Quinones, chromones and xanthones 11Figure 1.11: Puvinic acid derivatives 12Figure 1.12: Two common triterpenenoid skeletons 12Figure 1.13: Some N-containing compounds 13Figure 1.14: Usn
The work presented in this thesis was completed in the period of December
2017 to December 2021 under the co-supervision of Professor Nguyen Kim Phi Phung of the University of Science, Vietnam National University, Ho Chi Minh City, Vietnam, and Associate Professor Warinthorn Chavasiri of the Chulalongkorn University, Thailand.
In compliance with the university’s regulations, I declare that:
1 Except where due acknowledgment 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 postgraduate study program;
4 Ethics procedures and guidelines have been followed.
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Firstly, I offer my sincerest gratitude to my supervisor Prof Nguyen Kim Phi Phung, who has supported me throughout the writing of this thesis with her patience, kindness, invaluable advice, and guidance.
I would also like to acknowledge my second supervisor, Prof Dr Warinthor Chavasiri for his guidance, patience, and who has taught me the true spirit of research I am deeply indebted to Assoc Prof Dr Huynh Bui Linh Chi of Dong Nai University for her teachings, kindness, helpful suggestions, and valuable advice in this research
I want to express my sincere thanks to Dr Vo Thi Phi Giao at the 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 their expert assistance in the authenticity of this lichen.
Iam very grateful to thank Assoc Prof Dr Pham Nguyen Kim Tuyen, Assoc. Prof Dr Duong Thuc Huy, and Dr Nguyen Tan Phat for giving up their precious time to help me with proofreading some isolated compounds of the thesis.
Additionally, I would like to thank my teachers and friends from the Department of Organic Chemistry, Faculty of Chemistry, University of Science, Vietnam National University-Ho Chi Minh City, and students at Dong Nai University for their helpful assistance.
Besides, I am also very grateful to thank Vingroup company because I was funded by Vingroup Joint Stock Company and supported by the Domestic PhD Scholarship Programme of Vingroup Innovation Foundation (VINIF), Vingroup Big Data Institute (VINBIGDATA), code VINIF.2020.TS.41.
Finally, an honorable mention goes to my family, especially my parents and wife, for their understanding and support in completing this thesis.
LIST OF ABBREVIA TIONS Ánh ng HH Hàng HH nh vn
LIST OF SCHEMES - LH nh TH HT HH HH nh nh xii
CHAPTER 1: LITERATURE REVIEW HH HH HH re 1 1.1 PRESENTATION OF LICHENS - -Q- HH HH HH ie, 1 1.2 LICHEN SECONDARY METABOLTTTES - SG Series 3
1.2.2 Shikimic acid pathWAy ác TH TH HH HH 12
1.2.3 Mevalonic acid patWayV/ - án HH TT TH HH HH 12
1.2.4 N-containing COIPOUTIỞS - (+ c1 111011911 E910 19119 ng ng rry 13 1.3 GENERAL INFORMATION OF USNEA CERATINA ARCH, PARMOTREMA
TINCTORUM (DESPR EX NYL.) HALE -.- 5 5 cSxsssersersrrerrs 13 IESN/3//2.0212,.,.0 in 15
1.3.2 Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae) 15
1.3.3 Parmotrema tinctorum (Despr ex Nyl.) Hale .-4!
Dilatatone (195) and sernanderin (196) were isolated from P dilatum by
Duong Thuc Huy et al (2021) from this lichen in Vietnam.!*?!
Depside atranorin (55), depsidone salazinic acid (77), and xanthone lichexanthone (144) were isolated from the chloroform extract of P. lichexanthonicum by Ana C Micheletti et al (2009)."!
N K Honda et al (2015) isolated salazinic acid (77) and lichexanthone
> Parmotrema nilgherrense a-Alectoronic acid (96), a-collatolic acid (95), and dehydrocollatolic acid
(113) were isolated by Madam K Kharel et al (2000).'°°!
Depside atranorin (55) was isolated by Verma Neeraj et al (2011).18i
Atranorin (55), chloroatranorin (56), salazinic acid (77), and consalazinic acid (78) were isolated from the acetone extract by Fazio A T et al (2009)."4!
Gyrophoric acid (76) was also found in the P reticulatum (2010).9!
Reticulatin (183), a terpenoid, from the hexane extract of lichen P reticulatum as well as five previously-reported compounds as zeorin (153), leucotylin (156), lupeol (161), betulinic acid (162), and dihydroreynosin (184) were isolated by
Duong Thuc Huy et al (2021) from this lichen in Vietnam.!'*?!
Atranorin (55) and chloroatranorin (56) were isolated from the hexane extract of P saccatilobum by Bugni T S et al (2009).13
Orsellinic acid (29), methyl orsellinate (30), atranorin (55), and lecanoric acid (57) were isolated by Jayaprakasha G K et al (2000).'4°!
Depside atranorin (55) and two depsidones: salazinic acid (77) and consalazinic acid (78) were isolated from the acetone extract by O’Donovan D G. et al (1980).131
Isolecanoric acid (59) was isolated by Sakurai A et al (1987).!°!
Orcinol (25), methyl ỉ-orsellinate (32), and methyl haematommate (23) were isolated by Irma S Rojas (2002).'57!
Ethyl orsellinate (31) was isolated by Santos L C et al (2004) !°*!
Atranorin (55) and lecanoric acid (57) were isolated by Honda N K et al.
A Ganesan et al (2015) isolated atranorin (55), chloroatranorin (56), lecanoric acid (57), and salazinic acid (77).LH
E M K Wijeratne et al (2015) isolated oxaspirol A (191), oxaspirol B (192), oxaspirol C (193), and oxaspirol D (194).13!
S Z A Khader et al (2018) isolated atranorin (55), usnic acid (135), and 4- hydroxy-5-methyl-isophthalic acid 3-(3,4-dihydroxy-5-methylphenyl) ester (66).!77!
P P Kumar et al (2019) isolated orsellinic acid (29), methyl atrate (26), atranorin (55), lecanoric acid (57), salazinic acid (77), and norlobaridone (97).!!!
Consalazinic acid (78), salazinic acid (77), and atranorin (55) were isolated from the P clavuliferum (2010).9
> Parmotrema rampoddense a-Alectoronic acid (96), a-collatolic acid (95), atranorin (55), chloroatranorin
(56), and skyrin (182) were found from the P rampoddense (2010).!7°!
V P Rajan et al (2016) isolated atranorin (55), chloroatranorin (56), a- collatolic acid (95), ỉ-alectoronic acid (130), and usnic acid (135).1!49
Orcinol (25), orsellinic acid (29), atranol (49), methyl /- orsellinate (32), atranorin (57), diffractaic acid (64), lecanoric acid (57), bailesidone
(114), and tinctorinone (115) were found by Nguyen Thi Ngoc Van et al (2021).18
Cristiferides A-B (41-67) together with six known compounds: 2,4- dihydroxyphthalide (40), lecanoric acid (57), orsellinic acid (29), 5-chloroorsellinic acid (34), methyl haematommate (23), and methyl /-orsellinate (32) were isolated by Nguyen Kim Tuyen Pham et al (2021),!°°!
In 2012, from the lichen Usnea aciculifera Vain, Tuong Lam Truong et al. isolated many compounds such as (+)-(12R)-usnic acid (135), methyl orsellinate
(30), norstictic acid (81), atranorin (55), barbatinic acid (63), antranol (49), stictic acid (79), and diffractaic acid (64).!!7°
After that, a new depside: aciculiferin A (74) was isolated, together with eleven known compounds: (+)-(12R)-usnic acid (135), methyl haematommate (23), methyl f-orsellinate (32), methyl orsellinate (30), atranol (49), 7-hydroxy-5- methoxy-6-methylphthalide (39), norstictic acid (81), stictic acid (79), atranorin
(55), barbatinic acid (63), and diffractaic acid (64) were also isolated by Tuong Lam
Truong et al in 2014.HZ7
Usnic acid (135), norstictic acid (81), stictic acid (79), and caperatic acid (21) were isolated by J F Keeton and M F Keogh (1973).!4!
Nishanth K S et al found protolichesterinic acid (9) in 2014.1191
> Usnea aliphatica a-(15-hydroxyhexadecyl)itaconic acid (20), usnic acid (135), norstictic acid
(81), and salazinic acid (77) were found in 1977 by M F Keogh and M E. Zurita [78]
In 1972, from the lichen Usnea angulata Ach, M E Mendiondo and J D.
Coussio isolated two compounds: (+)-usnic acid (135) and norstictic acid (81).!7>!
(+)-usnic acid (135) and 5a,8q@-epidioxy-5 ứ-ergosta-6,22-diene-3/@'-ol (166) were isolated in 1978 from the lichen Usnea annulata (Mull Argo.).!®?!
In 2007, from the lichen Usnea articulata (L.), F L Devehat and J Boustie isolated fourteen compounds, including hypoprotocetraric acid (117) and cryptostictinolide (107), stictic acid (79), norstictic acid (81), fumarprotocetraric acid (116), peristictic acid (121), cryptostictic acid (106), constictic acid (80), menegazziaic acid (85), barbatinic acid (63), atranorin (55), methyl /-orsellinate
(+)-Usnic acid (135), salazinic acid (77), norstictic acid (81), atranorin (55) and protocetraric acid (89) were found in 2010 by L B Din et a/.15"
The U baileyi thalli were collected on tree barks at Lam Dong province, Vietnam In 2018, Kieu Van Nguyen et al found two new compounds, including a new bixanthone, bailexanthone (150) and a new depsidone, bailesidone (12) The known compounds were elucidated such as stictic acid (79), constictic acid (80), cryptostictic acid (106), hypoconstictic acid (86), menegazziaic acid (85), 8’-O-methylconstictic acid (123), methylstictic acid (118), 8'-O- methylmenegazziaic acid (122), virensic acid (90), 9'-O-methylprotocetraric acid
(93), protocetraric acid (89), barbatic acid (63), diffractaic acid (64), 4-Ó- demethylbarbatic acid (65), atranorin (55), (20R,24R)-ocotillone (163), (20S,24K)- ocotillone (164), betulonic acid (165), usnic acid (135), 7-hydroxy-5-methoxy-6- methylphthalide (39), methyl 4-O methylhaematomate (44), methyl /-orsellinate
Eight compounds, 8'-O-methylprotocetraric acid (88), protocetraric acid
(89), virensic acid (90), subvirensic acid (91), barbatic acid (63), diffractaic acid
(64), 4-O-demethylbabartic acid (65), and atranorin (55), were isolated using various chromatographic methods by Kieu Van Nguyen et al (2019).1“1
Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae)
Scientific name: Parmotrema praesorediosum (Nyl.) Hale Family: Parmeliaceae
Morphography: Thallus foliose, adnate to the substratum, 3—10 cm across.
Lobes round, 4-10 mm wide; margins entire or crenate, eciliate, and sorediate. Upper surface: pale grey to grey, smooth, dull, emaculate, weakly rugose,
15 lacking isidia, and sorediate Soralia marginal, linear to crescent shaped, granular. Medulla white Lower surface: black, minutely rugose, with shiny, mottled, ivory or brown, and erhizinate marginal zone Rhizines: sparse, simple, short.
Apothecia and pycnidia are not seen (Figure 1.15).'*4!
Figure 1.15: Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae).
Spotest: Cortex K+ (yellow), C—, KC—, P-; medulla K—, C—, KC-, P—
“+ K: 10% aqueous solution of potassium hydroxide, KOH. s*P: para-phenylenediamine solution, CsHsN2 (para-phenylenediamine: | g, sodium sulphite: 10 g, washing-up liquid: 0.5 mL). s+ C: Saturated solution of calcium hypochlorite solution, Ca(C]O)a.
“* KC: K is applied to the specimen After about 30 seconds it can be taken up on to a tissue or white filter paper A drop of C is then placed on the paper next to the K The colour change, which is often fleeting, occurs where they intermingle.
TLC: Atranorin, chloroatranorin, and fatty acids (protopraesorediosic acid, praesorediosic acid).
Parmotrema tinctorum (Despr ex Nyl.) Hale -<++ 17 1.4 CHEMICAL STUDIES ON THE GENUS PARMOTREMA AND USNEA
Scientific name: Parmotrema tinctorum (Despr ex Nyl.) Hale Family: Parmeliaceae
Morphography: Thallus: foliose, loosely attached to the substratum, membranaceous to coriaceous, up to 18 cm across Lobes: rotund to irregular, 5—15 mm wide; margins: entire to crenate, eciliate Upper surface: pale grey to grey green, shiny, becoming dull towards the thallus center, somewhat longitudinally folded in the marginal region, emaculate, without soredia, isidiate Isidia: sparse to dense, laminal and eventually marginal, granular to filiform, simple to coralloid branched, thin, brown tipped or concolorous Medulla: white Lower surface: black, minutely wrinkled, smooth, shiny, with a broad, erhizinate, pale brown to dark tan marginal zone Rhizines sparse, simple, and short Apothecia and pycnidia: not seen.H391
The upper surface The lower surface
Figure 1.16: Parmotrema tinctorum (Despr ex Nyl.) Hale.
Spotest: Cortex K+ (yellow), C-, KC-, P-; medulla
TLC: atranorin, chloroatranorin, and lecanoric acid.
1.4 CHEMICAL STUDIES ON THE GENUS PARMOTREMA AND USNEA
The lichen genus PArMotre ma 5 <1 vn key 18 1.4.2 The 00.20 0/11
A Torres-Benitez et al (2017) isolated many compounds such as tetrahydroxydocosanoic acid (12), pentahydroxyoxooctacosanoic acid (13), hydrox ydioxohenicosanoic acid (14), trioxohenicosanoic acid (15), dihydroxydioxononadecanoic acid (16), dihydroxyheptadecatrienoic acid (17), orsellinic acid (29), ethyl haematommate (24), atranol (49), haematommic acid (43), pentyldivaric acid (54), atranorin (55), lecanoric acid (57), decarboxythamnolic acid
(72), evernic acid (73), 2-O-methylstenosporic acid (75), barbatic acid (63), salazinic acid (77), stictic acid (79), connorstictic acid (83), substictic acid (84), hypoconstictic acid (86), a-collatolic acid (95), lobaric acid (99), furfuric acid
(112), /ỉ-collatolic acid (131), loxodinol (79), usnic acid (82), and lepraric acid
C Y Ragasa et al (2018) isolated atranorin (55) and zeorin (153).""®!
A Ganesan et al (2015) isolated atranorin (55), chloroatranorin (56), lecanoric acid (57) and salazinic acid (77).LH
Z R Guterres e al (2017) isolated salazinic acid (77).54
U V Mallavadhani et al (2018) isolated one depside atranorin (55).!°6)
U V Mallavadhani et al (2019) isolated methyl haematommate (23), orcinol
V Sieteiglesias et al (2019) isolated constictic acid (80), and stictic acid
Atranorin (55), salazinic acid (77), and consalazinic acid (78) were reported in the P clavuliferum by L F G Brandão et al (2010).!7*!
M I Choudhary et al (2011) isolated atraric acid (methyl orsellinate) (30), orsellinic acid (29), ethyl heamatommate (24), ethyl orsellinate (31), lecanoric acid
A Torres-Benitez et al (2017) isolated many compounds including tetrahydroxydocosanoic acid (12), pentahydroxyhexacosanoic acid (18), heptahydroxypentacosanoic acid (19), orsellinic acid (29), atranorin (55), chloroatranorin (56), lecanoric acid (57), evernic acid (73), barbatic acid (63), thamnolic acid (68), haemathamnolic acid (69), gyrophoric acid (76), pseudocyphellarin A (70), sekikaic acid (71), salazinic acid (77), constictic acid
(80), connorstictic acid (83), substictic acid (84), hypoconstictic acid (87), consalazinic acid (78), norstictic acid (81), conprotocetraric acid (92), a-alectoronic acid (96), lobaric acid (99), -alectoronic acid (130), (+)-usnic acid (135), and strepsilin (141).)
N K Honda et al (2016) isolated protolichesterinic acid (9), atranorin (55), norlobaridone (97) and loxodin (98).!%7!
(+)-Praesorediosic acid (1), (+)-protopraesorediosic acid (7), atranorin (55), and chloroatranorin (56) were isolated by Feeya David et al (1990).27)
Lecanoric acid (57) and stictic acid (79) were isolated from Parmelia praesorediosa (Nyl.) by Ramesh P et al (1994).TH]
Huynh Bui Linh Chi et al isolated zeorin (153) and 1/,3/-diacetoxyhopan-29- oic acid (154) (2011) from this lichen in Vietnam.'47 48!
Some phenolic compounds from the lichen P praesorediosum (Nyl.) Hale (Parmeliaceae) such as praesorether A (124), praesorether B (125), praesorether C
(126), praesorether D (127) and praesalide A (35), praesalide B (36), and praesalide
C (37) were isolated by Huynh Bui Linh Chi et al from this lichen in Vietnam.
New y-lactone carboxylic acids from the lichen P praesorediosum (Nyl.) Hale such as (+)-vinapraesorediosic acid A (3), (+)-6-methyl vinapraesorediosate A (4), (+)-vinapraesorediosic acid B (5), (+)-6-methyl praesorediosate (2), and (+)- vinapraesorediosic acid C (6) were isolated by Huynh Bui Linh Chi et al (2016) from this lichen in Vietnam.L
Praesorexanthone A (151) and praesorexanthone B (152) were isolated by
Huynh Bui Linh Chi er al (2020) from this lichen in Vietnam.!®!
In 2021, Bui-Linh-Chi Huynh et al also isolated vinapraesorediosic acids D
(11), E (38), and paresordin A (134) from this lichen in Vietnam L1: !7!
Atranorin (55), lecanoric acid (57), and a-collatolic acid (95) were isolated from P sancti-angelii by Verma Neeraj et al (2011).18'
In 2016, Nguyen Thi Thu Tram ef al isolated f-orcinolcarboxylate (33), salazinic acid (77), and atranorin (55).
Hopane-6a,16a,22-triol (155), leucotylin (156), 16/-acetoxyhopane-6z,22- diol (157), 6a-acetox yhopane-16/,22-diol (158), zeorin (153), 6a-acetox yhopane-
22-ol (159), ergosterol peroxide (168), brassicasterol (178), and atranorin (55) were isolated by Duong Thuc Huy et al in 2019 from this lichen in Vietnam.l8!: 3!
Huynh Tram et al isolated atranol (49), methyl haematomate (23), orsellinic acid (29), and lecanoric acid (57) in 2019.121
Duong Thuc Huy et al isolated three compounds, including sanctis A-C (197,
198, 199) in 2018 from this lichen in Vietnam.!!?”?l
Parmosidone A (100), parmosidone B (101), parmosidone C (103), parmosidone D (104), parmosidone E (102), parmoether A (132), and parmoether B
(133) were isolated from P tsavoense by Duong Thuc Huy et al (2015) from this lichen in Vietnam.9!
Parmosidone K (105) and barbatic acid (63) were also isolated by Hoai Thu
Nguyen et al in 2016 from this lichen in Vietnam.!'°?!
Duong Thuc Huy et al isolated methyl (E)-2,4-dihydroxy-6-methyl-3-(3- oxobut-1-en-1-yl)benzoate (51), atranol (49), and 2-O-methylatranol (50) in
T H Duong et al (2018) isolated tsavoenone A (187), tsavoenone B (188), and tsavoenone C (189) from this lichen in Vietnam.3!!
Parmosidones F (111) was isolated by Duong Thuc Huy et al in 2020 from this lichen in Vietnam !°7!
Orcinol (25), orsellinic acid (29), methyl orsellinate (30), methyl /ỉ- orsellinate (32), methyl haematommate (23), atranorin (55), gyrophoric acid (76), lecanoric acid (57), protocetraric acid (89), 9’-O-methylprotocetraric acid (93), and (+)-usnic acid (135) were isolated by Duong Thuc Huy et al (2011, 2012) from this lichen in Vietnam lZ* 28l
Lichesterinic acid (8), 8-[2,4-dihydroxy-6-(2-oxoheptyl)phenoxy]-6-methoxy- 3-pentyl-1H-isochromen-l-one (128), 8-[2,4-dihydroxy-6-(2-oxoheptyl)phenoxy]- 6-hydroxy-3-pentyl-1H-isochromen-l-one (129), /-collatolc acid (130), D- mannitol (185), and D-arabinitol (186) were isolated by Duong Thuc Huy et al.
(2014) from this lichen in Vietnam.1”!
Protocetraric acid (92), malonprotocetraric acid (94), and (+)-usnic acid (135) were isolated by Myles F Keogh et al (1977).
Depside atranorin (55), depsidones salazinic acid (77), norstictic acid
(81), hypostictic acid (82), and protocetraric acid (89) were isolated from P. dilatum by N.K Honda et al (2010).011
L F G Brandão et al (2013) isolated atranorin (55), diffractaic acid (64), divaricatic acid (61), perlatolic acid (62), norstictic acid (81), protocetraric acid
(89), psoromic acid (110), (+)-usnic acid (135), and lichexanthone (144) !!"71
Z R Guterres et al (2017) isolated protocetraric acid (89).!!>4!
Dilatatone (195) and sernanderin (196) were isolated from P dilatum by
Duong Thuc Huy et al (2021) from this lichen in Vietnam.!*?!
Depside atranorin (55), depsidone salazinic acid (77), and xanthone lichexanthone (144) were isolated from the chloroform extract of P. lichexanthonicum by Ana C Micheletti et al (2009)."!
N K Honda et al (2015) isolated salazinic acid (77) and lichexanthone
> Parmotrema nilgherrense a-Alectoronic acid (96), a-collatolic acid (95), and dehydrocollatolic acid
(113) were isolated by Madam K Kharel et al (2000).'°°!
Depside atranorin (55) was isolated by Verma Neeraj et al (2011).18i
Atranorin (55), chloroatranorin (56), salazinic acid (77), and consalazinic acid (78) were isolated from the acetone extract by Fazio A T et al (2009)."4!
Gyrophoric acid (76) was also found in the P reticulatum (2010).9!
Reticulatin (183), a terpenoid, from the hexane extract of lichen P reticulatum as well as five previously-reported compounds as zeorin (153), leucotylin (156), lupeol (161), betulinic acid (162), and dihydroreynosin (184) were isolated by
Duong Thuc Huy et al (2021) from this lichen in Vietnam.!'*?!
Atranorin (55) and chloroatranorin (56) were isolated from the hexane extract of P saccatilobum by Bugni T S et al (2009).13
Orsellinic acid (29), methyl orsellinate (30), atranorin (55), and lecanoric acid (57) were isolated by Jayaprakasha G K et al (2000).'4°!
Depside atranorin (55) and two depsidones: salazinic acid (77) and consalazinic acid (78) were isolated from the acetone extract by O’Donovan D G. et al (1980).131
Isolecanoric acid (59) was isolated by Sakurai A et al (1987).!°!
Orcinol (25), methyl ỉ-orsellinate (32), and methyl haematommate (23) were isolated by Irma S Rojas (2002).'57!
Ethyl orsellinate (31) was isolated by Santos L C et al (2004) !°*!
Atranorin (55) and lecanoric acid (57) were isolated by Honda N K et al.
A Ganesan et al (2015) isolated atranorin (55), chloroatranorin (56), lecanoric acid (57), and salazinic acid (77).LH
E M K Wijeratne et al (2015) isolated oxaspirol A (191), oxaspirol B (192), oxaspirol C (193), and oxaspirol D (194).13!
S Z A Khader et al (2018) isolated atranorin (55), usnic acid (135), and 4- hydroxy-5-methyl-isophthalic acid 3-(3,4-dihydroxy-5-methylphenyl) ester (66).!77!
P P Kumar et al (2019) isolated orsellinic acid (29), methyl atrate (26), atranorin (55), lecanoric acid (57), salazinic acid (77), and norlobaridone (97).!!!
Consalazinic acid (78), salazinic acid (77), and atranorin (55) were isolated from the P clavuliferum (2010).9
> Parmotrema rampoddense a-Alectoronic acid (96), a-collatolic acid (95), atranorin (55), chloroatranorin
(56), and skyrin (182) were found from the P rampoddense (2010).!7°!
V P Rajan et al (2016) isolated atranorin (55), chloroatranorin (56), a- collatolic acid (95), ỉ-alectoronic acid (130), and usnic acid (135).1!49
Orcinol (25), orsellinic acid (29), atranol (49), methyl /- orsellinate (32), atranorin (57), diffractaic acid (64), lecanoric acid (57), bailesidone
(114), and tinctorinone (115) were found by Nguyen Thi Ngoc Van et al (2021).18
Cristiferides A-B (41-67) together with six known compounds: 2,4- dihydroxyphthalide (40), lecanoric acid (57), orsellinic acid (29), 5-chloroorsellinic acid (34), methyl haematommate (23), and methyl /-orsellinate (32) were isolated by Nguyen Kim Tuyen Pham et al (2021),!°°!
In 2012, from the lichen Usnea aciculifera Vain, Tuong Lam Truong et al. isolated many compounds such as (+)-(12R)-usnic acid (135), methyl orsellinate
(30), norstictic acid (81), atranorin (55), barbatinic acid (63), antranol (49), stictic acid (79), and diffractaic acid (64).!!7°
After that, a new depside: aciculiferin A (74) was isolated, together with eleven known compounds: (+)-(12R)-usnic acid (135), methyl haematommate (23), methyl f-orsellinate (32), methyl orsellinate (30), atranol (49), 7-hydroxy-5- methoxy-6-methylphthalide (39), norstictic acid (81), stictic acid (79), atranorin
(55), barbatinic acid (63), and diffractaic acid (64) were also isolated by Tuong Lam
Truong et al in 2014.HZ7
Usnic acid (135), norstictic acid (81), stictic acid (79), and caperatic acid (21) were isolated by J F Keeton and M F Keogh (1973).!4!
Nishanth K S et al found protolichesterinic acid (9) in 2014.1191
> Usnea aliphatica a-(15-hydroxyhexadecyl)itaconic acid (20), usnic acid (135), norstictic acid
(81), and salazinic acid (77) were found in 1977 by M F Keogh and M E. Zurita [78]
In 1972, from the lichen Usnea angulata Ach, M E Mendiondo and J D.
Coussio isolated two compounds: (+)-usnic acid (135) and norstictic acid (81).!7>!
(+)-usnic acid (135) and 5a,8q@-epidioxy-5 ứ-ergosta-6,22-diene-3/@'-ol (166) were isolated in 1978 from the lichen Usnea annulata (Mull Argo.).!®?!
In 2007, from the lichen Usnea articulata (L.), F L Devehat and J Boustie isolated fourteen compounds, including hypoprotocetraric acid (117) and cryptostictinolide (107), stictic acid (79), norstictic acid (81), fumarprotocetraric acid (116), peristictic acid (121), cryptostictic acid (106), constictic acid (80), menegazziaic acid (85), barbatinic acid (63), atranorin (55), methyl /-orsellinate
(+)-Usnic acid (135), salazinic acid (77), norstictic acid (81), atranorin (55) and protocetraric acid (89) were found in 2010 by L B Din et a/.15"
The U baileyi thalli were collected on tree barks at Lam Dong province, Vietnam In 2018, Kieu Van Nguyen et al found two new compounds, including a new bixanthone, bailexanthone (150) and a new depsidone, bailesidone (12) The known compounds were elucidated such as stictic acid (79), constictic acid (80), cryptostictic acid (106), hypoconstictic acid (86), menegazziaic acid (85), 8’-O-methylconstictic acid (123), methylstictic acid (118), 8'-O- methylmenegazziaic acid (122), virensic acid (90), 9'-O-methylprotocetraric acid
(93), protocetraric acid (89), barbatic acid (63), diffractaic acid (64), 4-Ó- demethylbarbatic acid (65), atranorin (55), (20R,24R)-ocotillone (163), (20S,24K)- ocotillone (164), betulonic acid (165), usnic acid (135), 7-hydroxy-5-methoxy-6- methylphthalide (39), methyl 4-O methylhaematomate (44), methyl /-orsellinate
Eight compounds, 8'-O-methylprotocetraric acid (88), protocetraric acid
(89), virensic acid (90), subvirensic acid (91), barbatic acid (63), diffractaic acid
(64), 4-O-demethylbabartic acid (65), and atranorin (55), were isolated using various chromatographic methods by Kieu Van Nguyen et al (2019).1“1
Three new xanthone dimers, eumitrins C—E (147-148-149), along with a new depsidone, 3'-O-demethylcryptostictinolide (108) were isolated from the acetone extract of the whole thallus of this lichen collected in Vietnam by Kieu Van Nguyen et al (2019).
> Usnea campestris Sant và Usnea cladonia Stellaris (Ach.)
In 1972, from the lichen Usnea campestris, M E Mendiondo and J D.
In 1969, from the lichen Usnea cladonia Stellaris, C F Culberson isolated (-)- usnic acid (136).
Canarione(5,8-dihydroxy-2-methyl-4H-naphtho[2,3]-pyran-4,6,9(6H,9H)- trione) (181) was isolated in 1977 from the lichen Usnea canariensis by S Huneck et al."'91
In 2003, Marante et al found atranol (49), chloroatranol (50), heamatommic acid (43), methyl heamatommate (23), and ethyl heamatommate (24), '*?!
In 2007, from the lichen Usnea cavernosa, P A Paranagama and A L.
Gunatilaka found two heptaketides, 1-hydroxydehydroherbarin (180) and herbarin
(+)-Usnic acid (135), norstictic acid (81), and salazinic acid (77) were found in
1972 from the lichen Usnea densirostra by M E Mendiondo and J D Coussio.!7>!
In 2008, Huan Yang Qi et al isolated many compounds such as depsidone, diffractione A (120), together with six phenolic compounds, including excelsione
(119), atranol (49), orsellinic acid (29), methyl orsellinate (30), ethyl orsellinate
In 2006, M S M Rawat and Pant G isolated seven compounds, including (+)-usnic acid (135), barbatinic acid (63), ethyl 2-hydroxy-3-methoxy-4,6- dimethylbenzoate (46), ethyl 2,4-dihydroxy-3,6-dimethylbenzoate (48), 2-hydroxy- 4-methoxy-3,6-dimethylbenzoic acid (47), diffractaic acid (64), and evernic acid
2-Hydroxy-[ “-(4-hydroxy-5-methoxyphenyl)ethanone (45) and (-)-usnic acid
(136) were isolated by Maulidiyah et al (2011) from the lichen Usnea flexuosa
In 2007, T Rezanka and K Sigler isolated hirtusneanoside (146).!!?7!
In 1987, from the lichen Usnea longissima, Y Nishitoba and J Mizutani found many compounds such as longissiminone A (52), longissiminone B (53), glutinol (160), (+)-usnic acid (135), barbatinic acid (63), diffractaic acid (64), 4-O- demethylbarbatic acid (65), evernic acid (73), usnic acid (135), squamatic acid (60), atranorin (55), ergosterol-5 3,8 Z-peroxide (167), and (-)-placodiolic acid (139).!9!!
In 2004, U V Mallavadhani and J A Elix isolated many compounds, including ergosterol (ergosta-5,7,22-trien-3/-ol) (168), episterol [ergosta-7,24(28)- dien-3/ol] (169), fecosterol [ergosta-8,24(28)-dien-3/ỉol] (170), lichesterol
(ergosta-5,8,22-trien-3ol) (174), 24-methylcholesta-5,22-dien-3/@ol (176), 24- ethylcholesta-5,22-dien-3/&ol (177), cholest-5-en-30l (172), 24-methylcholest-5- en-3/-ol (173), 24-ethylcholest-5-en-3/-ol (175), 4-O-methylorsellinic acid (27), ethyl 4-Ó-methylorsellinate (28), methyl /@orsellinate (33), lecanoric acid (57), usenamine A (137), usenamine B (138), and isousone (140).L°ề
Laxinamujila Bai et al (2014) isolated a new benzofuranone as ethyl diethyl 2-[3,3-bis(7-acetyl-4,6-dihydroxy-3,5-dimethylbenzofuran-2-yl)acryloyl]malonate
(143), and seven known compounds: (+)—usnic acid (135), atranorin (55), barbatinic acid (63), evernic acid (73), diffractaic acid (64), isomuronic acid (10), and ethyl 4-
(7-acetyl-4,6-dihydroxy-3,5-dimethyl-2-oxo-2,3-dihydrobenzofuran-3-y])-4-(7- acetyl-4,6-dihydroxy-3,5-dimethylbenzofuran-2-yl)-3-oxobutanoate (142).
Nguyen Huu Tri et al (2022) reported the isolation of six known compounds, including two depsides lecanorin (58) and isolecanoric acid (59), two depsidones: norstictic acid (81) and methylstictic acid (118), an ergosterol, 22F,24R-5a,6a- epoxyergosta-8,22-diene-3f,7a -diol (171), and lupeol (161) from the lichen Usnea lapponica Vain.!°]
Methyl 3,4-dicarboxy-3-hydroxy-19-oxoeicosanoate (22) was isolated from the lichen Usnea meridensis by M F Keogh and I Duran (1977).!”?!
In 2015, Jose’ L Rojas et al isolated a new compound, 3-methoxycarbonyl-2- hydroxy-6-methoxy-4-methylbenzọc acid (42).!9!
Compounds such as norstictic acid (81), salazinic acid (77), usnic acid (135), atranorin (55), protocetraric acid (89), and virensic acid (90) were isolated by Din L.
B and Elix J A in 2010 from the lichen Usnea rubrotincta.'&"
In 2005, from the lichen Usnea sp., Selvaluxmy K et al found 8-O- methylstictic acid (118).”I
From the lichen Usnea undulata, J A Elix and U Eenkaninan isolated galbinic acid (acetylsalazinic acid) (109), (+)-usnic acid (135), norstictic acid (81), salazinic acid (7), and 2’-O-methylhypostictic acid (87) in 1975.131
Nasim Sultana and Anthony Jide Afolayan (2011) found a new depsidone, 2’- O-methylhypostictic acid (87), together with seven known compounds, methyl /- orsellinate (32), norstictic acid (81), menegazziaic acid (85), (+)-usnic acid (135), hypoconstictic acid (86), salazinic acid (77), and galbinic acid (109) '?!
Nguyen Thi Thu Tram ef al isolated four compounds, including (12R)-(+)- usnic acid (135), methyl orsellinate (30), methyl £-orsellinate (32), and 7-hydroxy-
ROOC CH; ROOC CH; HOOC CH;
(+)-6-Methyl praesorediosate (2) R=CH; (+)-6-Methyl vinapraesorediosate A (4) R = CH; acid B (5)
HOOC CH; HOOC CH; HOOC CH, v oO O
(+)-Vinapraesorediosic acid C (6) (+)-Protopraesorediosic acid (7) Lichesterinic acid (8)
HOOC CH, o HOOC CH; HO (0) HOOC CH, OH
HạC ? 0 HạC u > O ơ o oO ae CH,
Protolichesterinic acid (9) Isomuronic acid (10) Vinapraesorediosic acid D (11)
OH OH OH OH OH OH
OH OH OH OH OH
O oO Hydroxydioxohenicosanoic acid (14) Trioxohenicosanoic acid (15)
HạC H;ạC 3 WC hcoon MELAS coocns 3 ©\y⁄Sx^cooen,
OH H COOH HO COOH lạ) HO COOH a-(15-Hydroxyhexadecyl) Caperatic acid (21) Methyl 3,4-dicarboxy-3-hydroxy-1 itaconic acid (20) 9-oxoeicosanoate (22)
HO OH HO OH CH, H;ạCOC OH
Methyl haematommate (23): R=CH; Orcinol (25) Methyl atranolate (26) 4-O-Methylorsellinic acid (27)
Methyl orsellinate (30) H CH; H P (38) Praesalide A (35) CH; CHạ
Ethyl orsellinate 31) H C,H; H Praesalide B (36) C,Hs CH;
Methyl forsellinate (32) CH; CH; H Praesalide C (37) C,H; C,H;
HạC HO OH HO OH H;CO
7-Hydroxy-5-methoxy- 2,4-Dihydroxyphthalide Cristiferides A 3-Methoxycarbonyl-2-hydroxy-
CH; CH, CH, CH; O oom 5 COOH COOH CH,OH H
HạCOC OH HO OH H;CO OH HO
Ethyl 4-O-methylorsellinate Haematommic acid Methyl 4-O-methylhaematomate 2' -Hydroxy-]'-
HạC OH H;CO OH H;CO OH HO OR
Ethyl-2-hydroxy-3- 2-Hydroxy-4-metoxy- Ethyl 2,4-dihydroxy Atranol (49) R=H methoxy-4,6- 3,6-dimethylbenzoic acid (47) 3,6-dimethylbenzoate (48) 2-O-Methylatranol (50) R=CH; dimethylbenzoate (46)
OH OH CsHiy teen H;CO CHO Soeur
H;COC OH HạCOC OH cl
Longissiminone A (52) Longissiminone B (53) Pentyldivaric acid (54) Methyl (£)-2,4-dihydroxy-6-methyl-3
-(3-oxobut-1-en-1-yl)benzoate (51) e DEPSIDES
COOCH¿ ” coon OH cH, O CH; O CH; O
Chloroatranorin (56) R=Cl Isolecanoric acid (59) R=COOH
CH, OH CH; O COOH oO COOH oO OH COOH
Divaricatic acid (61) Perlatolic acid (62) Barbatic acid (63) R,;=OH R;=OMe
Diffractaic acid (64) Rị= OMe R;=OMe 4-O-Demethylbabartic acid (65) R=OH R;=OH
4-Hydroxy-5-methyl-isophthalic acid Cristiferides B (67) Thamnolic acid (68)
COOH CH; on, i OH on Hạc COOCH; on rạn
CHO OHC oO OH bao
HạCO on HO CH, CH H;ạCO
Haemathamnolic acid (69) Pseudocyphellarin A (70) Sekikaic acid (71)
“ " ou va OH ee oa CH
HạCO Hạ HạCO CH; HạCO OCH;
Decarboxythamnolic acid (72) Evernic acid (73) MS J csculiferin A (74)
2-O-Methylstenosporic acid (75) Gyrophoric acid (76) Squamatic acid (60) e DEPSIDONES
Salazinic acid (77) H CHO CH;OH Menegazziaic acid (85) R,=OH, R;=H, R;=OH
Consalazinic acid (78) H CH,OH CH,OH Hypoconstictic acid (86) R¡=CH;, R=OH, R3=OH
Stictic acid (799) CH; CHO CH, 2'-O-methylhy
Constictic acid (80) CH; CHO CH,OH P9Sfet acid (87) Ri-CH;, Rạ=H, Ry-OCH;
Hypostictic acid (82) CH; CH; CH;
CH;O CH, 0 oO R oO OR
HO re) OH HO = OH
8'-O-methylprotocetraric acid (88) R=CH;OMe Conprotocetraric acid (92) R=H, R=CH;OH
Protocetraric acid (89) R=CH,OH 9'-O-Methylprotocetraric acid (93) R=H, R=CHO
Virensic acid (90) R=Me Malonprotocetraric acid (94) R=COCH;COOH, R'=OCH;
R,O O 1 hy HO 0 COOH OH ° HC Rị OH Ry
Rị R; Ry Rạ R, Rạ a-Collatolicacid (95) CH; H Norlobaridone (97) H H Parmosidone A (100) CHO OH a-Alectoronic acid (96) HH Loxodin (98) COOCH; H Parmosidone B (101) OH OH
Lobaric acid (99) COOCH; CH; Parmosidone E (102) CHO H
HO CH, HạCO oO é) Chọn
R Ry Cryptostictic acid (106) R;=CH; R,=OH Galbinic acid (109)
Parmosidone C (103) CH; COOCH; Cryptostictinolide (107) R;=CH; R;ạ=H
CH;O COOCH; x ‘CH; CH;O O OH
Psoromic acid (110) Bailesidone (114) on COOH Í a XI, pee COOH jae H;ạCƠ
8-O-Methylstictic acid or Methylstictic acid (118)
= a fee ns H,CO o os H;CO
8-[2,4-Dihydroxy-6-(2- oxoheptyl)phenoxy- 8-[2,4-Dihydroxy-6-(2-oxoheptyl)phenoxy] O 6-methoxy-3-pentyl-1H-isochromen-1-one -6-hydroxy-3-pentyl-1H-isochromen-1-one
/ỉ-Alectoronic acid (130) H f#-Collatolic acid (131) CH;
CH, (+)-Usnic acid (135) (-)-Usnic acid (
HạCO (6) OCH, OCOCH; = y.cooCc OH CO `
Hopan-6œ,22-diol or Zeorin (153) 18,3 B-Diacetoxyhopan-29-oic acid (154)
R,;=H; R;=ứ-OH_ : Hopane-6a,16a,22-triol (155) R,=H; R,= OH: Leucotylin (156) R\=H; R;= # OAc : 16/ỉ-Acetoxyhopane-6 a,22-diol (157) Rị;R¿=/@-OH : 6a-Acetoxyhopane-16/,22-diol (158) R,; Rạ=H : 6a-Acetoxyhopane-22-ol (159)
HạCO pHs H,CO CH oO oO OH | HO O° ~CH;
OH O_ OH e COMPOUNDS OF OTHER TYPES
D-Arabinitol (186) Tsavoenone A (187) CH; Tsavoenone B (188) CH,
Oo CH; O OH O x AD HL oO HO O me H (6) ọ OCHS OHHC OH O-CH; CH,
BIOLOGICAL ACTIVITIES óc vn ng ng nnnưệp 44 1 Antibacterial activity 0.0 ae 44 2 Antifungal activity cọ TH HH HH HH re 46 3 Cytotoxic and antimutagenic aC{IVI{Y - - - ôsp 47
Research by scientists has shown that compounds produced from lichen species have quite effective antibacterial and antifungal properties.
P Balaji, Indian scientist, tested the antibacterial ability with 11 strains of human pathogenic bacteria, on 5 types of extract extracted with organic solvents of increasing polarity such as hexane, dichloromethane, ethyl acetate, acetone and methanol of the lichen Parmotrema praesoredium The results obtained show that ethyl acetate and dichloromethane extracts have antibacterial properties against bacterial strains such as: Bacillus cereus, Corynebacterium diptheriae, Shigella flexnerii, Staphylococcus aureus, Vibrio cholerae and Candida albicans."*!
HạC OH H;ạC OCH, HO CạH;
Muller K (2001) tested in vitro with protolichesterinic acid (9) and found that it has the ability to fight Helicobacter pylori bacteria In addition, with many other tests, the author has also published showing that a large number of compounds from lichens such as: alectosarmentin, pannarin, emodin, physcion, evernic acid (90), leprapinic acid also have the ability to inhibit growth of bacteria or fungi.!88!
The compounds sekikaic (71) acid and lobaric acid (99) had strong antibacterial activity against Gram-negative strains (E.coli, S typhi) and Gram- positive bacterial strains (B subii1is).!5?!
Atranorin (55) (from Physcia aipolia), fumarprotocetraric acid (116) (from Cladonia furcata), gyrophoric acid (76) (from Umbilicaria polyphylla), lecanoric acid (57) (from Ochrolechia androgyna), physodic acid (from Hypogymnia physodes), protocetraric acid (89) (from Flavoparmelia caperata), stictic acid (79)
(from Xanthoparmelia) and (+)-usnic acid (135, 136) (from Flavoparmelia caperata) had relatively strong antibacterial effects against 6 types of bacteria and
10 fungi, among which are human, animal, and plant pathogens, mycotoxins, and food spoilage organisms (+)-Usnic acid (135, 136) was found to be the most potent antibacterial compound (comparable to streptomycin), and fatty acids and stictic acid (79) were the weakest.!©!
; ; Gram (+), Bacteroides spp., Clostridium perfringens, Usnic acid
1 Bacillus subtilis, Staphylococcus aureus, Staphylococcus
(135, 136) spp., Enterococcus spp., Mycobacterium aurum.
Methyl haematommate (23) | Epidermophyton floccosum, Microsporum canis, M.
3 Methy] orsellinate (30) gypseum, Trichophyton rubrum, T mentagrophytes,
Ethyl orsellinate (31) Verticillium achliae, Bacillus subtilis, Staphylococcus Methyl f-orsellinate (32) aureus, Pseudomonas aeruginosa.
4 | Alectosarmentin Staphylococcus aureus, Mycobacterium smegmatitis.
7 | Pulvinic acid Drechslera rostrata, Alternaria alternata
Isodivaricatic acid, divaricatinic acid (61) and (+)-usnic acid (135, 136), isolated from the lichen Protousnea poeppigii, showed activity against
Microsporum gypseum, Tricho-phyton mentagrophytes and T rubrum.'*!
Whiton and Lawrey (1982) reported that evernic acid (90) and vulpinic acid significantly inhibited the germination of Sordaria fimicola."*"!
Specifically, some compounds isolated from the extracts of lichen species have antibacterial and antifungal properties as shown in Table 1.1.''>7!
As microorganisms have evolved resistance to many antibiotics, pharmaceutical researchers need to pursue new sources for antibacterial drugs All these research results show that lichens and their metabolites contain important bioactive substances for the treatment of various diseases caused by microorganisms Compounds in lichen may protect against the mold that causes canker sores !83!
Aqueous, ethanol, and ethyl acetate extracts of the lichens Alectoria sarmentosa and Cladonia rangiferina have been shown to have antifungal effects against various fungi, including human pathogens (Rankovic' and Mi8ic', 2007), In which ethanol extract has the highest activity.8!
In 2004, Halama and Van Haluwinreported that acetone extracts of Evernia prunastri and Hypogymnia physodes showed strong inhibitory effects on the growth of some plant-pathogenic fungi such as Phytophthora infestans, Pythium ultimum, and Ustilago maydis.#!l
The common (+)-usnic acid (135, 136) was a compound with well-known antifungal properties.5"! However, other than usnic acid, there are only a few reports on the antifungal activity of secondary metabolites Research on 6 fungal strains: T longifusis, A flavus, M canis, F solani, C albicans and C glabrata, the results were shown in Table 1.2.11“6
3 | Sekikaic acid (71) A flavus and F solani.
4 | Erythrin, Atranorin (55) There was no antifungal activity against any of the fungi tested.
5 | Methyl orsellinate (31) T longifusus, A fl avus, M canis and F solani
6 | Methyl ỉ-orsellinate (32) | Resistant to 6 types of fungi.
Over the years, scientists have published many results showing that a large portion of the compounds isolated from lichens are cytotoxic For example, usnic acid was tested for antitumor using the Lewis lung cancer testing system three decades ago by Kupchan and Kopperman.!*!
HạC OCH; HạCO OH COOH
(+)-Usnic acid (135) (isolated from Cladonia convoluta) induced apoptic cell death in fetal lymphocytic lymphoma cells and was cytotoxic in various cancer cell lines, such as Lewis lung carcinoma, murine leukemia, human brain metastasis, human prostate carcinoma, human mammary carcinoma, and human hemangioblastoma Usnic acid also reduced the proliferation of human breast cancer cells and human lung cancer cells without any DNA damage.°! Finding
47 cancer therapies that do not damage DNA and do not cause the development of secondary malignancies later in life is of great medical interest Therefore, (-)-usnic acid can be a premise for preparing non-toxic, natural anti-cancer drugs
In addition, (+)-usnic acid (135) is active against Junin virus (Arenaviridae), the causative agent of dengue fever in humans in Argentina, as well as against
Tacaribe virus, a non-pathogenic virus (Russell H Fazio, 2007).!! Parietin
(isolated from Teloschistes chrysophthalmus) showed viral efficacy against similar viruses, !©7]
In addition, compounds belonging to the depside and depsidone framework isolated from lichens also have relatively high cytotoxicity In 2011, Boustie J and colleagues announced that the compounds lobaric acid (99) and baeomyceic acid have the ability to inhibit the growth of 14 cancer cell lines with ICso values in the range of 12-65 g/mL Besides, the test also showed the ability to inhibit the growth of DU-145 prostate cancer cells with ICso in the range of 25-30 pg/mL (Table
HạCO OH COOH HạC COOH CHO
Table 1.3 Cytotoxic and antimutagenic activityl56: 88: !52]
No Compounds Active on all cell types
; - Anti-Lewis lung cancer, P388 leukemia, mitotic
1 | (-)-Usnic acid (136) ơ - - ; inhibitor, active against HaCaT keratinocytes.
Scabrosin ester and derivative, | Toxic against murine P815 mastocytoma cells and euplectin other cell lines.
Activity against K-562 leukemia cells and Ehrlich
4 Active in the cloning of rat hepatocytes. and stitic acid (79)
5 Toxic to the regeneration of lymphocytes. sphaerophorin
6 ơ Active against leukemia cells. emodin and derivative
Russo et al (2008) reported that sphaerophorin (depside) (isolated from Sphaerophorus globosus) and pannarin (depsidone) (isolated from Psoroma pholidotoides or from Psoroma reticulatum, P pulchrum, and P pallidum) has the ability to inhibit the growth of human M14 melanoma cells, causing apoptotic cell death The anticancer activities of these secondary metabolites show promise in the treatment of potent skin tumors.!!!2l
Compounds with anti-cancer and anti-mutation properties include: (-)-usnic acid, protolichesterinic acid, nephrosteranic acid, polyporic acid, derivatives, physodalic acid, glucans and lichenin derivatives.!!!6l
In another study of orsellinic acid esters (12) (isolated from Parmotrema tinctorum) on the cell lines Hep-2 laryngeal carcinoma, MCF-7 breast cancer, 786 renal carcinoma-0, and B16-F10 melanoma mouse cancer, giving the following results:12?1 e n-Butyl orsellinate was the most active against HEp-2 (ICso 7.2 ug/mL),
MCF-7 (ICso 14.0 ug/mL), 786-0 (ICso 12.6 ug/mL) and B16- cells F10 (ICso
11.4 ug/mL) In addition, n-butyl orsellinate was more active than cisplatin against B16-F10 cells (ICso 12.5 ug/mL). e Ethyl orsellinate was more active against HEp-2 than against MCF-7, 786-0 or B16-F10 cells. e n-Pentyl orsellinate is less active against HEp-2 cells than n-propyl orsellinate and n-butyl orsellinate. e Lecanoric acid and methyl orsellinate were less active against any of the cell lines used, with ICso values higher than 50 ug/mL. e Among the split chain esters, sec-butyl orsellinate and tert-butyl orsellinate showed similar activity against HEp-2, MCF-7 and B16-F10 cells and were more active than isopropyl orsellinate. e tert-Butyl orsellinate is more active than isopropyl orsellinate and sec-butyl orsellinate against 786-0 and B16-F10 cells.
The results of the test showed that orsellinate activity increased with chain elongation (from methyl to ứ-butyl]).!”?!
Another study, showed that O-alkyl salazinic acids from Parmotrema lichexantonicum, have cytotoxic potential against colon cancer (HCT-8), melanoma
(MDA-MB-435) and tumor cells brain tumor (SF-295) was examined by Micheletti et a[.Ì
1.5.4 Antiviral activity and inhibition of viral enzymes
VIETNAMESE LICHENS 0 eee eeceecesseeseesseeseceeesecaesseecesseeseesseesaeeeeesaeeneees 54
Vietnam has a tropical monsoon climate which is favorable for diverse tropical lichens, but the lichen flora of Vietnam has so far attracted little attention.?l
Previous studies on the Vietnamese lichens in the past focused mainly on their taxonomy but not on chemical constituents.
For the last 11 years, Vietnamese lichens have been interested in chemical constituents, this information is presented in Table 1.5.
Table 1.6: Studies on Vietnamese lichens
No RESEARCHERS LICHEN SPECIES YEAR
2 | Huynh Bui Linh Chi!**! Parmotrema prasorediosum 2014
3 | Ly Hoang Dieml”! Ramalina farinacea 2014
4 | Tuong Lam Truong!!! Usnea aciculifera 2014
7 | Nguyen Thi Thuy Linh!!! Ramalina peruviana 2020
This research contributed considerably to the lichen phytochemical studies in Vietnam For example, Huynh Bui Linh Chi isolated 21 new compounds, Nguyen Thi My Dung found seven new compounds, and Duong Thuc Huy detected 15 new compounds from the Vietnamese lichens in the past.
The hot climate of Vietnam affected the biosynthesis of lichen substances in
Vietnam This specific climate created lots of novel compounds possessing uncommon skeletons.'**! Chemical research on Vietnamese lichens needs to be improved and given more attention to finding new compounds to add to the understanding of the diversity of lichens in Vietnam.
Stemming from my interest in the diversity and biological activity of substances in lichens, I conducted phytochemical research on one lichen species collected in Vietnam and two lichen species in Laos The main goal of this thesis is to research substances from lichens to isolate new compounds to expand the diversity of lichens not only in Vietnam but also in neighboring countries with similar climates In addition, many compounds were tested for biological activity to find compounds having activities.
2.1 INSTRUMENTS AND CHEMICALS o 'H-, %C- and 2D-NMR spectra were acquired on Bruker Avance 500! (500
MHz for 'H-NMR and 125 MHz for !C-NMR) Chemical shift values are given in ppm. o Proton chemical shifts were referenced to the solvent residual signal of
(CD3)2CO at ôn 2.84, CDCl3 at ổn 7.24, CD30D at ổn 3.31, and DMSO-d6 at ou 2.50. o The °C NMR spectra were referenced to the central peak of (CD3)2CO at &
29.84, CDC]a at d¢ 77.23, CD30D at đ 49.15, and DMSO-4 at dc 39.51. o The HR-ESI-MS spectra were recorded on Bruker micrOTOF Q-II. o The ECD spectra were measured on Department of Chemistry, Faculty of
Science, Chulalongkorn University, Bangkok, 10330, Thailand. o Optical rotations were measured on a Kruss (P8000-TF, Germany) polarimeter with the length of tube of five cimetres. o Melting points were determined on Maquenne block (Kruss M5000, The
Center Analysis of the University of Science, National University- Ho Chi Minh City, Vietnam.) © All instruments are in the Central Laboratory for Analysis, University of
Science, National University — Ho Chi Minh City. o Thin-layer chromatography (TLC) and preparative TLC were performed on silica gel GEas4 (Merck) Chemical for visualizing TLC plates: 10% H2SO4 aqueous solution, or 5% vamillin/H›SOa, then followed by heating. o Column chromatography was performed on silica gel (Himedia) (230-400
Mesh) o Solvents: n-hexane, chloroform, ethyl acetate, acetone, ethanol, methanol, and acetic acid from Chemsol, and all were used as purchased.
56 o The solutions of different extracts were evaporated under reduced pressure until dryness by using rotary evaporator Buchi-111. o Four cell lines: MCF-7 (breast cancer cell line), HeLa (cervical cancer cell line),
NCI-H460 (human lung cancer cell line) and HepG2 (a human liver cancer cell line) were tested by sulforhodamine B colorimetric assay method (SRB assay), and camptothecin was the positive control. o With a-glucosidase enzyme, acarbose was the positive control.
U ceratina Arch (1) P praesorediosum (Nyl.) Hale (2) P tinctorum (Despr ex Nyl.) Hale (3)
Figure 2.1: The lichen U ceratina Arch (1), P praesorediosum (Nyl.) Hale (2),
P tinctorum (Despr ex Nyl.) Hale (3).
Lichen of P praesorediosum (Nyl.) Hale (Figure 2.1) 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 authenticated by Dr 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
> Usnea ceratina Arch and Parmotrema tinctorum (Despr ex Nyl.) Hale
Lichens U ceratina Arch (Figure 2.1) and P tinctorum (Despr ex Nyl.) Hale (Figure 2.1) were collected at Ban Paksong town, Paksong district, Champasak province, Laos in April, 2015 Scientific name authenticated by Dr Vo Thi Phi Giao, Faculty of Biology, University of Science, National University — Ho Chi Minh City and by Dr Harrie J M Sipman., Freie University, Berlin, Germany.
Voucher specimens (No US-B030) of U ceratina Arch (1) and (No US-B025) of P tinctorum (Despr ex Nyl.) Hale (3) were deposited in the Herbarium of the Department of Organic Chemistry, Faculty of Chemistry, University of Science, National University - Ho Chi Minh City-Vietnam.
2.3.1 Extraction and isolation on the lichen Usnea ceratina
The fresh lichen thalli of U ceratina (1.60 kg) were cleaned under running tap water and air-dried The ground powder (1.15 kg) was macerated with acetone (4 x
10 L) at room temperature during 24 hours After filtration, the solvent was evaporated under reduced pressure to dryness to afford the crude acetone residue (163.0 g) (Scheme 1).
This crude extract was subjected to silica gel solid phase extraction and eluted consecutively with chloroform, ethyl acetate, acetone, and methanol to provide chloroform (C, 60.0 g), ethyl acetate (EA, 12.0 g), acetone (Ac, 55.0 g), and methanol (M, 3.5 g) fractions, respectively The extracts were stored at 4°C in the refrigerator in the laboratory of the organic chemistry department.
The thesis was carried out in two chloroform (C, 60.0 g) and ethyl acetate (EA, 12.0 g) extracts According to previous research data, these two extracts contained many new compounds in lichen.
Fraction C (60.0 g) was applied to silica gel column chromatography (CC) and eluted with the solvent systems of n-hexane—chloroform (stepwise, 9:1 to 0:10) to give 5 subfractions, coded C1 (13.58 g), C2 (8.53 g), C3 (6.59 g), C4 (5.74 g), and C5 (5.05 g), then eluted with chloroform—methanol (stepwise, 9:1 to 0:10) to give 4 sub-fractions, namely C6 (4.83 g), C7 (3.82 g), C8 (1.98 g), and C9 (1.04 g).
Fraction C2 (8.5 g) was silica gel rechromatographed and eluted with hexane-—chloroform (10:0—5:5) to give three compounds, MT-28 (6.5 mg), MT-33 (8.0 mg), and MT-15 (7.0 mg).
Fraction C3 (66 g) was silica gel rechromatographed and _ eluted chloroform—methanol (10:0—5:5) to give two compounds, MT-29 (6.5 mg) and MT-
Fraction C4 (5.7 g) was applied to silica gel column chromatography (CC) and eluted with the solvent systems of n-hexane—chloroform—methanol (5:5:1) to give three compounds, MT-3 (10.0 mg), MT-5 (6.0 mg), and MT-15 (5.0 mg).
The sub-fraction C5 (5.05 g) was rechromatographed by silica gel CC, eluted with n-hexane—chloroform (8:2—7:3) and chloroform—methanol (95:5, v/v) to give three compounds, MT-16 (5.0 mg), MT-17 (11.0 mg), and MT-14 (5.0 mg).
The sub fraction C6 (4.83 g) was applied to silica gel CC, eluted with n- hexane—chloroform (7:3, v/v) to afford MT-18 (6.5 mg) and MT-19 (6.0 mg).
The same manner was applied to the sub fraction C7 (3.82 g), eluted with n- hexane-—chloroform (5:5, v/v) to afford MT-21 (6.0 mg).
The ethyl acetate extract (12.0 g) was subjected to silica gel column chromatography, eluted by the solvent system of chloroform—methanol with increasing methanol to get seven fractions, coded EA1—EA7.
The fraction EA3 (l5 g) was rechromatographed, eluted with chloroform—methanol (98:2, v/v) to deliver two compounds, MT-30 (5.0 mg) and MT-29 (6.5 mg).
The fraction EA4 (3.8 g) was separated by silica gel chromatographic column using chloroform—methanol-acetic acid (98:2:1 drop, v/v) to give three compounds MT-9 (6.0 mg), MT-3 (5.0 mg), and MT-4 (5.0 mg) The fraction EA6 (1.3 g) was rechromatographed, eluted with chloroform—ethyl acetate—methanol (6:3:1, v/v) to deliver MT-16 (5.5 mg) The fraction EA7 (1.3 g) was rechromatographed, eluted with chloroform—methanol (98:2, v/v) to deliver MT-22 (6.8 mg).
2.3.2 Extraction and isolation on the lichen Parmotrema praesorediosum
EXPERIMENTAL SECTION | cccccceeeteeeeeeneneeaes 56 2.1 INSTRUMENTS AND CHEMICALUS - 2Ă 5S Series 56 2.2 LICHEN MATERIALS c2 1S *S 1H HH TH HH ng HH 57 2.3 EXTRACTION AND ISOLATION PROCEDURES -© 5552 58 2.3.1 Extraction and isolation on the lichen Usned Ceratind - 5555 << <s+ 58 2.3.2 Extraction and isolation on the lichen Parmotrema praesorediosum
Extraction and isolation on the lichen Parmotrema fÌHICÍOTHIN
The fresh lichen thalli P tinctorum was washed, dried, and ground into powder (3.15 kg), and was macerated in acetone (4 x 10 L) at room temperature during 24 hours After filtration, the acetone solution was evaporated at a reduced pressure to provide the crude acetone extract (350.0 g) This crude extract was subjected to silica gel solid phase extraction and eluted consecutively with n-hexane, ethyl acetate, and
61 methanol to afford n-hexane extract (H, 15.0 g), chloroform extract (C, 16.43 g), ethyl acetate extract (EA, 140.0 g), and methanol extract (M, 163.0 g) (Scheme 3) The extracts were stored at 4 °C in the refrigerator in the laboratory of the organic chemistry department.
The thesis was conducted on the ethyl acetate extract sample because this extract had a big mass (140.0 g) Accordingly, from this extract, it is possible to isolate many new substances with small volumes that previous studies have missed.
The extract EA (140.0 g) was applied to silica gel column chromatography (CC) and eluted with the solvent systems of n-hexane-ethyl acetate (stepwise, 7:3, 5:5, 0:10, v/v) then methanol to give ten fractions, coded EA1—EA 10.
Fraction EAT (1.7 g) was divided in two sub-fractions EA1.1 (576.0 mg) and EA1.2 (487.0 mg) by silica gel CC, using the mobile phase as n-hexane—chloroform (7:3) Sub-fraction EA1.1 was rechromatographed by silica gel CC, eluted with n- hexane-chloroform (7:3-5:5) to provide two fractions EAl.la, EA1.1b, and a compound MT-1 (12.0 mg) Purification of fraction EA1.1a by silica gel CC using the solvent system of n-hexane—chloroform—ethyl acetate (9:2:1, v/v) such as an eluent resulted in two compounds, MT-10 (24.0 mg) and MT-20 (2.5 mg) The same procedure for subfraction EA1.2 was conducted, eluted with n-hexane—chloroform (5:5) to obtain four compounds, MT-2 (5.0 mg), MT-3 (14.3 mg), MT-4 (18.7 mg), and MT-6 (10.3 mg).
Fraction EA2 (16.2 g) was subjected to silica gel column chromatography and eluted by chloroform—methanol (98:2, 95:5, 9:1) to give three compounds, MT-12 (9.5 mg), MT-13 (11.2 mg), and 7 (7.0 mg).
Fraction EA3 (15.05 g) was subjected to silica gel column chromatography and eluted by n-hexane—chloroform (5:5, v/v) to give two compounds, MT-31 (8.0 mg) and MT-32 (7.5 mg).
| Cleaned and deed, ground, aoe
_ ~ Fluted with solvents of a: polarities
| Πmae MT-29 MT-8 (8.0 CMe | MT-2 (65 mg) |
” &@ssg e5 | ME-28 65mg).= uae S82” MT-30 (5.0 mg)
G Hcwe | MT-3 (100mg) CMe | MT-3 (5.0 mg)
6749) sa) MT-5 (60mg) |—+ a2) MT-4 (5.0 mg)
Be MT-18 (6.5 mg) EAs CcCraue MTIS
Lai G HC MT-21 | CMe MT-22 (3.82 g) 55 (6.0 mg) (1.30 pan "m82” (68mg)
Scheme 1: Procedure for isolation of
- Macareted with methanol, room temp.
- Eluted with solvents of different polarities, ~ CC with P;€ (9:1-0:10)
+ ý 4 Chloroform || Ethyl acetate Acetone Methanol Fraction
- C:Me EAa AC C:Ac:Acetic
MT+1 (67mg) [eSH*) vận - | qgagg) I99534np)| MT-28(62mg)
MT-26 (6.0 mg) C:Me EAi || | ACs €:Ác:Acet\
MT-27 (90mg) |“ 955 | (144g) | ng szseem°| MT-H 65 mg)
MT-3 (18.1 mg) | cme EAs |} Lạ ACs C:Ac:Acetic| MT-24 (6.5 mg)
MT-12 (7.3 mg) “gi (2.9 g) (9.09 g) 82:3 drops") MT-25 (7.0 mg)
Scheme 2: Procedure for isolation of compounds from Parmotrema praesorediosum (Nyl.) Hale.
(5.0 kg) iy Cleaned and dried, ground,
| ~ Macerated with acetone, room temp,
~ Solvent was evaporated n-Hexane _ Chloroform +
~ Eluted with solvents of different palarities. t 4
Scheme 3: Procedure for isolation of compounds
BIOLOGICAL ASSAYS LG HH HH HH HH HH TH Hy 66 1 Cytotoxic vn ốc
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, Ho Chi Minh City, Vietnam The person in charge of the unit is Assoc Prof Ho Huynh Thuy Duong The determination of cytotoxic activities against the HeLa (human epithelial carcinoma), MCF-7 (human breast cancer), NCI- H460 (human lung cancer), and HepG2 (human liver cancer) cell lines of isolated products was performed at a concentration of 100 uwg/mL using the Sulforhodamine
B (SRB) assay with camptothecin as the positive control !!7°!
All cells were cultured in E7>MEM medium (Eagle’s Minimal Essential Medium) supplemented with 10% foetal bovine serum (FBS), 1% of 2 mM L- glutamine, 50 IU/mL penicillin, and 50 wg/mL streptomycin, and maintained at 37 °C in a 5% CO; atmosphere with 95% humidity Viable cells were counted and inoculated in 96-well plate with density of 10* cells/100 wL/well After 24 h the cells were treated with a pure compound while the control wells were added only by 100 4L 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 wL of cold 50% (w/v) trichloroAcOH per well and incubated at 4 °C for 1 h The plates were washed five times with distilled water and then air-dried. After that, a solution of 50 L of SRB (0.4% w/v in 1% AcOH) was added to each well, and allowed to stay at room temperature for 30 mins The SRB solution was removed from the plates by rinsing four times with a 1% glacial AcOH solution (200 /L/well) The plates were air-dried for 12-24 h The bound SRB was dissolved to each well by adding 100 L 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
66 proportional to the cell number Cell survival was measured as the percentage absorbance compared to the control (non-treated cells) Evaluation of the result was based on the I% (at the concentration of 100 g/mL): 0-49% (inactive), 50-70%
(active), 70-90% (strong), and 90—100% (very strong).!!*?!
Where: OD = OD’ tested sample — OD’ blanck.
Samples were sent to be in vitro tested at Ho Chi Minh City Open University and the University of Science (the Faculty of Biology and Chemistry), Vietnam National University- Ho Chi Minh City, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam.
The a-glucosidase (0.1 U/mL) and the substrate (1 mM p-nitrophenyl-a-D- glucopyranoside) were dissolved in 0.1 M phosphate buffer (pH 6.9) The stock sample was dissolved in 50% DMSO A 10 uL test sample was pre-incubated with a-glucosidase (40 uL) at 37 °C for 10 mins A substrate solution (50 uL) was then added to the reaction mixture and incubated at 37 °C for an additional 20 mins, and terminated by adding 1 M Na2CO3 solution (100 uL) Enzymatic activity was quantified by measuring the absorbance at 405 nm (ALLSHENG AMR-100 microplate reader) The percentage inhibition of activity was calculated as follows:
% Inhibition = [(A0-A13/A0] x 100 where: A0 is the absorbance without the sample;
AI is the absorbance of the sample.
From the value of I% and sample concentration, draw a nonlinear curve Based on the nonlinear curve, calculate ICso (sample concentration at which 50% of a- glucosidase activity is inhibited) by substituting y = 50 into the logarithmic nonlinear curve equation of the form y = aln(x) +b The sample has stronger inhibitory activity when the ICs value is smaller Through ICs0, it is possible to evaluate and compare a-glucosidase inhibitory activity between extract samples and compared to the control positive The ICso value was deduced from the plot of % inhibition versus the concentration of the test sample Acarbose was used as a positive control and each experiment was performed in triplicate.!!!”!
From the lichens U ceratina, P praesorediosum, P tinctorum, 34 lichen substances were isolated, including 7 new compounds in nature and 27 known ones.
No Type of compound U ceratina P.praesorcdiosum P tinctorum
The chemical structure of all compounds was elucidated on the basis of NUR and MS spectroscopic analysis All new compounds were checked by Scifinder at Kobe Pharmaceutical University, Japan in December 2018 and the University of Rennes in 2019 However, the compound MT-22 was checked by SciFinder in Paris Saclay University in December 2023.
For better interpretation of the chemical structures of the isolated compounds, they were classified into six groups as listed below.
HO OH HO OH HO OH!
Oreinol (MT-)) Atranol (M12) Onsellime acid(ME3)
Methyl orsellinate (M‘I-4) Methyl /-orsellinate (M'I-5) Methyl haematommate (MI-6)
Methyl đ9- -2,4- dihydroxy- 6-methyl-3- Rhizonic acid (M'I-8) Usneaceratin H (M'I-9)
(3-oxobut-1-en- 1-y])benzoate (M'I-7) (New: compound)
Figure 3.1: Chemical structures of compounds (MT-1—MT-11).
3.1.1.1 Structure elucidation of compound orcinol (MT-1) se Colorless needles. s* Melting point: 107 °C. ằ Isolated from the lichens P praesorediosum and P tinctorum (Schemes 2 and 3).
+ Mass spectrum (Appendix 1.1): HR-ESI-MS (positive mode) m/z 125.0601
[M+H]*, calcd C7H9O2 for 125.0603, corresponding to the molecular formula of C7HsO2z, 124.0525. ằ The 'H and C-NMR data (acetone-de) (Appendies 1.2 and 1.3): see Table
Compound MT-1 was isolated as colorless needles The HR-ESI-MS (positive mode) displayed a pseudomolecular ion peak with m/z 125.0601 [M+HỊ”, corresponding to the molecular formula of C7HsQO2.
The 'H-NMR (acetone-ds) (Table 3.1) spectrum displayed signals of one methyl group at ổn 2.15 (3H, s, H-7), three aromatic methine protons at ổn 6.16
(3H, s, H-1, H-3 and H-5), and two hydroxyl protons at ổn 8.02 (2H, s, 2-OH and 4-
The C-NMR spectrum showed the resonances of seven carbons, including of one methyl group at dc 21.5 (C-7), three aromatic methine carbons at đc 100.7 (C-
3), 108.4 (C-1 and C-5), and three substituted aromatic carbons, and two of which were oxygenated at dc 140.6 (C-6) and 159.4 (C-2 and C-4) (Table 3.1).
These spectroscopic data were compatible with the published ones;!”l therefore compound MT-1 was orcinol.
Table 3.1: The NMR data of (MT-1-MT-6).
MT-I“ MT-2“ MT-3° MT-4* MT-5° MT-6° MT-1“ MT-2“ MT-3° MT-4* MT-5° MT-6"
^ recorded in Acetone-ds, ? recorded in CDC]:, ° recorded in DMSO-d¢
3.1.1.2 Structure elucidation of compound atranol (MT-2)
** Isolated from the lichen P tinctorum (Despr ex Nyl.) Hale (Scheme 3).
“* Mass spectrum (Appendix 2.1): HR-ESI-MS (negative mode) m/z 151.0397
[M-HỊ, calcd CsH703 for 151.0395, corresponding to the molecular formula of CsHsO3, M 152.0473.
The 'H and C-NMR data (acetone-ds) (Appendices 2.2 and 2.3): see Table
Compound MT-2 was isolated as yellow needles The HR-ESI-MS (negative mode) displayed a pseudomolecular ion peak with m/z 151.0397 [M-H], corresponding to the molecular formula of CsHsOs3.
The 'H-NMR spectrum data of compound MT-2 gave signals of one methyl group at ở; 2.23 (3H each, s, H-7), two aromatic methine protons at ổn 6.26 (2H, s,
H-1 and H-5), one formyl group at ổn 10.27 (1H, s), two hydroxyl protons at ổn
The C-NMR spectrum data showed the resonances of eight carbons, including one methyl group at dc 22.4 (C-7), six aromatic methine carbons at đc 108.5 (C-1 and C-5), 109.4 (C-3), 151.6 (C-6), and 163.1 (C-2 and C-4), and one formyl group at dc 194.3 (3-CHO).
When comparing the spectral data of MT-2 with MT-1 (orcinol), the results showed that MT-2 had the disappearance of the aromatic methine proton at ổn 6.16 (H-3), replaced by the appearance of the signal of the formyl group (ổn 10.27 and dc 194.3) These spectroscopic data were compatible with the published ones.'*7!
Accordingly, the structure of compound MT-2 was established as atranol, and this compound was also found in another lichen such as P tsavoense, U baileyi, U. hookeri, U diffracta Vain, and U aciculifera Vain, in advance.!> 42: 6 32 127]
3.1.1.3 Structure elucidation of compound orsellinic acid (MT-3) s* White needles. s* Melting point: 184 °C
+ Isolated from the lichens U ceratina, P praesorediosum (Nyl.) Hale, and P. tinctorum (Schemes 1, 2, and 3). ằằ Mass spectrum (Appendix 3.1): HR-ESI-MS (negative mode) m/z 167.0346
[M-H], calcd CạH;Ox for 167.0344, corresponding to the molecular formula of CsHsOa, M 168.0423.
The 'H and '3C-NMR data (DMSO-4) (Appendices 3.2 and 3.3): see Table
3.1. s* HMBC spectra (DMSO-ds) (Appendix 3.4).
Compound MT-3 was isolated as white needles The HR-ESI-MS (negative mode) displayed a pseudomolecular ion peak with m/z 167.0346 [M-H], corresponding to the molecular formula of CsHgOu.
The 'H-NMR spectrum displayed signals of one methyl group at ou 2.41 (3H, s, 6-CH3), two aromatic methine protons at 64 6.01 (1H, d, J = 2.5 Hz, H-3), and
6.04 (1H, d, J = 2.5 Hz, H-5) (Table 3.1) Moreover, the !C-NMR spectrum showed the resonances of eight carbons, including of one aromatic methyl group at dc 23.4 (C-7), two aromatic methine carbons at dc 100.3 (C-3), 109.5 (C-5), four substituted aromatic carbons at dc 107.0 (C-1), 142.5 (C-6), 160.4 (C-2), 165.3 (C-
4), and one carboxyl carbon at dc 173.0 (1-COOH) (Table 3.1).
Similarly, when comparing the spectral data of MT-3 with MT-1 (orcinol), the results showed that MT-3 had the disappearance of the aromatic methine proton at ổn 6.16 (H-1), the appearance of the presence of the carboxyl group (dc 173.0).
Besides, The position of the carboxyl group was determined via HMBC correlations from the methyl protons at ổn 2.41 (6-CH3) to carbon signals C-1 (dc 107.0), C-5 (dc 109.5) and C-6 (dc 142.5), and from the aromatic proton at ổn 6.02 (H-3) to carbon signals C-1 (đc 107.0) and C-2 (dc 160.4) (Figure 3.2).
Orselinic acid (MT-3) Figure 3.2: The chemical structure and HMBC correlations of (MT-3).
The NMR data of MT-3 showed good compatibility with those of orselinic acid in the literature;?! !!5Ì so the chemical structure of compound MT-3 was
RESULTS AND DISSCUSSIONS 0.0 eee eeeeseeteeteeneeeeneens 69 3.1 CHEMICAL STRUCTURE ELUCIDATION ccceecccccssscceesteceeteeeeeseeeeeseeeeeaes 69 3.1.1 Monocyclic COmpDOUTIđS - G5 3221321833118 18911 1811811 811111 3x re 70 3.1.1.1 Structure elucidation of compound orcinol (MẽT-]) -ô ô<< <=>s 71 3.1.1.2 Structure elucidation of compound atranol (MĨT-2) .-‹ -ô<+<<ô++s 72 3.1.1.3 Structure elucidation of compound orsellinic acid (M T-3)
Structure elucidation of compound methyl orsellinate (MT-4)
** Isolated from the lichens U ceratina and P tinctorum (Schemes 1 and 3). s* Mass spectrum (Appendix 4.1): HR-ESI-MS (positive mode) m/z 183.0668
[M+H]*, calcd CoHi104 for 183.0657, corresponding to the molecular formula of CsH¡oOa, 182.0579.
The 'H and C-NMR data (acetone-ds) (Appendices 4.2 and 4.3): see Table
Compound 4 was isolated as colorless needles The HR-ESI-MS (positive mode) displayed a pseudomolecular ion peak with m/z 183.0668 [M+H]*, corresponding to the molecular formula of CoH¡oOa The 'H-NMR spectrum displayed signals of one methyl group at ổn 2.46 (3H, s, 6-CHạ), one methoxy group at ổn 3.92 (3H, s, 1-COOCHS), two aromatic methine protons at dy 6.23 (1H, d, J= 2.5 Hz, H-3), 6.28 (1H, m, H-5), and two hydroxyl protons at dy 11.60 (1H, s, 2-OH) and ổn 9.15 (1H, s, 4-OH) The abnormality of the signal of the H-5 proton is explained that there may have been an additional signal of another compound with a very small concentration, a substance that only has the H-5 proton and no H-3 such as MT-5—MT-9 That hindered the separation signal of H-5 with
The '°C-NMR spectrum showed the resonances of nine carbons including of one aromatic methyl group at dc 24.2 (C-7), one methoxy at dc 52.2 (1-COOCHS), two aromatic methine carbons at dc 105.4 (C-3), 112.4 (C-5), four substituted aromatic carbons, two of which were oxygenated, at dc 101.7 (C-1), 144.4 (C-6),
163.4 (C-4), 166.3 (C-2), and one carboxyl carbon at dc 173.0 (1-COOCHsS).
The spectral data of MT-4 showed that the structure of MT-4 was similar to
MT-3, but MT-4 had the ester group (-COOCH3 at ốc 52.2 and dc 173.0) without the carboxyl group (-COOH) like MT-3 These spectroscopic data were suitable
(1281 therefore compound MT-4 was methyl with the published ones in the literature; orsellinate, and this compound was also found in another lichen such as P. planatilobatum, P stuppeum, U aciculifera Vain, U diffracta Vain, and U. undulata, in advance.l7? 8: 40, 126, 42, 99]
Structure elucidation of compound methyl ỉ-orsellinate (MT-5)
“+ Melting point: 143 °C s* Isolated from the lichens U ceratina (Schemes 1). s* Mass spectrum (Appendix 5.1): HR-ESI-MS (negative mode) m/z 195.0596
[M-HỊ, calcd CioH1104 for 195.0656, corresponding to the molecular formula of Cio0H1204, 196.0736. s The 'H and C-NMR data (DMSO-de) (Appendices 5.2 and 5.3): see Table
3.1. s* HMBC spectra (DMSO-ds) (Appendix 5.4).
Compound MT-5 was isolated as a white prism The HR-ESI-MS (negative mode) displayed a pseudomolecular ion peak with m/z 195.0596 [M-H], corresponding to the molecular formula of CioHi204 The 'H-NMR spectrum displayed signals of two methyl groups at ổn 1.87 (3H, s, 3-CHạ) and 2.29 (3H, s, 6- CH3), one methoxy group at ổn 3.77 (3H, s, 1-COOCHS), one aromatic methine proton at ổn 6.21 (1H, s, H-5), and one hydroxyl proton at ổn 11.54 (1H, s, 2-OH).
The '3C-NMR spectrum showed the resonances of ten carbons, including of two aromatic methyl groups at dc 8.0 (3-CHs3) and 23.5 (6-CH3), one methoxy at ốc 52.0
(1-COOCHg), one aromatic methine carbon at ốc 110.6 (C-5), five substituted aromatic carbons, two of which were oxygenated, at dc 104.2 (C-1), 108.2 (C-3),
138.8 (C-6), 160.1 (C-4), 161.7 (C-2), and one carboxyl carbon at đc 171.9 (1- COOCHS).
In the HMBC spectrum, there were correlations of the methoxy protons at ổn 3.77 with the carbonyl carbon at dc 171.9 (1-COOCHs), indicating the methoxy group attached at 1-COO- and the correlations of the hydroxyl proton at ổn 11.54
(2-OH) with carbon signals at dc 104.2 (C-1), 108.2 (C-3), 161.7 (C-2) and 160.1 (C-4), indicating the hydroxyl group attached at C-2 The correlations of two methyl protons at ổn 1.87 (3-CHạ) and 2.29 (6-CH3) with carbon signals at dc 108.2 (C-3), 160.1 (C-4), 161.7 (C-2) and 104.2 (C-1), 110.6 (C-5), 138.8 (C-6) indicated that two methyl groups were joined to C-3 and C-6, respectively.
The spectral data showed that MT-5 had lost a proton aromatic signal at ổn 6.21 (H-3) and had an additional methyl group signal (ổn 1.87 and & 23.5) compared to MT-4 These spectroscopic data were suitable with the published one, !!08è All these properties suggested that compound MT-5 was methyl ỉ- orsellinate, and this compound was also found in another lichen such as P. planatilobatum, P tinctorum, P cristiferum, U aciculifera Vain, U articulate (L.)
Hoff, U baileyi, and U undalata, in advance.?7: 78 52, 28 127, 36 64 99]
Methyl ỉ-orsellinate (MT-5) Figure 3.3: The chemical structure and HMBC correlations of (MT-5).
Structure elucidation of compound methyl haematommate (MT-6)
+ Isolated from the lichens P tinctorum (Scheme 3).
+ Mass spectrum (Appendix 6.1): HR-ESI-MS (negative mode) m/z 209.0449
[M-H], calcd CioHoOs for 209.0450, corresponding to the molecular formula of CioH100s, 210.0528.
% The !H and C-NMR data (CDCls) (Appendices 6.2 and 6.3): see Table 3.1.
HMBC spectra (CDC]:) (Appendix 6.4) Compound MT-6 was isolated as colorless needles The HR-ESI-MS
(negative mode) displayed a pseudomolecular ion peak with m/z 209.0449 [M-H], corresponding to the molecular formula of C¡oHoO:.
The 'H-NMR spectrum displayed signals of one methyl group at ou 2.52 (3H, s, 6-CH3), one methoxy group at ổn 3.96 (3H, s, 1-COOCHS), one aromatic methine proton at ổn 6.29 (1H, s, H-5), one formyl group at ổn 10.34 (1H, s), and two chelated hydroxyl protons at ổn 12.40 (1H, s, 4-OH) and 12.86 (1H, s, 2-OH).
The '3C-NMR spectrum showed the resonances of ten carbons, including of one aromatic methyl group at dc 25.3 (6-CH3), one methoxy at đ 52.4 (1- COOCHg), one aromatic methine carbon at dc 112.3 (C-5), five substituted aromatic carbons, two of which were oxygenated, at dc 104.0 (C-1), 108.4 (C-3), 152.5 (C-6), 158.1 (C-4), 166.8 (C-2), one carboxyl carbon at óc 172.2 (1- COOCH3), and one formyl group at dc 194.1 (3-CHO).
The substitution pattern of MT-6 was confirmed by HMBC correlations The methyl proton at ổn 2.52 (3H, s, 6-CH3) showed HMBC cross peaks with three aromatic carbons 104.0 (C-1), 112.3 (C-5), and 152.5 (C-6) The position of the methyl ester was determined by the HMBC correlation from ổn 3.96 (3H, s, 1-
COOCHS) to the one carboxyl carbon at dc 172.2 (1-COOCH:) The presence of a formyl group was also inferred through HMBC correlations from the formyl group
78 at ổn 10.34 (1H, s) to the three oxygenated aromatic carbons 108.4 (C-3), 166.8 (C-
4), and 168.4 (C-2) The correlations of two hydroxyl protons at ổn 12.40 (1H, s, 4- OH) and 12.86 (1H, s, 2-OH) with carbon signals at 108.4 (C-3), 112.3 (C-5), 166.8 (C-4), and 104.0 (C-1), 108.4 (C-3), 168.4 (C-2) indicated that two hydroxyl protons were joined to C-4 and C-2, respectively.
The spectral data showed that MT-6 had lost a proton aromatic signal at ổn 6.23 (H-3) and had an additional one formyl group at ổn 10.34 and dc dc 194.1 compared to MT-4 Analysis of 1D, 2D-NMR data (Figure 3.4) and the comparison of these data with the ones in the literature!!*®! suggested that compound
MT-6 is methyl haematommate (MT-6), , and this compound was also found in another lichen such as P mesotropum, P planatilobatum, P tinctorum, P. cristiferum, and U hookeri, in advance.Š7.?1: 28: 52 96 82] z7
Methyl haematommate (MT-6) Figure 3.4: The chemical structure and HMBC correlations of (MT-6).
3.1.1.7 Structure elucidation of compound methyl (£)-2,4-dihydroxy-6-methyl- 3-(3-oxobut-1-en-1-yl)benzoate (MT-7). s* White amorphous powder.
** Isolated from the lichens P tinctorum (Scheme 3). s* Mass spectrum (Appendix 7.1): HR-ESI-MS (positive mode) m/z 233.0811
[M-H20+H]"*, calcd C¡:H¡zO¿ for 233.0814, corresponding to the molecular formula of Ci3H14Os, 250.0842. s The 'H and '3C-NMR data (DMSO-ds) (Appendices 7.2 and 7.3): see Table
3.2. s* HSQC and HMBC spectra (DMSO-d¢) (Appendices 7.4 and 7.5).
Compound MT-7 was isolated as white amorphous powder The HR-ESI-MS (positive mode) displayed a pseudomolecular ion peak with m/z 233.0811 [M-
H20+H]", corresponding to the molecular formula of C13H14Os.
The 'H-NMR spectrum displayed signals of two methyl groups at ổi 2.26 (3H, s, H-11) and 2.41 (3H, s, 6-CHs3), one methoxy group at ổn 3.88 (3H, s, 1- COOCHS), one aromatic methine proton at dy 6.39 (1H, s, H-5), two olefin protons with coupling trans at ổn 7.13 (1H, d, J.5 Hz, H-9), and 7.81 (1H, d, J.5 Hz,
H-8), and one chelated hydroxyl proton at dy 12.45 (1H, brs, 2-OH).
The '3C-NMR spectrum showed the resonances of 13 carbons including of two methyl groups at ốc 23.8 (6-CHạ), 27.5 (C-11), one methyl ester at dc 52.3 (1- COOCH;), one aromatic methine carbon at dc 111.1 (C-5), five substituted aromatic carbons, two of which were oxygenated, at dc 104.7 (C-1), 107.3 (C-3), 144.3 (C-6), 162.0 (C-4), and 163.4 (C-2), two olefin carbons at óc 128.5 (C-9), 133.4 (C-8), one carboxyl carbon at dc 171.6 (1-COOCHs), and one carbonyl carbon at dc 198.6 (C-10).
In the HMBC spectrum, there were correlations of the methoxy protons at ổn
3.88 (3H, s, 1-COOCHs3) with the carbonyl carbon at óc 171.6 (1-COOCH), indicating the methoxy group attached at 1-COO-, and the correlations of the chelated hydroxyl proton at ổn 12.45 with carbon signals at dc 104.7 (C-1), 163.4 (C-2) indicating the hydroxyl group attached at C-2 The correlations of the methyl protons at ổn 2.41 (3H, s, 6-CH3) with carbon signals at dc 104.7 (C-1), 111.1 (C-
5), 144.3 (C-6) indicated that the methyl group was joined to C-6.
Besides, the large coupling constants of H-8 (ổn 7.81, d, J.5 Hz) and H-9 (ổn 7.13, d, J.5 Hz) proved that this double bond -CH=CH- possessing a trans configuration Proton H-8 shifted to the low field zone, indicating the conjugated system of the double bond at C-8/C-9 and a methylketone group at C-10 (dc 198.6). This finding was supported by HMBC correlations of H-8, H-9, and 11-CH3 to C-10 (Figure 3.5) Moreover, there were correlations of the olefin proton at ổn 7.81 (H-8)
80 with carbon signals at dc 107.3 (C-3), 163.4 (C-2), indicating the olefin group attached at C-3.
From the spectral data, it showed that there was a difference between MT-7 and MT-4 Specifically, the structure of MT-7 had the loss of the proton signal of H-3 and the appearance of many other signals of the branch line (-CH=CH-CO-
CH3) These spectroscopic data were suitable with the published ones in the literature of P tsavoense;*! therefore, compound MT-7 was methyl (£)-2,4- dihydroxy-6-methyl-3-(3-oxobut-1-en-1-yl)benzoate.
Methyl (£)-2,4-dihydroxy-6-methyl-3-(3-oxobut-1-en-1-yl)benzoate (MT-7)
Figure 3.5: The chemical structure and HMBC correlations of (MT-7).
Table 3.2: The NMR data of(MT-7-MT-11).
MT-7° MT-8“ MT-9“ MT-10° MT-I1I° MT-7° MT-8°* MT-9*“ MT-10° MT-11°
4 recorded in Acetone-2s„ P recorded in CDC]:, ° recorded in DMSO-d¢
3.1.1.8 Structure elucidation of compound rhizonic acid (MT-8)
“+ A white powder. s* Isolated from the lichen U ceratina (Scheme 1).
% The 'H and '3C-NMR data (acetone—ds) (Appendices 8.1 and 8.2): see Table
*“* HMBC spectra (acetone—ds) (Appendix 8.3).
Compound MT-8 was isolated as a white powder The 'H-NMR spectrum data of compound MT-8 gave signals of two methyl groups at dy 1.99 and 2.59 (3H each, s, 3-CH3 and H-7), one methoxy group at oy 3.86 (3H, s, 4-OCH3), one aromatic methine proton at ổn 6.42 (1H, s, H-5), no signals of hydroxyl group The
C-NMR spectrum data showed the resonances of ten carbons, including two methyl groups at ốc 8.1 (3-CH:) and 24.5 (C-7), one methoxy carbon at dc 55.9 (1- COOCHS), six aromatic methine carbons at dc 106.4 (C-5), 106.9 (C-1), 110.6 (C-
3), 141.7 (C-6), 162.0 (C-4), and 163.4 (C-2), two of which were oxygenated, and one carboxyl carbon at dc 169.5 (1-COOCHs) (Table 3.2) The HMBC correlations between the methyl protons at ổn 1.99 and 2.59 with carbon signals at dc 110.6 (C-
3), 162.0 (C-4), 163.4 (C-2) and 106.4 (C-5), 106.9 (C-1), and 141.7 (C-6) indicated that the methyl groups was joined to C-3 and C-6, respectively The methoxy group
(ổn 3.86) was linked at C-4 on their HMBC correlation with carbon signals at Ốc
Spectroscopic data showed that the structure of MT-8 was similar to MT-4, but MT-8 had an additional methoxy group signal (ổn 3.86 and dc 55.9) that was attached to the C-4 position These spectroscopic data were compatible with the published one of U emidotteries'’”!, All these properties suggested that compound
MT-8 was 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid or rhizonic acid and the first time known in the Usnea genus.
2-Hydroxy-4-methoxy-3,6-dimethylbenzoic acid or rhizonic acid (MT-8)
Figure 3.6: The chemical structure and HMBC correlations of (MT-8).
3.1.1.9 Structure elucidation of compound usneaceratin B (MT-9) s*+® A white solid. s* Isolated from the lichen U ceratina (Scheme 1).7
“+ Mass spectrum (Appendix 9.1): HR-APCI-MS (nagetive mode) m/z
197.0452 [M-H], calcd CoHoO; for 197.0444, corresponding to the molecular formula of CoHioOs, 198.0528. s The 'H and '3C-NMR data (acetone—ds) (Appendices 9.2 and 9.3): see Table
3.2. s* HMBC and NOESY spectra (acetone—ds) (Appendices 9.4 and 9.5). ©
Compound MT-9 was obtained as a white solid The molecular formula was identified as CoHi00s by HR-APCI-MS data ([M-HỊ m/z 197.0452, calcd for
The °C and 'H-NMR data of 9 (Table 3.2) detailed nine signals according to one carbonyl carbon at dc 162.6 (1-COOH), three oxygenated aromatic carbons at ốc 148.5 (C-2), 137.5 (C-3), and 153.4 (C-4), two quaternary aromatic carbons at Ốc 114.8 (C-1) and 133.2 C-6), one methine aromatic carbon at dc 113.1 (C-5)/ổn 6.82 (H-5), one methoxy carbon at dc 56.6 (4-OCHs3)/6n 3.87 (4-OCHg), and one methyl carbon at đc 20.5 (C-7)/ổn 2.39 (H-7) Those data of MT-9 confirmed a benzoic acid framework similar of orsellinic acid (MT-3), except for the difference in the appearance of two carbons at dc 137.6 (C-3), 56.6 (4-OCH:) The singlet proton at ou 6.82 (1H, s, H-5) in MT-9 and the disappearing of one methine aromatic carbon
84 at ốc 100.3 (C-3) in 3 (Table 3.1) attested a pentasubstituted benzene system bearing one carboxyl, two hydroxyls one methoxy and one methyl groups for MT- 9.
The HMBC spectrum of MT-9 (Figure 3.7) supported correlations between protons at ổn 2.39 (H3-7) and carbons at ốc 114.9 (C-1), 113.1 (C-5), and 162.6 (1- COOH), between proton at ổn 6.82 (H-5) and carbons at dc 114.9 (C-1), 100.3 (C-
3), 153.4 (C-4), between protons at ổn 3.87 (4-OCHS) and carbon at dc 153.4 (C-4), in addition, proton H-5 (ổn 6.82) was close in space to the methyl group H3-7 (ổn
2.39) and 4-OCH3 (64 3.87) in NOESY spectrum (Figure 3.7), were illustrated the arrangement of these substituents in the benzene nucleus.
From the spectroscopic data, it showed that there was a difference between MT-9 and MT-8 Specifically, the structure of MT-9 had the loss of the methyl signal (3-CH3) and the appearance of many other signals of the hydroxyl group compared to MT-8.
Thus, the chemical structure of MT-9 was assigned as 3-hydroxy-4-O- methylorsellinic acid, named usneaceratin B Compound MT-9 was a new natural product of the lichen U ceratina Arch, and this compound was a new compound in nature.
(New compound) Figure 3.7: The chemical structure, HMBC and NOESY correlations of (MT-9).
3.1.1.10 Structure elucidation of compound 2-ethylhexyl orsellinate (MT-10). ô+ A white amorphous powder.
+ [a]2° 0 (c=0.1, EtOH). oe** Isolated from the lichen P tinctorum (Scheme 3).
** Mass spectrum (Appendix 10.1): HR-ESI-MS (negative mode) m/z
279.1599 [M-H], calcd CisbH230O4 for 279.1596, corresponding to the molecular formula of C¡¿H›4Oa, 280.1676.
% The 'H and C-NMR data (DMSO-ds) (Appendices 10.2 and 10.3): see
** COSY and HMBC spectra (DMSO-d) (Appendices 10.4 and 10.5).
Compound MT-10, a white amorphous powder, had the molecular formula as C16H2404 on the basis of the HR-ESI-MS data with a deprotonated ion peak at m/z 279.1599 (calcd.for Ci6H2404-H, 279.1596).
The 'H-NMR spectrum of MT-10 showed the presence of one hydrogen-bond hydroxy group (ổn 11.01, 1H, brs, 2-OH), two meta-coupled protons [ổn 6.17 (1H, d, J=2.0 Hz, H-5) and 6.15 (1H, d, J= 2.0 Hz, H-3)], one oxygenated methylene [ổn 4.18 (1H, dd, J.0, 5.0 Hz, H-9a) and 4.15 (1H, dd, J.0, 5.5 Hz, H-9b)], one spÌ methine (du 1.63, 1H, m, H-10), three methyl groups [ổu 2.31 (3H, s, H-7), 0.87
(3H, ¢, J=7.5 Hz, H-16) and 0.86 (3H, ¢, J=7.5 Hz, H-14)], and other signals of methylene groups in the range of 1.20—1.40 ppm.
The !*C-NMR spectrum displayed the presence of 16 carbons, including one carbonyl ester carbon at dc 170.5 (C-8), three methines at dc 100.6 (C-3), 110.5 (C-
Structure elucidation of compound usneaceratin B (MT-9)
s*+® A white solid. s* Isolated from the lichen U ceratina (Scheme 1).7
“+ Mass spectrum (Appendix 9.1): HR-APCI-MS (nagetive mode) m/z
197.0452 [M-H], calcd CoHoO; for 197.0444, corresponding to the molecular formula of CoHioOs, 198.0528. s The 'H and '3C-NMR data (acetone—ds) (Appendices 9.2 and 9.3): see Table
3.2. s* HMBC and NOESY spectra (acetone—ds) (Appendices 9.4 and 9.5). ©
Compound MT-9 was obtained as a white solid The molecular formula was identified as CoHi00s by HR-APCI-MS data ([M-HỊ m/z 197.0452, calcd for
The °C and 'H-NMR data of 9 (Table 3.2) detailed nine signals according to one carbonyl carbon at dc 162.6 (1-COOH), three oxygenated aromatic carbons at ốc 148.5 (C-2), 137.5 (C-3), and 153.4 (C-4), two quaternary aromatic carbons at Ốc 114.8 (C-1) and 133.2 C-6), one methine aromatic carbon at dc 113.1 (C-5)/ổn 6.82 (H-5), one methoxy carbon at dc 56.6 (4-OCHs3)/6n 3.87 (4-OCHg), and one methyl carbon at đc 20.5 (C-7)/ổn 2.39 (H-7) Those data of MT-9 confirmed a benzoic acid framework similar of orsellinic acid (MT-3), except for the difference in the appearance of two carbons at dc 137.6 (C-3), 56.6 (4-OCH:) The singlet proton at ou 6.82 (1H, s, H-5) in MT-9 and the disappearing of one methine aromatic carbon
84 at ốc 100.3 (C-3) in 3 (Table 3.1) attested a pentasubstituted benzene system bearing one carboxyl, two hydroxyls one methoxy and one methyl groups for MT- 9.
The HMBC spectrum of MT-9 (Figure 3.7) supported correlations between protons at ổn 2.39 (H3-7) and carbons at ốc 114.9 (C-1), 113.1 (C-5), and 162.6 (1- COOH), between proton at ổn 6.82 (H-5) and carbons at dc 114.9 (C-1), 100.3 (C-
3), 153.4 (C-4), between protons at ổn 3.87 (4-OCHS) and carbon at dc 153.4 (C-4), in addition, proton H-5 (ổn 6.82) was close in space to the methyl group H3-7 (ổn
2.39) and 4-OCH3 (64 3.87) in NOESY spectrum (Figure 3.7), were illustrated the arrangement of these substituents in the benzene nucleus.
From the spectroscopic data, it showed that there was a difference between MT-9 and MT-8 Specifically, the structure of MT-9 had the loss of the methyl signal (3-CH3) and the appearance of many other signals of the hydroxyl group compared to MT-8.
Thus, the chemical structure of MT-9 was assigned as 3-hydroxy-4-O- methylorsellinic acid, named usneaceratin B Compound MT-9 was a new natural product of the lichen U ceratina Arch, and this compound was a new compound in nature.
(New compound) Figure 3.7: The chemical structure, HMBC and NOESY correlations of (MT-9).
Structure elucidation of compound 2-ethylhexyl orsellinate (MT-10)
+ [a]2° 0 (c=0.1, EtOH). oe** Isolated from the lichen P tinctorum (Scheme 3).
** Mass spectrum (Appendix 10.1): HR-ESI-MS (negative mode) m/z
279.1599 [M-H], calcd CisbH230O4 for 279.1596, corresponding to the molecular formula of C¡¿H›4Oa, 280.1676.
% The 'H and C-NMR data (DMSO-ds) (Appendices 10.2 and 10.3): see
** COSY and HMBC spectra (DMSO-d) (Appendices 10.4 and 10.5).
Compound MT-10, a white amorphous powder, had the molecular formula as C16H2404 on the basis of the HR-ESI-MS data with a deprotonated ion peak at m/z 279.1599 (calcd.for Ci6H2404-H, 279.1596).
The 'H-NMR spectrum of MT-10 showed the presence of one hydrogen-bond hydroxy group (ổn 11.01, 1H, brs, 2-OH), two meta-coupled protons [ổn 6.17 (1H, d, J=2.0 Hz, H-5) and 6.15 (1H, d, J= 2.0 Hz, H-3)], one oxygenated methylene [ổn 4.18 (1H, dd, J.0, 5.0 Hz, H-9a) and 4.15 (1H, dd, J.0, 5.5 Hz, H-9b)], one spÌ methine (du 1.63, 1H, m, H-10), three methyl groups [ổu 2.31 (3H, s, H-7), 0.87
(3H, ¢, J=7.5 Hz, H-16) and 0.86 (3H, ¢, J=7.5 Hz, H-14)], and other signals of methylene groups in the range of 1.20—1.40 ppm.
The !*C-NMR spectrum displayed the presence of 16 carbons, including one carbonyl ester carbon at dc 170.5 (C-8), three methines at dc 100.6 (C-3), 110.5 (C-
5), and 38.2 (C-10), three methyls [đc 22.5 (C-7), 13.9 (C-14), and 10.9 (C-16)], five methylenes (đc 66.7, 30.0, 28.4, 23.5, and 22.6), and four quaternary aromatic carbons (dc 162.1, 161.4, 141.1, and 106.8, two former being oxygenated) Detailed comparison of the 1D NMR data of compound MT-10 and methyl orsellinate (4) indicated that they shared the similar aromatic skeleton The difference was the replacement of methyl ester group in methyl orsellinate (MT-4) by the 2-ethylhexyl group in MT-10.
Figure 3.8: The HMBC and COSY correlations of (MT-10).
HMBC and COSY correlations supported this arrangement (Figure 3.8). Particularly, H2-9 and H-5 gave HMBC cross-peaks to C-8, indicating the ester linkage at C-8 HMBC correlations of H-9 to C-10 and C-15, of H2-15 to C-9, C-10, and C-11, and of H3-16 to C- 10 and C-15 indicated the position of the ethyl group at C-10 COSY correlations deduced the spin system through H3-16—H2-15—H-10- H2-9 and H-14/H-13/H-12/H-11/H-10 Consequently, compound MT-10 was elucidated as 2-ethylhexyl orsellinate (Figure 3.8).
Besides, compound MT-10 had the null specific optical rotation, and no Cotton effects in the ECD spectrum (data not shown), indicating that this compound was a racemic mixture.
Compound MT-10 was a new natural product of the lichen P. tinctorum (Despr ex Nyl.) Hale, and was a new compound in nature It is worth noting that the presence of the 2-ethylhexyl ester group is quite rare among lichen (Figure 3.8).
Structure elucidation of compound praesorediosic acid (MT-11)
+ White power. s* Isolated from the lichen P praesorediosum (Scheme 2).
* Mass spectrum (Appendix 11.1): HR-ESI-MS (positive mode) m/z 211.0549
[M+H]*, calcd CioHi1Os for 211.0607, corresponding to the molecular formula of CioH100s, 210.0528.
% The 'H and C-NMR data (DMSO-ds) (Appendices 11.2 and 11.3): see
Compound MT-11 was isolated as a white powder Its molecular formula was determined as C¡oH¡oOs through its pseudomolecular ion peak at m/z 211.0549
[M+H]* in the HR-ESI-MS spectrum The 'H-NMR spectrum data of compound 11 showed signals of two hydroxyl protons at ổn 12.09 (1H, s), and 8.27 (1H, s), a formyl group at ổn 10.44 (1H, s), an aromatic proton at ổn 6.87 (1H, s), an oxymethylene group at 64 4.62 (2H, s), and a methyl group at ổn 2.45 (3H, s) The
C-NMR spectra displayed ten carbons, including a formyl group (& 192.8), a carbon carboxyl (óc 164.0), an oxymethylene carbon (đc 52.6), a methyl carbon (dc 21.4), and six aromatic carbons in the zone of 110-161 ppm (Table 3.2).
The HMBC spectrum observed cross peak from the methyl protons at ổn 2.45 (H-7) to carbon signals C-1 (ức 112.0), C-6 (6c 152.3), and C-7 (đc 117.3), and from the oxymethylene protons at ổn 4.62 (1-CH2OH) to carbon signals C-2 (dc 160.3), and C-6 (6c 152.3), suggesting attachment of the hydroxymethyl group to the benzene ring at C-1 Moreover, the HMBC correlations from aromatic proton at ou 6.87 (H-5) to aromatic carbon C-4 (dc 110.6), methyl carbon C-7 (đ 21.4) and carboxyl carbon 1-COOH (óc 164.0), as well as the formyl proton at dy 10.44 (3- CHO) to aromatic carbons at C-4 (dc 110.6) and C-2 (dc 160.3), indicated that the carboxyl group was joined to C-4 (Figure 3.9).
Consequently, the structure of MT-11 was proposed to be 3-formyl-2-
88 hydroxy-1-(hydroxymethyl)-6-methylbenzoic acid Compound MT-11 was a new compound isolated from natural lichen and a new compound in nature, and was named praesorediosic acid.
HOOO OH HOO OH MỞ
(New compound) Figure 3.9: The chemical structure and HMBC correlations of (MT-11).
Figure 3.10: Chemical structures of compounds (MT-12, 13, 14 and 15).
Structure elucidation of compound lecanorin (MT-12)
+ White powder. s* Isolated from the lichens P praesorediosum and P tinctorum (Schemes 2 and 3). ằ Mass spectrum (Appendix 12.1): HR-ESI-MS (negative mode) m/z
273.0773 [M-H], calcd CisH3Os for 273.0763, corresponding to the molecular formula of CisH14Os, 274.0842. ® The 'H and °C-NMR data (CDC]:) (Appendices 12.2 and 12.3): see Table
** HSQC and HMBC spectra (CDC]:) (Appendices 12.4 and 12.5).
Compound MT-12 was isolated as a white powder The HR-ESI-MS (negative mode) displayed a pseudomolecular ion peak with m/z 273.0773 [M-HỊ, corresponding to the molecular formula of CisHi4Os The 'H-NMR spectrum displayed signals of two methyl groups at ổn 2.33 (3H, s, 6’-CH3) and 2.62 (3H, s, 6-CH3), two aromatic methine protons at ổn 6.31 (1H, s, H-3), 6.32 (1H, s, H-5), three aromatic methine protons at ổn 6.50 (1H, s, H-3'), 6.58 (1H, s, H-1’), and 6.59
(1H, s, H-5'), and one hydroxyl protons at d4 11.42 (1H, s, 2-OH) The "C-NMR and HSQC spectra showed the resonances of 15 carbons, including of two aromatic methyl groups at dc 21.6 (C-7'), 24.7 (C-8), five aromatic methine carbons at ốc 101.6 (C-3), 106.6 (C-3'), 112.0 (C-5), 114.4 (C-5'), and 114.8 (C-1'), seven substituted aromatic carbons, four of which were oxygenated at dc 105.0 (C-1), 141.1 (C-6'), 144.5 (C-6), 150.8 (C-2'), 156.6 (C-4'), 161.4 (C-4), and 166.2 (C-2), and one carbonyl carbon at dc 170.5 (C-7).
In the HMBC spectrum, the correlations of the hydroxyl proton at ổn 11.31 with carbon signals at dc 101.6 (C-3), 105.0 (C-1), 161.4 (C-4), and 166.2 (C-2) indicated that the hydroxyl group was joined to C-2 The two methyl groups (ổn 2.33 and 2.62) were linked at C-6’ and C-6 based on their HMBC correlations with carbon signals at dc 114.4 (C-5’), 114.8 (C-1’), 141.1 (C-6’), and 105.0 (C-1), 112.0
Analysis of spectroscopic data revealed that MT-12 was a depside consisting of one unit of MT-3 (orsenillic acid) linked to one unit of MT-1 (orcinol) These spectroscopic data were compatible with the published ones.! Accordingly, the structure of MT-12 was established as lecanorin, and this compound was also found in another lichen such as U diffracta Vain and U lapponica in advance.'*-°)
Table 3.3: The NMR data of (MT-12—MT-15).
MTI?” MTI3 MTIẾ MTIS MTI2P MTI“ MTI£ MT-15"
3 recorded in Acetone-, ° recorded in CDCl, ° recorded in DMSO-d¢
Structure elucidation of compound gyrophoric acid (MT-13)
+ Isolated from the lichens P tinctorum (Scheme 3). ® Mass spectrum (Appendix 13.1): HR-ESI-MS (negative mode) m/z
467.0989 [M-H] , calcd Cz4H¡sOio for 467.0978, corresponding to the molecular formula of C24H20010, 468.1057. ® The 'H and C-NMR data (acetone-ds) (Appendices 13.2 and 13.3): see
** HSQC and HMBC spectra (acetone—ds) (Appendices 13.4 and 13.5).
Compound MT-13 was isolated as colorless needles, mp 225 °C The HR- ESI-MS (negative mode) displayed a pseudomolecular ion peak with m/z 467.0989 [M-H], corresponding to the molecular formula of C24H20O10.
The 'H-NMR data (acetone-ds, 5 ppm, J in Hertz): 11.13 (1H, s, 2-OH), 6.87
6"-CH3) The !3C-NMR (acetone-ds) data were presented in Table 3.3.
Compound MT-13 was a depside The 1D and 2D NMR data of compound 13 displayed signals of three orselinic units with six aromatic protons, three methyl groups in the 'H-NMR spectrum, and 24 carbon signals in the !*C-NMR spectrum
Figure 3.12: The HMBC correlations of (MT-13).
Spectroscopic data analysis showed that MT-13 had a structure consisting of three MT-3 (orsellinic acid) units linked together through two ester linkages at dc
169.0 These spectroscopic data were suitable with the published data.?°
Accordingly, the chemical structure of MT-13 was established as gyrophoric acid and determined for the first time in this lichen, , and this compound was also found in another lichen such as P cooperi and P robustum, in advance.L'6: 7!
Structure elucidation of compound atrnorin (MT-14) -‹ -ôô+-s 94 3.1.2.4 Structure elucidation of compound diffractaic acid (MT-15)
+ Isolated from the lichen U ceratina (Scheme 1). ằ Mass spectrum (Appendix 14.1): HR-ESI-MS (negative mode) m/z
373.0830 [M-H], calcd CisHi7Os for 373.0924, corresponding to the molecular formula of Ci9HisOs, 374.1002. s The 'H and C-NMR data (DMSO-ds) (Appendices 14.2 and 14.3): see
Table 3.3. ằ HMBC spectra (DMSO-de) (Appendix 14.4).
Compound MT-14 was isolated as white prisms, mp 196 °C and its mass spectrum showed a quasi-molecular ion peak at m/z 373.0830 [M-H], corresponding to the molecular formula of Ci9HisOs.
The 'H-NMR spectrum displayed signals of three methyl groups at 67 2.03, 2.34 and 2.39 (3H each, s), one methoxy group at dy 3.87 (3H, s, H-10'), two
94 aromatic methine protons at 676.42 (1H, s, H-5) and 6.65 (1H, s, H-5'), one formyl group at 67 10.21 (1H, s, H-9), and one hydroxyl proton at 67 10.52 (1H, s, 2-OH).
The '3C-NMR spectrum showed the resonances of 19 carbons, including of three aromatic methyl groups at dc 9.3 (C-9’), 21.2 (C-8'), and 21.2 (C-8), one methoxy group at dc 52.4 (7'-OCHsS), two aromatic methine carbons at dc 109.1 (C-5) and 115.7 (C-5'), ten substituted aromatic carbons, four of which were oxygenated, at dc 107.9 (C-1), 110.7 (C-3), 115.2 (C-1'), 116.3 (C-3'), 136.7 (C-6'), 149.1 (C-6),
151.5 (C-4'), 157.5 (C-2'), 161.6 (C-4), 163.5 (C-2), two carboxyl carbons at ốc 164.6 (C-7) and 169.8 (C-7'), and one formyl group at dc 193.9 (C-9) (Table 3.3).
These findings implied that compound MT-14 was composed of two mono- aromatic units, haematommic acid and ỉ-orsellinic acid The substitution pattern was confirmed by HMBC correlations (Figure 3.13) Moreover, the position of functional groups of MT-14 was also determined by analysis of HMBC spectra
(Figure 3.13) The NMR data were compatible with the published ones!*’ ©! so the chemical structure of compound MT-14 was suggested to be atranorin, , and this compound was also found in another lichen such as P andinum, P austrocetratum,
P autrosinense, P mesotropum, P robustum, P screminiae, P praesorediosum, P. sancti-angelii, P planatilobatum, P dilatum, P lichexanthonicum, P nilgherrense,
3.1.2.4 Structure elucidation of compound diffractaic acid (MT-15)
Melting point: 190 °C + Isolated from the lichen U ceratina (Scheme 1).
“+ Mass spectrum (Appendix 15.1): HR-ESI-MS (negative mode) m/z
373.1281 [M-H], calcd C20H2107 for 373.1288, corresponding to the molecular formula of C20H2007, M 374.1366.
* The 'H and C-NMR data (acetone-ds) (Appendices 15.2 and 15.3): see
Table 3.3. s* HMBC spectrum (acetone—ds) (Appendix 15.5).
Compound MT-15 was isolated as colorless needles The HR-ESI-MS
(negative mode) displayed a pseudomolecular ion peak with m/z 373.1281 [M-H], corresponding to the molecular formula of C29H2007 The 'H-NMR spectrum data of compound MT-15 gave signals of four methyl groups at ở; 2.10, 2.13, 2.44 and 2.63 (3H each, s), three methoxy groups at ổn 3.83 and 3.89 (3H each, s), two aromatic methine protons at dy 6.73 (1H, s, H-5) and 6.46 (1H, s, H-5’) The !3C-
NMR spectrum data showed the resonances of 20 carbons, including four methyl groups at dc 9.0 (C-9’), 9.6 (C-9), 20.0 (C-8), and 23.8 (C-8'), twelve aromatic methine carbons at dc 109.0 (C-5), 115.1 (C-5’), 116.1 (C-3’), 117.8 (C-1’), 117.8 (C-3), 121.6 (C-1), 135.8 (C-6), 140.9 (C-6'), 152.4 (C-4'), 157.8 (C-2), 160.7 (C-
4), and 163.9 (C-2’), four of which were oxygenated, and two carboxyl carbons at
96 ốc 166.8 and 175.5 (Table 3.3) In the HMBC spectrum of MT-15, the correlations of the methoxy protons at 64 3.89 (H3-4) and 3.83 (H3-2) with carbon signals at dc 160.7 (C-4) and 157.8 (C-2) indicated that the methoxy protons were joined to C-4 and C-2, respectively In the A ring, the HMBC correlations between the methyl protons at ổn 2.44 and 2.13 with carbon signals at đc 109.0 (C-5), 121.6 (C-1), 135.8 (C-6), and 117.8 (C-3), 157.8 (C-2), 160.7 (C-4) indicated that two methyl groups were joined to C-6 and C-3, respectively The position of functional groups of B ring was also determined by analysis of HMBC spectra and NMR data were compatible with the published ones.!7*!
Figure 3.14: The HMBC correlations of (MT-15).
These spectral data indicated that MT-15 consisted of two units of f-orsellinic acid and HMBC spectra confirmed the proposed structure as shown in Figure 3.14. Accordingly, the chemical structure of compound MT-15 was established as diffractaic acid, and this compound was also found in another lichen such as P. dilatum, P cristiferum, U baileyi, U emidotteries, and U longissimi, in advance.[117 98, 141, 77, 151]
Stictic acid (MT-16) 8'-O-Methylstictic acid (MT-17) 8'-O-Ethylstictic acid (MT-18)
Ceratinalone (MT-19) Tinctorinone A (MT-20) (New compound) (New compound)
Figure 3.15: Chemical structures of compounds (MT-16—MT-24).
Depsid one oo ố ố e
** Isolated from the lichen U ceratina (Scheme 1).
** Mass spectrum (Appendix 16.1): HR-ESI-MS (negative mode) m/z
385.0557 [M-H], calcd CioHi309 for 385.0560, corresponding to the molecular formula of C¡oH¡4Oo, 386.0638. ¢ The 'H and °C-NMR data (DMSO-ds) (Appendices 16.2 and 16.3): see
Compound MT-16 was isolated as a white powder The HR-ESI-MS (negative mode) displayed a pseudomolecular ion peak with m/z 385.0557 [M-HỊ, corresponding to the molecular formula of C¡oH¡4Oo The 'H-NMR spectrum data of compound 16 gave signals of two methyl groups at 672.19 and 2.49 (3H each, s), one methoxy group at dy 3.91 (3H, s), one acetal proton at ou 6.60 (1H, br), one aromatic methine proton at ổn 7.08 (1H, s, H-5), one hydroxyl protons at ổn 8.21
(1H, s, 8'-OH), and one formyl proton at du 10.46 (1H, s) The C-NMR spectrum showed the resonances of 19 carbons, including two aromatic methyl groups at ốc 9.7 (C-9”) and 21.6 (C-9), one methoxy group at & 56.9 (4-OCHg), one acetal carbon at dc 95.2 (C-8’), twelve aromatic methine carbons at ốc 109-164 ppm with five of which were oxygenated, three carboxyl carbons at dc 160.8 (C-7), 166.5 (C-
7'), and 186.8 (C-8) Furthermore, compound MT-16 had the null specific optical rotation indicating that this compound was a racemic mixture The spectra of the compound MT-16 were compatible with the published ones.!**! Accordingly, the chemical structure of compound MT-16 was established as stictic acid.
Table 3.4: The NMR data of (MT-16—MT-21).
MT-16° MT-17“ MT-18° MT-19* MT-20° MT-21° MT-16° MT-17“ MT-18° MT-19* MT-20° MT-21*
4 recorded in Acetone-d¢, ? recorded in CDC]:, ° recorded in DMSO-d¢
3.1.3.2 Structure elucidation of compound 8’-O-methylstictic acid (MT-17)
+ White amorphous powder. s* Isolated from the lichen U ceratina (Scheme 1). ằ Mass spectrum (Appendix 17.1): HR-ESI-MS (positive mode) m/z 423.0651
[M+Na]”, calcd C2oHisOoNa for 423.0692, corresponding to the molecular formula of C20Hi6O9, 400.0795.
* The 'H and '3C-NMR data (Acetone-ds) (Appendices 17.2 and 17.3): see
%* NOESY, HSQC, HMBC and ECD spectra (Acetone—ds) (Appendices 17.4,
Compound MT-17 was isolated as a white amorphous powder The HR-ESI-
MS (positive mode) displayed a pseudomolecular ion peak with m/z 423.0651
[M+Na]”, corresponding to the molecular formula of C2Hi6Os.
The 'H-NMR spectrum data of compound MT-17 gave signals of two methyl groups at dy 2.26 and 2.54 (3H each, s, H-9’ and H-9), two methoxy groups at dy 3.63 and 4.01 (3H each, s), one acetal proton at ổn 6.53 (1H, s), one aromatic methine proton at ổn 7.11 (1H, s, H-5), and one formyl proton at ổn 10.52 (1H, s).
The '3C-NMR spectrum showed the resonances of 20 carbons, including of two aromatic methyl groups at dc 9.4 (C-9”) and 21.9 (C-9), two methoxy groups at ốc 57.2 (4-OCH3) and 57.5 (C-10'), one acetal carbon at dc 102.9 (C-8'), twelve aromatic methine carbons at dc 109.0 (C-1'), 113.5 (C-5), 115.0 (C-1), 115.9 (C-3),
2), and 164.4 (C-4) with five of which were oxygenated, three carboxyl carbons at ốc 158.9 (C-7), 168.3 (C-7'), and 187.3 (C-8).
In the HMBC spectrum, the correlations of two methyl groups at ổ; 2.26 (H-
9’) and 2.54 (H-9) with carbon signals at dc 121.9 (C-3'), 150.2 (C-4’), 151.9 (C-2") and 113.5 (C-5), 115.0 (C-1), and 151.9 (C-6) indicated that the methoxy protons were joined to C-3” and C-6, respectively The two methoxy groups (ổn 3.63 and
4.01) were linked at C-8’ and C-4 based on their HMBC correlations with carbon signals at dc 102.9 (C-8’) and 164.4 (C-4), respectively The proton at dy 10.52 (H-
8) showed the HMBC correlations with the carbon signal at dc 115.9 (C-3) indicated that the formyl group at dc 187.3 (C-8) was joined to C-3 In addition, proton H-5 (ổn 7.11) was close in space to protons H-9 (ổn 2.54) in NOESY (Figure 3.16), which was illuminated the arrangement of these substituents in the benzene nucleus.
Analysis of the spectroscopic data showed that compound MT-17 had a similar structure to compound MT-16 (stictic acid) However, compound MT-17 had the methoxy group that was attached to the C-8' position while compound MT-
16 (stictic acid) had the hydroxyl group.
Moreover, depsidones containing a B-ring fused with a y-butyrolactone moiety had the absolute configuration was determined by the electronic circular dichroism
(ECD) spectrum * '°] The similar negative cotton effects (CE) of compounds
MT-1I7-MT-20, indicated that they possessed the same S absolute configuration.
These CEs were reminiscent to those of lobarientalone A! or bailesidone 11, indicating the (8’S) configuration of MT-17.
From these findings, the chemical structure of compound MT-17 was elucidated as 8’-O-methylstictic acid.'°*! Besides, this compound was also found in another lichen such as U lapponica and the lichen Usnea sp from Sri Lanka, in
3.1.3.3 Structure elucidation of compound 8’-O-ethylstictic acid (MT-18) ằ White amorphous powder.
** Isolated from the lichen U ceratina (Scheme 1).
* Mass spectrum (Appendix 18.1): HR-ESI-MS (positive mode) m/z 437.0826
[M+Na]*, calcd C21HisOoNa for 437.0849, corresponding to the molecular formula of C21:His00, M 414.0951.
* The 'H and °C-NMR data (CDC]:) (Appendices 18.2 and 18.3): see Table
3.4. ® NOESY, HSQC, HMBC and ECD spectra (CDC]:) (Appendices 18.4, 18.5,
Compound MT-18 was isolated as a white amorphous powder The HR-ESI-
MS (positive mode) displayed a pseudomolecular ion peak with m/z 437.0826
[M+Na]*, corresponding to the molecular formula of C21HisOo The 'H-NMR spectrum data of compound MT-18 gave signals of three methyl groups at dy 1.28 (3H, ¢, 7.5 Hz, H-11'), 2.30 and 2.57 (3H each, s, H-9', and H-9), one methoxy group at 673.99 (3H, s), one oxymethylene group at 674.01 (2H, dd, 7.5Hz), one acetal proton at ổn 6.49 (1H, s), one aromatic methine proton at dy 6.75 (1H, s, H-
5), and one formyl proton at ổn 10.53 (1H, s) The !*C-NMR spectrum showed the resonances of 21 carbons, including of three methyl groups at dc 9.3 (C-9'), 15.1
(C-11'), and 22.5 (C-9), one methoxy group at dc 56.8 (4-OCHg), one ethoxy group at dc 15.1 and 66.9 (8'-O—CH,—CHs3), one acetal carbon at dc 102.3 (C-8'), twelve aromatic methine carbons at dc 108.0 (C-1'), 112.1 (C-5), 114.5 (C-1), 115.0 (C-3),
121.1 (C-3'), 132.5 (C-6'), 138.7 (C-5'), 149.7 (C-4'), 151.7 (C-6), 152.7 (C-2’), 163.1 (C-2), and 163.8 (C-4) with five of which were oxygenated, three carboxyl carbons at dc 160.9 (C-7), 169.9 (C-7'), and 186.9 (C-8).
In the HMBC spectrum, the correlations of two methyl groups at dy 2.30 (H-
9") and 2.57 (H-9) with carbon signals at dc 121.1 (C-3’), 149.7 (C-4'), 152.7 (C-2') and 112.1 (C-5), 114.5 (C-1), 151.7 (C-6) indicated that two methyl groups were joined to C-3’ and C-6, respectively The methoxy group (ổn 3.99) was linked at C-
4 based on its HMBC correlation with carbon signal at 163.8 (C-4) The proton at ồn 10.53 (H-8) showed the HMBC correlations with the carbon signal at dc 115.0 (C-3), 163.1 (C-1), and 163.8 (C-4) indicated that the formyl group at 6c 186.9 (C-
8) was joined to C-3 The difference was the HMBC correlations of the oxymethylene of the ethoxy group (du 4.01) with carbons signals at 15.1 (C-11’), 102.3 (C-8') indicated that the ethoxy group was joined to C-8’ In addition, proton
H-5 (ổn 6.75) was close in space to protons H3-9 (64 2.54), 4-OCH: (ổn 3.99), and proton H-8° was close to H2-10’ in NOESY (Figure 3.17), which were illuminated the arrangement of these substituents in the benzene nuclei and in the five- membered ring lactone.
Figure 3.17: The HMBC correlations of (MT-18).
Furthermore, the S configuration of call C-8’ was discussed in 3.1.3.2 The ECD spectrum of MT-18 showed the negative Cotton effect is very similar to that of MT-17 isolated from U ceratina Therefore, analysis of the spectroscopic data showed that compound MT-18 had a similar structure to compound MT-17 (8'-O- methylstictic acid) However, compound MT-18 had an ethoxy group that was attached to the C-8' position while compound MT-17 (8'-O-methylstictic acid) had a methoxy group These spectroscopic data were compatible with the published one.!l All these observations suggested that compound MT-18 was 8’-O- ethylstictic acid Besides, this compound was also found in another lichen as
3.1.3.4 Structure elucidation of compound ceratinalone (MT-19)
+ Isolated from the lichen U ceratina (Scheme 1).
+ Mass spectrum (Appendix 19.1): HR-ESI-MS (positive mode) m/z 425.0827
[M+Na]*, calcd C2oHisOoNa for 425.0849, corresponding to the molecular formula of CaoH:sÒo, 402.0951.
% The 'H and °C-NMR data (acetone-ds) (Appendices 19.2 and 19.3): see
Table 3.4. s* NOESY, HSQC, HMBC and ECD spectra (acetone-ds) (Appendices 19.4,
Compound MT-19 was obtained as white amorphous powder Its molecular formula was determined to be CaoHisOo from its HR-ESI-MS ion at m/z 425.0827
[M+Na]* (calcd for C2oHis09Na, 425.0849).
The !H NMR spectrum displayed signals for one aromatic proton (ổn 6.87, s), one acetal proton (ổn 6.72, s), a methoxy group (ổn 3.92, s), an ethoxy group (ổn
4.00, 2H, g, J =7.0 Hz and 1.32, 3H, ¿, J = 7.0 Hz), and two methyl groups at ổn
2.23 and 2.39 (3H each, s) The !C NMR and HSQC spectra in accordance with
HR-MS data exhibited 20 carbon signals including two carboxyl carbons (dc 161.9 and 168.9), one acetal carbon (ốc 102.5), one methoxy group (ốc 56.7), one ethoxy group (óc 67.0 and 15.3), two methyl groups (đc 9.2 and 20.3), and 12 aromatic carbons in the range 108—153 ppm.
HMBC cross-peaks of proton H-5 (ổn 6.87) with carbons C-1 (đc 114.8), C-3
(dc 136.1), and C-4 (đc 152.9), of the methyl protons at 64 2.39 (H3-9) with carbon signals at dc 114.8 (C-1), 112.3 (C-5), and 134.0 (C-6) defined the connectivity through C-1—C-6 and further validated the A-ring of f-orcinol depsidone scaffold
(Duong et al 2015; 2020)? !%l_ This was also supported by the NOESY correlations of H-5 with both 4-OCH3 and H3-9 In the B-ring, HMBC correlations from H-8' to carbon signals at dc 108.9 (C-1’), 140.0 (C-6’), 168.9 (C-7’), and 67.0 (C-10’) indicated the presence of a y-lactone moiety in MT-19 (Figures 3.18),
106 further comfirmed by NOESY correlation between H-8’ and H-10’.
Compounds of other types - c1 1 TH TH ng ng ng ngệt 130 1 Structure elucidation of compound sernanderin (MT-31)
Semanderin (MT-31) Hopane-6œl6/22-tiol(MT-32) Palmitic acid (MT-33)
Figure 3.30: Chemical structures of compounds (MT-31— MT-34).
3.1.6.1 Structure elucidation of compound sernanderin (MT-31)
+ White powder. s* Isolated from the lichen P tinctorum (Scheme 3). ằằ Mass spectrum (Appendix 31.1): HR-ESI-MS (positive mode) m/z 291.1230
M+H]*, calcd C¡¿HioO; for 291.1233, corresponding to the molecular formula of CisHisOs, 290.1154.
* The 'H and !C-NMR data (acetone-đs) (Appendices 31.2 and 31.3): see
Table 3.9. ® COSY, NOESY and HMBC spectra (acetone-#s) (Appendices 31.4, 31.5, and 31.6).
Compound MT-31 was isolated as a white amorphous powder The molecular formula was established to be CisHisOs on the basis of the positive-ion mode HR- ESI-MS data with a pseudomolecular ion peak at m/z 291.1230 (calcd for Cis6H19Os 291.1233).
The 'H NMR spectrum of MT-31 showed the presence of singlets for two methyl groups (dy 1.51 and 2.42), three diastereotopic methylene groups (64 2.50—
2.80), one sp? methine group (ở; 2.44-2.84, m), one methoxy group (ổn 3.93, s), and one aromatic methine proton (67 6.16 s).
The '3C NMR spectrum showed the presence of one isolated ketone carbonyl carbon (ốc 207.9), one ester carbonyl carbon (óc 173.4), three methylene groups (dc
53.9, 46.3, and 35.0), one methoxy group (dc 52.3), one sứ” methine carbon (dc 27.6) and two sp? methyl groups (ốc 28.7 and 24.1), one aromatic methine carbon
(ốc 112.4) and six quaternary aromatic carbons, three of which were oxygenated (dc 162.7, 158.5 and 79.2) In the HMBC spectrum, there were correlations of the methyl protons at 67 1.51 and 2.42 with the carbons at dc 79.2 (C-12), 53.9 (C-11), 46.3 (C-9) and 142.0 (C-6), 112.4 (C-5), 105.2 (C-1), indicating that two methyl groups attached at C-12 and C-6, respectively In addition, proton H-5 (ổn 6.16) was close in space to protons H3-15 (ổn 2.42) in NOESY (Figure 3.31), elucidating the arrangement of these substituents in the benzene nucleus.
The correlations of the protons at ổn 2.44-2.84 (H-8), 2.50-2.56 (H-13), and 2.70-2.80 (H2-9) with carbon signal at đc 207.9 (C-10) and of the protons at ổn 1.52
(H3-14) with carbon signals at đc 35.0 (C-13), 53.9 (C-11), 79.2 (C-12), indicating that the isolated ketone carbon at C-10 and defining the connectivity as C-0—C-8—C-
13 at C-12 Besides, the COSY spectra supported the identification of this links, analysis of the COSY spectra (Figure 3.31) for MT-31 revealed that H2-8 (ổn 2.44— 2.84) correlated with Ha-9 (2.70—2.80) as well as with the methylene group Ha-13. Further HMBC correlations of H-5 (ổn 6.16) to carbons at dc 158.5 (C-4), 111.0 (C-
3), 105.2 (C-1), and 24.1 (C-15) These spectroscopic data were compatible with the published ones in the literature from Lethariella sernanderi;!*! therefore, compound
MT-31 was sernanderin Besides, this compound was also found in another lichen as P dilatum, in advance.8!
Table 3.9: The NMR data of (MT-31—MT-34).
No On oc ồn oc ồn oc ồn oc
4 recorded in Acetone-đs, ằ recorded in CDC]:, ° recorded in DMSO-đ;¿
3.1.6.2 Structure elucidation of compound hopane-6a,16f,22-triol (MT-32) ô+ A white amorphous powder. s* Isolated from the lichens P tinctorum (Scheme 3) 7 oe ằ Mass spectrum (Appendix 32.1): HR-ESI-MS (positive mode) m/z 443.3893
[M-H20+H]*, calcd C30Hs102 for 443.3892, corresponding to the molecular formula of C30Hs203, 460.3919. s The 'H and 'C-NMR data (CDCl) (Appendices 32.2 and 32.3): see Table
The molecular formula of MT-32 was determined as C30Hs203 through the pseudomolecular ion peak at m/z 443.3893 [M-H20+H]* in the HR-ESI-MS spectrum The C-NMR spectrum of MT-32 displayed thirty carbon signals, including eight methyls, ten methylenes, six methines, and six quaternary carbons. This suggested that compound MT-32 was a triterpenoid.
In the HMBC spectrum, the correlations of the olefinic protons H-21 (dx
2.50) with carbon signals atf& 46.0 (C-18), 60.9 (C-17), and 74.7 (C-22) (Figure 3.32) The coupling constants of upfield methine H-5 (dy 0.85, d, J = 11.0 Hz) exhibited 1,2- diaxial 'H—'H coupling to a secondary oxymethine (ở; 4.00, dt, 10.5,
4.0 Hz, H-6) That mean the hydroxyl group at C-6 was at a-orientation In addition, this secondary alcohol methine H-16 exhibited two 1,2-diaxial coupling constant values Jaa = 9.0 Hz, indicating the /-orientation of 16-OH group.
These spectroscopic data were compatible with the published one of the lichen
Ramalina peruviana Arch.P?Ì All these properties suggested that compound MT-32 was hopane-6a,16/,22-triol or leucotylin Besides, this compound was also found in another lichen as P sancti-angelii, in advance.l3!: 51
3.1.6.3 Structure elucidation of compound palmitic acid (MT-33) s* White crystal. s* Melting point: 40 °C
** Isolated from the lichen U ceratina (Scheme 1). ằ Mass spectrum (Appendix 33.1): HR-ESI-MS (negative mode) m/z
255.2327 [M-H], calcd Ci6H3102 for 255.2326, corresponding to the molecular formula of C16H32O2, 256.2404.
* The !H and C-NMR data (CDC]:) (Appendices 33.2 and 33.3): see Table
Compound MT-33 was isolated as a white crystal The HR-ESI-MS (negative mode) displayed the [M—H] at m/z = 225.2327 (calcd 255.2326 for C16H3102), corresponding to the molecular formula of Ci6H3202 The 'H-NMR spectrum displayed signals of some methylene groups at 67 2.34 (2H, í, J= 7.5 Hz, H-2),
1.63 (2H, m, H-3) and 1.27 (rs), one methyl group at dy 0.87 (3H, t, J= 7.0 Hz, H-
16) The !*C-NMR spectrum showed the resonances of many carbons, including one methyl group at ốc 14.3 (C-16), fourteen methylene groups at dc 22.8-34.0, one carboxyl group at dc 179.0 The HR-MS spectrum and the NMR were compatible with the published ones.! Accordingly, the chemical structure of compound MT-
33 was established as palmitic acid and the first time known in the Usnea genus.
3.1.6.4 Structure elucidation of compound uracil (MT-34) © White power.
** Isolated from the lichen P praesorediosum (Scheme 2). s* Mass spectrum (Appendix 32.1): HR-ESI-MS (positive mode) m/z 135.0039
[M+Na]*, calcd CaHaN2O2Na for 135.0771, corresponding to the molecular formula of C4H4N2O2, 112.0273.
% The 'H and !'C-NMR data (DMSO-de) (Appendices 34.2 and 34.3): see
Table 3.9. s* HSQC and HMBC spectra (DMSO-d) (Appendices 34.4 and 34.5).
Compound MT-34 was isolated as a white power The HR-ESI-MS (positive mode) displayed the [M+Na]* ion at m/z 135.0039 (calcd 135.0771 for
C4H4N202Na), corresponding to the molecular formula of C4H4N2O>2 Its 'H-NMR spectrum showed a pair of signals of two olefinic protons at 647.38 (1H, d, J =7.5
Hz, H-6) va ổ; 5.41 (1H, d, J = 7.5 Hz, H-5) (Table 3.9) The “C-NMR and
HSQC spectra of 34 displayed four carbon signals, including two olefinic carbon signals at dc 142.8 (C—6) and 100.0 (C—5), two carbonyl carbons at dc 164.4 (C-4) and 151.8 (C-2).
Figure 3.33: The chemical structure and HMBC correlations of (MT-34).
In the HMBC spectrum, the correlations of the olefinic protons H-5 (61 5.41) and H-6 (ở; 7.38) with carbon signals at dc 164.4 (C—4), 142.8 (C—6) and 151.8 (C—
MT-34 was established as uracil.?°!
3.2.1 Cytotoxic activity against four cancer cell lines
Samples of one new and four known compounds (at the concentration of 100 ug/mL) were tested the cytotoxic activity against four cell lines: MCF-7 (breast cancer cell line), HeLa (cervical cancer cell line), NCI-H460 (human lung cancer cell line) and HepG2 (a human liver cancer cell line) by sulforhodamine B colorimetric assay method (SRB assay).!!7°!
Each sample was tested three times The cytotoxic activity of five compounds, expressed as percentage of cell growth inhibition (I%), was presented in Table 3.10. Furthermore, three compounds (diffrataic acid, ceratinalone and 8’-O-methylstictic acid) had ICso values determined in Table 3.12.
Table 3.10 % Inhibition of cytotoxic activity against four cancer cell lines of some isolated compounds
No Compound” MCF-7 HeLa NCI-H460 HepG2
(positive control)” a) The compounds were tested at the concentration of 100 g/mL. b) The presented data are means of three experiments + S.D. c) Camptothecin was tested at the concentration of 0.01 ug/mL for MCF-7 and NCI-H 460 and of 1 ug/mL for HeLa.
The results showed that the compound praesorediosic (MT-11) showed
137 potential inhibitive activity against MCF-7 cell line with %I about 56-61%, while virensic acid (MT-23) showed potential inhibitive activity against HeLa cell line with %I about 50-57%.
In Table 3.11, diphenyl ether compounds had good activity against the MCF-7 cancer cell line They were higher than compound praesorediosic (MT-11) including lobariether C (72.44+3.46), praesorether G (72.8+1.8), parmoether B (79.11+3.47), and parmosidone C (66.75+3.42) (the data were recorded in Duong
Thuc Huy's thesis) while the remaining compounds were less active than this compound.|41
Several depside compounds had very good activity against the HeLa cancer cell line and were higher than virensic acid (MT-23) including 4-Ó- demethylbarbatic acid (88.5+3.3), sekikaic acid (79.5+1.7) in Linh Chi's thesis.'*°!
Besides, (£)- methyl caffeate (monocyclic compound, in My Dung's thesis) had superior activity compared to compounds in lichen with %I about 91.07%.PTI
Therefore, (£)- methyl caffeate will be a potential substance to fight this type of cancer in Table 3.11.
Table 3.11 % Inhibition of cytotoxic activity against four cancer cell lines of some compounds in other lichens! 24][45][97]
No Compound” MCF-7 HeLa NCI-H460 HepG2
(positive control)? a) The compounds were tested at the concentration of 100 ug/mL. b) The presented data are means of three experiments + S.D. c) Camptothecin was tested at the concentration of 0.01 ug/mL for MCF-7 and NCI-H 460 and of 1 ug/mL for HeLa.
Hae COOH ois H;ạCO COOCH; HO 3
CHO CH, Lobariether C (E)-CH=CH-CO-CH;
Lobariether B CH;OH Lobariether D (E)-CH=CH-COOH Lobariether E (E)-CH=CH-COOCH; Praesorether G
The compounds (Table 3.10) had poor activity against the NCI-H460 cancer cell line In Table 3.11, there were many diphenyl ether compounds with very good activity including lobariether C (59.67+3.70), parmoether A (41.02+6.99), and parmoether B (85.51+0.98) Besides, two compounds also showed good activity in this cell line including parmosidone D (50.67+1.67) and methylstictic acid (68.11+5.08) Furthermore, (£)- methyl caffeate was also a potential substance to fight this type of cancer with %I about 84.61%.
Diphenyl ether compounds (Table 3.11) had very good activity against the HepG2 cancer cell line such as lobariether C (66.29+4.25), parmoether A (59.36+1.91), and parmoether B (84.73+40.68) Besides, two compounds also showed good activity in this cell line including parmosidone C (62.56+2.68) and (E)- methyl caffeate (71.25+2.60) In contrast, the compounds (table 3.10) did not show good results on this cancer cell line.
In summary, the information in Tables 3.10 and 3.11 showed that diphenyl ether compounds had very good activity against cancer cell lines, especially lobariether C and (£)- methyl caffeate (monocyclic compound).
Table 3.12 ICso of cytotoxic activity against four cancer cell lines of diffrataic acid, ceratinalone, and 8 -Ó-methylstictic acid.
No Compound MCF-7 HeLa NCI-H460 HepG2
The ICso values in three cell lines (HeLa, NCI-H460, and HepG2) of diffrataic acid (MT-15) were smaller than the ICso value of ceratinalone (MT-19), 8’-O- methylstictic acid (MT-17) (Table 3.12) It indicated that diffrataic acid (MT-15) could be a potential inhibitor against HeLa, NCI-H460, and HepG2 cell lines In addition, the ICso value on the MCF-7 cell line of 8’-O-methylstictic acid (MT-17) was 15.61 uwg/mL (Table 3.12) indicating an inhibitory potential against this cell line.
BIOSYNTHESIS OF ISOLATED COMPOUNDS IN LICHENS
The symbiosis between algae and fungi forms lichen Initially, lichen synthesizes sugar compounds for the system, then these sugar molecules are broken down into Acetyl CoA, and part of Acetyl CoA can be converted into Malonyl CoA Next, there are ring-closing reactions and substituent attachments from those CoA along the polyketide pathway to form Orsellinic acid, followed by depsides and the other compounds in the lichen (Figure 1.2)13!
Polyketide synthases (PKSs) are a family of enzyme complexes that produce polyketides, a class of secondary metabolites of bacteria, fungi, plants, and some animals The biosynthesis of these polyketides has some similarities with the biosynthesis process of fatty acids The first enzyme in the chain is an acetyl-CoA carboxylase, which requires ATP to produce malonyl-CoA, the first substrate of PKSs.!2!
CH;-CO-SCoA + HOOC-CH;-CO-SCoA ~
CHs 2 7 COOH orsellinate depside hydrolase COOH
| orsellinate decarboxylase Gyrophoric acid (MT-13)
HO OH dehydrogenase HO OH
Figure 3.34: Biosynthesis of monocyclic compoundslŠ7 !2!1
The coupling to an initial molecule of acetyl-CoA of successive malonyl- CoA molecules requires Claisen decarboxylative condensations until a polyketone chain of eight carbon atoms is obtained The biosynthesis of gyrophoric acid (MT-13), a tridepside of the orcinol series in Umbilicaria papulosa, was studied by supplying lichen thalli with '4C-labeled malonyl-CoA
(Figure 3.34) Subsequently, hydrolysis of gyrophoric acid (MT-13) produced
147 orsellinic acid (MT-3) Next were many products including orcinol (MT-1) and atranol (MT-2) from orsellinic acid (MT-3).H!?!!
CH¡-CO-SCoA ——> OH -CO-CH3-CO-CH-CO-CH,-CO-SCoA: r
CH: Hy ; CH a oon 2 COOH
COOCH, SAM COOCH, a oxydase