was allowed to sit in the cold for 1 hour the flask was decanted and the precipitate was washed with cold water then 200 ml of 15 acetic acid water was added and the mixture stirred for 2 hours the solution was filtered and the solute collected and ammonia hydroxide or sodium hydroxide 15 solution was added slowly with stirring until a precipitate formed the ppt was collected either by filtration or centrifugation the ppt was dissolved in acetonitrile with heating and filtered the solvent was evaporated and the solid dried under p 2 o 5 in a high vacuum this material was used in cytotoxicity assay and material was sent to midwest research institute for analysis of the structure and purity testing of compounds in the mtt assay in a 72 well plate the bot 2 human breast cancer cells were allowed to grow for 24 hours following this growth time dilutions of the drug was added the cells were checked for percentage of cell death at 24 and 48 hours this checking was done by visual appearance of the cells rounded up floating cells or disrupted cells indicating cell death cells flattened and attached to the bottom of the tissue culture flask indicated alive and healthy cells at 144 hours mtt visual inspection assay bot 2 cancer cells after treatment with an anthracycline or cytotoxic anthraquinone compounds the dead cells become rounded detach and float the compound is initially dissolved in 0 1 n hydrochloric acid or dilute acetic acid serial 1 to 4 dilutions of the compound are made with buffered tissue culture media and pipetted into a wells of a 72 well microtiter plate determination is made of 100 cell death at 24 48 and 144 hours for each well controls are always run.