JBIR-65, a new diterpene, isolated from a sponge-derived Actinomadura sp. SpB081030SC-15

Abstract

seed culture in test tubes was agitated on a reciprocal shaker (355 r.p.m.) at 271C for 2 days. Aliquots (2.5 ml) of the broth were transferred to 500-ml baffled Erlenmeyer flasks containing 100 ml of a production medium consisting of glycerin 2.0%, molasses 1.0%, casein 0.5% and CaCO3 0.4%, pH 7.2 (adjusted before sterilization) and cultured on a rotary shaker (180 r.p.m.) at 271C for 5 days. The mycelia in the fermentation broth (2 l) was separated by centrifugation and then extracted with 80% acetone. The extract was evaporated in vacuo to remove the acetone, and the aqueous residue was extracted with EtOAc. The organic layer was dried over Na2SO4 and evaporated to dryness. The residue (700 mg) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-60, Moritex, Tokyo, Japan) and eluted with a gradient system of n-hexane–EtOAc (0–30% EtOAc) and CHCl3-MeOH (0–50% MeOH), successively. The 3% MeOH-eluted fraction (50.1 mg) was further purified by preparative reverse-phase HPLC using an L-column 2 column (20 i.d.150 mm; Chemical Evaluation and Research Institute, Tokyo, Japan) with 60% MeOH-H2 Oc ontaining 0.1% formic acid (flow rate: 10 mlmin 1 ) to yield 1 (0.22 mg,