Basidiomycetes, as decomposers of forest litter, represent an ecologically important group of organisms in the environment, and are known to produce a large variety of secondary metabolites with unique chemical structures and interesting biological activities.1 The genus Xylaria has been known to produce a diverse class of bioactive compounds, including cytochalasin analogs with chemokine receptor antagonistic activity and cytotoxicity,2 multiplolides A, B and xylariamide A with antifungal activity,3, 4 xylarenals A and B with neuropeptide Y receptor antagonistic activity5 and xyloketals A–E, acetylcholinesterase inhibitors.6 Earlier, we reported two antifungal substances, xylarinic acids A and B, from the methanolic extract of Xylaria polymorpha.7 Our ongoing investigation for novel chemical constituents from X. polymorpha has resulted in the isolation of two new 2-benzoxepin derivatives with ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate)) radical scavenging activity. Benzoxepin is very rare in naturally occurring compounds. In this paper, we describe the isolation and structure determination of xylarinols A (1) and B (2), and their biological activity.

Xylarinols were isolated from the fruiting bodies of X. polymorpha, as shown in Figure 1. The collected fruiting bodies were ground and then extracted twice with methanol (MeOH) at room temperature for 2 days. After removal of MeOH under reduced pressure, the concentrate was partitioned between chloroform and water and then ethyl acetate and water. The ethyl acetate-soluble portion was chromatographed on a column of silica gel and eluted with increasing amounts (2.0, 5.0, 10, 20 and 50%, stepwise) of MeOH in CHCl3 to give two fractions, which exhibited moderate ABTS radical scavenging activity. One was purified by Sephadex LH-20 column chromatography with CHCl3–MeOH (1 : 1, v/v), followed by preparative reversed-phase HPLC with 40% aqueous MeOH at a flow rate of 6.0 ml min−1 to yield xylarinol A (1, 1.0 mg). The other fraction was purified by preparative reversed-phase HPLC with 30% aqueous MeOH at a flow rate of 6.0 ml min−1 to provide xylarinol B (2, 1.7 mg).

Figure 1
figure 1

Purification procedures of xylarinol A (1) and B (2).

Full size image

Xylarinol A was isolated as a white powder and showed a molecular ion peak at m/z 176 in the electron impact mass measurement. Its high-resolution electron impact mass measurement provided an accurate mass at m/z 176.0472 [M+, Δ−0.1 mmu], establishing its molecular formula as C10H8O3. The UV spectrum in MeOH exhibited absorption maxima at 205 (log ɛ 4.75), 217 (log ɛ 4.63), 277 (log ɛ 4.43) and 316 (log ɛ 4.07) nm. The IR spectrum suggested the presence of a hydroxyl group (3444 cm−1) and an α,β-unsaturated ester group (1651 cm−1). The 1H-NMR spectrum showed signals due to 1,2,3-trisubstituted benzene ring at δ 7.28, 6.95 and 6.94, two olefinic methine peaks assigned to a cis-1,2-disubstituted double bond unit at δ 7.31 (J=12.0 Hz) and 6.29 (J=12.0 Hz), and a methylene peak at δ 5.24. In the 13C-NMR spectrum, an ester carbonyl carbon at δ 169.6, an oxygen-bearing sp2 carbon at δ 154.9, five sp2 methines, two sp2 quaternary carbons and an oxymethylene at δ 61.4 were evident (Table 1). The 1H–1H COSY spectrum revealed two partial structures, and the heteronuclear multiple quantum correlation spectrum established the proton-bearing carbons, as shown in Figure 2. The structure of 1 was unambiguously determined by the heteronuclear multiple bond correlation spectrum. The long-range correlations from H-6 to C-8 and C-9a, and from H-8 to C-6 and C-9a revealed the presence of 2,3-disubstituted-phenol moiety in 1. The oxepinone ring system was determined by the heteronuclear multiple bond correlations of H-1 to C-3 (δ 169.6), C-5a (δ 137.3) and C-9a (δ 121.7), of H-4 to C-3 (δ 169.6) and C-5a (δ 137.3), and of H-5 to C-9a (δ 121.7). Finally, the heteronuclear multiple bond correlations from H-1 to C-9 (δ 154.9) and from H-5 to C-6 (δ 120.7) completed the structure of 1 as shown in Figure 2. Therefore, the structure of 1 was determined to be 9-hydroxy-1H-benzo[c]oxepin-3-one, a new benzoxepin derivative.

Table 1 1H- and 13C-NMR spectral data of xylarinols A (1) and B (2)
Full size table
Figure 2
figure 2

Structures of xylarinol A (1) and B (2) elucidated by 2-D NMR experiments.

Full size image

Xylarinol B was obtained as a yellow powder with a specific rotation value of −3.32 (c 0.1, MeOH). The molecular formula of 2 was established to be C12H16O4 by the high-resolution EI-MS providing a molecular ion peak at m/z 224.1050 [M+, Δ+0.1 mmu]. The UV spectrum in MeOH exhibited absorption maxima at 211 (log ɛ 3.93), 222 (log ɛ 3.87), 269 (log ɛ 3.29) and 276 (log ɛ 3.28) nm, and its IR spectrum suggested the presence of a hydroxyl group at 3433 cm−1. The 1H-NMR spectrum exhibited signals assignable to 1,2,3-trisubstituted benzene at δ 7.10, 6.67 and 6.64, three oxygenated methines at δ 5.41, 3.70 and 3.61, one nonequivalent oxygenated methylene at δ 5.05 and 4.95, one methylene at δ 1.82, and one methyl at δ 1.16. The 13C-NMR spectrum showed 12 carbons, which were identified as an oxygen-bearing sp2 carbon at δ 152.0, three sp2 methines, two sp2 quaternary carbons, three oxymethines, one oxymethylene, one methylene and one methyl by the DEPT spectrum. Two partial structures were established on the basis of the proton multiplicity and J values, as well as 1H–1H COSY spectral data, as shown in Figure 2. The proton at δ 5.05 (H-1a) of the nonequivalent oxygenated methylene protons was split as a doublet of doublet by the geminal coupling of 12.0 Hz and homoallylic coupling of 2.8 Hz to H-5, which was confirmed by the COSY data. These partial structures were unambiguously connected by the heteronuclear multiple bond correlation spectrum, which showed the long-range correlations of H-1 to C-5a and C-9a, of H-4 to C-3 and C-5a, of H-6 to C-5, C-8, and C-9a, and of H-8 to C-9a, establishing the presence of benzoxepin moiety. Therefore, the structure of 2 was determined as 1,3,4,5-tetrahydro-3-(1-hydroxyethyl)benzo[c]oxepin-5,9-diol, a new benzoxepin derivative. The relative configuration of oxepin ring was proposed by the NOE experiments. NOE enhancements of H-1a, H-3 and H-4 by irradiation of H-5 were observed, and H-1a showed NOE with H-5, suggesting that methine protons of H-1a, H-3 and H-5 were coplanar (Figure 2).

Benzoxepin is known to be very rare in naturally occurring compounds. To date, several 1-benzoxepin and 1-benzoxepinone derivatives have been isolated from Marsamiellus ramealis,8 Mycena galopus9 and Pterula species10, 11 as antibiotics or inhibitors of NADH. We evaluated the antimicrobial activity of compounds 1 and 2 by the conventional paper disk (Advantec, Tokyo, Japan; 8 mm in diameter) method7 at a concentration of 50 μg per disk. Fifteen test microorganisms, including 12 phytopathogenic fungi (Pythium ultinum, Fusarium oxysporium, Magnaporthe grisea, Aspergillus niger, Alternaria panax, Phytophthora capsici, Alternaria mali, Alternaria porri, Botrytis cinerea, Rhizoctonia solani, Fulvia fulva, Cylindrocarpon destructans) and three bacteria (Salmonella sendai, Staphylococcus aureus, Bacillus subtilis), were used. However, compounds 1 and 2 showed no antimicrobial activity against all test organisms. We also measured the ABTS radical scavenging activity of both compounds by using ABTS radical cation decolorization assay with minor modifications.12 As a result, these compounds were found to exhibit moderate ABTS radical scavenging activity with 40 and 45% inhibition, respectively, at 100 μM concentration. Compounds 1 and 2 were obtained in too small amounts for biological activity test, and so some synthetic effort is needed for further study.