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Lignans and other constituents of Zanthoxylum heitzii
Pergamon
PtWaknlmy.
Vol. 37. No. 3. w 867469.1!294 EkViUSCbXLtd Rinted in Great Britain 0031~9422f94 n.m+am
LIGNANS AND OTHER CONSTITUENTS OF ZANTHOXYLUA4 HEZTZZI SILV~RE NGOUELA, ETIENNE TSAMO* and JOSEPH D. CONNOLLY?
Department of Organic Chemistry, University of Yaoundt I, Box 812 Yaoundt, Cameroon; tDepartmcnt The University, Glasgow G12 8QQ, U.K.
of Chemistry,
(Received24 Janwry 1994)
Key Word Index-Zanthoxylum
heitzii; Rutaceae; bark; lignans; alkaloids; triterpenes.
Abstract-Two new natural lignans, meso-2,3-bis(3,4,5-trimethoxybenzyl)-1,4-butanediol and 4-acetoxy-2,3-bis(3,4,5trimethoxybenzyl)-1-butanol, four known lignans, two alkaloids and triterpenes were isolated from the bark of Zanthoxylum heitzii and identified on the basis of their spectral data.
IBTRODUCTION It
is current practice to consider Zanthoxylwn and Fag-
ara as one genus Zanthoxyltun L. [ 11. Zanthoxylum heitzii is a medicinal plant widely used in central Africa for the
treatment of many diseases [2, 33. Early work on the heartwood of Z. heitzii (as Fagara heitzii) [4] led to the isolation of the alkaloid, flindersine. However, other species of Zanthoxylum contain a wide array of secondary metabolites including terpenes, alkaloids and lignans [S-8]. In a continuation of our investigation of Cameroonian medicinal plants, particularly of this genus [9]. we report the isolation and characterization from Z. heitzii of two novel lignans, meso-2,3-bis(3,4,Strimethoxybenzyl)1,4-butanediol (1) and 4-acetoxy-2,3-bis(3,4,5-trimethoxybenzyl)-I-butanol (2), the known lignans, sesamin, savinin, artigenin methyl ether and 2-(3’,4’-dimethoxybenzyl)-3-(3”,4”,5”-trimethoxybenzyl)-~-butyrolactone, as well as the alkaloids, fagaramide and nitidine, the triterpenoid, lupeol, and a mixture of campesterol, sitosterol and stigmasterol.
bMe
R’
R*
R3
1
H
H
OMc
2
H
AC
OMe
3
H
H
H
t
RESULTS AND DISCUSSION
The ethyl acetate extract from the bark of Z. heitzii was repeatedly chromatographed on silica. Compound 1 was obtained as crystals, mp 142-146”. It had a [M]’ at m/z
Me0
450 and a molecular formula Cz4HJ40s which corresponds to eight double bond equivalents. Its mass fragmentation pattern showed a peak at m/z 432 [M - H,O]+ and peak fragments containing an aromatic ring (m/z 181 [C6H,(OMe),CH,]+) (m/z 208 [C,H,(OMe),CH,=CH,]+). The IR spectrum exhibited bands at 3300 (hydroxyl group), 1600 and 1500 cm-’ (aromatic nucleus). The 13CNMR spectrum (Table 1) showed the presence of only nine carbon atom signals
*Author to whom correspondence should lx addressed.
4
shared between four sp2 carbon atoms, three sp3 carbon atoms with one bearing an oxygen atom (660.4) and two methoxy groups at 660.8 and 56.0. The fact that only nine signals were observed in the 13C NMR spectrum of 1, in spite of its high molecular formula, was very significative and suggested that it was a symmetrical aromatic molecule. The aliphatic region of the ‘%NMR spectrum
867
S. NGOUELA et al.
868
each) corresponding to four benzylic protons. The four methylene protons of the two CH,OH groups were observed as two large doublets at 63.55 and 3.59. The two singlet signals at 63.79 and 3.81 integrating each for nine protons were assigned to six aromatic methoxy groups. The four proton singlet at 66.34 indicated that the two aromatic rings probably had a 2,4,6- or 3,4,5_trimethoxy substitution pattern. The mass spectral fragmentation of 1 confirmed the presence of a trimethoxybenzyl group (m/z 181, base peak). The location of the methoxyl groups on the 3,4 and 5 positions was based on the comparison of the carbon shifts of the aromatic region of 1 with those of the model compound, 1.2,3-trimethoxybenzene (4), and standard chemical shift theory [l 1, 123. Thus, compound 1 was characterized as 2,3-bis (3,4,5-trimethoxybenzyl)1,4-butanediol. Since 1 showed no optical rotation it is assumed to be the meso derivative. The (+) form has been described as the transformation product of the naturally lignan glycoside, hazaleanin A [13]. Compound 2 was also isolated as crystals, mp 121-122” with a [M]’ at m/z 492, molecular formula C26HJ,609. Its IR spectrum was very similar to that of 1 with additional bands for acetate (I 730 and 1250 cm - I). The 13C and ‘HNMR spectra of 2 confirmed the presence of one acetate group with the signals at 6170.9. 21.9 (-AC) and 2.06 (3H). Hydrolysis of 2 yielded a compound identical to 1 in all respects (IR, mp, NMR). Consequently, 2 could be deduced to bc 4-acetoxy-2,3bis(3,4,5-trimethoxybenzyl)-1-butanol. Lupeol, sesamin, savinin, artigenin methyl ether, fagaramide [9] and 2-(3’,4’-dimethoxybenzyl~3-(3”,4”,5”-trimethoxybenzyl)-y-butyrolactone [ 141, were also isolated and identified through comparison of their spectral data with published data. The quatemary alkaloid, nitidine [IS], was isolated from the methanol extract of the ethyl acetate-defatted bark.
Table 1. “CNMR spectral data for compounds l-3 (SO.32 MHz for 1 and 2, 25.2 MHz for 3, CDCI,, TMS) C 1 2 3 4 5 6 7 8 9 1’ 2 3 4 5’ 6 7 8’ 9 3.3’ (OMe) 5.5’ (OMe) 4,4’(OMe) COMe
1 136.3’ s 105.8 d 153.1 s 136.1’s 153.1 s 105.8 d 36.6 t 43.7 d 60.5 f
136.3’ s 105.8 d 153.1 s 136.1’s 153.1 s 105.8 d 36.6 t 43.7 d 60.4 t 56.0 s 56.0 s 60.8 s _
COMe
‘-‘Assignments DEPT.
may lx. reversed;
2
3
136.P s 105.6’ d 153.1 s 136.1’s 153.1 s 105.7c d 35.5b t 39.4 d 62.4 t 136.2’ s 105.6’ d 153.1 s 136.1’s 153.1 s 105.7’ d 35.8b t 42.9 d 64.6 t 56.0 s 56.0 s 60.8 s 170.9 s 21.9q s, d
132.9 111.V 148.7 147.0 1120 120.8 35.7 43.8 60.3 132.9 1110 148.7 147.0 112.~ 120.8 35.7 43.8 60.3 55.7 -. -
t, q multiplicities
from
(Table 1) of 1 was very similar to that of secoisolariciresinol methyl ether (3) [lo], thus suggesting the presence of the same dibenzyl-lA-butanediol system. This was evident in the ‘H NMR spectrum (Table 2) from the appearance of the multiplet at 61.90 attributed to two methines and the two doublets of doublets at 62.65 and 2.75 (2H
Table 2. ‘H NMR spectral data for compounds
l-3 (200 MHz for 1 and 2, 100 MHz
for 3, CDCI,) 2
H
1
3
8,8’
1.90 (2H, m)
1.70-2.15
7.7’
2.65 (2H, dd, J = 6; 10) 2.75 (2H, dd, J = 6; 10)
2.75 (4H, br d)
2.55-2.80
9 or 9
3.55 (ZH, br d) 3.59 (2H, br d)
3.50 (ZH, br d) 3.70 (2H, br d)
3.65 (2H-9’. br d) 4.10 (IH-9’. dd, 5=6;
(2H, m)
1.95 (2H, m) 2.2 (lH, m) (4H, m)
11) 4.3 (lH-9’.
11) 3.79 (12H, s) 3.81 (6H, s)
3.80 (6H, s) 3.83 (6H, s)
26 5.5’ and for
6.34 (4H, s)
6.60-6.90
7.6’ 3 MeCO
--
-
OMe
3.77 3.78 3.79 3.80 6.26 (2H, s)
(6H, m) 6.31 (2H, s) 2.06 (3H, s)
dd, J =6;
Constituents of Zantbxylum EXPERIMENTAL
General. Mps: uncorr. ‘H NMR: 200 MHz with TMS as int. standard. ‘%NMR: SO.32MHz. EI-MS: direct inlet system at 70 eV. Silica gel: 60 (70-230 mesh, Merck). Plant materials. Bark of Z. heitzii was collected at Hawae, Province du Centre, Cameroon, in 1988. A voucher specimen is deposited at the National Herbarium, Yaoundt. Extraction and isolation. Powdered bark (10 kg) was extracted with EtOAc at room temp. for 4 days, followed by MeOH. Concn of the solvent yielded 180 g and 150 g of EtOAc and MeOH extracts, respectively. The EtOAc extract (100 g) was chromatographed on a silica gel column and eluted with hexane and mixts with increasing polarity of hexane-EtOAc and EtOAc; 100 ml frs were collected. The following compounds were first obtained: lupeol (100 mg), mixt. of sterols (600 mg), sesamin (100 mg), savinin (500 mg), fagaramide (500 mg) and artigenin Me ether (766 mg) [9]. Frs 61-79 were rechromatographed to yield trans-2-[3’,4’dimethoxybenzyl]-3-[3”,4”,5”-trimethoxybenzyl]-~-butyrolactone (70 mg) [14] and frs 80-87, compounds 1 and 2. The quaternary alkaloid, nitidine [ 153, was isolated from the MeOH extract. All the previously known compounds were identified by their physical and spectroscopic data and by comparison with authentic samples. Meso-Z3-bis (3,4,5-nimelhoxybenry~l,~~~~~ (I). Crystalline solid, mp 142-146”. [alo +O. IR VKB’ cm-‘: 3500, 2880, 1600, 1500, 1250. 13C and ‘HNMR in Tables 1 and 2. El-MS m/z (rel. int.): 450 CM]’ (48.5), 181 (loo), 151 (18.8). 4-Acetoxy-2,3-bis (3,4,5-trimethoxybenzyl)-1-butanol (2). Crystalline solid, mp 121-122”. [a&,+0. IR vKBrcm- *: 3500, 2880, 1730, 1600, 1500, 1250. 13C and ‘H NMR in Tables 1 and 2. EI-MS m/z (rel. int.): 492 [Ml+ (79), 432 (4.6), 207 (4.4), 181 (lOO), 151 (13.2).
869
heittii
Acknowledgemenrs-We thank Dr David S. Rycroft, University of Glasgow, for running 1‘C and ‘H NMR spectra. REFERENCES
1. Waterman, P. G. (1975) Tuxon 24, 361. 2. Adjanohoon, E. J., Ahyi, A. M. R., Assi, L. A., Chibon, P. and Gusset, G. (1988) Medecine Tradition&e et Phannacopie. Contribution aux Ctudes ethnobotaniques et floristiques en Rtpublique Populaire du Congo. 3. Olatunji, 0. A. (1976) Bull. Sci. Assoc. Nigeria 2, 62. 4. Ahmad, S. (1984) J. Nat. Prod. 47, 391. 5. Fish, F. and Waterman, P. G. (1973) Taxon 22, 177. 6. Ayafor, J. F., Sondengam, B. L., Bilong, A. N., Tsamo, E., Kimbu, S. F. and Okogun, J. 1.(1982) J. Nat. Prod. 45, 714. 7. Ayafor, J. F., Sondengam, B. L. and Ngadjui, B. T. (1981) Plunto Med. 44, 139. 8. Ayafor, J. F., Sondengam, B. L., Kimbu, S. F., Tsamo, E. and Connolly, J. D. (1982) Phytochemistry 21, 2602. 9. Ayafor, J. F., Ngadjui, B. T., Sondengam, B. L. and Tsamo, E. (1984) Planta Med. 210. 10. Fonseca, S. F., Nielsen, L. T. and Rieveda, E. A. (1979) Phytochemistry 18, 1703. 11. Stothers, J. B. (1972) Carbon-13 NMR Spectroscopy. Academic Press, NewYork. 12. Ernst, L. (1974) Tetrahedron Letters 3079. 13. Shibuya, H., Takeda, Y., Zhang, R., Tanitame, A., Tsai, Y. L. and Kitagawa, I. (1992) Chem. Phurm. Bull. 40, 2639. 14. Takaoka, D., Imouka, M. and Hiroi, M. (1977) Bull.
Chem. Sot. Jpn 50,282l. 15. Fish, F. and Waterman, G. P. (1972) Phytochemistry 11, 3007.
PtWaknlmy.
Vol. 37. No. 3. w 867469.1!294 EkViUSCbXLtd Rinted in Great Britain 0031~9422f94 n.m+am
LIGNANS AND OTHER CONSTITUENTS OF ZANTHOXYLUA4 HEZTZZI SILV~RE NGOUELA, ETIENNE TSAMO* and JOSEPH D. CONNOLLY?
Department of Organic Chemistry, University of Yaoundt I, Box 812 Yaoundt, Cameroon; tDepartmcnt The University, Glasgow G12 8QQ, U.K.
of Chemistry,
(Received24 Janwry 1994)
Key Word Index-Zanthoxylum
heitzii; Rutaceae; bark; lignans; alkaloids; triterpenes.
Abstract-Two new natural lignans, meso-2,3-bis(3,4,5-trimethoxybenzyl)-1,4-butanediol and 4-acetoxy-2,3-bis(3,4,5trimethoxybenzyl)-1-butanol, four known lignans, two alkaloids and triterpenes were isolated from the bark of Zanthoxylum heitzii and identified on the basis of their spectral data.
IBTRODUCTION It
is current practice to consider Zanthoxylwn and Fag-
ara as one genus Zanthoxyltun L. [ 11. Zanthoxylum heitzii is a medicinal plant widely used in central Africa for the
treatment of many diseases [2, 33. Early work on the heartwood of Z. heitzii (as Fagara heitzii) [4] led to the isolation of the alkaloid, flindersine. However, other species of Zanthoxylum contain a wide array of secondary metabolites including terpenes, alkaloids and lignans [S-8]. In a continuation of our investigation of Cameroonian medicinal plants, particularly of this genus [9]. we report the isolation and characterization from Z. heitzii of two novel lignans, meso-2,3-bis(3,4,Strimethoxybenzyl)1,4-butanediol (1) and 4-acetoxy-2,3-bis(3,4,5-trimethoxybenzyl)-I-butanol (2), the known lignans, sesamin, savinin, artigenin methyl ether and 2-(3’,4’-dimethoxybenzyl)-3-(3”,4”,5”-trimethoxybenzyl)-~-butyrolactone, as well as the alkaloids, fagaramide and nitidine, the triterpenoid, lupeol, and a mixture of campesterol, sitosterol and stigmasterol.
bMe
R’
R*
R3
1
H
H
OMc
2
H
AC
OMe
3
H
H
H
t
RESULTS AND DISCUSSION
The ethyl acetate extract from the bark of Z. heitzii was repeatedly chromatographed on silica. Compound 1 was obtained as crystals, mp 142-146”. It had a [M]’ at m/z
Me0
450 and a molecular formula Cz4HJ40s which corresponds to eight double bond equivalents. Its mass fragmentation pattern showed a peak at m/z 432 [M - H,O]+ and peak fragments containing an aromatic ring (m/z 181 [C6H,(OMe),CH,]+) (m/z 208 [C,H,(OMe),CH,=CH,]+). The IR spectrum exhibited bands at 3300 (hydroxyl group), 1600 and 1500 cm-’ (aromatic nucleus). The 13CNMR spectrum (Table 1) showed the presence of only nine carbon atom signals
*Author to whom correspondence should lx addressed.
4
shared between four sp2 carbon atoms, three sp3 carbon atoms with one bearing an oxygen atom (660.4) and two methoxy groups at 660.8 and 56.0. The fact that only nine signals were observed in the 13C NMR spectrum of 1, in spite of its high molecular formula, was very significative and suggested that it was a symmetrical aromatic molecule. The aliphatic region of the ‘%NMR spectrum
867
S. NGOUELA et al.
868
each) corresponding to four benzylic protons. The four methylene protons of the two CH,OH groups were observed as two large doublets at 63.55 and 3.59. The two singlet signals at 63.79 and 3.81 integrating each for nine protons were assigned to six aromatic methoxy groups. The four proton singlet at 66.34 indicated that the two aromatic rings probably had a 2,4,6- or 3,4,5_trimethoxy substitution pattern. The mass spectral fragmentation of 1 confirmed the presence of a trimethoxybenzyl group (m/z 181, base peak). The location of the methoxyl groups on the 3,4 and 5 positions was based on the comparison of the carbon shifts of the aromatic region of 1 with those of the model compound, 1.2,3-trimethoxybenzene (4), and standard chemical shift theory [l 1, 123. Thus, compound 1 was characterized as 2,3-bis (3,4,5-trimethoxybenzyl)1,4-butanediol. Since 1 showed no optical rotation it is assumed to be the meso derivative. The (+) form has been described as the transformation product of the naturally lignan glycoside, hazaleanin A [13]. Compound 2 was also isolated as crystals, mp 121-122” with a [M]’ at m/z 492, molecular formula C26HJ,609. Its IR spectrum was very similar to that of 1 with additional bands for acetate (I 730 and 1250 cm - I). The 13C and ‘HNMR spectra of 2 confirmed the presence of one acetate group with the signals at 6170.9. 21.9 (-AC) and 2.06 (3H). Hydrolysis of 2 yielded a compound identical to 1 in all respects (IR, mp, NMR). Consequently, 2 could be deduced to bc 4-acetoxy-2,3bis(3,4,5-trimethoxybenzyl)-1-butanol. Lupeol, sesamin, savinin, artigenin methyl ether, fagaramide [9] and 2-(3’,4’-dimethoxybenzyl~3-(3”,4”,5”-trimethoxybenzyl)-y-butyrolactone [ 141, were also isolated and identified through comparison of their spectral data with published data. The quatemary alkaloid, nitidine [IS], was isolated from the methanol extract of the ethyl acetate-defatted bark.
Table 1. “CNMR spectral data for compounds l-3 (SO.32 MHz for 1 and 2, 25.2 MHz for 3, CDCI,, TMS) C 1 2 3 4 5 6 7 8 9 1’ 2 3 4 5’ 6 7 8’ 9 3.3’ (OMe) 5.5’ (OMe) 4,4’(OMe) COMe
1 136.3’ s 105.8 d 153.1 s 136.1’s 153.1 s 105.8 d 36.6 t 43.7 d 60.5 f
136.3’ s 105.8 d 153.1 s 136.1’s 153.1 s 105.8 d 36.6 t 43.7 d 60.4 t 56.0 s 56.0 s 60.8 s _
COMe
‘-‘Assignments DEPT.
may lx. reversed;
2
3
136.P s 105.6’ d 153.1 s 136.1’s 153.1 s 105.7c d 35.5b t 39.4 d 62.4 t 136.2’ s 105.6’ d 153.1 s 136.1’s 153.1 s 105.7’ d 35.8b t 42.9 d 64.6 t 56.0 s 56.0 s 60.8 s 170.9 s 21.9q s, d
132.9 111.V 148.7 147.0 1120 120.8 35.7 43.8 60.3 132.9 1110 148.7 147.0 112.~ 120.8 35.7 43.8 60.3 55.7 -. -
t, q multiplicities
from
(Table 1) of 1 was very similar to that of secoisolariciresinol methyl ether (3) [lo], thus suggesting the presence of the same dibenzyl-lA-butanediol system. This was evident in the ‘H NMR spectrum (Table 2) from the appearance of the multiplet at 61.90 attributed to two methines and the two doublets of doublets at 62.65 and 2.75 (2H
Table 2. ‘H NMR spectral data for compounds
l-3 (200 MHz for 1 and 2, 100 MHz
for 3, CDCI,) 2
H
1
3
8,8’
1.90 (2H, m)
1.70-2.15
7.7’
2.65 (2H, dd, J = 6; 10) 2.75 (2H, dd, J = 6; 10)
2.75 (4H, br d)
2.55-2.80
9 or 9
3.55 (ZH, br d) 3.59 (2H, br d)
3.50 (ZH, br d) 3.70 (2H, br d)
3.65 (2H-9’. br d) 4.10 (IH-9’. dd, 5=6;
(2H, m)
1.95 (2H, m) 2.2 (lH, m) (4H, m)
11) 4.3 (lH-9’.
11) 3.79 (12H, s) 3.81 (6H, s)
3.80 (6H, s) 3.83 (6H, s)
26 5.5’ and for
6.34 (4H, s)
6.60-6.90
7.6’ 3 MeCO
--
-
OMe
3.77 3.78 3.79 3.80 6.26 (2H, s)
(6H, m) 6.31 (2H, s) 2.06 (3H, s)
dd, J =6;
Constituents of Zantbxylum EXPERIMENTAL
General. Mps: uncorr. ‘H NMR: 200 MHz with TMS as int. standard. ‘%NMR: SO.32MHz. EI-MS: direct inlet system at 70 eV. Silica gel: 60 (70-230 mesh, Merck). Plant materials. Bark of Z. heitzii was collected at Hawae, Province du Centre, Cameroon, in 1988. A voucher specimen is deposited at the National Herbarium, Yaoundt. Extraction and isolation. Powdered bark (10 kg) was extracted with EtOAc at room temp. for 4 days, followed by MeOH. Concn of the solvent yielded 180 g and 150 g of EtOAc and MeOH extracts, respectively. The EtOAc extract (100 g) was chromatographed on a silica gel column and eluted with hexane and mixts with increasing polarity of hexane-EtOAc and EtOAc; 100 ml frs were collected. The following compounds were first obtained: lupeol (100 mg), mixt. of sterols (600 mg), sesamin (100 mg), savinin (500 mg), fagaramide (500 mg) and artigenin Me ether (766 mg) [9]. Frs 61-79 were rechromatographed to yield trans-2-[3’,4’dimethoxybenzyl]-3-[3”,4”,5”-trimethoxybenzyl]-~-butyrolactone (70 mg) [14] and frs 80-87, compounds 1 and 2. The quaternary alkaloid, nitidine [ 153, was isolated from the MeOH extract. All the previously known compounds were identified by their physical and spectroscopic data and by comparison with authentic samples. Meso-Z3-bis (3,4,5-nimelhoxybenry~l,~~~~~ (I). Crystalline solid, mp 142-146”. [alo +O. IR VKB’ cm-‘: 3500, 2880, 1600, 1500, 1250. 13C and ‘HNMR in Tables 1 and 2. El-MS m/z (rel. int.): 450 CM]’ (48.5), 181 (loo), 151 (18.8). 4-Acetoxy-2,3-bis (3,4,5-trimethoxybenzyl)-1-butanol (2). Crystalline solid, mp 121-122”. [a&,+0. IR vKBrcm- *: 3500, 2880, 1730, 1600, 1500, 1250. 13C and ‘H NMR in Tables 1 and 2. EI-MS m/z (rel. int.): 492 [Ml+ (79), 432 (4.6), 207 (4.4), 181 (lOO), 151 (13.2).
869
heittii
Acknowledgemenrs-We thank Dr David S. Rycroft, University of Glasgow, for running 1‘C and ‘H NMR spectra. REFERENCES
1. Waterman, P. G. (1975) Tuxon 24, 361. 2. Adjanohoon, E. J., Ahyi, A. M. R., Assi, L. A., Chibon, P. and Gusset, G. (1988) Medecine Tradition&e et Phannacopie. Contribution aux Ctudes ethnobotaniques et floristiques en Rtpublique Populaire du Congo. 3. Olatunji, 0. A. (1976) Bull. Sci. Assoc. Nigeria 2, 62. 4. Ahmad, S. (1984) J. Nat. Prod. 47, 391. 5. Fish, F. and Waterman, P. G. (1973) Taxon 22, 177. 6. Ayafor, J. F., Sondengam, B. L., Bilong, A. N., Tsamo, E., Kimbu, S. F. and Okogun, J. 1.(1982) J. Nat. Prod. 45, 714. 7. Ayafor, J. F., Sondengam, B. L. and Ngadjui, B. T. (1981) Plunto Med. 44, 139. 8. Ayafor, J. F., Sondengam, B. L., Kimbu, S. F., Tsamo, E. and Connolly, J. D. (1982) Phytochemistry 21, 2602. 9. Ayafor, J. F., Ngadjui, B. T., Sondengam, B. L. and Tsamo, E. (1984) Planta Med. 210. 10. Fonseca, S. F., Nielsen, L. T. and Rieveda, E. A. (1979) Phytochemistry 18, 1703. 11. Stothers, J. B. (1972) Carbon-13 NMR Spectroscopy. Academic Press, NewYork. 12. Ernst, L. (1974) Tetrahedron Letters 3079. 13. Shibuya, H., Takeda, Y., Zhang, R., Tanitame, A., Tsai, Y. L. and Kitagawa, I. (1992) Chem. Phurm. Bull. 40, 2639. 14. Takaoka, D., Imouka, M. and Hiroi, M. (1977) Bull.
Chem. Sot. Jpn 50,282l. 15. Fish, F. and Waterman, G. P. (1972) Phytochemistry 11, 3007.