Aust. J. Chem.

, 1975,28,1793-7

The Structure of Galbinic Acid.

A Depsidone from the Lichen
Usnea undulata

John A. Elix and Udomsri Engkaninan

Chemistry Department, Australian National University,
P.O. Box 4, Canberra, A.C.T. 2600.

Abstract
The depsidone, galbinic acid (or-acetylsalazinic acid) (4), has been isolated from the lichen Usnea
undulata together with usnic, norstictic and salazinic acids. The structure of the compound (4)followed
from a combination of spectroscopic and chemical data and was confirmed by comparison with a
synthetic sample. The acetylation of salazinic acid was further investigated.

The depsidones, that is the substituted derivatives of depsidone (2-(2'-hydroxy-

phenoxy)benzoic acid lactone or 11H-dibenzo[b,e][1,4]dioxepin-l l-one), are the
second largest group of lichen metabolites being found largely in these organisms with
several representatives also isolated from fungi.ls2
During a chemotaxonomic survey of some lichens of eastern A ~ s t r a l i a , ~it- was
~
observed that Usnea undulata Stirt. produced four major metabolites. This had been
noted previously7 and Asahina isolated the unknown 'galbinic a ~ i d 'from ~ , ~this lichen
but did not elucidate the structure of this compound. Subsequently we have under-
taken the large scale extraction of this lichen and followed this by fractional crystalliza-
tion and preparation layer chromatography of the extract. This led to the isolation
of the known derivatives, usnic acid (I), norstictic acid (2), salazinic acid (3) and 4 3 ' ) -
acetylsalazinic acid or galbinic acid (4).*
The structure of the last compound followed, initially, from the spectroscopic
properties. In particular, the p.m.r. spectrum was very similar to that of salazinic acid
but exhibited an additional C-methyl resonance (6 2.04) and a methylene signal

* Traditional names and numbering have been used for all the compounds mentioned here. Accord-
ing to IUPAC rules, norstictic acid should be called 1,4,10-trihydroxy-5,8-dimethyl-3,7-dioxo-1,3-
[1,4]benzodioxepn-l l-carbaldehyde.
dihydro-7H-isobenzofuro[4,5-b]
Dean, F.M., 'Naturally Occurring Oxygen Ring Compounds' (Butterworths: London 1962).
Culberson, C. F., 'Chemical and Botanical Guide to Lichen Products' (University of North Carolina
Press: Chapel Hill 1969); Supplement to Chemical and Botanical Guide to Lichen Products,
Bryologist, 1970,73, 177-377.
Cresp, T. M., Elix, J. A., Kurokawa, S., and Sargent, M. V., Aust. J. Chern., 1972,25, 2167.
Baker, C., Elix, J. A,, Murphy, D. P. H., Kurokawa, S., and Sargent, M. V., Aust. J. Bot., 1973,21,
137.
Elix, J. A., Ferguson, B. A., and Sargent, M. V., Aust. J. Chem., 1974,27,2403.
Elix, J . A,, Aust. J. Chern., 1975, 28, 849.
' Asahina, Y., J. Jap. Bot., 1963,38, 225; 1967,42, 321.
* Culberson, W.L., Arneu. J. Bot., 1961,48, 168.
 J. A. Elix and U. Engkaninan

(6 5.32), 0.59 p.p.m. downfield with respect to the corresponding signal of salazinic
acid.

HO' (2) R = X norsticfic acid

(3) R = OH salazinic acid
(4) R = OAc galbinic acid

Acetylation of (4) with acetic anhydride and a trace of sulphuric acid produced
hexaacetylsalazinic acid (5),9,10while acid-catalysed methanolysis of this depsidone
gave the diphenyl ether (6). The structure of these two derivatives was established by
comparison with authentic specimens obtained from salazinic acid (3) and thus verified
the overall substitution pattern of (4).

The structure of the depsidone (4) was eventually confirmed by its synthesis from
salazinic acid.ll Salazinic acid (3) has been acetylated under a variety of conditions
with varying degrees of s u c c e ~ s . ~Certainly,
-~~ the reaction of salazinic acid with
acetic anhydride and a trace of sulphuric acidg,10 proceeded readily, and led to the
formation of hexaacetylsalazinic acid (5). In several i n ~ t a n c e s , the
~ , ~treatment
~ of
salazinic acid with acetic anhydride alone, has been reported to give a triacetyl
derivative, tentatively formulated as cr(3'),2',4-triacetylsalazinic acid (7).12 We have
repeated this experiment and have shown that the major product is in fact cr(3'),cr(6'),4-
triacetylsalazinic acid (8). The low solubility of (8) in aqueous sodium hydrogen
carbonate solution and the downfield shift of the methine and methylene protons in
the p.m.r. spectrum of this compound were consistent with this structural assignment.
Moreover, (8) exhibited a significant peak at mle 221 in the mass spectrum, so given
the normal A-ringfragmentation of depsidones upon electron impact,13this established
the position of the third acetyl group.
Asahina, Y., and Asano, J., Ber. Deut. Chem. Ges., 1933, 66, 689.
lo Briner, G. P., Gream, G. E., and Riggs, N. V., Aust. J. Chem., 1960, 13, 277.
Asahina, Y., and Tukamoto, T., Ber. Deut. Chem. Ges., 1934, 67, 963.
" Asahina, Y., and Shibata, S., 'Chemistry of Lichen Substances' p. 131 (Japanese Society for the
Promotion of Science: Tokyo 1954).
l 3 Huneck, S., Djerassi, C., Becher, D., Barber, M., Ardenne, M. von, Steinfelder,K., and Tiimmler, R.,
Tetrahedron, 1968, 24, 2707.
Structure of Galbinic Acid

If salazinic acid was treated with acetic anhydride at a lower temperature or if

benzyl salazinate (9) (prepared by benzylation of salazinic acid with phenyldiazo-
methane) was boiled in acetic anhydride, the diacetyl compound, a(3'),a(b1)-diacetyl-
salazinic acid (lo), was obtained in addition to (8). The structure of the compound
(10) followed from a similar combination of evidence to that described for the triacetyl
compound (8).

R' R2 R3 R4
(7) Ac OAc H Ac
(8) H OAc Ac Ac
(10) H OAc Ac H
(11) H SMe H H

Galbinic acid (4) (as a-acetylsalazinic acid) has been prepared previously simply by
refluxing salazinic acid in glacial acetic acid.ll In our hands this proved highly in-
efficient due to the very low solubility of salazinic acid under these conditions. How-
ever, the addition of a portion of dimethylformamide or dimethyl sulphoxide overcame
this difficulty and galbinic acid (4) was readily obtained. The thiomethyl derivative
(11) was obtained as a byproduct when dimethyl sulphoxide was used as cosolvent.
The synthetic sample of galbinic acid (4) was shown to be identical with the natural
metabolite isolated from Usnea undulata.

Experimental
The general experimental details have been published previ~usly.~

Extraction of Usnea undulata Stirt, f. fruiticans Asahina7

The lichen material was collected on bark of Acacia trees in forest, Batehaven, N.S.W., J. A.
Elix-496 (CANB).
The dried thalli (50 g) were extracted with anhydrous ether in a Soxhlet extractor (30 h). Sub-
sequently the thalli were extracted with acetone for a similar period of time. The ethereal solution
was evaporated and the residue extracted with hot benzene (25 ml). The benzene-insoluble residue
(1.5 g) was shown by t.1.c. to consist of three major components: norstictic, salazinic and galbinic
acids. The benzene-soluble fraction was diluted with ethanol whereupon usnic acid (0.28 g, 0.6%)
crystallized and was identified by comparison with authentic material (m.p., mass spectrum, t.1.c.).
On concentration of the acetone extract salazinic acid (0.5 g) crystallized out and was filtered off
(identified by comparison with authentic sample). The residue (0.6 g) obtained on evaporation of the
filtrate was shown to contain usnic acid in addition to the three components mentioned above and was
combined with that material (total yield 2.1 g). A portion of this mixture (0.1 g) was applied to a
silica gel plate and eluted with 15% acetic acid-toluene. Four major bands developed. The initial
band contained traces of usnic acid, while the second band yielded norstictic acid (10 mg, overall
yield 0.4%) identified by comparison with authentic material (m.p., t.l.c., mass spectrum, p.m.r.).
The slowest moving band yielded a further quantity of salazinic acid (28 mg, overall yield 2.2%).
 J. A. Elix and U. Engkaninan

The third Sand yielded galbinic acid (59 mg, overall yield 2.6%) which crystallized from ethyl acetate-
light petroleum as colourless crystals which were identified by comparison with the authentic (syn-
thetic) material (infrared, p.m.r., mass spectra, RF in three independent solvent systems).

Synthesis of Galbinic Acid

(A) A mixture of salazinic acid (200 mg), dimethylformamide (1.0 ml) and glacial acetic acid
(4.0 ml) was stirred under reflux for 2.5 h. The mixture was then cooled, diluted with water and
extracted with ethyl acetate. The ethyl acetate extract was washed with water, dried (MgSO,) and
the solvent evaporated. The residue was adsorbed on a silica gel plate and eluted with 15% acetic
acid-toluene. One major band developed and this yielded galbinic acid (4) (120 mg, 54%) which
crystallized from ethyl acetate-light petroleum in colourless crystals which decomposed at 260-265"
without melting ( l k 7 discolours 160-22OC, dec. 260"; lit." dec. 275-276" with COz liberation)
(Found: C, 53.2; H, 3.8. Calc. for C20H14011,H,0: C, 53.6; H, 3.6%). v,, (Nujol) 3500
(broad OH), 1740, 1730, 1650, 1620 (C=O) cm-'; A,, 241 ( E 29000), 321 (8500) nm; p.m.r. [(CD3)2-
SO-CDCl,] 6 2.04 (3H, s, CH,CO), 2.56 (3H, s, ArCH,), 5.32 (2H, s, ArCH2), 6.76 (lH, s, ArH),
7.01 (IH, br s, OCHOH) and 10.59 (lH, s, CHO); mass spectrum mle 152 (29%), 151 (32), 60 (52),
45 (75),44 (loo), 43 (77). RF values:14 benzene (180), dioxan (45), acetic acid ( 9 , RF 0.31 ; n-hexane
(130), diethyl ether (80), formic acid (20), RF 0.12; toluene (200), acetic acid (30), RF 0' 17.
(B) A mixture of salazinic acid (320 mg), dimethyl sulphoxide (3 ml) and glacial acetic acid (6 ml)
was refluxed for 1 h, cooled, and diluted with water. The solid which separated out was filtered,
dissolved in a minimum amount of ethyl acetate and applied to a silica gel plate. On elution with 15%
acetic acid-toluene two major bands developed. The faster moving band was identified as the thio-
methyl derivative (1 1) (92 mg, 21%) which crystallized from ethyl acetate-light petroleum as colourless
crystals, m.p. 215" (dec.) (Found: C, 54.1; H, 3.9; S, 7.4. C19H1409Srequires C, 54.5; H, 3.4;
S, 7.7%). P.m.r. [(CD,),SO-CDC13] 6 2.08 (3H, s, SCH3),2.39 (3H, s, ArCH,), 3.88 (2H, s, CHZS),
6.78 (IH, s, ArH), 6.93 (lH, br s, OCHOH), 10.58 (IH, s, CHO); mass spectrum mle418 (M+,41%),
371 (100). The slower moving band yielded galbinic acid (4) (210 mg, 59%), identified by comparison
with authentic material.

Acetylation o f Galbinic Acid

A mixture of galbinic acid (76 mg), acetic anhydride (0.8 ml) and concentrated sulphuric acid
(1 drop) was stirred at room temperature for 16h. Water (10 ml) was then added and the mixture stirred
for a further 3 h. The solid which separated was filtered off and recrystallized from ethanol to give
hexaacetylsalazinic acid (5) (96 mg, 83%) as colourless crystals, m.p. 146-147" (lit.93'0 178") (Found:
C , 54.3; H, 4.4. Calc. for C30H26017: C, 54.7; H, 4.0%). v,,, (Nujol) 1780 (lactone C=O),
1752 (acetyl and depsidone C=O) cm-'; p.m.r.15 (CDCI,) 6 1.99,2.11,2.16,2.24,2.40,2.46,2.81
(each 3H, s, CCH,), 5.12,5.51 (each lH, d, ArCHJ, 7.04 (lH, s, ArH) and 7.94,8.05 (each lH, s,
OCHO); mass spectrum mle 658 (M+ <I%), 43 (100). Hexaacetylsalazinic acid (5) was also
obtained by treating salazinic acid with acetic anhydride and concentrated sulphuric acid under
similar conditions.

Methanolysis of Galbinic Acid (4) and Salazinic Acid (3)

A mixture of galbinic acid (4) (1 10 mg), methanol (2 ml) and concentrated sulphuric acid (1 drop)
was stirred under reflux for 24 h. The mixture was then cooled, diluted with ethyl acetate and poured
into water. The organic layer was separated, washed with water, dried (Na2S04) and the solvent
evaporated. The residue was chromatographed on a silica gel plate, 15% acetic acid-toluene being
used as the eluent. The major band yielded the diphenyl ether (6) (70 mg, 61%) which crystallized
from methanol to form colourless crystals, m.p. 139", which were unstable and decomposed on stand-
ing (no satisfactory microanalysis obtained); p.m.r. (CDCI,) 6 2.37 (3H, s, ArCH,), 3.23, 3.51,
3.53 (each 3H, s, OCH,), 4.81 (2H, s, ArCH2), 5.50 (lH, s, 0CHOCH3), 6.74 (lH, s, ArH) and
10.32 (lH, s, CHO). This diphenylether (6) was also obtained by treating salazinic acid under similar
conditions.

l4 Culberson, C. F., J. Chromatogr., 1972, 72, 113.

l5 Huneck, S., and Linscheid, P., 2. Naturforsch., B, 1968, 23, 717.
Structure of Galbinic Acid

~(3'),a(6'),4-TriacetylsalazinicAcid (8)
A mixture of salazinic acid (300 mg) and acetic anhydride (9 ml) was boiled under reflux for 0.5 h.
The cooled solution was diluted with 2.5% sulphuric acid and extracted with ethyl acetate. The
ethyl acetate extract was washed with water, dried (Na2S04)and the solvent evaporated. The residue
was applied to a silica gel plate and eluted with 3% acetone<hloroform. Two major bands developed.
The faster moving band yielded the triacetylsalazinic acid (8) (230 mg, 58%) which crystallized from
ethanol to form colourless crystals, m.p. 193-195" (dec.) (lk7s8205-206') (Found: C, 55.8; H, 3.7.
Calc. for C24H18013: C, 56.0; H, 3.5%). v, (Nujol) 1787 (lactone C=O), 1767, 1744 (acetyl and
depsidone C=O), 1642 (aldehyde C=O) cm-I; p.m.r. (CDCI,) G 1.96, 2.14, 2.46, 2.62 (each 3H, s,
CCH,), 5.32 (2H, br, ArCH2), 6.86 (IH, s, ArH), 7.55 (lH, s, OCHOAc) and 10.39 (lH, s, CHO);
mass spectrum mle 514 (M+ < 1%), 221 (5), 43 (100).

Benzyl Salazinate (9)

A suspension of salazinic acid (0.50 g) in acetone (50 ml) was treated with an excess of ethereal
phenyldiazomethane, and the mixture stood overnight. The solvent was then evaporated and the
residue extracted with hot ethyl acetate. The unchanged salazinic acid was filtered off and the filtrate
concentrated to give benzyl salazinate (9) (0.40 g, 65%) as a colourless solid, m.p. 191-194" (dec.)
(Found: C, 62.6; H, 3.7. Cz,HlsOlo requires C, 62.8; H, 3.8%). P.m.r. [(CD3)2SO-CDC13]6
2.52 (3H, s, ArCH,), 4.88 (2H, s, ArCH20H), 5.42 (2H, s, C02CH2), 6.76 (lH, s, H5), 7.40 (5H,
s, 0CH2C6H,) and 10.25, 10.36 (each lH, s, CHO); mass spectrum mle 478 (M+ < 1%), 91 (100).

a(3'),cr(6')-Diacetylsalazinic Acid (10)

(A) A solution of benzyl salazinate (300 mg) in acetic anhydride (15 ml) was stirred at 115" for
0 . 5 h. The cooled solution was diluted with 2.5O/, sulphuric acid and extracted with ethyl acetate.
The ethyl acetate solution was washed with water, dried (Na2S04) and evaporated. The residue
obtained was applied to a silica gel plate and eluted with ljO/, acetic acid-toluene. Two major bands
developed, the faster moving of which yielded traces of cr(3'),~(6'),4-triacetylsalazinic acid (8). The
slower band contained cr(3'),a(6')-diacetylsalazinic acid (10) (70 mg, 25%) which crystallized from ethyl
acetate-light petroleum to form colourless crystals, m.p. 183-185" (dec.) (Found: mol. wt, 472.0640.
C22H16012requires mol. wt, 472.0642). v,,, (hujol) 1780, 1748, 1650, 1622 (C=O) cm-l; p.m.r.
(CDCI,) G 1 .94,2.40,2.54 (each 3H, s, CCH,), 5.26 (2H, s, Arc&), 6.74 (1H, s, ArH), 7.68 (1H,
br s, OCHOAc), 10.56 (lH, s, CHO) and 12.63 (lH, br s, bonded OH); mass spectrum mle 472
(Mt < I % ) , 43 (100).
(B) A mixture of salazinic acid (200 mg), acetic anhydride (6 ml) and dimethylformamide (2 ml)
was stirred at 65" until all salazinic acid had dissolved (35 min). The mixture was then cooled, diluted
with water and extracted with ethyl acetate. The ethyl acetate extract was washed with water, dried
(Na2S04), concentrated, and the residue adsorbed on a silica gel plate. Elution with 15:; glacial
acetic acid-toluene gave 3 bands. The second band yielded a(3'),a(6')-diacetylsalazinic acid (10)
(39 mg, 16%). The first and the third bands contained a(3'),~(6'),4-triacetylsalazinic acid (8) (120 mg,
45%) and a(3')-acetylsalazinic acid (4) (50 mg, 22%), respectively.

Manuscript received 24 March 1975