Chemistry of calophyllum wallichianum, scapania undulata, plagiochila colorans and biotransformation of natural products

CHEMISTRY OF CALOPHYLLUM WALLICHIANUM, SCAPANIA UNDULATA, PLAGIOCHILA COLORANS AND BIOTRANSFORMATION OF NATURAL PRODUCTS HUANG MINGXING NATIONAL UNIVERSITY OF SINGAPORE 2007... Phytoc

CHEMISTRY OF CALOPHYLLUM WALLICHIANUM, SCAPANIA UNDULATA, PLAGIOCHILA COLORANS

AND BIOTRANSFORMATION OF NATURAL PRODUCTS

HUANG MINGXING

NATIONAL UNIVERSITY OF SINGAPORE

2007

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude and appreciation to my supervisor, Associate Professor Leslie J Harrison, for his guidance, patience and encouragement during the past two years It will be impossible for me to complete my project without your invaluable advice and support

I wish to thank Associate Professor Benito C Tan in the Department of Biological Science for his invaluable suggestions I will also thank Associate Professor Teck K Tan in the Department of Biological Science and Associate Professor Eng Soon Tok in the Department of Physics for every convenience and support they provided

I also would like to thank my friends, Yanmei, Xiaowei, Li Wei, Chiakwu, who help me a lot throughout my project

My appreciation will also give to the technical staff of Department of Chemistry for NMR, MS and XRD analysis, and Ms Chua Ling Lih for the technical support for the biological part of my project I also want to thank National University of Singapore for financial support and the research scholarship

Finally but not least, I really want to express my deep gratitude to my dear family members, my father, my mother, my brothers and my sister You always stand behind

me and support me I do want to say thank you to all of you, for your love, understanding and encouragement

5.2.1 Small-scale Screening Experiments 83

5.2.2 Biotransformation of (±)-Camphor by Mucor plumbeus 85 5.2.3 Biotransformation of (±)-Camphor by Aspergillus niger 87

5.3.1 General Procedure for Biotransformation 88

5.3.2 Biotransformation of (±)-Camphor by Mucor plumbeus 89 5.3.3 Biotransformation of (±)-Camphor by Aspergillus niger 90

References 93

Summary

This thesis deals with the phytochemistry of one ever green tree and two liverworts The isolation and purification were mainly achieved by the combination of various chromatographic techniques and the structural identifications were mainly dependent on the employment of spectroscopic techniques including Nuclear

Magnetic Resonance (1D and 2D NMR), Mass Spectrometry, X-ray Diffraction, etc

Chapter one gives a general background of natural products chemistry Chapter

two to chapter four concern the chemical studies of three plants: Calophyllum wallichianum, Scapania undulata and Plagiochila colorans The last chapter covers

the biotransformation of terpenoids

Phytochemical studies on Calophyllum wallichianum afforded two novel

coumarins (74 and 75) along with eight known compounds: cordatolide A (63), cordatolide B (64), 12-O-methylcordatolide B (69), 12-O-methylcalanolide B (70), trapezifolixanthone (71), pseudocordatolide C (72), carpachromene (73) and (+)-epiafzelechin (76)

Investigation of Scapania undulata led to the isolation of six novel compounds

(119-124) together with five known compounds: (-)-longiborneol (78),

(-)-ent-longipinanol (84), compound 110, diplophyllolide A (117),

ent-5β-hydroxydiplophyllolide (118) In addition, three compounds (125-127) were

also isolated and there have been many evidences indicating that they were degraded labdane-type diterpenoids, but their structural identification still requires further investigation

The last plant studied was Plagiochila colorans, and this research yielded two

known sesquiterpenoids: peculiaroxide (139) and gymnomitrol (140)

In the studies of terpenoids biotransformation, small scale trial experiments were developed to screen the fungi that are capable to transform the substrates to be

investigated, and the selected biotransformation of camphor by Mucor plumbeus and Aspergillus niger afforded three and four mono-hydroxylated products, respectively

Key words: Natural products, Calophyllum wallichianum, Scapania undulata, Plagiochila colorans, Biotransformation

List of Tables

Table 2-1 1H, 13C NMR Data and HMBC Correlations of 74

Table 3-1 1H, 13C NMR Data of 117 and 1H NMR data of ent-Diplophyllolide A

Table 3-2 1H, 13C NMR Data and HMBC Correlations of 119

Table 3-3 1H, 13C NMR Data and HMBC Correlations of 120

Table 3-4 1H, 13C NMR Data and HMBC Correlations of 121

Table 3-5 13C NMR Comparison of 122 and Scapanin B

Table 3-6 1H, 13C NMR Data and HMBC Correlations of 123

Table 3-7 1H, 13C NMR Data and HMBC Correlations of 124

Table 3-8 1H, 13C NMR Data and HMBC Correlations of 125

Table 3-9 Comparison of the NMR Data of 120, 125 and 126

Table 3-10 1H, 13C NMR Data and HMBC Correlations of 126

Table 5-1 GC Data of the Small Scale Trial Experiments

List of Figures Figure 2-1 Relative stereochemistry of ring D fragment of 63 Figure 2-2 Key HMBC correlations of compound 74

Figure 2-3 ORTEP diagram of compound 75

Figure 3-1 ORTEP diagram of compound 110

Figure 3-2 Octant projection diagrams of compound 104 Figure 3-3 Key HMBC correlations of compound 119

Figure 3-4 ORTEP diagram of compound 119

Figure 3-5 Key HMBC Correlations of compound 120 Figure 3-6 NOESY correlations of compound 120

Figure 3-7 ORTEP diagram of compound 120 Figure 3-8 Key HMBC Correlations of compound 121 Figure 3-9 NOESY correlations of compound 121

Figure 3-10 ORTEP diagram of compound 122

Figure 3-11 Key HMBC correlations of compound 123 Figure 3-12 ORTEP diagram of compound 123

Figure 3-13 Key HMBC Correlations of 124

Figure 3-14 ORTEP diagram of compound 124

Figure 3-15 Key HMBC correlations of compound 125 Figure 3-16 Key HMBC Correlations of compound 126

List of Schemes

Scheme 1-1 Biosynthesis of phloracetophenone (43)

Scheme 1-2 Biosynthesis of alternariol (44)

Scheme 3-1 Chemical transformation of scapanin A (91) to 110

Scheme 5-1 Hydroxylation of α-ionone (141) with Streptomyces strains

Scheme 5-2 Possible metabolic pathway of camphor (157) in the larva of Spodoptera

litura

Scheme 5-3 Scheme of the biotransformation of camphor with Mucor plumbeus Scheme 5-4 Scheme of the biotransformation of camphor with Aspergillus niger

Chapter 1 General Introduction

1.1 Introduction

There is a very long history of compounds from living organisms such as plants and animals being utilized by human beings The employment of these substances in the treatment of human diseases can be traced back to the Sumerian civilization In

ancient Egypt, India, China, etc., a large number of plants had been found to work well to treat some diseases For example, Ma Huang, or Ephedra spp (Ephedraceae)

was traditionally used as a diaphoretic, anti-asthmatic and diuretic drugi, and Qian

Ceng Ta, or Huperzia serrata (Huperziaceae) was employed to treat fever and

inflammationii,iii In addition, natural occurring compounds have also been used for thousands of years as dyes (e.g indigo, shikonin), flavours (e.g vanillin, capsaicin, mustard oils), fragrances (e.g various essential oils), stimulants (e.g caffeine, nicotine), hallucinogens (e.g morphine, cocaine), insecticides (e.g nicotine, piperine), and so on

1.2 Natural Products

Natural products consist of two categories: primary metabolites and secondary metabolites Primary metabolites are ubiquitous in living organisms where they play essential roles in the growth, development and reproduction Carbohydrates, amino acids, lipids, nucleic acids and proteins are typical examples of primary metabolites Secondary metabolites, however, are chemical compounds that are not strictly necessary for the survival of organisms However, they often play some essential roles from the perspective of ecology Many metabolites, for example, have been found to provide defense against predators, parasites and diseases Common examples of this category of metabolites include terpenoids, alkaloids, flavonoids, and so forth Compared with primary metabolites, the occurrence of secondary metabolites is rather restricted However, it is noteworthy that the boundary between primary metabolites

and secondary metabolites is sometimes blurred For example, some fatty acids and sugars are extremely rare and found only in several species, and at the same time, some sterols are found to play essential role for the survival of many organism and therefore must be considered to be primary metabolites Currently, the concept of natural products is widely considered to be secondary metabolites

Although natural products have been diversely utilized for thousands of years, their modern and systematic studies did not begin until late eighteenth century The development of modern separation methods, such as various analytical techniques and preparative chromatographic methods made it possible to isolate compounds present

in extremely small quantities, whilst the development of spectroscopic techniques such as UV, NMR, MS, CD, etc, leads to the rapid structural elucidation even with trace quantityiv

Natural products do not directly relate to the survival of creatures, their crucial roles in the evolutionary and ecological perspectives, however, have been widely recognized for a long time In plant kingdom, some natural products serve as attractants to ensure pollination and reproduction, some act to warn and defend against herbivores and some other compounds play significant role in the competitionwith surrounding plants for light, space and nutrients At the same time, secondary metabolites are also frequently employed by various animals to communicate, defend and hunt

Some secondary metabolites contribute to the color of flowers, which is a significant means to attract pollinators At the same time, the blend of different classes

of volatile secondary metabolites (mainly monoterpenoids) in plants produces a broad

spectrum of scents to behave as attractants For example, Nicotiana otophora use

volatile and fragrant compounds such as α-thujene (1), myrcene (2), limonene (3),

1,8-cineole (4), sabinene (5), etc to attract pollinating insects, whereas apple (Malus×domestica) uses 2-phenethyl alcohol (6), linalool (7), cis-3-hexenyl acetate (8)

and other compounds for the same purposev

(1) α-thujene (2) myrcene (3) limonene (4) 1,8-cineole

(5) sabinene (6) 2-phenethyl alcohol (7) linalool (8) cis-3-hexenyl acetate

On the other hand, many secondary metabolites in plants play essential roles in

deterring herbivores due to their bitter or pungent taste For example, quinine (9), strychnine (10), brucine (11), emetine (12) and sparteine (13) have bitter taste, whilst capsaicin (14) and piperine (15) are pungent alkaloidsvi

O

N H

O

H

R1

R2

(12) emetine (13) sparteine

(14) capsaicin (15) piperine

However, it should be noted that the taste properties are sometimes not identical for all animals, e.g some smelly compounds such as 2-thioethanol are strong repellents for humans, but food containing such compounds often attracts geesevii Furthermore, some secondary metabolites of plants display extreme toxicity,

which protects the hosts against the herbivores For example, Delphinium consolida

(Ranunculaceae) contains diterpene alkaloids such as delphinine (16), delcosine (17) and delsoline (18) that are very toxic to animals

H

HN O

(16) delphinine: R1 = OCH3, R2 = CH3, R3 = H, R4 = COOCH3, R5 = OCOC6H5, R6 = OH;

(17) delcosine: R1 = OH, R2 = CH2CH3, R3 = OH, R4 = OH, R5 = OH, R6 = H;

(18) delsoline: R1 = OH, R2 = CH2CH3, R3 = OH, R4 = OH, R5 = OCH3, R6 = H

In addition to defending against herbivores, plants also have to compete with surrounding plants to acquire more light, water, space and nutrients, and this competition is termed as allelopathy A range of secondary metabolites including phenolic acids, glucosinolates, terpenoids, flavonoids and alkaloids have allelopathic activity and these metabolites are capable to inhibit the germination or growth of other plants to ensure the advantageous position of the hosts in the competition For

example, vinblastine (19) and yohumbine (20) from Catharanthus roseus

(Apocynaceae) are able to cause temporary abnormalities in cell division of

neighboring Vicia faba viii Other compounds that also exhibit allelopathic activities

include cocaine (21) from Erythroxylum coca, strychnine (10) from Strychnos nux-vomica, physostigmine (22) from Physostigma venenosum ix and caffeine (23),

theobromine (24) and theophylline (25) from Coffea arabica x

(19) vinblastine (20) yohimbine

(21) cocaine (22) physostigmine

(23) caffeine (24) theobromine (25) theophylline

The employment of secondary metabolites by animals is rather frequent as well Some insects are able to excrete bitter tasty alkaloids through a reflex bleeding mechanism when they are attacked by predators For example, coccinellid beetles synthesize many types of defensive alkaloids The most typical alkaloids include

precoccinelline (26) from Coccinella septempunctata, hippodamine (27) from

HO COOCH3H

O N

O

CH 3

H3C

Hippodamia convergens, myrrhine (28) from Myrrha octodecimguttata, propyleine

(29) from Propylaea quatuordecimpunctataxi and chilocorine A (30) from Chilocorus

batrachotoxinin A (31) and batrachotoxin (32)xiii

H

CH3

N

H H

H

CH3

N

H H

H

CH3

N H

H

CH 3

N H

H3C

N

C O

CH3

H3C

On the other hand, many secondary metabolites are widely employed by animals such as ants and insects for communication with other members Chemicals that are released by an organism for transmission of a message to other members of the same species are normally regarded as pheromones The roles are not identical for different pheromones Some serve as alarm pheromones, and some other may serve as

trail pheromones or sex pheromones, etc For example, Myrmicine ants,

Pristomyrmex pungens excrete 6-n-pentyl-2-pyrone (33) from the poison gland to

mark their paths, and some other monoterpenoids released such as α-pinene (34), camphene (35), α-phellandrene (36), and α-terpinene (37) were found to slightly

increase the trail-following response of this species of antsxiv

(33) 6-n-pentyl-2-pyrone (34) α-pinene

(35) camphene (36) α-phellandrene (37) α-terpinene

Sex pheromones are often volatile, and sometimes the pheromones released by the female can be detected by its potential mate from as far away as 10 km Examples

of characteristic sex pheromones include anabaseine (38) and anabasine (39) from

Messor ants, Messor capensis and skatole (40) in Pheidole ants, Pheidole fallax xv

O O

(38) anabaseine (39) anabasine (40) skatole

In addition, secondary metabolites are also used by animals including spiders,

snakes, lizards, and so on, for hunting For example, many neuroactive

acylpolyamines have been discovered in the venom of a few spiders For example,

NSTX-3 (41) and CNS 2013 (42) were discovered in Nephila clavataxvi and

Dolomedes okefinokensisxvii, respectively Most spider acylpolyamines are believed to

function by blocking glutamate-sensitive calcium channels, but the exact mechanism

remains under investigation

(41) NSTX-3

(42) CNS 2013

1.3 Biosynthesis of Natural Products

There are a huge number of natural products in nature, and they differ greatly in

terms of their structures, characteristics and functions in living creatures However,

N

N

N

N H

N H

the number of building blocks required to biosynthesize these metabolites are surprisingly few Acetate pathways, shikimate pathways, mevalonate pathways and deoxyxylulose phosphate pathways cover the biosynthesis of most of the secondarymetabolites we encounter, and the building blocks of these pathways are acetyl coenzyme A (acetyl-CoA), shikimic acid, mevalonic acid and 1-deoxyxylulose 5-phosphate, respectively In other words, most of secondary metabolites including

terpenoids, alkaloids, phenols, etc are all based on these four compounds The acetate

pathway leads to the formation of phenols, prostaglandins, macrolide antibiotics as well as various fatty acids and their derivatives at the primary and secondary metabolism interface The shikimate pathway produces many phenols, cinnamic acid derivatives, lignans and alkaloids, whilst the mevalonate and deoxyxylulose phosphate pathways in combination afford a large number of terpenoid and steroid metabolites1

In most cases, the biological reactions involve coenzyme A esters such as acetyl-CoA，which forms part of a long alkyl chain as in a fatty acid or may be part of

an aromatic system such as phenols Fatty acids are a large group of secondary metabolites that could be found in almost all plants The fatty acid biosynthesis first

of all involves the initial conversion of acetyl-CoA into malonyl-CoA, a reaction involving ATP, CO2 and the coenzyme biotin as the carrier of CO2 The conversion of acetyl-CoA into malonyl-CoA increases the acidity of the α-hydrogens, and therefore provides a better nucleophile for the following Claisen condensation The successive incorporation of malonyl-CoA together with the following reduction will lead to the extension of the chain length by two carbons (C2 unit) for each cycle, until the required chain length is obtained For example, the combination of one acetate starter unit with seven malonates would give the C16 fatty acid, palmitic acid [CH3(CH2)7COOH]

However, in fatty acid biosynthesis, if reduction after each condensation step does not happen, the formed poly-β-keto ester may undergo cyclization to form aromatic compounds For example, one acetate starter group and three malonate chain extension units (C2 unit) could form a C8 polyketo ester, and a following Claisen

reaction and enolization will convert this intermediate into phloracetophenone (43)

Likewise, alternariol (44), a metabolite from the mould Alternaria tenuis, can be

established to be derived from a single C14 polyketide chain which is formed from seven C2 units

(43) phloracetophenone

Scheme 1-1 Biosynthesis of phloracetophenone (43)

(44) alternariol

Scheme 1-2 Biosynthesis of alternariol (44)

In contrast to the extension of C2 unit as stated in the above cases, the terpenoids are derived from C5 isoprene units, but the isoprene units really employed in the

biosynthesis are dimethylallyl diphosphate (DMAPP) (45) and isopenetenyl diphosphate (IPP) (46) The combination of DMAPP and IPP yields geranyl diphosphate (GPP) (47), and further extension of C5 unit leads to the formation of

farnesyl diphosphate (FPP) (48), geranylgeranyl diphosphate (GGPP) (49), etc GPP,

FPP and GGPP are precursors of monoterpenoids (C10), sesquiterpenoids (C15) and

O

O O

7

SCoA

O SCoA

diterpenoids (C20), respectively There are relatively few acylic compounds such as

farnesol (50), geranylgeraniol (51), etc., being derived from only the simple

condensation of these C5 units However, in most cases, these diphosphates precursors will undergo other reactions such as cyclization reactions to finally form the various structurally different terpenoids

1.4 Natural Products in Modern Drug Discovery

The potent applications of natural products in medicine and pharmacy have drawn people’s interest for a long time With the development of isolation and structural

identification techniques, it became possible to recognize which constituent accounts for a specific biological property, and this greatly accelerated the procedure of drug discovery Natural products traditionally have played an important role in drug discovery, but the improvement of alternative drug discovery methods such as rational drug design and combinatorial chemistry to some extent threatened natural products drug discovery However, natural products are still an essential part in drug discovery because they are able to provide a broad array of lead compounds, which is the basis

of modern drug discovery It was reported that at least 21 natural products and natural product-derived drugs have been launched onto the market in the United States, Europe or Japan from 1998 to 2004xviii For example, lobeline (52), an alkaloid from

Lobelia inflate, is the active constituent of anti-smoking products such as Cig-Ridettes

and Citotal, as well as of preparations against bronchial asthma, chronic bronchitis,

cough, vascular disorders and insomnia In addition, byrostatin-1 (53), a natural

product that was discovered from Bugula neritina (Bryozoan), is a protein kinase C

(PKC) and it has been granted Orphan Drug status by the FDA and designated an Orphan Medicinal Product in Europe for oesophageal cancerxix

(52) lobeline (53) byrostatin-1

N

OH O

CH3

O

HO OCH 3

OH

OH

OH H

H

H3CO

O

Chapter 2 Chemistry of Calophyllum wallichianum

2.1 Introduction

Calophyllum is a plant genus of tropical evergreen trees in the family Clusiaceae

with approximately 200 species The distribution of this genus of trees is rather wide, and they are found in Madagascar, eastern Africa, South and Southeast Asia, the Pacific islands, and the West Indies as well as South America Their growth environments vary greatly as well, and a large number of habitats including ridges in mountain forests, coastal swamps, lowland forest and coral cays are suitable for the growth and production of this genus

In terms of the phytochemistry, Calophyllum is a rich source of aromatic

compounds A wide spectrum of xanthone and coumarin derivatives have been reported from this genus, and many coumarin derivatives display anti-HIV activities which imply their potential application for treating HIVxx Research on C inophyllum

afforded a series of inophyllum type pyranocoumarins such as soulattrolide (54), inophyllum G-1 (55), inophyllum G-2 (56) and inophyllum P (57) Similar studies on

C lanigerrum led to the discovery of a series of calanolide type pyranocoumarins

such as calanolide A (58), calanolide E (59), calanolide E2 (60), calanolide F (61), and

calanone (62), and the research of C cordato-oblongum yielded a series of

cordatolide type pyranocoumarins including cordatolide A (63), cordatolide B (64) and oblongulide (65) as well as xanthones such as cordato-oblonguxanthone (66), jacareubin (67) and scriblitifolic acid (68)xxi

O O

OH

O O

OH

O O

OH

O O

OH

O O

O

HO

O O

O

O O

OH

O O

OH

O O

OH

(67) jacareubin (68) scriblitifolic acid

There have been extensive studies of the chemistry of the genus Calophyllum, and natural products from C wallichianum will be introduced in this chapter

2.2 Results and Discussions

Phytochemical studies of the hexane extract of C wallichianum led to the

isolation of seven known compounds, cordatolide A (63), cordatolide B (64), 12-O-methylcordatolide B (69), 12-O-methylcalanolide B (70), trapezifolixanthone

(71), pseudocordatolide C (72), carpachromene (73), and one novel compound 74

The EtOAc extract of C wallichianum afforded one known compound

(+)-epiafzelechin (76) and one novel compound 75

Cordatolide A (63)

Compound 63 was obtained as white needles C20H22O5; m.p 106.0–107.0˚C; the

1H and 13C NMR spectra indicated that this compound had an ester carbonyl group [δC 160.5 (C-2)] and a trisubstituted double bond [δH 5.92 (1H, s, H-3); δC 151.6 (C-4), 110.6 (C-3)] In addition, a 2,2-dimethyl-2H-pyrano-ring was indicated by two doublets at 6.60 ppm [1H, d, J = 10.1 Hz; δC 127.2 (C-8)] and 5.53 ppm [1H, d, J = 9.5 Hz; δC 116.4 (C-7)] and two singlets at 1.50 ppm [3H, s; δC 28.0 (C-14)] and 1.49 ppm [3H, s; δC 27.4 (C-15)] In addition, the doublet-quartet at 3.92 ppm [1H, dq, J = 8.8, 6.3 Hz, H-10; δC 67.1 (C-10)] and the doublet at 4.70 ppm [1H, d, J = 7.5 Hz,

O O

O OH

H-12; δC 74.0 (C-12] implied the CH3-CH-CH(CH3)-CH-OH fragment Meanwhile,

as shown in Fig 2-1, the coupling constant of 8.8 Hz (JH-10,11) and 7.5 Hz (JH-11,12) implied the di-axial relationships between H-10 and H-11 and between H-11 and H-12

in ring D This compound was identified as cordatolide A by comparison of the NMR data with those literature valuesxxii

(63) cordatolide A Fig 2-1 Relative stereochemistry of ring D fragment of 63

Cordatolide B (64)

Cordatolide B (64) was obtained as white needles; C20H22O5; m.p

217.0-218.0˚C; Similar to compound 63, the 1H and 13C NMR spectra of compound

64 also indicated an ester carbonyl group [δC 161.0 (C-2)], a trisubstituted double bond [δH 5.92 (1H, s, H-3); δC 151.8 (C-4), 110.8 (C-3)], a 2,2-dimethyl-2H-pyrano-ring [δH 6.62 (1H, d, J = 10.1 Hz), 5.52 (1H, d, J = 10.1 Hz), 1.48 (3H, s); 1.47 (3H, s); δC 127.0 (C-8), 116.5 (C-7), 27.8 (C-14), 27.7 (C-15)] and

an oxygen bonded tertiary carbon [δH 4.25 (1H, dq, J = 10.1, 5.7 Hz, H-10; δC 73.0

(C-10)] In addition, for the same reason, the coupling constant of 10.8 Hz (JH-10,11) also implied the di-axial relationship between H-10 and H-11 However, H-12 afforded a doublet at 4.95 ppm [1H, d, J = 3.2 Hz, H-12; δC 61.7 (C-12], whose coupling constant indicated the axial-equatorial relationship between H-11 and H-12, and H-12 therefore must be at equatorial position since H-11 was at axial position

Thus, compound 64 was identified as a diastereoisomer of compound 63 Careful

comparison of the NMR data with literature19 revealed that this compound was

O O

OH

2

4 4a 4b

13

14 15

δC 126.8 (C-8), 116.5 (C-7), δC 27.8 (C-14), δC 27.7 (C-15)] Furthermore, the doublet-quartet at 4.30 ppm [1H, dq, J = 10.8, 6.3 Hz, H-10; δC 73.4 (C-10)] and the doublet at 4.55 ppm [1H, d, J = 2.8 Hz, H-12; δC 70.7 (C-12)] also revealed the O-CH(CH3)-CH(CH3)-CH- fragment, and the relative structure of H10, H-11 and

H-12 could also be established to be identical to that of cordatolide B (65) based on

the coupling constants However, the singlet at 3.58 ppm [δH 3.58 (3H, s, H-18); δC

59.2 (C-18)] clearly indicated a OCH3 group, which could only be connected to C-12 Thus, the hydroxyl group at C-12 of cordatolide B was replaced by OCH3 group, and this compound was therefore established to be12-O-methylcordatolide B, which could

be further confirmed by the comparison of the NMR data with literature valuesxxiii

4b

6 8 8a 8b

10 12 12a 12b

13

14 15

12-O-methylcalanolide B (70)

12-O-methylcalanolide B (70) was obtained as yellow solid C23H28O5; the 1H

NMR spectrum was very similar to that of 12-O-methylcordatolide B (69) as

elucidated above, and it also revealed a 2,2-dimethyl-2H-pyrano-ring [δH 6.62 (d, J = 10.1 Hz, H-8), 5.51 (d, J = 10.1 Hz, H-7), δH 1.48 (3H, s, H-16), 1.47 (3H, s, H-17)], a tri-substituted double bond [δH 5.94 (s, H-3)], a OCH3 group [δH 3.60 (s, H-20)] and two oxygen bonded tertiary carbons [δH 4.56 (d, J = 2.5 Hz, H-12), 4.27 (dq, J = 10.7, 6.3 Hz, H-10)] The splitting pattern of H-10 and H-12 again revealed the di-axial relationship between H-10 and H-11 and axial-equatorial relationship between H-11 and H-12 and therefore led to the relative structure of this compound However,

different from 12-O-methylcordatolide B (69), a doublet at 2.56 ppm (3H, d, J = 0.7 Hz) was absent, and at the same time, compound 70 afforded an extra triplet at 1.02

ppm (3H, t, J = 7.5) and two multiplets at 2.89 ppm (2H, m) and 1.65 ppm (2H, m) This information clearly indicated that the methyl group at C-4 was replaced by a

n-propyl group compared with 69 Hence, compound 70 was determined to be

12-O-methylcalanolide B, and this was further confirmed by the NMR data comparison with literature valuesxxiv This compound was also found in C lanigerum

(70) 12-O-methylcalanolide B (71) trapezifolixanthone

Trapezifolixanthone (71)

This compound was obtained as yellow needle; C23H22O5; m.p 171.0-172.0˚C; the

1H and 13C NMR spectra showed a chelated hydroxyl group [δH 13.1 (s, OH-1)], a cis

O

OH

O OH

O

1 2 3 4 4a 4b 5 6

7 8 8a 9 9a 1'

2' 3' 4' 5' 1''

3'' 4'' 5''

O O

OCH3

1

3 4 4a 4b 5 6 8 8a 8b

10 12

12a 12b

13

15 16

17

18

19

20

double bond [δH 6.75 (d, J = 9.5 Hz), 5.61 (d, J = 9.5 Hz); δC 127.5 (C-2’), 115.7 (C-1’)], a 1,2,3-trisubstituted aromatic ring [δH 7.75 (dd, J = 7.9, 1.9 Hz, H-8), 7.30 (dd, J = 8.2, 1.9 Hz, H-6), 7.24 (t, J = 8.2 Hz, H-7); δC 124.0 (C-7), 119.7 (C-6), 116.8 (C-8)], a one carbonyl group [δC 181.0 (C-9)] In addition, two doublets at 6.75 ppm [1H, d, J = 9.5 Hz; δC 115.7 (C-1’)] and 5.61 [1H, d, J = 9.5 Hz; δC 127.5 (C-2’)] as well as a singlet at 1.49 ppm [6H, s; δC 28.3 (C-4’, C-5’)] implied a 2,2-dimethyl-2H-pyrano-ring, and the presence of a 2-methylbut-2-enyl group was indicated by two singlets at 1.87 ppm [3H, s; δC 17.9 (C-5’’)] and 1.73 ppm [3H, s; δC

25.6 (C-4’’)] and a broad triplet at 5.23 ppm [1H, br t, J = 6.9 Hz, H-2’’; δC 122.7

(C-2’’)] Comparison of the NMR data with reference showed that compound 71 was

trapezifolixanthone, a xanthone found also in C trapezifoliumxxv and Tovomita brevistamineaxxvi

Pseudocordatolide C (72)

This compound was obtained as yellow needles C20H22O5; The 1H and 13C NMR

spectra showed the structural similarity with cordatolide B (69) The groups as shown

below were indicated by the NMR data: a carbonyl group [δC 160.4 (C-2)], a trisubstituted double bond [δH 5.84 (1H, s); δC 111.1 (C-3), 154.7 (C-4)] and a 2,2-dimethyl-2H-pyrano-ring [δH 6.76 (1H, d, J = 10.1 Hz), 5.52 (1H, d, J = 10.1 Hz), 1.46 (3H, s), 1.41 (3H, s); δC 126.7 (C-11), 115.4 (C-12), 28.1 (C-16), 27.9 (C-17)] In addition, there were also two methyl groups [δH 1.34 (3H, d, J = 6.6 Hz), 1.02 (3H, d,

J = 7.0 Hz); δC 24.3 (C-14), 16.4 (C-15)] next to tertiary carbon [δC 75.3 (C-6), 34.8

(C-7)] based on their doublet splitting patterns However, compared with 69, the

alkene protons [δH 6.76 (1H, d, J = 10.1 Hz), 5.52 (1H, d, J = 9.8 Hz)] of 72 were

clearly more deshielded, and this implies the nearer position of this cis double bond

This compound therefore should be a member of pseudocordatolide class

With regard to the relative stereochemistry of H-6, H-7 and H-8, the coupling

constant of 2.5 Hz of JH-6,7 implied that at least one of H-6 and H-7 was at equatorial

position., and the coupling constant between H-7 and H-8 (JH-7,8 = 5.9 Hz) also implied one of them was at equatorial position Unfortunately, this information was

still insufficient for the establishment of the relative structure Comparison of the NMR data with literature values xxvii showed that compound 72 was

pseodocordatolide C, a known compound isolated also from Calophyllum lanigerum

(72) pseudocordatolide C

Carpachromene (73)

Carpachromene (73) was obtained as light yellow crystals C20H16O5; m.p 239.0-240.0˚C; the 1H NMR spectrum showed a chelated hydroxyl proton [δH 13.1 (1H, s)] and a 1,4-disubstrituted aromatic ring [δH 7.78 (2H, d, J = 8.2 Hz), 6.96 (2H,

d, J = 7.9 Hz)] In addition, two doublets at 6.72 ppm (1H, d, J = 10.1 Hz) and 5.61 ppm (1H, d, J = 10.1 Hz) together with the singlet at 1.47 ppm (6H, s) clearly implied

a 2,2-dimethyl-2H-pyrano-ring Comparison of the NMR data with those literature values revealed that this compound was carpachromene, a flavone observed also in

Flindersia leavicarpaxxviii,xxix

O O

HO

2

4 4a 4b

6 8 8a 8b

10 12

12a 12b 1

13

14 15

16 17

spectra revealed one ketone carbonyl group [δC 191.3 (C-8)], one ester carbonyl group

[δC 160.8 (C-2)], one trisubstituted double bond [δH 5.98 (1H, d, J = 1.3 Hz); δC 111.9

(C-3), 159.8 (C-4)] In addition, similar to other coumarin derivatives as identified

above, a 2,2-dimethyl-2H-pyrano ring was still present according to the two doublets

[δH 6.80 (1H, d, J = 10.1 Hz), 5.64 (1H, d, J = 10.1 Hz); δC 128.1 (C-11), 115.0 (C-12)]

and two singlets [δH 1.55 (3H, s), 1.50 (3H, s); δC 28.3 (C-18), 28.1 (C-19)] Two

methyl groups [δH 1.56 (3H, d, J = 6.9 Hz), 1.20 (3H, d, J = 6.9 Hz); δC 19.6 (C-14),

9.8 (C-15)] that were next to tertiary carbon could be recognized as well

Table 2-1: 1H, 13C-NMR Data and HMBC Correlations of 74

Similar to pseudocordatolide C (72) as described above, the deshielded alkene

proton at 6.80 ppm again indicated that this compound was also a pseudocordatolide

compound Careful comparison of the NMR data between compound 74 and

pseudocordatolide C revealed their close structural similarity, except the hydroxyl group had converted into carbonyl group according to the resonance at 191.3 ppm

HMBC correlations of this compound as shown in Table 2-1 and Fig 2-2 confirmed

this hypothesis and led to the establishment of the skeleton of the structure:

74 Fig 2-2: Key HMBC correlations of compound 74

The trans- relationship of CH3-14 and CH3-15 was based on the coupling

constant of JH-6, 7 The coupling constant of 11.9 Hz revealed that H-6 and H-7 must

have di-axial relationship as shown in cordatolide B (64), hence these two methyl

groups must be both at equatorial position and therefore had trans- configuration

Compound 75

This compound was obtained as white crystals C20H22O5; the 1H and 13C NMR

spectra were very similar to that of pseudocordatolide C (72) as described above, the characteristic groups belonging to courmarin datives were still present in 75, such as

an ester carbonyl group [δC 160.7 (C-2)], a trisubstituted double bond [δH 5.93 (1H, d,

J = 1.2 Hz); δC111.2 (C-3), 155.2 (C-4)] and a 2,2-dimethyl-2H-pyrano-ring [δH 6.83 (1H, d, J = 10.1 Hz), 5.57 (1H, d, J = 10.1 Hz), 1.51 (3H, s), 1.46 (3H, s); δC 127.1 (C-11), 115.6 (C-12), 28.4 (C-16), 28.0 (C-17)] Similarly, the chemical shift of the characteristic H-12 at 6.83 ppm again implied that this compound was a member of pseudocordatolide class as well There were also another two methyl groups [δH 1.43 (3H, d, J = 6.9 Hz), 0.82 (3H, d, J = 6.9 Hz); δC 24.4 (C-14), 17.5 (C-15)] next to tertiary carbon [δH 4.41 (1H, qd, J = 6.9, 1.9 Hz), 1.99 (1H, qt, J = 6.9, 1.9 Hz); δC

O

O O

O

O

2

4 4a 4b

6 8

8b 8a

10

12 12a 12b

13

14 15

16

17

O O

O

71.5 (C-6), 36.4 (C-7)] based on their doublet splitting patterns Despite of the high

similarity of the NMR spectrum between compound 75 and 72, their difference of the

resonances of H-6, H-7 and H-8 was also clear Compare with the 2.5 Hz of JH-6,7 and

5.9 Hz of JH-7,8 for 72, the coupling constants both became to 1.9 Hz for 75 Although

it was still insufficient to establish the relative stereochemistry of this compound from

the coupling constants, it definitely indicated that it had different structure with 72

75 Fig 2-3: ORTEP diagram of compound 75

To establish its stereochemistry, crystallization of compound 69 was carried out and the following X-ray analysis led to the diagram as shown in Fig 2-3, and the

structure of this compound therefore was established

It could be observed that the structure was almost identical to

pseudocordatolide C (72) except the stereochemistry of C-6, C-7 and C-8, and this

structure is novel Attempts of assignment of the 13C NMR peaks to the structure have been made by comparing with the NMR data of pseudocordatolide C, but it should be noted that this assignment definitely may not be completely correct due to the lack of 2D NMR data

(+)-Epiafzelechin (76)

This compound was obtained as yellow solid C15H14O5; m/z 274.0; [α]D = -27.3 (c=6.73 mg/mL, Acetone); 1H NMR spectra indicated a 1,4-disubstituted aromatic

O O

O

HO

O

3 1

4a 4b

6 8

8a

8b

10

12 12a 12b

13

14 15

16

17

ring [δH 7.35 (2H, d, J = 8.8 Hz), 6.81 (2H, dd, J = 8.8, 1.9 Hz)] and two meta-

aromatic protons [δH 6.02 (1H, d, J = 2.5 Hz), 5.92 (1H, d, J = 1.9 Hz)] In addition, the 13C NMR spectra showed that there were two tertiary carbons [δC 78.3 (C-2), 65.6 (C-3)] that were next to oxygen atoms and a secondary carbon [δH 2.86 (1H, dd, J = 16.4, 4.4 Hz), 2.75 (1H, dd, J = 17.0, 3.3 Hz); δC 28.3 (C-4)] that was next to a tertiary carbon (C-3) Furthermore, the two proton of the methylene group coupled to H-3 with the coupling constants of 4.4 Hz and 3.3 Hz, these small values implied that neither of them had di-axial relationship with H-3 Hence, the equatorial position of H-3 could be deduced Comparison of the NMR data with literature revealed that this

compound was (+)-epiafzelechin, a flavan derivative that also present in Cryptolestes pusillusxxx

C18 (25-40 μm, Merck) and DIOL (Merck, Lichroprep 40-63 μm) For Gel Permeation Column Chromatography (GPC), Sephadex LH-20 (MeOH:CH2Cl2 = 1:1) was

O

OH

OH OH

HO

2 1

4 4a 5

7 8 8a

1' 3' 5' 6'

utilized High Performance Liquid Chromatography (HPLC) was performed on a Shimadzu LC-8A system with RI detection or UV detection HPLC columns: Lichrosorb 10 DIOL, 250 × 4.60 mm; Luna 5μ C18, 250 × 4.60 mm and Phenomenex partisil 10 silica, 250 × 4.60 mm

Spectroscopy

Optical rotations were recorded on Perkin-Elmer 241 SCO 0937 Digital Polarimeter UV spectra were recorded on a SHIMADZU 1601PC UV-Visible spectrophotometer IR spectra were recorded on BIO-RAD Excalibur Series FTS

3000 Electron impact (EI) mass spectra were measured with a VG Micromass 7035 instrument NMR spectra were measured with Bruker DPX 300 [300 MHz (1H) and

75 MHz (13C)] or Bruker AMX 500 [500 MHz (1H) and 125 MHz (13C)] using CDCl3

as solvent unless otherwise stated Muptiplicities were determined by the DEPT pulse sequence or deduced from 2D HMQC Coupling constants (J) were measured in Hertz (Hz) X-Ray Diffraction (XRD) data for single crystal structures was recorded on a Bruker AXS SMART APEX CCD X-Ray Diffractometer Sadabs (Sheldrick 2001) was employed for absorption corrections, λ = 0.71073 Å Tables of atomic co-ordinates, bonds lengths and angles, anisotropic displacement parameters and hydrogen atom co-ordinates are deposited with the Cambridge Crystallographic Data Center

Melting Points

Melting points are recorded on BÜCHI B-540 instruments

2.7 g of hexane extract of Calophyllum wallichianum leaf was roughly separated

by gradient flash column chromatography (CC) (EtOAc:Hexane = 20:80, 50:50, 80:20, 100:0) to give two fractions

Fraction 1

Sephadex column procedure was employed at first to get rid of most of the fatty acid and chlorophyll, and three fractions were collected Fraction 1.1 was mainly fat based on the 1H NMR spectrum and therefore was not further studied Fraction 1.2 was separated by flash CC (EtOAc:Hexane = 12:88) and the following purification by

reversed phase HPLC (C18; MeOH:H2O = 85:15) yielded compound 70 (0.8 mg) and

69 (3.5 mg) Fraction 1.3 was separated by flash CC (EtOAc:Hexane = 10:90), and

the obtained fraction was purified by another flash CC (Acetone:Hexane = 20:80) to

afford compound 71 (5.3 mg)

Fraction 2

Sephadex column procedure was employed as well to remove most of the fat and chlorophyll, and the defatted mixture was applied to flash CC (Aectone:Hexane = 25:75) The obtained fraction 2.2 was separated by flash CC (EtOAc:Hexane = 20:80)

to afford compound 72 (180.2 mg) and another two mixtures, one was further purified

by flash CC (EtOAc:Hexane = 25:75) to yield compound 74 (0.7 mg) whilst the other

was further purified by reversed HPLC (C18; MeOH:H2O = 78:22) to yield 63 (5.3 mg) and 64 (4.3 mg) At the same time, the obtained fraction 2.3 was purified by normal phase HPLC (DIOL, EtOAc:Hexane = 30:70) to give compound73 (2.6 mg)

In addition, the ethyl acetate extract of C.wallichianum was investigated as well

3.0 g of crude extracts were roughly separated by gradient flash CC (Acetone:Hexane

= 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0), and the obtained fraction was defatted by a Sephadex column procedure to yield two fractions Fraction 1 was

purified by normal phase flash CC (EtOAc:Hexane = 25:75) to afford compound 75

(15.1 mg) Fraction 2 was purified by normal phase flash CC (Acetone:Hexane =

55:45) to afford compound 76 (76.8 mg)

Cordatolide A (63)

C20H22O5; m.p 106.0–107.0˚C; 1H NMR (CDCl3) δ 6.60 (1H, d, J = 10.1 Hz), 5.92 (1H, s), 5.53 (1H, d, J = 9.5 Hz), 4.70 (1H, d, J = 7.5 Hz), 3.92 (1H, dq, J = 8.8, 6.3 Hz), 2.57 (3H, s), 1.50 (3H, s), 1.49 (3H, s), 1.45 (3H, d, J = 5.1), 1.13 (3H, d, J = 6.9 Hz); 13C NMR (CDCl3) δ 160.5 (C-2), 155.2 (C-4b), 154.0 (C-12b), 153.2 (C-8b), 151.6 (C-4), 127.2 (C-8), 116.4 (C-7), 110.6 (C-3), 106.4 (C-8a), 106.3 (C-4a), 104.7 (C-12a), 77.6 (C-6), 74.0 (C-12), 67.1 (C-10), 40.4 (C-11), 28.0 (C-14), 27.4 (C-15), 24.5 (C-17), 18.9 (C-16), 15.1 (C-13)

Cordatolide B (64)

C20H22O5; m.p 217.0-218.0˚C; 1H NMR (CDCl3) δ 6.62 (1H, d, J = 10.1 Hz), 5.92 (1H, s), 5.52 (1H, d, J = 10.1 Hz), 4.95 (1H, d, J = 3.2 Hz), 4.25 (1H, dq, J = 10.1, 5.7 Hz), 2.56 (3H, s), 1.48 (3H, s), 1.47 (3H, s), 1.42 (3H, d, J = 6.3 Hz), 1.13 (3H, d, J = 6.9 Hz); 13C NMR (CDCl3) δ 161.0 (C-2), 155.0 (C-4b), 153.4 (C-12b), 153.2 (C-8b), 151.8 (C-4), 127.0 (C-8), 116.5 (C-7), 110.8 (C-3), 106.3 (C-8a), 106.2 (C-4a), 104.9 (C-12a), 77.6 (C-6), 73.0 (C-12), 61.7 (C-10), 38.2 (C-11), 27.8 (C-14), 27.7 (C-15), 24.4 (C-17), 18.8 (C-16), 12.5 (C-13)

12-O-methylcordatolide B (69)

C21H24O5; m.p 112.0-113.0˚C; 1H NMR (CDCl3) δ 6.62 (1H, d, J = 10.1 Hz, H-3), 5.92 (1H, d, J = 0.7 Hz), 5.51 (1H, d, J = 10.1 Hz), 4.55 (1H, d, J = 2.8 Hz), 4.30 (1H,

dq, J = 10.8, 6.3 Hz), 3.58 (3H, s), 2.56 (3H, d, J = 0.7 Hz), 1.71 (1H, m), 1.48 (3H, s), 1.47 (3H, s), 1.40 (3H, d, J = 6.3 Hz), 1.14 (3H, d, J = 7.0 Hz); 13C NMR (CDCl3) δ 160.7 (C-2), 154.7 (C-4b), 153.4 (C-12b), 153.3 (C-8b), 151.9 (C-4), 126.8 (C-8), 116.5 (C-7), 110.9 (C-3), 106.2 (C-8a), 104.5 (C-4a), 103.8 (C-12a), 77.5 (C-6), 73.4 (C-10), 70.7 (C-12), 59.2 (C-18), 38.6 (C-11), 27.8 (C-14), 27.7 (C-15), 24.4 (C-17), 19.1 (C-16), 13.2 (C-13)

12-O-methylcalanolide B (70)

C23H28O5; 1H NMR (CDCl3) δ 6.62 (1H, d, J = 10.1 Hz, H-8), 5.94 (1H, s, H-3), 5.51 (1H, d, J = 10.1 Hz, H-7), 4.56 (1H, d, J = 2.5 Hz, H-12), 4.27 (1H, dq, J = 10.7, 6.3

Hz, H-10), 3.60 (3H, s, OCH3), 2.89 (2H, m, H-13), 1.65 (2H, m, H-14), 1.48 (3H, s, H-16), 1.47 (3H, s, H-17), 1.40 (3H, d, J = 6.3 Hz, H-18), 1.15 (3H, d, J = 6.9 Hz, H-19), 1.02 (3H, t, J = 7.5 Hz, H-15); 13C NMR (CDCl3) δ 160.9 (C-2), 158.5 (C-4), 153.8 (C-8b), 153.1 (C-12b), 151.4 (C-4b), 126.6 (C-7), 116.6 (C-8), 110.3 (C-3), 106.0 (C-12a), 104.7 (C-8a), 103.2 (C-4a), 77.6 (C-6), 73.4 (C-10), 70.8 (C-12), 59.4 (C-20), 38.7 (C-11), 38.7 (C-13), 27.8 (C-16), 27.9 (C-17), 23.3 (C-14), 19.2 (C-18), 14.1 (C-15), 13.3 (C-19)

Trapezifolixanthone (71)

C23H22O5; m.p 171.0-172.0˚C; 1H NMR (CDCl3) δ 13.1 (1H, s), 7.75 (1H, dd, J = 7.9, 1.9 Hz), 7.30 (1H, dd, J = 8.2, 1.9 Hz), 7.24 (1H, t, J = 8.2 Hz), 6.75 (1H, d, J = 9.5 Hz), 5.61 (1H, d, J = 9.5 Hz), 5.23 (1H, br t, J = 6.9 Hz), 3.50 (2H, d, J = 6.9 Hz), 2.17 (1H, s), 1.87 (3H, s), 1.73 (3H, s), 1.59 (3H, s), 1.49 (6H, s); 13C NMR (CDCl3) δ 181.0 (C-9), 158.3 (C-3), 156.1 (C-1), 153.7 (C-4a), 144.4 (C-5), 144.2 (C-4b), 131.6 (C-3’’), 127.5 (C-2’), 124.0 (C-7), 122.7 (C-2’’), 120.9 (C-8a), 119.7 (C-6), 116.8 (C-8), 115.7 (C-1’), 107.0 (C-4), 104.8 (C-2), 103.4 (C-9a), 78.3 (C-3’), 28.3 (C-4’), 28.3 (C-5’), 25.6 (C-4’’), 21.7 (C-1’’), 17.9 (C-5’’)

Pseudocordatolide C (72)

C20H22O5; 1H NMR (CDCl3) δ 6.76 (1H, d, J = 10.1 Hz), 5.84 (1H, s), 5.52 (1H, d, J = 9.8 Hz), 4.96 (1H, d, J = 5.9 Hz), 4.28 (1H, dq, J = 2.5, 6.6 Hz), 2.45 (3H, s), 1.46 (3H, s), 1.41 (3H, s), 1.34 (1H, d, J = 6.6 Hz), 1.02 (1H, d, J = 7.0 Hz); 13C NMR (CDCl3)

δ 160.5 (C-2), 154.8 (C-4), 154.6 (C-8b), 152.8 (C-4b), 150.1 (C-12b), 126.8 (C-11), 115.5 (C-12), 111.2 (C-12a), 109.1 (C-3), 104.0 (C-8a), 102.4 (C-4a), 78.7 (C-10), 75.4 (C-6), 64.4 (C-8), 34.9 (C-7), 28.2 (C-16), 28.0 (C-17), 24.4 (C-13), 16.5 (C-14), 7.6 (C-15)

Carpachromene 73

C20H16O5; m.p 239.0-240.0˚C; 1H NMR (CDCl3) δ 13.1 (1H, s), 7.78 (2H, d, J = 8.2 Hz), 6.96 (2H, d, J = 7.9 Hz), 6.72 (1H, d, J = 10.1 Hz), 6.54 (1H, s), 6.40 (1H, s), 5.61 (1H, d, J = 10.1 Hz), 1.47 (6H, s)

Compound 74

C20H20O5; 1H NMR (CDCl3) δ 6.80 (1H, d, J = 10.1 Hz), 5.98 (1H, d, J = 1.3 Hz), 5.64 (1H, d, J = 10.1 Hz), 4.28 (1H, qd, J = 6.3, 11.9 Hz), 2.56 (3H, s), 2.53 (1H, m), 1.56 (3H, d, J = 6.9 Hz), 1.55 (3H, s), 1.50 (3H, s), 1.20 (3H, d, J = 6.9 Hz); 13C NMR (CDCl3) δ 191.3 (C-8), 160.8 (C-2), 159.8 (C-8), 154.2 (C-4b), 154.2 (C-12b), 152.1 (C-4), 128.1 (C-11), 115.0 (C-12), 111.9 (C-3), 107.3 (C-8a), 103.8 (C-12a), 103.2

(C-4a), 79.9 (C-6), 79.0 (C-10), 47.1 (C-7), 28.3 (C-16), 28.1 (17), 24.6 (C-13), 19.6 (C-14), 9.8 (C-15)

Compound 75

C20H22O5; 1H NMR (CDCl3) δ 6.83 (1H, d, J = 10.1 Hz), 5.93 (1H, d, J = 1.2 Hz), 5.57 (1H, d, J = 10.1 Hz), 4.62 (1H, d, J = 1.8 Hz), 4.41 (1H, qd, J = 6.9, 1.9 Hz), 2.54 (3H, s), 1.99 (1H, qt, J = 6.9, 1.9 Hz), 1.51 (3H, s), 1.46 (3H, s), 1.43 (3H, d, J = 6.9 Hz), 0.82 (3H, d, J = 6.9 Hz); 1H NMR (Acetone-d6) δ 6.73 (1H, d, J = 9.5 Hz), 5.89 (1H, s), 5.72 (1H, d, J = 10.1 Hz), 4.63 (1H, br s), 4.56 (1H, qd, J = 6.3, 1.9 Hz), 2.56 (3H, d, J = 1.2 Hz), 1.49 (3H, s), 1.45 (3H, s), 1.44 (3H, d, J = 6.3 Hz), 0.76 (3H, d, J

= 6.9 Hz); 13C NMR (CDCl3) δ 160.7 (C-2), 155.2 (C-4), 155.0 (C-8b), 153.9 (C-4b), 150.6 (C-12b), 127.1 (C-11), 115.6 (C-12), 111.2 (C-3), 107.8* (C-8a), 103.8* (C-4a), 102.5* (C-12a), 78.4 (C-10), 71.5 (C-6), 65.4 (C-8), 36.4 (C-7), 28.4 (C-16), 28.0 (C-17), 24.4 (C-14), 17.5 (C-15), 9.1 (C-13) (* means the assignment of these signals may be interchangeable)

(+)-Epiafzelechin (76)

C15H14O5; m/z 274.0; [α]D = -27.3 (c=6.73 mg/mL, Acetone); 1H NMR (Acetone-d6) δ 8.36 (1H, s), 8.21 (1H, s), 8.05 (1H, s), 7.29 (2H, d, J = 8.8 Hz), 6.78 (2H, dd, J = 8.8, 1.9 Hz), 5.99 (1H, d, J = 2.3 Hz), 5.89 (1H, d, J = 2.3 Hz), 4.87 (1H, s), 4.21 (1H, br t,

J = 4.3 Hz), 2.86 (1H, dd, J = 16.4, 4.4 Hz), 2.62 (1H, dd, J = 16.6, 3.6 Hz); 13C NMR (Acetone-d6) δ 156.4, 156.3 and 156.2 (C-5, C-7 and C-4’ interchangeable assignments), 155.8 (C-8a), 130.0 (C-1’), 128.0 (C-2’), 128.0 (C-6’), 114.4 (C-3’), 114.4 (C-5’), 98.7 (C-4a) 95.2 (C-6), 94.4 (C-8), 78.3 (C-2), 65.6 (C-3), 28.3 (C-4)

Crystallographic data for compound 75

C20H22O5; M.W 342.38; Monoclinic; Space group P2(1); a = 7.0289(9) Å, b = 24.394(3) Å, c = 10.8950(13) Å; α = 90°, β = 103.8°, γ = 90°; V = 1814.3(4) Å3; Z = 4; Density (calculated) = 1.253 Mg/m3; F(000) = 728; μ = 0.090 mm-1; Data were collected using a crystal size ca 0.70 × 0.30 × 0.28 mm3