- Email: [email protected]
Chromenes from Arnica sachalinensis and A. amplexicaulis
Short Reports
1070
278 (37), 222 (98), 191 (42), 181 (19), 163 (13), 151 (77); ‘HNMR (CDCI,):63,11(1H,d, J=~Hz),~.~~(IH,~,J=~Hz),S.~~(~H, dd. J=6, 8 Hz) 3.72, 3.86, 3.92(each 3H, s) 3.84(9H, s), 6.20, 7.91 (each lH, d, J= 16 Hz), 6.61-7.32 (9H, m). Hydrolysis of compound lb. Compound lb (40mg) was refluxed with 5 ml 10% KOH-MeOH for 1 hr. After acidification and work-up with EtOAc and H,O, the organic layer was coned yielding 19 mg of yellow crystals le. The mother liquor was purified by TLC using C,H,-EtOH-HCO,H (80:20: 1) as solvent, 5 mg of lc along with R-( +)-b-(3,4-dimethoxyphenyl)lactic acid were obtained. Compound lc. Yellow crystals, mp 178-180”. EI-MS m/z (rel. int.): 354 ([Ml’, 100). 339 (28). 309 (55), 278 (75). Acknowledgements -Financial support was provided by the Natural Sciences Foundation of China. We thank the Department of Instrumental Analysis of our Institute for UV, MS and
NMR measurements.
REFERENCES
1. Compilation of Chinese Herb Medicine (1975) Vol. 1, p. 242. People’s Publishing House, Beijing. 2. Li, L. N., Tan, R. and Chen, W. M. (1984) Planta Med. 227. 3. Ai, C. B. and Li, L. N. (1988) J. Nat. Prod. (Lloydia) 51, 145. 4. Ai, C. B. and Li, L. N. (1992) Planta Med. (in press). 5. Xu. L. N., Wang, J. P.. Tlan, Q. Y. and Han, Y. H. (1990) Proceedings of the 3rd National Symposium of Activating Blood Circulation and Removing Blood Stasis Research, p. 198. Lushan, China. 6. Xu, L. N., Li, D. Y. and Tian, Q. Y. (1990) Proceedings of the 3rd National Symposium of Actmatmg Blood Circulation and Removing Blood Stasis Research, p. 198. Lushan, China. 7. Anjaneyulu, A. S. R., Reddy, A. V. R. and Reddy, D. S. K (1984) Tetrahedron 40, 4245 8. AI, C. B. and Li, L. N. (1991) Chinese Chem. Letters 1, 17. 9. Pelletier, S. W.. Chokshi, H. P. and Desai, H. K. (1986) J. Nat. Prod. 49,892.
Phytochem~sfry, Vol. 31. No. 3, pp 1070-1072. 1992 Pnnted in Great Britain
CHROMENES
FROM
CLAUS M.
ARNICA
PAOREITER,
0
AND A. AMPLEXICAULIS
SACHALINENSIS
GUNTER WILLUHN, ALOIS STEIGEL* and
Institut fiir Pharmazeutische Biologie der Heinrich-Heine-Universitiit Organische und Makromolekulare Chemie der Heinrich-Heine-Universitiit Physlologische Chemie der Hemrich-Heine-Universitlt
0031~9422/92 $5.00+0.00 1992 Pergamon Press plc
UWE
MATTHIESEN~
Diisseldorf, 4000 Diisseldorf, Germany; Dlisseldorf, 4000 Diisscldorf, Germany; Diisseldorf, 4000 Diisseldorf, Germany
*Institut trnstitut
fiir fiir
(Received 8 July 1991) Key Word Index---Armca sachalinensis; A. amplexicaulis; Asteraceae; demethoxyencccalin; 12-isobutyryloxy-demethoxyencecalin.
chrometles
(benzopyransk
12-acetoxy-
Abstract-Flowerheads of Arnica sachalinensis yielded, in addition to the known chromenes acetovanillochromene, demethylencecalin and demethoxyencecalin, the new compounds 12-acetoxy-, 12-isobutyryloxy- and 12-isovaleryloxy-demethoxyencecalin. Demethoxy-and demethylencecalin are also constituents of A. amplexicaulis. The assignment of the 13C NMR signals of demethoxyencecalin is revised by 2D-13C, ‘H NMR experiments. Chemotaxonomic aspects are discussed. INTRODUCTION
Chromenes (benzopyrans) and benzofurans are prominent natural products of many genera of the Asteraceae and have become useful taxonomic markers within this family [l-3]. In a previous paper [4], we reported on the isolation of demethoxyencecalin (1) from the flowers of Arnica sachalinensis (Regl.) A. Gray, which was the first chromene found in the genus Arnica. The tribal position of the genus Arnica, traditionally within the tribe Senecioneae, has been the subject of considerable discussion [5,6]. On morphological, serological and chemical grounds, it has been argued that Arnica is more related to the Heliantheae than to the Senecioneae, thus Robinson [7] includes Arnica in this tribe, subtribe Chaenactidinae. In Bohlmann’s opinion, however, Arnica has more resemblance to the closely related subtribe Gaillardiinae
5
R
H
1 2 3
OiBut
4
OiValoc O-2-MeBut
OAc
0
6
Short Reports [8]. Other authors [9, lo] prefer the establishment of a provisional tribe Arniceae. We deal with the isolation and identification of the chromenes 2-6 from A. sachalinensis (subgenus Andropurpurea) and the chromenes 1 and 5 from A. amplexicaulk (subgenus Chamissonis). Compounds 2-4 are new naturally occurring esterified chromenes, whereas 5 and 6 have been previously found in other species of the Asteraceae Cl, 11,121.
RESULTS AND
DISCUSSION
Chromatography of the methylene chloride extract of the flowerheads of A. sachalinensis on a Sephadex LH-20 column yielded two fractions rich in chromenes l-4, and 5 and 6, respectively. These fractions were separated by repeated column chromatography on silica gel and further by TLC. The spots on TLC showed an intense blue fluorescence (5, yellow) in UV light (365 nm). After treatment with anisaldehyde-HzSO, and heating they gave a yellow colour, which characteristically changed to blue (1, violet). The structure of 2 clearly followed from the mass, ‘H and 13C NMR spectra, which were in part close to those of 1 [4]. The mass spectrum showed the molecular ion at m/z 260, the base peak at m/z 245 [M-Me] + and the characteristic fragmentation pattern of a 12-acyloxy-chromene [l l] with significant fragment ions at m/z 187 [M-MeCO-O-CHz]+, 159 [M - MeCO-O-CHzCO] ’ and 144 [M -(Me+ MeCO-0-CHz-CO)] +. All these signals correspond to the 1Zacetoxy derivative of 1. In the ‘H NMR spectrum, the additional 12-acetoxy group caused the expected changes. The two protons of the methylene group (H-12) appeared as a singlet at 65.28 and the acetyl group as a singlet at 62.23. The 13C NMR spectrum showed a downfield shift of the signal for C-12 to which the acetoxy group is attached and two additional carbon resonances of an acetyl group (Table 1). Table 1. ‘%NMR
spectral data of compounds l-3 (75 MHz, CDCl,, TMS as int. standard) 1
C 2 3 4 5 6 7 8 9 10 11 12 13 14 OR
77.542 131.194 121.679 126.904 130.315 130.233 116.125 157.425 120.637 196.718 26.288 28.364 28.364
2 77.812 131.468 121.412 126.406 127.172 129.521 116.473 158.094 120.963 190.513 65.727 28.422 28.422 170.539 20.657
OMe PAssignment interchangeable.
3 77.780 131.426 121.443 126.420 127.281 129.520 116.444 158.015 120.930 190.767 65.476 28.419 28.419 176.667 33.826 19.023 19.023
and 6
6 77.862 131.087 121.754 120.614 129.788 111.242 148.290’ 146.696’ 120.842 196.758 26.239 28.217 28.217
56.245
1071
The mass, ‘H and 13C NMR spectra of 3 showed that this compound is closely related to 2, only the ester group being different. The typical ‘H and 13C NMR signals indicated the presence of an isobutyryloxy group. The ‘H NMR data agreed with those of the isobutyrylation product of 12-hydroxydemethoxyencecalin, synthesized by Bohlmann et al. [ 133. The minor constituent 4 was not obtained in pure form. Its structure was determined by GC-MS analysis. The mass spectrum showed the same fragmentation pattern as 2 and 3 and clearly indicated the presence of an isovaleryloxy or 2-methylbutyryloxy group at C-12. The known chromenes 5 and 6 were identified by mass, ‘H and 13C NMR spectra [l 1,121 and by comparison (TLC, GC) with authentic samples. Since, as a rule, structure elucidation of chromenes is unequivocally possible by mass and ‘H NMR spectral analysis, only few ’ 3C NMR data of 2,2-dimethylchromenes are recorded in the literature. Moreover, the existing data have not been determined by special experiments. For this reason, some assignments of the carbon signals are contradictory or specified to be interchangeable [4, 14-161. Therefore, an unambiguous analysis of the chemical shift data for 1 (Table 1) was performed by 2D heteronuclear correlation experiments using direct (‘J,,) and long range (3Jcn) couplings. Recently, a short report has been given by Timar et al. [17] on the “C NMR assignments of a series of synthetic, substituted 2,2-dimethylchromenes related to the precocenes. The assignments of the carbon resonances of 2,3 and 6 were established by comparison with the data of 1. In the flowerheads of A. amplexicaulis the chromenes 1 and 5 were identified by direct comparison (TLC, GC, GC-MS) with the authentic samples. Compounds 2-4 are new naturally occurring chromenes to the best of our knowledge. 12-Hydroxy-demethoxyencecalin is a constituent of Calea species (Heliantheae) [lS, 191 and Baccharis glutinosa (Astereae) [20]. Up to now only six 12-acyloxy derivatives of other chromenes are described as constituents of species included to the Heliantheae and Eupatorieae [11,21-24-J. So far no benzofurans, which are widely distributed in the tribe Senecioneae [l], could be detected in either Arnica species. The occurrence of chromenes in the genus Arnica supports the integration of this genus into the tribe Heliantheae. EXPERIMENTAL
Plantmaterial. Arnica sackalinensis and A. amplexicauliswere grown from seed, originating from the Botanical garden of the Academy of Sciences, Moscow, Russia, and the University of Marburg/Lahn, Germany (Vouchers on deposit in the Herbaria of the Institute of Pharmaceutical Biology, University of Diisseldorf). Dried, powdered flowerheads (1.2 and 1.1 kg) were extracted with CH,Cl, and the extracts first separated by CC on Sephadex LH-20 (Pharmacia) with MeOH as eluent and further on silica gel 60 (70-230 mesh) with CHzClz (A. sachalinensis)or mixtures of petrol-Et,0 (initial 19: 1; A. amplexicaulis).Fractions of A. sachalinensisafter prep. TLC (silica gel 60 plates, Merck) with n-pentane-Et,0 (8: 17) gave 4 mg 2 (oil), 6 mg 3 (oil), 2 mg 4 (crude, oil), 4 mg 5 (amorph.) and 2 mg 6 (oil). TLC on silica gel 60 F,,, (Merck) with toluene-Et,0 (4: 1): R,: 10.52; 2 0.48; 3 0.52; 4 0.72; 5 0.68; 6 0.38. GC. Column OV-01 (Machemy & Nagel), 25 m x 0.25 mm; temp. programmed 150 to 270” at lo” min- ‘; inj./detector temp.
1072
Short Reports
3OO”, carrier N, at 1.2 ml min - t: R, (mitt): 1 7.0; 2 6.7; 3 8.7; 4 9.1; 5 4.6; 6 5.3. 12-Acetoxy-demethoxyenceculm (2). GC-MS 70 eV, m/z (ml. int.): 260 [M]’ (35) 245 [M--Me]+ (lOOj, 217 [M-Ac]+ (2). 203 [M-Me-CH,=C=OJ’ (43), 202 [M-Me-AC]+ (8). 187 CM-AC-OCH,]’ (99), 159 [M-Ac-OCH,-.CO] + (11) 144 CM-Me-AC-OCH,-COJ’ (50), 101 [AC-OCH,-CO]+(2), 73 [AC-OCH,] ’ (1). 43 [AC]’ (22): ‘H NMR (300 MHz, CD&): 6 1.463 (6H, s. H-13, H-14), 2.231 (3H, s. AC), 5.279 (2H, s, H-12). 5.684 (lH, d, J=lO Hz, H-3), 6.348 (lH, d, 5=9.89 Hz, H-4), 6.808 (lH, d, J=8.48 Hz, H-8), 7.574 (lH, d, 5=2.19 Hz, H-5). 7.698 (lH, dd, 5=8.41 and 2.0 Hz, H-7). 12-lsobutyryloxy-demethoxyencecalin (3). GC-MS 70 eV, m/z (rel. int.): 288 [M]’ (48), 273 [M-Me]+ (100) 217 [M - C,H,CO] + (1). 203 [M - Me - C,H,C=C=O] t (49) 202 [M-Me-C,H,CO’]+ (ll), 187 [M-CC,H,CO,CHZ]+ (99), 144 [M-Me 159 [M-C,H,CO-OCH,-CO] + (18) -C,H,CO-OCH,--CO] + (65). 129 [C,H,CO-OCH,CO] + (6), 101 [C,H,CO-OCH,]+ (l), 71 [C3H7C0]+ (15). 43 [71 -CO]+ (56); ‘H NMR (300 MHz, CD&): S 1.266 (6H, d, Me,CHCO,J=6.99 Hz), 1.461 (6H,s, H-13, H-14). 2.742(lH, m Me,CHCO), 5.263 (2H, s, H-12), 5.678 (lH, d, 5=9.88 Hz, H-3). 6.341 (IH, d. 5=9.58 Hz, H-4), 6.805 (lH, d, 5=8.53 Hz, H-8), 7.571 (lH, d, J=2.20 Hz, H-5), 7.694 (IH, dd, J=8.455 and 2.0 Hz, H-7). 12-lsovaleryloxy- or 2-methylbutyryloxy-demethoxyencecalin (4). GC-MS 70 eV, m/z (rel. int.): 302 [MJ + (13) 287 [M - Me] + (96), 203 [M-Me-C,Hs=C=O]+ (49) 187 [M -C,H,CO,CH,] + (100). 159 [M-CC,H,CO-OCH,-CO] + (3) 144 [M-Me-C,H,-CO-OCH,-CO]’ (20). 115 [C,H,CO-OCHr] + (8). 57 [C,H,] + (8).
REFERENCES
1. Proksch, 2335. 2. Proksch, 3. Proksch,
P. and
Rodriguez,
E. (1983) Phytochemistry 22,
P. (1985) Plant Syst. Euol. 150, 89. P. and Clark, C. (1987) Phytochemistry 26, 171.
4. Willuhn. G., Junior, I. and Wcndisch. D. (1987) Planta Med. 349. 5. Turner, B. I,. and Powell, A. M. (1977) in The Biology and Chemtstry ofthe Composrtae (Heywood. V. H., Harborne, J. B. and Turner, B. L., eds), pp. 699.. 737. Academic Press, London. 6. Nordenstarn, S. (1977) in The Biology and Chemistry of the Compositae (Heywood, V. H., Harborne, J. B. and Turner, B. L., eds), pp. 799-830. Academtc Press, London. 7. Robmson, H. (1981) Smithsonian Contrib. Botany 51, 1. 8. Bohlmann, F. (1990) Plant Spst. Eool. 4 (SuppI.), 67. 9. De Emerenctano, V. P., Kaplan, M. A. C. and Got&b, 0. R. (1985) Biochem. Syst. Ecol. 13, 145. 10. Seaman, F. C. (1982) Eat. Rev. 48, 121. 11. Anthonsen, T. (1969) Acra Chem. Stand 23. 3605. 12. Taylor, D. R. and Wrtght, J. A. (1971) Phytochemistry 10, 1665. 13. Bohlmann, F. and Stoehr, F. M. (1980) Liebigs Ann. Chem. 185. 14 Diaz, D. P. P., Anas, C. T. and Joseph-Nathan, P. (1987) Phytochemistry 26. 809. 15. Kulkarm, M. M., Nagasampagi, B. A., Deshpande, S. G. and Sharma. R. N. (1987) Phytochemlstry 26, 2669. 16. Samat, A. M., Gugliemelelmetti, R. J. and Martin, G. J. (1976) Org. Magn. Rrs. 8. 62. 17. Timar, T., Sebiik, P., Kover, K. E., Jaszberenyi, J. C. and Batta, G. (1989) Magn. Res. Chem. 27, 303. 18. Bohlmann, F. Gupta. R. K., Jakupovic, J., Kmg, R. M. and Robinson, H. (1982) Phytochemistry 21, 2899. 19. Castro, V.. Tamayo-Castillo, G. and Jakupovtc. J. (1989) Phytochemistrp 28, 2415. 20. Dominguez, X. A., El Dahmt, S. and Rombold, C. (1986) J. Nat. Prod. 49, 143. 21. Bohlmann, F. and Ftcdler. L (1978) Phytochemistry 17. 566. 22. Bohlmann, F., Frttz, U., Kmg. R. M. and Robinson, H. (198 1) Phytochemistry 20, 743. 23. Bohlmann. F.. Jakupovic. J. and Lonitz. M. (1977) Chem. Eer. 110, 331. 24. Quijano, L, Calderon. J. S. and Rios, T. (1977) Rev. Latmoam. Quim. 8, 90.
1070
278 (37), 222 (98), 191 (42), 181 (19), 163 (13), 151 (77); ‘HNMR (CDCI,):63,11(1H,d, J=~Hz),~.~~(IH,~,J=~Hz),S.~~(~H, dd. J=6, 8 Hz) 3.72, 3.86, 3.92(each 3H, s) 3.84(9H, s), 6.20, 7.91 (each lH, d, J= 16 Hz), 6.61-7.32 (9H, m). Hydrolysis of compound lb. Compound lb (40mg) was refluxed with 5 ml 10% KOH-MeOH for 1 hr. After acidification and work-up with EtOAc and H,O, the organic layer was coned yielding 19 mg of yellow crystals le. The mother liquor was purified by TLC using C,H,-EtOH-HCO,H (80:20: 1) as solvent, 5 mg of lc along with R-( +)-b-(3,4-dimethoxyphenyl)lactic acid were obtained. Compound lc. Yellow crystals, mp 178-180”. EI-MS m/z (rel. int.): 354 ([Ml’, 100). 339 (28). 309 (55), 278 (75). Acknowledgements -Financial support was provided by the Natural Sciences Foundation of China. We thank the Department of Instrumental Analysis of our Institute for UV, MS and
NMR measurements.
REFERENCES
1. Compilation of Chinese Herb Medicine (1975) Vol. 1, p. 242. People’s Publishing House, Beijing. 2. Li, L. N., Tan, R. and Chen, W. M. (1984) Planta Med. 227. 3. Ai, C. B. and Li, L. N. (1988) J. Nat. Prod. (Lloydia) 51, 145. 4. Ai, C. B. and Li, L. N. (1992) Planta Med. (in press). 5. Xu. L. N., Wang, J. P.. Tlan, Q. Y. and Han, Y. H. (1990) Proceedings of the 3rd National Symposium of Activating Blood Circulation and Removing Blood Stasis Research, p. 198. Lushan, China. 6. Xu, L. N., Li, D. Y. and Tian, Q. Y. (1990) Proceedings of the 3rd National Symposium of Actmatmg Blood Circulation and Removing Blood Stasis Research, p. 198. Lushan, China. 7. Anjaneyulu, A. S. R., Reddy, A. V. R. and Reddy, D. S. K (1984) Tetrahedron 40, 4245 8. AI, C. B. and Li, L. N. (1991) Chinese Chem. Letters 1, 17. 9. Pelletier, S. W.. Chokshi, H. P. and Desai, H. K. (1986) J. Nat. Prod. 49,892.
Phytochem~sfry, Vol. 31. No. 3, pp 1070-1072. 1992 Pnnted in Great Britain
CHROMENES
FROM
CLAUS M.
ARNICA
PAOREITER,
0
AND A. AMPLEXICAULIS
SACHALINENSIS
GUNTER WILLUHN, ALOIS STEIGEL* and
Institut fiir Pharmazeutische Biologie der Heinrich-Heine-Universitiit Organische und Makromolekulare Chemie der Heinrich-Heine-Universitiit Physlologische Chemie der Hemrich-Heine-Universitlt
0031~9422/92 $5.00+0.00 1992 Pergamon Press plc
UWE
MATTHIESEN~
Diisseldorf, 4000 Diisseldorf, Germany; Dlisseldorf, 4000 Diisscldorf, Germany; Diisseldorf, 4000 Diisseldorf, Germany
*Institut trnstitut
fiir fiir
(Received 8 July 1991) Key Word Index---Armca sachalinensis; A. amplexicaulis; Asteraceae; demethoxyencccalin; 12-isobutyryloxy-demethoxyencecalin.
chrometles
(benzopyransk
12-acetoxy-
Abstract-Flowerheads of Arnica sachalinensis yielded, in addition to the known chromenes acetovanillochromene, demethylencecalin and demethoxyencecalin, the new compounds 12-acetoxy-, 12-isobutyryloxy- and 12-isovaleryloxy-demethoxyencecalin. Demethoxy-and demethylencecalin are also constituents of A. amplexicaulis. The assignment of the 13C NMR signals of demethoxyencecalin is revised by 2D-13C, ‘H NMR experiments. Chemotaxonomic aspects are discussed. INTRODUCTION
Chromenes (benzopyrans) and benzofurans are prominent natural products of many genera of the Asteraceae and have become useful taxonomic markers within this family [l-3]. In a previous paper [4], we reported on the isolation of demethoxyencecalin (1) from the flowers of Arnica sachalinensis (Regl.) A. Gray, which was the first chromene found in the genus Arnica. The tribal position of the genus Arnica, traditionally within the tribe Senecioneae, has been the subject of considerable discussion [5,6]. On morphological, serological and chemical grounds, it has been argued that Arnica is more related to the Heliantheae than to the Senecioneae, thus Robinson [7] includes Arnica in this tribe, subtribe Chaenactidinae. In Bohlmann’s opinion, however, Arnica has more resemblance to the closely related subtribe Gaillardiinae
5
R
H
1 2 3
OiBut
4
OiValoc O-2-MeBut
OAc
0
6
Short Reports [8]. Other authors [9, lo] prefer the establishment of a provisional tribe Arniceae. We deal with the isolation and identification of the chromenes 2-6 from A. sachalinensis (subgenus Andropurpurea) and the chromenes 1 and 5 from A. amplexicaulk (subgenus Chamissonis). Compounds 2-4 are new naturally occurring esterified chromenes, whereas 5 and 6 have been previously found in other species of the Asteraceae Cl, 11,121.
RESULTS AND
DISCUSSION
Chromatography of the methylene chloride extract of the flowerheads of A. sachalinensis on a Sephadex LH-20 column yielded two fractions rich in chromenes l-4, and 5 and 6, respectively. These fractions were separated by repeated column chromatography on silica gel and further by TLC. The spots on TLC showed an intense blue fluorescence (5, yellow) in UV light (365 nm). After treatment with anisaldehyde-HzSO, and heating they gave a yellow colour, which characteristically changed to blue (1, violet). The structure of 2 clearly followed from the mass, ‘H and 13C NMR spectra, which were in part close to those of 1 [4]. The mass spectrum showed the molecular ion at m/z 260, the base peak at m/z 245 [M-Me] + and the characteristic fragmentation pattern of a 12-acyloxy-chromene [l l] with significant fragment ions at m/z 187 [M-MeCO-O-CHz]+, 159 [M - MeCO-O-CHzCO] ’ and 144 [M -(Me+ MeCO-0-CHz-CO)] +. All these signals correspond to the 1Zacetoxy derivative of 1. In the ‘H NMR spectrum, the additional 12-acetoxy group caused the expected changes. The two protons of the methylene group (H-12) appeared as a singlet at 65.28 and the acetyl group as a singlet at 62.23. The 13C NMR spectrum showed a downfield shift of the signal for C-12 to which the acetoxy group is attached and two additional carbon resonances of an acetyl group (Table 1). Table 1. ‘%NMR
spectral data of compounds l-3 (75 MHz, CDCl,, TMS as int. standard) 1
C 2 3 4 5 6 7 8 9 10 11 12 13 14 OR
77.542 131.194 121.679 126.904 130.315 130.233 116.125 157.425 120.637 196.718 26.288 28.364 28.364
2 77.812 131.468 121.412 126.406 127.172 129.521 116.473 158.094 120.963 190.513 65.727 28.422 28.422 170.539 20.657
OMe PAssignment interchangeable.
3 77.780 131.426 121.443 126.420 127.281 129.520 116.444 158.015 120.930 190.767 65.476 28.419 28.419 176.667 33.826 19.023 19.023
and 6
6 77.862 131.087 121.754 120.614 129.788 111.242 148.290’ 146.696’ 120.842 196.758 26.239 28.217 28.217
56.245
1071
The mass, ‘H and 13C NMR spectra of 3 showed that this compound is closely related to 2, only the ester group being different. The typical ‘H and 13C NMR signals indicated the presence of an isobutyryloxy group. The ‘H NMR data agreed with those of the isobutyrylation product of 12-hydroxydemethoxyencecalin, synthesized by Bohlmann et al. [ 133. The minor constituent 4 was not obtained in pure form. Its structure was determined by GC-MS analysis. The mass spectrum showed the same fragmentation pattern as 2 and 3 and clearly indicated the presence of an isovaleryloxy or 2-methylbutyryloxy group at C-12. The known chromenes 5 and 6 were identified by mass, ‘H and 13C NMR spectra [l 1,121 and by comparison (TLC, GC) with authentic samples. Since, as a rule, structure elucidation of chromenes is unequivocally possible by mass and ‘H NMR spectral analysis, only few ’ 3C NMR data of 2,2-dimethylchromenes are recorded in the literature. Moreover, the existing data have not been determined by special experiments. For this reason, some assignments of the carbon signals are contradictory or specified to be interchangeable [4, 14-161. Therefore, an unambiguous analysis of the chemical shift data for 1 (Table 1) was performed by 2D heteronuclear correlation experiments using direct (‘J,,) and long range (3Jcn) couplings. Recently, a short report has been given by Timar et al. [17] on the “C NMR assignments of a series of synthetic, substituted 2,2-dimethylchromenes related to the precocenes. The assignments of the carbon resonances of 2,3 and 6 were established by comparison with the data of 1. In the flowerheads of A. amplexicaulis the chromenes 1 and 5 were identified by direct comparison (TLC, GC, GC-MS) with the authentic samples. Compounds 2-4 are new naturally occurring chromenes to the best of our knowledge. 12-Hydroxy-demethoxyencecalin is a constituent of Calea species (Heliantheae) [lS, 191 and Baccharis glutinosa (Astereae) [20]. Up to now only six 12-acyloxy derivatives of other chromenes are described as constituents of species included to the Heliantheae and Eupatorieae [11,21-24-J. So far no benzofurans, which are widely distributed in the tribe Senecioneae [l], could be detected in either Arnica species. The occurrence of chromenes in the genus Arnica supports the integration of this genus into the tribe Heliantheae. EXPERIMENTAL
Plantmaterial. Arnica sackalinensis and A. amplexicauliswere grown from seed, originating from the Botanical garden of the Academy of Sciences, Moscow, Russia, and the University of Marburg/Lahn, Germany (Vouchers on deposit in the Herbaria of the Institute of Pharmaceutical Biology, University of Diisseldorf). Dried, powdered flowerheads (1.2 and 1.1 kg) were extracted with CH,Cl, and the extracts first separated by CC on Sephadex LH-20 (Pharmacia) with MeOH as eluent and further on silica gel 60 (70-230 mesh) with CHzClz (A. sachalinensis)or mixtures of petrol-Et,0 (initial 19: 1; A. amplexicaulis).Fractions of A. sachalinensisafter prep. TLC (silica gel 60 plates, Merck) with n-pentane-Et,0 (8: 17) gave 4 mg 2 (oil), 6 mg 3 (oil), 2 mg 4 (crude, oil), 4 mg 5 (amorph.) and 2 mg 6 (oil). TLC on silica gel 60 F,,, (Merck) with toluene-Et,0 (4: 1): R,: 10.52; 2 0.48; 3 0.52; 4 0.72; 5 0.68; 6 0.38. GC. Column OV-01 (Machemy & Nagel), 25 m x 0.25 mm; temp. programmed 150 to 270” at lo” min- ‘; inj./detector temp.
1072
Short Reports
3OO”, carrier N, at 1.2 ml min - t: R, (mitt): 1 7.0; 2 6.7; 3 8.7; 4 9.1; 5 4.6; 6 5.3. 12-Acetoxy-demethoxyenceculm (2). GC-MS 70 eV, m/z (ml. int.): 260 [M]’ (35) 245 [M--Me]+ (lOOj, 217 [M-Ac]+ (2). 203 [M-Me-CH,=C=OJ’ (43), 202 [M-Me-AC]+ (8). 187 CM-AC-OCH,]’ (99), 159 [M-Ac-OCH,-.CO] + (11) 144 CM-Me-AC-OCH,-COJ’ (50), 101 [AC-OCH,-CO]+(2), 73 [AC-OCH,] ’ (1). 43 [AC]’ (22): ‘H NMR (300 MHz, CD&): 6 1.463 (6H, s. H-13, H-14), 2.231 (3H, s. AC), 5.279 (2H, s, H-12). 5.684 (lH, d, J=lO Hz, H-3), 6.348 (lH, d, 5=9.89 Hz, H-4), 6.808 (lH, d, J=8.48 Hz, H-8), 7.574 (lH, d, 5=2.19 Hz, H-5). 7.698 (lH, dd, 5=8.41 and 2.0 Hz, H-7). 12-lsobutyryloxy-demethoxyencecalin (3). GC-MS 70 eV, m/z (rel. int.): 288 [M]’ (48), 273 [M-Me]+ (100) 217 [M - C,H,CO] + (1). 203 [M - Me - C,H,C=C=O] t (49) 202 [M-Me-C,H,CO’]+ (ll), 187 [M-CC,H,CO,CHZ]+ (99), 144 [M-Me 159 [M-C,H,CO-OCH,-CO] + (18) -C,H,CO-OCH,--CO] + (65). 129 [C,H,CO-OCH,CO] + (6), 101 [C,H,CO-OCH,]+ (l), 71 [C3H7C0]+ (15). 43 [71 -CO]+ (56); ‘H NMR (300 MHz, CD&): S 1.266 (6H, d, Me,CHCO,J=6.99 Hz), 1.461 (6H,s, H-13, H-14). 2.742(lH, m Me,CHCO), 5.263 (2H, s, H-12), 5.678 (lH, d, 5=9.88 Hz, H-3). 6.341 (IH, d. 5=9.58 Hz, H-4), 6.805 (lH, d, 5=8.53 Hz, H-8), 7.571 (lH, d, J=2.20 Hz, H-5), 7.694 (IH, dd, J=8.455 and 2.0 Hz, H-7). 12-lsovaleryloxy- or 2-methylbutyryloxy-demethoxyencecalin (4). GC-MS 70 eV, m/z (rel. int.): 302 [MJ + (13) 287 [M - Me] + (96), 203 [M-Me-C,Hs=C=O]+ (49) 187 [M -C,H,CO,CH,] + (100). 159 [M-CC,H,CO-OCH,-CO] + (3) 144 [M-Me-C,H,-CO-OCH,-CO]’ (20). 115 [C,H,CO-OCHr] + (8). 57 [C,H,] + (8).
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