Phytochemivtry, Vol. 34, No. 5, pp. 1436.1437, 1993 003 1 9422i93 $6.00 + 0.00 Printed in Great Britan. ,T: 1993 Pergamon Press Ltd

SESQUITERPENE LACTONES OF CORDIFOLZA, SUBGENUS AUSTROMONTANA

IRMGARD MERFORT* and DETLEF WENDISCH~

Institut fiir Pharmazeutische Biologie der Heinrich-Heine-Universitlit Diisseldorf, Geb. 26.23, Universitltsstrasse 1, D-40225 Diisseldorf 1, Germany; tzentralbereich Forschung und Entwicklung der Bayer AC. D-5090 Leverkusen-Bayerwerk, Germany

(Received in revised form 6 May 1993)

Key Word Index-h&a cordifolia; ; Compositae; sesquiterpene lactones; pseudoguaian- olide.

Abstract-From A. cordijblia, in addition to two known compounds, a sesquiterpene lactone, new as a natural compound, was identified. The structures were elucidated by spectroscopic methods.

INTRODUCTION those from 2 in the ‘HNMR spectrum, except for the signals of H-7, H-8, H-13a, H-13b, H-14 and H-15 (see Arnica cordifolia Hooker is probably the most wide- spread Arnica in western ranging from Table 2). The signals of H-8 and H-7 appeared at lower the Yukon Territory south to northern New Mexico and field than those of 2 leading to the conclusion that 3 has a central , with disjunct populations in Ontario cis-fused lactone ring [7]. This was confirmed by the value of J.,ar,801, and [ 11. According to refs [ 1, 21 it represents (7.9 Hz), which is 8 Hz or smaller in the ancestral species in subgenus Austromontana, one of pseudoguaianolides with cis-annelation of the lactone the five subgenera of the genus Arnica L., which gave rise ring [7, 81. Additionally, the allylic couplings between to the rest of the subgenus [2]. Morphological studies H-7 and the exocyclic methylene protons were smaller and the flavonoid profile have supported this hypothesis than 3 Hz, which is characteristic for a c&fused lactone [3]. We now report the identification of three sesquiter- ring according to the so-called Samek-rule for 7,8 fused pene lactones in leaves of A. cordijblia. These natural pseudoguaianolides [9, lo]. The 13C NMR data of 3 were compounds are valuable tools in chemotaxonomic stu- close to those of 2 (see Table 2) except for the signals of dies [4]. C-7, C-8 and C-9, thus confirming the changed stereo- chemistry at C-8. Compound 3, new as a natural product, has been earlier prepared synthetically [ 11, 121. RESULTS AND DISCUSSION The isomeric compounds 2 and 3 differ in their con- The methylene chloride extract from leaves of formations. According to ref. [6] compound 2 shows a A. cordifolia afforded the pseudoguaianolides l-3. Com- conformation belonging to the pseudorotational chair pound 1 was identified as carabrone, a component of a domain. However, the geometry fits neither a pure chain mixture, by GC, GC-MS and TLC analysis and com- nor a pure twist-chair pattern. The c&isomer (3) has a parison with the authentic compound. A second com- twist-boat conformation [6]. pound was identified as 2,3_dihydroaromaticin [S] by 2D Within the subgenus Austromontana, A. cordifolia is the COSY and NOESY experiments and the “CNMR second species containing pseudoguaianolides [ 133. spectrum. A HETCOR experiment showed that six car- Compounds 2 and 3 are found for the first time in the bon assignments given in the literature [6] should be genus Arnica, moreover, 3 is new as a natural compound. revised as shown in Table 1. Within the genus Arnica the xanthanolide 1 has only been Compound 3 was obtained as part of a mixture with 2. found in A. longiji%a [14] which is regarded as a highly The only separation possible was by GC. The mass individualized species with its own section in subgenus spectrum after GC-MS analysis from 3 ([Ml+ at m/z 248) was identical with that of 2, thus supporting the presence of two isomers. It was identified by NMR techniques as e. i-::tl, 2,3_dihydroaromatin. In the 13C/lH two dimensional correlated spectrum most signals of 3 overlapped with

*Author to whom correspondence should be addressed. 2 3

1436 Short Reports 1437

Table 1. ‘%NMR spectral data EXPERIMENTAL of compounds 2 and 3 (in CDCI,, material. were collected in July 1988 at 6 ppm) Rabbit Ears Pass, Colorado, U.S.A. A voucher specimen C 2* 3 is deposited at the herbarium of the University of Mis- souri, Biology Department at Kansas City, Missouri, 48.6 49.4 U.S.A. 2 24.1 22.7 General. NMR: 500 MHz, GC-MS: EI, 70 eV; GC: 3 35.2 35.5 25 m, 0.25 mm i.d., carrier gas N,, 1.3 ml min-‘, FID, 4 222.6 219.3 temp. program: 150-270”, rate 10“ min- l. 5 50.0 50.1 Isolation and identi$cation. Dried, powdered leaves 6 34.5 34.9 (46.Og) of A. cordijblia were extracted with CH,Cl,. The 7 44.7 39.3 8 80.8 77.1 MeOH-soluble part (1.6 g) of the extract (1.9 g) was first 9 44.0 37.1 sepd by CC (Sephadex LH-20) with MeOH. Further CC 10 29.6 28.9 (silica gel) using n-hexane with increasing amounts of 11 140.3 139.5 EtOAc from 5 to lOO%, next with Me&O from 20 to 12 169.8 169.6 lOO%, finally MeOH gave, among other fractions, one 13 120.0 123.3 containing l-3. Compound 2 was crystallized from this Me-5 22.0 19.3 fraction (22 mg). Further sepn followed by CC (silica gel) Me-10 20.0 17.8 with toluene and increasing amounts of EtOAc from 2 to 10 % yielded 51 mg of a mixture of 2 and 3 (25 % 3 by GC *Assignments are based on 13C- analysis). Repeated MPLC (RP-18), HPLC and crystal- DEPT and HETCOR. lization attempts did not lead to any sepn. A further fraction gave 8 mg of a mixture, containing 1, besides two further sesquiterpene lactones. Table 2. ‘HNMR spectral data of compounds 2 and 3 (in CDC&*, 6 ppm) Acknowledgements-We thank Mrs E. Miiller for tech-

H 2 3 nical assistance, Dr S. J. Wolf (University of Missouri, Biology Department, Kansas City, Missouri, U.S.A.) for 1 1.94 WI t collecting leaves of A. cordifolia and Dr U. Matthiesen 2u 2.10 m 2.07 m (Spurenelementelabor der Medizinischen Einrichtunaen 28 1.61 m 1.51 m der Heinrich-Heine-Universitiit Diisseldorf) for taking 3a 2.17 m t the mass spectra. 38 2.45 rn 6a 248 dd 5 6fi 1.51 dd t REFERENCES 7 2.82 m 3.06 m 1. Wolf, S. J. and Denford, K. E. (1983) Biochem. Syst. 8 4.28 ddd 4.76 ddd Ecol. 11, 111. 9a 1.42 dd 1.61 m 2. Maguire, B. (1943) Brittonia 4, 386. 98 2.45 m 2.42 m 10 1.94 m 1.94 m 3. Wolf, S. J. and Denford, K. E. (1984) Rhodora 86,239. 13a 6.17 d 6.27 d 4. Seaman, F. C. (1982) Bat. Rev. 48, 121. 13b 5.50 d 5.67 d 5. Bohlmann, F. and Mahanta, P. K. (1979) Phyto- 14 1.09 d 1.13 d chemistry 18, 887. 15 1.04 s 0.93 s 6. Appendino, G., Calleri, M., Chiari, G., Gariboldi, P. and Menichini, F. (1986) Gazz. Chim Ital. 116, 637. J(Hz): 2: 1,2a _ 5; 1,2/I: u 11.5; 2a,3c( = 9.2; 28,3a 7. Bohlmann, F., Mahanta, P. K., Jakupovic, J., = 1.4; 3a,3B = 19.8; 6a,68 = 14.9; 617 = 6.2; 6&7 Rastogi, R. C. and Natu, A. A. (1978) Phytochemistry =11.8; 7,8=9.3; 7,13a=3.6; 7,13b=3.2; 8,9a 17, 1165. =12.1; 8,9/?=3.1; 9a,Bj3= 12.5; 9a,lO= 11.8; lo,14 8. Herz, W., Aota, K., Holub, M. and Samek, Z. (1970) =6.1. 3: 7,8: 7.9; 7,13a=2.5; 7,13b: 2.0; 8,9a: 3.1; 8,9/?: 11.6; 10,14: 6.8. J. Org. Chem. 35, 2611. *Assignments confirmed by HETCOR (2 and 3) 9. Samek, Z. (1970) Tetrahedron Letters 671. and ‘H-H COSY (2). 10. Samek, Z. (1978) Collec. Czech. Chem. Commun. 43, tSignals overlapped\nth those from compound 2. 3210. 11. Lansbury, P. T. and Vacca, J. P. (1982) Tetrahedron 3fI, 2797. 12. Ortega, A., Romo de Vivar, A. and Romo, J. (1968) Chamissonis [2]. Before conclusions concerning sys- Can. J. Chem. 46, 1539. tematic and evolutionary relationships of Arnica species 13. Ebert, M. and Willuhn, G. (1987) Pharm. Weekbl. 9, within the subgenus Austromontana can be arrived at, 232. further studies of sesquiterpene lactones from other 14. Willuhn, G. and Herrmann, H.-D. (1976) Arch. Arnica species are necessary. Pharm. 309, 333.