Chemistry of the lichen type common in southern Vietnam

Chia sẻ: nhatro75

Lichen is a special form of life is formed by the symbiosis between algae and some fungi. The mycelium absorbs water and minerals provide the algae. Algae due to chlorophyll, use them to make organic matter to feed on both sides. In this common life of algae and fungi have a certain role, neither party relying solely on the real nao.Hinh is called symbiosis.

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Nội dung Text: Chemistry of the lichen type common in southern Vietnam

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
page
Content


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
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



-i-
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



-ii-
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




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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




-iv-
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
1 13
Table 1. H- and C-NMR spectroscopic data of 11, 11a and 11b in CDCl3 .......................... 14
Table 2. 1H- and 13C-NMR spectroscopic data of 12 and 12a in CDCl3 .................................. 15
Table 3. 1H- and 13C-NMR spectroscopic data of 15-17 in CDCl3 .......................................... 19
Table 4. 1H-NMR spectroscopic data of 19-22 in CDCl3 ........................................................ 21
Table 5. 13C-NMR spectroscopic data of 19-25 in CDCl3 ....................................................... 22
Table 6. 1H-NMR spectroscopic data of 23-25 in CDCl3 ........................................................ 27
Table 7. 1H- and 13C-NMR spectroscopic data of 42 and 42m ................................................ 43
Table 8. 13C-NMR spectroscopic data of 47, 48, 52-54 and 58 in CDCl3 ................................ 51
Table 9. 1H-NMR spectroscopic data of 48, 52-54 in CDCl3 ................................................... 52
Table 10. 1H-NMR spectroscopic data of 63-66 in CDCl3 ...................................................... 64
Table 11. 13C-NMR spectroscopic data of 63, 64 and related compounds in CDCl3 ................ 65
Table 12. 13C-NMR spectroscopic data of 65-70 in CDCl3 ..................................................... 70
Table 13. 1H-NMR spectroscopic data of 67-70 ...................................................................... 74




-v-
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)




Crustose Fruticose
Foliose
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)



-1-
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 19th 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 20th 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



-2-
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 spores15)
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



-3-
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



-4-
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.




-5-
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.




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



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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 (3 x 1 l)

Acetone extract
(8.41 g)
CC/CHCl3-MeOH
MeOH (0%) (1%) (2%) (3-50%)

Fr-I Fr-II Fr-III Fr-IV
(1.25 g) (3.27 g) (2.19 g) (1.62 g)
CC/CHCl3-MeOH CC/CHCl3-MeOH
CC/CHCl3-MeOH
pTLC A-C MeOH (0%) (0%) (1-2%)
11 (1.32 g)
3 (2.0 mg) Fr-IIa Fr-IIb Fr-IIc
13 (798.4 mg)
4 (4.6 mg) (417 mg) (539 mg) (2.17 g)
6 (42.7 mg)
pTLC B-D pTLC B-D pTLC B-D
7 (1.7 mg)
8 (6.8 mg) 11 (1.21 g)
15 (65.6 mg) 5 (10.2 mg)
9 (737 mg) 12 (365.7 mg)
18 (66.2 mg) 12 (327.2 mg)
10 (95.2 mg) 13 (21.3 mg)
25 (2.9 mg) 20 (21.6 mg)
19 (19.6 mg) 14 (139.0 mg)
21 (25.0 mg)
22 (22.0 mg) 16 (43.9 mg)
23 (9.1 mg)
26 (22.5 mg) 17 (13.7 mg)
24 (8.1 mg)
pTLC
27 (4.8 mg)
A CHCl3
B CHCl3 - MeOH (99:1), (85:5), (9:1)
C n-Hexane - Et2O (1:1), (3:7)
D Toluene - AcOH (20:3)

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




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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 C9H10O4. 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 1H-NMR spectral features indicated the presence of a pair of
meta-coupled aromatic protons at δH 6.22 and 6.27 (each 1H, d, J=2.5 Hz), a methoxyl
(δH 3.92) and a methyl (δH 2.49) groups, and a chelated phenolic hydroxyl group at δH
11.72. The 13C-NMR spectrum showed signals corresponding to nine carbons, including
a carbonyl (δC 172.1), two oxygen-bearing aromatic carbons (δC 160.3 and 165.4), two
quaternary aromatic carbons (δC 105.5 and 144.0), two CH (δC 101.3 and 111.3), a
methoxyl (δC 51.9) and a methyl (δC 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 C12H16O4, that 8 CH
3
7
is, C3H6 more than that of methyl orsellinate (3). The NMR 6 COOR
1
spectral features of 4 were similar to those of 3, but 4 showed
2
4
signals of n-butoxyl group instead of methoxyl group as seen in HO OH
4 : R = n-C4H9
3. This was confirmed by the HMBC correlation from
5 : R = C2H5
oxygenated methylene protons at δH 4.34 (t, J=6.5 Hz, H2-1′) to
carbonyl carbon at δC 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 [-CH3: δH 1.41 (t, J=7.0 Hz); -OCH2: δH 4.40 (q, J=7.0 Hz)] in place of
methoxyl group. Accordingly, 5 was elucidated as ethyl orsellinate.35)




-8-
Methyl β -orsellinate (6)
8
The HR-EIMS of 6 indicated the molecular formula of CH3
7
6
H COOCH3
C10H12O4, i.e. a CH2 group more than that of 3. The NMR
1
spectral features of 6 were similar to those of 3 except for the 3
HO OH
presence of an additional methyl group (δH 2.10, δC 7.7) and 9
CH3 HMBC
absence of an aromatic proton. Moreover, the HMBC NOESY
6
correlation from the methyl group (δH 2.10, H3-9) 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
C10H10O5 by HR-EIMS. The 1H-NMR spectrum of 7
showed six singlets for two hydrogen-bonded phenolic
hydroxyl groups at δH 12.42 and 12.89, a formyl proton at
δH 10.34, an aromatic proton at δH 6.30, a methoxyl and a
13
methyl group. Its 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 8
CH3 O
C11H11O5Cl. Its 1H-NMR spectral features showed the
6
Cl 7 O CH3
similarity to those of 7 except for the absence of the 1

signal due to an aromatic proton. In addition, the 1H- HO OH
13 9
CHO
and C-NMR spectra of 8 exhibited the signals COSY
HMBC
corresponding to an ethoxyl group [-CH3: δH 1.46 (t, 8
J=7.0 Hz), δC 14.1; -OCH2: δH 4.47 (q, J=7.0 Hz), δC 62.5] instead of methoxyl group




-9-
as seen in 7. The HMBC correlations from H3-8 (δH 2.72), 4-OH (δH 13.15) and H-9 (δH
10.36) to quaternary carbon C-5 (δC 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. 186-187oC and had a
molecular formula of C19H18O8 determined
1
by its HR-EIMS. Its H-NMR spectrum
indicated the presence of two aromatic
singlets at δH 6.40 and 6.52, three methyls at
δH 2.09, 2.55 and 2.69, one methoxy at δH
3.99, three phenolic hydroxyls at δH 11.94, 12.49 and 12.54, and a formyl group at δH
10.35. The 13C-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 δC 193.8, two ester carbonyl carbons at δC 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 C19H17O8Cl. 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 (δH 10.38) and methyl




-10-
group H3-8 (δH 2.87) to quaternary carbon C-5 (δC 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-Alectoronic acid (11)
C5H11
Compound 11 was isolated as a colorless solid with a NOESY
2" H
molecular composition of C28H32O9. Its UV spectrum HO
O 1"
showed maxima at 209, 266 and 314 nm. IR spectrum of 11 6
H O
7
1
exhibited absorption bands at 3361, 1714, 1676, 1613 and
4 O
1579 cm-1, indicating the presence of hydroxyl and carbonyl HO
groups, and aromatic ring. The 1H-NMR spectrum showed 11

signals for three aromatic protons, two β-keto alkyl C7 side chain (δH 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-CHN2 to yield two derivatives 11a
and 11b.


The HR-EIMS of 11a established
the molecular formula of C31H38O9. Its
1
H-NMR exhibited the signals due to
three aromatic protons at δH 6.09 (d,
J=2.5 Hz), 6.35 (d, J=2.5 Hz) and 6.54
1
(s). The H-NMR spectra showed
further signals for an olefinic proton at
δH 6.10 (s), three methoxyl groups, a β-
keto alkyl C7 side chain, a n-pentyl
13
group and a hydrogen-bonded phenolic proton [δH 11.68 (s)] (Table 1). The C-NMR
spectrum of 11a showed no carbon signals assignable to C-1′′ methylene and C-2′′
carbonyl carbons, but newly demonstrated two sp2 carbons at δC 102.7 (C-1′′) and 159.2




-11-
(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 H2-1′′′ to C-6′, C-2′′′; from
methylene H2-3′′′ of C7 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 C32H40O9, a CH2
group more than 11a. The spectral
features of 11b were similar to those of
11a except for the presence of an
additional methoxyl group [δH 3.88 (s)
and δC 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)
40)
These findings suggested 11 to be α-alectoronic acid 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 -40oC 39) and confirmed by Millot et al. 44) in similar experiment. At room temperature
the pseudo-acid form (11α and 11β ) is the predominant tautomer.39)




-12-
C5H11 C5H11 C5H11
O O O
O O O
O O O
OH OH OH
O O
HO HO HO
COOH
O O O
O O O
C5H11
HO C5H11 C5H11 OH
11 11

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


α-Collatolic acid (12)
Compound 12 was obtained as a colorless solid. Its HR-SIMS established the
molecular formula of C29H34O9, 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 [δH 3.76 (s) and δC 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-CHN2 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 -50oC demonstrated that α-
collatolic acid (12) could exit predominantly in the pseudo-acid tautomeric form as α-
alectoronic acid (11).39)
C3H7
H
C5H11 3" H
2"
H
O H
O O
1"
H3CO
7
6 OCH3
O H
OH
O
1
1'
O
4
O
O
H3CO H3CO
O 6' 7'
O
H
O 1"'
H
HO COSY H
C5H11 3"'
HMBC H
12 12a
NOESY C3H7




-13-
Table 1. 1H- and 13C-NMR spectroscopic data of 11, 11a and 11b in CDCl3

11 11a 11b
No. 1 13 1 13 1 13
H C H (J, Hz) C H (J, Hz) 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-OCH3 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'-OCH3 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'-OCH3 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'-OCH3 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




-14-
Table 2. 1H- and 13C-NMR spectroscopic data of 12 and 12a in CDCl3
12 12a
No. 1 13 1 13
H (J, Hz) C H (J, Hz) 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-OCH3 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'-OCH3 4.04 s 56.6
3' 6.60 s 108.0 6.52 s 94.9
4' 148.1 157.4
4'-OCH3 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




-15-
β-Alectoronic acid (13) and 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 1H- 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-CHN2 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-alectoronic acid (15) and 2′′′-O-methyl-α-alectoronic
acid (16)
Compound 15 appeared as a pale yellow solid and gave a molecular formula of
C30H36O9 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 1H- NMR spectrum indicated the presence of signals for three
aromatic protons at δH 6.36, 6.41 (each d, J=2.5 Hz) and 6.73 (s), a methylene at δH 3.07



-16-
and 3.46 (each 1H, d, J=17.0 Hz), a β-keto alkyl H
C4H9 H
3"
C7 side chain, a n-pentyl group, an ethoxyl H
H H
O
O
group and two phenolic protons at δH 7.87 (br s) 1"
H OH
O
13
and 11.05 (s). The C-NMR spectrum showed 7
1 4'
1' O
the signals for a β-keto alkyl C7 side chain, a n- O
4
HO 7'
H 1"'
pentyl, an ethoxyl groups and a ketal carbon, O
H 2"'
H
H
O 3"' H
besides the signals for a methylene, three COSY HCH
H 49
2 HMBC
aromatic CH, nine sp quaternary carbons, and H3C
15
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 H2-α of ethoxyl group to ketal carbon at δC 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.


2′′′-O-ethyl-β-
New isocoumarin 2′′′-O-methyl-β -alectoronic acid (17) and
alectoronic acid (18)
The mass spectra of compounds 17 and 18 displayed that these compounds
possessed molecular formulas of C29H34O9 and C30H36O9 which were identical with
those of 16 and 15, respectively. The 1H- and 13C-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.
39,46)
Detailed 2D NMR analysis and comparison with the reported data 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




-17-
product because EtOH was not used as solvent in this study. Compound 2′′′-O-methyl-
β-alectoronic acid (17) was identified as a new structure isolated from natural lichen.




Compounds 15-18 possessed an asymmetric center at ketal carbon C-2′′′, but these
compounds exhibited no optical activity ([α]D~0o). These compounds were analyzed by
chiral HPLC and each compound demonstrated two peaks in a ratio of approximately
1:1 (Fig. 8). These findings revealed that compounds 15-18 were racemic compounds.




Fig. 8: Chiral HPLC of 18
Column: CHIRALCEL OJ-RH 0.46 × 15 cm
Mobile phase: H2O-CH3CN (6:4)
Flow rate: 1.0 ml/min
Detection: 254 nm




-18-
Table 3. 1H- and 13C-NMR spectroscopic data of 15-17 in CDCl3

15 16 17
No.
1 13 1 13 1 13
H (J , Hz) C H (J , Hz) C H (J , Hz) C

1 112.5 112.4 102.5
162.1 a 162.1 d
2 163.5
3 6.36 d (2.5) 106.5 6.35 d (2.0) 106.5 6.27 brs 103.3
4 161.5 161.6 162.1
4-OH 7.87 br s
5 6.41 d (2.5) 117.9 6.41 d (2.0) 117.9 6.27 br s 103.3
6 140.9 140.9 142.1
162.4 a 162.4 d
7 160.8
1' 104.9 104.9 99.6
2' 160.2 160.2 163.5
2'-OH 11.05 s 11.01 s 11.12 br s
3' 6.73 s 107.7 6.73 s 107.7 6.42 s 105.6
4' 150.4 150.4 158.9
5' 139.9 140.0 133.4
6' 130.2 130.0 131.4
7' 168.4 168.2 168.8
1" 3.83 s 48.1 3.83 s 48.1 6.11 s 103.3
2" 210.1 210.2 159.0
3" 2.66 td (7.0, 2.5) 42.9 2.66 t (7.0) 42.9 2.43 m 33.1
4" 1.66 quint (7.0) 23.3 1.65 quint (7.0) 23.2 1.65 m 26.4
5" 1.34 m 31.3 1.34 m 31.3 1.34 m 31.2
22.4 b 22.4 e
6" 1.34 m 1.34 m 1.34 m 22.4
13.95 c 13.9 f
7" 0.93 t (7.0) 0.94 t (7.0) 0.91 t (7.0) 14.0
1"' 3.07 d (17.0) 30.5 3.06 d (17.0) 30.3 2.88 br s 30.8
3.46 d (17.0) 3.46 d (17.0) 3.01 br s
2"' 107.8 107.7 107.5
3"' 1.90 ddd (14.5, 12.0, 5.0) 35.8 1.92 m 35.1 1.78 m 34.9
2.05 ddd (14.5, 12.0, 5.0) 2.05 m 1.90 m
4"' 1.44 m 23.3 1.43 m 23.2 1.30 m 23.0
1.50 m 1.51 m
5"' 1.36 m 31.7 1.37 m 31.7 1.25 m 31.6
22.5 b 22.5 e
6"' 1.36 m 1.37 m 1.25 m 22.4
14.03 c 14.0 f
7"' 0.91 t (7.0) 0.91 t (7.0) 0.85 t (7.0) 14.0
3.64 dq (8.5, 7.0) 58.2 3.33 s 49.9 3.30 s 49.9
3.70 dq (8.5, 7.0)
1.07 t (7.0) 15.3

a, b, c, d, e, f
Assignments may be interchangeable




-19-
Dehydrocollatolic acid (19)
Compound 19 was obtained as a colorless
C5H11
H
crystalline solid. The HR-EIMS of 19 exhibited 2"
H
O 1'' H O
a peak at m/z 524.2062 [M+], indicating a H OH
O
molecular formula of C29H32O9. Its 1H-NMR
O
O
H3CO
spectrum showed two meta-coupled doublets at
H 1"' 2"' O
H
δH 6.58 and 6.61 (J=2.5 Hz), and a singlet at δH
HO 3"'
COSY
6.79 due to the aromatic protons, a hydrogen- 6"'
HMBC
H3C
NOESY
bonded hydroxyl signal at δH 11.08, a methoxyl 5"'
7"'

19
singlet at δH 3.85, a β-keto alkyl C7 side chain
and four sets of methylenes. Furthermore, the 1H-NMR spectrum of 19 exhibited a
methyl signal at δH 1.07 (d, J=6.5 Hz), which was coupled with a methine resonance at
δH 4.06 (1H, m) (Table 4). The 13C-NMR spectrum of 19 showed, besides signals due to
two methyl and a methoxyl groups, nine methylene carbons, a methine and three
aromatic sp2 CH carbons, and thirteen quaternary carbons including a carbonyl carbon
at δC 206.4, a ketal carbon at δC 104.1, two ester carbonyl carbons at δC 162.4 and 168.1,
and five oxygenated carbons (Table 5). COSY spectrum showed the sequence from H2-
3′′′ methylene to H3-7′′′ methyl; HMBC correlations from H2-1′′′ to C-3′′′, from H3-7′′′
to C-5′′′ and 6′′′; indicating the appearance of 6,6-spiro-ring system. These spectral
features were identical with those of dehydrocollatolic acid.47) This assignment was
supported by the detailed 2D NMR studies as shown.




-20-
Table 4. 1H-NMR spectroscopic data of 19-22 in CDCl3 (J , Hz)

21a
19 20 22
No.

3 6.61 d (2.5) 6.39 d (2.0) 6.06 br s 6.55 br s
3.85 s 3.79 s
4-OCH3
5 6.58 d (2.5) 6.40 d (2.0) 6.429 d (2.0) 6.47 d (2.0)
2'-OH 11.08 s 11.07 s 11.20 s
3' 6.79 s 6.74 s 6.433 s 6.50 s
4'-OH 8.92 br s
1" 3.93 d (17.0) 3.81 d (17.0) 6.31 s 6.22 s
4.04 d (17.0) 3.86 d (17.0)
3" 2.55 t (7.0) 2.63 td (7.5, 2.0) 2.50 t (7.0) 2.52 t (7.5)
4" 1.62 quint (7.0) 1.64 quint (7.5) 1.73 m 1.71 m
5" 1.30 m 1.32 m 1.42 m 1.37 m




-21-
6" 1.31 m 1.35 m 1.42 m 1.37 m
7" 0.90 t (7.0) 0.90 t (7.0) 0.96 t (7.0) 0.92 t (7.0)
1"' 3.09 d (17.0) 3.05 d (17.0) 2.95 m 2.96 d (16.5)
3.49 d (17.0) 3.42 d (17.0) 3.10 m 3.13 d (16.5)
3"' 1.70 m 1.69 m 1.59 td (13.5, 4.5) 1.59 td (13.5, 4.5)
2.10 m 2.06 m 1.99 br d (13.5) 2.04 br d (13.5)
4"' 1.75 m 1.75 m 1.70 m 1.71 m
2.10 m 2.08 m 2.02 m 2.12 qt (13.5, 4.0)
5"' 1.31 m 1.30 m 1.25 m 1.26 m
1.70 m 1.70 m 1.69 m 1.70 m
6"' 4.06 m 4.03 m 3.98 m 4.07 m
7"' 1.07 d (6.5) 1.04 d (7.0) 1.05 d (6.5) 1.09 d (6.5)
a
Measured in CD3OD
Table 5. 13C-NMR spectroscopic data of 19-25 in CDCl3
21a
No. 19 20 22 23 24 25
1 114.1 112.5 102.2 103.8 112.8 112.9 113.4
2 161.8 162.1 162.2 162.4 162.1 162.1 162.1
3 104.9 106.5 102.0 104.0 106.2 106.3 106.1
4 163.5 161.4 166.1 166.0 161.3 161.2 160.8
4-OCH3 55.9 55.8
5 115.1 117.7 105.5 102.5 117.7 117.7 117.3
6 141.4 141.0 143.9 142.1 141.0 141.1 141.3
7 162.4 162.4 163.2 160.4 162.3 162.4 162.2
1' 104.8 104.9 100.9 100.0 104.9 105.1 104.8
2' 160.3 160.2 162.9 162.1 160.2 160.2 160.3
3' 107.9 107.7 103.4 103.4 108.0 108.0 108.1
4' 150.4 150.4 159.3 156.8 150.4 150.3 150.5
5' 139.9 139.8 133.3 133.9 139.7 139.7 139.7
6' 129.6 129.8 ND 131.1 129.7 129.8 129.6
7' 168.1 168.2 170.2 168.4 168.1 168.3 168.0
1" 48.0 48.1 104.4 103.3 48.0 48.0 47.9
2" 206.4 209.7 159.9 159.5 209.2 209.2 207.9
3" 42.8 42.8 34.1 33.3 42.9 42.9 42.9
4" 23.4 23.2 27.7 26.5 23.3 23.3 23.3
5" 31.4 31.3 32.4 31.2 31.3 31.3 31.3
6" 22.5 22.4 23.5 22.4 22.4 22.4 22.4
7" 13.9 13.9 14.4 14.0 14.0 14.0 13.9
1"' 33.5 33.3 ND 34.3 31.9 31.4 32.0
2"' 104.1 104.2 105.2 103.6 111.9 111.7 111.9
3"' 33.7 33.6 34.4 33.6 35.5 36.4 35.6
4"' 18.1 18.1 19.3 18.0 30.4 30.8 30.4
5"' 31.7 31.7 32.9 31.8 106.1 106.7 106.3
6"' 68.8 68.9 70.0 68.7
7"' 21.6 21.5 21.9 21.5
64.0 64.2 68.4
α
15.1 15.0 31.5
β
19.2
γ
13.8
δ
a
Measured in CD3OD ND: not detected




-22-
New depsidone dehydroalectoronic acid (20)
Compound 20 was isolated as a colorless
solid and the molecular formula was determined
as C28H30O9 by HR-EIMS, that is, a CH2 group
less than that of 19. The UV spectrum of 20
displayed the maxima at 216, 254 and 316.5 nm.
The IR spectrum of 20 had absorptions at 3322,
1738, 1682, 1614 and 1478 cm-1. The 1H- and
13
C-NMR spectra of 20 indicated the signals due
to three aromatic CH carbons, a hydrogen-
bonded phenolic hydroxyl group, a β-keto alkyl C7 side chain and a 6,6-spiro-ring
system (Tables 4 and 5). These spectral features were closely similar to those of the co-
occuring unique depsidone dehydrocollatolic acid (19), except for the absence of 4-
methoxyl signal, suggesting 20 to be demethylated compound of 19. The proposed
structure of 20 was fully coincident with its 2D NMR spectral data. Accordingly, the
planar structure of compound 20 was determined as shown and designated
dehydroalectoronic acid.


New isocoumarin 21
C3H7
Compound 21 was obtained as a colorless
H
4"
solid. The HR-EIMS spectrum established the H
3"
H
molecular formula of C28H30O9 which is the
H
O
2"
1"
1 13
same composition of 20. The H- and C- HO
7
6 OH
H 1
O
5
NMR spectra of 21 closely resembled those of 5' O
HO O
20. The marked differences were the presence
1"'
O
H
of an additional olefinic proton at δH 6.31 (s,
COSY O 3"'
HMBC
H-1′′) and oxygenated quaternary carbon at 6"'
H3C
21
δC 159.9 (C-2′′) in 21 instead of methylene
group and carbonyl carbon as seen in 20, respectively (Tables 4 and 5). In addition, the
HMBC spectrum of 21 showed the correlations from olefinic proton H-1′′ to carbon
signals at δC 105.5 (C-5), 143.9 (C-6), 159.9 (C-2′′) and from aromatic proton H-5 to C-




-23-
3 (δC 102.0), C-4 (δC 166.1) and C-1′′ (δC 104.4) suggested an isocoumarin ring system.
The proposed structure was confirmed by analysis of 2D NMR.


New isocoumarin 22
The HR-EIMS of compound 22 showed C3H7
H
4"
molecular formular of C29H32O9, i.e. 14 H
3"
mass units more than that of 21. The 1H- H
H
O
2"
13
and C-NMR spectral features of 22 were HO
7
6 OH
H 1
O
similar to those of 21 (Tables 4 and 5), the 4 5' O
difference was only observed for the H3CO O
H 1"'
OH
H
additional methoxyl group at δH 3.79 (s) H
COSY O 3"' H
and δC 55.8. The HMBC spectrum of 22 HMBC 6"'
H3C
exhibited the cross peak of methoxyl 22
proton at δH 3.79 to oxygenated quaternary carbon at δC 166.0 (C-4). Accordingly,
compound 22 was elucidated to be 4-O-methylated compound of 21. From their
structures, 21 and 22 were supposed to be the isocoumarin compounds derived from
depsidones 20 and 19, respectively. It is likely that the lichen species containing
depsidones with oxo-alkyl substituents may also be found to contain the isocoumarin
forms isomeric with them.40)


Compounds 19-22 possessed two asymmetric carbons C-2′′′ and C-6′′′ of the spiro-
ring system. The relative configuration at spiro-ring of 22 could be deduced from
analysis of 1H-1H coupling constants and NOESY correlations. The detailed inspection
of the coupling constants J3′′′ax/4′′′ax (13.5 Hz), J4′′′ax/5′′′ax (13.5 Hz), J4′′′ax/3′′′eq (4.0 Hz),
J4′′′ax/5′′′eq (4.0 Hz) enabled assignment of the signals for methylene protons at δH 1.26,
1.59, 1.70, 1.71, 2.04 and 2.12 to H-5′′′ax, H-3′′′ax, H-5′′′eq, H-4′′′eq, H-3′′′eq and H-
4′′′ax, respectively. The NOESY correlations of 22 were observed between H-3′′′ax/H-
1′′′ (δH 3.13), H-3′′′eq/H-1′′′ (δH 2.96), H-4′′′ax/H-6′′′, H2-5′′′/H3-7′′′ indicated the axial
orientation of H-6′′′ and equatorial orientation of H3-7′′′ (Fig. 9). On the other hand,
compound 22 ([α]D -121o) was subjected to chiral HPLC to analyze their stereoisomers.
A minor peak and a major peak were observed at 14.5 min and 16 min in a ratio of 1:6,



-24-
respectively. In addition, its NMR spectral features demonstrated signals for a single
compound. Accordingly, compound 22 was a mixture of enantiomeric isomers.
However, the absolute stereochemistry could not be determined. The similar NOESY
correlations of compounds 19-22 suggested that these compounds possessed the same
relative configurations at spiro-ring system. Compounds 19-21 could be a mixture of
enantiomeric isomers, although chiral HPLC analyses of 19-21 failed to separate their
isomers.




Fig. 9. Proposed stereochemistry of spiro-ring system of 22


New depsidones parmosidones A (23), B (24) and C (25)
C5H11
Compound 23, named parmosidone A, was
H
isolated as a pale yellow solid. The MS spectrum H
O 1" H O
H 6
OH
7O
of compound 23 established the molecular
1
formula of C28H30O10. The 1H-NMR spectrum 4
O
O
HO
exhibited signals for a pair of meta-coupled H
O
H 2"'
H
23
aromatic protons at δH 6.40 and 6.44 (each d, O
H
COSY H
J=2.0 Hz), an aromatic proton at δH 6.75 (s), two H
5"'
HMBC H O H
methylene groups at δH 3.81 and 3.92 (each 1H, d, NOESY

J=17.0 Hz) and at δH 3.32 and 3.49 (each 1H, d, J=17.0 Hz), and n-pentyl group. It
showed further signals due to an ethoxyl group and a doublet for an acetal methine
proton H-5′′′ at δH 5.28 (J=5.0 Hz), which was connected in sequence in the 1H-1H
COSY spectrum with two methylene signals at δH 2.34 and 2.38 (each 1H, m) and at δH



-25-
2.03 and 2.40 (each 1H, m) (Table 6). These 1H-NMR spectral data, together with 13C-
NMR spectral features (Table 5) revealed that 23 had a similar depsidone structure as 20,
but differed from 20 in the spiro-ring system. The NOESY spectrum of 23 showed the
correlations between H-5′′′ and methylene protons at δH 3.37 and 3.60 of the ethoxyl
group. In the HMBC spectrum of 23, a significant correlation was observed from H-5′′′
to the ketal carbon at δC 111.9 (C-2′′′). These findings indicated 23 possessed a six-five-
member spiro-ring instead of a six-six-member ring as seen in 20.


Compound 24 was isomeric with 23. The 1H- C5H11
H
13
and C-NMR spectra of 24 (Tables 6 and 5) H
O 1" H O
H 6
7O OH
demonstrated the depsidone skeleton. The
1
differences between these compounds could be 4
O
O
HO
only accounted for by a ketal proton at δH 5.29 H O
H 2"'
H
24 H
(H-5′′′) of 24 due to a doublet of doublets signal O H
H
COSY
HH
(J=5.5, 3.5 Hz) instead of doublet signal of H-5′′′ 5"'
HMBC O
NOESY
(J=5.0 Hz) as seen in 23. Accordingly, the
structure of 24 was elucidated and designated parmosidone B.

C5H11
The HR-SIMS established the composition H
H
O 1" H O
of 25 as C30H34O10, i.e. having two methylene
H 6
OH
7O
groups more than that of 23. The NMR spectral 1
4
O
O
features of 25 (Tables 5 and 6), named HO
H
O
H 2"'
parmosidone C, were also similar to those of 23, H
25 O
COSY
except for the presence of n-butoxyl group at C- H
H
HMBC H
5"'
H
5′′′. Detail NMR studies of this compound led O H
NOESY
us to determine 25 as to be shown.


Compounds 23-25 were representative for 6,5-spiroketal natural products and
possessed two asymmetric carbons C-2′′′ and C-5′′′. In 1H-NMR spectra, compound 24
merely differed from 23 and 25 in the coupling constant of ketal proton H-5′′′, implied
that 24 was distinct from 23 and 25 at the stereochemistry of asymmetric carbon C-5′′′.



-26-
The ketal proton H-5′′′ of 24 resonated at δH 5.29 ppm as doublet of doublets (J=5.5,
3.5 Hz) due to the coupling to the vicinal methylene protons H2-4′′′. On the other hand,
the ketal proton H-5′′′ of 23 and 25 resonated as doublet (J=5.0 and 5.5 Hz,
respectively). These differences indicated that the methine proton H-5′′′ of 23 and 25
had coupling with only one of the vicinal methylene protons H2-4′′′. The coupling
between H-5′′′ and the other proton of H2-4′′′ was not observed implying that the
dihedral angle between H-5′′′ and one of protons H-4′′′ was close to 90o.48) Although
the coupling constants of the ketal proton H-5′′′ of compounds 23-25 have been
recognized, the stereochemistry of the spiro-ring systems still remains to be elucidated.


Table 6. 1H-NMR spectroscopic data of 23-25 in CDCl3 (J, Hz)
No. 23 24 25
3 6.40 d (2.0) 6.39 d (2.5) 6.51 d (2.5)
4-OH 7.58 br s 7.50 br s 6.89 br s
5 6.44 d (2.0) 6.47 d (2.5) 6.48 d (2.5)
2'-OH 10.94 s 11.05 s 10.96 s
3' 6.75 s 6.77 s 6.79 s
1" 3.81 d (17.0) 3.85 d (17.0) 3.86 d (17.0)
3.92 d (17.0) 3.90 d (17.0) 3.96 d (17.0)
3" 2.64 t (7.5) 2.63 td (7.0, 2.0) 2.60 t (7.5)
4" 1.65 quint (7.5) 1.65 quint (7.0) 1.65 m
5" 1.33 m 1.33 m 1.31 m
6" 1.33 m 1.33 m 1.34 m
7" 0.91 t (7.0) 0.91 t (7.0) 0.91 t (7.0)
1"' 3.32 d (17.0) 3.25 d (17.0) 3.35 d (16.5)
3.49 d (17.0) 3.46 d (17.0) 3.53 d (16.5)
3"' 2.34 m 2.16 m 2.34 m
2.38 m 2.59 m 2.38 m
4"' 2.03 m 2.25 m 2.03 m
2.40 m 2.35 m 2.41 m
5"' 5.28 d (5.0) 5.29 dd (5.5, 3.5) 5.28 d (5.5)
3.37 dq (10.0, 7.0) 3.41 dq (10.0, 7.0) 3.32 dt (10.0, 7.0)
α
3.60 dq (10.0, 7.0) 3.60 dq (10.0, 7.0) 3.56 dt (10.0, 7.0)
1.16 t (7.0) 1.15 t (7.0) 1.51 quint (7.0)
β
1.34 m
γ
0.89 t (7.5)
δ




-27-
2.1.4. Other lichen substances


(+)-Usnic acid (26)
Compound 26 was obtained as light yellow
needles, mp. 184-185oC, [α]D +455o. The molecular
formula of C18H16O7 was established by HR-EIMS.
The 1H-NMR spectrum exhibited the resonances
for an aromatic protons at δH 5.99 (s), four methyl
groups at δH 1.77, 2.12, 2.67 and 2.69 (each 3H, s),
and three hydrogen-bonded phenolic protons at δH
13
11.04, 13.33 and 18.84 (each 1H, s). The C-NMR spectrum revealed the resonances
for 18 carbons which were assigned for four methyl groups, a sp3 quaternary, five sp2
quaternary, an aromatic CH, five sp2 oxygenated quaternary and three carbonyl carbons
at δC 198.1, 200.4 and 201.8. These spectral features together with 2D NMR analysis of
26 led to identify 26 as (+)-usnic acid.13,49) Usnic acid is a characteristic lichen
substance, and is especially abundant in genera such as Alectoria, Cladonia, Usnea,
Lecanora, Ramalina and Evernia.50)


Skyrin (27)




Compound 27 was isolated as a red crystalline solid. The UV spectrum showed the
maxima at 219.5, 259, 303.5, 341.5 and 465.5 nm and the IR spectrum exhibited the
absorption bands at 3466, 3262, 1674, 1625, 1602, 1552 and 1483 cm-1, indicating the



-28-
presence of hydroxyl and carbonyl groups, and aromatic ring. The 1H-NMR spectrum
revealed the signals of three aromatic protons at δH 6.80 (s), 7.10 and 7.31 (each 1H, br
s), a methyl group at δH 2.38 (3H, s) and two hydrogen-bonded hydroxyl groups at δH
13
12.12 and 12.86 (each 1H, s). The C-NMR spectrum showed the signals for 15
carbons including a methyl group, 12 aromatic carbons and two carbonyl groups at δC
183.1 and 191.7. These findings, together with 2D NMR experiments suggested that
compound 27 could be an anthraquinone derivative with a substitution at C-5 such as 5-
chloroemodin (28).51) However, the HR-ESIMS established the molecular formula of 27
to be C30H18O10, indicating a symmetrical structure. Hence, compound 27 was identified
as a bianthraquinone skyrin.52)




-29-
2.2. Chemical investigation of the lichen thalli of Rimelia clavulifera
Purification of acetone and MeOH extracts of the foliose lichen R. clavulifera gave
fifteen known lichen substances: lecanoric acid (2), methyl orsellinate (3), methyl β-
orsellinate (6), methyl haematommate (7), atranorin (9), chloroatranorin (10), α-
alectoronic acid (11), α-collatolic acid (12), β-alectoronic acid (13), β-collatolic acid
(14), dehydrocollatolic (19), (+)-usnic acid (26), skyrin (27), gyrophoric acid (29) and
salazinic acid (30) (Fig. 10). These known compounds were identified with isolated
compounds from the lichen thalli of Parmotrema mellissii, except for lecanoric acid (2),
gyrophoric acid (29) and salazinic acid (30).




Fig. 10. Extraction and isolation procedure for R. clavulifera




-30-
Lecanoric acid (2)
Compound 2 was obtained as a white
powder. The HR-SIMS of 2 gave the molecular
formula of C16H14O7. Its 1H-NMR spectrum
showed two singlets at δH 2.61 (3H) and 2.66
(3H) for methyl groups, a pair of doublets for
two aromatic protons at δH 6.31 and 6.39 (each
1H, J=2.5 Hz) and a pair of broad singlets at δH
6.66 and 6.72 (each 1H) for protons H-5′ and H-3′, respectively. The 13C-NMR showed
the signals due to 16 carbons corresponding to two methyls, four aromatic methines and
ten quaternary carbons including four oxygenated, a carbonyl carbon at δC 170.5 and a
carboxyl group at δC 174.2. These spectral data suggested that 2 consisted of two sets of
orsellinic acid units and HMBC spectra confirmed the proposed structure as shown.
Accordingly, the structure of 2 was established as lecanoric acid.38,53)


Gyrophoric acid (29)
The HR-SIMS spectrum of
29 established a molecular
formula of C24H20O10. Its 1H-
NMR spectrum showed
singlets for three methyl groups
at δH 2.36 (6H) and 2.50 (3H)
and signals for three pairs of
meta-coupling aromatic protons and three broad singlets for phenolic groups. The 13C-
NMR spectrum of 29 revealed 24 lines attributable to three sp3 methyls, six sp2
methines, and fifteen non-protonated carbons including six oxygen-bearing, two
carbonyl and a carboxyl carbons. These spectral data indicated that compound 29
possessed an orsellinic acid unit more than 2. Hence, the structure of 29 was determined
to be tridepside gyrophoric acid.53)




-31-
Salazinic acid (30)
Compound 30 was obtained as a major 9'
8
CH3 O CH2OH
component of the lichen R. clavulifera and had a H O OH
7
61 3'
molecular formula of C18H12O10 as indicated by 1'
O
HR-ESIMS. Its IR spectrum showed absorption HO O 6'
3 7'
9CHO 8' O
bands at 1772, 1742 and 1661 cm-1 correspoding
HO
to depsidone ring, butyrolactone and aldehyde NOESY
HMBC
54) 1
30
group, respectively. Its H-NMR showed the
signals due to an aromatic proton at δH 6.87 (s), a hemiacetal proton at δH 6.80 (br s), an
aldehyde proton (δH 10.46), a hydroxymethyl (δH 4.66), a methyl (δH 2.45) and a
hydrogen-bonded hydroxyl (δH 12.10) groups. The 13C-NMR spectrum of 30 exhibited
18 carbons including 13 quaternary carbons (two carbonyls, five oxygen-bearing
aromatic and six aromatic carbons), three methines (an aldehyde, an aromatic and an
oxygenated), an oxygenated methylene and a methyl group. These spectral features
together with 2D NMR analysis suggested that 30 was salazinic acid.54)


In conclusion, two foliose lichen species Parmotrema mellissii and Rimelia
clavulifera collected in the South of Vietnam were chemically investigated. From the
thalli of P. mellissii, twenty five lichen substances including five new depsidones and
three new isocoumarins were isolated. Chemical investigation of the thalli of R.
clavulifera yielded fifteen known lichen substances. The depsidones α-alectoronic acid
(11) and α-collatolic acid (12), common lichen substances in the lichen genus
Parmotrema,47) were isolated as major metabolites from the lichen thalli of P. mellissii.
Dehydroalectoronic acid (20) and parmosidones A (23), B (24) and C (25) were new
depsidones with unique spiro-ring system. This type of depsidone has not been reported
with single exception of dehydrocollatolic acid (19).




-32-
Chapter 3: Secondary metabolites from the cultured lichen mycobionts


3.1. Chemical investigation of the cultured mycobionts of Graphis vestitoides
Graphidaceae is the second largest family of lichenized fungi and the most
important element of lichen communities in tropical regions, with over 50 genera and
1,500 species. The lichen genus Graphis (family of Graphidaceae), the so-called Script
Lichens, is a genus comprising about 300 species.55,56) The chemistry of the thalli of
Graphis species is relatively simple, such as lecanoric acid (2), norstitic acid (31), stictic
acid (32), psoromic acid (33) and protocetraric acid (34).55,57) Previous chemical
investigations on the cultured lichen mycobionts of various Graphis species [G.
awaensis, G. apriens, G. cognata, G. desquamescens, G. handellii, G. proserpens, G.
prunicola, G. rikuzensis, G. scripta, G. scripta var. pulverulenta and G. tetralocularis]
led to the isolation of quinone,58) coumarins,59-61) chromones,62) 6H-dibenzo[b,d]pyran-
6-ones 25,63,64) and other aromatic derivatives 61,65-67) (Fig. 11).
O O O CH2OH
O OH O OCH3 O OH

O
RO O HO O HO O CO2H
CHO CHO COOH CHO
O
HO
norstictic acid (31) : R = H psoromic acid (33) protocetraric acid (34)
stictic acid (32) : R = CH3
O H3CO O OH O
H3CO
O OH
Cl
O
H3CO H3CO H3CO O
O Cl 5-hydroxy-2,3-dimethyl-7-
graphisquinone 8-methyldichlorodiaportin -methoxychromone
OR O O H3CO O
H3CO
O
O O
OH
R
H3CO H3CO2C
H3CO
OH
O
HO
OCH3
OCH3
HO
R = H : graphislactone A R = H : proserin A
graphislactone D
R = CH3 : graphislactone B R = OH : proserin B
R = OCH3 : proserin C

Fig. 11. Selected secondary metabolites from the thalli and cultured mycobionts of
Graphis species




-33-
Graphis vestitoides Cultured mycobionts
Photo 3. G. vestitoides thalli and its cultured mycobionts


Specimens of Graphis vetitoides (Fink) Staiger were collected from the tree bark in
Dong Nai Province, Vietnam in 2008 (Photo 3). The polyspore-derived mycobionts
were cultivated on a malt-yeast extract medium supplemented with 10% sucrose
(MY10) at 18oC in the dark. After three months, the cultures were harvested and
extracted with Et2O, acetone and then with MeOH at rt. Subsequent purification of the
extracts by a combination of column chromatography, prep. TLC and prep. HPLC gave
a phenolic compound (35), five known isocoumarin derivatives (36-40), a new
isocoumarin (41) and a novel 14-membered macrolide (42) (Fig. 12).


2-Acetyl-3,5-dihydroxybenzoic acid (35)
Compound 35 was obtained as a white solid. The HR-
H
APCIMS of compound 35 showed a pseudo-molecular ion
HO COOH
1
([M-H]-) at m/z 195.0291 (calcd for C9H7O5, 195.0294). The 5
CH3
H
UV spectrum of 35 displayed maxima at 210.5, 241 and 286.5 3
OH O
nm. The IR spectrum of 35 showed strong absorption bands
COSY HMBC
due to phenolic hydroxyl and carboxyl groups (3481 cm-1,
35
broad) and conjugated carbonyl groups (1701 and 1602 cm-1).
Its 1H-NMR exhibited two meta-coupled aromatic protons at δH 6.19 and 6.32 (each 1H,
13
d, J=2.0 Hz) and one methyl singlet at δH 2.38. The C-NMR, DEPT and HSQC
indicated that 35 had one methyl, two methine and six quaternary carbons including two




-34-
oxygenated carbons at δ 162.5 and 163.7, a carboxyl and carbonyl groups at δ 175.3 and
197.8, respectively. The carboxyl and hydroxyl groups could be inferred from the
13
combination of MS, C-NMR and HMBC spectra. These spectral features led us to
identify the structure of 35 to be 2-acetyl-3,5-dihydroxybenzoic acid.
The polyspore-derived mycobionts of
the lichen Graphis vestitoides
Cultivated on MY10 medium f or 3 months
Harvested and free-dried colonies
(33.5 g)
Extracted at rt

Acetone (3 x 100 ml)
Et2O (3 x 100 ml) MeOH (3 x 100 ml)
Et2O extract Acetone extract MeOH extract
(362.1 mg) (8.15 g)
(122.1 mg)
pTLC A pTLC A n-BuOH
Visualized band Visualized band Water layer n-BuOH extract
(48.9 mg) (42.5 mg) (5.32 g) (2.46 g)
CC/CHCl3-MeOH
pTLC B-D MeOH (0%) (1%) (1-2%) (10-50%)
38 (29.3 mg) Fr-I Fr-II Fr-IV
Fr-III
39 (41.4 mg) (97.6 mg) (658.5 mg) (82.3 mg)
(125 mg)
pTLC
Fatty comp.
A n-Hexane - Et2O - AcOH (10:15:1)
pTLC D, E, F
pTLC E, F
B n-Hexane - Et2O (1:9) pHPLC
C n-Hexane - Et2O - AcOH (40:60:1)
35 (8.3 mg) 36 (24.7 mg)
D CHCl3 - MeOH (9:1)
38 (173.3 mg) 37 (7.7 mg)
E Et2O pHPLC
39 (169.7 mg) 39 (7.3 mg)
F CHCl3 - MeOH (8:2), (6:4) Column: C18 Bondasphere
40 (2.3 mg) 41 (20.3 mg)
Detection: 254 nm
42 (57.0 mg)
MeOH - H2O (4:6)

Fig. 12. Extraction and isolation procedure for cultured mycobionts of G. vestitides


trans-5,7-Dihydroxy-3-(1-hydroxyethyl)phthalide (36)
Compound 36 was obtained as colorless plates. Its HR- OH O
7
H
ESIMS indicated a molecular formula of C10H10O5. Its UV 6
1
O
spectrum showed maxima at 217.5, 257.5 and 293 nm. The 5 3
HO H
4
IR spectrum showed absorption bands at 3304, 3127, 1713, 8
CH3
H HO
1616 and 1482 cm-1, representing the hydroxyl, a carbonyl H
1 COSY HMBC
group and substituted aromatic system. The H-NMR
36


-35-
spectrum of 36 revealed the presence of two meta-coupled aromatic protons at δH 6.31
(d, J=2.0 Hz) and 6.47 (dd, J=2.0, 1.0 Hz), two sp3 oxygenated methine protons at δH
3.98 (qd, J=6.5, 5.0 Hz) and 5.22 (br d, J=5.0 Hz), and a methyl doublet at δH 1.16
13
C-NMR spectrum showed signals for a methyl carbon, two sp3
(J=6.5 Hz). Its
oxygenated methine carbons, two sp2 CH carbons and five quaternary carbons including
two oxygenated and one carbonyl carbons. The position of functional groups of 36 was
determined by analysis of 2D NMR spectra (COSY, NOESY, HSQC and HMBC). The
COSY correlation was observed between an aromatic proton at δH 6.47 and a methine
proton at δH 5.22. The latter was correlated with a methine proton at δH 3.98 which was
connected to methyl group. Furthermore, the HMBC correlation from the methine
proton at δH 5.22 to a carbonyl carbon at δC 172.1 implied a lactone ring. These findings
suggested to be 5,7-dihydroxy-3-(1-hydroxyethyl)phthalide. The relative
36
configuration of C-3 and C-8 was determined to be trans based on the coupling constant
of 5.0 Hz.68) Thus, compound 36 was elucidated to be trans-5,7-dihydroxy-3-(1-
hydroxyethyl)phthalide. This compound has been previously synthesized as a racemic
compound,68) but their absolute configurations were not determined. The absolute
stereochemistry of two chiral centers C-3 and C-8 of 36 still remains to be elucidated.


cis-5,7-Dihydroxy-3-(1-hydroxyethyl)phthalide (37)
Compound 37 was isomeric with 36. The spectral OH O
H
features (UV, IR, 1D and 2D NMR) demonstrated the planar
1
O
structure of 37 was the same as 36. The difference was 3
H
HO
ascribed to the cis configuration of C-3 and C-8, which 8
CH3
H H
could be accounted for by the coupling constant of 3.5 Hz HO
COSY HMBC
between two methine protons at δΗ 4.13 (qd, J=6.5, 3.5 Hz,
37
H-8) and 5.23 (br d, J=3.5 Hz, H-3). Accordingly, the
structure of 37 was identified as cis-5,7-dihydroxy-3-(1-hydroxyethyl)phthalide.68) The
specific optical rotations ([α]D) of (3R,8R)- and (3S,8S)-isomer of 37 were -62.0o and
+60.7o, respectively.68) The isolated compound 37 could be a mixture of enantiomeric
isomers on the basis of its [α]D +6.2o.




-36-
4,6-Dihydroxy-3,9-dehydromellein (38)
Compound 38 was obtained as a white crystalline solid. OH O
H
Its molecular formula was determined by HR-ESIMS as 7 8a
1
O
C10H8O5. It showed UV maxima at 217.5, 271.5 and 307 3
4 9
H
4a
HO 5
-1
nm, and IR bands at 3202, 1664, 1628 and 1498 cm , H OH H
H
representing the hydroxyl and conjugated lactone COSY HMBC
1
functional groups. Its H-NMR spectrum showed signals
38
for two meta-coupled aromatic protons at δH 6.30 (d, J=2.0
Hz) and 6.54 (dd, J=2.0, 0.5 Hz); the latter was coupled with a sp3 methine proton at δH
5.21 (br). It exhibited signals for a pair of exo-methylene protons at δH 4.91 (dd, J=1.5,
13
0.5 Hz) and 4.93 (t, J=1.5 Hz). The C-NMR spectrum in combination with DEPT
experiments of 38 showed signals for a sp3 oxygenated methine, a sp2 methylene carbon
and eight sp2 quaternary carbons including an ester carbonyl and three oxygenated
carbons. The HMBC correlations from the sp3 methine proton (H-4) to four carbon
signals at δC 99.3 (C-8a), 106.8 (C-5), 144.8 (C-4a) and 157.5 (C-3), from an aromatic
proton (H-5) to carbon signals at δC 66.5 (C-4), 167.1 (C-6) and C-8a, and from another
aromatic proton (H-7) to C-6, C-8 (δC 165.3) and C-8a suggested 6,8-substituted
dihydroisocoumarin ring system. Further HMBC correlations from two sp2 methylene
protons (H2-9) to C-3, C-4 and C-1 (δC 167.3) indicated the substitution of the exo-
methylene group at C-3. The molecular formula of 38 and chemical shifts of 13C-NMR
spectrum indicated three hydroxyl groups at C-4, C-6 and C-8. Accordingly, 38 was
determined to be 4,6-dihydroxy-3,9-dehydromellein.67)
Compound 38 was previously isolated from the cultured mycobionts of Graphis
proserpens. The absolute configuration of C-4 in 4,6-dihydroxy-3,9-dehydromellein
(38) could not be determined by modified Mosher’s method, since its esterification with
MTPA failed to yield a desired product, but afforded 38a (Fig. 13).67) Thus, the
configuration at C-4 still remains to be elucidated.




-37-
Fig. 13. MTPA ester of 38


6,8-Dihydroxy-3-(hydroxymethyl)isocoumarin (39)
COSY
Compound 39 was isolated as colorless needles.
HMBC
OH O
The HR-ESIMS of 39 exhibited a peak at m/z NOESY
H 7 8a
O
207.0292 [M-H]-, indicating a molecular formula of
3
OH
9
C10H8O5. It showed UV maxima at 237, 277.5, 289.5 4a
HO 4
5
HH
H H
and 328 nm, and IR bands at 3237, 1678, 1620, 1579
and 1488 cm-1, suggesting the presence of hydroxyl
39
group(s), carbonyl group and substituted aromatic
system. The 1H-NMR spectrum exhibited signals for a pair of meta-coupled aromatic
protons, an olefinic proton and a hydroxymethyl group. The 13C-NMR spectrum showed
ten carbon signals for a sp3 oxygenated methylene, three sp2 CH carbons, and six sp2
quaternary carbons including three oxygenated carbons and an ester carbonyl carbon.
The HMBC correlations from an olefinic proton (H-4) to C-3, C-4a and C-5, from H-5
to C-4, C-6 and C-8a, and from H-7 to C-6, C-8 and C-8a suggested 6,8-substituted
isocoumarin ring system. Further HMBC correlations from methylene protons (H2-9) to
C-3, C-4 and C-4a indicated the substitution of the hydroxymethyl group at C-3. On the
basis of these spectral features, the structure of 39 was determined to be 6,8-dihydroxy-
3-(hydroxymethyl)isocoumarin.69)


cis-4,6-Dihydroxymellein (40)
The HR-ESIMS of 40 exhibited a molecular formula of
C10H10O5, two mass units more than that of 38. Its 1H-NMR
spectrum displayed its structural similarity to 38. The only
difference between 38 and 40 was accounted for by the hydrogenation of C-3/C-9 in 40.



-38-
The 1H-NMR spectrum of 40 showed signals for an oxygenated methine proton at δH
4.63 (qd, J=6.6, 2.4 Hz, H-3) and a methyl group at δ 1.47 (d, J=6.6 Hz, H3-9) instead
of two sp2 methylene protons as seen in 38. The relative configuration at C-3 and C-4
was suggested to be cis by the comparison of J3,4 (2.4 Hz) of 40 with those of similar
dihydroisocoumarin.70) Accordingly, compound 40 was determined to be cis-4,6-
dihydroxymellein.71,72)


New isocoumarin 6,8-dihydroxyisocoumarin-3-carboxylic acid (41)
COSY
Compound 41 was isolated as a white crystalline
OR3 O HMBC
solid, mp > 300oC. The HR-ESIMS of 41 established NOESY
H 8a
7
O
1
the molecular formula of C10H6O6. It showed UV 39
2
COOR1
4a
maxima at 251.5, 289, 300, 324.5 and 336.5 nm, and R O 4
5
H
H
IR bands at 3393 (br), 3245 (br), 1679, 1637, 1609
and 1498 cm-1, representing the hydroxyl group(s), 41 : R1 = R2 = R3 = H
41a : R1 = R2 = R3 = CH3
1
carbonyl group and aromatic ring. Its H-NMR
43 : R1 = R3 = H, R2 = CH3
spectrum showed the signals for meta-coupled
aromatic protons at δH 6.43 and 6.59 (each 1H, d, J=2.0 Hz) and an olefinic proton at δH
13
C-NMR spectrum exhibited the signals for three sp2 CH carbons and
7.23 (s). The
seven sp2 quaternary carbons including three oxygenated carbons and two carbonyl
carbons. These spectral features suggested that 41 possessed a carboxyl group instead of
a hydroxymethyl group as in 39. This was confirmed by the HMBC spectrum. The
correlations from an olefinic proton (H-4) to three carbon signals at δC 99.0 (C-8a),
138.8 (C-4a) and 150.0 (C-3), and from H-5 to carbon signals at δC 108.5 (C-4), 165.8
(C-6) and C-8a, and from H-7 to C-6, C-8 (δC 162.5) and C-8a suggested that 41
possessed isocoumarin ring system as 39. The HMBC correlation observed from the
olefinic proton (H-4) to carboxyl carbon at δC 161.8 (C-9) indicated the substitution of
the carboxyl group at C-3. These spectral features were similar to those of 6-methoxy-8-
hydroxyisocoumarin-3-carboxylic acid (43),73) except for the absence of methoxyl
group in 41. Methylation of 41 with TMS-CHN2 afforded methyl ester 41a whose 1H-
NMR spectrum demonstrated the signals for three additional methoxyl groups at δH




-39-
3.93, 3.94 and 3.99. Accordingly, the structure of 41 was identified as 6,8-
dihydroxyisocoumarin-3-carboxylic acid.


New 14-membered macrolide 42
Compound 42 was obtained as a colorless solid.
The elemental composition of 42 was established by
HR-ESIMS as C14H18O5, implying six degrees of
unsaturation. The presence of hydroxyl and carbonyl
groups as well as double bonds was shown by IR
absorption bands at 3423 (br), 1710 and 1598 cm-1. The 1H-NMR spectrum of 42
exhibited the signals for eight methylene protons, six olefinic methine protons and two
oxygenated methine protons at δH 4.70 (br d, J=9.0 Hz) and 5.24 (m) (Table 7). The 13C-
NMR spectrum of 42 showed 14 signals, which were assigned by DEPT as four
methylenes, two oxygenated methines and eight sp2 carbons including six CH carbons
and two carbonyl carbons at δC 168.2 and 177.5 (Table 7). The sequence from olefinic
proton at δH 5.83 (H-3) to oxygenated methine proton at δH 4.70 (H-14) was revealed by
the 1H-1H COSY. The sequence was further defined by HMBC correlations as shown.
Further significant HMBC correlations from olefinic protons H-3 and H-4 (δH 7.42), and
from oxygenated methine proton H-14 to carbonyl carbon C-2 (δC 168.2), and from H-
14 to carboxyl C-15 (δC 177.5) established a 14-membered lactone ring with a carboxyl
group at C-14. Furthermore, the presence of a hydroxyl at C-5 was proposed based on
the molecular formula and the characteristic chemical shifts of H-5 (δH 5.24) and C-5
(δC 68.8). The geometry of the double bonds C-3/C-4, C-6/C-7 and C-9/C-10 was
assigned as E, Z and E based on 1H-1H coupling constants of 15.5, 11.0 and 15.0 Hz,
respectively. These spectral data of 42 closely resembled those of mutolide (44), a
fungal macrolide isolated from the culture broth of the fungus Sphaeropsidales sp.
(strain F-24′707),74) except that 44 showed the presence of a hydroxyl group at C-8,
methyl group at C-14 and E configuration of double bond C-6/C-7. Accordingly, the
planar structure of 42 was elucidated as shown.




-40-
The absolute configuration of C-14 of 42 was determined by PGME method.75) This
method is based on the principle as follows: A chiral α,α-disubstituted acetic acid is
condensed with (R)- and (S)-PGME. In the diastereomeric pair of PGME amides, the
protons of R2 residue of the (S)-isomer will have the more upfield chemical shifts than
those of the (R)-isomer owing to the diamagnetic anisotropic effect of the benzene ring.
The same will stand for the protons of R1 (Fig. 14).76) Compound 42 possessing a
carboxyl group at chiral center C-14 was condensed with (R)- and (S)-PGME to yield
42a and 42b, respectively. Analysis the ∆δ (δ(S) - δ(R)) values of the PGME amides of 42
led to assign the S configuration of C-14 (Fig. 14).




Fig. 14. PGME method for determination of absolute configuration of carboxylic acid


To determine the absolute configuration at C-5 by
modified Mosher’s method, the carboxyl group C-15
was methylated. Treatment of 42 with TMS-CHN2
unexpectedly yielded a methylated compound 42m.




-41-
The HR-APCI mass spectrum of 42m established the composition of C15H20O5, that was
14 mass units more than that of 42. The spectral features of 42m and 42 showed the
similarity, but 42m exhibited an additional methoxyl group, a methylene carbon (C-3)
and an oxygenated methine carbon (C-4) instead of carboxyl group and a double bond
(C-3/C-4) as seen in 42 (Table 7). The HMBC spectrum of 42m showed the interactions
from methoxyl group (δH 3.75) and oxygenated methine proton H-14 (δH 4.74) to
carbonyl carbon C-15 (δC 170.2) confirmed the occurrence of carboxyl group in 42.
Further HMBC correlations from methylene protons H2-3 (δH 3.73 and 4.10) to C-4 (δC
49.2) and C-5 (δC 65.1), from oxygenated methine proton H-4 (δH 3.71) to C-5, and
from H-5 (δH 5.00) to C-4 suggested the presence of an epoxy ring between C-4 and C-
5. The 1H-1H coupling constants between olefinic protons H-6/H-7, H-9/H-10 were 11.0,
15.5 Hz, respectively, clearly revealed that the same geometry of these double bonds
had occurred as in 42. The epoxide ring was assigned to be cis based on the coupling
constant 4.0 Hz of H-4/H-5.
The absolute configuration of C-5 of 42 could not be determined, since its
methylation with TMS-CHN2 failed to yield a desired ester with 5-hydroxyl group.
Hence, the stereochemistry at C-5 still remains to be elucidated.


In conclusion, the metabolites of the cultivated colonies of G. vestitoides were
chemically investigated to isolate a new isocoumarin (41), a novel 14-membered
macrolide (42) and six known substances. Several 14-membered macrolides were
isolated from marine original sources77-79) and fungal origins,74,80,81) but have not been
isolated from the lichen thalli or cultured mycobionts. This is the first instance of
isolation of 14-membered macrolide from lichen mycobionts.




-42-
Table 7. 1H- and 13C-NMR spectroscopic data of 42 and 42m

42a 42mb

No. (J, Hz) (J, Hz)
δH δC δH δC
2 168.2 161.4
3 5.83 dd (15.5, 2.0) 116.8 4.10 dd (9.0, 2.0) 50.1
3.73 m
4 7.42 dd (15.5, 4.0) 153.5 3.71 m 49.2
5 5.24 m 68.8 5.00 dd (9.5, 4.0) 65.1
6 5.46 dd (11.0, 4.0) 133.6 5.71 br dd (11.0, 9.5) 127.6
7 5.50 m 128.8 5.75 td (11.0, 5.5) 133.5
8a 2.74 br d (15.0) 33.1 2.59 br dt (16.5, 5.0) 30.0
8b 2.91 m 3.05 m
9 5.45 m 128.4 5.17 m 129.3
10 5.63 br dt (15.0, 7.5) 132.3 5.41 br dt (15.5, 4.5) 129.8
11a 2.05 m 34.2 2.13 m 30.8
11b 1.96 m 1.86 m
12a 1.22 m 26.4 1.44 m 23.5
12b 1.86 m 1.76 m
13a 1.80 m 31.5 2.07 m 26.3
13b 2.01 m
14 4.70 br d (9.0) 76.8 4.74 dd (7.0, 5.0) 74.6
177.5c
15 170.2
OCH3 3.75 s 52.4
a b c
Measured in CD3OD Measured in CDCl3 Broad signal.




-43-
3.2. Chemical investigation of the cultured mycobionts of Sacographa tricosa
Sarcographa, family of Graphidaceae, is a
widely distributed, tropical and subtropical
crustose lichen genus. Norstictic acid (31) and
stictic acid (32) were reported for the chemical
constituent of the thalli of Sarcographa genus.82,83)
The cultured mycobionts of this lichen genus have
never been chemically investigated. The thalli of
Sarcographa tricosa (Ach.) Müll. Arg. collected in Korea and Australia were detected
no chemical compounds.82,84) The polyspore-derived mycobionts of S. tricosa collected
from the tree bark in Dong Nai Province, Vietnam were cultivated on MY10 medium at
18oC in the dark (Photo 4). After several months, the cultures were harvested and
extracted with n-hexane and Et2O. These extracts were separated by chromatographic
procedures to afford ergosterol peroxide (45), six known (46-51) and three new (52-54)
eremophilane-type sesquiterpenes (Fig. 15).




Sarcographa tricosa Cultured mycobionts
Photo 4. S. tricosa thalli and its cultured mycobionts




-44-
The polyspore-derived mycobionts of
the lichen Sarcographa tricosa
Cultivated on MY10 medium
f or 4-8 months
Harvested and freeze-dried colonies
(185.5 g)
Extracted at rt

n-Hexane (2 x 800 ml) Et2O (3 x 400 ml)
n-Hexane extract Et2O extract
(0.23 g) (0.92 g)
CC/n-Hexane-Et2O
pTLC A, B
pHPLC Et2O (0-10%) (20-30%) (50-70%) (70-100%)
45 (30.3 mg)
Fr-I Fr-II Fr-IV
Fr-III
46 (9.3 mg)
(170 mg) (135 mg) (245 mg)
(284 mg)
47 (12.6 mg)
Fatty comp.
48 (49.3 mg) pTLC B pTLC B pTLC C, D
52 (1.2 mg)
Fatty comp. (58.2 mg) 50 (2.3 mg)
47 (119.8 mg)
45 (10.5 mg)
51 (4.9 mg)
48 (83.7 mg)
52 (2.1 mg)
pTLC 53 (6.9 mg)
49 (8.0 mg)
A Et2O - AcOEt (10:1) 54 (2.6 mg)
pHPLC
B Toluene - AcOEt (3:2), (1:1), (1:2)
Column: Sunfire
C n-Hexane - AcOEt (1:4) Detection: 254 nm
D Toluene - AcOEt - AcOH (5:5:0.2) n-Hexane - 2-Propanol (7:3)

Fig. 15. Extraction and isolation procedure for cultured mycobionts of S. tricosa


Ergosterol peroxide (45)
Compound 45 was isolated as a
colorless crystalline solid, [α]D -34o. The
HR-ESIMS established the composition of
C28H44O3. The IR spectrum showed
absorption bands at 3376, 1458, 1378,
1044 and 967 cm-1. The 1
H-NMR
spectrum exhibited signals for six methyl groups at δH 0.82 (s), 0.82 (d, J=6.5 Hz), 0.83
(d, J=7.0 Hz), 0.88 (s), 0.91 (d, J=7.0 Hz) and 1.00 (d, J=6.5 Hz), four olefinic protons
at δH 5.14 (dd, J=15.5, 7.5 Hz), 5.22 (dd, J=15.5, 8.0 Hz), 6.24 (d, J=8.5 Hz) and 6.50
(d, J=8.5 Hz), oxygenated methine at δH 3.97 (tt, J=12.0, 5.0 Hz), and twenty protons at




-45-
δH 1.23-2.10. The 13C-NMR spectrum showed the presence of 28 carbons, including 6 ×
CH3, 7 × CH2, 11 × CH (one bearing oxygen, four olefinic carbons) and four quaternary
carbons (two bearing oxygen). The proton and carbon signals of 45 were assigned by
analysis of COSY, HSQC and HMBC spectra.




The relative configuration of 45 could be deduced from the coupling constants and
NOESY correlations. The geometry of two double bonds C6/C7 and C-22/C-23 was
determined as Z and E on the basis of the coupling constants of 8.5 Hz and 15.5 Hz,
respectively. The coupling constant (J=12.0 Hz) of H-3 with H-2ax (δH 1.84) and H-4ax
(δH 1.93), and NOESY correlation of H-3 and H-1ax (δH 1.95) implied the axial
orientation of H-3 and thus equatorial orientation of the hydroxyl group. The NOESY
correlations observed between H-2ax/H3-18, H2-4/H-6, H-4ax/H3-18, H-7/H-15, H-
7/H3-19, H-11/H3-18, H-11/H3-19 and H3-19/H3-21 indicated the β-orientation of H3-18,
H3-19 and H3-21, 5α,8α-peroxide and the α-orientation of H-9, H-14, H-17. These
spectral data were in good agreement with those of ergosterol peroxide.85,86)


Sporogen-AO 1 (46)
The HR-ESIMS of 46 suggested the elementary 9
1
O
composition of C15H20O3. Its UV spectrum showed a 10
12
7
5
maximum at 242.5 nm and its IR spectrum revealed the HO 3
11
O
absorption bands at 3434 and 1672 cm-1 due to 14
13
15
1
hydroxyl and carbonyl groups. The H-NMR COSY HMBC
46
demonstrated signals of an isopropenyl group, one
olefinic proton, a methine proton, two oxygenated methines, two methylenes and two
13
methyl groups. The C-NMR showed the presence of 15 carbons, comprising three
methyls, 3 × CH2 (one olefinic), 4 × CH (one olefinic, two bearing oxygen) and five
quaternary carbons (two olefinic, one bearing oxygen, one carbonyl). COSY and



-46-
HMBC data demonstrated that 46 is composed of a bicyclic sesquiterpene core. The
decalin unit of the sesquiterpene was deduced by HMBC correlations from methyl
protons (H3-14, s) to quaternary carbons at δC 41.0 (C-5) and 162.9 (C-10), methine
carbons at δC 44.4 (C-4) and 68.3 (C-6). The HMBC spectrum showed the correlations
from methyl protons (H3-15, d) to C-3 (δC 71.0), C-4 and C-5, and from H2-12 and H3-
13 to C-7 (δC 63.5), suggesting the eremophilane skeletone. Oxygenated methine proton
H-6 (δH 3.33) showed HMBC correlations to quaternary carbons C-5 and C-10, and
oxygenated quaternary carbon C-7 characterized for an epoxide ring at C-6/C-7. These
spectral features determined 46 to be eremophilane-type sesquiterpene sporogen-AO
1.87,88)
The coupling constants J3,2ax (11.0 Hz) and J3,4
(11.0 Hz) and NOESY correlations of H-1eq/H-9, H-
3/H3-14, H-6/H3-14 and H-6/H3-15 established the
relative configuration of 46. The similar optical
rotation ([α]D +221o, lit. +236o 87)
and +214o 88)
)
suggested that the isolated compound 46 possessed the
same absolute configuration with those of reported
compound sporogen-AO 1 which was determined by CD spectrum.88)


Dihydrosporogen-AO 1 (47)
Compound 47 was obtained as colorless needles.
Molecular formula of compound 47 was determined to be
C15H22O3 by HR-ESIMS, two mass units more than that
of 46. The UV spectrum showed end absorption. The IR
spectrum exhibited absorption bands for hydroxyl group
(3348 cm-1) and double bond (1675 cm-1). The 1H-NMR
spectrum of 47 showed the presence of three methyl groups, two methylenes, a methine
13
proton, three oxygenated methine protons and three olefinic protons. The C-NMR
spectrum showed the presence of 15 carbons (Table 8). These spectral features of 47
were similar to those of 46, except for the presence of an oxygenated methine proton at
δH 4.52 (t, J=3.0 Hz, H-8) and the absence of carbonyl carbon as seen in 46, indicating




-47-
compound 47 was a hydrogenated compound 46. Accordingly, compound 47 was
determined to be dihydrosporogen-AO 1.89)
Dihydrosporogen-AO 1 (47) was isolated from the
culture broth of fungus Alternaria citri and the
absolute configuration was previously determined by
the CD spectrum of its dibenzoate derivative.89) As the
NMR spectral data of 47 have not been fully reported,
detailed spectroscopic analysis including 2D NMR
experiments was performed to assign all proton signals of 47. The relative configuration
of 47 was assigned based on the 1H-1H coupling constants of J3,2ax (10.5 Hz), J3,4 (10.5
Hz) and J8,9 (3.0 Hz) and the NOESY correlations of H-1ax/H3-14, H-1eq/H-9, H-3/H3-
14, H-6/H3-14, H-6/H3-15 and H-8/H3-13. Furthermore, the absolute stereochemistry of
47 was re-examined by the 1H-NMR analyses on the corresponding MPA esters.90) The
principle of this method is following: a chiral secondary alcohol is esterified with (R)-
and (S)-MPAs. In the diastereomeric pair of MPA esters, the protons of L1 are shielded
by the aromatic ring and will resonate in the (R)-MPA ester at higher field than in the
(S)-MPA ester. The protons of L2 residues on the other side of the molecule are
unaffected by the shielding of the aromatic ring and therefore resonate in the (S)-MPA
ester at higher field than in (R)-MPA ester (Fig. 16).90) Analysis of the 1H-NMR data
differences between (R)- and (S)-MPA esters (47a and 47b) prepared from 47 led us to
determine that 47 possessed 3R and 8S (Fig. 16). Consequently, the stereochemistry of
47 was confirmed as 3R, 4R, 5R, 6R, 7S, 8S.




Fig. 16. Determination of absolute configuration by MPA esters




-48-
Petasol (48)
Compound 48, was obtained as a colorless oil. HR-
ESIMS showed that 48 had the molecular formula of
C15H22O2. The UV spectrum exhibited the maxima at
236.5 nm. The IR spectrum showed the presence of
hydroxyl (3433 cm-1) and α,β-unsaturated carbonyl
(1669 cm-1) groups. The 1H- and 13
C-NMR spectra of
48 (Tables 9 and 8) suggested that 48 possessed an eremophilane skeleton closely
related to 46. Differences were accounted for by the absence of 6,7-epoxide and the
presence of a methylene and a methine. This assumption was supported by detailed
analysis of COSY and HMBC spectra, suggesting 48 to be petasol.91)




Petasol (48) was first isolated from Petasites fragrans in a free form,91) although its
esters such as petasin (55) and S-petasin (56) had now been isolated. Since alkaline
hydrolysis of the esters failed to yield petasol (48) but afforded isopetasol (49), the
absolute stereostructure was investigated on its esters but not on petasol (48) itself.92-94)
The relative stereochemistry of 48 was determined based on the 1H-1H coupling
constants J3,2ax (11.0 Hz), J3,4 (11.0 Hz), J6ax,7 (14.0 Hz), J6eq,7 (4.5 Hz) and the NOESY
correlations H-1ax/H-3, H-1ax/H3-14, H-1eq/H-9, H-4/H-6ax, H-3/H3-14, H-6eq/H3-14,
H-6eq/H3-15, H-7/H3-14 (Fig. 17). Therefore, the absolute stereochemistry of 48 was
re-examined in this study. The absolute configuration of C-3 of 48 was elucidated by
the 1H-NMR analyses of the corresponding MPA esters 48a and 48b. The 1H-NMR data
differences between (R)- and (S)-MPA ester derivatives of 48 indicated that 48
possessed 3R configuration (Fig. 17). Therefore, the absolute stereochemistry of 48 was
established as 3R, 4R, 5R, 7S.




-49-
Fig. 17. Configuration of compound 48


Isopetasol (49)
Compound 49 was obtained as a colorless
crystalline solid. The HR-ESIMS showed that 49
possessed the molecular formula of C15H22O2, which
was the same composition of petasol (48). The UV
spectrum exhibited the maxima at 246 and 278 nm. The
IR spectrum showed the presence of hydroxyl (3504 cm-1) and carbonyl (1655 cm-1)
groups. The 1H- and 13
C-NMR spectra of 49 revealed the similarity to those of 48,
except that the vinyl group was replaced by a methyl group [δH 2.10 (d, J=2.1 Hz) and
δC 22.1] in 49. The 13C-NMR spectrum of 49 showed an additional olefinic quaternary
carbon. These findings led us to determine 49 to be isopetasol.94)


JBIR-27 (50)
Compound 50 was isolated as a colorless solid. Its O
8
10
HR-ESIMS established the molecular formula of
3 5
6
C15H22O3. The IR spectrum showed absorption bands for HO
a hydroxyl (3398 cm-1) and an α,β-unsaturated carbonyl 14
OH
-1 1 13 COSY HMBC
(1656 cm ) groups. The H- and C-NMR of 50
50
resembled those of petasol (48), except for the absence
of a methyl singlet, but showed the additional oxygenated methylene group at δH 3.94
and 3.97 (each 1H, d, J=11.0 Hz, H2-14) and δC 65.9 (C-14). These findings implied
that the methyl group C-14 in 48 was replaced by a hydroxymethyl group in 50. This



-50-
assignment was confirmed by the HMBC correlations from H2-14 to C-6 and C-10, and
from H2-6 to C-14, and the NOESY interactions of H-1ax/H2-14, H-3/H2-14 and H-
7/H2-14. Accordingly, compound 50 was elucidated as JBIR-27.95)


1β-Hydroxypetasol (51)
Analysis of compound 51 by HR-ESIMS established
a molecular formula of C15H22O3. Its IR spectra showed
the presence of hydroxyl and α,β-unsaturated carbonyl
groups at 3401 and 1665 cm-1. The 1H- and 13
C-NMR
spectral features of 51 exhibited the similarity to those
of petasol (48). However, the 1H-NMR spectrum of 51
showed a signal at δH 4.48 (t, J=3.0 Hz), which was not
observed in that of 48. The COSY cross peaks between H-1 (δH 4.48) and two
methylene protons H2-2 (δH 1.65 and 2.35), and HMBC correlation from olefinic proton
H-9 (δH 5.87) to C-1 (δC 73.7) suggested the structure of 51 to be 1β-hydroxypetasol.96)


Table 8. 13C-NMR spectroscopic data of 47, 48, 52-54 and 58 in CDCl3
C 47 48 52 53 54 58
1 30.2 31.0 27.3 30.5 30.7 30.3
2 35.7 35.1 33.8 36.2 35.7 36.6
3 71.9 71.0 71.4 71.9 72.0 72.0
4 43.6 50.2 46.4 50.8 50.1 50.8
5 38.8 39.8 39.4 39.3 38.4 39.1
6 67.9 41.7 42.2 35.4 36.2 41.3
7 65.6 50.3 50.5 42.0 44.9 47.3
8 65.3 198.8 199.0 63.6 74.4 68.7
9 119.5 124.4 124.1 121.0 118.6 123.1
10 140.4 167.9 170.2 147.9 148.0 146.3
11 142.1 143.4 143.7 146.3 81.9 144.6
12 113.4 114.4 114.3 111.9 77.9 112.6
13 19.6 20.0 20.0 22.8 20.4 19.4
14 17.6 17.3 18.9 18.1 18.1 18.8
15 11.3 10.4 11.9 10.8 11.1 10.4




-51-
Table 9. 1H-NMR spectroscopic data of 48, 52-54 in CDCl3 (J , Hz)

52 53 54
H 48

1ax 2.45 tdd (15.0, 4.5, 2.0) 2.83 tdd (14.5, 5.0, 1.5) 2.29 tdt (14.5, 4.5, 1.5) 2.29 tdt (14.5, 4.5, 2.0)
1eq 2.34 ddd (15.0, 4.5, 2.5) 2.14 ddd (14.0, 4.0, 2.5) 2.18 ddd (14.5, 5.0, 3.0) 2.23 ddd (14.5, 4.5, 3.0)
2ax 1.45 dddd (15.0, 12.0, 11.0, 2.5) 1.72 tdd (14.5, 4.0, 2.5) 1.36 dddd (14.5, 12.0, 11.0, 5.0) 1.36 m
2eq 2.16 dtd (12.0, 4.5, 2.5) 2.03 ddt (14.5, 5.0, 2.5) 2.07 dtd (12.0, 4.5, 3.0) 2.03 dtd (12.0, 4.5, 3.0)
3 3.62 td (11.0, 4.5) 3.95 q (2.5) 3.54 td (11.0, 4.5) 3.53 td (11.0, 4.5)
4 1.34 dq (11.0, 6.5) 1.52 qd (7.0, 2.5) 1.25 m 1.18 dq (11.0, 6.5)
6ax 1.88 t (14.0) 1.83 br t (13.5) 1.57 m 0.92 t (13.5)




-52-
6eq 2.02 dd (14.0, 4.5) 1.97 dd (13.0, 4.5) 1.60 m 1.76 dd (13.5, 5.0)
7 3.11 dd (14.0, 4.5) 3.17 dd (14.0, 4.5) 2.25 br dt (11.5, 3.5) 2.00 dt (13.5, 5.0)
8 4.09 br 4.52 td (5.0, 2.0)
9 5.78 d (2.0) 5.80 d (1.5) 5.63 dd (5.5, 1.5) 5.59 dd (5.0, 2.0)
12 4.82 br s 4.82 br s 4.83 br s 3.73 d (10.0)
4.98 quint (1.5) 4.98 quint (1.5) 5.03 br s 3.83 d (10.0)
13 1.74 br s 1.74 br s 1.84 br s 1.34 s
14 1.18 s 1.39 s 0.96 s 0.93 s
15 1.08 d (6.5) 1.11 d (7.0) 1.06 d (6.5) 1.06 d (6.5)
New eremophilane 3-epi-petasol (52)
Compound 52 was obtained as colorless needles and
the molecular formula was established as C15H22O2 by
HR-ESIMS. Its UV spectrum showed a maximum at
240 nm, and its IR spectrum exhibited the absorption
bands at 3479 and 1657 cm-1, indicating the presence of
hydroxyl and α,β-unsaturated carbonyl groups. The 1H-NMR spectrum of 52 showed
signals for three methyls at δH 1.11 (d, J=7.0 Hz), 1.39 (s), and 1.74 (br s), one olefinic
proton at δH 5.80 (d, J=1.5 Hz), two vinyl protons at δH 4.82 (br s) and 4.98 (quint,
J=1.5 Hz), three methines at δH 1.52 (qd, J=7.0, 2.5 Hz), 3.17 (dd, J=14.0, 4.5 Hz) and
3.95 (q, J=2.5 Hz), and three sets of methylene protons at δH 1.72-2.83 (Table 9). The
13
C-NMR and DEPT spectra of 52 revealed 15 carbon signals due to three methyls,
three sp3 methylenes, one sp2 methylene, three sp3 methines, one sp2 methine and four
quaternary carbons, including two olefinic and one carbonyl carbons (Table 8). These
spectral features of 52 closely resembled those of petasol (48).91) The only remarked
difference in their 1H-NMR spectra was that the oxygenated methine proton signal of 52
was observed at δH 3.95 as quartet (J=2.5 Hz) instead of a triplet of doublets at δH 3.62
(J=11.0, 4.5 Hz) as seen in petasol (48). The 1H-1H COSY and HMBC experiments
implied that compound 52 possessed the same eremophilane backbone as petasol (48)
and differed from 52 only in the configuration at C-3.
The detailed inspection of the coupling
constants enabled assignment of the signals for
methylene protons at δH 1.72, 1.83, 1.97, 2.03,
2.14 and 2.83 to H-2ax, H-6ax, H-6eq, H-2eq, H-
1eq and H-1ax, respectively. The NOESY
correlations observed between H-1ax/H3-14, H3-
14/H3-15, H-4/H-6ax, H-6eq/H3-14, H-6eq/H3-15
and H-7/H3-14 indicated the β-orientation of H-1ax, H-6eq, H-7, H3-14 and H3-15 and
α-orientation of H-4, H-6ax, the isopropenyl group as in petasol (48). The 1H-1H
coupling constant (J=2.5 Hz) of H-3 with H-2ax, H-2eq and H-4 implied the equatorial



-53-
orientation of H-3 and thus axial orientation of the hydroxyl group on the upward face
of the ring system. Accordingly, compound 52 was formulated as shown and designated
as 3-epi-petasol.


New eremophilane dihydropetasol (53)
Compound 53 was isolated as a colorless crystalline
solid. The HR-ESIMS established a molecular formula
of C15H24O2, that is, two mass units more than that of
petasol (48). The IR spectrum showed absorption bands
at 3341, 1450, 1022 and 884 cm-1, and UV spectrum
exhibited end absorption, indicating the presence of hydroxyl group(s) but the absence
of an α,β-unsaturated carbonyl group. The 1H- and 13
C-NMR spectra of 53 (Tables 9
and 8) together with DEPT and HMBC experiments suggested that the structure of 53
was closely related to petasol (48). Differences between two isolates were ascribable to
the absence of the carbonyl carbon and the presence of an additional oxygenated
methine [δH 4.09 and δC 63.6] in 53. These findings implied that the carbonyl group in
48 was reduced to a hydroxyl group in 53. The substitution of the hydroxyl group at C-8
was evidenced from the COSY correlations between H-7 (δH 2.25) and an oxygenated
methine at δH 4.09 (H-8), and between H-8 and an olefinic proton at δH 5.63 (H-9), as
well as HMBC correlation from H-9 to an oxygenated methine carbon at δC 63.6 (C-8).
The relative configuration of 53 was deduced H H
H 1
from the coupling constants and NOESY 3
H
HO 14 CH3
correlations. The NOESY cross-peaks observed
H
H3C 4
5
between H-1ax/H3-14, H-3/H3-14, and H3-14/H-7 9
H
H
H
H
indicated that H-1ax, H-3, H3-14 and H-7 were H 6 8
7
OH
axially oriented on the same face on the ring NOESY H CH3
system. The coupling constants (J=11.0 Hz or 11.5
53
Hz) of H-3/H-2ax, H-3/H-4 and H-6ax/H-7 implied a trans-diaxial relationship between
each pairs of protons and indicated that compound 53 had the same configurations at C-
3, C-4, C-5 and C-7 as petasol (48). The coupling constant (J=3.5 Hz) of H-7/H-8 was




-54-
different from that of petasinol (57) (J=9.6 Hz),97) indicating a cis-relationship of H-7
and H-8 and furthermore a quasi-equatorial orientation of H-8.
Finally, the absolute stereochemistry of 53
was determined by its chemical correlation
with petasol (48). Reduction of petasol (48)
with NaBH4-CeCl398) yielded 53 as a minor
product along with its 8-epimer 58, whose
structure was confirmed by spectroscopic
means (Table 8) and comparison with the reported data for petasinol (57).97) Thus, the
structure of 53 was established as shown and designated dihydropetasol.


New eremophilane sarcographol (54)
Compound 54, named sarcographol, was obtained
as a crystalline solid, which was shown to have a
molecular formula of C15H24O3 by HR-ESIMS. Its IR
spectrum exhibited absorption bands at 3441, 3375
and 1659 cm-1, characteristic of a hydroxyl group(s)
and a double bond. The 1H- and 13
C-NMR spectra of
54 (Tables 9 and 8) closely resembled those of dihydropetasol (53) except that the
double bond C-11/C-12 was replaced by an oxygenated quaternary carbon at δC 81.9
(C-11) and an oxygenated methylene at δH 3.73 and 3.83 (each d, J=10.0 Hz, H2-12),
and δC 77.9 (C-12). The HMBC correlations from H-7 (δH 2.00) to C-11, from H2-12 to
C-7 (δC 44.9), C-8 (δC 74.4) and C-11, from H3-13 (δH 1.34) to C-7, C-11 and C-12
suggested that an ether linkage between C-8 and C-12 forms a tetrahydrofuran ring with
methyl and hydroxyl groups at C-11 as in cyclodebneyol (59).99) Furthermore, the
molecular formula of 54 and chemical shifts of 1H- and 13C-NMR spectra indicated the
location of a hydroxyl group at C-3 [δH 3.53 (td, J=11.0, 4.5 Hz) and δC 72.0].




-55-
The relative configuration of 54 could be H H
H
1 1 1
deduced from analysis of H- H coupling 3
H
14CH3
HO
constants and NOESY correlations. The 15
H
H3C 10
4
5 9 H
coupling constants J2ax,3 (11.0 Hz), J3,4 (11.0
H
H H
8
Hz), and J6ax,7 (13.5 Hz) as well as NOESY 7
6
H
correlations of H-1ax/H3-14, H-3/H3-14, H3- OH O
13
H
NOESY H3C 11
14/H3-15 and H-7/H3-14 indicated that the
H
H 12
54
relative configurations at C-3, C-4, C-5 and C-7
of 54 were consistent with those of 53. Moreover, from the coupling constants J7,8 (5.0
Hz) and J8,9 (5.0 Hz), and the magnitudes of allylic coupling J9,1ax (2.0 Hz) and
homoallylic coupling J8,1ax (2.0 Hz), the dihedral angles between H-1ax and the C1-
C10-C9 plane,100) and between H-8 and the plane formed by C8-C9-C10101) were
estimated as ca. 90o and ca. 140o, respectively. These findings established quasi-
equatorial orientation of H-8 and a cis-fused tetrahydrofuran ring. Furthermore, the
NOESY spectrum showed significant cross peaks of H-6eq/H3-13 and H-6ax/H3-13,
demonstrating that H3-13 was in close proximity to H2-6, hence the configuration of C-
11 was assigned as S*. Accordingly, the structure of sarcographol was thereby
elucidated as shown.
The absolute stereochemistry of new eremophilanes 52 and 54 could not been
chemically determined owing to the minute amount of the isolated compounds.
Compounds 48-54 isolated in this study could be expected to have a close biosynthetic
relationship. Compounds 49-54 were different from 48 only in the oxidation pattern.
Since the absolute configuration of 48 was determined, it is reasonable to assign the
same absolute configurations for the chiral centers C-4, C-5 and C-7 within molecules
48-54 on the basis of biogenetic considerations.




-56-
In conclusions, diverse eremophilane-type sesquiterpenes have been isolated from
91,97,102)
and fungi,87-89,96) and exhibited a variety of biological activities, such as
plants
cytotoxic,95,102) anti-HIV 103)
and antibacterial 104) activities. This type of sesquiterpenes
has never been isolated from lichen thalli or lichen mycobionts. This investigation of
cultured lichen mycobionts of S. tricosa yielded ergosterol peroxide (45), six known
(46-51) and three new (52–54) eremophilane-type sesquiterpenes. This is the first
instance of isolation of eremophilane-type sesquiterpenes from lichen mycobionts in
culture. It is also noteworthy that the metabolic ability was expressed only in the
isolated mycobionts.




-57-
3.3. Chemical investigation of the cultured mycobionts of Pyrenula sp.
Pyrenula belongs to the family of Pyrenulaceae in the order Pyrenulales. The genus
has a widespread distribution, especially in tropical regions, and contains about 200
species.105) In 1970, Santesson reported the presence of the anthraquinone in Pyrenula
cerina.106) Anthraquinones, xanthones, isocoumarins, benzofuran, azaphilone and
napthopyrones (Fig. 18) were previously isolated from the cultured mycobionts of
Pyrenula species, such as P. japonica and P. pseudobufonia.107-110)




Fig. 18. Metabolites from thalli and cultured mycobionts of Pyrenula species


The crustose lichen Pyrenula sp. was collected in Dalat city, Vietnam and its
polyspore-derived mycobionts were cultivated on MY10 medium (Photo 5). After
cultivation for 3 months, the colonies were harvested and extracted with Et2O, acetone
and then MeOH. The extracts were separated by a combination of chromatographic
procedures to afford two anthraquinones (60 and 61), a xanthone (62) and eight new
polyketides (63-70) (Fig. 19).




-58-
Pyrenula sp. Cultured mycobionts
Photo 5. Pyrenula sp. thalli and its cultured mycobionts




Fig. 19. Extraction and isolation procedure for cultured mycobionts of Pyrenula sp.



-59-
Chrysophanol (60)
Compound 60 was isolated as orange
184-185oC.
prism, mp. The MS
spectrum showed a pseudo-molecular
ion peaks at m/z 253.0515 [M-H]-,
indicating the molecular formula of
C15H10O4. Its IR spectrum showed the
absorption bands at 3431 (hydroxyl group), 1677, 1629 and 1606 characteristic of
anthraquinone. The 1H-NMR spectrum exhibited the signals due to two hydrogen-
bonded hydroxyl groups at δH 12.02 and 12.13, five aromatic protons and a methyl
group at δH 2.47. Five aromatic protons were analyzed by COSY correlations as a pair
of meta-coupled doublets at δH 7.11 and 7.66 (each 1H, d, J=1.0 Hz) and an aromatic
AMX system at δH 7.30 (1H, dd, J=8.5, 1.0 Hz), 7.68 (1H, dd, J=8.5, 8.0 Hz) and 7.83
13
(1H, dd, J=8.0, 1.0 Hz). The C-NMR spectrum showed 15 carbon atoms, which
included two carbonyl downfield signals at δC 192.6 and 182.1, indicative of the
presence of the chelated and nonchelated carbonyl, respectively. The exact location of
the aromatic protons and the substituted functional groups was established based on 2D
NMR (COSY, HSQC, HMBC and NOESY). Thus, 60 was identified as 1,8-dihydroxy-
3-methylanthraquinone (chrysophanol).111)


Emodin (61)
Compound 61 was obtained as
orange crystal. HR-ESIMS of the
compound exhibited a strong peak at m/z
269.0459 [M-H]-, an increase of 16 mass
units with respect to 60. The 1H-NMR
spectral features of 61 were similar to
those of 60, except for the presence of a pair of meta-coupled protons at δH 6.68 and
7.28 (each 1H, d, J=2.5 Hz) in stead of an aromatic AMX system as seen in 60.
13
Furthermore, its C-NMR spectral data demonstrated the low field signal at δC 158.8
(C-6), indicating the presence of hydroxyl group at C-6 which was in accord with its




-60-
molecular formula. This was confirmed by the 2D NMR spectra analysis (COSY,
HSQC, HMBC and NOESY). Consequently, 61 was elucidated as 1,6,8-trihydroxy-3-
methylanthraquinone (emodin).111)


1,5,8-Trihydroxy-3-methylxanthone (62)
Compound 62 was isolated as yellow
needles. The molecular formula of 62
was found to be C14H10O5 by HR-
ESIMS. Its 1H-NMR spectrum showed
the presence of a hydroxyl group at δH
8.53 (s), two hydrogen-bonded hydroxyl
groups at δH 11.09 and 11.74 (each 1H, s) and a methyl group at δH 2.46 (3H, s). Further
analysis 1H-NMR spectrum of 62 demonstrated the signals due to a pair of meta-
coupled broad singlets at δH 6.67 and 6.87, and a pair of ortho-coupled doublets at δH
13
6.68 and 7.35 (each 1H, d, J=7.5 Hz). The C-NMR spectrum exhibited 14 carbon
atoms including a carbonyl carbon at δC 187.0, five oxygenated sp2 carbons, four
aromatic CH, three quaternary aromatic carbons and a methyl group. These spectral
features together with analysis of 2D NMR suggested 62 to be 1,5,8-trihydroxy-3-
methylxanthone, which was previously isolated from the cultured mycobionts of the
lichen Pyrenula pseudobufonia.108)


New polyketide pyrenulic acid A (63)
Compound 63 was isolated as a colorless
solid. The molecular formula of 63 was
established as C26H36O3 by HR-ESIMS,
implying nine units of unsaturation. Its UV
spectrum showed the strong absorption at
300 nm and IR spectrum displayed
absorption bands at 3431 (O-H), 1687 (C=O)
cm-1. The 1H-NMR spectrum of 63 exhibited
the signals for eight olefinic protons, six




-61-
methine protons including an oxygenated methine, six methylene protons and five
13
methyl groups (Table 10). The C-NMR spectrum of 63 showed the signals for a
carboxyl carbon at δC 172.1, eight olefinic methines and two quaternary olefinic carbons.
Furthermore, 13C-NMR spectrum indicated the presence of six sp3 methine carbons, an
oxygenated quaternary carbon, three methylenes and five methyl carbons (Table 11).
All proton and carbon signals were assigned by COSY and HMBC experiments to
formulate the partial structures. The 1H-1H COSY spectrum showed the sequence from
olefinic doublet at δH 5.81 (J=15.0 Hz, H-2) to olefinic proton at δH 6.20 (dd, J=15.0,
10.0 Hz, H-7) throughout four other olefinic protons in turn at δH 7.37 (H-3), 6.23 (H-4),
6.71 (H-5) and 6.12 (H-6). HMBC correlations from two olefinic protons at δH 5.81 (H-
2) and 7.37 (H-3) to the carboxyl group at δC 172.1 (C-1) suggested a trienoic acid
moiety with all trans configurations.
The 1H-1H COSY experiments also revealed the spin system from a methyl signal at
δH 1.67 (H3-26) to methine proton at δH 2.32 (H-8), which was further connected to a
methine proton at δH 1.79 (H-17), and from a methyl signal at δH 1.73 (H3-25) to
olefinic signal at δH 5.45 (H-15). Further COSY correlations from H-17 to H2-13 as well
as HMBC correlations from olefinic proton (H-15) to C-9, 13, 17, from a methyl group
(H3-25) to C-15, 16, 17, and from another methyl (H3-26) to C-11, 12, 13 established a
decalin system with two methyl groups and two trisubstituted double bonds at C-11/C-
12 and C-15/C-16. Further analysis of COSY and HMBC experiments in a combination
of 1H-, 13C-NMR and molecular formula defined a sec-butyl group with a methyl group
and a trisubtituted epoxy ring. The connectivity of these partial structures was
determined by COSY and HMBC correlations. These spectral features of 63 were
closely similar to those of cladobotric acids A (71) and C (72) isolated from
fermentation broth of fungus Cladobotryum species.112) The only difference was that
oxygenated quaternary carbon C-17 in cladobotric acid A (71) was replaced by a
methine carbon in 63. This was confirmed by a COSY correlation between methine
protons H-8 and H-17, and HMBC correlations from H-19, H3-24 and H3-25 to a
methine carbon at δC 53.4 (C-17). Accordingly, the planar structure of 63 was
elucidated as shown.




-62-
The 1H-1H coupling constants between H-8 and 1 COOH

H-9, and between H-8 and H-17 were 12.0 and 6.0 3

Hz, respectively. NOESY correlations were
observed between H-9 and H3-24, and between H- 6
23
24
1
17 and H-19 suggested that the relative H R 22
18
11 8 20
configuration of C-8, 9, 14 and 17 of 63 was 9 17 19
O
14
26
R2
presumed to be the same as cladobotric acids. The 16
25
H
13
differences were reasonably accounted for by the 63 : R1 = H, R2 = CH3
71 : R1 = OH, R2 = CH3
hydroxylation at C-17 or C-26 of 63. This
72 : R1 = H, R2 = CH2OH
presumption was further supported by the
comparison of 13C-NMR spectral data of 63 with those of cladobotric acids A (71) and
C (72) (Table 11). Consequently, the structure of 63 was elucidated and designed as
pyrenulic acid A.




-63-
Table 10. 1H-NMR spectroscopic data of 63-66 in CDCl3 (J , Hz)

64 66
H 63 65
2 5.81 d (15.0) 5.84 d (15.0) 5.91 d (15.5) 5.89 d (15.0)
3 7.37 dd (15.0, 11.0) 7.38 dd (15.0, 11.0) 7.40 dd (15.5, 11.0) 7.35 dd (15.0, 11.0)
4 6.23 dd (15.0, 11.0) 6.24 dd (15.0, 11.0) 6.57 ddd (15.5, 11.0, 1.0) 6.51 dd (15.0, 11.0)
5 6.71 dd (15.0, 10.0) 6.54 dd (15.0, 11.0) 6.38 dd (15.5, 4.5) 6.31 dd (15.0, 5.0)
6 6.12 dd (15.0, 10.0) 6.13 dd (15.0, 11.0) 4.37 br dd (10.0, 4.5) 4.34 br dd (10.0, 5.0)
7 6.20 dd (15.0, 10.0) 5.71 dd (15.0, 11.5) 4.12 d (10.0) 4.24 br d (10.0)
8 2.32 ddd (12.0, 10.0, 6.0) 2.25 td (11.5, 6.0) --- 2.09 m
9 1.68 m 1.50 m 2.04 m 2.01 m
10 1.47 br t (15.0) 1.46 br t (13.0) 2.13 m 2.00 m
1.99 m 1.89 br d (15.5) 2.27 br d (17.0) 2.33 m
11 5.37 br d (3.0) 5.35 br d (3.0) 5.39 br d (4.0) 5.38 br s
13 1.76 m 1.77 br t (14.0) 1.92 br t (15.0) 1.82 br t (16.0)




-64-
2.02 br dd (17.0, 3.0) 2.01 br dd (16.0, 4.5) 2.04 m 1.96 br d (16.5)
14 1.96 m 1.96 m 2.22 m 2.05 m
15 5.45 br s 5.39 br s 5.60 br s 5.38 br s
17 1.79 d (6.0) 2.61 br d (6.0) 2.16 br s 2.17 br s
19 2.48 d (8.5) 4.93 br d (9.5) 3.57 d (6.5) 3.46 d (6.0)
20 1.31 m 2.29 m 2.15 m 2.12 m
21 1.29 m 1.17 m 1.40 br d (13.0) 1.38 br d (13.0)
1.66 m 1.33 m 1.86 m 1.89 m
22 0.94 t (7.5) 0.83 t (7.5) 3.44 dd (12.5, 5.0) 3.42 dd (12.0, 5.0)
3.63 td (12.5, 2.0) 3.64 br t (12.0)
23 0.96 d (7.0) 0.91 d (6.5) 1.11 d (7.5) 1.09 d (7.5)
24 1.30 s 1.56 s 1.19 s 1.22 s
25 1.73 br s 1.57 br s 1.80 br s 1.77 br s
26 1.67 br s 1.66 br s 1.66 br s 1.66 br s
Table 11. 13C-NMR spectroscopic data of 63, 64 and
related compounds in CDCl3

72112)
71112)
C 63 64

1 172.1 172.3 169.7 171.7
2 119.0 118.9 120.4 119.1
3 147.2 147.3 146.2 147.0
4 127.9 127.6 129.2 128.0
5 142.2 142.3 140.5 142.1
6 130.1 129.9 133.6 130.2
7 142.2 144.2 136.8 141.8
8 49.0 48.7 59.0 48.8
9 36.3 35.0 38.0 36.2
10 31.9 31.6 31.8 31.4
11 121.3 121.3 121.2 123.3
12 134.0 134.2 134.0 137.5
13 37.7 37.9 37.1 33.1
14 38.6 38.5 38.3 37.9
15 129.6 127.0 132.2 129.3
16 132.1 131.9 133.9 132.4
17 53.4 54.8 75.6 53.2
18 61.3 134.4 65.1 61.5
19 68.9 136.8 63.8 68.8
20 34.8 34.4 34.2 34.6
21 27.7 30.3 27.7 27.5
22 11.2 12.2 11.2 11.1
23 15.4 21.1 15.3 15.3
24 15.7 17.8 15.7 15.5
25 23.0 22.0 18.1 23.0
26 23.4 23.5 23.4 67.1




-65-
New polyketide pyrenulic acid B (64)
Compound 64, pyrenulic acid B, had a
molecular formula of C26H36O2, i.e. one
oxygen atom less than that of pyrenulic acid
A (63). The NMR spectroscopic features of
64 closely resembled those of 63. However,
compound 64 showed an olefinic proton
signal at δH 4.93 and two sp2 carbon signals
at δC 134.4 (C-18) and 136.8 (C-19) instead
of the oxygenated sp3 carbon signals due to
C-18 and C-19 as seen in 63 (Tables 10 and 11). 1H-1H COSY spectrum showed a
correlation between an olefinic proton at δH 4.93 (H-19) and methine proton at δH 2.29
(H-20), which was correlated with a methyl signal at δH 0.91 (d, J=6.5 Hz, H3-23), and
HMBC interactions were observed from a methine proton at δH 2.61 assignable to H-17
to C-18 and 19, from H-19 to C-17, 18, 20 and 21, and from H3-24 to C-17, 18 and 19.
These findings demonstrated that 64 possessed a double bond at C-18/C-19 instead of
an epoxy ring as seen in 63. The geometry of the double bond C-18/C-19 was
determined to be E by the NOESY cross-peak observed between H-17 and H-19.
Relative configuration of asymmetric centers in 64 was deduced to be the same as in 63
from the similarity of the coupling constants of 11.5 Hz (H-8/H-9) and 6.0 Hz (H-8/H-
17), and 13C-NMR spectral data (Tables 10 and 11).


New polyketide pyrenulic acid C (65) 1
HOOC
The HR-ESIMS established the molecular
formula of pyrenulic acid C (65) to be C26H36O6, COSY
HMBC
that is, three oxygen atoms more than that of 23
H CH3
pyrenulic acid A (63). The UV spectrum of 65 O
H 6
HO 20
7 19
HO
showed the maximum at 254 nm, implying the 8 18
22
1
modification of a trienoic acid moiety. The H- 9 17 O H
24
H
CH3
26
NMR spectrum of 65 showed the resemblance to 14
H3C CH3
25
that of 63, however lacked the signals due to 65



-66-
methine proton H-8, 6,7-olefinic protons and 22-methyl group, but demonstrated the
signals for an oxymethylene at δH 3.44 and 3.63 (H2-22), and two oxygenated methine
13
protons at δH 4.37 (H-6) and 4.12 (H-7) (Table 10). The C-NMR spectrum of 65
showed six oxygenated sp3 carbons, which were classified to one methylene, three
methine and two quaternary carbons by DEPT experiments (Table 12). Three CH
carbons at δC 76.02, 77.6 and 74.6, and a methylene carbon at δC 54.8 were assigned to
C-6, C-7, C-19 and C-22 from the COSY sequence starting from an olefinic proton at
δH 6.38 (H-5) and from a methyl signal at δC 1.11 (d, J=7.5 Hz, H3-23). HMBC
interactions from H-6 and a methyl group at δH 1.19 (H3-24) enabled to assign a
quaternary carbon to C-8 and C-18. And the key HMBC interactions from H-6 to C-8
and C-19 (δC 74.6), and from H2-22 to C-18 (δC 78.4) allowed to make up 2,3,5-
trioxygenated oxepane and a 3-oxygenated-4-methyltetrahydro-2H-pyran ring.


HO
The relative configuration
R H
7
of 65 was determined by its H
H
H H
20
6 19
coupling constants and H O
H HO H
H H3C 23
8
H
significant NOESY cross-peaks. 9
10
18
17
The coupling constants J6,7 O
H H
H
22
H3C
(10.0 Hz) suggested that H-6 H C H
3 24
14
and H-7 were located in anti H NOESY
CH3
H H
arrangement. Moreover, the
COOH
R=
coupling constant J19,20 (6.5 Hz)
65
and the NOESY correlations
observed H-6/H-10eq (δH 2.13), H-7/H-17, H-7/H-19, H-17/H-19, H-9/H3-24, H-22ax
(δH 3.63)/H3-24 and H3-23/H3-24 indicated that H-7, H-17 and H-19 oriented axially on
the upward face. On the other hand, H-9, H3-23 and H3-24 were directed downward.




-67-
Further informations were obtained from the 1H- R1OOC 1
NMR spectrum of 65a, which was prepared from 65.113)
The coupling constant J9,14 (11.5 Hz) and NOESY
correlation 8-OAc/H-14 indicated trans decalin system H 23
6
R2O OH
and anti arrangement of 8-OAc/H-9. In addition, the
R2O 7 20
H8
coupling constants J6,7 (10.0 Hz) and J19,20 (6.0 Hz) and 18
17 O
9
the NOESY correlations of H-6/H-10eq, H-7/H-19, H- 22
H 24
14
26
17/H-19, H-22ax/H3-24 and H3-23/H3-24 suggested the 25
H
65 : R1 = R2 = H
plausible conformation of 65a. Consequently, the
65a : R1 = CH3; R2 = Ac
relative stereochemistry of pyrenulic acid C was
determined to be 6R*, 7R*, 8R*, 9R*, 14S*, 17R*, 18S*, 19R*, 20R*.


New polyketide pyrenulic acid D (66)
The molecular formula of compound 66, 1
HOOC
pyrenulic acid D, was determined by HR-ESIMS
as C26H36O5, that was 16 mass units less than that COSY
HMBC
of pyrenulic acid C (65). Its UV, IR and NMR 23
H
H CH3
H 6O
spectroscopic features were similar to those of 65 HO 7 20
H
except that the oxygenated quaternary carbon C-8 8 18
22
17 24
9 O H
(δC 75.97) in 65 was replaced by a methine carbon
H
CH3
26
14
H3C CH3
in 66 (δH 2.09 and δC 49.6) (Tables 10 and 12). 25

66
These findings, together with its molecular
formula, implied that 66 possessed a proton instead of the hydroxyl group at C-8. This
assignment was confirmed by the COSY cross-peaks of H-8 (δH 2.09) and H-7 (δH 4.24),
H-8 and H-17 (δH 2.17), and HMBC interactions from H-8 to C-7 (δC 75.5) and C-18
(δC 80.0). The coupling constants J6,7 (10.0 Hz), J7,8 (< 1.0 Hz), J8,17 (< 1.0 Hz), J19,20
(6.0 Hz) and the similar NOESY correlations in 65 and 66 suggested that 66 owned the
same configuration as 65.




-68-
To determine the absolute OCH3
H
configuration at C-7, 66 was Ph O
subjected to methylation and
O
H
R
followed by esterification with
H
7
H
H
(S)-MPAs.90) H6
(R)- and The 20
19
O
H
H H H
relative configuration of (R)- and H
H H
22
9 H3C
8
10
17
(S)-MPA ester derivatives (66a H 18
H
O
H
H3C
and 66b) was identified based on
H3C 14
CH3
the 1H-1H coupling constants and NOESY
H H H
1
NOESY correlations. The H-
COOCH3
R=
NMR spectrum of 66a exhibited
66a
the coupling constants of 10.0 Hz
(H-6/H-7), 2.0 Hz (H-7/H-8), 12.0 Hz (H-8/H-9), 4.0 Hz (H-8/H-17) and 6.0 Hz (H-
19/H-20). In the NOESY spectrum of 66a, the correlations of H-6/H-10eq, H-7/H-17,
H-7/H-19, H-17/H-19 and H-22ax/H3-24 were observed. These findings indicated that
66a adopted the same conformation as 66. From the differences in the proton NMR
chemical shifts of both esters (Fig. 20), the absolute configuration of C-7 was
determined to be S. Consequently, the structure of 66 was elucidated to be 6R, 7S, 8S,
9R, 14R, 17S, 18S, 19R, 20R.




Fig. 20. Determination of absolute configuration of 66




-69-
Table 12. 13C-NMR spectroscopic data of 65-70 in CDCl3

70a
68a
C 66 69
65 67

1 171.6 171.8 170.2 171.0 171.0
171.1
2 120.8 121.4 121.1 121.0 123.4
124.5
3 146.3 145.8 145.8 145.8 145.5
145.0
4 129.4 129.9 131.1 131.0 131.6
132.8
5 143.1 142.7 142.4 139.5 143.2
142.5
6 76.02 77.4 77.2 77.7 78.9
77.8
7 77.6 75.5 75.5 73.1 70.8
78.2
8 75.97 49.6 49.3 43.6 57.1
76.7
37.6 b
9 33.8 33.8 31.9 37.0
39.4
10 29.0 32.9 32.7 30.7 32.6
29.9
11 122.2 121.6 121.5 121.4 122.9
123.0
12 133.4 134.2 134.2 133.5 134.0
134.6
13 36.7 37.3 37.3 38.0 38.3
38.1
37.7 b
14 42.2 42.2 38.1 41.6
39.4
15 128.7 128.4 128.9 128.7 130.6
129.0
16 133.7 135.5 135.1 132.7 138.0
134.6
17 64.7 56.2 56.5 50.5 78.6
65.2
18 78.4 80.0 78.4 147.3 149.8
77.7
19 74.6 74.7 82.2 81.9 75.7
83.0
20 32.1 32.4 35.1 36.2 37.7
35.9
21 31.6 31.8 25.5 25.3 27.1
26.7
22 54.8 54.6 12.6 10.8 11.6
13.0
20.8 c
23 13.6 13.6 16.3 16.4
21.2
21.0 c
24 15.3 16.2 115.1 115.6
19.6
25 26.1 25.7 26.2 21.8 19.1
26.9
26 23.2 23.4 23.3 23.3 23.6
23.6
a b, c
Measured in CD3OD assignments may be interchangeable.




-70-
New poliketides pyrenulic acids E (67) and F (68)
Another set of closely related compounds HOOC 1
comprised pyrenulic acid E (67) and pyrenulic
COSY
acid F (68). The HRMS measurements of 67
HMBC
and 68 revealed the molecular formulas of H 23
CH3
H 6O
C26H38O5 and C26H38O6, which were two HO 22
19
R7 CH3
20
protons more than that of pyrenulic acid D 18
OH
9 17 24
CH3
(66) and pyrenulic acid C (65), respectively.
26 14
H3C CH3
1 13
The H- and C-NMR spectra of 67 and 68 25

were similar to those of 66 and 65 except for 67 : R = H
68 : R = OH
the presence of a methyl group [67: δH 0.87
and δC 12.6, 68: δH 0.87 and δC 13.0] instead of an oxymethylene C-22 in 66 and 65
(Tables 12 and 13). These findings were explained by the structures of 67 and 68 with a
hydroxyl group at C-18 and a sec-butyl group at C-19 instead of a 3-oxygenated-4-
methyltetrahydro-2H-pyran ring as seen in 66 and 65. This was supported by the
analysis of their 2D NMR (COSY, HSQC, HMBC) spectral features. The similar
coupling constants and ROESY correlations demonstrated that 67 and 68 possessed the
same configurations as 66 and 65.


HOOC 1 HO
H
R
7
H
H6 H
19
H O
H R1 20
H H CH CH
CH3 2 3
H 10
23 8
18
9
OH
6 OH
17
HO H
20
R 7
H
22 H3C
H ROESY
18
H3C 14
OH
9 CH3
17
H H H
24
14 H
COOH
26 25 R=
H
67 : R1 = H
67 : R = H
68 : R1 = OH
68 : R = OH




-71-
New polyketide pyrenulic acid G (69)
Compound 69, pyrenulic acid G, had a HOOC1
molecular formula of C26H36O4, which was
COSY
consistent with the loss of H2O from pyrenulic
HMBC
13
acid E (67). In the C-NMR spectrum of 69, H
O H CH3
H
2 6
the signals of two sp carbons were observed at HO 22
19
CH3
20
δC 147.3 (C-18) and 115.1 (C-24) instead of an 8 18
9 24
CH2
17
3
oxygenated sp carbon (C-18) and a methyl 25
26
H3C CH3
14
carbon (C-24) signals as seen in 67 (Table 12).
The marked differences were accounted for by 69
the dehydration of C-18 hydroxyl group in 67 to an exomethylene group in 69. These
assignments were clarified by the HMBC interactions observed from two olefinic
protons (H2-24) to C-17 (δC 50.5), C-18 and C-19 (δC 81.9), and from proton H-19 (δH
3.63) to C-18 and C-24. The relative stereochemistry of 69 resembled those of 67 based
on the 1H-1H coupling constants and NOESY correlations.
To identify the absolute configuration of C-7 of pyrenulic acid G (69), (R)- and (S)-
MPA esters 69a and 69b were prepared in the same procedure for 66. The differences of
the proton NMR chemical shifts between two ester derivatives let us to determine the
absolute configuration of C-7 to be S (Fig. 21). Consequently, the structure of 69 was
determined to be 6R, 7S, 8S, 9R, 14R, 17S, 19R. The configuration at C-20 of 69 was
presumed to be the same as that of 66 based on the biosynthetic origin of the same
lichen species.




-72-
Fig. 21. Determination of absolute configuration of 69


New polyketide pyrenulic acid H (70)
Compound 70, pyrenulic acid H, was HOOC1
isolated as colorless needles. Its HR-ESIMS
established the molecular formula of C26H36O5,
H
that was one oxygen atom more than that of
O H CH3
H
pyrenulic acid G (69). Its UV, IR, 1H- and 13C- HO 6
22
HH 19
CH3
NMR (Tables 12 and 13) spectra displayed its 8
CH
9 17
13
OH 24 2
structural similarity to 69. However, the C- 14
26
H3C CH3
NMR spectrum of 70 showed an oxygenated H 25

quaternary carbon at δC 78.6 (C-17) instead of a 70
methine carbon in 69. The structure of 70 was confirmed by the HMBC correlations
from protons H-7, H-19, H2-24 and H3-25 to C-17. The stereochemistry of 70 was
proposed to be similar to those of pyrenulic acids E (67) and F (68) by analysis of 1H-1H
coupling constants and NOESY correlations.




-73-
Table 13. 1H-NMR spectroscopic data of 67-70 (J , Hz)

H 67a 68b 69a 70b
2 5.94 d (15.0) 5.96 d (15.5) 5.93 d (15.5) 5.91 d (15.0)
3 7.39 dd (15.0, 11.0) 7.27 dd (15.5, 11.0) 7.40 dd (15.5, 11.0) 7.27 dd (15.0, 11.0)
4 6.50 dd (15.5, 11.0) 6.53 dd (15.0, 11.0) 6.50 ddd (15.5, 11.0, 1.0) 6.42 dd (15.0, 11.0)
5 6.26 dd (15.5, 7.5) 6.25 dd (15.0, 7.0) 6.36 dd (15.5, 6.0) 6.24 dd (15.0, 8.0)
6 4.30 dd (10.0, 7.5) 4.18 dd (10.0, 7.0) 4.68 br t (6.0) 4.20 br t (8.0)
7 4.16 dd (10.0, 1.5) 3.90 d (10.0) 4.08 dd (6.0, 5.0) 4.67 br d (8.0)
8 2.08 ddd (11.0, 5.5, 1.5) --- 1.88 ddd (11.0, 7.0, 5.0) 2.05 br d (11.0)
9 1.99 m 1.96 td (11.0, 4.0) 1.70 br qd (11.0, 4.0) 1.82 m
10 2.04 m 2.09 m 1.80 m 2.12 m
2.35 br d (13.0) 2.18 br d (16.0) 2.41 br d (15.5) 2.28 br d (17.5)
11 5.39 br d (4.5) 5.40 br s 5.41 br s 5.39 br s
13 1.80 br t (16.0) 1.84 m 1.77 m 1.72 m




-74-
1.97 br d (16.0) 2.00 br d (16.0, 4.0) 2.02 m 1.96 br d (15.0)
14 2.06 m 2.25 br t (14.0) 2.04 m 2.07 br t (14.0)
15 5.43 br s 5.47 br s 5.42 br s 5.46 br s
17 2.17 br d (3.5) 2.10 br s 2.68 br d (7.0)
19 3.22 d (3.0) 3.36 d (2.0) 3.63 d (9.0) 4.06 br d (10.0)
20 1.72 m 1.80 m 1.90 m 1.75 m
21 0.96 m 0.96 m 1.19 m 1.10 m
1.64 m 1.63 m 1.93 m 1.86 m
22 0.87 t (7.0) 0.87 t (7.5) 0.93 t (7.0) 0.92 t (7.5)
23 0.99 d (6.5) 0.98 d (7.5) 0.88 d (6.5) 0.80 d (6.5)
24 1.19 s 1.07 s 5.09 br s 5.14 s
5.29 br s 5.24 s
25 1.88 br s 1.89 br s 1.56 br s 1.69 br s
26 1.66 br s 1.65 br s 1.67 br s 1.63 br s
a Measured b measured
in CDCl3 in CD3OD.
Pyrenulic acids A-H (63-70) possess an unusual carbon skeleton arising from the
closure of the polyketide chain. This is the first instance for the isolation of this type of
metabolite from the cultured mycobionts of lichens. Pyrenulic acids are related to the
cytotoxic polyketides cladobotric acids (71, 72) which were isolated from the
fermentation broth of fungus Cladobotryum species.112) The biosynthesis of cladobotric
13
C-labeled precursors.112)
acids were investigated by incorporation experiments with
Pyrenulic acids and cladobotric acids only differ in the degree of oxidation. Therefore,
the biosynthesis of pyrenulic acids and cladobotric acids could be proposed as follow.
CoAS O
O
O
X
X
O Me
Me
O O
O OO
X Me
Me X
O O
X Me = S-adenosylmethionine

COOH COOH COOH




R1

O O
R2
71 : R1 = OH, R2 = CH3
64 63
72 : R1 = H, R2 = CH2OH
Cladobotryum sp.
HOOC HOOC HOOC




HO HO
HO O O O
R
R
OH
O R

65 : R = OH 67 : R = H 69 : R = H
66 : R = H 68 : R = OH 70 : R = OH

Fig. 22. Proposed biosynthesis of pyrenulic acids and related compounds



-75-
In conclusion, the investigation of the cultured mycobionts of the crustose lichen
Pyrenula sp. collected in Vietnam led to isolate two anthraquinones (60 and 61), a
xanthone (62) and eight new polyketides, pyrenulic acids A-H (63-70). This type of
polyketides has not been isolated from either lichen thalli or cultured lichen mycobionts.
The related known compounds cladobotric acids isolated from the fungus Cladopotryum
sp. exhibited cytotoxicity and antimicrobial activity.112) Moreover, the biosynthesis of
cladobotric acids was found that the closure of the polyketide chain did not follow the
usual folding pattern in fungal polyketides.112)




-76-
Chapter 4: Biological activity of isolated compounds


Lichen substances exhibit a great diversity of biological effects, including
antimicrobial, anti-inflammatory, antioxidant, antiproliferative and cytotoxic activities.
Lobaric acid (73) showed antiproliferative114,115) and antimitotic116) activities. α-
Alectoronic acid (11) had cytotoxicity.44) Atranorin (9) and chloroatranorin (10)
inhibited COX-1 and COX-2 enzymes.33) Compounds sporogen-AO 1 (46),
cyclodebneyol (59), cladobotric acids A (71) and C (72), which were possessed the
same skeleton with isolated compounds from cultured Vietnamese lichen mycobionts
(eremophilanes, polyketides), exhibited antifungal,87,99) cytotoxic activities.112) From our
interest in the biological activities of these compounds, selected compounds isolated
from Vietnamese lichens were tested to evaluate their biological actions on mammalian
DNA polymerases activity and cancer cell growth.




Fig. 23. Structure of reported bioactive metabolites


4.1. Inhibitory effect on mammalian DNA polymerase activity
The human genome encodes at least 15 DNA polymerases that participate in cellular
DNA synthesis.117,118) Eukaryotic cells contain 3 replicative polymerases (α, β and ε), 1
mitochrondrial polymerase (γ) and at least 11 non-replicative polymerases (β, ζ, η, θ, ι,
κ, λ, µ, ν, terminal deoxynucleotidyl transferase and REV1).119,120) The studies on DNA
polymerases were initiated in a search for specific inhibitors, because these enzymes are



-77-
essential for DNA replication, repair and cell divisions. Selective inhibitors of
mammalian DNA polymerases are considered a group of potentially useful cancer
chemotherapy agents, because some DNA polymerases inhibitors suppress human
cancer cell proliferation and have cytotoxicity.121)




Fig. 24. Selected compounds for bio-assay


Selected compounds (Fig. 24) isolated from the lichen thalli and cultured
mycobionts were investigated for their in vitro biochemical action. The inhibition of
three mammalian DNA polymerases, namely calf polymerase α, rat polymerase β and
human polymerase κ, by 500 µM and 100 µM of each compound was evaluated (Figs.




-78-
25-27). Compounds 4,6-dihydroxy-3,9-dehydromellein (38), 6,8-dihydroxy-3-
(hydroxymethyl)isocoumarin (39), dihydroxysporogen-AO 1 (47) and petasol (48) at
100 µM had no effect on the activities of DNA polymerases tested. The depsides
atranorin (9) and chloroatranorin (10) showed slightly inhibitory activity against
polymerases α and κ with IC50 less than 100 µM.
Depsidone salazinic acid (30) at 100 µM exhibited the 50% inhibition on the all
tested polymerases α, β and κ. Depsidones α-alectoronic acid (11), α-collatolic acid
(12), 2′′′-O-ethyl-α-alectoronic acid (15) and isocoumarin derivatives β-alectoronic acid
(13), β-collatolic acid (14) were more potent polymerase inhibitors than the others.
Novel polyketides pyrenulic acids C (65) and D (66) isolated from the cultured
mycobionts of Pyrenula sp. showed the inhibitory effect on polymerases α, β and κ at
100 µM. These results suggested that the depsidones and polyketides with aliphatic side
chain maybe play an important role in the polymerases inhibition.


9

10

11

12 100 µM
13
500 µM
14
Compounds




15

30

38

39

47

48

65

66

0 20 40 60 80 100
Polymerase α relative activity (%)


Fig. 25. Inhibitory effects of isolated compounds on the activity of calf DNA
polymerase α. Each compound (500 µM and 100 µM) was incubated with calf
polymerase α (0.05 units). Polymerase activity in the absence of the compound was
taken as 100%, and the relative activity is shown.




-79-
9

10

11

12
100 µM
13
500 µM
14
Compounds




15
30

38

39

47

48

65
66

0 20 40 60 80 100
Polymerase β relative activity (%)


Fig. 26. Inhibitory effects of isolated compounds on the activity of rat DNA polymerase
β. Each compound (500 µM and 100 µM) was incubated with rat polymerase β (0.05
units). Polymerase activity in the absence of the compound was taken as 100%, and the
relative activity is shown.


9

10

11
100 µM
12
13 500 µM
14
Compounds




15

30

38

39

47

48

65

66

0 20 40 60 80 100
Polymerase κ relative activity (%)


Fig. 27. Inhibitory effects of isolated compounds on the activity of human DNA
polymerase κ. Each compound (500 µM and 100 µM) was incubated with human
polymerase κ (0.05 units). Polymerase activity in the absence of the compound was
taken as 100%, and the relative activity is shown.



-80-
4.2. Inhibitory effect on cancer cell growth
Polymerases have recently emerged as important cellular targets for chemical
intervention in the development of anti-cancer agent.121) Isolated compounds from
Vietnamese lichens could therefore be useful in chemotherapy, and we investigated the
cytotoxic effect of selected compounds against HCT116 human colon carcinoma
cultured cell line. As shown in Fig. 28, 100 µM chloroatranorin (10), α-alectoronic acid
(11), α-collatolic acid (12), β-alectoronic acid (13), β-collatolic acid (14), 2′′′-O-ethyl-
α-alectoronic acid (15), salazinic acid (30), pyrenulic acids C (65) and D (66)
suppressed cell growth with LD50 value less than 100 µM, whereas atranorin (9), 4,6-
dihydroxy-3,9-dehydromellein (38), 6,8-dihydroxy-3-(hydroxymethyl)isocoumarin (39),
dihydroxysporogen-AO 1 (47) and petasol (48) had effect approximately 10-20% on
cell growth. The influence of depsidones and polyketides on HCT116 cell growth
showed the same tendency as that on the inhibition of polymerase α (Fig. 25),
suggesting that cancer cell growth prevention by these compounds may be related to
inhibition of the activities of DNA replicative polymerases, such as polymerase α.


9
10
11
12
100 µM
13
14
Compounds




15
30
38
39
47
48
65
66

0 20 40 60 80 100
Rate of HCT116 cell growth (%)


Fig. 28. Inhibitory effects of isolated compounds on the proliferation of HCT116 human
colon carcinoma cultured cell growth. Each compound (100 µM) was added to the
cultured HCT 116 cells. The cells were incubated for 24 h and the rate of cultured cell
growth inhibition was determined by WST-1 assay.122) Cell growth inhibition of the
cancer cells in the absence of the compound was taken as 100%.



-81-
Chapter 5. Conclusions


In conclusion, the chemical investigation of the thalli of two foliose lichens and the
cultured mycobionts of three crustose lichens species collected in the South of Vietnam
led to isolation of 21 novel secondary metabolites and 36 known substances. The
structures of isolated compounds were determined by a combination of spectroscopic
and chemical methods.
From the thalli of the foliose lichen Parmotrema mellissii, twenty five lichen
substances (3-27) including five new depsidones (15, 20, 23-25) and three new
isocoumarins (17, 21 and 22) were isolated. With the single exception of
dehydrocollatolic acid (19), the depsidones (20, 23-25) with a spiro-ring system have
not been reported. Chemical investigation of the air-dried thalli of Rimelia clavulifera
gave fifteen known lichen substances (2, 3, 6, 7, 9, 10, 11-14, 19, 26, 27, 29 and 30).
Salazinic acid (30) was obtained as a major lichen substance from the thalli of R.
clavulifera.
From the cultures of the lichen mycobionts of Graphis vestitoides, a phenolic
compound (35), five known isocoumarin derivatives (36-40), a new isocoumarin (41)
and a novel 14-membered macrolide (42) were obtained. The cultured mycobionts of
Graphis species have been recognized as a source of isocoumarins. However, 14-
membered macrolides have not been reported from the lichen thalli or cultured
mycobionts.
The investigation of cultured fungal partners of Sarcographa tricosa yielded
ergosterol peroxide (45), six known (46-51) and three new (52-54) eremophilane-type
sesquiterpenes. This type of sesquiterpenes was isolated from plants or fungi, but not
from the lichen thalli or cultured lichen mycobionts.
Two anthraquinones (60 and 61), a xanthone (62) and eight new polyketides (63-70)
were isolated from the cultured mycobionts of Pyrenula species. This type of
polyketides has never been isolated from the lichen thalli or cultured lichen mycobionts.




-82-
Parmotrema mellissii & Rimelia clavulif era

O
R1 CO2R2
CO2R CO2CH3
O OH
HO OH HO OH HO OH
HO OH CO2H
CHO
3 : R = CH3
7 : R1 = H; R2 = CH3
4 : R = n-C4H9
2 6
8 : R1 = Cl; R2 = C2H5
5 : R = C2H5

C5H11 C5H11
O
R O OH
O O
O
HO OH
O OH O
HO OH CO2CH3
O
CHO O RO O
RO O
O
O
9:R=H 11 : R = H 13 : R = H
HO C5H11 HO C5H11
10 : R = Cl 12 : R = CH3 14 : R = CH3


C5H11 C5H11 C5H11
O O O
O O
HO OH
O O
OH OH
O
O
O O
O
HO
HO RO
O O
O
O O
O
15 : R = C2H5 RO C5H11 17 : R = CH3 RO C5H11 19 : R = CH3
16 : R = CH3 18 : R = C2H5 20 : R = H

C5H11 C5H11 O
O O HO O OH
O
HO OH O OH
O O
O O
O
RO OH O
HO O
O O
26
O O
H
OH O OH
RO
21 : R = H 23, 24 : R = C2H5
22 : R = CH3 25 : R = n-C4H9
HO
O
O O CH2OH
O
CO2H
O OH O OH
HO
O
OH
HO OH O HO O
O CHO O OH O OH
HO
29 30 27
New compound




-83-
Except for isocoumarin, the other skeletons including 14-membered macrolide,
eremophilane-type sesquiterpene and a type of polyketide were reported herein for the
first instance from the lichen cultured lichen mycobionts. These results suggested that
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. It is also noteworthy that the metabolic ability was expressed only in
the isolated mycobionts.
Depsidones 11, 12 and 15, isocoumarin derivatives 13 and 14, and polyketides 65
and 66 suppressed the mammalian DNA polymerases α, β and κ, and HCT116 human
colon carcinoma cell growth. Since these bioactive compounds possess the aliphatic
side chains, these structures must be important for the abovementioned activities.
This is the first research taken on chemical investigation of lichens growing in
Vietnam. These results pointed out that the Vietnamese lichens could be new sources of
bioactive compounds with novel skeletons.




-84-
Sarcographa tricosa
OH
O

HO HO
O
O
47
46
O
O
HO 45 O O

HO
HO
R1
O 52
49
HO
CH2R2 H
OH O
48 : R1 = R2 = H
50 : R1 = H, R2 = OH HO
HO OH
H
51 : R1 = OH, R2 = H
53 54




-85-
ACKNOWLEDGMENTS
I am grateful to Vietnamese Government (Project 322) for providing the fellowship.
I would like to thank Quang Ngai Province People’s Committee and Pham Van Dong
University, Vietnam for their support and encouragement. Financial supports from Kobe
Pharmaceutical University (KPU), Japan are also gratefully acknowledged.
I am very grateful to my supervisor, Prof. Dr. Takao Tanahashi (KPU), for his
acceptance, kind advice, guidance, patience, encouragement and correction throughout
this study.
I would like to acknowledge Dr. Yukiko Takenaka, Dr. Katsumi Nishimura and Ms.
Sachiko Kozaki (KPU) for their kind advice, guidance, support, help and correction
during my study in Japan.
I am thankful to Dr. Nobuo Hamada (Osaka City Institute of Public Health and
Environmental Sciences, Japan) for cultivation the lichen mycobionts.
I am grateful to Prof. Dr. Akimori Wada, Prof. Dr. Okiko Miyata and Prof. Dr.
Masataka Moriyasu (KPU) for their reviews, comments and revisions the manuscript.
I am gratefully indebted to Dr. Isao Yoshimura (Hattori Botanical Laboratory,
Japan), Prof. Dr. Hiromi Miyawaki (Saga University, Japan) and Ms. Vo Thi Phi Giao
(University of Science, VNU-HCM) for identification the lichen specimens.
Thanks are also due to Drs. Makiko Sugiura and Chisato Tode (KPU) for NMR
spectral, and to Dr. Atsuko Takeuchi (KPU) for mass spectral measurements. Thanks
are given to Prof. Dr. Yoshiyuki Mizushina (Kobe-Gakuin University) for bio-assay.
I would like to thank my previous supervisor, Prof. Dr. Nguyen Kim Phi Phung
(University of Science, VNU-HCM), for her warm support and encouragement.
I express my gratitude to Ms. Ngo Thi Tinh, Ms. Nguyen Ngoc Uyen, Mr. Le
Hoang Da and Mr. Ngo Quang Thoi from Vietnam for their help in collecting and
sending the lichen samples to Japan.
I would like to thank all of my family and friends for their support and
encouragement. Many thanks are due to MDs Tran Van Hung and Pham Thi Van
Huyen for their kind help and advice. Special thanks are given to my parents and my
wife for their support, love and encouragement all the time.
Japan, March 2012
Le Hoang Duy



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EXPERIMENTAL SECTION
EXPERIMENTAL SECTION


1. General
Melting points were measured on a Yanaco micro melting apparatus and are
uncorrected.
UV spectra were recorded on a Shimadzu UV-240 spectrophotometer.
IR spectra were recorded on a Shimadzu FTIR-8200 infrared spectrophotometer.
Optical rotations were measured on a Jasco DIP-370 digital polarimeter.
Mass spectral data were obtained with a Hitachi M-4100 mass spectrometer.
NMR experiments were performed with Varian VXR-500, Varian UNITY INOVA
and Gemini-300 spectrometers, with TMS as internal standard.
HPLC was performed using a Waters system (600E Multisolvent Delivery System,
2487 Dual λ Absorbance Detector).
Chiral HPLC analysis [column: CHIRALCEL OJ-RH (0.46 cm φ × 15 cm, Daicel
Chemical Industries, Ltd.); flow rate: 1.0 ml/min; detection: 254 nm].
Silica gel 60 (Merck) was used for column chromatography.
Thin-layer chromatography was performed on precoated Kieselgel 60F254 plates
(Merck) and spots were visualized under UV light or sprayed with phosphomolybdic
acid solution (H2O 500 ml, H3PO4 85% 7.5 ml, concentrated H2SO4 25 ml,
H3(PMo12O40)•nH2O 12.1 g), then heated.


2. Cultivation lichen mycobionts
All lichen mycobionts were cultivated at Osaka City Institute of Public Health and
Environmental Sciences, Japan by Dr. Nobuo Hamada.
Discharge spores: A small fresh thallus was soaked in water for about 15 mins. The
thallus fragment was blotted to remove excess water and fastened with a petroleum jelly
to the upper half of a Petri dish. Then the upper half of the dish was inverted over the
lower half which contained an agar layer. The spores discharged on the agar surface
could be observed by a microscope. After spore germination had occurred, a block of
agar with spores was cut out by a sterilized spear needle and transferred to a test tube
that contained nutrient medium for cultivation (Fig. 3).15)




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Culture medium: malt extract 10 g, yeast extract 4 g, sucrose 100 g, agar 15 g, H2O
1 l, pH 7 (MY10).
Culture condition: at 18oC in the dark for 3-8 months.


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


Plant material
The lichens P. mellissii and R. clavulifera were collected from the pine trees bark in
Dalat City, Vietnam (ca. 1,520 m alt.) in 2008. The voucher specimens were identified
by Dr. Isao Yoshimura, Hattori Botanical Laboratory, Japan and Ms. Giao T.P. Vo,
University of Science Ho Chi Minh City, Vietnam and were deposited at Kobe
Pharmaceutical University. The thalli of the lichens were cleaned and dried at rt before
extraction.


2.1. Extraction and isolation of lichen substances from P. mellissii
The dried thalli of the lichen P. mellissii (60.0 g) were extracted with acetone at rt (3
× 1.0 l), and the combined extracts were concentrated under reduced pressure to give a
residue (8.41 g). This extract was subjected to CC and eluted by solvent system CHCl3-
MeOH with increasing MeOH ratios to obtain four fractions, fr-I (0% MeOH, 1.25 g),
fr-II (1% MeOH, 3.27 g), fr-III (2% MeOH, 2.19 g) and fr-IV (3-50% MeOH, 1.62 g).
Fr-I was purified by CC (CHCl3-MeOH) and prep. TLC (CHCl3; CHCl3-MeOH, 99:1,
85:5, 9:1; n-hexane-Et2O, 1:1), giving 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), 22 (22.0 mg), 26 (22.5 mg) and 27
(4.8 mg). Fr-II was separated by CC to obtain three sub-fractions, fr-IIa (0% MeOH,
417 mg), fr-IIb (0% MeOH, 539 mg), fr-IIc (1-2% MeOH, 2.17 g). These sub-fractions
were continuously purified by CC (CHCl3-MeOH) and prep. TLC (CHCl3-MeOH, 99:1,
9:1; n-hexane-Et2O, 1:1; toluene-AcOH, 20:3) to yield lichen substances. Fr-IIa gave 15
(65.6 mg), 18 (66.2 mg) and 25 (2.9 mg). Fr-IIb yielded 5 (10.2 mg), 12 (327.2 mg), 20
(21.6 mg), 21 (25.0 mg), 23 (9.1 mg) and 24 (8.1 mg). Fr-IIc afforded 11 (1.21 g), 12




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(365.7 mg), 13 (21.3 mg), 14 (139.0 mg), 16 (43.9 mg) and 17 (13.7 mg). Purification
of fr-III by CC (CHCl3-MeOH) gave 11 (1.32 g) and 13 (798.4 mg).


2.1.1. Mono-aromatic compounds


Methyl orsellinate (3)
Colorless crystalline solid.
mp. 136-137oC (CHCl3).
UV λmax (EtOH) nm (log ε): 215 (4.09), 263 (3.84), 304 (3.48).
IR (KBr) νmax cm-1: 3366, 2956, 1654, 1620, 1450, 1379, 1321, 1263, 1201.
H-NMR (CDCl3): δ 2.49 (3H, br s, H3-8), 3.92 (3H, s, -OCH3), 6.22 (1H, br d, J=2.5
1


Hz, H-5), 6.27 (1H, d, J=2.5 Hz, H-3), 11.72 (1H, s, 2-OH).
C-NMR (CDCl3): δ 24.3 (C-8), 51.9 (-OCH3), 101.3 (C-3), 105.5 (C-1), 111.3 (C-5),
13


144.0 (C-6), 160.3 (C-4), 165.4 (C-2), 172.1 (C-7).
EIMS m/z (rel. int.): 182 (30.8) [M+], 150 (100), 122 (36.8), 84 (39.6), 49 (77.6).
HR-EIMS m/z: 182.0573 (calcd for C9H10O4: 182.0579 [M+]).


n-Butyl orsellinate (4)
Colorless crystalline solid.
mp. 64-65oC (CHCl3-MeOH).
UV λmax (EtOH) nm (log ε): 216 (3.93), 264 (3.72), 300 (3.29).
IR (KBr) νmax cm-1: 3365, 2919, 1707, 1640, 1584, 1504, 1472, 1318, 1276, 1262, 1216.
H-NMR (CDCl3): δ 0.98 (3H, t, J=7.5 Hz, H3-4′), 1.48 (2H, m, H2-3′), 1.76 (2H, m,
1


H2-2′), 2.50 (3H, br s, H3-8), 4.34 (2H, t, J=6.5 Hz, H2-1′), 6.22 (1H, br d, J=2.5 Hz, H-
5), 6.28 (1H, d, J=2.5 Hz, H-3), 11.85 (1H, s, 2-OH).
C-NMR (CDCl3): δ 13.7 (C-4′), 19.4 (C-3′), 24.4 (C-8), 30.6 (C-2′), 65.3 (C-1′), 101.3
13


(C-3), 105.8 (C-1), 111.3 (C-5), 144.0 (C-6), 160.2 (C-4), 165.5 (C-2), 171.8 (C-7).
NOESY: H-5/H3-8.
HMBC: 2-OH→C-1, 2, 3; H-3→C-1, 2, 4, 5; H-5→C-1, 3; H3-8→C-1, 5, 6; H2-1′→C-
7, 2′, 3′; H2-2′→C-1′, 3′, 4′; H2-3′→C-1′, 2′, 4′; H3-4′→C-2′, 3′.
EIMS m/z (rel. int.): 224 (14.3) [M+], 150 (100), 122 (8.8).



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HR-EIMS m/z: 224.1049 (calcd for C12H16O4: 224.1049 [M+]).


Ethyl orsellinate (5)
Colorless needles.
mp. 129-130oC (CHCl3-MeOH).
IR (KBr) νmax cm-1: 3475, 3364, 2918, 1705, 1644, 1585, 1506, 1475.
H-NMR (CDCl3): δ 1.41 (3H, t, J=7.0 Hz, H3-2′), 2.51 (3H, br s, H3-8), 4.40 (2H, q,
1


J=7.0 Hz, H2-1′), 5.25 (1H, br s, 4-OH), 6.22 (1H, dd, J=2.7, 0.6 Hz, H-5), 6.27 (1H, d,
J=2.7 Hz, H-3), 11.82 (1H, s, 2-OH).
EIMS m/z (rel. int.): 196 (5.7) [M+], 150 (15.9), 84 (100).
HR-EIMS m/z: 196.0745 (calcd for C10H12O4: 196.0736 [M+]).


Methyl β -orsellinate (6)
Colorless crystalline solid.
mp. 142-143oC (CHCl3-MeOH).
UV λmax (EtOH) nm (log ε): 218 (4.33), 269.5 (4.16), 303 (3.60).
IR (KBr) νmax cm-1: 3406, 3086, 2984, 1628, 1500, 1446, 1368, 1317, 1275, 1199.
H-NMR (CDCl3): δ 2.10 (3H, s, H3-9), 2.46 (3H, br s, H3-8), 3.92 (3H, s, -OCH3), 5.20
1


(1H, br s, 4-OH), 6.21 (1H, br s, H-5), 12.02 (1H, s, 2-OH).
C-NMR (CDCl3): δ 7.7 (C-9), 24.1 (C-8), 51.8 (-OCH3), 105.7 (C-1), 108.5 (C-3),
13


110.5 (C-5), 140.1 (C-6), 158.1 (C-4), 163.2 (C-2), 172.6 (C-7).
EIMS m/z (rel. int.): 196 (53.6) [M+], 164 (100), 136 (83).
HR-EIMS m/z: 196.0734 (calcd for C10H12O4: 196.0736 [M+]).
HR-CIMS m/z: 197.0825 (calcd for C10H13O4: 197.0814 [M+H]+).


Methyl haematommate (7)
Colorless crystalline solid.
mp. 142-143oC (CHCl3-MeOH).
UV λmax (EtOH) nm (log ε): 241 (4.15), 261 (4.05), 335 (3.42).
IR (KBr) νmax cm-1: 2962, 1718, 1645, 1577, 1477, 1458, 1439, 1325, 1265, 1201.




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H-NMR (CDCl3): δ 2.53 (3H, d, J=0.5 Hz, H3-8), 3.96 (3H, s, -OCH3), 6.30 (1H, br s,
1


H-5), 10.34 (1H, s, H-9), 12.42 (1H, s, 4-OH), 12.89 (1H, s, 2-OH).
C-NMR (CDCl3): δ 25.2 (C-8), 52.3 (-OCH3), 103.9 (C-1), 108.4 (C-3), 112.1 (C-5),
13


152.3 (C-6), 166.7 (C-4), 168.3 (C-2), 172.0 (C-7), 193.9 (C-9).
EIMS m/z (rel. int.): 210 (49) [M+], 178 (22), 150 (100), 122 (16.5).
HR-EIMS m/z: 210.0542 (calcd for C10H10O5: 210.0529 [M+]).


Ethyl chlorohaematommate (8)
Colorless crystalline solid.
mp. 97-98oC (CHCl3-MeOH).
UV λmax (EtOH) nm (log ε): 238 (4.17), 272.5 (4.09), 353.5 (3.51), 400 (3.13).
IR (KBr) νmax cm-1: 2988, 1700, 1657, 1554, 1467, 1439, 1409, 1365, 1315, 1249, 1202,
1014.
H-NMR (CDCl3): δ 1.46 (3H, t, J=7.0 Hz, H3-2′), 2.72 (3H, s, H3-8), 4.47 (2H, q,
1


J=7.0 Hz, H2-1′), 10.36 (1H, s, H-9), 12.74 (1H, s, 2-OH), 13.15 (1H, s, 4-OH).
C-NMR (CDCl3): δ 14.1 (C-2′), 20.8 (C-8), 62.5 (C-1′), 105.5 (C-1), 108.6 (C-3),
13


114.9 (C-5), 149.1 (C-6), 162.5 (C-4), 164.6 (C-2), 171.0 (C-7), 193.8 (C-9).
HR-ESIMS m/z: 257.0218 (calcd for C11H10O535Cl: 257.0217 [M-H]-).


2.1.2. Depsides


Atranorin (9)
Light yellow crystal.
mp. 186-187o C (CHCl3).
UV λmax (EtOH) nm (log ε): 213 (3.87), 255.5 sh (3.60), 280 (3.68), 300.5 (3.57), 382.5
(2.88).
IR (KBr) νmax cm-1: 2955, 2932, 1655, 1584, 1472, 1453, 1410, 1380, 1354, 1286, 1215.
H-NMR (CDCl3): δ 2.09 (3H, s, H3-9′), 2.55 (3H, s, H3-8′), 2.69 (3H, s, H3-8), 3.99
1


(3H, s, -OCH3), 6.40 (1H, s, H-5), 6.52 (1H, s, H-5′), 10.35 (1H, s, H-9), 11.94 (1H, s,
2′-OH), 12.49 (1H, s, H-2), 12.54 (1H, s, 4-OH).




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C-NMR (CDCl3): δ 9.4 (C-9′), 24.0 (C-8′), 25.6 (C-8), 52.3 (-OCH3), 102.8 (C-1),
13


108.5 (C-3), 110.3 (C-1′), 112.8 (C-5), 116.0 (C-5′), 116.8 (C-3′), 139.9 (C-6′), 152.0
(C-4′), 152.4 (C-6), 162.9 (C-2′), 167.5 (C-4), 169.1 (C-2), 169.7 (C-7), 172.2 (C-7′),
193.8 (C-9).
EIMS m/z (rel. int.): 374 (6.7) [M+], 196 (65.2), 164 (100), 136 (75.8).
HR-EIMS m/z: 374.1014 (calcd for C19H18O8: 374.1002 [M+]).


Chloroatranorin (10)
Light yellow crystal.
mp. 207-208oC (CHCl3-MeOH).
UV λmax (EtOH) nm (log ε): 214 (3.65), 252 (3.31), 286.5 (3.42), 303.5 sh (3.36), 395
(2.74).
IR (KBr) νmax cm-1: 2956, 1651, 1587, 1446, 1403, 1381, 1360, 1081, 1027, 844, 806.
H-NMR (CDCl3): δ 2.09 (3H, s, H3-9′), 2.55 (3H, s, H3-8′), 2.87 (3H, s, H3-8), 3.99
1


(3H, s, -OCH3), 6.52 (1H, s, H-5′), 10.38 (1H, s, H-9), 11.95 (1H, s, 2′-OH), 12.33 (1H,
br s, 2-OH), 13.24 (1H, br s, 4-OH).
C-NMR (CDCl3): δ 9.4 (C-9′), 21.2 (C-8), 24.0 (C-8′), 52.4 (-OCH3), 104.3 (C-1),
13


108.7 (C-3), 110.4 (C-1′), 115.7 (C-5), 115.8 (C-5′), 116.7 (C-3′), 140.0 (C-6′), 149.0
(C-6), 151.9 (C-4′), 162.9 (C-2′), 163.4 (C-4), 166.3 (C-2), 169.2 (C-7), 172.2 (C-7′),
193.7 (C-9).
HR-ESIMS m/z: 407.0537 (calcd for C19H16O835Cl: 407.0534 [M-H]-).


2.1.3. Depsidones and Isocoumarin derivatives


α-Alectoronic acid (11)
Colorless solid.
UV λmax (EtOH) nm (log ε): 209 (4.60), 266 sh (4.02), 314 (3.62).
IR (KBr) νmax cm-1: 3361, 2956, 1714, 1676, 1613, 1579, 1480, 1453, 1362.
1
H-NMR: Table 1.
13
C-NMR: Table 1.
HR-SIMS m/z: 511.1983 (calcd for C28H31O9: 511.1969 [M-H]-).



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Methylation of 11
To a solution of 11 (10 mg) in MeOH (3 ml) was added an excess of TMS-CHN2 in
n-hexane, and the whole was stirred at rt for 4 hrs. After quenching by diluted AcOH in
MeOH, the reaction mixture was concentrated and the residue was purified by prep.
TLC (n-hexane-Et2O, 3:7) to yield methylated compounds 11a (5.7 mg) and 11b (3.4
mg).


Compound 11a
1
H-NMR: Table 1.
13
C-NMR: Table 1.
EIMS m/z (rel. int.): 554 (8.2) [M+], 456 (6.3), 424 (9.4), 279 (7.8), 149 (40.5), 97
(26.2), 73 (100).
HR-EIMS m/z: 554.2535 (calcd for C31H38O9: 554.2517 [M+]).


Compound 11b
1
H-NMR: Table 1.
13
C-NMR: Table 1.
EIMS m/z (rel. int.): 568 (14.5) [M+], 470 (25), 394 (33), 147 (15.1), 97 (34.5), 73 (100).
HR-EIMS m/z: 568.2681 (calcd for C32H40O9: 568.2674 [M+]).


α-Collatolic acid (12)
Colorless solid.
UV λmax (EtOH) nm (log ε): 209 (4.64), 247 sh (4.12), 264 sh (4.04), 314 sh (3.61).
IR (KBr) νmax cm-1: 3445, 2956, 1718, 1672, 1610, 1572, 1479, 1432, 1363.
1
H-NMR: Table 2.
13
C-NMR: Table 2.
HR-SIMS m/z: 525.2144 (calcd for C29H33O9: 525.2126 [M-H]-).

Methylation of 12
To a solution of 12 (7 mg) in Et2O (3 ml) and MeOH (2 ml) was treated with TMS-
CHN2 in n-hexane as mentioned above. The reaction mixture was purified by prep. TLC
(CHCl3-MeOH, 99:1) to yield methylated compounds 12a (6.2 mg)



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Compound 12a
1
H-NMR: Table 2.
13
C-NMR: Table 2.
EIMS m/z (rel. int.): 536 (9.5) [M+], 173 (9.5), 149 (15.5), 97 (22.5), 73 (100).
HR-EIMS m/z: 536.2418 (calcd for C31H36O8: 536.2412 [M+]).


β-Alectoronic acid (13)
Colorless solid.
UV λmax (EtOH) nm (log ε): 212 (4.54), 245.5 (4.76), 278 sh (3.95), 316 (3.96).
IR (KBr) νmax cm-1: 3245, 2956, 1697, 1659, 1601, 1487, 1463, 1363, 1257.
H-NMR (CD3OD): δ 0.78 (3H, t, J=7.0 Hz, H3-7′′′), 0.93 (3H, t, J=7.0 Hz, H3-7′′),
1


1.03 (2H, m, H2-5′′′), 1.15 (2H, m, H2-6′′′), 1.37 (6H, m, H2-5′′, H2-6′′ & H2-4′′′), 1.70
(2H, m, H2-4′′), 2.35, 2.43 (each 1H, m, H2-3′′′), 2.50 (2H, t, J=7.5 Hz, H2-3′′), 4.18
(2H, br s, H2-1′′′), 6.10 (1H, br s, H-3), 6.29 (1H, s, H-1′′), 6.40 (1H, br s, H-5), 6.43
(1H, s, H-3′).
C-NMR (CD3OD): δ 14.2 (C-7′′′), 14.3 (C-7′′), 23.2 (C-6′′), 23.3 (C-5′′′, C-6′′′), 27.5
13


(C-4′′), 32.1 (C-5′′), 32.2 (C-5′′′), 33.9 (C-3′′), 42.1 (C-1′′′), 43.1 (C-3′′′), 101.9 (C-1),
102.9 (C-3), 103.8 (C-3′), 104.5 (C-1′′), 105.2 (C-5), 111.3 (C-1′), 133.2 (C-6′), 134.4
(C-5′), 143.5 (C-6), 154.0 (C-4′), 159.5 (C-2′′), 162.5 (C-7), 162.8 (C-2′), 163.6 (C-2),
165.8 (C-4), 175.1 (C-7′).
HMBC: H-3→C-1, 2, 4, 5; H-5→C-1, 4, 1′′; H-3′→C-1′, 2′, 4′, 5′; H-1′′→C-1, 5, 6, 3′′;
H2-3′′→C-2′′, 4′′, 5′′; H2-4′′→C-2′′, 3′′, 5′′; H2-5′′→C-3′′, 4′′, 6′′; H2-6′′→C-4′′, 5′′, 7′′;
H3-7′′→C-5′′, 6′′; H2-1′′′→C-1′, 5′; H2-4′′′→C-3′′′, 6′′′; H2-5′′′→C-3′′′, 4′′′, 6′′′, 7′′′;
H2-6′′′→C-5′′′, 7′′′; H3-7′′′→C-5′′′, 6′′′.
HR-SIMS m/z: 511.1958 (calcd for C28H31O9: 511.1969 [M-H]-).


β-Collatolic acid (14)
Colorless solid.
UV λmax (EtOH) nm (log ε): 214 (4.50), 245 (4.74), 278 sh (3.95), 322.5 (3.95).
IR (KBr) νmax cm-1: 3391, 2956, 1703, 1660, 1604, 1567, 1507, 1470, 1372, 1248.




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H-NMR (acetone-d6): δ 0.83 (3H, t, J=7.0 Hz, H3-7′′′), 0.92 (3H, t, J=7.0 Hz, H3-7′′),
1


1.24 (4H, m, H2-5′′′ & H2-6′′′), 1.38 (4H, m, H2-5′′ & H2-6′′), 1.45 (2H, m, H2-4′′′),
2.05 (2H, m, H2-3′′′), 1.70 (2H, quint, J=7.0 Hz, H-4′′), 2.51 (2H, t, J=7.0 Hz, H2-3′′),
3.40 (2H, br s, H2-1′′′), 3.79 (3H, s, 4-OCH3), 6.29 (1H, br s, H-3), 6.39 (1H, s, H-1′′),
6.47 (1H, s, H-3′), 6.65 (1H, d, J=2.0 Hz, H-5).
C-NMR (acetone-d6): δ 14.2 (C-7′′ & C-7′′′), 23.1 (C-6′′ & C-6′′′), 24.0 (C-4′′′), 27.3
13


(C-4′′), 31.9 (C-5′′), 32.3 (C-5′′′), 33.8 (C-3′′), 41.6 (C-3′′′), 56.2 (4-OCH3), 101.6 (C-3),
103.0 (C-5), 103.4 (C-1′ & C-3′), 103.6 (C-1′′), 103.8 (C-1), 133.3 (C-6′), 133.6 (C-5′),
143.1 (C-6), 157.1 (C-4′), 159.6 (C-7), 159.9 (C-2′′), 162.3 (C-2), 162.8 (C-2′), 166.3
(C-4), 171.2 (C-7′).
HMBC: H-3→C-2, 4, 5; 4-OCH3→C-4; H-5→C-3, 4, 1′′; H-3′→C-1′, 2′, 4′, 5′; H-
1′′→C-1, 5, 6, 2′′, 3′′; H2-3′′→C-1′′, 2′′, 4′′, 5′′; H2-4′′→C-2′′, 3′′, 5′′, 6′′; H2-5′′→C-3′′,
4′′, 6′′, 7′′; H2-6′′→C-4′′, 5′′, 7′′; H3-7′′→C-5′′, 6′′; H2-5′′′→C-3′′′, 4′′′, 6′′′, 7′′′; H2-
6′′′→C-5′′′, 7′′′; H3-7′′′→C-5′′′, 6′′′.
HR-SIMS m/z: 525.2136 (calcd for C29H33O9: 525.2126 [M-H]-).


New depsidone 2′′′-O-ethyl-α-alectoronic acid (15)
Pale yellow solid.
[α]D27 +2.6o (c=0.24, CHCl3).
UV λmax (EtOH) nm (log ε): 247 (4.41), 317.5 (4.04).
IR (KBr) νmax cm-1: 3391, 2956, 1730, 1682, 1613, 1478.
1
H-NMR: Table 3.
13
C-NMR: Table 3.
EIMS m/z (rel. int.): 540 (13.1) [M+], 524 (24.5), 494 (100), 476 (40), 450 (50).
HR-EIMS m/z: 540.2346 (calcd for C30H36O9: 540.2361 [M+]).
Chiral HPLC analysis [column: CHIRALCEL OJ-RH 0.46 × 15 cm; mobile phase:
H2O-CH3CN (1:1), flow rate: 1.0 ml/min; detection: 254 nm]: two peaks were observed
at 19.8 min and 23.2 min in a ratio of approximately 1:1.




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2′′′-O-methyl-α-alectoronic acid (16)
Compound 16 was isolated in a mixture with 15 in a ratio of 2:1.
Colorless solid.
1
H-NMR (CDCl3): Table 3.
13
C-NMR (CDCl3): Table 3.
HMBC: H-3→C-1, 2, 4, 5; H-5→C-1, 3, 4, 1′′; H-3′→C-1′, 2′, 4′, 5′, 7′; H2-1′′→C-1, 5,
6, 2′′; H2-3′′→C-2′′, 4′′, 5′′; H2-4′′→C-2′′, 3′′, 5′′, 6′′; H2-5′′→C-4′′, 6′′, 7′′; H2-6′′→C-
5′′, 7′′; H3-7′′→C-5′′, 6′′; H2-1′′′→C-1′, 5′, 6′, 3′′′; 2′′′-OCH3→C-2′′′; H2-3′′′→C-2′′′,
4′′′, 5′′′; H2-4′′′→C-5′′′, 6′′′; H2-5′′′→C-6′′′, 7′′′; H2-6′′′→C-5′′′, 7′′′; H3-7′′′→C-5′′′,
6′′′.
HR-EIMS m/z: 526.2222 (calcd for C29H34O9: 526.2204 [M+]).


New isocoumarin 2′′′-O-methyl-β -alectoronic acid (17)
Colorless crystalline solid.
mp. 139-140oC (CHCl3-MeOH).
[α]D18 0o (c=0.72, MeOH).
UV λmax (EtOH) nm (log ε): 217 (4.32), 245.5 (4.50), 318 (4.03).
IR (KBr) νmax cm-1: 3398, 2957, 1720, 1662, 1600.
1
H-NMR: Table 3.
13
C-NMR: Table 3.
EIMS m/z (rel. int.): 526 (0.4) [M+], 494 (13), 450 (100), 394 (5.9), 352 (12.2), 44
(43.9).
HR-EIMS m/z: 526.2221 (calcd for C29H34O9: 526.2204 [M+]).
Chiral HPLC analysis [column: CHIRALCEL OJ-RH 0.46 × 15 cm; mobile phase:
H2O-CH3CN (1:1); flow rate: 1.0 ml/min; detection: 254 nm]: two peaks were observed
at 9.5 min and 10.3 min in a ratio of approximately 1:1.


2′′′-O-ethyl-β -alectoronic acid (18)
Colorless crystalline solid.
mp. 180-181oC (CHCl3-MeOH).
[α]D25 0o (c=0.98, MeOH).



-96-
UV λmax (EtOH) nm (log ε): 216 (4.38), 245 (4.72), 271 sh (4.25), 318 (4.16).
IR (KBr) νmax cm-1: 3290, 2956, 1795, 1600, 1601, 1483, 1464, 1360, 1260.
H-NMR (CDCl3): δ 0.87 (3H, m, H3-7′′′), 0.91 (3H, m, H3-7′′), 1.11 (3H, t, J=7.0 Hz,
1


H3-β), 1.26 (4H, m, H2-5′′′ & H2-6′′′), 1.29 (2H, m, H2-4′′′), 1.36 (4H, m, H2-5′′ & H2-
6′′), 1.70 (2H, m, H2-4′′), 1.82, 1.96 (each 1H, m, H2-3′′′), 2.50 (2H, t, J=7.5 Hz, H2-3′′),
2.95, 3.21 (each 1H, d, J=16.0 Hz, H2-1′′′), 3.62, 3.67 (each 1H, m, H2-α), 6.18 (1H, s,
H-1′′), 6.48 (2H, br s, H-3 & H-5), 6.49 (1H, s, H-3′), 11.05 (1H, s, 2′-OH).
C-NMR (CDCl3): δ 14.0 (C-7′′ & C-7′′′), 15.2 (C-β), 22.4 (C-6′′ & C-6′′′), 23.1 (C-
13


4′′′), 26.5 (C-4′′), 31.2 (C-5′′), 31.5 (C-1′′′), 31.6 (C-5′′′), 33.2 (C-3′′), 35.5 (C-3′′′),
58.1 (C-α), 99.8 (C-1′), 103.1 (C-1′′), 103.3 (C-3′), 103.5 (C-1), 104.1 (C-3), 105.6 (C-
5), 107.5 (C-2′′′), 131.4 (C-6′), 134.1 (C-5′), 142.1 (C-6), 157.0 (C-4′), 160.9 (C-7),
159.4 (C-2′′), 162.0 (C-2′), 162.6 (C-4), 163.1 (C-2), 168.9 (C-7′).
HMBC: H-3→C-2, 4; H-5→C-4, 1′′; 2′-OH→C-1′, 2′, 3′; H-3′→C-1′, 2′, 4′, 5′, 7′; H-
1′′→C-1, 5, 6, 2′′, 3′′; H2-3′′→C-1′′, 2′′, 4′′, 5′′; H2-4′′→C-2′′, 3′′, 5′′, 6′′; H2-5′′→C-4′′,
6′′; H2-6′′→C-5′′, 7′′; H3-7′′→C-5′′, 6′′; H2-1′′′→C-1′, 5′, 6′, 2′′′; H2-3′′′→C-2′′′, 4′′′,
5′′′; H2-4′′′→C-5′′′, 6′′′; H2-5′′′→C-6′′′, 7′′′; H2-6′′′→C-5′′′, 7′′′; H3-7′′′→C-5′′′, 6′′′;
H2-α→C-2′′′, β; H3-β→C-α;
HR-EIMS m/z: 540.2369 (calcd for C30H36O9: 540.2361 [M+]).
Chiral HPLC analysis [column: CHIRALCEL OJ-RH 0.46 × 15 cm; mobile phase:
H2O-CH3CN (6:4); flow rate: 1.0 ml/min; detection: 254 nm]: two peaks were observed
at 34.5 min and 37.5 min in a ratio of approximately 1:1.


Dehydrocollatolic acid (19)
Colorless crystalline solid.
mp. 157-158oC (CHCl3-MeOH).
[α]D28 +56o (c=0.53, CHCl3).
UV λmax (EtOH) nm (log ε): 248.5 (4.22), 257 sh (4.20), 322.5 (3.73).
IR (KBr) νmax cm-1: 3420, 2935, 2873, 1741, 1719, 1680, 1616, 1571, 1475, 1439, 1390,
1370, 1336, 1295, 1260, 1199, 1147, 1084, 1019.
1
H-NMR: Table 4.




-97-
13
C-NMR: Table 5.
EIMS m/z (rel. int.): 524 (0.6) [M+], 481 (0.7), 344 (64), 260 (53.7), 233 (100), 57
(51.5).
HR-EIMS m/z: 524.2062 (calcd for C29H32O6: 524.2048 [M+]).


New depsidone dehydroalectoronic acid (20)
Colorless solid.
mp. 180-181oC (CHCl3).
[α]D19 -35o (c=0.96, CHCl3).
UV λmax (EtOH) nm (log ε): 216 (4.46), 254 (4.15), 316.5 (3.83).
IR (KBr) νmax cm-1: 3322, 2934, 1738, 1682, 1614, 1478, 1367, 1333, 1250, 1209.
1
H-NMR: Table 4.
13
C-NMR: Table 5.
EIMS m/z (rel. int.): 510 (60) [M+], 466 (100), 432 (42.2), 406 (55.7), 370 (56), 249
(29.4), 219 (42.2), 192 (18.2), 163 (35.7), 95 (34.6), 69 (82.1).
HR-EIMS m/z: 510.1913 (calcd for C28H30O9: 510.1891 [M+]).


New isocoumarin 21
Colorless solid.
mp. 225-226oC (CHCl3).
[α]D18 -23o (c=0.41, MeOH).
UV λmax (EtOH) nm (log ε): 216.5 (4.36), 245.5 (4.69), 318.5 (4.12).
IR (KBr) νmax cm-1: 3199, 2931, 1730, 1662, 1601, 1483, 1464, 1366, 1331, 1252.
1
H-NMR: Table 4.
13
C-NMR: Table 5.
EIMS m/z (rel. int.): 510 (80.9) [M+], 466 (100), 406 (57.2), 370 (68), 249 (35.2), 219
(43.3), 163 (39.2), 97 (35.3), 69 (75.2).
HR-EIMS m/z: 510.1884 (calcd for C28H30O9: 510.1891 [M+]).


New isocoumarin 22
Colorless solid.




-98-
mp. 203-204oC (MeOH).
[α]D25 -121o (c=0.48, CHCl3).
UV λmax (EtOH) nm (log ε): 217.5 (4.29), 245 (4.64), 272.5 sh (4.11), 322.5 (4.13).
IR (KBr) νmax cm-1: 3209, 2932, 1698, 1671, 1628, 1604, 1567, 1474, 1374, 1332, 1250.
1
H-NMR: Table 4.
13
C-NMR: Table 5.
EIMS m/z (rel. int.): 524 (70.2) [M+], 480 (100), 446 (36.3), 420 (56.8), 384 (46.2), 262
(27.6), 219 (24.6), 164 (29.6), 97 (41.7), 69 (80.8).
HR-EIMS m/z: 524.2056 (calcd for C29H32O9: 524.2048 [M+]).
Chiral HPLC analysis [column: CHIRALCEL OJ-RH 0.46 × 15 cm; mobile phase:
CH3CN-H2O (1:1), flow rate: 1.0 ml/min; detection: 254 nm]: minor peak at 14.5 min
and a major peak at 16.0 min were observed in a ratio of 1:6, respectively.


New depsidone parmosidone A (23)
Pale yellow solid.
[α]D20 0o (c=0.72, CHCl3).
UV λmax (EtOH) nm (log ε): 215.5 (4.51), 252 (4.18), 317 (3.81).
IR (KBr) νmax cm-1: 3273, 2957, 1739, 1685, 1613, 1579, 1479, 1359, 1257.
1
H-NMR: Table 6.
13
C-NMR: Table 5.
HR-SIMS m/z: 525.1773 (calcd for C28H29O10: 525.1762 [M-H]-).


New depsidone parmosidone B (24)
Pale yellow solid.
[α]D21 0o (c=0.28, CHCl3).
UV λmax (EtOH) nm (log ε): 200 (4.50), 246 (4.28), 317 (3.86).
IR (KBr) νmax cm-1: 3272, 2928, 2855, 1730, 1684, 1613, 1479, 1360, 1257.
1
H-NMR: Table 6.
13
C-NMR: Table 5.
HR-SIMS m/z: 525.1756 (calcd for C28H29O10: 525.1762 [M-H]-).




-99-
New depsidone parmosidone C (25)
Pale yellow solid.
[α]D21 0o (c=0.29, CHCl3).
UV λmax (EtOH) nm (log ε): 216 (4.60), 252 (4.27), 317.5 (3.90).
IR (KBr) νmax cm-1: 3267, 2927, 1739, 1685, 1613, 1479, 1359, 1257.
1
H-NMR: Table 6.
13
C-NMR: Table 5.
HR-SIMS m/z: 553.2078 (calcd for C30H33O10: 553.2075 [M-H]-).


2.1.4. Other lichen substances


(+)-Usnic acid (26)
Light yellow needles.
mp. 184-185oC (CHCl3-MeOH).
[α]D18 +455o (c=0.80, CHCl3).
UV λmax (EtOH) nm (log ε): 200.5 (4.75), 233 sh (4.33), 288 (4.27), 336.5 sh (3.68).
IR (KBr) νmax cm-1: 3093, 2930, 1692, 1634, 1613, 1544, 1456, 1359, 1336, 1317, 1222,
840, 820.
H-NMR (CDCl3): δ 1.77 (3H, s, H3-13), 2.12 (3H, s, H3-16), 2.67 (3H, s, H3-15), 2.69
1


(3H, s, H3-18), 5.99 (1H, s, H-4), 11.04 (1H, s, 10-OH), 13.33 (1H, s, 8-OH), 18.84 (1H,
s, 3-OH).
C-NMR (CDCl3): δ 7.6 (C-16), 27.9 (C-15), 31.3 (C-18), 32.1 (C-13), 59.0 (C-12),
13


98.4 (C-4), 101.7 (C-7), 104.0 (C-11), 105.3 (C-2), 109.4 (C-9), 155.2 (C-6), 157.5 (C-
10), 163.9 (C-8), 179.4 (C-5), 191.7 (C-3), 198.1 (C-1), 200.4 (C-17), 201.8 (C-14).
EIMS m/z (rel. int.): 344 (77.9) [M+], 260 (72.3), 233 (100), 149 (35.7), 95 (23.8).
HR-EIMS m/z: 344.0885 (calcd for C18H16O7: 344.0897 [M+]).


Skyrin (27)
Red crystalline solid.
mp. 212oC (acetone) (dec.).




-100-
UV λmax (EtOH) nm (log ε): 219.5 (4.20), 259 (4.11), 303.5 (3.87), 341.5 sh (3.52),
465.5 (3.69).
IR (KBr) νmax cm-1: 3466, 3262, 1674, 1625, 1602, 1552, 1483, 1457, 1393, 1363, 1154,
1135, 1112, 1046, 1024.
H-NMR (acetone-d6): δ 2.38 (6H, s, 3-CH3 & 3′-CH3), 6.80 (2H, s, H-7 & H-7′), 7.10
1


(2H, br s, H-2 & H-2′), 7.31 (2H, br s, H-4 & H-4′), 12.12 (2H, s, 1-OH & 1′-OH),
12.86 (2H, s, 8-OH & 8′-OH).
C-NMR (acetone-d6): δ 22.0 (3-CH3 & 3′-CH3), 108.5 (C-7 & C-7′), 110.8 (C-12 &
13


C-12′), 114.3 (C-13 & C-13′), 121.3 (C-4 & C-4′), 123.8 (C-5 & C-5′), 124.3 (C-2 & C-
2′), 133.1 (C-11 & C-11′), 134.8 (C-14 & C-14′), 149.4 (C-3 & C-3′), 162.9 (C-1 & C-
1′), 165.3 (C-6 & C-6′), 166.3 (C-8 & C-8′), 183.1 (C-10 & C-10′), 191.7 (C-9 & C-9′).
HR-ESIMS m/z: 537.08257 (calcd for C30H17O10: 537.08221 [M-H]-).


2.2. Extraction and isolation of lichen substances from R. clavulifera
The dried thalli of the lichen R. clavulifera (30.0 g) were extracted with acetone,
MeOH at rt and hot MeOH (3 × 200 ml) at 800C, the combined extracts were
concentrated under reduced pressure to give residues (1.80 g, 1.16 g and 2.08 g,
respectively). These extracts were dissolved in acetone and filtered to obtain insoluble
and soluble fractions. The soluble fraction of acetone extract (908 mg) was separated by
prep. TLC (CHCl3), affording four bands a (148.9 mg), b (33.0 mg), c (92.2 mg) and d
(396.5 mg). Band a was subjected to prep. TLC (n-hexane-Et2O, 3:7; benzene-AcOEt,
4:6), giving 9 (115.1 mg), 10 (3.0 mg) and 19 (4.0 mg). Band b was purified prep. TLC
(CHCl3-MeOH, 95:5), giving 6 (2.9 mg) and 26 (2.8 mg). Purification of band c by prep.
TLC (n-hexane-Et2O, 3:7; CHCl3-MeOH, 9:1 or 8:2) yielded 12 (34.0 mg), 14 (16.2
mg) and 27 (2.5 mg). Band d was separated by prep. TLC (CHCl3-MeOH, 9:1; n-
hexane-Et2O-AcOH, 5:4:1 or 4:4:1; toluene-AcOH, 85:15), yielding 2 (6.2 mg), 3 (8.6
mg), 11 (77.5 mg), 12 (23.6 mg), 13 (11.0 mg), 27 (3.3 mg) and 29 (44.0 mg). The
soluble fraction of MeOH extract (862 mg) was purified by prep. TLC (toluene-
dioxane-AcOH, 18:5:1) yielded 6 (8.2 mg) and 9 (28.2 mg). The soluble fraction of hot
MeOH extract (901 mg) was separated by prep. TLC (toluene-dioxane-AcOH, 18:5:1 or
n-hexane-Et2O, 1:1), giving 6 (3.3 mg), 9 (21.8 mg), 10 (11.5 mg) and 27 (4.1 mg). The



-101-
insoluble fractions of acetone (726 mg), MeOH (250 mg) and hot MeOH (1.17 g)
extracts were combined and purified by recrystalization to give 30 (1.89 g).


Lecanoric acid (2)
White powder.
IR (KBr) νmax cm-1: 3535, 3459, 2923, 1657, 1614, 1593, 1464, 1320, 1257.
H-NMR (acetone-d6): δ 2.61 (3H, s, H3-8), 2.66 (3H, s, H3-8′), 6.31 (1H, d, J=2.5 Hz,
1


H-3), 6.39 (1H, d, J=2.5 Hz, H-5), 6.66 (1H, br s, H-5′), 6.72 (1H, br s, H-3′).
C-NMR (acetone-d6): δ 24.0 (C-8′), 24.4 (C-8), 101.8 (C-3), 104.9 (C-1), 109.1 (C-3′),
13


112.9 (C-5), 113.2 (C-1′), 116.5 (C-5′), 144.7 (C-6′), 144.8 (C-6), 154.3 (C-4′), 164.1
(C-4), 165.7 (C-2′), 166.7 (C-2), 170.5 (C-7), 174.2 (C-7′).
HR-SIMS m/z: 317.0657 (calcd for C16H13O7: 317.0662 [M-H]-).


Gyrophoric acid (29)
White solid.
mp. 215-218o C.
UV λmax (EtOH) nm (log ε): 212.5 (4.96), 245 sh (4.13), 271 (4.27), 309 (4.08).
IR (KBr) νmax cm-1: 3324, 3094, 2935, 1672, 1661, 1644, 1610, 1586, 1464, 1446, 1420,
1382.
H-NMR (DMSO-d6): δ 2.36 (6H, s, H3-8 & H3-8′), 2.50 (3H, s, H3-8′′), 6.23 (1H, br s,
1


H-5), 6.24 (1H, br s, H-3), 6.35 (1H, br s, H-5′′), 6.41 (1H, br s, H-3′′), 6.66 (1H, br s,
H-5′), 6.67 (1H, br s, H-3′), 10.02 (1H, br s, 4-OH), 10.32 (1H, br s, 2-OH), 10.45 (1H,
br s, 2′-OH).
C-NMR (DMSO-d6): δ 19.3 (C-8′), 21.2 (C-8), 22.3 (C-8′′), 100.4 (C-3), 107.0 (C-3′′),
13


107.1 (C-3′), 108.4 (C-1), 109.7 (C-5), 112.6 (C-5′′), 114.1 (C-5′), 116.7 (C-1′′), 118.3
(C-1′), 137.8 (C-6′), 140.1 (C-6), 141.3 (C-6′′), 151.5 (C-4′′), 151.9 (C-4′), 156.1 (C-2′),
159.9 (C-2), 161.0 (C-4), 164.0 (C-2′′), 165.7 (C-7′), 167.0 (C-7), 171.0 (C-7′′).
HR-SIMS m/z: 467.0989 (calcd for C24H19O10: 467.0979 [M-H]-).


Salazinic acid (30)
White solid.



-102-
mp. 268-270oC (dec.).
IR (KBr) νmax cm-1: 3575, 3300, 1772, 1742, 1661, 1558, 1439, 1380, 1297, 1261.
H-NMR (DMSO-d6): δ 2.45 (3H, s, H3-8), 4.66 (2H, s, H2-9′), 6.80 (1H, br s, H-8′),
1


6.87 (1H, s, H-5), 8.30 (1H, br s, 8′-OH), 10.46 (1H, s, H-9), 12.10 (1H, br s, 4-OH).
C-NMR (DMSO-d6): δ 21.3 (C-8), 52.6 (C-9′), 94.8 (C-8′), 109.6 (C-1′), 110.6 (C-3),
13


111.9 (C-1), 117.3 (C-5), 123.4 (C-3′), 137.2 (C-5′), 138.0 (C-6′), 148.1 (C-4′), 152.2
(C-6), 152.7 (C-2′), 160.2 (C-7), 163.5 (C-2), 163.9 (C-4), 165.8 (C-7′), 192.7 (C-9).
HR-ESIMS m/z: 387.0358 (calcd for C18H11O10: 387.0352 [M-H]-).


Chapter 3: Secondary metabolites from the cultured lichen mycobionts


3.1. Chemical investigation of the cultured mycobionts of Graphis vestitoides


Plant material
Specimens of Graphis vestitoides (Fink) Staiger were collected from the tree bark in
Tan Phu forest, Dong Nai Province, Vietnam (126 m alt.) in October 2008. The voucher
specimen was identified by Prof. H. Miyawaki, Saga University, Japan and deposited in
Saga University, Japan (registration No. LHD174). Mycobionts were obtained from the
spores discharged from apothecia of a thallus, and were cultivated in test tubes
containing modified MY10 medium at 18oC in the dark. After cultivation for three
months, the colonies were harvested.


Extraction and isolation
The harvested colonies (121 test tubes, dry weight 33.5 g) were extracted with Et2O,
acetone and then MeOH (3 × 100 ml) at rt, and the combined extracts were concentrated
under reduced pressure to give Et2O (122.1 mg), acetone (362.1 mg) and MeOH (8.15
g) residues, respectively. MeOH residue was dissolved in water and then separated by
n-BuOH to obtain n-BuOH extract (2.46 g).
Et2O and acetone extracts were separated by prep. TLC (n-hexane-Et2O-AcOH,
10:15:1). The visualized bands under UV light 254 nm of Et2O extract (48.9 mg) and
acetone extract (42.5 mg) were combined and repeatly purified by prep. TLC (n-hexane-




-103-
Et2O, 1:9; n-hexane-Et2O-AcOH, 40:60:1; CHCl3-MeOH, 9:1), giving 38 (29.3 mg) and
39 (41.4 mg).
n-BuOH extract was subjected to CC and eluted by solvent system CHCl3-MeOH
with increasing MeOH ratios to obtain four fractions, fr-I (97.6 mg), fr-II (658.5 mg,
1% MeOH), fr-III (125.0 mg, 1-2% MeOH) and fr-IV (82.3 mg, 10-50% MeOH). Fr-I
was determined as mixture of fatty compounds. Fr-II was separated by prep. TLC
(Et2O; CHCl3-MeOH, 8:2, 6:4), giving 35 (8.3 mg), 38 (173.3 mg), 39 (169.7 mg), 40
(2.3 mg) and 42 (57.0 mg). Fr-III was separated by prep. TLC (Et2O; CHCl3-MeOH, 9:1,
8:2) and prep. HPLC (µBondasphere C18 5µm, H2O-MeOH, 6:4; H2O-CH3CN, 9:1),
giving 36 (24.7 mg), 37 (7.7 mg), 39 (7.3 mg) and 41 (20.3 mg).


2-Acetyl-3,5-dihydroxybenzoic acid (35)
White solid (MeOH).
mp. 196oC (dec.).
UV λmax (EtOH) nm (log ε): 210.5 (4.29), 241 sh (3.86), 286.5 sh (3.38).
IR (KBr) νmax cm-1: 3481, 2558, 1701, 1602, 1558, 1488, 1383, 1359, 1289, 1255, 1174.
H-NMR (CD3OD): δ 2.38 (3H, s, -COCH3), 6.19 (1H, d, J=2.0 Hz, H-6), 6.32 (1H, d,
1


J=2.0 Hz, H-4).
C-NMR (CD3OD): δ 24.0 (-COCH3), 105.1 (C-4), 107.2 (C-6), 111.7 (C-2), 143.1 (C-
13


1), 162.5 (C-5), 163.7 (C-3), 175.3 (-COOH), 197.8 (-COCH3).
HR-APCIMS m/z: 195.0291 (calcd for C9H7O5: 195.0294 [M-H]-).


trans-5,7-Dihydroxy-3-(1-hydroxyethyl)phthalide (36)
Colorless plates.
mp. 212-213oC (CHCl3-MeOH).
[α]D16 -60o (c=0.93, MeOH).
UV λmax (EtOH) nm (log ε): 217.5 (4.44), 257.5 (4.08), 293 (3.71).
IR (KBr) νmax cm-1: 3304, 3127, 1713, 1616, 1482, 1357, 1317, 1223, 1166.
H-NMR (CD3OD): δ 1.16 (3H, d, J=6.5 Hz, H3-9), 3.98 (1H, qd, J=6.5, 5.0 Hz, H-8),
1


5.22 (1H, br d, J=5.0 Hz, H-3), 6.31 (1H, d, J=2.0 Hz, H-6), 6.47 (1H, dd, J=2.0, 1.0 Hz,
H-4).



-104-
C-NMR (CD3OD): δ 17.8 (C-9), 69.9 (C-8), 85.2 (C-3), 102.7 (C-4), 103.7 (C-6),
13


105.0 (C-7a), 152.8 (C-3a), 159.7 (C-7), 166.9 (C-5), 172.1 (C-1).
HR-ESIMS m/z: 211.0600 (calcd for C10H11O5: 211.0607 [M+H]+).


cis-5,7-Dihydroxy-3-(1-hydroxyethyl)phthalide (37)
Colorless solid.
[α]D17 +6.2o (c=0.55, MeOH).
UV λmax (EtOH) nm (log ε): 217.0 (4.39), 257.5 (4.02), 293 (3.65).
IR (KBr) νmax cm-1: 3188, 1720, 1616, 1479, 1356, 1291, 1216, 1167, 1065.
H-NMR (CD3OD): δ 1.23 (3H, d, J=6.5 Hz, H3-9), 4.13 (1H, qd, J=6.5, 3.5 Hz, H-8),
1


5.23 (1H, br d, J=3.5 Hz, H-3), 6.29 (1H, d, J=2.0 Hz, H-6), 6.45 (1H, dd, J=2.0, 1.0 Hz,
H-4).
C-NMR (CD3OD): δ 18.7 (C-9), 68.8 (C-8), 84.8 (C-3), 102.5 (C-4), 103.7 (C-6),
13


105.6 (C-7a), 153.1 (C-3a), 159.6 (C-7), 166.9 (C-5), 172.2 (C-1).
HR-ESIMS m/z: 211.0599 (calcd for C10H11O5: 211.0607 [M+H]+).


4,6-Dihydroxy-3,9-dehydromellein (38)
White crystalline solid.
mp. 171-172oC (CHCl3-MeOH).
[α]D16 +4.3o (c=1.03, MeOH).
UV λmax (EtOH) nm (log ε): 217.5 (4.28), 271.5 (4.04), 307 (3.87).
IR (KBr) νmax cm-1: 3202, 1664, 1628, 1498, 1475, 1367, 1269, 1188, 1173, 1126, 1017.
H-NMR (CD3OD): δ 4.91 (1H, dd, J=1.5, 0.5 Hz, H-9), 4.93 (1H, t, J=1.5 Hz, H-9),
1


5.21 (1H, br, H-4), 6.30 (1H, d, J=2.0 Hz, H-7), 6.54 (1H, dd, J=2.0, 0.5 Hz, H-5).
C-NMR (CD3OD): δ 66.5 (C-4), 97.0 (C-9), 99.3 (C-8a), 103.3 (C-7), 106.8 (C-5),
13


144.8 (C-4a), 157.5 (C-3), 165.3 (C-8), 167.1 (C-6), 167.3 (C-1).
HR-ESIMS m/z: 207.0292 (calcd for C10H7O5: 207.0294 [M-H]-).


6,8-Dihydroxy-3-(hydroxymethyl)isocoumarin (39)
Colorless needles.
mp. 244-245oC (MeOH).



-105-
UV λmax (EtOH) nm (log ε): 237 (4.44), 277.5 sh (3.79), 289.5 sh (3.67), 328 (3.77).
IR (KBr) νmax cm-1: 3237, 1678, 1620, 1579, 1488, 1388, 1249, 1180, 1155, 1103, 1072,
1028, 867, 716.
H-NMR (CD3OD): δ 4.34 (2H, s, H2-9), 6.32 (1H, d, J=2.0 Hz, H-7), 6.35 (1H, d,
1


J=2.0 Hz, H-5), 6.51 (1H, s, H-4).
C-NMR (CD3OD): δ 61.4 (C-9), 99.8 (C-8a), 103.1 (C-7), 104.5 (C-5), 104.9 (C-4),
13


140.8 (C-4a), 157.5 (C-3), 164.9 (C-8), 167.5 (C-1), 167.8 (C-6).
HR-ESIMS m/z: 207.0292 (calcd for C10H7O5: 207.0294 [M-H]-).


cis-4,6-Dihydroxymellein (40)
White solid.
[α]D18 -6.1o (c=0.18, MeOH).
UV λmax (EtOH) nm (log ε): 213.5 (4.00), 228 sh (3.74), 267.5 (3.70), 306.5 (3.54).
IR (KBr) νmax cm-1: 3386, 1655, 1632, 1626, 1507, 1383, 1248, 1170, 1118.
H-NMR (CD3OD): δ 1.47 (3H, d, J=6.6 Hz, H3-9), 4.44 (1H, d, J=2.4 Hz, H-4), 4.63
1


(1H, qd, J=6.6, 2.4 Hz, H-3), 6.28 (1H, d, J=2.4 Hz, H-7), 6.40 (1H, d, J=2.4 Hz, H-5).
HR-ESIMS m/z: 209.0448 (calcd for C10H9O5: 209.0294 [M-H]-).


New isocoumarin 6,8-dihydroxyisocoumarin-3-carboxylic acid (41)
White crystalline solid.
mp. >300oC (MeOH).
UV λmax (EtOH) nm (log ε): 251.5 (4.48), 289 (3.67), 300 (3.67), 324.5 (3.70), 336.5
(3.75).
IR (KBr) νmax cm-1: 3393, 3245, 1679, 1637, 1609, 1498, 1391, 1327, 1240, 1181, 801,
689.
H-NMR (DMSO-d6): δ 6.43 (1H, d, J=2.0 Hz, H-7), 6.59 (1H, d, J=2.0 Hz, H-5), 7.23
1


(1H, s, H-4); (CD3OD): δ 6.44 (1H, d, J=2.1 Hz, H-7), 6.53 (1H, d, J=2.1 Hz, H-5),
7.32 (1H, s, H-4).
C-NMR (DMSO-d6): δ 99.0 (C-8a), 102.6 (C-7), 104.4 (C-5), 108.5 (C-4), 138.8 (C-
13


4a), 150.0 (C-3), 161.8 (C-9), 162.5 (C-8), 165.5 (C-1), 165.8 (C-6).
HR-ESIMS m/z: 221.0085 (calcd for C10H5O6: 221.0086 [M-H]-).



-106-
Methylation of 41
To a solution of 41 (3.0 mg) in MeOH (1.0 ml) was added TMS-CHN2 in n-hexane
(0.2 ml), and the whole was stirred at rt for 70 mins. After quenching by diluted acetic
acid in MeOH, the reaction mixture was concentrated in vacuo and the residue was
purified by prep. TLC (CHCl3-MeOH, 95:5) to yield methylated compound 41a (1.8
mg).


Compound 41a
H-NMR (CDCl3): δ 3.93 (3H, s, -OCH3), 3.94 (3H, s, -OCH3), 3.99 (3H, s, -COOCH3),
1


6.56 (1H, d, J=2.1 Hz, H-7), 6.60 (1H, d, J=2.1 Hz, H-5), 7.31 (1H, s, H-4).
HR-ESIMS m/z: 265.0704 (calcd for C13H13O6: 265.0713 [M+H]+).


New 14-membered macrolide (42)
Colorless solid.
[α]D22 +121o (c=1.06, MeOH).
UV λmax (EtOH) nm (log ε): 215 sh (3.91).
IR (KBr) νmax cm-1: 3423, 2930, 1710, 1598, 1414, 1268.
1
H-NMR: Table 7.
13
C-NMR: Table 7.
HR-ESIMS m/z: 265.1084 (calcd for C14H17O5: 265.1077 [M-H]-).


Preparation of (R) and (S)-PGME amides of 42
To a stirred solution of a mixture of 42 (2.0 mg) and (R)-PGME (5.0 mg) in DMF
(1.0 ml) were successively added PyBOP (12.0 mg), HOBT (3.0 mg) and triethylamin
(10 µl) at 0oC. After the mixture was stirred at rt for 3 hrs, CHCl3 (15 ml) was added
and resulting diluted solution was successively washed with 1N HCl. The organic phase
was dried (Na2SO4) and concentrated to give a residue which was subjected to prep.
TLC (Et2O-AcOEt, 5:1) to afford the amide 42a (0.89 mg).
Compound 42 (2.0 mg) was treated with (S)-PGME (5.0 mg) as described above to
yield 42b (1.01 mg).




-107-
Compound 42a
H-NMR (CDCl3): δ 1.287 (1H, m, H-12a), 1.607 (1H, m, H-12b), 1.879 (2H, m, H2-
1


13), 1.955 (2H, m, H2-11), 2.814 (2H, m, H2-8), 3.729 (3H, s, -OCH3), 5.173 (1H, dd,
J=6.0, 4.5 Hz, H-14), 5.252 (1H, m, H-5), 5.380-5.460 (2H, m, H-7, H-10), 5.553 (1H,
d, J=7.0 Hz, PGME-CH), 5.546-5.610 (2H, m, H-6, H-9), 6.041 (1H, dd, J=15.5, 2.0 Hz,
H-3), 7.102 (1H, br d, J=7.0 Hz, PGME-NH), 7.240 (1H, dd, J=15.5, 4.0 Hz, H-4),
7.331-7.371 (5H, m, PGME-Ph).
NOESY: H-4/H-8, H-5/H-8, H-10/H-13, H-12a/H-14, H-14/PGME-NH.
HR-ESIMS m/z: 414.1908 (calcd for C23H28NO6: 414.1918 [M+H]+).


Compound 42b
H-NMR (CDCl3): δ 1.401 (1H, m, H-12a), 1.658 (1H, m, H-12b), 1.937 (2H, m, H2-
1


13), 1.992 (2H, m, H2-11), 2.822 (2H, m, H2-8), 3.744 (3H, s, -OCH3), 5.182 (1H, dd,
J=7.0, 3.0 Hz, H-14), 5.250 (1H, m, H-5), 5.393-5.472 (2H, m, H-7, H-10), 5.563 (1H,
d, J=7.0 Hz, -PGME-CH), 5.556-5.621 (2H, m, H-6, H-9), 6.042 (1H, dd, J=15.5, 2.0
Hz, H-3), 7.046 (1H, br d, J=7.0 Hz, PGME-NH), 7.247 (1H, dd, J=15.5, 4.0 Hz, H-4),
7.316-7.373 (5H, m, PGME- Ph).
NOESY: H-4/H-8, H-5/H-8, H-10/H-13, H-12a/H-14, H-14/PGME-NH.
HR-ESIMS m/z: 414.1906 (calcd for C23H28NO6: 414.1918 [M+H]+).


Methylation of 42
To a solution of 42 (6.5 mg) in MeOH (2.0 ml) was added TMS-CHN2 in n-hexane
(0.3 ml), and the whole was stirred at rt for 50 mins. After quenching by diluted acetic
acid in MeOH, the reaction mixture was concentrated in vacuo and the residue was
purified by prep. TLC (CHCl3-MeOH, 9:1) and prep. HPLC (5SLII-waters, n-hexane-2-
propanol, 8:2) to yield methylated compound 42m (4.2 mg).


Compound 42m
IR (KBr) νmax cm-1: 3446, 2365, 1734, 1636, 1388.
1
H-NMR: Table 7.




-108-
13
C-NMR: Table 7.
HR-APCIMS m/z: 281.1381 (calcd for C15H21O5: 281.1390 [M+H]+).


3.2. Chemical investigation of the cultured mycobionts of Sarcographa tricosa


Plant material
Specimens of Sarcographa tricosa (Ach.) Müll. Arg. were collected from tree bark
in Ma Da forest, Dong Nai Province, Vietnam (94 m alt.) in September 2008. The
voucher specimen was identified by Prof. H. Miyawaki, Saga University, Japan and was
deposited in Saga University, Japan (registration No. LHD068). Mycobionts were
obtained from the spores discharged from apothecia of a thallus, and were cultivated in
test tubes containing modified MY10 medium at 18oC in the dark. After cultivation for
4-8 months, the colonies were harvested.


Extraction and isolation
The harvested colonies (285 test tubes, dry weight 185.5 g) were extracted with n-
hexane (2 × 800 ml) and Et2O (3 × 400 ml) at rt, and the combined extracts were
concentrated under reduced pressure to give n-hexane (0.23 g) and Et2O (0.92 g)
extracts, respectively. The n-hexane extract was separated by prep. TLC (Et2O-AcOEt,
10:1; toluene-AcOEt, 3:2, 1:1 or 1:2) and by prep. HPLC (Sunfire, n-hexane-2-propanol,
9:1), giving 45 (30.3 mg), 46 (9.3 mg), 47 (12.6 mg), 48 (49.3 mg), 52 (1.2 mg) and
fatty compounds (58.2 mg).
The Et2O extract was subjected to CC and eluted by solvent system n-hexane-Et2O
with increasing Et2O ratios to obtain four fractions, fr-I (0-10% Et2O, 170 mg), fr-II
(20-30% Et2O, 135 mg), fr-III (50-70% Et2O, 284 mg) and fr-IV (70-100% Et2O, 245
mg). Fr-I was mixture of fatty compounds (168.9 mg). Fr-II was purified by prep. TLC
(toluene-AcOEt, 3:2, 1:1), giving 45 (10.5 mg) and 52 (2.1 mg). Fr-III was separated by
prep. TLC (toluene-AcOEt, 1:1, 1:2) to yield 47 (119.8 mg), 48 (83.7 mg) and 49 (8.0
mg). Purification of fr-IV by prep. TLC (n-hexane-AcOEt, 1:4; toluene-AcOEt-AcOH,
5:5:0.2) afforded 50 (2.3 mg), 51 (4.9 mg), 53 (6.9 mg) and 54 (2.6 mg).




-109-
Ergosterol peroxide (5α,8α-epidioxy-24-norcholesta-6,22-dien-3β -ol) (45)
Colorless crystalline solid.
mp. 174-175oC (CHCl3).
[α]D22 -34o (c=1.07, CHCl3).
IR (KBr) νmax cm-1: 3376, 2956, 2872, 1458, 1378, 1044, 967.
H-NMR (CDCl3): δ 0.82 (3H, s, H3-19), 0.82 (3H, d, J=6.5 Hz, H3-26), 0.83 (3H, d,
1


J=7.0 Hz, H3-27), 0.88 (3H, s, H3-18), 0.91 (3H, d, J=7.0 Hz, H3-28), 1.00 (3H, d, J=6.5
Hz, H3-21), 1.23 (2H, m, H-11, H-17), 1.25 (1H, m, H-12), 1.36 (1H, m, H-16), 1.40
(1H, m, H-15), 1.49 (1H, m, H-25), 1.51 (1H, m, H-9), 1.52 (1H, m, H-11), 1.55 (1H, m,
H-2), 1.58 (1H, m, H-14), 1.60 (1H, m, H-15), 1.70 (1H, dt, J=13.5, 3.5 Hz, H-1eq),
1.84 (1H, m, H-2), 1.85 (2H, m, H-16, H-24), 1.93 (1H, m, H-4ax), 1.95 (2H, m, H-1ax,
H-12), 2.02 (1H, m, H-20), 2.10 (1H, ddd, J=13.5, 5.0, 2.0 Hz, H-4eq), 3.97 (1H, tt,
J=12.0, 5.0 Hz, H-3ax), 5.14 (1H, dd, J=15.5, 7.5 Hz, H-22), 5.22 (1H, dd, J=15.5, 8.0
Hz, H-23), 6.24 (1H, d, J=8.5 Hz, H-6), 6.50 (1H, d, J=8.5 Hz, H-7).
C-NMR (CDCl3): δ 12.9 (C-19), 17.5 (C-28), 18.2 (C-18), 19.6 (C-26), 19.9 (C-27),
13


20.6 (C-15), 20.9 (C-21), 23.4 (C-11), 28.6 (C-16), 30.1 (C-2), 33.1 (C-25), 34.7 (C-1),
36.92 (C-4), 36.95 (C-10), 39.3 (C-12), 39.7 (C-20), 42.8 (C-24), 44.6 (C-13), 51.1 (C-
9), 51.7 (C-14), 56.2 (C-17), 66.5 (C-3), 79.4 (C-8), 82.1 (C-5), 130.7 (C-7), 132.3 (C-
23), 135.2 (C-22), 135.4 (C-6).
HR-ESIMS m/z: 429.3369 (calcd for C28H45O3: 429.3371 [M+H]+).


Sporogen-AO 1 (46)
Colorless oil.
[α]D22 +221o (c=0.80, MeOH).
UV λmax (EtOH) nm (log ε): 242.5 (4.03).
IR (KBr) νmax cm-1: 3434, 2942, 1672, 1455, 1113, 1028, 881.
H-NMR (CDCl3): δ 1.23 (3H, s, H3-14), 1.27 (3H, d, J=7.0 Hz, H3-15), 1.45 (1H, m,
1


H-2ax), 1.82 (1H, dq, J=11.0, 7.0 Hz, H-4), 1.87 (3H, t, J=1.5 Hz, H3-13), 2.16 (1H, dtd,
J=12.5, 4.5, 2.5 Hz, H-2eq), 2.34 (1H, ddd, J=14.5, 4.5, 2.5 Hz, H-1eq), 2.52 (1H, tdd,
J=14.5, 4.5, 2.0 Hz, H-1ax), 3.33 (1H, s, H-6), 3.62 (1H, td, J=11.0, 4.5 Hz, H-3), 5.11




-110-
(1H, quint, J=1.5 Hz, H-12), 5.12 (1H, quint, J=1.5 Hz, H-12), 5.77 (1H, d, J=2.0 Hz,
H-9).
C-NMR (CDCl3): δ 11.3 (C-15), 18.8 (C-14), 19.8 (C-13), 30.9 (C-1), 35.2 (C-2), 41.0
13


(C-5), 44.4 (C-4), 63.5 (C-7), 68.3 (C-6), 71.0 (C-3), 114.5 (C-12), 121.2 (C-9), 139.1
(C-11), 162.9 (C-10), 192.8 (C-8).
HR-ESIMS m/z: 249.1487 (calcd for C15H21O3: 249.1492 [M+H]+).


Dihydrosporogen-AO 1 (47)
Colorless needles.
mp. 124-125oC (n-hexane-CH2Cl2).
[α]D24 +104o (c=0.84, MeOH).
IR (KBr) νmax cm-1: 3348, 2968, 2935, 2872, 1675, 1648, 1455, 1380, 1264, 1138, 1037,
958, 917, 882, 841.
H-NMR (CDCl3): δ 1.01 (3H, s, H3-14), 1.16 (3H, d, J=6.5 Hz, H3-15), 1.33 (1H, dddd,
1


J=14.0, 11.5, 10.5, 5.0 Hz, H-2ax), 1.62 (1H, dq, J=10.5, 6.5 Hz, H-4), 1.87 (3H, br s,
H3-13), 2.04 (1H, dtd, J=11.5, 5.0, 2.5 Hz, H-2eq), 2.17 (1H, ddd, J=14.0, 5.0, 2.5 Hz,
H-1eq), 2.29 (1H, tddd, J=14.0, 5.0, 3.0, 2.0 Hz, H-1ax), 3.08 (1H, s, H-6), 3.52 (1H, td,
J=10.5, 5.0 Hz, H-3), 4.52 (1H, t, J=3.0 Hz, H-8), 5.06 (1H, quint, J=1.5 Hz, H-12),
5.17 (1H, br s, H-12), 5.25 (1H, br t, J=2.0 Hz, H-9).
13
C-NMR: Table 8.
HR-ESIMS m/z: 273.1463 (calcd for C15H22O3Na: 273.1468 [M+Na]+).


Preparation of (R)- and (S)-MPA esters of 47
To a solution of 47 (5.5 mg) in dry CH2Cl2 (1 ml) was added (R)-MPA (36.5 mg),
EDC (57.6 mg) and a few crystals of 4-pyrrolidinopyridine, and the whole was stirred at
rt for 15 hrs. The reaction mixture was poured into 1 N HCl and extracted with CHCl3.
The CHCl3 layer was dried over MgSO4 and concentrated in vacuo. The residue was
purified by prep. TLC (n-hexane-Et2O-AcOH, 3:7:0.5) to yield 47a (10.7 mg).
Treatment of 47 (5.8 mg) with (S)-MPA (38.5 mg) followed by prep. TLC gave 47b
(11.7 mg).




-111-
Compound 47a
H-NMR (CDCl3): δ 0.530 (3H, d, J=6.9 Hz, H3-15), 0.966 (3H, s, H3-14), 1.360 (1H,
1


m, H-2ax), 1.782 (3H, br s, H3-13), 1.816 (1H, dq, J=11.1, 6.6 Hz, H-4), 2.005 (1H, m,
H-2eq), 2.086 (1H, br dt, J=14.4, 4.2 Hz, H-1eq), 2.247 (1H, br tt, J=14.4, 4.2 Hz, H-
1ax), 2.862 (1H, s, H-6), 3.392, 3.398 (each 3H, s, 3-MPA-OCH3 and 8-MPA-OCH3),
4.721, 4.801 (each 1H, s, 3-MPA-CH and 8-MPA-CH), 4.757 (1H, td, J=11.1, 4.5 Hz,
H-3), 4.848 (1H, t, J=2.1 Hz, H-9), 4.899 (1H, quint, J=1.5 Hz, H-12), 5.056 (1H, br s,
H-12), 5.953 (1H, t, J=2.1 Hz, H-8), 7.280-7.481 (10H, m, 3-MPA-Ph and 8-MPA-Ph).
HR-ESIMS m/z: 569.2510 (calcd for C33H38O7Na: 569.2517 [M+Na]+).


Compound 47b
H-NMR (CDCl3): δ 0.962 (3H, d, J=6.6 Hz, H3-15), 1.013 (3H, s, H3-14), 1.192 (1H,
1


m, H-2ax), 1.460 (3H, br s, H3-13), 1.822 (1H, m, H-2eq), 1.940 (1H, dq, J=10.5, 6.6
Hz, H-4), 2.109 (1H, br d, J=14.4 Hz, H-1eq), 2.255 (1H, br t, J=14.4 Hz, H-1ax), 2.894
(1H, s, H-6), 3.401, 3.415 (each 3H, s, 3-MPA-OCH3 and 8-MPA-OCH3), 4.552, 4.677
(each 1H, br s, H2-12), 4.751, 4.816 (each 1H, s, 3-MPA-CH and 8-MPA-CH), 4.805
(1H, td, J=10.5, 4.5 Hz, H-3), 5.156 (1H, br s, H-9), 5.830 (1H, br t, J=2.4 Hz, H-8),
7.285-7.440 (10H, m, 3-MPA-Ph and 8-MPA-Ph).
HR-ESIMS m/z: 569.2512 (calcd for C33H38O7Na: 569.2517 [M+Na]+).


Petasol (48)
Colorless oil.
[α]D20 +130o (c=0.82, CHCl3).
UV λmax (EtOH) nm (log ε): 236.5 (4.12).
IR (KBr) νmax cm-1: 3433, 2971, 2942, 1669, 1625, 1444, 1374, 1330, 1252, 1216, 1195,
1070, 1038, 898.
1
H-NMR: Table 9.
13
C-NMR: Table 8.
HR-ESIMS m/z: 235.1695 (calcd for C15H23O2: 235.1699 [M+H]+).




-112-
Preparation of (R)- and (S)-MPA esters of 48
Compound 48 (6.9 mg) was treated with (R)-MPA (24.5 mg) as described above and
the crude product was purified by prep. TLC (CHCl3-AcOEt, 9:1) to yield 48a ester (6.4
mg). Treatment of 48 (8.4 mg) with (S)-MPA (29.8 mg) followed by prep. TLC gave
48b (8.6 mg).


Compound 48a
H-NMR (CDCl3): δ 0.504 (3H, d, J=6.9 Hz, H3-15), 1.149 (3H, s, H3-14), 1.478 (1H,
1


m, H-2ax), 1.490 (1H, m, H-4), 1.712 (3H, br s, H3-13), 1.809 (1H, t, J=13.5 Hz, H-6ax),
1.930 (1H, dd, J=13.5, 4.8 Hz, H-6eq), 2.194 (1H, dtd, J=12.3, 4.5, 2.4 Hz, H-2eq),
2.335 (1H, ddd, J=15.3, 4.2, 2.4 Hz, H-1eq), 2.484 (1H, tdd, J=15.3, 4.5, 1.5 Hz, H-1ax),
3.068 (1H, dd, J=13.5, 4.8 Hz, H-7), 3.410 (3H, s, MPA-OCH3), 4.744 (1H, s, MPA-
CH), 4.787 (1H, d, J=1.0 Hz, H-12), 4.859 (1H, td, J=10.8, 4.5 Hz, H-3), 4.966 (1H,
quint, J=1.5 Hz, H-12), 5.764 (1H, d, J=1.5 Hz, H-9), 7.329-7.454 (5H, m, MPA-Ph).
HR-ESIMS m/z: 383.2220 (calcd for C24H31O4: 383.2224 [M+H]+).


Compound 48b
H-NMR (CDCl3): δ 0.895 (3H, d, J=6.9 Hz, H3-15), 1.199 (3H, s, H3-14), 1.306 (1H,
1


m, H-2ax), 1.601 (1H, dq, J=10.8, 4.5 Hz, H-4), 1.727 (3H, br s, H3-13), 1.866 (1H, t,
J=14.1 Hz, H-6ax), 1.978 (1H, m, H-2eq), 2.022 (1H, dd, J=14.1, 4.8 Hz, H-6eq), 2.271
(1H, ddd, J=15.0, 4.5, 2.7 Hz, H-1eq), 2.436 (1H, tdd, J=15.0, 4.8, 1.5 Hz, H-1ax),
3.093 (1H, dd, J=14.1, 4.8 Hz, H-7), 3.425 (3H, s, MPA-OCH3), 4.773 (1H, s, MPA-
CH), 4.811 (1H, br s, H-12), 4.914 (1H, td, J=10.8, 4.5 Hz, H-3), 4.984 (1H, quint,
J=1.0 Hz, H-12), 5.741 (1H, d, J=1.5 Hz, H-9), 7.198-7.448 (5H, m, MPA-Ph).
HR-ESIMS m/z: 383.2219 (calcd for C24H31O4: 383.2224 [M+H]+).


Isopetasol (49)
Colorless crystalline solid.
mp. 120-121oC (n-hexane-CHCl3).
[α]D20 +106o (c=0.25, CHCl3).
UV λmax (EtOH) nm (log ε): 246 (4.00), 278 sh (3.76).




-113-
IR (KBr) νmax cm-1: 3504, 2937, 1655, 1619, 1297, 1222, 1034.
H-NMR (CDCl3): δ 0.99 (3H, s, H3-14), 1.12 (3H, d, J=6.6 Hz, H3-15), 1.34-1.52 (2H,
1


m, H-2ax & H-4), 1.85 (3H, d, J=1.2 Hz, H3-13), 2.10 (3H, d, J=2.1 Hz, H3-12), 2.10-
2.19 (2H, m, H-2eq & H-6), 2.34 (1H, m, H-1), 2.41 (1H, m, H-1), 2.91 (1H, d, J=13.8
Hz, H-6), 3.59 (1H, td, J=10.5, 4.5 Hz, H-3), 5.77 (1H, d, J=1.8 Hz, H-9).
C-NMR (CDCl3): δ 10.8 (C-15), 17.3 (C-14), 22.1 (C-12), 22.6 (C-13), 30.5 (C-1),
13


35.3 (C-2), 41.3 (C-6), 42.0 (C-5), 49.2 (C-4), 71.4 (C-3), 126.6 (C-9), 127.3 (C-7),
143.3 (C-11), 166.1 (C-10), 191.8 (C-8).
HR-ESIMS m/z: 235.1692 (calcd for C15H23O2: 235.1699 [M+H]+).


JBIR-27 (50)
Colorless solid.
[α]D22 +166o (c=0.13, MeOH).
UV λmax (EtOH) nm (log ε): 239.5 (4.00).
IR (KBr) νmax cm-1: 3398, 2939, 1656, 1445, 1253, 1056.
H-NMR (CDCl3): δ 1.15 (3H, d, J=6.5 Hz, H3-15), 1.43 (1H, dq, J=11.0, 6.5 Hz, H-4),
1


1.49 (1H, m, H-2ax), 1.72 (3H, br s, H3-13), 1.94 (1H, t, J=14.0 Hz, H-6ax), 2.20 (1H,
dtd, J=12.0, 4.5, 2.5 Hz, H-2eq), 2.32 (1H, dd, J=13.5, 5.0 Hz, H-6eq), 2.43 (1H, ddd,
J=14.5, 4.5, 2.5 Hz, H-1eq), 2.52 (1H, tdd, J=14.5, 4.5, 1.5 Hz, H-1ax), 3.51 (1H, dd,
J=14.0, 5.0 Hz, H-7), 3.75 (1H, td, J=11.0, 4.5 Hz, H-3), 3.94 (1H, d, J=11.0 Hz, H-14),
3.97 (1H, d, J=11.0 Hz, H-14), 4.83 (1H, br s, H-12), 4.98 (1H, quint, J=1.5 Hz, H-12),
6.00 (1H, d, J=1.5 Hz, H-9).
C-NMR (CDCl3): δ 10.8 (C-15), 19.7 (C-13), 31.8 (C-1), 35.1 (C-2), 40.1 (C-6), 44.9
13


(C-5), 50.4 (C-4), 51.5 (C-7), 65.9 (C-14), 71.4 (C-3), 114.6 (C-12), 127.4 (C-9), 143.6
(C-11), 163.6 (C-10), 199.1 (C-8).
NOESY: H-1eq/H-9, H-1ax/H2-14, H-3/H2-14, H-6eq/H2-14, H-6eq/H3-15, H-7/H2-14.
HR-ESIMS m/z: 251.1643 (calcd for C15H23O3: 251.1648 [M+H]+).


1β-Hydroxypetasol (51)
Colorless oil.
[α]D23 +48o (c=0.42, MeOH).



-114-
UV λmax (EtOH) nm (log ε): 233 (3.98).
IR (KBr) νmax cm-1: 3401, 2946, 1665, 1028.
H-NMR (CDCl3): δ 1.11 (3H, d, J=7.0 Hz, H3-15), 1.35 (1H, m, H-4), 1.38 (3H, s, H3-
1


14), 1.65 (1H, ddd, J=14.5, 11.0, 3.0 Hz, H-2ax), 1.73 (3H, dd, J=1.0, 0.5 Hz, H3-13),
1.92 (1H, t, J=14.0 Hz, H-6ax), 2.03 (1H, dd, J=13.0, 5.0 Hz, H-6eq), 2.35 (1H, ddd,
J=14.5, 4.5, 3.0 Hz, H-2eq), 3.22 (1H, dd, J=14.0, 5.0 Hz, H-7), 4.03 (1H, td, J=11.0,
4.5 Hz, H-3), 4.48 (1H, t, J=3.0 Hz, H-1), 4.84 (1H, br t, J=1.0 Hz, H-12), 5.00 (1H,
quint, J=1.0 Hz, H-12), 5.87 (1H, s, H-9).
C-NMR (CDCl3): δ 10.4 (C-15), 19.5 (C-14), 20.0 (C-13), 39.2 (C-5), 41.6 (C-2), 43.1
13


(C-6), 50.0 (C-4), 50.8 (C-7), 67.2 (C-3), 73.7 (C-1), 114.6 (C-12), 126.8 (C-9), 143.1
(C-11), 165.4 (C-10), 199.5 (C-8).
NOESY: H-1eq/H-9, H-3/H3-14, H-6eq/H3-14, H-6eq/H3-15, H-7/H3-14.
HR-ESIMS m/z: 251.1643 (calcd for C15H23O3: 251.1648 [M+H]+).


New eremophilane 3-epi-petasol (52)
Colorless needles.
mp. 130-131oC (n-hexane-CHCl3).
[α]D23 +114o (c=0.17, CHCl3).
UV λmax (EtOH) nm (log ε): 240 (4.02).
IR (KBr) νmax cm-1: 3479, 2937, 1657, 1444, 1250, 1208, 949, 900.
1
H-NMR: Table 9.
13
C-NMR: Table 8.
HR-ESIMS m/z: 235.1691 (calcd for C15H23O2: 235.1699 [M+H]+).


New eremophilane dihydropetasol (53)
Colorless crystalline solid.
mp. 138-140oC (n-hexane-CHCl3).
[α]D21 +208o (c=0.38, CHCl3).
IR (KBr) νmax cm-1: 3341, 2934, 1450, 1022, 884.
1
H-NMR: Table 9.
13
C-NMR: Table 8.



-115-
HR-ESIMS m/z: 259.1669 (calcd for C15H24O2Na: 259.1675 [M+Na]+).


Reduction of 48
To a solution of 48 (7.5 mg) in MeOH (0.4 ml) was added CeCl3•7H2O (12.0 mg)
and then NaBH4 (1.2 mg), and the mixture was stirred at rt for 1 hr. The reaction
mixture was diluted with H2O and extracted with CHCl3 (3 × 10 ml). The CHCl3 layer
was dried over Na2SO4 and concentrated in vacou. The crude product was subjected to
prep. TLC (n-hexane-Et2O-AcOEt, 1:1:0.3) to give 53 (0.2 mg), 58 (2.8 mg) and 48 (1.3
mg). Compound 53 was identified with the isolated compound from the culture (1H-
NMR, [α]D).


8-Epi-dihydropetasol (58)
Colorless crystalline solid.
mp. 126-127oC (CHCl3).
[α]D26 -36o (c=0.26, CHCl3).
H-NMR (CDCl3): δ 1.00 (3H, d, J=6.5 Hz, H3-15), 1.02 (3H, d, J=1.0 Hz, H3-14), 1.14
1


(1H, dq, J=10.5, 6.5 Hz, H-4), 1.30 (1H, m, H-2ax), 1.32 (1H, br t, J=13.0 Hz, H-6ax),
1.68 (1H, dd, J=13.0, 3.0 Hz, H-6eq), 1.74 (3H, dd, J=1.5, 1.0 Hz, H3-13), 2.08 (1H, dtd,
J=12.0, 4.5, 2.0 Hz, H-2eq), 2.14 (1H, ddd, J=14.0, 5.0, 2.5 Hz, H-1eq), 2.19 (1H, ddd,
J=13.0, 9.5, 3.0 Hz, H-7), 2.26 (1H, tdt, J=14.0, 4.5, 1.5, H-1ax), 3.53 (1H, td, J=10.5,
4.5 Hz, H-3), 4.08 (1H, br d, J=9.5 Hz, H-8), 4.89 (1H, br s, H-12), 4.92 (1H, quint,
J=1.5 Hz, H-12), 5.39 (1H, t, J=1.5 Hz, H-9).
13
C-NMR: Table 8.
NOESY: H-1ax/H3-14, H-1eq/H-9, H-3/H3-14, H-4/H-6ax, H-6eq/H3-15, H-7/H3-14.
HMBC: H2-1→C-2, 5, 9, 10; H2-2→C-3; H-4→C-3, 5, 15; H2-6→C-4, 5, 7, 8, 11, 14;
H-7→C-8; H-9→C-1, 5, 7; H2-12→C-7, 11, 13; H3-13→C-7, 11, 12; H3-14→C-4, 5, 6,
10; H3-15→C-3, 4, 5.
HR-ESIMS m/z: 259.16625 (calcd for C15H24O2Na: 259.16751 [M+Na]+).


New eremophilane sarcographol (54)
Colorless crystalline solid.




-116-
mp. 224-225oC (n-hexane-CHCl3).
[α]D23 +102o (c=0.11, CHCl3).
IR (KBr) νmax cm-1: 3441, 3375, 2953, 2878, 1659, 1443, 1378, 1262, 1120, 1036, 1012,
918, 881.
1
H-NMR: Table 9.
13
C-NMR: Table 8.
HR-ESIMS m/z: 253.1800 (calcd for C15H25O3: 253.1805 [M+H]+).


3.3. Chemical investigation of the cultured mycobionts of Pyrenula sp.


Plant material
Specimens of Pyrenula sp. were collected from the tree bark in BiDoup-Nui Ba,
Dalat City, Vietnam (ca. 1,500 m alt.) in Nov. 2008. The voucher specimen was
determined by Prof. H. Miyawaki (Saga University, Japan) and deposited at Saga
University, Japan (registration No. LHD210). Mycobionts were obtained from the
spores discharged from apothecia of a thallus, and were cultivated in test tubes
containing modified MY10 medium at 18oC in the dark. After cultivation for three
months, the colonies were harvested.


Extraction and isolation
The harvested colonies (111 test tubes, dry weight 31.7 g) were extracted with Et2O,
acetone and then MeOH (3 × 100 ml) at rt, and the combined extracts were concentrated
under reduced pressure to give Et2O (2.64 g), acetone (1.23 g) and MeOH (5.76 g)
residues, respectively. MeOH residue was dissolved in water and then extracted with n-
BuOH to obtain n-BuOH extract (2.70 g). Et2O, acetone and n-BuOH extracts were
subjected to CC and eluted by solvent system CHCl3-MeOH with increasing MeOH
ratios. Et2O extract gave six fractions, E1 (132.0 mg), E2 (201.7 mg), E3 (183.0 mg),
E4 (1.91 g, 1% MeOH), E5 (202.6 mg, 2-5% MeOH), E6 (48.5 mg, 10-50% MeOH).
From acetone extract, six fractions, A1 (67.8 mg), A2 (99.6 mg), A3 (44.6 mg), A4
(760.5 mg, 1% MeOH), A5 (109.2 mg, 2-5% MeOH), A6 (149.2 mg, 10-50% MeOH)
were obtained. n-BuOH extract led us to collect five fractions, B1 (196.3 mg), B2




-117-
(320.4 mg), B3 (1.28 g, 1% MeOH), B4 (204.7 mg, 2-5% MeOH), B5 (412.0 mg, 10-
50% MeOH).
From TLC analysis, these fractions were combined to obtain six fractions, a (396.1
mg, E1, A1, B1), b (621.7 mg, E2, A2, B2), c (227.6 mg, E3, A3), d (3.95 g, E4, A4,
B3), e (516.5 mg, E5, A5, B4) and f (609.7 mg, E6, A6, B5).
Fraction a was separated by prep. TLC (n-hexane-Et2O, 8:2, 1:1; n-hexane-Et2O-
AcOH, 75:75:1), giving 60 (3.9 mg) and fatty compounds (214.3 mg).
Fraction b was separated by prep. TLC (n-hexane-Et2O, 1:1; n-hexane-Et2O-AcOH,
75:75:1, 75:75:2; CHCl3-MeOH, 95:5; CHCl3-MeOH-AcOH, 95:5:0.5, 9:1:0.1) and
prep. HPLC (Waters 5SL-II 20 × 250 mm, n-hexane-AcOEt, 7:3), giving 61 (4.0 mg),
62 (2.2 mg), 63 (38.1 mg), 64 (10.2 mg) and fatty compounds (194.0 mg).
Fraction c was separated by prep. TLC (CHCl3-MeOH-AcOH, 95:5:0.5, 9:1:0.1)
and prep. HPLC (Waters 5SL-II 20 × 250 mm, n-hexane-Et2O, 6:4), giving 69 (32.7
mg).
Fraction d was subjected to CC and eluted by solvent system CHCl3-MeOH with
increasing MeOH ratios to give two sub-fractions. Compound 65 (2.65 g) was obtained
from the sub-fraction eluted with 1-2% MeOH. The other sub-fraction, eluted with 0-
1% MeOH, was subjected to prep. TLC (n-hexane-Et2O-AcOH, 5:5:0.5, 4:6:0.5), giving
65 (721.0 mg) and 66 (205.9 mg).
Fraction e was separated by prep. TLC (n-hexane-Et2O-AcOH, 4:6:0.5, 3:7:0.5) and
prep. HPLC (Waters 5SL-II 20 × 250 mm, n-hexane- Et2O, 7:3), giving 65 (15.4 mg),
67 (3.5 mg), 68 (95.3 mg) and 70 (30.4 mg).


Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) (60)
Orange prism.
mp. 184-185oC (CHCl3-MeOH).
UV λmax (EtOH) nm (log ε): 225.5 (4.22), 255.5 (3.94), 277.5 sh (3.66), 287.5 (3.68),
430.5 (3.64).
IR (KBr) νmax cm-1: 3431, 1677, 1629, 1606, 1475, 1454, 1374, 1271, 1208.




-118-
H-NMR (CDCl3): δ 2.47 (3H, s, 3-CH3), 7.11 (1H, d, J=1.0 Hz, H-2), 7.30 (1H, dd,
1


J=8.5, 1.0 Hz, H-7), 7.66 (1H, d, J=1.0 Hz, H-4), 7.68 (1H, dd, J=8.5, 8.0 Hz, H-6),
7.83 (1H, dd, J=8.0, 1.0 Hz, H-5), 12.02 (1H, s, 1-OH), 12.13 (1H, s, 8-OH).
C-NMR (CDCl3): δ 22.3 (3-CH3), 113.8 (C-11), 115.9 (C-14), 120.0 (C-5), 121.4 (C-
13


4), 124.4 (C-2), 124.6 (C-7), 133.3 (C-13), 133.7 (C-12), 137.0 (C-6), 149.4 (C-3),
162.5 (C-8), 162.8 (C-1), 182.1 (C-10), 192.6 (C-9).
HR-ESIMS m/z: 253.0515 (calcd for C15H9O4: 253.0501 [M-H]-).


Emodin (1,6,8-trihydroxy-3-methylanthraquinone) (61)
Orange crystal.
mp. 251-252oC (acetone).
UV λmax (EtOH) nm (log ε): 220.5 (4.00), 253.5 (3.77), 291 (3.81), 438 (3.48).
IR (KBr) νmax cm-1: 3421, 2926, 1629, 1270.
H-NMR (acetone-d6): δ 2.48 (3H, s, 3-CH3), 6.68 (1H, d, J=2.5 Hz, H-7), 7.16 (1H, d,
1


J=1.0 Hz, H-2), 7.28 (1H, d, J=2.5 Hz, H-5), 7.59 (1H, d, J=1.0 Hz, H-4), 12.08 (1H, s,
1-OH), 12.20 (1H, s, 8-OH).
C-NMR (acetone-d6): δ 22.0 (3-CH3), 108.9 (C-7), 109.7 (C-5), 110.5 (C-14), 114.5
13


(C-11), 121.5 (C-4), 125.0 (C-2), 134.3 (C-13), 135.7 (C-12), 149.6 (C-3), 158.8 (C-6),
163.3 (C-1), 166.3 (C-8), 182.3 (C-10), 191.8 (C-9).
HR-ESIMS m/z: 269.0459 (calcd for C15H9O5: 269.0450 [M-H]-).


1,5,8-Trihydroxy-3-methylxanthone (62)
Yellow needles.
mp. 267-268oC (acetone).
UV λmax (EtOH) nm (log ε): 236.5 sh (4.07), 256 (4.22), 273.5 sh (4.07), 342.5 (3.75),
405 sh (3.28).
IR (KBr) νmax cm-1: 3447, 1660, 1635, 1609, 1591, 1504, 1290, 1246, 1209.
H-NMR (acetone-d6): δ 2.46 (3H, s, 3-CH3), 6.67 (1H, br s, H-2), 6.68 (1H, d, J=7.5
1


Hz, H-7), 6.87 (1H, br s, H-4), 7.35 (1H, d, J=7.5 Hz, H-6), 8.53 (1H, s, 5-OH), 11.09
(1H, s, 8-OH), 11.74 (1H, s, 1-OH).




-119-
C-NMR (acetone-d6): δ 22.5 (3-CH3), 106.6 (C-9a), 108.5 (C-4), 108.8 (C-8a), 110.4
13


(C-7), 112.3 (C-2), 125.2 (C-6), 138.2 (C-5), 144.7 (C-4b), 151.3 (C-3), 154.2 (C-8),
157.1 (C-4a), 162.0 (C-1), 187.0 (C-9).
HR-ESIMS m/z: 257.0455 (calcd for C14H9O5: 257.0450 [M-H]-).


New polyketide pyrenulic acid A (63)
Colorless solid.
[α]D22 -82o (c=0.85, CHCl3).
UV λmax (EtOH) nm (log ε): 300 (4.29).
IR (KBr) νmax cm-1: 3431, 2963, 2927, 1687, 1636, 1615, 1434, 1380, 1303, 1270.
1
H-NMR: Table 10.
13
C-NMR: Table 11.
HR-ESIMS m/z: 395.2601 (calcd for C26H35O3: 395.2588 [M-H]-).


New polyketide pyrenulic acid B (64)
Colorless solid.
[α]D21 -225o (c=0.95, CHCl3).
UV λmax (EtOH) nm (log ε): 300 (4.21).
IR (KBr) νmax cm-1: 3423, 2961, 2926, 1688, 1614, 1378, 1272.
1
H-NMR: Table 10.
13
C-NMR: Table 11.
NOESY: H-2/H-4, H-3/H-5, H-6/H-8, H-17/H-19.
HR-ESIMS m/z: 379.2651 (calcd for C26H35O2: 379.2639 [M-H]-).


New polyketide pyrenulic acid C (65)
Colorless solid.
[α]D22 -10.7o (c=0.92, CHCl3).
UV λmax (EtOH) nm (log ε): 254 (4.36).
IR (KBr) νmax cm-1: 3446, 2923, 1692, 1641, 1614, 1434, 1383, 1246.
1
H-NMR: Table 10.
13
C-NMR: Table 12.



-120-
NOESY: H-2/H-4, H-3/H-5, H-6/H-10eq, H-7/H-17, H-7/H-19, H-9/H3-24, H-17/H-19,
H-22ax/H3-24, H3-23/H3-24.
HR-ESIMS m/z: 443.2445 (calcd for C26H35O6: 443.2435 [M-H]-).


Methylation and acetylation of 65
To a solution of 65 (60.2 mg) in MeOH (2.0 ml) was added an excess of TMS-
CHN2 in n-hexane, and the whole was stirred at rt for 45 mins. After quenching by
diluted acetic acid in MeOH, the reaction mixture was concentrated in vacuo and the
residue was purified by prep. TLC (n-hexane-acetone, 3:2) to yield methyl ester of 65
(50.3 mg). To a stirred mixture of the methyl ester (7.4 mg) and isopropenyl acetate
(0.12 ml) at 85-90oC was added iodine (2.1 mg) with continued stirring at the same
temperature for 5 mins.113) After completion of the reaction, the mixture was cooled to rt,
then added 5% aq. solution of Na2S2O3 (4 ml) and extracted with CHCl3. The CHCl3
layer was washed with a saturated solution of NaHCO3 (6 ml), dried over MgSO4 and
concentrated in vacuo. The residue was purified by prep. TLC (CHCl3-AcOEt, 9:1) to
yield 65a (4.3 mg).


Compound 65a
H-NMR (CDCl3): δ 1.05 (3H, d, J=7.5 Hz, H3-23), 1.20 (3H, s, H3-24), 1.38 (1H, br dd,
1


J=13.5, 1.5, H-21eq), 1.69 (3H, br s, H3-26), 1.72 (3H, br s, H3-25), 1.86 (3H, s, 8-
OCOCH3), 1.91 (1H, m, H-13), 1.95 (1H, m, H-21ax), 1.97 (3H, s, 7-OCOCH3), 2.02
(1H, m, H-13), 2.13 (1H, m, H-20), 2.17 (1H, m, H-9), 2.25 - 2.27 (2H, m, H2-10), 2.35
(1H, br t, J=11.5 Hz, H-14), 3.43 (1H, br dd, J=12.5, 5.0 Hz, H-22eq), 3.59 (1H, td,
J=12.5, 2.0 Hz, H-22ax), 3.746 (3H, s, COOCH3), 3.752 (1H, br s, H-17), 3.87 (1H, d,
J=6.0 Hz, H-19), 4.32 (1H, dd, J=10.0, 7.5 Hz, H-6), 5.40 (1H, br s, H-11), 5.47 (1H, q,
J=1.5 Hz, H-15), 5.87 (1H, d, J=15.0 Hz, H-2), 6.16 (1H, dd, J=15.0, 7.5 Hz, H-5), 6.32
(1H, dd, J=15.0, 11.0 Hz, H-4), 6.38 (1H, d, J=10.0 Hz, H-7), 7.24 (1H, dd, J=15.0,
11.0 Hz, H-3).
13
C-NMR (CDCl3): 13.6 (C-23), 15.6 (C-24), 20.9 (8-OCOCH3), 21.8 (7-OCOCH3),
23.3 (C-26), 25.4 (C-25), 28.9 (C-10), 31.4 (C-21), 32.1 (C-20), 36.8 (C-13), 38.3 (C-9),
38.4 (C-14), 51.6 (COOCH3), 54.7 (C-22), 56.8 (C-17), 73.1 (C-7), 74.5 (C-19), 76.3
(C-6), 78.3 (C-18), 86.5 (C-8), 121.4 (C-11), 122.0 (C-2), 127.5 (C-15), 130.6 (C-4),



-121-
132.2 (C-16), 134.4 (C-12), 139.5 (C-5), 143.7 (C-3), 167.1 (C-1), 168.6 (7-OCOCH3),
169.4 (8-OCOCH3).
HMBC: COOCH3→C-1; H-2→C-1, 4; H-3→C-1; H-4→C-5, 6; H-5→C-3, 4, 6; H-
6→C-4, 5, 7, 8, 19; H-7→C-6, 8, 9, 7-OCOCH3; 7-OCOCH3→7-OCOCH3; 8-
OCOCH3→8-OCOCH3; H-15→C-9, 14, 17, 25; H-17→C-7, 8, 9, 15, 16, 18, 24, 25; H-
19→C-6, 17, 18, 20, 23, 24; H2-21→C-19, 20, 23; H-22eq→C-18, 20; H3-23→C-19, 20,
21; H3-24→C-17, 18, 19; H3-25→C-15, 16, 17; H3-26→C-11, 12, 13.
HR-ESIMS m/z: 565.2773 (calcd for C31H42O8Na: 565.2779 [M+Na]+).


New polyketide pyrenulic acid D (66)
Colorless solid.
[α]D23 -7.9o (c=0.86, CHCl3).
UV λmax (EtOH) nm (log ε): 254.5 (4.38).
IR (KBr) νmax cm-1: 3442, 2926, 1693, 1641, 1614, 1433, 1381, 1246.
1
H-NMR: Table 10.
13
C-NMR: Table 12.
NOESY: H-2/H-4, H-3/H-5, H-6/H-9, H-6/H-10eq, H-7/H-17, H-7/H-19, H-9/H3-24, H-
17/H-19, H3-23/H3-24.
HR-ESIMS m/z: 427.2493 (calcd for C26H35O5: 427.2486 [M-H]-).


Preparation of (R)- and (S)-MPA esters of 66
Compound 66 (10.0 mg) was treated with TMS-CHN2 as described above to yield
methyl ester compound (8.2 mg). To a solution of methyl ester of 66 (4.5 mg) in dry
CH2Cl2 (2.0 ml) was added (R)-MPA (7.7 mg), EDC (12.4 mg) and a few crystals of 4-
pyrrolidinopyridine, and the whole was stirred at rt for 23 hrs. The reaction mixture was
poured into 1 N HCl and extracted with CHCl3. The CHCl3 layer was dried over MgSO4
and concentrated in vacuo. The residue was purified by prep. TLC (n-hexane-Et2O-
AcOH, 4:6:0.4) and prep. HPLC (n-hexane-Et2O, 3:2), yielding 66a (3.3 mg). Methyl
ester of 66 (3.6 mg) was treated with (S)-MPA (8.0 mg) as described above to yield 66b
(1.8 mg).




-122-
Compound 66a
H-NMR (CDCl3): δ 1.052 (3H, d, J=7.5 Hz, H3-23), 1.150 (1H, m, H-10ax), 1.161 (3H,
1


s, H3-24), 1.380 (1H, br d, J=13.0 Hz, H-21eq), 1.616 (3H, br s, H3-26), 1.728 (3H, br s,
H3-25), 1.731 (1H, m, H-14), 1.747 (1H, m, H-13), 1.809 (1H, ddd, J=12.0, 4.0, 2.0 Hz,
H-8), 1.850 (1H, m, H-13), 1.882 (1H, m, H-21ax), 1.871 (1H, m, H-9), 1.997 (1H, br d,
J=16.5 Hz, H-10eq), 2.063 (1H, br q, J=6.0 Hz, H-20), 2.254 (1H, d, J=4.0 Hz, H-17),
3.347 (3H, s, MPA-OCH3), 3.406 (1H, dd, J=12.5, 4.5 Hz, H-22eq), 3.531 (1H, d, J=6.0
Hz, H-19), 3.604 (1H, td, J=12.5, 2.0 Hz, H-22ax), 3.760 (3H, s, COOCH3), 4.366 (1H,
dd, J=10.0, 6.0 Hz, H-6), 4.629 (1H, s, MPA-CH), 5.222 (1H, br d, J=5.5 Hz, H-11),
5.285 (1H, br s, H-15), 5.525 (1H, dd, J=10.0, 2.0 Hz, H-7), 5.804 (1H, d, J=15.5 Hz,
H-2), 5.995 (1H, dd, J=15.5, 6.0 Hz, H-5), 6.195 (1H, dd, J=15.5, 11.0 Hz, H-4), 7.162
(1H, dd, J=15.5, 11.0 Hz, H-3), 7.311-7.366 (5H, m, MPA-Ph).
C-NMR (CDCl3): δ 13.6 (C-23), 16.1 (C-24), 23.3 (C-26), 25.6 (C-25), 31.8 (C-21),
13


32.1 (C-10), 32.3 (C-20), 33.3 (C-9), 37.3 (C-13), 41.8 (C-14), 46.1 (C-8), 51.6
(COOCH3), 54.5 (C-22), 55.7 (C-17), 57.4 (MPA-OCH3), 74.7 (C-19), 75.2 (C-6), 76.0
(C-7), 79.6 (C-18), 82.9 (MPA-CH), 121.6 (C-11), 121.9 (C-2), 127.5 (MPA-Ph), 128.1
(C-15), 128.7, 129.0 (MPA-Ph), 130.2 (C-4), 133.8 (C-12), 135.3 (C-16), 135.6 (MPA-
Ph), 139.6 (C-5), 143.6 (C-3), 167.1 (C-1), 169.5 (MPA-CO).
HMBC: COOCH3→C-1; H-2→C-1, 4; H-3→C-1, 4, 5; H-4→C-2, 3, 5; H-5→C-3, 6;
H-6→C-4, 5, 7, 19; H-7→C-6, 8, 9, 7-MPA-CO; H-11→C-26; H-15→C-9, 13, 14, 17,
25; H-17→C-8, 9, 15, 16, 18, 24, 25; H-19→C-17, 18, 20, 23, 24; H-20→C-18, 19, 21,
22, 23; H2-21→C-19, 20, 22, 23; H-22eq→C-18, 20; H3-23→C-19, 20, 21; H3-24→C-
17, 18, 19; H3-25→C-15, 16, 17; H3-26→C-11, 12, 13.
HR-ESIMS m/z: 613.3132 (calcd for C36H46O7Na: 613.3143 [M+Na]+).


Compound 66b
H-NMR (CDCl3): δ 1.031 (3H, d, J=7.0 Hz, H3-23), 1.177 (3H, s, H3-24), 1.355 (1H,
1


br d, J=14.0 Hz, H-21eq), 1.674 (3H, br s, H3-26), 1.754 (3H, br s, H3-25), 1.797 (1H, m,
H-10ax), 1.820 (1H, m, H-13), 1.850 (1H, m, H-21ax), 1.960 (1H, m, H-13), 1.990 (1H,
m, H-14), 2.010 (1H, m, H-9), 2.031 (1H, m, H-20), 2.080 (1H, ddd, J=12.0, 4.0, 2.0 Hz,
H-8), 2.230 (1H, br d, J=15.0 Hz, H-10eq), 2.300 (1H, d, J=4.0 Hz, H-17), 3.373 (3H, s,




-123-
MPA-OCH3), 3.380 (1H, m, H-22eq), 3.479 (1H, d, J=6.0 Hz, H-19), 3.601 (1H, td,
J=13.0, 2.0 Hz, H-22ax), 3.760 (3H, s, COOCH3), 4.245 (1H, dd, J=10.0, 6.0 Hz, H-6),
4.654 (1H, s, MPA-CH), 5.354 (1H, br s, H-15), 5.400 (1H, br d, J=5.0 Hz, H-11),
5.442 (1H, dd, J=10.0, 2.0 Hz, H-7), 5.604 (1H, d, J=15.0 Hz, H-2), 5.652 (1H, dd,
J=15.5, 6.0 Hz, H-5), 5.787 (1H, dd, J=15.5, 11.0 Hz, H-4), 6.914 (1H, dd, J=15.5, 11.0
Hz, H-3), 7.320-7.352 (5H, m, MPA-Ph).
C-NMR (CDCl3): δ 13.5 (C-23), 16.1 (C-24), 23.3 (C-26), 25.6 (C-25), 31.8 (C-21),
13


32.3 (C-20), 32.9 (C-10), 33.5 (C-9), 37.3 (C-13), 42.0 (C-14), 46.4 (C-8), 51.6
(COOCH3), 54.5 (C-22), 55.6 (C-17), 57.4 (MPA-OCH3), 74.7 (C-19), 75.2 (C-6), 76.3
(C-7), 79.6 (C-18), 82.7 (MPA-CH), 121.4 (C-11), 121.7 (C-2), 127.2 (MPA-Ph), 128.0
(C-15), 128.7, 128.9 (MPA-Ph), 129.9 (C-4), 134.5 (C-12), 135.5 (C-16), 135.8 (MPA-
Ph), 139.3 (C-5), 143.6 (C-3), 167.1 (C-1), 169.4 (MPA-CO).
NOESY: H-2/H-4, H-3/H-5, H-6/H-10eq, H-7/H-17, H-7/H-19, H-9/H3-24, H-17/H-19,
H-22ax/H3-24, H3-23/H3-24.
HMBC: COOCH3→C-1; H-2→C-1, 4; H-3→C-1; H-4→C-5; H-5→C-3, 4; H-6→C-4,
7, 19; H-7→C-5, 6, 9, MPA-CO; H-11→C-9, 26; H-15→C-9, 17, 25; H-17→C-15, 16,
18, 24, 25; H-19→C-6, 17, 18, 20, 23, 24; H-20→C-19, 23; H2-21→C-19, 22, 23; H-
22eq→C-18, 20; H3-23→C-19, 20, 21; H3-24→C-17, 18, 19; H3-25→C-15, 16, 17; H3-
26→C-11, 12, 13.
HR-ESIMS m/z: 613.3135 (calcd for C36H46O7Na: 613.3143 [M+Na]+).


New polyketide pyrenulic acid E (67)
Colorless solid.
ROESY: H-2/H-4, H-3/H-5, H-5/H-7, H-6/H-10eq, H-7/H-17, H-7/H-19, H-9/H3-24, H-
17/H-19.
1
H-NMR: Table 13.
13
C-NMR: Table 12.
HR-ESIMS m/z: 429.2660 (calcd for C26H37O5: 429.2643 [M-H]-).


New polyketide pyrenulic acid F (68)
Colorless solid.




-124-
[α]D24 -11.2o (c=0.85, CHCl3).
UV λmax (EtOH) nm (log ε): 252.5 (4.38).
IR (KBr) νmax cm-1: 3439, 2962, 2927, 1694, 1644, 1620, 1434, 1380, 1269.
1
H-NMR: Table 13.
13
C-NMR: Table 12.
ROESY: H-2/H-4, H-3/H-5, H-5/H-7, H-6/H-10eq, H-7/H-17, H-7/H-19, H-9/H3-24, H-
17/H-19.
HR-ESIMS m/z: 445.2612 (calcd for C26H37O6: 445.2592 [M-H]-).


New polyketide pyrenulic acid G (69)
Colorless solid.
[α]D23 -8.2o (c=0.60, CHCl3).
UV λmax (EtOH) nm (log ε): 256.5 (4.37).
IR (KBr) νmax cm-1: 3420, 2964, 1692, 1639, 1614, 1434, 1380, 1237.
1
H-NMR: Table 13.
13
C-NMR: Table 12.
NOESY: H-2/H-4, H-3/H-5, H-7/H-10eq, H-7/H-17, H-7/H-19, H-17/H-19.
HR-ESIMS m/z: 411.2548 (calcd for C26H35O4: 411.2537 [M-H]-).


Preparation of (R)- and (S)-MPA esters of 69
Compound 69 (8.2 mg) was methylated by TMS-CHN2 as described above to yield
a methyl ester (6.4 mg). Portion of methyl ester of 69 (3.2 mg) and (2.3 mg) was
esterified to (R)-MPA ester 69a (1.7 mg) and (S)-MPA ester 69b (1.2 mg), respectively.


Compound 69a
H-NMR (CDCl3): δ 0.812 (3H, d, J=6.5 Hz, H3-23), 0.878 (3H, t, J=7.0 Hz, H3-22),
1


1.089 (1H, m, H-21), 1.150 (1H, m, H-10), 1.590 (3H, br s, H3-25), 1.609 (3H, br s, H3-
26), 1.671 (1H, m, H-13), 1.750 (1H, m, H-9), 1.750 (1H, m, H-20), 1.770 (1H, m, H-
10), 1.770 (1H, m, H-14), 1.781 (1H, m, H-21), 1.854 (1H, m, H-8), 1.891 (1H, m, H-
13), 2.763 (1H, d, J=5.0 Hz, H-17), 3.351 (3H, s, MPA-OCH3), 3.667 (1H, d, J=9.0 Hz,
H-19), 3.768 (3H, s, COOCH3), 4.333 (1H, m, H-6), 4.602 (1H, s, MPA-CH), 5.041




-125-
(1H, s, H-24), 5.188 (1H, br s, H-11), 5.245 (1H, s, H-24), 5.330 (1H, br s, H-15), 5.417
(1H, br d, J=8.0 Hz, H-7), 5.852 (1H, d, J=15.5 Hz, H-2), 5.982 (1H, dd, J=15.5, 7.0 Hz,
H-5), 6.245 (1H, dd, J=15.5, 11.0 Hz, H-4), 7.158 (1H, dd, J=15.5, 11.0 Hz, H-3),
7.312-7.348 (5H, m, MPA-Ph).
C-NMR (CDCl3): δ 10.9 (C-22), 16.2 (C-23), 22.1 (C-25), 23.3 (C-26), 25.3 (C-21),
13


30.8 (C-10), 32.0 (C-9), 37.1 (C-20), 37.6 (C-13), 39.2 (C-14), 43.7 (C-8), 51.0 (C-17),
51.6 (COOCH3), 57.5 (MPA-OCH3), 74.3 (C-6), 76.1 (C-7), 82.3 (C-19), 82.9 (MPA-
CH), 117.0 (C-24), 121.45 (C-11), 121.50 (C-2), 127.4 (MPA-Ph), 128.0 (C-15), 128.6,
128.9 (MPA-Ph), 129.9 (C-4), 132.8 (C-16), 133.2 (C-12), 135.6 (MPA-Ph), 139.7 (C-
5), 143.9 (C-3), 145.6 (C-18), 167.3 (C-1), 169.4 (MPA-CO).
NOESY: H-2/H-4, H-3/H-5, H-6/H-10eq, H-7/H-17, H-7/H-19, H-17/H-19.
HMBC: COOCH3→C-1; H-2→C-1, 4; H-3→C-1; H-5→C-3; H-15→C-9, 14, 17, 25;
H-17→C-16, 18; H-19→C-17, 18, 20, 23, 24; H2-21→C-19, 20, 22, 23; H3-22→C-20,
21; H3-23→C-19, 20, 21; H2-24→C-17, 18, 19; H3-25→C-15, 16, 17; H3-26→C-11, 12,
13.
HR-ESIMS m/z: 597.3187 (calcd for C36H46O6Na: 597.3194 [M+Na]+).


Compound 69b
H-NMR (CDCl3): δ 0.802 (3H, d, J=6.5 Hz, H3-23), 0.849 (3H, t, J=7.0 Hz, H3-22),
1


1.040 (1H, m, H-21), 1.617 (3H, br s, H3-25), 1.669 (3H, br s, H3-26), 1.726 (1H, m, H-
21), 1.738 (1H, m, H-20), 1.760 (1H, m, H-13), 1.800 (1H, m, H-10), 1.850 (1H, m, H-
9), 1.980 (1H, m, H-14), 1.990 (1H, br d, J=14.0 Hz, H-13), 2.080 (1H, br d, J=14.0 Hz,
H-10), 2.110 (1H, ddd, J=11.0, 5.5, 2.5 Hz, H-8), 2.812 (1H, d, J=5.5 Hz, H-17), 3.362
(3H, s, MPA-OCH3), 3.654 (1H, d, J=9.0 Hz, H-19), 3.779 (3H, s, COOCH3), 4.213
(1H, t, J=7.5 Hz, H-6), 4.630 (1H, s, MPA-CH), 5.074 (1H, s, H-24), 5.249 (1H, s, H-
24), 5.346 (1H, dd, J=7.5, 2.5 Hz, H-7), 5.393 (1H, br s, H-11), 5.400 (1H, br s, H-15),
5.594 (1H, dd, J=15.5, 7.5 Hz, H-5), 5.681 (1H, d, J=15.5 Hz, H-2), 5.977 (1H, dd,
J=15.5, 11.0 Hz, H-4), 6.836 (1H, dd, J=15.5, 11.0 Hz, H-3), 7.325-7.345 (5H, m,
MPA-Ph).
C-NMR (CDCl3): δ 10.9 (C-22), 16.2 (C-23), 22.0 (C-25), 23.3 (C-26), 25.2 (C-21),
13


31.5 (C-10), 32.2 (C-9), 37.2 (C-20), 37.7 (C-13), 39.3 (C-14), 43.8 (C-8), 51.0 (C-17),




-126-
51.5 (COOCH3), 57.3 (MPA-OCH3), 73.9 (C-6), 76.6 (C-7), 82.6 (C-19), 82.7 (MPA-
CH), 117.1 (C-24), 121.2 (C-2), 121.9 (C-11), 127.2 (MPA-Ph), 127.9 (C-15), 127.9,
128.7 (MPA-Ph), 129.5 (C-4), 133.0 (C-16), 133.9 (C-12), 135.7 (MPA-Ph), 139.3 (C-
5), 144.1 (C-3), 145.4 (C-18), 167.3 (C-1), 169.2 (MPA-CO).
NOESY: H-2/H-4, H-3/H-5, H-6/H-10eq, H-7/H-17, H-7/H-19, H-17/H-19.
HMBC: COOCH3→C-1; H-2→C-1, 4; H-5→C-3; H-6→C-4, 5, 7,19; H-7→C-6, 8, 9;
H-11→C-9, 13; H-15→C-14, 25; H-17→C-8, 9, 15, 16, 18; H-19→C-17, 18, 20, 21,
24; H2-21→C-20; H3-22→C-20, 21; H3-23→C-19, 20, 21; H2-24→C-17, 18, 19; H3-
25→C-15, 16, 17; H3-26→C-11, 12, 13.
HR-ESIMS m/z: 597.3191 (calcd for C36H46O6Na: 597.3194 [M+Na]+).


New polyketide pyrenulic acid H (70)
Colorless needles.
mp. 146-147oC (CHCl3-MeOH).
[α]D22 +86o (c=1.03, MeOH).
UV λmax (EtOH) nm (log ε): 256 (4.39).
IR (KBr) νmax cm-1: 3423, 2695, 1693, 1643, 1614, 1434, 1382, 1309, 1264.
1
H-NMR: Table 13.
13
C-NMR: Table 12.
NOESY: H-2/H-4, H-3/H-5, H-5/H-7, H-6/H-10eq, H-7/H-19.
HR-ESIMS m/z: 427.2503 (calcd for C26H35O5: 427.2486 [M-H]-).


Chapter 4: Biological activity of isolated compounds
The bio-assay was taken at Kobe-Gakuin University, Japan by Dr. Y. Mizushina.


4.1. Enzymes
DNA polymerase α was purified from thymus by immuno-affinity column
chromatography as described previously.123) Recombinant rat DNA polymerase β was
purified from E. coli JMpβ5, as described by Date et al.124) A truncated form of
polymerase κ (residues 1-560) with 6 × His-tags attached at the C-terminus was
overproduced in E. coli and purified as described previously.125)



-127-
4.2. DNA polymerase assays
The standard reaction mixture for polymerase α (24 µl final volume) contained 50
mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM MgCl2, 5 µM poly(dA)/oligo(dT)18
(= 2/1), 10 µM [3H]dTTP (100 cpm/pmol), 15% (v/v) glycerol and 8 µl of an enzyme
inhibitor solution. The standard reaction mixture for polymerase β was the same, except
that it also contained 150 mM KCl. The reaction mixture for polymerase κ was the same
as for polymerase α.
The compounds were dissolved in distilled DMSO at various concentrations and
sonicated for 30 s. Aliquots of 4 µl sonicated samples were mixed with 16 µl of each
enzyme (final amount 0.05 units) in 50 mM Tris-HCl (pH 7.5) containing 1 mM
dithiothreitol, 50% glycerol and 0.1 mM EDTA, and kept at 0oC for 10 min. These
inhibitor-enzyme mixtures (8 µl) were added to 16 µl of each of the enzyme standard
reaction mixtures and incubation was carried out at 37oC for 60 min. Activity without
the inhibitor was considered 100% and the remaining activity at each concentration of
the inhibitor was determined relative to this value. One unit of polymerase activity was
defined as the amount of enzyme that catalyzed the incorporation of 1 nmol dTTP into
synthetic DNA template-primers in 60 min at 37oC under the normal reaction conditions
for each enzyme.


4.3. Cell culture and measurement of cell viability
The cell human cancer cell line, HCT116 (colon carcinoma cells), was obtained
from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human
cancer cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine
serum, penicillin (100 units/ml) and streptomycin (100 µg/ml). HTC116 cells were
cultured at 37oC in a humid atmosphere of 5% CO2/95% air. For the cell growth assay,
the cells were plated at 1 × 104 cells into each well 96-well microplates with various
concentrations of the isolated compounds. These compounds were dissolved in DMSO
at a concentration of 10 mM as a stock solution. The stock solutions were diluted to the
appropriate final concentrations with growth medium as 0.5% DMSO just before use.
Cell viability was determined by WST-1 assay.122)




-128-
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List of compounds


No. Name Lichen species
vulpinic acid
1
lecanoric acid RC
2
methyl orsellinate RC, PM
3
n-butyl orsellinate PM
4
ethyl orsellinate PM
5
methyl β-orsellinate RC, PM
6
methyl haematommate RC, PM
7
ethyl chlorohaematommate PM
8
atranorin RC, PM
9
chloroatranorin RC, PM
10
α-alectoronic acid RC, PM
11
α-collatolic acid RC, PM
12
β-alectoronic acid RC, PM
13
β-collatolic acid RC, PM
14
2′′′-O-ethyl-α-alectoronic acid PM
15
2′′′-O-methyl-α-alectoronic acid PM
16
2′′′-O-methyl-β-alectoronic acid PM
17
2′′′-O-ethyl-β-alectoronic acid PM
18
dehydrocollatolic acid RC, PM
19
dehydroalectoronic acid PM
20
new isocoumarin PM
21
new isocoumarin PM
22
parmosidone A PM
23
parmosidone B PM
24
parmosidone C PM
25
(+)-usnic acid RC, PM
26
skyrin RC, PM
27
5-chloroemodin
28
gyrophoric acid RC
29
salazinic acid RC
30
norstictic acid
31
stictic acid
32
psoromic acid
33
protocetraric acid
34
2-acetyl-3,5-dihydroxybenzoic acid GV
35
trans-5,7-dihydroxy-3-(1-hydroxyethyl)phtalide GV
36
cis-5,7-dihydroxy-3-(1-hydroxyethyl)phtalide GV
37
4,6-dihydroxy-3,9-dehydromellein GV
38
6,8-dihydroxy-3-(hydroxymethyl)isocoumarin GV
39
cis-4,6-dihydroxymellein GV
40
6,8-dihydroxyisocoumarin-3-carboxylic acid GV
41
14-membered macrolide GV
42
6-methoxy-8-hydroxyisocoumarin-3-carboxylic acid
43
mutolide
44
ergosterol peroxide ST
45
sporogen-AO 1 ST
46
dihydrosporogen-AO 1 ST
47
petasol ST
48
isopetasol ST
49
JBIR-27 ST
50
1β-hydroxypetasol ST
51
3-epi-petasol ST
52
dihydropetasol ST
53
sarcographol ST
54
petasin
55
S-petasin
56
petasinol
57
8-epi-dihydropetasol
58
cyclodebneyol
59
chrysophanol PS
60
emodin PS
61
1,5,8-trihydroxy-3-methylxanthone PS
62
pyrenulic acid A PS
63
pyrenulic acid B PS
64
pyrenulic acid C PS
65
pyrenulic acid D PS
66
pyrenulic acid E PS
67
pyrenulic acid F PS
68
pyrenulic acid G PS
69
pyrenulic acid H PS
70
cladobotric acid A
71
cladobotric acid C
72
lobaric acid
73


RC: Rimelia clavulifera PM: Parmotrema mellissii
GV: Graphis vestitoides ST: Sarcographa tricosa
PS: Pyrenula sp.
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