Antibiotic R2, a new angucyclinone compound from Streptosporangium sp. Sg3

Abstract

Angucyclinones are a subclass of angucyclines, which are defined as microbial quinone natural products related to tetracyclines and anthracyclines and bearing, as characteristic structural feature, a tetracyclic benz[a]anthracene ring system assembled in an angular manner.1 This structural moiety is biosynthetically derived from a decaketide chain formed via the polyketide biosynthetic pathway. The term ‘angucycline’ includes molecules with hydrolysable sugar moieties, whereas ‘angucyclinone’ refers to a sugarless compound or a compound with a C-glycosidic linked sugar moiety.1,2 Angucyclines are isolated from the fermentation broth of actinomycetes, mostly from the genus Streptomyces, but also from Actinomadura, Nocardia and Streptosporangium genera.1 Angucyclines show a broad spectrum of biological activities including antitumor,1,2 antibacterial, antifungal, antiviral,3,4 enzyme inhibitory5,6 and platelet aggregation inhibitory properties.7,8 In a continuous search for new bioactive compounds from actinomycetes other than the genus Streptomyces, several Saharan soil samples collected in arid ecosystems were explored.9–11 Among the isolates, we were interested by a new actinomycete strain belonging to the genus Streptosporangium and producing a new compound (R2) identified as a new angucyclinone. This paper describes the isolation, structure elucidation and antimicrobial activities of compound R2. Details on the isolation and the taxonomy of the producing organism Streptosporangium sp. Sg3 were described in a previous paper.11 To isolate compound R2, Streptosporangium sp. Sg3 was cultivated at 30 1C for 9 days in Erlenmeyer flasks (500 ml) containing 100 ml of ISP2 broth (yeast extract 4 g, malt extract 10 g, glucose 4 g, in 1 l distilled water, pH 7.2) on a rotary shaker (250 r.p.m.). The cultures (8 l) were centrifuged and filtered to remove mycelium. The culture filtrate was extracted with an equal volume of n-butanol to generate a crude extract (1.9 g). The latter was fractionated by size exclusion chromatography on Sephadex LH-20 (75% MeOH in H2O), resulting in five fractions, I–V. Fraction IV including R2, was of red color and exhibited an antibacterial activity. It was subjected to semipreparative reversed-phase HPLC using an Interchim UP5ODB column (250 7.8 mm) (Interchim, Montlucon, France) and developed using a continuous grade from 20 to 75% MeOH in H2O (UV detection at 220 nm), yielding three active fractions (1, 2, 3). The major peak 2 with the main antibacterial activity was subjected to repeated HPLC to yield 3.5 mg of compound R2. Compound R2 was obtained as a red powder (optical rotation [a]D +56 (c 0.11, MeOH). The ESI-MS spectrum contained an ion peak at m/z 475.1 [M–H] , and its molecular formula was determined by HRESI-MS analysis as C26H19O9 (calcd 475.43 for (M–H) , found 475.43). The UV absorption maximum at 535 nm was because of the red color and suggested a quinone chromophore. Absorptions at 3259, 2949 and 1721 cm 1 in the IR spectrum of R2 were characteristic of hydroxy, methyl and carbonyl groups, respectively. R2 was soluble in MeOH and DMSO, and insoluble in chloroform, n-hexane and H2O. The structure of R2 (Figure 1) was determined by 1H and 13C NMR spectroscopy and by using 1H–1H COSY45, 1H–13C HMQC and 1H–13C HMBC experiments. The 1H and 13C chemical shifts of compound R2 are given in Table 1. The 13C and Heteronuclear SingleQuantum Correlation spectra showed 26 carbon signals with a large number of quaternary carbons (17 out of 26). From the 13C data, it was possible to discern two keto-carbonyl groups (dc 188.4 and 186.5), one carboxylic acid group (dc 174.1), 19 sp2-hybridized carbons (dc from 160.2 to 114.7), two sp3-hybridized carbons bearing an electronegative heteroatom (dc 73.0 and 72.5), one sp3-hybridized carbon (dc 22.6) and two methyl groups (dc 22.4 and 12.4). In CD3OD, the 1H NMR spectrum revealed two ortho-coupled aromatic protons (dH 7.82 and 7.66, 2H, J1⁄47.5 Hz), two aromatic protons (dH 8.24 and 7.02, 2H, s), an AB system (dH 4.45 and 4.36, JAB1⁄410.7 Hz), an ethyl group (dH 2.83 and 1.32, 5H, J1⁄47.4 Hz) and a methyl group (dH 2.70, 3H, s). In DMSO, NMR signals were significantly broader but the