Metabolic removal of sulfur from 1-methylisatin 3-thiosemicarbazone.

The efficacy of 1-methylisatin 3-thiosemicarbazone for the inhibition of neurovaccinia virus in mice has been documented by D. J. Bauer and P. W. Sadler (Brit. J. Pharmacol. 15:101, 1960). The inhibition of adenovirus multiplication by this compound has been reported by D. J. Bauer and K. Apostolov (Science 154:796, 1966). The present communication pertains to the microbial conversion of 1-methylisatin 3-thiosemicarbazone to the syn and anti isomers of 1-methylisatin 3-semicarbazone. Cladosporium resinae ATCC 11273 was grown in a complex medium containing (grams per liter): glucose monohydrate (Cerelose, sterilized separately), 50.0; soy flour (Soyafluff), 5.0; yeast extract (Difco), 5.0; malt extract (Difco), 5.0; NaCl, 1.0; KH2PO4, 4.0; K2HPO4, 2.0. Inoculated medium (100 ml) in 500-mi Erlenmeyer flasks was shaken for 2 days at 28 C on a Gump rotary shaker at 250 rev/min; 100 mg of the 3-thiosemicarbazone (Aldrich) was added, and the incubation was continued for 8 days. The contents from 200 flasks were pooled, adjusted to pH 1 with HCl, and extracted with 6 volumes of an acetonechloroform (1:1) mixture. The lower phase was evaporated at 30 C to approximately 5 liters and treated with 200 g of anhydrous Na2SO4. The filtered solvent was evaporated until 26 g of crude amorphous solid remained. For analytical purposes, the necessary samples were examined by means of thin-layer chromatography (TLC), employing silica gel (Brinkman) developed with benzene-methanol (8:2). Compound detection was achieved under ultraviolet light (2,537 A). The substrate and its apparently isomeric metabolites migrated with the following approximate RF values: 1-methylisatin 3-thiosemicarbazone, 0.50; 1-methylisatin 3-semicarbazone (isomer X1379), 0.35; 1-methylisatin 3-semicarbazone (isomer X1378), 0.25. The products were separated in silica gel (Davison 923) columns, with chloroform as the suspending solvent. The sample was added as a solid, after adsorption to powdered silica gel, and column fractions were monitored by TLC. The relative order in which the compounds were removed from the column was directly related to their RF values. The separation and elution of isomer X1379 was achieved with 2% (v/v) acetone in chloroform. After solvent removal at 30 C, isomer X1379 (1,498 mg) was recrystallized from water containing a trace of dimethyl sulfoxide, yielding orange needles which melted (Kofler) at 214 to 216 C. After the elution of metabolite X1379, the solvent was changed to 2% methanol in chloroform, and isomer X1378 was eluted. Minor impurities