Four new ss-orcinol meta-depsides from Pertusaria and Siphula lichens

Article abstract of DOI:10.1071/CH99085

The new depsides decarboxyhypothamnolic acid [3-(2′,4′-dihydroxy-3′,6′-dimethylphenyloxycarbonyl)-2- hydroxy-6-methoxy-4-methylbenzoic acid] (4) and cryptothamnolic acid [3-(3′-formyl-2′-hydroxy-4′-methoxy6′-methylbenzoyloxy)- 4,6-dihydroxy-2,5-dimethylbe

Full text of DOI:10.1071/CH99085

C S I R O P U B L I S H I N G
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Aust. J. Chem., 1999, 52, 837-840
b
Four New -Orcinol meta-Depsides from
Pertusaria and Siphula Lichens
John A. Elix,A Caroline E. Barclay,A Judith H. Wardlaw,A
Alan W. Archer, B Sen-hua YuC and Gintaras Kantvilas D
A Department of Chemistry, The Faculties, Australian National University, Canberra, A.C.T. 0200.
B National Herbarium of New South Wales, Royal Botanic Gardens, Sydney, N.S.W. 2000.
C Department of Biology, Nanjing Normal University, Nanjing 210097, People’s Republic of China.
D Tasmanian Herbarium, G.P.O. Box 252-04, Hobart, Tas. 7001.
The new depsides decarboxyhypothamnolic acid [3-(2Ј,4Ј-dihydroxy-3Ј,6Ј-dimethylphenyloxycarbonyl)-2-
hydroxy-6-methoxy-4-methylbenzoic acid] (4) and cryptothamnolic acid [3-(3Ј-formyl-2Ј-hydroxy-4Ј-methoxy-
6Ј-methylbenzoyloxy)-4,6-dihydroxy-2,5-dimethylbenzoic acid] (6) have been detected in extracts of a Chinese
Pertusaria species together with hypothamnolic acid (3) and their structures implied by means of chromatographic
and spectroscopic comparisons with synthetic materials. Two further new depsides, neothamnolic acid [3-(5Ј,7Ј-
dihydroxy-6Ј-methyl-1Ј-oxo-1Ј,3Ј-dihydroisobenzofuran-4Ј-yloxycarbonyl)-2-hydroxy-6-methoxy-4-methylben-
zoic acid] (8) and lactothamnolic acid [3-(6Ј-formyl-5Ј,7Ј-dihydroxy-1Ј-oxo-1Ј,3Ј-dihydroisobenzofuran-4Ј-
yloxycarbonyl)-2-hydroxy-6-methoxy-4-methylbenzoic acid] (9), have been isolated from the lichen Siphula
ramalinoides, and the structure of these compounds followed from a combination of spectroscopic data, derivati-
zation and degradation experiments.
Introduction
New Depsides Present in the Pertusaria Species
para-Depsides and depsidones derived biosynthetically
from b-orsellinic acid moieties are common lichen metabo-
lites, but the related meta-depsides are much more restricted
in number with only five representatives known, namely
thamnolic acid (1),1 decarboxythamnolic acid (2),1
hypothamnolic acid (3),1 haemathamnolic acid (5)1 and dis-
sectic acid (7).2
In this paper, we describe the characterization of four such
new depsides, cryptothamnolic acid (6) and decarboxy-
hypothamnolic acid (4), present in a Chinese Pertusaria
species, and neothamnolic acid (8) and lactothamnolic acid
(9), present in the South American lichen Siphula
ramalinoides Nyl. ex Crombie.
Thin-layer chromatographic (t.l.c.), high-performance
liquid chromatographic (h.p.l.c.) and mass spectrometric
analysis of the total acetone extract of an undescribed
Chinese Pertusaria species indicated the presence of
hypothamnolic acid (3) as the major constituent together
with minor quantities of two related depsides. Subsequently
we have undertaken the syntheses of decarboxyhypotham-
nolic acid (4) and cryptothamnolic acid (6) and found the
chromatographic behaviour of these compounds to be iden-
tical with that of the minor depsides present in the lichen.
The successful synthesis of the depsides (4) and (6) was
achieved by direct condensation of the appropriately substi-
tuted aromatic carboxylic acids and phenols in the presence
© CSIRO 1999
Manuscript received 14 July 1999
0004-9425/99/090837
10.1071/CH99085

838
J. A. Elix et al.
of dicyclohexylcarbodiimide (Scheme 1). Potentially reac-
tive phenol and carboxy groups were protected by O-benz-
zylcryptothamnolate (16) respectively. Hydrogenolysis of
the esters (13) and (16) over palladized carbon produced the
depsides decarboxyhypothamnolic acid (4) and cryptotham-
nolic acid (6) in high yield. The isolation of (4) and (6) from
the lichen was not practicable given the minute quantities
available.
ylation.
We have successfully employed this route
previously in the synthesis of similarly substituted natural
depsides.1,2 The mononuclear precursors (10),1* (14)3† and
(15)1‡ had been synthesized previously, whereas 2,4-
dibenzyloxy-3,6-dimethylphenol (11) was obtained from the
corresponding aldehyde (12) by oxidation with m-chloroper-
benzoic acid (Scheme 1). Depside ester formation between
the acid (10) and phenol (11), and between the carboxylic
acid (14) and the phenol (15) was achieved by treatment with
dicyclohexylcarbodiimide and yielded benzyl 2Ј,4Ј-di-O-
benzyldecarboxyhypothamnolate (13) and benzyl 4Ј-O-ben-
New Depsides Present in Siphula
ramalinoides Nyl. ex Crombie
A preliminary chromatographic (t.l.c. and h.p.l.c.) and
mass spectrometric examination of an acetone extract of the
lichen Siphula ramalinoides indicated that this lichen con-
tained two depsides of unknown structure. A subsequent
preparative-scale extraction of this species followed.
However, fractional crystallization and preparative-layer
chromatography were unsuccessful in separating the two
compounds present. The 1H n.m.r. spectrum of this mixture
exhibited three C-methyl resonances (d 2.17, 2.47, 2.49),
two O-methyl resonances (d 4.05, 4.12), two methylene
signals (d 5.31, 5.32), two aromatic signals (d 6.62, 6.47), a
broad hydroxy resonance (d 8.31) and an aldehyde reso-
nance (d 10.37). This mixture of compounds did not exhibit
molecular ions in the positive-ion electron impact mass spec-
trum, but did exhibit fragment ions at m/z 226 and 209 as has
been observed for thamnolic acid (1)1 and hypothamnolic
acid (3),1 indicating that all four compounds probably con-
tained the same A-ring moiety. This was confirmed by
methanolysis of the mixture, whereupon the acid (17)§ was
formed but the B-ring derivatives could not be isolated. On
the basis of this spectroscopic and degradative data the ten-
tative structures of neothamnolic acid and lactothamnolic
acid were formulated as (8) and (9) respectively.
Although we were unable to separate the naturally occur-
ring mixture of (8) and (9), we were successful in separating
and characterizing the corresponding permethyl and acetyl
derivatives and in so doing confirming their assigned struc-
tures. Thus methylation of the mixture of (8) and (9) by
treatment with excess ethereal diazomethane for a period of
3 days afforded a mixture of the corresponding permethyl
derivatives which were separated by preparative-layer chro-
matography.
The faster moving band afforded the diester (18),¶
derived by cleavage of the A-ring moiety prior to, or during,
diazomethylation. The second band contained methyl
2,4Ј,6Ј-tri-O-methylneothamnolate (19), and the third band
methyl
2,4Ј,6Ј-tri-O-methyl-5Ј-C-methyllactothamnolate
(20). The structure of the permethyl derivatives (19) and
1
(20) followed from their respective H n.m.r. and mass
spectra. It is interesting that under these reaction conditions
the 5Ј-formyl group of lactothamnolic acid (9) underwent C-
methylation to give the corresponding methyl ketone (20).
The natural mixture of neothamnolic acid (8) and lac-
tothamnolic acid (9) was acetylated by treatment with acetic
* 3-Benzyloxycarbonyl-2-hydroxy-4-methoxy-6-methylbenzoic acid.
† 3-Formyl-2-hydroxy-4-methoxy-6-methylbenzoic acid.
‡ Benzyl 4-benzyloxy-2,5-dihydroxy-3,6-dimethylbenzoate.
§ 2-Hydroxy-6-methoxy-3-methoxycarbonyl-4-methylbenzoic acid.
¶ Dimethyl 2,4-dimethoxy-6-methylbenzene-1,3-dicarboxylate.

b-Orcinol meta-Depsides from Pertusaria and Siphula
839
Detection of New Depsides (4) and (6) by Comparative
Chromatography
The undescribed Pertusaria species was collected on bark, Lijiang,
Mt. Yulongshan, Yunnan Province, China, 3300 m, Jinong Wu &Aitang
Liu 82-195 (NNU). Comparative h.p.l.c. and t.l.c. indicated the pres-
ence of hypothamnolic acid (3) (major), decarboxyhypothamnolic acid
(4) (minor) [standard t.l.c. RF values:5,8 RF(A) 0.05; RF(B) 0.15; RF(C)
0.13; standard h.p.l.c.9,10 RI 0.15], and cryptothamnolic acid (6) (minor)
[standard t.l.c. RF values:5,8 RF(A) 0.38; RF(B) 0.32; RF(C) 0.40; standard
h.p.l.c.9,10 RI 0.25]. The chromatographic behaviour of the latter two
compounds was identical with that of the synthetic samples prepared
below. The h.p.l.c. apparatus was coupled to a photodiode array detec-
tor for ultraviolet spectroscopic comparisons. By this means the spectra
of the components eluting from the chromatogram were recorded and
computer-matched against a library of ultraviolet spectra recorded for
the authentic lichen metabolites under identical conditions. For the
above substances the correlation of the ultraviolet spectra was greater
than 99.9%. This composition was also consistent with the observed
lichen mass spectrum with prominent fragment peaks observed at m/z
362, 346, 209, 194, 193, 192, 191, 180, 168, 165 (Found: m/z (mol. wt)
362.0999. C18H18O8 requires mol. wt 362.1001. Found: m/z (mol. wt)
346.1051. C18H18O7 requires mol. wt 346.1052).
2,4-Dibenzyloxy-3,6-dimethylphenol (11)
A solution of 2,4-dibenzyloxy-3,6-dimethylbenzaldehyde (12)11 (0.5
g, 1.44 mmol) in anhydrous dichloromethane (50 ml) was added over 30
min to a stirred solution of m-chloroperbenzoic acid (3 mmol) in anhy-
drous dichloromethane (30 ml) at room temperature. The solution was
stirred for a further 1 h and then the solvent was evaporated under
reduced pressure. The residue was dissolved in ethyl acetate and the
solution washed repeatedly with 10% sodium hydrogen carbonate solu-
tion, with saturated brine and then dried (MgSO4). The crude formate
obtained on evaporation of the solvent was dissolved in methanol (30
ml) and the solution cooled to 0°. A solution of 10% aqueous potassium
hydroxide (20 ml) was then added with stirring under an atmosphere of
nitrogen. After stirring was continued for a further 45 min at this tem-
perature, the reaction mixture was poured into cold, dilute hydrochloric
acid, extracted with ethyl acetate and the extract washed with water,
brine and dried (MgSO4). The residue obtained on evaporation of the
solvent was purified by chromatography over silica gel using 10% ethyl
acetate/light petroleum as eluent. The major band afforded the phenol
(11) (0.24 g, 50%) as a colourless oil (Found: mol. wt 334.1570.
C22H22O3 requires mol. wt 334.1566). 1H n.m.r. (CDCl3) d 2.23, 2.25,
2s, Me; 4.88, 5.01, 2s, CH2; 5.24, s, OH; 6.55, s, H 5; 7.25–7.56, m, Ph.
Mass spectrum m/z 334 (M, 3%), 91 (100).
anhydride and concentrated sulfuric acid, and the mixture of
acetates so obtained was then methylated by reaction with
ethereal diazomethane. Preparative-layer chromatography
of the crude product afforded two major bands. The faster
moving band yielded methyl 2,4Ј,6Ј-tri-O-acetylneotham-
nolate (21) and the slower band methyl pentaacetyllacto-
thamnolate (22). Again the structures of these two
derivatives followed from their spectroscopic properties.
Neothamnolic acid (8) and lactothamnolic acid (9) are
further representatives of the b-orcinol meta-depsides, and
may arise biosynthetically from the more common deriva-
tives hypothamnolic acid (3) and thamnolic acid (1) by oxi-
dation of the 2Ј-methyl group and subsequent lactonization.
3-(2Ј,4Ј-Dihydroxy-3Ј,6Ј-dimethylphenyloxycarbonyl)-2-hydroxy-
6-methoxy-4-methylbenzoic Acid (Decarboxyhypothamnolic Acid) (4)
Experimental
A
solution of 3-benzyloxycarbonyl-2-hydroxy-4-methoxy-6-
The general experimental details have been described previously.4
methylbenzoic acid (10)1 (170 mg, 0.54 mmol), 2,4-dibenzyloxy-3,6-
dimethylphenol (11) (150 mg, 0.45 mmol) and dicyclohexylcarbodiimide
(120 mg, 0.58 mmol) in anhydrous toluene (3 ml) and 1,2-
dimethoxyethane (1 ml) was stirred at room temperature for 18 h. The
solid was filtered off, the filtrate concentrated and purified by radial
chromatography over silica gel using 2–10% ethyl acetate/light
petroleum as eluent. The major band afforded the depside ester (13)
(170 mg, 50%) as a colourless gum. 1H n.m.r. (CDCl3) d 2.20, 2.21,
2.54, 3s, ArMe; 3.87, s, OMe; 4.85, 5.07, 2s, ArOCH2; 5.41, s, CO2CH2;
6.32, 6.63, 2s, H 5,5Ј; 7.20–7.48, m, Ph.
Chromatography
The lichen fragments were freed as far as possible from obvious
organic substrate material and extracted with warm acetone for thin-
layer chromatography (t.l.c.) or with warm methanol for high-perfor-
mance liquid chromatography (h.p.l.c.). Compounds were identified
by t.l.c. by using the methods standardized for lichen products5–8 and by
h.p.l.c. with retention index values (RI) calculated from benzoic acid
and solorinic acid controls.9 For t.l.c. standard RF values were deter-
mined in three independent t.l.c. solvent systems: (A)
toluene/dioxan/acetic acid (180:45:5); (B) hexane/t-butyl methyl
ether/formic acid (140:72:18); (C) toluene/acetic acid (170:30). For
h.p.l.c. a Spectra System, a Phenomenex Hypersil 5C18 column (250
by 4.6 mm) and a spectrometric detector operating at 254 nm with a
flow rate of 1 ml/min were used. Two solvent systems were used: 1%
aqueous orthophosphoric acid and methanol in the ratio 3:7 (A) and
methanol (B). The run started with 100% A and was raised to 58% B
within 15 min, then to 100% B within a further 15 min, followed by iso-
cratic elution in 100% B for a further 10 min.
Asolution of the depside ester (13) (170 mg) in ethyl acetate (20 ml)
was stirred in an atmosphere of hydrogen with 10% palladized carbon
(25 mg) for 3 h. The catalyst was then filtered off and the solvent evap-
orated. The residue was crystallized from ethyl acetate to give decar-
boxyhypothamnolic acid (4) (85 mg, 87%) as colourless microcrystals,
m.p. 270° (dec.) (Found: C, 59.5; H, 5.2. C18H18O8 requires C, 59.7;
1
H, 5.0%). H n.m.r. (CDCl3) d 2.05, 2.16, 2.54, 3s, ArMe; 4.15, s, OMe;
6.27, 6.50, 2s, H 5,5Ј; 6.40, 7.30, 2s, 2Ј,4Ј-OH; 13.14, s, 2-OH. Mass
spectrum m/z 362 (M, 0.8%), 209 (18), 192 (14), 191 (100), 165 (15),
152 (14), 149 (31), 105 (13).

840
J. A. Elix et al.
5-(3Ј-Formyl-2Ј-hydroxy-4Ј-methoxy-6Ј-methylbenzoyloxy)-2,4-
The second band yielded methyl 2,4Ј,6Ј-tri-O-methylneothamnolate
dihydroxy-3,6-dimethylbenzoic Acid (Cryptothamnolic Acid) (6)
(19) (5.0 mg, 12%) as a colourless oil (Found: C, 60.4; H, 5.0.
C23H24O10 requires C, 60.0; H, 5.2%). H n.m.r. (CD3COCD3) d 2.27,
1
Asolution of 3-formyl-2-hydroxy-4-methoxy-6-methylbenzoic acid
(14)3 (80 mg, 0.38 mmol), benzyl 4-benzyloxy-2,5-dihydroxy-3,6-
dimethylbenzoate (15)1 (120 mg, 0.32 mmol) and dicyclohexylcarbodi-
imide (80 mg, 0.39 mmol) in anhydrous toluene (5 ml) and
1,2-dimethoxyethane (1 ml) was stirred at room temperature for 18 h.
The precipitate was filtered off, the filtrate concentrated and the residue
purified by radial chromatography over silica gel using 2–10% ethyl
acetate/light petroleum as eluent. The major band afforded the depside
2.51, 2s, ArMe; 3.84, 3.88, 3.91, 3.95, 4s, OMe; 4.09, s, CO2Me; 5.18,
s, CH2; 6.61, s, H 5. Found: mol. wt 459.1294. C23H23O10 requires mol.
wt 459.1291. Mass spectrum m/z 459 (M –1, 0.7%), 429 (15), 238 (29),
237 (100), 191 (22), 177 (10).
The third major band afforded methyl 2,4Ј,6Ј-tri-O-methyl-5Ј-C-
methyllactothamnolate (20) (2.1 mg, 5%) as a colourless gum (Found:
C, 59.3; H, 5.0. C24H24O11 requires C, 59.0; H, 4.9%). 1H n.m.r.
(CD3COCD3) d 2.49, 2.55, 2s, ArMe, COMe; 3.88, 3.91, 3.92, 3.96, 4s,
OMe; 4.16, s, CO2Me; 5.21, s, CH2; 6.61, s, H 5. Mass spectrum m/z
457 (M –OMe, 47%), 238 (100).
1
ester (16) (67 mg, 37%) as a colourless gum. H n.m.r. (CDCl3) d 2.16,
2.24, 2.53, 3s, ArMe; 3.94, s, OMe; 4.87, s, ArOCH2; 5.42, s, CO2CH2;
6.22, s, H 5Ј; 7.25–7.52, m, Ph; 10.28, s, CHO; 11.74, 12.78, 2s, bonded
OH.
Sequential Acetylation and Methylation of Mixture of (8) and (9)
A solution of the depside ester (16) (67 mg) in ethyl acetate (15 ml)
was stirred in an atmosphere of hydrogen with 10% palladized carbon
(25 mg) for 3 h. The catalyst was then filtered off and the solvent evap-
orated. The residue was crystallized from ethyl acetate to give cryp-
tothamnolic acid (6) (32 mg, 70%) as cream microcrystals, m.p.
211–213° (Found: C, 58.2; H, 5.0. C19H18O9 requires C, 58.5; H,
4.6%). 1H n.m.r. (CDCl3) d 2.10, 2.18, 2.55, 3s, ArMe; 3.99, s, OMe;
6.42, s, H 5Ј; 8.05, br, OH, CO2H; 10.29, s, CHO; 11.84, s, OH. Mass
spectrum m/z 346 (0.06%), 210 (10), 194 (11), 193 (100), 191 (10).
A mixture of neothamnolic acid (8) and lactothamnolic acid (9) (30
mg) was dissolved in acetic anhydride (5 ml), concentrated sulfuric acid
(1 drop) added and the solution stored at room temperature for 16 h.
Water (25 ml) was added and the solution stirred for 2.5 h. The mixture
was extracted with ethyl acetate and the combined extracts were
washed with water, brine and dried (MgSO4). Excess ethereal dia-
zomethane was added to the filtrate and the solution stored at room tem-
perature for 16 h. The solvent was then evaporated and the residue
applied to a silica gel plate and eluted with 50% ethyl acetate/light
petroleum. Two major bands developed.
The faster moving band afforded methyl 2,4Ј,6Ј-tri-O-acetyl-
neothamnolate (21) (2.0 mg, 5%) as a colourless gum (Found: C, 57.6;
H, 4.0. C26H24O13 requires C, 57.4; H, 4.4%). 1H n.m.r. (CD3COCD3)
d 2.12, 2.18, 2.29, 2.46, 2.59, 5s, ArMe, COMe; 3.90. 3.93, 2s, OMe;
5.19, s, CH2; 6.77, s, H 5. Mass spectrum m/z 513 (M –OMe, 0.5%),
471 (2.3), 191 (100).
Extraction of Siphula ramalinoides Nyl. ex Crombie
The lichen material was collected on soil, Isla Clarence, Tierra del
Fuego, Chile, S. Stenroos 2587, 18 January 1987 (H).
The dried lichen thallus (0.77 g) was extracted with anhydrous
acetone (100 ml) in a Soxhlet extractor for 36 h. The acetone extract
was then concentrated to give a mixture of neothamnolic acid (8) and
lactothamnolic acid (9) as a yellow oil (0.134 g, 17%). 1H n.m.r.
(CD3COCD3) d 2.17, 2.47, 2.49, 3s, ArMe; 4.05, 4.12, 2s, OMe; 5.31,
5.32, 2s, CH2; 6.62, 6.74, 2s, ArH; 8.31, br s, OH; 10.37, s, CHO. Mass
specrum m/z 226 (1%), 209 (12), 198 (2), 191 (32). This mixture could
not be resolved by t.l.c. Standard t.l.c. RF values:5,8 RF(A) 0.05; RF(B)
0.20; RF(C) 0.16. Standard h.p.l.c.9,10 for neothamnolic acid (8) RI 0.13;
lactothamnolic acid (9) RI 0.12.
The second band contained methyl pentaacetyllactothamnolate (22)
(3.2 mg, 7%) as a colourless gum (Found: C, 54.4; H, 4.0. C30H28O17
requires C, 54.5; H, 4.2%). 1H n.m.r. (CD3COCD3) d 2.07 (6H), 2.20,
2.31, 2.48, 2.58, 5s, ArMe, COMe; 3.90. 3.93, 2s, OMe; 5.22, s, CH2;
6.77, s, H 5; 8.02, s, ArCH. Mass spectrum m/z 629 (M –OMe, 15%),
628 (33), 615 (30), 614 (100).
Methanolysis of Mixture of (8) and (9)
Acknowledgment
A mixture of neothamnolic acid (8) and lactothamnolic acid (9) (29
mg) was boiled under reflux in anhydrous methanol for 2 h. The solvent
was evaporated and the residue applied to a silica gel plate
(20 by 20 by 0.1 cm) and eluted with 15% acetic acid/toluene. The
faster moving band afforded 2-hydroxy-6-methoxy-3-methoxycar-
bonyl-4-methylbenzoic acid (17) (5.1 mg, 30%) as colourless micro-
crystals, identical with authentic (synthetic)11 material (t.l.c., h.p.l.c., 1H
n.m.r., mass spectrum). 1H n.m.r. (CD3COCD3) d 2.32, s, ArMe; 3.84,
4.04, 2s, OMe; 6.57, s, H 5. Subsequent bands yielded mixtures of
products and could not be resolved.
We wish to thank the Australian Research Council for
generous financial support.
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6
7
A solution of a mixture of neothamnolic acid (8) and lactothamnolic
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diazomethane and the solution stored at room temperature for 3 days.
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9
petroleum.
Three major bands developed.
The faster
Feige, G. B., Lumbsch, H. T., Huneck, S., and Elix, J. A., J.
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n.m.r. (CD3COCD3) d 2.34, s, ArMe; 3.81, 3.83, 3.90, 3.91, 4s, OMe;
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