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Bibenzyl derivatives from the liverwort Ricciocarpos natans
0031~9422(!M)lMO39-U
Pergamon
BIBENZYL
DERIVATIVES
FROM THE LIVERWORT NATANS*
Phyrochemutry. Vol. 36, No. 3, pi 675-677. 1994 Copyrighl 0 1994 Ekvk Science Ltd Printed I” Great Bntain. All righlr memd 0031.-9422/w 57.CO+O.a)
RZCCIOCARPOS
SIEGMUND KUNZ and HANS BEcKERt Fachrichtung 12.3, Pharmakognosie und Analytische Phytochemie Universitlt des Saarlandes, D-66041 Saarbriicken, F.R.G.
(Receioed25 October 1993) Key Word Index-Ricciocarpos lunularin; liquid culture.
natans; Ricciaceae; Hepaticae; bibenzyls; dimeric bisbibenzyls;
pre-
Abstract-A new bibenzyl(2,5,4’-trihydroxybibenzyl), a dimeric bisbibenzyl(6’,6”‘-his-riccardin C) and a phenylethylcyclohexenone (prelunularin) were isolated from the liverwort Ricciocarpos natans grown in axenic culture. All structures were established on the basis of spectral data and chemical evidence.
INTRODUCTION Bryophytes are known as a rich source for bibenzyl derivatives some of which, e.g. bisbibenzyls, have only been detected in liverworts [I]. Previously we reported bibenzyl glycosides from Ricciocarpos natans [2]. This report presents additional bibenzylic compounds (2,5,4’trihydroxybibenzyl, 6’,6”‘-his-riccardin C) and a phenylethylcyclohexenone derivative (prelunularin) isolated from this liverwort species. As R. natans is quite rare in Central Europe, the plant material for this phytochemical study was obtained by means of an axenic culture. RESULTSANDDlSCUSSION
Fractionation of the acetone extract of the freeze-dried plant material on RPl&silica gel followed by separation on DIOL-silica resulted in the isolation of three new phenolic compounds. Compound 1 showed a [M + H]+ at m/z 847 in the positive CI-mass spectrum. The permethylated derivative la exhibited a [M +H]+ at m/z 931 indicating six hydroxyls for 1. As the signals displayed in the ‘H NMR spectrum (Table l), however, integrated to only 20 protons and the spectrum of la revealed only three methoxyls, the compound was concluded lo be a symmetrical dimer. The chemical shifts indicated four benzylic methylenes (62.65 and 2.88, respectively 4H, br s) and four benzene rings (12 aromatic protons in the range from 6 5.39 to 6.98) suggesting a bisbibenzyl structure as monomeric partial structure. Comparison of the ‘H NMR data with those of known bisbibenzyls showed almost identical resonances compared lo riccardin C
*Publication Nr. 74 of ‘Arbeitskreis Chemie und Biologie der Moose’. tAuthor to whom correspondence should be addressed.
[2,3] revealing differences only for H-S and H-6’. As the signal of H-6’ was lacking and the resonance of H-S’ was shifted downfield, the structure 6’,6”‘-his-riccardin C was assigned to the compound. Other dimeric bisbibenzyls have recently been reported by Hashimoto et al. [4] and Asakawa (personal communication). The [M + H] + peak of 2 was observed at m/z 23 1. The ‘H NMR spectrum (Table 2) revealed two benzylic methylenes at 62.71 (4H, s) and seven aromatic protons on two benzene rings. The spin systems observed indicated a ring with a 4’-substitution (4H, AA’BB’) and a ring with a 2,4-, 2,5- or 3,4-disubstitution pattern. NOE experiments were in favour of a 2,5disubstitution of ring A leading to the conclusion that 2 was 2,5,4’-trihydroxybibenzyl. This assignment was confirmed by comparison of the data with those of the aglycone of 5,4’-dihydroxybibenzyl-2-0-/?-D-glucopyranoside [2] obtained by acid hydrolysis of this glycoside. Compound 3 gave a [M + H] + peak at m/z 233 in the positive CI-mass spectrum. The ‘HNMR spectrum (Table 2) displayed the signals of a l&substituted phenolic ring (67.01, 6.69), the singlet signal of a proton at a double-bond (H-2, 65.82), besides resonances of four methylenes in the range from 62.37 to 2.75 and the multiplet of an oxygen-geminal aliphatic proton at 64.17 (H-5). The 13C NMR further indicated a carbonyl function (6201.1) that was conjugated to a double-bonding as revealed by the IR spectrum (v166Ocm-‘). As NOE effects were observed between H-a and H-2 (3. I %), as well as between H-b and H_4’/H-6’ (4.6%) and selective decoupling experiments showed H-5 lo be vicinal to two methylenes (H-4 eq/ax and H-6 eq/ax), the structure l(4’-hydroxyphenylethyl)-5-hydroxy-cyclohex-l-en-3-one was assigned to the compound. Moreover, the hydroxyl at C-5 was concluded to be in an equatorial position as indicated by the constants of proton-couplings (4,7 and 4, 8.5 Hz between H-5 and the vicinal methylene protons,
675
676
S. KUNZ and H.
BECKER
a
2
‘G 3
/
0
5
3
2
Table
I. ‘HNMR
spectral data of compounds (Me,CO-d,)
1 and
la
Table 2. ‘H NMR data of compounds and 3 (MeOH-d,)
H
I
In
H
2j2” 313” 515” 6/6”
6.77-6.85’ 6.77-6.85’ 6.77-6.R5’ 6.77-6.85*
7!7”/8/8” IO/lo” 12jlZ’
2.88 6.93 6.74 6.98 5.39 6.87 2.65 6.25
6.79-6.84” 6.79-6.84b 6.79-6.84” 6.79-6%tb 2.90 br s 7.02 d (2.7) 6.82 dd (8.4, 2.7) 7.00 d (8.4) 5.47 d (2. I) 6.84 d (2.1) 2.69 6.28 dd (7.6, 1.5) 6.88 d (7.6) 6.58 d ( I .5) 3.80 s 3.86 s 3.68 .s
2 3 4-aq. 4-ax. 5 6-aq.
13/13” 3’/‘3”’ 5’i5”’ 7’;7”‘/g’/g”’ 1UjlU” 1 I’/1 1”’ 14’/14” 0Mel”l”’ OMel ;/I 1” OMe13’/13”’ ‘Yomplex
br s d (2.6) dd (8.3, 2.6) d (8.3) d (2.0) d (2.0) dd (7.7, 1.6)
6.82 d (7.7) 6.43 d ( 1.6)
_. multiplet.
deduced from J-resolved-COSY spectrum). The evaluation of the absolute configuration at C-5, however, needs further investigations. Due to structural analogy to prelunularic acid [S], the precursor of lunularic acid, 3 was named prelunularin. Corresponding to prelunuaric acid forming lunularic acid in alkaline aqueous solution, prelunularin is converted into lunularin as shown by chromatographic analysis of an alkaline aqueous solution of 3. It was earlier shown that lunularin is formed by decarboxylation of lunularic acid [6,7]. The existence of
2
3 5.82 .s
6.64 d (8.5) 6.45 dd (8.5, 3.0) -6.55 d (3.0)
6-ax.
2 3’ 5’ 6’
2 (Me&O-d,)
7.01 6.71 6.71 7.01 2.71 2.71 ‘.bAssignments
d d d d s s
(8.6) (8.6) (8.6) (8.6)
2.58 2.37 4. I7 2.65 2.39 7.01 6.69 6.69 7.01 2.53 2.75
dd (16.0, dd (16.0. “I dd (I 8.0, dd (18.0, d (8.5) d (8.5) d (8.5) d (8.5) m m
4.0) 8.5)” 4 0) 7 O)b
may be exchanged,
prelunularin and its conversion to lunularin, however, lead to the suggestion that 3 is the genuine precursor of the bibenzyl. lunularin.
EXPERIMENTAL
General. Optical rotation: MeOH. CD MeOH. UV: MeOH. IR: KBr. ‘HNMR (400MHz) and 13C NMR (100 MHz): Me,CO-d, (1, la, 2) or MeOH-d, (3). CI-MS: isobutane. Awenic culture. The culture was grown in 200 ml conical flasks with 70 ml liquid BS medium according to ref. [SJ for 3 weeks, followed by cultivation for 2 weeks on 70 ml B5 medium with 10% of the original nitrate and
Bibenzyls from Ricciocarposnatans phosphate concn. Both media contained 20 g I- ’ sucrose. The flasks were kept under constant illumination (5000 lux) at 22”. Extraction and isolation. Freeze-dried plant material (250 g) was milled and successively extracted with CH,CI, and Me&O. The Me&O extract was coned in OUCIJO and applied to vacuum-liquid chromatography [9] on Europrep Cl8 (15-25 pm) with a H,O-MeOH gradient to yield 5 frs. HPLC of fr. 1 (LiChrospher DIOL, 5 pm; n-hexane-t-BuMeether, 1:9) afforded 2 (2 mg) and 3 (3 mg). HPLC of fr. 5 (LiChrospher DIOL, 5 pm; nhexane-BuMeether, 1:19) yielded 10 mg 1. 6’6”-Bis-riccardin C (1). IR vii: cm - ‘: 3500-3100, 3030, 2930,2860, 1620, 1510, 1440, 1380, 1225, 860, 815. MS(CI+): m/z 847 [M+H]+. ‘jCNMR: 636.1 (t), 38.1 (t), 38.6 (t), 38.9 (t), 114.1 (d), 116.5 (d), 117.1 (d), 117.5 (d), 121.5(d), 123.1*(d), 125.2(d), 126.8(s), 127.5(s), 130.3*(d), 133.2 (d), 133.5 (d), 141.0 (s), 141.9 (s), 142.5 (s), 144.3 (s), 148.8 (s), 154.2 (s), 154.5 (s), 157.9 (s) (* overlapping signals of 2 carbons). Pet-methyl-6’,6”‘-his-riccardin C (la). IR vi:: cm - ‘: 3030,2930,2860,1610,1580,1510,1470,1420,1270,1225, 1170,1125,1040,855,815.MS(C1+):m/z931 [M+H]+. “CNMR: 636.4 (t), 38.1 (t), 38.7 (r), 38.9 (r), 55.5 (q), 112.3*(d), 115.9 (d), 118.4 (d), 122.4 (d), 122.9*(d), 125.2 (d), 128.7 (s), 130.3* (d), 131.5 (s), 133.2 (d), 133.3 (d), 134.2 (s), 137.0(s), 140.7(s), 142.1 (s), 144.3(s), 153.1 (s), 154.5(s), 157.2 (s), 160.3 (s) (* overlapping signals of 2 carbons). 2,5,4’-Trihydroxybibenzyl (2). UV jzg” nm: 284, 223. IR vi;; cm- ‘: 3500-3100, 3030, 2930, 2860, 1620, 1520, 1500,1455,1380,1210,I145,840,805,725. MS(CI+): m/z 231 [M+H]+. Prelunulurin (3). [cc];’ : + 24”. CD: A&,,,_- 0.1 (320 nm). UV i::FH nm: 272,225. IR vi:: cn- ‘: 3400, 3010, 2930, 2870,1745,1660,1600,1510,1450,1390,1250,1175,1060, 890, 830. MS(C1’): m/z 233 [M+H]‘. 13CNMR: 633.2 (t. Cx), 39.5 (t, CD), 41.1 (t, C-6)*, 46.7 (t, C-4)*, 67.4 (d, C5). 116.3 (d, C-3’/C-5’), 126.6 (d, C-2), 130.3 (d, C-2’/C-6’), 156.8 (s, C-4’). 165.6 (s, C-l), 201.1 (s, C-3). (* assignment may be exchanged). Methylation of compound 1. Compound 1 (5 mg) was dissolved in 5 ml Me&O and I ml MeI, and 2 g K&O,
611
were added and the soln refluxed for 2 hr. After removal of K&O, and evapn to dryness, the product was purified by VLC on silica using a n-hexane-EtOAc gradient to yield 3.5 mg la. Chromutographic analysis. TLC: HPTLC-RP18 F,,,, plates (Merck) MeOH-H,O (13:7), detection: UV,,, or spraying with anisaldehyde-H,SO, reagent [lo] or Echtblausalz B-soln [lo], R,(lunularin) = 0.35, R,(3) = 0.65. Acknowledgements-The
authors wish to thank Drs U. Langenbahn and J. Zapp (Universitlt des Saarlandes, Fachrichtung Pharmakognosie, Saarbriicken) for measuring NMR spectra and M. C. Schweigert (Universite Louis Pasteur, Department de Chimie, Strasbourg) for accurate mass measurements.
REFERENCES
Mues, R. and Zinsmeister, H. D. (1988) J. Hattori Bot. Lab. 64, 109.
Kunz, S. and Becker, H. (1992) Phytochemistry 31, 3981.
Asakawa, Y. and Matsuda, R. (1982) Phytochemistry 21, 2143.
Hashimoto, T., Yoshida, T., Tori, M., Takaoka, S. and Asakawa, Y. (1993) Abstract 4047, XV International Botanical Congress. Yokahama, Japan. Ohta, Y., Abe, S., Komura, H. and Kobayashi, M. (1984) J. Hattori Bot. Lab. 56, 249. Pryce, R. J. and Linton, L. (1974) Phytochemistry 13, 2497.
Gorham, J. (1977) Phytochemistry 16, 915. Gamborg, 0. L. (1982) in Plant Tissue Culture (Wetter, L. R. and Constabel, F., eds), p. 1. National Research Council of Canada, Saskatoon. 9. Pelletier, S. W., Chokshi, H. P. and Desai, H. K. (1986) J. Nat. Prod. 49, 892. 10. Stahl, E. and Schild, W. (198 1) Pharmuzeutische Biologic, Drogenonalyse II: Inhaltsstofle und Isolierungen. Gustav Fischer, Stuttgart.
Pergamon
BIBENZYL
DERIVATIVES
FROM THE LIVERWORT NATANS*
Phyrochemutry. Vol. 36, No. 3, pi 675-677. 1994 Copyrighl 0 1994 Ekvk Science Ltd Printed I” Great Bntain. All righlr memd 0031.-9422/w 57.CO+O.a)
RZCCIOCARPOS
SIEGMUND KUNZ and HANS BEcKERt Fachrichtung 12.3, Pharmakognosie und Analytische Phytochemie Universitlt des Saarlandes, D-66041 Saarbriicken, F.R.G.
(Receioed25 October 1993) Key Word Index-Ricciocarpos lunularin; liquid culture.
natans; Ricciaceae; Hepaticae; bibenzyls; dimeric bisbibenzyls;
pre-
Abstract-A new bibenzyl(2,5,4’-trihydroxybibenzyl), a dimeric bisbibenzyl(6’,6”‘-his-riccardin C) and a phenylethylcyclohexenone (prelunularin) were isolated from the liverwort Ricciocarpos natans grown in axenic culture. All structures were established on the basis of spectral data and chemical evidence.
INTRODUCTION Bryophytes are known as a rich source for bibenzyl derivatives some of which, e.g. bisbibenzyls, have only been detected in liverworts [I]. Previously we reported bibenzyl glycosides from Ricciocarpos natans [2]. This report presents additional bibenzylic compounds (2,5,4’trihydroxybibenzyl, 6’,6”‘-his-riccardin C) and a phenylethylcyclohexenone derivative (prelunularin) isolated from this liverwort species. As R. natans is quite rare in Central Europe, the plant material for this phytochemical study was obtained by means of an axenic culture. RESULTSANDDlSCUSSION
Fractionation of the acetone extract of the freeze-dried plant material on RPl&silica gel followed by separation on DIOL-silica resulted in the isolation of three new phenolic compounds. Compound 1 showed a [M + H]+ at m/z 847 in the positive CI-mass spectrum. The permethylated derivative la exhibited a [M +H]+ at m/z 931 indicating six hydroxyls for 1. As the signals displayed in the ‘H NMR spectrum (Table l), however, integrated to only 20 protons and the spectrum of la revealed only three methoxyls, the compound was concluded lo be a symmetrical dimer. The chemical shifts indicated four benzylic methylenes (62.65 and 2.88, respectively 4H, br s) and four benzene rings (12 aromatic protons in the range from 6 5.39 to 6.98) suggesting a bisbibenzyl structure as monomeric partial structure. Comparison of the ‘H NMR data with those of known bisbibenzyls showed almost identical resonances compared lo riccardin C
*Publication Nr. 74 of ‘Arbeitskreis Chemie und Biologie der Moose’. tAuthor to whom correspondence should be addressed.
[2,3] revealing differences only for H-S and H-6’. As the signal of H-6’ was lacking and the resonance of H-S’ was shifted downfield, the structure 6’,6”‘-his-riccardin C was assigned to the compound. Other dimeric bisbibenzyls have recently been reported by Hashimoto et al. [4] and Asakawa (personal communication). The [M + H] + peak of 2 was observed at m/z 23 1. The ‘H NMR spectrum (Table 2) revealed two benzylic methylenes at 62.71 (4H, s) and seven aromatic protons on two benzene rings. The spin systems observed indicated a ring with a 4’-substitution (4H, AA’BB’) and a ring with a 2,4-, 2,5- or 3,4-disubstitution pattern. NOE experiments were in favour of a 2,5disubstitution of ring A leading to the conclusion that 2 was 2,5,4’-trihydroxybibenzyl. This assignment was confirmed by comparison of the data with those of the aglycone of 5,4’-dihydroxybibenzyl-2-0-/?-D-glucopyranoside [2] obtained by acid hydrolysis of this glycoside. Compound 3 gave a [M + H] + peak at m/z 233 in the positive CI-mass spectrum. The ‘HNMR spectrum (Table 2) displayed the signals of a l&substituted phenolic ring (67.01, 6.69), the singlet signal of a proton at a double-bond (H-2, 65.82), besides resonances of four methylenes in the range from 62.37 to 2.75 and the multiplet of an oxygen-geminal aliphatic proton at 64.17 (H-5). The 13C NMR further indicated a carbonyl function (6201.1) that was conjugated to a double-bonding as revealed by the IR spectrum (v166Ocm-‘). As NOE effects were observed between H-a and H-2 (3. I %), as well as between H-b and H_4’/H-6’ (4.6%) and selective decoupling experiments showed H-5 lo be vicinal to two methylenes (H-4 eq/ax and H-6 eq/ax), the structure l(4’-hydroxyphenylethyl)-5-hydroxy-cyclohex-l-en-3-one was assigned to the compound. Moreover, the hydroxyl at C-5 was concluded to be in an equatorial position as indicated by the constants of proton-couplings (4,7 and 4, 8.5 Hz between H-5 and the vicinal methylene protons,
675
676
S. KUNZ and H.
BECKER
a
2
‘G 3
/
0
5
3
2
Table
I. ‘HNMR
spectral data of compounds (Me,CO-d,)
1 and
la
Table 2. ‘H NMR data of compounds and 3 (MeOH-d,)
H
I
In
H
2j2” 313” 515” 6/6”
6.77-6.85’ 6.77-6.85’ 6.77-6.R5’ 6.77-6.85*
7!7”/8/8” IO/lo” 12jlZ’
2.88 6.93 6.74 6.98 5.39 6.87 2.65 6.25
6.79-6.84” 6.79-6.84b 6.79-6.84” 6.79-6%tb 2.90 br s 7.02 d (2.7) 6.82 dd (8.4, 2.7) 7.00 d (8.4) 5.47 d (2. I) 6.84 d (2.1) 2.69 6.28 dd (7.6, 1.5) 6.88 d (7.6) 6.58 d ( I .5) 3.80 s 3.86 s 3.68 .s
2 3 4-aq. 4-ax. 5 6-aq.
13/13” 3’/‘3”’ 5’i5”’ 7’;7”‘/g’/g”’ 1UjlU” 1 I’/1 1”’ 14’/14” 0Mel”l”’ OMel ;/I 1” OMe13’/13”’ ‘Yomplex
br s d (2.6) dd (8.3, 2.6) d (8.3) d (2.0) d (2.0) dd (7.7, 1.6)
6.82 d (7.7) 6.43 d ( 1.6)
_. multiplet.
deduced from J-resolved-COSY spectrum). The evaluation of the absolute configuration at C-5, however, needs further investigations. Due to structural analogy to prelunularic acid [S], the precursor of lunularic acid, 3 was named prelunularin. Corresponding to prelunuaric acid forming lunularic acid in alkaline aqueous solution, prelunularin is converted into lunularin as shown by chromatographic analysis of an alkaline aqueous solution of 3. It was earlier shown that lunularin is formed by decarboxylation of lunularic acid [6,7]. The existence of
2
3 5.82 .s
6.64 d (8.5) 6.45 dd (8.5, 3.0) -6.55 d (3.0)
6-ax.
2 3’ 5’ 6’
2 (Me&O-d,)
7.01 6.71 6.71 7.01 2.71 2.71 ‘.bAssignments
d d d d s s
(8.6) (8.6) (8.6) (8.6)
2.58 2.37 4. I7 2.65 2.39 7.01 6.69 6.69 7.01 2.53 2.75
dd (16.0, dd (16.0. “I dd (I 8.0, dd (18.0, d (8.5) d (8.5) d (8.5) d (8.5) m m
4.0) 8.5)” 4 0) 7 O)b
may be exchanged,
prelunularin and its conversion to lunularin, however, lead to the suggestion that 3 is the genuine precursor of the bibenzyl. lunularin.
EXPERIMENTAL
General. Optical rotation: MeOH. CD MeOH. UV: MeOH. IR: KBr. ‘HNMR (400MHz) and 13C NMR (100 MHz): Me,CO-d, (1, la, 2) or MeOH-d, (3). CI-MS: isobutane. Awenic culture. The culture was grown in 200 ml conical flasks with 70 ml liquid BS medium according to ref. [SJ for 3 weeks, followed by cultivation for 2 weeks on 70 ml B5 medium with 10% of the original nitrate and
Bibenzyls from Ricciocarposnatans phosphate concn. Both media contained 20 g I- ’ sucrose. The flasks were kept under constant illumination (5000 lux) at 22”. Extraction and isolation. Freeze-dried plant material (250 g) was milled and successively extracted with CH,CI, and Me&O. The Me&O extract was coned in OUCIJO and applied to vacuum-liquid chromatography [9] on Europrep Cl8 (15-25 pm) with a H,O-MeOH gradient to yield 5 frs. HPLC of fr. 1 (LiChrospher DIOL, 5 pm; n-hexane-t-BuMeether, 1:9) afforded 2 (2 mg) and 3 (3 mg). HPLC of fr. 5 (LiChrospher DIOL, 5 pm; nhexane-BuMeether, 1:19) yielded 10 mg 1. 6’6”-Bis-riccardin C (1). IR vii: cm - ‘: 3500-3100, 3030, 2930,2860, 1620, 1510, 1440, 1380, 1225, 860, 815. MS(CI+): m/z 847 [M+H]+. ‘jCNMR: 636.1 (t), 38.1 (t), 38.6 (t), 38.9 (t), 114.1 (d), 116.5 (d), 117.1 (d), 117.5 (d), 121.5(d), 123.1*(d), 125.2(d), 126.8(s), 127.5(s), 130.3*(d), 133.2 (d), 133.5 (d), 141.0 (s), 141.9 (s), 142.5 (s), 144.3 (s), 148.8 (s), 154.2 (s), 154.5 (s), 157.9 (s) (* overlapping signals of 2 carbons). Pet-methyl-6’,6”‘-his-riccardin C (la). IR vi:: cm - ‘: 3030,2930,2860,1610,1580,1510,1470,1420,1270,1225, 1170,1125,1040,855,815.MS(C1+):m/z931 [M+H]+. “CNMR: 636.4 (t), 38.1 (t), 38.7 (r), 38.9 (r), 55.5 (q), 112.3*(d), 115.9 (d), 118.4 (d), 122.4 (d), 122.9*(d), 125.2 (d), 128.7 (s), 130.3* (d), 131.5 (s), 133.2 (d), 133.3 (d), 134.2 (s), 137.0(s), 140.7(s), 142.1 (s), 144.3(s), 153.1 (s), 154.5(s), 157.2 (s), 160.3 (s) (* overlapping signals of 2 carbons). 2,5,4’-Trihydroxybibenzyl (2). UV jzg” nm: 284, 223. IR vi;; cm- ‘: 3500-3100, 3030, 2930, 2860, 1620, 1520, 1500,1455,1380,1210,I145,840,805,725. MS(CI+): m/z 231 [M+H]+. Prelunulurin (3). [cc];’ : + 24”. CD: A&,,,_- 0.1 (320 nm). UV i::FH nm: 272,225. IR vi:: cn- ‘: 3400, 3010, 2930, 2870,1745,1660,1600,1510,1450,1390,1250,1175,1060, 890, 830. MS(C1’): m/z 233 [M+H]‘. 13CNMR: 633.2 (t. Cx), 39.5 (t, CD), 41.1 (t, C-6)*, 46.7 (t, C-4)*, 67.4 (d, C5). 116.3 (d, C-3’/C-5’), 126.6 (d, C-2), 130.3 (d, C-2’/C-6’), 156.8 (s, C-4’). 165.6 (s, C-l), 201.1 (s, C-3). (* assignment may be exchanged). Methylation of compound 1. Compound 1 (5 mg) was dissolved in 5 ml Me&O and I ml MeI, and 2 g K&O,
611
were added and the soln refluxed for 2 hr. After removal of K&O, and evapn to dryness, the product was purified by VLC on silica using a n-hexane-EtOAc gradient to yield 3.5 mg la. Chromutographic analysis. TLC: HPTLC-RP18 F,,,, plates (Merck) MeOH-H,O (13:7), detection: UV,,, or spraying with anisaldehyde-H,SO, reagent [lo] or Echtblausalz B-soln [lo], R,(lunularin) = 0.35, R,(3) = 0.65. Acknowledgements-The
authors wish to thank Drs U. Langenbahn and J. Zapp (Universitlt des Saarlandes, Fachrichtung Pharmakognosie, Saarbriicken) for measuring NMR spectra and M. C. Schweigert (Universite Louis Pasteur, Department de Chimie, Strasbourg) for accurate mass measurements.
REFERENCES
Mues, R. and Zinsmeister, H. D. (1988) J. Hattori Bot. Lab. 64, 109.
Kunz, S. and Becker, H. (1992) Phytochemistry 31, 3981.
Asakawa, Y. and Matsuda, R. (1982) Phytochemistry 21, 2143.
Hashimoto, T., Yoshida, T., Tori, M., Takaoka, S. and Asakawa, Y. (1993) Abstract 4047, XV International Botanical Congress. Yokahama, Japan. Ohta, Y., Abe, S., Komura, H. and Kobayashi, M. (1984) J. Hattori Bot. Lab. 56, 249. Pryce, R. J. and Linton, L. (1974) Phytochemistry 13, 2497.
Gorham, J. (1977) Phytochemistry 16, 915. Gamborg, 0. L. (1982) in Plant Tissue Culture (Wetter, L. R. and Constabel, F., eds), p. 1. National Research Council of Canada, Saskatoon. 9. Pelletier, S. W., Chokshi, H. P. and Desai, H. K. (1986) J. Nat. Prod. 49, 892. 10. Stahl, E. and Schild, W. (198 1) Pharmuzeutische Biologic, Drogenonalyse II: Inhaltsstofle und Isolierungen. Gustav Fischer, Stuttgart.