Gymnopilin K: a new cytotoxic gymnopilin from Gymnopilus spectabilis

Abstract

In our continuing search for structurally interesting and bioactive metabolites from Korean wild mushrooms,1–5 we have collected scores of endemic Korean mushroom species in the mountainous areas during the hot humid summer and prepared MeOH extracts from them for antitumor activity. Among the collected wild mushrooms, the extract of Gymnopilus spectabilis showed significant cytotoxicity against some human tumor cell lines using a sulforhodamine B bioassay. The hallucinogenic mushroom G. spectabilis (Cortinariaceae) is widely known as big laughter mushroom (Ohwaraitake in Japanese) as it can cause excessive laughing in those who consume it.6 This hallucinogenic mushroom has been a rich source of unique metabolites. Chemical constituents of this mushroom have been reported to have gymnoprenols possessing the structure of a novel type of polyisoprenepolyol with 45–60 carbon atoms as major substances.7–9 Some gymnopilins showing depolarizing activity as bitter principles, were also isolated.10 In addition, a cytotoxic fatty acid (ostopanic acid),11 antioxidant phenolics (bisnoryangonin and hispidin)12 and a hallucinogenic alkaloid (psilocybin)13 have been isolated from this source. Column chromatographic separation of its MeOH extract resulted in the isolation of a new gymnopilin named gymnopilin K (1), together with four known compounds, including gymnopilin A9 (2),10 gymnopilin A10 (3),10 gymnopilene (4)9 and gymnoprenol A9 (5)10 (Figure 1). The structure of 1 was elucidated on the basis of 1D and 2D NMR spectroscopic data analysis as well as chemical reactions, and the known compounds 2–5 were identified by comparison of physical and spectroscopic data with literature values. Here, we describe the isolation and structural elucidation of 1 as well as the cytotoxic activities of compounds 1–5. The air-dried and powdered fruiting bodies of G. spectabilis (153 g) were extracted with 80% aqueous MeOH two times at room temperature and then filtered. The filtrate was evaporated under vacuum to afford a MeOH extract (20 g), which was partitioned with n-hexane, CHCl3 and n-BuOH subsequently using H2O, yielding n-hexane (100 mg), CHCl3 (2.3 g) and n-BuOH fractions (1.9 g). Each fraction was evaluated for cytotoxicity against A549, SK-OV-3, SK-MEL-2 and HCT-15 cell lines using a sulforhodamine B bioassay. We selected the CHCl3 soluble fraction for current phytochemical investigation because the CHCl3 soluble fraction had significant cytotoxicity against the tested tumor cell lines. The active CHCl3 soluble fraction (2.3 g) was separated on a silica gel column with CHCl3-MeOH (5:1) to yield seven fractions (G1–G7). Fraction G3 (250 mg) was separated on a Sephadex LH-20 (Pharmacia, Uppsala, Sweden) column with CH2Cl2MeOH (1:1) and then purified by RP-C18 preparative HPLC (Econosil RP-18 10m column (Alltech, Nicholasville, KY, USA), 250 10 mm) using a solvent of MeCN-MeOH-H2O (9:1:0.3) to yield compounds 4 (4 mg) and 5 (5 mg). Fraction G5 (420 mg) was separated on a Sephadex LH-20 column with CH2Cl2-MeOH (1:1) to afford two subfractions (G51–G52). Fraction G51 (170 mg) was subjected to passage over a Waters Sep-Pak Vac 6 cc (Waters, Milford, MA, USA) (100% MeOH) and then purified by RP-C18 preparative HPLC (80% MeOH) to yield compound 3 (5 mg). Fraction G6 (780 mg) was separated on a Sephadex LH-20 column with CH2Cl2-MeOH (1:1) to obtain two subfractions (G61–G62). Fraction G62 (450 mg) was separated on a RP-C18 silica gel column (75% MeOH) to yield six subfractions (G621–G626). Fraction G621 (27 mg) was purified by RP-C18 preparative HPLC (80% MeOH) to yield compound 2 (4 mg) and fraction G622 (35 mg) was purified by RP-C18 preparative HPLC (70% MeOH) to afford compound 1 (7 mg). Compound 1 was obtained as a colorless gum with a negative specific rotation value [a]25 D 8.7 (c 0.35, MeOH). Its molecular formula was determined to be C51H98O14 from the [M+Na] + peak at m/z 957.6859 (calcd for C51H98NaO14, 957.6854) in the positive-ion high resolution (HR)-ESI-MS spectrum. The IR spectrum of 1 showed a broad hydroxyl band at 3440 cm 1 and a carbonyl absorption band at 1715 cm 1. The physicochemical properties of 1 are summarized in Supplementary Information. The 1Hand 13C-NMR spectral data of 1 are shown in Table 1. The 1Hand 13C-NMR spectra (Table 1) of 1 were very similar to those of 2, with an apparent difference being the absence of signals for cis-vinyl methyls at dH 1.63 (3H, s, H-18) and 1.62 (3H, s, H-18); dC