The fungus T. eximius was collected at Nagano, Japan in 2010. The fresh fruit body (300 g) was extracted with methanol (500 ml) for 2 months. After this extract being evaporated to aqueous solution, the residue was partitioned between water and EtOAc (ethyl ethanoate) to yield the tylopilusin mixture (1.1 g) after evaporation of the EtOAc fraction. The crude extract was dissolved in a small volume of chloroform, applied on a silica gel column (22 g, 28 × 14 cm2, 0.04–0.063 mm, MERCK, Darmstadt, Germany) and eluted stepwise with 100% chloroform, 20:1, 10:1, 5:1 and 1:1 (v/v) chloroform–methanol and 100% methanol (200 ml each). Tylopilusin C was observed in the fraction eluted with 1:1 chloroform–methanol. This fraction was further purified by reversed-phase C18 HPLC (20 × 250 mm2, PEGASIL ODS, Senshu Scientific Co., Tokyo, Japan) under the following conditions; solvent, a 30-min linear gradient from 20–40% acetonitril in 0.05% trifluoroacetic acid; a flow rate of 8.0 ml min−1; UV detection at 340 nm. Under these conditions, tylopilusin C was eluted as a peak with a retention time of 20.1 min. This fraction was collected and concentrated to yield pure tylopilusin C (6.0 mg) as a pale yellow solid.

The structure of 1 was mainly elucidated by analysis of NMR spectra, including 2D NMR. The molecular formula for 1 was established as C18H12O7 ([M+H]+ m/z 341.0659) on the basis of high resolution ESI-MS measurement, indicating that 1 contained 13 degrees of unsaturation. (Table 1) The IR spectrum of 1 showed characteristic absorption at 3259 and 1685 cm−1, suggesting the presence of a carbonic acid moiety. The 1H NMR spectrum of 1 (in DMSO-d6 (dimethyl sulfoxide-D6)) showed four types of aromatic methine signals. (Table 2) Analysis of the 1H NMR data using decoupling methods suggested two types of 1,4-disubstituted benzene rings. The 13C NMR spectrum (in DMSO-d6) showed 18 resolved signals, which were classified into 8 sp2 methine carbons and 10 quaternary carbons, including 2 carbonyl carbons (C-9 and C-12) and 5 oxygenated sp2 carbons (C-4, C-7, C-8, C-11 and C-16) (Table 2). Among them, C-1 to C-6 and C-13 to C-18 were identified as 1, 4-disubstituted benzene ring carbons. Furthermore, the chemical shifts of C-7 (δ 145.0), C–8 (δ 141.7), C-9 (δ 173.3), C–10 (δ 125.5) and C-11 (δ 159.1) suggested that a 3-hydroxy-4-pyranone ring should exist.5, 6 In additional HMBC NMR experiment, the cross peaks from 2-H and 6-H (δ 7.93) to C-7 indicated that one benzene is linked at C-7 of the pyranone ring. Furthermore, correlations from 14-H and 18-H (δ 7.06) to C-10 allowed the other benzene and the pyranone functionalities to be joined at C-10 as shown in Figure 1. Taking into consideration the molecular formula and the IR data, a carbonyl acid moiety was connected at C-11. Based on all the data, the total structure of 1 was elucidated as shown in Figure 1. Unfortunately, signals of 8-OH and 12-OH were not obserbed in 1H NMR experiments. To confirm the structure, 1 was methylated with trimethylsilyl diazomethane,7 and the derivative 2 was analyzed by HI-ESI-MS measurements. (Methylation of tylopilusin C; Tylopilusin C (1.8 mg) was disolved in MeOH (200 μl) and benzene (800 μl) and trimethylsilyl diazomethane was added (500 μl, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) at 25 °C for 30 min. The reaction mixture was evaporated in vacuo to obtain the products (1.8 mg). The products were purified by reversed-phase C-18 HPLC (10 × 250 mm2, PEGASIL ODS), a 70% acetonitril in 0.05% trifluoroacetic acid, at a flow rate 2.5 ml min−1 with UV detection at 340 nm. Under this condition, tetramethyltylopilusin C (2) was eluted as a peak with a retention time of 13.2 min. This peak was collected and concentrated to yield 1.0 mg as pale yellow solid.) (Tetramethyltylopilusin C (2): Pale yellow solid; 1H NMR (600 MHz, CDCl3) δH 8.07 (2H, d, J=9.0 Hz), 7.24 (2H, d, J=9.0 Hz), 7.02 (2H, d, J=9.0 Hz), 6.96 (2H, d, J=9.0 Hz), 3.92 (3H, s), 3.89 (3H, s), 3.84 (3H, s) and 3.75 (3H, s); HR-ESI-MS [M+Na]+ m/z 419.11098 (C22H20O7Na, calcd 419.1107).) The molecular formula of 2 was established as C22H20O7, indicating that four methyl residues were incorporated into 1. Furthermore, four methoxyl methyl signals (δ 3.92, 3.89, 3.84 and 3.75) were observed in 1H NMR experiments. Thus, the structure of the derivative was elucidated as 2, supporting the structure of 1.

Table 1 Physico-chemical properties of tylopilusin C
Full size table
Table 2 NMR spectroscopic data in DMSO-d6 for tylopilusin C
Full size table

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