ĐẠI HỌC QUỐC GIA THÀNH PHỐ HỒ CHÍ MINH

Transcription

VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY
UNIVERSITY OF SCIENCE

HUYNH BUI LINH CHI
STUDY ON CHEMICAL
CONSTITUENTS AND BIOLOGICAL
ACTIVITIES OF THE LICHEN
PARMOTREMA PRAESOREDIOSUM
(NYL.) HALE
(PARMELIACEAE)
DOCTORAL THESIS IN CHEMISTRY
Ho Chi Minh City, 2014
VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY
UNIVERSITY OF SCIENCE

HUYNH BUI LINH CHI
STUDY ON CHEMICAL CONSTITUENTS AND
BIOLOGICAL ACTIVITIES OF THE LICHEN
PARMOTREMA PRAESOREDIOSUM (NYL.) HALE
(PARMELIACEAE)
Subject: Organic Chemistry
Code number: 62 44 27 01
Examination Board:
Prof. Dr. Nguyen Minh Duc
(1st Reviewer)
Assoc. Prof. Dr. Tran Cong Luan
(2nd Reviewer)
Assoc. Prof. Dr. Pham Dinh Hung
(3rd Reviewer)
Assoc. Prof. Dr. Le Thi Hong Nhan
(1st Independent Reviewer)
Dr. Le Tien Dung
(2nd Independent Reviewer)
SUPERVISORS: PROF. DR. NGUYEN KIM PHI PHUNG
PROF. DR. TAKAO TANAHASHI
Ho Chi Minh City, 2014
i
SOCIALIST REPUBLIC OF VIETNAM
INDEPENDENCE-FREEDOM-HAPPINESS
DECLARATION
The work presented in this thesis was completed in the period of November
2009 to November 2013 under the co-supervision of Professor Nguyen Kim Phi
Phung of the University of Science, Vietnam National University, Ho Chi Minh
City, Vietnam and Professor Takao Tanahashi of the Kobe Pharmaceutical
University, Japan.
In compliance with the university regulations, I declare that:
1. Except where due acknowledgement has been made, the work is that of the
author alone;
2. The work has not been submitted previously, in whole or in part, to qualify for
any other academic award;
3. The content of the thesis is the result of the work which has been carried out
since the official commencement date of the approved doctoral research program;
4. Ethics procedures and guidelines have been followed.
Ho Chi Minh City, Sept 30, 2014
PhD student
HUYNH BUI LINH CHI
ii
ACKNOWLEDGEMENTS
There are many individuals without whom the work described in this thesis
might not have been possible, and to whom I am greatly indebted.
Firstly, I wish to thank my supervisor, Prof. Dr. Nguyen Kim Phi Phung for
her knowledge, support, and guidance, hundreds of meetings/emails and for always
keeping me on my toes, from the very beginning to the very end of my PhD.
I would also like to acknowledge my second supervisor, Prof. Dr. Takao
Tanahashi for his guidance, patience and who has taught me the true spirit of
research. I am deeply indebted to Dr. Yukiko Takenaka at Kobe Pharmaceutical
University, Japan for her teachings, kindness, helpful suggestion and valuable
advice in this research.
I would also like to express my sincere thanks to PhD Vo Thi Phi Giao at
University of Science, Vietnam National University, Ho Chi Minh City and Dr.
Harrie J. M. Sipman, Botanic Garden and Botany Museum Berlin-Dahlem, Freie
University, Berlin, Germany for his expertise in the identification of lichen.
I am very grateful to thank Prof. Dr. Shigeki Yamamoto, Prof. Dr. Hitoshi
Watarai at Osaka University, Japan and PhD. Do Thi My Lien for giving up their
precious time to help me with CD spectra, sample preparation and proof reading of
some isolated compounds of the thesis.
A special thanks to Dr. Le Hoang Duy for his helpful assistance and
friendship during my work at Kobe Pharmaceutical University, Japan.
I would like to acknowledge the encouragement, insightful comments of the
rest of examination board: Prof. Dr. Nguyen Cong Hao, Prof. Dr. Nguyen Minh
Duc, Assoc. Prof. Dr. Tran Cong Luan, Assoc. Prof. Dr. Pham Dinh Hung, Assoc.
Prof. Dr. Nguyen Trung Nhan, Dr. Pham Nguyen Kim Tuyen and Dr. Le Tien
Dung.
iii
Similarly, I would also like to thank my teachers, friends and students in the
Department of Organic Chemistry, Faculty of Chemistry, University of Science,
Vietnam National University-Ho Chi Minh City.
Most importantly, I would like to thank my husband, for being the most
patient and supportive witness to my academic journey over the past four years.
Without his support, love and encouragement, this study would not have been
possible.
Finally, I would like to thank my parents for believing in me and for being
proud of me. Their unconditional love and support has given me the strength and
courage while I am far from home.
THANK YOU
iv
TABLE OF CONTENTS
DECLARATION ....................................................................................................... i
ACKNOWLEDGEMENTS ...................................................................................... ii
TABLE OF CONTENTS ......................................................................................... iv
LIST OF ABBREVIATIONS.................................................................................. vi
LIST OF TABLES ................................................................................................... xi
LIST OF FIGURES ............................................................................................... xiii
LIST OF APPENDICES......................................................................................... xv
INTRODUCTION ..................................................................................................... 1
CHAPTER 1: LITERATURE REVIEW ................................................................ 3
1.1. GENERIC DESCRIPTION ............................................................................... 3
1.1.1. The lichen .......................................................................................................... 3
1.1.2. Parmotrema praesorediosum (Nyl.) Hale ......................................................... 4
1.2. CHEMICAL STUDIES ON THE LICHEN GENUS PARMOTREMA ........... 6
1.2.1. Lichen secondary metabolites ........................................................................... 6
1.2.2. Chemical studies on the lichen genus Parmotrema .......................................... 8
1.3. BIOLOGICAL ACTIVITIES ......................................................................... 14
1.3.1. The biological significance of lichen metabolites ........................................... 14
1.3.2. The biological significance of the lichen Parmotrema ................................... 15
CHAPTER 2: EXPERIMENTAL ......................................................................... 20
2.1. MATERIALS AND ANALYSIS METHODS ............................................... 20
2.2. LICHEN MATERIALS .................................................................................. 22
v
2.3. EXTRACTION AND ISOLATION PROCEDURES .................................... 22
2.3.1. Isolating compounds from the methanol precipitate ....................................... 22
2.3.2. Isolating compounds from the petroleum ether E1 extract ............................ 23
2.3.3. Isolating compounds from the petroleum ether E2 extract ............................ 23
2.3.4. Isolating compounds from the chloroform extract ......................................... 24
2.4. PREPARATION OF SOME DERIVATIVES ................................................ 28
2.4.1. Esterification of PRAES-C2 ............................................................................ 28
2.4.2. Methylation of PRAES-C25 ............................................................................ 29
2.5. BIOLOGICAL ASSAY .................................................................................. 29
2.5.1. Cytotoxicity ..................................................................................................... 29
2.5.2. In vitro acetylcholinesterase (AChE) inhibition assay .................................... 30
CHAPTER 3: RESULTS AND DISSCUSSION .................................................. 32
3.1. CHEMICAL STRUCTURE ELUCIDATION .............................................. 32
3.1.1. Chemical structure of aliphatic acids .............................................................. 33
3.1.1.1. Structure elucidation of compound PRAES-C1........................................... 33
3.1.1.2. Structure elucidation of compound PRAES-E14 ......................................... 34
3.1.1.3. Structure elucidation of compound PRAES-C10 ........................................ 35
3.1.2. Chemical structure of mononuclear phenolic compounds .............................. 45
3.1.2.1. Structure elucidation of compound PRAES-T1 ........................................... 45
3.1.2.2. Structure elucidation of compound PRAES-E1 ........................................... 46
vi
3.1.2.10. Structure elucidation of compound PRAES-C24 ...................................... 56
3.1.3. Chemical structure of depsides ....................................................................... 66
3.1.4. Chemical structure of depsidones ................................................................... 70
3.1.5. Chemical structure of diphenyl ethers ............................................................ 74
vii
3.1.6. Chemical structure of dibenzofurans .............................................................. 97
3.1.6.3. Structure elucidation of compound PRAES-C8......................................... 100
3.1.7. Chemical structure of xanthones ................................................................... 103
3.1.8. Chemical structure of triterpenoids ............................................................... 111
3.1.8.1. Structure elucidation of compound PRAES-E17 ....................................... 111
3.1.9. Chemical structure of a macrocylic compound............................................. 117
3.2. BIOLOGICAL ASSAY ................................................................................ 120
3.2.1. Cytotoxicity activitivy................................................................................... 120
3.2.2. Acetylcholinesterase inhibitory activity ....................................................... 121
CHAPTER 4: CONCLUSION ............................................................................. 124
4.1. CONSTITUENTS OF PARMOTREMA PRAESOREDIOSUM ................... 124
4.2. BIOLOGICAL ASSAY ................................................................................ 132
FUTURE OUTLOOK ........................................................................................... 133
viii
LIST OF PUBLICATIONS .................................................................................. 134
REFERENCES ...................................................................................................... 135
APPENDICES ....................................................................................................... 145
ix
LIST OF ABBREVIATIONS
1D
One dimensional
2D
Two dimensional
Ac
Acetone
AcOH
Acetic acid
br
Broad
C
Chloroform
calcd
Calculated
CC
Column chromatography
CD
Circular dichroism
COSY
Homonuclear shift correlation spectroscopy
CPCM
Conductor-like polarized continuum model
d
Doublet
dd
Doublet of doublets
DEPT
Distortionless enhancement by polarisation transfer
DMSO
Dimethyl sulfoxide
EA
Ethyl acetate
EI-MS
Electron-impact ionization mass spectrum
EtOH
Ethanol
H
n-Hexane
HMBC
Heteronuclear multiple bond correlation spectroscopy
HPLC
High performance liquid chromatography
HR-EIMS
High resolution electron-impact ionization mass spectrum
HR-ESIMS
High resolution electrospray ionization mass spectrum
x
HSQC
Heteronuclear single quantum correlation spectroscopy
IR
Infrared spectrophotometry
m
Multiplet
M
Methanol
MeOH
Methanol
min
Minutes
MS
Mass spectrum
NMR
Nuclear magnetic resonance
NOESY
Nuclear overhauser enhancement spectroscopy
P
Petroleum ether
ppm
Parts per million (chemical shift value)
pre TLC
Preparative thin-layer chromatography
q
Quartet
quint
Quintet
ROESY
Rotating-frame overhauser enhancement spectroscopy
s
Singlet
sext
Sextet
t
Triplet
TD-DFT
Time dependent density functional theory
TLC
Thin-layer chromatography
TMS
Tetramethylsilane
UV
Ultraviolet
xi
LIST OF TABLES
Table 1.1.
In vitro biological activities of the lichen genus Parmotrema
17
Table 3.1.
Isolated compounds from Parmotrema praesorediosum
32
Table 3.2.
1
38
Table 3.3.
13
Table 3.4.
NMR data of PRAES-T1, PRAES-E1, PRAES-T2
47
Table 3.5.
NMR data of PRAES-E11, PRAES-T4, PRAES-T6, PRAES-E2
52
Table 3.6.
NMR data of PRAES-C22, PRAES-C23, PRAES-C24
58
Table 3.7.
NMR data of PRAES-C25, PRAES-C25M, PRAES-C26
65
Table 3.8.
NMR data of PRAES-T3, PRAES-C7, PRAES-C9, PRAES-E18
68
Table 3.9.
NMR data of PRAES-C14 and PRAES-C12
72
Table 3.10.
1
H NMR data of PRAES-C5 and Lecanorol
76
Table 3.11.
1
H NMR data of PRAES-C15, PRAES-C16, PRAES-C20,
H NMR of aliphatic compounds
C NMR of aliphatic compounds
PRAES-C18, PRAES-C3 and PRAES-C4
Table 3.12
13
39
82
C NMR data of PRAES-C15, PRAES-C16, PRAES-C20,
PRAES-C18, PRAES-C3 and PRAES-C4
83
Table 3.13. NMR data of PRAES-C21
96
Table 3.14. NMR data of PRAES-E5 and PRAES-E3 (CDCl3)
98
Table 3.15. NMR data of PRAES-C8, PRAES-E5 and Usimine A
102
Table 3.16. NMR data of PRAES-C27, Blennolide G, Blennolide B and
Chromone lactone (CDCl3)
Table 3.17. NMR data of PRAES-C27 and PRAES-C28 (CDCl3)
106
110
xii
Table 3.18. NMR data of PRAES-E17, PRAES-E6, PRAES-E13 and 1β,3βDiacetoxyhopan-22-ol
Table 3.19. NMR data of PRAES-E15
115
120
Table 3.20. % Inhibition of cytotoxic activity against three cancer cell lines of
isolated compounds
122
Table 3.21. IC50 value of cytotoxic activity against three cancer cell lines of
isolated compounds
122
Table 3.22. Acetylcholinesterase inhibition of some extracts and isolated
compounds
123
xiii
LIST OF FIGURES
Figure 1.1. Types of the lichen
3
Figure 1.2. Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae)
5
Figure 1.3. Biosynthetic pathways of the major groups of lichen substances
7
Figure 2.1: Isolation of compounds from the prepicitate and petroleum ether
extracts of Parmotrema praesorediosum (Nyl.) Hale
26
Figure 2.2: Isolation of compounds from the chloroform extract of Parmotrema
praesorediosum (Nyl.) Hale
27
Figure 3.1. HMBC correlations of PRAES-C1 and PRAES-E14
36
Figure 3.2. HMBC correlations of PRAES-C10
37
Figure 3.3. HMBC correlations of PRAES-E19
41
42
Figure 3.5. Comparison of experimental CD spectrum of PRAES-C2Me and
theoretical calculated one.
44
Figure 3.6. CD spectra of isolated aliphatic compounds
45
Figure 3.7. HMBC correlations of PRAES-E1 and PRAES-T2
48
Figure 3.8. HMBC correlations of PRAES-E11, PRAES-T4 and PRAES-E2
52
Figure 3.9. HMBC and NOESY correlations of PRAES-C22
54
56
57
Figure 3.12. COSY, HMBC and NOESY correlations of PRAES-C25M
61
Figure 3.13. Mechanism for the methylation of PRAES-C25
62
Figure 3.14. HMBC and NOESY correlations of PRAES-C25 and PRAES-C26 63
xiv
Figure 3.15. HMBC correlations of PRAES-C9 and PRAES-C7
67
Figure 3.16. COSY and HMBC correlations of PRAES-E18
70
73
75
78
Figure 3.20. HMBC and ROESY correlations of PRAES-C16
80
85
Figure 3.22. 1H NMR data of PRAES-C18 and diphenyl ether
87
88
90
93
94
Figure 3.27. ROESY correlations of PRAES-C21
95
97
99
101
Figure 3.31. The structure of Usimine A
102
Figure 3.32. COSY, HMBC and ROESY correlations of PRAES-C27
104
109
Figure 3.34. COSY and HMBC correlations of PRAES-C28
111
112
114
118
xv
LIST OF APPENDICES
Appendices 1-7: IR, MS and NMR spectra of PRAES-C1
146
Appendices 8-15: IR, MS and NMR spectra of PRAES-E14
149
Appendices 16-21: MS and NMR spectra of PRAES-C10
153
156
Appendices 27-33: IR, MS and NMR spectra of PRAES-E19
159
162
Appendices 41-44: NMR spectra of PRAES-T1
166
Appendices 45-49: MS and NMR spectra of PRAES-E1
168
170
Appendices 55-58: NMR spectra of PRAES-E11
173
175
Appendices 63-66: MS and NMR spectra of PRAES-T6
177
179
181
185
189
193
Appendices 98-106: IR, MS and NMR spectra of PRAES-C25M
194
199
203
Appendices 120-124: NMR spectra of PRAES-C7
205
xvi
208
211
Appendices 137-141: NMR spectra of PRAES-C12
214
216
219
223
227
232
236
239
242
246
248
249
252
256
261
263
264
268
INTRODUCTION
Lichens are by definition symbiotic organisms composed of a fungal partner
(mycobiont) and one or more photosynthetic partners (photobiont/s). The photobiont
can be either a green alga or a cyanobacterium. Morphologically lichens can be
classified into three major groups. They are foliose, fruticose and crustose. Growing
rates of lichens are extremely slow. More than twenty thousand species of lichens
have been found. They can tolerate very drastic weather conditions and are resistant
to insects and other microbial attacks. Lichens produce a variety of secondary
compounds. They play an important role in protection and maintenance of the
symbiotic relationship [1].
Many lichen secondary metabolites exhibited antibiotic, antitumour,
antimutagenic, allergenic, antifungal, antiviral, enzyme inhibitory and plant growth
inhibitory properties [5, 12]. In 2007, Balaji. P. et al. [3] indicated that
dichloromethane, ethyl acetate and acetone methanol extracts of Parmotrema
praesorediosum showed antimicrobial activity against ten bacterial (Gram + and -)
(Bacillus cereus, Corynebacterium diptheriae, Proteus mirabilis, Proteus vulgari,
Pseudomonas aeruginosa, Salmonella typhi, Shigella flexnerii, Staphylococcus
aureus, Streptococcus pyogenes and Vibrio cholera) and one fungal Candida albicans
by using standard dics diffusion method. This lichen could therefore be a potential
source in the search for pharmaceutical useful chemicals.
The primary goal of the present work was to isolate secondary metabolites
on the lichen Parmotrema praesorediosum (Nyl.) Hale. The chemical structure of
isolated compounds was characterized by spectroscopic methods (1D-, 2D-NMR,
HRMS, CD). Finally, the purified substances from this source were assayed for the
cytotoxic activities against three cell lines: MCF-7 (breast cancer cell line), HeLa
(cervical cancer cell line) and NCI-H460 (human lung cancer cell line) by
1
sulforhodamine B colorimetric assay method (SRB assay) [56] and the inhibition
against acetylcholinesterase in vitro.
Based on spectroscopic evidence and their physical properties, the chemical
structures were attributed for be forty compounds, including six aliphatic acids,
twelve mononuclear phenolic acids, three depsides, two depsidones, eight diphenyl
ethers, three dibenzofurans, two xanthones, three triterpenoids and a macrocyclic
compound. The latter twenty two compounds appeared to be new and among
eighteen known compounds, twelve compounds were known for the first time from
the genus Parmotrema. These results pointed out that the Vietnamese lichens could
be new sources of bioactive compounds with novel skeletons
2
CHAPTER 1
LITERATURE REVIEW
1.1. GENERIC DESCRIPTION
1.1.1. The lichen
Lichens are by definition symbiotic organisms, usually composed of a fungal
which is most often either a green alga or cyanobacterium. The photobionts produce
carbohydrates by photosynthesis for themselves and for their dominant fungal
counterparts (mycobionts), which provide physical protection, water and mineral
supply [73]. Overall the lichen symbiosis is a very successful one, as lichens are
found in almost all terrestrial habitats from the tropics and deserts to polar regions.
As the results of the relationship, both the fungus and algae/cyanobacterium
partners, which mostly thrive in relatively moist and moderate environments in free
living form, have expanded into many extreme terrestrial habitats, where they
would separately be rare or non-existent [52]. On the basis of their forms and
habitats, lichens are traditionally divided into three main morphological groups:
crustose, foliose and fructicose (Figure 1.1) [42].
Crustose
Foliose
Figure 1.1. Types of the lichen
3
Fructicose
The lichen symbiosis is different other than kinds of symbiosis because the
lichen takes on a new body shape that neither the fungus nor the alga had
independently [73]. About 17,000 different lichen taxa, including 16,750 lichenized
Ascomycetes, 200 Deuteromycetes, and 50 Basidiomycetes have been described
world-wide. A thallus consists of a cortex and a medulla, both made up of fungal
tissue and a photobiont layer in which the alga and cyanobacterial cells are
endeveloped by fungal hyphae.
1.1.2. Parmotrema praesorediosum (Nyl.) Hale
The Parmeliaceae is a large and diverse family of Lecanoromycetes. With
over 2000 species in roughly 87 genera, it is regarded as the largest family of lichen
forming fungi [39]. The most speciose genera in the family are the well-known
groups: Xanthoparmelia (800+ species), Usnea (500+ species), Parmotrema (350+
species), and Hypotrachyna (190+ species) [39]. Nearly all members of the family
have a symbiotic association with a green alga (most often Trebouxia spp., but
Asterochloris spp. are known to associate with some species) [73]. The majority of
Parmeliaceae species have a foliose, fruticose, or subfruticose growth form. The
family has a cosmopolitan distribution, and can be found in a wide range of habitats
and climatic regions [73]. Members of the Parmeliaceae can be found in most
terrestrial environments
Parmotrema A. Massal. (previously known as Parmelia s.lat.) is one of the
largest genera of parmelioid core in the family Parmeliaceae [39]. The Parmotrema
genus is characterized by foliose thalli forming short and broad, rarely elongated,
often ciliate lobes, a pored epicortex, cylindrical conidia and the intermediate type
of lichenan between Cetraria-type lichenan and Xanthoparmelia-type lichenan. The
lower surface of the thallus is white to black, usually sparingly rhizinate with a wide
bare marginal zone, sometimes irregularly rhizinate or finely short-rhizinate with
scattered much longer rhizines mixed without an erhizinate margin or with a very
narrow one [72].
4
The upper surface
The lower surface
Figure 1.2. Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae)
Scientific name:
Parmotrema praesorediosum (Nyl.) Hale
Parmelia praesorediosa Nyl.
Family: Parmeliaceae
Morphography: Thallus foliose, adnate to the substratum, 3~10 cm across.
Lobes round, 4~10 mm wide; margins entire or crenate, eciliate, sorediate. Upper
surface pale grey to grey, smooth, dull, emaculate, weakly rugose, lacking isidia,
sorediate. Soralia marginal, linear to crescent shaped, granular. Medulla white.
Lower surface black, minutely rugose, with shiny, mottled, ivory or brown,
erhizinate marginal zone. Rhizines sparse, simple, short. Apothecia and pycnidia is
not seen [49].
Spotest: Cortex K+ (yellow), C−, KC−, P−; medulla K−, C−, KC−, P−
TLC: atranorin, chloroatranorin, fatty acids (protopraesorediosic acid,
praesorediosic acid).
5
1.2. CHEMICAL STUDIES ON THE LICHEN GENUS PARMOTREMA
1.2.1. Lichen secondary metabolites
Primary metabolites of lichens, which are intracellular, are proteins, amino
acids, polyols, carotenoids, polysaccharides and vitamins. Lichens produce a wide
array of secondary metabolites (intracellular). There are over 700 lichen substances
reported to date and many are restricted to the lichenised state. Broadly speaking,
there are three types of lichen substances based on their biosynthetic origin [43]
(Figure 1.2).
 The acetate-malonate pathway produces depsides, depsidones and
dibenzofurans. The most important of these are the esters and the oxidative
coupling products of simple phenolic units related to orcinol and 3-orcinol.
Most depsides and depsidones are colorless compounds which occure in the
medulla of the lichen. However, usnic acids, yellow cortical compounds
formed by the oxidative coupling of methylphloroacetophenone units are
found in the cortex of many lichen species. Anthraquinones, xanthones and
chromones, are all pigmented compounds which occur in the cortex. They
are also produced by the acetate-malonate pathway, but their biosynthesis
results from intramolecular condensation of long, folded polyketide units
rather than the coupling of phenolic units.
 The shikimic acid pathway produces two major groups of pigmented
compounds, which occur in the cortex: pulvinic acid derivatives and
terphenylquinones. Although most pulvinic acid derivatives lack nitrogen,
they are biosynthesized through phenylalanine. Nitrogen is strongly limited
to metabolic activities in most lichens, and nitrogen rich metabolites such as
alkaloids are unknown among lichen substances.
 The mevalonic acid pathway produces terpenoids and steroids. These
compounds are found in lichens and many of them occur in higher plants as
well.
6
Alga
Fungus
glucose
erythritol
ribitol
mannitol
Usnic acids Anthraquinones
Poly saccharides
Sugars
Methylphloroacetophenone/
acetylmethylphloroglucinol
Pentose phosphate cycle
Polyketide
Malonyl-CoA
Amino acids
Mevalonic
acid
Shikimic
acid
-Orsellinic acid
Depsones
Squalenes
Geranylgeranyl-p-p
Triterpens
Phenylalanine Terphenylquinones
Diterpenes
Pulvinic acid
derivatives
Secondary
aliphatic acids,
esters and related
derivatives
Glucolysis
Acetyl CoA
Phenylpyruvic acid
Xanthones,
Chromones
Orsellinic acid
and homologues
para-
meta-Depsides
Benzyl esters
Tridepsides
Dibenzofuran
Depsidones
Diphenyl
ethers
Steroids
Carotenoids
Figure 1.3. Biosynthetic pathways of the major groups of lichen substances [43].
7
1.2.2. Chemical studies on the lichen genus Parmotrema
 Parmotrema praesorediosum
(+)-Praesorediosic acid (1), (+)-protopraesorediosic acid (2), atranorin (11)
and chloroatranorin (12) were isolated by David F. et al. (1990) [20].
Lecanoric acid (14) and stictic acid (18) were isolated from Parmelia
praesorediosa (Nyl.) by Ramesh P. et al. (1994) [62].
 Parmotrema sancti-angelii
Atranorin (11), lecanoric acid (14) and α-collatolic acid (25) were isolated by
Neeraj V. et al. (2011) [55].
 Parmotrema conformatum
Protocetraric acid (21), malonprotocetraric acid (23) and (+)-usnic acid (40)
were isolated by Keogh M. F. (1977) [44].
 Parmotrema dilatum
Depside atranorin (11), depsidones salazinic acid (16), norstictic acid (19),
hypostictic acid (20) and protocetraric acid (21) were isolated from Parmotrema
dilatum by Honda N. K. et al. (2010) [32].
 Pamotrema lichexanthonicum
Depside atranorin (11), depsidone salazinic acid (16) and xanthone
lichexanthone (41) were isolated from the chloroform extract of Pamotrema
lichexanthonicum by Ana C. M. et al. (2009) [3].
 Parmotrema mellissii
Methyl orsellinate (5), ethyl orsellinate (6), n-butyl orsellinate (7), methyl βorsellinate (8), methyl haematommate (9), ethyl chlorohaematommate (10),
atranorin (11), chloroatranorin (12), α-alectoronic acid (24), α-collatolic acid (25),
2′′′-O-methyl-α-alectoronic
acid
(26),
2′′′-O-ethyl-α-alectoronic
acid
(27),
dehydroalectoronic acid (28), dehydrocollatolic acid (29), parmosidone A (30),
8
parmosidone B (31), parmosidone C (32), isocoumarin A (33), isocoumarin B (34),
β-alectoronic acid (36), β-collatolic acid (37), 2′′′-O-methyl-β-alectoronic acid (38),
2′′′-O-ethyl-β-alectoronic acid (39), (+)-usnic acid (40) and skyrin (42) were
isolated from Parmotrema mellissii that was collected at Da Lat city, Vietnam by
Lê Hoàng Duy et al. (2012) [52].
 Parmotrema nilgherrense
α-Alectoronic acid (24), α-collatolic acid (25) and dehydrocollatolic acid
(29) were isolated by Kharel M. K. et al (2000) [45].
Depside atranorin (11) were isolated by Neeraj V. et al. (2011) [55].
 Parmotrema planatilobatum
Orcinol (3), orsellinic acid (4), methyl orsellinate (5), methyl β-orsellinate
(8), methyl haematommate (9), atranorin (11), gyrophoric acid (13), lecanoric acid
(14), protocetraric acid (21), 9-methylprotocetraric acid (22), methyl 2-[3-(2,6dihydroxy-4-methylbenzyl)-2,4-dihydroxy-6-methylphenoxy]-3-formyl-4-hydroxy6-methylbenzoate (35) and usnic acid (40), were isolated by Duong T. H. et al.
(2011, 2012) [22, 23].
 Parmotrema reticulatum
Atranorin
(11),
chloroatranorin
(12),
salazinic
acid
(16)
and
consalazinic acid (17) were isolated from the acetone extract by Fazio A. T. et al.
(2009) [25].
 Parmotrema saccatilobum
Atranorin (11) and chloroatranorin (12) were isolated from the hexane
extract of Parmotrema saccatilobum by Bugni T. S. et al. (2009) [12].
 Parmotrema stuppeum
Orsellinic acid (4), methyl orsellinate (5), atranorin (11) and lecanoric acid
(14) were isolated by Javaprakasha G. K. et al. (2000) [40].
9
 Parmotrema subisidiosum
Depside atranorin (11) and two depsidones salazinic acid (16) and
consalazinic acid (17) were isolated from the acetone extract by O‟Donovan D. G.
et al. (1980) [57].
 Parmotrema tinctorum
Isolecanoric acid (15) was isolated by Sakurai A. et al. (1987) [63].
Ethyl orsellinate (6) was isolated by Santos L. C. et al. (2004) [64].
Atranorin (11) and lecanoric acid (14) were isolated by Honda N. K. et al.
(2010) [32].
Chemical structure of the compounds isolated from different species of
genus Parmotrema
Aliphatic acids
Mononuclear phenolic compounds
10
Depsides
Depsidones
11
12
Diphenylethers
13
Quinone
1. 3. BIOLOGICAL ACTIVITIES
1.3.1. The biological significance of lichen metabolites
Production of secondary metabolites is costly to the organisms in terms of
nutrient and energy, therefore one would expect that the plethora of metabolites
produced by lichens would have biological significance to the organisms. Recent
field and laboratory studies have shown that many of these compounds are indeed
involved in important ecological roles. Some of the possible biological functions of
lichen metabolites, are summarized as below [43]:
 Antibiotic activities – provide protection against microorganisms.
 Photoprotective activities – aromatic substances absorb UV light to protect
algae (photobionts) against intensive irradiation.
14
 Promote symbiotic equilibrium by affecting the cell wall permeability of
photobionts.
 Chelating agents – capture and supply important minerals from the substrate.
 Antifeedant/ antiherbivory activities – protect the lichens from insect and
animal feedings.
 Hydrophobic properties – prevent saturation of the medulla with water and
allow continuous gas exchange.
 Stress metabolites – metabolites secreted under extreme conditions.
1.3.2. The biological significance of the lichen genus Parmotrema
1.3.2.1. Antimicrobial activities
The lichen Parmotrema species were observed a marked dose dependent
inhibition of test bacteria by lichen extracts. It has been found that lichens of the
genus Parmotrema are promising antimicrobial agents. Balaji P. et al. [41] reported
marked antimicrobial efficacy of dichloromethane extract of P. praesorediosum
collected from silicious rocks of Western Ghats of Tamil Nadu. Kumar et al. [50]
showed the antibacterial activity of methanol extract of P. pseudotinctorum from
the Western Ghats of Karnataka. Sinha and Biswas [69] reported the antibacterial
efficacy of solvent extracts of P. reticulatum from Sikkim, India. Neeraj V. et al.
[44] found antibacterial efficacy of solvent extracts of P. nilgherrensis and P.
sancti-angelii collected from Karnataka, India. Chauhan and Abraham [14] showed
the inhibitory effect of methanol extract of Parmotrema sp. collected from
Kodaikanal forest, India against clinical isolates of bacteria. Javeria et al. [3]
showed the inhibitory efficacy of solvent extracts of P. nilgherrense collected from
Nainital, India against drug resistant bacteria.
1.3.2.2. Antioxidant activities
Lichens have been shown promising as they possess various bioactivities
including antioxidant activity. The DPPH free radical scavenging assay is one of the
most widely used assays to evaluate the antioxidant activity of several kinds of
15
samples including lichen extracts. The method is simple, rapid, sensitive and
requires small amount of samples. It has been found that Parmotrema species
possess radical scavenging activity. Kekuda et al. [50] observed dose dependent
DPPH radical scavenging activity in the lichen P. pseudotinctorum. Though the
scavenging of free radicals by lichen extracts was lesser than ascorbic acid, it is
evident that the extracts showed hydrogen donating ability and therefore the
extracts could serve as free radical scavengers, acting possibly as primary
antioxidants. Extract of P. grayanum showed high scavenging activity followed by
P. praesorediosum and P. tinctorum as indicated by lower IC50 value [76].
Methanol and ethanol extract of P. reticulatum have shown DPPH radical
scavenging activity [68] (Table 1.1).
1.3.2.3. Antitumor activities
The action of lichen-derived compounds on tumor cells has been a focus of
evaluations for some decades. Lichexanthone and protocetraric acid isolated from
the lichens Parmotrema dilatatum (Vain.) Hale and Parmotrema lichexanthonicum
Eliasaro & Adler were evaluated against UACC-62 and B16-F10 melanoma cells
and 3T3 normal cells by Sulforhodamine B assay [7]. A cytotoxicity assay was
carried out in vitro with sulforhodamine B (SRB) using HEp-2 larynx carcinoma,
MCF-7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 murine melanoma
cell lines, in addition to a normal (Vero) cell line in order to calculate the selectivity
index of the compounds from the lichen Parmotrema tinctorum [8]. The
relationship between O-alkyl salazinic acids from Parmotrema lichexantonicum
Eliasaro and Alder and potentially cytotoxic against human colon carcinoma (HCT8), melanoma (MDA-MB-435), and brain (SF-295) tumor cell was investigated by
Micheletti A. C. et al. [54].
16
Table 1.1. In vitro biological activities of the lichen genus Parmotrema.
Activity
Content
Source
Refferences
Against ten bacterial (Gram + and -) (Bacillus cereus, The dichloromethane, ethyl Balaji. P. et
Corynebacterium diptheriae, Proteus mirabilis, Proteus vulgari, acetate, acetone and methanol al. [3]
Pseudomonas aeruginosa, Salmonella typhi, Shigella flexnerii, extracts of P.praesorediosum
Staphylococcus aureus, Streptococcus pyogenes and Vibrio cholera)
and one fungal Candida albicans
Against eight bacterial (Gram + and -) Staphylococcus aureus, S. The methanol extracts of P. Vivek M. N.
epidermidis, Bacillus cereus, Klebsiella pneumoniae, Enterobacter tinctorum, P. grayanum and et al. [76]
aerogenes, Shigella flexneri, Salmonella typhi and Escherichia coli P. praesorediosum.
by Agar well diffusion assay.
Antimicrobial
Antibacterial
Antifungal
Against Bacillus subtilis, Erwinia chrysanthemi, Escherichia coli, The
methanol,
ethanol, Sati S. C. et
Agrobacterium tumefaciens and Xanthomonas phaseoli
chloroform extract of the al [65]
lichen P. nilgherrense
Against Pseudomonas aeruginosa, P. fluorescens, Proteus vulgaris, The acetone, methanol, ethyl Javeria S. et
Shegilla flexneri, Klebsiella pneumoniae and Salmonella typhi.
acetate and benzene extract al. [41]
of the lichen P. nilgherrense
Against Staphylococcus aureus, Clostridium perfringens, Escherichia The methanol extract of P.
coli and Pseudomonas aeruginosa by Agar well diffusion method
pseudotinctorum
Against Staphylococcus aureus and Pseudomonas aeruginosa by The methanol extract
Agar well diffusion method
Parmotrema sp.
Againts the bacterial strains Bacillus cereus, Bacillus subtilis,
Escherichia coli, Klebsiella pneumonia, Micrococcus luteus, Proteus
vulgaris, Staphylococcus aureus, Streptococcus faecalis, Sarcina
lutea and yeast strains Candida albicans, Cryptococcus var. diffluens.
17
Kumar S. V.
P et al. [50]
of Chauhan R.
et al. [14]
The ethyl acetate extract of Neeraj V. et
Parmotrema
nilgherrensis al. [44]
and
Parmotrema
sanctiangelii
Against Staphylococcus aureus and Escherichia coli
Parmotrema
lichexantonicum
Micheletti
A. C. [54]
Against five bacterial strains viz. Staphylococcus aureus, Escherichia The acetone and methanol Sinha S. N.
coli, Vibrio cholerae, Shigella dysenteriae. Shigella flexneri
extracts
of Parmotrema et al. [69]
reticulatum
DPPH radical scavenging activity
The benzene and acetone Javaprakash
extracts
of Parmotrema a G. K. et al.
stuppeum
[33]
The methanol extract exhibited marked antioxidant activity by The methanol extract of P.
scavenging DPPH* (free radical) and converting into DPPHH.
pseudotinctorum
Antioxidant
Kumar
S.V.P et al.
[50]
P. grayanum (IC50148.39μg/ml) showed higher scavenging potential The methanol extracts of P. Vivek M. N.
followed by P. praesorediosum (IC50 179.81μg/ml) and P. tinctorum tinctorum, P. grayanum and et al. [76]
(IC50 439.06μg/ml) by scavenging of DPPH radicals.
P.praesorediosum.
Determined by Malondialdehyde (MDA) assay and ABTS radical Parmotrema austrosinese and Vattem D.
quenching assay
Parmotrema perforatum
A. et al. [75]
DPPH radical scavenging activity.
The ethanol and methanol Sharma B.
extracts of the lichen C. et al. [68]
Parmotrema reticulatum
Active in superoxide radical (SOR), DPPH, and nitric oxid Parmotrema grayana Hue
scavenging activity.
Active in hydroxyl and hypochlorous radical scavenging, DPPH,
superoxide, singlet oxygen, nitric oxide and peroxynitrite scavenging
activity.
Yousuf S. et
al. [77]
The methanol extract of the Ghate N. B.
lichen
Parmotrema et al. [29]
reticulatum
Antioxidant activities by using DPPH, ABTS, superoxide, and The ethyl acetate extract of Raj P. S. et
hydroxyl radical scavenging assay.
Parmotrema tinctorum
al [61]
18
Activity
Content
Source
Refferences
Antitumor activity against malignant cell lines of erythro leukemia.
Parmotrema
dilatatum Yousuf S. et
(Vain.)
Hale
and
Parmotrema
al. [77]
Antiproliferative against capan-1 and -2, PANC-1 (parcrease), NCIH1415 (lung cell), PC-3 (prostate), T47-D (breast), AGS (stomach), tinctorum (Nyl.) Hale
NTH: OVCAR-3 (ovaries) and JURKAT (acute promyelocytic, Tcell and erythrocell leukemina cell lines).
Toxicity test of against Artemia salina with BSLT method
Cytotoxicity
Anticancer
Antitumor
The dichloromethane extract
and phenolic compounds of
the lichen P. tinctorum
Kusumaning
rum I. K.. et
al. [51]
Gomes A.
T. et al. [30]
Cytotoxic against MCF-7 cells with an IC50 value 130.03±3.11 g/ml
The methanol extracts of the Ghate N. B.
lichen
Parmotrema et al. [29]
reticulatum
Against human colon carcinoma (HCT-8), melanoma (MDA-MB435), and brain (SF-295) tumor cell
Parmotrema
Micheletti
lichexantonicum Eliasaro and A. C. [54]
Alder
Cytotoxic against on B16-F10 murine melanoma, UACC-62 human
melanoma cells and NIH/3T3fibroblasts by sulforhodamine B (SRB)
assay
Parmotrema
dilatatum Brandão L.
(Vain.) Hale and Parmotrema F. G. et al.
lichexantonicum Eliasaro and [7]
Alder
In vitro with sulforhodamine B (SRB) using HEp-2 larynx arcinoma, Parmotrema tinctorum (Nyl.) Bogo D. et
MCF7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 urine Hale
al. [8]
melanoma cell lines
19
CHAPTER 2
EXPERIMENTAL
2.1. MATERIALS AND ANALYSIS METHODS
 TLC was carried out on precoated silica gel 60 F254 (Merck) and precoated
Kieselgel 60F254 plates (Merck).
 Gravity column chromatography was performed with silica gel 60 (Merck)
and silica gel 60 (0.040 – 0.063 mm, Himedia).
 TLC spots were detected under ultraviolet (UV254) irradiation or visualized
by spraying with a solution of 5% vanillin in ethanol, followed by heating at
100 oC.
 Solvents: Hexane, diethyl ether, petroleum ether (60-90
o
C), toluene,
chloroform, ethyl acetate, acetone, methanol, acetic acid.
 Melting points were determined on Maquenne block(a).
 The NMR experiments using residual solvent signal as internal reference:
chloroform-d H 7.24, C 77.23 and acetone-d6 H 2.09, C 206.31, 30.6 were
performed with:
 Bruker Avance 500III (500 MHz for 1H and 125 MHz for 13C-NMR(a, b).
 Varian VXR-500 spectrometers, with tetramethylsilane as internal
standard(c).
 The HR–ESI–MS were recorded on
 HR–ESI–MS MicroOTOF–Q mass spectrometer(a).
 Hitachi M-4100 mass spectrometer(c).
20
 The IR spectra were obtained with
 Bruker Vector 22 infrared spectrophotometer(a).
 Shimadzu FTIR-8200 infrared spectrophotometer(c).
 Optical rotations were measured on
 Kruss (German) digital polarimeter(a).
 Jasco DIP-370 digital polarimeter(c).
 Absorption and CD spectra were measured on
 Jasco V-570 spectrophotometer(d).
 Jasco J-820E spectropolarimeter(d).
 TD-DFT calculations of the CD spectra were optimized at the level of
B3LYP/6-311++G** in vacuo and in CPCM solvent model of methanol. The
populations of the two stable conformers were calculated based on the
relative energies with the Boltzmann distribution at 300 K. The optimization
under the CPCM solvent model of methanol did not change significantly
these geometries or populations. The electronic CD spectra of the stable
conformers were calculated at the TD-DFT theory with the same basis sets as
the optimizations by using Gaussian09 program, fitted by Gaussian curves
with 0.30 eV line width, and then weighted-averaged based on the
Boltzamann population.
(a)
The Center Analysis of the University of Science, National University- Ho
Chi Minh City, Vietnam.
(b)
The Institute of Chemistry, Vietnam Academy of Science and Technology,
Hanoi, Vietnam.
(c)
Life Science Center, Kobe Pharmaceutical University, Japan.
(d)
Osaka University, Japan.
21
2.2. LICHEN MATERIALS
The lichen Parmotrema praesorediosum (Nyl.) Hale was collected at Nam
Cat Tien National Forest Reserve and Intermediate Zones, Nam Cat Tien Village,
Tan Phu District, Dong Nai Province, Vietnam in January-July 2009. The scientific
name of the lichen was determined by MSc. Vo Thi Phi Giao, Faculty of Biology,
University of Science, National University – Ho Chi Minh city.
A voucher specimen (No US-B020) was deposited in the Herbarium of The
Department of Organic Chemistry, Faculty of Chemistry, University of Science,
National University - Ho Chi Minh City-Vietnam.
2.3. EXTRACTION AND ISOLATION PROCEDURES
The fresh lichen thalli (5.0 kg) were cleaned under running tap water and airdried. The ground powder sample (3.0 kg) was extracted with methanol at room
temperature by method of maceration. After filtration, the solvent was evaporated at
the reduced pressure. While the methanolic solution was evaporated, a precipitate
occurred and was filtered off, then the solution was continued evaporated to
dryness. The resulting was the precipitate (9.0 g) and the crude methanolic residue
(450.0 g).
The methanolic residue (450.0 g) was subjected to silica gel solid phase
extraction and eluted consecutively with petroleum ether, chloroform, ethyl acetate,
acetone and methanol in turn at room temperature to afford petroleum ether E1
extract (25.0 g), petroleum ether E2 extract (15.0 g), chloroform extract (105.0 g),
ethyl acetate extract (50.0 g), acetone extract (45.0 g) and methanol extract (37.0 g)
(Figure 2.1).
2.3.1. Isolating compounds from the methanol precipitate (Figure 2.1)
The precipitate (9.0 g) was silica gel chromatographed, eluted with
petroleum ether–chloroform to give 5 fractions (symboled as fraction T1 to fraction
T5).
22
 Fraction T2 (1.0 g) was silica gel rechromatographed and eluted with
hexane–chloroform (8:2) to give five compounds: PRAES-T1 (9.0 mg),
PRAES-T2 (7.0 mg), PRAES-T3 (5.0 mg), PRAES-T4 (45.0 mg) and
PRAES-T6 (40.0 mg).
2.3.2. Isolating compounds from the petroleum ether E1 extract (Figure 2.1)
The petroleum ether E1 (25.0 g) was applied to silica gel column
chromatography, eluted with petroleum etherethyl acetate (10:0-5:5) to give 8
fractions (symboled as fraction E1.1 to fraction E1.8)
 Fraction E1.3 (4.5 g) was separated by silica gel column chromatography,
eluted with petroleum ether–ethyl acetate (98:2) to give compound PRAESE2 (5.0 mg).
 Fraction E1.4 (3.7 g) was silica gel rechromatographed, eluted with
petroleum ether–ethyl acetate (98:2) to give two compounds, coded PRAESE1 (200.0 mg) and PREAS-E6 (5.0 mg).
eluted with petroleum ether–ethyl acetate (95:5) to give PRAES-E3 (4.0 mg).
eluted with petroleum ether–ethyl acetate (95:5) to give PREAS-E5 (1.0 g).
2.3.3. Isolating compounds from the petroleum ether E2 extract (Figure 2.1)
The petroleum ether E2 (15.0 g) was applied to silica gel column
chromatography, eluted with petroleum ether–ethyl acetate (9:1-5:5) to give 9
fractions (symboled as fraction E2.1 to fraction E2.9).
 Fraction E2.4 (2.8 g) was silica gel rechromatographed, eluted with
petroleum ether–ethyl acetate (95:5) to give compound PRAES-E11 (about
1.5 g).
23
 Fraction E2.5 (4.5 g) was silica gel rechromatographed and eluted with
petroleum ether–ethyl acetate (9:1) to give two compounds PRAES-E13 (5.0
mg) and PRAES-E14 (25.0 mg).
 The same manner was applied on fraction E2.6 (3.7 g) eluted with petroleum
ether–ethyl acetate (9:1) to afford two compounds, coded PRAES-E15
(30.0 mg) and PRAES-E17 (50.0 mg) and on fraction E2.7 (2.5 g) eluted
with petroleum ether–ethyl acetate (9:1) to afford two compounds
PRAES-E18 (15.0 mg) and PRAES-E19 (250.0 mg).
2.3.4. Isolating compounds from the chloroform extract
The chloroform extract (105.0 g) was subjected to silica gel column
chromatography and eluted by the solvent system of petroleum ether–ethyl acetate
with increasing ethyl acetate ratios to obtain twenty three fractions from C1 to C23
(Figure 2.2).
 Fraction C13 (5.7 g) was silica gel rechromatographed, eluted with
petroleum ether–chloroform (8:2) to give three compounds: PRAES-C2
(about 1 g), PRAES-C4 (50.0 mg) and PRAES-C5 (5.0 mg).
petroleum ether–chloroform (8:2) to give two compounds: PRAES-C1 (200.0
mg) and PRAES-C3 (5.0 mg).
petroleum ether–chloroform (5:5) to give three compounds: PRAES-C7 (7.0
mg), PRAES-C8 (15.0 mg) and PRAES-C11 (295.0 mg).
chloroform–methanol (95:5) to give three compounds: PRAES-C10 (15.0
mg), PRAES-C12 (15.0 mg) and PRAES-C14 (5.0 mg).
24
 Fraction C19 (6.1 g) was applied on silica gel column and eluted with a
gradient solvent system of chloroform–acetone (95:5) to give three fractions
(C19a, C19b and C19c).
 Fraction C19a (1.0 g) was silica gel rechromatographed, eluted with
chloroform–acetone (98:2) and subjected to pre TLC using chloroform–
methanol (9:1 and 95:5) as eluent to afford PRAES-C15 (5.0 mg).
 Fraction C19b (3.2 g) was silica gel rechromatographed, eluted with
chloroform–acetone (98:2) to give six fractions (C19ba to C19bf).
Fraction C19ba (169.6 mg) was subjected to pre TLC (chloroform–
methanol, 95:5, 9:1 and n-hexane–diethyl ether, 5:5) to afford three
compounds PRAES-C18 (7.0 mg), PRAES-C20 (10.5 mg) and PRAESC21 (18.7 mg). Fraction C19bc (454.3 mg) was subjected to pre TLC
(chloroform–methanol, 95:5) to afford PRAES-C16 (28.1 mg).
 Fractions C20 (23.9 g) was repeatedly subjected to silica gel column
chromatography, eluted with chloroform–methanol (10:0-9:1) to obtain eight
fractions (from C20a to C20h). The fraction C20c (5.8 g) was subjected to
silica gel chromatography with solvent of chloroform–methanol to get six
fractions (from C20ca to C20cf) (Figure 2.2).
 Fractions C20cb (979.3 mg) was silica gel rechromatographed, eluted
with n-hexane–diethyl ether and continuously subjected to pre TLC
(n-hexane–diethyl ether (2:8) and chloroform–methanol (98:2) to afford
five compound PRAES-C22 (8.0 mg), PRAES-C23 (71.7 mg), PRAESC24 (6.2 mg), PRAES-C25 (15.7 mg) and PRAES-C26 (6.3 mg).
 Fraction C20ce (839.5 mg) was silica gel rechromatographed, eluted with
chloroform–methanol and subjected to pre TLC with different kinds of
solvents (chloroform–methanol, 98:2 and toluene–acetone, 8:2) to afford
two compounds PRAES-C27 (61.9 mg) and PRAES-C28 (21.4 mg).
25
Fresh lichen (5.0 kg)
0) - Cleaned and dried, ground.
Air-dried lichen powder (3.0 kg)
- Macerated with methanol, room temp.
- Solvent was partly evaporated.
- Evaporated to dryness.
Precipitate
(9.0 g)
Crude methanolic residue (450.0 g)
- Solid phase extraction.
- Eluted with solvents of different polarities.
Petroleum ether
E1 (25.0 g)
Petroleum ether
E2 (15.0 g)
- CC with P:EA (10:0-5:5).
E1.3 (4.5 g)
E1.4 (3.7 g)
P:EA (98:2)
PRAES-E2 (5.0 mg)
PRAES-E10)(200.0 mg)
(98:2)
0) (5.0 mg)
PRAES-E6
0)
P:EA (95:5)
E1.6 (4.7 g)
Ethyl acetate
(50.0 g)
- CC with P:EA (9:1-5:5).
P:EA
0)
P:EA (95:5)
E1.5 (2.5 g)
0)
Chloroform
(105.0 g)
0) (4.0 mg)
PRAES-E3
E2.4 (2.8 g)
E2.5 (4.5 g)
P:EA (95:5)
P:EA
(9:1)
0)
E2.6 (3.7 g)
P:EA
(9:1)
0)
0) (1.0 g)
PRAES-E5
0)
Acetone
(45.0 g)
E2.7 (2.5 g)
Figure 2.1. Isolation of compounds from the precipitate and
0)
petroleum ether extracts of Parmotrema praesorediosum (Nyl.) Hale
26
- CC with P:C (9:1-0:10).
Methanol
(37.0 g)
PRAES-E11 (1.5 g)
Fraction T2
(1.0 g)
- CC.
- Eluted with H:C (8:2).
0) (5.0 mg)
PRAES-E13
PRAES-T1 (9.0 mg)
0) (25.0 mg)
PRAES-E14
0) (7.0 mg)
PRAES-T2
0) (30.0 mg)
PRAES-E15
0) (50.0 g)
PRAES-E17
P:EA
0) (15.0 mg)
PRAES-E18
(9:1)
0)(250.0 mg)
PRAES-E19
0)
0) (5.0 mg)
PRAES-T3
0)(45.0 mg)
PRAES-T4
0)(40.0 mg)
PRAES-T6
0)
Chloroform extract (105.0 g)
CC with P:EA (10:0-0:10).
C13 (5.7 g)
C15 (3.4 g)
C16 (4.2 g)
P:C
(8:2)
P:C (8:2)
PRAES-C2 (1.0 g)
PRAES-C1 (200.0 mg)
PRAES-C4 (50.0 mg)
0)
PRAES-C3 (5.0 mg)
0)
PRAES-C5 (5.0 mg)
0)
PRAES-C7 (7.0 mg)
0)
0)
PRAES-C8 (15.0 mg)
0)
PRAES-C11 (295.0 mg)
P:C (5:5)
PRAES-C10 (15.0 mg)
C17 (6.1 g)
C19 (6.1 g)
C:M
(95:5)
C:Ac
10:0-0:10
0)
PRAES-C12 (15.0 mg)
PRAES-C15 (5.0 mg)
0)
PRAES-C14 (5.0 mg)
0)
PRAES-C16 (28.1 mg)
0)
0)
PRAES-C18 (7.0 mg)
C19a (1.0 g)
C19b (3.2 g)
C:Ac
(98:2)
C:Ac
(98:2)
0)
PRAES-C20 (10.5 mg)
PRAES-C21 (18.7 mg)
PRAES-C22 (8.0 mg)
PRAES-C23 (71.7 mg)
C20 (23.9 g)
C:M
10:0-9:1
C20c (5.8 g)
C:M
(95:5)
0)
PRAES-C24 (6.2 mg)
0)
PRAES-C25 (15.7 mg)
0)
PRAES-C26 (6.3 mg)
0)
PRAES-C27 (61.9 mg)
PRAES-C28 (21.4 mg)
Figure 2.2. Isolation of compounds from the chloroform extract of
Parmotrema praesorediosum (Nyl.) Hale
27
2.4. PREPARATION OF SOME DERIVATIVES
2.4.1. Esterification of PRAES-C2
The esterification of PRAES-C2 with methanol, ethanol, n-propanol, nbutanol and isopentanol in the presence of concentrated H2SO4 as a catalyst was
prepared by using the following procedure:
 Praes-C2 (x mg) was completely dissolved in the chosen alcohol (ml) which
was acidified with concentrated H2SO4 for pH control of 0-1.
 The reaction mixture was stirred and refluxed.
 The progress of the reaction was monitored by thin layer chromatography.
 The resulting mixture was concentrated in vacuo to give the crude product,
which was then purified by preparative silica gel TLC.
Methylation of PRAES-C2: PRAES-C2 (20 mg) in 10 ml methanol was
acidified with H2SO4 98% to get pH = 0-1. This solution was stirred for 8 hours at a
temperature of 80 oC. The reaction mixture was concentrated at the reduced pressure
and the residue was purified by preparative TLC (petroleum ether– chloroform, 5 :
5) to yield PRAES-C2Me (4 mg).
Ethylation of PRAES-C2: PRAES-C2 (20 mg) in 10 ml ethanol was
acidified with H2SO4 98% to get pH = 0-1. This solution was stirred for 8 hours at a
temperature of 80 oC. The reaction mixture was concentrated under vacuum
condition and the residue was purified by preparative TLC (petroleum ether–
chloroform, 5 : 5) to yield PRAES-C2Et (6.0 mg).
n-Propylation of PRAES-C2: PRAES-C2 (20 mg) in 10 ml n-propanol
which was acidified with H2SO4 98% to get pH = 0-1. The solution was stirred for 9
hours at a temperature of 80 oC. The reaction mixture was concentrated under the
reduced pressure and the residue was purified by preparative TLC (petroleum
ether–chloroform, 5 : 5) to yield PRAES-C2Pro (8.2 mg).
28
2.4.2. Methylation of PRAES-C25
The methylation of PRAES-C25 with TMS-CH2N2 was prepared as the
following procedure: To a solution of PRAES-C25 (12.0 mg) in Et2O (1 ml) and
MeOH (0.5 ml), TMS-CH2N2 in n-hexane were added. The solution was stirred at
room temperature for 1 hour and 15 mins. After termination by diluted acetic acid in
MeOH, the reaction mixture was concentrated in vacuo and the residue was purified
by preparative TLC (n-Hexane-Et2O, 2:8) to yield PRAES-C25M (4.1 mg).
2.5. BIOLOGICAL ASSAYS
2.5.1. Cytotoxicity
Determination of cytotoxic activities against the HeLa (human epithelial
carcinoma), MCF–7 (human breast cancer) and NCI-H460 (human lung cancer) cell
lines of tested samples was performed at the concentration of 100 g/mL using the
Sulforhodamine B (SRB) assay with camptothecin as the positive control [71].
Samples were sent to be in vitro tested at the Faculty of Biology, University of
Science, Vietnam National University- Ho Chi Minh City, 227 Nguyen Van Cu
Street, District 5, 784355 Ho Chi Minh City, Vietnam. The person in charge of the
unit: Assoc. Prof. Ho Huynh Thuy Duong.
All cells were cultured in E‟MEM medium (Eagle‟s Minimal Essential
Medium) supplemented with 10% foetal bovine serum (FBS), 1% of 2 mM Lglutamine, 50 IU/mL penicillin, 50 μg/mL streptomycin and maintained at 37 °C in
a 5% CO2 atmosphere with 95% humidity. Viable cells were counted and
inoculated in 96-well plate with density of 104 cells/100 μL/well. After 24 h the
cells were treated with pure compound while the control wells were added only by
100 μL medium.
All experiments were performed in triplicate. The plates were incubated in an
atmosphere of 5% CO2, 95% humidity at 37°C for 48 h. Adherent cell cultures were
fixed by adding 50 μL of cold 50% (w/v) trichloroacetic acid per well and incubated
29
at 4°C for 1 h. The plates were washed five times with distilled water and air dried.
Then a solution of 50 μL of SRB (0.4% w/v in 1% acetic acid) was added to each
well and allow staining at room temperature for 30 mins. The SRB solution was
removed out of plates by rinsing 4 times with a 1% glacial acetic acid solution (200
μL/well). The plates were air-dried for 12–24 h. The bound SRB was dissolved to
each well by adding 100 μL of 10 mM Tris Base (pH 10.5). The plates were shaken
gently for 20 mins and the optical density of each well was read using a scanning
multiwall spectrophotometer at a test wavelength of 492 nm and a reference
wavelength of 620 nm.
The optical density (OD) of SRB in each well is directly proportional to the
cell number. Cell survival was measured as the percentage absorbance compared to
the control (non-treated cells). Evaluation of the result based on the I% (at the
concentration of 100 g/mL): 0–49% (inactive), 50–70% (active), 70–90% (strong),
90–100% (very strong) [78].
% Inhibitive activity 
With:
OD
ODc  ODs
x100
ODc
= OD‟ tested sample – OD‟ blanck
OD‟ = OD492 – OD620
ODc = OD of 0.25% DMSO
ODs = OD tested sample
2.5.2. In vitro acetylcholinesterase (AChE) inhibition assay
The isolated compounds from Parmotrema praesorediosum (Nyl.) Hale were
screened for their acetylcholinesterase inhibitory activity at the concentration of 100
µg/ml through Ellman‟s colorimetric method [28]. All the experiments were done in
Department of Pharmacology, Ho Chi Minh City Medicine and Pharmacy
University, 41 Dinh Tien Hoang Str., Dist. 1, Ho Chi Minh City, Vietnam. The
person in charge of the unit: Assoc. Prof. Vo Phung Nguyen.
30
The principle of the method is the enzyme acetylcholinesterase (AchE)
hydrolyzes the substrate (Acetylthiocholine is used as the substrate) to give a
compound. This compound will further react with Ellman reagent (5,5‟-dithio-bis2-nitrobenzoate ion or DTNB) to give 2-nitrobenzoate-5-mecaptothiocholine and 5thio-2-nitrobenzoate. The later possesses a yellow color and therefore, the rate of
color production is measured at 412 nm by a spectrophotometer. The reaction with
the thiol has been shown to be sufficiently rapid so as not to be rate limiting in the
measurement of the enzyme, and the used concentrations do not inhibit the enzymic
hydrolysis. By recording the output of the photometer continuously, records of the
complete assay can be obtained. All samples were tested in triplicate.
Absorbance was measured at 412 nm, and the percent inhibitive activity was
determined by comparison with the negative control as the following formula:
% Inhibitive activity 
With:
A N  AT
x100 %
AN
AN
= OD412 of negative control
AT
= OD412 of tested sample
Evaluation of the result based on the I% (at the concentration of 1.0 mg/ml):
0–30% (very weak), 30–50% (weak), 50–70% (average), 70–90% (strong), 90–
100% (very strong).
31
CHAPTER 3
RESULTS AND DISSCUSSION
3.1. CHEMICAL STRUCTURE ELUCIDATION
From the lichen Parmotrema praesorediosum (Nyl.) Hale, 40 lichen
substances were isolated including 22 new compounds along with 18 known ones.
The structure of all compounds was elucidated on the basis of NMR and MS
spectroscopies. All new compounds were checked by Scifinder in Kobe
Pharmaceutical University, Japan in March 2012. In order to well interpretation,
they were divided into nine groups as listed in Table 3.1.
Table 3.1. Isolated compounds from Parmotrema praesorediosum
Group
Type of compound
Total
New
Known
compounds compounds
1
Aliphatic acids
6
5
1
2
Mononuclear phenolic compounds
12
5
7
3
Depsides
3
0
4
4
Depsidones
2
0
2
5
Diphenyl ethers
8
7
1
6
Dibenzofurans
3
1
2
7
Xanthones
2
2
0
8
Triterpenoids
3
1
2
9
Macrocyclic compound
1
1
0
32
3.1.1. Chemical structure elucidation of aliphatic acids
3.1.1.1. Structure elucidation of compound PRAES-C1
 White needles (acetone).
 Melting point: 104–105 C.
  D + 518 (c= 0.006, EtOH).
23

 IR spectrum (Appendix 1): IR (KBr) max cm-1: 3444 (OH), 1740 (C=O
lactone), 1706 (C=O carboxyl), 1217 (CO).
 CD spectrum (Figure 3.6).
 Mass spectrum (Appendix 2): HR-ESI-MS m/z 395.2781 [M+H]+ (calcd. for
C23H38O5 + H, 395.2797)
 1H NMR spectrum (CDCl3) (Appendix 3): see Table 3.2.

13
C NMR and DEPT spectra (CDCl3) (Appendix 4, 5): see Table 3.3.
 HSQC and HMBC spectra (CDCl3) (Appendix 6, 7).
Compound PRAES-C1 was obtained as white needles. The HR-ESI-MS
showed the pseudomolecular ion peak at m/z 395.2781 [M+H]+ corresponding to the
molecular formula C23H38O5 which implied five degrees of unsaturation. Its IR
spectrum displayed two intense absorptions at 1740 cm-1 and 1706 cm-1 that were
assigned to the lactone and the conjugated carboxyl functional group.
The NMR spectra (Tables 3.2 and 3.3) exhibited signals attributable to an methyl-,-unsaturated––lactone moiety [H 2.23 (3H, d, J=2.0 Hz, H-5), 5.12
(1H, m, H-4); C 11.1 (C-5), 81.6 (C-4), 139.3 (C-2), 147.2 (C-3), 172.9 (C-1)] and
an aliphatic side chain [H 1.25–1.28 (m, -CH2-), 1.58 (m, H-7), 2.11 (m, H-7), 2.15
(s, terminal -CH3), 2.43 (t, J=7.5 Hz, H-21); C 24.0, 24.8, 29.3–29.9, 32.9, 43.9
(-CH2-), 30.0 (terminal -CH3)].
33
Despite the absence of the signal at C 210.6 in the
13
C NMR spectrum of
compound PRAES-C1, the HMBC correlations from H-21 (H 2.43) and H-23 (H
2.15) to a carbon signal at C 210.6 confirmed the presence of a ketone group at C22 of the aliphatic side chain of PRAES-C1. Complete analysis of the HSQC and
HMBC data as well as combining the HR-ESI-MS for PRAES-C1 resulted in its
planar
structure
as
4-methyl-5-oxo-2-(16-oxoheptadecyl)-2,5-dihydrofuran-3-
carboxylic acid. The absolute stereochemistry of the sole stereogenic centre C-4 in
PRAES-C1 was determined to be R by comparison of its ultraviolet CD spectral
data (Figure 3.6) with that reported for isomuronic acid isolated from Neuropogon
trachycarpus [7]. PRAES-C1 was dextrorotatory therefore it was a homologous
compound of isomuronic acid with the side chain containing two more methylene
units. Therefore, the structure of PRAES-C1 was determined as (+)-4-methyl-5oxo-2-(16-oxoheptadecyl)-2,5-dihydrofuran-3-carboxylic
acid
or
(+)-
vinapraesorediosic acid A.
3.1.1.2. Structure elucidation of compound PRAES-E14
  D + 837 (c= 0.006, EtOH).
23

34
 Mass spectrum (Appendix 9): HR-ESI-MS m/z 409.2988 [M+H]+ (calcd. for
C24H40O5 + H, 409.2954).

13
 COSY, HSQC and HMBC spectra (CDCl3) (Appendix 13, 14, 15).
Compound PRAES-E14 was isolated as white needles and its molecular
formula was determined as C24H40O5 through its pseudomolecular ion peak at m/z
409.2988 [M+H]+ in the HR-ESI-MS spectrum.
The comparison of spectral data, i.e. NMR, IR, CD of PRAES-C1 and
PRAES-E14 showed the similarity (Table 3.2 and 3.3), except for the presence of
an additional methoxy group in PRAES-E14. The 1H and
13
C NMR spectra of
PRAES-E14 showed signals at H 3.89 (3H, s, OCH3) and C 52.3 (OCH3),
characteristics of a methyl ester. In the HMBC spectrum of PRAES-E14, signal of
this methoxy group (H 3.89) gave cross peak to C-6 (C 162.7) indicating that the
methoxy group was at C-6.
The 2D NMR experiments proved the proposed structure to be correct and
allowed the completed characterization of compound PRAES-E14 as (+)-methyl 4methyl-5-oxo-2-(16-oxoheptadecyl)-2,5-dihydrofuran-3-carboxylate
methyl vinapraesorediosate A.
35
or
(+)-6-
  D + 22 (c= 0.0013, CHCl3).
23

 Mass spectrum (Appendix 16): HR-ESI-MS m/z 419.2754 [M+Na]+ (calcd.
for C23H40O5 + Na, 419.2775).
 1H NMR spectrum (Acetone-d6) (Appendix 17): see Table 3.2.

13
C and DEPT NMR spectra (Acetone-d6) (Appendix 18, 19): see Table 3.3.
 HSQC and HMBC spectra (Acetone-d6) (Appendix 20, 21).
The 1H and
13
C NMR spectra as well as CD spectrum of PRAES-C10 were
similar to those of PRAES-C1, including signals of the α-methyl-α,β-unsaturated-lactone and the aliphatic side chain which were presented in Tables 3.2 and 3.3.
However, some chemical shift differences were observed due to a modification
36
of the terminal side chain. A sharp singlet (3H, H-23) at H 2.15 as well as a
triplet (2H, J = 7.5 Hz, H-21) at H 2.43 corresponding to the terminal 2oxopropyl group of the aliphatic side chain of
compound PRAES-C1
were
replaced by signals at H 1.10 (3H, d, J = 6.5 Hz), 3.70 (1H, sext, J = 6.5 Hz) and
2.05 (2H, m) for a terminal 2-hydroxypropyl group of the side chain of compound
PRAES-C10.
As expected, the signal at C 210.6 (C-22) of the carbonyl carbon in
compound PRAES-C1 was replaced by the signal at C 68.0 (C-22) for a
hydroxylated secondary carbon in compound PRAES-C10. This point was further
corroborated by the HR-ESI-MS of PRAES-C10 which showed a typical quasi–
molecular ion peak at m/z 419.2754 [M+Na]+ (calcd. for C23H40O5Na, 419.2775).
On the basis of these observations, (+)-2-(16-hydroxyheptadecyl)-4-methyl-5-oxo2,5-dihydrofuran-3-carboxylic acid or vinapraesorediosic acid B proved to be a new
natural compound.
37
Table 3.2.1H NMR of aliphatic compounds
No
PRAES-C1(a)
PRAES-E14(a)
PRAES-C10(b)
H, J (Hz)
H, J (Hz)
H, J (Hz)
No
PRAES-C11(b)
PRAES-E19(a)
PRAES-C2(a)
H, J (Hz)
H, J (Hz)
H, J (Hz)
4
5.12
m
5.09
m
5.14
m
4
5.14
m
5.09
m
5.12
m
5
2.23
d (2.0)
2.18
d (2.0)
2.12
d (2.0)
5
2.12
d (2.0)
2.18
d (2.5)
2.24
d (2.0)
3.89
s
3.88
s
1.56
m
1.39
m
1.55
m
1.60
m
2.07
m
1.62
m
2.05
m
2.14
m
br s
1.29
br s
(-CH2-)n
br s
1.25
br s
t (7.5)
2.05
m
19
1.60
m
1.64
m
2.32
t (7.5)
3.70
sext (6.5)
20
2.27
t (7.5)
2.34
t (7.5)
1.10
d (6.5)
20-OCH3
6-OCH3
7
(-CH2-)n
–
1.58
m
2.11
m
1.25–1.28
21
2.43
22
–
23
2.15
br s
t (7.5)
1.25–1.27
2.41
–
s
2.13
s
–
6-OCH3
7
a) Measured in chloroform-d. b) Measured in acetone-d6.
38
–
1.35
m
2.05
m
1.29–1.38
–
br s
1.23–1.30
–
–
–
3.68
s
Table 3.3.13C NMR of aliphatic compounds
No
PRAESC1(a)
PRAESE14(a)
PRAESC10(b)
C
C
C
No
PRAESC11(b)
PRAESE19(a)
PRAESC2(a)
C
C
C
1
172.9
172.9
173.8 1
173.4
173.1
172.9
2
139.3
137.5
137.5 2
137.3
137.6
139.6
3
147.2
147.6
150.0 3
149.4
147.8
147.3
4
81.6
81.4
82.4 4
81.8
81.6
81.5
5
11.1
10.8
11.2 5
10.8
11.0
11.1
6
164.9
162.7
164.4 6
163.9
162.8
165.9
7
32.9
32.8
33.8 7
33.4
33.0
32.9
8
24.8
24.7
27.1 8
25.5
24.8
25.1
29.3–29.9
29.2–29.6
29.4–30.4
29.2–29.7
29.3–29.7
20
24.0
23.9
25.9 18
21
43.9
43.8
40.7 19
25.7
24.9
34.3
22
210.6
209.3
68.0 20
34.3
34.1
175.0
30.0
–
29.8
52.3
174.7
–
179.5
52.5
–
–
9–19
23
6-OCH3
29.9–30.8 9–17
24.4 21
– 6-OCH3
24.9
20-OCH3
51.7
  D + 513 (c= 0.001, MeOH).
23

for C21H34O6 + Na, 405.2253).

13
C NMR spectra (Acetone-d6) (Appendix 24): see Table 3.3.
39
Examination of NMR, IR, MS, and physical data of compound PRAES-C11
showed that they were in good agreement with those reported for (+)-praesorediosic
acid, isolated by David et al. from this lichen [20]. Detailed 2D-NMR analysis
and comparison with the reported data [22] led us to determine the structure of
compound
PRAES-C11
as
(+)-2-(14-carboxyltetradecyl)-4-methyl-5-oxo-2,5-
dihydrofuran-3-carboxylic acid or (+)-praesorediosic acid.
23
  D + 450 (c= 0.001, EtOH).

 Mass spectrum (Appendix 28): HR-ESI-MS m/z 397.2596 [M+H]+ (calcd.
for C22H36O6 + H, 397.2590).

13
C and DEPT NMR spectra (CDCl3) (Appendix 30, 31): see Table 3.3.
40
The NMR spectra of PRAES-E19 were similar to those of PRAES-C11,
except for the presence of an additional methoxy group in PRAES-E19. The HMBC
correlations between the methoxy group at H 3.88 (s) to signal at C 162.8 (C-6) in
compound PRAES-E19 indicated the location of a methoxy group at C-6. These
spectral features were identical with those of (+)-methyl 2-(14-carboxyltetradecyl)4-methyl-5-oxo-2,5-dihydrofuran-3-carboxylate or 6-methyl praesorediosate. This
assignment was supported by the detailed IR, MS, 2D NMR studies as shown.
 D + 130 (c= 0.001, EtOH).
23


41
for C21H34O6 + Na, 405.2254).

13
Compound PRAES-C2 was isolated as white needles and its high resolution
mass spectrum showed a quasi–molecular ion peak at m/z 405.2200 [M+Na]+
corresponding to the molecular formula of C21H34O6 which was identical to
PRAES-C11. The 1H and 13C NMR spectra of PRAES-C2 were also similar to those
of PRAES-C11, including signals of the α,β-unsaturated--lactone and the aliphatic
side chain which were presented in Table 3.2 and 3.3.
However, compound PRAES-C2 differed from PRAES-C11 in the aliphatic
side chain. The terminal carboxyl group in the side chain of PRAES-C11 was
replaced by a methoxycarbonyl group and the length of the chain of PRAES-C2 is
less one methylene group comparing to PRAES-C11. This was proved by the
presence of an additional singlet signal at H 3.68 (3H, OCH3) in the 1H spectrum as
well as a further signal at C 51.7 in the 13C spectrum of PRAES-C2. This methoxy
group was at C-20 by the HMBC correlations between signal at H 3.68 to the
carboxyl carbon at C 175.0 (C-20). The HMBC experiments confirmed the
presence of a carboxyl group at C-3 of the lactone ring as normal by correlations of
42
the methyl signal at H 2.24 (H-5) to signals at C 165.9 (carboxyl carbon), 172.9
(C-1), 139.6 (C-2) and 147.3 (C-3).
Compound PRAES-C2 was esterified with methanol, ethanol and n-propanol
to give PRAES-C2Me, PRAES-C2Et, PRAES-C2Pro, respectively, proved the
proposed structure of PRAES-C2 to be correct. This showed that under the acidic
condition, the lactone ring in PRAES-C2 could not be opened, therefore, the
obtained product possessed two ester functional groups. These data identified
compound PRAES-C2 as (+)-4-methyl-2-(13-methoxycarbonyltridecyl)-5-oxo-2,5dihydrofuran-3-carboxylic acid or (+)-vinapraesorediosic acid C.
Determining the absolute configuration at the C-4 position of aliphatic
compounds (Figure 3.5 and 3.6)
The depicted relative stereochemistry of the dihydrofuranone ring was
established on the basic of the CD spectrum. PRAES-C2 showed a negative Cotton
effect at ~225 nm associated to the n   transition of lactone chromophore. In
view of the more intense CD, the assignment of the absolute configuration of the
methylated derivative PRAES-C2-Me was confirmed by circular dichroism
calculations between the experimental spectrum and the theoretical calculation the
assignment of the absolute configuration. Theoretical calculations of the CD
spectrum of a model molecule of PRAES-C2-Me were executed with timedependent density functional theory (TD-DFT) of the B3LYP functional and the 643
311++G** basis set. The simulated spectrum under vacuum (Figure 3.5, center)
well reproduced the experimental / CD couplet at 225/260 nm by the
simulated couplet at 240/270 nm. The similar result was obtained by the
calculation under a CPCM solvent model of methanol (Figure 3.5, center).
The CPCM solvent model increased the absorption and CD intensities, but
the ratio (/) of the couplet was similar to that in vacuo. The agreements between
the experiment and the calculations indicated that PRAES-C2-Me possessed the R
chirality at the C-4 position. The absolute configuration at the C-4 position in the
dihydrofuranone ring of compounds PRAES-C1, PRAES-C10, PRAES-C11,
PRAES-E19, PRAES-C2, PRAES-C2Et and PRAES-C2Pro was suggested to be R
by comparison of their CD spectra with that of compound PRAES-C2Me (Figure
3.6) as well as with data in the literature [7].
As the biosynthetic aspect, the assignment of the chiral carbon C-4 of
PRAES-E14, a methyl ester derivative of PRAES-C1, was also proposed to be R, as
they were isolated from the same material and possessing similar positive optical
rotation.
Figure 3.5. Comparison of experimental CD spectrum of PRAES-C2Me in
methanol (left), theoretical CD spectra (center) calculated in vacuo (dotted line) and
in CPCM solvent model of methanol (solid), and two stable conformers (S1 and S2)
of a model molecule of PRAES-C2Me with 4R configuration with simulated
populations at 300 K. The calculation conditions: TD-DFT/B3LYP/6-311++G**
and /CPCM(MeOH). The calculated spectra were weighted-averages of calculated
spectra of S1 and S2.
44
Figure 3.6. CD spectra of isolating aliphatic compounds
3.1.2. Monoaromatic compounds
3.1.2.1. Structure elucidation of compound PRAES-T1
 Colorless needles (Methanol)
 Melting point: 145-146 C.
 1H and 13C NMR spectra (CDCl3) (Appendix 41, 42): see Table 3.4.
Compound PRAES-T1 was obtained as colorless needles. The 1H NMR
spectrum displayed signals for a methoxy group at H 3.96 (3H, s), a formyl group
at H 10.34 (H-8), a methyl group at H 2.53 (3H, s), an aromatic methine proton at
H 6.29 (s, H-5), and two chelated hydroxyl protons (H 12.86 and 12.40). The 13C,
DEPT NMR spectra showed the resonances of 10 carbons including one aromatic
methyl (C 25.2), a methyl ester group [C 172.0 (C-7), 52.3 (C-10)], one formyl
45
group (C 193.9) and six substituted aromatic carbons, two of which were
oxygenated. The exact location of the aromatic protons and the substituted
functional groups was established based on 2D NMR (HSQC and HMBC). In the
HMBC spectrum, the aldehydic proton H-8 (H 10.34) showed correlations with C3 (C 108.5) and the oxygenated carbon C-4 (C 166.7). The methoxy proton (H
3.96) showed correlations with carboxyl carbon (C 172.0). Two chelated hydroxyl
protons (H 12.86 and 12.40) jointed to C-2 and C-4, based on their HMBC
correlations with carbon signals at C 168.3 and 166.7, respectively. Similarly, the
aromatic methine proton at H 6.29 (H-5) showed HMBC correlations with C-3 (C
108.5) and C-4 (C 166.7). These spectroscopic data were suitable with the
published data [74], therefore compound PRAES-T1 was methyl haematommate.
 Yellow needles (Ethanol).
for C10H9O5Cl+Na, 267.0036).
Compound PRAES-E1 was isolated as yellow needles. The spectral data of
PRAES-E1 and PRAES-T1 were similar, with a remark that PRAES-E1 lacked the
aromatic proton signal. The HR-ESI-MS (positive mode) displayed the
46
pseudomolecular ion peak at m/z = 266.9874 (calcd. 267.0036 for C10H9O5ClNa),
corresponding to the molecular formula of C10H9O5Cl. Moreover, the HR-ESI-MS
(positive mode) showed two ion peaks at m/z 266.9874 and 268.9839. These two
peaks separated by 2 mass atomic units and with a ratio of 3 : 1 in their peak
heights,
confirming that the molecule contained one chlorine atom.
This
observation, together with the presence of chlorine in the molecule, suggested that
compound PRAES-E1 was methyl chlorohaematommate [38].
Table 3.4. NMR data of PRAES-T1, PRAES-E1, PRAES-T2 (CDCl3)
No
PRAES-T1
H, J (Hz)
PRAES-E1
C
H, J (Hz)
PRAES-T2
C
H, J (Hz)
C
1
103.9
105.2
104.0
2
3
168.3
108.5
165.4
108.5
168.4
108.5
4
166.7
162.4
166.6
112.1
152.3
114.9
148.8
172.0
171.4
5
6
6.29 s
7
6.28 s
112.1
152.3
171.7
8
10.34 s
193.9
10.31 s
193.6
10.34 s
193.9
9
2.53 s
25.2
2.66 s
20.7
2.54 s
25.2
10
3.96 s
52.3
3.99 s
52.7
4.38 t (6.5)
65.8
11
1.78 dt (7.5, 7.0)
30.5
12
1.49 sext (7.5)
19.4
13
0.99 t (7.5)
13.6
2-OH
12.86 s
12.61 s
12.98 s
4-OH
12.40 s
13.07 s
12.39 s
47
 White paraffin oil.
 1H,
13
C and DEPT NMR spectra (CDCl3) (Appendix 50, 51, 52): see Table
3.4.
Compound PRAES-T2 was obtained as white paraffin oil. The spectral data
of compounds PRAES-T1 and PRAES-T2 were similar. The 1H NMR spectrum
displayed signals for a butyl group [H 4.38 (2H, t, J=6.5 Hz), 1.78 (2H, dt, J=7.5
Hz, 7.0 Hz), 1.49 (2H, sext, J=7.5 Hz), 0.99 (3H, t, J=7.5 Hz)]. The
13
C NMR of
compound PRAES-T2 also showed signals for a butyl group (C 65.8, 30.5, 19.4,
13.6). The exact location of the aromatic proton and the substituted functional group
were established based on 2D NMR (Figure 3.7). These spectroscopic data
proposed that compound PRAES-T2 was n-butyl haematommate.
48
 Yellow needles (Methanol).
 1H and 13C NMR spectra (Acetone-d6) (Appendix 55, 56): see Table 3.5.
Compound PRAES-E11 was obtained as yellow needles. The 1H NMR
spectrum displayed signals for a methoxy group at H 3.91 (3H, s), methyl group at
H 2.45 (3H, s), two aromatic methine protons [H 6.23 (d, J=2.0 Hz, H-3) and 6.27
(d, J =2.0 Hz, H-5)], and two hydroxyl protons at H 11.58, 9.26. The
13
C, DEPT
NMR spectra showed the resonances of 9 carbons including one aromatic methyl
(C 24.2), a methyl ester [C 172.9 (C=O), 52.1 (OCH3)], and six substituted
aromatic carbons, two of which were oxygenated.
In the HMBC spectrum, the methoxy protons (H 3.91) showed the
correlation with the carbonyl carbon (C 172.9). Two hydroxyl protons (H 11.58
and 9.26) jointed to C-2 and C-4, based on their HMBC correlations with carbon
signals at C 166.3 and 163.4, respectively (Figure 3.8).
Similarly, the C-3 aromatic methine proton (H 6.23) showed HMBC
correlation with C-2 (C 166.3) and C-4 (C 163.4). The methyl group (H 2.53)
jointed to C-6 on the basis of its HMBC correlation with carbon signal at C 144.3
and C-5 (C 112.3). These spectroscopic data were suitable with the published one
[6], compound PRAES-E11 was methyl orsellinate.
49
 Colorless scale.
The NMR spectral features of PRAES-T4 were similar to those of PRAESE11 except for the presence of an additional methyl group (δH 2.10, δC 7.6) and the
absence of an aromatic proton. Moreover, the HMBC correlation from the methyl
group (δH 2.10) to C-2, C-3 and C-4 suggested the structure of PRAES-T4 to be
methyl β-orsellinate (Figure 3.8) [38].
 Yellow needles.
 Mass spectrum (Appendix 63): HR-ESI-MS (positive mode) m/z 153.0516
[M+H]+ (calcd. for C8H8O3+H, 153.0552).
 1H and 13C NMR spectra (Acetone-d6) (Appendix 64, 65): see Table 3.5.
 HMBC spectra (Acetone-d6) (Appendix 66).
The molecular formula of PRAES-T6 was established as C8H8O3 by HRESI-MS. The 1H NMR spectrum of PRAES-T6 showed six singlets for two chelated
hydroxyl groups at δH 10.67, a formyl proton at δH 10.26, two aromatic protons at
δH 6.25 and a methyl group at δH 2.23. Its
50
13
C NMR spectrum showed 8 carbon
signals including a methyl group, two aromatic methines, a formyl group, and four
quaternary aromatic carbon signals. The exact location of the aromatic protons and
the substituted functional groups was established based on 2D NMR (HSQC and
HMBC). These spectroscopic data were suitable with the published atranol [38].
Therefore, PRAES-T6 was identified as atranol.
 Colorless oil.
The NMR spectral features of compound PRAES-E2 resembled those of
PRAES-E11. The differences were the presence of a n-propyl group [-CH2: δH 2.83
(quint, J=6.5, 1.5 Hz), δC 38.9; -CH2: δH 1.55 (sext, J= 8.0, 7.5, 5.0 Hz), δC 24.9 and
-CH3: δH 0.95 (t, J=7.5 Hz), δC 14.2] instead of a methyl group as in PRAES-E11
and the addition of a methoxy group. The substitution pattern was confirmed by
HMBC correlations (Figure 3.8). Accordingly, the structure of PRAES-E2 was
established as methyl divaricatinate [38].
51
Table 3.5. NMR data of PRAES-E11, PRAES-T4, PRAES-T6 and PRAES-E2
No
PRAES-E11(a)
H, J (Hz)
PRAES-T4(b)
C
H, J (Hz)
PRAES-T6(a)
C
H, J (Hz)
1
105.3
105.3
2
166.3
3
6.23
d (2.0)
4
5
6.27
d (2.0)
H, J (Hz)
C
104.7
163.1
163.1
165.6
101.6
108.6
109.3
163.4
158.1
163.1
6.20
s
110.6
6
144.3
140.2
7
172.9
172.6
3-CH3
2.10
s
6.25
s
C
108.5
112.3
6.25
PRAES-E2(b)
s
108.5
6.34
d (2.5)
98.9
164.0
6.29
d (2.5)
151.5
110.7
147.7
172.0
7.6
3-CHO
10.26
s
194.2
4-OCH3
3.80
s
55.3
3.92
s
51.8
2.83
quint (6.5, 1.5)
38.9
2
1.55
sext (8.0, 7.5, 5.0)
24.9
3
0.95
t (7.5)
14.2
7-OCH3
3.91
s
52.1
3.92
s
51.8
1
2.45
s
24.2
2.45
s
24.0
2.23
s
2-OH
11.58
s
12.02
s
10.67
s
4-OH
9.26
s
5.22
s
10.67
s
22.4
a) Measured in acetone-d6. b) Measured in chloroform-d.
52
11.68
s
 White amorphous solid.
lactone), 1630 (C=C), 1363 (CO).
 Mass spectrum (Appendix 72): HR-ESI-MS (positive mode) m/z: 255.0862
[M+H]+ (calcd. for C12H14O6+H, 255.0869).
 1H, 13C and DEPT NMR spectra (CDCl3) (Appendix 73, 74, 75): Table 3.6.
 HSQC, HMBC and NOESY spectra (CDCl3) (Appendix 76, 77, 78).
Compound PRAES-C22 was obtained as a white amorphous solid. The
molecular formula C12H14O6 was deduced from a pseudomolecular ion [M+H]+ at
m/z 255.0862 in the HR-ESI-MS. The IR spectrum showed characteristic
absorptions for a hydroxyl group (3243 cm−1) and a lactone group (1785 cm−1).
The 1H NMR spectrum displayed signals for three methoxy groups at H
3.53, 3.62 and 3.86 (each 3H, s), two methylene protons at H 4.84 and 4.88 (each
1H, d, J = 14.0 Hz), an aromatic proton at H 6.88 (s), an acetalic methine proton at
H 6.33 (s) and a hydroxyl phenolic proton at H 9.08 (s). The 13C NMR spectrum
showed the signals for three methoxy groups (C 56.1, 56.3 and 59.3), a sp3
methylene (C 70.0), an aromatic methine (C 97.6), an acetalic methine carbon (C
102.2), a carboxyl group (C 168.8) and one penta-substituted benzene ring (Table
3.6). The IR, 1H NMR and
13
C NMR data indicated the presence of an α,-
unsaturared--lactone.
The position of functional groups of PRAES-C22 was determined by
analysis of 2D NMR spectra (NOESY, HSQC and HMBC). The HMBC correlation
from methoxy groups at H 3.62 (H3-1), 3.53 (H3-1), 3.86 (H3-9) to an acetalic
methine at C 102.2 (C-3), to the oxygenated methylene carbon at C 70.0 (C-8) and
53
to an oxygenated carbon at C 159.7 (C-6) suggested that three methoxy groups
were at C-3, C-6 and C-8, respectively.
This was confirmed by the NOESY experiment which showed the
correlations between the methoxy group at H 3.53 (H3-1) and methylene protons
at H 4.84 and 4.88 (H2-8), between the methoxy group at H 3.62 (H3-1) with the
acetalic methine proton at H 6.33 (H-3), and between the methoxy group at H 3.86
(H3-9) with the aromatic proton at H 6.88 (H-7), respectively.
The HMBC correlations from signal of an aromatic proton at H 6.88 (H-7)
to carboxyl carbon at C 168.8 (C-1). The NOESY cross peaks between hydroxyl
phenolic group at H 9.08 and methoxy protons at H 3.62 (H3-1') indicated that the
hydroxyl group located at C-4 (Figure 3.9).
Accordingly, the structure of PRAES-C22 was determined as praesalide A.
The stereochemistry of the sole chiral center C-3 was not determined.
54
lactone), 1625 (C=C), 1341 (CO).
[M+H]+ (calcd. for C13H16O6+H, 269.1026).
 1H,
13
C and DEPT NMR spectra (CDCl3) (Appendix 81, 82, 83): see Table
3.6.
Compound PRAES-C23 was obtained as a white amorphous solid and the
molecular formula was established as C13H16O6 by HR-ESI-MS, with 14 mass
atomic units more than that of PRAES-C22.
The IR,
1
H and
13
C NMR spectra of PRAES-C23 revealed a close
relationship to those of PRAES-C22, except for the presence of an ethoxy group at
C-3 instead of the methoxy group as in PRAES-C22.
The HMBC correlations from an acetalic methine at H 6.40 (H-3) to the
oxygenated methylene carbon at δC 65.3 (C-1) and from methylene protons at H
3.86 and 3.94 (H2-1) to the acetalic carbon at δC 101.5 (C-3) indicated that this
ethoxy group located at C-3. This was confirmed by the NOESY cross peaks which
showed the proximity in space between oxygenated methylene protons at H 3.86
and 3.94 (H2-1) with methyl protons at H 1.33 (H3-2) and with the acetalic
methine at H 6.40 (H-3).
The substitution pattern was confirmed by HSQC, HMBC and NOESY
correlations (Figure 3.10).
55
These results revealed the structure of PRAES-C23 as 3-ethoxy-4-hydroxy6-methoxy-5-methoxymethylphthalide or praesalide B. The absolute configuration
of C-3 was not established. Praesalide B was identified as a new structure isolated
from natural lichen.
lactone), 1624 (C=C), 1339 (CO).
[M+H]+ (calcd. for C14H18O6+H, 283.1182).
56
 1H, 13C and DEPT NMR spectra (CDCl3) (Appendix 89, 90, 91): see Table
3.6.
The HR-ESI-MS of PRAES-C24 indicated the molecular formula of
C14H18O6. The IR and NMR spectral features of PRAES-C24 were similar to those
of PRAES-C23 but PRAES-C24 showed signals of an ethoxy group at C-8 instead
of the methoxy group as seen in PRAES-C23 (Table 3.6).
The HMBC correlations from methylene protons at H 3.70 (H2-1) to the
oxygenated methylene carbon at δC 68.1 (C-8) and methylene protons at H 4.86
and 4.92 (H2-8) to the carbon at δC 67.6 (C-1) indicated that this ethoxy group
located at C-8. This was supported by the NOESY cross peaks between H2-1 at H
3.70 and H2-8 at H 4.86 and 4.92 (Figure 3.11).
allowed the completed characterization of compound PRAES-C23 as 3-ethoxy-4hydroxy-6-methoxy-5-ethoxymethylphthalide, although the absolute configuration
of the sole carbon at C-3 was not established.
57
Compound
3-ethoxy-4-hydroxy-6-methoxy-5-ethoxymethylphthalide
or
praesalide C was identified as a new structure isolated from natural lichen.
Table 3.6. NMR data of PRAES-C22, PRAES-C23 and PRAES-C24 (CDCl3)
No.
PRAES-C22
δH
J (Hz)
1
3
PRAES-C23
δC
δH
J (Hz)
168.8
6.33 s
102.2
PRAES-C24
δC
δH
J (Hz)
169.0
6.40 s
101.5
δC
169.0
6.40 s
101.5
3a
124.5
124.9
124.8
4
153.3
153.2
153.3
5
116.1
116.0
116.2
6
159.7
159.6
159.5
7
6.88 s
7a
8
97.6
6.87 s
97.5
128.6
4.84 d (14.0)
70.0
4.88 d (14.0)
6.86 s
128.6
4.83 d (14.0)
70.0
4.88 d (14.0)
97.4
128.5
4.86 d (14.0)
68.1
4.92 d (14.0)
9
3.86 s
56.1
3.85 s
56.1
3.85 s
56.1
1'
3.62 s
56.3
3.86 dq (9.5, 7.0)
65.3
3.86 dq (9.5, 7.0)
65.3
3.94 dq (9.5, 7.0)
2'
1''
3.53 s
59.3
1.33 t (7.0)
15.1
1.33 t (7.0)
15.1
3.53 s
59.3
3.70 q (7.0)
67.6
1.32 t (7.0)
15.0
2''
4-OH
9.08 s
3.94 dq (9.5, 7.0)
9.03 br s
58
9.33 s
 Yellow solid.
[M+Na]+ (calcd. for C11H12O6+Na, 263.0532).
 1H NMR spectra (DMSO-d6) (Appendix 96): see Table 3.7.
 HMBC spectra (DMSO-d6) (Appendix 97).
 Structure elucidation of compound PRAES-C25M
 Yellow solid.
carboxyl), 1626 (C=C), 1238 (CO).
[M+H]+ (calcd. for C13H16O6+H, 269.1026).
 1H,
13
C and DEPT NMR spectra (CDCl3) (Appendix 100, 101, 102): see
Table 3.7.
 COSY, HSQC, HMBC and NOESY spectra (CDCl3) (Appendix 103, 104,
105, 106).
The 1H NMR spectrum of PRAES-C25 showed signals for two methoxy
groups at H 3.21 and 3.87 (each 3H, s), a methylene group at H 4.40 (s), an
aromatic proton at H 6.86 (s), an aldehydic proton at H 10.42 (br s) and a chelated
hydroxyl group at H 12.47 (br s).
However, the
13
C NMR spectrum only displayed signals for two methoxy
groups (C 56.0 and 57.3), a methylene group (C 61.1) and two carboxyl carbons
(C 162.9 and 168.4). The chemical structure of PRAES-C25 could not be
59
determined by the spectral data, therefore, compound PRAES-C25 was methylated
with an excess of TMS-CH2N2 to yield the PRAES-C25M derivative.
The HR-ESI-MS of PRAES-C25M established the molecular formula of
C13H16O6. Its 1H NMR spectrum exhibited signals for three methoxy groups at H
3.37, 3.87 and 3.90 (each 3H, s), two methylene groups [H 3.37 (1H, m) and
3.57 (1H, dd, J = 18.0, 6.5 Hz)] and [H 4.52 and 4.56 (1H each, d, J = 10.5 Hz)],
a hemiacetalic proton at H 6.15 (1H, dd, J = 6.5, 2.5 Hz) and an aromatic proton at
H 7.09 (1H, s). The 13C NMR revealed the resonances for 13 carbons which were
assigned for three methoxy groups (C 52.1, 56.2 and 58.2), two methylene groups
(C 38.9 and 63.1), a hemiacetalic carbon (C 101.8), a carboxyl carbon (C 166.6)
and six aromatic carbons.
The hemiacetalic proton H-11 of PRAES-C25M resonated at H 6.15 as
doublet of doublets (J = 6.5, 2.5 Hz) due to the coupling to the vicinal methylene
protons H2-12 (H 3.37 and 3.57). The HMBC correlations from this proton at
H 6.15 (H-11) to two aromatic carbons at C 120.4 (C-2) and C 158.5 (C-3) and
the COSY spectrum showed the sequence from the methylene (H2-12) to the
oxygenated methine (H-11) suggested the appearance of a benzofuran skeleton.
The position of the oxygenated methylene group (C-8) was determined by the
HMBC correlations from signal at H 4.52 and 4.56 (H2-8) to signals of two
oxygenated carbons at C 158.5 (C-3) and 158.8 (C-5).
The HMBC correlations between the aromatic proton at H 7.09 (H-6) with
signals of the carboxyl carbon at C 166.6 (C-7), with four aromatic carbons at
C 158.5 (C-5), 126.5 (C-1), 120.4 (C-2), 113.1 (C-4) as well as the correlations of
the two methoxy signals at H 3.90 (H3-13), 3.87 (H3-9) with signals at C 158.5
(C-5), 166.6 (C-7) indicated that the ester group located at C-1 (C 166.6). The
NOESY interaction from H-6 to the methoxy group H 3.87 helped to support the
60
position of these substituents. The substitution pattern was confirmed by HSQC,
HMBC and NOESY correlations (Figure 3.12).
Consequently, the structure of PRAES-C25M was elucidated as methyl 2hydroxy-6-methoxy-7-methoxymethyl-2,3-dihydrobenzofuran-4-carboxylate
or
praesalide D. The stereochemistry of the sole chiral center C-2 was not determined.
Prolonged treatment of PRAES-C25 with an excess of diazomethane in
methanol proceeded with the methylation of the carboxyl group and cyclization to
form benzofuranol skeleton (Figure 3.13). This specific mechanism is supported by
several observations. The first was the reaction of aldehyde PRAES-C25 with
61
diazomethane to form homoligated aldehyde in a modification of the Buchner–
Curtius–Schlotterbeck reaction [10, 58, 66].
The reaction has since been extended to form hemiacetal by acid-initiated
nucleophilic addition of aldehyde with alcohol. This result with benzofuranol
cyclization had been reported by Franko-Filipasic B. R. et al [26, 70].
Compound PRAES-C25 was isolated as a yellow solid and showed a
pseudomolecular ion peak at m/z 263.0523 in its ESI-MS spectrum indicative the
molecular formula of C11H12O6Na. Further analysis of HMBC spectra for PRAESC25 revealed that H-6 (δH 6.86) correlated with a carbon signal at δC 113.3 and the
methoxy group H3-9 (δH 3.87) as well as the methylene protons H2-8 (δH 4.40)
correlated with a oxygenated carbon signal at δC 162.9, confirming the presence of
aromatic carbon C-4 (δC 113.3) and C-5 (δC 162.9) (Figure 3.14).
62
Complete analysis of the HMBC data as well as combining the HR-MS for
PRAES-C25
showed
that
it
was
2-formyl-3-hydroxy-5-methoxy-4-
(methoxymethyl)benzoic acid or praesalide E. Compound PRAES-C25 was a new
structure isolated from natural lichen.
 Yellow solid.
carboxyl), 1615 (C=C), 1286 (CO).
63
 1H,
13
Table 3.7.
Compound PRAES-C26 was isolated as a yellow solid. The HR-ESI-MS
(positive mode) displayed a pseudomolecular ion peak with m/z 307.0787 [M+Na]+,
corresponding to the molecular formula of C13H16O7. The 1H NMR spectrum of
PRAES-C26 showed signals of two methoxy groups at H 3.39 and 3.75 (each 3H,
s), one methylene group at H 4.57 (s), a chelated hydroxyl group at H 10.20 (s), an
ethoxy group at H 4.24 (d, J = 7.0 Hz) and 1.21 (t, J = 7.0 Hz) and an aromatic
proton at H 6.62 (s).
The
13
C NMR revealed the resonant signals for 13 carbons, including two
methoxy groups (C 56.0 and 58.4), a methylene carbon (C 63.7), two carboxyl
carbons (C 169.1 and 173.6), an ethoxy group (C 62.1 and 13.7) and six aromatic
carbons.
The substitution pattern of PRAES-C26 was confirmed by HMBC and
NOESY correlations. The methylene protons at H 4.57 (H2-8) showed HMBC
cross peaks with two oxygenated aromatic carbons C-3 (C 159.3) and C-5 (C
161.5). The presence of a carboxyl group at C-1 (C 106.5) was also inferred
through HMBC correlations from the aromatic proton at H 6.62 (H-6) to the
carboxyl carbon C-7 (C 173.6). The position of the methoxy group (C-9) was
determined by the HMBC correlation from H3-11 (H 3.75) to the oxygenated
aromatic carbon C 161.5 (C-5). This was supported by the NOESY cross peaks of
the methoxy protons H3-9 at H 3.75 to the aromatic proton H-6 at H 6.62 and to
the methylene protons H2-8 at H 4.57 (Figure 3.14). Consequently, the structure of
PRAES-C26 was proposed to be 2-ethoxycarbonyl-3-hydroxy-5-methoxy-464
methoxymethylbenzoic acid. PRAES-C26 was a new compound isolated from
natural lichen and was named as praesalide F
Table 3.7. NMR of PRAES-C25, PRAES-C25M and PRAES-C26
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
3-OH
PRAES-C25(a)
δH J (Hz)
δC
δH
PRAES-C25M(b)
J (Hz)
113.3
162.9
6.86
4.40
3.87
3.21
10.42
12.47
s
s
s
s
br s
168.4
61.1
56.0
57.3
7.09
s
4.56
4.52
3.87
3.37
6.15
3.37
3.57
3.90
d (10.5)
d (10.5)
s
s
dd (6.5, 2.5)
m
dd (18.0, 6.5)
s
δC
126.5
120.4
158.5
113.1
158.8
104.1
166.6
63.1
6.62
s
4.57
s
56.2
58.2
101.8
38.9
3.75
3.39
s
s
4.24
d (7.0)
52.1
1.21
10.20
t (7.0)
s
s
a) Measured in DMSO-d6 b) Measured in chloroform-d.
65
δH
PRAES-C26(b)
J (Hz)
δC
106.5
113.9
159.3
113.9
161.5
102.6
173.6
63.7
56.0
58.4
169.1
62.1
13.7
3.1.3. Depsides
 White powder.
 1H,
13
Table 3.8.
Compound PRAES-T3 was a depside. In the 1H NMR spectrum, all of the
ten resonances are singlets. It displayed a methoxy group at H 3.98 (s), a formyl
group at H 10.36 (s), three methyl groups at H 2.69 (s), 2.54 (s), 2.09 (s), two
isolated aromatic methine protons at H 6.51 and 6.40 (1H each, s) and three
chelated hydroxyl protons at H 12.52, 12.48 and 11.91 (1H each, s).
The
13
C, DEPT-NMR spectra displayed two carboxyl groups (C 172.2 and
169.1), an aldehyde carbon (C 193.8), a methoxy group (C 52.3), three methyl
groups (C 25.5, 23.9 and 9.3) and twelve aromatic carbons. These findings implied
that compound PRAES-T3 was composed of two mono-aromatic units,
haematommic acid and β-orsellinic acid. The substitution pattern was confirmed by
HSQC and HMBC correlations.
These spectroscopic data were suitable with the published one [38, 74]. All
these properties suggested that compound PRAES-T3 was atranorin.
66
Table 3.8. NMR data of PRAES-T3, PRAES-C7 and PRAES-E18
No
PRAES-T3(b)
δH J (Hz)
PRAES-C7(a)
δC
δH J (Hz)
PRAES-E18(b)
δC
δH J (Hz)
1
2
102.9
169.1
103.9
161.5
3
4
5
6
7
8
9
10
11
2-OH
4-OH
108.6
167.5
112.8
152.4
169.7
193.8
25.5
109.5
164.3
111.8
140.8
171.1
7.8
24.3
6.40 s
10.36 s
2.69 s
6.48 s
2.08 s
2.60 s
δC
104.6
164.4
6.38
s
6.38
s
2.98
1.74
0.94
3.83
m
sext (7.5)
t (7.5)
s
98.9
165.5
110.9
148.8
168.9
38.8
24.8
14.3
55.4
12.48 s
12.52 s
1
110.3
115.3
104.5
2
162.9
163.7
156.9
3
116.8
115.3
124.9
4
152.0
151.0
156.0
5
6.51 s
116.0
6.41 s
114.5
6.43
s
106.2
6
139.8
140.5
146.8
7
172.2
174.8
175.5
8
2.09 s
9.3
2.00 s
9.4
2.98
m
39.0
9
2.54 s
23.9
2.62 s
23.4
1.66
sext (7.5)
25.2
10
3.98 s
52.3
1.00
t (7.5)
14.3
3.89
s
56.0
11
2-OH
11.91 s
4-OH
a) Measured in acetone-d6. b) Measured in chloroform-d.
 Colorless needles.
67
 1H, 13C and DEPT NMR spectra (Acetone-d6) (Appendix 120, 121, 122): see
Table 3.8.
Compound PRAES-C7 was obtained as colorless needles. The 1H NMR
spectrum showed singlets for four methyl groups at δH 2.00 (s), 2.08 (s), 2.60 (s)
and 2.62 (s) and two aromatic protons at δH 6.41 (s) and 6.48 (s). The 13C and DEPT
NMR exhibited 18 carbons corresponding to four methyl groups (C 24.3, 23.4, 9.4
and 7.8), two aromatic methines (C 111.8 and 114.5) and twelve quaternary
carbons including two carboxyl carbons (C 171.1 and 174.8) and four oxygenated
carbons (C 164.3, 163.7, 161.5 and 151.0). These spectral data indicated that
PRAES-C7 consisted of two units of -orsellinic acid and HMBC spectra
confirmed the proposed structure as shown in Figure 3.15.
These spectroscopic data were suitable with the published data [31, 38].
Accordingly, the structure of PRAES-C7 was established as 4--demethylbarbatic
acid. This compound was also found in Ramalina subdecipiens [38].
68
[M+H]+ (calcd. for C22H27O8, 419.1701).
 1H,
13
Table 3.8.
Compound PRAES-E18 was a meta-depside and showed a pseudomolecular
ion peak at m/z 419.1702 in its HR-ESI-MS spectrum indicative the molecular
formula of C22H27O8.
The 1H NMR and HSQC spectra of PRAES-E18 showed three aromatic
protons at δH 6.43 (1H, s) and 6.38 (2H, s), two methoxy groups at δH 3.89 (s) and
3.83 (s), four methylene groups at δH 2.98 (4H, m), 1.74 (2H, sext, 7.5 Hz), 1.66
(2H, sext, 7.5 Hz) and two methyl groups at δH 1.00 (t, 7.5 Hz), 0.94 (t, 7.5 Hz).
69
The 13C NMR spectrum revealed twenty two carbons including two carboxyl
groups (δC 175.5 and 168.9), two methoxy groups (δ 56.0 and 55.4), three aromatic
methine carbons (δC 110.9, 106.2, 98.9) and two n-propyl groups (δC 38.8, 24.8,
14.3, 39.0, 25.2 and 14.3).
In the COSY spectrum, there were correlations of H-8 and H-9, of H-9 and
H-10, of H-8 and H-9, and of H-9 and H-10. Moreover, the DEPT spectrum
showed the presence of four methylene carbons and two methyl carbons, therefore,
compound PRAES-E18 possessed two n-propyl groups.
The exact location of the aromatic protons and the substituted functional
groups was established based on 2D NMR (Figure 3.16).Detailed 2D NMR analysis
and comparison with the reported data led us to determine the structure of PRAESE18 was sekikaic acid, respectively [38]. This compound was also found in
Ramalina boulhautiana [38].
3.1.4. Depsidones
 Melting point: 244250 C.
 Mass spectrum (Appendix 132):
(calcd. for C19H16O9+Na, 411.0692).
70
HR-ESI-MS m/z 411.0681 [M+Na]+,
 1H and 13C NMR spectra (DMSO-d6) (Appendix 133, 134): see Table 3.9.
 HSQC and HMBC spectra (DMSO-d6) (Appendix 135, 136).
Compound PRAES-C14 was isolated as colorless needles. The HR-ESI-MS
(positive mode) displayed a pseudomolecular ion peak with m/z 411.0681 [M+Na]+,
corresponding to the molecular formula of C19H16O9. The 1H NMR spectrum of
PRAES-C14 was simple. Its
1
H NMR spectrum displayed six singlets
corresponding to two methyl groups at H 2.64 (H-9, s) and 2.47 (H-9, s), an
oxygenated methylene at H 4.58 (H-8, s), an aromatic proton at H 6.83 (H-5, s),
an aldehyde proton at H 10.76 (H-8, s) and a methoxy group at H 3.31 (H-10, s).
The 13C and DEPT NMR spectra indicated that the molecule of PRAES-C14
contained two methyl groups [δC 21.2 (C-9) and 15.4 (C-9)], one oxygenated
methylene [δC 62.9 (C-8)], one aldehyde group [δC 193.4 (C-8)], an aromatic
methine [δC 117.9 (C-5)], a methoxy group [δC 57.9 (C-10)], two carbonyl groups
[δC 161.4 (C-7) and 172.9 (C-7)] and 11 quaternary carbons [δC 113.0 (C-1), 165.5
(C-2) , 112.0 (C-3), 165.3 (C-4), 153.4 (C-6), 113.1 (C-1), 159.0 (C-2), 116.7
(C-3), 147.5 (C-4), 143.0 (C-5) and 133.5 (C-6)].
The position of the methoxy group (C-10) was determined by the HMBC
correlation from the methoxy protons at H 3.31 (H3-10) to the methylene carbon
C-8 (C 62.9). Analysis of 1D, 2D-NMR data (Figure 3.17) and the comparison of
these data with the ones in the literature [9] suggested that compound PRAES-C14
is 8-O-methylprotocetraric acid.
71
Table 3.9. NMR data of PRAES-C14, PRAES-C12
No
PRAES-C14(b)
δH J (Hz)
δC
PRAES-C12(a)
δH J (Hz)
δC
PRAES-C12(a)
No
δH J (Hz)
δC
1
113.0
114.6
1
106.4
2
165.5
164.7
2
162.8
3
112.0
111.8
3
110.2
4
165.3
164.7
4
161.4
117.8
5
119.8
5
6.83 s
117.9
6.71 s
6
153.4
153.7
6
137.9
7
161.4
160.8
7
173.6
8
10.73 s
193.4
10.76 s
194.6
8
2.69 s
19.9
9
2.47 s
21.2
2.32 s
21.2
9
1.99 s
8.9
10
3.90 s
52.0
4-OH
12.16
12.16 s
1
113.1
113.6
2
159.0
160.4
3
116.7
119.1
4
147.5
145.9
5
143.0
141.5
6
133.5
132.5
7
172.9
166.4
8
4.58 s
62.9
4.06 s
22.2
9
2.64 s
15.4
2.74 s
14.9
10
3.31 s
57.9
2-OH
11.19 s
a) Measured in acetone-d6. b) Measured in Acetone-d6. and DMSO-d6.
72
 Melting point: 250254 C.
Table 3.9.
The 1H and
13
C NMR spectra of PRAES-C12 revealed a close relationship
with those of PRAES-C14. The 1H NMR spectrum of PRAES-C12 displayed
singlet signals for four methyl groups at H 2.74 (s), 2.69 (s), 2.32 (s) and 1.99 (s),
two chelated hydroxyl protons at H 12.16 (s) and 11.19 (s), a methylene group at
H 4.06 (s), an aromatic proton at H 6.71 (s), an aldehyde proton at H 10.76 (s)
and a methoxy group at H 3.90 (s).
The 13C and DEPT NMR spectra showed resonances of 28 carbons including
four methyl groups [δC 21.2 (C-9), 19.9 (C-9), 14.9 (C-9) and 8.9 (C-8)], three
carboxyl carbons [δC 173.6 (C-7), 166.4 (C-7) and 160.8 (C-7)], one methylene
carbon [δC 22.2 (C-8)], an aldehyde group [δC 194.6 (C-8)], a methoxy group [δC
52.0 (C-10)], an aromatic methine and 17 quaternary carbons.
73
These spectral data indicated that compound PRAES-C12 possessed one
-orsellinic acid unit more than PRAES-C14. The -orsellinic acid unit was jointed
to C-3 (δC 119.1) by a methylene group, on the base of HMBC correlations of these
methylene protons δH 3.97 (H2-8) to aromatic carbons at δC 160.4 (C-2), 119.1 (C3), 145.9 (C-4), 161.4 (C-4), 119.8 (C-5), and 137.9 (C-6) (Figure 3.17).
The exact location of the aromatic proton and the substituted functional
groups was established based on 2D NMR (HSQC and HMBC). Accordingly, the
structure of PRAES-C12 was determined to be furfuric acid. This compound was
also found in Pseudevernia furfuracea (L.) Zopf [38].
3.1.5. Diphenyl ethers
 Mass spectrum (Appendix 142):
HR-ESI-MS m/z 297.0752 ([M+Na]+,
(calcd. for C15H14O5+Na, 297.0733).
Table 3.10.
74
Compound PRAES-C5 was isolated as white amorphous solid. The HR-ESIMS (positive mode) displayed a pseudomolecular ion peak with m/z 297.0752
[M+Na]+, corresponding to the molecular formula of C15H14O5.
The 1H NMR spectrum exhibited signals for five aromatic methine protons at
H 6.29, 6.38, 6.58, 6.63 (5H, H-3, 5, 1‟, 3‟, 5‟) and these resonant signals
concentrated in a narrow zone within 2.5 Hz, therefore these aromatic protons were
meta coupled. The proton spectrum also showed signals corresponding to two
methyl groups (H 2.29, 2.59). The 13C-NMR spectrum revealed the presence of 15
carbons including two methyl groups (C 21.4, 24.4), one carboxyl group (C 171.0)
and 12 aromatic carbons including four oxygenated carbons (C 166.7, 164.1, 159.1
and 152.0).
The NMR data suggested the presence of two aromatic rings. The first ring
was similar to orsellinic acid. The second ring contained three meta protons at
H 6.58 (H-3, H-5) and 6.63 (H-1) as well as a methyl group at H 2.29. The
HMBC spectrum showed the correlations of the methyl group with C-6‟ (C 141.2)
and the aromatic methine carbon C-5 (C 114.5). These findings implied that
compound PRAES-C5 was composed of two mono-aromatic units, an orsellinic
acid unit and an orcinol unit (Figure 3.18).
Clear observation showed that PRAES-C5 did not contain a chelated
hydroxyl group. Up to this point, the 1H NMR spectral features PRAES-C5 were
somewhat different comparing to the corresponding ones of the depside lecanorol
although these two compounds were measured in the same deuterated solvent
75
(acetone-d6) (Table 3.10). The chemical shift value (H 6.38, H-5, acetone-d6) of the
aromatic proton in the 1H NMR spectrum of PRAES-C5 was somewhat at higher
field than the similar proton (H 6.63, H-5, acetone-d6) in lecanorol [36]. This
evidence suggested that PRAES-C5 was a tri-ortho-substituted diphenyl ether since
these compounds are known to adopt an “H-inside conformation” in which the
single ortho aromatic proton is shielded by the adjacent aromatic ring [15, 24].
Table 3.10. 1H NMR data of compound PRAES-C5 and Lecanorol
No.
Lecanorol[36] (Acetone-d6)
PRAES-C5 (Acetone-d6)
δH
J (Hz)
δH
J (Hz)
3
6.29 d (2.5)
6.35 d (2.5)
5
6.38 dd (0.5, 2.5)
6.63 s
8
2.59 s
2.63 s
2-OH
11.33 s
4-OH
9.47 s
1
6.63 t (0.5, 1.0)
6.63 s
3
6.58 t (0.5, 1.0)
6.44 d (2.5)
5
6.58 t (0.5, 1.0)
6.67 d (2.5)
7
2.29 s
2.30 s
8.71 s
2-OH
Therefore a proposed diphenyl ether structure as shown in Figure 3.18 could
meet all the mentioned criteria. The structure of PRAES-C5 was elucidated as 4-
76
hydroxy-2-(3-hydroxy-5-methylphenoxy)-6-methylbenzoic acid or praesorether A.
Praesorether A was identified as a new compound isolated from natural lichen.
 Yellow solid.
carboxyl), 1645 (C=C),
[M+H]+, (calcd. for C17H16O7+H, 333.0975).

13
Compound PRAES-C15 was obtained as a yellow solid and possessed a
molecular formula of
C17H16O7, as determined from HR-ESI-MS, with the a
pseudomolecular ion peak at m/z 333.0970. The 1H NMR spectrum exhibited
signals corresponding to two meta-coupled protons at H 6.32 (d, J = 2.5 Hz) and
6.17 (d, J = 2.5 Hz), and a singlet at H 6.55 due to one aromatic proton, a
hydrogen-bonded hydroxyl group at H 12.06 (s), a formyl group at H 10.39 (s), a
methoxy group at H 3.50 (s) and two methyl groups at H 2.22 (s) and 2.00 (d, J =
0.5 Hz). The
13
C-NMR spectrum showed the resonances of 17 carbons including
two methyl groups (C 20.8 and 17.0), a methoxy groups (C 52.4), a carboxyl
77
carbon (C 167.0), a formyl group (C 193.7) and twelve substituted aromatic
carbons, five of which were oxygenated.
The HMBC spectrum of PRAES-C15 indicated that the first A ring
contained a methyl group (H 2.22, C-9) and a chelated hydroxyl group (H 12.06),
which correlated with a methine aromatic carbon at C 113.9 (C-5). Furthemore,
this hydroxyl proton and the aldehyde proton correlated with the same carbon
bearing an oxygen at C 164.0 (C-4) (Figure 3.19). The assignment of substituents
on the first ring showed that PRAES-C15 possessed a methyl heamatommate unit
(9), a monophenolic compound that was also isolated in this species.
The B ring was substituted with two meta coupled proton at δH 6.32 (H-3)
and 6.17 (H-1) as well as a methyl group at δH 2.00. In the HMBC spectrum, this
methyl group showed correlations with the first methine aromatic carbon at δC
109.5 (C-1) and two carbons at δC 136.3 (C-5) and 131.1 (C-6). Therefore, the
methyl group at δC 17.0 and the second methine aromatic carbon at δC 131.1 were in
para positions together. The NOESY spectrum of compound PRAES-C15 showed
the correlations between the formyl proton H-8 of A-ring and the methyl protons
H3-7 at δH 2.00 of B-ring as well as the methoxy protons H3-10 at H 3.50 of A-ring
and the aromatic proton H-3 at H 6.32 of B-ring. The proximity in space of these
substituents implied that the two rings A and B are not co-planar. Via the ether
bridge, the two rings are in two different planes that formed a certain angel, as
proposed in Figure 3.19.
78
Accordingly, the structure of PRAES-C15 was elucidated as shown.
Compound
PRAES-C15
was
designated
as
methyl
methylphenoxy)-3-formyl-4-hydroxy-6-methylbenzoate.
2-(2,4-dihydroxy-6PRAES-C15
was
identified as a new compound isolated from natural lichen and was named as
praesorether B.
 Yellow solid.
carboxyl), 1646 (C=C), 1263 (CO).
[M+H]+ (calcd. for C27H26O11+H, 527.1554).

13
C and DEPT NMR spectra (Acetone-d6) (Appendix 159, 160): see Table
3.12.
 HSQC, HMBC and ROESY spectra (Acetone-d6) (Appendix 161, 162, 163).
Compound PRAES-C16 was isolated as a yellow solid. The HR-ESI-MS
established a molecular formula of C27H26O11. The 1H NMR spectrum of PRAESC16 exhibited signals for three methyl groups at H 2.58, 2.15 and 1.99 (each 3H,
79
s), two methoxy groups at H 3.88 and 3.18 (each 3H, s), a methylene group at H
3.97 and 3.98 (each 1H, br s), three aromatic protons at H 6.52 (d, J = 0.5 Hz), 6.33
and 6.24 (each 1H, s), a formyl group at  10.44 (s) and two hydroxyl protons at H
12.00 (s) and 10.56 (s).
The 13C NMR spectrum of compound PRAES-C16 revealed the presence of
27 carbons, including three methyl groups (C 20.5, 19.1 and 16.6), two methoxy
groups (C 52.0 and 52.2), a formyl group (C 195.6), a methylene group (C 20.7),
two carboxyl carbons (C 166.6 and 172.4) and three methine carbons (C 112.9,
108.9 and 101.5) and fifteen quaternary aromatic carbons.
These spectroscopic data proposed that compound PRAES-C16 had three
phenolic rings (A, B and C), each ring had one aromatic methine proton and one
80
methyl group. Analysis of 1D and 2D-NMR data indicated that compound PRAESC16 ressembled PRAES-C15 and it contained a further orsellinic acid unit.
The orsellinic acid unit was jointed to B-ring at C-3 (δC 118.1) by a
methylene carbon. This methylene carbon connected B and C rings on the base of
HMBC correlations from this methylene protons δH 3.97 and 3.98 (H2-8) to
aromatic carbons of B-ring at δC 153.3 (C-2), 113.1 (C-3), 149.2 (C-4), and to
aromatic C-ring carbons at δC 160.0 (C-4), 119.5 (C-5) and 142.5 (C-6). This
connection was confirmed by the NOESY cross peaks between methylene protons
at δH 3.97 and 3.98 (H2-8) and methyl protons at δH 2.58 (H3-8) of C-ring (Figure
3.20).
Therefore, the structure of PRAES-C16 was determined as shown.
Compound PRAES-C16 was designated as methyl 2-[3-(4,6-dihydroxy-3methoxycarbonyl-2-methylbenzyl)-2,4-dihydroxy-6-methylphenoxy]-3-formyl-4hydroxy-6-methylbenzoate. Compound PRAES-C16 is a new one in the nature and
was named praesorether C.
81
Table 3.11. 1H NMR data of PRAES-C15, PRAES-C16, PRAES-C20, PRAESC18, PRAES-C3 and PRAES-C4.
No
PRAES-C15(a)
PRAES-C16(b)
PRAES-C20(b)
PRAESC18(a)
PRAESC3(b)
PRAESC4(a)
H J (Hz)
H J (Hz)
H J (Hz)
H J (Hz)
H J (Hz)
H J (Hz)
6.48
5
6.55
s
6.52
8
10.39
s
10.44
s
10.44
9
2.22
s
2.15
s
2.13
12.06
s
12.00
s
7-OCH3
3.50
s
3.18
s
3.17
s
1
6.17
d (2.5)
6.24
s
6.19
d (1.0)
3
6.32
d (2.5)
7
2.00
d (0.5)
1.99
s
1.98
s
5.20
3.97
br s
3.97
br s
3.98
br s
3.98
br s
4-OH
8
d (0.5)
d (0.5)
s
d (0.5)
7-OCH3
6.71
s
6.46
s
6.54
s
10.12
s
10.42
s
10.32
s
2.31
s
2.07
s
2.21
s
11.92
s
12.03
s
12.05
s
3.23
s
2.94
s
3.28
s
6.27
s
6.31
s
s
2.11
s
1.99
s
4.05
s
3.76
s
3.88
s
3.14
s
6.29
s
6.29
s
6.38
s
2.45
s
13.44
s
3.94
s
1
3
6.33
s
6.31
s
6.39
s
5
2.06
7
8
2.58
s
2.61
s
2.61
s
9
3.88
s
3.86
s
3.91
s
10.56
s
2-OH
7-OCH3
a) Measured in chloroform-d. b) Measured in acetone-d6
82
s
Table 3.12.
13
C NMR data of PRAES-C15, PRAES-C16, PRAES-C20, PRAESC18, PRAES-C3 and PRAES-C4.
No
PRAESC15(a)
PRAESC16(b)
PRAESC20(b)
PRAESC18(a)
PRAESC3(b)
PRAESC4(a)
C
C
C
C
C
C
1
114.9
115.5
115.7
116.5
116.1
115.3
2
158.1
158.9
159.2
156.4
159.4
158.3
3
110.6
111.0
111.0
111.8
111.5
110.9
4
164.0
164.3
164.2
163.6
164.3
164.1
5
113.9
112.9
112.7
116.2
112.8
114.0
6
147.7
148.2
148.1
148.1
148.1
147.6
7
167.0
166.6
166.8
165.8
167.0
167.5
52.4
52.0
52.0
52.2
51.9
52.3
8
193.7
195.6
195.6
193.7
195.6
193.8
9
1
20.8
109.5
20.5
108.9
20.5
108.5
21.2
103.4
20.6
109.6
20.9
111.1
2
153.3
153.3
153.9
153.1
152.5
152.3
3
102.1
113.1
113.7
117.6
114.1
112.2
4
148.8
149.2
149.8
151.7
148.4
145.5
5
136.3
135.8
135.7
134.2
138.3
136.3
6
131.1
129.9
129.6
129.2
136.4
129.5
7
17.0
16.6
16.7
101.6
16.6
16.8
20.7
21.1
20.0
18.2
17.3
7-OCH3
8
169.6
9
56.8
7-OCH3
1
109.4
107.1
107.5
109.6
105.0
2
161.5
162.3
162.4
131.4
160.0
3
101.5
102.0
102.0
111.2
111.0
4
160.0
163.5
159.5
155.7
111.0
5
119.5
120.9
117.1
109.6
113.4
6
142.5
142.1
142.6
136.4
142.0
7
172.4
172.9
172.0
21.3
173.1
7-OCH3
52.2
51.9
52.0
52.4
8
19.1
19.5
19.3
24.1
161.0
9
83
 Yellow solid.
carboxyl), 1647 (C=C), 1272 (CO).
[M+H]+, (calcd. for C27H24O11+H, 525.1398).

13
C and DEPT NMR spectra (Acetone-d6) (Appendix 167, 168): see Table
3.12.
 COSY, HSQC, HMBC and ROESY spectra (Acetone-d6) (Appendix 169,
170, 171, 172).
Compound PRAES-C20 was obtained as a yellow solid. The IR, 1H, 13C and
DEPT NMR spectral features of PRAES-C20 were closely similar to those of
PRAES-C16, except for the lack of two chelated hydroxyl protons. The NMR
spectra also revealed the signals of three methyl groups (H/C 2.13/20.5, 2.61/19.5
and 1.98/16.7), two methoxy groups (H/C 3.17/52.0 and 3.86/51.9), a formyl group
(H/C 10.44/195.6), a methylene group (H/C 3.97 and 3.98/21.1), three aromatic
methine carbons (H/C 6.48/112.7, 6.19/108.5 and 6.31/102.0) and two carboxyl
carbons (C 166.8 and 172.9), and fifteen quaternary aromatic carbons.
Analysis of 2D NMR spectra (COSY, HSQC, HMBC and ROESY) showed
that the structure of PRAES-C20 was similar with that of praesorether C (PRAESC16) (Table 3.12). Moreover the ROESY spectrum of compound PRAES-C20
showed the correlations between the formyl proton H-8 at δH 10.44 of A-ring and
the methyl protons H3-7 at δH 1.98 of B-ring, as well as the methylene protons H-8
and the methyl protons H3-8 of C-ring (Figure 3.21).
84
However, the HR-ESI-MS (positive mode) showed a pseudomolecular ion
peak at m/z 525.1396 [M+H]+ (calcd. 525.1398), corresponding to the molecular
formula of C27H24O11, two atomic mass units less than the one of PRAES-C16. The
two compounds PRAES-C16 and PRAES-C20 were measured in the same
deuterated solvent (acetone-d6), but the proton spectrum of PRAES-C16 showed
two chelated hydroxyl protons at δH 12.00 (4-OH) and 10.56 (2-OH), but the
proton spectrum of PRAES-C20 did not.
Combination of the disappearance of the two chelated hydroxyl protons and
the less of two atomic mass unit of PRAES-C20 comparing to the one of PRAESC16, revealed that there was a peroxy bridge between A-ring and C-ring, via the
hydroxyl group at C-4 of A-ring and the hydroxyl group at C-2 of C-ring.
85
Therefore, the structure of PRAES-C20 was as shown. It is a new compound in the
nature and was named praesorether D.
 Yellow solid.
lactone), 1651 (C=C), 1276 (CO).
[M+H]+ (calcd. for C29H26O14+H, 599.1402).

13
C and DEPT NMR spectra (CDCl3) (Appendix 176, 177): see Table 3.12
 HSQC, HMBC and ROESY spectra (CDCl3) (Appendix 178, 179, 180).
Compound PRAES-C18 was isolated as a yellow solid, which was shown to
have the molecular formula of C29H26O14 by the HR-ESI-MS. The 1H NMR
spectrum of PRAES-C18 displayed signals for two methyl groups at H 2.61 and
2.31 (each 3H, s), three methoxy groups at H 3.91, 3.23 and 3.14 (each 3H, s), a
methylene group at H 4.05 (s), an acetalic proton at H 5.20 (s), two aromatic
86
protons at H 6.71 and 6.39 (each 1H, s), a formyl group at H 10.12 (s) and a
chelated hydroxyl proton at H 11.92 (s).
The 13C NMR spectrum of compound PRAES-C18 revealed the presence of
29 carbons including two methyl groups (C 21.2 and 19.3), three methoxy groups
(C 56.8, 52.2 and 52.0), a formyl group (C 193.7), a methylene group (C 20.0), an
acetalic carbon (C 101.6), three carboxyl carbons (C 165.8, 169.6 and 172.0) and
two methine carbons (C 116.2 and 102.0).
The IR spectrum showed characteristic absorption for a hydroxyl group
(3394 cm-1) and a lactone group (1732 cm-1). These spectral features were closely
similar to those of PRAES-C16, except for the disappearance of one aromatic
proton and one methyl group, and the appearance of an acetalic proton at H 5.20,
one more methoxy group at H 3.14, an acetalic carbon at C 101.6 and a carboxyl
carbon at C 169.6.
This observation, together with analysis of HMBC spectrum, suggested the
presence of a -lactone ring in PRAES-C18. The NOESY correlations between
methoxy protons at H 3.14 (C7-OCH3) and the formyl proton at H 10.12 (H-8)
87
indicated that the -lactone ring jointed to the B-ring. This was confirmed by the
comparison of the 1H NMR spectrum of PRAES-C18 and the one of a diphenyl
ether in the literature that were measured in the same deuterated solvent (CDCl3-d)
(Figure 3.22) [37].
The methylene group jointed B and C-rings at C 117.6 (C-3) and 117.1 (C5) which was deduced from HMBC correlations of H2-8 (H 4.05) to C-2 (C
153.1), C-4 (C 151.7), C-4 (C 159.5), C-5 (C 117.1) and C-6 (C 142.6). This
was supported by NOESY cross peaks between methylene protons at H 4.05 (H28) and methyl proton at H 2.61 (H3-8) (Figure 3.23).
88
allowed the completed structure of compound PRAES-C18 as show in Figure 3.23.
PRAES-C18 was identified as a new compound isolated from the nature and was
named as praesorether E.
1.5.6. Structure elucidation of compound PRAES-C3
 Yellow solid.
[M+Na]+ (calcd. for C25H24O9+Na, 491.1313)

13
C and DEPT NMR spectra (Acetone-d6) (Appendix 183, 184): Table 3.12.
The HR-ESI-MS of compound PRAES-C3 showed a pseudomolecular ion
peak at m/z 491.1301 [M+Na]+ (calcd. 491.1313 for C25H24O9Na), corresponding
to the molecular formula of C25H24O9.
The 1H NMR spectrum of PRAES-C3 revealed the signals of three methyl
groups at H 2.11, 2.07 and 2.06 (each 3H, s), one methoxy group at H 2.94 (s), a
89
methylene group at H 3.80 (2H, s), one chelated hydroxyl group at H 12.03 (s),
one formyl group at H 10.42 (s), and four aromatic protons at H 6.49, 6.27 (each
1H, s) and 6.29 (2H, s).
The
13
C NMR spectrum indicated 25 carbon signals due to three methyl
carbons (C 21.3, 20.6 and 16.6), four aromatic methine carbons [C 116.1, 112.8
and 109.6 (two carbons)], one methoxy group (C 51.9), a methylene carbon (C
18.2), a formyl group (C 195.6), one carboxyl carbon (C 167.0) and 14 quaternary
aromatic carbons.
These findings, together with 2D NMR experiments implied that compound
PRAES-C3 was composed of a PRAES-C15 unit and an orcinol unit. These two
units were linked together via a methylene group. The methylene group jointed the
orcinol unit C-ring to PRAES-C15 at its C-3. This was deduced from HMBC
correlations of methylene protons H2-8 (H 3.80) to carbons of B-ring at C 114.1
(C-3), 148.4 (C-4), 152.5 (C-2), 111.2 (C-3) and to C-ring at C 155.7 (C-4)
(Figure 3.24).
The 2D NMR experiments and the comparison of these data with the ones in
the literature proved the proposed structure to be correct [23]. All these findings
allowed the completed characterization of compound PRAES-C3 depicted as
90
methyl
2-[3-(2,6-dihydroxy-4-methylbenzyl)-2,4-dihydroxy-6-methylphenoxy]-3-
formyl-4-hydroxy-6-methylbenzoate or praesorether F.
 Yellow solid.
[M+Na]+, (calcd. for C28H26O12+Na, 577.1323).

13
Compound PRAES-C4 was isolated as a yellow solid. The HR-ESI-MS of
compound PRAES-C4 showed a pseudomolecular ion peak at 577.1316 [M+Na]+
(calcd. 577.1323), corresponding to the molecular formula of C28H26O12.
Analysis of 1D and 2D NMR data for PRAES-C4 revealed a relationship to
those of PRAES-C3 espescially the A and B-rings. The marked differences were at
the C-ring with the presence of an additional methyl ester group [δC 173.1 (C-7),
52.4 (7-OCH3)] and a carboxyl group at δC 161.0 (C-9) in the 13C NMR spectrum
and in the 1H NMR spectrum the appearance of a hydroxyl chelated proton at δH
91
13.44 (2-OH) and the lack of one aromatic proton comparing to the one of
PRAES-C3.
In the HMBC spectrum, the correlations of an aromatic proton δH 6.38
(H-5) to carbon signals at C-4 (δC 111.0), C-3 (δC 111.0), C-1 (δC 105.0), C-8
(δC 24.1) and C-9 (δC 161.0) indicated that the carboxyl group jointed to C-4.
The location of the methoxycarbonyl group at C-1 was confirmed by HMBC cross
peaks between the methyl protons at δH 2.45 (H3-8) to carbon signals at C-1 (δC
105.0), C-5 (δC 113.4) and C-6 (δC 142.0) (Figure 3.25).
Consequently, the structure of PRAES-C4 was elucidated as show in.
Compound PRAES-C4 was designated as methyl 2-[3-(2-carboxyl-6-hydroxy-5methoxycarbonyl-4-methylphenylmethyl)-2,4-dihydroxy-6-methylphenoxy]-3formyl-4-hydroxy-6-methylbenzoat. PRAES-C4 was identified as a new compound
isolated from the nature and was named as praesorether G.
92
 Yellow solid.
carboxyl), 1645 (C=C), 1268 (CO).
[M+H]+, (calcd. for C43H40O16+H, 813.2396).
Table 3.13.
 HSQC, HMBC and ROESY spectra (Acetone-d6) (Appendix 198, 199, 200).
Compound PRAES-C21 was isolated as a yellow solid and the molecula of
C43H40O16 which was confirmed by HR-ESI-MS with m/z 813.2394 ([M+H]+,
calcd. 813.2400).
The 1H NMR spectrum of PRAES-C21 revealed the signals of two methoxy
groups at H 3.00 (s), five methyl groups at H 1.97, 2.08, 2.12, 2.13 and 2.38 (each
3H, s), two methylene groups at H 3.81 (2H, s), 3.88 and 3.89 (each 1H, s), two
hydrogen-bonded hydroxyl groups at H 12.09 (s), two formyl groups at H 10.43
and 10.45 (each 1H, s), and five aromatic protons at H 6.16, 6.22, 6.24, 6.47 and
6.48 (each 1H, s).
93
The
13
C NMR spectrum indicated 43 carbon signals due to two methoxy
groups (C 51.9 and 51.8), five methyl carbons (C 16.6, 16.7, 20.5, 20.5 and 20.8),
five aromatic methine carbons (C 108.2, 108.2, 108.9, 112.4 and 112.7), two sp3
methylene (C 19.3 and 21.6), two carboxyl carbons (C 166.8 and 167.0), two
aldehydic carbons (C 195.7 and 195.7) and 25 aromatic quaternary carbons.
These findings, together with 2D NMR experiments and the HR-ESI-MS
revealed that compound PRAES-C21 was composed of two PRAES-C15 units and
an orcinol unit and this orcinol unit linked to two PRAES-C15 units via two
methylene groups.
The HMBC correlations of methylene protons H2-8b (H 3.88 and 3.89) to
carbon signals at C 114.1 (C-3b), 150.0 (C-4b), 153.6 (C-2b) of the Bb-ring,
119.4 (C-4), 137.8 (C-5) and 156.5 (C-3) of the C-ring indicated that the
methylene group C-8b (C 21.6) jointed the two Bb and C rings at their C-3b
(C 114.1) and C-4 (C 119.4), respectively. This was comfirmed by NOESY
cross peaks between H2-8b (H 3.88 and 3.89) and H3-7 (H 2.38) (Figure 3.27).
The second methylene group H2-8a (H 3.81) jointed Ba and C-rings at their
C-3a (C 114.9)
and C-2 (C 112.5), respectively, on the base of HMBC
correlations from the methylene protons H2-8a (H 3.81) to carbon signals at
94
C 114.9 (C-3a), 149.1 (C-4a), 152.7 (C-2a) of Ba-ring and to C 112.5 (C-2), and
152.9 (C-1) (Figure 3.26).
Therefore, the structure of PRAES-C21 was determined as 2,6-bis-[3-(6formyl-5-hydroxy-2-methoxycarbonyl-3-methylphenoxy)-2,6-dihydroxy-4methylphenylmethyl]-1,3-dihydroxy-5-methylbenzene as shown in Figure 3.26.
Compound PRAES-C21 was a new natural compound of lichens and was named as
praesorether H.
95
Table 3.13. NMR data of compound PRAES-C21 (Acetone-d6)
No.
PRAES-C21
δH J (Hz)
No.
δC
PRAES-C21
δH J (Hz)
δC
1a
116.0
1b
115.6
2a
159.2
2b
159.6
3a
111.1
3b
111.0
4a
164.1
4b
164.2
112.4
5b
6a
148.0
6b
148.2
7a
167.0
7b
166.8
5a
6.48 s
6.47 s
112.7
8a
10.45 s
195.7
8b
10.43 s
195.7
9a
2.12 s
20.5
9b
2.13 s
20.5
7a-OCH3
3.00 s
51.9
7b-OCH3
3.00 s
51.8
4a-OH
1'a
12.09 s
6.22 s
4b-OH
12.09 s
108.9
1'b
6.16 s
108.2
2a
152.7
2b
153.6
3'a
114.9
3'b
114.1
4a
149.1
4b
150.0
5a
136.3
5b
135.8
6a
130.5
6b
129.4
7a
2.08 s
16.7
7b
1.97 s
16.7
8a
3.81 s
19.3
8b
3.88 s
21.6
3.89 s
1''
152.9
2''
112.5
3
156.5
4
119.4
5
137.8
6
6.24 s
108.2
7
2.38 s
20.8
96
3.1.6. Dibenzofurans
 Yellow prisms (chloroform).
  D + 74 (c= 0.001, EtOH).
23

Compound PRAES-E5 was isolated as yellow prisms. The proton spectrum
of compound PRAES-E5 was very simple in which all of the proton resonances
were singlets and all of the hydroxyl protons were sharp singlets indicating that they
formed H-bonds with the oxygen of neighbouring keto groups.
The 1H NMR spectrum exhibited signals for two chelated hydroxyl groups at
H 13.29 (s) and 11.01 (s), two methoxy groups at H 2.68 and 2.66, two methyl
groups at H 2.11 and 1.76 and an aromatic methine proton at H 5.97. The
13
C,
DEPT-NMR spectra showed signals for three carbonyl carbons [ C 201.7 (C-14),
200.3 (C-17) and 198.1 (C-1)], four methyl groups [C 32.1 (C-13), 31.2 (C-18),
27.8 (C-15) and 7.5 (C-16)].
97
Table 3.14. NMR data of PRAES-E5 and PRAES-E3 (CDCl3)
No
PRAES-E5
δH J (Hz)
Usnic acid[38]
PRAES-E3
δC
δH J (Hz)
δC
δH J (Hz)
δC
1
198.1
198.7
198.1
2
105.3
105.4
105.3
3
191.7
191.8
191.8
4
5.97
s
98.3
5.95
s
98.1
5.92
s
98.4
5
179.4
179.9
179.4
6
155.2
159.2
155.2
7
101.6
101.9
101.6
8
163.9
166.5
163.9
9
109.4
107.8
109.4
10
157.5
154.7
157.6
11
103.9
102.2
104.0
12
59.1
59.7
59.1
13
1.76
s
14
32.1
1.76
s
201.7
31.8
1.75
s
201.8
32.2
201.8
15
2.66
s
27.8
2.66
s
28.0
2.66
s
27.9
16
2.11
s
7.5
2.12
s
7.6
2.10
s
7.6
18
2.68
s
2.77
s
2.67
s
8-OH
13.29
s
14.38
s
13.31
s
10-OH
11.01
s
11.30
s
11.02
s
18.84
s
17
200.3
31.2
204.6
3-OH
33.1
200.4
31.3
In the HMBC spectrum, the methyl protons at H 2.11 (H3-16) showed cross
peaks with C-8 (C 163.9), C-9 (C 109.4) and C-10 (C 157.5), therefore this
methyl group jointed to C-9 (C 109.4).
The location of the two acetyl groups was confirmed by HMBC correlations
from the methyl protons at H 2.66 (H3-15) to C-2 (C 105.3) and the methyl protons
at H 2.68 (H3-18) to C-7 (C 101.6) (Figure 3.28). The comparison of these
spectroscopic data of PRAES-E5 with those of usnic acid in the literature showed
good compatibility [27, 38, 80]. Furthermore, compound PRAES-E5 was
98
dextrorotatory,  D + 74 (c= 0.001, EtOH), therefore, PRAES-E5 was determined
23
to be (+)-(12R)-usnic acid.

 Yellow prisms (chloroform).
  D + 80 (c= 0.001, EtOH).
23

 HMBC spectrum (CDCl3) (Appendix 207).
Compound PRAES-E3 was isolated as yellow prisms. The NMR spectra of
compound PRAES-E3 were quite similar to those of compound PRAES-E5. The
only difference was just observed in the HMBC spectrum of PRAES-E3. The
HMBC spectrum exhibited cross peaks of the methyl protons at H 2.12 (H3-16) to
carbon signals at C-8 (C 166.5), C-6 (C 159.2), C-7 (C 101.9) and of the methyl
protons at H 2.77 (H3-18) to carbon signal at C-9 (C 107.8).
99
This result suggested that the methyl group jointed to C-7 (C 101.9) and the
acetyl group located at C-9 (C 107.8) (Figure 3.29). Compound PRAES-C3 was
also dextrorotatory, therefore the structure of compound PRAES-E3 was suggested
to be (+)-(12R)-isousnic acid [27, 38]. This compound was also found in Cladonia
mitis Sandst) [38].
 Yellow solid.
23
  D + 852 (c= 0.001, MeOH).

[M+Na]+, (calcd. for C18H17O6N+Na, 366.0954).
 1H,
13
Table 3.15.
Compound PRAES-C8 was obtained as a yellow solid. The HR-ESI-MS
(positive mode) showed a pseudomolecular ion peak at m/z 366.0938 [M+Na]+
(calcd. 366.0954), corresponding to the molecular formula of C18H17O6N.
Analysis of 1H and 13C NMR data, and comparison with those of PRAES-E5,
enabled the identification of PRAES-C8 possessing the 9bH-dibenzofurandione
moiety. The carbon numeration for PRAES-C8 was chosen as the one previously
used for PRAES-E5. In the 13C NMR spectrum, there was no signal at 200-205 ppm
for a ketone group, as in PRAES-E3 or PRAES-E5, but there appeared a new signal
100
at 180-175 ppm for a carboxyl group or an imino group. This observation, together
with the presence of a nitrogen atom in the molecule, suggested the assignment of
C-14 as the carbon of an imino group (C=N). This assignment was confirmed by the
similarity of the remaining NMR data with those of Usimine A [67] (Figure 3.31 ).
The 1H and 13C NMR assignments for this moiety of the molecule were confirmed
by analysis of HMQC and HMBC data. The connection of the iminoethyl group to
C-2 was approved by the HMBC correlations of H3-15 (δH 2.66) to signals at δC
175.5 (C-14) and 103.7 (C-2).
The absolute configuration of the sole chiral center C-12 of PRAES-C8 was
determined by comparison of its specific rotation value. In the biosynthetic aspect,
the assignment of the chiral carbon C-12 of PRAES-C8 was also proposed to be R
as in PRAES-E3 and PRAES-E5 because they were isolated from the same material
23
and possessing similar positive optical rotation,  D + 852 (c= 0.001, MeOH).
These data identified compound PRAES-C8 as show. PRAES-C8 was a new natural

compound and was named as (+)-(12R)-praesousimine..
101
Table 3.15. NMR data of PRAES-C8, PRAES-E5 and Usimine A
No
PRAES-C8(a)
δH J (Hz)
δC
PRAES-E5(a)
δH J (Hz)
δC
Usimine A(b)[67]
δH J (Hz)
δC
1
198.9
198.1
198.9
2
103.7
105.3
103.1
3
190.8
191.7
190.3
4
5.82 s
102.4
5.97 s
98.3
5.84 s
102.3
5
174.9
179.4
175.1
6
155.9
155.2
155.9
7
101.6
101.6
101.5
8
163.7
163.9
163.7
9
108.4
109.4
108.4
10
153.8
157.5
152.8
11
105.0
103.9
106.0
12
57.4
59.1
57.6
13
1.70 s
14
32.1
1.76 s
175.5
32.1
1.71 s
201.7
31.1
175.1
15
2.62 s
26.6
2.66 s
27.8
2.66 s
31.8
16
2.09 s
7.6
2.11 s
7.5
2.40 s
7.6
17
200.8
31.9
200.3
2.68 s
31.2
200.8
18
2.67 s
2.67 s
8-OH
13.35 s
13.29 s
13.33 s
10-OH
11.76 s
11.01 s
11.67 s
a) Measured in chloroform-d. b) Measured in pyridine-d5.
102
31.4
3.1.7. Xanthones
 Light yellow gum.
20
  D + 1.07 (c= 2.152, CHCl3).
lactone), 1648 (C=C), 1270 (CO).
[M+H]+ (calcd. for C32H30O14+H, 639.1715).
 1H,
13
Table 3.16.
 COSY, HSQC, HMBC and ROESY spectra (CDCl3) (Appendix 219, 220,
221, 222).
Compound PRAES-C27 was obtained as light yellow gum. The HR-ESI-MS
(positive mode) m/z 639.1710 [M+H]+ (calcd. 639.1715, corresponding to the
molecular formula of C32H30O14). Analysis of 1H,
13
C, DEPT and HSQC spectra
revealed that PRAES-C27 possessed two methoxy groups (C 53.3 and 53.7), two
methyl carbons (C 14.9 and 18.0), four sp3 methines (C 29.3, 33.5, 77.0 and 82.7),
three sp3 methylenes (C 36.3, 36.7 and 39.8), five carbonyl carbons (C 169.1,
170.3, 174.9, 187.2 and 194.1), two oxygenated quaternary carbons and fourteen
olefinic or aromatic carbons.
A comparison of the 1H and
13
C NMR data of PRAES-C27 with those of
blennolide G [67] (Table 3.16) revealed a good compatibility, except for the 1H and
13
C chemical shift values and the coupling constant of some carbons, such as at C-5
(C 77.0), C-6 (C 29.3), C-7 (C 36.3), C-9 (C 82.7), C-10 (C 33.5) and
C-11 (C 36.7) in PRAES-C27. All proton and carbon signals were assigned by
COSY and HMBC experiments to formulate the planar structure of PRAES-C27
103
(Figure 3.32). These findings implied that PRAES-C27 was a planar isomer of
blennolide G.
The stereochemistry of PRAES-C27 was proposed based on the ROESY
experiments as well as the comparison of the H, C values and the coupling
constants of some protons. In the F ring of PRAES-C27, the diaxial conformation
between the carbinolic hydrogen H-5 (H 3.92) and the methine hydrogen H-6
(H 2.42) was confirmed by the large coupling constant of J5,6 11.0 Hz. This value
was also observed in eumitrin A2 [80].
Figure 3.32. COSY, HMBC and ROESY correlations of PRAES-C27 (CDCl3)
104
The ROESY correlations of H-5/H3-11 also supported the anti arrangement
of H-5 and H-6. Moreover, the comparison of the chemical shift values of the C-5
(C 77.0) with another xanthone such as blennolide B, possessing a β-hydroxyl
group at C-5 (C 77.0) showed good compatibility (Table 3.16) [79]. These
comparisons led to the establishment of the orientations of the hydroxyl group at C5 and the methyl connected to C-6 in an anti arrangement (Figure 3.32).
In the C ring of PRAES-C27, the comparison of H, C values and the
coupling constants of protons showed that the spatial structure of PRAES-C27 did
not suit to the one of Blennolide G but well resembled the one of chromone lactone
44 (Table 3.16) (Figure 3.32) [60]. Furthermore, the ROESY correlations of H-9
and H-10 supported that H-9 and H3-13 were located in an anti arrangement
(Figure 3.32). These findings suggested that the stereochemistry of this moiety of
PRAES-C27 was similar to the one of chromone lactone 44.
Consequently, the relative stereochemistry of PRAES-C27 was proposed as
shown. Compound PRAES-C27 was proved to be a new natural compound. This
20
compound was dextrorotatory,  D + 1.07 (c= 2.152, CHCl3), therefore it was
named (+)-praesorexanthone A.
105
Table 3.16. NMR data for PRAES-C27, Blennolide G, Blennolide B and Chromone lactone 44 (all in CDCl3)
No.
δH
1
2
3
4
4a
5
6
7
J (Hz)
7.52
6.63
d (8.5)
d (8.5)
3.92
2.42
2.32
dd (11.0, 2.0)
m
dd (19.0,
10.5)
dd (19.0, 6.0)
2.74
8
8a
9
9a
10a
11
Blennolide G[79]
PRAES-C27
1.17
d (6.5)
δC
159.4
117.8
141.3
107.7
158.4
77.0
29.3
36.3
177.7
101.6
187.2
106.9
84.8
18.0
δH
J (Hz)
7.43
6.57
d (8.5)
d (8.5)
4.13
2.12
2.40
s
m
dd (19.0, 6.2)
2.53
dd (19.0, 11.3)
1.18
d (6.9)
Blennolide B[79]
δC
159.4
118.3
139.6
107.6
157.3
71.3
28.5
32.6
179.9
99.9
187.6
107.0
84.8
17.5
106
δH
J (Hz)
6.53
7.36
6.55
dd (8.3, 8.0)
t (8.3)
dd (8.2, 8.0)
3.92
2.41
2.30
dd (11.2, 2.6)
m
dd (19.1, 10.6)
2.74
dd (19.1, 6.2)
1.17
d (6.5)
Chromone lactone 44[60]
δC
162.1
110.7
138.0
107.9
158.8
77.0
29.3
36.3
177.5
101.7
187.1
107.2
84.7
18.0
δH
J (Hz)
δC
No.
δH
12
13
1-OH
5-OH
8-OH
2'
3'
4'
4'a
5'
6'
7'
8'
8'a
9'
10'
11'
12'
13'
14'
15'
5'-OH
Blennolide G[79]
PRAES-C27
3.73
11.75
2.92
13.77
3.20
3.28
J (Hz)
s
s
br s
s
d (17.5)
d (17.5)
7.45
6.62
d (8.5)
d (8.5)
4.82
2.98
2.71
2.48
d (6.5)
m
dd (17.0, 8.0)
dd (17.0, 7.5)
1.34
3.77
11.93
d (7.5)
s
s
δC
170.3
53.3
84.4
39.8
194.1
107.5
159.2
118.1
140.2
107.3
158.3
82.7
33.5
36.7
174.9
14.9
169.1
53.7
δH
3.72
11.84
2.52
13.94
3.05
3.21
J (Hz)
s
s
s
s
d (17.0)
d (17.0)
7.53
6.62
d (8.5)
d (8.5)
4.45
2.85
2.91
2.23
d (3.9)
m
dd (17.5, 9.4)
dd (17.5, 4.3)
1.29
3.76
11.87
d (6.8)
s
s
Blennolide B[79]
δC
171.2
53.5
84.2
39.7
194.1
107.5
159.2
118.1
141.3
107.3
158.6
87.6
30.0
36.1
175.0
20.8
168.8
53.6
107
δH
J (Hz)
3.69
11.22
s
s
13.80
s
Chromone lactone 44[60]
δC
δH
J (Hz)
δC
170.3
53.1
3.18
3.25
d (17.3)
d (17.3)
6.54
7.41
6.55
dd (8.2, 0.9)
t (8.3)
dd (8.3, 0.8)
4.79
2.96
2.69
2.46
d (6.8)
m
dd (17.3, 8.3)
dd (17.3, 7.9)
1.32
3.73
11.44
d (7.2)
s
s
84.3
39.8
193.9
107.5
161.9
110.5
139.0
107.5
158.8
82.7
33.4
36.7
174.8
14.8
169.0
53.6
 Yellow gum.
21
  D -2.17 (c= 1.861, CHCl3).
lactone), 1624 (C=C), 1248 (CO).
[M+H]+ (calcd. for C32H30O15+H, 655.1664).
 1H,
13
Table 3.17.
 COSY, HSQC, HMBC and ROESY spectra (CDCl3) (Appendix 228, 229,
230, 231).
Compound PRAES-C28 was obtained as light yellow gum. The HR-ESI-MS
with a pseudomolecular ion peak at m/z
655.1660 [M+H]+ (calcd. 655.1664,
corresponding to the molecular formula of C32H30O15), showed that it possessed 16
atomic mass units more than the one of PRAES-C27.
The spectral features of PRAES-C28 resembled those of PRAES-C27,
suggesting it possessed the same polycyclic skeleton. The differences were
accounted for the presence of a carbonyl carbon (C 198.8) instead of an oxygenated
carbon at C-8 and the lack of a chelated hydroxyl proton of C8-OH as in
PRAES-C27. These findings suggested that PRAES-C28 was a derivative of
PRAES-C27. The substitution pattern of PRAES-C28 was confirmed by HMBC
and ROESY correlations (Figure 3.33 and 3.34).
In the F ring of PRAES-C28, there were marked differences in the chemical
shift values of C-5, C-6, C-7, C-8, C-8a and C-9 comparing to the corresponding
ones of PRAES-C27. The carbon C-8 was a carbonyl carbon and C-8a was a
quaternary oxygenated carbon. However, the large coupling constants J5,6 10.5 Hz
and significant ROESY cross peaks between H-5/H3-11 suggested that H-5 and H-6
108
were located in an anti arrangement but the configurations of C-5 and C-6 were
reversed comparing to the corresponding ones in PRAES-C27 (Figure 3.33).
In the B moiety, the comparison of the C values of chiral centers at C-9 (C
82.8), C-10 (C 33.5) and C-11 (C 36.8) of PRAES-C28 with the one
of
PRAES-C27 showed good compatibility (Table 3.17).
These findings suggested the stereochemistry of PRAES-C28 as shown in
Figure 3.33. The absolute configuration of C-8a was not established. Based on the
negative specific rotation and the 2D NMR data, the stereochemistry of PRAESC28 was proposed as shown. PRAES-C28 was a new natural compound and was
named (-)-praesorexanthone B.
109
Table 3.17. NMR data for PRAES-C27, PRAES-C28 (CDCl3)
No.
1
2
3
4
4a
5
6
7
8
8a
9
9a
10a
11
12
13
1-OH
5-OH
8-OH
8a-OH
2'
3'
4'
4'a
5'
6'
7'
8'
8'a
9'
10'
11'
12'
13'
14'
15'
5'-OH
PRAES-C27
δH
J (Hz)
7.52 d (8.5)
6.63 d (8.5)
3.92
2.42
2.32
2.74
dd (11.0, 2.0)
m
dd (19.0, 10.5)
dd (19.0, 6.0)
1.17 d (6.5)
3.73
11.75
2.92
13.77
s
s
br s
s
3.20 d (17.5)
3.28 d (17.5)
7.45 d (8.5)
6.62 d (8.5)
4.82
2.98
2.71
2.48
d (6.5)
m
dd (17.0, 8.0)
dd (17.0, 7.5)
1.34 d (7.5)
3.77 s
11.93 s
δC
159.4
117.8
141.3
107.7
158.4
77.0
29.3
36.3
177.7
101.6
187.2
106.9
84.8
18.0
170.3
53.3
84.4
39.8
194.1
107.5
159.2
118.1
140.2
107.3
158.3
82.7
33.5
36.7
174.9
14.9
169.1
53.7
110
PRAES-C28
δH
J (Hz)
7.48 d (8.5)
6.67 d (8.5)
4.51
2.06
2.49
2.97
br d (10.5)
m
dd (15.0, 5.0)
m
1.22 d (6.5)
3.67 s
11.68 s
2.80 br s
4.94 br s
3.19 d (17.0)
3.28 d (17.0)
7.54 d (8.5)
6.58 d (8.5)
4.82
2.97
2.72
2.48
d (7.0)
m
dd (17.0, 8.0)
dd (17.0, 7.5)
1.32 d (7.5)
3.75 s
11.88 s
δC
160.6
118.4
141.2
107.4
157.6
74.0
32.1
43.5
198.8
89.5
191.9
106.6
71.9
18.5
167.9
53.5
84.4
39.7
194.2
107.5
159.1
117.5
141.4
107.3
158.5
82.8
33.5
36.8
175.1
14.9
169.0
53.7
Figure 3.35. COSY and HMBC correlations of PRAES-C28
3.1.8. Triterpenoids
 White powder.
 1H and 13C NMR spectra (pyridine-d5) (Appendix 232, 233): see Table 3.18.
 HSQC and HMBC spectra (pyridine-d5) (Appendix 234, 235).
Compound PRAES-E17 was a hopane skeleton triterpenoid. The 13C, DEPT
NMR spectra permitted differentiating the 30 carbons of PRAES-E17 into eight
methyls, ten methylenes, six methines and six quaternary carbons.
Characteristic NMR data (Table 3.19) for eight tertiary methyls were [ H/C
0.91/16.4 (C-28), 0.93/17.5 (C-25), 1.00/17.3 (C-27), 1.09/18.5 (C-26), 1.29/22.6
111
(C-24), 1.35/29.8 (C-29), 1.39/31.4 (C-30), 1.56/37.3 (C-23)]. On the base of the
HMBC and HSQC spectra, the proton signal at H 2.38 (H-21) showed cross-peaks
with the signals at C 54.7 (C-17), 44.3 (C-18), 27.0 (C-20), and 72.5 (C-22).
The exact location of all substituted groups were established based on 2D
NMR (Figure 3.35). The comparison of these spectroscopic data of compound
PRAES-E17 with those of zeorin in the literature [19] showed good compatibility.
Therefore, PRAES-E17 was hopan-6,22-diol or zeorin.
 White powder.
112
The spectral data of compound PRAES-E17 and PRAES-E6 were similar,
with a remark that PRAES-E6 lacked the hydroxyl group at C-6. Analysis of the 1D
NMR spectral data and the comparison of these data with the ones in the literature
suggested that compound PRAES-E6 was hopan-22-ol [33].
 White amorphous powder.
([M+Na]+, (calcd. for C34H54O6+Na, 581.3813).
 1H,
13
Table 3.18.
Compound PRAES-E13 was a white amophous powder. The HR-ESI-MS
(positive mode) showed a molecular ion peak at m/z 581.3763 [M+Na]+ indicative
of a molecular formula of C34H54O6. The comparison of NMR data of PRAES-E13
and PRAES-E6 showed good similarity suggesting that both compounds possessed
the same triterpenoic framework.
However, the comparison of the 13C NMR data of PRAES-E13 with the ones
of PRAES-E6 showed that PRAES-E13 exhibited two further acetoxy groups at
C 170.1 (C-1‟), 21.7 (C-2‟), 170.4 (C-1‟‟), 21.1 (C-2‟‟), one carboxyl group at
C 182.1 but lacked a methyl group. This was supported by the HMBC experiments
113
with the correlations of the protons H-1 (H 4.67) to C-1‟ (C 170.1) and of H-3
(H 4.59) to C-1‟‟ (C 170.4). The HMBC spectrum also showed that the doublet
methyl proton signal at H 1.14 (H-30) and the methine proton signal H 2.33
(H-22) correlated with the carboxyl carbon C-29 (C 182.1) and C-30 (C 17.7),
respectively. The exact location of all substituted functional groups were
established based on 2D NMR (Figure 3.36).
In the 1H NMR spectrum, the large coupling constants of H-1 and H-3 as
well as the comparison of
13
C spectral features for PRAES-E13 with the one of
1,3-diacetoxyhopan-22-ol in the literature [35] suggested that the two acetoxy
groups at C-1 and C-3 were -orientated (Table 3.18). These spectroscopic data
proposed that compound PRAES-E13 was 1,3-diacetoxyhopan-29-oic acid and
was identified as a new natural compound from the lichens.
114
Table 3.18. NMR data of PRAES-E17, PRAES-E6, PRAES-E13 and 1,3-diacetoxyhopan-22-ol
No
.
PRAES-E17(b)
δH
J (Hz)
PRAES-E6(a)
1
δC
40.7
2
19.0
3
4
5
6
7
8
9
10
11
44.4
34.1
61.2
67.9
45.8
43.0
50.3
39.4
21.4
4.20 dt (4.5, 10.0)
1.80 m
δH
1.64
0.96
1.66
1.47
1.30
J (Hz)
m
m
m
m
m
0.78 bd(14.0)
1.38 m
1.42 m
1,57 m
PRAES-E13(a)
δC
41.4
δH
J (Hz)
4.67 dd (11.0, 4.5)
δC
80.3
1,3-diacetoxyhopan22-ol(a)
δC
80.3
20.1
1.89 m
1.65 m
4.59 dd (12.5, 4.5)
30.0
30.0
76.4
37.8
52.8
17.8
33.0
42.2
50.7
42.2
22.9
76.4
37.8
52.8
17.7
32.9
42.3
50.7
42.2
23.0
45.4
32.7
56.3
18.9
33.4
42.1
51.6
38.0
24.3
115
0.77 m
1.49 m
12
13
14
15
16
17
18
19
24.4
49.8
42.2
34.7
22.3
54.7
44.3
41.7
20
21
22
23
24
25
26
27
28
29
30
1‟
2‟
1‟‟
2‟‟
27.0
51.6
72.5
37.3
22.6
17.5
18.5
17.3
16.4
29.8
31.4
2.38
q (11.0)
1.56
1.29
0.93
1.09
1.00
0.91
1.35
1.39
s
s
s
s
s
s
s
s
1.40 m
1.49 m
1.45 m
1.93 bd(12.5)
1.47 m
1.64
0.96
1.77
2.22
m
m
m
m
0.89
0.96
0.87
0.96
0.96
0.87
1.18
1.21
s
s
s
s
s
s
s
s
24.6
50.5
42.1
34.6
22.1
54.1
44.3
41.5
26.7
51.3
74.1
33.6
22.1
16.4
17.1
17.1
16.4
31.2
28.9
1.32 m
1.25 m
1.91
2.37
2.33
0.84
0.84
1.00
0.95
0.91
0.79
m
m
m
s
s
s
s
s
s
1.14 d (6.0)
1.97 s
2.02
a) Measured in chloroform-d b) Measured in pyridine-d5
116
s
23.8
48.7
42.0
33.4
19.8
53.7
44.3
40.9
24.0
49.3
41.9
34.5
21.9
53.9
43.9
41.2
26.6
42.6
42.0
27.9
16.1
12.9
16.9
16.5
15.7
182.1
17.7
170.1
21.7
170.4
21.1
26.6
51.1
73.9
27.8
16.9
12.8
16.0
16.9
16.1
28.7
30.9
170.0
21.0
170.2
21.7
3.1.9. MACROCYCLIC COMPOUND
 Colorless oil.
 1H,
13
Table 3.21.
Compound PRAES-E15 was isolated as a colorless oil. The HR-ESI-MS
spectrum of compound PRAES-E15 showed a pseudomolecular ion peak at m/z
695.3667 [M+Na]+ (calcd. 695.3621), corresponding to the molecular formula of
C34H56O13.
The 1H NMR spectrum displayed signals for eight methyl doublets at H 0.73
(14-CH3), 0.85 (4-CH3), 0.87 (6-CH3), 0.92 (16-CH3), 1.01 (12-CH3), 1.04 (2-CH3),
1.20 (19-CH3) and 1.35 (9-CH3), four acetoxy groups at H 2.03, 2.04, 2.06 and 2.10
(3H each, s), six methylene protons H 0.96, 1.76 (m, H2-5), 0.98, 1.50 (m, H2-15)
and 1.55, 1.62 (m, H2-17) and thirteen methine protons at H 1.55, 1.81, 1.88, 2.00,
2.59, 2.95, 3.78, 4.70, 4.86, 5.02, 5.09, 5.25, 5.34 (1H each) (Table 3.21).
The 13C, DEPT NMR spectra showed the resonances of 34 carbons including
eight methyl groups (C 13.5, 13.8, 14.0, 14.5, 15.2, 15.8, 17.7 and 20.7), four
methyl ester groups [C 20.8, 170.7 (7-OCOCH3), 21.1, 170.5 (8-OCOCH3), 20.9,
170.1 (13-OCOCH3) and 20.8, 170.2 (18-OCOCH3)], two carboxyl carbons group
(C 172.7 and 177.2), three methylene carbons (C 35.0, 36.8 and 40.5) and thirteen
methine carbons, seven of which were oxygenated.
Analysis of the COSY spectra (Figure 3.37) for PRAES-E15 revealed that H-2
(δH 2.59) correlated with H-3 (δH 3.78) as well as with the methyl group 2-CH3
117
(δH 1.04). The COSY data, in combination with HSQC data, also revealed
correlations of 4-CH3 (δH 0.85) with H-4 (δH 1.81), H-4 with H-5 (δH 0.96 and 1.76),
H-5 with H-6 (δH 2.00), H-6 with H-7 (δH 4.86) as well as with the methyl protons
6-CH3 (δH 0.87), H-7 with H-8 (δH 5.34), H-8 with H-9 (δH 5.25) and H-9 with 9CH3 (δH 1.35).
The observation of the COSY spectrum also revealed the connectivity from
12-CH3 to 19-CH3. Briefly, the COSY spectrum revealed the correlations from 12CH3 (δH 1.01) with H-12 (δH 2.95), H-12 with H-13 (δH 4.70), H-13 with H-14 (δH
1.88), H-14 with H-15 (δH 0.98 and 1.50), H-15 with H-16 (δH 1.55), H-16 with H17 (δH 1.62), H-17 with H-18 (δH 5.09), H-18 with H-19 (δH 5.02) and H-19 with
19-CH3 (δH 1.20).
These spectroscopy data (Table 3.19) as well as comparison of these data with
those previously reported by Polborn et al. [59] proposed that compound
PRAES-E15 was a macrocyclic bis-lactone. The position of functional groups of
compound PRAES-E15 was determined by analysis of 2D NMR spectra (COSY,
HSQC and HMBC) as shown in Figure 3.37.
118
The HMBC spectrum for compound PRAES-E15 revealed correlations from
methyl protons 7-OCOCH3 (δH 2.10) to carboxyl carbon at δC 170.7 (7-OCOCH3)
and methine carbon C-7 (δC 77.0), from 8-OCOCH3 (δH 2.03) to carboxyl carbon at
δC 170.5 (8-OCOCH3) and C-8 (δC 72.6), from 13-OCOCH3 (δH 2.04) to carboxyl
carbon at δC 170.1 (13-OCOCH3) and C-13 (δC 80.0), and from 18-OCOCH3
(δH 2.06) to carboxyl carbon at δC 170.2 (18-OCOCH3) and C-18 (δC 73.5),
indicated that four acetoxy groups located at C-7 (C 80.8), C-8 (C 72.6), C-13 (C
80.0) and C-18 (C 73.5). HMBC observations from H-9 (δH 5.25) to C-11 (δC
172.7) as well as from H-19 (δH 5.02) to C-1 (δC 177.2) suggested that the structure
of compound PRAES-E15 was as shown.
Consequently, the structure of compound PRAES-E15 was assigned to be
7,8,13,18-tetraacetoxy-3-hydroxy-2,4,6,9,12,14,16,19-octamethyl-10,20-dioxa1,11-dioxoycloicosane or praesbislactone. The absolute configuration of PRAESE15 has not yet been determined.
119
Table 3.19: NMR data of compound PREAS-E15 (CDCl3)
PRAES-E15
No.
δH J (Hz)
1
PRAES-E15
No.
δC
177.2
11
δH
J (Hz)
δC
172.7
2
2.59
dq (10.0, 7.0)
44.0
12
2.95
qd (7.0, 2.5)
41.0
3
3.78
dd (10.0, 4.0)
73.1
13
4.70
brd (8.5)
80.0
4
1.81
m
30.6
14
1.88
m
32.0
5
0.96
m
36.8
15
0.98
m
40.5
1.76
m
1.50
m
6
2.00
m
31.1
16
1.55
m
25.7
7
4.86
dd (10.0, 3.0)
77.0
17
1.55
m
35.0
1.62
m
8
5.34
brs
72.6
18
5.09
td ( 9.0, 2.5)
73.5
9
5.25
qd (6.5, 2.0)
69.7
19
5.02
dq ( 9.0, 6.5)
70.9
9-CH3
1.35
d (6.5)
15.8
19-CH3
1.20
d (6.5)
17.7
2-CH3
1.04
d (7.0)
13.8
12-CH3
1.01
d (7.0)
14.0
4-CH3
0.85
d (6.5)
13.5
14-CH3
0.73
d (6.5)
14.5
6-CH3
0.87
d (6.5)
15.2
16-CH3
0.92
d (6.5)
20.7
7-OCOCH3
7-OCOCH3
2.10
s
8-OCOCH3
8-OCOCH3
2.03
s
170.7
13-OCOCH3
20.8
13-OCOCH3
170.5
18-OCOCH3
21.1
18-OCOCH3
170.1
2.04
s
20.9
170.2
2.06
s
20.8
3.2. BIOLOGICAL ASSAY
3.2.1. Cytotoxic activity against three cancer cell lines
Samples of 12 new and 3 known compounds (at the concentration of 100
µg/mL) were tested the cytotoxic activity against three cell lines: MCF-7 (breast
cancer cell line), HeLa (cervical cancer cell line) and NCI-H460 (human lung
cancer cell line) by sulforhodamine B colorimetric assay method (SRB assay) [71].
120
Every sample was tested three times. The cytotoxic activity of these
compounds, expressed as a percentage of cell growth inhibition (I%), was presented
in Table 3.22. In general, any tested compound with a percentage of inhibition
higher than 50% may be potential anticarcinogen and was then be determined the
IC50 value.
The results showed that 4-O-demethylbarbatic acid (58), sekikaic acid (59)
and praesorether G (67) performed good inhibitive activity on all three cell lines
with %I about 7080%. The IC50 values on three cell lines of praesorether G (67)
were smaller than the one of sekikaic acid (59) (Table 3.20 and 3.21).
Especially, the IC50 value of praesorether G (67) against MCF-7 cells was
17.9 µg/mL (Table 3.23). It indicated that praesorether G (67) may be potential
inhibitor against MCF-7, HeLa and NCI-H460 cell lines
3.2.2. Acetylcholinesterase inhibitory activity
Anti–AChE activity of isolated compounds was determined by Ellman‟s
method with galanthamine as the positive control [28]. The results were presented
in Table 3.22.
The tested results showed that all the extracts and isolated compounds from
the lichen Parmotrema praesorediosum had no effect on acetylcholinesterase.
121
Table 3.20 : % Inhibition of cytotoxic activity against three cancer cell lines of
isolated compounds
Compounda)
No.
Inhibition of Cell Growth (I %)
HeLa
NCIH460
MCF-7
40.30.5b)
39.34.0
52.52.3
1
Vinapraesorediosic acid A (43)
2
6-Methyl vinapraesorediosate A (44)
13.56.7
22.02.4
25.310.9
3
Vinapraesorediosic acid B (45)
17.01.1
-4.11.3
26.84.9
4
Praesorediosic acid (1)
0.30.1
-1.90.9
-3.71.4
5
6-Methyl praesorediosate (46)
2.32.0
4.92.5
5.70.2
6
Vinapraesorediosic acid C (47)
-6.41.8
-12.95.1
-3.33.7
7
-3.82.2
-5.54.9
9.73.9
8
1,3-Diacetoxyhopan-29-oic acid (75)
Praesbislactone (76)
-1.60.7
1.70.3
1.90.3
9
Praesalide B (53)
6.91.4
16.03.2
8.74.1
10
Praesoreusimine (70)
18.93.4
6.00.1
3.71.2
11
Praesorether A (62)
38.51.0
9.32.0
25.02.3
12
Praesorether F (35)
29.092.4
36.46.4
25.10.9
13
Praesorether G (67)
79.91.4
74.02.5
72.81.8
14
Sekikaic acid (59)
79.51.7
77.04.1
81.51.6
15
4-O-Demethylbarbatic acid (58)
88.53.3
79.11.1
81.51.4
58.23.3
77.60.6
41.22.4
Camptothecin (positive control)
c)
a) The compounds were tested at the concentration of 100 μg/mL.
b) The presented data are means of three experiments ± S.D.
c) Camptothecin was tested at the concentration of 0.01 μg/mL for MCF-7 and NCI-H 460
and of 1 μg/mL for HeLa.
Table 3.21 : IC50 of cytotoxic activity against three cancer cell lines of
Praesorether G (67) and Sekikaic acid (59)
No.
Compound
IC50 (µg/mL)
HeLa
NCI-H460
MCF-7
1
Praesorether G (67)
22.54.7
21.42.1
17.90.8
2
Sekikaic acid (59)
53.44.1
48.85.4
44.15.4
122
Table 3.22. : Acetylcholinesterase inhibition of isolated compounds on
acetylcholinesterase
No.
Compound
Concentration (mg/mL)
1.0
0.5
0.25
1
Methyl haematommate (9)
-7.5
-7.2
-5.3
2
Butyl haematommate (49)
-7.9
-6.8
-5.7
3
Atranorin (11)
-8.7
-7.9
-6.9
4
Methyl -orsellinate (8)
-7.2
-7.0
-5.1
5
Atranol (50)
-6.4
-6.6
-4.6
6
Methyl chlorohaematommate (48)
-7.4
-6.5
-5.1
7
Methyl divaricatinate (51)
-7.7
-6.6
-5.4
8
(+)-(12R)-Isousnic acid (69)
-7.8
-7.7
-6.5
9
(+)-(12R)-Usnic acid (40)
-8.3
-7.6
-6.2
10
Methyl orsellinate (8)
-7.0
-6.4
-5.1
11
1,3-Diacetoxyhopan-29-oic acid (75)
-7.9
-7.9
-7.4
12
6-Methyl vinapraesorediosate A (44)
-8.5
-5.7
-5.7
13
Zeorin (Hopan-6α,22-diol) (74)
-9.9
-8.8
-7.6
14
Praesorether A (62)
-8.3
-6.7
-6.8
15
6-Methyl praesorediosate (46)
-8.1
-5.9
-5.6
16
Vinapraesorediosate A (43)
-8.6
-6.1
-5.5
17
Vinapraesorediosic acid C (47)
-8.3
-5.9
-5.6
123
CHAPTER 4
CONCLUSION
4.1. CONSTITUENTS OF PARMOTREMA PRAESOREDIOSUM
The chemical investigation of the lichen Parmotrema praesorediosum
growing in Vietnam led to the isolation of forty compounds, including twenty two
new compounds. Among eighteen known compounds, twelve compounds were
known for the first time from the genus Parmotrema. The chemical structure of
isolated compounds was determined by a combination of spectroscopic and
chemical methods, as well as comparing with the ones in the literature.
 Aliphatic acids: Except for (+)-praesorediosic acid (1), the other five
aliphatic acids (43-47) have not been reported. The five new aliphatic acids
(43-47) possessed the same γ-lactone skeleton as (+)-praesorediosic acid (1).
These compounds were obtained as major lichen substances from the thallis
of Parmotrema praesorediosum. These results were suitable for previous
phytochemical studies on this lichen by Krog H. [40].
 Mononuclear phenolic compounds: Twelve compounds (5, 8-9, 48-55, 57)
were isolated including seven known (5, 8-9, 48-51), three new phtalic acids
(52-54) and two new mononuclear phenolic compounds (55, 57). The phtalic
acids derived from polyketide pathway constituted a relatively rare group of
lichen, only one product known prior to the present work.
 Depsides: Four known depsides were isolated (11, 15, 58, 59). They are the
most typical aromatic polyketides.
 Depsidones: Two known compounds (60-61) was also isolated.
 Diphenyl ether: Although diphenyl ethers was a relatively rare group of
lichens, the investigation of the chloroform extract of the lichen Parmotrema
praesorediosum yielded eight compounds (35, 62-68) with only one known
(35) and seven new compounds (62-68). With the single exception of
124
PRAES-C5 (62), the other diphenyl ethers (35, 63-68) possessed the same
skeleton with the two isolated depsidones (60-61). These results suited to the
biosynthesis pathway [13]. Huneck S. [27] revealed that the diphenyl ethers
were sometimes referred as „pseudodepsidones‟ due to their apparent
biosynthetic relationship therefore they could contain the same or similar
monoaromatic units.
 Dibenzofurans:
Dibenzofurans were the third most abundant group of
coupled phenolics in lichens after depsides and depsidones. The chemistry
and biosynthesis of usnic acid and its derivative have been well-studied due
to their interesting structure, high yeild lichens and possessed various
biological activities. Up to this point, this present work also obtained three
compounds of dibenzofuran group (40, 69, 70), including two known (40,
69) and one new compound (70).
 Xanthones: From the chloroform extract of Parmotrema praesorediosum,
two new xanthones (71-72) were isolated. Xanthones were the most typical
compound in Usnea genus, but not from the lichen Parmotrema genus.
 Triterpenoids Three hopan skeleton triterpenoids (73-75), including one
new (75) and two known compounds (73, 74) were isolated from this lichen.
They are the first triterpenoids reported in Parmotrema genus.
 Macrocyclic compound: One new macrocyclic compound (75) was isolated
from the petroleum ether extract of the thallis lichen Parmotrema
praesorediosum.
125
 Group 1: Aliphatic acids (six compounds, five new compounds and one
known compound)
126
 Group 2: Mononuclear phenolic compounds (twelve compounds, five new
compounds and seven known compounds).
127
 Group 3: Depsides (four known compounds).
 Group 4: Depsidones (two known compounds).
 Group 5: Diphenyl ether (eight compounds, seven new compounds and one
known compound).
128
129
 Group 6: Dibenzofurans (three compounds, one new compound and two
known compounds).
 Group 7: Xanthones (two new compounds).
130
 Group 8: Triterpenoids (three compounds, one new compound and two
known compounds).
 Group 9: Macrocyclic compound (one new compound).
131
4.2. BIOLOGICAL ASSAY
4.2.1. Cytotoxicity
Fifteen compounds isolated from Parmotrema praesorediosum were tested
the cytotoxic activity against three cancer cell lines: MCF-7, HeLa and NCI-H460
by SRB assay method. The result showed that phenolic compounds exhibited
antiproliferative effect against several lympho cell lines. Example for two depsides
4-O-demethylbarbatic acid (58), sekikaic acid (59) and one diphenyl ether
praesorether G (67) performed strong inhibitive activities on all three cell lines. The
other skeletons as aliphatic acids, macrocyclic compound or triterpenoids showed
no cytotoxic activity against three surveyed cancer cell lines.
This results has been previously reported that numerous lichens contain
various phenolic components with anticancer activity including usnic acid,
lecanoric acid, gyrophoric acid, salazinic acid, lobaric acid, evernic acid, and
vulpinic acid [11, 13]. Further, it has also been suitable for phenolics arrest the cell
cycle and activate apoptotic signal transduction pathways in cancerous cells [1].
4.2.2. In vitro acetylcholinesterase inhibition activity
Seventeen pure compounds isolated from Parmotrema praesorediosum were
in vitro tested the inhibition against acetylcholinesterase. The results showed that all
the tested samples had no effect on acetylcholinesterase
132
FUTURE OUTLOOK
 Studying the chemical constituents of the remaining extracts of Parmotrema
praesorediosum, including ethyl acetate extract (50.0 g), acetone extract (45.0
g) and methanol extract (37.0 g) (Figure 4.1).
 Preparation of some derivatives from isolated aliphatic acids
 Testing the biological activity on other types of cancer cell lines and some other
inhibitory activities of isolated compounds and derivatives.
133
LIST OF PUBLICATIONS
1. HUYNH BUI LINH CHI, DUONG THUC HUY, TAKAO TANAHASHI,
NGUYEN KIM PHI PHUNG, Contribution to the study on chemical constituents of
the lichen Parmotrema praesorediosum (Nyl.) Hale, Parmeliaceae, Vienam Journal
of Chemistry, 48(4B), 332-337 (2010).
2. HUYNH BUI LINH CHI, DUONG THUC HUY, HA XUAN PHONG, TAKAO
TANAHASHI, NGUYEN KIM PHI PHUNG, Two new compounds from the
lichen Parmotrema praesorediosum (Nyl.) Hale, Parmeliaceae, Journal of Science
and Technology. 49(5B), 430-435 (2011).
3. HUYNH BUI LINH CHI, DUONG THUC HUY, TUONG LAM TRUONG, HA
XUAN PHONG, TAKAO TANAHASHI, NGUYEN KIM PHI PHUNG, A new
diphenyl ether from the lichen Parmotrema praesorediosum (Nyl.) Hale,
Parmeliaceae, Proceeding of the 3rd International Conference on Analytical Sciences
and Life Science, 309 – 312 (2013).
4. HUYNH BUI LINH CHI, TUONG LAM TRUONG, TAKAO TANAHASHI,
NGUYEN KIM PHI PHUNG, A new macrocylic compound from the lichen
Parmotrema praesorediosum (Nyl.) Hale, Parmeliaceae, Journal of Science and
Technology. 52(5A), 150-155 (2014).
134
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APPENDICES
145
6
HOOC
5
CH3
3
H
23
H3C
22
C
21
H2C
20
H2C
H2C
11
8
H2C
O
Appendix 1. IR spectrum for PRAES-C1
[M+Na]+
[M+H]+
Appendix 2. MS spectrum for PRAES-C1
146
7
H2C
2
4
1
O
O
23
4
5
21
7
Appendix 3. 1H NMR spectrum for PRAES-C1
6
HOOC
5
CH3
3
H
23
H3C
22
C
21
H2C
20
H2C
H2C
11
8
H2C
7
H2C
2
4
1
O
O
O
7
4
1
6
3
21
2
Appendix 4. 13C NMR spectrum for PRAES-C1
147
20
5
Appendix 5. DEPT spectrum for PRAES-C1
6
HOOC
5
CH3
3
H
23
H3C
22
C
21
H2C
20
H2C
H2C
11
8
H2C
7
H2C
2
4
1
O
O
Appendix 6. HSQC spectrum for PRAES-C1
148
O
6
HOOC
5
CH3
3
H
23
H3C
22
C
21
H2C
20
H2C
H2C
11
8
H2C
7
H2C
2
4
1
O
O
O
Appendix 7. HMBC spectrum for PRAES-C1
6
H3COOC
5
CH3
3
H
23
H3C
22
C
21
H2C
20
H2C
H2C
11
8
H2C
7
H2C
2
4
1
O
O
O
Appendix 8. IR spectrum for PRAES-E14
149
[M+H]+
6
H3COOC
5
CH3
3
H
23
H3C
22
C
21
H2C
20
H2C
H2C
11
8
H2C
7
H2C
2
4
1
O
O
O
Appendix 9. MS spectrum for PRAES-E14
6-OCH3
23
5
21
4
Appendix 10. 1H NMR spectrum for PRAES-E14
150
6
H3COOC
5
CH3
3
H
23
H3C
22
C
21
H2C
20
H2C
H2C
11
8
H2C
7
H2C
2
4
1
O
O
O
21
4
7
6-OCH3
22
1
6
3
2
Appendix 11.13C NMR spectrum for PRAES-E14
Appendix 12. DEPT spectrum for PRAES-E14
151
20
5
6
H3COOC
5
CH3
3
H
23
H3C
22
C
21
H2C
20
H2C
H2C
11
8
H2C
7
H2C
O
Appendix 13. COSY spectrum for PRAES-E14
Appendix 14. HSQC spectrum for PRAES-E14
152
2
4
1
O
O
6
H3COOC
5
CH3
3
H
23
H3C
22
C
21
H2C
20
H2C
H2C
11
8
H2C
7
H2C
2
4
1
O
O
Appendix 15. HMBC spectrum for PRAES-E14
[M+Na]+
153
O
23
5
22
4
21
21 7
20
8
4
22
1
6
3
2
154
5
155
6
HOOC
5
CH3
3
H
HO
21
C
20
H2C
19
H2C
8
H2C
H2C
7
H2C
10
O
[M+Na]+
156
2
4
1
O
O
6
HOOC
5
CH3
3
H
21
C
HO
20
H2C
19
H2C
8
H2C
H2C
7
H2C
2
4
1
O
O
10
5
O
20
7
4
7
20
19
4
5
21
1
6
3
2
157
6
HOOC
5
CH3
3
H
HO
21
C
20
H2C
19
H2C
7
H2C
8
H2C
H2C
2
4
1
O
O
10
O
6
HOOC
5
CH3
3
H
HO
21
C
20
H2C
19
H2C
8
H2C
H2C
7
H2C
2
4
1
O
O
10
O
158
6
H3COOC
5
CH3
3
H
HO
21
C
20
H2C
19
H2C
8
H2C
H2C
7
H2C
2
4
1
O
O
10
O
C=O lactone
Appendix 27. IR spectrum for PRAES-E19
[M+H]+
159
6
H3COOC
5
CH3
3
H
21
C
HO
20
H2C
19
H2C
7
H2C
8
H2C
H2C
2
4
1
O
O
5
10
O
6-OCH3
20
7
4
6
H3COOC
5
CH3
3
H
21
C
HO
20
H2C
19
H2C
8
H2C
H2C
7
H2C
2
4
1
O
O
10
7
O
19
4
6-OCH3
3
21
1
6
20
2
Appendix 30. 13C NMR spectrum for PRAES-E19
160
5
6
H3COOC
5
CH3
3
H
HO
21
C
20
H2C
19
H2C
8
H2C
H2C
7
H2C
2
4
1
O
O
10
O
161
6
H3COOC
5
CH3
3
H
HO
21
C
20
H2C
19
H2C
8
H2C
H2C
7
H2C
2
4
1
O
O
10
O
162
[M+Na]+
6
HOOC
5
CH3
3
H
21
H3CO
20
C
19
H2C
18
H2C
8
H2C
H2C
7
H2C
9
O
6
HOOC
5
CH3
3
H
21
H3CO
20
C
19
H2C
18
H2C
7
H2C
8
H2C
H2C
2
4
1
O
5
O
20-OCH3
9
O
19
4
7
Appendix 36. H1 NMR spectrum for PRAES-C2
163
2
4
1
O
O
18
19
4
20
1
3
6
7
20-OCH3
2
Appendix 37. C13 NMR spectrum for PRAES-C2
6
HOOC
5
CH3
3
H
21
H3CO
20
C
19
H2C
18
H2C
8
H2C
H2C
7
H2C
2
4
1
O
O
9
O
164
5
6
HOOC
5
CH3
3
H
21
H3CO
20
C
19
H2C
18
H2C
H2C
9
8
H2C
7
H2C
2
4
1
O
O
O
165
9
CH3 O
5
7
10
1
3
HO
8
OCH3
OH
CHO
Appendix 41. 1H NMR spectrum for PRAES-T1
9
CH3 O
5
7
10
1
3
HO
8
OCH3
OH
CHO
Appendix 42. 13C NMR spectrum for PRAES-T1
166
9
CH3 O
5
7
10
1
3
HO
8
OCH3
OH
CHO
Appendix 43. HSQC spectrum for PRAES-T1
Appendix 44. HMBC spectrum for PRAES-T1
167
[M+Na]+
Cl
9
CH3 O
5
7
10
1
3
HO
8
OCH3
OH
CHO
168
A
Cl
9
CH3 O
5
7
10
1
3
HO
8
OCH3
OH
CHO
169
Cl
9
CH3 O
5
7
10
1
3
HO
8
OCH3
OH
CHO
9
CH3 O
5
7
10
1
3
HO
8
11
12
13
OCH2CH2CH2CH3
OH
CHO
170
9
CH3 O
5
7
10
1
3
HO
8
11
12
13
OCH2CH2CH2CH3
OH
CHO
Appendix 52. DEPT spectrum for PRAES-T2
171
9
CH3 O
10
5
1
3
HO
8
7
11
12
13
OCH2CH2CH2CH3
OH
CHO
172
1'
CH3 O
5
1
3
HO
7
OCH3
OH
1'
CH3 O
5
HO
1
3
7
OCH3
OH
173
1'
CH3 O
5
HO
1
3
7
OCH3
OH
174
1'
CH3 O
5
HO
1
3
7
OCH3
OH
CH3
175
1'
CH3 O
5
7
1
3
HO
OCH3
OH
CH3
1'
CH3 O
5
HO
1
3
7
OCH3
OH
CH3
176
[M+H]+
Appendix 63. MS spectrum for PRAES-T6
1'
CH3
5
HO
1
3
OH
CHO
177
1'
CH3
5
HO
1
3
OH
CHO
178
3'
CH3
2'
1'
5
H3CO
3
O
7
1
OCH3
OH
3'
CH3
2'
1'
5
H3CO
3
1
O
7
OCH3
OH
179
3'
CH3
2'
1'
5
3
H3CO
O
7
1
OCH3
OH
3'
CH3
2'
1'
5
H3CO
3
1
O
7
OCH3
OH
180
C=O (lactone)
1785 cm-1
[M+Na]+
[M+H]+
181
1
1
9
7
4-OH
3
8
3
7
8
9
1
1
6
4
7a 3a
5
1
182
183
9
4-OH
8
7
3
8
9
11
7
3
5
3a
7a
4
6
1
Appendix 78. NOESY spectrum for PRAES-C22
184
1 1
C=O (lactone)
1789 cm-1
[M+H]+
185
9
1
2
3
7
8
1
4-OH
3
9
8
7
1
1
6
4
7a 3a
1
5
186
2
187
9
7
3
1
2
8
1
2
9
1
1
8
7
3
5
3a
7a
4
6
1
188
C=O (lactone)
1766 cm-1
[M+Na]+
[M+H]+
189
2, 2
9
4-OH
3
7
1
8
1
9
7
3
1
8
6
1
4
1
5
7a 3a
2, 2
190
191
9
4-OH
7
3
8
1
2, 2
1
2, 2
9
8
1
7
3
5
3a
7a
4
6
1
192
[M+Na]+
193
Appendix 98. IR spectrum for PRAES-C25M
194
[M+Na]+
[M+H]+
Appendix 99. MS spectrum for PRAES-C25M
13
10
9
6
8
11
Appendix 100. 1H NMR spectrum for PRAES-C25M
195
12
12
8
6 11
7
5, 3
1
2
10
9
13
4
Appendix 101. 13C NMR spectrum for PRAES-C25M
Appendix 102. DEPT spectrum for PRAES-C25M
196
12
Appendix 103. COSY spectrum for PRAES-C25M
Appendix 104. HSQC spectrum for PRAES-C25M
197
6
8
11
13 9
10
12
12
13
9
10
8
11
6
4
2
1
5, 3
7
Appendix 105. HMBC spectrum for PRAES-C25M
Appendix 106. NOESY spectrum for PRAES-C25M
198
[M+Na]+
199
10
9
8
13
12
6
3-OH
Appendix 109. `1H NMR spectrum for PRAES-C26
10
9
12
8
11 5
2, 4
3
7
6
1
200
13
201
10
8
6
12
9
13
9
10
12
8
6
1
2, 4
5
11
7
3
202
13
203
Appendix 117. DEPT spectrum for PRAES-T3
204
205
206
207
[M+H]+
Appendix 144. H1 NMR spectrum for PRAES-E18 Appendix 144. H1 NMR
spectrum for PRAES-E18
208
209
210
211
212
213
214
215
[M+Na]+
216
217
218
OH
C=O
C=C
219
[M+H]+
A
220
221
222
OH
C=O
C=C
223
[M+H]+ [M+Na]
224
+
225
226
Appendix 163. ROESY spectrum for PRAES-C16
C=O
OH
C=C
227
[M+Na]+
[M+H]+
228
229
Appendix 169. COSY spectrum for PRAES-C20
230
231
OH
C=O
C=C
[M+Na]+
[M+H]+
232
233
234
235
[M+Na]+
236
237
238
[M+Na]+
239
240
241
C=O
OH
C=C
[M+Na]+
[M+H]+
242
243
244
245
246
247
248
[M+Na]+
249
250
251
OH
C=O
C=C
252
[M+H]+
253
254
255
256
OH
C=O
C=C
257
[M+H]+
258
259
260
261
262
263
[M+Na]+
264
Appendix 240.13C NMR spectrum for PRAES-E13
265
266
267
[M+Na]+
268
269
270
Appendix 252. 1H NMR spectrum for PRAES-C2Me
271
Appendix 253. 13C NMR spectrum for PRAES-C2Me
Appendix 254. 1H NMR spectrum for PRAES-C2Et
272
Appendix 255. 13C NMR spectrum for PRAES-C2Et
Appendix 256. 1H NMR spectrum for PRAES-C2Pro
273
Appendix 257. 13C NMR spectrum for PRAES-C2Pro
Appendix 258. HSQC spectrum for PRAES-C2Pro
274
Appendix 259. HMBC spectrum for PRAES-C2Pro
275

ĐẠI HỌC QUỐC GIA THÀNH PHỐ HỒ CHÍ MINH

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