 (brs, 1H), 5.29 (t, J = 6.9 Hz, 1H), 5.20 (s, 2H), 4.37 (d, J = 5.7 Hz, 2H), 3.77 (s, 3H), 3.40 (d, J = 6.9 Hz, 2H), 2.36 (s, 4H), 2.15 (s, 3H), 1.83 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 172.90, 172.55, 163.68,163.61, 153.58, 144.05, 134.57, 134.23, 129.24, 129.14, 123.10, 121.94, 116.80, 115.52, 115.24, 106.39, 70.05, 61.01, 42.70, 35.24, 34.99, 22.64, 16.10, 11.53. ESI-HRMS (m/z) calcd for C24H27FNO5+ [M+H]+: 428.18678, found: 428.18616.
4.1.3 (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methyl-N-(4-methylbenzyl)hex-4-enamide (A3)
White powder; yield 75%; m.p. 116-118℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.68 (s, 1H), 7.12 (s, 4H), 5.75 (brs, 1H), 5.28 (t, J = 6.6 Hz, 1H), 5.19 (s, 2H), 4.35 (d, J = 5.7 Hz, 2H), 3.77 (s, 3H), 3.40 (d, J = 6.6 Hz, 2H), 2.34 (s, 7H), 2.15 (s, 3H), 1.82 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 172.90, 172.41, 163.64, 153.60, 144.03, 137.07, 135.29, 134.61, 129.28 (2C), 127.60 (2C), 122.99, 122.03, 116.75, 106.39, 70.03, 61.00, 43.25, 35.29, 35.14, 22.62, 21.07, 16.12, 11.55. ESI-HRMS (m/z) calcd for C25H30NO5+ [M+H]+: 424.21185, found: 424.2115.
4.1.4 (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methyl-N-phenethylhex-4-enamide (A4)
White powder; yield 70%; m.p. 102-104℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.69 (brs, 1H), 7.34-7.29 (m, 2H), 7.25 (d, J = 7.2 Hz, 1H), 7.21-7.12 (m, 2H), 5.49 (brs, 1H), 5.25 (t, J = 6.9 Hz, 1H), 5.13 (s, 2H), 3.78 (s, 3H), 3.48-3.38 (m, 4H), 2.70 (t, J = 6.9 Hz, 2H), 2.35-2.22 (m, 4H), 2.14 (s, 3H), 1.81 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 172.92, 172.55, 163.66, 153.59, 144.09, 138.92,134.57, 128.67 (2C), 128.59 (2C), 126.46, 122.81, 122.05, 116.77, 106.38, 70.02, 61.01, 40.53, 35.66, 35.22, 35.05, 22.61, 16.13, 11.55. ESI-HRMS (m/z) calcd for C25H30NO5+ [M+H]+: 424.21185, found: 424.21115.
4.1.5 (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-N-(4-hydroxyphenethyl)-4-methylhex-4-enamide (A5)
White powder; yield 70%; m.p. 112-114℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.60 (brs, 2H), 6.92 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 7.2 Hz, 2H), 5.85 (t, J = 5.7 Hz, 1H), 5.24 (t, J = 6.9 Hz, 1H), 5.09 (s, 2H), 3.76 (s, 3H), 3.40-3.34 (m, 4H), 2.56 (t, J = 7.2 Hz, 2H), 2.28 (s, 4H), 2.11 (s, 3H), 1.79 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 173.35, 173.07, 163.67, 155.19, 153.53, 144.20, 134.36, 129.83, 129.58(2C), 123.01, 122.03, 116.85, 115.57(2C), 106.33, 70.12, 61.03, 40.95, 35.22, 34.94, 34.65, 22.62, 16.06, 11.54. ESI-HRMS (m/z) calcd for C25H30NO6+ [M+H]+: 440.20676, found: 440.20621.
4.1.6 (E)-N-butyl-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide (B1)
White powder; yield 58%; m.p. 76-78℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.70 (s, 1H), 5.46 (brs, 1H), 5.28 (t, J = 7.2 Hz, 1H), 5.22 (s, 2H), 3.79 (s, 3H), 3.41 (d, J = 6.9 Hz, 2H), 3.20 (dd, J = 12.6, 6.0 Hz, 2H), 2.35-2.23 (m, 4H), 2.17 (s, 3H), 1.83 (s, 3H), 1.47-1.25 (m, 4H), 0.91 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 172.93, 172.56, 163.69, 153.62, 144.03, 134.72, 122.80, 122.10, 116.79, 106.37, 70.07, 61.02, 39.18, 35.33, 35.18, 31.68, 22.62, 20.04, 16.13, 13.72, 11.57. ESI-HRMS (m/z) calcd for C21H30NO5+ [M+H]+: 376.21185, found: 376.21127.
4.1.7 (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methyl-N-pentylhex-4-enamide (B2)
White powder; yield 65%; m.p. 108-110℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.70 (s, 1H), 5.47 (brs, 1H), 5.27 (t, J = 6.9Hz, 1H), 5.22 (s, 2H), 3.78 (s, 3H), 3.41 (d, J = 6.9 Hz, 2H), 3.22-3.15 (m, 2H), 2.36-2.26 (m, 4H), 2.17 (s, 3H), 1.83 (s, 3H), 1.48-1.39 (m, 2H), 1.35-1.24 (m, 4H), 0.90 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 172.93, 172.51, 163.68, 153.63, 144.02, 134.74, 122.79, 122.10, 116.79, 106.38, 70.06, 61.02, 39.45, 35.33, 35.21, 29.31, 29.05, 22.62, 22.33, 16.14, 13.97, 11.57. ESI-HRMS (m/z) calcd for C22H32NO5+ [M+H]+: 390.22750, found: 390.22690.
4.1.8 (E)-N-hexyl-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide (B3)
White powder; yield 63%; m.p. 106-108℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.70 (s, 1H), 5.47 (brs, 1H), 5.27 (t, J = 6.9Hz, 1H), 5.22 (s, 2H), 3.79 (s, 3H), 3.41 (d, J = 6.9 Hz, 2H), 3.22-3.15 (m, 2H), 2.35-2.24 (m, 4H), 2.17 (s, 3H), 1.83 (s, 3H), 1.47-1.37 (m, 2H), 1.28 (s, 6H), 0.90 (t, J = 6.3 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 172.92, 172.52, 163.68, 153.63, 144.02, 134.74, 122.80, 122.10, 116.78, 106.38, 70.06, 61.02, 39.49, 35.34, 35.21, 31.47, 29.59, 26.58, 22.62, 22.55, 16.14, 14.01, 11.57. ESI-HRMS (m/z) calcd for C23H34NO5+ [M+H]+: 404.24315, found: 404.24274.
4.1.9
Methyl (E)-(6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoyl)glycinate (C1)
White powder; yield 70%; m.p. 98-100℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.71 (brs, 1H), 6.01 (brs, 1H), 5.29 (t, J = 6.9 Hz, 1H), 5.22 (s, 2H), 3.99 (d, J = 5.1 Hz, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 3.41 (d, J = 6.9 Hz, 2H), 2.35 (s, 4H), 2.16 (s, 3H), 1.83 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 172.90, 172.82, 170.48, 163.66, 153.61, 144.05, 134.39, 123.08, 122.09, 116.77, 106.39, 70.05, 61.02, 52.34, 41.13, 35.05, 34.80, 22.62, 16.11, 11.56. ESI-HRMS (m/z) calcd for C20H26NO7+ [M+H]+: 392.17038, found: 392.17004.
4.1.10
methyl (E)-(6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoyl)phenylalaninate (C2)
White powder; yield 67%; m.p. 82-84℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.69 (s, 1H), 7.32-7.29 (m, 1H), 7.28-7.24 (m, 2H), 7.07 (dd, J = 7.8 Hz, 2.1Hz, 2H), 5.92 (d, J = 8.7 Hz, 1H), 5.26 (t, J = 6.9 Hz, 1H), 5.17 (s, 2H), 4.90-4.83 (m, 1H), 3.78 (s, 3H), 3.72 (s, 3H), 3.40 (d, J = 6.9 Hz, 2H), 3.05 (t, J = 5.4 Hz, 2H), 2.30 (s, 4H), 2.15 (s, 3H), 1.81 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 172.90, 172.09 (2C), 163.66, 153.60, 144.06, 135.90, 134.38, 129.20 (2C), 128.54 (2C), 127.09, 122.83, 122.05, 116.73, 106.38, 70.02, 61.01, 53.00, 52.28, 37.95, 35.00, 34.91, 22.61, 16.15, 11.57. ESI-HRMS (m/z) calcd for C27H32NO7+ [M+H]+: 482.21733, found: 482.21692.
4.1.11 (E)-N-(2-(dimethylamino)ethyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide (D1)
White powder; yield 79%; m.p. 76-78℃. 1H NMR (300 MHz, CDCl3, ppm) δ 7.44 (brs, 1H), 5.30-5.24 (m, 1H), 5.21 (s, 2H), 3.78 (s, 3H), 3.57 (dd, J = 10.2 Hz, 5.3Hz, 2H), 3.40 (d, J = 6.7 Hz, 2H), 3.01 (t, J = 5.1 Hz, 2H), 2.72 (s, 6H), 2.36 (brs, 4H), 2.16 (s, 3H), 1.83 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 173.55, 172.82, 163.62, 153.82, 144.16, 134.66, 122.73, 122.32, 116.62, 106.44, 69.95, 61.05, 58.11, 44.05(2C), 35.13, 35.11, 34.98, 22.68, 16.24, 11.55. ESI-HRMS (m/z) calcd for C21H31N2O5+ [M+H]+: 391.22275, found: 391.22235.
4.1.12 (E)-N-(2-(diethylamino)ethyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide (D2)
White powder; yield 73%; m.p. 60-62℃. 1H NMR (300 MHz, CDCl3) δ 6.76 (brs, 1H), 5.26 (t, J = 6.5 Hz, 1H), 5.21 (s, 2H), 3.78 (s, 3H), 3.40 (d, J = 6.9 Hz, 4H), 2.74 (d, J = 7.1 Hz, 6H), 2.32 (s, 4H), 2.16 (s, 3H), 1.83 (s, 3H), 1.13 (t, J = 7.1 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 172.97, 172.78, 163.60, 154.61, 144.11, 134.53, 123.06, 122.37, 116.04, 106.41, 69.89, 61.01, 51.62, 46.75 (2C), 36.62, 35.28 (2C), 22.74, 16.21, 11.54, 11.18 (2C). ESI-HRMS (m/z) calcd for C23H35N2O5+ [M+H]+: 419.25405, found: 419.25369.
4.1.13 (E)-N-(2-(ethyl(isopropyl)amino)ethyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide (D3)
White powder; yield 66%; m.p. 58-60℃. 1H NMR (300 MHz, CDCl3) δ 6.14 (brs, 1H), 5.25 (t, J = 5.8 Hz, 1H), 5.21 (s, 2H), 3.77 (s, 3H), 3.40 (d, J = 6.8 Hz, 2H), 3.21 (d, J = 5.3 Hz, 2H), 3.02 (t, J = 6.0 Hz, 2H), 2.57 (brs, 2H), 2.41 – 2.19 (m, 5H), 2.16 (s, 3H), 1.82 (s, 3H), 1.02 (d, J = 6.4 Hz, 12H). 13C NMR (75 MHz, CDCl3): δ 172.94, 172.38, 163.69, 153.70, 144.01, 134.66, 122.50, 122.18, 116.68, 106.36, 70.04, 61.02 (2C), 47.68, 43.01, 37.90, 35.41, 35.37, 22.62, 20.78 (2C), 16.21 (2C), 11.57. ESI-HRMS (m/z) calcd for C25H39N2O5+ [M+H]+: 447.28535, found: 447.28503.
4.1.14 (E)-N-(2-acetamidoethyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide (D4)
White powder; yield 72%; m.p. 98-100℃. 1H NMR (300 MHz, CDCl3) δ 7.71 (s, 1H), 6.26 (d, J = 23.9 Hz, 2H), 5.27 (dd, J = 15.0, 8.2 Hz, 1H), 5.22 (s, 2H), 3.78 (s, 3H), 3.41 (d, J = 6.9 Hz, 2H), 3.32 (t, J = 2.4 Hz, 4H), 2.31 (s, 3H), 2.17 (s, 2H), 1.98 (s, 2H), 1.82 (s, 3H), 1.66 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 174.05, 172.92, 171.37, 163.60, 153.57, 144.18, 134.46, 122.92, 122.07, 116.84, 106.44, 70.09, 61.04, 40.31, 40.04, 35.22, 35.00, 23.18, 22.64, 16.13, 11.58. ESI-HRMS (m/z) calcd for C21H29N2O6+ [M+H]+: 405.20201, found: 405.20178.
4.1.15 (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methyl-N-(2-(piperidin-1-yl)ethyl)hex-4-enamide (E1)
White powder; yield 65%; m.p. 122-124℃. 1H NMR (300 MHz, CDCl3) δ 6.49 (s, 1H), 5.27 (t, J = 6.5 Hz, 1H), 5.18 (s, 2H), 3.78 (s, 3H), 3.46 – 3.29 (m, 4H), 2.71 – 2.41 (m, 6H), 2.32 (s, 4H), 2.15 (s, 3H), 1.83 (s, 3H), 1.64 (dt, J = 10.9, 5.6 Hz, 4H), 1.48 (d, J = 5.2 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 173.01, 172.83, 163.45, 156.03, 144.27, 134.21, 123.96, 122.60, 114.97, 106.41, 69.64, 60.99, 57.20, 54.75(2C), 36.19, 35.24, 34.98, 25.45(2C), 23.94, 22.96, 15.97, 11.48. ESI-HRMS (m/z) calcd for C24H35N2O5+ [M+H]+: 431.25045, found: 431.25381.
4.1.16 (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methyl-N-(2-(pyrrolidin-1-yl)ethyl)hex-4-enamide (E2)
White powder; yield 59%; m.p. 130-132℃. 1H NMR (300 MHz, CDCl3) δ 6.69 (brs, 1H), 5.27 (t, J = 6.9 Hz, 1H), 5.18 (s, 2H), 3.80 (s, 3H), 3.41 (dd, J = 11.7 Hz, 5.4 Hz, 4H), 2.91 – 2.71 (m, 6H), 2.34 (s, 4H), 2.15 (s, 3H), 1.93-1.85 (m, 4H), 1.83 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 173.07(2C), 163.40, 156.58, 144.34, 134.18, 124.39, 122.74, 114.62, 106.47, 69.55, 60.99, 54.98, 54.48(2C), 37.76, 35.22, 34.88, 23.37(2C), 23.00, 15.99, 11.46. ESI-HRMS (m/z) calcd for C23H33N2O5+ [M+H]+: 417.23840, found: 417.23795.
4.1.17
(E)-N-(2-(1H-indol-3-yl)ethyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide (E3)
White powder; yield 51%; m.p. 116-118℃. 1H NMR (300 MHz, CDCl3) δ 8.39 (s, 1H), 7.69 (s, 1H), 7.54 (d, J = 7.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.25 – 7.16 (m, 1H), 7.15 – 7.05 (m, 1H), 6.98 (d, J = 2.1 Hz, 1H), 5.67 (t, J = 5.4 Hz, 1H), 5.25 (t, J = 6.9 Hz, 1H), 5.00 (s, 2H), 3.77 (d, J = 6.3 Hz, 3H), 3.52 (dd, J = 12.9, 6.8 Hz, 2H), 3.38 (d, J = 6.9 Hz, 2H), 2.82 (t, J = 6.9 Hz, 2H), 2.36 – 2.20 (m, 4H), 2.09 (s, 3H), 1.80 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 173.00, 172.87, 163.59, 153.46, 144.21, 136.41, 134.51, 127.27, 122.83, 122.16, 122.00, 121.90, 119.19, 118.54, 116.83, 112.62, 111.36, 106.30, 70.04, 61.03, 39.78, 35.28, 35.04, 25.27, 22.62, 16.12, 11.50. ESI-HRMS (m/z) calcd for C27H31N2O5+ [M+H]+: 463.22275, found: 463.22253.
4.1.18 (E)-6-(6-(4-benzylpiperazin-1-yl)-3-methyl-6-oxohex-2-en-1-yl)-7-hydroxy-5-methoxy-4-methylisobenzofuran-1(3H)-one (E4)
White powder; yield 73%; m.p. 121-123℃. 1H NMR (300 MHz, CDCl3) δ 7.36 – 7.29 (m, 4H), 7.25 (dd, J = 8.8, 3.8 Hz, 1H), 5.27 – 5.22 (m, 1H), 5.20 (s, 2H), 3.77 (s, 3H), 3.59 (t, J = 4.8 Hz, 2H), 3.51 (s, 2H), 3.44 (t, J = 4.8 Hz, 2H), 3.39 (d, J = 6.8 Hz, 2H), 2.50 – 2.35 (m, 6H), 2.32-2.25 (m, 2H), 2.15 (s, 3H), 1.82 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 172.88, 171.18, 163.65, 153.57, 144.07, 137.47, 134.77, 129.15(2C), 128.31(2C), 127.28, 122.41, 122.19, 116.75, 106.36, 70.02, 62.82, 61.02, 53.11, 52.73, 45.54, 41.48, 35.02, 31.94, 22.63, 16.39, 11.57. ESI-HRMS (m/z) calcd for C28H35N2O5+ [M+H]+: 479.25405, found: 479.25369.
4.1.19 (E)-N-(3-(1H-imidazol-1-yl)propyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enamide(E5)
White powder; yield 63%; m.p. 116-118℃. 1H NMR (300 MHz, CDCl3) δ 7.47 (s, 1H), 6.99 (d, J = 38.6 Hz, 2H), 5.69 (s, 1H), 5.28 (t, J = 11.6 Hz, 1H), 5.21 (s, 2H), 3.95 (t, J = 6.9 Hz, 2H), 3.77 (s, 3H), 3.44 (t, J = 16.4 Hz, 2H), 3.22 (dd, J = 12.8, 6.4 Hz, 2H), 2.30 (s, 4H), 2.15 (s, 3H), 2.02 – 1.89 (m, 2H), 1.83 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 173.15, 172.89, 163.61, 153.60, 144.17, 134.57(2C), 132.34, 122.90(2C), 122.12, 116.84, 106.44, 70.05, 61.04, 44.61, 36.58, 35.16, 34.91, 31.24, 22.67, 16.18, 11.57. ESI-HRMS (m/z) calcd for C23H30N3O5+ [M+H]+: 428.21800, found: 428.21771.
4.2 Materials needed for cell and animal experiments
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). HFF-1 cell (Human Foreskin Fibroblasts-1) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Toxoplasma gondii is a highly virulent Toxoplasma gondii RH strain, which was donated by the Zoonoses Research Center, Won Kwang University School of Medicine, South Korea. The experimental animals were provided by the Animal Experiment Center of Yanbian University (license number SCXK 2011-0007), and are raised in the animal laboratory of the School of Pharmacy, Yanbian University, the experimental conditions meet the national laboratory animal requirements.
4.3 In vitro anti-T. gondii experiment
The cytotoxicity and anti-T. gondii activity of the compounds were tested by the classic MTT method. HFF-1 cells what in the logarithmic growth phase was collected, and inoculated in a 96-well plate (1×104 cells per well), after 24 h of incubation, adding Toxoplasma gondii tachyzoites at a ratio of cells per well: Toxoplasma gondii = 1:5, continue to cultivate for 24 h, and then added different concentrations of test drugs (10-1000 μM), in addition, spiramycin and DMSO were used as a positive control and negative control respectively, the final concentration of DMSO solvent did not exceed 0.1%. After 24 h of incubation, 15 μL of MTT solution (0.5 mg/mL) was added to each well, the OD value (absorbance) was read on a microplate reader at a wavelength of 492 nm. The cytotoxicity of the tested drugs was determined under the same experimental conditions, except that the cells were not infected with Toxoplasma gondii, and the other operating methods were the same. After determining the OD value, the IC50 of each group of cells and the selectivity index (SI) were calculated.
4.4 In vivo anti-T. gondii experiment
Thirty female KM mice were used for in vivo experiments. Six mice were randomly selected as the normal group without any treatment. The remaining 24 mice were used to establish an acute Toxoplasma infection animal model (Toxoplasma gondii was injected intraperitoneally in mice (2×103/mouse)), which was then randomly divided into four groups (infected but not treatment group, Spiramycin group, MPA group, E5 group), each group has six animals. Four hours after infection, each test compound was given to mice by gavage at 100 mg/kg once a day for four consecutive days. The untreated group was given the same dose of normal saline. On the 5th day, the mice's eye blood was collected and sacrificed by cervical dislocation. Their abdominal cavity was rinsed with sterile physiological saline to collect the parasites/tachyzoites. Those were counted under an optical microscope (BDS200 inverted microscope) to calculate the inhibitory rate of the drug on Toxoplasma gondii. At the same time, the liver and spleen were dissected, and the liver and spleen index, serum alanine aminotransferase (ALT), aspartate aminotransferase AST, liver homogenate glutathione GSH, and malondialdehyde MDA were determined. Moreover record the weight change of mice during the administration period.
4.4.1 The liver and spleen index assay
After the mice of each group were sacrificed by neck dissection, they were dissected, the liver and kidney of the mice were separated, and the weight of the liver and spleen of each mouse was weighed and recorded, and then the liver and spleen index was obtained. The dissected liver and spleen were stored at low temperature, and the unused internal organs were temporarily stored in a refrigerator at -80 ℃.
Liver (spleen) index (%) = [wet weight of liver/spleen (g) / body weight of mice (g) ] × 100%
4.4.2 Determination of ALT and AST in serum
The determination of AST and ALT in serum is based on Lai's method. An appropriate amount of serum was mixed with 5 times the amount of ALT or AST matrix buffer, then it was placed at 37℃ for 30 min, and 1 mmol/L 2,4-dinitrophenylhydrazine solution equal in volume was added to the matrix buffer and mixed well, after 20 min, 0.4 mol/L sodium hydroxide solution was added and placed at room temperature for 5 min. Adjusting the zero with distilled water at a wavelength of 505 nm, the absorbance of each sample was read and calculated, and obtained the concentration of ALT and AST in the serum (U/L) through the standard curve[52].
ALT matrix buffer: 1.79 g of DL-alanine and 29.2 mg of α-ketoglutarate were accurately weighed, and dissolved using an appropriate amount of 0.1 mol/L phosphate buffer. After that, 1 mol/L of sodium hydroxide solution was added to adjust the pH to 7.4. Finally, additional phosphate buffer was added to adjust the volume to 100 ml to obtain the ALT matrix buffer (stored at 4℃).
AST matrix buffer: 24.2 mg α-ketoglutarate and 2.66g DL-aspartic acid were accurately weighed, and dissolved using appropriate amount of 0.1 mo/L phosphate buffer. After that, 1 mol/L of sodium hydroxide solution was added to adjust the pH to 7.4. Finally, Finally, additional phosphate buffer was added to adjust the volume to 100 ml to obtain the AST matrix buffer (stored at 4℃).
4.4.3 Determination of GSH and MDA in liver homogenate
Preparation of liver homogenate: A certain amount of liver tissue was washed using pre-cooled physiological saline, and then an electric homogeniser was used to prepare a 20% tissue homogenate in 0.85% normal saline after drying. Followed by centrifugation (4000 rpm, 10 min), the supernatant was taken as a liquid reserve. The whole process should be carried out at low temperature as much as possible.
Protein content determination: The protein content of each group of mouse liver homogenate was tested according to the BCA protein concentration determination kit (Shanghai Biyuntian Biotechnology Co., Ltd.).
GSH determination: An appropriate amount of liver homogenate supernatant was mixed according to the supernatant: 20% trichloroacetic acid = 2:1, followed by centrifugation (4000 rpm, 10 min). Subsequently, the prepared 0.3 mol/L disodium hydrogen phosphate buffer and 0.04% DTNB reagent were added in sequence and mixed thoroughly, and then the absorbance value was measured at 412 nm. In the experiment, the GSH standard curve was drawn with glutathione standard substance and the regression equation was obtained. Substituting the measured absorbance value of the experimental sample into the regression equation, the concentration of GSH in each sample solution can be calculated. Finally, the GSH content in the liver homogenate (mg/gprot) = the measured GSH concentration in the sample solution (mg/ml) / the liver homogenate protein concentration (gprot/ml)[53].
MDA determination: MDA is determined by the thiobarbituric acid method. A certain amount of liver homogenate supernatant was mixed with 0.5% thiobarbituric acid, and then it was boiled in a water bath for 1 h, centrifuged after cooling (6000 rpm, 10 min). Moreover the upper pink clear liquid was measured absorbance value at 532 nm. In addition, 1,1,2,2-tetraethoxypropane was used as a standard sample to draw a standard curve, and the absorbance value of the experimental sample was substituted into the standard curve to obtain the MDA concentration of each sample solution. Finally, the MDA content in the liver homogenate (μmol/gprot) = the measured MDA concentration in the sample solution (μmol/ml) / the liver homogenate protein concentration (gprot/ml)[54].