Structures and biological activities of indocarbazostain, new inhibitors of NGF-induced neurite outgrowth in PC12 cells.

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During the course of screening for modulators of signal transudation of mammalian cells, we have discovered novel inhibitors, indocarbazostain and indocarbazostatin B from a culture broth of a Streptomyces sp. In our assay conditions. Among 4671 extracts, only one culture produced active compounds of interest. The producing strain identified as Streptomyces sp. TA-0403 was cultivated at 30℃ for 3 days in 500ml flasks each containing 70 ml of the medium. The fermentation broth (35 L) was centrifuged and the mycelial cake was extracted with acetone. After the removal of acetone, pure compounds were isolated. The HR FAB-MS, UV, ^1H and ^<13>C NMR data indicate the chromophore system of these compounds to be heteroatom substituted indolo [2,3-a] pyrrolo [3,4-c] carbazole-5.7(6H)-dione. The structure of indocarbazostain was deduced ^1H-^1H COSY, PFG HMBC, PFG HMQC AND DIF NOE data. The relative and absolute configurations of the sugar moiety of indocarbazostatin were clarified by MM2 and MOPAC calculations and CD analyses. The compounds, indocarbazostain and indocarbazostatin B inhibited NGF-induced neurite outgrowth in PC12 cells at 6nM and 24 nM respectively, whereas K252a inhibited at 200 nM under our assay conditions. K252a showed cytotoxicity at a concentration three times higher than minimal effective concentration. However, indocarbazostatin did not show obvious cytotoxicity to PC12 cells at a concentration nine times higher than the minimal effective concentration.