Synthesis and identification of flavone analogs
All reagents and solvents were obtained from Sigma–Aldrich (St. Louis, MO, USA), TCI chemicals (Tokyo, Japan) and Merck (Darmstadt, Germany). All solvents for column chromatography from RCI Labscan (Samutsakorn, Thailand) were distilled before use. Reactions were monitored by thin–layer chromatography (TLC) using aluminium Merck TLC plates coated with silica gel 60 F254. Normal phase column chromatography was performed using silica gel 60 (0.063–0.200mm, 70–230 mesh ASTM, Merck, Darmstadt, Germany). Proton and carbon nuclear magnetic resonance (1H and 13C NMR) spectra were recorded on a Jeol JNM–ECZ500/S1 (500MHz). Chemical shifts were expressed in parts per million (ppm), J values were in Hertz (Hz). High-resolution mass spectra (HRMS) data were obtained with Micro-TOF mass spectrometer. IR spectra were recorded using the Thermo Scientific™ Nicolet™ iS50 FTIR spectrometer with ATR module and are reported in wave number (cm−1). Melting points were measured using a melting point apparatus (Griffin) and are uncorrected.
General procedure A21
A mixture of flavone, alkyl halide, K2CO3 and dry DMF or dry acetone was stirred at 60°C overnight. After completion, the reaction was quenched with DI water and extracted with EtOAc (3 times). The combined organic layers were washed with brine, dried with anh. Na2SO4, filtered and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography.
General procedure B22
A mixture of flavone, propionyl chloride, K2CO3 and dry DMF was stirred at room temperature overnight. The reaction was quenched with DI water and extracted with EtOAc (3 times). The combined organic layers were washed with brine, dried with anh. Na2SO4, filtered and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography.
General procedure C23
A mixture of flavone, acid anhyhydride and pyridine was stirred at room temperature overnight. The reaction was quenched with DI water and extracted with CH2Cl2. The combined organic layers were washed with 10% NaOH, 1M HCl and brine, dried with anh. Na2SO4, filtered and concentrated in vacuo.
General procedure D27
BF3⋅Et2O was added into a mixture of cinnamoyl chloride and 3,4,5-trimethoxyphenol. After refluxing at 90°C for an hour, the reaction was quenched with DI water and extracted with EtOAc (3 times). The combined organic layers were dried with anh. Na2SO4, filtered and concentrated in vacuo.
General procedure E28
The mixture of 6-hydroxy-2,3,4-trimethoxychalcone and I2 was dissolved in DMSO. After refluxing at 90°C for 3h, the reaction was quenched with DI water and extracted with CH2Cl2 (3 times). The combined organic layers were dried with anh. Na2SO4, filtered and concentrated in vacuo.
General procedure F37
To a solution of 5,6,7-trimethoxyflavone in CH2Cl2 at 0 ºC was added BBr3. After stirring at room temperature for 6h, the reaction was quenched with 1M HCl and extracted with EtOAc (3 times). The combined organic layers were dried with anh. Na2SO4, filtered and concentrated in vacuo.
General Procedure H38
To a solution of 5,6,7-trimethoxyflavone in toluene was added AlCl3. After stirring at 100°C for 3h, the reaction was quenched with 1M HCl and left stirring for an hour. The mixture was extracted with EtOAc (3 times). The combined organic layers were dried with anh. Na2SO4, filtered and concentrated in vacuo.
6-Methoxyflavone (1b)
The title compound was synthesized following the General Procedure A using 6-hydroxyflavone (48mg, 0.20mmol), MeI (66µL, 1.0mmol), K2CO3 (140mg, 1.0mmol) and dry DMF (0.50mL). The crude product was purified by silica gel column chromatography (eluent: EtOAc/hexanes = 1:2) to give to give a white powder of 1b (45mg, 89%). TLC (EtOAc:hexanes 2:1): Rf = 0.49; 1H-NMR (500MHz, acetone-d6) δ 8.06 (dd, J = 7.1, 2.9, 2H), 7.67 (d, J = 9.6Hz, 1H), 7.55–7.59 (m, 3H), 7.49 (d, J = 3.1Hz, 1H), 7.36 (dd, J = 8.9, 2.9Hz, 1H), 6.81 (s, 1H), 3.90 (s, 3H); 13C-NMR (126MHz, acetone-d6) δ 176.9, 162.8, 157.2, 151.0, 132.0, 131.5, 129.1, 126.3, 124.7, 123.1, 119.9, 106.3, 105.0, 55.4. 1H and 13C data are consistent with literature values39.
6-Propionoxyflavone (1c)
The title compound was synthesized following the General Procedure B using 6-hydroxyflavone (48mg, 0.20mmol), propionyl chloride (42µL, 0.48mmol), K2CO3 (56mg, 0.40mmol) and dry DMF (0.50mL). The crude product was purified by silica gel column chromatography (eluent: EtOAc/hexanes = 1:1) to give a pale-yellow powder of 1c (51mg, 78%). TLC (EtOAc:hexanes 2:1): Rf = 0.27; 1H-NMR (500MHz, DMSO-d6) δ 8.12 (dd, J = 5.7, 3.1Hz, 2H), 7.88 (d, J = 9.9Hz, 1H), 7.76 (d, J = 2.9 H, 1H), 7.64 (d, J = 2.3Hz, 1H), 7.62–7.58 (m, 3H), 7.07 (s, 1H), 2.66 (q, J = 7.7Hz, 2H), 1.16 (t, J = 7.2Hz, 3H); 13C-NMR (126MHz, DMSO-d6) δ 177.2, 173.2, 163.4, 153.7, 148.1, 132.5, 131.6, 129.7, 129.1, 127.0, 124.5, 120.7, 117.4, 107.0, 27.4, 9.3. 1H and 13C data are consistent with literature values40.
7-Methoxyflavone (2b)
The title compound was synthesized following the General Procedure A using 7-hydroxyflavone (97mg, 0.40mmol), MeI (50µL, 0.80mmol), K2CO3 (111mg, 0.80mmol) and dry DMF (0.50mL). The crude product was purified by silica gel column chromatography (eluent: EtOAc/hexanes = 2:1) to give a white powder of 2b (102mg, 99%). TLC (EtOAc:hexanes 2:1): Rf = 0.52; 1H-NMR (500MHz, CDCl3) δ 8.14 (d, J = 8.7Hz, 1H), 7.94–7.88 (m, 2H), 7.57–7.48 (m, 3H), 7.07–6.94 (m, 2H), 6.77 (s, 1H), 3.93 (s, 3H); 13C-NMR (126MHz, CDCl3) δ 176.8, 164.3, 163.1, 158.1, 132.0, 131.5, 129.1, 127.2, 126.3, 118.0, 114.5, 107.7, 100.5, 56.0. 1H and 13C data are consistent with literature values40.
7-Propionoxyflavone (2c)
The title compound was synthesized following the General Procedure B using 7-hydroxyflavone (96mg, 0.40mmol), propionyl chloride (84µL, 0.96mmol), K2CO3 (111mg, 0.80mmol) and dry DMF (0.50mL). The crude product was purified by silica gel column chromatography (eluent: EtOAc/hexanes = 1:2) to give a white powder of 2c (31mg, 22%). TLC (EtOAc:hexanes 2:1): Rf = 0.24; 1H-NMR (500MHz, acetone-d6) δ 8.10 (d, J = 8.6Hz, 1H), 8.08–8.02 (m, 2H), 7.62–7.50 (m, 4H), 7.23 (d, J = 8.6Hz, 1H), 6.82 (s, 1H), 2.66 (q, J = 7.5Hz, 2H), 1.20 (t, J = 7.4Hz, 3H). 13C-NMR (126MHz, acetone-d6) δ 176.4, 171.9, 163.3, 156.7, 155.2, 131.7, 131.7 129.1, 126.4, 126.4, 121.6, 119.8, 111.5, 107.2, 27.2, 8.3. 1H and 13C data are consistent with literature values22.
7,8-Dioxoloflavone (3b)
The title compound was synthesized following the General Procedure A using 7,8-dihydroxyflavone (51mg, 0.20mmol), dibromomethane (17µL, 0.24mmol), K2CO3 (138mg, 1.0mmol) and dry DMF (0.50mL) at 100°C. The crude product was purified by silica gel column chromatography (eluent: EtOAc/hexanes = 2:1) to give a pale-yellow powder of 3b (38mg, 71%). TLC (EtOAc:hexanes 2:1): Rf = 0.63; 1H-NMR (500MHz, CDCl3) δ 7.92 (dd, J = 7.9, 2.2Hz, 2H), 7.81 (d, J = 8.4Hz, 1H), 7.49–7.56 (m, 3H), 6.96 (d, J = 8.4Hz, 1H), 6.74 (s, 1H), 6.22 (s, 2H); 13C-NMR (126MHz, CDCl3) δ 177.5, 162.7, 152.5, 141.3, 134.9, 131.7, 131.6, 129.1, 126.3, 120.3, 120.1, 107.2, 107.1, 103.3. 1H data are consistent with literature values41.
5-Hydroxy-7-methoxyflavone (4b)
The title compound was synthesized following the General Procedure A using chrysin (51mg, 0.20mmol), MeI (63µL, 1.0mmol), K2CO3 (138mg, 1.0mmol) and anhydrous DMF (1.0mL). The crude product was purified by silica gel column chromatography (eluent: EtOAc/hexanes = 1:9) to give a pale-yellow powder of 4b (76mg, 67%). TLC (EtOAc:hexanes 1:4): Rf = 0.21; 1H-NMR (500MHz, CDCl3) δ 12.72 (br s, 1H), 7.86 (dd, J = 8.0Hz, 2H), 7.56–7.48 (m, 3H), 6.64 (s, 1H), 6.48 (d, J = 2.3Hz, 1H), 6.35 (d, J = 2.3Hz, 1H), 3.87 (s, 3H); 13C-NMR (126MHz, CDCl3) δ 182.5, 165.7, 164.0, 162.2, 157.8, 131.9, 131.3, 129.2, 126.4, 105.9, 105.8, 98.3, 92.7, 55.9. 1H and 13C data are consistent with literature values42.
7-Benzyloxy-5-hydroxyflavone (4c)
The title compound was synthesized following the General Procedure A using chrysin (254mg, 1.0mmol), BnBr (178µL, 1.5mmol), K2CO3 (276mg, 2.0mmol) and acetone (10mL). After the combined organic layers were concentrated in vacuo, a pale yellow powder of 4c (310mg, 90%) was obtained without further purification. TLC (EtOAc:hexanes 1:2): Rf = 0.60; 1H-NMR (500MHz, CDCl3) δ 12.64 (br s, 1H), 7.88 (d, J = 6.5Hz, 2H), 7.51–7.57 (m, 3H), 7.37–7.48 (m, 5H), 6.68 (s, 1H), 6.59 (d, J = 2.1Hz, 1H), 6.46 (d, J = 2.2Hz, 1H), 5.15 (s, 2H); 13C-NMR (126MHz, CDCl3) δ 182.6, 164.7, 164.1, 162.3, 157.8, 135.8, 131.9, 131.4, 129.2, 128.8, 128.5, 127.6, 126.4, 106.0, 99.0, 93.6, 70.5. 1H and 13C data are consistent with literature values43.
6,8-Dibromo-5,7-dihydroxyflavone (4d)
A mixture of chrysin (254mg, 1.0mmol), NBS (196mg, 1.1mmol), dry THF (4.0mL) and conc. H2SO4 (50µL) was stirred at 60°C for 2h. After completion, the reaction was quenched with DI water. The mixture was extracted with EtOAc (3 × 20mL). The combined organic layers were washed with brine, dried with anh. Na2SO4, filtered and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (eluent: EtOAc/hexanes = 1:2) to give a light brown powder of 4d (128mg, 31%). TLC (EtOAc:hexanes 1:2): Rf = 0.17; 1H-NMR (500MHz, DMSO-d6) δ 13.65 (br s, 1H), 10.97 (br s, 1H), 8.06 (d, J = 6.8Hz, 2H), 7.57–7.50 (m, 3H), 7.13 (s, 1H); 13C-NMR (126MHz, DMSO-d6) δ 182.2, 174.1, 164.1, 157.9, 157.6, 152.9, 133.1, 130.8, 129.9, 127.1, 105.7, 95.1, 89.0. 1H and 13C data are consistent with literature values26.
5,7-Dihydroxy-8-nitroflavone (4e)
The mixture of chrysin (254mg, 1.0mmol), conc. HNO3 (0.10mL), and glacial acetic acid (3.0mL) was stirred at 60°C for an hour. After completion, the reaction was quenched with DI water and kept at 0°C. The precipitate was collected by suction filtration. The crude mixture was purified by silica gel column chromatography (eluent: EtOAc/hexanes = 2:1) to give a white powder of 4e (72mg, 24%). TLC (EtOAc:hexanes 2:1): Rf = 0.42; 1H-NMR (500MHz, CDCl3) δ 12.02 (br s, 1H), 8.10 (d, J = 6.8Hz, 2H), 7.57–7.64 (m, 3H), 6.92 (s, 1H), 6.49, (s, 1H); 13C-NMR (126MHz, CDCl3) δ 186.7, 168.5, 167.9, 163.2, 155.2, 138.4, 135.2, 134.6, 131.7, 111.3, 108.4, 104.1, 101.6. 1H and 13C data are consistent with literature values24.
8-Amino-5,7-dihydroxyflavone (4f)
A mixture of 4e (154mg, 0.26mmol), Sn powder (309mg, 2.6mmol), 12M HCl (10mL) and EtOH (10mL) was mixed at 0°C and left stirring at room temperature for 72h. After completion, the reaction was quenched with DI water. The mixture was neutralized by sat. NaHCO3(aq) and extracted with EtOAc (3 × 20mL). The combined organic layers were washed with brine, dried with anh. Na2SO4, filtered and concentrated in vacuo to give a brown powder of 4f (81mg, 58%). TLC (EtOAc:hexanes 1:1): Rf = 0.35; 1H-NMR (500MHz, DMSO-d6) δ 12.06 (br s, 1H), 8.21 (d, J = 7.2Hz, 2H), 7.54–7.61 (m, 3H), 6.90 (s, 1H), 6.29 (s, 1H); 13C-NMR (126MHz, DMSO-d6) δ 182.9, 163.5, 151.9, 151.8, 143.8, 132.5, 131.4, 129.6, 127.2, 116.9, 105.0, 104.1, 98.9. 1H and 13C data are consistent with literature values44.
5-Hydroxy-6,7-dimethoxyflavone (5b)
The title compound was synthesized using the previously described method21. Light yellow solid; Yield: 19%; TLC (EtOAc:hexanes 3:2): Rf = 0.17; 1H NMR (500MHz, acetone-d6): δ 12.80 (br s, 1H), 8.08–8.06 (m, 2H), 7.60–7.57 (m, 3H), 6.88 (s, 1H), 6.81 (s, 1H), 3.97 (s, 3H), 3.77 (s, 3H); 13C NMR (126MHz, CDCl3): δ 182.9, 164.1, 159.0, 153.4, 153.1, 132.8, 132.0, 131.4, 129.2, 126.4, 106.4, 105.7, 90.7, 61.0, 56.4. 1H and 13C NMR data are consistent with literature values21.
5,6,7-Triacetoxyflavone (5c)
The title compound was synthesized following the General Procedure C using baicalein (20mg, 0.074mmol), pyridine (60µL), and acetic anhydride (2.2mL). The reaction was stirred at room temperature for 4h. The reaction was quenched with DI water and extracted with EtOAc (3 × 10mL). The combined organic layers were concentrated in vacuo yielding a pale-yellow solid of 5c (29mg, 99%). TLC (EtOAc:hexanes 2:3): Rf = 0.26; 1H NMR (500MHz, CDCl3): δ 7.84 (dd, J = 8.3, 1.8Hz, 2H), 7.54–7.50 (m, 3H), 7.49 (s, 1H), 6.64 (s, 1H), 2.43 (s, 3H), 2.34 (s, 3H), 2.33 (s, 3H); 13C NMR (126MHz, CDCl3): δ 176.3, 168.4, 167.3, 167.1, 162.9, 154.3, 147.0, 142.2, 131.9, 131.1, 129.3, 129.2, 126.3, 110.4, 108.3, 105.9, 20.9, 20.8, 20.2. 1H and 13C NMR data are consistent with literature values23.
5,6,7-Tripropionoxyflavone (5d)
Propionic anhydride (2.4mL, 15mmol) was added into the solution of baicalein (1.0g, 3.7mmol) in CH2Cl2 (50mL). NEt3 (2.0mL, 3.7mmol) was added into the mixture. After stirring at room temperature for 2h, the reaction was quenched with DI water and extracted with CH2Cl2. The combined organic layers were washed with 10% NaOH, 1M HCl and brine. The crude mixture was concentrated in vacuo to give an off-white solid of 5d (1.3g, 80%). TLC (EtOAc:hexanes 1:3): Rf = 0.21; 1H NMR (500MHz, CDCl3): δ 7.84 (dd, J = 8.1, 1.5Hz, 2H), 7.54–7.47 (m, 4H), 6.63 (s, 1H), 2.75 (q, J = 7.5Hz, 2H), 2.61 (q, J = 7.6Hz, 2H), 2.60 (q, J = 7.6Hz, 2H), 1.32–1.25 (m, 9H); 13C NMR (126MHz, CDCl3):δ 176.4, 171.8, 170.9, 170.7, 162.7, 154.2, 147.1, 142.3, 132.8, 131.9, 131.1, 129.2, 126.3, 115.7, 110.3, 108.3, 27.7, 27.6, 27.2 9.4, 9.0, 8.9. 1H and 13C NMR data are consistent with literature values23.
5-Hydroxy-6,7-dipropionoxyflavone (5e)
To a solution of 5d (200mg, 0.46mmol) in acetic acid (2.76mL), conc. HNO3 (0.46mL) was added dropwise at 0°C. The reaction was stirred at 65°C for 2h, quenched with DI water and filtered. The residue was recrystallized in EtOH to give a pale-yellow solid of 5e (105mg, 60%). m.p.: 132–134°C; TLC (EtOAc:hexanes 1:4): Rf = 0.21; 1H NMR (500MHz, CDCl3): δ 12.91 (s, 1H), 7.88 (d, J = 7.1Hz, 2H), 7.60–7.50 (m, 3H), 6.98 (s, 1H), 6.73 (s, 1H), 2.69–2.59 (m, 4H), 1.33–1.27 (m, 6H); 13C NMR (126MHz, CDCl3): δ 178.8, 171.3, 171.1, 165.1, 158.11, 153.5, 153.4, 148.6, 132.4, 131.0, 129.3, 126.5, 111.5, 105.9, 101.7, 27.7, 27.2, 9.3, 9.1; IR (neat): 3017 (C–H), 2990 (C–H), 2847 (O–H), 1812 (C=O), 1793 (C=O) 1642 (C=O), 1560 (C=C), 1432 (C=C), 1315 (C=C), 1244 (C=C), 1159 (C–O), 1090, (C–O) cm-1; HRMS (m/z): [M + H]+ calcd. for C21H19O7, 383.1131; found 383.1135.
8-Bromo-5,6,7-trihydroxyflavone (5f)
NBS (54mg, 0.30mmol) was added into a solution of baicalein (54mg, 0.20mmol) in THF (6.2mL). The reaction was stirred at room temperature for 3h, quenched with DI water and extracted with EtOAc (3 times). The combined organic layers were concentrated in vacuo to give a light-yellow solid of 5f (65mg, 93%). TLC (EtOAc:hexanes 1:3): Rf = 0.10; 1H NMR (500MHz, acetone-d6): δ 12.82 (br s, 1H), 8.18 (dd, J = 7.6, 2.1Hz, 2H), 7.67–7.62 (m, 3H), 6.90 (s, 1H); 13C NMR (126MHz, acetone-d6): δ 183.0, 163.8, 150.8, 147.4, 146.6, 132.1, 131.3, 130.4, 129.3, 126.5, 105.3, 104.6, 86.6. 1H and 13C NMR data are consistent with literature values45.
5,6,7-Triacetoxy-8-bromoflavone (5g)
The title compound was synthesized following the General Procedure C using 5f (17.5mg, 0.050mmol), pyridine (40.5µL), and acetic anhydride (1.5mL) to give a pale beige solid of 5g (15mg, 63%). TLC (EtOAc:hexanes 1:1): Rf = 0.24; 1H NMR (500MHz, CDCl3): δ 7.95 (dd, J = 8.1, 1.5Hz, 2H), 7.57–7.51 (m, 3H), 6.70 (s, 1H), 2.42 (s, 3H), 2.41 (s, 3H), 2.34 (s, 3H); 13C NMR (126MHz, CDCl3): δ 181.1, 169.5, 165.5, 169.2, 162.8, 151.7, 151.0, 141.5, 133.2, 132.3, 129.4, 129.3, 126.5, 112.4, 105.6, 104.6, 20.9, 20.4, 20.1. 1H and 13C NMR data are consistent with literature values46.
8-Bromo-5,6,7-tripropionoxyflavone (5h)
The title compound was synthesized following the General Procedure C using 5f (94mg., 0.27mmol), pyridine (22µL, 0.27mmol), and propionic anhydride (1.1mL, 8.3mmol) to give a pale beige solid of 5h (43mg, 59%). m.p. 113–115 ºC; TLC (EtOAc:hexanes = 2:3): Rf = 0.18; 1H NMR (500MHz, CDCl3): δ 8.01–7.90 (m, 2H), 7.61–7.47 (m, 3H), 6.70 (s, 1H), 2.78–2.65 (m, 4H), 2.60 (q, J = 7.4Hz, 2H), 1.39–1.24 (m, 9H).; 13C NMR (126MHz, CDCl3): δ 176.0, 171.4, 170.5, 169.8, 162.7, 151.7, 146.5, 141.6, 134.3, 132.2, 130.7, 129.3, 126.5, 116.5, 108.0, 105.3, 27.6, 27.4, 27.2, 9.4, 9.2, 8.9; IR (neat): 3059 (C–H), 1817 (C=O), 1803 (C=O), 1704 (C=O), 1630 (C=C) 1415 (C=C), 1357 (C=C), 1165 (C–O), 1121 (C–O), 1075 (C–O) cm-1; HRMS (m/z): [M + H]+ calcd. for C24H22Br79O8, 517.0498; found 517.0490.
5,6,7-Trimethoxyflavone (5i)
General Procedure D was followed using BF3⋅Et2O (5.0mL), cinnamoyl chloride (866mg, 5.0mmol) and 3,4,5-trimethoxyphenol (958mg, 5.0mmol) to give an orange-brown solid of 6-hydroxy-2,3,4-trimethoxychalcone (6i, 2.7g, 100%). Then, the title compound was synthesized following General Procedure E using 6i (314mg, 1.0mmol) and I2 (20mg, 0.080mmol) and DMSO (12mL) to give a dark yellow solid of 5i (126mg, 40%). TLC (EtOAc:hexanes 1:1): Rf = 0.28; 1H NMR (500MHz, CDCl3): δ 7.86 (dd, J = 7.9, 1.8Hz, 2H), 7.51–7.47 (m, 3H), 6.80 (s, 1H), 6.66 (s, 1H), 3.98 (s, 3H), 3.97 (s, 3H), 3.90 (s, 3H); 13C NMR (126MHz, CDCl3): δ 177.1, 160.1, 158.0, 154.5, 152.7, 140.6, 132.4, 130.6, 127.5, 126.0, 113.0, 108.6, 96.3, 62.3, 61.7, 56.4. 1H and 13C NMR data are consistent with literature values47.
4′-Bromo-5,6,7-trimethoxylflavone (5j)
General Procedure D was followed using BF3⋅Et2O (8.2mL), 4′-bromocinnamoyl chloride (1.23g, 5.0mmol) and 3,4,5-trimethoxyphenol (958mg, 5.0mmol) to give a dark-orange solids of 4′-bromo-6-hydroxy-2,3,4-trimethoxychalcone (6j, 702mg, 36%). Then, the title compound was synthesized following General Procedure E using 6j (650mg, 1.7mmol), I2 (34mg, 0.14mmol) and DMSO (18mL) to give a dark green solid of 5j (419mg, 65%). TLC (EtOAc:hexanes 1:1): Rf = 0.27; 1H NMR (500MHz, CDCl3): δ 7.72 (d, J = 8.8Hz, 2H), 7.62 (d, J = 8.8Hz, 2H), 6.79 (s, 1H), 6.63 (s, 1H), 3.97 (s, 6H), 3.90 (s, 3H); 13C NMR (126MHz, CDCl3): δ 177.1, 160.1, 158.0, 154.5, 152.7, 140.6, 132.4, 130.6, 127.5, 126.0, 113.0, 108.6, 96.3, 62.3, 61.7, 56.4. 1H and 13C NMR data are consistent with literature values28.
5,6,7-Trimethoxy-4′-nitroflavone (5k)
General Procedure D was followed using BF3⋅Et2O (7.4mL), 4′-nitrocinnamoyl chloride (1.3g, 6.2mmol) and 3,4,5-trimethoxyphenol (1.14g, 6.2mmol) to give an orange-brown solid of 6-hydroxy-2,3,4-trimethoxy-4′-nitrochalcone (6k, 675mg, 31%). Then, the title compound was synthesized following General Procedure E using 6k (620mg, 1.7mmol), I2 (34mg, 0.14mmol) and DMSO (18mL) to give a dark orange solid of 5k (366mg, 60%). TLC (EtOAc:hexanes 1:1): Rf = 0.31; 1H NMR (500MHz, CDCl3): δ 8.35 (d, J = 8.6Hz, 2H), 8.04 (d, J = 8.7Hz, 2H), 6.82 (s, 1H), 6.75 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.92 (s, 3H); 13C NMR (126MHz, CDCl3): δ 176.8, 162.9, 158.5, 154.5, 152.7, 149.3, 140.9, 137.6, 128.9, 123.5, 113.1, 110.6, 96.3, 62.3, 61.7, 56.5. 1H and 13C NMR data are consistent with literature values47.
4′-Amino-5,6,7-trimethoxyflavone (5l)
To a solution of 5k (60mg, 0.17mmol) in EtOH (2.7mL) at 0°C was slowly added 12M HCl (2.7mL), followed by Sn powder (100mg, 0.84mmol). The reaction was stirred at room temperature for 1.5h, quenched with sat. NaHCO3 and extracted with EtOAc (3 times). The combined organic layers were concentrated in vacuo to give a dark orange solid of 5l (54mg, 100%). TLC (5% MeOH in CH2Cl2): Rf = 0.32; 1H NMR (500MHz, CDCl3): δ 7.68 (d, J = 8.6Hz, 2H), 6.76 (s, 1H), 6.73 (d, J = 8.6Hz, 2H), 6.52 (s, 1H), 3.97 (s, 3H), 3.96 (s, 3H), 3.90 (s, 3H); 13C NMR (126MHz, CDCl3): δ 177.4, 161.8, 157.5, 154.5, 152.6, 149.6, 140.3, 127.7, 121.1, 114.8, 112.9, 106.0, 96.3, 62.3, 61.6, 56.3. 1H and 13C NMR data are consistent with literature values47.
4′-Bromo-5,6,7-trihydroxyflavone (5m)
The title compound was synthesized following the General Procedure F using 5j (50mg, 0.13mmol), CH2Cl2 (1.0mL), and BBr3 (36µL, 0.38mmol) to give a dark green solid of 5m (42mg, 94%). TLC (5% MeOH in CH2Cl2): Rf = 0.10; 1H NMR (500MHz, DMSO-d6): δ 8.01 (d, J = 8.6Hz, 2H), 7.77 (d, J = 8.6Hz, 2H), 6.98 (s, 1H), 6.62 (s, 1H); 13C NMR (126MHz, DMSO-d6): δ 182.6, 162.3, 154.2, 150.3, 147.4, 132.7, 130.7, 129.9, 128.8, 126.1, 105.3, 104.8, 94.6. 1H and 13C NMR data are consistent with literature values27.
5,6,7-Trihydroxy-4′-nitroflavone (5n)
The title compound was synthesized following the General Procedure F using 5k (20mg, 0.056mmol), CH2Cl2 (1mL), and BBr3 (50µL, 0.53mmol) to give a dark orange solid of 5n (13mg, 74%). TLC (5% MeOH in CH2Cl2): Rf = 0.06; 1H NMR (500MHz, DMSO-d6): δ 12.45 (s, 1H), 10.69 (s, 1H), 8.88 (s, 1H), 8.35 (d, J = 7.1Hz, 2H), 8.30 (d, J = 6.9Hz, 2H), 7.11 (s, 1H), 6.62 (s, 1H); 13C NMR (126MHz, DMSO-d6): δ 184.1, 162.6, 158.7, 154.3, 151.3, 147.5, 145.2, 136.5, 128.0, 124.6, 107.5, 104.9, 94.7. 1H and 13C NMR data are consistent with literature values48.
4′-Amino-5,6,7-trihydroxyflavone (5o)
The title compound was synthesized following the General Procedure F using 5l (40mg, 0.12mmol), CH2Cl2 (1.0mL), and BBr3 (50µL, 0.53mmol) to give a yellow solid of 5o (30mg, 86%). m.p. > 250 ºC (decompose); TLC (5% MeOH in CH2Cl2): Rf = 0.28; 1H NMR (500MHz, DMSO-d6): δ 7.70 (d, J = 9.0Hz, 2H), 6.72 (d, J = 8.9Hz, 2H), 6.53 (s, 1H), 6.48 (s, 1H); 13C NMR (126MHz, DMSO-d6): δ 182.3, 167.5, 158.9, 154.0, 150.0, 147.3, 143.2, 128.5, 123.4, 114.0, 106.2, 104.3, 94.2; IR (neat): 3512 (O–H), 3497 (N–H), 3479 (N–H), 3077 (C–H), 2922 (C–H), 2879 (O–H), 1678 (C=O), 1497 (C=C), 1470 (C=C), 1381 (C=C), 1336 (C=C) cm−1; HRMS (m/z): [M + H]+ calcd. for C15H12NO5, 286.0715; found 286.0719.
4′-Bromo-5-hydroxy-6,7-dimethoxyflavone (5p)
The title compound was synthesized following the General Procedure H using 5j (30mg, 0.077mmol), toluene (5.6mL), and AlCl3 (52mg, 0.39mmol) to give a brown solid of 5p (63mg, 100%). TLC (5% MeOH in CH2Cl2): Rf = 0.70; 1H NMR (500MHz, CDCl3): δ 7.75 (d, J = 8.7Hz, 2H), 7.65 (d, J = 8.7Hz, 2H), 6.65 (s, 1H), 6.55 (s, 1H), 3.96 (s, 3H), 3.92 (s, 3H); 13C NMR (126MHz, CDCl3): δ 182.7, 162.9, 159.1, 153.3, 153.1, 132.9, 132.5, 130.3, 127.8, 126.7, 106.4, 105.9, 90.8, 61.0, 56.5. 1H and 13C NMR data are consistent with literature values27.
5-Hydroxy-6,7-dimethoxy-4′-nitroflavone (5q)
The title compound was synthesized following the General Procedure H using 5k (30mg, 0.092mmol), toluene (6.7mL), and AlCl3 (61mg, 0.46mmol) to give a brown solid of 5q (59mg, 81%). m.p. 145–150ºC; TLC (2% MeOH in CH2Cl2): Rf = 0.59; 1H NMR (500MHz, CDCl3): δ 8.38 (d, J = 9.0Hz, 2H), 8.07 (d, J = 8.9Hz, 2H), 6.77 (s, 1H), 6.59 (s, 1H), 3.98 (s, 3H), 3.93 (s, 3H); 13C NMR (126MHz, CDCl3): δ 182.4, 161.1, 159.5, 157.6, 155.7, 153.1, 137.2, 133.1, 127.3, 124.4, 107.9, 104.1, 90.9, 61.0, 56.6; IR (neat): 3442 (O–H), 3013 (C–H), 2988 (C–H), 1697 (C = O), 1586 (N–O), 1449 (C = C), 1360 (C = C), 1345 (N–O), 1244 (C = C) cm-1; HRMS (m/z): [M + H]+ calcd. for C17H14NO7, 344.0770; found 344.0779.
5,6-Dihydroxy-7-methoxy-4′-nitroflavone (5r)
To a solution of 5q (30mg, 0.087mmol) in acetic acid (1.36mL) was added 47% HBr (0.68mL). After refluxing at 120ºC for 3h, the mixture was quenched with sat. NaHCO3 and extracted with EtOAc (3 times). The combined organic layers were concentrated in vacuo to give a dark brown solid of 5r (25mg, 89%). m.p. 174–178 ºC; TLC (EtOAc:hexanes 1:1): Rf = 0.25; 1H NMR (500MHz, CDCl3): δ 8.37 (d, J = 8.9Hz, 2H), 8.06 (d, J = 8.9Hz, 2H), 6.77 (s, 1H), 6.65 (s, 1H), 4.02 (s, 3H); 13C NMR (126MHz, CDCl3): δ 181.7, 162.3, 160.2, 152.6, 151.6, 147.4, 135.3, 129.1, 127.3, 123.4, 107.1, 103.6, 95.4, 64.0; IR (neat): 3454 (O–H), 3045 (C–H), 2950 (C–H), 2935 (O–H), 1684 (N–O), 1651 (C=O), 1604 (C=C), 1451 (C=C), 1342 (N–O), 1324 (C=C) cm−1; HRMS (m/z): [M + H]+ calcd. for C16H12NO7, 330.0614; found 330.0610.
Anti-dengue activity and cellular toxicity
Cells and viruses
The cell lines of LLC/MK2 (ATCC®CCL-7) and C6/36 (ATCC®CRL-1660) were maintained in minimal essential medium (MEM) (Gibco®, Langley, USA) supplemented with 10% fetal bovine serum (Gibco®, Langley, USA); 100 I.U./mL penicillin, and 100μg/mL streptomycin (Bio Basic Canada, Ontario, Canada); 10mM HEPES (4-(2-hydroxyethyl)-1-piperazine-ethane-sulfonic acid) (Sigma Aldrich, St. Louis, USA) at 37°C under condition of 5% CO2 and 28°C, respectively18. Reference strain of DENV2 (New Guinea C strain, accession number NC_001474.2) was propagated in C6/36 and LLC/MK2 cell line with MEM medium added with 1% FBS, 100 I.U./mL penicillin, 100μg/mL streptomycin, and 10mM HEPES at 37°C in 5% CO2 incubator18.
Screening antiviral efficacy
LLC/MK2 cells (5 × 104) were seeded in 24-well plate and incubate at 37°C under 5% CO2 overnight. Cells were infected with DENV2 NGC at a multiplicity of infection (M.O.I. of 0.1) and compounds at 10µM were added and DMSO at 1% was used as control. Cultured plates were incubated for 1h with gentle rocking every 15min. Cells were washed with PBS, MEM supplemented with 1% FBS, 100 I.U./mL penicillin, and 100µg/mL streptomycin, 10mM HEPES was added in a presence of a compound at 10µM. Cells were incubated at 37°C with 5% CO2 for 3days. Supernatants were collected to determine viral titers by plaque titration assay as previously described49. The selected compounds were further analyzed for an effective concentration (EC50).
Screening cellular toxicity
Cytotoxicity of the compounds was also accessed at the concentration of 10µM in parallel with the viral inhibition screening. LLC/MK2 cells (1 × 104) were seeded in 96-well plate and incubated at 37°C under 5% CO2 overnight; compounds were added after 24h, and then incubated for 2days. DMSO at 1% was used as mock treatment. Cytotoxicity was measured using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) kit (Promega, Wisconsin-Madison, WI, USA) according to the manufacturer’s instruction and analyzed by EnSight Multimode Plate Reader spectrophotometry at A450nm. (Perkin Elmer, Waltham, MA, USA).
Antiviral efficacy
LLC/MK2 (ATCC® CCL-7), and C6/36 (ATCC® CRL-1660) cell lines were propagated and maintained as previously described18,29,31. Effective concentration (EC50) of the compounds against the DENV2 were tested using LLC/MK2 cells18,29. Briefly, cells were seeded overnight and infected with each virus at the multiplicity of infection (M.O.I.) of 0.1 for 1h. The compound was added during and after infection, and cells were incubated for 72h. Supernatants were collected for plaque titration31. EC50 results were means and standard errors of three independent experiments.
Molecular docking studies
To predict the possible viral protein target for flavone analogs, the crystal structures of dengue viral proteins were retrieved from the protein databank as follows; envelope (E) protein, the allosteric site of NS2B/NS3 protease (NS2B/NS3 pro), the SAM binding site of NS5 methyltransferases (NS5 MTase), and NS5 RNA-dependent RNA polymerase (NS5 RdRp), with PDB entry 1OKE18, 3U1I50, 6KR251, and 3VWS52, respectively. The native inhibitors for E protein, NS2B/NS3 pro, NS5 MTase, and NS5 RdRp are 3-100-2253, SYC-130754, Sinefungine55, and NITD-10752 were used as a reference data for the molecular docking study. The protein structures were considered a receptor prepared as a standard protocol29. The native inhibitors and ligands selected based on the antiviral efficacy were constructed and optimized using Gaussview 6 and Gaussian 16 at HF/6-31g* basis set56. The binding energy and pose were predicted using Autodock VinaXB57. The respective reference ligands were redocked to their crystal structure to validate the molecular docking protocol. The best interaction energy score (kcal/mol) of each flavone analog was ranked and plotted versus the native inhibitor. By considering each protein target of DENV, the binding affinity difference (ΔD) between native ligand (Do) and flavone analog (Di) calculated from Autodock VinaXB called likelihood ratios was applied to determine the potent compound (Eq.2). The promising interaction of a ligand and its target with a greater ΔD value suggested a more optimistic interaction of ligand. The binding pose and interaction were visualized using the UCSF ChimeraX58 and BIOVIA Discovery Studio Visualizer V21.1.059.
$$\Delta D = D_{{\text{o}}} - D_{{\text{i}}}$$
(2)
Physicochemical properties prediction
A total of 33 flavone analogs were analyzed in terms of physicochemical descriptors, drug-likeness, and the ADME properties, which are absorption, distribution, metabolism, and excretion, using the SwissADME webserver33 and ADMETlab 2.034. The Lipinski parameters are commonly used to estimate the drug-likeness properties by considering the following criteria; molecular weight ≤ 500, ≤ 5 hydrogen bond donors (HBDs), ≤ 10 hydrogen bond acceptors (HBAs), ≤ 5 = log of octanol to water partition coefficient (Log P), and the number of rotational bonds ≤ 535.
QSAR moodelling
The optimized structures of all flavone analogs and 36 descriptors obtained from ADMETlab were imported into the QSAR module of Materials Studio software to derive the relationship between physicochemical properties and anti-dengue activities. First, it should be noted that the Genetic Algorithm program in Materials Studio software was applied to select the significant descriptors. Next, the correlation coefficients between each pair of descriptors were calculated to avoid the overfitting equation. Finally, multiple linear regression was applied to obtain the QSAR models.
