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Compound 3b. Reactants: 2c (0.060 g, 0.15 mmol) and diethylamine (2 mL, 19.4 mmol);
elution solvent for column chromatography, chloroform:methanol, 100:4 (v/v).
Compound 3c: Reactants: 2c (0.060 g, 0.15 mmol) and pyrrolidine (0.25 mL, 3 mmol);
elution solvent for column chromatography, chloroform:methanol, 100:6 (v/v).
Compound 3d. Reactants: 2c (0.060 g, 0.15 mmol) and piperidine (0.25 mL, 2.52 mmol);
elution solvent for column chromatography, chloroform:methanol, 100:1 (v/v).
Compound 3e. Reactants: 2c (0.060 g, 0.15 mmol) and morpholine (0.25 mL, 2.88
mmol); elution solvent for column chromatography, chloroform:methanol, 100:1 (v/v).
Compound 3f: Reactants: 2a (0.060 g, 0.16 mmol) and an aqueous solution of dimethyl-
amine (30 %, 2 mL, 9.97 mmol); elution solvent for column chromatography, chloro-
form:methanol, 100:3 (v/v).
Compound 3g. Reactants: 2b (0.060 g, 0.15 mmol) and dimethylamine aqueous solution
(2 mL, 30%, 9.97 mmol); elution solvent for column chromatography, chloroform:methanol,
100:8 (v/v).
Compound 3h. Reactants: 2d (0.060 g, 0.14 mmol) and dimethylamine aqueous solution
(30%, 2 mL, 9.97 mmol); elution solvent for column chromatography, chloroform:methanol,
100:8 (v/v).
Compound 3i. Reactants: 2e (0.060 g, 0.14 mmol) and dimethylamine aqueous solution
(30%, 2 mL, 9.97 mmol); elution solvent for column chromatography, chloroform:methanol,
100:8 (v/v).
Growth of cell and chemicals
The human hepatocarcinoma cell lines (Hep-G2 and BEL-7402), human cervix tumor
cell line (HeLa), human gastric cancer cell line (MGC-803) and human nasopharyngeal
carcinoma cell line (CNE) were obtained from the Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained in RPMI
1640 medium supplemented with 10 % fetal bovine serum and 100 U mL-1 penicillin and 100
μg mL-1 streptomycin. The cells were kept at 37 °C in a humidified atmosphere containing 5
% CO2. The nine benzo[b]xanthone derivatives 3a–i were applied in DMSO to 10 mM and
stored at −80 °C. MTT (2-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-2H-tetrazolium bromide)
was obtained from Amresco Chemical Corp (USA) and was dissolved in 0.01 M PBS to 5 mg
mL-1. Deionized water was used in all experiments.
Cell proliferation assay (MTT assay)
Inhibition of cell proliferation by the benzo[b]xanthone derivatives was measured using
the MTT assay. Briefly, cells were plated in 96-well culture plates at the density of 5000 cells
per well in RPMI 1640 medium in 200 μL aliquots. After 24 h incubation, the cells were
treated with the benzo[b]xanthone derivatives (0.3, 1, 3, 10 and 30 μM, four wells per
concentration) for 48 h. Then 20 μL of a 5 mg mL-1 MTT solution was added to each well and
the cells were further incubated at 37 °C for another 4 h. The supernatant was discarded, 150
μL DMSO per well was added and the absorbance (A) was measured at 490 nm. The cell
inhibition (IR) was calculated using the following equation:
A − A
(
)
c
t
IR (%) =
×100
A
c
where Ac and At are the absorption of the control and treated samples, respectively.
The IC50, values, calculated by the logit method,19 were taken as the concentrations of
the benzo[b]xanthone derivatives causing 50 % inhibition of cell viabilities.
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