10.1002/cbic.201800431
ChemBioChem
FULL PAPER
= 6.1, 9.8 Hz, 1H), 3.94-3.99 (m, 4H), 4.77 (d, J = 1.5 Hz, 1H), 5.03 (dd, J
= 10.0, 10.0 Hz, 1H), 5.14 (dd, J = 9.3, 9.8 Hz, 1H), 5.20-5.34 (m, 5H),
6.29 (s, 1H), 6.41 (d, J = 2.2 Hz, 1H), 6.53 (d, J = 2.2 Hz, 1H), 6.58 (s,1H),
6.97 (d, J = 8.6 Hz,1H), 7.41 (dd, J = 2.2, 8.6 Hz, 1H), 7.46 (d, J = 2.2 Hz,
1H), 12.83 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ: 17.1, 20.5, 20.5, 20.5,
20.5, 20.6, 20.6, 55.9, 66.1, 66.5, 68.6, 69. 0, 69.3, 70.7, 70.8, 72.4, 73.1,
94.9, 97.6, 97.9, 99.6, 104.2, 106.4, 110.8, 112.5, 119.1, 123.5, 146.0,
150.2, 157.0, 161.7, 161.9, 164.2, 169.2, 169.3, 169.8, 169.9, 170.1, 170.1,
182.1. HR-MS [ESI+, (M+Na)+]: calculated for C40H44NaO21, 883.2273;
found, 883.2275.
chloroform-methanol (3:1) furnished 16 as a yellow solid (45.1 mg, 19%)
and 6 (69.3 mg, 19%) as yellow amorphous solid.
Further purification for biological study was performed with reverse-phase
HPLC. Elution with water-acetonitrile (70:30) furnished pure 6: 1H-NMR
(500 MHz, DMSO-d6) δ: 3.14-3.16 (m, 1H), 3.24-3.27 (m, 2H), 3.42-3.46
(m, 2H), 3.70 (dd, J = 5.3, 10.0 Hz, 1H), 3.84 (s, 3H), 3.87 (s, 3H), 4.61
(dd, J = 5.3, 5.8 Hz, 1H), 5.04-5.06 (m, 2H), 5.12 (d, J = 4.8 Hz, 1H), 5.39
(d, J = 4.9 Hz, 1H), 6.44 (d, J = 2.2 Hz, 1H), 6.87 (d, J = 2.2 Hz, 1H), 7.05
(s, 1H), 7.13 (d, J = 8.5 Hz,1H), 7.58 (d, J = 2.2 Hz, 1H), 7.70 (dd, J = 2.2,
8.5 Hz, 1H), 12.89 (s, 1H). 13C-NMR (125 MHz, DMSO-d6) δ: 56.2, 56.3,
61.1, 70.0, 73.6, 76.9, 77.7, 95.6, 100.0, 100.5, 104.6, 105.9, 110.0, 112.2,
120.6, 123.2, 149.5, 152.8, 157.4, 161.6, 163.5, 164.3, 182.6. HR-MS
[ESI+, (M+Na)+]: calculated for C23H24NaO11, 499.1216; found, 499.1207.
HPLC water/acetonitrile = 70/30, flow rate 0.5 mL/min, tR = 13.0 min. The
purity was >99%, judged by the absorption at 200 nm and 254 nm.
During the process of HPLC separation, elution with water-acetonitrile
(50:50) furnished pure 16: 1H-NMR (500 MHz, DMSO-d6) δ: 3.84 (s, 3H),
3.87 (s, 3H), 6.19 (d, J = 2.1 Hz, 1H), 6.51 (d, J = 2.1 Hz, 1H), 6.96 (s, 1H),
7.11 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.67 (dd, J = 2.2, 8.8 Hz,
1H), 10.84 (br s, 1H), 12.91 (s, 1H). Its 1H-NMR spectrum was identical
with that reported previously.[49] 13C-NMR (125 MHz, DMSO-d6) δ:56.2,
56.3, 94.6, 99.3, 104.2, 104.3, 109.9, 112.1, 120.5, 123.3, 149.5, 152.6,
157.8, 161.9, 163.8, 164.7, 182.3. HR-MS [ESI+, (M+Na)+]: calculated for
C17H14NaO6, 337.0688; found, 337.0694. HPLC water/acetonitrile = 50/50,
flow rate 0.5 mL/min, tR = 15.1 min. The purity was >99%, judged by the
absorption at 200 nm and 254 nm.
5-Hydroxy-7-[hexa-O-acetyl-(6-O-α-L-rhamnopyranosyl-β-D-
glucopyranosyl)oxy]-3’,4’-dimethoxyflavone (15c): To a mixture of 15b
(4.17 g, 4.84 mmol) and potassium carbonate (868 mg, 6.28 mmol) in
anhydrous DMF (70 mL) was added iodomethane (500 µL, 8.00 mmol)
and stirred for 10 h at 50 °C. The mixture was poured into water and the
precipitates were collected by filtration and dried in vacuo. The residue
was purified by silica gel column chromatography (100 g). Elution with
hexane-ethyl acetate (1:2) furnished 15c as a yellow amorphous solid
(2.99 g, 82%). 1H-NMR (500 MHz, CDCl3) δ: 1.15 (d, J = 6.3 Hz, 3H), 1.94
(s, 3H), 2.03 (s, 3H), 2.05 (s, 3H), 2.07 (s, 3H), 2.07 (s, 3H), 2.09 (s, 3H),
3.68 (dd, J = 5.3, 11.8 Hz, 1H), 3.81-3.93 (m, 3H), 3.97 (s, 3H), 3.99 (s,
3H), 4.72 (br s, 1H), 5.02 (dd, J = 9.5, 9.5 Hz, 1H), 5.17 (dd, J = 9.3, 9.3
Hz, 1H), 5.23-5.35 (m, 5H), 6.43 (d, J = 2.2 Hz, 1H), 6.58 (d, J = 2.2 Hz,
1H), 6.61 (s,1H), 7.00 (d, J = 8.6 Hz,1H), 7.34 (d, J = 2.2 Hz, 1H), 7.53 (dd,
J = 2.2, 8.6 Hz, 1H), 12.82 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ: 17.2,
20.4, 20.5, 20.5, 20.5, 20.6, 20.6, 55.9, 55.9, 66.2, 66.5, 68.6, 68.8, 69.3,
70.8, 70.9, 72.4, 73.1, 95.2, 97.6, 98.0, 99.4, 104.4, 106.5, 108.7, 111.1,
120.1, 123.3, 149.1, 152.3, 157.0, 161.6, 162.1, 164.1, 169.1, 169.3, 169.6,
169.6, 169.8, 170.0, 182.1. HR-MS [ESI+, (M+Na)+]: calculated for
C41H46NaO21, 897.2429; found, 897.2422.
3’,5-Diacetoxy-7-[nona-O-acetyl-(6-O-α-L-rhamnopyranosyl-(4-O-α-
D-glucopyranosyl)-β-D-glucopyranosyl)oxy]-4’-methoxyflavone
(18a): To a solution of 17 (521 mg, 0.674 mmol) in pyridine (2 mL) was
added iodine (190 mg, 0.749 mmol) and the mixture was stirred for 23 h at
90 °C. Then, acetic anhydride (3 mL) and pyridine (2 mL) were added to
the reaction and the mixture was stirred for 30 h at the same temperature.
After cooling, the mixture was poured into water. The precipitates were
collected by filtration and the residue was purified by silica gel column
chromatography (10 g). Elution with hexane-ethyl acetate (1:4) furnished
18a as a yellow amorphous solid (823 mg, 99%). 1H-NMR (500 MHz,
CDCl3) δ: 1.03 (d, J = 6.4 Hz, 3H), 1.94 (s, 3H), 2.01 (s, 3H), 2,03 (s, 3H),
2.03 (s, 3H), 2.06 (s, 3H), 2.07 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.09 (s,
3H), 2.37 (s, 3H), 2.43 (s, 3H), 3.88-3.94 (m, 6H), 4.00-4.04 (m, 2H), 4.08
(dd, J = 2.4, 12.4 Hz, 1H), 4.16 (dd, J = 9.1, 9.1 Hz, 1H), 4.36 (dd, J = 3.9,
12.4 Hz 1H), 4.76 (d, J = 1.5 Hz, 1H), 4.87 (dd, J = 3.9, 10.5 Hz, 1H), 5.02
(dd, J = 10.0, 10.0 Hz, 1H), 5.07 (dd, J = 10.0, 10.0 Hz, 1H), 5.14-5.18 (m,
2H), 5.22 (dd, J = 3.4, 10.3 Hz, 1H), 5.32-5.40 (m, 3H), 5.46 (d, J = 3.6 Hz,
1H), 6.51 (s, 1H), 6.65 (d, J = 2.4 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 7.08
(d, J = 8.8 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.72 (dd, J = 2.2, 8.8 Hz, 1H).
13C-NMR (125 MHz, CDCl3) δ: 17.0, 20.5, 20.5, 20.6, 20.6, 20.6, 20.6, 20.7,
20.8, 20.8, 20.9, 21.1, 56.1, 61.6, 65.5, 66.7, 68.2, 68.5, 68.7, 69.2, 69.6,
70.2, 70.6, 71.3, 71.5, 74.0, 74.6, 95.2, 97.4, 97.5, 102.2, 107.5, 109.1,
112.5, 112.7, 120.9, 123.7, 125.3, 140.1, 150.7, 154.0, 158.3, 159.8, 161.3,
168.7, 169.4, 169.4, 169.5, 169.7, 169.8, 169.9, 169.9, 170.1, 170.4, 170.4,
176.0. HR-MS [ESI+, (M+Na)+]: calculated for C56H64NaO31, 1255.3329;
found, 1255.3310.
5-Hydroxy-7-(6-O-α-L-rhamnopyranosyl-β-D-glucopyranosyl)oxy-
3’,4’-dimethoxyflavone (7): To a solution of 15c (4.17 g, 4.84 mmol) in
methanol (160 mL) was added triethylamine (32 mL) and stirred for 13 h
at 70 °C. The precipitates were collected by filtration and dried in vacuo
to give 7 as a slightly yellow powder (1.28 g, 60%). Further purification for
biological study was performed with reverse-phase HPLC. Elution with
water-acetonitrile (75:25) furnished pure 7. 1H-NMR (500 MHz, DMSO-d6)
δ: 1.06 (d, J = 6.1 Hz, 3H), 3.10-3.16 (m, 2H), 3.24-3.30 (m, 2H), 3.38-3.46
(m, 3H), 3.57-3.61 (m, 1H), 3.63-3.65 (m, 1H), 3.81-3.86 (m, 4H), 3.87 (s,
3H), 4.44 (d, J = 6.1 Hz, 1H), 4.53 (d, J = 1.2 Hz, 1H), 4.58 (d, J = 4.4 Hz,
1H), 4.68 (d, J = 5.7 Hz, 1H), 5.05 (d, J = 7.5 Hz, 1H), 5.18 (d, J = 4.9 Hz,
1H), 5.21 (d, J = 5.6 Hz, 1H), 5.43 (d, J = 4.9 Hz, 1H), 6.45 (d, J = 2.2 Hz,
1H), 6.80 (d, J = 2.2 Hz, 1H), 7.05 (s,1H), 7.17 (d, J = 8.5 Hz,1H), 7.57 (d,
J = 2.2 Hz, 1H), 7.70 (dd, J = 2.2, 8.5 Hz, 1H), 12.90 (s, 1H). 13C-NMR
(125 MHz, DMSO-d6) δ: 18.3, 56.2, 56.3, 66.5, 68.8, 70.0, 70.8, 71.2, 72.5,
73.5, 76.1, 76.7, 95.4, 100.0, 100.4, 101.0, 104.5, 105.9, 109.9, 112.3,
120.7, 123.2, 149.4, 152.7, 157.4, 161.6, 163.4, 164.3, 182.5. HR-MS
[ESI+, (M+Na)+]: calculated for C29H34NaO15, 645.1795; found, 645.1814.
HPLC water/acetonitrile = 75/25, flow rate 0.5 mL/min, tR = 11.2 min. The
purity was >99%, judged by the absorption at 200 nm and 254 nm.
5-Hydroxy-7-β-D-glucopyranosyloxy-3’,4’-dimethoxyflavone (6) and
5,7-Dihydroxy-3’,4’-dimethoxyflavone (16): A suspension of 7 (486 mg,
0.753 mmol in sulfuric acid (0.75% w/v, 9.4 mL) was heated for 30 min in
a high-pressure steam sterilizer. After cooling down to 70 °C, the reaction
flask was taken up and placed in a refrigerator at 5 °C, overnight. The
supernatant solution was removed by decantation and the yellow
precipitates were suspended in acetone and collected by filtration, washed
with water dried in vacuo to give 6 as a slightly yellow powder (59.1 mg,
16%). The acetone solution was neutralized with AmberlystⓇ A26 and
dried over anhydrous sodium sulfate, concentrated in vacuo. The residue
was purified by silica gel column chromatography (10 g). Elution with
3’,5-Dihydroxy-7-[nona-O-acetyl-(6-O-α-L-rhamnopyranosyl-(4-O-α-
D-glucopyranosyl)-β-D-glucopyranosyl)oxy]-4’-methoxyflavone
(18b): To a solution of 18a (823 mg, 0.667 mmol) in methanol (6.5 mL)
was added sodium perborate tetrahydrate (418 mg, 2.72 mmol) and the
mixture was stirred for 17 h at 0 °C. The reaction mixture was filtered
through silica gel column with ethyl acetate as eluent. The filtrate was
concentrated in vacuo and the residue was purified by silica gel column
chromatography (10 g). Elution with hexane-ethyl acetate (1:4) furnished
18b as a yellow amorphous solid (622 mg, 81%). 1H-NMR (500 MHz,
CDCl3) δ: 1.05 (d, J = 6.3 Hz, 3H), 1.95 (s, 3H), 2.01 (s, 3H), 2,03 (s, 3H),
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