Journ . Haltori Bot. Lab. No. 63 : 351- 355 (Dec. 1987)

STEROIDS AND NORTRITERPENOIDS FROM LICHENS'

R. TABACCHI\ G. TSOUPRAS2 AND S. HUNECK3

ABSTRACT. The following lichens have been analysed for and nortriterpenoids by GCI MS : Evernia mesomorpha, Lecanora stenotropa, Leptogium saturninum, Nephroma he/veticum, Par­ melia omphalodes, Ramalina terebrata, Rhizoplaea ehrysoleuea, Umbilicaria deellssata, and Usnea antare/iea. Fourteen steroids and three nortriterpenoids have been found.

I NTRODUCTION Only relatively few lichens were analysed for steroids, triterpenoids and simple aliphatic compounds, contrary to the numerous phenolic compounds. Reasons for this are a) the low concentration of these metabolites which ranges in the case of steroids about 0.01 ~;.; , b) the small amount of plant material available, and c) that these metabolites are mostly complex mixtures. The following steroids and nortriterpenoids with known structure have been iso­ lated from lichens: cholestan-3,B-ol, , , , , lichesterol, ergost-5-en-3,B-ol, , ergosterol peroxide, 24-ethyIcholestan-3,B-ol, cIionasterol, sitosterol, 24-ethyIcholest-7-en-3,B-ol, 24-methyIcholestan-3,B-ol, cam­ pesterol, 24-methyIcholest-7-en-3,B-ol (Culberson et al. 1977), peroxyergosteryl di­ varicatinate (Bruun & Motzfeldt 1974), 24-methyIcholesta-5, 22-dien-3,B-ol (Safe et at. 1975), (Nicollier et al. 1979), 21 a H-30-nor-hopan-3, 22-dione (Nicollier et al. 1979), and 21a H-30-nor-hopan-22-one (Hveding-Bergseth et al. 1979). We investigated the "" fraction of 9 lichens by gas liquid chromatography­ mass spectrometry (GC-MS) and report here on the results.

R ESULTS AND DISCUSSION The neutral parts of the lichen extracts were chromatographed on Silica Gel and the "steroid" fractions anlysed by GC-MS or MS. Table 1 shows the compounds detected in the lichen species investigated. 9(l1)-Dehydroergosterolperoxide and ergosterol peroxide have been separated by

1 Contribution No. 148 of the series "Lichen Substances". No. 147 : Huneck, S., A. Morales Men­ dez & K. Kalb. 1987. The chemistry of Dirinaria and Pyxine species (Pyxinaceae) from South Ame­ rica. Journ. Hattori Bot. Lab. 62 : 331-338. 2 Institute of Chemistry, University of Neuchatel, Avenue de Bellevaux 51, CH-2000 Neuchiitel, Switzerland. 3 Institute of Plant Biochemistry of the Academy of Sciences of the GDR, GDR-4050 HallejSaale, Weinberg, German Democratic Republic. 352 Journ. Hattori Bot. Lab. No. 63 198 7

TABLE 1. Steroids, nor-triterpenoids and n-nonacosan-lO-ol from lichens.

Lichen

Compound

Brassicasterol + + + + + + Campesterol + + + + + + + + Cholesterol + + + + + 9{l1)-Dehydroergosterolperoxide + Episterol + + + Ergosta-7,22-dien-3p-ol + + + Ergosta-5, 7, 9(1 I), 22-tetraen-3,, -ol + + + + + + + Ergosterol + + + + + + + + Ergosterolperoxide + 24-Ethylcholesta-5, 22-dien-2p-ol + + + + + + + + 24-Ethylcholest-7 -en-3p-ol + Lichesterol + + + + + + + 19-nor-Ergosta-5, 7, 9(1\), 22-tetraen-3,3-ol + Sitosterol + + + + + + + + 21 a H-30-nor-Hopan-3, 22-dione I + 21 pH-30-nor-Hopan-3,22-dione II + 22, 29, 30-trinor-Hopan-3, 21-dione [J[ + n-Nonacosan-lO-ol +

HPLC (column: Nucleosil-RP 18, eluent: methanol: water= 94 : 6) and identified by NMR and MS (Gunalitaka et al. 1981). The steroids have been identified on the basis of their mass spectra and GLC retentions time. with LJ7 double bond exhibits typical fragments due to the loss of the side chain. The shorter GLC retention time of LJ5 than of .:17 sterols made it possible to distinguish between both types (Knights 1967, Itoh et al. 1983). nor-Triterpenoids I, II and III have been separated by HPLC (column: Nucleosil RP-18, eluent: gradient methanol- water, 80 to 100 %) and identified by NMR and MS (Ageta & Arai 1983, Nicollier et al. 1979, Lehn & Ourisson 1962). The mass spectra of I, 11 and III are similar (M+ : 426, 426, 398) and suggest the structure of hopane or lupane derivatives. The NMR spectra show only differences in the region of the methyl groups (Table 2). The comparison with the lH-NMR spectra of 30-nor-lupan-3, 20-dione (IV), ob­ tained from lupenone shows differences only for the C-27 and C-28 methyl groups. We did not succeed in the synthesis, from 20-hydroxy-hopan-3-one, of the analogous hopane derivative, probably because the configuration of the molecule hinders the formation of the complex with osmium tetroxide - sodium periodate. 9(1l)-Dehydroergosterolperoxide. MS, m/z 426 (M+, 10 %), 411 (7), 408 (5), R. TABACCHI et al.: Steroids and nortriterpenoids from lichens 353

TABLE 2. 'H-NMR chemical shifts of the methyl groups of the nor-triterpenoids I, n, Ill, and IV. Chemical shift of the methyl groups Compound C-23 C-24 C-25 C-26 C-27 C-28 C-29 I 0.935 1.028 1.077 1.021 0 .953 0 .703 2 . 175 II 0.935 1.028 1.075 1.010 0 .940 0.585 1.150 III 0.935 1.027 1.077 1.027 0 .935 1.077 IV 0.933 1.026 1.072 1.030 0 .965 0.790 2.170

394 (36),376 (34),361 (8),299 (3), 251 (14), 107 (12), 95 (14), 81 (30),69 (90),55 (lOO). 'N-NMR (0 of the methyl groups compared to ergosterolperoxide): C-1 8: 0.738 (0.825), C-19: 1.1 (0.895), C-21: 0.920 (0.914), C-26: 0.822 (0.822), C-27: 0.840 (0.840), C-28: 1.009 (l.008). Ergosta-7,22-dien-3,B-ol. MS, m/z 398 (M+, 32 %),380 (5), 383 (12), 300 (7),271 (100), 255 (12), 213 (10), 119 (16), 105 (22), 91 (28), 69 (30), 55 (33). Ergosta-5, 7, 9(11), 22-tetraen-3,B-yl-trimethylsilylether. MS, m/z 466 (M+, 18 %), 451 (5),376 (57),361 (14), 325 (5), 277 (7), 251 (100),249 (20), 230 (7), 209 (27), 195 (23), 157 (14), 129 (18), 69 (70), 55 (50). 19-nor-Ergosta-5, 7, 9(10), 22-tetraen-3,B-ol. MS, m/z 380 (M+, 68 %), 365 (5), 362 (28), 347 (3), 319 (8), 282 (5), 253 (22), 238 (25), 237 (100), 228 (15), 213 (27), 195 (47), 183 (35), 181 (45), 167 (27), 165 (30), 141 (32), 128 (20). 19-nor-Ergosta-5, 7, 9(10), 22-tetraen-3,B -yl-trimethylsilylether. MS, m/z 452 (M+, 10 %), 437 (5), 362 (lOO), 237 (92), 221 (12), 210 (52), 195 (55), 183 (47), 181 (30), 169 (27), 155 (20), 141 (40), 128 (12),109 (11). 21a H-30-nor-Hopan-3, 22-dione (I). MS, m/z 426 (M+, 60 %), 411 (30), 393

(10),383 (20, [M-CHa-CO-] +), 205 (30, oW), 191 (100, ~o), 173 (15),

:! O ~.: .....?o . H H I : ; o~ / 0 / II

III IV 354 Journ. Hattori Bot. Lab. No. 63 1 987

163 (30), 149 (28), 121 (25), 107 (30), 95 (45),81 (44),67 (50), 55 (35), 43 (80). 21p H-30-nor-Hopan-3, 22-dione (Il). MS, m/z 426 (M+, 35 %), 411 (10), 393

(8), 383 (50, [M-CH3- CO-] +), 205 (30, o.o=r ), 191 (100, :Q1rO), 173 (55), 163 (30), 149 (28), 121 (25), 107 (28), 95 (45), 81 (44),67 (50),55 (30), 43 (80). 22,29, 30-trinor-Hopan-3, 21-dione (Ill). MS, mj z 398 (M+, 100 %), 383 (20), 365 (5), 355 (25), 220 (5), 205 (80), 191 (20), 177 (28), 163 (30), 149 (25), 107 (30), 81 (60), 67 (80), 55 (75). 30-nor-lupan-3, 20-dione (IV). MS, m j z 426 (M +, 80 %), 411 (8),408 (16),393 (7),383 (18), 365 (4), 340 (20),231 (17),205 (54),191 (20),177 (26),163 (37),135 (30), 135 (30), 121 (44), 107 (65), 95 (80), 93 (69), 81 (100),69 (65), 67 (75), 55 (60), 43 (90). Table 1 shows that brassicasterol, campesterol, ergosta-5, 7, 9(11), 22-tetraen-3j3- 01, ergosterol, 24-ethy1cholesta-5, 22-dien-3j3-01, lichesterol, and sitosterol are the more abundant sterols in the lichens investigated. Remarkable is the occurrence of the nor­ triterpenoids I, 11, and III in Rhizoplaca chrysoleuca and it would be of interest to look for these or similar compounds in the other Rhizoplaca species.

EXPERIMENTAL

Instruments used for the analyses: lH-NMR: Bruker WP-200, 200 MHz, solvent: CDCI 3• MS: NERMAG R-30-10 MS - MS triple-quadrupole with SIDAR-data-system; spectra at 70 eV. GLC: DANI 6500, column: SE 54 fused silica, 25 m, 0.23 mmg). The air dried and pulverized lichens were extracted with diethyl ether, the neutral parts of the extracts chromatographed on Silica Gel, the "steroid" fractions eluted with a n-hexane - diethyl ether gradient and crystallized from CHCl3-methanol. Evernia mesomorpha Nyl. Mongolian Peoples Republic, Chovsgol Aimak, Alag Erdene Somon, at the west bank of the Chovsgollake on Larix sihirica; leg. et det. S. Huneck, 14. 7. 1983 . From 365 g lichen 5 mg (0.0013%) steroid mixture in plates of m.p. 124-126°. Lecanora stenotropa Nyl. German Democratic Republic, on copper slate near Eisleben; leg. et det. S. Huneck, 17. 6. 1984. From 890 g lichen 0.02 g (0.002%) steroid mixture of m.p. 120-126°. Leptogium saturninum (Dickson) Nyl. Mongolian Peoples Republic, Bulgan Aimak, Gurvanbulag Somon, Chogno-tarna-uul (Cecerleg Mountains), on rocks; leg. S. Huneck, 26. 6. 1983, det. T. Ahti, 20. 12. 1984. From 52 g lichen 0.034 g (0.06 %) steroid mixture of m.p. 120-160°. Nephroma helveticum Ach. Mongolian Peoples Republic, ChovsgOl Aimak, Alag Erdene Somon, on rocks at the northern slope of the Sumber-uul; leg. et det. S. Huneck, 19. 7. 1983. From 93 g lichen 0.Ql g (0.01 %) steroid mixture of m.p. 130-134°. Parmelia omphalodes (L.) Ach. Mongolian Peoples Republic, Tov Aimak, Mongon­ mort Somon, Chentej, on rocks; leg. S. Huneck, 6. 8. 1983, det. T. Ahti, 20. 12. 1984. From

365 g lichen after chromatography and elution with n-hexane: Et 20 = 49: 1 0.01 g (0.0027%) n-nonacosan-1O-01 in needles of m.p. 76-78° (from n-hexane). Oxidation with lones' reagent in gave n-nonacosan-lO-one in plates of m.p. 70-72° (from acetone), IR, ~~; cm-I:

1700. Further elution of the column with n-hexane: Et 20 = 1 : 1 yielded the "steroid" fraction R. T ABACCH et al.: Steroids and nortriterpenoids from lichens 355

(0.005 g, 0.0013 %) of m.p. 124-126°. Elution with Et20 gave finally a mixture (0.003 g, 0.0008 %) of ergosterol peroxide and 9(l1)-dehydroergosterolperoxide in needles of m.p. 164- 166° (from methanol). Ramalina ferebrafa Hook. f. et Tay!. South Orkney Islands, Signy Island, North Point; leg. et del. R.I. Lewis Smith, Febr. 1981. From 50 g lichen 0.002 g (0.004 %) steroid mixture of m.p. 122-130°. Rhizoplaca chrysoleuca (Smith) Zopf. Mongolian Peoples Republic, Archangai Aimak, Ondor Dlan Somon, on rocks near the canon of the Tschulut gol; leg. et det. S. Huneck, 30. 6. 1983. From 189 g lichen a mixture of the nortriterpenoids J, IJ, and III (0.014 g, 0.007 %) in needles of m.p. 230- 236° (from CHCJ 3-methanol). Umbilicaria decussafa (ViII.) Zahlbr. Antarctic Peninsula, Adelaide Island, Rothera Point; leg. et det. R. 1. Lewis Smith, March 1981. From 233 g lichen 0.1 g (0.0042%) steroid mixture of m.p. 139- 141 °. Usnea anfarctica Du Rietz. South Orkney Islands, Signy Island, Berntsen Point; leg. et det. R. 1. Lewis Smith, Febr. 1981. From 200 g lichen 0.02 (0.01 %) steroid mixture of m.p. 120-126°.

ACKNOWLEDGEMENT. R. T. and G. T. thank the Swiss National Science Foundation (Project No. 2.241-0.84) for financial support.

REFERENCES

Ageta, H. & Y. Arai. 1983. Fern constitutents : pentacyclic tritcrpenoids isolated from Polypodium niponicllm and P. formosanulIl. Phytochemistry 22 : 1801-1808. Bruun, T. & A.-M. Motzfeld. 1974. 5a, 8a-Peroxyergosteryl divaricatinate from Haematomma ventosum. Acta Chem. Scand. B 29 : 274-275. Culberson, C. F., W. L. Culberson & A. Johnson. 1977. Second Supplement to "Chemical and Botanical Guide to Lichen Products". The American Bryological and Lichenological Society, SI. Louis. Gunalitaka, A. A. L., Y. Gopichand, F. G. Schmitz & c. Djerassi. 1981. Minor and trace sterols in marine Invertebrates. 26. Isolation and structure elucidation of nine new 5a, 8a-epidioxy sterols from four marine organisms. J. org. Chem. 46 : 3860-3866. Hveding-Bergseth, N., T. Bruun & H. Kj9sen . 1983. Isolation of 30-nor-21a-H-hopan-22-one (iso­ adiantone) from the lichen Platismatia glauca. Phytochemistry 22: 1826-1827. Itoh, T. , D. Sica & C. Djerassi. 1983. Minor and trace sterols in marine Invertebrates. Part 35 . Isola­ tion and structure elucidation of seventy-four sterols from the sponge Axinella cannabina. J. Chem. Soc., Perkin I: 147-153. Knights, B. A. 1967. Identificat ion of plant sterols by combined GLC/mass spectrometry. J. Gas Chromatog. 5: 273-282. Lehn, J.-M. & G. Ourisson. 1962. Resonance magnetique nucleaire de produits naturels. I. Introduc­ tion general. Triterpenes de la serie du lupane: les groupes methyles. Bull. Soc. chim. France : 1137-1142. Nicollier, G., R. Tabacchi, J. Gavin, J. L. Breton & A. G. Gonzalez. 1979. Triterpenes de la "mousse de chene" (Evernia prunaslri. (L.) Ach.). Helv. chim. Acta 62 : 807-810. Safe, S. , L. M. Safe & W. S. G. Maass. 1975. Sterols of three lichen species: , Lobaria scrobiculata and Usnea longissima. Phytochemistry 14: 1821-1823.