Preparative-Scale Metabolism of Imipramine Using A. flavipes (ATCC 16795)-A. flavipes was grown in 4.0 liters of medium contained in 40 500-ml erlenmeyer flasks. After a 24-hr incubation of the Stage I1 cultures, a total of 800 mg of 1-HCl was distributed evenly among the cultures; incubation was continued for 13 days (200 rpm, room temperature). The cultures were harvested by homogenization of the whole culture, followed by filtration (through buchner funnel). The aqueous culture filtrate (pH 8) was extracted with 10 500-ml portions of chloroform, and the combined chloroform layers were dried (sodium sulfate) and evaporated in uacuo to leave 663 mg of a brown oily residue.
Preparative TLC of 330 mg of the residue was carried out using silica gel G plates (2.0 mm thick, 20 X 20 cm) developetl in ethyl acetatemethanol-ammonium hydroxide (81:15:4). The major band, corresponding to the N-oxide, was located by UV light and scraped off, and the silica gel was extracted exhaustively with 10% methanol-chloroform. Evaporation of the solvent afforded 112 mg of crude V, which was crystallized from benzene-hexane as white needles, mp 76-78O [lit. ( 24) mp 75-79OI. Direct comparison of V with an authentic sample of imipramine-N-oxide showed the two samples to be identical (melting point, mixed melting point, TLC, co-TLC, and superimposable IR spectra).
Preparative-Scale Metabolism of Imipramine (I) Using M. griseo-cyanus (ATCC 1207a)-A total of 1 g of I-HCl was fed to M. griseo-cyanus as described for C. blakesleeana and worked up as described for F. orysporum. The chloroform residue (664 mg) was chromatographed over aluminas (90 g) using chloroform followed by an increasing percentage of methanol in chloroform. A total of 496 mg of I and 45 mg