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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