AT 2433 A1

The indolo[2,3-a]carbazole glycosides are potent antitumor antibiotics currently undergoing clinical trials for the treatment of numerous types of cancer. AT2433-A1 is the most complex member of this family of compounds possessing a unique disaccharide with a sensitive aminodeoxysugar and an unsymmetric aglycon. The synthesis of this natural product requires a method for glycosylation that sets the stereochemistry of the anomeric center and the regiochemistry of the aglycon. These goals were accomplished by carrying out the Mannich cyclization of a bis-3, 4-(3-indolyl)succinimide to give a key class of indoline intermediates that could be glycosylated stereoselectively with complex carbohydrates without hydroxyl protection or activation. The regiochemistry of the Mannich cyclization was precisely controlled by choosing between kinetic or thermodynamic conditions. This strategy culminated in the first synthesis of the antitumor antibiotic AT2433-A1.

The antibiotics AT2433-A1 and AT2433-B1 are two indolocarbazole diglycosides related to the antitumor drug rebeccamycin known to stabilize topoisomerase I-DNA complexes. This structural analogy prompted us to explore the binding of four indolocarbazole diglycosides with DNA and their capacity to interfere with the DNA cleavage-reunion reaction catalyzed by topoisomerase I. The molecular basis of the drug interaction with double-stranded DNA and with purified chromatin, with particular emphasis on the role of the carbohydrate moiety, was investigated by means of complementary spectroscopic techniques, including surface plasmon resonance and electric linear dichroism. We compared the DNA binding properties, sequence recognition, and effects on topoisomerase I-mediated DNA relaxation and cleavage of AT2433-A1 bearing a 2,4-dideoxy-4-methylamino-L-xylose residue, its dechlorinated analog AT2433-B1, the diastereoisomer iso-AT2433-B1 with an inverted aminosugar residue, and compounds 5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, 12-beta-D-glucopyranosyl-12,13-dihydro-6-methyl (JDC-108) and 5H-indolo[2,3-a]pyrrolo[3, 4-c]carbazole-5,7(6H)-dione, 12-(6-O-alpha-D-galacto-pyranosyl-beta-D-glucopyranosyl)-12,13-dihydro-6-methyl (JDC-277) with an uncharged mono- and disaccharide, respectively. The two antibiotics AT2433-A1 and AT2433-B1 proved to be highly cytotoxic to leukemia cells and this may be a consequence of their tight intercalative binding to DNA, preferentially into GC-rich sequences as inferred from DNase I footprinting studies and surface plasmon resonance measurements. Like the diastereoisomer iso-AT2433-B1, they have no inhibitory effect on topoisomerase I, in contrast to the uncharged diglycoside JDC-277, which stimulates DNA cleavage by the enzyme mainly at TG sites, as observed with camptothecin. Cytotoxicity measurements with CEM and CEM/C2 human leukemia cell lines sensitive and resistant to camptothecin, respectively, also suggested that topoisomerase I contributes, at least partially, to the mechanism of action of the neutral diglycoside JDC-277 but not to that of the cationic AT2433 compounds. Together, the results indicate that sequence-selective DNA interaction and topoisomerase I inhibition is controlled to a large extent by the stereochemistry of the diglycoside moiety.

Compounds AT2433-A1 (A1), AT2433-A2 (A2), AT2433-B1 (B1), and AT2433-B2 (B2) were isolated from the cultured broth of Actinomadura melliaura sp. nov. (SCC 1655). Structurally these materials are closely related to rebeccamycin (1), an indolocarbazole antitumor antibiotic. A1, A2, B1, and B2 were active against Staphylococcus aureus A9537, Streptococcus faecalis A20688, Streptococcus faecium (ATCC 9790), Micrococcus lutea (ATCC 9341), Bacillus subtilis (ATCC 6633). A1 and B1 were active against P388 leukemia in mice.