376 Planta Mcd. 57(1991)
New Perylenequinones from Shiraia bambusicola TadashiKishi"5, Satoshi Tahara2, Naoki Taniguchi3. Mitsuya Tsuda4, Chihiro Tanaka4. and Shozo Takahashi4 Kyoto Municipal Junior College of Nursing, Higashi-Takada-cho 1—2, Mibu, Nakagyo-ku, Kyoto 604, Japan 2 Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060, Japan Kyoto City Institute of Public Health, Higashi-Takada-cho 1—2, Mibu, Nakagyo-ku, Kyoto 604, Japan Pesticide Research Institute, Kyoto University, Sakyo-ku, Kyoto 606, Japan Address for correspondence
Isolation and purification
Abstract Two new perylenequinones, named hypocrellin B and C, together with the known hypocrellin A were isolated from the stromatal tissues of Shiraia barnbusicola. The structures of these compounds, including the absolute stereochemistry, were determined.
evaporated to dryness in vacuo (31.2 g), after which the extract was partitioned between chloroform and water. The chloroform-soluble fraction (10.4 g) ws chromatographed on silica gel (250 g) with 3.5% oxalic acid column (0 4.5 cm x 60cm). Chloroform-EtOH (99: 1) was used as the elution solvent. The resulting red pigment fraction (613.3 mg) was purified by multiple development by pre-
Key words
parative TLC on silica gel with 3.5% oxalic acid and using the chloroform-EtOH (99: 1). Crystallization from acetone yielded
Shiraia bambusicola, perylenequinones,
30.8mg of 1, 31.1mg of2, and 30.4mg of3, respectively, from the middle, lower, and upper bands.
hypocrellins A, B, C, structure elucidation, parasitic fungus.
Introduction
Shirala bambusicola P. Hennigs, a conspicuous fungus parasitic on Phyllostachys spp., is re-
corded only from Japan and China (1). It has been used as medicinal fungi under the name of "zhu huang" in China (2). The active principle(s) of this fungus, however, has not been analyzed.
During our study of the life-history of this fungus, we have observed that its stromatal tissues have an-
tibacterial activity against Bacillus subtilis (Ehrenberg) Cohn. We here report the isolation and structural forms of the active perylenequinones from the stromatal tissues of S. bambusicola.
Materials and Methods Melting points were determined by the micro hot-plate method and are uncorrected. UV spectra were recorded
with a Hitachi U-3210 spectrophotometer. IR spectra were recorded with a Hitachi 285 spectrometer as KBr discs or in CC14. FD-
MS spectra were measured with a JEOL JMS-O1SG-2 mass spectrometer. 1H-, 13C-NMR and COLOC spectra were recorded with a Bruker AM 500 (500 MHz, FT) in CDCI3. CD spectra were recorded
with a JASCO J-20 automatic recording spectropolarimeter in MeOH at 23°C.
Ninety grams of air-dried stomata of S. barnbusicola were treated with about 41 of MeOH. The extract was
Reactions A 3.12mg sample of I and a 3.42mg sample of2 were each dissolved in 10.0 ml of toluene. A 0.5 ml portion of each solution was heated at 120°C for 80 mm in a sealed ampule. The resulting solutions were concentrated and then analyzed by TLC and CD spectroscopy. After addition of 2 &l of conc. H2S04 to 9.5 ml each
of solutions I and 2, 0.5 ml samples of each solution were heated at
50°C for 60mm and at 105°C for 30mm, respectively. Reaction mixtures were treated with 20 ml of ethyl acetate, then washed with 5% NaHCO3 and saturated NaC1. The extract was concentrated to dryness in vacuo. The FD-MS, CD, and UV spectra of each
product were measured.
Hypocrellin A (1) Blood-red crystals, m.p.: softens at 214°C, melts at 245—250°C. Molecular weight: FD—MS rn/z 546 (Mt, 100%) C30H26010. UV Amax (MeOH) nm (log ): 581 (4.10), 540 (4.08), 464 (4.38), 340 (3.71), 280sh (4.44), 266 (4.50), 214 (4.66). IR max in CCJ4 (0.22mM): 3495 (-OH'O) cm1. JR max in KBr: 3470 (-OH),
1700 (>CO), 1600 (aromatic ring) cm. CD: Fig.2. 1H-NMR b: 15.955 (1H, s, -OHO-), 15.912 (1H, s, -OHO-), 6.566 (111, s, 8-CH=), 6.550 (1H, s, 5-CH=), 4.112 (3H x 2, s, -OMe x 2), 4.074 (311 x 2, s, -OMe x 2), 3.514 (1H, d, JAB = 12.0Hz, 13-CHA), 2.632 (1H, d, J 12.0 Hz, 13-CHB), 3.448 (1H, s, 15-CH), 1.892 (1H, s, 18-Me), 1.705 (1H, s, 16-Me), 1.633 (1H, hr. s, -OH). 13C-NMR b: 207.4 (C-17), 180.3 (C-4 or C-9), 179.9 (C-9 or C-4), 171.8 (C-3 or C-b), 170.9 (C-b or C-3), 167.5 (C-6 or C-7), 167.5 (C-7 or C-6), 150.9 (C-2 or C-il), 150.6 (C-il or C-2), 134.0 (C-12), 133.2 (C-i), 128.5 (C-12a), 127.6 (C-la), 125.0 (C-3b or C-7b), 125.0 (C-7b or C-3b), 118.2 (C-6a or C-7a), 117.7 (C-7a or C-6a), 106.9 (C-3a or C-9a), 106.7 (C-9a or C-3a), 102.1 (C-5 or C-8), 102.0 (C-8 or C-5), 78.8 (C-l4), 62.1 (2-OMe or 11-OMe), 60.8 (11-OMe or 2-OMe), 61.7 (C-15), 56.6 (6-OMe or 7-OMe), 56.5 (7-OMe or 6-OMe), 41.9 (C-13), 30.1 (C-18), 27.0 (C-16).
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Received: April23, 1990
Planta Med. 57(1991) 377
New Perylenequinones from Shirala bambasicola
O OH
Hypocrellin B (2)
Black red powdery crystals, m.p.: softens at 195°C, melts at 215—217°C. Molecular weight: FD-MS m/z 546 (Mt, 100%) C,0H26010. u .& (MeOH) nm (log ): 581 (4.05), 540
H3
H3CO
(4.03), 467 (4.32), 340 (3.65), 282sh (4.37), 266 (4.44), 261sh (4.44), 220 sh (4.60), 210 (4.61). JR 'max in CC14 (0.23mM): 3543 (OH'O) CIfl1. JR Vms,, in KBr: 3440 (-OH), 1700 (>CO), 1600 (aromatic ring) cm1. CD: Fig. 2. 'H-NMR ô: 16.062 (1H, s, -OHO-), 15.963(1H, s, -OH•O-), 6.566(1H, s, 8-CH=), 6.557(1H, s, 5-CH=),
I
O OH
OC H3
2
1
4.274 (3H, s, -OMe), 4.185 (3H, s, -OMe), 4.076 (3H, s, -OMe), 4.072 (3H, s, -OMe), 3.798 (1H, s, -OH), 3.726 (1H, s, 15-CH), 3.673 (1H, d, J = 13.9 Hz, 13-CHA), 2.347 (1H, d, J = 13.9 Hz, 13-CHB),
1-OH
1.834 (1H, s, 18-Me), 1.787 (1H, s, 16-Me). 13C-NMR b: 206.6 (C-17), 180.0 (C-4 or C-9), 179.5 (C-9 or C-4), 171.8 (C-3 or C-b), 171.7 (C-b or C-3), 167.6 (C-6 or C-7), 167.2 (C-7 or C-6), 151.8 (C-2 or C-li), 149.1 (C-li orC-2), 135.1 (C-i orC-12), 131.2 (C-12 or C-i), 128.3 (C-la or C-12a), 127.7 (C-12a or C-la), 125.3 (C-3b or C-7b), 124.4 (C-7b or C-3b), 117.9 (C-6a or C-7a), 117.4 (C-7a or C-8), 101.7 (C-8 orC-5), 78.6 (C-14), 61.9 (2-OMe or 11-OMe), 61.0
3
(li-OMe or 2-OMe), 64.2 (C-iS), 56.5 (6-OMe or 7-OMe), 56.5 (7-OMe or 6-OMe), 42.5 (C-13), 28.4 (C-18), 24.8 (C-16).
Hypocrellin C (3) Black red crystals, m.p.: softens at 270°C, melts at 279—281°C. Molecular weight: FD-MS m/z 528 (Mt 100%) C30H,409. IN Amax (MeOH) nm (log ): 584sh (3.71), 549 (3.90), 460 (4.20), 335 (3.70), 244sh (4.43), 222 (4.52), 212 (4.52). 'max in KBr: 1680 (a,/-unsaturated >CO), 1600 (aromatic ring) cm1. CD:
Fig. 1 Perylenequinones isolated from Shiraia bambusicola; 1 = hypocrellin A fP(S), 14S, 15R1; 2 hypocrellin B [M(R), 14S, 15R]; 3 = hypocrellin C [MP]. P(or S), M (or R): axial chirality. Hypocrellin C is a racemate with respect to axial chirality.
5.
Fig. 2. 1H-NMR h: 16.028 (1H, s, -OHO-), 16.006 (1H, S. 6.432 (1H, s, 8-CH=), 6.418 (1H, s, 5-CH=), 4.148 (3H, s, -OMe), 4.087 (3H, s, OMe), 4.048 (3H x 2, s, -OMe x 2), 4.038 (1H, d, J = 11.6Hz, 13-CHA), 3.224 (1H, d, J = 11.6Hz, 13-CHB), 2.373 (1H, s, 18-Me), 1.841 (1H, s, 16-Me). 13C-NMR: h: 200.2 (C-17), 185.9 (C-4 or C-9), 185.8 (C-9 or C-4), 168.4 (C-3 or C-b), 168.0 (C-b or C-3), 164.9 (C-6 or C-7), 163.4 (C-7 or C-6), 149.5 (C-2 or C-il),
146.8 (C-il or C-2), 134.3* (C-i or C-12), 134.1* (C-12 or C-i), 124.2 (C-la or C-12a), 124.1 (C-i2a or C-ia), 124.0 (C-3b or C-7b), 123.2 (C-7b or C-3b), 122.0 (C-6a or C-7a), 121.2 (C-7a or C-6a), 108.7 (C-3aorC-9a), 107.4 (C-9a or C-3a), 103.3 (C-5 or C-8), 103.2 (C-8 or C-5), 144.8 (C-14), 61.3 (2-OMe or 11-OMe), 61.2 (11-OMe or 2-OMe), 134.5* (C-15), 56.6 (6-OMe or 7-OMe), 56.5 (7-OMe or 6-OMe). 34.8 (C-13), 29.5 (C-i8). 20.8 (C-16) (* interchangeable).
—5
X/nm
Results and Discussion A methanol extract from air-dried stromata of S. bambusicola was partitioned between chloroform and water. Three perylenequinones 1—3 were isolated from the chloroform fraction. Comparisons of the spectroscopic data from the field desorption mass (FD-MS), 'H-, and 13C-NMR spectrometry indicated that the molecular weight of the compounds 1, 2, and 3 agreed with the formulae C,0H260,0, C,0H,60,0, and C30H,409, respectively. The structural elucidation of compounds 1—3 is based on the hH and '3C-NMR studies as well as chemical evidence. Comparison of the 'Hand 13C-NMR spectra of compounds 1—3 suggested a common carbon skeleton (3) (Fig. 1). Compound I The 'H- and '3C-NMR spectra of 1 were almost coincident with those of hypocrellin with some excep-
tions; therefore, both compounds should have a similar chemical structure. The NMR chemical shifts of hypocrellin
were presented with some confusions in the literature (3). Arnone et al. (4) have previously pointed out these prob-
Fig. 2 CD spectra of perylenequinones isolated from Shiraia bambusicola in methanol.
lems. The reasons for the appearance of two extra signals in
the region of h = 182—171 ppm and the absence of two sig-
nals around ô =
118 ppm [should be assigned to 6a and 7a-C, see reference (5)1 in the literature (3) are not clear. However, the remaining 13C-NMR chemical shifts for hypocrellin are quite close to those of our compound 1. The relative configuration of hypocrellin was already confirmed
by X-ray analysis (3). However the absolute configuration of
the compound was not elucidated (6). The circular di-
chroism (CD) spectrum of compound I (Fig. 2) resembled that of isocercosporin. As the absorption is mainly due to
the inherently dissymmetrical chromophore of the
perylenequinone ring (4, 6), it follows that 1 has the same axial chirality as that of isocercosporin [P(S)1 (8). Together with the established relative stereochemistry of hypocrellin, the axial chirality [P(S)] of I showed that the respective absolute configurations at C-14 and C-15 in this molecule
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C-6a). 107.0 (C-3a or C-9a), 106.6 (C-9a or C-3a), 102.1 (C-S or
378 Planta Mcd. 57(1997)
Compound 2 The 1H- and 13C-NMR spectra of 2 were very
similar to those of 1; therefore, the relative configurations at the alicyclic carbons seem to be identical with those of 1. The CD curves of 2, however, were opposite to those of 1, and were of the cercosporin type (Fig. 2). The CD absorption
of a 1 : 1 mixture of compounds I and 2 was counterbalanced and there being none of wavelengths longer than 300 nm. The axial chirality therefore was considered to be M(R). Compounds I and 2 are easily separated by TLC, an indication that they are diastereoisomers. These facts require that the absolute configuration of the asymmetric carbons of the alicyclic ring of 2 be the same as those of 1; i.e. 145 and 15R. Compound 3 On the basis of the bathochromic shift in the absorption maxima of the liv spectrum of 3, its shifted JR
absorption maximum at 1680 cm1 (a,-unsaturated ketone) differs from the maxima of compounds I and 2 in
that it has an absorption band at 1700 cm1 (isolated ketone); moreover, the absence of signals assignable to 14-OH and 15-methine protons in the 1H-NMR spectrum suggests that the third compound is a dehydration product of compounds 1 and 2 at C-14 and C-iS. The appearance of two sp2 carbons at about (5 = 145 and 135 ppm, and the disappearance of two sp3 carbons at (5 = 79 (compounds 1 and 2) and 64 (compound 2) or 62 (compound 1) ppm in the 13CNMR spectrum also confirm this conclusion.
There was no CD absorption which indicates that 3 is a mixture of dehydrated products from I and
2. Thus, I and 2 were dehydrated by heating them separately with conc. H2SO4Itoluene at 50°C for 60 mm. A spot
corresponding to 3 was clearly seen in the mixture from 1 but was poorly detected from 2 on TLC. When the reaction temperature was raised to 105 °C, 30mm of heating the re-
spective reaction mixtures of 1 and 2 gave spots corresponding to 3. The UV-absorption spectra of the dehydration products from 1 and 2 clearly coincided with the spectrum of 3. In the FD-MS spectrum, each dehydration product afforded the molecular ion at m/z = 528 (100%). The dehydration product showed no CD absorption, presumably because of isomerization of each axially chiral
OH and C-i 5-H in compound I was favored. In addition, the C- 15-H in 2 was detected at a lower magnetic field than that of 1, presumably because of the coplanarity of C-iS-H and
the aromatic ring D. The conformation of the alicyclic moiety in hypocrellin-type compounds depends on the twisting of the
perylenequinone ring. The characteristic behaviors of 1 and 2 on acid-catalyzed dehydration (rate [11> [21) and the 1H-NMR deshielding of C-15-H in 2 are very compatible with the less hindered stereostructures; [P(S), 14S, 15R] for compound 1 and [M(R), 145, 15R] for compound 2.
The absolute stereochemistries of compounds 1 and 2 found from CD spectroscopic (axial chiral-
ity) and X-ray crystallographic (relative configuration) analyses are thus well supported by the chemical and 1HNMR spectroscopic properties.
Cercosporin is reported to isomerize by reflux in toluene for 25 mm (7), giving an equilibrium mixture with diastereoisomeric isocercosporin that has the opposite axial chirality; whereas, elsinochrome A is very thermostable (9). In the latter, axial chirality is stabilized by the alicyclic side structure (4, 8, 9). Compounds 1 and 2 were heated
at 120°C for 80mm in toluene, but only the parent compound was detected on TLC, and the CD curve was unchanged in each case. These results provide circumstantial evidence that these compounds are stabilized by the additional aliphatic ring in their molecules.
We isolated two hypocrellin analogues in addition to hypocrellmn and propose that compound 1 (hypocrellin) be called hypocrellin A, compound 2 hypocrel-
lin B, and compound 3 hypocrellin C. The first compound has been isolated from Hypocrella bambusae (B. et Br.) Sacc., a member of the Clavicipitaceae. Unfortunately, S. bambusicola is not a clavicipitaceous fungus but belongs to the Loculoascomycetes (i). In terms of fungal taxonomy "hypocrellin" could be considered to be an inappropriate name for the latter two compounds. However, we choose to use "hypocrellin" for all 3 compounds and distinguish them by alphabetical letters avoid confusion.
It is very interesting that the two different fungi have the same perylenequinone. Both are parasitic on
bamboo twigs and form relatively large stromata (2). Biological activities of these compounds and their roles in the life-history of the fungus will be reported elsewhere.
product into an almost equimolar mixture of the two axial stereomers.
Acknowledgements We thank Mr. Kenji Watanabe, for FD-MS and
The IR spectra of compounds 1 and 2 in dilute CC14 solution showed absorption maxima attributable to the H-bonded OH group at 3495 cm1 (compound 1) and 3543 cm (compound 2), which is probably due to the cisconfiguration of C-14-OH and C-15-C(°=O)Me, as in hypocrellin (3).
Other chemical and spectroscopic properties of these two compounds were examined. Dehydration of I took place more easily than that of 2; therefore, an antiperiplanar (or pseudo-trans-diaxial) orientation of C-14-
1H-NMR analyses. We are grateful to Dr. Teruhiko Yoshihara, Dept. of Agric. Chem., Hokkaido University for his invaluable suggestions
during this study.
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areS andR. The 1H- and 13C-NMR spectroscopic and chemical properties discussed hereafter correlated well with this configuration.
TadashiKishi et al.
Planta Med. 57(1991) 379
New Perylenequinones from Shiraia bambusicola
References
2
Tsuda, M.. Ueyama, A. (1987) in: Pleomorphic fungi: the diversity
and its taxonomic implications, (Sugiyama, J., ed.), pp. 181—199, Kodansha/Elsevier, Tokyo/Amsterdam. Liu, B. (1978) Chinese medical fungi, 2nd ed., Shanshi People's Press, Shanshi, (in Chinese). Chen, W.-S., Chen, Y.-T., Wan, X.-Y., Friedrichs, B., Puff, H., Breitmaier, E. (1981) Liebigs. Ann. Chem. 1880—1885.
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organic natural products. 52, 1—71. Yamazaki, S., Ogawa, T. (1972) Agric. Biol. Chem. 36, 1707—1727. Nasini, G., Merlini, L., Andreetti, G. D., Bocelli, G., Sgarabotto, P. (1982) Tetrahedron 38. 2787—2796.
Lousberg, R. J. J. Ch., Salemink, C. A., Weiss, U., Batterham, 1. J. (1969) J. Chem. Soc. (C) 1219—1227.
Arnone, A., Camarda, L., Nasini, G., Merlini, L. (1985) J. Chem. Soc., Perkin Trans. 1,1387—1392.
Downloaded by: National University of Singapore. Copyrighted material.
1
Kurobane, I., Vining, L. C., Mclnnes, A. G., Smith, D. G., Walter, J. A. (1981) Can. J. Chem. 59, 422—430. Weiss, U., Merlini, L., Nasini, G. (1987) Progress in the chemistry of
New Perylenequinones from Shiraia bambusicola TadashiKishi"5, Satoshi Tahara2, Naoki Taniguchi3. Mitsuya Tsuda4, Chihiro Tanaka4. and Shozo Takahashi4 Kyoto Municipal Junior College of Nursing, Higashi-Takada-cho 1—2, Mibu, Nakagyo-ku, Kyoto 604, Japan 2 Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060, Japan Kyoto City Institute of Public Health, Higashi-Takada-cho 1—2, Mibu, Nakagyo-ku, Kyoto 604, Japan Pesticide Research Institute, Kyoto University, Sakyo-ku, Kyoto 606, Japan Address for correspondence
Isolation and purification
Abstract Two new perylenequinones, named hypocrellin B and C, together with the known hypocrellin A were isolated from the stromatal tissues of Shiraia barnbusicola. The structures of these compounds, including the absolute stereochemistry, were determined.
evaporated to dryness in vacuo (31.2 g), after which the extract was partitioned between chloroform and water. The chloroform-soluble fraction (10.4 g) ws chromatographed on silica gel (250 g) with 3.5% oxalic acid column (0 4.5 cm x 60cm). Chloroform-EtOH (99: 1) was used as the elution solvent. The resulting red pigment fraction (613.3 mg) was purified by multiple development by pre-
Key words
parative TLC on silica gel with 3.5% oxalic acid and using the chloroform-EtOH (99: 1). Crystallization from acetone yielded
Shiraia bambusicola, perylenequinones,
30.8mg of 1, 31.1mg of2, and 30.4mg of3, respectively, from the middle, lower, and upper bands.
hypocrellins A, B, C, structure elucidation, parasitic fungus.
Introduction
Shirala bambusicola P. Hennigs, a conspicuous fungus parasitic on Phyllostachys spp., is re-
corded only from Japan and China (1). It has been used as medicinal fungi under the name of "zhu huang" in China (2). The active principle(s) of this fungus, however, has not been analyzed.
During our study of the life-history of this fungus, we have observed that its stromatal tissues have an-
tibacterial activity against Bacillus subtilis (Ehrenberg) Cohn. We here report the isolation and structural forms of the active perylenequinones from the stromatal tissues of S. bambusicola.
Materials and Methods Melting points were determined by the micro hot-plate method and are uncorrected. UV spectra were recorded
with a Hitachi U-3210 spectrophotometer. IR spectra were recorded with a Hitachi 285 spectrometer as KBr discs or in CC14. FD-
MS spectra were measured with a JEOL JMS-O1SG-2 mass spectrometer. 1H-, 13C-NMR and COLOC spectra were recorded with a Bruker AM 500 (500 MHz, FT) in CDCI3. CD spectra were recorded
with a JASCO J-20 automatic recording spectropolarimeter in MeOH at 23°C.
Ninety grams of air-dried stomata of S. barnbusicola were treated with about 41 of MeOH. The extract was
Reactions A 3.12mg sample of I and a 3.42mg sample of2 were each dissolved in 10.0 ml of toluene. A 0.5 ml portion of each solution was heated at 120°C for 80 mm in a sealed ampule. The resulting solutions were concentrated and then analyzed by TLC and CD spectroscopy. After addition of 2 &l of conc. H2S04 to 9.5 ml each
of solutions I and 2, 0.5 ml samples of each solution were heated at
50°C for 60mm and at 105°C for 30mm, respectively. Reaction mixtures were treated with 20 ml of ethyl acetate, then washed with 5% NaHCO3 and saturated NaC1. The extract was concentrated to dryness in vacuo. The FD-MS, CD, and UV spectra of each
product were measured.
Hypocrellin A (1) Blood-red crystals, m.p.: softens at 214°C, melts at 245—250°C. Molecular weight: FD—MS rn/z 546 (Mt, 100%) C30H26010. UV Amax (MeOH) nm (log ): 581 (4.10), 540 (4.08), 464 (4.38), 340 (3.71), 280sh (4.44), 266 (4.50), 214 (4.66). IR max in CCJ4 (0.22mM): 3495 (-OH'O) cm1. JR max in KBr: 3470 (-OH),
1700 (>CO), 1600 (aromatic ring) cm. CD: Fig.2. 1H-NMR b: 15.955 (1H, s, -OHO-), 15.912 (1H, s, -OHO-), 6.566 (111, s, 8-CH=), 6.550 (1H, s, 5-CH=), 4.112 (3H x 2, s, -OMe x 2), 4.074 (311 x 2, s, -OMe x 2), 3.514 (1H, d, JAB = 12.0Hz, 13-CHA), 2.632 (1H, d, J 12.0 Hz, 13-CHB), 3.448 (1H, s, 15-CH), 1.892 (1H, s, 18-Me), 1.705 (1H, s, 16-Me), 1.633 (1H, hr. s, -OH). 13C-NMR b: 207.4 (C-17), 180.3 (C-4 or C-9), 179.9 (C-9 or C-4), 171.8 (C-3 or C-b), 170.9 (C-b or C-3), 167.5 (C-6 or C-7), 167.5 (C-7 or C-6), 150.9 (C-2 or C-il), 150.6 (C-il or C-2), 134.0 (C-12), 133.2 (C-i), 128.5 (C-12a), 127.6 (C-la), 125.0 (C-3b or C-7b), 125.0 (C-7b or C-3b), 118.2 (C-6a or C-7a), 117.7 (C-7a or C-6a), 106.9 (C-3a or C-9a), 106.7 (C-9a or C-3a), 102.1 (C-5 or C-8), 102.0 (C-8 or C-5), 78.8 (C-l4), 62.1 (2-OMe or 11-OMe), 60.8 (11-OMe or 2-OMe), 61.7 (C-15), 56.6 (6-OMe or 7-OMe), 56.5 (7-OMe or 6-OMe), 41.9 (C-13), 30.1 (C-18), 27.0 (C-16).
Downloaded by: National University of Singapore. Copyrighted material.
Received: April23, 1990
Planta Med. 57(1991) 377
New Perylenequinones from Shirala bambasicola
O OH
Hypocrellin B (2)
Black red powdery crystals, m.p.: softens at 195°C, melts at 215—217°C. Molecular weight: FD-MS m/z 546 (Mt, 100%) C,0H26010. u .& (MeOH) nm (log ): 581 (4.05), 540
H3
H3CO
(4.03), 467 (4.32), 340 (3.65), 282sh (4.37), 266 (4.44), 261sh (4.44), 220 sh (4.60), 210 (4.61). JR 'max in CC14 (0.23mM): 3543 (OH'O) CIfl1. JR Vms,, in KBr: 3440 (-OH), 1700 (>CO), 1600 (aromatic ring) cm1. CD: Fig. 2. 'H-NMR ô: 16.062 (1H, s, -OHO-), 15.963(1H, s, -OH•O-), 6.566(1H, s, 8-CH=), 6.557(1H, s, 5-CH=),
I
O OH
OC H3
2
1
4.274 (3H, s, -OMe), 4.185 (3H, s, -OMe), 4.076 (3H, s, -OMe), 4.072 (3H, s, -OMe), 3.798 (1H, s, -OH), 3.726 (1H, s, 15-CH), 3.673 (1H, d, J = 13.9 Hz, 13-CHA), 2.347 (1H, d, J = 13.9 Hz, 13-CHB),
1-OH
1.834 (1H, s, 18-Me), 1.787 (1H, s, 16-Me). 13C-NMR b: 206.6 (C-17), 180.0 (C-4 or C-9), 179.5 (C-9 or C-4), 171.8 (C-3 or C-b), 171.7 (C-b or C-3), 167.6 (C-6 or C-7), 167.2 (C-7 or C-6), 151.8 (C-2 or C-li), 149.1 (C-li orC-2), 135.1 (C-i orC-12), 131.2 (C-12 or C-i), 128.3 (C-la or C-12a), 127.7 (C-12a or C-la), 125.3 (C-3b or C-7b), 124.4 (C-7b or C-3b), 117.9 (C-6a or C-7a), 117.4 (C-7a or C-8), 101.7 (C-8 orC-5), 78.6 (C-14), 61.9 (2-OMe or 11-OMe), 61.0
3
(li-OMe or 2-OMe), 64.2 (C-iS), 56.5 (6-OMe or 7-OMe), 56.5 (7-OMe or 6-OMe), 42.5 (C-13), 28.4 (C-18), 24.8 (C-16).
Hypocrellin C (3) Black red crystals, m.p.: softens at 270°C, melts at 279—281°C. Molecular weight: FD-MS m/z 528 (Mt 100%) C30H,409. IN Amax (MeOH) nm (log ): 584sh (3.71), 549 (3.90), 460 (4.20), 335 (3.70), 244sh (4.43), 222 (4.52), 212 (4.52). 'max in KBr: 1680 (a,/-unsaturated >CO), 1600 (aromatic ring) cm1. CD:
Fig. 1 Perylenequinones isolated from Shiraia bambusicola; 1 = hypocrellin A fP(S), 14S, 15R1; 2 hypocrellin B [M(R), 14S, 15R]; 3 = hypocrellin C [MP]. P(or S), M (or R): axial chirality. Hypocrellin C is a racemate with respect to axial chirality.
5.
Fig. 2. 1H-NMR h: 16.028 (1H, s, -OHO-), 16.006 (1H, S. 6.432 (1H, s, 8-CH=), 6.418 (1H, s, 5-CH=), 4.148 (3H, s, -OMe), 4.087 (3H, s, OMe), 4.048 (3H x 2, s, -OMe x 2), 4.038 (1H, d, J = 11.6Hz, 13-CHA), 3.224 (1H, d, J = 11.6Hz, 13-CHB), 2.373 (1H, s, 18-Me), 1.841 (1H, s, 16-Me). 13C-NMR: h: 200.2 (C-17), 185.9 (C-4 or C-9), 185.8 (C-9 or C-4), 168.4 (C-3 or C-b), 168.0 (C-b or C-3), 164.9 (C-6 or C-7), 163.4 (C-7 or C-6), 149.5 (C-2 or C-il),
146.8 (C-il or C-2), 134.3* (C-i or C-12), 134.1* (C-12 or C-i), 124.2 (C-la or C-12a), 124.1 (C-i2a or C-ia), 124.0 (C-3b or C-7b), 123.2 (C-7b or C-3b), 122.0 (C-6a or C-7a), 121.2 (C-7a or C-6a), 108.7 (C-3aorC-9a), 107.4 (C-9a or C-3a), 103.3 (C-5 or C-8), 103.2 (C-8 or C-5), 144.8 (C-14), 61.3 (2-OMe or 11-OMe), 61.2 (11-OMe or 2-OMe), 134.5* (C-15), 56.6 (6-OMe or 7-OMe), 56.5 (7-OMe or 6-OMe). 34.8 (C-13), 29.5 (C-i8). 20.8 (C-16) (* interchangeable).
—5
X/nm
Results and Discussion A methanol extract from air-dried stromata of S. bambusicola was partitioned between chloroform and water. Three perylenequinones 1—3 were isolated from the chloroform fraction. Comparisons of the spectroscopic data from the field desorption mass (FD-MS), 'H-, and 13C-NMR spectrometry indicated that the molecular weight of the compounds 1, 2, and 3 agreed with the formulae C,0H260,0, C,0H,60,0, and C30H,409, respectively. The structural elucidation of compounds 1—3 is based on the hH and '3C-NMR studies as well as chemical evidence. Comparison of the 'Hand 13C-NMR spectra of compounds 1—3 suggested a common carbon skeleton (3) (Fig. 1). Compound I The 'H- and '3C-NMR spectra of 1 were almost coincident with those of hypocrellin with some excep-
tions; therefore, both compounds should have a similar chemical structure. The NMR chemical shifts of hypocrellin
were presented with some confusions in the literature (3). Arnone et al. (4) have previously pointed out these prob-
Fig. 2 CD spectra of perylenequinones isolated from Shiraia bambusicola in methanol.
lems. The reasons for the appearance of two extra signals in
the region of h = 182—171 ppm and the absence of two sig-
nals around ô =
118 ppm [should be assigned to 6a and 7a-C, see reference (5)1 in the literature (3) are not clear. However, the remaining 13C-NMR chemical shifts for hypocrellin are quite close to those of our compound 1. The relative configuration of hypocrellin was already confirmed
by X-ray analysis (3). However the absolute configuration of
the compound was not elucidated (6). The circular di-
chroism (CD) spectrum of compound I (Fig. 2) resembled that of isocercosporin. As the absorption is mainly due to
the inherently dissymmetrical chromophore of the
perylenequinone ring (4, 6), it follows that 1 has the same axial chirality as that of isocercosporin [P(S)1 (8). Together with the established relative stereochemistry of hypocrellin, the axial chirality [P(S)] of I showed that the respective absolute configurations at C-14 and C-15 in this molecule
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C-6a). 107.0 (C-3a or C-9a), 106.6 (C-9a or C-3a), 102.1 (C-S or
378 Planta Mcd. 57(1997)
Compound 2 The 1H- and 13C-NMR spectra of 2 were very
similar to those of 1; therefore, the relative configurations at the alicyclic carbons seem to be identical with those of 1. The CD curves of 2, however, were opposite to those of 1, and were of the cercosporin type (Fig. 2). The CD absorption
of a 1 : 1 mixture of compounds I and 2 was counterbalanced and there being none of wavelengths longer than 300 nm. The axial chirality therefore was considered to be M(R). Compounds I and 2 are easily separated by TLC, an indication that they are diastereoisomers. These facts require that the absolute configuration of the asymmetric carbons of the alicyclic ring of 2 be the same as those of 1; i.e. 145 and 15R. Compound 3 On the basis of the bathochromic shift in the absorption maxima of the liv spectrum of 3, its shifted JR
absorption maximum at 1680 cm1 (a,-unsaturated ketone) differs from the maxima of compounds I and 2 in
that it has an absorption band at 1700 cm1 (isolated ketone); moreover, the absence of signals assignable to 14-OH and 15-methine protons in the 1H-NMR spectrum suggests that the third compound is a dehydration product of compounds 1 and 2 at C-14 and C-iS. The appearance of two sp2 carbons at about (5 = 145 and 135 ppm, and the disappearance of two sp3 carbons at (5 = 79 (compounds 1 and 2) and 64 (compound 2) or 62 (compound 1) ppm in the 13CNMR spectrum also confirm this conclusion.
There was no CD absorption which indicates that 3 is a mixture of dehydrated products from I and
2. Thus, I and 2 were dehydrated by heating them separately with conc. H2SO4Itoluene at 50°C for 60 mm. A spot
corresponding to 3 was clearly seen in the mixture from 1 but was poorly detected from 2 on TLC. When the reaction temperature was raised to 105 °C, 30mm of heating the re-
spective reaction mixtures of 1 and 2 gave spots corresponding to 3. The UV-absorption spectra of the dehydration products from 1 and 2 clearly coincided with the spectrum of 3. In the FD-MS spectrum, each dehydration product afforded the molecular ion at m/z = 528 (100%). The dehydration product showed no CD absorption, presumably because of isomerization of each axially chiral
OH and C-i 5-H in compound I was favored. In addition, the C- 15-H in 2 was detected at a lower magnetic field than that of 1, presumably because of the coplanarity of C-iS-H and
the aromatic ring D. The conformation of the alicyclic moiety in hypocrellin-type compounds depends on the twisting of the
perylenequinone ring. The characteristic behaviors of 1 and 2 on acid-catalyzed dehydration (rate [11> [21) and the 1H-NMR deshielding of C-15-H in 2 are very compatible with the less hindered stereostructures; [P(S), 14S, 15R] for compound 1 and [M(R), 145, 15R] for compound 2.
The absolute stereochemistries of compounds 1 and 2 found from CD spectroscopic (axial chiral-
ity) and X-ray crystallographic (relative configuration) analyses are thus well supported by the chemical and 1HNMR spectroscopic properties.
Cercosporin is reported to isomerize by reflux in toluene for 25 mm (7), giving an equilibrium mixture with diastereoisomeric isocercosporin that has the opposite axial chirality; whereas, elsinochrome A is very thermostable (9). In the latter, axial chirality is stabilized by the alicyclic side structure (4, 8, 9). Compounds 1 and 2 were heated
at 120°C for 80mm in toluene, but only the parent compound was detected on TLC, and the CD curve was unchanged in each case. These results provide circumstantial evidence that these compounds are stabilized by the additional aliphatic ring in their molecules.
We isolated two hypocrellin analogues in addition to hypocrellmn and propose that compound 1 (hypocrellin) be called hypocrellin A, compound 2 hypocrel-
lin B, and compound 3 hypocrellin C. The first compound has been isolated from Hypocrella bambusae (B. et Br.) Sacc., a member of the Clavicipitaceae. Unfortunately, S. bambusicola is not a clavicipitaceous fungus but belongs to the Loculoascomycetes (i). In terms of fungal taxonomy "hypocrellin" could be considered to be an inappropriate name for the latter two compounds. However, we choose to use "hypocrellin" for all 3 compounds and distinguish them by alphabetical letters avoid confusion.
It is very interesting that the two different fungi have the same perylenequinone. Both are parasitic on
bamboo twigs and form relatively large stromata (2). Biological activities of these compounds and their roles in the life-history of the fungus will be reported elsewhere.
product into an almost equimolar mixture of the two axial stereomers.
Acknowledgements We thank Mr. Kenji Watanabe, for FD-MS and
The IR spectra of compounds 1 and 2 in dilute CC14 solution showed absorption maxima attributable to the H-bonded OH group at 3495 cm1 (compound 1) and 3543 cm (compound 2), which is probably due to the cisconfiguration of C-14-OH and C-15-C(°=O)Me, as in hypocrellin (3).
Other chemical and spectroscopic properties of these two compounds were examined. Dehydration of I took place more easily than that of 2; therefore, an antiperiplanar (or pseudo-trans-diaxial) orientation of C-14-
1H-NMR analyses. We are grateful to Dr. Teruhiko Yoshihara, Dept. of Agric. Chem., Hokkaido University for his invaluable suggestions
during this study.
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areS andR. The 1H- and 13C-NMR spectroscopic and chemical properties discussed hereafter correlated well with this configuration.
TadashiKishi et al.
Planta Med. 57(1991) 379
New Perylenequinones from Shiraia bambusicola
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