166
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
[a]2D0 ¼ ꢀ13.4 (c ¼ 0.5, pyridine). IR (KBr): 3426 (OH), 1738 (C¼O), 1631 (C¼C), 1266, 1215 (SO3Na).
1H- and 13C-NMR: Tables 4 and 2. ESI-MS (pos.): 1325 ([MþNa]þ ). ESI-MS (neg.): 1279 ([MꢀNa]ꢀ ).
HR-ESI-MS (pos.): 1325.6123 ([MþNa]þ , C55H84Na3O28S2þ ; calc. 1325.6128).
Acid Hydrolysis. The glycoside 1 or 2 (1 mg) was heated in 2m CF3COOH (1 ml) at 1208 for 2 h. The
mixture was evaporated to dryness, and the residue was partitioned between CH2Cl2 and H2O. The aq.
phase was concentrated in vacuo. Then, pyridine (1 ml) and NH2OH·HCl (2 mg) were added to the dried
residue, and the mixture was stirred at 908 for 30 min. After that time, 1 ml of Ac2O was added, and the
heating at 908 was continued for another hour. The soln. was concentrated, and the resulting aldononitrile
peracetates were analyzed by GC/MS. The carbohydrates were determined by comparing the retention
times and MS behavior with standard aldononitrile peracetates prepared from authentic sugars by the
same procedure performed for the sample. Xylose, quinovose, and 3-O-methyl-glucose were identified in
a ratio of 2 :1:1.
Desulfation of Pentactaside B (1) and Pentactaside C (2). Glycoside 1 or 2 (10 mg) was dissolved in
pyridine/dioxane 1:1 (3.0 ml) and heated under reflux for 4 h. The mixture was partitioned between H2O
and BuOH. The BuOH extract was evaporated and the residue purified by HPLC eluting with 80%
MeOH at a rate of 1.5 ml/min to yield the pure 1a (8 mg) or 2a (7.5 mg).
Didesulfopentactaside B (¼(3b,9b,16b)-3-{[3-O-Methyl-b-d-glucopyranosyl-(1!3)-b-d-xylopyra-
nosyl-(1!4)-6-deoxy-b-d-glucopyranosyl-(1!2)-b-d-xylopyranosyl]oxy}-18-oxo-18,20-epoxylanosta-
7,24-dien-16-yl Acetate; 1a). White amorphous powder. M.p. 214–2168. [a]2D0 ¼ ꢀ30.8 (c¼0.5, pyridine).
1H- and 13C-NMR: Tables 3 and 5. ESI-MS (pos.): 1121 ([MþNa]þ ). ESI-MS (neg.): 1097 ([MꢀH]ꢀ ).
Didesulfopentactaside C (¼(3b,9b,16b)-3-{[3-O-Methyl-b-d-glucopyranosyl-(1!3)-b-d-xylopyra-
nosyl-(1!4)-6-deoxy-b-d-glucopyranosyl-(1!2)-b-d-xylopyranosyl]oxy}-18-oxo-18,20-epoxylanosta-
7,25-dien-16-yl Acetate; 2a). White amorphous powder. M.p. 222–2258. [a]2D0 ¼ ꢀ26.7 (c ¼ 0.5, pyridine).
1H- and 13C-NMR: Tables 3 and 5. ESI-MS (pos.): 1121 ([MþNa]þ ). ESI-MS (neg.): 1097 ([MꢀH]ꢀ ).
Cytotoxicity Tests. The cytotoxicities of 1 and 2 against leukemic cells (P388), human colon cancer
(HCT-116), human breast cancer (MCF-7), human gastric cancer (MKN-28), and human lung cancer
(A-549) cells were evaluated by the sulforhodamine-B (SRB) assay, as described in [12], with the
anticancer agent 10-hydroxycamptothecin as a positive control. Briefly, the target tumor cells were grown
to log phase in RPMI-1640 medium containing 10% fetal bovine serum (FBS). After dilution to 4ꢁ104
cells/ml with complete medium, 90 ml of the obtained cell suspension was added to each well of a 96-well
culture plate. Cultures were pre-incubated for 24 h in a humidified 5% CO2 atmosphere at 378. Then,
control or test soln. (DMSO as solvent; 10 ml) was put into each well, and the plate was incubated for
additional 72 h. At the end of exposure, the cells were fixed by addition of 50 ml of cold 50% CCl3COOH
at 488 for 1 h, and the plates were washed with tap water (5ꢁ ), and air-dried. Then, 0.4% SRB soln.
(50 ml) in 1% AcOH was added, and staining was allowed to proceed for 30 min. The residual dye was
washed out with 1% AcOH (4ꢁ ), and the plates were air-dried. To each well, 10 mm non-buffered Tris
soln. (150 ml) was added, and the optical density of each well was measured with a microplate reader at
520 nm. The activities of 1, 2, and the positive control were determined at 100, 10, 1, 0.1, and 0.01 mm (each
concentration being tested in triplicate). Data were calculated as percent inhibition of I according to the
formula: I¼(100ꢀ(ODt/ODs)ꢁ100), where ODt and ODs are the mean optical densities of the test
compounds and the solvent control, resp. The concentration inducing 50% inhibition of cell growth (IC50)
was determined graphically for each experiment by curve fitting using the Prism 4.0 software (GraphPad)
and the equation derived by DeLean et al. [13]. IC50 Values represent the mean of three independent
experiments, and are expressed as meanꢂS.D. using Studentꢂs t-test.
REFERENCES
[1] V. A. Stonik, G. B. Elyakov, in ꢃBioorganic Marine Chemistryꢂ, Ed. P. J. Scheuer, Springer, Berlin,
1988, pp. 43–86.
[2] A. P. Murray, C. Muniaꢄn, A. M. Seldes, M. S. Maier, Tetrahedron 2001, 57, 9563.
[3] Z.-R. Zou, Y.-H. Yi, H.-M. Wu, J.-H. Wu, C.-C. Liaw, K.-H. Lee, J. Nat. Prod. 2003, 66, 1055.