In a previous paper1*, we reported that Streptomyces sp. RK-286 produced a novel inhibitor of protein kinase C (PKC), RK-286C (4'-demethylamino-4/-hydroxystaurosporine). In the course of the fermentation, a new PKC inhibitor designated RK-286D was isolated as a minor component from the mycelial cake of Streptomyces sp. RK-286. In this paper, we report that RK-286D is a novel indolocarbazole antibiotic structurally related to RK-286C. The producing strain was inoculated into a 500-ml cylindrical flask containing 70ml of seed medium (glucose 2%, soybeanmeal 2.5%, soluble starch 1%, meat extract 0.1%, dried yeast 0.4%, K2HPO4 0.005%, and NaCl 0.2%, adjusted to pH 7.3), and cultured at 28°C for 3 days on a rotary shaker (250rpm). The seed culture was transferred to the production medium (18 liters, glucose 0.1%, soybean meal 1%, malt extract 1%, oatmeal 0.5%, RPMI-1640 medium Nissui 0.25%, K2HPO4 0.1%, and MgSO4 0.01%, adjusted to pH 7.2) in a 30-liter jar fermenter. The fermentation was carried out at 28°C for 7 days under agitation (250rpm) andaeration (1 5 liters/minute). The acetone extract of mycelial cake was concentrated under reduced pressure and the resulting aqueous solution was extracted with EtOAc. After concentration of the EtOAc layer, the oily residue was applied to a silica gel column (packed in CHC13 - MeOH, 20 : 1). Active fractions were eluted with CHCl3-MeOH (10:1). The fractions contained staurosporine2), RK-286C3) and RK-286D. RK-286D enriched fractions were collected and concentrated under reduced pressure. The residue was added to a Sephadex LH-20 column equilibrated with 100% MeOH, and developedwith the same solvent system. Active fractions were combined and concentrated to yield a yellow powder. The powder was finally purified by HPLC on a reverse-phase column (Capcell Pak C18, MeOH-0.1% NH4OH-H2O, 80:10:10). Four mg of pure RK-286Dwere obtained from 18-literfermentation broth. RK-286D is a pale yellow powder with mp 225 ~230°C (dec). It is optically active, [a]£° -60.0° (c 0.13, MeOH). The molecular formula was established as C26H23N3O4 based on HREI-MS (M+ m/z 441.1691, zl+0.4mmu). FD-MS gave a parent peak at 442 (M+H)+. UV data ofRK-286D are similar to those of RK-286C; UYl^Hnm (e) 364 (ll,466), 347 (13,230), 335 (20,286), 320 (sh), 290 (98,785), 278 (sh), 233 (39,690). RK-286D showed positive color reactions to Rydon-Smith and anisaldehyde - H2SO4tests, and negative to ninhy-drin and ferric chloride reagents. *H NMRdata are summarized in Table 1 and the assignments are in accord with those for RK-286C and staurosporine3). Decoupling experiment (Fig. 1) and the NOE difference spectrum revealed that the sugar moiety of RK-286D is digitoxose, attached to N-13 of the chromophore (Fig. 2). The digitoxopyranosyl moiety is in the lC4 conformation, which is presumably due to the steric effect of the bulky aglycone moiety. The absolute stereochemistry remains to be determined. However, it is considered that RK-286Dis a shunt metabolite in the biosynthesis of staurosporine and RK-286C (4'-demethylamino-4'-hydroxystaurosporine). Inhibitory activities of RK-286D against bleb formation40 induced by PDBu and in vitro PKC activity were compared with RK-286Cand staurosporine (Table 2). Staurosporine showed the strongest inhibiton in both assays, and RK-286D showed the weakest inhibition. Since the chromophore is the same in the three compounds, the results suggest that the sugar moiety of indolocarbazole group antibiotics is important for their biological activity. Antimicrobial activity of RK-286D was not observed at a concentration of