Cadinane sesquiterpenes from the mushroom Lyophyllum transforme

Phytochemistry 93 (2013) 192–198

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Cadinane sesquiterpenes from the mushroom Lyophyllum transforme Marco Clericuzio a, Roberto Negri b, Maurizio Cossi a, Gianluca Gilardoni c, Davide Gozzini c, Giovanni Vidari c,⇑ a

Università del Piemonte Orientale, Dipartimento di Scienze e Innovazione Tecnologica, Via T. Michel 11, 15121 Alessandria, Italy Università del Piemonte Orientale, Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Largo Donegani 2, 28100 Novara, Italy c Università degli Studi di Pavia, Dipartimento di Chimica, V.le Taramelli 12, 27100 Pavia, Italy b

a r t i c l e

i n f o

Article history: Received 8 January 2013 Received in revised form 21 March 2013 Available online 2 May 2013 Keywords: Lyophyllum transforme Lyophyllaceae Structure elucidation Absolute configuration TD-DFT calculations CD Sesquiterpenes Cadinane

a b s t r a c t Two rare cadinane-type sesquiterpenes, lyophyllone A (1) and lyophyllanetriol A (2), were isolated from the mushroom Lyophyllum transforme. The structures were elucidated on the basis of exhaustive NMR techniques, together with MS, UV–Vis and molecular modelling. The absolute configuration of lyophyllone A was determined by ab initio theoretical CD calculation performed by Density Functional Theory (DFT) using the B3PW91/6-31G(d,p) basis set. The experimental CD were found to be in good agreement with the corresponding population-weighted theoretical CD spectra, allowing for the determination of the absolute stereochemistry of the compound. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Mushrooms (Basidiomycota) have been used for food, medicinal, and spiritual purposes in diverse human societies from the very beginning of civilisation. For example, the 5300-year-old Ice-Man who was discovered in a Tyrolean glacier was found carrying pieces of the birch polypore, Piptoporus betulinus (Bull.: Fr.) P. Karst., though its actual use is still debated (Pöder, 2005). In recent years, mushrooms have attracted the increasing attention for the abundance of bioactive natural products contained in fruiting bodies or in culture broths; for this reason, they represent a potential valuable source for new natural drugs (Barros et al., 2008; Gao, 2006; Wasser and Weiss, 1999; Zjawiony, 2004). Lyophyllum P. Karst. is a relatively small genus of fungi, belonging to the Lyophyllaceae family and comprising about 40 species, that are mostly widespread in the northern hemisphere temperate regions (Kirk et al., 2008). Antidiabetic (Miura et al., 2002), anti-dermatitis (Ukawa et al., 2007), radioprotective and antitumor effects (Gu et al., 2005), ACE inhibitory activity (Gao et al., 2011),

⇑ Corresponding author. Tel.: +39 0382 987322; fax: +39 0382 987323. E-mail address: [email protected] (G. Vidari). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.03.019

serum lipid level lowering properties (Ukawa et al., 2002) have been attributed to Lyophyllum decastes, while anti-inflammatory properties have been found for a Lyophyllum connatum extract (Yasukawa et al., 1996). Phytochemical investigations include the isolation of polysaccharides with antitumor effects from L. decastes (Ukawa et al., 2000), and a new ceramide (Yaoita et al., 2003), lyophillin (Fugmann and Steglich, 1984), b-hydroxyergothioneine, ergothioneine, N-hydroxy-N0 ,N0 -dimethylurea, and connatin from L. connatum (Kimura et al., 2005). All the urea derivatives displayed the ability to scavenge free radicals; moreover, both ergothioneine and b-hydroxyergothioneine showed protective properties against carbon tetrachloride-induced injury in rat primary culture hepatocytes (Kimura et al., 2005). Given the limited phytochemical investigation of Lyophyllum species, we decided to initiate a systematic study of the species in this genus, continuing our investigation on the chemical constituents of wild Italian mushrooms. In this report, we describe the isolation and structural elucidation of two new cadinane sesquiterpenes, named lyophyllone A (1) and lyophyllanetriol A (2) from the fresh fruiting bodies of Lyophyllum transforme (Britzelm.) Singer. This is a rather rare species, which grows solitary, rarely fasciculate, in deciduous forests of Apennines, from late summer to autumn.

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Table 1 NMR spectroscopic data (300 MHz, CD2Cl2) for lyophyllone A (1) and lyophyllanetriol A (2). Position

2. Results and discussion

Lyophyllone A (1)

Lyophyllanetriol A (2)

dC, type

dH (J in Hz)

dC, type

dH (J in Hz)

1

42.4, CH2

26.3, CH2

2 3 4 5 6 7 8 9 10 11 12 13

199.3, C 136.5, C 145.3, CH 47.0, CH 77.3, C 105.4, C 75.7, CH 40.6, CH 40.2, CH 146.4, C 112.0, CH2 68.3, CH2

14 15

14.7, CH3 16.1, CH3

2.08, dd (16.0, 13.5) 2.73, dd (16.0, 3.7) – – 7.18, br s 2.66, br d (10.6) – – 3.23, d (10.3) 1.49–1.60, m 1.61–1.75, m – 5.15, t (2.0) 5.31, t (2.0) 4.28, dd (13.4, 2.0) 4.69, dd (13.4, 2.0) 1.03, d (6.4) 1.84, br s

1.10–1.25, m 1.90–2.10, ma 1.90–2.10, ma – 5.76, br s 2.28, br d (10.4) – – 3.18, d (10.0) 1.30–1.40, m 1.04–1.12, mb – 5.50, t (2.4) 5.07, t (2.0) 4.25, dt (13.2, 2.0) 4.63, dt (13.2, 2.4) 1.04, d (6.5)b 1.75, br s

2.1. Structures of new compounds Fresh fruiting bodies of L. transforme were frozen at 20 °C, minced, and extracted with acetone. Extraction of mushrooms at low temperature in aprotic solvents is highly recommended to avoid the formation of chemical artefacts, which may occur when mushroom are ground and cells are disrupted at room temperature, while enzymes are not inactivated (Daniewski and Vidari, 1999). Indeed, the raw extract obtained according to this procedure was identical (TLC) to an extract obtained by maceration in acetone of an intact mushroom specimen, indicating that the main detectable metabolites remained unchanged. After acetone evaporation, the remaining aqueous phase was extracted with EtOAc which, after removal in vacuo, left a brownish oily residue. This was dissolved in acetonitrile and the solution was extracted with hexane to remove apolar components. TLC analysis of the residue obtained by evaporation of MeCN indicated the presence of different compounds detected as deep blue-green spots by spraying with the vanillin sulfuric acid reagent (Wagner and Bladt, 1996). They were fractioned by multiple preparative chromatographic separations on silica gel and RP-18 columns which eventually yielded compounds 1 and 2. The common sterols ergosterol and 3b,5a,22E,24R)-5,8-epidioxyergosta-6,22-dien-3-ol (ergosterol peroxide) were identified in the hexane fraction, by comparing the spectroscopic data with the literature (Liu et al., 2010). The molecular formula of lyophyllone A (1) was determined as C15H20O5 from the MW 280 inferred from the ESI MS spectrum ((MH) m/z 279), HRESI MS (see Section 4), and from the H/C atoms counting in the NMR spectra (Table 1). The 1H NMR spectrum showed three olefinic hydrogens; two of them (d 5.31 (t, J = 2.0) and 5.15 (t, J = 2.0)) were attributed to an exo-CH2 group (H2-12) by HSQC correlation with an sp2 carbon (dC 112.0), while the third one (d 7.18, br s, H-4)) was typical of a CH in b-position to an a,b-unsaturated carbonyl group. The presence of the conjugated ketone was confirmed by the C-atom system in the 13C NMR spectrum (dC 199.3 (s, C-2), 145.3 (d, C-4), 136.5 (s, C-3)), by the 1675 cm1 absorption in the IR spectrum, and by the 238 nm (log e = 4.01) band in the UV spectrum. In addition, the 1 H NMR spectrum of 1 (Table 1) revealed signals assigned to a vinyl Me group (d 1.84, br s, H3-15), a secondary Me group (d 1.03, d, J = 6.4, H3-14), an oxygenated methylene group forming an AB system (d 4.69 (dd, J = 13.4, 2.0) and d 4.28 (dd, J = 13.4, 2.0), H2-13), an oxygenated methine (d 3.23, d, J = 10.3, H-8), and a CH2 group flanking the CO (d 2.73 (dd, J = 16.0, 3.7) and d 2.08 (dd, J = 16.0, 13.5), H2-1). Moreover, the 13C NMR spectrum of 1 (Table 1) displayed one sp2 quaternary C-atom (dC 146.4, C-11), three sp3 CH atoms (dC 47.0, 40.6, and 40.2, C-5, C-9, and C-10, respectively), which were connected to the corresponding bonded protons by the HSQC spectrum, and two oxygenated sp3 quaternary C-atoms (dC 105.4 (C-7) and dC 77.3 (C-6)), including an acetal one. Since

a,b

30.5, CH2 135.1, C 119.9, CH 45.9, CH 76.9, C 105.1, C 75.8, CH 40.0, CH 38.1, CH 146.5, C 110.8, CH2 67.5, CH2 14.5, CH3 23.0, CH3

Overlapping signals.

three out of six unsaturation degrees were accounted for by the presence of the C@O and the two C@C bonds, compound 1 was inferred to be a tricyclic system. The 1H–1H COSY spectrum of 1 indicated the two isolated spin systems a and b (Fig. 1a), which were connected to each other by HSQC and HMBC spectra. Thus, correlations (Fig. 1b) of H3-15 with C-2 and C-4, H-1 with C-3, C-5, and C-10, H-10 with C-2, C-4, and C-6, H-8 with C-7 were detected, implying a bicyclo(4.4.0)decane moiety. HMBC correlations from the exo-methylene protons (H2-12) to C-6 and from H2-13 to C-6, C-7, C-11, and C-12 defined the presence and the location of a tetrahydrofuran ring with the exo-CH2 group (fragment b). The whole of these data, considered together, indicated a cadinane-type sesquiterpenoid structure for lyophyllone A, in accordance with the constitutional formula 1 shown above. The relative stereochemistry of 1 was established by the analysis of the NOESY spectrum (Fig. 1c) and 3J coupling constants. In fact, in the 1H NMR spectrum of 1, the large coupling constants of H-5 (J5,10 = 10.6) and H-8 (J7,8 = 10.3), characteristic for a diaxial relationship with H-10 and H-9, respectively, demonstrated the trans-juncture of the decalin system and the diequatorial orientation of OH-8 and Me-9. Moreover, NOESY correlations between H-5 and H-9, and between H-8, H-10, and Me-9 confirmed the trans-juncture of the decalin ring system and the relative stereochemistry of stereocenters H-5, H-8, H-9, and H-10. Finally, a cisfusion between the five- and six-membered rings with a cis-diol group at C-6 and C-7, was established by the correlations of Ha12 with H-10 and Ha-13 with H-8. The molecular formula of lyophyllanetriol A (2) was determined as C15H22O4 from the MW 266, inferred from the ESI+-MS spectrum ((M+Na)+ m/z 289), HRESI+ MS (see Section 4), and from the H/C atoms counting in the NMR spectra (Table 1), that implied one unsaturation and one O-atom less than in 1. The 1H and 13C NMR spectroscopic data of 2 (Table 1) indicated many signals common to compound 1, such as one vinylic (d 1.75, H3-15) and one secondary (d 1.04 (3H, d, J = 6.5), H3-14) Me groups, an exo-CH2 vinyl moiety (d 5.50 (1H, t, J = 2.4) and d 5.07 (1H, t, J = 2.0), H2-12), an oxygenated methine (d 3.18 (1H, d, J = 10.0), H-8), and two oxygenated sp3 quaternary C-atoms (dC 105.1 (C-7) and dC 76.9 (C-6)), including an acetal one. However, the spectroscopic data for the carbonyl group at C-2 were absent; instead, the signal for the vinylic proton H-4 (d 5.76 (1H, br s)) was upfield shifted and an additional CH2 group (d 1.10–1.25 (1H, m) and d 1.90–2.10 (1H,

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14

O

1

a

2

OH OH

b

OH

H

OH

O

4

3

9

9 10

OH

5

15

8

OH

7

8

6

O

H

11

5 10

HO

O

O

H Ha

13

Ha

Hb

13

4

OH

H

12

(a)

O

H OH

12

Hb

(c)

(b)

Fig. 1. Important 1H–1H COSY (a), HMBC (b), and NOESY (c) correlations observed for 1.

14 1

OH

OH OH

b

OH

5

3 15

4

9

8

2

a

H

OH

9

10

OH

7

O

6

O 13

5

10

HO 8

H Ha

11

H OH

O

OH

4

H

H

Ha

13 12

Hb

Hb

12

(a)

(b)

(c)

Fig. 2. Important 1H–1H COSY (a), HMBC (b), and NOESY (c) correlations observed for 2.

m); dC 26.3 (t)) was detected. The 1H–1H COSY spectrum of 2 showed that the C-bonded protons constituted two isolated spin systems (Fig. 2a), one (a) comprising the two Me groups and all the hydrogens located at the periphery of the decalin ring-system, while the other one (b) comprised the protons H2-12 and H2-13. The two moieties were connected to each other by HSQC and HMBC correlations (Fig. 2b), which allowed us to firmly establish the entire tricyclic structure of 2. The relative stereochemistry of 2 was determined to be identical to the stereostructure of 1, on the basis of the large 3J coupling constants of H-5 (J5,10 = 10.4) and H-8 (J8,9 = 10.0), and the NOESY correlations between H-5 and H-9, H-8, and H-13a, and between H-12a and H-4 and H-10. In conclusion, spectroscopic data indicated that lyophyllanetriol A (2) was the 2-deoxo derivative of 1. Compounds 1 and 2 showed no inhibitory activity against the growth in vitro of several human tumor cell lines. 2.2. Absolute stereochemistry of compounds 1 and 2 Lyophyllone A (1) and lyophyllanetriol A (2) are members of a small group of rare fungal cadinane sesquiterpenes, including strobilols A–M and stereumins A–G (Hiramatsu et al., 2011; Liu et al., 2010), featuring a cis-diol group on the bridgehead carbons of the juncture between a decalin and a methylidene-substituted tetrahydrofuran ring. Strobilols were isolated from the fruiting bodies and culture broth of Strobilurus ohshimae (Tricholomataceae) (Hiramatsu et al., 2011), while stereumins were found in the culture broth and the mycelium of a Stereum strain (Stereaceae) (Liu et al., 2010). Notably, it appears that the fungal enzymes regulating the biosynthesis of the two groups of sesquiterpenes, have opposite enantioselectivity, as shown by the enantiomeric stereochemistry proposed for stereumin B and strobilol B on the basis of the opposite signs of molecular rotations (Fraga, 2009). Thus, it appears important to determine the absolute configuration (AC) of compounds 1 and 2 on a sound basis. At the onset of such investigation, we excluded the possibility to use the classical

Mosher esters, not only for the paucity of isolated compounds, but also for the foreseeable difficulties in achieving a clean regioselective monoesterification of one of the three vicinal hydroxyl groups in 1 and 2. Alternatively, we undertook a study of the AC of lyophyllone A (1) on the basis of its chiroptical properties. The CD curve of 1, recorded in MeOH (Fig. 5, solid line), showed a weak negative dichroic absorption (De = 0.3) at about 330–340 nm, in the region of the enone n ? p⁄ transition, and, at shorter wavelengths, a positive band at about 240 nm (De  +1), and a negative, more intense band at about 205 nm (De  6.5), both attributable to p ? p⁄ transitions of the enone chromophore. For a twisted transoid a,b-unsaturated enone, the signs of the CD maxima at longer wavelengths are usually determined by the sense of helicity of this chromophore and may lead to the assignment of the absolute configuration of a compound on the basis of semiempirical ‘‘chirality’’ rules (Gawronski, 1982; Kirk, 1986). Application of such rules to lyophyllone A (1) thus required preliminary estimation of the dihedral angle of the enone moiety by molecular modelling. The lowest energy conformations of 1 were investigated by ab initio computational methods, using the model B3LYP and the 631+g(d,p) basis set. Initially, the structures were minimized in vacuo. Two low-energy conformers, differing by 2.06 kcal/mol, were found (A and B in Fig. 3), that showed an almost identical distorted half chair–chair geometry of the decalin-ring system, and a different conformation of the tetrahydrofuran ring, which was twisted in A and an envelope in B. A third high energy geometry (C in Fig. 3) was also located, 3.49 kcal/mol above the minimum. Molecular geometries resulting from the different puckering of five-membered rings, in particular of THF rings, have been extensively studied in the literature, both theoretically and experimentally: the two lowest energy conformations, the twist C2, and the envelope Cs symmetry geometries, are typically very close in energy (0.3–0.5 kcal/mol) and mutually related by ring pseudorotation (Han and Kang, 1996; Ning et al., 2008). We presumed that the calculated higher energy separation between the conformations A and B of compound 1 was reasonably due to an increased rigidity of the THF ring, caused by the fusion with the trans-decalin system.

M. Clericuzio et al. / Phytochemistry 93 (2013) 192–198

195

Fig. 3. B3LYP-calculated lowest energy conformations of lyophyllone A (1).

Fig. 4. CD spectra calculated for conformations A (solid line) and B (dotted line) of lyophyllone A 1 in MeOH.

Subsequently, the above geometries were minimized in different solvents, using the conductor-like polarizable continuum model (Cossi et al., 2003). Interestingly enough, the energy gap between conformations A and B dropped considerably, whereas the energy of conformation C remained considerably higher (3.5 kcal/mol). Conformation C was therefore discarded in the subsequent analysis. Notably, the calculated energy difference between conformations A and B was merely 0.32 kcal/mol in MeOH. In each of the minimized geometries of 1, the a,b-conjugated carbonyl system appeared to be almost planar, a feature that had also been observed in earlier molecular mechanics calculations (data not shown). In fact, the O2–C2–C3–C4 dihedral angle in 1 was calculated to be about +177–178°. This made the application of simple CD

‘‘helicity rules’’ (Gawronski, 1982; Kirk, 1986) unreliable for shedding light on the absolute configuration of lyophyllone A. Indeed, we supposed that the contribution of the small skewness of the chromophore to the chirality of the electronic transitions in compound 1 could be overwhelmed by the chiral arrangement of substituents in the molecule. Ab initio calculation of the optical activity of lyophyllone A, employing the TD-DFT theoretical model (Stephens et al., 2006), was thus the method of choice for achieving evidences about the absolute stereochemistry of compound 1 from the CD spectrum (see Calculation Details in Section 4). At first, the electronic transitions of 1 were calculated up to 190 nm. The calculated UV spectrum showed an intense

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Fig. 5. Experimental CD (solid line) and Bolztmann averaged calculated CD spectra (dotted line) of lyophyllone A 1 in MeOH.

absorption band at 238 (conformation A)/235 (conformation B) nm, and a second band at 228–229 nm (both conformations), about 75% less intense than the other one (Supporting information). Subsequently, we calculated the electronic CD spectra for each of the two lowest energy geometries A and B of lyophyllone A, minimized in MeOH, assuming the absolute configuration written as 1. The two graphics, shown in Fig. 4, were rather different between each other, in accordance with the different puckering of the THF ring, which causes a different spatial orientation of the C(11)– C(12) double bond with respect to the enone system. In fact, the interaction between these two chromophores is likely to dominate the observed Cotton Effect (CE) of compound 1, according to an interpretation based on a (chromophore) independent-systems frame (Tinoco, 1962), while ether and alcoholic groups hardly contribute to the observed CE at shorter frequencies, as they are very little absorptive at wavelengths >190 nm (Snyder and Johnson, 1978). The Boltzmann-averaged CD curve shown in Fig. 5 (dotted line) was computed weighting the two conformations according to their calculated population at 298 K. Superimposition of the calculated and the experimental CD spectra (Fig. 5, solid line) appears good at the two longest-wavelength transitions (from 350 to about 240 nm), both in sign and intensity. On the other hand, superimposition of the two curves is less good at wavelengths <230 nm. In particular, our calculations predict a series of low-intensity negative bands in this region, while the experimental curve shows, instead, only one, rather intense, negative band. We attribute this discrepancy to the approximation implied in the calculations and the conformation uncertainty. TD-DFT CD calculations indicate, therefore, the absolute configuration 5R,6S,7R,8S,9R,10R for compound 1. Biosynthetic considerations suggest a homochiral configuration for lyophyllanetriol A (2). 3. Conclusions In this paper we have reported the first phytochemical investigation of the fruiting bodies of L. transforme. Two new representatives of a rare group of cadinane sesquiterpenes, lyophyllone A (1) and lyophyllanetriol A (2), have been isolated, for the first time, from a fungal species of the Lyophyllaceae family. The two

compounds are structurally related to stereumins (Liu et al., 2010) and strobilols (Hiramatsu et al., 2011) found in two species belonging, respectively, to the Stereaceae and the Tricholomataceae families of Basidiomycetes; however, a special feature of structures 1 and 2 is an unprecedented hydroxylic group on the C-8 of the tricyclic skeleton, indicating the presence of a regiospecific oxygenase in the fruiting bodies of L. transforme. The assignment of the absolute stereochemistry to lyophyllone A (1) could not be performed on an experimental basis (i.e. transformation to a diastereomeric pair, or to a compound of known AC) but was performed by ab initio calculations of its CD curve. In our case, the presence of two conformations having almost equal energy, and leading to different calculated CD curves, requires a word of caution about the output of the theoretical approach. Anyway, as it is evident from Fig. 4, the spectra calculated for conformations A and B of 1 are not mutually specular, so the potential dominance of conformation B would not lead to the enantiomeric AC of 1, but only to a worse agreement between calculated and experimental CD curves. It may be of taxonomic relevance to note that the stereostructures assigned to compounds 1 and 2 belong to the enantiomeric series of stereumins isolated from a Stereum culture, instead of the antipodal group of strobilols, typical of the fruiting bodies of a Strobilurus species. 4. Experimental 4.1. General experimental procedures Optical rotations were measured on a Perkin–Elmer 241 polarimeter. CD spectra were measured on a Jasco J-710 spectropolarimeter. Mass spectra were obtained with a Thermo Scientific LTQ XL Linear Ion Trap mass spectrometer, equipped with an ESI source; both direct infusion and HPLC–MS were performed with this instrument. High Resolution mass spectra were measured on a FT-ICR Apex II mass spectrometer of Bruker Daltonics. UV spectra were recorded on a Perkin–Elmer Lambda 5 instrument. IR spectra were recorded on an FT-IR Perkin–Elmer BX spectrometer. NMR spectra were recorded on a Bruker Avance III 500 MHz instrument, operating at 499.802 MHz (1H) and 125.687 MHz (13C) or, alternatively, at 300 MHz on a Bruker CXP 300 spectrometer. Chemical

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shifts are reported in d units, and solvent peaks were used as internal standards. The multiplicity of each carbon atom was determined by DEPT experiments. Normal pressure preparative column chromatography was carried out on Merck LiChroprep RP-18 (25–40 lm) or Merck silicagel (230–400 mesh), for reversed and direct phase elution modes, respectively. Medium pressure liquid chromatography was performed by means of a MPLC Biotage Isolera system, using home-made preparative cartouches. Semipreparative HPLC was carried out on a Jasco PU-2080 PLUS dual pump HPLC instrument, equipped with a UV detector and a Hypersil BDS C18 5 lm column, operating with an eluent flow of 4.5 mL min1. TLC was performed over Merck F254 glass plates (RP-18), or aluminum supported (0.25 mm) sheets (silicagel). Spots were detected under UV light (254 and 366 nm) and, additionally, they were stained by exposure to a 0.5% solution of vanillin in H2SO4–EtOH (4:1), followed by heating at 100 °C. 4.2. Fungal material Fresh fruiting bodies of L. transforme (340 g) were collected in October 2010 in a mixed conifer and beech forest at Brallo, at about 1100 m on Apennine Mountains south of Pavia (NW Italy). The mushroom species was identified by Alfredo Gatti of the Voghera Mycological Group. A voucher specimen is deposited at the Department of Chemistry with the accession number LF1. 4.3. Extraction and isolation Freshly collected fungal material was frozen at 20 °C and extracted three times with acetone. After solvent evaporation under vacuum, the resulting aqueous phase was extracted with EtOAc and the separated organic layer was dried over Na2SO4 and evaporated to give 522 mg of crude extract. This residue was then partitioned between hexane and MeCN to afford, after solvent removal, an apolar (A, 378 mg) and a polar (B, 133 mg) fraction, respectively. Ergosterol and ergosterol peroxide were identified in fraction A along with an uninvestigated mixture of fatty acid esters. Separation of fraction B was achieved by chromatography on, in the order: (a) a reversed-phase column, eluted with an initial mixture of H2O–MeOH, 2:1, and then increasing the MeOH content in the solvent; (b) repetitive silica gel columns, eluted with different gradients of hexane–EtOAc; (c) a reversed-phase column, eluted under medium pressure with H2O–MeCN, 10:3; (d) a semipreparative HPLC C18 column, eluted with a gradient of MeCN in H2O. Eventually, lyophyllone A (1, 5.3 mg) and lyophyllanetriol A (2, 4.3 mg) were isolated in a pure form. 4.4. Lyophyllone A (1) (3aR,4S,5R,5aR,9aR,9bS)-1,2,4,5,5a,6,9a,9bOctahydro-3a,4,9b-trihydroxy-5,8-dimethyl-1methylidenenaphto(2,1-b)furan-7(3aH)-one) Colorless oil; [a]D23 11.3 (CH2Cl2, c = 0.5); CD, see Fig. 5; UV (CH2Cl2) kmax (log e) 238 (4.01); IR (thin film) mmax 3400, 1650, 1376, 1260, 1020, 922 cm1; 1H and 13C NMR data are reported in Table 1; MS (ESI) m/z 279 [MH], 261 [MHH2O], 243 [MHH2O]; negative mode HR-ESIMS m/z: 279.12331 [MH], C15H19O5 requires 279.12325. 4.5. Lyophyllanetriol A (2) (3aR,4S,5R,5aR,9aR,9bS)1,2,3a,4,5,5a,6,7,9a,9b-Decahydro-5,8-dimethyl-1methylidenenaphto(2,1-b)furane-3a,4,9b-triol) Colorless oil, [a]D23 -42.7 (CH2Cl2, c = 0.3); UV (CH2Cl2) end absorption; IR (thin film) mmax 3390, 1451, 1378, 1242, 1087, 1036, 998, 914, 776 cm1; 1H and 13C NMR data are reported in Table 1; MS (ESI+) m/z 555 [2M+Na]+, 289 [M+Na]+; positive mode

197

HR-ESIMS+ m/z: 289.14155 [M+Na]+, C15H22NaO4 requires 289.14158. MS (ESI) m/z 265 [MH], 247 [MH-H2O]; negative mode HR-ESIMS m/z: 265.14394 [MH], C15H21O4 requires 265.14398. 4.6. Calculation details Theoretical calculations were performed at the Density Functional Theory (DFT) level, using the hybrid B3LYP functional and the 6-31+G(d,p) basis set (Becke, 1988, 1993; Lee et al., 1988), based on the standard Pople’s 6-31G set (Hariharan and Pople, 1973), extended with diffuse and polarization functions (Dunning, 1989). Solvent effects were included with the conductor-like polarizable continuum model (C-PCM) (Cossi et al., 2003), implemented in Gaussian03 suite (Frisch et al., 2004). After the geometry optimization of the various conformers, the electronic absorptions and CD spectra were computed using a Time Dependent-DFT procedure, including in the calculations the 20 lowest-energy electronic transitions. In order to compare calculated R values (in the velocity form) with experimental De data, a Gaussian function with a 7.5 nm band-width was associated with all the transitions. Acknowledgments We thank Prof. Mariella Mella, the University of Pavia, for NMR measurements and the Italian MIUR (Funds PRIN 2009) for partial financial support. All data concerning the reported compounds are included in LIBIOMOL, a chemical library of natural and synthetic bioactive molecules, accessible at the website http://www. libiomol.unina.it. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 03.019. References Barros, L., Cruz, T., Baptista, P., Estevinho, L.M., Ferreira, I.C.F.R., 2008. Wild and commercial mushrooms as source of nutrients and nutraceuticals. Food Chem. Toxicol. 46, 2742–2747. Becke, A.D., 1988. Density-functional exchange-energy approximation with correct asymptotic-behavior. Phys. Rev. A 38, 3098–3100. Becke, A.D., 1993. A new mixing of Hartree–Fock and local density-functional theories. J. Chem. Phys. 98, 1372–1377. Cossi, M., Rega, N., Scalmani, G., Barone, V., 2003. Energies, structures, and electronic properties of molecules in solution with the C-PCM solvation model. J. Comput. Chem. 24, 669–681. Daniewski, W.M., Vidari, G., 1999. Constituents of Lactarius (mushrooms). In: Herz, W., Falk, H., Kirby, G.W., Moore, R.E., Tamm, Ch. (Eds.), Fortschritte der Chemie organischer Naturstoffe, Springer-Verlag, Wien, vol. 77, pp. 69–171. Dunning Jr., T.H., 1989. J. Chem. Phys. 90, 1007–1023. Fraga, B.M., 2009. Natural sesquiterpenoids. Nat. Prod. Rep. 26, 1125–1155. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Montgomery Jr., J.A., Vreven, T., Kudin, K.N., Burant, J.C., Millam, J.M., Iyengar, S.S., Tomasi, J., Barone, V., Mennucci, B., Cossi, M., Scalmani, G., Rega, N., Petersson, G.A., Nakatsuji, H., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Klene, M., Li, X., Knox, J.E., Hratchian, H.P., Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Ayala, P.Y., Morokuma, K., Voth, G.A., Salvador, P., Dannenberg, J.J., Zakrzewski, V.G., Dapprich, S., Daniels, A.D., Strain, M.C., Farkas, O., Malick, D.K., Rabuck, A.D., Raghavachari, K., Foresman, J.B., Ortiz, J.V., Cui, Q., Baboul, A.G., Clifford, S., Cioslowski, J., Stefanov, B.B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Martin, R.L., Fox, D.J., Keith, T., Al-Laham, M.A., Peng, C.Y., Nanayakkara, A., Challacombe, M., Gill, P.M.W., Johnson, B., Chen, W., Wong, M.W., Gonzalez, C., Pople, J.A., 2004. Gaussian 03, Revision C.02. Gaussian, Inc., Wallingford, CT. Fugmann, B., Steglich, W., 1984. Unusual components of the toadstool Lyophyllum connatum (Agaricales). Angew. Chem. 23, 72–73. Gao, J.M., 2006. New biologically active metabolites from Chinese higher fungi. Curr. Org. Chem. 10, 849–871.

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M. Clericuzio et al. / Phytochemistry 93 (2013) 192–198

Gao, X.J., Yan, P.S., Wang, J.B., Yu, J.J., 2011. ACE inhibitory, antitumor and antioxidant activities of submerged culture materials of three medicinal mushrooms. Appl. Mech. Mater. 145, 179–183. Gawronski, J.K., 1982. Circular dichroism and stereochemistry of chiral conjugated cyclohexenones. Tetrahedron 38, 3–26. Gu, Y., Ukawa, Y., Oshima, M., Suzuki, I., Maenaka, T., Choi, I.S., 2005. Radioprotection and antitumor effect by Lyophyllum decastes Singer and Propolis in mice. Int. J. Med. Mushrooms 7, 407. Han, S.J., Kang, Y.K., 1996. Pseudorotation in heterocyclic five-membered rings: tetrahydrofuran and pyrrolidine. J. Mol. Struct. (Theochem) 369, 157–165, and references therein. Hariharan, P.C., Pople, J.A., 1973. The influence of polarization functions on molecular orbital hydrogenation energies. Theor. Chim. Acta 28, 213–222. Hiramatsu, F., Murayama, T., Koseki, T., Funakoshi, T., Shiono, Y., 2011. Cadinane sesquiterpenoids, strobilols L and M from Strobilurus ohshimae. Nat. Prod. Res. 25, 781–788, and references therein. Kimura, C., Nukina, M., Igarashi, K., Sugawara, Y., 2005. b-Hydroxyergothioneine, a new ergothioneine derivative from the mushroom Lyophyllum connatum, and its protective activity against carbon tetrachloride-induced injury in primary culture hepatocytes. Biosci. Biotechnol. Biochem. 69, 357–363. Kirk, D.N., 1986. The chiroptical properties of carbonyl compounds. Tetrahedron 42, 777–818. Kirk, P.M., Cannon, P.F., Minter, D.W., Stalpers, J.A., 2008. Dictionary of the fungi, 10th ed. CAB International, Wallingford, UK, ISBN 978-0-85199-826-8, p. 395. Lee, C., Yang, W., Parr, R.G., 1988. Development of the Colle–Salvetti correlationenergy formula into a functional of the electron-density. Phys. Rev. B 37, 785– 789. Liu, F.F., Li, G.H., Yang, Z.S., Zheng, X., Yang, Y., Zhang, K.Q., 2010. Two new sesquiterpenes from the fungus Stereum sp. Helv. Chim. Acta 93, 1737–1741. Miura, T., Kubo, M., Itoh, Y., Iwamoto, N., Kato, M., Park, S.R., Ukawa, Y., Kita, Y., Suzuki, I., 2002. Antidiabetic activity of Lyophyllum decastes in genetically Ttype 2 diabetic mice. Biol. Pharm. Bull. 25, 1234–1237. Ning, C.G., Huang, Y.R., Zhang, S.F., Deng, J.K., Liu, K., Wang, F., 2008. Experimental and theoretical structure of tetrahydrofuran under pseudorotation. J. Phys. Chem. A 112, 11078–11087.

Pöder, R., 2005. The Ice Man’s fungi: facts and mysteries. Int. J. Med. Mushrooms 7, 357–359. Snyder, P.A., Johnson Jr., W., 1978. Circular dichroism of l-borneol. J. Am. Chem. Soc. 100, 2939–2944. Stephens, P.J., McCann, D.M., Devlin, F.J., Smith, A.B., 2006. Determination of the absolute configurations of natural products via density functional theory calculations of optical rotation, electronic circular dichroism and vibrational circular dichroism: the cytotoxic sesquiterpene natural products quadrone, suberosenone, suberosanone, and suberosenol A acetate. J. Nat. Prod. 69, 1055– 1064, and references therein. Tinoco Jr., I., 1962. Theoretical aspects of optical activity part two: polymers. Adv. Chem. Phys. 4, 113–160. Ukawa, Y., Ito, H., Hisamatsu, M., 2000. Antitumor effects of (1 ? 3)-b-D-glucan and (1 ? 6)-b-D-glucan purified from newly cultivated mushroom, hatakeshimeji (Lyophyllum decastes Sing.). J. Biosci. Bioeng. 90, 98–104. Ukawa, Y., Furuichi, Y., Kokean, Y., Nishi, T., Hisamatsu, M., 2002. Effect of hatakeshimeji (Lyophyllum decastes Sing.) mushroom on serum lipid levels in rats. J. Nutr. Sci. Vitaminol. 48, 73–76. Ukawa, Y., Izumi, Y., Ohbuchi, T., Takahashi, T., Ikemizu, S., Kojima, Y., 2007. Oral administration of the extract from hatakeshimeji (Lyophyllum decastes Sing.) mushroom inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice. J. Nutr. Sci. Vitaminol. 53, 293–296. Yaoita, Y., Hosokawa, M., Okabe, S., Kikuchi, M., 2003. A new ceramide from two edible mushrooms, Grifola frondosa and Lyophyllum connatum. Nat. Med. 57, 189–191. Yasukawa, K., Kanno, H., Kaminaga, T., Takido, M., Kasahara, Y., Kumaki, K., 1996. Inhibitory effect of methanol extracts from edible mushroom on TPA-induced ear oedema and tumour promotion in mouse skin. Phytother. Res. 10, 367–369. Wagner, H., Bladt, S., 1996. Plant Drug Analysis. A Thin Layer Chromatography Atlas, second ed. Springer-Verlag, Berlin. Wasser, S.P., Weiss, A.L., 1999. Medicinal properties of substances occurring in higher Basidiomycetes mushrooms: current perspective (review). Int. J. Med. Mushrooms 1, 31–62. Zjawiony, J.K., 2004. Biologically active compounds from Aphyllophorales (Polypore) fungi. J. Nat. Prod. 67, 800–810.