An unusual spirocyclic isopimarane diterpenoid and other isopimarane diterpenoids from fruiting bodies of Xylaria polymorpha

Phytochemistry Letters 6 (2013) 439–443

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An unusual spirocyclic isopimarane diterpenoid and other isopimarane diterpenoids from fruiting bodies of Xylaria polymorpha Yoshihito Shiono a,*, Norika Matsui a, Takayuki Imaizumi a, Takuya Koseki a, Tetsuya Murayama a, Eunsang Kwon b, Tomomi Abe c, Ken-ichi Kimura c a b c

Department of Food, Life, and Environmental Science, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan Research and Analytical Center for Giant Molecules, Graduate School of Science, Tohoku University, Sendai, Miyagi 980-8578, Japan Department of Biological Chemistry and Food Science, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 February 2013 Received in revised form 17 May 2013 Accepted 18 May 2013 Available online 5 June 2013

Two new isopimarane-type diterpenes, spiropolin A (1) and myrocin E (3), were isolated from Xylaria polymorpha together with the known compound, myrocin D (2), in the course of a screening of the fruiting bodies of X. polymorpha. Their structures were determined on the basis of spectroscopic analysis, chemical conversion and X-ray analysis. Spiropolin A (1) restored the growth inhibition caused by the hyperactivated Ca2+-signaling in mutant yeast. ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Xylaria polymorpha Fruiting body Ca2+ signaling

1. Introduction We focused on fungi as a resource for novel biologically active compounds, and studied the biodiversity of their secondary metabolites (Shiono et al., 2012). Fungi belonging to the genus Xylaria are saprophytic fungi, which live preferentially on dead trees as decay fungi, and inside the host plant as endophytic fungi (Chareprasert et al., 2012). Previous investigations have revealed that this genus is a rich source of structurally novel and pharmacologically active secondary metabolites. Examples include cytotoxic (Pittayakhajonwut et al., 2005), antimicrobial (Tarman et al., 2012; Healy et al., 2004), anti-HIV (Singh et al., 1999), and antioxidant (Liu et al., 2007) metabolites. Members of the ascomycete genus Xylaria have been found as fruiting bodies on the surfaces of dead trees. The fruiting bodies are hard and black, and cover the white mycelia, stromata, and synnema. Although many compounds were reported from the mycelial culture of this fungus, the chemical constituents of the fruiting body have not been extensively studied (Lee et al., 2009; Shiono et al., 2009a). In our continuing study of the fruiting bodies of this genus, the strain was isolated from the wild mushroom Xylaria polymorpha, collected in Yamagata, Japan. This strain was then cultured on the

* Corresponding author at: Department of Food, Life, and Environmental Science, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan. Tel.: +81 235 28 2873; fax: +81 235 28 2873. E-mail address: [email protected] (Y. Shiono).

steamed unpolished rice medium for four weeks. After cultivation, the fruiting bodies were harvested and extracted with methanol. The organic extract was concentrated, and the aqueous residue was extracted with ethyl acetate. This extract was subjected to silica gel and octadecyl silica gel (ODS) column chromatography, and finally purified by HPLC using an ODS column to afford two new compounds, spiropolin A (1) and myrocin E (3), together with the known compound, myrocin D (2). This paper reports the isolation, structural elucidation, and biological activities of the compounds encountered in this study.

2. Results and discussion Compound 2 was determined to be myrocin D through the analysis of the spectral data (MS, UV, [a]D, IR, 1H and 13C NMR), which were indistinguishable from those of myrocin D (Fig. 1) (Tsukada et al., 2011). Compound 1 was assigned the molecular formula C20H28O6 on the basis of the [MH] peak in HRESITOFMS. The IR spectrum of 1 revealed absorption bands at 3463 (hydroxy group), 1732 (glactone), and 1698 cm1 (a,b-unsaturated carboxylic acid). Close inspection of the 1H and 13C NMR spectra (Table 1), DEPT and HMQC data revealed the presence of three methyl carbons (C-17, C-18, and C-20), five sp3 methylene carbons (C-2, C-3, C-11, C-12, and C-19) including an oxygen-bearing carbon (C-19), two sp3 methine carbons (C-1 and C-5), an sp2 methylene carbon (C-16), two sp2 methine carbons (C-14 and C-15), four sp3 quaternary

1874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2013.05.008

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Fig. 1. Structures of compounds 1, 2 and 3.

carbons (C-4, C-9, C-10, and C-13) including an oxygen-bearing carbon (C-9), an sp2 quaternary carbon (C-8), and g-lactone and carboxyl carbonyl carbons (C-6 and C-7). The 1H NMR spectral data of 1 (Table 1) showed signals due to the terminal vinyl group [dH 5.72 (1H, dd, J = 17.0, 10.7 Hz, H-15), 4.99 (1H, d, J = 10.7 Hz, H-16) and 4.70 (1H, d, J = 17.0 Hz, H-16)], one primary alcohol [dH 3.58 (1H, d, J = 10.2 Hz, H-19) and 4.46 (1H, d, J = 10.2 Hz, H-19)], three tertiary methyl groups [dH 1.19 (3H, s, Me-18), 1.23 (3H, s, Me-17)

and 1.89 (3H, s, Me-20)], and one olefinic proton [dH 7.15 (1H, s, H14)]. Because the two carbonyl carbons and four olefinic carbons account for four degrees of unsaturation, 1 must possess three rings. Further structural information was obtained from analysis of the 1H–1H COSY correlations, which showed the connectivity between C-1, C-2, and C-3, between C-11 and C-12, and between C15 and C-16. The data from HMBC experiments allowed all the protons and carbons to be assigned. The key HMBC correlations summarized in Fig. 2 revealed the diterpene moiety to have a spiro[perhydro-2-benzofuranone-3(3H),10 -20 -cyclohexene] (6/5/6 rings) skeleton. In addition, the HMBC correlations from H-14 to C7 of 1 and from the methoxy group, [dH 3.75 (3H, s, 7-OMe)] to C-7 of the monomethyl ester (1a) suggested the presence of the carboxyl group at C-8. These data established that 1 has a planar structure, as shown in Fig. 2. Conclusive evidence of the structure and absolute stereochemistry of 1 was obtained from X-ray crystallographic analysis; the ORTEP drawing is shown in Fig. 3. The molecular formula of compound 3 was determined by HRESITOFMS to be C20H25O6. Preliminary spectroscopic investigation indicated that some structural features had already been observed in 2. The IR and 1H and 13C NMR spectra of 3 resembled those of 2. Detailed comparison of the chemical shifts and coupling constants between the 13C NMR spectra of 3 and 2 revealed differences in the signals due to C-3, C-4, C-5, C-6, C-14, C-18, C-19, and C-20 (Table 1). The planar structure of 3 was deduced on the basis of HMBC experiments, which revealed key correlations resembling those exhibited by 2. The stereochemical assignments of C-5, C-6 and C-7 were analyzed by interpretation of 1H NMR coupling constants. Both H-5 and H-6 were determined to be transdiaxial on the basis of coupling constant of 3JH-5,H-6 = 10.7 Hz. The large coupling constant of H-6 (3JH-6,H-7 = 9.3 Hz) showed that H-7 was also in an axial configuration. The relative stereochemistry of the methyl group at C-4 had a-configuration based upon an NOE correlation from H-5. On the basis of the close structural similarity and the MS data of 3, this compound was determined to be the opened g-lactone ring of 2, formed through hydrolysis of the ester. In addition, acid hydrolysis of 2 with 10% HCl yielded 3. We next examined the biological activities of the isolated compounds. A mutant yeast screening system was used to search for the inhibitors of Ca2+-signaling of isolated compounds (Miyakawa and Mizunuma, 2007; Shiono et al., 2009b). The mutant strain, S. cerevisiae (zds1D erg3D pdr1D pdr3D: YNS17 strain), used in this study cannot grow at high CaCl2 concentrations because the growth is arrested on the G2 phase by the hyperactivation of the cellular Ca2+-signal. The inhibitors of Ca2+-signal transduction are detected by their ability to stimulate cell growth as a growth zone around a paper disk containing the active compound (Shitamukai et al., 2000). The Ca2+-signaling pathways for growth regulation (cell cycle) are composed of several signaling molecules such as the Ca2+ channel, calcineurin, Pkc1 protein kinase C, Mpk1 MAPK, and Mck1 GSK-3. Under this screening assay system, 1 showed dose-dependent growth-restoring activity against mutant yeast involving Ca2+-signal transduction (from 25 mg/spot to 1.56 mg/spot) (Fig. 4). However, 2 and 3 exhibited no activity even at a dose of 25 mg/spot (data not shown). Myrocin D (2) has weak cytotoxic activity against the human promyelocytic cell line HL60 at IC50 288 mM (Kimura et al., 2012). However, 1 and 3 did not show any cytotoxic effect at concentrations up to 500 mM. Compound 1 was also evaluated for its antifungal activity against filamentous fungus (Aspergillus clavatus), as well as for its antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. However, 1 did not show antimicrobial activity. Although further study of the mechanism of the action by 1 may be needed, 1 might be a new lead compound as a Ca2+-signal transduction inhibitor. According to the literature, no diterpenes with one spiro[perhydro-2-benzofuranone-3(3H),10 -20 -cyclohexene] skeleton have

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Table 1 NMR spectroscopic data (400 MHz, d in ppm) for 1 and 3. 1a

No

3b

dC

dH

HMBC

dC

dH

1 2

71.4 d 29.1 t

2, 3, 5, 9, 10

15.7 d 20.9 t

3

32.9t

4.25 dd (9.5, 4.9) 1.92–2.12c 2.09 m 1.39 br. d (12.1) 1.92-2.12c

1, 5

35.5 t

1.90 1.59 1.72 1.11 1.25

4 5 6 7 8 9 10 11

30.6 s 46.5 d 174.4 s 169.4 s 132.9 s 87.8 s 52.2 s 26.6 t

3.44 s

1, 4, 6, 9, 10, 18, 19

12

30.5 t

1.92–2.12b 2.63 m 1.61 br d (15.1) 2.66 m

9, 13, 14, 15, 17 9, 13, 14, 15

13 14 15 16

38.6 s 148.9 d 143.9 d 112.6 t

17 18 19

26.4 q 18.1 q 68.5 t

20

14.6 q

7.15 5.72 4.70 4.99 1.23 1.19 3.58 4.46 1.89

7, 9, 12 12, 14, 17 13 13 12, 13, 14, 15 3, 4, 5, 19 3, 4, 5, 18 3, 4, 5, 18 1, 5, 9, 10

a b c

s dd (17.0, 10.7) d (17.0) d (10.7) s s d (10.2) d (10.2) s

44.2 s 45.7 d 79.1 d 75.7 d 140.9 s 78.1 s 29.6 s 221.8 s 54.4 t 42.2 s 130.0 d 144.9 d 113.6 t 28.7 q 31.5 q 181.2 s 12.5 t

HMBC m m m m m

2, 4, 5, 18, 19

2.52 d (10.7) 3.88 dd (10.7, 9.3) 4.21 dd (9.3, 2.0)

7, 9, 18, 19 4, 8, 10 5, 9, 14

2.36 dd (13.1, 2.0) 2.56 d (13.1)

9, 13, 17 9, 13, 15

6.00 5.64 4.87 4.94 1.25 1.42

7, 9, 12, 17 12, 14, 17 13, 15 13, 15 12, 13, 14, 15 3, 4, 5, 19

t (2.0) dd (17.1, 10.2) d (17.1) d (10.2) s s

0.93 t (6.3) 0.24 dd (8.3, 6.3)

1, 5, 9, 10

In C5D5N. In CD3OD. Overlapped signals.

been isolated from nature, but a compound with this skeleton was synthesized from isopimarane diterpene (Sparapano et al., 2004). Thus, we can conclude that 1 is the first naturally occurring diterpene containing a spirocycle with the C6/C5/C6 skeleton. 3. Experimental procedures 3.1. Instrumentation Optical rotation values were measured with a Horiba SEPA-300 polarimeter, and IR, and UV spectra were respectively recorded

with Jasco J-20A, Shimadzu UV mini-1240, and Jasco J-20A spectrophotometers. Mass spectra were obtained with a Synapt G2 mass spectrometer instruments. NMR data were recorded on an a Jeol EX-400 spectrometer at 400 MHz for 1H and 100 MHz for 13 C. Chemical shifts are given on a d (ppm) scale with TMS as an internal standard. 1H, 13C, DEPT, COSY, HMQC and HMBC spectra were recorded using standard Jeol standard pulse sequences. X-ray crystallographic analysis was made on a Rigaku VariMax with RAPID imaging plate diffractometer with graphite monochromated Cu Ka radiation (l = 1.54187 A˚). 3.2. Biological material The wild mushroom X. polymorpha was collected at the foot of Mt. Gassan, Yamagata Prefecture, Japan in Sep. 2008 and identified by Y.S. This strain has been deposited at our laboratory of the Faculty of Agriculture, Yamagata University, Yamagata, Japan.

Fig. 2. 1H–1H COSY and HMBC (arrows) correlations observed for 1.

Fig. 3. ORTEP view of compound 1.

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3.5. X-ray structure determination of 1 Crystal data for 1, C20H28O6 ˙ H2O, formula weight = 382.45, colorless block, monoclinic, space group P21 (#4); a = 7.3115 (2) A˚, b = 12.5358 (3) A˚, c = 10.7405 (3) A˚, b = 99.970 (2)8, V = 969.56 (4) A˚3; Z = 2, Dc = 1.310 g/cm3, F (000) = 412.00, m 1 (CuKa) = 8.163 cm , VariMax with RAPID diffractometer, 10724 reflection measured, 3184 unique (Rint = 0.0671), final R1 = 0.0541 and wR (all data) = 0.553, GOF = 1.095. Single crystals suitable for X-ray structure analysis were obtained by recrystallization from acetonitrile. X-ray diffraction data were collected at 93 K on a Rigaku VariMax with RAPID diffractometer using filtered Cu-Ka radiation. The structure was solved by direct methods (Sheldrick, 2008) and expanded using Fourier techniques. Complete crystallographic data, as a CIF file, have been deposited with Cambridge Crystallographic Data Center (CCDC No. 919720). Copies can be obtained free of charge from: The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK ([email protected]). Fig. 4. Growth restored activity of 1 against the mutant strain, Saccharomyces cerevisiae YNS17. Growth restored activities of 1 against S. cerevisiae YNS17 (zds1D erg3D pdr1D pdr3D) in the presence of 0.3 M CaCl2. 1: 25 mg/spot, 2: 12.5 mg/spot, 3: 6.25 mg/spot, 4. 3.13 mg/spot, 5: 1.56 mg/spot, 6: 0.78 mg/spot, 7: 2.5 ng/spot (FK506).

3.3. Fermentation Pieces of fruiting bodies were surface-sterilized with 70% EtOH and placed on potato dextrose agar plates. Plates were incubated at 25 8C for 7 days and colonies appearing on the plates were isolated. The strain X. polymorpha grew on slants of potato agar. A loopful of the culture was transferred on sterilized unpolished rice (20 g/ Petri dish  50: total 1000 g) at 25 8C for 4 weeks. 3.4. Extraction and isolation The fruiting bodies of X. polymorpha was extracted with MeOH, and the MeOH extract was concentrated. The resulting aqueous concentrate was partitioned into n-hexane and EtOAc layers. The purification of the EtOAc layer was guided by the intense blue characteristic coloration with vanillin–sulfuric acid solution on TLC plates. The EtOAc layer (4.0 g) was chromatographed on silica gel column using a gradient of n-hexane–EtOAc (100:0 to 0:100) and EtOAc–MeOH (50:50 and 0:100) to give fractions 1–13 (Fr. 1-1 to 1-13). Fr. 1-12 (EtOAc:MeOH = 50:50, 240 mg) was subjected to ODS column chromatography by eluting with H2O and an increasing ratio of MeOH to afford eleven fractions (Fr. 2-1 to 2-11). Fr. 2-7 (90.6 mg) was subject to semi-preparative ODS HPLC (MeOH–H2O, 80:20) to obtain spiropolin A (1) (25.1 mg; tR = 10.5 min) and myrocin E (3) (10.0 mg; tR = 12 min). Fr. 2-6 (50.6 mg) was then subjected to semi-preparative ODS HPLC (MeOH–H2O, 70:30) to afford myrocin D (2) (17.0 mg; tR = 12 min). 3.4.1. Spiropolin A (1) Colorless crystal; [a]D20–127.68 (c 0.71, MeOH); IR (KBr) nmax cm1; 3463, 3021, 2962, 1732, 1698, 1209, 1049; HRESITOFMS m/z [MH]: 363.1830, calcd. for C20H27O6, 363.1808; FABMS m/z: 363 [MH]. For 1H and 13C NMR, see Table 1. 3.4.2. Myrocin E (3) White amorphous solid; [a]D20 181.18 (c 1.1, MeOH); IR (KBr) nmax cm1; 3397, 3077, 3014, 1704, 1280, 1114; HRESITOFMS m/z [MH]: 361.1689, calcd. for C20H25O6, 361.1651. For 1H and 13C NMR see Table 1.

3.6. Methyl ester derivative of spiropolin A (1) Compound 1 (8.0 mg) was dissolved in a solution of MeOH, and trimethylsilyldiazomethane (2.0 M in diethylether, 0.05 ml), was added to the solution. The mixture was stirred at 0 8C for 5 min and evaporated to dryness. The residue (13.4 mg) was subjected to silica gel column chromatography with mixtures of n-hexane– EtOAc to afford a mono methyl ester (1a, 5.0 mg). 1a: HRESITOFMS (positive ion mode) m/z 379.2135 [M+H]+ (calculated for C21H31O6, 379.2121); 1H NMR (400 MHz, C5D5N): dH 4.13 (1H, m, H-1), 1.63 (1H, br d, J = 16.0 Hz, H-2), 1.78 (2H, m, H-2 and 3), 1.36 (2H, m, H-3 and 12), 2.60 (H, s, H-5), 1.90 (1H, m, H-11), 2.30 (1H, br d, J = 14.5 Hz, H-11), 2.13 (1H, dd, J = 16.1, 14.0 Hz, H12), 3.75 (3H, OMe), 6.75 (1H, s, H-14), 5.70 (1H, dd, J = 17.1, 11.0 Hz, H-15), 4.92 (1H, d, J = 17.1 Hz, H-16), 5.05 (1H, d, J = 11.0 Hz, H-16), 1.20 (3H, s, Me-17), 1.11 (3H, s, Me-18), 3.36 (1H, d, J = 10.0 Hz, H-19), 3.57 (1H, d, J = 10.0 Hz, H-19), 1.23 (3H, s, Me-20); 13C NMR (100 MHz, C5D5N): dc 73.3 (d, C-1), 29.1 (t, C-2), 34.0 (t, C-3), 36.5 (s, C-4), 51.3 (s, C-5), 175.4 (s, C-6), 168.0 (s, C-7), 130.9 (s, C-8), 89.0 (s, C-9), 52.2 (s, C-10), 27.1 (t, C-11), 31.0 (t, C12), 39.2 (s, C-13), 151.6 (d, C-14), 143.9 (d, C-15), 114.4 (t, C-16), 26.6 (q, C-17), 14.5 (q, C-18), 72.4 (t, C-19), 14.0 (q, C-20). 3.7. Acid hydrolysis of myrocin D (2) Solution of 2 (5 mg) in 10% HCl (5 mL) was stirred for 6 h at room temperature. The reaction mixture was extracted with ethyl acetate to afford hydrolysate (3.5 mg), which was identical with 3 on the basis of comparison of 1H, 13C NMR data and TLC. 3.8. Growth restored activity of samples against YNS17 strain Screening was performed according to previous described method (Kimura et al., 2012). Each sample was dissolved in MeOH and two-fold dilutions of them were used. Difco1 yeast-peptonedextrose (YPD) broth and YPD agar were purchased from Becton Dickinson Biosciences (Franklin Lakes, NJ, USA). The mutant yeast, YNS17 (MATa zds1::TRP1 erg3::HIS3 pdr1::hisG-URA3-hisG pdr3::hisG) yeast strains was derivatives of strain W303-1A (Chanklan et al., 2008). A 5 ml aliquot of samples was spotted on a YPD agar medium containing YNS17 strain and 0.3 M CaCl2. After 3 days of incubation at 28 8C, the intensity of the growth spot were observed as the result of inhibition of Ca2+-signal transduction. FK506 (2.5 ng/spot) was used as a positive control. FK506 was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (the present Astellas Pharma Inc., Tokyo Japan).

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