New cadinane sesquiterpenoids from the basidiomycetous fungus Pholiota sp.

Abstract

Five new cadinane sesquiterpenoids, pholiotins A–E (1–5), and three known compounds, 11-hydroxy-1(10)-valencen-2-one (6), 8,11-dihydroxy-1(10)-eremophilen-2-one (7) and durgamone (8), were isolated from the crude extract of Pholiota sp. Their structures were established on the basis of extensive spectroscopic analysis. Their absolute configurations were determined by X-ray diffraction, the Snatzke's method and electronic circular dichroism (ECD) calculations. All isolates were tested for antifungal activity.

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Introduction

Fungi are well-known producers of bioactive secondary metabolites.^1–5 Based on this consideration and the documented success in finding new bioactive natural products from special types of fungi,⁶ we initiated chemical studies of the Cordyceps-colonizing fungi,^7–10 and those that were isolated from the soil samples surrounding Cordyceps sinensis.^11,12 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. Some species of Pholiota are edible, but some are toxic.^13–16 The isolated fungus Pholiota sp. (SCK05-7-ZP19) was grown in a solid-substrate fermentation culture. An organic solvent extract of the culture was active against Aspergillus flavus (CGMCC 3.0951). Bioassay-guided fractionation of the extract led to the isolation of five new metabolites, pholiotins A–E (1–5), and three known compounds, 11-hydroxy-1(10)-valencen-2-one (6),¹⁷ 8,11-dihydroxy-1(10)-eremophilen-2-one (7),¹⁸ and durgamone (8)^19,20 (Fig. 1). Details of the isolation, structure elucidation, and antifungal activity of these metabolites are reported herein.

Fig. 1 Chemical structure of 1–8.

Results and discussion

Pholiotin A (1) was assigned the molecular formula C₁₅H₂₄O₅ (four degrees of unsaturation) on the basis of HRESIMS. The ¹H and ¹³C NMR spectra (Table 1) of 1 showed resonances for two methyl groups, four methylenes (one oxygenated), five methines (one oxygenated), two olefinic carbons (one protonated), one quaternary carbon (δ_C 74.9) and one carboxylic carbon (δ_C 168.7). These data accounted for all ¹H and ¹³C resonances except for four exchangeable protons, and suggested that 1 was a bicyclic compound. Interpretation of the ¹H–¹H COSY NMR data established two isolated proton spin-systems (Fig. 2), which were C-3–C-2–C-1–C-6–C-7–C-8–C-9 and C-7–C-12 (including C-13 and C-14). HMBC correlations (Fig. 2) from H-1, and H₂-8 to C-10, and from H₂-15 to C-1, C-9, and C-10 implied that C-1, C-9 and C-15 were all attached to C-10 directly, completing the cyclohexane moiety. Correlations from H₂-2 to C-4, H₂-3 to C-5, and from H-5 to C-1, C-3 and C-7 revealed the connections of the C-4/C-5 olefin to C-3, and C-6, respectively, establishing the cyclohexene ring, which fused to the cyclohexane subunit at C-1/C-6 to the completion of the octahydronaphthalene core structure. Other correlations from H-3b and H-5 to the carboxylic carbon C-11 (δ_C 168.7) showed that C-11 was connected to the C-4/C-5 olefin at C-4. Considering the chemical shifts for C-9 (δ_C 69.5), C-10 (δ_C 74.9), C-11 (δ_C 168.7) and C-15 (δ_C 62.8), as well as the molecular formula requirement for 1, the remaining four exchangeable protons were assigned as C-9-, C-10-, C-11- and C-15-OH, respectively. Therefore, the gross structure of pholiotin A was established as 1. Finally, the structure of 1 was confirmed by single-crystal X-ray crystallographic analysis, and a perspective ORTEP plot is shown in Fig. 3. The X-ray data also allowed assignment of its relative configuration.

Table 1 NMR data for compounds 1–3

Pos.	1		2		3
	δ C ^a	δ H ^b (J in Hz)	δ C ^a	δ H ^b (J in Hz)	δ C ^a	δ H ^b (J in Hz)
a Recorded at 125 MHz. b Recorded at 500 MHz.
1	42.1, CH	1.80, m	43.0, CH	1.80, m	43.0, CH	1.80, m
2a	22.4, CH2	2.24, m	22.2, CH2	2.25, m	22.3, CH2	2.24, m
2b		1.28, m		1.28, m		1.28, m
3a	26.4, CH2	2.47, m	26.3, CH2	2.47, m	26.4, CH2	2.50, m
3b		2.18, m		2.18, m		2.18, m
4	131.7, qC		131.9, qC		131.7, qC
5	140.6, CH	7.07, s	140.3, CH	7.10, s	140.4, CH	7.11, s
6	40.2, CH	2.04, m	40.4, CH	2.04, m	40.4, CH	2.04, m
7	39.5, CH	1.66, m	40.1, CH	1.66, m	40.1, CH	1.66, m
8a	28.4, CH2	1.83, m	26.6, CH2	1.81, m	28.5, CH2	1.81, m
8b		1.52, m		1.52, m		1.52, m
9	69.5, CH	4.07, br s	73.1, CH	5.37, br s	69.5, CH	4.02, br s
10	74.9, qC		74.2, qC		73.7, qC
11	168.7, qC		168.5, qC		168.5, qC
12	26.5, CH	2.12, m	26.5, CH	2.12, m	26.5, CH	2.12, m
13	21.5, CH3	0.92, d (6.9)	21.5, CH3	0.95, d (6.9)	21.5, CH3	0.97, d (6.9)
14	15.4, CH3	0.82, d (6.9)	15.2, CH3	0.87, d (6.9)	15.4, CH3	0.88, d (6.9)
15a	62.8, CH2	3.63, br s	63.1, CH2	3.71, d (12)	62.8, CH2	4.22, d (12)
15b				3.65, d (12)		4.16, d (12)
16					171.0, qC
17					20.8, CH3	2.15, s
18			170.9, qC
19			21.2, CH3	2.12, s

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Fig. 2 Key ¹H–¹H COSY, HMBC correlations for 1.

Fig. 3 Thermal ellipsoid representations of 1 and 5.

The absolute configuration of 1 was initially assigned by application of the modified Mosher method.^21,22 Treatment of 1 with (S)- and (R)-MTPACl afforded the (R)-MTPA ester 1a and the (S)-MTPA esters 1b, respectively. The selectivity for the acylation of C-9 hydroxy group was not achieved, but the ester 1a acylation of C-15 hydroxy group was obtained by reversed-phase HPLC purification. The absolute configuration of the sec/tert-9,10-diol moiety in 1a was assigned using the in situ dimolybdenum CD method developed by Frelek.^23,24 Upon addition of dimolybdenum tetraacetate [Mo₂(OAc)₄] to 1a in DMSO solution, a metal complex of chiral vic-diol with the achiral Mo₂(OAc)₄ was generated as an auxiliary chromophore. Since 1a has an inherent CD resulting from the C-11 carboxylic chromophore, this contribution was subtracted to give the induced CD of the metal complex to avoid its overlap (>250 nm) with those generated after addition of Mo₂(OAc)₄. Therefore, the observed sign of the Cotton effect in the induced spectrum originates solely from the chirality of the vic-diol moiety expressed by the sign of the O–C–C–O torsion angle. The positive Cotton effects observed at around 310 and 400 nm, respectively, in the induced CD spectrum permitted assignment of the 9S and 10S absolute configuration in 1a on the basis of the empirical rule proposed by Snatzke (Fig. 4 and 5). The absolute configuration of 9,10-diol moiety in 1 was deduced as 9S and 10S by analogy to 1a. Considering the relative configuration established by single-crystal X-ray crystallographic, 1 was assigned the 1S,6S,7R,9S, and 10S.

Fig. 4 CD spectrum of 1a in DMSO containing Mo₂(OAc)₄.

Fig. 5 The sterical configurations for the cottonogenic derivative of 1a.

The absolute configuration of 1 was further confirmed by comparison of the experimental and the simulated circular dichroism (CD) spectra (Fig. 6) generated by the time-dependent density functional theory (TDDFT).²⁵ Compound 1 was used to calculate two enantiomers, (1S,6S,7R,9S,10S)-1 (1c) and (1R,6R,7S,9R,10R)-1 (1d). MMFF94 conformational search and DFT re-optimization at the B3LYP/6-31+G(d) level yielded 6 lowest energy conformers for 1c (Fig. S25†). The overall calculated ECD spectra of 1c and 1d were then generated by Boltzmann weighting of the conformers (Fig. 6). The experimental ECD curve of 1 was nearly identical to the calculated ECD spectrum of 1c, suggested the 1S,6S,7R,9S,10S absolute configuration for 1, which was consistent with the result deduced from the Snatzke's method.

Fig. 6 Experimental ECD spectrum of 1 in MeOH and the calculated ECD spectra of 1c and 1d, after a UV correction of 10 nm.

The molecular formula of pholiotin B (2) was determined to be C₁₇H₂₆O₆ (five degrees of unsaturation) by HRESIMS, 42 mass units higher than that of 1. Analysis of the NMR spectroscopic data (Table 1) of 2 revealed nearly identical structural features to those found in 1, except that the oxymethine proton signal (H-9) was shifted downfield (δ_H 4.07 in 1 vs. 5.37 in 2). In addition, NMR resonances corresponding to an acetyl group (δ_H 2.12; δ_C 21.2 and 170.9) were observed, indicating that the C-9 oxygen of 2 was acylated, which was confirmed by an HMBC correlation from H-9 to the carboxylic carbon (δ_C 170.9) of the acetyl group. Therefore, 2 was determined as the C-9 monoacetate of 1 and its relative configuration was deduced by analogy to 1. The CD spectra of 1 and 2 (Fig. S20 and S21; ESI†) both showed positive Cotton effects at 220 nm, and negative Cotton effects at 249 nm, indicating that the absolute configuration of 2 was the same as that of 1.

Pholiotin C (3) was assigned the same molecular formula C₁₇H₂₆O₆ as 2 by HRESIMS. Interpretation of the NMR data (Table 1) of 3 showed similar resonances to those of 2, indicating that 3 is also a monoacetate of 1 but with a different position for acetylation. Specifically, the oxymethene proton signals of H₂-15 in 3 (δ_H 4.16, 4.22) were significantly downfield compared to 1 (δ_H 3.63), suggesting that OH-15 was acetylated, which was supported by an HMBC correlation from H₂-15 to the carboxylic carbon of the acetyl unit (δ_C 171.0). On the basis of these data, 3 was determined as the C-15 monoacetate of 1. The relative and absolute configurations of 3 were deduced as shown by analogy to 1, which was further confirmed by comparison of its CD data with those of 1 (Fig. S20 and S22; ESI†).

Pholiotin D (4) gave a pseudomolecular ion [M + Na]⁺ peak, consistent with a molecular formula of C₁₉H₂₈O₇ (6 degrees of unsaturation). Analysis of the ¹H and ¹³C NMR data of 4 (Table 2) revealed that 4 is a diacetate of 1. The esterification shifts (Δ = 1.19 ppm for H-9, Δ = 0.62/0.57 ppm for H₂-15) were observed, which allowed the acetyl groups to locate at C-9 and C-15, respectively. On the basis of these data, 4 was determined as the C-9 and C-15 diacetate of 1. This observation was supported by relevant HMBC correlations from H-9 to C-18 (δ_C 171.4) and from H₂-15 to C-16 (δ_C 171.1). The relative and absolute configurations of 4 were deduced as shown by analogy to 1, which was further confirmed by comparison of its CD data with those of 1 (Fig. S20 and S23; ESI†).

Table 2 NMR data for compounds 4 and 5

Pos.	4		5
	δ C ^a	δ H ^b (J in Hz)	δ C ^a	δ H ^b (J in Hz)
a Recorded at 125 MHz. b Recorded at 500 MHz.
1	41.6, CH	1.83, m	46.4, CH	1.97, dt (12, 3.2)
2a	21.3, CH2	2.24, m	34.1, CH2	2.28, m
2b		1.26, m		1.49, m
3a	25.4, CH2	2.49, m	25.5, CH2	2.53, dd (18, 4.6)
3b		2.18, m		2.01, m
4	131.1, qC		131.6, qC
5	139.2, CH	7.11, s	140.0, qC	7.16, s
6	39.4, CH	2.04, m	40.3, CH	2.42, dt (10, 1.2)
7	39.3, CH	1.59, m	38.4, CH	1.80, m
8a	25.7, CH2	1.86, m	26.7, CH2	1.90, dt (12, 3.3)
8b		1.46, m		1.36, dt (3.0, 13)
9	72.1, CH	5.26, br s	72.9, CH	4.40, br s
10	73.6, qC		155.1, qC
11	169.9, qC		168.5, qC
12	25.6, CH	2.13, m	25.5, CH	2.28, m
13	20.6, CH3	0.95, d (6.9)	21.5, CH3	0.98, d (6.9)
14	14.4, CH3	0.89, d (6.9)	15.3, CH3	0.82, d (6.9)
15a	64.7, CH2	4.25, d (12)	106.0, CH2	4.88, s
15b		4.21, d (12)		4.66, s
16	171.1, qC
17	21.1, CH3	2.15, s
18	171.4, qC
19	21.4, CH3	2.12, s

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The elemental composition of pholiotin E (5) was established as C₁₅H₂₂O₃ (five degrees of unsaturation) by HRESIMS. Analysis of the ¹H and ¹³C NMR data (Table 2) of 5 revealed almost identical with those found of 1, except that the sp³ quaternary carbon (δ_C 74.9) and the oxymethylene (δ_H 3.63; δ_C 62.8) were replaced by the exocyclic olefin C-10/C-15 (δ_H 4.66 and 4.88; δ_C 106.0 and 155.1, respectively), which was confirmed by HMBC cross-peaks from H-6 to C-10, H-9 to C-15, and from H₂-15 to C-1, C-9, and C-10. Therefore, the structure of 5 was determined and the relative configuration was confirmed by X-ray crystallography (Fig. 3).

The absolute configuration of 5 was deduced by comparison of the experimental and the simulated circular dichroism (CD) spectra (Fig. 7) generated by the time-dependent density functional theory (TDDFT).²⁵ Compound 5 was used to calculate two enantiomers, (1R,6R,7S,9R)-5 (5a) and (1S,6S,7R,9S)-5 (5b). MMFF94 conformational search and DFT re-optimization at the B3LYP/6-31+G(d) level yielded 4 lowest energy conformers for 5a (Fig. S26†). The overall calculated ECD spectra of 5a and 5b were then generated by Boltzmann weighting of the conformers (Fig. 7). The experimental ECD curve of 5 was nearly identical to the calculated ECD spectrum of 5b, suggested the 1S,6S,7R,9S absolute configuration for 5.

Fig. 7 Experimental ECD spectrum of 5 in MeOH and the calculated ECD spectra of 5a and 5b, after a UV correction of 10 nm.

The known compounds 6–8 isolated from the crude extract were identified as 11-hydroxy-1(10)-valencen-2-one (6),¹⁷ 8,11-dihydroxy-1(10)-eremophilen-2-one (7)¹⁸ and durgamone (8),^19,20 respectively, by comparison of their NMR and MS data with those reported.

Compounds 1–8 were tested for antifungal activity of Aspergillus flavus (CGMCC 3.0951), Fusarium nivale (CGMCC 3.4600) and Piricularia oryzae (CGMCC 3.3283). Compound 4 showed antifungal effects against Aspergillus flavus (CGMCC 3.0951), with an IC₅₀ value 25.1 µM, while the positive control amphotericin B showed an IC₅₀ value of 3.3 µM. Other compounds proved to be inactive in antifungal assays even at 200 µg mL⁻¹.

Conclusions

In conclusion, five new cadinane sesquiterpenoids, pholiotins A–E (1–5) have been isolated from the crude extract of Pholiota sp. Their structures were elucidated based on NMR spectroscopic data and electronic circular dichroism (ECD) calculations. The activity experiments exhibited that compound 4 showed antifungal activities against Aspergillus flavus (CGMCC 3.0951). Pholiotins A–E (1–5) are new cadinane sequiterpenoids. Pholiotin A (1) is structurally related to the known fungal metabolite dysodensiol D,²⁶ but differs by having two more hydroxy groups at C-9 and C-15, respectively. Compound 2 is an acetylation product of 1 at C-9, whereas 3 is an acetylation product of 1 at C-15. Pholiotin D (4) is the C-9 and C-15 diacetylation product of 1. Pholiotin E is a new analogue of 4,10(14)-cadinadien-15-oic acid, differing by having a hydroxy group attached to C-9.²⁷ This was the first report that cadinane sesquiterpenoids were isolated and identified from the genus Pholiota.

Experimental

General experimental procedures

Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter using MeOH as solvent. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. ¹H and ¹³C NMR data were acquired with Varian Mercury-500 spectrometer using solvent signals (acetone-d₆: δ_H 2.05/δ_C 29.8, 206.1; CDCl₃; δ_H 7.26/δ_C 77.7) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS data were recorded on a Bruker Esquire 3000^plus spectrometer, and HRESIMS data were obtained using Bruker APEX III 7.0 T and APEX II FT-ICR spectrometers, respectively.

Fungal material

The culture of Pholiota sp. was isolated from a soil sample on the surface of the fruiting body of C. sinensis (Berk.) Sacc. collected in Kangding, Sichuan, People's Republic of China. The isolate was identified by one of the authors (X. L.) 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 in X. L's culture collection at the Institute of Microbiology, Chinese Academy of Sciences, Beijing. The fungal 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 × 0.5 × 0.5 cm³) under aseptic conditions, 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 H₂O to give a final spore/cell suspension of 1 × 10⁶ mL. Distilled H₂O (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 × 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. The fraction (62 mg) eluted with 50% EtOAc was separated by Sephadex LH-20 column chromatography (CC) eluting with 1 : 1 CH₂Cl₂–MeOH. The resulting subfractions were combined and further purified by RP HPLC (Agilent Zorbax SB-C₁₈ column; 5 µm; 9.4 × 250 mm; 42% MeOH in H₂O for 5 min, followed by 45–100% for 30 min; 2 mL min⁻¹) to afford 1 (5.2 mg, t_R 17.32 min) and 4 (2.0 mg, t_R 26.21 min). The fraction (32 mg) eluted with 60% EtOAc was also separated by RP HPLC (60% MeOH in H₂O for 5 min, followed by 80–90% for 25 min) to afford 5 (2.1 mg, t_R 15.23 min). The fraction (98 mg) eluted with 65% EtOAc was also separated by RP HPLC (50% MeOH in H₂O for 5 min, followed by 80–90% for 35 min) to afford 6 (3.2 mg, t_R 17.28 min), 7 (2.8 mg, t_R 28.20 min) and 8 (1.5 mg, t_R 20.30 min). The fraction (90 mg) eluted with 70% EtOAc was purified by RP HPLC (42% MeOH in H₂O for 5 min, followed by 45–100% for 30 min) to afford 2 (4.5 mg, t_R 18.94 min) and 3 (3.6 mg, t_R 21.12 min).

Pholiotin A (1)

White needles, (acetone–H₂O), mp 230–235 °C; [α]²⁵_D +15.7 (c 0.6, MeOH); UV (MeOH) λ_max (log ε) 212 (3.39), 227 (3.40) nm; CD (c 1.0 × 10⁻³ M, MeOH) λ_max (Δε) 220 (+2.7), 249 (−1.9); IR (neat) ν_max 3334, 2953, 1710, 1640, 1408, 1278, 1069, 1040 cm⁻¹; ¹H and ¹³C NMR data see Table 1; HMBC data (acetone-d₆, 500 MHz) H-1 → C-2, 3, 6, 7, 10, 15; H₂-2 → C-1, 3, 4, 6; H-3b → C-11; H-5 → C-1, 3, 4, 7, 11; H-6 → C-2, 4, 8, 12; H-7 → C-8; H₂-8 → C-6, 10, 12; H-12 → C-7, 8, 13, 14; H₃-13 → C-7, 12, 14; H₃-14 → C-7, 12, 13; H₂-15 → C-1, 9, 10; HREIMS m/z 307.1513 (calcd for C₁₅H₂₄O₅Na 307.1516).

X-ray crystallographic analysis of pholiotin A (1)²⁸

Upon crystallization from acetone–H₂O (5 : 1) using the vapor diffusion method, colorless crystals were obtained for 1, a crystal (0.23 × 0.14 × 0.12 mm) was separated from the sample and mounted on a glass fiber, and data were collected using a Rigaku RAPID IP diffractometer with graphite-monochromated Mo Kα radiation, λ = 0.71073 Å at 173(2) K. Crystal data: C₁₅H₁₄O₅, M = 284.34, space group orthorhombic, P2(1)2(1)2(1); unit cell dimensions a = 5.5709 (11) Å, b = 15.994(3) Å, c = 16.547(3) Å, V = 1474.4(5) ų, Z = 4, D_calcd = 1.281 mg m⁻³, µ = 0.095 mm⁻¹, F(000) = 616. The structure was solved by direct methods using SHELXL-977 (ref. 29) and refined by using full-matrix least-squares difference Fourier techniques. All non-hydrogen atoms were refined with anisotropic displacement parameters and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Absorption corrections were performed using the Siemens Area Detector Absorption Program (SADABS).³⁰ The 13 237 measurements yielded 1947 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R₁ = 0.0450 and wR₂ = 0.1089 [I > 2σ(I)].

Preparation of (R)-(1a)

A sample of 1 (1.0 mg, 0.004 mmol), (S)-MTPA Cl (2.0 µL, 0.011 mmol), and pyridine-d₅ (0.5 mL) were allowed to react in an NMR tube at ambient temperature for 24 h, with the ¹H NMR data of the R-MTPA ester derivative (1a) were obtained directly on the reaction mixture: ¹H NMR (pyridine-d₅, 600 MHz) δ 6.99 (1H, s, H-5), 4.25 (1H, d, J = 10 Hz, H-15a), 4.21 (1H, d, J = 10 Hz, H-15b), 3.82 (1H, s, H-9), 2.37 (1H, dd, J = 15, 5.7 Hz, H-3a), 2.13 (1H, td, J = 5.5, 1.0 Hz, H-2a), 2.11 (1H, m, H-12), 2.07 (1H, m, H-3b), 2.00 (1H, m, H-6), 1.73 (1H, t, J = 11 Hz, H-8a), 1.68 (1H, dt, J = 12, 3.3 Hz, H-1), 1.52 (1H, m, H-7), 1.33 (1H, dt, J = 11, 1.5 Hz, H-8b), 1.13 (1H, m, H-2b), 0.90 (3H, d, J = 6 Hz, H₃-13), 0.76 (3H, d, J = 5.5 Hz, H₃-14).

Absolute configuration of the 9,10-diol moiety in 1a^23,24

HPLC grade DMSO was dried with 4 Å molecular sieves. According to a published procedure, a mixture of 1 : 1.3 diol/Mo₂(OAc)₄ for 1a was subjected to CD measurements at a concentration of 1.0 mg mL⁻¹. The first CD spectrum was recorded immediately after mixing, and its time evolution was monitored until stationary (about 10 min after mixing). The inherent CD was subtracted. The observed signs of the diagnostic bands at around 310 and 400 nm in the induced CD spectrum were correlated to the absolute configuration of the 9,10-diol moiety.

Pholiotin B (2)

White powder; [α]²⁵_D +20.5 (c 0.4, MeOH); UV (MeOH) λ_max (log ε) 216 (3.39), 220 (3.38) nm; CD (c 1.0 × 10⁻³ M, MeOH) λ_max (Δε) 222 (+2.2), 249 (−0.8); IR (neat) ν_max 3413, 2957, 1692, 1641, 1371, 1243, 1039 cm⁻¹; ¹H and ¹³C NMR data see Table 1; HMBC data (acetone-d₆, 500 MHz) H-1 → C-2, 3, 6, 9; H₂-2 → C-1, 3, 6; H₂-3 → C-1, 2, 4, 5; H-5 → C-1, 3, 7, 11; H-6 → C-1, 2, 4, 8, 12; H-7 → C-6, 8; H₂-8 → C-6, 7, 12; H-9 → C-1, 7, 10, 18; H-12 → C-7, 13, 14; H₃-13 → C-7, 12, 14; H₃-14 → C-7, 12, 13; H₂-15 → C-1, 9, 10; H₃-19 → C-18; HRESIMS m/z 349.1619 (calcd for C₁₇H₂₆O₆Na, 349.1622).

Pholiotin C (3)

White powder; [α]²⁵_D +26.9 (c 0.4, MeOH); UV (MeOH) λ_max (log ε) 216 (3.55), 220 (3.62) nm; CD (c 1.3 × 10⁻³ M, MeOH) λ_max (Δε) 219 (+2.7), 250 (−0.8); IR (neat) ν_max 3422, 2956, 1690, 1641, 1371, 1241, 1040 cm⁻¹; ¹H and ¹³C NMR data see Table 1; HMBC data (acetone-d₆, 500 MHz) H-1 → C-3, 15; H-2a → C-1, 6; H-2b →C-1, 3; H₂-3 → C-1, 2, 4, 5; H-5 → C-3, 4, 6, 7, 11; H-7 → C-8; H₂-8 → C-6, 10; H-9 → C-7; H-12 → C-7, 13, 14; H₃-13 → C-7, 12, 14; H₃-14 → C-7, 12, 13; H₂-15 → C-1, 9, 10, 16; H₃-17 → C-16; HREIMS m/z 349.1623 (calcd for C₁₇H₂₆O₆Na 349.1622).

Pholiotin D (4)

White powder; [α]²⁵_D +15.5 (c 0.2, MeOH); UV (MeOH) λ_max (log ε) 212 (3.14), 220 (3.13) nm; CD (c 0.8 × 10⁻³ M, MeOH) λ_max (Δε) 218 (+0.5), 259 (−0.2); IR (neat) ν_max 3440, 2931, 1719, 1372, 1239, 1037 cm⁻¹; ¹H and ¹³C NMR data see Table 1; HMBC data (acetone-d₆, 500 MHz) H-1 → C-3, 15; H₂-2 → C-6; H₂-3 → C-1; H-5 → C-1, 11; H-6 → C-2; H-7 → C-8; H₂-8 → C-6, 10, 12; H-9 → C-1, 7, 10, 18; H-12 → C-7, 13, 14; H₃-13 → C-7, 12, 14; H₃-14 → C-7, 12, 13; H₂-15 → C- 9, 16; H₃-17 → C-16; H₃-19 → C-18; HRESIMS m/z 391.1728 (calcd for C₁₉H₂₈O₇Na, 391.1727).

Pholiotin E (5)

White needle; mp 130–135 °C; [α]²⁵_D −16.9 (c 0.2, MeOH); UV (MeOH) λ_max (log ε) 214 (3.94) nm; CD (c 1.0 × 10⁻³ M, MeOH) λ_max (Δε) 217 (−20); IR (neat) ν_max 3322, 2950, 2921, 2624, 1689, 1643, 1390, 1259, 1057 cm⁻¹; ¹H and ¹³C NMR data see Table 1; HMBC data (acetone-d₆, 500 MHz) H₂-2 → C-4; H₂-3 → C-1, 5; H-5 → C-1, 3, 11; H-6 → C-2, 10; H-7 → C-1; H-9 → C-1, 7, 15; H-12 → C-7, 13, 14; H₃-13 → C-7, 12, 14; H₃-14 → C-7, 12, 13; H₂-15 → C-1, 9, 10; HRESIMS m/z 249.1498 (calcd for C₁₅H₂₁O₃, 249.1496).

X-ray crystallographic analysis of pholiotin E (5)³¹

Upon crystallization from acetone–H₂O (5 : 1) using the vapor diffusion method, colorless crystals were obtained for 5, a crystal (0.20 × 0.15 × 0.10 mm) was separated from the sample and mounted on a glass fiber, and data were collected using a Rigaku RAPID IP diffractometer with graphite-monochromated Mo Kα radiation, λ = 0.71073 Å at 173(2) K. Crystal data: C₃₀H₄₆O₇, M = 518.67, space group orthorhombic, P2(1)2(1)2; unit cell dimensions a = 16.315 (3) Å, b = 17.050 (3) Å, c = 5.2386 (10) Å, V = 1457.3 (5) ų, Z = 2, D_calcd = 1.182 mg m⁻³, µ = 0.083 mm⁻¹, F(000) = 564. The structure was solved by direct methods using SHELXL-97 (ref. 29) and refined by using full-matrix least-squares difference Fourier techniques. All non-hydrogen atoms were refined with anisotropic displacement parameters and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Absorption corrections were performed using the Siemens Area Detector Absorption Program (SADABS).³⁰ The 12 011 measurements yielded 1929 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R₁ = 0.0430 and wR₂ = 0.1026 [I > 2σ(I)].

11-Hydroxy-1(10)-valencen-2-one (6)

¹H, ¹³C NMR, and the MS data were consistent with literature values.¹⁷

8,11-Dihydroxy-1(10)-eremophilen-2-one (7)

¹H, ¹³C NMR, and the MS data were consistent with literature values.¹⁸

Durgamone (8)

¹H, ¹³C NMR, and the MS data were consistent with literature values.^19,20

Computational details

Systematic conformational analyses for 1c and 1d, 5a and 5b were performed via the Molecular Operating Environment (MOE) ver. 2009.10. (Chemical Computing Group, Canada) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational analyses were further optimized using DFT at the B3LYP/6-31+G(d) or cam-B3LYP/6-31G(d) basis set level. The stationary points have been checked as the true minima of the potential energy surface by verifying they do not exhibit vibrational imaginary frequencies. The 50 lowest electronic transitions were calculated at the Cam-B3LYP/6-31+G(d) level, and the rotational strengths of each electronic excitation were given using both dipole length and dipole velocity representations. ECD spectra were stimulated using a Gaussian function with a half-bandwidth of 0.3 eV. Equilibrium populations of conformers at 298.15 K were calculated from their relative free energies (ΔG) using Boltzmann statistics. The overall ECD spectra were then generated according to Boltzmann weighting of each conformer. The systematic errors in the prediction of the wavelength and excited-state energies are compensated for by employing UV correction.²⁵

Antifungal assays

Antifungal assays were conducted in triplicate following the National Center for Clinical Laboratory Standards (NCCLS) recommendations.³² The fungi, Aspergillus flavus (CGMCC 3.0951), Piricularia oryzae (CGMCC 3.3283), and Fusarium 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 10⁴ hyphae per mL (in PDB medium). Test samples (4 mg mL⁻¹ as 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 µL. Alamar blue (10 µL 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 E_x/E_m = 544/590 nm. The inhibition was calculated and plotted versus test concentrations to afford the IC₅₀.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21372004), the Program of the Excellent Young Scientists of Chinese Academy of Sciences, the Youth Innovation Promotion Association of Chinese Academy of Sciences (2011083) and Natural Science Fund of Colleges and Universities in Jiangsu Province (16KJA180005, 16KJB180006).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures, NMR spectra of compounds 1–5. CCDC 861573 and 861574. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra22448b

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