Tài liệu 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 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 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 -iii- 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 Foliose Fruticose 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 antiinflmammatory 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 -6- 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%) Fr-I (1.25 g) Fr-II (3.27 g) CC/CHCl3-MeOH pTLC A-C MeOH (0%) 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) 27 (4.8 mg) Fr-IIa (417 mg) pTLC B-D 15 (65.6 mg) 18 (66.2 mg) 25 (2.9 mg) (2%) (3-50%) Fr-III (2.19 g) Fr-IV (1.62 g) CC/CHCl3-MeOH (0%) (1-2%) Fr-IIb (539 mg) CC/CHCl3-MeOH Fr-IIc (2.17 g) 11 (1.32 g) 13 (798.4 mg) pTLC B-D pTLC B-D 5 (10.2 mg) 12 (327.2 mg) 20 (21.6 mg) 21 (25.0 mg) 23 (9.1 mg) 24 (8.1 mg) 11 (1.21 g) 12 (365.7 mg) 13 (21.3 mg) 14 (139.0 mg) 16 (43.9 mg) 17 (13.7 mg) pTLC 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 -7- 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 2,4dihydroxy-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 8 CH 3 6 7 COOR 1 2 4 OH signals of n-butoxyl group instead of methoxyl group as seen in HO 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 nbutyl 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 6 H 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 presence of an additional methyl group (δH 2.10, δC 7.7) and 7 COOCH3 3 HO OH 9 CH3 HMBC NOESY absence of an aromatic proton. Moreover, the HMBC 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 methyl group. Its 13 C-NMR spectrum showed 10 carbon signals including a methyl, a methoxyl, a methine, a formyl, a carbonyl and five quaternary carbon signals. These spectral features resembled those of 6, except for the presence of formyl group at the C-3 position instead of a methyl group. This was supported by the HMBC correlation observed from formyl proton (H-9) to C-2, 3 and 4. Consequently, the structure of 7 was elucidated as methyl haematommate.36) Ethyl chlorohaematommate (8) The HR-ESIMS of 8 established the composition of 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 1Hand 13 C-NMR spectra of 8 exhibited the signals corresponding to an ethoxyl group [-CH3: δH 1.46 (t, 8 CH3 O Cl 6 7 1 HO O CH3 OH 9 CHO COSY HMBC 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 by its HR-EIMS. Its 1 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) 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 C5H11 NOESY 2" H H O O 1" 6 H O 7 1 exhibited absorption bands at 3361, 1714, 1676, 1613 and 1579 cm-1, indicating the presence of hydroxyl and carbonyl HO groups, and aromatic ring. The 1H-NMR spectrum showed 4 O 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 (s). The 1 H-NMR spectra showed further signals for an olefinic proton at δH 6.10 (s), three methoxyl groups, a βketo alkyl C7 side chain, a n-pentyl group and a hydrogen-bonded phenolic proton [δH 11.68 (s)] (Table 1). The 13 C-NMR spectrum of 11a showed no carbon signals assignable to C-1′′ methylene and C-2′′ carbonyl carbons, but newly demonstrated two 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-Omethyl-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 recyclization 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 α and 11β β ) is the predominant tautomer.39) -12-