Z.-Y. Zhu et al. / European Journal of Medicinal Chemistry 75 (2014) 297e300
299
Table 2
silica gel, eluting with DCM/EtOAc (50:1) to give 2 [13] a light
yellow solid (2.20 g, 74%). Mp: 168e169 ꢀC. 1H NMR (400 MHz,
CDCl3, d ppm): 2.345 (s, 3H); 6.575 (d, J ¼ 2.0 Hz, 1H); 6.729 (s, 1H);
Percentages of cell cycle phases analyzed by FCM.
G1 (%)
S (%)
G2/M (%)
7.54 (q, J ¼ 6.4 Hz, 4H); 7.873 (d, J ¼ 6.8 Hz, 2H); 12.731 (s, 1H). 13
C
H22 control
Chrysin
Compound 3
14.38 ꢁ 0.54
12.97 ꢁ 0.45
6.22 ꢁ 0.37*
70.21 ꢁ 0.38
69.91 ꢁ 0.21
57.44 ꢁ 0.49*
15.42 ꢁ 0.33
17.13 ꢁ 0.42
36.34 ꢁ 0.52*
NMR (100 MHz, CDCl3, d ppm): 21.21, 101.04, 105.47, 106.12, 108.92,
126.40, 129.18, 130.94, 132.18, 155.93, 156.74, 161.90, 164.70, 168.40,
182.86. MS (ESI): 297.11 ([M þ H]þ).
*P < 0.01 compared with the H22 control group.
6.1.2. 5,7-Di-O-acetyl chrysin (3)
concentration which causes a 50% decrease in cell growth, obtained
by calculated given values.
Acetic anhydride (2.06 g, 0.02 mol) was added dropwise to a
solution of chrysin (2.54 g, 0.01 mol) in 50 ml of pyridine under
stirring, and the reaction mixture was continued to stir overnight at
room temperature. The solvent was removed by rotary evaporator.
The residue was dissolved in dichloromethane, washed by 1 M HCl
for 3 times, then with saturated sodium bicarbonate solution and
water to neutral. The organic phase was separated, dried over
MgSO4, concentrated in vacuo. The residue was purified by column
chromatography on silica gel, eluting with DCM/EtOAc (50:1) to
give 3 [13] a white solid (2.70 g, 80%). Mp: 193e195 ꢀC. 1H NMR
(400 MHz, CDCl3, d ppm): 2.354 (s, 3H); 2.449 (s, 3H); 6.660 (s, 1H);
6.850 (s, 1H); 7.358 (s, 1H); 7.527 (q, J ¼ 6.4 Hz, 3H); 7.856 (d,
5. Cell cycle phases analysis by FCM
H22 cells was treated with chrysin and compounds 3, 1 mmol/l
for 24 h, then cells were collected and washed three times with ice-
cold PBS (pH 7.2), be cautious. After fixed with 70% ice ethanol at
4
ꢀC for 1 h, the cells were stained with propidium iodide (PI;
100 mg/ml). The stained cells were then transferred to flow tubes
by passing through a nylon mesh with a pore size of 40 m. Flow
m
cytometric analysis was performed using a flow cytometer (Becton
Dickinson, CA, USA). Percentages of cells in G1, S and G2/M were
determined by use of MODFIT and Cell Quest software.
J ¼ 6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3,
d ppm): 21.12, 21.21,
108.61, 109.08, 113.66, 114.98, 126.23, 129.11, 131.07, 131.80, 150.22,
153.93, 157.70, 162.58, 168.04, 169.46, 176.43. MS (ESI): 339.14
([M þ H]þ).
6. Experimental section
6.1.3. 5,7-Di-O-(ethoxycarbonyl)methyl chrysin (4)
6.1. Chemistry
Bromoacetate (3.68 g, 0.022 mol) was added dropwise to a
mixture of chrysin (2.54 g, 0.01 mol) and anhydrous potassium
carbonate (1.66 g, 0.011 mol) in 50 ml of acetone. The reaction
mixture was stirred for 8 h at 50 ꢀC. After filtration, the filtrate was
concentrated under reduced pressure, and the residue was purified
by column chromatography on silica gel, eluting with PE/EtOAc
(1:3) to give a white solid (3.62 g, 85%). Mp: 123e124 ꢀC. 1H NMR
All chemicals (reagent grade) used were purchased from
SigmaeAldrich (U.S.A) and Aladdin-Reagent Co., Ltd (China). TLC
was run on the silica gel coated aluminum sheets (Silica Gel 60
GF254, E. Merck, Germany). 1H NMR and 13C NMR were recorded on
a Bruker AVANCE instrument (400 MHz). Melting points were
determined on a X-4 MP apparatus (YALIEN Institute of Mechanical
and Electrical Technology, Beijing, China). ESI mass spectra were
obtained on an LCQ-Advantayc-MAX (LAM10188, Finnigan, Co., Ltd,
USA).
(400 MHz, CDCl3,
d ppm): 1.309 (m, 6H); 4.300 (m, 4H); 4.696 (s,
2H); 4.791 (s, 2H); 6.575 (d, J ¼ 2.0 Hz, 1H), 6.678 (s, 1H), 7.536 (m,
4H), 7.850 (d, J ¼ 6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3,
d ppm):
9.32, 9.38, 56.72, 57.00, 60.69, 62.18, 90.42, 94.55, 104.39, 105.86,
121.24, 121.57, 124.20, 126.67, 154.46, 154.84, 156.97, 162.98, 163.38,
177.72. HRESIMS m/z:449.1225[M þ Na]þ (calcd for C23H22O8Na,
449.1212).
6.1.1. 7-O-Acetyl chrysin (2)
Acetic anhydride (1.03 g, 0.01 1 mol) was added dropwise to a
solution of chrysin (2.54 g, 0.01 mol) in 50 ml of pyridine under
stirring. After 2 h of reaction at room temperature, the solvent was
removed by rotary evaporator. The residue was dissolved in
dichloromethane, washed by 1 M HCl for 3 times, then with satu-
rated sodium bicarbonate solution and water to neutral. The
organic phase was separated, dried over MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
6.1.4. 5,7-Di-O-carboxymethyl chrysin (5)
A mixture of compound 4 (2.13 g, 0.005 mol) and potassium
hydroxide (1.68 g, 0.003 mol) in 120 ml of methanol was stirred at
40 ꢀC for 2 h. After filtration, the residue was washed by methanol,
then dissolved in water, adjusted to pH 1e2 by using HCl solution,
filtered, the residue was dried in vacuum to give a light yellow solid
(1.24 g, 67%). Mp: 268e270 ꢀC. 1H NMR (400 MHz, DMSO-d6,
d
ppm): 4.855 (d, J ¼ 3.2 Hz, 4H); 6.497 (s, 1H); 6.837 (s, 1H); 6.939
(s, 1H); 7.582 (m, 3H); 8.060 (d, J ¼ 6.0 Hz, 2H), 13.171 (s, 2H). 13
C
NMR (100 MHz, DMSO-d6,
d ppm): 65.42, 66.61, 95.50, 99.29,
108.59, 109.46, 126.48, 129.51, 131.23, 131.99, 159.10, 159.37, 160.54,
162.51, 169.84, 170.11, 176.32. HRESIMS m/z:371.0779, [M þ H]þ
(calcd for C19H15O8, 371.0767).
6.1.5. 7-O-Benzoyl chrysin (6)
Benzoyl chloride (1.55 g, 0.011 mol) was added dropwise to a
solution of chrysin (2.54 g, 0.01 mol) in 50 ml of pyridine with
stirring. After 2 h at room temperature, the solvent was removed by
rotary evaporator. The residue was dissolved in dichloromethane,
washed by 1 M HCl for 3 times, then with saturated sodium bi-
carbonate solution and water to neutral. The organic phase was
separated, dried over MgSO4, concentrated in vacuo. The residue
was purified by column chromatography on silica gel, eluting with
Scheme 1. Synthesis of chrysin derivatives.