Penipratynolene, a novel nematicide from Penicillium bilaiae Chalabuda

Article abstract of DOI:10.1271/bbb.68.257

New acetylenic nematicidal compound, penipratynolene (1), methy (2′R)-4-(2e-hydroxy-3e-butynoxy)benzoate, together with two known compounds, 6-methoxycarbonylpicolinic acid (2) and 2,6-pyridinedicarboxylic acid (3), were isolated from the culture filtrate of Penicillium bilaiae Chalabuda. The structures of 1-3 were established by spectroscopic methods. The absolute configuration of 1 was confirmed by using a modified version of Mosher's method. Compounds 1-3 showed nematicidal activity of 77%, 52%, and 98%, respectively, by a bioassay at 300 mg/l with the root-lesion nematode Pratylenchus penetrans.

Full text of DOI:10.1271/bbb.68.257

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Penipratynolene, a Novel Nematicide from
Penicillium bilaiae Chalabuda
d
a
b
c
Satoshi NAKAHARA , Miyako KUSANO , Shozo FUJIOKA , Atsumi SHIMADA & Yasuo
a
KIMURA
a
Department of Biological and Environmental Chemistry, Faculty of Agriculture, Tottori
University
b
Umea Plant Science Centre, Department of Forest Genetics and Plant Physiology, The
Swedish University of Agricultural Sciences
c
The Institute of Physical and Chemical Research (RIKEN)
d
Department of Environmental Chemistry, Faculty of Engineering, Kyushu Kyoritsu
University
Published online: 22 May 2014.
To cite this article: Satoshi NAKAHARA, Miyako KUSANO, Shozo FUJIOKA, Atsumi SHIMADA & Yasuo KIMURA (2004)
Penipratynolene, a Novel Nematicide from Penicillium bilaiae Chalabuda, Bioscience, Biotechnology, and Biochemistry,
68:1, 257-259, DOI: 10.1271/bbb.68.257
To link to this article: http://dx.doi.org/10.1271/bbb.68.257
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Biosci. Biotechnol. Biochem., 68 (1), 257–259, 2004
Note
Penipratynolene, a Novel Nematicide from Penicillium bilaiae Chalabuda
1
2
3
4
Satoshi NAKAHARA, Miyako KUSANO, Shozo FUJIOKA, Atsumi SHIMADA,
1
;y
and Yasuo KIMURA
1
Department of Biological and Environmental Chemistry, Faculty of Agriculture,
Tottori University, Tottori-shi 680-8553, Japan
2
Umea Plant Science Centre, Department of Forest Genetics and Plant Physiology,
The Swedish University of Agricultural Sciences, SE-901 83 Umea, Sweden
3
The Institute of Physical and Chemical Research (RIKEN), Wako-shi 351-0198, Japan
4
Department of Environmental Chemistry, Faculty of Engineering, Kyushu Kyoritsu University,
Kitakyushu-shi 807-8585, Japan
Received August 22, 2003; Accepted October 4, 2003
New acetylenic nematicidal compound, penipratyno-
column chromatography on silica gel with mixtures of
n-hexane and acetone.
0
0
0
lene (1), methy (2 R)-4-(2 -hydroxy-3 -butynoxy)ben-
zoate, together with two known compounds, 6-methoxy-
carbonylpicolinic acid (2) and 2,6-pyridinedicarboxylic
acid (3), were isolated from the culture filtrate of
Penicillium bilaiae Chalabuda. The structures of 1–3
were established by spectroscopic methods. The abso-
lute configuration of 1 was confirmed by using a
modified version of Mosher’s method. Compounds 1–3
showed nematicidal activity of 77%, 52%, and 98%,
respectively, by a bioassay at 300 mg/l with the root-
lesion nematode Pratylenchus penetrans.
1) The fraction eluted with 40% acetone in n-hexane
(1.78 g) was further fractionated on silica gel with
mixtures of n-hexane and EtOAc. The active fraction,
obtained with 15% EtOAc in n-hexane (0.12 g), was
purified by preparative TLC developed with benzene-
EtOAc (6:4) and recrystallized from benzene to afford 1
(14 mg).
2) The fraction eluted with 50% acetone in n-hexane
(13.60 g) was fractionated on silica gel with mixtures of
n-hexane and EtOAc. The fraction eluted with 35%
EtOAc in n-hexane (0.41 g) was further fractionated on
silica gel with mixtures of benzene and acetone. The
active fraction, obtained with 5% acetone (40 mg), was
recrystallized from benzene to afford 2 (15 mg).
Key words: penipratynolene; nematicide; Penicillium
bilaiae Chalabuda; root-lesion nematode;
Pratylenchus penetrans
3)
The fraction eluted with 90% acetone in n-hexane
(2.12 g) was fractionated on silica gel with mixtures of
We have previously investigated such fungal metab-
1
)
2)
olites as aspyrone, peniprequinolone, penigequino-
)
CHCl3 and MeOH. The active fraction, obtained with
5% MeOH in CHCl (232 mg), was recrystallized from
lones2 and 3-methoxy-4,6-dihydroxy-4-(4 -methoxy-
0
3
2
)
phenyl)quinolinone, ꢀꢁ-dehydrocurvularin and related
curvularines3 for their potential as nematicides against
the root-lesion nematode Pratylenchus penetrans, which
is a parasite of many crop plants and causes root
CHCl to afford 3 (15 mg).
3
)
The physicochemical properties of penipratynolene
ꢀ
(1) are as follows: colorless needles, mp 246–248 C,
2
0
ꢀ
½ꢂꢁ
ꢂ11:2 (c ¼ 0:2, EtOH). UV ꢃmax (EtOH) nm ("):
D
necrosis.4 Our screening program for novel naturally
occurring nematicides led to the investigation of the
metabolites of Penicilium bilaiae Chalabuda. These
studies led to the isolation, structural determination, and
biological activity evaluation of 1–3.
–7)
254 (10,700), 271 (sh, 4600). IR ꢄmax (KBr) cm : 3460,
3238, 2952, 2120, 1692, 1607, 1512, 1437, 1323, 1292.
H-NMR (acetone-d6, 270.05 MHz) ꢅ: 2.97 (1H, d,
ꢂ1
1
0
J ¼ 1:5 Hz, 4 -H), 3.84 (3H, s, 8-H), 4.19 (2H, dd,
0
J ¼ 3:5, 6.7 Hz, 1 -H), 4.74 (1H, ddd, J ¼ 1:5, 3.5,
0
0
P. bilaiae was cultured in a malt extract medium
ꢀ
6.7 Hz, 2 -H), 4.89 (1H, br.s, 2 -OH), 7.07 (2H, d,
J ¼ 8:8 Hz, 3-H, 5-H), 7.96 (2H, d, J ¼ 8:8 Hz, 2-H, 6-
(
40 l) at 25 C for 23 days without shaking. The culture
1
3
broth was filtered to separate the mycelium, and the
filtrate adjusted to pH 2.0 with a 2 N HCl solution. The
filtrate was successively extracted with EtOAc and
evaporated to dryness under reduced pressure. The
resulting residue (36.2 g) was first fractionated by
H). C-NMR (acetone-d6, 67.80 MHz) ꢅ: 51.9 (q, C-8),
0
0
0
61.1 (d, C-2 ), 71.4 (t, C-1 ), 74.6 (d, C-4 ), 80.8 (s, C-
0
3 ), 114.3 (d, C-3, C-5), 123.5 (s, C-1), 131.7 (d, C-2, C-
þ
6), 161.9 (s, C-4), 166.7 (s, C-7). EIMS m=z: 220 (M ),
189, 165, 121. Anal. Found: C, 65.30 %; H, 5.37. Calcd.
y
To whom correspondence should be addressed. Fax: +81-857-31-5363; E-mail: kimura@muses.tottori-u.ac.jp

258
S. NAKAHARA et al.
for C H O : C, 65.43; H, 5.50%.
1
2
12
4
Compound 1 was obtained as colorless needles, and
its molecular formula was determined to be C H O
4
12
12
by MS and elemental analysis. The IR absorption band
ꢂ1
at 2120 cm and two signals at ꢅ74.6 and 80.8 ppm in
1
3
the C-NMR spectrum indicated the presence of two
ꢂ1
13
acetylenic carbons. A band at 1692 cm and one C-
NMR signal at ꢅ166.7 ppm indicated the presence of a
ꢂ1
carbonyl carbon. A band at 3460 cm and one proton
1
signal at ꢅ4.89 ppm in the H-NMR spectrum indicated
the presence of a D O exchangeable hydroxyl group.
2
1
3
1
The C- and H-NMR spectra of 1 indicated the
presence of one methoxy, one O-substituted aliphatic
methylene, one O-substituted aliphatic methine, and one
,4-di-substituted phenyl group. In the 13C- H COLOC
1
1
spectrum of 1, a cross peak between the methoxy proton
signal at ꢅ3.84 ppm and the carbonyl carbon was
observed, and this carbonyl carbon had a cross peak
Fig. 2. Effects of Compounds 1–4 on the Nematode, Pratylenchus
penetrans.
Each bar represents ꢃSE of 3 experiments.
0
with 2-H. The partial structure corresponding to C-1 to
0
C-4 was derived from their coupling constants in the
1
0
Table 1. Nematicidal Activities of Compounds 1–3 and Aspyrone
against Pratylenchus penetrains
H-NMR spectrum, and NOE between 1 -H and 3-H was
observed. Thus, the planar structure of 1 was established
ꢄ
as shown in Fig. 1.
The absolute stereochemistry of C-2 was determined
by using a modification of Mosher’s method. 1 was
treated with (R)-(ꢂ)- and (S)-(þ)-2-methoxy-2-trifluoro-
methyl-2-phenylacetyl chlorides (MTPA Cls) to afford
Compound
Nematicidal activity (LD50
)
0
Compound 1
Compound 2
Compound 3
Aspyrone
10–30
100–300
<1
8)
100–300
0
0
the C-2 -(S)- and C-2 -(R)- MTPA esters of 1. Positive
1
ꢄ
Concentration (mg/l) causing over 50% of nematodes to be immobile after
3 days.
ꢀꢅ values (ꢅS-ꢅR) in the H NMR spectra were observed
for 2 and 4 -H, while negative ꢀꢅ values were located at
1 , 2, 3 and 6-H (Fig. 2). These results revealed the
0
absolute configuration of C-2 to be R.
0
0
0
6
-Methoxycarbonylpicolinic acid (2) and 2,6-pyridi-
nedicarboxylic acid (3) were identified by comparing
their physicochemical properties with those reported
9
–11)
(
Fig. 1).
In addition, methylation of 2 and 3 with
diazomethane gave the same dimethyl-2,6-pyridinedi-
carboxylate (4).
The nematicidal activities of 1–4 against the root-
lesion nematode, P. penetrans are shown in Fig. 3 and
Table 1. Compounds 1–3 promoted nematicidal activity
proportional to its concentration from 1 mg/l to
1000 mg/l, but 4 showed weak nematicidal activity.
Compounds 1, 2, 3 and 4 had nematicidal activities
against P. penetrans of 77%, 52%, 98% and 36% at a
concentration of 300 mg/l, respectively. In addition, 1
and 3 had more effective nematicidal activities than that
1–3)
of aspyrone as a positive control.
The nematicidal
activities of 2–4 suggested that the carboxy groups in the
molecule played an important role in this activity. The
presence of acetylenic carbons may play an important
role in nematicidal activity of 1, since many naturally
occurring acetylene compounds are known as nematici-
1
2,13)
dal substances.
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ꢀꢅ (ꢅS-ꢅR) values, obtained for the MTPA esters of 1-a, are
expressed in ppm.

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