Three eremophilane derivatives, MBJ-0011, MBJ-0012 and MBJ-0013, from an endophytic fungus Apiognomonia sp. f24023

The producing microorganism, Apiognomonia sp. f24023, classified based on partial sequence of the D1/D2 region of the 28S ribosomal RNA gene (data not shown), was isolated from a plant growing in Iwata, Shizuoka Prefecture, Japan. The strain was cultivated in 250-ml Erlenmeyer flasks, each containing 25 ml of a seed medium consisting of potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc., Hokkaido, Japan) 2%, glucose (Junsei Chemical, Tokyo, Japan) 2%, soybean powder (Honen SoyPro, J-Oil Mills, Tokyo, Japan) 2%, KH₂PO₄ 0.1%, and MgSO₄·7H₂O 0.05%. The flasks were shaken on a rotary shaker (220 r.p.m.) at 25 °C for 3 days. Aliquots (0.5 ml) of the broth were transferred to 500-ml Erlenmeyer flasks containing 50 ml of a production medium consisting of fructose (Junsei Chemical) 1.5%, xylitol (Junsei Chemical) 1.5%, maltose monohydrate (Junsei Chemical) 1.5%, galactose (Wako Pure Chemical Industries, Osaka, Japan) 1.5%, soybean powder 2%, gluten meal (Nihon Shokuhin Kako, Tokyo, Japan) 1%, yeast extract powder (Oriental Yeast, Tokyo, Japan) 0.5%, ZnSO₄·7H₂O 0.02%, MgSO₄·7H₂O 0.02%, FeSO₄·7H₂O 0.02%, and 3-indoleacetic acid 0.002%, pH 6.0 (adjusted before sterilization), and were cultured on a rotary shaker (220 r.p.m.) at 25 °C for 4 days. After fermentation, the whole culture broth (2 l) was extracted with an equal volume of n-BuOH. The n-BuOH extract was partitioned between CHCl₃ (350 ml × 3) and brine (350 ml). The organic layer was evaporated and the concentrate (868 mg) was fractionated using medium-pressure silica gel chromatography (Purif-Pack SI-30, Shoko Scientific, Yokohama, Japan) with the 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). Compound 1 (2.5 mg) was obtained after purification of the 2% MeOH fraction (21 mg) by 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, retention time (Rt)=19.5 min). The 5% MeOH fraction (102 mg) was applied to reversed-phase medium-pressure column (Purif-Pack ODS-30, Shoko Scientific, Yokohama, Japan) and developed with the stepwise H₂O–MeOH solvent system (70, 80 and 100% MeOH). The 80% MeOH eluate (25.6 mg) was subjected to HPLC (CAPCELL PAK C18 MGII column, 70% aqueous MeOH containing 0.1% formic acid, flow rate: 10 ml min⁻¹) to yield 2 (17.2 mg, Rt=14.6 mim) and crude 3 (8.3 mg, Rt=17.0 min). The crude material containing 3 was then re-chromatographed with the same system to give pure 3 (1.2 mg, Rt=17.0 min).

The structure determination of 1 was carried out by dividing it into two partial moieties on the basis of DQF-COSY and CT-HMBC as follows. The sequence from an oxymethine proton H-3 (δ_H 5.38) to a methyl proton H₃-14 (δ_H 0.99) through a methine proton H-4 (δ_H 1.88) was observed in the DQF-COSY. Long-range ¹H–¹³C couplings from the methyl proton H₃-14 to an oxymethine carbon C-3 (δ_C 74.0), a methine carbon C-4 (δ_C 43.7) and a quaternary carbon C-5 (δ_C 37.9), and from a methyl proton H₃-15 (δ_H 1.21) to C-4, C-5, a methylene carbon C-6 (δ_C 39.6) and an olefinic quaternary carbon C-10 (δ_C 160.1) revealed the sequence from C-3 to C-6 as shown in Figure 2a. HMBC correlations from an olefinic methine proton H-2 (δ_H 6.01), which was spin coupled to an olefinic methine proton H-1 (δ_H 6.22), to C-4 and the olefinic quaternary carbon C-10, and from the olefinic proton H-1 to C-3, C-5 and C-10 established the six-membered cyclic moiety. Long-range ¹H–¹³C coupling from methylene protons H₂-6 (δ_H 2.01 and 1.93) to an α,β-unsaturated ketone carbonyl carbon C-8 (δ_C 198.5), which in turn long-range coupled to a methine proton H-7 (δ_H 3.25), was observed. Together with these HMBC correlations, long-range couplings from an olefinic proton H-9 (δ_H 5.80) to C-5, a methine carbon C-7 (δ_C 50.6) and an olefinic carbon C-1 (δ_C 129.6) revealed an 2-decalone moiety. Furthermore, long-range couplings from a methyl proton H₃-13 (δ_H 1.72) to C-7, an olefinic quaternary carbon C-11 (δ_C 143.3) and an exomethylene carbon C-12 (δ_C 115.0) established a 6-oxygenated 4,4a,5,6-tetrahydro-4a,5-dimethyl-3-(1-methylethenyl)-2(3H)-naphthalenone moiety (an eremophilane skeleton) as shown in Figure 2a.

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