Pholiotone A, a new polyketide derivative from Pholiota sp

Abstract Pholiotone A (1), a new polyketide derivative, with tetrahydrobenzofuran-4(2H)-one skeleton, together with four known compounds, trichodermatides A (2) and B (3) and koninginins B (4) and E (5), were isolated from the crude extract of Pholiota sp. The structures of all the isolated compounds were determined mainly by NMR experiments, the modified Mosher method and electronic circular dichroism (ECD) calculations. The antifungal and cytotoxicity of all isolates were evaluated. Graphical Abstract


Introduction
Fungi commonly thrive in competitive environments, and it is often hypothesized that their secondary metabolic capabilities have been influenced by selection pressures exerted by other organisms. Indeed, fungal metabolites have been implicated in diseases of plants and insects, animal poisonings or intoxications, biocontrol of other fungi, and interspecies antagonism (Gloer 1995;B erdy 2005;Davis et al. 2010). Based on this consideration and the documented success in finding new bioactive natural products from special types of fungi (Gooday 1997;Senadeera et al. 2012), we initiated chemical studies of the Cordyceps-colonizing fungi (Guo et al. 2007;Zhang Y et al. 2007;Ma et al. 2011), and those that were isolated from the soil samples surrounding Cordyceps sinensis (Lin et al. 2010;Lin et al. 2011). Little was known about biological activities of compounds from the medicinal mushroom of the genus Pholiota (Becker et al. 1994;Yang et al. 2018;Zhu et al. 2018). In the current work, the basidiomycetous fungus Pholiota sp. (SCK05-7-ZP19) was isolated from a soil sample on the surface of C. sinensis collected in Kangding, Sichuan, People's Republic of China. In the course of our search for bioactive compounds from Pholiota sp., a new polyketide derivative, pholiotone A (1) (Figure 1), together with four known compounds, trichodermatides A (2) and B (3) (Sun et al. 2008), and koninginins B (4) and E (5) (Parker et al. 1995), were isolated from the EtOAc extract of solid-substrate fermentation culture. Details of the isolation, structure elucidation, and bioactivities are reported herein.

Results and discussion
Pholiotone A (1) was assigned the molecular formula C 16 H 26 O 4 (four degrees of unsaturation) on the basis of HRESIMS. Its UV spectrum (k max 273 nm) and IR spectrum (m max 1588 cm À1 ) suggested the presence of an a,b-unsaturated ketone group. The 1 H and 13 C NMR spectra (Table S1) of 1 showed resonances for one methyl group, nine methylenes, three oxygenated methines, two olefinic carbons, and a,b-unsaturated ketone carbon (d C 194.8). 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. Analysis of the 1 H-1 H COSY NMR data for 1 ( Figure S7) defined two isolated spin-systems of C-2-C-4 and C-7-C-16. In the HMBC spectrum ( Figure S7), correlations from H 2 -2 [d H 2.56 (1H, dt, J ¼ 13.9, 4.9 Hz) and 2.36 (1H, m)] to the a,b-unsaturated ketone carbon C-1 (d C 194.8), H 2 -3 [d H 2.34 (1H, m) and 2.03 (1H, m)] to C-1 (d C 194.8) and C-5 (d C 174.8), and from H-4 [d H 4.59 (1H, m)] to C-5 (d C 174.8) indicated that C-2 (d C 34.7) is connected C-1 (d C 194.8), and C-4 (d C 63.8) is attached the C-5/C-6 olefin at C-5 (d C 174.8), completing the a,b-unsaturated cyclohexenone moiety. HMBC correlations from H 2 -7 [d H 3.03 (1H, m) and 2.53 (1H, m)] to the C-5 (d C 174.8) and C-6 (d C 113.2) olefinic carbons established the connection from C-7 (d C 32.3) to the C-5/C-6 olefin at C-6 (d C 113.2). Considering the chemical shifts for C-5 (d C 174.8) and C-8 (d C 85.8), as well as the bicyclic nature of 1, the two carbons were attached to the same oxygen atom to form a dihydrofuran, which fused to the cyclohexenone moiety at C-5/C-6 to complete the tetrahydrobenzofuran-4(2H)-one skeleton of 1. The two exchangeable protons were located at C-4 and C-10, respectively, by default, which were partially supported by the chemical shift values for C-4 (d C 63.8), and C-10 (d C 69.8). Collectively, the planar structure of 1 was assigned as shown.
The relative configuration of 1 was proposed by analysis of NOESY data ( Figure S9). NOESY correlations of H-8 with H-10 revealed their proximity in space. However, the relative configuration of C-4 could not be determined due to the lack of relevant NOESY correlations.
The absolute configuration of C-10 in 1 was determined using the modified Mosher method (Dale and Mosher 1973;Ohtani et al. 1991). Treatment of 1 with (S)-and (R)-MTPA Cl afforded the major products, the R-(1a) and S-MTPA (1b) di-esters, respectively. The difference in chemical shift values (Dd ¼ d Sd R ) for the diastereomeric esters 1b and 1a was calculated to assign the 10S absolute configuration. Therefore, the 10S configuration was proposed for 1 on the basis of the Dd results summarized in Figure S2. The absolute configuration of C-4 in 1 was assigned by application of the CD exciton chirality method. The CD spectrum of 1 ( Figure S3) showed a positive Cotton effect at 299 (De -0.75) nm, which is similar to that of trichodermaketones A (Song et al. 2010), correlating with 4R absolute configuration. Combining the relative configuration established above, the 4R and 10S absolute configuration was assigned for 1.
The absolute configuration of 1 was further confirmed by comparison of the experimental and the simulated circular dichroism (CD) spectra ( Figure S4) generated by the time-dependent density functional theory (TDDFT) (Berova et al. 2007). Due to insignificant contribution to the ECD property of 1 by the aliphatic chain, a simplified structure 1' (Figure S4) was used to calculate the four enantiomers, (4R,8R)-1' (1'a), (4S,8S)-1 (1'b), (4S,8R)-1' (1'c) and (4R,8S)-1' (1'd). MMFF94 conformational search and DFT reoptimization at the B3LYP/6-31 þ G(d,p) level yielded 27 and 26 lowest energy conformers for 1'a and 1'c, respectively. The overall calculated ECD spectra of 1'a-1'd were then generated by Boltzmann weighting of the conformers. The experimental ECD curve of 1 was nearly identical to the calculated ECD spectrum of 1'a, suggested the 4R,8R absolute configuration for 1, which was consistent with the result deduced from the CD exciton chirality method.
The known compounds 2-5 isolated from the crude extract were identified as trichodermatides A (2) and B (3), and koninginins B (4) and E (5) by comparison of their NMR and MS data with those reported (Parker et al. 1995).
All five compounds (1-5) were evaluated for activity against microorganism and cytotoxicity against eight human tumor cell lines. But none of them exhibited positive effect even at 200 lg/ml (B erdy 2005;Jeong et al. 2009;Zhang N et al. 2011).

Conclusions
In conclusion, a new polyketide derivative, pholiotone A (1) has been isolated from the crude extract of Pholiota sp. The conformation of compound 1 was confirmed by elucidated based on NMR spectroscopic data, the modified Mosher method and by comparison of the experimental CD spectrum with calculated spectra. Pholiotone A (1) is a new polyketide derivative with tetrahydrobenzofuran-4(2H)-one skeleton. Pholiotone A shares the same skeleton as that of trichodermaketone C, but differs in having a methine at C-9 and a hydroxy group at C-10, instead of a trans-double bond at C-9 and C-10. To our knowledge, pholiotone A (1) is the first polyketide with tetrahydrobenzofuran-4(2H)-one skeleton identified from Pholiota sp. The discovery of 1 further expanded the structural diversity of this rare class of natural products and provided an alternative way for their configurational assignment.