of Novel Antimicrobial Diterpenes, Isolated from Healthy Leaves of a Bacterial Leaf Blight-resistant Cultivar of Rice Plant. Yoshiki Kono, Jun Uzawa, Kimiko ...
Agric.
Biol.
Chem.,
55 (3),
803-811,
1991
803
Structures of Oryzalides A and B, and Oryzalic Acid A, a Group
of Novel Antimicrobial Diterpenes, Isolated from Healthy Leaves of a Bacterial Leaf Blight-resistant
Cultivar of Rice Plant
Yoshiki Kono, Jun Uzawa, Kimiko Kobayashi, Yoshikatsu Suzuki, Masakazu Uramoto, Akira Sakurai, Minoru Watanabe,* Tohru Teraoka,* Daijiro Hosokawa,* Manabu Watanabe* and Hideaki Kondo** The Institute of Physical and Chemical Research, Wako-shi, Saitama 351-01, Japan * Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183, Japan ** Taisho Pharmaceutical Co., Omiya-shi, Saitama 330, Japan Received September 17, 1990 Oryzalides A and B, and oryzalic acid A, which were isolated from healthy flag leaves of a bacterial leaf blight-resistant cultivar of rice plant as a group of novel antimicrobial compounds, were confirmed by chemical and spectroscopic studies to be £«f-kaurane (C19 and C20) or e/if-kaurene (C1 9) analogues, i.e., oryzalide A, e/if-15a,16-epoxy-l/Mrydroxy-2-oxa-kauran-3-one; B, e/if-l/J,15adihydroxy-2-oxa-16-kauren-3-one; and oryzalic acid A, e/if-15a,16-epoxy-2,3-secokauran-2,3-dioic acid.
Momilactones^
A and B, and oryzalex-
bacterium, suggesting a minor contribution to the resistance. chemically characterized phytoalexins from In the course of a study on resistance mechanisms in rice cultivars to bacterial leaf infected rice leaves pretreated with WL-28325 (2,2-dichloro-3,3-dimethylcyclopropane carblight, Watanabe et al. have reported that boxylic acid; the former) prior to infection with lesion enlargement by the compatible strain of the rice blast fungus, Pyricularia oryzae, or the causal bacterium, Xanthomonas campestris from lesions infected with the fungus (the pv. oryzae, was markedly inhibited as a result latter). The existence and enhanced production of inducing resistance by a preliminary of such prohibitins (preformed antimicrobial inoculation with incompatible strains of the substances), as a-linolenic acid and its related same bacterium12); there was subsequently an
ins2"4) A, B, C and D were isolated
as
metabolites,5"7) azelaic acid and oxygencontaining unsaturated fatty acids including correlated lipoxygenase activity8 ~ 10) were also found in rice blast leaves infected with P.
abundant production of ethyl acetate-extractable
acidic
antibacterial
resistance-induced
substances
in the
leaves.13) It was presumed,
infected with bacterial pathogens Sato11} has not has yet been documented. Although
therefore, that these antibacterial substances were closely involved in the appearance of resistance. Similar antibacterial substances were also detected in small quantities from healthy rice leaves, and purification and isolation of the active substances were achieved
reported the existence of such antibacterial substances as syringaldehyde and others
by using large amounts of healthy rice leaves, because of the difficulty in obtaining inoculated
oryzae.
Onthe contrary, the production or increase
of phytoalexins
or prohibitins
in rice plant
against the causal agent of bacterial leaf blight of rice
plant
in healthy
rice
leaves,
their
quantity was decreased by infection with the
leaves in sufficient quantity.
As has already
been reported,14) during the course of isolating these antibacterial substances from healthy
804
Y. Kono et
Fig. 1. Structures with the Absolute Configuration and Their Derivatives.
of Oryzalides A (I) and B (II),
leaves, we isolated and determined a structure with
the relative
configuration
al.
of a novel
diterpene analogue, oryzalide A (I). Further-
more, during an investigation of related products, we succeeded in isolating two new
Oryzalic Acid A (III)
in the experimental section in detail. The filtrate of an 80%methanol extract was concentrated to aqueoussolution and then extracted with ethyl acetate or ether at pH 8.5 (the neutral fraction).
The remaining
aqueous phase was
antibacterial compounds, oryzalide B (II)beingand re-extracted with ethyl acetate or ether after oryzalic acid A (III), their structures determined e/2/-kaurane
to be a novel C19-e«/-kaurene or analogue (Fig. 1). In this paper,
adjusting to pH 2.5 with 6 n HC1 (the first acidic fraction).
The neutral
extract,
which
was
contaminated with a small amount of acidic we present details of the structural assignment compounds, was re-extracted with ethyl acetate and absolute configuration of oryzalide A, as at pH 8.5 and then at pH 2.5 (the second acidic well as the structures oryzalic acid A.
of oryzalide
B and
fraction).
The
two
acidic
extracts
were
chromatographically purified and fractionated
to obtain active oryzalides A and B from the first, and oryzalic acid A from the second fraction in pure form. Isolated oryzalides A
Results and Discussion The
successful
isolation
procedure
for
(4.4mg, isolated from the extract of 20kg of oryzalides Aand B, and oryzalic acid A from leaves) and B (22.4mg), and oryzalic acid A healthy flag leaves of the rice plant is described (4.7mg) were examined by UV, IR and NMR
805
Structures of Oryzalides A and B, and Oryzalic Acid A
methods, and a single crystal of oryzalide A calcd.).
wasThefurther analyzed by X-ray. EI-MS spectra of oryzalide M+ at mjz 320,
oryzalic 302
and
and
A gave B and
of oryzalide
acid A gave M+ -18 (H2O) at m/z 332,
respectively.
The
molecular
formula of oryzalide A was(320.1977 determined found, to be C19H28O4 by HR-MS 320. 1985 calcd.).14) The molecular formulae of oryzalide B and oryzalic acid A were determined to be C19H28O4 and C2oH3005,
respectively, by HR-MSof their methylated derivatives (oryzalide calcd.;B methyloryzalic ester: 334.2158 fround, 334.2145 acid A dimethyl
ester:
378.2408
fround,
378.2404
The UVabsorption maxima of all three
compounds were found at 212 of 213nm, suggesting no conjugated group in their
molecules. The IR spectra of the three showed adsorptions attributable to carbonyl function between 1700 and 1710cm" x. Oryzalides A and B showed broad absorptions of hydroxyl functions at around 3460 and 3430cm"1,
respectively. The chemical shifts of the 1H- and 13C-NMRspectra of oryzalides A and B, and of oryzalic acid A are shown in Table I. A single crystal of oryzalide A was obtained by recrystallizing acetate-77-hexane.
from a solvent of ethyl The X-ray analysis was
carried out on this single crystal to determine
Table I. Assignments of Signals in the Proton and Carbon-13 NMRSpectra of Oryzalides A and B, Their Derivatives and Oryzalic Acid A I1)
iH*)
13Cd)
Hfl
Oryzalide
A
IT1)
Ia2)
Oryzalide
B
Oryzalic acid A III2)
IIc2)
iH«)
13CC)
1H«)
13Cc) 1H«
13Cc)
2
2
5.382) 100.42) 9.13
41.4
101.23) 204.5
5.35
100.5 9.ll
204.8
C-2
1.97 1.35 1.53 1.57 1.77
C-5 C-6
C-7
43.9 19.5 33.9
2.ll 1.ll.7 1.l-
1.87
C-9 C-10
1.46 1.66 1.58 1.68 2.17 1.13 1.47 2.75
C-ll
C-12 C-13 C-14 C-15 C-16
C-17 Me C-18
C-19Me C-20Me OMe
39.3
42.9* 18.0 26.5
38.9 31.9
67.5 61.3 14.3
1.19 1.17 -
1.80
2.00
20.6
1.4 1.59 1.49 1.77
33.8
28.1
23.4 14.3 -
42.5 37.8
1.78
1.ll.7 1.ll.7 2.16 1.ll.7 2.75
[1.19
[1.10 1.17 3.61
18.5
26 38 31
-41.9 19.7
.4
.7 .4
67. .0 61.
.3
14.
.4*
25.3]**
23.7]** 13.2* 51.6
1.2 1.41 1.50 1.64 2.77 1.44 1.84 3.88
178.1 46.1* 48.3
20.9 33.2
1.74 46.8
46.6*
42.7
41.2 56.0 19.1
17.9
32.1
41.9 35.8
1.16 1.56 1.52 1.64 2.88 1.66 1.89 5.57
50 0~ 7 0~ 7
177.7 186.4 46.0 47.9 20.7 34.2 43.5
19
41.8 42.9
.0~
18.2
.7
32.2
.0-
27.1
.7
42.3 36.8
.13 .0~
38.8 31.8
.7
82.2 159.0
5.10 5.23 1.25 1.19 1.16
2.ll 1.47 1.57 1.48 1.79
33.4
40.8*
55.4
1.43
1.44
Me 1.27
48.3
l.7
41.4*
C-8
40.3*
46.4
42.9*
C-4
179.6
177.1
179.4
C-3
108.8
28.9 23.4 15.8
5.13 5.20 [1.17 [1.08 1.22 3.58
-82.7 154.0 111.1
.74
67.5
.42
14.3
61.3
25.4]** [1.27 23.7]** [1.25 13.4 1.10 51.7
* & ** exchangeable. 1} in CDCI3+CD3OD, 2) CDC13+D2O, 3) CD3OD; a) 500MHz, b) 400MHz, c) 125MHz, d) 100MHz.
806
Y. Kono et al.
(x 2), 61.3 and 179.4. The ^-NMR spectrum of oryzalide A showed some changes of sig-
nals due to its concentration, e.g., a higher concentration (approximately 20mg/ml) of oryzalide A in CDC13-D2O gave unusually broad signals at around 3 1.9 (1.85-2.05; H-5 and H-9) and 5.7 (5.40-6.00; H-l), while a
lower concentration (approximately 2 mg/ml) Fig. 2. Perspective Viewof the Molecular Structure of Oryzalide A. the structure with the relative configuration of oryzalide A.14) The crystal data for cryzalide A (C19H28O4, 320.41) indicated the following dimensions: orthorhombic, P212121; a= 1 1.222 (3), b=23.507 V=1685.3 (8)A3;
(6),
c=6.389 (2) Dc=1.263nig-m"3,
A;
Z=4, \i (Cu-
Ka)=0.662mm"1, 2=1.54184A. All the nonhydrogen atoms were refined with anisotropic thermal
parameters.
Twenty-eight
H atoms
were found on a difference Fourier mapand refined isotropically, the final R being 0.059 and Rw being 0.055. All computations were performed by a FACOMM-780 computer using the UNICS-III program system.15) The resulting structure for oryzalide A is shown in Fig. 2 with figurations.
the perspective
view of all con-
(H-20),
1.27
(H-18),
and their
1.19
respective
(H-19)
and
carbons
at 3 14.3,
1.17
28.1, 23.4 and 14.3. Ten methylene protons were observed (H-7),
1.13/1.47 carbons
at 3 1.35/1.53
1.46/1.66
(H-ll),
(H-6),
1.58/1.68
(H-14), and their were at 3 19.5, 33.9,
1.57/1.77
(H-12)
and
five respective 18.0, 26.5 and
at 3 5.38 (H-l, s), 2.01 (H-5,
dd) and 1.96 (H-9, 13C-NMR spectrum
br. d). Although the of oryzalide A in the
foregoing solvent gave a very weak signal at 3 100.4, it gave a broad singlet at 3 101.2 in 12CD3OD. These observations
suggested
the
presence of an additional methine proton and carbon due to an acetal group in the molecule. Methylation of oryzalide A with CH2N2gave a monomethylated compound which showed O-methyl and aldehyde signals 9.13
by
iH-NMR,
and
at 3 3.61 and
351.6
and
204.5
by
13C-NMR, respectively, confirming the existence of a lactol group in the molecule. Furthermore, the results of 1H(3 61.3, 2.75, singlet) and 13C-NMR (367.5, CH; =C=) suggested epoxide
that oryzalide A contained group. From these results,
an the
unsaturated degree of 6 (two of them were attributable to both a carbonyl and an epoxide group)
In addition to the result by X-ray analysis, the structural assignment of oryzalide A was madefrom the NMRspectra in order to use the results for a structural analysis of the related compounds. The CH-COSYspectrum (500MHz, CDCI3+CD3OD) of oryzalide A showed four singlets ofmethyl protons at 3 1.44 (H-17),
gave sharp signals
suggested
that
oryzalide
A was
composed of four rings. The HMBC(heteronuclear multiple-bond
correlation)
spectrum
(400 MHz, CDC13 + D2O) oforyzalide A/orzalide A methyl ester showed the following long-range (H-l9) and £28.6/25.3
respectively, (C-5)
and
coupling: 1.29/1.19 (C-18)
methyls at (H-l8) with and 23.5/23.7
and at 42.8/46.4
178.9/177.1
3 1.17/1.17 (H-20) with (C-10), 43.1/48.3 (C-5) aldehyde of the methyl with carbon at 355.4 3 1.44/1.42 (H-17) with
43.1/48.3
a methyl
at
carbons at 3 42.8/55.4 and 39.9/37.8 (C-9); an ester at 3 9.13 (H-l) (C-10); a methyl at carbons at 3 62.3/61.3
(C-16),
2.75 (H-15), and their four respective
a methine of the methyl ester at 3 2.75 (H-l5) also showed coupling with the carbon at 3 42.5 (C-8). These results support partial structures
(H-5),
were at 3 43.9,
1.87
39.3,
(H-9,
brs),
2.17
38.9 and 67.5.
(H-13)
and
carbons
Five qua-
ternary carbons were observed at (541.4, 42.9
(C-15)
(C-4),
31.9. Four methine protons were observed at 3 1.97
68.5/67.0
(C-3);
3
