Lichen Substances VII. Identification of Orsellinate Derivatives from Lobaria linita

Tenuiorin, methyl gyrophorate (pseudocyphellarin) and methyl evernate were isolated from Lobaria linita. In addition, evidence (two-directional thin layer chromatography) was obtained for the presence of methyl lecanorate and methyl orsellinate, as well as of their respective parent compounds 4-0methylgyrophoric acid, gyrophoric acid, evernic acid, lecanoric acid and orsellinic acid in the same lichen. Several independent studies have been made recently on the chemical constituents of Lobaria linita (Culberson, 1969a; Yoshimura, 1971; Jordan, 1973). The only compound positively identified as a natural product was tenuiorin (Asahina & Yanagita, 1933; Culberson, 1969a). In addition, a number of chromatographically detectable phenolic spots were reported. The present paper deals with the identification of some of these constituents. MATERIAL AND METHODS Mass spectra (70 ev) were recorded on a Bell and Howell C.E.C. 21-110 instrument using a direct introduction probe, n.m.r. spectra on a Varian A-60A instrument, and i.r. spectra on a Perkin-Elmer 237 spectrophotometer. Two-directional thin layer chromatography (TLC) with the solvent systems benzene-dioxane acetic acid 90:24:4 (1st direction) and hexane-ether-formic acid (2nd direction) was carried out as described previously (Maass, 1975a). Either hand-coated silica gel G plates (0.8 mm thick) or Merck silica gel 60 F-254 plates (0.25 mm thick) were used (activated by heating in an oven at 1150 for at least 2 hrs.). After having been developed in the 1st direction, the chromatograms were exposed for 1 hr. to a stream of air of low relative humidity (30%7 or less) to drive off the solvent and avoid undue uptake of water which would interfere with separation of compounds in the 2nd solvent system. For identification of linitin A, linitin B and linitin C of Yoshimura (1971), one-directional chromatograms were developed in n-hexaneethyl acetate-formic acid 50:40:7, using ether extracts of Lobaria linita and reference compounds. Phenols were reacted with a diazotized reagent, and either Fast Bordeaux BD Salt (FB) or Fast Blue Salt B (FBL) were used. Colors were developed with a spray of aqueous sodium carbonate and were, for the individual compounds, as follows (FBL, Rf values in brackets): methyl evernate (0.71) dirty purple turning pink, tenuiorin (0.70) dirty purple turning orangish-pink, methyl orsellinate (0.65) dull reddish-brown to purplish-brown, methyl lecanorate (0.62) dirty purplish-red, evernic acid (0.59) cherry-red, methyl gyrophorate (0.58) dirty grayish-violet turning dirty purple, 4-0-methylgyrophoric acid (0.58) pinkish-red, orsellinic acid (0.51) dull reddish-brown to dirty brownish-red, lecanoric acid (0.49) dull reddish-brown to purplish and gyrophoric acid (0.49) dull brownish-red to purplish. Relative mobilities of linitin 1 National Research Council No. 14727. 2 Atlantic Regional Laboratory, National Research Council of Canada, Halifax, Nova Scotia, Canada B3H 3Z1 This content downloaded from 207.46.13.193 on Thu, 08 Sep 2016 04:19:23 UTC All use subject to http://about.jstor.org/terms 1975] MAASS: LICHEN SUBSTANCES 179 A, tenuiorin, and gyrophoric acid (see Yoshimura, 1971, table 2) identify linitin A with methyl gyrophorate. Linitins B and C were reported as having higher Rf values than tenuiorin. They do not give a positive color reaction with diazotized coupling reagents and thus are unlikely to be phenolic constituents. The main phenolic constituents of L. linita (tenuiorin and methyl gyrophorate) may also be detected by their strong violet-gray fluorescence under shortwave fluorescent light. In addition, a strongly grayish-violet fluorescent spot was sometimes found at an Rf of 0.92, and this is believed to be identical to linitin C. It gave a gray to grayish-violet color reaction when sprayed with 10%o sulfuric acid and heated but did not give a positive Liebermann reaction. The only material in extracts of L. linita that gave a color reaction after spraying with acetic anhydride and concentrated sulfuric acid in succession was associated with the methyl gyrophorate spot in this one-directional system. Specimens of Lobaria linita (Ach.) Rabh. from the following localities were investigated by two-directional TLC: CENTRAL JAPAN, Prov. Sinano, Yatugatake Mts., Mt. Takami-isi, Kurokawa 58287 (var. tenuior (Hue) Asah., us). USA, ALASKA, Umiat, along northern ridge of Colville River, Thomson & Shushan 5980 (us). ALASKA, Sitka, Mt. Cross, Evermann 205 det. Hale (us). CANADA, B.C., USA border near Skagway, Haynes Road, in sloping alpine meadows opposite a glacier, Maass 21.VI.1966. B.C., Cassiar, 1550 m alt., in dense moist forest of Abies lasiocarpa over mosses, Szczawinski 687-B (us). ALBERTA, Jasper Nat. Pk., Canadian zone on ridge opposite Angel Glacier, 1800 m alt., Imshaug 7043 (us). SWITZERLAND, Canton of Vallais, Zermatt, Gorner Gorge, Thomson & Frey 9394 (us). Lobaria linita (29.4 g air-dried) collected near Skagway was extracted with absolute ether to give ca. 560 mg of extractive. This was chromatographed on silicic acid with methylene chloride according to Maass (1975a) and yielded methyl evernate (18 mg), tenuiorin (300 mg) and methyl gyrophorate (27.2 mg after recrystallization from acetone). These compounds showed the following characteristics: Methyl evernate (M = 346), m.p. 143.5-144.50 (mixed m.p. with authentic methyl evernate undepressed); .max.EtOH 214 (e 47600), 269 (e 24200), 307 (E 14300); VmaxCHCl3 1694, 1665 s, 1615 s, 1573 m-s; 8 (DMSO-d6) 2.25, 2.39 (2 arom. CH3), 3.76 (1 OCH3), 3.82 (1 COOCH.), 6.37 (2 arom. H), 6.60 (2 arom. H), 10.27 (2 OH); m/e 346 (M+), 182, 165, 150, 122. Tenuiorin (M = 496), m.p. 1750 and 235-2400 dec.; XmaxEtOH 214 (e 13500), 268 (E 5800), 307 (e 3600); VmaxCHC13 1695, 1670 s, 1607 s, 1583 (sh), 1575 (sh), 1300 w; 8 (DMSO-d6) 2.27, 2.38, 2.40 (3 arom. CH3), 3.76 (1 OCH3), 3.82 (1 COOCH3), 6.38 (2 arom. H), 6.66 (multiplet, 4 arom. H), 11.33 (3 OH), M+ not observed; m/e 346, 182, 165, 150, 122. Methyl gyrophorate ( pseudocyphellarin, M = 482), m.p. 3000d. (with some carbonization and formation of a whitish sublimate), .maxEtOH 214 (e 62700), 272 (E 27300), 307 (E 17800); VnmaxCHC'3 1695, 1667, 1600 s; (DMSO-d6) 2.26 (1 arom. CH3), 2.38 (2 arom. CH3), 3.82 (1 COOCH3), 6.24 (2 arom. H), 6.66 (multiplet, 4 arom. H), 10.19 (3 hydrogen-bonded OH, the non-bonded OH was not observed), M+ not observed; m/e 332, 182, 151, 150, 122. RESULTS AND DISCUSSION By means of the two-directional TLC system adopted for the rapid identification of lichen substances (see Maass, 1975a) the following orsellinate type of compounds (Fig. 1) were detected in the ether extract of Lobaria linita: tenuiorin, methyl evernate, methyl gyrophorate (pseudocyphellarin), methyl lecanorate and traces of methyl orsellinate, as well as gyrophoric acid and traces of 4-0-methylgyrophoric acid, evernic acid, lecanoric acid and orsellinic aid. Tenuiorin, methyl gyrophorate and methyl evernate were subsequently obtained in a pure form and fully agreed with authentic specimens with regard to u.v., i.r., n.m.r. and mass spectra. These compounds cannot be derived by simple hydrolysis of any of the other constituents in the extract and thus are genuine natural products. The same applies to 4-0-methylgyrophoric acid and gyrophoric acid. Whereas under unfavorable conditions of work-up, evernic acid and methyl lecanorate might arise by hydrolytic decomposition of the methylated tridepsides, there is evidence to indicate their natural occurrence in L. linita. On one hand none of the methylated tridepsides nor, as a matter of fact, evernic acid and methyl lecanorate showed any sign This content downloaded from 207.46.13.193 on Thu, 08 Sep 2016 04:19:23 UTC All use subject to http://about.jstor.org/terms 180 THE BRYOLOGIST [Volume 78