Chemistry of the lichen type common in southern Vietnam pptx

Doctoral Thesis

CHEMICAL STUDY OF COMMON LICHENS IN
THE SOUTH OF VIETNAM



















Le Hoang Duy

Department of Organic Chemistry

Graduate School of Kobe Pharmaceutical University
Kobe Pharmaceutical University


March 2012


Content page

List of abbreviations i
List of figures iv
List of photos v
List of tables v
Chapter 1: General introduction 1
1.1. The lichen and usage of lichens 1
1.2. Lichen substances 2
1.3. Cultivation of lichen mycobionts 3
1.4. Vietnamese lichen 5
1.5. Research scope and objectives 5
Chapter 2: Lichen substances from the lichen thalli of Parmotrema mellissii and
Rimelia clavulifera 6
2.1. Chemical investigation of the lichen thalli of P. mellissii 7
2.1.1. Mono-aromatic compounds 8
2.1.2. Depsides 10
2.1.3. Depsidones and Isocoumarin derivatives 11
2.1.4. Other lichen substances 28
2.2. Chemical investigation of the lichen thalli of R. clavulifera 30
Chapter 3: Secondary metabolites from the cultured lichen mycobionts 33
3.1. Chemical investigation of the cultured mycobionts of Graphis vestitoides 33
3.2. Chemical investigation of the cultured mycobionts of Sacographa tricosa 44

3.3. Chemical investigation of the cultured mycobionts of Pyrenula sp. 58
Chapter 4: Biological activity of isolated compounds 77
4.1. Inhibitory effect on mammalian DNA polymerase activity 77
4.2. Inhibitory effect on cancer cell growth 81
Chapter 5: Conclusions 82
Acknowledgment 86
Experimental section 87
References 129
List of compounds

-i-

List of abbreviations

1D one dimensional
2D two dimensional
Ac acetyl
alt. altitude
aq. aqueous
ax axial
br broad
calcd calculated
CC silica gel column chromatography
CD circular dichroism
COSY homonuclear shift correlation spectroscopy
d doublet
dd doublet of doublets
ddd doublet of doublets of doublets
dddd doublet of doublets of doublets of doublets
dec. decomposed

DEPT distortionless enhancement by polarisation transfer
DMF N,N-dimethyl formamide
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
dq doublet of quartets
dt doublet of triplets
dtd doublet of triplets of doublets
dTTP 2'-deoxythymidine 5'-triphosphate
EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
EDTA ethylenediaminetetraacetic acid
eq equatorial
EI-MS electron-impact ionization mass spectrum
HMBC heteronuclear multiple bond correlation spectroscopy
HOBT 1-hydroxybenzotriazole

-ii-

HPLC high performance liquid chromatography
hr hour
HR-APCIMS high resolution atmospheric pressure chemical ionization mass
spectrum
HR-EIMS high resolution electron-impact ionization mass spectrum
HR-ESIMS high resolution electrospray ionization mass spectrum
HR-SIMS high resolution secondary ion mass spectrum
HSQC heteronuclear single quantum correlation spectroscopy
IR infrared spectrophotometry
lit. literature
m multiplet
Me methyl
min minutes

mp. melting point
MPA methoxyphenylacetic acid
MS mass spectrum
MTPA 2-methoxy-2-trifluoromethylphenylacetic acid
NMR nuclear magnetic resonance
NOESY nuclear Overhauser enhancement spectroscopy
PGME phenylglycine methyl ester
ppm parts per million (chemical shift value)
Prep. HPLC preparative high performance liquid chromatography
Prep. TLC preparative thin-layer chromatography
PyBOP benzotriazolyloxytri(pyrrolidinyl)phosphonium hexafluorophosphate
rel. int. relative intensity
rt room temperature
q quartet
qd quartet of doublets
quint quintet
ROESY rotating-frame Overhauser enhancement spectroscopy
s singlet
sh shoulder

-iii-

SIMS secondary ion mass spectrum
t triplet
td triplet of doublets
tdd triplet of doublets of doublets
tdt triplet of doublets of triplets
TLC thin-layer chromatography
TMS tetramethylsilane
UV ultraviolet

-iv-

List of figures
Page
Fig. 1. The structure of first known lichen substances 2
Fig. 2. Characteristic lichen substances 3
Fig. 3. Ahmadjian’s method for isolating lichen mycobionts by means of spores 3
Fig. 4. Selected metabolites isolated from the cultured lichen mycobionts 4
Fig. 5. Selected bioactive lichen substances isolated from Parmotrema species 6
Fig. 6. Extraction and isolation procedure for P. mellissii 7
Fig. 7. Tautomeric interchange of α-alectoronic acid (11) 13
Fig. 8. Chiral HPLC of 18 18
Fig. 9. Proposed stereochemistry of spiro-ring system of 22 25
Fig. 10. Extraction and isolation procedure for R. clavulifera 30
Fig. 11. Selected secondary metabolites from the thalli and cultured mycobionts of Graphis
species 33
Fig. 12. Extraction and isolation procedure for cultured mycobionts of G. vestitoides 35
Fig. 13. MTPA ester of 38 38
Fig. 14. PGME method for determination of absolute configuration of carboxylic acid 41
Fig. 15. Extraction and isolation procedure for cultured mycobionts of S. tricosa 45
Fig. 16. Determination of absolute configuration by MPA esters 48
Fig. 17. Configuration of compound 48 50
Fig. 18. Metabolites from thalli and cultured mycobionts of Pyrenula species 58
Fig. 19. Extraction and isolation procedure for cultured mycobionts of Pyrenula sp. 59
Fig. 20. Determination of absolute configuration of 66 69
Fig. 21. Determination of absolute configuration of 69 73
Fig. 22. Proposed biosynthesis of pyrenulic acids and related compounds 75
Fig. 23. Structure of reported bioactive metabolites 77
Fig. 24. Selected compounds for bio-assay 78

Fig. 25. Inhibitory effects of isolated compounds on calf DNA polymerase α 79
Fig. 26. Inhibitory effects of isolated compounds on rat DNA polymerase β 80
Fig. 27. Inhibitory effects of isolated compounds on human DNA polymerase κ 80
Fig. 28. Inhibitory effects of isolated compounds on HCT116 cultured cell growth 81

-v-

List of photos

Page
Photo 1. Growth forms of lichen 1
Photo 2. The thalli of foliose lichens P. mellissii and R. clavulifera 6
Photo 3. G. vestitoides thalli and its cultured mycobionts 34
Photo 4. S. tricosa thalli and its cultured mycobionts 44
Photo 5. Pyrenula sp. thalli and its cultured mycobionts 59



List of tables

Page
Table 1.
1
H- and
13
C-NMR spectroscopic data of 11, 11a and 11b in CDCl
3
14
Table 2.
1

H- and
13
C-NMR spectroscopic data of 12 and 12a in CDCl
3
15
Table 3.
1
H- and
13
C-NMR spectroscopic data of 15-17 in CDCl
3
19
Table 4.
1
H-NMR spectroscopic data of 19-22 in CDCl
3
21
Table 5.
13
C-NMR spectroscopic data of 19-25 in CDCl
3
22
Table 6.
1
H-NMR spectroscopic data of 23-25 in CDCl
3
27
Table 7.
1
H- and

13
C-NMR spectroscopic data of 42 and 42m 43
Table 8.
13
C-NMR spectroscopic data of 47, 48, 52-54 and 58 in CDCl
3
51
Table 9.
1
H-NMR spectroscopic data of 48, 52-54 in CDCl
3
52
Table 10.
1
H-NMR spectroscopic data of 63-66 in CDCl
3
64
Table 11.
13
C-NMR spectroscopic data of 63, 64 and related compounds in CDCl
3
65
Table 12.
13
C-NMR spectroscopic data of 65-70 in CDCl
3
70
Table 13.
1
H-NMR spectroscopic data of 67-70 74

-1-
Chapter 1: General introduction

1.1. The lichens and usage of lichens
The lichens are symbiotic organisms, usually composed of a fungal partner
(mycobiont) and one or more photosynthetic partners (photobionts), which is most often
either a green alga or cyanobacterium. About 17,000 different lichen taxa, including
16,750 lichenized Ascomycetes, 200 Deuteromycetes, and 50 Basidiomycetes have
been described world-wide.

The photobionts produce carbohydrates by photosynthesis
for themselves and for their dominant fungal counterparts (mycobionts), which provide
physical protection, water and mineral supply. Based on this association, lichens have
adapted to extreme ecological conditions, being dominant at high altitudes, in Arctic
boreal and also tropical habitats, and colonized a wide range of different substrata, such
as rocks, bare ground, leaves, bark, metal, glass. Lichens are traditionally divided into
three growth morphological forms: these are the crustose, foliose and fruticose types
(Photo 1).
1-3)







Photo 1. Growth forms of lichen
Lichens have been used by humans for centuries as food,

4)
as source of dye,
5)
as raw
materials in perfumery and for therapeutic properties in folk medicine. The fragrance
industry uses two species of lichen Evernia prunastri var. prunastri (oakmoss) and
Pseudevernia furfuracea (treemoss). About 700 tons of oakmoss are currently processed
every year by French producers.
6,7)
Several lichen extracts have been used for various
remedies in folk medicine, such as Lobaria pulmonaria for lung troubles, Xanthoria
parientina for jaundice, Usnea spp. for strengthening hair, Cetraria islandica (Iceland
moss) for tuberculosis, chronic bronchitis and diarrhea.
4,8)
The screening tests with
lichens have indicated the frequent occurrence of metabolites with antioxidant,
antibiotic, antimicrobial, antiviral, antitumor, analgestic and antipyretic properties.
9-11)

Crustose
Foliose
Fruticose

-2-
These usages of lichen are limited to folk medicine, perfume and dying industry,
although manifold biological activities of lichen metabolites have been recognized with
potential applications in medicine, agriculture and cosmetics industry.
11,12)

1.2. Lichen substances
Lichens are one of the most important sources of biologically active compounds
other than plants. The chemistry of lichen was attractive the chemists from the early
time of organic chemistry. The chemical aspect of lichen substances was published by
Zopf in early 19
th
century. The lichen substances first known in their structure were
vulpinic acid (1) and lecanoric acid (2) (Fig. 1). The structure of most lichen substances
remained unknown till the studies of Asahina and Shibata in early 20
th
century. The
development of TLC and HPLC in 1960s, together with modern spectroscopic methods
led to the isolation and identification of many new lichen substances.
13)


Fig. 1. The structure of first known lichen substances

Recently, over 800 lichen substances were isolated and classified in many classes:
aliphatic acids, γ-, δ- and macrocyclic lactones, monocyclic aromatic compounds,
quinones, chromones, xanthones, dibenzofurans, depsides, depsidones, depsones,
terpenoids, steroids, carotenoids and diphenyl ethers.
3,13,14)
Among them, depsides,
depsidones and dibenzofurans are unique to lichens (Fig. 2). Depsides are formed by
condensation of two or more hydroxybenzoic acids whereby the carboxyl group of one
molecule is esterified with a phenolic hydroxyl group of a second molecule. Depsidones
have an ether linkage in addition to the ester linkage of the depsides, resulting in a rigrid
polycyclic system.
12)

The main natural roles of lichen substances, although they are not all well
understood yet, include: protection against a large spectrum of viral, baterial and
protozoan parasites, against animal predators such as insects and nematodes and against

-3-
plant competitors; defence against environmental stress factors such as ultraviolet rays
and excessive dryness; physiological regulation of lichen metabolism, such as the ability
to increase the algae cell wall permeability for increasing the flux of nutrients to the
fungal component.
3,14)


Fig. 2. Characteristic lichen substances

1.3. Cultivation of lichen mycobionts
Lichens are often immersed in rock or bark substrata and grow very slowly, so it is
difficult to collect large scale of lichen biomass. Even though the manifold activities of
lichen metabolites have now been recognized, their therapeutic potential has not been
fully exploited yet and thus remains pharmaceutically unexploited. Therefore,
laboratory cultures of lichen mycobionts provide a means by which lichen secondary
metabolites can be produced for pharmaceutical purposes.

Fig. 3. Ahmadjian’s method for isolating lichen mycobionts by means of spores
15)
1, 2) Discharg spores from the lichen. 3, 4) Transfer a block agar with germinated
spores to a culture tube.

Numerous lichens and lichen mycobionts have been cultivated over the past 30
years. The method for isolating lichen mycobionts into culture by means of spores was

-4-
developed in the 1960’s by Ahmadjian (Fig. 3). Lichen-forming fungi have gained a
notoriety for being difficult to isolate and grow in pure culture; their slow growth rates
in particular have presented a major obstacle to physiological investigations of axenic
states. The majority of studies on isolated mycobionts have been undertaken with the
aim of investigating either lichen resynthesis and thallus development under laboratory
conditions or secondary metabolite production.
15)


Fig. 4. Selected metabolites isolated from the cultured lichen mycobionts

Lichen-forming fungi have been shown to retain in axenic the capacity to
biosynthesize secondary products found in the lichenised state.
16)
In some cases, the
metabolites produced in the greatest abundance might differ from those found in the
lichen.
17)
Crittenden et al. reported on the isolation of 1,183 species of mycobionts from
lichens.
18)

The application of tissue cultures of lichens and the cultivation of lichen thalli
in vitro have been described by Yamamoto et al.
19)
and Yoshimura et al.
20)
Härmälä et al.
cultivated the photobionts of some species of Cetraria, Cladina and Cladonia, but
detected no phenol carboxylic acids.
21)
It is generally thought that only the mycobionts
are able to synthesize typical lichen substances. However, the mycobiontic cultures do
not always synthesize the same metabolites as the lichen themselves, but have an ability
to produce substances which are structurally related to fungal metabolites (Fig. 4).
22-28)

From the view-point of evolution, the origin of isolated mycobionts might be the same

-5-
as that of free-living fungi. In the symbiotic state with photobionts, the original
metabolic ability of mycobionts might be suppressed by any action of the photobiont,
but expressed in the isolated mycobionts.

1.4. Vietnamese lichen
Vietnam has a tropical monsoon climate which is favorable for diverse tropical
lichens, but the lichen flora of Vietnam has so far attracted little attention. Taxa reported
previously from Vietnam were mostly collected in the north (Tokin) and in central
Vietnam (Annam). In 2006, the total lichen flora of Vietnam was reported 275 species,
122 of which are remarked as new records from Vietnam. The lichen flora of Vietnam
was estimated at least 1,000 species.
29)

Recently, Giao published a survey of the lichens
collected in Western Highlands of central Vietnam and reported a list of 83 macrolichen
species, including 61 species new records for Vietnam.
30)
Previous studies on the
Vietnamese lichens focused mainly on their taxonomy but not on chemical constituents.

1.5. Research scope and objectives
From my interest in the diversity and biological activities of lichen substances,
phytochemical studies on Vietnamese lichens were undertaken.
The major aim of this thesis is to
1. Investigate the lichen substances from the macrolichens collected in the Western
Highlands of Vietnam (ca. 1,500 m alt.) to isolate novel and/or bioactive
compounds.
2. Investigate the chemical constituents of cultured mycobionts which were
discharged from the crustose lichens collected in different habitats (ca. 90 –
1,500 m alt.) in the South of Vietnam.
3. Evaluate the bioactive action of isolated metabolites on mammalian DNA
polymerases activity and cancer cell growth.


-6-
Chapter 2: Lichen substances from the lichen thalli of Parmotrema mellissii and
Rimelia clavulifera

The family of Parmeliaceae comprises more than 2,400 species in about 85 genera,
are foliose lichens widely distributed in tropical regions of the world.
31,32)
Some of the
genera Parmotrema and Rimelia of this family have been studied on phyto-biochemical

properties and showed satisfactory results. Depside, depsidone and xanthone dimer
derivative (Fig. 5) isolated from various species of Parmotrema exhibited anti-
inflmammatory and anti-tubercular activities.
33,34)


Fig. 5. Selected bioactive lichen substances isolated from Parmotrema species
The lichen species Parmotrema mellissii (C.W. Dodge) Hale and Rimelia
clavulifera (Räsänen) Kurok. (Photo 2), which are widespread in the Langbiang Plateau
(Dalat city, Vietnam) and have not been studied on their chemical constituents, were
chemically investigated.

P. mellissii R. clavulifera
Photo 2. The thalli of foliose lichens P. mellissii and R. clavulifera
1 cm


-7-

2.1. Chemical investigation of the lichen thalli of P. mellissii
The air-dried thalli of the foliose lichen P. mellissii were extracted with acetone.
The acetone extract was then separated and purified by column chromatography and
prep. TLC to yield twenty five lichen substances (3-27) (Fig. 6). Among them, five
depsidones (15, 20, 23-25) and three isocoumarins (17, 21 and 22) were new
compounds.

The lichen Parmotrema mellissii
(60 g dry thallus)
Acetone extract
(8.41 g)

Acetone (3
x 1 l)
CC/CHCl
3
-MeOH
MeOH (0%)
Fr-IV
(1.62 g)
Fr-III
(2.19 g)
(3-50%)
11 (1.32 g)
13 (798.4 mg)
p
TLC
A CHCl
3
B CHCl
3
- MeOH (99:1), (85:5), (9:1)
C n-Hexane - Et
2
O (1:1), (3:7)
D Toluene - AcOH (20:3)
Fr-I
(1.25 g)
Fr-II
(3.27 g)
(1%)
3 (2.0 mg)

4 (4.6 mg)
6 (42.7 mg)
7 (1.7 mg)
8 (6.8 mg)
9 (737 mg)
10 (95.2 mg)
19 (19.6 mg)
2
2 (22.0 mg)
26 (22.5 mg)
27 (4.8 mg)
1
5 (65.6 mg)
18 (66.2 mg)
2
5 (2.9 mg)
CC/CHCl
3
-MeOH
pTLC A-C
CC/CHCl
3
-MeOH
CC/CHCl
3
-MeOH
MeOH (0%)
Fr-IIc
(2.17 g)
(1-2%)

Fr-IIa
(417 mg)
Fr-IIb
(539 mg)
(0%)
pTLC B-D
5 (10.2 mg)
12 (327.2 mg)
2
0 (21.6 mg)
2
1 (25.0 mg)
2
3 (9.1 mg)
2
4 (8.1 mg)
pTLC B-D
11 (1.21 g)
12 (365.7 mg)
13 (21.3 mg)
14 (139.0 mg)
16 (43.9 mg)
1
7 (13.7 mg)
pTLC B-D
(2%)

Fig. 6. Extraction and isolation procedure for P. mellissii

-8-

2.1.1. Mono-aromatic compounds

Methyl orsellinate (3)
Compound 3 was isolated as a colorless
crystalline solid. Its HR-EIMS exhibited molecular
formula of C
9
H
10
O
4
. The UV spectrum showed
maxima at 215, 263 and 304 nm. The IR spectrum
showed absorption bands at 3366 (broad, hydroxyl
groups), 1700 (carbonyl group), 1654 and 1620
(aromatic ring) cm
-1
. Its
1
H-NMR spectral features indicated the presence of a pair of
meta-coupled aromatic protons at δ
H
6.22 and 6.27 (each 1H, d, J=2.5 Hz), a methoxyl
(δ
H
3.92) and a methyl (δ
H
2.49) groups, and a chelated phenolic hydroxyl group at δ
H

11.72. The
13
C-NMR spectrum showed signals corresponding to nine carbons, including
a carbonyl (δ
C
172.1), two oxygen-bearing aromatic carbons (δ
C
160.3 and 165.4), two
quaternary aromatic carbons (δ
C
105.5 and 144.0), two CH (δ
C
101.3 and 111.3), a
methoxyl (δ
C
51.9) and a methyl (δ
C
24.3) carbon. The position of substituted functional
groups was determined by the combination of 2D NMR spectra (COSY, NOESY,
HSQC and HMBC). Accordingly, the structure of 3 was determined as methyl 2,4-
dihydroxy-6-methylbenzoate which has the trivial name of methyl orsellinate.
35)

n-Butyl orsellinate (4) and ethyl orsellinate (5)
The molecular formula of 4 was identified as C
12
H
16
O
4

, that
is, C
3
H
6
more than that of methyl orsellinate (3). The NMR
spectral features of 4 were similar to those of 3, but 4 showed
signals of n-butoxyl group instead of methoxyl group as seen in
3. This was confirmed by the HMBC correlation from
oxygenated methylene protons at δ
H
4.34 (t, J=6.5 Hz, H
2
-1′) to
carbonyl carbon at δ
C
171.8 (C-7). Therefore, the structure of 4 was determined to be n-
butyl orsellinate.
35)
Similarly, the structure of 5 differed from 3 in the presence of
ethoxyl group [-CH
3
: δ
H
1.41 (t, J=7.0 Hz); -OCH
2
: δ
H
4.40 (q, J=7.0 Hz)] in place of
methoxyl group. Accordingly, 5 was elucidated as ethyl orsellinate.

35)

CH
3
COOR
OHHO
4 : R = n-C
4
H
9
5 : R = C
2
H
5
1
2
6
7
8
4

-9-

Methyl β
ββ
β-orsellinate (6)
The HR-EIMS of 6 indicated the molecular formula of
C
10
H

12
O
4
, i.e. a CH
2
group more than that of 3. The NMR
spectral features of 6 were similar to those of 3 except for the
presence of an additional methyl group (δ
H
2.10, δ
C
7.7) and
absence of an aromatic proton. Moreover, the HMBC
correlation from the methyl group (δ
H
2.10, H
3
-9) to C-2, 3 and 4 suggested the
structure of 6 to be methyl β-orsellinate.
13)


Methyl haematommate (7)
The molecular formula of 7 was established as
C
10
H
10
O
5

by HR-EIMS. The
1
H-NMR spectrum of 7
showed six singlets for two hydrogen-bonded phenolic
hydroxyl groups at δ
H
12.42 and 12.89, a formyl proton at
δ
H
10.34, an aromatic proton at δ
H
6.30, a methoxyl and a
methyl group. Its
13
C-NMR spectrum showed 10 carbon
signals including a methyl, a methoxyl, a methine, a formyl, a carbonyl and five
quaternary carbon signals. These spectral features resembled those of 6, except for the
presence of formyl group at the C-3 position instead of a methyl group. This was
supported by the HMBC correlation observed from formyl proton (H-9) to C-2, 3 and 4.
Consequently, the structure of 7 was elucidated as methyl haematommate.
36)


Ethyl chlorohaematommate (8)
The HR-ESIMS of 8 established the composition of
C
11
H
11
O

5
Cl. Its
1
H-NMR spectral features showed the
similarity to those of 7 except for the absence of the
signal due to an aromatic proton. In addition, the
1
H-
and
13
C-NMR spectra of 8 exhibited the signals
corresponding to an ethoxyl group [-CH
3
: δ
H
1.46 (t,
J=7.0 Hz), δ
C
14.1; -OCH
2
: δ
H
4.47 (q, J=7.0 Hz), δ
C
62.5] instead of methoxyl group
COOCH
3
CH
3
OHHO

CH
3
H
1
3
6
7
8
HMBC
NOESY
9
6
CH
3
OHHO
CHO
Cl
1
6
7
8
9
8
O
O
CH
3
COSY
HMBC

-10-
as seen in 7. The HMBC correlations from H
3
-8 (δ
H
2.72), 4-OH (δ
H
13.15) and H-9 (δ
H

10.36) to quaternary carbon C-5 (δ
C
114.9) suggested the substitution of chlorine at C-5.
Thus, the structure of 8 was determined as ethyl chlorohaematommate.
37)


2.1.2. Depsides

Atranorin (9)
Compound 9 was isolated as light
yellow crystal, mp. 186-187
o
C and had a
molecular formula of C
19
H
18
O
8

determined
by its HR-EIMS. Its
1
H-NMR spectrum
indicated the presence of two aromatic
singlets at δ
H
6.40 and 6.52, three methyls at
δ
H
2.09, 2.55 and 2.69, one methoxy at δ
H

3.99, three phenolic hydroxyls at δ
H
11.94, 12.49 and 12.54, and a formyl group at δ
H

10.35. The
13
C-NMR spectrum of 9 showed, besides signals due to three methyl and one
methoxyl groups, two aromatic CH carbons and thirteen quaternary carbons including a
formyl carbon at δ
C
193.8, two ester carbonyl carbons at δ
C
169.7 and 172.2, and four
oxygenated carbons. These findings implied that compound 9 was composed of two
mono-aromatic units, haematommic acid unit and β-orsellinic acid unit. The substitution
pattern was confirmed by HMBC and NOESY correlations. Thus, compound 9 was

elucidated as a typical depside, atranorin.
37,38)


Chloroatranorin (10)
The HR-ESIMS of 10 indicated the
molecular formula of C
19
H
17
O
8
Cl. The NMR
spectral features of compound 10 resembled
those of atranorin (9). The only difference
was that the signal for the aromatic methine
carbon in 9 was replaced by a quaternary
carbon in 10. HMBC correlations from aldehyde proton H-9 (δ
H
10.38) and methyl

-11-
group H
3
-8 (δ
H
2.87) to quaternary carbon C-5 (δ
C
115.7) suggested the location of
chlorine atom at C-5. Accordingly, the structure of 10 was established as

chloroatranorin.
13)

2.1.3. Depsidones and Isocoumarin derivatives

α
αα
α-Alectoronic acid (11)
Compound 11 was isolated as a colorless solid with a
molecular composition of C
28
H
32
O
9
. Its UV spectrum
showed maxima at 209, 266 and 314 nm. IR spectrum of 11
exhibited absorption bands at 3361, 1714, 1676, 1613 and
1579 cm
-1
, indicating the presence of hydroxyl and carbonyl
groups, and aromatic ring. The
1
H-NMR spectrum showed
signals for three aromatic protons, two
β
-keto alkyl C
7
side chain (δ
H

value from 0.84 to
3.85) (Table 1). The chemical structure of 11 couldn’t be determined except for a partial
structure as shown because of the broadness of signals in its NMR spectra. Therefore,
compound 11 was treated with an excess of TMS-CHN
2
to yield two derivatives 11a
and 11b.

The HR-EIMS of 11a established
the molecular formula of C
31
H
38
O
9
. Its
1
H-NMR exhibited the signals due to
three aromatic protons at δ
H
6.09 (d,
J=2.5 Hz), 6.35 (d, J=2.5 Hz) and 6.54
(s). The
1
H-NMR spectra showed
further signals for an olefinic proton at
δ
H
6.10 (s), three methoxyl groups, a
β

-
keto alkyl C
7
side chain, a n-pentyl
group and a hydrogen-bonded phenolic proton [δ
H
11.68 (s)] (Table 1). The
13
C-NMR
spectrum of 11a showed no carbon signals assignable to C-1′′ methylene and C-2′′
carbonyl carbons, but newly demonstrated two sp
2
carbons at δ
C
102.7 (C-1′′) and 159.2
C
5
H
11
HO
4
6
1"
2"
O
H
O
O
O
1

NOESY
7
1
1
H
H

-12-
(C-2′′), and HMBC correlations from H-5 to C-1′′, from H-1′′ to C-5, 6, 2′′ and 3′′,
indicating the formation of isocoumarin skeleton. HMBC observations from 2′-OH to
C-1′, 2′, 3′; from H-3′ to C-1′, 2′, 4′, 5′, 7′; from methylene H
2
-1′′′ to C-6′, C-2′′′; from
methylene H
2
-3′′′ of C
7
side chain to C-2′′′ suggested the structure of second aromatic
ring of 11a. The position of three methoxyl groups at C-4, 4′ and 7′ were assigned by
HMBC and NOESY correlations. Accordingly, the structure of 11a was determined to
be methyl 4′-O-methyl-β-collatolate.
39)


The HR-EIMS of 11b indicated the
molecular formula of C
32
H
40
O

9
, a CH
2

group more than 11a. The spectral
features of 11b were similar to those of
11a except for the presence of an
additional methoxyl group [δ
H
3.88 (s)
and δ
C
56.5] at C-2′ (Table 1).
Consequently, the structure of 11b was
assigned to be methyl 2′,4′-di-O-
methyl-β-collatolate.
39)

These findings suggested 11 to be α-alectoronic acid
40)
which was first isolated
from the lichen Alectoria japonica by Asahina and co-workers.
41-43)

It is well known that prolonged treatment of α-alectoronic acid (11) with an excess
of diazomethane proceeded with partial cleavage of depsidone-ester linkage and re-
cyclization to form isocoumarin skeleton. The very broad signals in NMR spetra of
compound 11 were arising from the rapid tautomeric interchange (Fig. 7).
39)
The

pseudo-acid tautomer with two cyclized forms has been reported by a NMR experiment
at -40
o
C
39)
and confirmed by Millot et al.
44)
in similar experiment. At room temperature
the pseudo-acid form (11α
αα
α and 11β
ββ
β) is the predominant tautomer.
39)


-13-
O
C
5
H
11
C
5
H
11
OH
COOH
HO
O

O
O
O
O
C
5
H
11
OH
HO
O
O
O
O
O
C
5
H
11
OH
O
C
5
H
11
OH
HO
O
O
O

O
O
HO
C
5
H
11
11 11

Fig. 7. Tautomeric interchange of α-alectoronic acid (11)
39)

α
αα
α-Collatolic acid (12)
Compound 12 was obtained as a colorless solid. Its HR-SIMS established the
molecular formula of C
29
H
34
O
9
, that is, 14 mass units more than that of 11. The UV, IR
and NMR spectral features of 12 resembled those of 11. The difference between
compounds was that the NMR spectra of 12 showed signal for an additional methoxyl
group [δ
H
3.76 (s) and δ
C
55.7] (Table 2). The location of methoxyl group at C-4 was

deduced from NOESY cross peaks between the methoxyl group and two aromatic
protons. Methylation of 12 with TMS-CHN
2
yielded 12a whose structure was
determined by 1D, 2D NMR and mass spectra. Thus, 12 was established as α-collatolic
acid.
45)
The NMR measurements at room temperature and at -50
o
C demonstrated that α-
collatolic acid (12) could exit predominantly in the pseudo-acid tautomeric form as α-
alectoronic acid (11).
39)
O
C
3
H
7
OCH
3
H
3
CO
O
H
H
H
H
H
3

CO
H
O
O
C
3
H
7
O
12a
COSY
HMBC
NOESY
3"
1"'
3"'
2"
1"
6
1
7
6'
1'
7'
4
O
C
5
H
11

OH
H
3
CO
O
O
O
O
O
HO
C
5
H
11
12
H
H
H
H


-14-

Table 1.
1
H- and
13
C-NMR spectroscopic data of 11, 11a and 11b in CDCl
3

No.
11 11a 11b
1
H
13
C

1
H (J, Hz)
13
C

1
H (J, Hz)
13
C
1

112.6

102.5

102.6

2

161.4

161.7

161.3

3 6.40

br s 106.4

6.09

d (2.5) 100.1

6.07

d (2.5) 100.5

4

162.7

165.0

165.0

4-OCH
3




3.74

s 55.6

3.74

s 55.6

5 6.40

br s 117.7

6.35

d (2.5) 102.2

6.34

d (2.5) 101.7

6

140.9

141.8

141.9

7

162.0

159.3

159.2

1'

ND

104.8

116.3

2'

160.1

162.8

156.0

2'-OH



11.68

s
2'-OCH
3

3.88

s 56.5

3' 6.67

s 108.0

6.54

s 100.2

6.52

s 96.4

4'

149.8

157.5

154.1

4'-OCH
3

3.76

s 56.1

3.78

s 56.1

5'

ND

134.9

134.8

6'

129.6

131.4

129.7

7'

ND

170.9

167.3

7'-OCH
3




3.84

s 52.1

3.82

s 52.1

1" 3.85

br s 48.0

6.10

s 102.7

6.09

s 102.7

2"

207.5

159.2

159.1

3" 2.58

m 42.9

2.48

t (7.5) 33.3

2.47

t (7.5) 33.4

4" 1.50-1.61

m 23.3

1.71

m 26.5

1.70

m 26.5

5" 1.23-1.30

m 31.3

1.37

m 31.2

1.36

m 31.2

6" 1.23-1.30

m 22.4

1.37

m 22.4

1.36

m 22.4

7" 0.84-0.88

m 13.9

0.92

t (7.0) 14.0

0.91

t (7.0) 13.9

1"' 3.48-3.50

br s 41.1

4.08

d (16.5) 42.6

3.68

m 41.6

4.15

d (16.5) 3.86

m
2"'

ND

207.1

206.7

3"' 2.07

m 42.9

2.33

t (7.0) 42.1

2.32

m 42.0





2.35

t (7.0) 2.36

m
4"' 1.50-1.61

m 23.3

1.40

m 23.4

1.38

m 23.3

5"' 1.23-1.30

m 31.4

1.09

m 31.1

1.09

quint (7.5) 31.1

6"' 1.23-1.30

m 23.0

1.18

m 22.4

1.17

m 22.4

7"' 0.84-0.88

m 13.9



0.80

t (7.0) 14.0



0.79

t (7.0) 13.9

ND: not detected

-15-


Table 2.
1
H- and
13
C-NMR spectroscopic data of 12 and 12a in CDCl
3

No.

12

12a
1
H (J, Hz)
13
C

1
H (J, Hz)
13
C
1 113.7



103.0

2 162.1



161.6

3 6.52

br s 104.7


5.93

d (2.5) 100.4

4 163.4



165.1

4-OCH
3
3.76

br s 55.7


3.76

s 55.6

5 6.52

br s 115.4


6.39

d (2.5) 101.0

6 141.5

142.5

7 162.7



159.2

1' ND



102.3

2' 159.8



161.3

2'-OCH
3



4.04

s 56.6

3' 6.60

s 108.0


6.52

s 94.9

4' 148.1



157.4

4'-OCH
3



3.88

s 56.3

5' 140.8

128.5

6' 129.4



134.8

7' 172.2



159.3

1" 3.89

br s 48.1


6.15

s 102.8

2" 207.0



160.0

3" 2.52

t (7.5) 42.7


2.51

t (7.5) 33.6

4" 1.59

quint (7.5) 23.3


1.73

m 26.5

5" 1.29

m 31.4


1.38

m 31.2

6" 1.29

m 22.4

1.38

m 22.3

7" 0.88

t (7.0) 13.9


0.93

t (7.0) 14.0

1"' 3.71

br s ND


6.25

s 96.6

2"' ND



159.6

3"' 2.20

m 41.6


2.38

t (7.5) 33.4

4"' 1.43

m 23.2


1.60

quint (7.5) 26.6

5"' 1.14

m 31.4


1.25

m 31.2

6"' 1.14

m 22.4

1.25

m 22.4

7"' 0.77

m 13.9



0.83

t (7.0) 13.9

ND: not detected

-16-
β
ββ
β-Alectoronic acid (13) and β
ββ
β-collatolic acid (14)
The HR-MS established that compounds
13 and 14 were isomeric with α-alectoronic
acid (11) and α-collatolic acid (12),
respectively. The
1
H- and
13

C-NMR spectra
of 13 and 14 closely related to those of 11
and 12, respectively, but showed signals for
an additional olefinic proton/carbon and
oxygenated quaternary carbon instead of C-
1′′ methylene group and C-2′′ carbonyl carbon as seen in 11 and 12, indicating an
isocoumarin core. In addition, compound 14 treated with excess TMS-CHN
2
in MeOH
yielded 11b. These findings together with analysis 2D NMR suggested the structure of
13 and 14 to be β-alectoronic acid
40)
and β-collatolic acid
45)
, respectively.
The broadness of signals of β-alectoronic acid (13) and β-collatolic acid (14) was
arising from the pseudo-acid tautomerism which had previously been described for
related compounds, α-alectoronic acid (11) and α-collatolic acid (12). This
phenomenon of β-collatolic acid (14) was confirmed by the NMR experiments taken at
different temperature conditions.
45)
Compound β-collatolic acid (14) was clearly
recognized as an artifact formed during the extraction process as a result of trans-
esterification of α-collatolic acid (12).
45)
However, the TLC investigations of extracts
from fresh lichen samples and the use of neutral solvents and mild conditions of
extraction confirmed the presence of the phenoxyisocoumarins β-alectoronic acid (13)
and β-collatolic acid (14) in the natural material.
40)

New depsidone 2′
′′
′′
′′
′′
′′
′-O-ethyl-α
αα
α-alectoronic acid

(15) and 2′
′′
′′
′′
′′
′′
′-O-methyl-α
αα
α-alectoronic
acid

(16)
Compound 15 appeared as a pale yellow solid and gave a molecular formula of
C
30
H
36
O

9
as determined by HR-EIMS. The UV spectrum exhibited the maxima at 247
and 317.5 nm. The IR spectrum showed the absorption bands at 3391, 1730, 1682, 1613
and 1478 cm
-1
. The
1
H- NMR spectrum indicated the presence of signals for three
aromatic protons at δ
H
6.36, 6.41 (each d, J=2.5 Hz) and 6.73 (s), a methylene at δ
H
3.07

-17-
and 3.46 (each 1H, d, J=17.0 Hz), a
β
-keto alkyl
C
7
side chain, a n-pentyl group, an ethoxyl
group and two phenolic protons at δ
H
7.87 (br s)
and 11.05 (s). The
13
C-NMR spectrum showed
the signals for a
β
-keto alkyl C

7
side chain, a n-
pentyl, an ethoxyl groups and a ketal carbon,
besides the signals for a methylene, three
aromatic CH, nine sp
2
quaternary carbons, and
two carbonyls due to a common depsidone core
(Table 3). These spectral features of 15 were remarkably similar to those of 2′′′-O-
methyl-α-alectoronic acid (16).
39)
The only difference between these compounds was
the substitution of ethoxyl group at C-2′′′ in 15 instead of methoxyl group as seen in 16.
The location of the ethoxyl group at C-2′′′ was confirmed by the HMBC correlations
between H
2
-α of ethoxyl group to ketal carbon at δ
C
107.8 (C-2′′′). Accordingly, 15 was
identified and designed 2′′′-O-ethyl-α-alectoronic acid. All other 2D NMR spectral data
were fully consisted with the proposed structure. Compound 16 was obtained as a
mixture with compound 15 in a ratio 2:1.


New isocoumarin 2′
′′
′′
′′
′′
′′

′-O-methyl-β
ββ
β-alectoronic acid

(17) and 2′
′′
′′
′′
′′
′′
′-O-ethyl-β
ββ
β-
alectoronic acid

(18)
The mass spectra of compounds 17 and 18 displayed that these compounds
possessed molecular formulas of C
29
H
34
O
9
and C
30
H
36
O
9
which were identical with

those of 16 and 15, respectively. The
1
H- and
13
C-NMR spectra of 17 (Table 3) and 18
indicated the signals for an olefinic proton H-1′′ and oxygenated quaternary carbon C-
2′′ which were characteristic signals for isocoumarin derivatives such as 13 and 14.
Detailed 2D NMR analysis and comparison with the reported data
39,46)
led us to
determine the structure of 17 and 18 was 2′′′-O-methyl-β-alectoronic acid and 2′′′-O-
ethyl-β-alectoronic acid, respectively. Compound 18 was isolated from the lichen
Alectoria sarmentosa and reported as an artifact formed from β-alectoronic acid (13)
during the treatment with EtOH.
46)
The compound 18 could possibly be a natural
15
3"'
O
O
OH
HO
O
O
O
C
4
H
9
H

3
C
H
H
H
O
O
C
4
H
9
3"
1"'
1"
1
7
4'
1'
7'
4
COSY
HMBC
H
H
H
H
H
H
H
H

2"'
H
H