May 2011
Oplopanpheside B (2): Colorless gum, [a]D ꢀ15 (cꢁ0.4, MeOH). UV
679
2
0
Logan, UT, U.S.A.), added with 10% (v/v) fetal bovine serum (Hyclone,
ꢀ
1
l
(MeOH) nm (log e): 217 (2.71), 220 (2.77), 358 (0.03). IR (KBr) cm
:
U.S.A.) in a humidified atmosphere with 5% CO
2
at 37 °C. Cell viability was
max
3
7
440, 2935, 1720, 1602, 1515, 1465, 1422, 1384, 1271, 1224, 1074, 818, determined by recording colorimetric measurements of the amount of insol-
1
13
46. H- and C-NMR spectrometric data are given in Table 1. HR-ESI-MS
uble formazan formed in living cells based on the reduction of MTT (Sigma,
St. Louis, MO, U.S.A.). In brief, before drug addition, 100 ml adherent cells
ꢂ
m/z: 541.1548 [MꢂNa] (Calcd for C H O Na: 541.1528).
2
2
30 14
2
0
Oplopanpheside C (3): Colorless amorphous powder, [a]D ꢀ10.7 (cꢁ were seeded into each well of a 96-well cell culture plate and allowed to ad-
0
.5, MeOH). UV lmax (MeOH) nm (log e): 198 (0.60), 215 (2.84), 219 here for 12 h, while suspended cells were seeded each with initial density of
ꢀ
1
5
(
1
2.87), 261 (1.55). IR (KBr) cm : 3400, 2924, 1714, 1587, 1507, 1455, 1ꢆ10 cells/ml in 100 ml medium. Each tumor cell line was exposed to the
1
420, 1382, 1356, 1333, 1244, 1195, 1127, 1080, 1019, 909, 843, 702. H-
tested compound at various concentrations (0.1, 1.0, 10, 100 mM/l) in tripli-
and C-NMR spectrometric data are given in Table 1. HR-ESI-MS m/z: cates for 48 h, with cis-platin (Sigma, U.S.A.) as positive control. After the
13
ꢂ
5
39.1741 [MꢂNa] (Calcd for C H O Na: 539.1735).
incubation, MTT (100 mg) was added to each well, and the incubation was
stopped after 4 h at 37 °C. The cells were lysed with 100 ml 20% sodium do-
decyl sulfate (SDS)–50% N,N-dimethylformamide (DMF) after removal of
100 ml medium. The optical densities (A) of 570 nm were measured using a
Bio-Rad 580 enzyme-linked immunosorbent assay microplate reader. The
cytotoxicity was calculated as cytotoxicity (%)ꢁ[(A570 of untreated
cellsꢀA570 of treated cells)/A570 of untreated cells]ꢆ100%.
2
3
32 13
Acid Hydrolysis Compounds 1 (2 mg) and 2 (2 mg) were hydrolyzed
ꢀ1
with 2 mol·l TFA (1 ml) in a sealed glass tube with screw cap which were
filled with N at 100 °C for 2 h, respectively. The hydrolyzed solution was
2
evaporated to dryness under 50 °C and then methanol (2 ml) was added for
further evaporation and complete removal of trifluoroacetic acid (TFA). The
hydrolysate was used for derivatization.
Sugar Analysis The stock solution of standard monosaccharides (1 ml)
was treated with hydroxylamine hydrochloride-pyridine solution (1 ml) in a
sealed glass tube at 90 °C for 30 min. Acetic anhydride (1 ml) was added and
continuing heated for another 30 min after the solution cooling to room tem-
perature. The cooled solution was evaporated to dryness under diminished
pressure at 50 °C. The residue was dissolved in dry methanol (2 ml) was
added. The mixture was filtered through 0.45 mm syringe filter (Agilent
Technologies) prior to injection into GC-MS system. The hydrolysate was
reacted with hydroxylamine hydrochloride and acetic anhydride to form the
derivatives directly as in the procedures mentioned above for sugar determi-
nation. GC-MS was performed on an Agilent 6890 gas chromatography
instrument coupled with an Agilent 5973 mass spectrometer (Agilent Tech-
nologies, Palo Alto, CA, U.S.A.). A HP-5MS capillary column (30 mꢆ
Acknowledgements The authors are grateful to Prof. Wen-Cai Ye for
HR-ESI-MS measurement. This work was supported in part by the
NIH/NCCAM (AT004418 and AT005362 to C. S. Yuan) and University of
Macau (UL015/09-Y1 to S. P. Li) grants.
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ml·min . The mass spectrometer was operated in electron-impact (EI)
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solution. The control sample was the mixture of the test sample with solvent
instead. The sample and control blanks were the mixtures of sample and
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S
SB
C
CB
S
ASB, A , and A are the absorbance of sample, sample blank, control, and
C
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Huang, Wei-Hua
