Chromatographic purification of compounds was carried out using column chromatography on Acros silica gel (60-200 mesh). Reaction progress and purity of compounds were monitored by TLC on Sorbfil PTLC-AF-A-UF plates. Melting points of the products were determined using a Stanford Research Systems MPA-100 OptiMelt appliance. 1H, 13C, HSQC, COSY NMR spectra were recorded on a Bruker Avance 400 spectrometer (400.17 and 100.62 MHz) of Spectral-Analytical Center of FRC Kazan Scientific Center of the RAS. Signals of chloroform-d (δH 7.24, δC 77.23), dimethyl sulfoxide-d6 (δH 2.50, δC 39.51) were used as references in the 1H and 13C NMR spectra. Coupling constants (J) are reported in Hz (splitting abbreviations: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad; and combinations thereof). HPLC/MS-experiment was carried out using a TripleTOF 5600, AB Sciex superhigh resolution mass spectrometer (Germany) from the solution in methanol by turboionic spray (TIS) ionization method with the collision energy with nitrogen molecules of 10 eV. For analyzing optically active substances, “ADP 440” (B + S) polarimeter was used.
Synthesis
(6-(Hydroxymethyl)-3,3,8-trimethyl-1,5-dihydro-[1, 3]dioxepino[5,6-c]pyridin-9-yl)-β-D-glucopyranoside (3): NaBH4 (0.05 g, 1.25 mmol) was added portion wise to a solution of compound 2 (0.50 g, 1.25 mmol) in 30 mL of methanol at 0°C. After stirring at room temperature for 20 min, the solution was neutralized with an aqueous solution of hydrochloric acid to pH = 7. Then the solvent was removed in vacuo, and the residue was purified by column chromatography (eluent ethanol – methylene chloride 1:3, v/v). It was obtained as a white amorphous solid, yield 89% (0.45 g); [α]25D + 10.5 (c 1.0, methanol); 1H NMR (DMSO-d6, 400 MHz) δ 1.41 (6H, s, С(CH3)2), 2.45 (3H, s, CH3), 3.05-3.16 (2H, m, 2CHO), 3.20-3.30 (2H, m, 2CHO), 3.42 (1H, dt, J = 11.5, 5.8 Hz, CH2OH), 3.64 (1H, ddd, J = 11.5, 4.6, 1.8 Hz, CH2OH), 4.39 (1H, dd, J = 5.8, 4.6 Hz, CH2OН), 4.41 (1Н, d, J = 7.2 Hz, ОCHO), 4.46 (2Н, d, J = 5.5 Hz, CH2OH), 4.84 (1H, d, J = 16.0 Hz, CH2О), 4.86 (1H, d, J = 16.8 Hz, CH2О), 4.89 (1H, d, J = 16.0 Hz, CH2О), 4.99 (1Н, d, J = 4.9 Hz, CHOН), 5.09 (1Н, d, J = 4.6 Hz, CHOН), 5.10 (1Н, t, J = 5.5 Hz, CH2OН), 5.11 (1H, d, J = 16.8 Hz, CH2О), 5.56 (1H, d, J = 4.8 Hz, CHOН); 13С NMR (DMSO-d6, 101 MHz) δ 19.3 (CH3), 23.6 (С(СН3)2), 23.7 (С(СН3)2), 59.2 (CH2O), 59.8 (CH2O), 61.2 (CH2O), 63.5 (CH2O), 70.0 (CHO), 73.8 (CHO), 76.2 (CHO), 76.8 (CHO), 101.9 (С(СН3)2), 105.0 (OCHO), 131.4 (CPyr), 142.1 (CPyr), 147.4 (CPyr), 148.6 (CPyr), 151.3 (CPyr); HRMS (ESI+) m/z calcd for C18H28NO9 402.1759, found 402.1764 (М+H+).
(4,5,6-Tris(hydroxymethyl)-2-methylpyridin-3-yl)-β-D-glucopyranoside (4): Concentrated HCl (0.80 mL) was added to a solution of compound 3 (0.40 g; 1.00 mmol) in water (30 mL). After stirring at room temperature for 3 hours, the solution was neutralized with NaHCO3. Then the solvents were evaporated under reduced pressure at 45 oC. The product was recrystallized from water. It was obtained as a white solid, yield 95% (0.34 g); mp 204°C; [α]25D - 8.0 (c 0.5, H2O); 1H NMR (DMSO-d6, 400 MHz) δ 2.52 (3H, s, CH3), 3.05-3.12 (2H, m, 2CHO), 3.21-3.27 (1H, m, CHO), 3.29-3.35 (1H, m, CHO), 3.39 (1H, m, CH2OH), 3.66 (1H, m, CH2OH), 4.51 (1Н, d, J = 7.7 Hz, ОCHO), 4.56-4.80 (7H, m, 2CH2OН+CH2OН), 4.93 (1Н, dd, J = 7.8, 4.5 Hz, CH2OH), 4.98 (1Н, t, J = 5.6 Hz, CH2OН), 5.03 (1Н, d, J = 5.0 Hz, CHOН), 5.07 (1Н, t, J = 5.4 Hz, CH2OН), 5.13 (1Н, d, J = 4.9 Hz, CHOН), 5.64 (1H, d, J = 4.9 Hz, CHOН); 13С NMR (DMSO-d6, 101 MHz) δ 19.7 (CH3), 54.4 (CH2O), 56.0 (CH2O), 61.4 (CH2O), 62.8 (CH2O), 70.4 (CHO), 73.9 (CHO), 76.3 (CHO), 76.6 (CHO), 104.5 (OCHO), 132.7 (CPyr), 142.4 (CPyr), 148.2 (CPyr), 150.5 (CPyr), 154.3 (CPyr); HRMS (ESI+) m/z calcd for C15H24NO9 362.1446, found 362.1451 (М+H+).
(7-Hydroxy-4-(hydroxymethyl)-2-methyl-5,7-dihydrofuro[3,4-b]pyridin-3-yl)-β-D-glucopyranoside (a mixture of two diastereomers) (5): сoncentrated HCl (0.5 mL) was added to a solution of compound 2 (0.40 g; 1.00 mmol) in water (20 mL). After stirring at room temperature for 3 hours, the solution was neutralized with NaHCO3. Then the solvents were evaporated under reduced pressure at 45 oC. The dry residue was dissolved in ethanol and the precipitate was filtered off. The filtrate was evaporated under reduced pressure. The dry residue was dissolved in water and purified using preparative reverse phase liquid chromatography (gradient elution with water–methanol). It was obtained as a white amorphous solid; yield 97% (0.35 g); 1H NMR (DMSO-d6, 400 MHz) δ 2.51 (3H, s, CH3), 2.52 (3H, s, CH3), 3.03-3.12 (4H, m, 4CHO), 3.19-3.30 (4H, m, 4CHO), 3.36-3.42 (2H, m, 2CH2OH), 3.62-3.66 (2H, m, 2CH2OH), 4.45 (1H, d, J = 7.4 Hz, OCHO), 4.47 (1H, d, J = 7.4 Hz, OCHO), 4.53-4.83 (6H, m, 2CH2OH + 2CH2OH), 4.96-5.15 (10H, m, 2CH2O + 4CHOH + 2CH2OH), 5.61 (2H, d, J = 4.5 Hz, 2CHOH), 6.02 (1H, s, ОСНОН), 6.03 (1H, s, ОСНОН), 6.76 (2H, br s, 2ОСНОН); 13С NMR (DMSO-d6, 101 MHz) δ 19.79 (CH3), 19.86 (CH3), 56.92 (CH2OH), 61.21 (CH2OH), 61.26 (CH2OH), 68.55 (CH2O), 68.62 (CH2O), 70.10 (CHO), 70.15 (CHO), 73.83 (CHO), 76.29 (CHO), 76.74 (CHO), 76.79 (CHO), 97.92 (OCHOH), 97.96 (OCHOH), 104.57 (OCHO), 104.69 (OCHO), 130.33 (CPyr), 139.12 (CPyr), 147.79 (CPyr), 152.52 (CPyr), 152.59 (CPyr), 154.43 (CPyr); HRMS (ESI+) m/z calcd for C15H22NO9 360.1289, found 360.1295 (М+H+).
(3,3,8-Trimethyl-1,5-dihydro-[1, 3]dioxepino[5,6-c]pyridin-9-yl)-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (7): phase transfer catalyst tetrabutylammonium bromide (TBAB) (0.92 g, 2.87 mmol), α-D-acetobromoglucose (2.90 g, 7.06 mmol) and a solution of K2CO3 (0.97 g, 7.06 mmol) in 15 mL of water were added sequentially to a solution of compound 6 (1.20 g, 5.74 mmol) in 15 mL of CHCl3. The reaction mixture was stirred for 3 hours at 45 oС. Then the organic part was separated and dried in vacuo. The dry residue was dissolved in methylene chloride and purified using column chromatography (eluent ethyl acetate). It was obtained as a white solid; yield 56% (1.74 g); mp 88-89°C; [α]25D + 16.0 (c 2.0, methanol); 1H NMR (CDCl3, 400 MHz) δ 1.45 (3H, s, С(CH3)2), 1.48 (3H, s, С(CH3)2), 2.03 (3H, s, CH3С(О)O), 2.04 (6H, s, 2CH3С(О)O), 2.14 (3H, s, CH3С(О)O), 2.47 (3H, s, CH3), 3.58 (1H, ddd, J = 12.3, 5.5, 2.3 Hz, CHCH2O), 4.08 (1H, dd, J = 12.3, 2.3 Hz, CHCH2O), 4.19 (1H, dd, J = 12.3, 5.5 Hz, CHCH2O), 4.68 (1H, d, J = 15.1 Hz, CH2О), 4.72 (1Н, d, J = 7.8 Hz, OCHO), 4.89 (1H, d, J = 16.8 Hz, CH2О), 4.92 (1H, d, J = 15.1 Hz, CH2О), 4.98 (1H, d, J = 16.8 Hz, CH2О), 5.13 (1H, t, J = 9.5 Hz, CHO), 5.25 (1H, t, J = 9.5 Hz, CHO), 5.31 (1H, dd, J = 9.5, 7.8 Hz, CHO), 8.05 (1Н, s, CHPyr); 13С NMR (CDCl3, 101 MHz) δ 19.3 (CH3), 20.7 (CH3С(О)O), 20.9 (CH3С(О)O), 23.5 (С(СН3)2), 23.9 (С(СН3)2), 59.8 (CH2O), 61.8 (CH2O), 61.9 (CH2O), 68.3 (CHO), 71.6 (CHO), 72.0 (CHO), 72.8 (CHO), 101.72 (С(СН3)2), 102.75 (OCHO), 133.7 (CPyr), 142.6 (CPyr), 143.3 (CPyr), 147.3 (CPyr), 150.6 (CPyr), 169.3 (CH3С(О)O), 169.6 (CH3С(О)O), 170.4 (CH3С(О)O), 170.7 (CH3С(О)O); HRMS (ESI+) m/z calcd for C25H34NO12 540.2076, found 540.2081 (М+H+).
(3,3,8-Trimethyl-1,5-dihydro-[1, 3]dioxepino[5,6-c]pyridin-9-yl)-β-D-glucopyranoside (8): a solution of K2CO3 (0.70 g, 5.04 mmol) in 1.5 mL of water was added to a solution of compound 7 (1.36 g, 2.52 mmol) in 15 mL of methanol. The reaction mixture was stirred at room temperature for 1.5 h. Then the solvents were evaporated under reduced pressure at 45 oC, and the product was purified by column chromatography (eluent ethanol – methylene chloride 1:4, v/v). It was obtained as a white amorphous solid; yield 92% (0.86 g); [α]25D + 13.9 (c 3.9, methanol); 1H NMR (DMSO-d6, 400 MHz) δ 1.41 (6H, s, С(CH3)2), 2.46 (3H, s, CH3), 3.06-3.15 (2H, m, 2CHO), 3.20-3.29 (2H, m, 2CHO), 3.41 (1H, br m, CH2OH), 3.65 (1H, ddd, J = 11.9, 4.7, 1.6 Hz, CH2OH), 4.40 (1H, dd, J = 5.8, 4.7 Hz, CH2OН), 4.44 (1Н, d, J = 7.3 Hz, ОCHO), 4.70 (1H, d, J = 15.2 Hz, CH2О), 4.81 (1H, d, J = 15.2 Hz, CH2О), 4.88 (1H, d, J = 17.0 Hz, CH2О), 4.98 (1Н, d, J = 4.8 Hz, CHOН), 5.07 (1Н, d, J = 4.7 Hz, CHOН), 5.12 (1H, d, J = 17.0 Hz, CH2О), 5.54 (1H, d, J = 4.7 Hz, CHOН), 8.00 (1Н, s, CHPyr); 13С NMR (DMSO-d6, 101 MHz) δ 19.4 (CH3), 23.4 (С(СН3)2), 23.6 (С(СН3)2), 59.3 (CH2O), 60.8 (CH2O), 61.2 (CH2O), 69.9 (CHO), 73.8 (CHO), 76.2 (CHO), 76.7 (CHO), 102.0 (С(СН3)2), 104.7 (OCHO), 133.0 (CPyr), 141.0 (CPyr), 142.1 (CPyr), 147.7 (CPyr), 150.9 (CPyr); HRMS (ESI+) m/z calcd for C17H26NO8 372.1653, found 372.1658 (М+H+).
(3,3,8-Trimethyl-1,5-dihydro-[1, 3]dioxepino[5,6-c]pyridin-9-yl)-6-O-(4-methylbenzoyl)-β-D-glucopyranoside (9): Me2SnCl2 (0.029 g, 0.13 mmol) was added to a solution of compound 8 (0.50 g, 1.35 mmol) in 33 mL of THF and 3 mL of water. The reaction mixture was stirred for 10 min at room temperature, then DIPEA (0.47 mL, 2.70 mmol) and p-toluoyl chloride (0.18 mL, 1.35 mmol) were sequentially added at 0 оC. After stirring at 0 оC for 1 h and then at room temperature for 1 h, the reaction was quenched with methanol, and then the resulting mixture was evaporated under reduced pressure. The product was purified by column chromatography (eluent methanol – methylene chloride 1:12, v/v). It was obtained as a white amorphous solid; yield 53% (0.35 g); [α]25D - 23.9 (c 1.0, methanol); 1H NMR (DMSO-d6, 400 MHz) δ 1.23 (3H, s, С(CH3)2), 1.35 (3H, s, С(CH3)2), 2.38 (3Н, s, СН3), 2.42 (3Н, s, СН3), 3.28-3.32 (3H, m, 3СНО), 3.45 (1H, m, CHO), 4.17 (1H, dd, J = 11.8, 6.3 Hz, СНCH2O), 4.57 (1Н, dd, J = 11.8, 1.5 Hz, СНCH2O), 4.59 (1Н, d, J = 7.0 Hz, ОCHO), 4.70 (1H, d, J = 15.3 Hz, CH2О), 4.79 (1H, d, J = 15.3 Hz, CH2О), 4.88 (1H, d, J = 16.8 Hz, CH2О), 5.01 (1H, d, J = 16.8 Hz, CH2О), 5.24 (1H, d, J = 3.3 Hz, CHOH), 5.37 (1H, d, J = 4.6 Hz, CHOH), 5.66 (1H, d, J = 4.4 Hz, CHOH), 7.29 (2H, d, J = 8.1 Hz, 2CНAr), 7.75 (2H, d, J = 8.1 Hz, 2CНAr), 8.02 (1Н, s, CHPyr); 13С NMR (DMSO-d6, 101 MHz) δ 19.4 (CH3), 21.2 (CH3), 23.3 (С(CH3)2), 23.5 (С(CH3)2), 59.0 (CH2O), 60.9 (CH2O), 63.8 (CH2O), 69.9 (CHO), 73.6 (CHO), 73.9 (CHO), 75.9 (CHO), 102.0 (С(CH3)2), 104.4 (OCHO), 126.9, 129.2, 133.1, 141.0, 142.3, 143.6, 147.4, 150.9 (5CPyr + 6CAr), 165.6 (C(O)O); HRMS (ESI+) m/z calcd for C25H32NO9 490.2072, found 490.2077 (М+H+).
(2-Methylpyridin-3-yl)-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (11): phase transfer catalyst tetrabutylammonium bromide (TBAB) (1.57 g, 4.86 mmol), α-D-acetobromoglucose (6.00 g, 14.6 mmol) and a solution of K2CO3 (2.01 g, 14.6 mmol) in 30 mL of water were added sequentially to a solution of compound 10 (1.06 g, 9.72 mmol) in 30 mL of CHCl3. The reaction mixture was stirred for 3 hours at 45 oС. Then the organic part was separated and dried in vacuo. The dry residue was dissolved in methylene chloride and purified using column chromatography (eluent ethyl acetate). It was obtained as a white solid; yield 74% (3.16 g); mp 129-130°C; [α]25D - 35.3 (c 1.0, methanol); 1H NMR (CDCl3, 400 MHz) δ 2.03 (3H, s, CH3С(О)O), 2.04 (3H, s, CH3С(О)O), 2.06 (3H, s, CH3С(О)O), 2.07 (3H, s, CH3С(О)O), 2.41 (3H, s, CH3), 3.85 (1H, ddd, J = 12.3, 5.3, 2.5 Hz, CHCH2O), 4.17 (1H, dd, J = 12.3, 2.5 Hz, CHCH2O), 4.27 (1H, dd, J = 12.3, 5.3 Hz, CHCH2O), 4.99 (1Н, d, J = 7.6 Hz, OCHO), 5.17 (1H, m, CHO), 5.27-5.36 (2H, m, 2CHO), 7.08 (1Н, dd, J = 8.2, 4.8 Hz, CHPyr), 7.27 (1Н, dd, J = 8.2, 1.5 Hz, CHPyr), 8.21 (1Н, dd, J = 4.8, 1.5 Hz, CHPyr); 13С NMR (CDCl3, 101 MHz) δ 19.3 (CH3), 20.7 (3CH3С(О)O), 20.8 (CH3С(О)O), 62.0 (CH2O), 68.3 (CHO), 71.1 (CHO), 72.3 (CHO), 72.6 (CHO), 99.5 (OCHO), 121.9 (CPyr), 122.5 (CPyr), 143.8 (CPyr), 150.1 (CPyr), 151.3 (CPyr), 169.2 (CH3С(О)O), 169.5 (CH3С(О)O), 170.3 (CH3С(О)O), 170.6 (CH3С(О)O); HRMS (ESI+) m/z calcd for C20H26NO10 440.1551, found 440.1559 (М+H+).
(2-Methylpyridin-3-yl)-β-D-glucopyranoside (12): a solution of K2CO3 (0.48 g, 3.46 mmol) in 2.0 mL of water was added to a solution of compound 11 (0.76 g, 1.73 mmol) in 20 mL of methanol. The reaction mixture was stirred at room temperature for 1.5 h. The formed precipitate was filtered off, washed twice with water and dried in vacuo. It was obtained as a white solid; yield 91% (0.43 g); mp 248°C (decomp.); [α]25D - 53.0 (c 0.5, DMSO); 1H NMR (DMSO-d6, 400 MHz) δ 2.40 (3H, s, CH3), 3.14-3.20 (1H, m, CHO), 3.24-3.30 (2H, m, 2CHO), 3.31-3.36 (1H, m, CHO), 3.41 (1H, dt, J = 11.6, 5.7 Hz, CH2OH), 3.69 (1H, ddd, J = 11.6, 5.7, 1.8 Hz, CH2OH), 4.56 (1H, t, J = 5.7 Hz, CH2OН), 4.85 (1Н, d, J = 7.5 Hz, ОCHO), 5.04 (1Н, d, J = 5.2 Hz, CHOН), 5.10 (1Н, d, J = 4.3 Hz, CHOН), 5.36 (1H, d, J = 3.5 Hz, CHOН), 7.17 (1Н, dd, J = 8.2, 4.9 Hz, CHPyr), 7.42 (1Н, dd, J = 8.2, 1.2 Hz, CHPyr), 8.21 (1Н, dd, J = 4.9, 1.2 Hz, CHPyr); 13С NMR (DMSO-d6, 101 MHz) δ 19.2 (CH3), 60.7 (CH2O), 69.7 (CHO), 73.2 (CHO), 76.5 (CHO), 77.1 (CHO), 100.7 (OCHO), 121.3 (CPyr), 122.0 (CPyr), 141.6 (CPyr), 148.3 (CPyr), 151.6 (CPyr); HRMS (ESI+) m/z calcd for C12H18NO6 272.1129, found 272.1134 (М+H+).
(2-Methylpyridin-3-yl)-2,6-di-O-(4-methylbenzoyl)-β-D-glucopyranoside (14): Me2SnCl2 (0.024 g, 0.11 mmol) was added to a suspension of compound 12 (0.30 g, 1.11 mmol) in 20 mL of THF. The reaction was stirred for 10 min at room temperature, then DIPEA (0.38 mL, 2.21 mmol) and p-toluoyl chloride (0.15 mL, 1.11 mmol) were sequentially added at 0 оC. After stirring at 0 оC for 1 h and then at room temperature for 1 month, the reaction was quenched with methanol, and then the resulting mixture was evaporated under reduced pressure. The product was purified by column chromatography (eluent methanol – methylene chloride 1:12, v/v). It was obtained as a white amorphous solid; yield 82% (0.23 g); [α]25D - 8.3 (c 0.3, methanol); 1H NMR (DMSO-d6, 400 MHz) δ 2.06 (3Н, s, СН3), 2.37 (3Н, s, СН3), 2.41 (3Н, s, СН3), 3.49 (1H, td, J = 9.1, 5.7 Hz, CHO), 3.74 (1H, td, J = 9.1, 5.9 Hz, CHO), 3.97 (1H, ddd, J = 9.1, 7.2, 1.4 Hz, CHO), 4.35 (1H, dd, J = 11.9, 7.2 Hz, СНCH2O), 4.67 (1Н, dd, J = 11.9, 1.4 Hz, СНCH2O), 5.14 (1H, dd, J = 9.1, 8.1 Hz, CHO), 5.36 (1Н, d, J = 8.1 Hz, ОCHO), 5.66 (1H, d, J = 5.9 Hz, CHOH), 5.73 (1H, d, J = 5.7 Hz, CHOH), 7.34 (2H, d, J = 8.1 Hz, 2CНAr), 7.39 (2H, d, J = 8.1 Hz, 2CНAr), 7.89 (4H, d, J = 8.1 Hz, 4CНAr), 6.99 (1Н, dd, J = 8.3, 4.8 Hz, CHPyr), 7.43 (1Н, d, J = 8.3 Hz, CHPyr), 8.04 (1Н, d, J = 4.8 Hz, CHPyr); 13С NMR (DMSO-d6, 101 MHz) δ 18.6 (CH3), 21.16 (CH3), 21.19 (CH3), 63.8 (CH2O), 70.2 (CHO), 73.5 (CHO), 73.8 (CHO), 74.0 (CHO), 98.3 (OCHO), 121.5, 121.8, 126.9, 127.0, 129.2, 129.3, 129.4, 142.3, 143.7, 143.8, 147.9, 150.9 (5CPyr + 12CAr), 165.0 (C(O)O), 165.5 (C(O)O); HRMS (ESI+) m/z calcd for C28H30NO8 508.1966, found 508.1971 (М+H+).
4-(Allyloxy)-3-methoxybenzoic acid (19): K2CO3 (11.04 g, 80.0 mmol) and allyl bromide (5.3 mL, 60.0 mmol) were sequentially added to a solution of 4-hydroxy-3-methoxybenzoic acid (3.36 g, 20.0 mmol) in 100 mL DMF. The reaction mixture was stirred for 3 hours at 40 oС. Then the precipitate was filtered off, and the filtrate was evaporated under reduced pressure. The dry residue was dissolved in 100 ml of EtOH/H2O (3:1, w/w), and NaOH (1.60 g, 40.0 mmol) was added. The resulting reaction mixture was refluxed for 1 h. The solution was then evaporated to dryness, then the solid residue was dissolved in water (100 mL), and the resulting solution was acidified slowly with 50% H2SO4 (6 mL) under stirring. Then the crystalline precipitate was filtered off, washed with water and dried in vacuo. It was obtained as a white solid; yield 92% (3.82 g); mp 174°C; 1H NMR (DMSO-d6, 400 MHz) δ 3.81 (3H, s, CH3O), 4.62 (2H, d, J = 5.3 Hz, CH2=CH-CH2O), 5.27 (1H, d, J = 10.5 Hz, CH2=CH-CH2O), 5.40 (1H, dd, J = 17.2, 1.1 Hz, CH2=CH-CH2O), 6.04 (1H, ddd, J = 22.4, 10.5, 5.3 Hz, CH2=CH-CH2O), 7.04 (1H, d, J = 8.5 Hz, CHAr), 7.45 (1H, d, J = 1.6 Hz, CHAr), 7.54 (1H, dd, J = 8.5, 1.6 Hz, CHAr), 12.69 (1Н, s, C(O)OH); 13С NMR (DMSO-d6, 101 MHz) δ 55.5 (CH3O), 68.9 (CH2=CH-CH2O), 112.1, 112.3, 118.0, 123.0, 123.1, 133.3, 148.5, 151.5, (CH2=CH-CH2O + 6CAr), 167.1 (C(O)OH).
4-(Allyloxy)-3-methoxybenzoyl chloride (20): 4-(Allyloxy)-3-methoxybenzoic acid (3.00 g) was dissolved in 12 mL of SOCl2, and the resulting mixture was refluxed for 2 h. The excess of SOCl2 was removed under vacuum, and the obtained residue was used at the next step without further purification.
(4-Formyl-2-methoxyphenyl)-6-O-(4-(allyloxy)-3-methoxybenzoyl)-β-D-glucopyranoside (21): Me2SnCl2 (0.05 g, 0.22 mmol) was added to a solution of compound 17 (0.68 g, 2.17 mmol) in 30 mL of THF and 3 mL of water. The reaction was stirred for 10 min at room temperature, then DIPEA (0.75 mL, 4.33 mmol) and 4-(allyloxy)-3-methoxybenzoyl chloride 20 (0.49 g, 2.17 mmol) were sequentially added at 0 оC. After stirring at 0 оC for 1 h and then at room temperature for 4 h, the reaction was quenched with methanol, and then the resulting mixture was evaporated under reduced pressure. The product was purified by column chromatography (eluent methanol – methylene chloride 1:15, v/v). It was obtained as a white amorphous solid; yield 73% (0.80 g); [α]25D - 40.4 (c 0.5, methanol); 1H NMR (DMSO-d6, 400 MHz) δ 3.22 - 3.28 (1H, m, CHO), 3.30 - 3.38 (2H, m, 2CHO), 3.77 (3H, s, CH3O), 3.83 (3H, s, CH3O), 3.81-3.87 (1H, m, CHO), 4.20 (1H, dd, J = 11.8, 7.7 Hz, CHCH2O), 4.58 (1H, dd, J = 11.8, 1.3 Hz, CHCH2O), 4.67 (2H, d, J = 5.3 Hz, CH2=CH-CH2O), 5.20 (1H, d, J = 7.2 Hz, OCHO), 5.28-5.31 (2H, m, CH2=CH-CH2O+CHOH), 5.41-5.45 (2H, m, CH2=CH-CH2O+CHOH), 5.49 (1H, d, J = 4.2 Hz, CHOH), 6.07 (1H, ddd, J = 22.5, 10.6, 5.3 Hz, CH2=CH-CH2O), 7.08 (1H, d, J = 8.5 Hz, CHAr), 7.26 (2H, br s, 2CHAr), 7.41 (2H, m, 2CHAr), 7.54 (1H, dd, J = 8.4, 1.8 Hz, CHAr), 9.81 (1Н, s, C(O)H); 13С NMR (DMSO-d6, 101 MHz) δ 55.57 (CH3O), 55.60 (CH3O), 64.0 (CH2O), 68.9 (CH2=CH-CH2O), 70.1 (CHO), 72.9 (CHO), 73.9 (CHO), 76.6 (CHO), 98.9 (OCHO), 110.8, 112.2, 112.4, 114.5, 118.1, 122.0, 123.1, 124.6, 130.5, 133.2, 148.6, 149.2, 151.3, 151.9 (CH2=CH-CH2O + 12CAr), 165.2 (C(O)O), 191.4 (C(O)H); HRMS (ESI+) m/z calcd for C25H28NaO11 527.1524, found 527.1529 (М+Na+).
(4-(Hydroxymethyl)-2-methoxyphenyl)-6-O-(4-hydroxy-3-methoxybenzoyl)-β-D-glucopyranoside (SACCHARUMOSIDE-B): Pd(PPh3)4 (0.03 g, 0.025 mmol) was added to a solution of compound 21 (0.25 g, 0.50 mmol) in 12 mL of CH3OH. The reaction was stirred for 5 min at room temperature, then NaBH4 (0.06 g, 1.49 mmol) was added. After stirring at room temperature for 1 h, the solution was neutralized with concentrated hydrochloric acid, and the resulting mixture was evaporated under reduced pressure. The product was purified by column chromatography (eluent methanol – methylene chloride 1:9, v/v). It was obtained as a white amorphous solid; yield 91% (0.21 g); [α]25D - 38.0 (c 0.5, methanol); 1H NMR (DMSO-d6, 400 MHz) δ 3.20 - 3.35 (3H, m, 3CHO), 3.69-3.76 (1H, m, CHO), 3.73 (3H, s, CH3O), 3.78 (3H, s, CH3O), 4.14 (1H, dd, J = 11.8, 7.4 Hz, CHCH2O), 4.38 (2H, d, J = 5.7 Hz, CH2OH), 4.56 (1H, dd, J = 11.8, 1.2 Hz, CHCH2O), 4.94 (1H, d, J = 7.1 Hz, OCHO), 5.09 (1H, t, J = 5.7 Hz, CH2OH), 5.22 (1H, d, J = 4.1 Hz, CHOH), 5.32 (1H, d, J = 4.7 Hz, CHOH), 5.36 (1H, d, J = 5.3 Hz, CHOH), 6.62 (1H, dd, J = 8.3, 1.3 Hz, CHAr), 6.88 (1H, d, J = 8.3 Hz, CHAr), 6.92 (1H, d, J = 1.3 Hz, CHAr), 7.01 (1H, d, J = 8.3 Hz, CHAr), 7.41 (1H, d, J = 1.8 Hz, CHAr), 7.45 (1H, dd, J = 8.3, 1.8 Hz, CHAr), 10.00 (1Н, s, OH); 13С NMR (DMSO-d6, 101 MHz) δ 55.5 (CH3O), 55.7 (CH3O), 62.7 (CH2O), 63.9 (CH2O), 70.2 (CHO), 73.2 (CHO), 73.8 (CHO), 76.7 (CHO), 99.8 (OCHO), 111.0, 112.7, 114.9, 115.2, 118.3, 120.4, 123.5, 136.4, 145.0, 147.4, 148.7, 151.7 (12CAr), 165.4 (C(O)O); HRMS (ESI−) m/z calcd for C22H25NO11 465.1402, found 465.1397 (М-H+).
Cytotoxicity evaluation
The cytotoxicity was evaluated on MCF-7 (human breast cancer cells), MDA-MB-231 (human breast cancer cells), A-498 (human kidney cancer cell), SNB-19 (human glioblastoma cell), M-14 (melanoma cell), NCI-H322M (lung cancer cell), HCT-15 (colon cancer cell), HCT-116 (colon cancer cell), PC-3 (prostate cancer), HEK 293 (human embryonic kidney), Chang Liver (human liver), MSC (human mesenchymal stem cells) by using MTT assay. Cells were cultured in a-MEM (minimum essential medium Eagle) / DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS, 2 mM L-glutamine, 100 µg/mL penicillin and 100 µg/mL streptomycin. Cells were seeded in 96-well plates at the density of 10,000 cells per well and grown overnight at 37°C and 5% CO2 in humidified atmosphere. Then the medium was changed to the fresh one containing compounds to be tested in concentration of 0.1-100 µg/mL. After 72 h of cultivation, the cultural fluid was discarded, and MTT solution (in DPBS) was added to the fresh media until final concentration of 0.5 mg/mL. After 2 h the liquid was replaced with dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) to dissolve formazan crystals, and absorption was measured on Tecan Infinite 200Pro at 557 nm with reference 700 nm. Based on data obtained, the CC50 values (concentrations decreasing the metabolic activity by 2-fold) were calculated. Experiments were carried out in biological triplicates (i.e. newly prepared cultures and medium) with independent repeats in each one. The IC50 and CC50 were calculated by plotting the log doses vs inhibition (expressed as a percentage relative to the control) – variable slope in Prism 6 (GraphPad Software Inc.).