Armillariols A to C from the culture broth of Armillaria sp.

Article abstract of DOI:10.1016/j.tetlet.2013.07.131

Three novel compounds were isolated from the culture broth of Armillaria sp. Their structures were elucidated mainly by spectroscopic data analyses. All the compounds regulated hypocotyl and root growth of lettuce.

Full text of DOI:10.1016/j.tetlet.2013.07.131

Tetrahedron Letters 54 (2013) 5481–5483
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Armillariols A to C from the culture broth of Armillaria sp.
Hajime Kobori a, Atsushi Sekiya b, Nobuhiro Yasuda c, Keiichi Noguchi d, Tomohiro Suzuki e
Jae-Hoon Choi e, Hirofumi Hirai f, Hirokazu Kawagishi a,e,f,
⇑
a Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
b Kyushu Research Center, Forestry & Forest Products Research Institute, 4-11-16 Kurokami, Kumamoto, Kumamoto 860-0862, Japan
c Japan Synchrotron Radiation Research Institute, SPring-8, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan
d Instrumentation Analysis Center, Tokyo University of Agriculture and Technology Koganei, Tokyo 184-8588, Japan
e Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
f Graduate School of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Three novel compounds were isolated from the culture broth of Armillaria sp. Their structures were
elucidated mainly by spectroscopic data analyses. All the compounds regulated hypocotyl and root
growth of lettuce.
Received 14 June 2013
Revised 23 July 2013
Accepted 26 July 2013
Available online 2 August 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Culture broth
Fungus
Armillaria sp.
Lettuce growth inhibition
Structure determination
Introduction
Therefore, we tried to isolate compounds with plant growth regu-
latory activity from the fungus.
The genus Armillaria (English name, Honey Fungus; Japanese
name, Naratake) belonging to the family physalacriaceae is a well
known group of edible mushroom throughout the world. People
have utilized it for its medicinal properties and edible qualities
for a long time. On the other hand, the genus has been also known
as a serious plant pathogen which causes root rot in various plant
species1 and the phenomenon is called ‘Armillaria root disease’.2,3
The rot is one of the most serious diseases of plants, which occurs
in many broadleaf trees, conifers, and several herbaceous plants
including Alchemilla mollis, Beta vulgaris, and so on.4 Furthermore,
it is known that the penetration of Armillaria mycelia into the fungi
Entoloma abortivum and Wynnea americana induces spherical
deformity of the fruiting bodies of those mushrooms.5 These facts
mean that Armillaria produces allelopathic substance(s). Protoillu-
dane sesquiterpene aryl esters have been isolated from the genus
Armillaria mushrooms and Clitocybe illudens.6–12 Those compounds
showed antimicrobial activity7,13,14 and cytotoxicity against hu-
man cancer cell lines.15,16 However, there are no evidences that
the compounds are toxic principles for ‘Armillaria root disease’.
Here, we describe the isolation, structural determination, and
biological activity of three novel compounds from the culture broth
of a strain of the genus.
Results and discussion
Isolation of the active compounds from Armillaria sp. was
guided by their growth-regulating activity on lettuce. Since culture
broth of the fungus inhibited the growth, the broth was extracted
O
1
OH
*
1'
OH
4
4'
*
O
OH
2
6'
7
O
OH
1'
5'
1
1''
7''
2
1
O1'
OH
OH
OH
3
1
⇑
Corresponding author. Tel./fax: +81 54 238 4885.
E-mail address: achkawa@ipc.shizuoka.ac.jp (H. Kawagishi).
Figure 1. Structure of 1–3.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.07.131

5482
H. Kobori et al. / Tetrahedron Letters 54 (2013) 5481–5483
supposed to be (Z)-2-methyl-6-(4-methylcyclohex-3-enyl)hept-5-
O
OH
ene-2,3-diol (Fig. 1). 2-Methyl-6-(4-methylcyclohex-3-en-1-yl)-
hept-6-ene-2,3-diol (10,11-dihydroxy-10,11H-b-bisabolene) that
has the same skeleton as 1 has been isolated from the aerial parts
of the plant Acritopappus confertus.17
OH
O
OH
2
Armillariol B (2) was purified as pale yellow oil. Its molecular
formula was determined as C9H18O5 by HR-ESI-MS m/z 229.1066
[M+Na]+ (calcd for C9H18NaO5, 229.1052), indicating the presence
of one degree of unsaturation in the molecule. Structure of 2 was
elucidated by interpretation of NMR spectra including DEPT, COSY,
HMQC, and HMBC (Fig. 2). The one unsaturation degree was ex-
plained by the presence of an ester (dC 173.6). The acid part,
3-methylbutanoate, was elucidated by the COSY correlations
(H2/H3; H3/H4, H3-CH3) and the HMBC correlations (H2/C1, C3,
C4, C3-CH3; H3/C2, C4, C3-CH3, H4/C2, C3, C3-CH3, H3-CH3/C2,
C3, C4) (Fig. 2). The COSY correlations (H10/H20, H20/H30, H30/H40)
and the HMBC correlations (H10/C20, C30, C1; H20/C10, C30, C40;
H30/C10, C20; H40/C20, C30) indicated the structure of the alcohol part
and its linkage position to the acid part. As a consequence, 2 was
determined as 2,3,4-trihydroxybutyl 3-methylbutanoate (Fig. 1).
Armillariol C (3) was purified as pale yellow oil. Its molecular
O
OH
O
OH
OH
OH
3
1
COSY
HMBC
Figure 2. COSY and HMBC correlations in 1–3.
with hexane, EtOAc, and 1-BuOH, successively. The active fractions,
the hexane soluble-, and the EtOAc soluble-fractions, were sub-
jected to repeated chromatography respectively to afford armillari-
ol A to C (1–3) (Fig. 1).
Armillariol A (1) was purified as colorless oil. The molecular for-
mula was determined as C15H26O2 by HR-ESI-MS m/z 261.1826
[M+Na]+ (calcd for C15H26NaO2, 261.1830), indicating the presence
of three degrees of unsaturation in the molecule. Structure of 1 was
elucidated by interpretation of NMR spectra including DEPT, COSY,
HMQC, and HMBC (Fig. 2). The DEPT experiment indicated the pres-
ence of four methyls, four methylenes, four methines, and three qua-
ternary carbons. The molecular formula, the unsaturation degrees,
13C NMR data (dC 72.6, 77.9, 120.8, 120.9, 133.8, 143.5, Table 1),
and the DEPT data indicated the presence of a ring, two double bonds,
and two hydroxy groups in the molecule. The complete assignment
of the protons and carbons of NMR was accomplished as shown in
Table 1. The structure of the alkyl chain (C1–C7) was elucidated by
the COSY correlations (H3/H4; H4/H5) and the HMBC correlations
(H1/C2, C2-CH3, C3; H2-CH3/C1, C2, C3; H3/C2-CH3, C4, C5; H4/C5,
C6; H5/C4, C7; H7/C5, C6) (Fig. 2). The presence of the cyclohexene
moiety (C10–C60) and its linking positions to a methyl and the chain
were confirmed by the COSY correlations (H10/H20, H60; H20/H30;
H50/H60) and the HMBC correlations (H5/C10, H7/C10, H10/C50; H40-
CH3 /C30, C40, C50) (Fig. 2). NOE was observed between H4 and H10
in the NOESY and NOE-difference experiments, indicating that the
geometry of the double bond was Z. As a result, structure of 1 was
formula was determined as
C13H20O4 by HR-ESI-MS m/z
263.1224 [M+Na]+ (calcd for C13H20NaO4, 263.1259), indicating
that the unsaturation degree in the molecule was four. Structure
of 1 was elucidated by interpretation of NMR spectra including
DEPT, COSY, HMQC, and HMBC (Fig. 2). The DEPT experiment indi-
cated the presence of two methyls, four methylenes, four methines,
and three quaternary carbons. The single carbon chain from C100 to
C700 was constructed by the 1H and 13C NMR (Table 1) spectra along
with COSY correlations (H30/H40, H100/H200, H200/H300, H300/H400, H400/
H500, H600/H700) and HMBC ones (H2/C1, C20; H30/C-1, C20, C40, C50;
H40/C20, C30, C50; H100/C40, C50, C200; H200/C300, C400; H300/C100, C200,
C400, C500; H400/C200, C500, C600; H500/C400, C600, C700; H600/C400, C500, C700;
H700/H500, H600). The molecular formula, the presence of a car-
bonyl-conjugated diene (C1, dC 185.5; C20, dC 152.0; C30, dC 118.6,
dH 7.12 (d, J = 3.7 Hz); C40, dC 109.8, dH 6.47 (d, J = 3.7 Hz); C50, dC
159.6) including two quaternary sp2 carbons, and the unsaturation
degree indicated that this compound possessed a furan. As a result,
structure of 3 was determined to be 1-(5-(1,2-dihydroxyhep-
tyl)furan-2-yl)ethanone (Fig. 1).
Confirmation of the planar structures and determination of ste-
reochemistry of 2 and 3 were performed by X-ray crystallography
Table 1
1H and 13C NMR data for 1–3 (1 and 3 in CDCl3, 2 in CD3OD)
Position
1
Position
2
Position
3
1H
13C
1H
13C
1H
13C
d (mult., J in Hz)
d
d (mult., J in Hz)
d
d (mult., J in Hz)
d
1
2
3
4
1.22 (s)
26.6
1
2
3
4
174.8
44.1
26.8
22.7
22.7
66.6
1
2
186.5
25.9
72.6
77.9
29.8
2.22 (d, 7.0)
2.08 (m)
0.95 (d, 6.7)
0.95 (d, 6.7)
4.13 (m)
4.16 (m)
3.82 (m)
3.60 (m)
3.60 (m)
2.43 (s)
3.36 (dd, 8.9, 4.0)
2.16 (m)
2.20 (m)
20
30
40
50
100
200
300
152.0
118.6
109.8
159.6
71.1
7.12 (d, 3.7)
6.47 (d, 3.7)
3-CH3
10
5
6
7
10
20
5.18 (dd, 7.3, 7.0)
120.9
143.5
19.5
35.4
29.4
4.56 (br s)
3.92 (m)
1.45 (m)
1.52 (m)
1.28 (m)
1.47 (m)
1.27 (m)
1.27 (m)
0.86 (t, 6.8)
1.66 (br. s)
2.60 (m)
1.80 (m)
2.02 (m)
5.38 (m)
20
30
40
70.4
73.0
64.1
73.2
33.0
400
25.2
30
40
50
120.8
133.8
30.5
500
600
700
31.7
22.5
14.0
1.92 (m)
2.05 (m)
1.53 (m)
1.58 (m)
1.16 (s)
60
27.5
2-CH3
40-CH3
23.7
23.6
1.64 (br s)

H. Kobori et al. / Tetrahedron Letters 54 (2013) 5481–5483
5483
Figure 3. ORTEP drawings of p-bromobenzoate of 2 and 3 with ellipsoids at the 30% probability level. Hydrogen atoms are shown spheres of arbitary radii.
analysis of their (p-bromo)benzoates (Fig. 3). The Flack parameter
(X = 0.16 (3)) of 2 in the analysis indicated that its absolute config-
uration might be as shown in Figure 1. However, the possibility of
the opposite configuration could not be excluded. Therefore, the
relative configuration of 2 and absolute configuration of 3 were
determined to be as shown in Figure 1. The stereochemistry at
the asymmetric carbons in 1 remains undetermined.
Research on Innovative Areas ‘Chemical Biology of Natural Prod-
ucts’ from MEXT.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.07.
131.
In order to examine plant growth regulatory activity of these
compounds, lettuce was used as the test plant. All the compounds
inhibited hypocotyl and root growth of the plant at 1 lmol/paper
References and notes
with significant differences (rate of growth length compared with
control standard deviation: 1, root 81.2 11.9%, hypocotyl
55.5 21.9%; 2, root 68.0 24.6%, hypocotyl 66.3 21.5%; 3, root
68.3 25.9%, hypocotyl 66.5 21.8%). 1 promoted the hypocotyl
1. Roll-Hansen, F. Eur. J. For. Pathol. 1985, 15, 22–31.
2. Cox, K. D.; Scherm, H. Biol. Control 2006, 37, 291–300.
3. Thomidis, T.; Exadaktylou, E. Crop Prot. 2012, 36, 49–51.
4. Robinson-Bax, C.; Fox, R. T. V. Mycologist 2002, 16, 21–22.
5. Ando, Y. Chiba Mycol. Club Bull. 2000, 16–17, 19–25.
6. Ayer, W. A.; Browne, L. M. Tetrahedron 1981, 37, 2197–2248.
7. Midland, S. L.; Izak, R. R.; Wing, R. M.; Zaki, A. I.; Munnecke, D. E.; Sims, J. J.
Tetrahedron Lett. 1982, 23, 2515–2518.
8. Arnone, A.; Cardillo, R.; Nasini, G. Phytochemistry 1986, 25, 471–474.
9. Donnelly, D. M. X.; Quigley, P. F.; Coveney, J. D.; Polonsky, J. Phytochemistry
1987, 26, 3075–3077.
10. Donnelly, D. M. X.; Hutchinson, R. M.; Coveney, J. D.; Yonemitsu, M.
Phytochemistry 1990, 29, 2569–2572.
growth at 10ꢀ2
lmol/paper (114 9.15%).
Since Armillaria species mainly infect woody plants and then
cause ‘Armillaria root disease’ to them,2,3 woody plants should
have been used in the bioassay. However, such an assay is very dif-
ficult for us because of low quantities of the compounds. Occasion-
ally several herbaceous plants are also damaged by those
pathogens.4 Therefore, the activity of the compounds toward let-
tuce suggested that armillariols A to C might play some roles in
the Armillaria root disease.
11. Yang, J.-S.; Su, Y.-L.; Yu, D.-Q.; Liang, X.-T. J. Chinese Pharm. Sci. 1993, 2, 10–17.
12. Donnelly, D. M. X.; Konishi, T.; Dunne, O.; Cremin, P. Phytochemistry 1997, 44,
1473–1478.
13. Oduro, K. A.; Munnecke, D. E.; Sims, J. J.; Keen, N. T. Trans. Br. Mycol. Soc. 1976,
66, 195–199.
Acknowledgment
14. Peipp, H.; Sonnenbichler, J. Biol. Chem. Hoppe–Seyler 1992, 373, 675–683.
15. Misiek, M.; Williams, J.; Schmich, K.; Hüttel, W.; Merfort, I.; Salomon, C. E.;
Aldrich, C. C.; Hoffmeister, D. J. Nat. Prod. 2009, 72, 1888–1891.
16. Bohnert, M.; Miethbauer, S.; Dahse, H.-M.; Ziemen, J.; Nett, M.; Hoffmeister, D.
Bioorg. Med. Chem. Lett. 2011, 21, 2003–2006.
We thank V.K. Deo (Shizuoka University) for valuable discus-
sion and the synchrotron radiation experiment was performed at
the BL02B1 of SPring-8 with the approval of the Japan Synchrotron
Radiation Research Institute (JASRI) (Proposal No. 2012B1109).
This work was partially supported by a Grant-in-Aid for Scientific
17. Bohlmann, F.; Zdero, C.; Jakupovic, J.; King, R. M.; Robinson, H. Phytochemistry
1983, 22, 2243–2252.