J.M e d . Chem. 1987,30, 1928-1934
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particle counter (Coulter Electronics, Inc., Hialeah, FL). The L-1210 cells do not adhere to the culture dish and can be separated from one another by aspirating the culture through the tip of a pipet. Thus dispersed, the cells were suspended in a total volume of 10 mL with a balanced salts solution (Isoton, Coulter Diagnostics, Hialeah, FL) and counted. The B16 cells do attach t o the plastic culture surface. The medium was removed from these cultures, and 0.2 mL of 0.01% crystalline porcine trypsin, 0.1% EDTA in divalent cation-free phosphate-buffered saline was added to each well. After 5 min a t 37 "C, the culture plates were chilled on ice and 0.1 mL of phosphate-buffered saline was added t o each well. The number of cells in the suspensions was then determined. The number of cells per well was plotted as a function of the concentration of the test agent, and the dose that reduced the cell count to 50% of the untreated controls was determined and is reported as the ID50. The IC50(Fg/mL) values for P388 (mouse leukemia, 9PS) and 9KB (human epidermoid carcinoma of the nasopharynx) cytotoxic cell culture activity were determined by following the established protocols of the National Institutes of Health, National Cancer Institute,78 a t the Purdue Cancer Center Cell Culture Laboratory. The KB cell line, derived from a human epidermoid carcinoma (KB-ADL; oral cavity), was supplied by the NCI contractor. The cell culture screen was performed according to the standard protoc01.'~ Samples were dissolved in dimethyl sulfoxide before final dilutions to 100, 10, 0.1, 0.01, 0.001 Wg/mL in the growth
medium. Samples were run in duplicate. The IDE0is the dose of sample that inhibib cell growth to 50% of the untreated control values. The ID, values were obtained by extrapolation from the least-squares fit of the dose-response curve. The PS cell line, derived from a mouse lymphocytic leukemia (P388; spleen and lymph nodes), was supplied by the NCI contractor. The cell culture screen was performed as described above. Each IC,, determination was run in duplicate and was repeated with samples la, l b , 5a, 5b, and 7-10. The IC50values (
