Fungal secondary metabolites are considered to be good sources for the screening of lead compounds of clinical drugs. Among fungal species, ∼6400 compounds are obtained from various filamentous fungi.1 Among them ∼950, 900 and 350 compounds are isolated from Aspergillus, Penicillium and Fusarium species, respectively. Moreover, several other members of filamentous and endophytic genus (Trichoderma, Phoma, Alternaria, Acremonium and Stachybotrys) also produce several hundreds of bioactive compounds. However, the rate of discovery of novel compounds from these fungi has decreased significantly. To increase the chances of discovering novel compounds, we chose to examine rare endophytic fungi. During our screening program, we isolated a novel cytotoxic compound, MBJ-0005 (1), consisting of a highly oxygenated ergostane-based skeleton, from the culture of Anthostomella eucalyptorum f25427. Anthostomella sp. are endophytic fungi, and notably only a few succinic acid derivatives from Anthostomella sp. have been reported.2 Herein, we report the isolation, structure determination and brief biological activity of 1.

The strain A. eucalyptorum f25427 was isolated from a plant collected in Kochi Prefecture, Japan. The strain was cultivated in 250-ml Erlenmeyer flasks, each containing 25 ml of a seed medium consisting of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc., Hokkaido, Japan), 1% glucose (Junsei Chemical, Tokyo, Japan), 2% soybean powder (SoyPro, J-Oil Mills, Tokyo, Japan), 0.1% KH2PO4 and 0.05% MgSO4·7H2O. 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 containing 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc.), 1% glucose (Junsei Chemical), 2% soybean powder (SoyPro, J-Oil Mills), 0.1% KH2PO4 and 0.05% MgSO4·7H2O, and were cultured on a rotary shaker (220 r.p.m.) at 25 °C for 4 days.

The entire broth (2 l) was extracted using an equal volume of n-BuOH. After concentrating in vacuo, the residual aqueous concentrate was extracted using EtOAc (100 ml × 3). The separated organic layer was dried over Na2SO4 and concentrated in vacuo. The dried residue (320 mg) was applied on a normal-phase medium-pressure liquid column (Purif-Pack SI-30; Shoko Scientific, Yokohama, Japan), and the column was successively eluted using a hexane–EtOAc solvent system (0, 5, 10, 20 and 25% EtOAc) followed by a CHCl3–MeOH solvent system (0, 2, 5, 10 and 20% MeOH). The target eluate (10% MeOH, 35 mg) was further 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 a 2996 photodiode array detector (Waters, Milford, MA, USA) and a 3100 mass detector (Waters) developed with 70% aqueous MeOH containing 0.1% formic acid (flow rate, 10 ml min−1) to yield 1 (7.3 mg, retention time=24.1 min).

Compound 1 was obtained as a colorless amorphous powder: [α]D26 −5.8 (c 0.6, in MeOH); UV λmax (ɛ) in MeOH: 247 (12 800) nm. The molecular formula was determined by high resolution-ESI-MS to be C28H42O7 (found: 489.2856 [M− H]−, calcd for C28H41O7: 489.2852). The presence of hydroxy and α,β-unsaturated carbonyl functionalities was established from its IR spectrum (νmax (attenuated total reflection) 3417, 1678 and 1619 cm−1). The direct connectivity between the protons and carbons was established from the heteronuclear single-quantum coherence spectrum, and the 13C and 1H NMR spectroscopic data for 1 are listed in Table 1. The structure of 1 was established on the basis of its DQF-COSY, TOCSY and constant-time HMBC (CT-HMBC)3 spectra as follows.

Table 1 13C (150 MHz) and 1H (600 MHz) NMR data for 1
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Three substructures were established through analyses of the DQF-COSY and TOCSY spectra, and the connectivity of those three units was established by the CT-HMBC spectrum. The sequence from methylene protons H2-1 (δH 2.41, 1.75) to methylene protons H2-4 (δH 1.99, 1.84) through two oxymethine protons H-2 (δH 4.08) and H-3 (δH 3.99) was revealed by the DQF-COSY spectrum. The 1H–13C long-range couplings from a singlet methyl proton H3-19 (δH 1.15) to a methylene carbon C-1 (δC 32.9), oxygenated quaternary carbons C-5 (δC 80.7) and C-9 (δC 76.3), and a quaternary carbon C-10 (δC 42.5), and from oxymethine proton H-3 and methylene protons H2-4 to the oxygenated quaternary carbon C-5 observed in the CT-HMBC spectrum of 1 revealed a 5-methylcyclohexane-1,2,4-triol substructure (ring A, Figure 1b). The sequence from a methine proton H-14 (δH 2.77) to a doublet methyl proton H3-21 (δH 1.00) through methylene protons H2-15 (δH 1.67, 1.58) and H2-16 (δH 2.05, 1.47), two methine protons H-17 (δH 1.49), and H-20 (δH 1.62), along with a sequence from H-20 to an olefinic proton H-23 (δH 6.66) through methylene protons H2-22 (δH 2.31, 2.08), established a 3-methylhept-1,4,7-yl substructure (Figure 1b). The 1H–13C long-range couplings from a singlet methyl proton H3-18 (δH 0.68) to a methine carbon C-14 (δC 52.7), quaternary carbon C-13 (δC 46.4) and methine carbon C-17 (δC 57.4), along with the HMBC correlations from methine proton H-14 to quaternary carbon C-13 and methine carbon C-17 established the cyclopentane moiety (ring D).

Figure 1
figure 1

(a) Structure of MBJ-0005 (1). (b) Correlations in DQF-COSY, TOCSY (bold lines) and CT-HMBC (arrows) spectra of 1. (c) Key NOESY correlations of 1.

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The 1H–13C long-range couplings from methylene protons H2-11 (δH 2.00, 1.73), which were 1H–1H spin-coupled with methylene protons H2-12 (δH 1.92, 1.72), to olefinic quaternary carbon C-8 (δC 164.3) and quaternary carbon C-13; from methylene protons H2-12 to oxygenated quaternary carbon C-9; from methine proton H-14 to a methylene carbon C-12 (δC 36.2); from methylene protons H2-15 to olefinic quaternary carbon C-8; from methylene protons H2-16 to quaternary carbon C-13; and from singlet methyl proton H3-18 to methylene carbon C-12 established the cyclohexane–cyclopentane moiety (rings C and D).

The 1H–13C long-range couplings from olefinic methine proton H-7 (δH 5.60) to two oxygenated quaternary carbons, C-5 and C-9, were observed. Moreover, the 1H–13C long-range couplings from methylene protons H2-4 to carbonyl carbon C-6 (δC 199.9), from methine proton H-14 to olefinic methine carbon C-7 (δC 121.1), and from singlet methyl proton H3-19 to oxygenated quaternary carbon C-9 established the 7-en-6-one system in the steroid nucleus.

As described previously, the sequence C21–C23 was established by DQF-COSY spectrum. The sequence from doublet methyl proton H3-26 (δH 1.18) to another doublet methyl proton H3-27 (δH 1.17) through methine proton H-25 (δH 2.92), which was in turn long-range coupled to olefinic quaternary carbon C-24 (δC 139.9) and carbonyl carbon C-28 (δC 171.0), was observed in the DQF-COSY spectrum. Finally, this sequence was determined to be attached to C-23 (δC 141.2) at C-25 (δC 28.5) through olefinic quaternary carbon C-24, as revealed by the 1H–13C long-range couplings from H-23 to C-24, C-25 and C-28. Moreover, carbonyl carbon C-28 was determined to be a carboxylic acid carbon based on the molecular formula. The NOESY between H-23 and H-25 established the geometry at C-23 as being Z. Thus, the planar structure including the stereochemistry at C-23 of 1 was determined to be a steroid derivative, as shown in Figure 1a.

The relative configuration was assigned on the basis of coupling constants and the analysis of the differential NOESY experiment. The large coupling constants for J3H,4Hax (11.1 Hz) (Figure 1c) established that H-3 is in axial orientation. The small coupling constant between H-3 and H-2 (2.7 Hz) proved that H-2 is in equatorial orientation. The NOESY correlations between H-3/Hax-1 (δH 1.75) and between Hax-4 (δH 1.84)/H3-19 indicated that this cyclohexane ring (ring A) should be a chair conformation. Therefore, the methyl group (C-19) attributed to be in axial orientation, and the hydroxy groups at C-2 and C-3 should be in axial and equatorial orientations, respectively. The strong NOESY correlations between Hax-11 (δH 1.73)/H3-19 and between Hax-11/H3-18 revealed that Hax-11, H3-19 and H3-18 are in the upper side of ring moieties. Furthermore, the NOE between Hax-12 (δH 1.72)/H-14 proved that the cyclohexane ring (ring C) should form the chair conformation. Therefore, the 9-OH group is elucidated to be in the lower side of the ring C. Further, the NOESY correlations between H-14/H-17 and between H3-18/H3-21 revealed that the side chain was located in the upper side of the cyclopentane moiety (ring D). Thus, the structure of 1 was established as (23Z)-24-carboxy-2β,3β,5α9α-tetrahydroxyergosta-7,23-dien-6-one (Figure 1). Although many ergostane derivatives have been reported, a 7-en-6-one system4, 5 is rare in fungal secondary metabolites.6, 7 This is the first report of an ergostane isolated from Anthostomella sp.

The cytotoxic activities of 1 against human embryonic kidney (HEK) 293 cells and human cervical carcinoma HeLa cells were determined using a colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, MO, USA) for 48 h. Compound 1 exhibited weak cytotoxic activities against HEK293 and HeLa cells with IC50 values of 51 and 26 μM, respectively.