SpI080624G1f01 was cultivated in 50-ml test tubes containing 15 ml of potato dextrose broth (24 g l−1 potato dextrose; BD Biosciences, San Jose, CA, USA). The test tubes were shaken on a reciprocal shaker (320 r.p.m.) at 27 °C for 2 days. Aliquots (5 ml) of the culture were transferred to 100-ml Erlenmeyer flasks containing 3 g of brown rice (Hitomebore, Miyagi, Japan), 6 mg of Bacto-yeast extract (BD Biosciences), 3 mg of sodium tartarate, 3 mg of potassium hydrogen phosphate and 9 ml of water and were incubated in static culture at 27 °C for 14 days.

The mycelia (gathered from 50 flasks) was extracted with 80% aqueous Me2CO (1400 ml) and filtered. After removal of Me2CO from the extract, the aqueous concentrate was extracted with EtOAc (1 liter × 3). The obtained organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The dried residue (4.9 g) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-30, Shoko Scientific Co., Yokohama, Japan) and eluted with a gradient system of n-hexane–EtOAc (0–25% EtOAc) followed by the stepwise solvent system of CHCl3–MeOH (0, 2, 5, 10, 20, 30 and 100% MeOH). The 5% MeOH eluate (534 mg) was applied on a Sephadex LH-20 column (GE Healthcare BioSciences AB, Uppsala, Sweden) and eluted with CHCl3–MeOH (1:1) to obtain crude 1 (143 mg). The active eluate was purified by preparative reversed-phase HPLC using a CAPCELL PAK C18 MGII column (5.0 μm, 20 i.d. × 150 mm; Shiseido, Tokyo, Japan) with 60% aqueous MeOH containing 0.1% formic acid (flow rate, 10 ml min−1) to yield 1 (2.9 mg, retention time (Rt), 27.2 min), along with JBIR-59 (14.6 mg, Rt, 19.3 min) and bisorbibutenolide4 (2.6 mg, Rt, 32.8 min).

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