New Aminophenoxazinones from a Marine Halomonas sp.: Fermentation, Structure Elucidation, and Biological Activity

Abstract

The addition of anthranilic acid to the culture medium of the marine derived Halomonas sp. strain GWS-BW-H8hM completely altered the secondary metabolite pattern relative to the standard conditions. The red-orange color of the culture filtrate extract was the result of the production of 2-aminophenoxazin-3-one (1), chandrananimycin C (5) and three new derivatives of 1 with a previously unknown substitution pattern: 2-amino-, 2-amino-8-benzoyl-, and 2-amino-8-(4-hydroxybenzoyl)-6-hydroxyphenoxazin-3-one (2ϳ4). The compounds were determined to have antibacterial and cytotoxic activities; a mode of action other than DNA intercalation is discussed.Keywords aminophenoxazones, structure elucidation, marine bacteria, Halomonas, antibiotics IntroductionThe strain GWS-BW-H8hM belongs to the genus Halomonas and hence to the g -proteobacteria. It was isolated from a water sample collected in the East Frisian Wadden Sea and its identification is described in related studies [1]. The strain was found to produce 3-(4Ј-hydroxyphenyl)-4-phenylpyrrole-2,5-dicarboxylic acid (HPPD-1), 3,4-di-(4Ј-hydroxy-phenyl)pyrrole-2,5-dicarboxylic acid (HPPD-2) and the literature-known indole derivatives 3-(hydroxyacetyl)-indole, indole-3-carboxylic acid, indole-3-carboxaldehyde, and indole-3-acetic acid. Both HPPD-1 and HPPD-2 show potent antitumor-promoting activities. Their production could be improved by optimization of the fermentation conditions [1].Addition of anthranilic acid to the medium caused a black color of both cells and culture broth. Besides nonsoluble pigments-probably melanin-and known colorless diketopiperazines (6ϳ10) orange-colored metabolites were produced. The latter were soluble in organic solvents and showed a typical UV absorption at 430 nm. From the colored fraction, five 2-aminophenoxazin-3-one derivatives (1ϳ5, Fig. 1) were isolated. The new metabolites 2, 3 and 4 exhibit a previously unknown substitution pattern of the core structure (1). The present work focuses on the production, isolation and structure elucidation of the new aminophenoxazinones and their biological activities. Results Production and IsolationHalomonas sp. strain GWS-BW-H8hM produces pigments, which cannot be obtained under standard conditions [1], when grown in artificial seawater containing peptone, yeast extract, and anthranilic acid as carbon and nitrogen sources.A medium containing tryptone 1%, yeast extract 0.5%, glycine 0.05%, and anthranilic acid 0.05% was applied in a bioreactor cultivation. Fig. 2 presents typical data of the cultivation: The maximum for biomass of 1.6 g/liter was reached after 8 hours. During cultivation, a significant increase of OD 540 for the cell suspension was observed. Compared with the small values of biomass, this increase is due to the pigment formation, which goes parallel to the consumption of anthranilic acid. The physiological activity, indicated by the pO 2 electrode data, the oxygen consumption rate (Q O 2 ), and the carbon dioxide production rate (Q CO 2 ), was connected with cell growth. Fr...

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