The marine environment has become an attractive new source of chemical diversity for drug discovery, as new bioactive substances are isolated from marine organisms, including phytoplankton, algae, sponges, tunicates and mollusks. 1 Our group has reported the isolation of azaspiracid-22 2 and JBIR-44 3 from marine sponges. Marine microorganisms, especially fungi, 4 are an untapped resource of novel bioactive substances such as diketopiperazine alkaloids, 5 trichodermatides 6 and carbonarones. 7 We have also reported the discovery of new sesquiterpenes, JBIR-27 and JBIR-28; 8 a new aspochracin derivative, JBIR-15; 9 new glycosyl benzenediols, JBIR-37 and JBIR-38; 10 and a new sorbicillinoid, JBIR-59 11 from marine-derived fungi. The Calcarea class of sponges has been recognized as a reliable source of richly bioactive metabolites possessing a 2-aminoimidazole skeleton. [12][13][14] However, the isolation of fungi from calcareous marine sponges has not been reported. Therefore, we attempted to isolate marine fungi from an unidentified calcareous marine sponge and then searched for secondary metabolites in the cultures of the isolated strains. In this study, we report the isolation of Aspergillus sp. fS14 from this marine sponge, and the fermentation, isolation and structure elucidation of two new compounds, designated JBIR-74 (1) and JBIR-75 (2) ( Figure 1a).The producing fungus, Aspergillus sp. fS14, was isolated from the unidentified marine sponge (class, Calcarea) collected offshore of Taketomikita, Ishigaki Island, Okinawa Prefecture, Japan, according to the previously reported method using jewfish (Argyrosomus argentatus) extract agar. 15,16 The sponge was rinsed with sterilized seawater, finely minced with scissors and resuspended in sterilized seawater. A 100 ml aliquot of this suspension was spread on jewfish extract agar plates prepared in 50% (v/v) artificial seawater (Marine art SF-1; Tomita Pharmaceutical, Tokyo, Japan) and supplemented with 35 mg ml À1 of nalidixic acid and 50 mg ml À1 of cycloheximide. Cycloheximide-resistant fungal colonies grew on the agar plates and were transferred to potato dextrose agar slants, where individual strains were maintained. Strain fS14 was cultivated in a 50 ml test tube containing 15 ml of seed medium (2.4 g l À1 Potato Dextrose Broth; BD Biosciences, San Jose, CA, USA). The test tube was shaken on a reciprocal shaker (355 r.p.m.) at 27 1C for 3 days. A 5 ml aliquot of the seed culture was inoculated into a 500 ml Erlenmeyer flask containing 15 g brown rice (Hitomebore, Miyagi, Japan), 30 mg Bacto Yeast Extract (BD Biosciences), 15 mg sodium tartrate, 15 mg potassium hydrogen phosphate and 45 ml water, and incubated in static culture at 27 1C for 14 days.The culture (one flask) was extracted with 80% aq. Me 2 CO (100 ml) and concentrated in vacuo. The aqueous concentrate was extracted with EtOAc (50 mlÂ3). The organic layer was evaporated after drying over Na 2 SO 4 . The residue (0.31 g) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pac...
