New polyketides from the basidiomycetous fungus Pholiota sp.

Abstract Two new polyketides, pholiotones B and C (1 and 2), and four known compounds, trichodermatide D (3), vermistatin (4), dehydroaltenuene A (5) and terpestacin (6) were isolated from the crude extract of Pholiota sp. Their structures were identified by NMR and MS spectroscopic data. The absolute configurations of compounds 1 and 2 were elucidated by modified Mosher’s method, electronic circular dichroism (ECD) calculations and 13C NMR calculations as well as DP4+ probability analyses. All the compounds were evaluated for their antifungal and cytotoxicity. Graphical Abstract


Introduction
Fungi are one of the most important sources of bioactive secondary metabolites, which played a vital role in drug discovery [1][2][3][4]. However, with the deepening of research, many strains from traditionally investigated habitats have been repeatedly studied, resulting in the rediscovery of known compounds. Fungi from special ecological niches have proven to be a prolific source of structurally unique secondary metabolites with diverse biological effects, presumably due to their highly evolved metabolic systems adapted during the natural selection process [5][6][7][8]. In the course of our search for bioactive compounds from fungi inhabiting unique environment, a strain of Pholiota sp. isolated from the soil sample surrounding Cordyceps sinensis in high altitude and cold area (Kangding, Sichuan, China), was chemically investigated, leading to the discovery of five new cadinane sesquiterpenoids, pholiotins A-E [9], and new polyketide derivative, pholiotone A [10]. Subsequent chemical investigations of the remaining fractions led to the isolation of two new polyketide derivatives, pholiotones B and C (1 and 2) (Figure 1), together with four known compounds, trichodermatide D (3) [11], vermistatinz (4) [12], dehydroaltenuene A (5) [13] and terpestacin (6) [14]. Details of the isolation, structure elucidation, and bioactivities are reported herein.

Results and discussion
Pholiotone B (1) was assigned the molecular formula C 16 H 26 O 4 (four degrees of unsaturation) based on HRESIMS. Its UV spectrum (k max 273 nm) and IR spectrum ( max 1620 cm À1 ) bands suggested the presence of an a,b-unsaturated ketone group. The 1 H and 13 C NMR spectra of 1 showed resonances for one methyl group, nine methylenes, three oxygenated methines, two olefinic carbons, and a,b-unsaturated ketone carbon (d C 195.1). These data accounted for all the 1 H and 13 C resonances except for two exchangeable protons, and suggested that 1 was a bicyclic compound. Based on these structural features, including the molecular formula and scrutiny of the literature revealed that 13 C NMR data of 1 closely matched with the previously published 13   attached to C-8. The two exchangeable protons were located at C-2 and C-10, respectively, by default, which were partially supported by the chemical shift values for C-2 (d C 71.4) and C-10 (d C 69.8). Collectively, the planar structure of 1 was assigned as shown. However, the optical rotation value ([a] 25 D À31.1, c 0.4, MeOH) of 1 was different from those of 10-deacetylkoningiopisin D [15], indicating that 1 was the isomer of 10-deacetylkoningiopisin D.
The relative configuration of 1 could not be determined due to the lack of relevant NOESY correlations. The absolute configuration of C-10 in 1 was established by the modified Mosher's method [16].  (Table S1). The calculation results indicated that 1a and 1d showed the higher DP4þ probability (68.28% and 25.09%, respectively) among the calculated stereoisomers, indicating that 1a and 1d were more possible configuration for 1 ( Figure 3). The absolute configuration of 1 was further determined by comparison of the experimental and calculated ECD spectra for two stereoisomers (2R,8S,10S)-1a and (2S,8R,10S)-1d using the TDDFT method at the M062X/6-31G(d) level ( Figure 4). The experimental ECD spectrum of 1 matched well with the calculated one of (2R,8S,10S)-1a, suggesting the absolute configuration of 2R,8S,10S for 1. Finally, the structure of compound 1 was determined and named as pholiotone B.
The molecular formula of pholiotone C (2), deduced by HRESIMS, was the same as that for 1 (C 16 H 26 O 4 ). The NMR spectroscopic data (Table 1) of 2 were almost similar to those of 1, indicating that they were also isomers. The absolute configuration of C-10 in 2 was also established by the modified Mosher's method [16].  Figure 4). On the basis of the above data, the structure of 2 was determined, and was named as pholiotone C.

General experimental procedures
Optical rotations were measured on an Anton Paar MCP 200 Automatic Polarimeter (Anton Paar, Graz, Austria) and UV data were obtained on a Thermo Genesys-10S UV/Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The ECD spectra were measured by JASCO J-815 spectropolarimeter (JASCO, Tsukuba, Japan). IR data were recorded using a Nicolet IS5 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 1 H and 13 C NMR data were acquired with Bruker Avance-500 spectrometer (Bruker, Bremen, Germany) using solvent signals (CDCl 3 ; d H 7.26/d C 77.7) as references. ESIMS data were recorded on a Bruker Esquire 3000 plus spectrometer (Bruker, Bremen, Germany), and HRESIMS data were obtained using Bruker APEX III 7.0 T and APEX II FT-ICR spectrometers (Bruker, Bremen, Germany), respectively.

Fungal material
The fungal strain Pholiota sp. was isolated from the soil samples surrounding C. sinensis (Berk.) Sacc. collected in Kangding, Sichuan, China, in May 2005. The isolate was identified based on morphology and sequence (Genbank Accession No. JQ411813) analysis of the ITS region of the rDNA and assigned the accession number SCK05-7-ZP19 at the Institute of Microbiology, Chinese Academy of Sciences, Beijing. The strain was cultured on slants of potato dextrose agar (PDA) at 25 C for 10 days. Agar plugs were cut into small pieces (about 0.5 Â 0.5 Â 0.5 cm 3 ) under aseptic conditions, and 15 pieces were used to inoculate three Erlenmeyer flasks (250 ml), each containing 50 ml of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract); the final pH of the media was adjusted to 6.5 and sterilized by autoclave. Three flasks of the inoculated media were incubated at 25 C on a rotary shaker at 170 rpm for five days to prepare the seed culture. Fermentation was carried out in eight Fernbach flasks (500 ml), each containing 80 g of rice. Spore inoculum was prepared by suspension in sterile, distilled H 2 O to give a final spore/cell suspension of 1 Â 10 6 /ml. Distilled H 2 O (120 ml) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 ml of the spore inoculum and incubated at 25 C for 40 days.

Extraction and isolation
The fermented material was extracted repeatedly with EtOAc (4 Â 1.0 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (3.1 g), which was fractionated by silica gel VLC using petroleum ether-EtOAc gradient elution.

Antifungal assays
Antifungal assays were conducted in triplicate following the National Center for Clinical Laboratory Standards (NCCLS) recommendations [17]. The fungi, A. flavus (CGMCC 3.0951), P. oryzae (CGMCC 3.3283), and F. nivale (CGMCC 3.4600) were obtained from China General Microbial Culture Collection (CGMCC) and were grown on PDA. Targeted fungi (3-4 colonies) were prepared from broth culture (A. flavus: 28 C for 36 h; the plant pathogens: 28 C for 48 h) and the final suspensions contained 10 4 hyphae/ml (in PDB medium). Test samples (4 mg/ml as a stock solution in DMSO and serial dilutions) were transferred to 96-well clear plate in triplicate, and the suspensions of the test organisms were added to each well, achieving a final volume of 200 ml. Alamar blue (10 ml of 10% solution) was added to each well as an indicator and amphotericin B and carbendazim were used as the positive controls. After incubation (A. flavus: 28 C for 36 h; the plant pathogens: 28 C for 48 h), the fluorescence intensity was measured at Ex/Em ¼ 544/590 nm. The inhibition was calculated and plotted versus test concentrations to afford the IC 50 .

MTS sssay
In vitro cytotoxicity was determined by the MTS method [18,19]. In a 96-well plate, each well was plated with 2 $ 5 Â 10 3 cells (It depends on the cell multiplication rate). After cell attachment overnight, the medium was removed, and each well was treated with 100 ml of medium containing 0.1% DMSO, or appropriate concentrations of the test compounds and the positive control paclitaxel (Sigma) (100 mM as a stock solution of a compound in DMSO and serial dilutions; the test compounds showed good solubility in DMSO and did not precipitate when added to the cells). The plate was incubated for 72 h at 37 C in a humidified, 5% CO 2 atmosphere. Proliferation was assessed by adding 20 ml of MTS (Promega) to each well in dark followed by a 90 min incubation at 37 C. The assay plate was read at 490 nm using a microplate reader.

13 C NMR calculation
The plausible conformers of compounds 1a-1d were performed by using the MMFF94 molecular mechanics force field. All the obtained conformers were subsequently optimized at the B3LYP/6-311 þ G(2d,p) level using Gaussian 09 software. The optimized conformers in an energy window of 3 kcal/mol (with no imaginary frequency) were further subjected to 13 C NMR calculation by using the GIAO method [20] at mPW1PW91/6-31G(d) level. The calculated chemical shifts (d calcd ) for each compound were weighted according to the Boltzmann distributions over the conformers.

ECD calculation
Conformational analyses for compound 1 was performed using OpenBabel in the MMFF94 molecular mechanics force-field within an energy window of 5.0 or 3.0 kcal/ mol. The conformers were then further optimized with the software package Gaussian 09 at the M062/6-31G(d) level for compound 1 [21], and the harmonic vibrational frequencies were also calculated to confirm their stability. The timedependent density functional theory (TD-DFT) methods at the M062X/6-31G(d) were applied to calculate the 60 lowest electronic transitions which obtained conformers in vacuum, respectively. The Gaussian function was applied to simulate the ECD spectrum of the conformers.