Penicillium sp. fS36 was isolated from an unidentified marine sponge collected at a depth of 195 m from a position (i.e., 28° 53′ 35.8″ N, 129° 29′ 57.0″ E) near Takarajima Island, Kagoshima Prefecture, Japan. The sponge was ground using a mortar and pestle. These pieces were suspended in sterile seawater and the suspension spread on manila clam extract agar.11 The isolated strain was maintained on potato dextrose agar medium (BD Biosciences, San Jose, CA, USA). The sequence analysis of ribosomal DNA and internal transcribed spacer region of the producing fungus showed high sequence similarities with Penicillium namyslowskii (AB028190, 100%). The strain was cultivated in 50 ml test tubes containing 15 ml of potato dextrose medium (24 g l−1 potato dextrose; BD Biosciences). The test tubes were shaken using a reciprocal shaker (320 r.p.m.) at 27 °C for 2 days. Aliquots (5 ml) of the culture were transferred to 500 ml Erlenmeyer flasks containing brown rice (15 g, Hitomebore, Miyagi, Japan), Bacto-yeast extract (30 mg, BD Biosciences), sodium tartarate (15 mg), dipotassium hydrogenphosphate (15 mg) and water (45 ml) and were incubated in a static culture at 27 °C for 14 days.

The culture (10 flasks) was extracted with 80% aqueous Me2CO (1 l) and the extract was filtered. After concentration in vacuo, water (600 ml) was added to the residual aqueous solution followed by extraction with EtOAc (700 ml × 3). The organic layer was dried over anhydrous Na2SO4 and evaporated. The residual portion (2.5 g) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-30, Shoko Scientific Co., Yokohama, Japan) and developed with a gradient system of n-hexane–EtOAc (0–25% EtOAc) followed by a stepwise solvent system comprising CHCl3–MeOH (0, 2, 5, 10, 20, 30 and 100% MeOH). The fractions including 1–3 were collected by LC-MS monitoring. The 5% MeOH eluate (230 mg) was applied on a Sephadex LH-20 column (GE Healthcare BioSciences AB, Uppsala, Sweden) and developed with CHCl3–MeOH (1:1) to obtain the crude material (38.1 mg). Compounds 1 (9.9 mg), 2 (1.7 mg) and 3 (1.7 mg) were obtained by preparative reversed-phase HPLC on a CAPCELL PAK C18 MGII column (5.0 μm, internal diameter 20 × 150 mm; Shiseido, Tokyo, Japan) with 55% aqueous MeOH containing 0.1% formic acid (flow rate: 10 ml min−1; retention times: 25.4 min for 1; 17.4 min for 2; 12.8 min for 3).

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