Isobenzofuranone derivatives are an important class of compounds displaying a variety of biological effects, such as antioxidant, antimicrobial, antiplatelet, cytotoxic activity and antiarrhythmic effects [1,2,3,4]. As sources of bioactive substance, fungi are now considered as efficient producers of biologically active and chemically novel compounds. Some of the more important sources of naturally occurring isobenzofuranone derivatives are fungi [5,6] and higher plants [7]. In our ongoing research on biologically active metabolites from fungi, we investigated the secondary metabolites produced in culture by Cephalosporium sp. AL031. This organism is a fungus that was isolated from Sinarundinaria nitida grown at Ailao Mountain (Yunnan, China). Previous phytochemical investigation on the fungus revealed a number of phenolic acids [8], dihydroisocoumarin glucosides [9,10] and pyrone derivatives [11,12]. In the present study, we describe the isolation and structure elucidation of a novel isobenzofuranone derivative, namely 4,6-dihydroxy-5-methoxy-7-methylphthalide (1), together with three known ones: 4,5,6-trihydroxy-7-methyl-1,3-dihydro-isobenzofuran (2), 4,6-dihydroxy-5-methoxy-7-methyl-1,3-dihydroisobenzofuran (3) and 4,5,6-trihydroxy-7-methylphthalide (4) (Figure 1), from the culture of the fungus Cephalosporium sp. AL031. The structure of the new compound 1 was determined based on MS, IR, 1D and 2D NMR spectral data, and the known ones 2–4 were identified by comparing their NMR data with those in the literature. The antioxidant activity of compounds 1–4 was evaluated by DPPH radical-scavenging assay.

Compound 1 was obtained as colorless needle-like crystals that gave a positive reaction in the 10% H₂SO₄-EtOH test. The molecular formula C₁₀H₁₀O₅ was deduced from a pseudomolecular ion [M + H]⁺ at m/z 211.0628 in the HR-ESI-MS (calcd. for C₁₀H₁₁O₅, 211.0607), which was in agreement with the ¹H- and ¹³C-NMR spectra. The IR spectrum showed characteristic absorption bonds for hydroxyl groups (3365 cm⁻¹), saturated methylene (2930 cm⁻¹) and a carbonyl group (1685 cm⁻¹).

The ¹H-NMR spectrum of 1(Table 1) exhibited signals attributable to a methoxyl group at δ 3.87 (3H, s), an oxymethylene moiety at δ 5.12 (2H, brs) and a methyl group at δ 2.42 (3H, s). The ¹³C-NMR and DEPT spectra of 1(Table 1) also showed the occurrence of three sp³-carbon signals related to a methoxyl group (δ 59.6), an oxymethylene moiety (δ 66.8) and a methyl group (δ 8.2). Including one quaternary sp²-carbon signal which was attributed to a carbonyl moiety at δ 172.7, compound 1 also indicated six quaternary sp²-carbon signals at δ 125.8, 142.1, 140.8, 149.6, 116.5 and 117.7. The ¹H-NMR and ¹³C-NMR data was assigned to an isobenzofuranone skeleton.

The ¹H-NMR and ¹³C-NMR data of 1 are very similar to those of 4,5,6-trihydroxy-7-methylphthalide [1] that has been isolated from the fungus Epicoccum sp. The only difference between these two compounds is the methoxyl group present in compound 1. HSQC and HMBC experiments revealed all significant connectivities. The correlation of the methyl group (δ 2.42) with C-6 (δ 149.6) and C-7a (δ 116.5) in the HMBC spectrum indicated the methyl unit connected to C-7. Similarly, the linkage of the methoxyl moiety at C-5 was deduced from the correlation between the methoxyl group (δ 3.87) and C-5 (δ 140.8). Furthermore, no correlations were observed between the methyl group (δ 2.42) or oxymethylene moiety (δ 5.12) and C-5, which unambiguously established the linkage of methoxyl moiety. All NMR data of compound 1 were assigned on the basis of DEPT, HSQC and HMBC experiments. Taken together, compound 1 was determined to be a new isobenzofuranone and named as 4,6-dihydroxy-5-methoxy-7-methylphthalide with the structure shown in Figure 1.

Together with the new compound 1, three known compounds 2–4 were identified by comparison of their spectroscopic data [1,13,14] with published values. We evaluated the antioxidant activity of isolated compounds 1–4 by DPPH radical-scavenging assay, which showed potent activity with the EC₅₀ values of 10, 7, 22 and 5 μM, respectively.

Repeated column chromatography (including normal-phase silica gel and Sephadex LH-20) of the EtOAc extract of the fermentation of the fungus Cephalosporium sp. AL031 has led to the isolation of a novel isobenzofuranone derivative, namely 4,6-dihydroxy-5-methoxy-7-methylphthalide (1), together with three known ones: 4,5,6-trihydroxy-7-methyl-1,3-dihydroisobenzofuran (2), 4,6-dihydroxy-5-methoxy-7-methyl-1,3-dihydroisobenzofuran (3), and 4,5,6-trihydroxy-7-methylphthalide (4). The structure of the new compound 1 was determined by spectroscopic methods, including 1D NMR, 2D NMR and MS experiments, and the structures of the known compounds including 2~4 were identified by comparing their NMR data with those of ones in the literature. All four compounds showed potent anti-oxidative activity by DPPH radical-scavenging assay, which are potential anti-oxidative agents. Comparing the antioxidant activity of compounds 1 and 4, 3 and 2, the latter containing three phenolic hydroxyl groups showed potent activity, which indicated the antioxidant activity correlates with the number of hydroxyl groups in the structure.