Chemistry of the lichen type common in southern vietnam

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, monocy

Doctoral Thesis

CHEMICAL STUDY OF COMMON LICHENS IN

THE SOUTH OF VIETNAM

Content page

List of abbreviations i

List of figures iv

List of photos v

List of tables v

Chapter 1: General introduction 1

1.1 The lichen and usage of lichens 1

1.2 Lichen substances 2

1.3 Cultivation of lichen mycobionts 3

1.4 Vietnamese lichen 5

1.5 Research scope and objectives 5

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

2.1 Chemical investigation of the lichen thalli of P mellissii 7

2.1.1 Mono-aromatic compounds 8

2.1.2 Depsides 10

2.1.3 Depsidones and Isocoumarin derivatives 11

2.1.4 Other lichen substances 28

2.2 Chemical investigation of the lichen thalli of R clavulifera 30

Chapter 3: Secondary metabolites from the cultured lichen mycobionts 33

3.1 Chemical investigation of the cultured mycobionts of Graphis vestitoides 33

3.2 Chemical investigation of the cultured mycobionts of Sacographa tricosa 44

3.3 Chemical investigation of the cultured mycobionts of Pyrenula sp 58

Chapter 4: Biological activity of isolated compounds 77

4.1 Inhibitory effect on mammalian DNA polymerase activity 77

4.2 Inhibitory effect on cancer cell growth 81

Chapter 5: Conclusions 82

Acknowledgment 86

Experimental section 87

References 129 List of compounds

EI-MS electron-impact ionization mass spectrum HMBC heteronuclear multiple bond correlation spectroscopy HOBT 1-hydroxybenzotriazole

HPLC high performance liquid chromatography

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

PyBOP benzotriazolyloxytri(pyrrolidinyl)phosphonium hexafluorophosphate rel int relative intensity

SIMS secondary ion mass spectrum

tdd triplet of doublets of doublets tdt triplet of doublets of triplets TLC thin-layer chromatography TMS tetramethylsilane

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

List of photos

Page

Photo 1 Growth forms of lichen 1

Photo 2 The thalli of foliose lichens P mellissii and R clavulifera 6

Photo 3 G vestitoides thalli and its cultured mycobionts 34

Photo 4 S tricosa thalli and its cultured mycobionts 44

Photo 5 Pyrenula sp thalli and its cultured mycobionts 59

List of tables Page Table 1 1H- and 13C-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

Chapter 1: General introduction

1.1 The lichens and usage of lichens

The lichens are symbiotic organisms, usually composed of a fungal partner (mycobiont) and one or more photosynthetic partners (photobionts), which is most often either a green alga or cyanobacterium About 17,000 different lichen taxa, including 16,750 lichenized Ascomycetes, 200 Deuteromycetes, and 50 Basidiomycetes have been described world-wide.The photobionts produce carbohydrates by photosynthesis for themselves and for their dominant fungal counterparts (mycobionts), which provide physical protection, water and mineral supply Based on this association, lichens have adapted to extreme ecological conditions, being dominant at high altitudes, in Arctic boreal and also tropical habitats, and colonized a wide range of different substrata, such

as rocks, bare ground, leaves, bark, metal, glass Lichens are traditionally divided into three growth morphological forms: these are the crustose, foliose and fruticose types (Photo 1).1-3)

Photo 1 Growth forms of lichen Lichens have been used by humans for centuries as food,4) as source of dye,5) as raw materials in perfumery and for therapeutic properties in folk medicine The fragrance

industry uses two species of lichen Evernia prunastri var prunastri (oakmoss) and

Pseudevernia furfuracea (treemoss) About 700 tons of oakmoss are currently processed

every year by French producers.6,7) Several lichen extracts have been used for various

remedies in folk medicine, such as Lobaria pulmonaria for lung troubles, Xanthoria

parientina for jaundice, Usnea spp for strengthening hair, Cetraria islandica (Iceland

moss) for tuberculosis, chronic bronchitis and diarrhea.4,8) The screening tests with lichens have indicated the frequent occurrence of metabolites with antioxidant, antibiotic, antimicrobial, antiviral, antitumor, analgestic and antipyretic properties.9-11)

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

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

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

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

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

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

CC/CHCl3-MeOH

CC/CHCl3-MeOH MeOH (0%)

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 δH11.72 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

is, C3H6 more than that of methyl orsellinate (3) The NMR spectral features of 4 were similar to those of 3, but 4 showed

signals of n-butoxyl group instead of methoxyl group as seen in

3 This was confirmed by the HMBC correlation from

oxygenated methylene protons at 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)

CH3COOR

OHHO

4 : R = n-C4H9

5 : R = C2H5

1 2

8

4

Methyl βββ-orsellinate (6) The HR-EIMS of 6 indicated the molecular formula of

C10H12O4, i.e a CH2 group more than that of 3 The NMR spectral features of 6 were similar to those of 3 except for the

presence of an additional methyl group (δH 2.10, δC 7.7) and absence of an aromatic proton Moreover, the HMBC 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 methyl group Its 13C-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

C11H11O5Cl Its 1H-NMR spectral features showed the

similarity to those of 7 except for the absence of the

signal due to an aromatic proton In addition, the 1H- and 13C-NMR spectra of 8 exhibited the signals

corresponding to an ethoxyl group [-CH3: δH 1.46 (t,

J=7.0 Hz), δC 14.1; -OCH2: δH 4.47 (q, J=7.0 Hz), δC 62.5] instead of methoxyl group

COOCH3

CH3

OHHO

CH3

H

1 3

8

HMBC NOESY

9

6

CH3

OHHO

CHO

Cl

1

6 7 8

as seen in 7 The HMBC correlations from H3-8 (δH 2.72), 4-OH (δH 13.15) and H-9 (δH10.36) 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

by its HR-EIMS Its 1H-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 δH3.99, three phenolic hydroxyls at δH 11.94, 12.49 and 12.54, and a formyl group at δH10.35 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

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

α-Alectoronic acid (11) Compound 11 was isolated as a colorless solid with a

molecular composition of C28H32O9 Its UV spectrum

showed maxima at 209, 266 and 314 nm IR spectrum of 11

exhibited absorption bands at 3361, 1714, 1676, 1613 and

1579 cm-1, indicating the presence of hydroxyl and carbonyl groups, and aromatic ring The 1H-NMR spectrum showed 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,

β-group and a hydrogen-bonded phenolic proton [δH 11.68 (s)] (Table 1) The 13C-NMR

spectrum of 11a showed no carbon signals assignable to C-1′′ methylene and C-2′′

carbonyl carbons, but newly demonstrated two sp 2 carbons at δC 102.7 (C-1′′) and 159.2

C5H11

HO 4

6 1"

NOESY

7

11

HH

(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

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)

These findings suggested 11 to be α-alectoronic acid 40) which was first isolated

from the lichen Alectoria japonica by Asahina and co-workers.41-43)

It is well known that prolonged treatment of α-alectoronic acid (11) with an excess

of diazomethane proceeded with partial cleavage of depsidone-ester linkage and cyclization to form isocoumarin skeleton The very broad signals in NMR spetra of

re-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 (11ααα and 11βββ) is the predominant tautomer.39)

C5H11

C5H11

OHCOOHHO

O O

O

OO

C5H11OHO

C5H11

OHHO

O O

O

OO

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

collatolic acid (12) could exit predominantly in the pseudo-acid tautomeric form as alectoronic acid (11).39)

6' 1' 7'

5H11

12

HH

HH

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

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

Table 2 1H- and 13C-NMR spectroscopic data of 12 and 12a in CDCl3

ND: not detected

β-Alectoronic acid (13) and βββ-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 13C-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-ααα-alectoronic acid (15) and 2′′′′′′′′′′′′-O-methyl-ααα-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 δ 6.36, 6.41 (each d, J=2.5 Hz) and 6.73 (s), a methylene at δ 3.07

and 3.46 (each 1H, d, J=17.0 Hz), a β-keto alkyl

C7 side chain, a n-pentyl group, an ethoxyl

group and two phenolic protons at δH 7.87 (br s) and 11.05 (s) The 13C-NMR spectrum showed the signals for a β-keto alkyl C7 side chain, a n-

pentyl, an ethoxyl groups and a ketal carbon, besides the signals for a methylene, three

aromatic CH, nine sp 2 quaternary carbons, and two carbonyls due to a common depsidone core

(Table 3) These spectral features of 15 were remarkably similar to those of

2′′′-O-methyl-α-alectoronic acid (16).39) The only difference between these compounds was

the substitution of ethoxyl group at C-2′′′ in 15 instead of methoxyl group as seen in 16

The location of the ethoxyl group at C-2′′′ was confirmed by the HMBC correlations between 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

New isocoumarin 2′′′′′′′′′′′′-O-methyl-βββ-alectoronic acid (17) and 2′′′′′′′′′′′′-O-ethyl-ββ alectoronic acid (18)

β-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

Detailed 2D NMR analysis and comparison with the reported data 39,46) led us to

determine the structure of 17 and 18 was methyl-β-alectoronic acid and

2′′′-O-ethyl-β-alectoronic acid, respectively Compound 18 was isolated from the lichen

Alectoria sarmentosa and reported as an artifact formed from β-alectoronic acid (13)

during the treatment with EtOH.46) The compound 18 could possibly be a natural

15

3"'

OOOH

O

C4H93"

1"'

1"

1 7 4'

1' 7' 4

COSY HMBC

HH

HHHH

HH

2"'

HH

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

5"'6"'7"'

112.5162.1106.5161.5

117.9140.9162.4104.9160.2

107.7150.4139.9130.2168.448.1210.142.923.331.322.413.9530.5

107.835.823.3

31.722.514.0358.215.3

a

a

b c

b c

102.5163.5103.3162.1

103.3142.1160.899.6163.5

105.6158.9133.4131.4168.8103.3159.033.126.431.222.414.030.8

107.534.923.0

31.622.414.049.9

6.36

7.876.41

11.056.73

3.83

2.661.661.341.340.933.073.46

1.902.051.441.501.361.360.913.643.701.07

d (2.5)

br s

d (2.5)

ss

s

td (7.0, 2.5)quint (7.0)m

mmm

6.11

2.431.651.341.340.912.883.01

1.781.901.30

1.251.250.853.30

brs

br s

br ss

s

mmmm

t (7.0)

br s

br s

mmm

mm

t (7.0)sTable 3.1H- and13C-NMR spectroscopic data of 15-17 in CDCl3

a, b, c, d, e, fAssignments may be interchangeable

3.83

2.661.651.341.340.943.063.46

1.922.051.431.511.371.370.913.33

d (2.0)

d (2.0)

ss

s

t (7.0)quint (7.0)m

t (7.0)s

112.4162.1106.5161.6

117.9140.9162.4104.9160.2

107.7150.4140.0130.0168.248.1210.242.923.231.322.413.930.3

107.735.123.2

31.722.514.049.9

e f

Dehydrocollatolic acid (19) Compound 19 was obtained as a colorless crystalline solid The HR-EIMS of 19 exhibited

a peak at m/z 524.2062 [M+], indicating a molecular formula of C29H32O9 Its 1H-NMR

spectrum showed two meta-coupled doublets at

δH 6.58 and 6.61 (J=2.5 Hz), and a singlet at δH

6.79 due to the aromatic protons, a bonded hydroxyl signal at δH 11.08, a methoxyl 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

hydrogen-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 sp 2 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

2"'

6"'

OOOH

O

19

COSY HMBC NOESY

HH

HH

1"' 3"'

5"' 7"' 1''

2"

Table 5 13C-NMR spectroscopic data of 19-25 in CDCl3

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 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-

hydrogen-occuring unique depsidone dehydrocollatolic acid (19), except for the absence of 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

4-dehydroalectoronic acid

New isocoumarin 21

Compound 21 was obtained as a colorless

solid The HR-EIMS spectrum established the molecular formula of C28H30O9 which is the

same composition of 20 The 1H- and 13C-

NMR spectra of 21 closely resembled those of

20 The marked differences were the presence

of an additional olefinic proton at δH 6.31 (s, H-1′′) and oxygenated quaternary carbon at

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 (5), 143.9 (6), 159.9 (2′′) and from aromatic proton H-5 to

C-1"'

3"' 6"'

OO

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

molecular formular of C29H32O9, i.e 14

mass units more than that of 21 The 1H- and 13C-NMR spectral features of 22 were similar to those of 21 (Tables 4 and 5), the

difference was only observed for the additional methoxyl group at δH 3.79 (s) and δC 55.8 The HMBC spectrum of 22

exhibited the cross peak of methoxyl 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 ring system The relative configuration at spiro-ring of 22 could be deduced from

spiro-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,

1"' 3"' 6"'

OO

O

H3C

HH

H

H3"

4

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) Compound 23, named parmosidone A, was

isolated as a pale yellow solid The MS spectrum

of compound 23 established the molecular

formula of C28H30O10 The 1H-NMR spectrum

exhibited signals for a pair of meta-coupled

aromatic protons at δH 6.40 and 6.44 (each d,

J=2.0 Hz), an aromatic proton at δH 6.75 (s), two methylene groups at δH 3.81 and 3.92 (each 1H, d,

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

23

2"'

5"'OOOH

C5H11O

O

H

H

HO

O

O

H

COSY HMBC NOESY

1"

6

1 74

HH

HH

HHH

H

2.03 and 2.40 (each 1H, m) (Table 6) These 1H-NMR spectral data, together with 13

C-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 member spiro-ring instead of a six-six-member ring as seen in 20

six-five-Compound 24 was isomeric with 23 The 1H- and 13C-NMR spectra of 24 (Tables 6 and 5)

demonstrated the depsidone skeleton The differences between these compounds could be only accounted for by a ketal proton at δH 5.29

(H-5′′′) of 24 due to a doublet of doublets signal

(J=5.5, 3.5 Hz) instead of doublet signal of H-5′′′

(J=5.0 Hz) as seen in 23 Accordingly, the

structure of 24 was elucidated and designated parmosidone B

The HR-SIMS established the composition

of 25 as C30H34O10, i.e having two methylene

groups more than that of 23 The NMR spectral features of 25 (Tables 5 and 6), named parmosidone C, were also similar to those of 23,

except for the presence of n-butoxyl group at

C-5′′′ Detail NMR studies of this compound led

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′′′

24 2"'

5"'OOOH

C5H11O

O

H

H

HO

O

O

H

COSY HMBC NOESY

1"

6

1 74

HH

H

HH

H

25

2"'

5"'OOOH

C5H11O

O

H

H

HO

O

O

H

COSY HMBC NOESY

1"

6

1 74

HH

HH

HHH

H

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)

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)

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 δH11.04, 13.33 and 18.84 (each 1H, s) The 13C-NMR spectrum revealed the resonances

for 18 carbons which were assigned for four methyl groups, a sp 3 quaternary, five sp 2 quaternary, an aromatic CH, five sp 2 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

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 δH12.12 and 12.86 (each 1H, s) The 13C-NMR spectrum showed the signals for 15 carbons including a methyl group, 12 aromatic carbons and two carbonyl groups at δC183.1 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 chloroemodin (28).51) However, the HR-ESIMS established the molecular formula of 27

5-to be C30H18O10, indicating a symmetrical structure Hence, compound 27 was identified

as a bianthraquinone skyrin.52)

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 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

β-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

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 13

C-NMR spectrum of 29 revealed 24 lines attributable to three sp 3 methyls, six sp 2

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)

Salazinic acid (30) Compound 30 was obtained as a major

component of the lichen R clavulifera and had a

molecular formula of C18H12O10 as indicated by HR-ESIMS Its IR spectrum showed absorption bands at 1772, 1742 and 1661 cm-1 correspoding

to depsidone ring, butyrolactone and aldehyde group, respectively.54) Its 1H-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)

O

1 3

8

9

1' 3'

6' 7'8'

9'H

30