Studies on differentiation inducing substances of animal cells. I. Differenol A, a differentiation inducing substance against mouse leukemia cells.

Sir: We have established a screening system for differentiation inducing substances of animal cells1). Using this system, we have recently isolated a new substance from culture filtrate of a streptomycete, which induces differentiation of mouse leukemia cells. The active substance was named differenol A. Streptomyces sp. ANS-127, the differenol Aproducing organism was isolated from a soil sample collected on our campus in Wako-shi, Saitama and the taxonomic study indicated that it resembles Streptomyces antibioticus. This strain was cultivated in jar fermentors for 90 hours at 28°C with a medium containing 2% glucose, 1% starch, 0.1% meat extract, 0.4% dried yeast, 0.2% NaCl, 0.005% K2HPO4 and 2.5% soy bean meal in tap water. Differenol A was extracted from the culture filtrate (28 liters, pH 8.2) with ethyl acetate at pH 7. The ethyl acetate layer was concentrated under reduced pressure and subjected to silica gel column chromatography with methanol in chloroform (0->100%). Further purification was carried out with HPLC (Nucleosil, 5C18), which was developed with acetonitrile water (55:45). One of the active substances, differenol A, was recrystallized from methanol water to give colorless needles (50 mg). Differenol A is a weakly acidic compound with melting point of 305~3100C. It has two titrable groups with pKa' of 8.6 and 10.7 in 66.7% dioxane. Elementary analysis and FD-mass spectroscopic determination of molecular weight (M+ 270) gave the formula, C15H10O5.