Alliacane sesquiterpenoids from submerged cultures of the basidiomycete Inonotus sp. BCC 22670

Alliacane sesquiterpenoids from submerged cultures of the basidiomycete Inonotus sp. BCC 22670

Phytochemistry xxx (2017) 1e7 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Alliacan...

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Phytochemistry xxx (2017) 1e7

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Alliacane sesquiterpenoids from submerged cultures of the basidiomycete Inonotus sp. BCC 22670 Masahiko Isaka*, Malipan Sappan, Sumalee Supothina, Kitlada Srichomthong, Somjit Komwijit, Thitiya Boonpratuang National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 November 2016 Received in revised form 22 January 2017 Accepted 28 January 2017 Available online xxx

Nine alliacane sesquiterpenoids, inonoalliacanes AeI, were isolated from culture broth of the basidiomycete Inonotus sp. BCC 22670. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. The absolute configuration of inonoalliacane F was determined by application of the modified Mosher's method. Inonoalliacane A, the most abundant sesquiterpene constituent, exhibited moderate antibacterial activity against Bacillus cereus, whereas inonoalliacane B showed antiviral activity against herpes simplex virus type 1. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Inonotus Hymenochaetaceae Alliacane Antibacterial activity Antiviral activity

1. Introduction Basidiomycetes have been valued as rich sources of biologically active compounds (Alves et al., 2012; De Silva et al., 2013; Gao, 2006). While some medicinal species have been well investigated for both fruiting bodies and mycelial cultures, many species still remain chemically unexplored or poorly studied. Inonotus P. Karst. (Hymenochaetaceae) is a genus of wood-rotting basidiomycete composed of more than 250 described species/subspecies. Inonotus obliquus, a popular medicinal mushroom, is used as a traditional medicine for the treatment of cancer and various other diseases. Chemical analysis of this fungus led to the isolation of two major groups of low-molecular-weight metabolites, lanostane-type triterpenoids (Ríos et al., 2012) and styrylpyrone-class phenolic polyketides (Lee and Yun, 2011; Zheng et al., 2010). Styrylpyrones have also been isolated from I. xeranticus (Lee and Yun, 2006). However, little is known about other classes of compounds from this genus, and there have been only a few reports of the chemical investigation of other Inonotus species. Bisabolane and drimane sesquiterpenoids were previously isolated from fruiting bodies of I. rickii

* Corresponding author. E-mail address: [email protected] (M. Isaka).

(Chen et al., 2014) and a bisabolane was isolated from I. vaninii (Yang et al., 2013). As part of our research into novel bioactive specialized metabolites from fungal sources in Thailand, we have recently reported the isolation of aromadendrane and cyclofarnesane sesquiterpenoids from Inonotus sp. BCC 23706 (Isaka et al., 2015). In a continuation of the research, we have investigated another strain, Inonotus sp. BCC 22670. The study led to the isolation and structure elucidation of nine alliacane sesquiterpenoids, named inonoalliacanes AeI (1a/1be6a/6b, 7e9), from the fermentation broth (Fig. 1). 2. Results and discussion The molecular formula of inonoalliacane A was determined by HRESIMS as C19H28O6. The 1H and 13C NMR spectra indicated that this compound exists as a linear form (1a) and a hemiketal form (1b). The 1a/1b ratio in CDCl3 was ca. 1.5:1 (1H NMR spectroscopy). On the basis of the DEPT-135 experiment and 2D NMR spectroscopic data (COSY, HMQC, and HMBC), the nineteen 13C NMR peaks assigned for 1a were categorized as a ketone (dC 206.8), an ester carbonyl (dC 177.2), an exomethylene group (dC 140.0, qC; dC 117.3, CH2), three sp3 quaternary carbons, five methines, two methylenes, and five methyl groups. The planar structure was deduced from COSY, HMQC, and HMBC data to be an alliacane isobutylate (Fig. 2).

http://dx.doi.org/10.1016/j.phytochem.2017.01.018 0031-9422/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Isaka, M., et al., Alliacane sesquiterpenoids from submerged cultures of the basidiomycete Inonotus sp. BCC 22670, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.018

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M. Isaka et al. / Phytochemistry xxx (2017) 1e7

Fig. 1. Structures of the compounds isolated from cultures of Inonotus sp. BCC 22670 (1a/1be6a/6b, 7e9) and semi-synthesis derivatives (10e13).

Fig. 2. COSY and HMBC correlations for 1a/1b.

The COSY data indicated the linkage of C-8eC-9eC-1eC-2 and connection of a methyl group (CH3-10) to C-1. The presence of geminal methyl groups (CH3-14 and CH3-15) was revealed by the HMBC correlations of these methyl protons to the other methyl carbon to each other (H3-14 to C-15, and H3-15 to C-14), and the correlations from both methyl groups (H3-14 and H3-15) to C-6 (CH, dC 76.0), C-7 (qC, dC 42.2), and C-8 (CH2). The location of an aliphatic ketone was assigned by the HMBC correlations from H-1 and H-2 to this carbon (C-3). The bicyclic ring was deduced from the HMBC correlations from H-6, H-8, and H-9 to an oxygenated quaternary carbon at dC 76.4 (C-5), and the correlations from H-6 and H-9 to another oxygenated quaternary carbon at dC 64.0 (C-4). The connection of an allylic alcohol unit (C-12eC-11eC-13) to C-4 was evident from the HMBC correlations from H2-12 and H2-13 to this oxygenated carbon. The C-6 isobutyrate was revealed by the HMBC correlations from H-6, H-20 , H3-30 , and 20 -CH3 to the ester carbonyl carbon C-10. The C-4/C-5 epoxide functionality, instead of a 4,5-diol,

was required based on the molecular formula (HRESIMS). The hemiketal form 1b was also deduced from similar NMR spectroscopic analysis. The significant difference with 1a was the replacement of the ketone (C-3) in 1a by a hemiketal carbon (dC 100.5). Other differences were the downfield shifts of C-4 (dC 67.4) and C-12 (dC 69.6) and the upfield shift of H-2 (dH 3.57) when compared with 1a. The location of the hemiketal carbon (C-3) was further confirmed by the HMBC correlations from H-2 and Hb-12 (dH 4.60) to this quaternary carbon. The relative configuration of 1a/1b was determined on the basis of the NOESY correlations (Fig. 3). The NOESY correlations for 1a, H2/H-1, H-2/H-9, H-1/H-9, H-9/Hb-8, H-9/H3-15, and Hb-8/H3-15, revealed their co-facial (b) relations. Key NOESY correlations to assign a-oriented protons were H3-10/Ha-8, Ha-8/H3-14, Ha-8/H-6, and H-6/H3-14, and these data also suggested a cis ring junction at C-5/C-9. NOESY correlations for 1b, H-2/H-1, H-1/H-9, H3-10/Ha-8, Ha-8/H3-14, and H3-14/H-6, were consistent with the data for 1a. Additional intense NOESY correlation Ha-13/H-6 for 1b indicated the configurations of C-4 and C-5 to be a b-epoxide. The configuration of the hemiketal carbon (C-3), b-orientation of OH, was evident from the requirement of cis ring junction at C-3/C-4. NMR spectroscopic and HRESIMS data for inonoalliacanes BeE (2a/2be5a/5b) suggested that they have the same alliacane sesquiterpene structure, but, differ only in the acyl group attached to C-6 (see Supporting Information for NMR data). Inonoalliacane B (2a/2b) possessed an acetyl group in place of the isobutyryl group in 1a/1b. The location of the acetyl group was confirmed by the HMBC correlations from H-6 (2a, dH 5.14; 2b, dH 4.89) and H3-20 (2a, dH 1.99; 2b, dH 2.14) to C-10 (2a, dC 171.5; 2b, dC 170.4). Inonoalliacane C (3a/3b) was a 2-hydroxyisobutyryl derivative. The acyl group was elucidated by the HMBC correlations from H-6 (3a, dH 5.19; 3b, dH 4.90), H3-30 (3a, dH 1.43; 3b, dH 1.50), and 20 -CH3 (3a, dH 1.39; 3b, dH 1.50) to C-10 (3a, dC 176.9; 3b, dC 176.9). Inonoalliacane E

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M. Isaka et al. / Phytochemistry xxx (2017) 1e7

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Fig. 3. Key NOESY correlations for 1a/1b.

(4a/4b) was identified as a 2-hydroxyisovaleryl derivative. The acyl group was elucidated on the basis of the COSY and HMBC data. The absolute configuration of C-20 remains unassigned. The acyl group of inonoalliacane E (5a/5b) was determined to be (E)-2-methyl-2butenoyl by analysis of the 2D NMR data. Key HMBC correlations for the acyl group were those from H-30 to C-10, C-40 , and 20 -CH3, from H3-40 to C-20 and C-30 , and from 20 -CH3 to C-10, C-20 , and C-30 . The E-configuration of the double bond was assigned by similarity of the chemical shifts of carbons and protons with those of ethyl (E)-2-methyl-2-buenoate: 5a, C-10 (dC 169.9), C-20 (dC 128.7), C-30 (dC 138.9), C-40 (dC 14.4), 20 -CH3 (dC 11.8), H-30 (dH 6.88), H-40 (dH 1.80), 20 -CH3 (dH 1.79); ethyl (E)-2-methyl-2-buenoate, C-1 (dC 168.1), C-2 (dC 129.0), C-3 (dC 136.8), C-4 (dC 14.3), 2-CH3 (dC 12.0), H-3 (dH 6.85), H-4 (dH 1.79), 2-CH3 (dH 1.82) (SDBSWeb: http://sdbs. db.aist.go.jp, National Institute of Advanced Industrial Science and Technology, accessed on October 9, 2016). The NOESY spectrum of 5a/5b lacked the cross-peak for H-3/20 -CH3. The molecular formula of inonoalliacane F (6a/6b) was determined by HRESIMS as C15H24O5. The 1H and 13C NMR spectra suggested alliacane skeleton similar to 1a/1b. The differences were the absence of C-2 hydroxyl group and the acyl group at C-6. The oxygenated methine proton H-6 (6a, dH 3.61; 6b, dH 3.48) was upfield shifted when compared to 1a/1b. There was additional methylene group (6a, dC 43.7, dH 2.82 and 2.21; 6b, dC 39.2, dH 1.70e1.70, 2H) in 6a/6b replacing the C-2 oxygenated methine in 1a/1b. The relative configuration of 6a/6b was confirmed by conversion to a methyl ketal derivative 10. Treatment of 6a/6b with pTsOH$H2O in dry MeOH at room temperature for 30 min gave 10 as a sole reaction product. The NOESY correlations Ha-2/H3-10, H3-10/ Ha-8, Ha-8/H-6, and H-6/H-13 demonstrated the co-facial (a) relation of these protons, and these data suggested a cis ring

junction at C-4/C-9 (Fig. 4). Key NOESY correlations to assign b-face protons were 3-OCH3/Hb-2, Hb-2/H-1, H-1/H-9, and H-9/Hb-8. It should be noted that this derivatization method was unsuccessful for 1a/1b. The absolute configuration of 6a/6b was determined by application of the modified Mosher's method (Ohtani et al., 1991). A pbromobenzoate derivative 11 was obtained by treatment of 6a/6b with p-bromobenzoyl chloride in pyridine. Secondary alcohol 11 was used for the preparation of Mosher ester derivatives. The Dd values of the (S)- and (R)-MTPA esters 12 and 13 indicated the 6S configuration (Fig. 5). Inonoalliacane G (7) was assigned the same molecular formula (C19H28O6) as 1a/1b. Interpretation of 2D NMR spectroscopic data revealed that it exists only as a hemiketal form in CDCl3 and its planar structure is the same as 1b. Significant differences of the NMR spectroscopic data with 1b were the chemical shifts of protons and carbons of the six-membered ring. The relative configuration of 7 was determined by analysis of NOESY data. The NOESY correlations H-1/H-9, H-9/Hb-8, and Hb-8/H3-15 revealed b-orientations of these protons. The NOESY correlations of the a-oriented protons were H-2/H3-10, H3-10/Ha-8, Ha-8/H3-14, and H3-14/H-6. Intense NOESY correlation of H-6/H-13 demonstrated the configurations of C-4/C-5 to be a b-epoxide. Consequently, inonoalliacane G (7) was identified as the C-2 epimer of 1a/1b. The molecular formula of inonoalliacane H (8) was determined by HRESIMS to be C19H30O6. Interpretation of NMR spectroscopic data suggested that its structure is closely related to 1a/1b, possessing an isobutyrate moiety. Significant difference was the presence of an additional oxymethine (dC 71.5; dH 3.93), replacing the C3 ketone in 1a. A tetrahydrofuran ring by ether linkage of C-3 and C-

Fig. 4. Key NOESY correlations for 10.

Fig. 5. Dd-Values (dSedR) of the Mosher esters 12 and 13.

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12 was ruled out, due to the similarity of the chemical shifts of carbons and protons for the allylic alcohol substituent (C-10eC-12) with those of 1b, and the requirement from the molecular formula (HRMS) to be a 2,3,12-triol derivative. The relative configuration of the six-membered ring carbons was determined on the basis of the NOESY data. NOESY correlations H-3/H-1, H-2/H-1, H-2/H-9, and H1/H-9 indicated b-orientations of these protons. Inonoalliacane I (9) was isolated in a small quantity (1.4 mg). The molecular formula was assigned to be C19H26O6 by HRESIMS. Its NMR data suggested a structure close to 1a, possessing an isobutyrate moiety and C-3 ketone functionality. The only structural difference was the presence of a formyl group (dC 191.0; dH 9.56, s), replacing the C-12 hydroxymethyl group in 1a. Location of the formyl group was revealed by the HMBC correlations from the formyl proton (H-12) to C-4, C-10, and C-11, and correlations from exomethylene protons (H2-11) to the formyl carbon (C-12). Alliacane-type sesquiterpenoids are rare from fungal sources. Several compounds have been isolated from basidiomycetes: alliacolide (Bradshaw et al., 1981) and alliacols A and B (Anke et al., 1981) from Marasmius alliaceus, purpuracolide from Gomphus purpuraceus (Jiang et al., 2008), and clitocybulols from Clitocybula oculus (Ayer et al., 1998). Compounds with sufficient sample amounts, 1a/1b, 2a/2b, 6a/ 6b, 7, and 8, were subjected to a panel of biological assays: antibacterial activity (Bacillus cereus and Enterococcus faecium), antiviral activity (herpes simplex virus type 1, HSV-1), antifungal activity (Candida albicans), antitubercular activity (Mcobacterium tuberculosis H37Ra), antimalarial activity (Plasmodium falciparum K1), and cytotoxicity to cancer cell-lines (KB, MCF-7, and NCI-H187) and nonmalignant Vero cells. Inonoalliacane A (1a/1b), the most abundant sesquiterpene constituent, exhibited activity against Bacillus cereus with an MIC value of 25 mg/mL, while all other compounds were inactive at a concentration of 50 mg/ml. It was also cytotoxic to Vero cells (IC50 17 mg/ml), but was inactive in all other assays. On the other hand, only inonoalliacane (2a/2b) showed activity against HSV-1 (IC50 17 mg/ml), and it was inactive in all other assays. Compounds 6a/6b, 7, and 8 were inactive in all tested assays. 3. Experimental 3.1. General experimental procedures Optical rotations were measured with a JASCO P-1030 digital polarimeter. UV spectra were recorded on a GBC Cintra 404 spectrophotometer. IR spectra were taken on a Bruker ALPHA spectrometer. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured with a Bruker micrOTOF mass spectrometer. Merck Silica gel 60H (particle size, 90% < 45 mM) was used for column chromatography. 3.2. Fungal material The fungus used in this study was collected from a bark of dead hardwood tree in Nong Pak Chi nature trail, Khao Yai National Park, Nakhon Ratchasima Province, Thailand on August 6, 2006. The natural mushroom specimen was deposited in the BIOTEC Bangkok Herbarium as BBH 17076. The living culture was deposited in the BIOTEC Culture Collection as BCC 22670. Basidiome morphology, Pileus: 5e12 mm diam, whitish, convex when young, plan to convex when age, mostly flatten disc but rarely umbilicate, uplifted margin, dry, smooth, 1e0.5 mm context, whitish, moist. Lamellae: slightly decurrent, 10e12 lamellulae with 1 series, thick, narrow, whitish with lamellae face and edge. Stipe: 0.5e1.0 mm wide  5e15 mm high, central, cylindrical, fistulose, smooth, dry,

basal disc. Substrate: rotten log. Habit: Lignicolous. Habitat: Casipitose. On the basis of the ITS rDNA sequence data (GenBank accession number: KT800054) this strain (BCC 22670) was identified as the genus Inonotus P. Karst. (Hymenochaetaceae), but it was not assignable to the species level. 3.3. Fermentation, extraction, and isolation The fungus BCC 22670 was maintained on potato dextrose agar at 25  C. The agar was cut into small plugs and inoculated into four 250-ml Erlenmeyer flasks containing 25 ml of malt extract broth (MEB; malt extract 6.0 g/l, yeast extract 1.2 g/l, maltose 1.8 g/l, dextrose 6.0 g/l). After incubation at 25  C for 7 days on a rotary shaker (200 rpm), each primary culture was transferred into a 1000-ml Erlenmeyer flask containing 250 ml of the same liquid medium (MEB), and incubated at 25  C for 7 days on a rotary shaker (200 rpm). The secondary cultures were pooled, and each 25 ml portion was transferred into one of 40 1000-ml Erlenmeyer flasks containing 250 ml of MEB. The final fermentation was carried out at 25  C for 97 days under static conditions. The cultures were filtered to separate broth and mycelia (residue). The broth was extracted with EtOAc (3  9 l) and concentrated under reduced pressure to obtain a brown gum (8.20 g). The broth extract was passed through a Sephadex LH-20 column chromatography (CC) (4.0  50 cm) eluted with acetone to obtain five pooled fractions (Fr-1 e Fr-5) wherein Fr-1 (3.80 g) and Fr-2 (1.78 g) contained sesquiterpenoids. Fr-1 was subjected to silica gel CC (4.0  15 cm, acetone/ CH2Cl2, step gradient elution from 0:100 to 20:80) to obtain seven fractions, Fr-1-1 e Fr-1-7. Fr-1-3 (206 mg) was separated by preparative HPLC using a reversed phase column (SunFire Prep C18 OBD, 19.0  250 mm, 10 mm; mobile phase MeCN/(0.05% formic acid in H2O) ¼ 50:50, flow rate 15 ml/min) to yield pure compounds 1a/1b (16 mg) and 9 (1.4 mg). Fr-1-4 (1.37 g) was also subjected to fractionation by preparative HPLC (mobile phase MeCN/(0.05% formic acid in H2O) ¼ 50:50) to furnish pure compounds 2a/2b (52 mg), 1a/1b (355 mg), and 5a/5b (13 mg) in the order of elution. Similarly, Fr-1-5 (1.24 g) was fractionated by preparative HPLC (mobile phase MeCN/(0.05% formic acid in H2O) ¼ 40:60) to yield 6a/6b (93 mg), 3a/3b (67 mg), 4a/4b (50 mg), and 7 (13 mg) in the order of elution. Fr-2 was subjected to silica gel CC (2.5  15 cm, acetone/CH2Cl2, step gradient elution from 0:100 to 20:80) to obtain seven fractions, Fr-2-1 e Fr-2-7. Fr-2-5 (883 mg) was separated by preparative HPLC (mobile phase MeCN/(0.05% formic acid in H2O) ¼ 40:60) to yield 6a/6b (165 mg) and 1a/1b (134 mg). Fr-2-6 was also separated by preparative HPLC (mobile phase MeCN/ (0.05% formic acid in H2O) ¼ 35:65) to yield 1a/1b (44 mg) and 8 (17 mg). 3.3.1. Inonoalliacane A (1a/1b) Colorless solid; [a]25D þ32 (c 0.10, MeOH); UV lMeOH (nm) max (log ε): 216 (3.76), 281 (3.28), 361 (3.25); IR nmax ATR (cm1): 3452, 1738, 1367, 1229, 1217, 1205; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Tables 1 and 2; HRESIMS (m/z): 375.1768 [MþNa]þ (calc. for C19H28O6Na, 375.1778). 3.3.2. Inonoalliacane B (2a/2b) Colorless solid; [a]24D þ31 (c 0.10, MeOH); UV lMeOH (nm) max (log ε): 217 (3.67), 283 (3.29); IR nmax ATR (cm1): 3438, 1723, 1666, 1372, 1240, 1043; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Supporting Information, Table S1; HRESIMS (m/z): 347.1437 [MþNa]þ (calc. for C17H24O6Na, 347.1465). 3.3.3. Inonoalliacane C (3a/3b) Colorless solid; [a]25D þ39 (c 0.10, MeOH); UV lMeOH (nm) max (log ε): 215 (3.38), 280 (3.04); IR nmax ATR (cm1): 3450, 1728, 1466,

Please cite this article in press as: Isaka, M., et al., Alliacane sesquiterpenoids from submerged cultures of the basidiomycete Inonotus sp. BCC 22670, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.018

M. Isaka et al. / Phytochemistry xxx (2017) 1e7 Table 1 NMR spectroscopic data for inonoalliacane A (1a/1b) in CDCl3. No.

1a

1b

dC, mult.

dH, mult. (J in Hz)

dC

dH, mult. (J in Hz)

1 2 3 4 5 6 7 8

39.4, CH 74.6, CH 206.6, qC 64.0, qC 76.4, qC 76.0, CH 42.2, qC 35.9, CH2

2.37, m 4.59, d (4.0)

27.6 74.8 100.5 67.4 76.4 78.3 39.1 38.1

1.88, m 3.57, d (1.6)

9 10 11 12

41.4, CH 8.6, CH3 140.0, qC 64.5, CH2

38.8 15.1 139.8 69.6

2.71, m 1.14, d (7.0)

13 14 15 10 20 30 20 -CH3

117.3, CH2 26.5, CH3 21.2, CH3 177.2, qC 34.5, CH 19.3, CH3 18.3, CH3

5.14, s

a 1.49, t (12.8) b 1.66, m 2.90, m 0.85, d (7.1) 4.30, 4.22, 5.26, 1.02, 1.15,

d (13.3) d (13.3) s; 5.19, d (1.2) s s

2.45, m 1.17, d (7.1) 1.10, d (6.9)

111.5 29.2 24.4 176.4 34.3 19.1 19.1

4.88, s

a 2.07, dd (12.8, 10.6) b 1.65, t (12.8)

4.82, 4.60, 5.36, 1.01, 0.99,

dt (13.2, 2.1) dt (13.2, 2.4) m; 5.35, m s s

2.65, m 1.22, d (7.0) 1.22, d (7.0)

Table 2 NMR spectroscopic data for inonoalliacane F (6a/6b) in CDCl3. No.

6a

6b

dC, mult.

dH, mult. (J in Hz)

dC

dH, mult. (J in Hz)

1 2

31.3, CH 43.7, CH2

2.27, m a 2.21, dd (14.2, 3.7) b 2.82, dd (14.2, 3.5)

25.8 39.2

1.89, m 1.72e1.70 (2H), m

3 4 5 6 7 8

206.6, qC 64.1, qC 77.1, qC 77.5, CH 41.7, qC 36.6, CH2

9 10 11 12

41.5, CH 14.9, CH3 140.4, qC 64.0, CH2

13 14 15

117.8, CH2 27.1, CH3 20.3, CH3

3.61, s

a 1.28, t (12.9) b 1.58, m 2.69, m 0.93, d (7.1) 4.25, 4.22, 5.52, 1.14, 1.04,

d (13.0) d (13.0) d (0.8); 5.27, s s s

100.2 68.4 76.91 76.88 40.2 35.8 40.6 16.8 140.2 68.2 110.4 28.2 22.0

3.48, s

a 1.22, t (12.6) b 1.56, m 2.54, m 0.88, d (7.2) 4.71, dt (13.2, 2.3) 4.51, dt (13.2, 2.3) 5.30e5.28 (2H), m 1.02, s 1.06, s

1385, 1264, 1153; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Supporting Information, Table S2; HRESIMS (m/z): 391.1721 [MþNa]þ (calc. for C19H28O7Na, 391.1727).

3.3.4. Inonoalliacane D (4a/4b) (nm) Colorless solid; [a]24D þ49 (c 0.10, MeOH); UV lMeOH max (log ε): 215 (3.51); IR nmax ATR (cm1): 3459, 1738, 1367, 1228, 1216; 1 H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, Supporting Information, Table S3; HRESIMS (m/z): 405.1880 [MþNa]þ (calc. for C20H30O7Na, 405.1884).

3.3.5. Inonoalliacane E (5a/5b) Colorless solid; [a]24D þ53 (c 0.10, MeOH); UV lMeOH (nm) max (log ε): 219 (3.80); IR nmax ATR (cm1): 3439, 1710, 1650, 1263, 1137, 1079; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, Supporting Information, Table S4; HRESIMS (m/z): 387.1768 [MþNa]þ (calc. for C20H28O6Na, 387.1778).

5

3.3.6. Inonoalliacane F (6a/6b) Colorless solid; [a]25D þ7 (c 0.10, MeOH); UV lMeOH max (nm) (log ε): 215 (3.31); IR nmax ATR (cm1):3302, 1715, 1705, 1389, 1127; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Tables 1 and 3; HRESIMS (m/z): 289.1403 [MþNa]þ (calc. for C15H22O4Na, 289.1410). 3.3.7. Inonoalliacane G (7) Colorless solid; [a]25D þ29 (c 0.10, MeOH); UV lMeOH (nm) max (log ε): 216 (3.52), 273 (3.15); IR nmax ATR (cm1): 3461, 1737, 1367, 1216, 1204; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, Tables 1 and 3; HRESIMS (m/z): 375.1781 [MþNa]þ (calc. for C19H28O6Na, 375.1778). 3.3.8. Inonoalliacane H (8) Colorless solid; [a]25D þ41 (c 0.10, MeOH); UV lMeOH (nm) max (log ε): 217 (3.42), 273 (3.15); IR nmax ATR (cm1): 3433, 1736, 1468, 1389, 1196, 1157, 1116, 1022; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, Tables 1 and 3; HRESIMS (m/z): 377.1939 [MþNa]þ (calc. for C19H30O6Na, 377.1935). 3.3.9. Inonoalliacane I (9) (nm) Colorless solid; [a]24D þ26 (c 0.10, MeOH); UV lMeOH max (log ε): 215 (3.42); IR nmax ATR (cm1): 3410, 1734, 1467, 1390, 1157; 1 H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, Tables 1 and 3; HRESIMS (m/z): 373.1612 [MþNa]þ (calc. for C19H26O6Na, 373.1622). 3.4. Synthesis of inonoalliacane F methyl ketal 10 To a solution of 6a/6b (1.0 mg) in MeOH (0.3 ml) was added pTsOH$H2O (1 mg) and the mixture was stirred at room temperature for 30 min. The reaction was terminated by addition of 1 M NaHCO3, and the mixture was partially concentrated by evaporation. The residual aqueous solution was extracted with EtOAc and the organic phase was concentrated under reduced pressure to obtain a methyl ketal derivative 10 (1.0 mg). 3.4.1. Inonoalliacane F methyl ketal (10) Colorless solid; UV lMeOH max (nm) (log ε): 215 (3.49); IR nmax ATR (cm1): 3465, 2953, 1373, 1212, 1020; 1H NMR (500 MHz, CDCl3) d 5.29 (1H, t, J ¼ 2.2 Hz, Ha-13), 5.23 (1H, t, J ¼ 2.2 Hz, Hb-13), 4.62 (1H, dt, J ¼ 13.1, 2.2 Hz, Ha-12), 4.48 (1H, dt, J ¼ 13.1, 2.2 Hz, Hb-12), 3.46 (1H, br d, J ¼ 5.5 Hz, H-6), 3.31 (3H, s, 3-OCH3), 2.47 (1H, m, H9), 1.95 (1H, bd d, J ¼ 5.5 Hz, 6-OH), 1.91 (1H, m, H-1), 1.72 (1H, dd, J ¼ 13.9, 3.8 Hz, Hb-2), 1.68 (1H, dd, J ¼ 13.9, 7.7 Hz, Ha-2), 1.57 (1H, dd, J ¼ 13.0, 8.8 Hz, Hb-8), 1.22 (1H, t, J ¼ 13.0 Hz, Ha-8), 1.03 (6H, s, H-14 and H-15), 0.86 (3H, d, J ¼ 7.2 Hz, H-10); 13C NMR (125 MHz, CDCl3) 140.9 (C, C-11), 109.2 (CH2, C-13), 103.1 (C, C-3), 77.1 (CH, C6), 75.7 (C, C-5), 68.6 (CH2, C-12), 67.7 (C, C-4), 49.0 (CH3, 3-OCH3), 40.7 (CH, C-9), 40.4 (C, C-7), 36.0 (CH2, C-8), 35.7 (CH2, C-2), 28.0 (CH3, C-14), 26.0 (CH, C-1), 21.7 (CH3, C-15), 16.7 (CH3, C-10); HRESIMS (m/z): 303.1575 [MþNa]þ (calc. for C16H24O4Na, 303.1567). 3.5. Synthesis of a p-bromobenzoate derivative 11 and application of the modified Mosher's method A mixture of 6a/6b (10 mg) and p-bromobenzoyl chloride (23 mg) in pyridine (0.3 ml) was stirred for 17 h. The mixture was diluted with EtOAc and washed with H2O and 1M NaHCO3, and the organic layer was concentrated in vacuo. The residue was separated by column chromatography on silica gel (EtOAc/CH2Cl2) to afford a p-bromobenzoate derivative 11 (9.2 mg). A portion of compound 11 (1.0 mg) was treated with (-)-(R)-MTPA-Cl (10 ml) and N,N-

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M. Isaka et al. / Phytochemistry xxx (2017) 1e7

Table 3 NMR spectroscopic data for inonoalliacanes G (7), H (8), and I (9) in CDCl3. No.

7

8

dC, mult.

dH, mult. (J in Hz)

dC

1 2 3 4 5 6 7 8

29.2, 74.0, 99.3, 68.9, 73.6, 78.0, 38.9, 37.1,

1.77, m 3.49, d (11.1)

32.4, 71.7, 71.5, 65.7, 70.6, 77.0, 40.4, 38.1,

9 10 11 12

40.9, CH 13.8, CH3 140.7, qC 68.0, CH2

13 14 15 10 20 30 20 -CH3

110.4, CH2 29.4, CH3 24.2, CH3 176.2, qC 34.4, CH 19.12, CH3 19.05, CH3

CH CH qC qC qC CH qC CH2

4.81, s

a 1.33, dd (13.2, 11.2) b 1.73, dd (13.2, 10.4) 2.89, m 0.97, d (7.0) 4.72, d (13.3) 4.50, d (13.3) 5.30e5.28 (2H), m 0.95, s 1.01, s 2.63, m 1.21, d (7.0) 1.21, d (7.0)

9

dH, mult. (J in Hz)

dC, mult.

dH, mult. (J in Hz)

CH CH CH qC qC CH qC CH2

1.87, m 3.74, t (3.4) 3.93, d (4.0)

39.9, CH 74.2, CH 204.7 62.7, qC 76.7, qC 75.4, CH 42.2, qC 36.5, CH2

2.42, m 4.64, d (4.6)

40.5, CH 12.4, CH3 143.3, qC 64.4, CH2

2.48, m 1.00, d (7.0)

40.8, CH 8.6, CH3 142.3, qC 191.0, CH

2.94, m 1.02, d (7.1)

136.3, CH2 26.7, CH3 21.6, CH3 175.3, qC 34.2, CH 19.1, CH3 18.5, CH3

6.42, s; 6.27, s 1.01, s 1.10, s

117.3, CH2 27.8, CH3 22.2, CH2 176.3, qC 34.3, CH 19.2, CH3 18.7, CH3

dimethylaminopyridine (DMAP, 10 mg) in CH2Cl2 (0.3 ml) at room temperature for 17 h. The mixture was diluted with EtOAc and washed with H2O and 1M NaHCO3, and the organic layer was concentrated in vacuo. The residue was purified by preparative HPLC (MeCN/H2O ¼ 80:20) to furnish a (S)-MTPA ester derivative 12 (0.6 mg). Similarly, (R)-MTPA ester derivative 13 (0.5 mg) was prepared from 11 (1.0 mg) and (þ)-(S)-MTPA-Cl. 3.5.1. Compound 11 Colorless gum; 1H NMR (400 MHz, CDCl3) d 7.94 (2H, d, J ¼ 8.5 Hz, H-300 and H-700 ), 7.74 (2H, d, J ¼ 8.5 Hz, H-400 and H-600 ), 5.58 (1H, s, Ha-13), 5.36 (1H, s, Hb-13), 4.94 (2H, s, H-12), 3.69 (1H, d, J ¼ 7.2 Hz, H-6), 3.10 (1H, d, J ¼ 7.2 Hz, 6-OH), 2.82 (1H, m, Hb-2), 2.70 (1H, m, H-9), 2.27 (1H, m, H-1), 2.10 (1H, m, Ha-2), 1.59 (1H, dd, J ¼ 12.6, 7.9 Hz, Hb-8), 1.31 (1H, t, J ¼ 12.6 Hz, Ha-8), 1.05 (3H, s, H-14 or H-15), 1.02 (3H, s, H-15 or H-14), 0.89 (3H, d, J ¼ 7.2 Hz, H-10); HRESIMS (m/z): 471.0798 and 473.0780 [MþNa]þ (calc. for C22H25BrNaO5, 471.0778 and 473.0760). 3.5.2. (S)-MTPA ester 12 Colorless gum; 1H NMR (400 MHz, CDCl3) d 7.92 (2H, d, J ¼ 8.5 Hz, H-300 and H-700 ), 7.58 (2H, d, J ¼ 8.5 Hz, H-400 and H-600 ), 7.58 (2H, m, phenyl of MTPA), 7.43e7.40 (3H, m, phenyl of MTPA), 5.52 (1H, s, Ha-13), 5.30 (1H, s, Hb-13), 5.22 (1H, s, H-6), 4.98 (1H, d, J ¼ 13.5 Hz, Ha-12), 4.85 (1H, d, J ¼ 13.5 Hz, Hb-12), 3.58 (3H, s, OCH3 of MTPA), 2.82 (1H, m, H-9), 2.81 (1H, dd, J ¼ 14.2, 3.8 Hz, Hb-2), 2.25 (1H, m, H-1), 2.16 (1H, dd, J ¼ 14.2, 4.4 Hz, Ha-2), 1.70 (1H, m, Hb-8), 1.41 (1H, dd, J ¼ 12.4, 12.2 Hz, Ha-8), 1.07 (3H, s, H-14), 0.93 (3H, s, H-15), 0.89 (3H, d, J ¼ 7.1 Hz, H-10); ESIMS (m/z): 687.1 and 689.1 [MþNa]þ. 3.5.3. (R)-MTPA ester 13 Colorless gum; 1H NMR (400 MHz, CDCl3) d 7.91 (2H, d, J ¼ 8.5 Hz, H-300 and H-700 ), 7.58 (2H, d, J ¼ 8.5 Hz, H-400 and H-600 ), 7.52 (2H, m, phenyl of MTPA), 7.40e7.38 (3H, m, phenyl of MTPA), 5.35 (1H, s, Ha-13), 5.22 (1H, s, Hb-13), 5.20 (1H, s, H-6), 4.89 (1H, d, J ¼ 12.8 Hz, Ha-12), 4.77 (1H, d, J ¼ 12.8 Hz, Hb-12), 3.51 (3H, s, OCH3 of MTPA), 2.82 (1H, m, H-9), 2.80 (1H, m, Hb-2), 2.25 (1H, m, H-1), 2.15 (1H, dd, J ¼ 14.2, 4.6 Hz, Ha-2), 1.71 (1H, dd, J ¼ 12.8, 8.6 Hz, Hb8), 1.41 (1H, dd, J ¼ 13.1, 11.7 Hz, Ha-8), 1.07 (3H, s, H-14), 1.05 (3H, s,

4.90, s

a 1.61, m b 1.59, m

4.26, 4.18, 5.35, 1.05, 1.01,

d (13.1) d (13.1) s; 5.21, s s s

2.49, m 1.15, d (7.0) 1.11, d (6.9)

4.81, s

a 1.58, t (12.7) b 1.68, dd (13.1, 8.3)

9.56, s

2.42, m 1.14, d (7.0) 1.04, d (7.0)

H-15), 0.89 (3H, d, J ¼ 7.2 Hz, H-10); ESIMS (m/z): 687.1 and 689.1 [MþNa]þ. 3.6. Biological assays Antibacterial activities against Bacillus cereus and Enterococcus faecium were performed using the resazurin microplate assay and the optical density microplate assay (OD600), respectively (CLSI, 2006). Vancomycin hydrochloride was the standard compound for B. cereus (MIC 2.00 mg/ml), and tetracycline hydrochloride was used as standard for E. faecium (MIC 0.0976 mg/ml). Antiviral activity against herpes simplex virus type 1 (HSV-1) and cytotoxicity to Vero cells (African green monkey kidney fibroblasts; host cells in the antiviral assay) were performed using the green fluorescent protein (GFP)-based microplate assay (Hunt et al., 1999). Acyclovir was used as the standard compound for the anti-HSV-1 assay (IC50 4.67 mg/ml). Ellipticine was the standard compound for the cytotoxicity to Vero cells (IC50 1.59 mg/ml). Acknowledgements Financial support from the Thailand Research Fund (grant No. DBG5980002) is gratefully acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.01.018. References Alves, M.J., Ferreira, S.C.F.R., Dias, J., Teixeira, V., Martins, A., Pintado, D., 2012. A review on antimicrobial activity of (basidiomycetes): extracts and isolated compounds. Planta Med. 78, 1707e1718. Anke, T., Watson, W.H., Giannetti, B.M., Steglich, W., 1981. Antibiotics from basidiomycetes: XIII The alliacols A and B from Marasmius alliaceus (JACQ. Ex. Fr.) Fr. J. Antibiot. 34, 1271e1277. Ayer, W.A., Shan, R., Trifonov, L.S., Hutchison, L.J., 1998. Sesquiterpenes from the nematicidal fungus Clitocybula oculus. Phytochemistry 49, 589e592. Bradshaw, A.P., Hanson, J.R., Sadler, I.H., 1981. The isoprene units of the sesquiterpenoid, alliacolide. J. Chem. Soc. Chem. Commun. 631e632. Chen, H.-P., Dong, W.-B., Feng, T., Yin, X., Li, Z.-H., Dong, Z.-J., Li, Y., Liu, J.-K., 2014. Four new sesquiterpenoids from fruiting bodies of the fungus Inonotus rickii. J. Asian Nat. Prod. Res. 16, 581e586.

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