Bioactive Sesquiterpene Aryl Esters from the Culture Broth of

Dec 19, 2014 - armillaridin (8), and armillarikin (9), were isolated from the culture broth of Armillaria sp. Their structures were determined by spec...
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Bioactive Sesquiterpene Aryl Esters from the Culture Broth of Armillaria sp. Hajime Kobori,† Atsushi Sekiya,‡ Tomohiro Suzuki,§ Jae-Hoon Choi,⊥ Hirofumi Hirai,§,⊥ and Hirokazu Kawagishi*,†,§,⊥ †

Graduate School of Science and Technology, §Research Institute of Green Science and Technology, and ⊥Graduate School of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan ‡ Kyushu Research Center, Forestry & Forest Products Research Institute, 4-11-16 Kurokami, Kumamoto, Kumamoto, 860-0862, Japan S Supporting Information *

ABSTRACT: Two new compounds, 10-dehydroxymelleolide D (1) and 13-hydroxymelleolide K (2), along with seven known compounds, 5′-O-methylmelledonal (3), melleolide D (4), 13-hydroxydihydromelleolide (5), melleolide (6), armillarinin (7), armillaridin (8), and armillarikin (9), were isolated from the culture broth of Armillaria sp. Their structures were determined by spectroscopic data analysis. All the compounds inhibited plant growth of lettuce. Melleolide (6) and armillarikin (9) inhibited mycelial growth of Coprinopsis cinerea and/or Flammulina velutipes.

T

he genus Armillaria (English name, Honey Fungus; Japanese name, Naratake), belonging to the family Physalacriaceae, is a well-known edible mushroom throughout the world. This mushroom is delicious, and people also have utilized it for its medicinal properties. On the other hand, the genus has been known as a serious plant pathogen that causes root rot in various plant species,1 and the phenomenon is called Armillaria root disease.2,3 Root rot is one of the most serious diseases of plants and occurs in many broadleaf trees and conifers and several herbaceous plants.4 Furthermore, it is known that penetration of Armillaria mycelia to the fungi Entoloma abortivum and Wynnea americana induces spherical deformity of the fruiting bodies of those mushrooms.5 These facts indicate that Armillaria produces allelopathic substance(s). Protoilludane sesquiterpene aryl esters have been isolated from Armillaria mushrooms and Clitocybe illudens.6−12 Those compounds showed antimicrobial activity10,13,14 and cytotoxicity against human cancer cell lines.15,16 In addition, armillariols A−C have been isolated from the culture broth of Armillaria sp. as plant growth regulators by our group.17 Until now, the toxic compounds resulting in Armillaria root disease and the inducers of deformity in other fungi have not been identified. Here, we describe the isolation, structural determination, and growth regulatory activity against plants and fungi of two new compounds and seven analogues from the culture broth of a strain of the genus. © XXXX American Chemical Society and American Society of Pharmacognosy

Isolation of the compounds from Armillaria sp. was guided by their growth-regulating activity on lettuce. Liquid culture of the fungus inhibiting the growth of lettuce was extracted successively with n-hexane, EtOAc, and n-BuOH. The active fractions, the n-hexane-soluble and the EtOAc-soluble fractions, were subjected to repetitive chromatography, respectively, to afford compounds 1−9. Received: April 18, 2014

A

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Table 1. 1H and 13C NMR Data for 1 and 2 (in CDCl3)

Compound 1 was purified as a white, amorphous material. The molecular formula was determined as C24H31ClO7 by HRESIMS (m/z 489.1661 [M + Na] + ; calcd for C24H31ClNaO7, 489.1656), indicating the presence of nine degrees of unsaturation in the molecule. The structure of 1 was elucidated by interpretation of NMR spectra including DEPT, COSY, HMQC, and HMBC (Figure 1). The DEPT experiment

1 position

δC, type

1

65.6, CH2

2 3 4 5

Figure 1. COSY and crucial HMBC correlations of 1 and 2.

indicated the presence of five methyls, four methylenes, four methines, and 11 quaternary carbons. The presence of a sesquiterpene skelton (C-1 to C-15) was constructed by the COSY correlations (H-5/H-6; H-9/H-10) and the HMBC correlations (H-1/C-2, C-3, C-4; H-3/C-1, C-2, C-4, C-12, C13; H-5/C-4, C-6; H-6/C-4, C-5, C-7, C-9; H-8/C-4, C-6, C-7, C-9; H-9/C-8, C-10, C-13; H-10/C-8, C-9, C-11, C-13, C-14, C-15; H-12/C-3, C-9, C-10, C-11, C-13, C-14, C-15; H-14/C10, C-11, C-12, C-15; H-15/C-10, C-11, C-12, C-14) (Figure 1). The presence of the other moiety, the benzene ring (C-2′ to C-7′), was suggested by the characteristic 13C NMR chemical shifts at δC 98.6, 106.3, 115.6, 140.0, 159.8, and 162.8. The HMBC correlations (H-4′/C-2′, C-3′, C-5′, C-6′; H-8′/C-2′, C-6′, C-7′; H-5′-OCH3/C-5′) indicated that the phenyl moiety has a methoxy at C-5′, a hydroxy at C-3′, and a methyl at C-7′ (Figure 1). The HMBC cross-peak (H-5/C-1′) and characteristic chemical shifts at δC 76.7 at C-5 and δC 170.7 at C-1′ indicated that the sesquiterpene moiety was connected to the phenyl group via an ester bond. The complete assignment of all the proton and carbon signals of NMR was accomplished as shown in Table 1. Compound 2 was purified as a white, amorphous material. Its molecular formula was determined as C 23 H27ClO7 by HRESIMS (m/z 473.1351 [M + Na] + ; calcd for C23H27ClNaO7, 473.1343), indicating the presence of 10 degrees of unsaturation in the molecule. The structure of 2 was elucidated by interpretation of NMR spectra including DEPT, COSY, HMQC, and HMBC (Figure 1). The NMR data of 2 were very similar to those of 1 (Table 1). The DEPT experiment indicated the presence of four methyls, three methylenes, five methines, and 11 quaternary carbons. The molecular formula, the unsaturation degrees, the 13C NMR data (δC 20.0, 21.4, 30.8, 30.9, 31.7, 34.6, 37.6, 43.2, 50.3, 58.1, 75.2, 75.3, 77.8, 102.1, 107.0, 113.8, 136.7, 139.0, 153.2, 156.0, 162.7, 169.9, 196.3, Table 1), and the DEPT data indicated that 2 had the same skeleton as that of 1. However, this compound possessed a formyl and a phenol instead of the hydroxymethyl and the methoxy in 1. The chemical shifts and coupling constants in the 1H and 13C NMR spectra of 1 and 2 were very similar to those of melleolide D, whose absolute configuration has been determined, indicating that their relative configuration is the same as that of melleolide D (Table 1 and see Experimantal

135.7, 133.8, 77.1, 76.7,

C CH C CH

6

31.2, CH2

7 8 9

38.8, C 21.7, CH3 49.5, CH

10

43.2, CH2

11 12

33.7, C 58.7, CH2

13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 5′OCH3 3′-OH

78.0, 31.3, 31.7, 170.7, 106.3, 162.8, 98.6, 159.8, 115.6, 140.0, 19.6, 56.3,

C CH3 CH3 C C C CH C C C CH3 CH3

2 δH, mult (J in Hz) 4.09, d (12.1) 4.34, d (12.1) 5.88, s 5.42, dd (8.9, 8.9) 1.91, dd (11.3, 8.9) 2.50, dd (11.3, 8.9) 1.24, s 2.21, dd (12.8, 5.2) 1.26, dd (12.8, 12.8) 1.63, dd (12.8, 5.2) 1.86, d (13.7) 1.89, d (13.7) 0.95, s 1.08, s

6.40, s

2.50, s 3.88, s 11.2, s

position

δC, type

1

196.3, CH

2 3 4 5

136.7, 153.2, 75.2, 75.3,

C CH C CH

6

31.7, CH2

7 8 9

37.6, C 21.4, CH3 50.3, CH

10

43.2, CH2

11 12

34.6, C 58.1, CH2

13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′

3′-OH

77.8, 30.8, 30.9, 169.9, 107.0, 162.7, 102.1, 156.0, 113.8, 139.0, 20.0,

C CH3 CH3 C C C CH C C C CH3

δH, mult (J in Hz) 9.53, s

6.71, s 5.56, dd (8.9, 8.9) 2.00, dd (11.3, 8.9) 2.29, dd (11.3, 8.9) 1.28, s 2.31, m 1.27, dd (13.7, 13.4) 1.67, dd (13.4, 7.3) 1.93, s

0.93, s 1.12, s

6.47, s

2.42, s

11.1, s

Section).18 Furthermore, The CD data (λext (Δε): 202 (−16.0), 216 (+16.6), 234 (−5.54), 258 (+5.99) nm) for 1 and (λext (Δε): 206 (−4.91), 221 (+7.16), 236 (−7.74), 267 (+6.02) nm) for 2 were similar to those of melleolide D (see Experimantal Section). These results showed the absolute configuration of 1 and 2 was (2R,2aS,4aR,7aR,7bR) and (2R,2aS,4aR,7aR,7bR), respectively. They were named 10dehydroxymelleolide D (1) and 13-hydroxymelleolide K (2), respectively, after their known analogues, melleolides D and K.8,19 Compounds 3 to 9 were identified as 5′-O-methylmelledonal,7 melleolide D,8 13-hydroxydihydromelleolide,9 melleolide,10 armillarinin,20 armillaridin,21 and armillarikin,22 respectively. They have been isolated from the mushroom genus Armillaria as antimicrobial compounds. The plant growth regulatory activity of the isolated compounds was determined using lettuce. All compounds tested inhibited the growth of lettuce using 1 μmol/paper (Figure 2). B

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Figure 2. Plant growth regulating activity of compounds 1−9 against (A) root or (B) hypocotyl of lettuce. Results are the mean ± standard deviation (n = 6). 2,4-Dichlorophenoxyacetic acid (2,4-D) was used as positive control. The asterisk indicates significant difference from control (Student’s t test; *, p < 0.05; **, p < 0.01).

Figure 3. Mycelial growth-regulating activity of melleolide (6) and armillarikin (9) against F. velutipes (A) and C. cinerea (B) (n = 3). The upper disks contained 1 μmol and the lower paper disks contained 0.1 μmol of compound. Amphotericin B was used as positive control.

All the compounds were subjected to the mycelial growth assay against Coprinopsis cinerea and Flammulina velutipes. Melleolide (6) inhibited the mycelial growth of C. cinerea and F. velutipes, and armillarikin (9) inhibited the mycelial growth of C. cinerea, each giving radially shaped clear zone (Figure 3). The activity of melleolide (6) was stronger than the positive control, amphotericin B. The protoilludane skeleton itself is important for growth inhibitory activity against lettuce. The formyl group at C-1 and the absence of a hydroxy at C-13 in the molecule were important for the antifungal activities. In a previous study, Peipp and Sonnenbichler reported the structure−activity relationship of similar sesquiterpene aryl esters. They have described that “it can be derived that a) both the oresllinate and sesquiterpene moiety contribute to the antibiotic activity, and

b) a decrease of the toxicity can be observed with an increase of the hydrophobic nature of the metabolites”.14 However, opposite results were obtained in this study; the sesquiterpene part of 6 is the most hydrophobic among the compounds in this study.

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EXPERIMENTAL SECTION

General Experimental Procedures. 1H NMR spectra (one- and two-dimensional) were recorded on a Jeol lambda-500 spectrometer at 500 and 270 MHz, while 13C NMR spectra were recorded on the same instrument at 125 MHz. The HRESIMS spectra were measured on a JMS-T100LC mass spectrometer. A Jasco grating infrared spectrophotometer was used to record the IR spectra. Specific rotation values were measured with a Jasco DIP-1000 polarimeter, and CD spectra were recorded on a Jasco J-820 CD spectrometer. HPLC separations were performed with a Jasco Gulliver system using a normal-phase C

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3.97 (C-1, d, 12.7), 4.25 (C-1, d, 12.7), 5.54 (C-5, dd, 8.9, 8.5), 5.94 (C-3, s), 6.36 (C-4′, s); 13C NMR (CDCl3) δ 19.5 (C-8′), 21.2 (C-8), 23.4 (C-14), 28.7 (C-15), 32.1 (C-6), 36.6 (C-7), 40.6 (C-11), 53.9 (C-9), 55.5 (C-12), 56.3 (C-5′-OCH3), 64.6 (C-1), 75.1 (C-5), 76.1 (C-4), 77.0 (C-13), 82.6 (C-10), 98.5 (C-4′), 106.5 (C-2′), 115.6 (C6′), 134.2 (C-3), 134.4 (C-2), 139.3 (C-7′), 159.6 (C-5′), 162.5 (C3′), 170.3 (C-1′); EIMS data were consistent with those of previously published reports.8 13-Hydroxydihydromelleolide (5): white, amorphous; [α]26D +32 (c 0.45, CHCl3); CD (MeOH) λext (Δε) 201 (−20.5), 215 (+16.2), 230 (−0.57), 256 (+7.88) nm; IR (neat) νmax 3648, 1733 cm−1; 1H NMR (CD3OD) δ 0.95 (C-14, s), 1.08 (C-15, s), 1.20 (C-8, s), 1.34 (C-10, dd, 12.8, 12.8), 1.56 (C-10, dd, 12.8, 5.5), 1.80 (C-6, dd, 11.0, 8.9), 1.85 (C-12, d, 13.7), 1.89 (C-12, d, 13.7), 2.18 (C-9, dd, 12.8, 5.5), 2.38 (C-8′, s), 2.60 (C-6, dd, 11.0, 8.9), 4.02 (C-1, d, 14.2), 4.31 (C-1, d, 14.2), 5.47 (C-5, dd, 8.9, 8.9), 5.90 (C-3, d, 1.2), 6.14 (C4′, d, 2.4), 6.17 (C-6′, d, 2.4); 13C NMR and FABMS data were consistent with those of previously published reports.9 However, there are no 1H NMR data in the report. Melleolide (6): colorless, cubic crystal (MeOH); CD (MeOH) λext (Δε) 211 (−8.67), 223 (+7.81), 236 (−8.24), 262 (+11.0) nm; NMR data were consistent with those of previously published reports;10 ESIMS m/z 423 [M + Na]+. Armillarinin (7): colorless, amorphous; NMR data were consistent with those of previously published reports;20 ESIMS m/z 488 [M + Na]+. Armillaridin (8): white needle crystal (MeOH); NMR data were consistent with those of previously published reports;21 ESIMS m/z 472 [M + Na]+. Armillarikin (9): white, amorphous; NMR data were consistent with those of previously published reports;22 ESIMS m/z 488 [M + Na]+. Bioassay. Growth activity against lettuce was examined as follows. Lettuce seeds were put on filter paper (Advantec No. 2, ⦶ 55 mm; Toyo Roshi Kaisha, Ltd., Japan), soaked in distilled water in a Petri dish (⦶ 60 × 20 mm), and incubated in a growth chamber in the dark at 25 °C for 1 day. On the other hand, test samples and 2,4dichlorophenoxyacetic acid (positive control) were dissolved in 1 mL of methanol (1, 10−2, 10−4, 10−6, and 10−8 μmol/mL), and then each solution was placed on filter paper (⦶ 55 mm) in a Petri dish (⦶ 60 × 20 mm). After the solvent was air-dried, 1 mL of distilled water was poured on the sample-loaded paper or intact filter paper (control). The preincubated lettuces (n = 6 in each Petri dish) were transferred onto the filter paper and incubated in a growth chamber in the dark at 25 °C for 3 days. The lengths of the hypocotyl and the root were measured using a ruler. Growth activity against fungus was examined as follows. The mycelia of each fungus were placed onto the center of a potato dextrose agar (PDA) plate and incubated at 25 °C for 3 days in an incubator. Meanwhile, the test compounds or amphotericin B (positive control) solution was added to autoclaved paper disks (Advantec ⦶ 8 mm; Toyo Roshi Kaisha, Ltd., Japan) and then air-dried. Each air-dried paper disk containing 1, 0.1, or 0 μmol/disk (control) of the compounds or amphotericin B was placed directly onto the incubated plate. Plates were further incubated at room temperature for 1 week (C. cinerea) or 2 weeks (F. velutipes). After the incubation, the inhibitory activity was evaluated by observation of clear zones due to growth inhibition of mycelia. The observation was repeated in duplicate for all of the test plates over a period of 2 weeks.

HPLC column (Senshu PAK AQ, Senshu Scientific Co., Ltd., Japan) and reversed-phase HPLC columns (COSMOSIL Cholester Waters, Nacalai Tesque, Japan). Silica gel plate (Merck F254) and silica gel 60N (Merck 100−200 mesh) were used for analytical TLC and for flash column chromatography, respectively. Fungal and Plant Materials. Armillaria sp. strain 543 was deposited at the culture collection of the Forestry and Forest Products Research Institute. Lettuce seeds (Lactuca sativa L. cv. Cisco; Takii Co., Ltd., Japan) were used in this study. The voucher specimen of the Coprinus cinereus (NBRC 30627) is located in NBRC (Biological Resource Center, NITE). Incubation. The culture medium (24 g/L) of Armillaria sp. was prepared containing potato dextrose broth (Difco). The medium was packed in each glass bottle (6 g/500 mL shake flask) and autoclaved (120 °C, 1.2 atm, 15 min). The preincubated mycelia of Armillaria sp. strain 543 (10 mL) were inoculated to the bottle and incubated under the conditions (22 °C, shaking with 120 rpm) for 30 days in an incubator (NR-30, Tietech, Japan). Extraction and Isolation. The culture broth of Armillaria sp. strain 543 (19 L) was filtered and concentrated under reduced pressure. The concentrated filtrate was successively partitioned between n-hexane and water (twice), EtOAc and water (twice), and then n-BuOH and water (twice). The n-hexane-soluble part (295 mg) was fractionated by normal-phase HPLC (Senshu PAK AQ, hexane− CHCl3, 30:70) to obtain eight fractions, and fraction 5 (38.5 mg) was further separated by reversed-phase HPLC (COSMOSIL Cholester Waters, 60% MeOH), giving 6 (18.3 mg). Fraction 1 (57.5 mg) was separated by normal-phase HPLC (Senshu PAK AQ, hexane−CHCl3, 20:80) to obtain seven fractions, and fractions 1-2 (12.6 mg), 1-3 (8.7 mg), and 1-4 (16.7 mg) were further separated by reversed-phase HPLC (COSMOSIL Cholester Waters, 70% MeOH), giving 7 (4.5 mg), 8 (3.3 mg), and 9 (5.7 mg), respectively. The EtOAc-soluble part (2.69 g) was fractionated by silica gel flash column chromatography (CH2Cl2; CH2Cl2−MeOH, 80:20, 60:40, 40:60, 20:80; and MeOH) to obtain six fractions. Fraction 2 (2.04 g) was fractionated by silica gel flash column chromatography (hexane−CH2Cl2, 10:90; CH2Cl2; CH2Cl2−MeOH, 90:10, 80:20; and MeOH) to obtain six fractions, and fraction 2-2 (657 mg) was further separated by normal-phase HPLC (Senshu PAK AQ, hexane−CHCl3, 30:70) to obtain 13 fractions. Fraction 2-2-4 (55.5 mg) was separated by reversed-phase HPLC (COSMOSIL Cholester Waters, 60% MeOH), giving 3 (12.8 mg) and 2 (15.6 mg). Compound 1 (5.4 mg) was obtained from fraction 2-2-4-6 (13.9 mg) by normal-phase HPLC (Senshu PAK AQ, hexane−CHCl3, 20:80). Fraction 2-2-8 (82.3 mg) was separated by reversed-phase HPLC (COSMOSIL Cholester Waters, 60% MeOH), affording 4 (26.2 mg). Fraction 2-2-12 (55.4 mg) was separated by reversed-phase HPLC (COSMOSIL Cholester Waters, 60% MeOH), affording 5 (14.8 mg). 10-Dehydroxymelleolide D (1): white, amorphous; mp 77−79 °C; [α]26D +41 (c 0.25, MeOH); CD (MeOH) λext (Δε) 202 (−16.0), 216 (+16.6), 234 (−5.54), 258 (+5.99) nm; IR (neat) νmax 3397, 1601, 1241 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 489 [M + Na]+; HRESIMS m/z 489.1661 [M + Na]+ (calcd for C24H31ClNaO7, 489.1656). 13-Hydroxymelleolide K (2): white, amorphous; mp 78−80 °C; [α]24D +26 (c 1.56, MeOH); CD (MeOH) λext (Δε) 206 (−4.91), 221 (+7.16), 236 (−7.74), 267 (+6.02) nm; IR (neat) νmax 3429, 1654, 1240 cm−1; 1H and 13C NMR, see Table 1; ESIMS m/z 473 [M + Na]+; HRESIMS m/z 473.1365 [M + Na]+ (calcd for C23H27ClNaO7, 473.1343). 5′-O-Methylmelledonal (3): colorless, amorphous; [α]26D +73 (c 0.34, MeOH); CD (MeOH) λext (Δε) 206 (−51.6), 220 (+11.7), 235 (−53.7), 262 (+46.1) nm; IR, NMR, and FABMS data were consistent with those of previously published reports.7,23 Melleolide D (4): white, amorphous; [α]26D +41 (c 1.3, CHCl3); CD (MeOH) λext (Δε) 202 (−3.81), 212 (+22.5), 234 (−5.23), 258 (+7.02) nm; IR (neat) νmax 3445, 1771 cm−1; 1H NMR (CDCl3) δ 0.93 (C-14, s), 1.11 (C-15, s), 1.31 (C-8, s), 1.79 (C-12, d, 14.3), 1.95 (C-12, d, 14.3), 1.98 (C-6, dd, 10.7, 8.5), 2.16 (C-6, dd, 10.7, 8.9), 2.39 (C-9, d, 4.0), 2.46 (C-8′, s), 3.67 (C-10, d, 3.7), 3.85 (C-5′-OCH3, s),

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ASSOCIATED CONTENT

S Supporting Information *

1

H, 13C, and 2D NMR spectra of compounds 1 and 2 are given. This material is available free of charge via the Internet at http://pubs.acs.org. D

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +81-54-238-4885. E-mail: [email protected]. jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemical Biology of Natural Products” from MEXT (Grant No. 24102513).

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