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Abstract

Identification of Secondary Metabolites from Marine

Microorganisms

Jihye Lee

School of Earth and Environmental Sciences

Marine Natural Products Chemistry Major

The Graduate School

Seoul National University

The marine environment has become a rich source of secondary

metabolites which are structurally unique and biologically active. Marine

microorganisms living in the diverse environment have been considered as

new sources of pharmaceutical leads and actual producers of bioactive

natural products.

In our search for new bioactive compounds from marine actinobacteria, we

obtained novel compounds, anmindenols A-B, marinopyrones A-D and

11A020-3A from the actinomycetes. Their structures were elucidated by the

interpretation of 1D, 2D NMR and MS spectroscopic data. Anmindenols A-

B and marinopyrone D were examined for their inhibition of nitric oxide

production in LPS-stimulated RAW 264.7 macrophage cells and exhibited

weak inhibitory activity on nitric oxide production with the IC50 value of 23

μM, 19 μM and 13 μM, respectively. 11A020-3A showed antibacterial

activity with IC50 value of 0.1 μM, 64 μM, 8 μM and 64 μM against Bacillus

subtilis, Kocuria rhizophila, Staphylococcus aureus and Escherichia coli,

respectively.

In the course of our research for the chemical investigation of secondary

metabolites from marine fungi, F8015-2B-2B, 2C, 2E and 2I were obtained

from the fungus Mycosphaerella nawae. The structures of the compounds

were determined by comparison of NMR and MS data.

keyword : Marine natural products, Marine microorganisms, Secondary

metabolites, Actinomycetes, Fungi, Spectroscopy, NMR

Student Number : 2013-30105

iii

Table of Contents

Chapter 1. Introduction of Marine Natural Products

1.1 Marine Natural Products

1.1.1 Marine Natural Products in General ............................................... 1

1.1.2 Drug Development from Marine Natural Products ...................... 9

1.2 Marine Microorganisms as a Source for Natural Products

1.2.1 Marine Microorganisms ............................................................. 12

1.2.2 Bioactive Metabolites from Marine Bacteria ............................. 14

1.2.3 Bioactive Metabolites from Marine Fungi ................................. 18

Chapter 2. Marinopyrones A-D, -Pyrones from Marine-

Derived Actinomycetes of the Family Nocardiopsaceae

2.1 Introduction ..................................................................................... 26

2.2 Results and Discussion

2.2.1 Structural Elucidation of Marinopyrones

2.2.1.1 Marinopyrone A (1) ............................................................... 29

2.2.1.2 Marinopyrone B (2) ............................................................... 33

iv

2.2.1.3 Marinopyrone C (3) ............................................................... 35

2.2.1.4 Marinopyrone D (4) ............................................................... 38

2.2.2 Biological Activities

2.2.2.1 Antibacterial Activity ............................................................. 41

2.2.2.2 Cytotoxicity ........................................................................... 41

2.2.2.3 Anti-Inflammatory Activity ................................................... 41

2.3 Experimental Section

2.3.1 Instruments and Data Collection ................................................ 44

2.3.2 Bacterial Material ....................................................................... 45

2.3.3 Cultivation, Extraction and Isolation .......................................... 46

2.3.4 Determination of the Absolute Configuration for compound 4 .. 47

2.3.5 Bioassays

2.3.5.1 MIC Assay ............................................................................. 48

2.3.5.2 MTT Assay ............................................................................ 49

2.3.5.3 Nitric Oxide Assay ................................................................ 49

Chapter 3. Anmindenols A and B, sesquiterpenoids from a

Marine-derived Streptomyces sp.

3.1 Introduction ..................................................................................... 51

v


3.2.1 Structural Elucidation of Anmindenols

3.2.1.1 Anmindenol A (5) .................................................................. 57

3.2.1.2 Anmindenol B (6) .................................................................. 60



3.2.2.2 Cytotoxicity ........................................................................... 63

3.2.2.3 Anti-Inflammatory Activity ................................................... 63





3.3.4 Electronic Circular Dichroism of a Dimolybdenum Complex ... 68

3.3.5 Bioassays

3.3.5.1 MIC Assay ............................................................................. 69

3.3.5.2 MTT Assay ............................................................................ 69

3.3.5.3 Nitric Oxide Assay ................................................................ 69

vi

Chapter 4. 11A020-3A from mudflat derived

Actinoalloteichus Hymeniacidonis sp.

4.1 Introduction ..................................................................................... 70


4.2.1 Structural Elucidation of 11A020-3A

4.2.1.1 11A020-3A (7) ....................................................................... 73







4.3.4 Bioassays

4.3.4.1 MIC assays ............................................................................ 80

Chapter 5. Usnic Acid Derivatives from Mycosphaerella

nawae

5.1 Introduction ..................................................................................... 81


vii

5.2.1 Structural Elucidation of Usnic Acid Derivatives

5.2.1.1 F8015-2B-2E (8) ................................................................... 83

5.2.1.2 F8015-2B-2I (9) ..................................................................... 86

5.2.1.3 F8015-2B-2B (10) ................................................................. 88

5.2.1.4 F8015-2B-2C (11) .................................................................. 90



5.3.2 Fungal Material .......................................................................... 92


Appendix A .............................................................................................. 95

Appendix B ............................................................................................ 120

Appendix C ............................................................................................ 135

Appendix D ........................................................................................... 142

한글초록 ............................................................................................... 161

List of Figures

Figure 1.1 Spongouridine (1.1), spongothymidine (1.2), ara-A (1.3) and ara-

C (1.4) .......................................................................................................... 2

Figure 1.2 Variation in number of new marine natural products

for 1985-2012 ............................................................................................ 3

Figure 1.3 The quantity and proportion of bioactive compounds in each

category of chemical compounds .............................................................. 3

Figure 1.4 Ziconotide ................................................................................ 4

Figure 1.5 Number and proportion of bioactive/non-bioactive compounds

from marine organisms (*PHVD: Prevention of heart and vascular disease,

**PN/NT: Protection of neurons/neurotoxicity) ......................................... 6

Figure 1.6 Halichondrin B (1.5) and Eribulin mesylate (1.6) ................... 8

Figure 1.7 Chemical structure of marine drugs on the market divided by

therapeutic area ........................................................................................ 10

Figure 1.8 Number of bioactive compounds isolated from the three groups

of marine organisms ................................................................................ 13

Figure 1.9 Bioactive metabolites from marine cyanobacteria ................. 14

Figure 1.10 Antimicrobial secondary metabolites from marine bacteria 16

Figure 1.11 Linezolid ............................................................................... 16

Figure 1.12 Cytotoxic secondary metabolites from marine bacteria ....... 18

Figure 1.13 Antimicrobial secondary metabolites from marine fungi .... 20

Figure 1.14 Cytotoxic secondary metabolites from marine fungi ........... 24

Figure 2.1 Structure of marinopyrone A (1) with COSY and key HMBC

correlations .............................................................................................. 30

Figure 2.2 Structures of synthetic compounds possessing �-pyrone moiety

................................................................................................................... 31

Figure 2.3 Structure of marinopyrone B (2) with COSY and key HMBC

correlations .......................................................................................... 33

Figure 2.4 Structure of marinopyrone C (3) with COSY and key HMBC

correlations .......................................................................................... 35

Figure 2.5 CD spectra for 1-3 .................................................................. 37

Figure 2.6 Structure of marinopyrone D (4) with COSY and key HMBC

correlations .......................................................................................... 38

Figure 2.7 NO assay of 1-4 ...................................................................... 42

Figure 2.8 IC50 value of 4 ........................................................................ 43

Figure 2.9 Strain CNQ-082 (left) and CNQ-675 (right) .......................... 45

Figure 3.1 Sesquiterpenoids from marine-derived actinobacteria ........... 55

Figure 3.2 Sesquiterpenoids from Streptomyces sp. ................................ 55

Figure 3.3 Indene-containing sesquiterpenoids ....................................... 56

Figure 3.4 Sesquiterpenoids with a saturated 6,5 ring system ................ 56

Figure 3.5 Structure of anmindenol A (5) with COSY, key HMBC and

NOESY correlations ................................................................................ 58

Figure 3.6 Structure of anmindenol B (6) with COSY and key HMBC

correlations .............................................................................................. 60



Figure 3.9 Strain CMDD10D111 ............................................................. 65

Figure 4.1 Secondary metabolites from the genus Actinoalloteichus ..... 72

Figure 4.2 Nocardiopsins A and B ........................................................... 73

Figure 4.3 Structure of 11A020-3A (7) with COSY and key HMBC

correlations .............................................................................................. 75

Figure 4.4 Strain CMDD11A020 ............................................................ 79

Figure 5.1 Structures of known compounds from fungal strain F8015-2B 82

Figure 5.2 Structure of F8015-2B-2E (8) with key HMBC correlations . 84

Figure 5.3 Structure of F8015-2B-2I (9) with key HMBC correlations .. 86

Figure 5.4 Structure of F8015-2B-2B (10) with key HMBC correlations 89

Figure 5.5 Structure of F8015-2B-2C (11) with key HMBC correlations 92

Figure 5.6 Fungal strain F8015-2B .......................................................... 93

List of Tables

Table 1.1 The marine pharmaceutical clinical pipelin ............................. 11

Table 1.2 Natural product antibiotics from marine fungi, listing all available

literature until March 2016 ...................................................................... 21

Table 1.3 Antitumors from marine fungi, listing all available literature until

March 2016 .............................................................................................. 25

Table 2.1 NMR data for marinopyrone A (1, CDCl3) .............................. 32

Table 2.2 NMR data for marinopyrone B (2, CDCl3) ............................. 34

Table 2.3 NMR Data for Marinopyrone C (3, CDCl3) ............................ 36

Table 2.4 NMR Data for marinopyrone D (4, MeOH-d4) ....................... 39

Table 3.1. NMR data for anmindenol A (5, CDCl3) ................................ 59

Table 3.2. NMR data for anmindenol B (6, CDCl3) ................................ 61

Table 4.1 NMR data for 11A020-3A (7, MeOH-d4) ................................ 76

Table 5.1 NMR data for (-)-mycousnine (5.1, CDCl3) a and F8015-2B-2E (8,

CDCl3)a .................................................................................................... 85

Table 5.2 NMR data for F8015-2B-2I (9, CDCl3) ................................... 87

Table 5.3 NMR data for F8015-2B-2B (10, MeOH-d4) .......................... 89

Table 5.4 NMR data for F8015-2B-2C (11, MeOH-d4) .......................... 91

List of Scheme

Scheme 2.1 Determination of the absolute configuration of 4 ................ 40

1

Chapter 1.

General Introduction of Marine Natural Products

1.1 Marine Natural Products

1.1.1 Marine Natural Products in General

Natural products have been a significant source of use in traditional

medicine, especially those from terrestrial materials like morphine from

poppies.1 Most of the current drug derived natural products have terrestrial

origins.2 Nevertheless, increasing of intensive research into the discovery of

novel pharmacologically active compounds with unique chemical structures

have leaded interest in marine natural products.

Oceans, covering more than 70% of the earth’s surface, have peculiar

environment such as high pressure, high saline density, low nutrient

concentration, low dissolved oxygen and controlled sunlight. Additionally,

considering that 32 of the 33 animal phyla are represented in aquatic

environments, 3 oceans possess biodiversity of marine organisms. This

enormous number of marine animal phyla and species indicates marine

organisms are potential resource to discover chemotherapeutic agents. 1 Molinski, T.F.; Dalisay, D.S.; Lievens, S.L.; Saludes, J.P. Nat. Rev. Drug Discov. 2009, 8, 69–85. 2 Rana Montaser, Hendrik Luesch Future Med. Chem. 2011,3, 1475–1489 3 L. Margulis, K.V. Schwartz, Five kingdoms, an illustrated guide to the phyla of life on earth, 2nd ed. W. H. Freeman & Co., New York, 1988.

2

The first reported marine natural products were spongouridine (1.1) and

spongothymidine (1.2), unusual aribino- and ribo- pentosyl nucleosides

isolated from the Caribbean sponge Tethya crypta. The compounds served

lead structures for anticancer drugs, ara-A (1.3, vidarabine, Vira-A®, FDA

approval 1976) and ara-C (1.4, cytarabine, Cytosar-U®, FDA approval

1969) (Figure 1.1).4

Figure 1.1 Spongouridine (1.1), spongothymidine (1.2), ara-A (1.3) and

ara-C (1.4)

The number of novel marine natural products has increased since the mid-

1980s (Figure 1.2) whereas the novel products reported annually were less

than 100 before 1985 with the developments of FT-NMR (1970) and 2D-

NMR (1980).5 More than 15,000 chemical substances including 4,196

4 Bergmann, W.; Feeney, R. J. J. Org. Chem. 1951, 16, 981-987. 5 Hu, G.P.; Yuan, J.; Sun, L.; She, Z.G.; Wu, J.H.; Lan, X.J.; Zhu, X.; Lin, Y.C.; Chen, S.P. Mar. Drugs 2011, 9, 514–525.

3

bioactive compounds were discovered since 1985.6

Figure 1.2 Variation in number of new marine natural products

for 1985-2012.6

6 Hu, Y.; Chen, J.; Hu, G.; Yu, J.; Zhu, X.; Lin, Y.; Chen, S.; Yuan, J. Mar. Drugs 2015, 13, 201–221.

4

Figure 1.3 The quantity and proportion of bioactive compounds in each

category of chemical compounds.6

Marine natural products are classified into eight categories based on types

of chemical structures: terpenoids, steroids (including steroidal saponins),

alkaloids, ethers (including ketals), phenols (including quinones),

strigolactones, peptides, and others (those that cannot be classified into the

above seven classes). As shown in Figure 1.3, peptides have the highest

proportion if bioactive compounds.6 The representative bioactive peptide in

marine natural products is ziconotide (Figure 1.4, Prialt®, FDA approval

2004), isolated from the venom of the cone snail Conus magnus. Ziconotide

is an analgesic agent for severe chronic pain.7

7 Jain, K.K. Expert Opin. Investig. Drugs 2000, 9, 2403–2410.

5

Figure 1.4 Ziconotide

6

Producers of marine natural products are divided into three groups of

marine organisms: marine microorganisms, marine algae and marine

invertebrates. Marine invertebrates form approximately 75% portion of

isolated compounds.5 56.89% of the total bioactive compounds were from

Porifera (mostly sponge) and Cnidaria (mostly coral) due to ease of

collection as their size abundance, color and benthic habitat (Figure 1.5).

Halichondrin B (1.5 in Figure 1.6) is the represent cytotoxic compound

isolated from the marine sponge Halichondria okadai in 1986. This

compound, a simplified macrocyclic ketone analogue, is the key for total

synthetic pathway of Eribulin mesylate (1.6 in Figure 1.6, E7389,

HalavenTM, FDA approval 2010), an anticancer drug for breast cancer and

liposarcoma,8

8 Huyck, T. K.; Gradishar, W.; Manuguid, F.; Kirkpatrick, P. Nat. Rev. Drug. Discov. 2011, 10, 173-174.

7

Figure 1.5 Number and proportion of

bioactive/non-bioactive compounds6 from

marine organisms (*PHVD: Prevention of

heart and vascular disease, **PN/NT:

Protection of neurons/neurotoxicity).6

8

Figure 1.6 Halichondrin B (1.5) and Eribulin mesylate (1.6)

The marine algae (seaweeds) were one of the major researched group for

natural products chemistry even though the portion (19.38%) of bioactive

compounds is the lowest as shown in Figure 1.5 because study of

microorganisms and marine invertebrates have been focused along with the

biodiscovery trends. The marine algae are regarded as the symbiont of

marine microbial organisms as marine fungi or bacteria associated with

algae are investigated.9 Further study of marine natural products from algae

and symbiotic relationship between algae and microbial organisms are

required.

Marine microorganisms, one of the potential sources for the discovery of

novel drugs, have been focused in the last decades. As shown in Figure 1.5,

9 Konig, G. M.; Kehraus, S.; Seibert, S. F.; Abdel-Lateff, A.; Muller, D. ChemBioChem, 2006, 7, 229-238.

9

the portion of novel bioactive compounds from marine microorganisms,

especially actinomycetes (47.01%) and bacteria (46.38%), are higher than

the average proportion and (28.39%). More details of natural products of

marine microorganisms will be descripted in the following section 1.2.

1.1.2 Drug Development from Marine Natural Products

Marine organisms are potential producers for bioactive metabolites as their

biodiversity, at least a million of marine species and hundreds of microbial

species, with unique conditions of the marine environment. Currently, there

are eight US Food and Drug Administration (FDA) and European

Medicines Agency (EMEA)-approved marine or marine-derived drugs.

While three marine drugs (Prialt® 1.12, Yondelis® 1.8 and Carragelose®

1.10) became drugs with their original chemical structures without any

modification, others took lead optimization in several steps of their

development to solve the supply and absorption, distribution, metabolism

and excretion/toxicity in pharmacokinetics (ADMET) properties. According

to Figure 1.7 and Table 1.1, eight marine drugs were obtained from marine

invertebrates, fish and red algae and used to treat cancer, virus, neuropathic

pain and hypertriglyceridemia.10

10 Martins, A.; Vieira, H.; Gaspar, H.; Santos, S. Mar. Drugs 2014, 12, 1066-1101.

10

Figure 1.7 Chemical structure of marine drugs on the market divided by

therapeutic area10

11

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12

1.2 Marine Microorganisms as a Source for Natural Products

1.2.1 Marine Microorganisms

Marine microorganisms, mostly bacteria but also fungi, are prolific

producers of bioactive compounds. The microbial biodiversity of marine

environments has not been identified taxonomically and chemically. It has

been regarded that 0.1% of the marine microbial diversity was studied.

As shown in Figure 1.8, the portion of novel bioactive compounds for

marine microorganisms is higher (37.13%) than invertebrates and algae.

Marine microorganisms are mainly associated with soft-bodied marine

organisms and play a role of higher hosts. Therefore, they produce chemical

defense agents to survive in their habitats.11 In the last decades, the number

of isolated secondary metabolites from marine microorganisms has

increased, thus 677 compounds has been reported until 2014.12

11 Debbab, A.; Aly, A.H.; Lin, W. H. Proksch, P. J. Microbial. Biotech. 2010, 3, 544-563. 12 Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep., 2016, 33, 382-431

13

Figure 1.8 Number of bioactive compounds isolated from the three groups

of marine organisms6

Compared with invertebrates and algaes, microorganisms are easy

fermentative and multiplied faster. This advantage and their living forms as

associated with marine organisms might suggest a solution for supply

problems for drug development. For example, Yondelis® (1.8, Figure 1.7),

isolated form marine tunicate Ecteinascidia turbinate and approved as an

anticancer drug, was obtained from sea squirt at extremely low yield. To

solve this supply issue, cyanosafracin B isolated from bacterium

Pseudomonas fluorescens was used for devised semisynthetic process.13

13 Cuevas, C.; Francesch, A. Nat. Prod. Rep. 2009, 26, 332-337.

14

Recently, the Sherman group at the University of Michigan reported

uncultivated bacterium produces the ecteinascidins with the identification

via metagenomic techniques.14

1.2.2 Bioactive Metabolites from Marine Bacteria

Bioactive metabolites from marine cyanobacteria

Marine cyanobacteria are important source of bioactive metabolites.

Dolastatin 10 (1.13) and curacin A (1.14), entered preclinal and clinical

trials and served as lead structures for synthetic process as antitumor agents.

Largazole (1.15) and somocystinamide A (1.16) were potent cytotoxic

moleculres for cancer therapy.15

Figure 1.9 Bioactive metabolites from marine cyanobacteria

14 Schofield, M. M.; Jain, S.; Porat, D.; Dick, G. J.; Sherman, D. H. Environ. Microbiol. 2015, 17, 3964–3975. 15 Moore, B. S.; Gulder, T. AM. Curr. Opin. Microbial. 2009, 12, 252-260.

15

Antimicrobial secondary metabolites

A new lipopeptide containing two D amino acids and acylated at the N-

terminus named tauramamide (1.17) was isolated from Brevibacillus

laterosporus PNG276 from Papua New Guinea. It showed potent minimum

inhibitory concentration (MIC) values of 0.11 mM against Gram-positive

human pathogen Enterococcus sp.16 Lipoxazolidinone A (1.18), B (1.191)

and C (1.20) were obtained from genus Marinispora (strain NPS008920)

isolated from a sediment collected in Cocos Lagoon, Guam. Compounds

1.18 1.20 showed broad spectrum antimicrobial activities similar to those of

the commercial antibiotic linezolid (Zyvox, Figure 1.11).17 Marinopyrroles

A (1.21) and B (1.22) were isolated from the actinomycetes strain CNQ-418

from a marine sediment sample (collected near La Jolla, CA) at a depth of

51 m strong antibiotic activity. Compound 1.21 and 1.22 displayed

noteworthy activity against methicillin-resistant S. aureus with MIC90 of

less than 2 mM.18

16 Desjardine, K.; Pereira, A.; Wright, H.; Matainaho, T.; Kelly, M.; Andersen, R. J. J. Nat. Prod. 2007, 70, 1850-1853. 17 Macherla, V. R.; Liu, J.; Sunga, M.; White, D. J.; Grodberg, J.; Teisan, S. J. Nat. Prod. 2007, 70, 1454-1457. 18 Hughes, C. C.; Prieto-Davo, A.; Jensen, P. R.; Fenical, W. Org. Lett. 2008, 10, 629-631.

16

Figure 1.10 Antimicrobial secondary metabolites from marine bacteria

Figure 1.11 Linezolid

17

Cytotoxic secondary metabolites

Salinosporamid A (1.23) was obtained from Salinispora tropica belong to

genus Salinispora, the first marine obligate actinomycete isolated from

ocean sediment.19 Salinosporamide A is a novel β-lactone-γ-lactam and

potential anticancer agent.20 Phase I human clinical trials were under way

for it as the treatment of multiple myeloma. Marinopyrroles A (1.21) and B

(1.22) were isolated from the actinomycetes strain CNQ-418 from a marine

sediment sample (collected near La Jolla, CA) at a depth of 51 m strong

antibiotic activity. Compound 1.21 and 1.242 displayed noteworthy activity

against methicillin-resistant S. aureus with MIC90 of less than 2 mM and

cytotoxicity against a human cancer cell line (HCT-116, colon carcinoma)

with 8.8 and 9.0 μM, respectively.21

19 Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666-673. 20 Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.; Jensen, P. R.; Fenical, W. Angew. Chem. Int. Ed. Engl. 2003, 42, 335-357. 21 Hughes, C. C.; Prieto-Davo, A.; Jensen, P. R.; Fenical, W. Org. Lett. 2008, 10, 629-631.

18

Figure 1.12 Cytotoxic secondary metabolites from marine bacteria

1.2.3 Bioactive Metabolites from Marine Fungi

The marine fungi are rich source for secondary metabolites with diverse

bioactivities. They are easily obtained from the inner tissues of marine

invertebrates, algae and sediments.

Antimicrobial secondary metabolites

In the 1950s, cephalosporin C (1.24), a -lactam type fungal antibiotic,

was discovered from a marine environment. 22 Gliotoxin (1.275) from

Aspergillus sp. Isolated from marine mud of the Seto Inland Sea was a new

antibiotic diketopiperazines in the late 1970s.23 The number of novel

bioactive fungal natural products is increasing steadily and has been

22 Newton, G. G. F.; Abraham, E. P. Nature 1955, 175, 548-548. 23 Okutani, K. Bull. Jap. Soc. Sci. Fish. 1977, 43, 995-1000.

19

identified. Particularly, 318 new compounds from marine-sourced fungi

have been reported until 2014.12 Fungal marine natural products have

diverse structural classes with predominant antibacterial and other

bioactivities. Trichodermamides A, A1, and B (1.26-1.28) from a marine

Trichoderma sp. inhibited the growth of Mycobacterium smegmatis, M.

bovis and M. tuberculosis with MICs range of 0.02-2.0 g/mL.24 Pestalone

(1.29), a chlorinated benzophenone, was obtained from the mixed

cultivation of a marine Pestalotia sp. with an unidentified marine bacterium.

Pestalone exhibited highly potent activity against methicillin-resistant

Staphylococcus aureus and vancomycin-resistant Enterococcus faecium

(MIC values of 37 ng/mL and 78 ng/mL, respectively).25 Other fungal

antibiotics are described in Figure 1.13, Table 1.2 with their chemical

structures and activities.

24 Pruksakorn, P.; Arai, M.; Kotoku, N.; Vilcheze, C.; Baughn, A.D.; Moodley, P.; Jacobs,W.R.; Kobayashi, M. Bioorg. Med. Chem. Lett. 2010, 20, 3658-3663. 25 Cueto, M.; Jensen, P.R.; Kauffman, C.; Fenical, W.; Lobkovsky, E.; Clardy, J. J. Nat. Prod. 2001, 64, 1444-1446.

20

Figure 1.13 Antimicrobial secondary metabolites from marine fungi

21

Tab

le 1

.2 N

atur

al p

rodu

ct a

ntib

iotic

s fro

m m

arin

e fu

ngi,

listin

g al

l ava

ilabl

e lit

erat

ure

until

Mar

ch 2

01626

Com

poun

d C

hem

ical

Cla

ss

Prod

ucer

, Ori

gin

Ant

ibio

tic A

ctiv

ity A

gain

st

Che

mic

al

Stur

ctur

e

15G

265α

,β,γ

m

acro

cycl

ic

poly

lact

ones

and

lip

odep

sipe

ptid

e

Hyp

oxyl

on o

cean

icum

, m

angr

ove

Stap

hylo

cocc

us e

pide

rmid

is,

Xant

hom

onas

cam

pest

ris

Prop

ioni

bact

eriu

m a

cnes

1.

30

Asc

ochy

tatin

sp

irodi

oxyn

apht

hale

ne

Asco

chyt

a sp

. NG

B4,

flo

atin

g sc

rap

of fe

ster

ing

rope

co

llect

ed a

t a fi

shin

g po

rt

Bact

eria

l tw

o-co

mpo

nent

re

gula

tory

syst

em

1.31

Asc

oset

in,

tetra

mic

aci

d Li

ndgo

myc

etac

eae,

H

alic

hond

ria

pani

cea

(s

pong

e fr

om B

altic

Sea

)

S. e

pide

rmid

is, S

. aur

eus,

M

R S.

aure

us, P

. acn

es, X

. cam

pest

ris,

Sept

oria

triti

ci

1.32

Bis

(2-

ethy

lhex

yl)p

htha

late

ph

thal

ate

* C

lado

spor

ium

sp.,

sea

wat

er in

man

grov

e ar

ea

Lokt

anel

la h

ongk

onge

nsis

, M. l

uteu

s, Rh

odov

ulum

sp.,

Rueg

eria

sp.,

Pseu

doal

tero

mon

as p

isci

da,

Vibr

io h

arve

yi

1.33

Cal

cari

des A

m

acro

cycl

ic a

nd

linea

r pol

yest

ers

Cal

cari

spor

ium

sp.,

Wad

den

sea

wat

er

Mac

rocy

clic

com

poun

ds: S

. epi

derm

idis

, Li

near

pol

yest

ers:

no

antib

iotic

act

ivity

1.

34

Cep

halo

spor

in

β-la

ctam

Aspe

rgill

us c

hrys

ogen

um,

sew

age

wat

er

Cep

halo

spor

ium

chr

ysog

enum

, se

a w

ater

Bro

ad sp

ectru

m

1.24

26 S

ilber

, J.;

Kra

mer

, A.;

Labe

s, A

.; Ta

sdem

ir, D

. Mar

. Dru

gs 2

016,

14,

137

-156

.

22

3-C

hlor

o-2,

5-di

hydr

oxy

benz

yl

alco

hol

benz

ene

deriv

ativ

e Am

pelo

myc

es sp

.,

mar

ine

biof

ilm

Mic

roco

ccus

sp.,

Vibr

io sp

., Ps

eudo

alte

rom

onas

sp.,

S. a

ureu

s,

S. h

aem

olyt

icus

1.

35

Cor

ollo

spor

in

phth

alid

e de

rivat

ives

C

orol

losp

ora

mar

itim

a,

mar

ine

drift

woo

d

Can

dida

mal

tosa

, Esc

heri

chia

col

i, Ps

eudo

mon

as a

erug

inos

a, B

acill

us

subt

ilis,

S. a

ureu

s, S.

aur

eus N

orth

G

erm

an e

pide

mic

stra

in,

S. e

pide

rmid

is, S

. hae

mol

ytic

us

1.36

Cyc

lo-(

Pro-

Phe)

di

keto

pipe

razi

ne

Uni

dent

ified

mar

ine

fung

us

mar

ine

biof

ilm

Antib

acte

rial

ant

ibio

film

: M

icro

cocc

us sp

., V i

brio

sp.,

Pseu

doal

tero

mon

as, S

. aur

eus,

S. h

aem

olyt

icus

1.37

Exo

phili

n A

3,

5-di

hydr

oxy-

deca

noic

pol

yest

er

Exop

hial

a pi

scip

hila

, M

ycal

e ad

haer

ens (

spon

ge)

E. fa

cium

, E. f

aeca

lis, S

. aur

eus,

M

R S.

aur

eus

1.38

Lin

dgom

ycin

, te

tram

ic a

cid

Lind

gom

ycet

acea

e,

Hal

icho

ndri

a pa

nice

a

(spo

nge

from

Bal

tic S

ea)

MR

S. a

ureu

s, S.

epi

derm

idis

, P. a

cnes

, X.

cam

pest

ris, S

. tri

tici

1.39

(+)-

Ter

rein

, cy

clop

ente

none

As

perg

illus

terr

eus P

F-26

, Ph

akel

lia fu

sca

(spo

nge)

B.

subt

ilis

1.40

23

Cytotoxic secondary metabolites

Verrucarin A (1.41) was isolated from the Palauan marine fungus

Myrothecium roridum. Verrucarin A inhibited interleukin-8 production from

human promyelocytic leukemia cells by inhibition of the activation of the

mitogen activated kinases c-JUN and p38.27 Zygosporamide (1.42) is a

cyclic depsipeptide isolated from a marine-derived fungus Zygosporium

masonii. Zygosporamide was tested for cytotoxicity against 60 cancer cell

lines in NCI.28 Cephalimysin A (1.43) was isolated from a strain of

Aspergillus fumigatus OPUST106B-5 originally separated from the marine

fish Mugilcephalus. Cephalimysin A exhibited significant cytotoxic activity

against the murine P-388 leukemia cell line and the human HL-60 leukemia

cell line.29 Other fungal cytotoxic compounds are described in Figure 1.14,

Table 1.3 with their chemical structures and activities.

27 Oda T et al. Marine Drugs. 2005, 3, 64-. 28 Oh, D. -C.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 2006, 47, 8625-8628. 29 Zhou, Y.; Debbab, A.; Mandi, A.; Wray, V.; Schulz, B.; Muller, Kassack, M.; Lin, W.; Kurtan, T.; Proksch, P.; Aly, A. H. Eur. J. Org. Chem. 2013, 5, 894-906.

24

Figure 1.14 Cytotoxic secondary metabolites from marine fungi

25

Table 1.3 Antitumors from marine fungi, listing all available literature until

March 201626,30

Compound Chemical Class Producer, Origin Cytotoxicity Against

Chemical Structure

Trichodermamides A-B

Trichoderma virens, ascidian

Didemnum molle

human colon carcinoma

1.26 1.28

Leptosins M, M1, N

epipolysulfanyldioxopiperazine

Leptosphaeria sp., marine algae

Sargassum tortile

Broad spectrum

1.44 1.45 1.46

Conidiogenone C

Penicillium sp. marine red algae genus Laurencia.

HL-60 1.47

Fusaranthraquinone,

fusarnaphthoquinones

naphtoquinone anhydrofusarubi

n

Fusarium spp. sea fan Annella sp.

MCF-7 cells (breast) HCT-8 (colon),

MDA-MB-435

(melanoma) and SF-295

(brain)

1.48 1.49

Cryptosphaerolide

ester-substituted sesquiterpenoid

Cryptosphaeria sp. (unidentified

ascidian, Bahamas) HCT-116 1.50

Paeciloxocins A-B

Paecilomyces sp. the mangrove fungus

collected from the Taiwan Strait.

HepG2 cell line (liver)

1.51 1.52

30 Hasan, S.; Ansari, M. I.; Mishra, M. Bioinformation 2015, 11, 176-181.

26

Chapter 2.

Marinopyrones A-D, α-Pyrones from Marine-Derived

Actinomycetes of the family Nocardiopsaceae31

2.1 Introduction

The actinomycetes, gram-positive bacteria that are common on land and

in the sea, are the major group of bacteria that produce bioactive secondary

metabolites. Over 10,000 bioactive molecules have been derived from

cultured actinomycetes, and a significant percentage of these have been

isolated from members of the very commonly encountered genus

Streptomyces.32 Due to the increasing the rate of rediscovery of known

bioactive natural products from terrestrial actinomycetes, there has been an

increased focus on marine microbial sources of bioactive natural products.33

Over the past two decades, the biodiversity of actinomycetes in marine

environments has been investigated yielding new strains and significant new

metabolites from these marine microbes.34 Based on phylogenetic analysis

of their 16S rRNA genes marine microbes have consistently been

31 This chapter is based on the following published paper by the author. Lee, J.; Han, C.; Lee, T. G.; Chin, J.; Choi, H.; Lee, W.; Paik, M. J.; Won, D. H.; Jeong, G.; Ko. J.; Yoon, Y. J.; Nam, S. –J.; Fenical, W.; Kang, H. Tetrahedron Lett. 2016, 57, 1997-2000 32 Bérdy, J. J. Antibiot. 2005, 58, 1–26. 33 Mincer, T. J.; Jensen, P. R.; Kauffman, C. A.; Fenical, W. Appl. Environ. Microbiol. 2002, 68, 5005–5011. 34 Jensen, P. R.; Mincer, T. J.; Williams, P. G.; Fenical, W. Antonie van Leeuwenhoek, 2005, 87, 43–48.

27

differentiated from their terrestrial relatives. 35 In previous studies, 13

groups of marine-derived actinomycetes have been identified and classified

as MAR groups. 36 Intensive chemical investigation of MAR group

representatives has led to the discovery of various structurally

unprecedentedly natural products. The most well-studied strain in the MAR

groups is Salinispora (MAR 1), which was also the first obligate marine

actinomycete taxon to be described. Salinosporamide A, a characteristic

secondary metabolite produced by S. tropica, is currently being investigated

as a potential anticancer agent in phase I clinical trials.37 Our strains CNQ-

675 and CNQ-082 appeared unique and shared 99.6% and 98.6% of the 16S

rRNA gene sequence identities with its nearest neighbor, Nocardiopsis sp.

and Streptomonospora sp. Members of the genus Streptomonospora were

first isolated from a saline lake and proposed as a new genus in the family

Nocardiopsaceae (suborder Streptosporangineae) in 2001.38 Studies of the

genus Streptomonospora have been mainly focused on the description of

strains,39a,b however, chemical studies have been rarely reported. The first

reported natural product from this genus is streptomonomicin, an antibiotic

35 Prieto-Davo, A.; Fenical, W.; Jensen, P. R. Aquat. Microb. Ecol. 2008, 52, 1–11. 36 a. Fenical, W.; Jensen, P. R. Nature Chem. Biol. 2006, 2, 666–673. b. Mincer, T. J.; Jensen, P. R.; Kauffman, C. A.; Fenical, W. Appl. Environ. Microbiol. 2002, 68, 5005–5011. 37 Niewerth, D.; Jansen, G.; Riethoff, L. F.; van Meerloo, J.; Kale, A. J.; Moore, B. S.; Assaraf, Y. G.; Anderl, J. L.; Zweegman, S.; Kaspers, G. J.; Cloos, J. Mol. Pharmacol. 2014, 86, 12–19. 38 Cui, X. -L.; Mao, P. -H.; Zeng, M.; Li, W. -J.; Zhang, L. -P.; Xu, L. -H.; Jiang, C. -L. Int. J. Syst. Evol. Microbiol. 2001, 51, 357–363. 39 a. Zhang, D. -F.; Pan, H. -Q. P.; Zhang, X. -M.; Zhang, Y. -G.; Klenk, H. -P.; Hu, J. -C.; Li, W. -J. Int. J. Syst. Evol. Microbiol. 2013, 63, 4447–4455. b. Sun, W.; Zhang, F.; He, L.; Karthik, L.; Li, Z. Front. Microbiol. 2015, 6, 1–15.

28

lasso peptide produced by Streptomonospora alba.40

In our studies, we examined the culture extract of the two halophile

strains of the family Nocardiopsaceae which led to the isolation of the

marinopyrones A-D (1-4). 1-4 are α-pyrone polyketides that appear to be

derived from propionate and acetate precursors. α-Pyrones are common

natural products that show uncommon, but diverse biological activities.41

Polyketides of a mixed acetate/propionate origin include aplysiopsenes A-D

from the sacoglossan sea slug Aplysiopsis formosa,42 stemphypyrone from

the endophytic fungus Stemphylium globuliferum,43 salinipyrones A and B

from the marine actinomycete Salinispora pacifica,44 phomapyrones C

from the blackleg fungus Phoma lingam, 45 xylarone and 8,9-

dehydroxylarone from Xylaria hypoxylon, 46 placidenes C-F from the

sacoglossan Placida dendritica47 as well as trichopyrone from the marine

sponge-derived fungus Trichoderma sp.48

40 Metelev, M.; Tietz, J. I.; Melby, J. O.; Blair, P. M.; Zhu, L.; Livnat, I.; Severinov, K.; Mitchell, D. A. Chemistry & Biology, 2015, 22, 241–250. 41 McGlacken, G. P.; Fairlamb, I. J. S. Nat. Prod. Rep. 2005, 22, 369–385 42 Ciavatta, M. L.; Manzo, E.; Nuzzo, G.; Villani, G.; Cimino, G.; Cervera, J. L.; Malaquias, M. A. E.; Gavagnin, M. Tetrahedron Lett., 2009, 50, 527–529. 43 Debbab, A.; Aly, A. H.; Edrada-Ebel, R.; Wray, V.; Müller, W. E. G.; Totzke, F.; Zirrgiebel, U.; Schächtele, C.; Kubbutat, M. H. G.; Lin, W. H.; Mosaddak, M.; Hakiki, A.; Proksch. P.; Ebel, R. J. Nat. Prod. 2009, 72, 626–631. 44 Oh, D. -C.; Gontang, E. A.; Kauffman, C. A.; Jensen, P. R.; Fenical. W. J. Nat. Prod. 2008, 71, 570–575. 45 Pedras, M. S. C.; Erosa-López, C. C. Quail, W.; Taylor, J. L. Bioorg. Med. Chem. Lett. 1999, 9, 3291–3294. 46 Schüfflera, A.; Sternerb, O.; Ankea, H. Z. Naturforsch. C. 2007, 62, 169–172. 47 Cutignano, A.; Fontana, A.; Renzulli, L.; Cimino, G. J. Nat. Prod. 2003, 66, 1399–1401. 48 Abdel-Lateff, A.; Fisch, K.; Wright, A. D. Z. Naturforsch. C. 2009, 64, 186–192.

29


2.2.1 Structural Elucidation of Marinopyrones

2.2.1.1 Marinopyrone A (1)

Marinopyrone A (1) was isolated as a colorless amorphous solid and its

molecular formula was determined to be C13H18O4, a formula indicating five

degrees of unsaturation, based on HRFABMS data. The 1H NMR spectrum

of 1 showed two olefinic protons [δH 6.35 (d, J = 15.5 Hz), 6.58 (dd, J =

15.5, 8.2 Hz)], one methine [δH 2.58, m], one methylene [δH 3.63 (dd, J =

10.2, 5.5 Hz) 3.58 (dd, J = 10.2, 7.6 Hz)], two methyl singlets [δH 2.02,

2.08], one methoxy singlet [δH 3.83], and one methyl doublet [δH 1.12 (d, J

= 6.8 Hz)]. The 13C NMR and HSQC spectroscopic data for 1 displayed four

methyl, one methylene, three methine, and five quaternary carbons (Table

2.1). The chemical shifts of H2-10 and C-10 [δC 67.0] indicated that 1 had a

hydroxy group at C-10, which was also supported by the IR absorption at

3442 cm–1. The E-geometry was assigned on the basis of JH-7, H-8 being 15.5

Hz. The remaining part of the alkyl chain was determined on the basis of

COSY correlations linking H-8/H-9/H2-10/H3-11. The downfield-shifted

carbon signals for C-2 [δC 165.2] and C-6 [δC 152.0] and HMBC

correlations from H3-14 to C-4, C-6 and H3-12 to C-2, C-4 supported an α-

pyrone ring moiety. A long-range HMBC correlation from H3-13 to C-4

30

suggested that the methoxy group was attached to C-4. Lastly, HMBC

correlations observed from H-7 and H-8 to C-6 permitted the complete

planar structure of 1 to be assigned (Figure 2.1).

Figure 2.1 Structure of marinopyrone A (1) with COSY and key HMBC

correlations.

To determine the absolute configuration at C-9 for 1, we compared its

specific rotation to structural similar synthetic compounds because natural

products possessing the �-pyrone moiety with the primary alcohol in the

terminal aliphatic chain have never been reported. The values of the specific

rotations of 1 ([α]D = +64.00) was proposed that the absolute configurations

of 1 at C-9 would be [1a, (R)-(E)-2-Methyl-4-phenylbut-3-en-1-ol; [α]D =

+46.8, c 0.75, CHCl3, 1b, (S)-(E)-2-Methyl-4-phenylbut-3-en-1-ol; [α]D = -

31

47, c 0.8, CHCl3]49 (Figure 2.2).

Figure 2.2 Structures of synthetic compounds possessing �-pyrone moiety

49 Ohfune, Yasufumi; Tomita, Masako J. Am. Chem. Soc. 1982, 104, 3511-3513.

32

Table 2.1 NMR data for marinopyrone A (1, CDCl3)a

Colorless, amorphous solid

Molecular formula : C13H18O4

HRFABMS : m/z 239.1286 [M+H]+

(calcd for C13H19O4, 239.1284)

LRESIMS : m/z 239.2 [M+H]+

IR (film) νmax : 3444, 1633 cm-1

UV (MeOH) λmax : 225, 330 nm

marinopyrone Aa No δC, mult.b δH, (J in Hz) COSY HMBC 2 165.2, C 3 109.7, C 4 168.2, C 5 111.2, C 6 152.0, C 7 119.5, CH 6.35, d (15.5) 8 6 8 139.9, CH 6.58, dd (15.5, 8.2) 9 6 9 40.4, CH 2.58, m 11 8, 10, 11

10 67.0, CH2 3.63, dd (10.2, 5.5) 3.58, dd (10.2, 7.6)

9 9

8, 9, 11 8, 9, 11

11 16.2, CH3 1.12, d (6.8) 8, 9, 10 12 10.4, CH3 2.08, s 2, 4, 5, 6 13 60.3, CH3 3.83, s 4 14 9.6, CH3 2.02, s 3, 4, 6, 7, 8

a700 MHz for 1H NMR and 175 MHz for 13C NMR in CDCl3. bNumbers of attached protons were determined by analysis of 2D NMR spectroscopic data.

33

2.2.1.2 Marinopyrone B (2)

Marinopyrone B (2) was isolated as a colorless amorphous solid, which

analyzed for the molecular formula C14H20O4 based on HRFABMS and

interpretation of combined NMR spectroscopic data (Table 2.2). The 1H

NMR spectroscopic data of 2 were almost identical to those of 1. However,

an additional methylene proton H2-11 [δH 1.57 (m), 1.38 (m)], which

correlated with a terminal methyl proton H3-12 [δC 11.7, δH 0.95 (t, J = 7.5

Hz)] suggested that 2 was a C-11 methylated derivative of 1 (Figure 2.3).

Figure 2.3 Structure of marinopyrone B (2) with COSY and key HMBC

correlations.

To determine the absolute configuration at C-9 for 2, we performed the

same experiment with the analysis method of the absolute configuration for

1. The values of the specific rotations of 2 ([α]D = +6.00) was proposed that

34

the absolute configurations of 2 at C-9 would be R compared to synthetic

compounds (Figure 2.2).

Table 2.2 NMR data for marinopyrone B (2, CDCl3)a



HRFABMS : m/z 253.1435 [M+H]+

(calcd for C14H21O4, 253.1440)


IR (film) νmax : 3442, 1642 cm-1


marinopyrone Ba No δC, mult.b δH, (J in Hz) COSY HMBC 2 165.2, C 3 109.6, C 4 168.3, C 5 111.2, C 6 151.9, C 7 120.9, CH 6.36, d (15.4) 8 8 139.0, CH 6.48, dd (15.4, 9.3) 9 3, 7, 9, 11, 15 9 48.4, CH 2.35, m 11 3, 8, 9, 13, 15

10 65.6, CH2 3.68, dd (11.2, 5.1), 3.55, dd (11.2, 8.2)

9 9

7, 8, 9, 11 7, 8, 9, 11

11 23.8, CH2 1.57, m, 1.38, m 12 7, 8, 9, 11 12 11.7, CH3 0.95, t (7.5) 9, 11 13 10.4, CH3 2.08, s 2, 3, 4, 5, 6 14 60.3, CH3 3.85, s 14 15 9.6, CH3 2.02, s 3, 4, 5, 6


35

2.2.1.3 Marinopyrone C (3)

Marinopyrone C (3) was also obtained as a colorless amorphous solid.

The molecular formula of 3 was assigned as C14H20O4 based on

interpretation of HRFABMS data. The 1H and 13C NMR spectra of 3 were

similar to those of 2 except that 3 possessed a methyl group at C-10 [δC 20.4,

δH 1.08 (d, J = 6.7 Hz) and a downfield shifted methylene carbon at C-12

[δC 60.8, δH 3.65 (m)]. Interpretation of 2D NMR spectroscopic data showed

that 3 had a hydroxy group at C-12 (Figure 2.4, Table 2.3).

Figure 2.4 Structure of marinopyrone C (3) with COSY and key HMBC

correlations.

36

Table 2.3 NMR Data for Marinopyrone C (3, CDCl3)a



HRFABMS : m/z 253.1432 [M+H]+

(calcd for C14H21O4, 253.1440)


IR (film) νmax : 3400, 1681 cm-1


marinopyrone Ca No δC, mult.b δH, (J in Hz) COSY HMBC 2 165.3, C 3 109.3, C 4 168.4, C 5 110.9, C 6 152.4, C 7 117.5, CH 6.22, d (15.4) 8 6, 8, 9 8 143.2, CH 6.53, dd (15.4, 8.5) 9 6, 9, 10, 12 9 34.2, CH 2.51, m 12 7, 8, 10, 12

10 20.4, CH3 1.08, d (6.7) 9 9

7, 8, 9, 12 7, 8, 9, 12

11 39.3, CH2 1.64, m 10 12 60.8, CH2 3.65, m 8, 9, 10 13 10.4, CH3 2.03, s 2, 3, 5 14 60.3, CH3 3.78, s 4 15 9.6, CH3 1.97, s 3, 4, 6


37

The absolute configuration at C-9 for 3 was proposed to S configuration

as the value of the specific rotation of 3 ([α]D = -35.0) was negative

compared with those of structural similar synthetic compounds (Figure 2.2).

We also obtained circular dichroism spectra of 1-3 to clarify their absolute

configurations. Compounds 1 and 2 showed positive ECD bands, whereas 3

illustrated a negative band at 350 nm, also supporting that 1 and 2 have R

configurations and 3 has an S configuration (Figure 2.5).

Figure 2.5 CD spectra for 1-3

38

2.2.1.4 Marinopyrone D (4)

Marinopyrone D (4) was isolated as a colorless amorphous solid. Its

molecular formula, indicating five degrees of unsaturation, was assigned as

C14H20O3 based upon analysis of HRFABMS data. The 1H and 13C NMR

data of 4 were almost identical to those of 3 except for the absence of the

hydroxy group at C-12 [δC 60.8] and presence of a methyl triplet (Figure 2.6,

Table 2.4).

Figure 2.6 Structure of marinopyrone D (4) with COSY and key HMBC

correlations.

39

Table 2.4 NMR Data for marinopyrone D (4, MeOH-d4)a



HRFABMS : m/z 237.1494 [M+H]+

(calcd for C14H21O3, 237.1491)


IR (film) νmax : 1671 cm-1


marinopyrone Da No δC, mult.b δH, (J in Hz) COSY HMBC 2 165.9, C 3 109.7, C 4 169.3, C 5 109.6, C 6 152.6, C 7 117.2, CH 6.38, d (15.4) 8 2, 5, 6, 8, 9 8 143.6, CH 6.50,dd (15.4, 8.3) 9 6, 7, 9 9 38.9, CH 2.29, m 10, 12 7, 8

10 18.7, CH3 1.10, d (6.7) 11 11

7 7

11 29.1, CH2 1.46, m 9, 10 12 10.7, CH3 0.93, t (7.4) 7, 9, 10 13 8.9, CH3 2.04, s 2, 3, 4, 5, 6 14 59.7, CH3 3.87, s 4 15 8.2, CH3 2.04, s 2, 3, 4, 5, 6

a700 MHz for 1H NMR and 175 MHz for 13C NMR in MeOH-d4. bNumbers of attached protons were determined by analysis of 2D NMR spectroscopic data.

40

In order to confirm the absolute configuration at C-9, the 2-methylbutyric

acid (4a) obtained from ozonolysis and oxidative work-up, was derivatized

as the α-naphthyl amide (Scheme 2.1). The α-naphthyl amide (4b)

derivative of authentic (2S)-4a was detected at 22.5 min, while authentic

(2R)-4a was detected at 27.0 min, respectively. The α-naphthyl amide

derivatives of 2-methylbutyric acid derived from 4 were observed at 22.5

min and 27.0 min, with a ratio of 1:3. Thus, absolute configuration of C-9 in

4 would be predominantly R (4b), but we could not confidently exclude that

compound 4 exists as a racemic mixture. Further, it is clearly possible that

partial racemization occurred during derivatization or isolation of 4.

Scheme 2.1 Determination of the absolute configuration of 4

41


2.2.2.1 Antibacterial Activity

Compounds 1-4 had no significant antibacterial activity against gram-

positive strains including Staphylococcus aureus ATCC 6538, Bacillus

subtilis ATCC 6633, Kocuria rhizophila ATCC 9341, S. epidermidis

ATCC12228 and gram-negative strains including Klebsiella pneumoniae

ATCC 4352, Escherichia coli ATCC 11775, and Salmonella typhimurium

ATCC 14028.

2.2.2.2 Cytotoxicity

Compounds 1-4 were not toxic to the human pancreatic cell lines (PANC-

1 and MIA-PaCa) and the human renal carcinoma cell line (A498) at

concentrations up to 100 μM.

2.2.2.3 Nitric Oxide Activity

Compounds 1-4 were tested for their inhibition of NO production in LPS-

stimulated RAW 264.7 macrophage cells. Marinopyrone D (4) was found to

inhibit NO production with an IC50 value of 13 μM, while 1-3 did not

display any significant activity (Figure 2.7, Figure 2.8). This result

illustrated that the functionality of the side chain plays an important role in

defining bioactivity. Marinopyrone D (4) possesses a hydrophobic terminal

42

methyl group while 1-3 contain a hydrophilic group at the terminal position

in the molecules.

Abbreviation : LPS = lipopolysaccharide

Figure 2.7 NO assay of 1-4

43

Figure 2.8 IC50 value of 4

44


2.3.1 Instruments and Data Collection

Optical rotations were measured using a Rudolph Research Autopol III

polarimeter with a 5 cm cell. UV spectra were recorded on a Scinco UVS-

2100 with a path length of 1 cm. CD spectra were collected in an Applied

Photophysics Chirascan plus CD spectrometer with a 0.5 mm path-length

rectangular cuvette. Infrared spectra were obtained using a Thermo Electron

Corporation spectrometer. NMR spectroscopic data for marinopyrones A-D

were obtained using a Bruker Avance 700 MHz spectrometer [CDCl3 (�H

7.26; �C 77.0) was used as an internal standard]. EI-MS and FAB-MS

spectra were measured on a JEOL, JMS-AX505WA mass spectrometer.

Low resolution LC-MS data were measured using an Agilent Technologies

6120 quadrupole LC/MS system with a reversed-phase C18 column

(Phenomenex luna, column (4.6 mm × 50 mm, 5μm) at a flow rate of 1.0

mL/min. HPLC separations were achieved using a WATERS 1525 binary

HPLC pump, WATERS 2489 UV/visible detector. Compounds 1-3 were

separated using a MG2 C18 (250 mm × 10 mm, 5 μm, flow rate = 2.0

mL/min) reversed-phase HPLC column, while 4 was purified using a

Phenomenex luna C18 column (250 mm × 10 mm, 5 μm, flow rate = 2.0

mL/min). Chiral HPLC analysis were performed with a Hewlett-Packard

45

series 1100 HPLC with G1310A Iso-pump using a Chiralpak IA column

(250 mm L × 4.6 mm, Daicel company, Japan).

2.3.2 Bacterial Material

Strain CNQ-082 and 675 were isolated from marine sediment collected at

La Jolla, California (Figure 2.9). The 16S rRNA gene sequence obtained by

using primers 27f and 1492r for CNQ-082 showed the 97.2% of sequence

similarity to the type strain for Streptomonospora halophila (EF423989.2),

and the sequence of CNQ-082 has been deposited with GenBank (accession

number EU214933.1). Strain CNQ-675 (GenBank entry KU293681) is most

similar to Nocardiopsis sp. (99.6% identical).

Figure 2.9 Strain CNQ-082 (left) and CNQ-675 (right)

46

2.3.3 Cultivation, Extraction and Isolation

CNQ-082 was cultured in 40 4 L Pyrex flasks each containing 1 L of the

medium SYP (10 g soluble starch, 4 g yeast extract, 2 g peptone, seawater

dissolved in 75 % 1 L natural seawater) at 27 °C with shaking at 150 rpm.

After 8 days, the broth was extracted with EtOAc and evaporated to yield

the organic extract (2.5 g). CNQ-675 was cultured at 27 °C with shaking at

130 rpm in 12 4 L Pyrex flask each containing 1 L of the same broth media

as CNQ-082. After 8 days, the broth was extracted with ethyl acetate and

evaporated to yield an organic extract (2.1 g).

The crude extract (2.5 g) from cultivation of strain CNQ-082 was

subjected to medium pressure liquid chromatographic separation using a

step-gradient elution with MeOH in dichloromethane (0%, 1%, 2%, 5%,

10%, 50%, 100%) to afford 7 fractions (Fr 1-Fr 7). Fr 1 (658.1 mg) was

further purified by C18 HPLC using 33% CH3CN in H2O to obtain

marinopyrone A (1, 3.4 mg), marinopyrone B (2, 1.2 mg) and marinopyrone

C (3, 4.5 mg). The organic extract (2.1 g) of CNQ-675 was

chromatographed on an open silica gel column with CH2Cl2-MeOH gradient

mixtures (100:0, 100:1, 50:1, 20:1. 10:1, 1:1, 0:100). Fr 1 (20.9 mg) was

further fractionated by C18 HPLC using 70% CH3CN in H2O to yield

marinopyrone D (4, 1.4 mg).

47

2.3.4 Determination of the Absolute Configuration for

compound 4

Marinopyrone D (4, 1.0 mg) was treated with excess ozone (1.5 mL

CH2Cl2, 20 min, -78 °C) followed by oxidative workup (6 drops of 30%

H2O2) and then dried (0.9 mg). Authentic α-naphthyl amide derivatives of

commercially available racemic mixtures and (S)-2-methylbutyric acid were

prepared using the coupling reagent 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (144 mg) and excess α-naphthyl amine

(180 mg) in 2-propanol (3 mL). The α-naphthyl amide derivative of 2-

methylbutyric acid from 4 was also prepared in the same manner. Liquid

chromatographic chiral separation of the α-naphthyl amide derivative of 2-

methylbutyric acid was carried out by isocratic HPLC with 5% 2-

propanol/hexane (v/v) as a mobile phase under UV 215 nm detection.50

2.3.5 Bioassays

2.3.5.1 MIC Assay

Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633,

Kocuria rhizophila ATCC 9341, S. epidermidis ATCC12228 and gram-

negative strains including Klebsiella pneumoniae ATCC 4352, Escherichia

50 Jin, J. Y; Bae, S. K.; Lee, W. Chirality 2009, 21, 871–877.

48

coli ATCC 11775, and Salmonella typhimurium ATCC 14028 were used.

These bacteria were inoculated in Mueller-Hinton agar media for 24 h at

36 °C. The bacterial colony was cultivated into 15 mL round tube

containing 6 mL of Mueller-Hinton broth (MHB) media at 36 °C and 200

rpm for 24 h. 1-4 and two positive controls (vancomycin and linezolid) with

128 μg/mL dissolved in DMSO were added 100 μL of each to 96-well

microtiter plate having 50 μL of MHB in rest well. Test compounds were

serially diluted and 50 μL of bacterial MHB media adjusted to concentration

of 1/100 diluted McFarland 0.5% standard. The 96-well was incubated for

24 h at 36 °C. After then, the minimum inhibitory values were determined

as the concentration of compounds at transparent well which inhibit the

growth of bacteria.

2.3.5.2 MTT Assay

A-498 (renal cancer cell), Panc-1 (human pancreatic carcinoma), Mia-

PaCa (human pancreatic carcinoma) were prepared. These cells were

cultured several times in petridish containing Dulbecco’s modified Eagle’s

medium (DMEM) added 1% penicillin-streptomycin and 10% fetal bovine

serum (FBS) at 37 °C in the incubator containing 5% CO2 and 80%

humidity. 50 μL of suspended cells were put into wells at a density of 1.2 ×

105 cells/mL for A-498, Panc-1, Mia-PaCa-2 cell lines. Seeded plates were

49

incubated at 37 °C in the incubator containing 5% CO2 and 80% humidity

durning one day. 1-4 and three positive controls (temsirolimus,

sunitinbmalate and 5-fluorouracil) were prepared to 10 mM in DMSO.

Samples were diluted to 100 μM in media and serially diluted to final

concentration 0.78 μM. 50 μL of diluted compounds were put into each well.

Each compound was tested in triplicate. The plates were incubated at 37 °C

in the incubator containing 5% CO2 and 80% humidity for 24 h. After then,

20 μL of MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide) reagent was added to each well. The plates were incubated at the

same incubator with identical conditions. After MTT reagent was removed

from the wells, 100 μL DMSO was added to each well. The plates were

incubated at the same incubator with identical conditions for 1 h. The cell

viability was determined by measurement of optical density using a

microplate ELISA reader at 560 nm. IC50 values were determined by using

SimaPlot 8.0 Notebook and excel.

2.3.5.3 Nitric Oxide Assay

The NO assay was performed for 1-4 by measuring NO production in

RAW 264.7 LPS-induced mouse macrophage cells. The cells were

maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented

with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37 °C,

50

in 5% CO2 humidified air. To evaluate the inhibitory activity of test

materials on LPS-induced NO production, the cells in 10% FBS DMEM

were plated in 96-well plates (2 × 105 cells/mL) and then incubated for 24

hours. After incubation, the cells were incubated in the medium with 100

ng/mL of LPS in the presence or absence of test samples. After an

additional 20 hours incubation, the media were collected and analyzed for

nitrite accumulation as an indicator of NO production by the Griess reaction.

Briefly, 100 μL of Griess reagent [0.1% N-(1-naphthyl)ethylenediamine

dihydrochloride in H2O and 1% sulfanilamide in 5% H3PO4] was added to

100 μL of each supernatant from LPS or sample-treated cells in 96-well

plates. The absorbance was measured at 540 nm using a microplate reader,

and nitrite concentration was determined by comparison with a sodium

nitrite standard curve. The percentage inhibition was expressed as [1 − (NO

level of test samples/NO level of vehicle − treated control)] × 100.

51

Chapter 3.

Anmindenols A and B, sesquiterpenoids from a

Marine-derived Streptomyces sp.51

3.1 Introduction

Tidal flats are spread widely over the west and southwest coasts of the

Korean peninsula.52 The physical and chemical properties of these tidal

flats are unpredictable due to water exchange caused by tidal cycles.53 The

shallow water column in tidal flats with strong tidal currents and high winds

stimulates transportation, dispersion and mixing of nutrients for biological

production.54 These physical vectors, along with the variability of salinity,

temperature, pH, and nutrient composition, contribute to the great diversity

of microbial communities in the tidal flats.55 The phylogenetic analysis

based on 16S rRNA sequences of bacteria from tidal flat sediments

51 This chapter is based on following references by the author.

a) Lee, J. Identification of secondary metabolites from marine-derived bacteria. Master’s Thesis, Seoul National University, February 2013.

b) Lee, J.; Kim, H.; Lee, T. G.; Yang, I.; Won, D. H.; Choi, H.; Nam, S. –J.; Kang, H. J. Nat. Prod. 2014, 77, 1528−1531.

Elucidation of planar structures and bioactivities for anmindenol A and B refer to a), the introduction and determination of the absolute configuration for anmindenol B refer to b). 52 Lee, T. L.; Lee, J.; Sul, W. J.; Iwai, S.; Chai, B.; Tiedje, J. M.; Park, J. Appl. Environ. Microbiol. 2011, 77, 3888–3891. 53 Wilms, R.; Sass, H.; Köpke, B.; Köster, J.; Cypionka, H.; Engelen, B. Appl. Environ. Microbiol. 2006, 72, 2756–2764. 54 Poremba, K.; Tillmann, U.;Hesse, K. J. Helgol. Mar. Res.1999, 53, 19–27. 55 Pierre, G.; Graber, M.; Rafiliposon, B. A.; Dupuy, C.; Orvain, F.; Cringnis, M. D.; Maugard, T. Microb. Ecol. 2012, 63, 157–169.

52

collected at Dongmak, located on the west cost of Korea, showed an average

sequence similarity to bacterial strains with sequences in GenBank of only

88.4%, ranging from 74.9 to 97.6%.56 These data suggested that tidal flat

sediments could be a great resource for discovering new microorganisms

that produce unique secondary metabolites.

During the course of our screening program designed to discover bacterial

secondary metabolites as inhibitors of inflammation, inducible nitric oxide

synthase (iNOS) was targeted. Nitric oxide (NO) is a free radical gas with

diverse physiological and pathological functions for neurotransmission, host

defense, and cardiovascular function in mammals.57 It is also important for

regulating immune cell function and as an activator in the cell-mediated

rejection of allergenic transplants.58 NO is produced by three isoforms of

NO synthases (NOS), neuronal (nNOS), inducible (iNOS), and endothelial

(eNOS), which catalyze the conversion of arginine to citrulline.27 However,

NO overproduction leads to numerous human diseases, such as asthma,

diabetes, inflammation, septic shock and chronic inflammatory diseases. 27

Thus, the specific control of NO production offers great therapeutic value.

In particular, the use of selective iNOS inhibitors could be beneficial in the

inflammatory process.28

56 Kim, B. S.; Oh, H. M.; Kang, H.; Park, S. S.; Chun, J. J. Microbiol. Biotechnol. 2004, 14, 205–211. 57 Surup, F.; Wagner, O.; Frieling, J. v.; Schleicher, M.; Oess, S.; Müller, P.; Grond, S. J. Org. Chem. 2007, 72, 5085–5090. 58 Hobbs, A.; Higgs, A.; Moncada, S. Annu. Rev. Pharmacol. Toxicol. 1999, 39, 191–220.

53

We investigated marine-derived bacterial strains isolated from tidal flat

sediments at Anmyeon Island on the west coast of Korea to discover novel

iNOS inhibitors, by evaluating the strain CMDD10D111. This strain shares

97.4% 16S rRNA gene sequence identity with Streptomyces

phaeopurpureus, indicating it could be a new Streptomyces sp. LC-MS

analysis of an extract of CMDD10D111 growing in Mar4 media to which

was added 3% DMSO revealed the presence of m/z peaks at 215.1 and 237.0

with an unusual chromophore. Large-scale fermentation and flash

chromatography followed by HPLC yielded anmindenols A (5) and B (6).

Anmindenols A (5) and B (6) are sesquiterpenoids possessing a unique

indene moiety. Terpenoids consist of important cell components such as

steroids, carotenoids, and vitamins, and also have been reported to possess a

broad variety of biological activities with a diversity of structures. 59

Traditionally, the producers of terpenoid secondary metabolite are plants,

insects, fungi and some marine invertebrates.60 Actinomycetes are also now

known as a source of producing terpenoid natural products including

sesquiterpenoids. In particular, nitropyrrolins A-E (3.1 – 3.5) 61 and

neomarinone(3.6)62 are meroterpenoids bearing a sesquiterpenoid moiety

59 Kuzuyama, T.; Seto, H. Nat. Prod. Rep. 2003, 20, 171–183 60 Gallagher, K. A.; Fenical, W.; Jensen, P. R. Curr. Opin. Biotechnol. 2010, 21, 794–800. 61 Kwon, H. C.; Espindola A. P. D. M.; Park, J. S.; Prieto-Davó, A; Rose, M.; Jensen, P. R.; Fenical, W. J. Nat. Prod. 2010, 73, 2047–2052. 62 Hardt, I. H.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 2000, 41, 2073–2076.

54

isolated from marine-derived actinomycetes (Figure 3.1) and caryolane-

1,7α-diol(3.7) 63 , 1,6,11-eudesmanetriols(3.8)33 and 11-eudesmene-1,6-

diol(3.9)33 are sesquiterpenoids discovered from Streptomyces sp. (Figure

3.2). However, indene-containing sesquiterpenoids, such as gloeophyllols

A-C (3.10-3.12) isolated from the mushroom Gloeophyllum sp. 97022, are

rare (Figure 3.3).64 In addition, there are other reported sesquiterpenoids

with a saturated 6,5 ring system. 2-Octahydro-3a,7,7,7a-tetramethyl-1-

methylene-1-H-indenemethanol (3.13), its ferulic ester (3.14) and 3b,4,4,7a-

tetramethyl-1-H-decahydroindeno[1,2-c]furan-3-ol (3.15) are hydroindenes

that have been isolated from the plant Thapsia Villosa. 65 Calenzanol

(3.16)66 and illudins S (3.17)37 and M (3.18)67 were discovered from the

red alga Laurencia microcladia and from the mushroom Clitocybe illudens,

respectively (Figure 3.4). Anmindenols A and B are the first sesquiterpenoid

natural products containing an indene moiety discovered from

actinomycetes.

63 Yang, Z.; Yang, Y.; Yang, X.; Zhang, Y.; Zhao, L.; Xu, L.; Ding, Z. Chem. Pharm. Bull. 2011, 59, 1430–1433. 64 Rasser, F.; Anke, T.; Sterner, O. Phytochemistry 2000, 54, 511–516. 65 Smitt, U. W.; Cornett, C.; Norup, E.; Christensen, S. B. Phytochemistry 1990, 29, 873–875 66 Guella, G.; Skropeta, D.; Breuils, S.; Mancini, I.; Pietra, F. Tetrahedron Lett. 2001, 42, 723–725. 67 McMorris, T. C.; Anchel, M. J. Am. Chem. Soc. 1965, 87, 1594–1600.

55

Figure 3.1 Sesquiterpenoids from marine-derived actinobacteria

Figure 3.2 Sesquiterpenoids from Streptomyces sp.

56

Figure 3.3 Indene-containing sesquiterpenoids

Figure 3.4 Sesquiterpenoids with a saturated 6,5 ring system

57


3.2.1 Structural Elucidation of Anmindenols

3.2.1.1 Anmindenol A (5)

Anmindenol A (5) was isolated as a yellow amorphous solid. Its

molecular formula was determined to be C15H18O based on HRFABMS

with seven degrees of unsaturation. The 1H NMR spectrum of 5 displayed

three aromatic protons [δH 7.18 (d, J = 7.6 Hz), 7.03 (d, J = 7.6 Hz), 7.38

(s)], two olefinic protons [δH 6.67 (s), 6.36 (d, J = 10.0 Hz)], and one methyl

singlet [δH 2.37]. The 1H NMR spectrum also showed a methyl doublet

integrating for six hydrogens [δH 1.13, (d, J = 6.7 Hz)]. The COSY cross

peaks of H3-12 and H3-13 to H-11 indicated that these methyls were coupled

to a methine proton (Table 3.1). In addition, the 13C NMR and HSQC

spectroscopic data revealed five olefinic carbons and five downfield shifted

quaternary carbons. These data and the molecular formula indicated that 5

contained two rings. The presence of 15 carbon signals, including of one

methylene carbon [δC 60.2], one upfield shifted quaternary carbon [δC 29.8]

and three methyl carbon signals [δC 23.5, 23.5, 21.8], in the 13C NMR

spectrum suggested that 5 was a sesquiterpenoid.

The COSY correlations of H-9 and H-6 to H-7, and the long-range

HMBC correlations from a methyl singlet H3-15 to carbons C-7, C-8 and C-

58

9 allowed the establishment of a trisubstituted-benzene moiety bearing a

methyl group at position C-8. The remaining unknown part of the structure

with the indene moiety was identified by interpreting the COSY and HMBC

spectroscopic data. An olefinic proton H-10 coupled to the methine proton

H-11 in the COSY spectrum was observed to exhibit HMBC correlations to

the carbons C-1, C-2 and C-3, which permitted the C-1/C-2/C-3 attachment.

Lastly, HMBC correlations from the methylene protons H2-14 to the

carbons C-3, C-4 and C-5 and from H-3 to the carbons C-1, C-2 and C-4

allowed the assignment of anmindenol A to be completed, as shown in

Figure 3.5. The 10E double-bond geometry was assigned by NOESY

correlations between H-3 and H-11.

Figure 3.5 Structure of anmindenol A (5) with COSY, key

HMBC and NOESY correlations.

COSY HMBC NOESY

59

Table 3-1. NMR data for anmindenol A (5, CDCl3) a

Amorphous yellow solid

Molecular formula : C15H18O

HRFABMS : m/z 213.2179 [M-H]-

(calcd for C15H17O, 213.1280)

LRESIMS : m/z 215.1[M+H]+

IR (film) νmax : 3444, 1633 cm-1

UV (MeOH) λmax : 202, 262, 305 nm

anmindenol Aa

No δC, mult.b δH, (J in Hz) COSY HMBC NOESY

1 138.1, C

2 137.1, C

3 120.6, CH 6.67, s 10 1, 2, 4, 5, 14 11, 14

4 144.4, C

5 138.5, C

6 118.9, CH 7.18, d (7.6) 1, 2, 4, 7, 8 14

7 127.8, CH 7.03, d (7.6) 6, 9 1, 5, 6, 9, 15 15

8 135.3, C

9 120.1, CH 7.38, s 1, 2, 5, 7, 15 10, 15

10 138.8, CH 6.36, d (10.0) 1, 3, 11, 12, 13 12, 13

11 29.8, CH 3.00, m 10 2, 5, 10, 12, 13 12, 13

12 23.5, CH3 1.13, d (6.7) 11 10, 11

13 23.5, CH3 1.13, d (6.7) 11 10, 11

14 60.2, CH2 4.73, s 3, 10 1, 3, 4, 5

15 21.8, CH3 2.37, s 7, 9 1, 7, 8, 9 a600 MHz for 1H NMR and 150 MHz 13C NMR. bNumbers of attached protons were determined by analysis of 2D spectroscopic data.

60

3.2.1.2 Anmindenol B (6)

Anmindenol B (6) was isolated as a yellow amorphous solid and the

molecular formula of 6 was determined to be C15H20O2 based on

HRFABMS. The 1H NMR spectrum of 6 was almost identical to that of 5

except for the presence of one additional methylene resonance. The 13C

NMR data of 6 were also similar to those of 5 except for the upfield-shifted

carbon signals for C-3 [δC 41.3] and C-4 [δC 81.1]. HMBC correlations from

H-3 to carbons C-4, C-5 and C-14 and the carbon chemical shift for C-4 [δC

81.1] indicated that 6 had a hydroxy group at C-4. The interpretation of 2D

NMR spectroscopic data permitted the identification of the structure of 6

(Figure 3.6, Table 3.2).

Figure 3.6 Structure of anmindenol B (6) with COSY and

key HMBC correlations.

COSY HMBC

61

Table 3.2. NMR data for anmindenol B (6, CDCl3) a

Amorphous yellow solid


HRFABMS : m/z 231.1377 [M-H]-

(calcd for C15H19O2, 231.1386)

LRESIMS : m/z 233.1[M+H]+

IR (film) νmax : 3444, 1633 cm-1


anmindenol Ba No δC, mult.b δH, (J in Hz) COSY HMBC 1 134.6, C 2 141.5, C

3 41.3, CH2 3.00, d (16.6) 2.62, d (16.9) 1, 2, 4, 5, 10, 14

1, 2, 4, 5, 10, 14

4 81.1, C 5 142.9, C 6 123.7, CH 7.24, d (7.4) 7 2, 4, 8, 9 7 129.0, CH 6.99, d (7.2) 6 5, 9, 15 8 139.1, C 9 120.5, CH 7.19, d (3.1) 1, 5, 7, 10, 15

10 129.0, CH 5.75, d (9.3) 3, 11 2, 3, 12, 13 11 29.0, CH 2.48, m 1, 10 12 22.9, CH3 0.98, d (4.8) 10, 11 13 22.8, CH3 0.98, d (5.5) 10, 11

14 69.1, CH2 3.67, d (10.7) 3.53, d (10.2) 4, 5

4, 5

15 21.5, CH3 2.29, s 5, 7, 8, 9 a700 MHz for 1H NMR and 175 MHz 13C NMR. bNumbers of attached protons were determined by analysis of 2D spectroscopic data.

62

To determine the absolute configuration for C-4, we conducted an ECD

experiment by using the Mo2(OAc)4 circular dichroism (CD) method.68 A

metal complex of a vicinal diol (cyclic and acyclic 1,2-diols) and

Mo2(OAc)4 acts as an auxiliary chromophore. As a consequence, the

conformational freedom of the flexible molecule gives an induced CD curve

at 305 nm. The observed sign of the Cotton effect induced by the O-C-C-O

torsion angle allows to assign the absolute configuration.69 Based on the

empirical rule, an (R)-1,2-diol with Mo2(OAc)4 gives rise to a negative CD

band at 305 nm, whereas a complex having the S configuration gives a

positive CD band at 305 nm.38 The negative cotton effect at 305 nm

observed in the CD spectrum of the metal complex of 6 (6-Mo2(OAc)4)

establishes the R-configuration for C-4.

Dehydration of 6 will form the more highly conjugated 5. It is possible

that 5 is an artifact formed during the extraction and isolation procedure,

although 5 was detected in the original extract by LC-MS and there was no

indication that 6 was converted to 5 during characterization of 6.

68 Frelek, J.; Ikekawa, N.; Takatsuto, S.; Snatzke, G. Chirality 1997, 9, 578–582. 69 Liu, H. -B.; Zhang, C. -R.; Dong, S. -H.; Yang, S. -P.; Sun, Q.; Geng, M. -Y.; Yue, J. -M. J. Asian Nat. Prod. Res. 2012, 14, 224–234.

63



Compounds 5-6 had no significant antibacterial activity against gram-

positive strains including Staphylococcus aureus ATCC 6538, Bacillus

subtilis ATCC 6633, Kocuria rhizophila ATCC 9341, S. epidermidis

ATCC12228 and gram-negative strains including Klebsiella pneumoniae

ATCC 4352, Escherichia coli ATCC 11775, and Salmonella typhimurium

ATCC 14028.

3.2.2.2 Cytotoxicity

Compounds 5-6 did not display any significant cytotoxicities against a

human renal cancer cell line (A498) and two human pancreatic cancer cell

lines (MIA-paca and PANC-1) up to a compound concentration of 100 μM.

3.2.2.3 Anti-Inflammatory Activity

Compounds 5-6 were tested for their effects against NO production in

lipopolysaccharide (LPS)-activated mouse macrophage RAW264.7 cells. 5

and 6 inhibited NO production with IC50 values of 23 μM and 19 μM,

respectively (Figure 3.6, Figure 3.7).

64


65


66



The optical rotation was measured using a Rudolph Research Autopol III

polarimeter with a 5 cm cell. The UV spectrum was recorded in a Scinco

UVS-2100 with a path length of 1 cm. CD spectra were collected in an

Applied Photophysics Chirascan plus CD spectrometer with a 0.5 mm path-

length rectangular cuvette. Infrared spectra were recorded on a Thermo

Electron Corporation spectrometer. NMR spectral spectroscopic data of

anmindenols A and B were obtained using Bruker Avance 600 MHz and

700 MHz spectrometers, respectively [CDCl3 (�H 7.26; �C 77.0) was used as

an internal standard]. EI-MS and FAB-MS spectra were measured on a

JEOL, JMS-AX505WA mass spectrometer. Low resolution LC-MS data

were measured using an Agilent Technologies 6120 quadrupole LC/MS

system with a reversed-phase C18 column (Phenomenex luna 5u (2), 4.6 mm

× 50 mm, 5μm) at a flow rate of 1.0 mL/min. The extracts were separated by

HPLC WATERS 1525 binary HPLC pump, WATERS 2489 UV visible

detector using an MG2 C18 (250 mm × 10 mm, 5 μm) reversed-phase HPLC

column.

67


Strain CMDD10D111 was isolated from marine sediment from Anmyeon

Island, Chungcheongnam-do, South Korea in 2010 (Figure 3.8). The 16S

rRNA gene sequence using primers 27f and 1492r for this strain has been

deposited with GenBank (accession number KC136293). It shares 97.4%

sequence identity with the type strain for Streptomyces phaeopurpureus

(EU274371.1).

Figure 3.9 Strain CMDD10D111


Strain CMDD10D111 was cultured in 40 4 L Pyrex flasks each containing

1 L of the medium Mar 4 (2 g kelp meal, 2 g D-mannitol, 1 g fish meal, KBr

20 g/L, Fe2(SO4)3·4H2O 8 g/L, 30 mL DMSO dissolved in 970 mL natural

68

seawater) at 25 °C with shaking at 150 rpm. After 10 days, the broth was

extracted with EtOAc and evaporated to yield an organic extract of

CMDD10D111 (3.8 g).

The extract (3.8 g) was subjected to silica flash column chromatography

using step-gradient elution of MeOH in CH2Cl2 (0%, 1%, 2%, 5%, 10%,

50%, 100%) to afford seven fractions (Fr 1-Fr 7). Fr 1 (740.9 mg), which

contained the mixture of anmindenols, was further purified by C18 HPLC

using 55% CH3CN in H2O to obtain anmindenols A (5, 3.2 mg) and B (6,

1.5 mg).

3.3.4 Electronic Circular Dichroism of a Dimolybdenum

Complex

Anmindenol B (0.5 mg) was dissolved in 250 μL of DMSO and the

solution was divided into two 125 μL aliquots. A 125 μL aliquot of 8.62

mM DMSO solution of Mo2(OAc)4 was added to 125 μL of prepared

anmindenol B solution to make 4.32 mM anmindenol B and Mo2(OAc)4

mixture solution. The mixture was kept for 30 min to form a stable metal

complex, after which the ECD spectrum was recorded for induced CD. The

observed band of the ECD curve at 305 nm was used to determine the

absolute configuration of C-4.

69

3.3.5 Bioassays

3.3.5.1 MIC Assay

The MIC assay was performed with a same protocol according to 2.3.5.1

(p. 46) against Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC

6633, Kocuria rhizophila ATCC 9341, S. epidermidis ATCC12228 and

gram-negative strains including Klebsiella pneumoniae ATCC 4352,

Escherichia coli ATCC 11775, and Salmonella typhimurium ATCC 14028.

3.3.5.2 MTT Assay

The MTT assay was performed with a same protocol according to 2.3.5.2

(p. 47) with human renal cancer cell line (A498) and two human pancreatic

cancer cell lines (MIA-paca and PANC-1).

3.3.5.3 Nitric Oxide Assay

The NO assay was performed for compounds by measuring NO

production in LPS-induced RAW 264.7 mouse macrophage cells according

to a previously protocol (2.3.5.3., p.48).

70

Chapter 4.

11A020-3A from mudflat derived Actinoalloteichus

hymeniacidonis

4.1 Introduction

Terrestrial bacteria and fungi have been screened to develop

pharmaceutical agents over the last decades. However, recent studies to

discover microbial metabolites have been focusing on investigation of

marine-derived bacteria and fungi as a new source of metabolites.

The genus Actinoalloteichus belongs to Actinomycetes, the most

significant producer of structurally new and biologically active substances,70

and is known as halophilic and salt-tolerant strains. The genus currently

contains only for species, Actinoalloteichus cyanogriseus, Actinoalloteichus

spitiensis, Actinoalloteichus hymeniacidonis and Actinoalloteichus

nanshanensis. Chemical studies of the genus Actinoalloteichus have been

rarely reported. Caerulomycins F-K (4.1-4.6)71, cyanogrisides A-D (4.7-

4.10)72 and cyanogramide (4.11)73 were isolated from cyanogriseus sp.

70 Berdy, J. J. Antibiot. 2005, 58, 1–26. 71 Fu, P.; Wang, S.; Hong, K.; Li, X.; Liu, P.; Wang, Y.; Zhu, W. J. Nat. Prod. 2011, 74, 1751–1756. 72 Fu, P.; Liu, P.; Li, X.; Wang, Y.; Wang, S.; Hong, K.; Zhu, W. Org. Lett. 2011, 13, 5948-5951. 73 Fu, P.; Kong,F.; Li, X.; Wang, Y.; Zhu, W. Org. Lett. 2014, 16, 3708−3711.

71

Neomaclafungins A I (4.12-4.20),74 26-membered antifungal macrolides,

were the first reported secondary metabolites from hymeniacidonis sp. and

cyclopentenone derivatives (4.21-4.27)75,76 with low cytotoxicites against

human lung adenocarcinoma cell line A549, human leukemia cell line K562,

and human renal carcinoma cell line ACHN were isolated from

Nanshanensis sp. (Figure 4.1).

During the course of a screening program to search for structurally unique

metabolites from rare halophilic strain, a novel compound, 11A020-3A (7),

was isolated from Actinoalloteichus hymeniacidonis sp. with two known

compounds, nocardiopsins A (4.28) and B (4.29), FKBP12-binding

macrolide polyketides (Figure 4.2).77 Herein, we describe the cultivation,

isolation, structure elucidation of compound 7.

74 Sato, S.; Iwata, F.; Yamada, S.; Katayama, M. J. Nat. Prod. 2012, 75, 1974-1982. 75 Wang, X.-J.; Zhang, J.; Qian, P. –T.; Wang, J. –D.; Liua, C. –X.; Xiang, W. –S. J. Asian. Nat. Prod. Res. 2014, 16, 587-592. 76 Wang, X.-J.; Zhang, J.; Wang, J. –D.; Qian, P. –T.; Liua, C. –X.; Xiang, W. Nat. Prod. Res. 2013, 27, 1863-1869. 77 Raju, R.; Piggott, A. M.; Conte, M.; Tnimov, S.; Alexandrov, K.; Capon, R. J. Chem. Eur. J. 2010, 16, 3194-3200.

72

Figure 4.1 Secondary metabolites from the genus Actinoalloteichus

73

Figure 4.2 Nocardiopsins A and B


4.2.1 Structural Elucidation of 11A020-3A

11A020-3A (7) was obtained as a yellow amorphous solid. The molecular

formula of 7 was deduced as C24H38O7 based on the HRFABMS data. The

13C NMR spectrum of 7 (Table 4.1) resolved 24 carbon signals that were

classified by phase sensitive HSQC spectra as five methyls, nine methines

including five olefinic, five methylenes and five quaternary carbons

including one ketone group (δC 203.6, C-11), one ester carbonyl group (δC

175.3, C-1). The coupled 1H NMR signals at H-7 (δH 5.96, dd, J = 15.4, 1.4)

and H-8(δH 5.81, dd, J = 6.3) suggested the presence of an E-1,2-

disubstitued moiety. The COSY cross peaks of H-8 to H-7 and H-9 (δH 4.33,

sext, J = 6.3), H-9 to H3-10 (δH 1.28, d, J = 6.3) established the first partial

structure. Second partial structure was identified by the COSY cross peaks

74

of H-4 (δH 2.44, m) to H2-3(δH 1.87, m), H-5(δH 3.28, d, J = 6.3) and H3-

22(δH 0.99, d, J = 7.7), H2-3 to H-2(δH 5.62, m). The COSY cross peak of H-

16 (δH 2.68, m, δH 1.94, m) to H2-17 (δH 6.27, q, J = 8.4) and H2-15 (δH 1.62,

t, J = 13.3), H-18 (δH 6.47, t, J = 10.5) to H2-17 and H-19 (δH 7.37, d, J =

11.9) established the rest partial structure. HMBC correlation from H3-21

(δH 1.35, s) to C-5 (δC 79.1), C-6 (δC 75.0) and C-7 (δC 134.2), indicated the

first partial structure was connected with the second partial structure.

HMBC correlation from H3-24 (δH 9.3, s) to C-11 (δC 203.6), C-19 (δC

135.3) and C-20 (δC 133.2), from H2-13 (δH 2.44, m) to C-23 (δC 25.7) and

C-14 (δC 74.8), from H2-16 to C-14 formed a 11-membered ring structure.

H2-13, H3-23 (δH 1.47, s) and H-2 showed HMBC correlations to a carbonyl

C-1 (δC 175.3), then the assignment of compound 7 were completed as

shown in Figure 4.3.

75

Figure 4.3 Structure of 11A020-3A (7) with COSY and key HMBC

correlations

76

Table 4.1 NMR data for 11A020-3A (7, MeOH-d4)a

Yellow, amorphous solid


HRFABMS : m/z 439.2564 [M+H]+

(calcd for C24H39O7, 439.2697)

LRESIMS : m/z 461.2 [M+Na]+

IR (film) νmax : 1671 cm-1


11A020-3Aa

No δC, major, mult.b δH, major (J in Hz) COSY HMBC δC, minor,

mult.b δH, minor (J in Hz)C

1 175.3, C 175.4, C 2 74.9, CH 5.62, md 3, 4 1, 3, 4, 22 74.8, CH 5.62, md 3 27.1, CH2 1.87, me 2 4 36.6, CH 2.44, mf 5, 22 2, 3, 5, 6, 22 5 79.1, CH 3.28, d (6.3) 4 2, 4, 6, 7, 21, 22 6 75.0, C 74.9, C 7 134.2, CH 5.96, dd (15.4, 1.4) 8 6, 9, 21 134.4, CH 5.98, dd (15.4, 1.4) 8 132.4, CH 5.81, dd (15.4, 6.3) 9 6, 7, 9, 10 132.3, CH 5.79, dd (15.4, 6.3) 9 67.8, CH 4.33, sext (6.3) 10 7, 8, 10 68.0, CH 4.33, sext (6.3)

10 22.1, CH3 1.28, d (6.3) 9 8, 9 11 203.6, C

12 33.0, CH2 3.06, m 2.43, mf

12b, 13a 12a, 13a, 13b

11, 13, 14 11, 20

13 39.2, CH2 1.99, dt (14, 2.1) 1.91, me

12b 12a

1, 11, 12 1, 12 ,14, 23

14 73.8, C 15 26.3, CH2 1.62, t (13.3) 16a 16, 17

16 25.7, CH2 2.68, m 1.94, m

15, 16b, 17 16a, 17

15, 17, 18 14, 15, 17, 18, 23

17 140.8, CH 6.27, q (8.4) 16a, 16b, 18 16, 19 18 124.7, CH 6.47, t (10.5) 17, 19 16, 19, 20 19 135.3, CH 7.37, d (11.9) 18 11, 17, 18, 20, 24 20 133.2, C 21 24.2, CH3 1.35, s 5, 6, 7, 24.4, CH3 1.34, s 22 11.3, CH3 0.99, d (7.7) 4 2, 4, 5, 6 11.2, CH3 0.99, d, 7.7 23 25.7, CH3 1.47, s 1, 12, 13, 14, 16 24 9.3, CH3 1.82, s 11, 17, 18, 19, 20

a700 MHz for 1H NMR and 175 MHz for 13C NMR in MeOH-d4. bNumbers of attached protons were determined by analysis of 2D NMR spectroscopic data. cOnly selected resonances were observed for the minor isomer due to overlapping with the major isomer. dOverlapping singals with major and minor isomers e,f Overlapping signals.

77



Compounds 7 showed antibacterial activity with IC50 values of 0.1 μM,

64 μM, 8 μM and 64 μM against Bacillus subtilis ATCC 6633, Kocuria

rhizophila ATCC 9341, Staphylococcus aureus ATCC 6538 and

Escherichia coli ATCC 11775, respectively.

78



NMR spectral spectroscopic data of compound 7 were obtained using a

Bruker Avance 700 MHz spectrometers. HRESIMS data were measured on

a SCIEX, Q-TOF 5600 mass spectrometer. Low resolution LC-MS data

were measured using an Agilent Technologies 6120 quadrupole LC/MS

system with a reversed-phase C18 column (Phenomenex luna 5u (2), 4.6 mm

× 50 mm, 5μm) at a flow rate of 1.0 mL/min. The extracts were separated by

HPLC WATERS 1525 binary HPLC pump, WATERS 2489 UV visible

detector using a Phenomenex luna C18 column (250 mm × 10 mm, 5 μm,

flow rate = 2.0 mL/min) reversed-phase HPLC column.


Strain CMDD11A020 was obtained from a marine sediment from Boreum-

Island, Incheon-si, South Korea in 2011. Mud sediments were dried by air

for 24 hours in a clean bench and given a heat shock at 55 °C for 5 minutes

in a low temperature incubator. Dried samples were lightly mortared by

glass rod and suspended in sterilized sea water, then spread on variously

prepared solid agar medium using L-shaped cell spreaders. These plates

were placed in 27 °C chamber and monitored for 1 to 3 months to obtain

79

colonies considered as actinomycetes. Strain CMDD11A020 was peaked

from ISP2 media agar plate showing brownish-black, branched hyphae

(Figure 4.4). The 16S rRNA gene was cloned using universal primers 27F

and 1492R and showed 99.5% (1406/1413) similarity to Actinoalloteichus

hymeniacidonis strain HPA177.

Figure 4.4 Strain CMDD11A020


The bacterial strain CMDD11A020 was cultured at 27°C with shaking at

140 rpm in 20 L Pyrex flask each containing 1 L of the medium SYP (10 g

soluble starch, 4 g yeast extract, 2 g peptone, 1 g CaCO3 seawater dissolved

in 1 L artificial seawater). After 10 days, the broth was extracted two times

with ethyl acetate and evaporated to yield 2.13 g of crude organic extract.

The extract (2.13 g) was separated by silica normal phase column MPLC

80

using step gradient elution of MeOH in dichloromethane (0%, 1%, 2%, 5%,

7.5%, 10%, 20%, 50% and 100%). Fraction 3 was further purified using

reversed-phase HPLC (Phenomenex luna C18 column, 250 mm × 10 mm, 5

μm, 2.0 mL/min, UV = 280 nm and 310 nm; CH3CN : H2O = 45 : 55) to

obtain 3.5 mg of compound 7.

4.3.4 Bioassays

4.3.4.1 MIC assays

The MIC assay was performed for a compound 7 against Bacillus subtilis

ATCC 6633, Kocuria rhizophila ATCC 9341, Staphylococcus aureus

ATCC 6538 and Escherichia coli ATCC 11775 according to a previously

protocol (2.3.5.1, p. 46).

81

Chapter 5. Usnic Acid Derivatives from

Mycosphaerella nawae

5.1 Introduction

Mycosphaerella nawae is the represent agent of circular leaf spot of

persimmon.78 (-)-Mycousnine (5.1) and (+)-isomycousnine (5.2), potent

phytotoxins and antimicrobial compounds, were reported as the first

secondary metabolites from Mycosphaerella nawae.79 These compounds

have similar backbone with (+)-usnic acid (5.3).

Usinic acid has been interested in chemists and pharmacologist since it

was first isolated in 1844.80 Usnic acid have been mainly isolated from

lichen genera such as Usnea (Usneaceae), Cladonia (Cladoniaceae),

Lecanora (Lecanoraceae). 81 Biological activities of usnic acid were

antitumor, antiviral, antimicrobial, anti-inflammatory and insecticidal

effects. Despite of these effects, particularly several liver damage limit its

use in medical application.82 Over the past few decades, significant efforts

for reducing the side effects of usnic acid have been performed by searching

78 Abe, Y.; Abe, M.; Hayashi, S.; Ogata, Annu. Rep. Soc. Plant Prot. North Jpn. 1996, 85-87. 79 Sassa, T.; Igarashi, M. Agric. Biol. Chem. 1990, 54, 2231-2237. 80 Ingolfsdottir, K. Phytochemistry 2002, 61, 729-736. 81 Reyim, M.; Adiljan; Abdulla, A. China Brewing 2010, 11, 122-124. 82 Guo, L.; Shi, Q.; Fang, J. L.; Mei, N.; Ali, A. A.; Lewis, S. M.; Leakey, J. E.; Frankos, V. H. J. Environ. Sci. Heal. 2008, 26, 317-338.

82

for its derivatives from natural resources.83

During the course of chemical studies of fungal strain isolated from

marine sediment, we obtained novel usnic acid derivatives with known

compounds: (-)-mycousnine (5.1), (+)-isomycousnine (5.2), (+)-usnic acid

(5.3) and (+)-9-O-methylplacodiolic acid (5.4) 84 from the extract of

Mycosphaerella nawae (Figure 5.1). Herein, the isolation, structural

elucidation of new compounds are discussed.

Figure 5.1 Structures of known compounds from fungal strain F8015-2B

83 Seo, C.; Sohn, J. H.; Park, S. M.; Yim, J. H.; Lee, H. K.; Oh, H. J. Nat. Prod. 2008, 71, 710-712. 84 Millot, M.; Kaouadji, M.; Champavier, Y.; Gamond, A.; Simon, A.; Chulia, A. J. Phytochem. Lett. 2013, 6, 31-35.

83


5.2.1 Structural Elucidation of Usnic Acid Derivatives

5.2.1.1 F8015-2B-2E (8)

Compound 8 was obtained as yellow amorphous. Its molecular formula,

C18H20O7, was deduced from LC/MS (m/z 371.1 [M + H]+) and 13C NMR

data. The 1H NMR spectrum displayed signals for five methyl groups (δH

1.62, 2.04, 2.61, 3.49 and 3.81) including two methoxys, two hydroxy-group

protons (δH 13.34, 9.34), one methylene protons (δH 3.15, d, 2.96, d, J =

17.5) and an olefinic proton at δH 5.55 (H-4). The 13C NMR spectrum

showed 18 carbon resonances, two ketocarbonyls, four oxygenated olefinics,

five olefinics, five methyls, and one sp3 quaternary carbon at δC 57.9 (C-9b).

The HMBCs from H3-10 to C-1, C-4a, and C-9a suggested that the angular

methyl group was located at C-9b, while those from H3-13 to C-12 and C-6

indicated that C-13 was connected to C-6. 8 was revealed its structure had

similarity, especially of the A and B rings with 5.1 with comparing of the

NMR data (Table 5.1). This was supported by cross-peaks in the HMBC

spectrum, from H3-11 to C-9 and C-7, from H-4 to C-2, C-4a, and C-9b,

from 7-OH to C-5a and C-8, and from 9-OH to C-9a and C-8 (Figure 5.2).

84

Figure 5.2 Structure of F8015-2B-2E (8) with key HMBC correlations

85

Table 5.1 NMR data for (-)-mycousnine (5.1, CDCl3) a and F8015-2B-2E (8,

CDCl3) a



HRFABMS : m/z 349.1302 [M+H]+

(calcd for C18H21O7, 349.1287)


UV (MeOH) λmax : 215, 295

(-)-mycousnine F8015-2B-2E Position δC, type (δH) δC, type δH, multb (J in Hz) HMBC

1 197.7, C 200.5, C 2 110.7, C 100.6, CH 5.55, s 3 194.6, C 175.4, C

4 37.9, CH2 (3.31, 3.17) 34.3, CH2

3.15, d (17.5) 2.96, d (17.5) 2, 4a

4a 109.6, C 111.6, C 5a 156.6, C 157.1, C 6 101.8, C 102.0, C 7 163.4, C 163.3, C 8 107.2, C 107.5, C 9 159.1, C 159.6, C 9a 105.3, C 106.5, C 9b 59.8, C 57.9, C

10 (9b-Me) 17.3, CH3 (1.63) 16.6, CH3 1.62, s 1, 4a, 9a, 9b 11 (8-Me) 7.2, CH3 (2.04) 7.4, CH3 2.04, s 5a, 6, 8 12 (6-CO-) 201 201.2, C 13 (6-COMe) 31.2, CH3 (2.59) 31.3, CH3 2.61, s 6, 13 14 (4a-OMe) 51.3, CH3 (3.54) 50.9, CH3 3.49, s 15 (3-OMe) 56.9, CH3 3.81, s

2-CO- 202.4, C 2-COMe 27.4, CH3 (2.56) 7-OH 13.36 OH 13.34, s 6, 7, 8, 9 9-OH 9.4 OH 9.34, s 7, 8, 9a a700 MHz for 1H NMR and 175 MHz for 13C NMR in CDCl3. bNumbers of attached

protons were determined by analysis of 2D NMR spectroscopic data.

86

5.2.1.2 F8015-2B-2I (9)

Compound 9 was obtained as yellow amorphous. Its molecular formula

was determined to be C18H20O7 based on LC/MS (m/z 371.2 [M + H]+) and

13C NMR data. Interpretation of the NMR data revealed that the structure of

9 was almost identical with that of 8 except the exchange of the C-11

methyl and C-12 acetyl groups in 9 compared with 8. In the result, these

differences of major chemical shifts at C-5a, C-9, C-12, and C-13 may be

shown presumably. The HMBC correlations from H3-11 to C-6, C-7, and C-

5a and from H3-15 to C-8 supported this exchange. These differences may

be demonstrated by deshielding of the resonances as the location of the

acetyl group in the anisotropic deshielding zone of ring A. This supported

the shielded C-6 resonance (δC 100.4) compared with 8. (Figure 5.3, Table

5.2).

Figure 5.3 Structure of F8015-2B-2I (9) with key HMBC correlations

87

Table 5.2 NMR data for F8015-2B-2I (9, CDCl3) a



HRFABMS : m/z 349.1305 [M+H]+

(calcd for C18H21O7, 349.1287)



F8015-2B-2I Position δC, type δH, multb (J in Hz) HMBC

1 201.0.5, C 2 100.2, CH 5.55, s 1, 3, 4, 9b 3 175.9, C

4 34.2, CH2 3.20, d (17.5) 2.94, d (17.5)

2, 3, 4a, 9b 2, 3, 4a, 9b

4a 110.6, C 5a 165.5, C 6 100.4, C 7 160.7, C 8 107.1, C 9 156.4, C 9a 106.1, C 9b 58.93, C

10 (9b-Me) 16.2, CH3 1.66, s 1, 4a, 9a, 9b 11 (8-Me) 7.5, CH3 2.02, s 5a, 6, 8 12 (6-CO-) 203.8, C 13 (6-COMe) 32.9, CH3 2.73, s 8, 12 14 (4a-OMe) 50.3, CH3 3.47, s 4a 15 (3-OMe) 56.8, CH3 3.84, s 3

7-OH OH 9.61, s 7, 8, 9 9-OH OH 14.32, s 7, 8, 9a a700 MHz for 1H NMR and 175 MHz for 13C NMR in CDCl3. bNumbers of attached


88

5.2.1.3 F8015-2B-2B (10)

Compound 10 was subjected as yellow amorphous. Its molecular formula

was determined to be C16H18N2O6 from HRFABMS and LC/MS (m/z 357.1

[M + Na]+). Interpretation of the NMR data revealed that the structure of 10

(Figure 5.4, Table 5.3) had a similar chemical backbone of 8 and

mycousnine, but suggested the presence of nitrogen in aromatic backbone

and two nitrogen protons based on HRFABMS with nitrogen rule, 13C NMR

spectrum and 1H NMR spectrum.

Figure 5.4 Structure of F8015-2B-2B (10) with key HMBC correlations

89

Table 5.3 NMR data for F8015-2B-2B (10, MeOH-d4) a


Molecular formula : C16H18N2O6

HRFABMS : m/z 334.1206 [M]+

(calcd for C16H18O6, 334.1165)



F8015-2B-2B Position δC, type δH, multb (J in Hz) HMBC

1 195.7, C 2 101.3, C 3 184.6, C

4 36.1, CH2 3.18, d (17.5) 3.02, d (17.5)

3, 4a, 9b 3, 4a, 9b

4a 112.6, C 5a 157.4, C 6 7 162.1, C 8 105.8, C 9 160.1, C 9a 108.2, C 9b 56.2, C

10 (9b-Me) 15.5, CH3 1.63, s 1, 4a, 9a, 9b 11 (8-Me) 6.1, CH3 1.99, s 7, 8, 9 12 (4a-OMe) 49.6, CH3 3.52, s 4a 13 (2-COMe) 201.6, C 14 (13-OMe) 29.9, CH3 2.63, s 2, 13

7-OH OH 13.37, s 7, 8 a700 MHz for 1H NMR and 175 MHz for 13C NMR in MeOH-d4. bNumbers of attached


90

5.2.1.4 F8015-2B-2C (11)

Compound 11 was isolated as yellow amorphous. Its molecular formula

was determined to be C16H18N2O6 from LC/MS (m/z 357.1 [M + Na]+).

Interpretation of the NMR data, comparing retention time of LC/MS with

that of 10 supported that 11 (Figure 5.5, Table 5.4) was an isomer of 10 as

same as compound 8 was an isomer of 9.

Figure 5.5 Structure of F8015-2B-2C (11) with key HMBC correlations

91

Table 5.4 NMR data for F8015-2B-2C (11, MeOH-d4) a


Molecular formula : C16H18N2O6



F8015-2B-2C Position δC, type δH, multb (J in Hz) HMBC

1 198.0, C 2 99.7, C 3 181.4, C

4 34.6, CH2 3.21, d (17.5) 3.05, d (17.5)

3, 4a, 9b 3, 4a, 9b

4a 111.4, C 5a 156.7, C 6 106.5, C 7 164.4, C 8 9 161.2, C 9a 107.5, C 9b 56.8, C

10 (9b-Me) 15.1, CH3 1.65, s 1, 4a, 9a, 9b 11 (8-Me) 6.1, CH3 1.98, s 7, 9 12 (4a-OMe) 49.2, CH3 3.47, s 4a 13 (2-COMe) 203.8, C 14 (13-OMe) 31.6, CH3 2.68, s 2, 13

7-OH OH 14.08, s 6, 7 a700 MHz for 1H NMR and 175 MHz for 13C NMR in MeOH-d4. bNumbers of attached


92



NMR spectral spectroscopic data were obtained using Bruker Avance 700

MHz spectrometers, respectively [CDCl3 (�H 7.26; �C 77.0) was used as an

internal standard]. Low resolution LC-MS data were measured using an

Agilent Technologies 6120 quadrupole LC/MS system with a reversed-

phase C18 column (Phenomenex luna 5u (2), 4.6 mm × 50 mm, 5μm) at a

flow rate of 1.0 mL/min. The extracts were separated by HPLC WATERS

1525 binary HPLC pump, WATERS 2489 UV visible detector using an

Phenomenex luna C18 column (250 mm × 10 mm, 5 μm, flow rate = 2.0

mL/min).

5.3.2 Fungal Material

F8015-2B was isolated from marine sediment in 5 m depth from Donghae-

si, Gangwon-do, South Korea in 2008. Collected sediment was dried in a

clean bench during 24 h and then crushed using sterile spoon. Powder were

stamped onto 1/3 marine agar media and incubated at 27 °C. After two

weeks, fungal spore with bacterial colonies were observed. This spore was

cultured with repeated inoculation on PDA media plate. F8015-2B was

identified having 99.6% (496/498) similarity to Mycosphaerella nawae

93

strain MY3 (Figure 5.4)

Figure 5.6 Fungal strain F8015-2B


F8015-2B was cultured in 6 L potato dextrose broth (PDB) dissolved in

seawater. The fungus was cultivated with seed agar blocks into 2.5 L plastic

culture flasks containing 1 L of PDB media at 27 °C and 140 rpm in shaking

incubator for a week.

The mycelium was filtered from broth fraction using gauze filtration. The

broth was extracted with EtOAc and the crude extract was evaporated (4.01

g).

The extract of broth fraction was divided in eight fractions with normal

phase silica gel open column chromatography using step-gradient solvent

mixture of dichloromethane and MeOH. Fraction 1 (974.9 mg), 2 (280.1

94

mg) and 3 (420.1 mg) were subjected to reversed phase HPLC

(Phenomenex luna C18 column, 250 mm × 10 mm, 5 μm, flow rate = 2.0

mL/min) with 65 % CH3CN in distilled water. As a result, 8 (7.5 mg), 9 (3.3

mg), 10 (2.2 mg), 11 (1.8 mg), 5.1 (56.3 mg), 5.2 (28.7 mg), 5.3 (6.8 mg)

and 5.4 (2.3 mg) were obtained.

95

Appendix A.

A.1 NMR Spectra of Marinopyrones A-D

A.2 Determination of the Absolute Configuration for Marinopyrone D

96

A.1

NM

R S

pect

ra o

f Mar

inop

yron

es A

-D

Figu

re A

.1.1

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e A (1

)

Figu

re A

.1.2

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of m

arin

opyr

one A

(1)

Figu

re A

.1.3

CO

SY sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one A

(1)

Figu

re A

.1.4

HSQ

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one A

(1)

Figu

re A

.1.5

HM

BC

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e A (1

)

Figu

re A

.1.6

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e B

(2)

Figu

re A

.1.7

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

B (2

)

Figu

re A

.1.8

CO

SY sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

B (2

)

Figu

re A

.1.9

HSQ

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

B (2

)

Figu

re A

.1.1

0 H

MB

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

B (2

)

Figu

re A

.1.1

1 1 H

NM

R sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

C (3

)

Figu

re A

.1.1

2 13

C N

MR

spec

trum

(175

MH

z, C

DC

l 3) o

f mar

inop

yron

e C

(3)

97

Figu

re A

.1.1

3 C

OSY

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e C

(3)

Figu

re A

.1.1

4 H

SQC

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e C

(3)

Figu

re A

.1.1

5 H

MB

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

C (3

)

Figu

re A

.1.1

6 1 H

NM

R sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f mar

inop

yron

e D

(4)

Figu

re A

.1.1

7 13

C N

MR

spec

trum

(175

MH

z, M

eOH

-d4)

of m

arin

opyr

one

D (4

)

Figu

re A

.1.1

8 C

OSY

spec

trum

(700

MH

z, M

eOH

-d4)

of m

arin

opyr

one

D (4

)

Figu

re A

.1.1

9 H

SQC

spec

trum

(700

MH

z, M

eOH

-d4)

of m

arin

opyr

one

D (4

)

Figu

re A

.1.2

0 H

MB

C sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f mar

inop

yron

e D

(4)

A.2

Det

erm

inat

ion

of th

e Abs

olut

e Co

nfig

urat

ion

for M

arin

opyr

one

D

Figu

re A

.2.1

Chi

ral a

naly

sis f

or ra

cem

ic st

anda

rd a

nd m

arin

opyr

one

D (4

) of 2

-met

hylb

utyr

ic a

cid

as 1

-nap

hthy

l am

ide

deriv

ativ

e

Figu

re A

.2.2

CD

spec

trum

of m

arin

opyr

one

D (4

)

98

Figu

re A

.1.1

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e A (1

)

99

Figu

re A

.1.2

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of m

arin

opyr

one A

(1)

100

Figu

re A

.1.3

CO

SY sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one A

(1)

101

Figu

re A

.1.4

HSQ

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one A

(1)

102

Figu

re A

.1.5

HM

BC

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e A

(1)

103

Figu

re A

.1.6

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e B

(2)

104

Figu

re A

.1.7

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

B (2

)

105

Figu

re A

.1.8

CO

SY sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

B (2

)

106

Figu

re A

.1.9

HSQ

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

B (2

)

107

Figu

re A

.1.1

0 H

MB

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

B (2

)

108

Figu

re A

.1.1

1 1 H

NM

R sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

C (3

)

109

Figu

re A

.1.1

2 13

C N

MR

spec

trum

(175

MH

z, C

DC

l 3) o

f mar

inop

yron

e C

(3)

110

Figu

re A

.1.1

3 C

OSY

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e C

(3)

111

Figu

re A

.1.1

4 H

SQC

spec

trum

(700

MH

z, C

DC

l 3) o

f mar

inop

yron

e C

(3)

112

Figu

re A

.1.1

5 H

MB

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of m

arin

opyr

one

C (3

)

113

Figu

re A

.1.1

6 1 H

NM

R sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f mar

inop

yron

e D

(4)

114

Figu

re A

.1.1

7 13

C N

MR

spec

trum

(175

MH

z, M

eOH

-d4)

of m

arin

opyr

one

D (4

)

115

Figu

re A

.1.1

8 C

OSY

spec

trum

(700

MH

z, M

eOH

-d4)

of m

arin

opyr

one

D (4

)

116

Figu

re A

.1.1

9 H

SQC

spec

trum

(700

MH

z, M

eOH

-d4)

of m

arin

opyr

one

D (4

)

117

Figu

re A

.1.2

0 H

MB

C sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f mar

inop

yron

e D

(4)

118

Figu

re A

.2.1

Chi

ral a

naly

sis f

or ra

cem

ic st

anda

rd a

nd m

arin

opyr

one

D (4

) of 2

-met

hylb

utyr

ic a

cid

as 1

-nap

hthy

l am

ide

deriv

ativ

e

119

Figu

re A

.2.2

CD

spec

trum

of m

arin

opyr

one

D (4

)

120

Appendix B.

B.1 NMR Spectra of Anmindenol A and B

B.2 Determination of the Absolute Configuration for Anmindenol B

12

1

B.1

NM

R S

pect

ra o

f Anm

inde

nol A

and

B

Figu

re B

.1.1

1 H N

MR

spec

trum

(600

MH

z, C

DC

l 3) o

f anm

inde

nol A

(5)

Figu

re B

.1.2

13C

NM

R sp

ectru

m (1

50 M

Hz,

CD

Cl 3)

of a

nmin

deno

l A (5

)

Figu

re B

.1.3

CO

SY sp

ectru

m (6

00 M

Hz,

CD

Cl 3)

of a

nmin

deno

l A (5

)

Figu

re B

.1.4

HSQ

C sp

ectru

m (6

00 M

Hz,

CD

Cl 3)

of a

nmin

deno

l A (5

)

Figu

re B

.1.5

HM

BC

spec

trum

(600

MH

z, C

DC

l 3) o

f anm

inde

nol A

(5)

Figu

re B

.1.6

NO

ESY

spec

trum

(600

MH

z, C

DC

l 3) o

f anm

inde

nol A

(5)

Figu

re B

.1.7

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f anm

inde

nol B

(6)

Figu

re B

.1.8

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of a

nmin

deno

l B (6

)

Figu

re B

.1.9

CO

SY sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of a

nmin

deno

l B (6

)

Figu

re B

.1.1

0 H

SQC

spec

trum

(700

MH

z, C

DC

l 3) o

f anm

inde

nol B

(6)

Figu

re B

.1.1

1 H

MB

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of a

nmin

deno

l B (6

)

12

2

B.2

Det

erm

inat

ion

of th

e Abs

olut

e Co

nfig

urat

ion

for A

nmin

deno

l B

Figu

re B

.2.1

Indu

ced

CD

spec

trum

by

anm

inde

nol B

(6) a

nd th

e M

o 2(O

Ac)

4

12

3

Figu

re B

.1.1

1 H N

MR

spec

trum

(600

MH

z, C

DC

l 3) o

f anm

inde

nol A

(5)

12

4

Figu

re B

.1.2

13C

NM

R sp

ectru

m (1

50 M

Hz,

CD

Cl 3)

of a

nmin

deno

l A (5

)

12

5

Figu

re B

.1.3

CO

SY sp

ectru

m (6

00 M

Hz,

CD

Cl 3)

of a

nmin

deno

l A (5

)

12

6

Figu

re B

.1.4

HSQ

C sp

ectru

m (6

00 M

Hz,

CD

Cl 3)

of a

nmin

deno

l A (5

)

12

7

Figu

re B

.1.5

HM

BC

spec

trum

(600

MH

z, C

DC

l 3) o

f anm

inde

nol A

(5)

12

8

Figu

re B

.1.6

NO

ESY

spec

trum

(600

MH

z, C

DC

l 3) o

f anm

inde

nol A

(5)

12

9

Figu

re B

.1.7

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f anm

inde

nol B

(6)

13

0

Figu

re B

.1.8

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of a

nmin

deno

l B (6

)

13

1

Figu

re B

.1.9

CO

SY sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of a

nmin

deno

l B (6

)

13

2

Figu

re B

.1.1

0 H

SQC

spec

trum

(700

MH

z, C

DC

l 3) o

f anm

inde

nol B

(6)

13

3

Figu

re B

.1.1

1 H

MB

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of a

nmin

deno

l B (6

)

13

4

Figu

re B

.2.1

Indu

ced

CD

spec

trum

by

anm

inde

nol B

(6) a

nd th

e M

o 2(O

Ac)

4

135

Appendix C.

C.1 NMR Spectra of 11A020-3A

13

6

C.1

NM

R S

pect

ra o

f 11A

020-

3A

Figu

re C

.1.1

1 H N

MR

spec

trum

(700

MH

z, M

eOH

-d4)

of 1

1A02

0-3A

(7)

Figu

re C

.1.2

13C

NM

R sp

ectru

m (1

75 M

Hz,

MeO

H-d

4 of 1

1A02

0-3A

(7)

Figu

re C

.1.3

CO

SY sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f 11A

020-

3A (7

)

Figu

re C

.1.4

Pha

se se

nsiti

ve H

SQC

spec

trum

(700

MH

z, M

eOH

-d4)

of 1

1A02

0-3A

(7)

Figu

re C

.1.5

HM

BC

spec

trum

(700

MH

z, M

eOH

-d4)

of 1

1A02

0-3A

(7)

13

7

Figu

re C

.1.1

1 H N

MR

spec

trum

(700

MH

z, M

eOH

-d4)

of 1

1A02

0-3A

(7)

13

8

Figu

re C

.1.2

13C

NM

R sp

ectru

m (1

75 M

Hz,

MeO

H-d

4) o

f 11A

020-

3A (7

)

13

9

Figu

re C

.1.3

CO

SY sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f 11A

020-

3A (7

)

14

0

Figu

re C

.1.4

Pha

se se

nsiti

ve H

SQC

spec

trum

(700

MH

z, M

eOH

-d4)

of 1

1A02

0-3A

(7)

14

1

Figu

re C

.1.5

HM

BC

spec

trum

(700

MH

z, M

eOH

-d4)

of 1

1A02

0-3A

(7)

142

Appendix D.

D.1 NMR Spectra of Usnic Acid Derivatives

14

3

D.1

NM

R S

pect

ra o

f Usn

ic A

cid

Der

ivat

ives

Figu

re D

.1.1

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f F80

15-2

B-2

E (8

)

Figu

re D

.1.2

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of F

8015

-2B

-2E

(8)

Figu

re D

.1.3

HSQ

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of F

8015

-2B

-2E

(8)

Figu

re D

.1.4

HM

BC

spec

trum

(700

MH

z, C

DC

l 3) o

f F80

15-2

B-2

E (8

)

Figu

re D

.1.5

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f F80

15-2

B-2

I (9)

Figu

re D

.1.6

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of F

8015

-2B

-2I (

9)

Figu

re D

.1.7

HSQ

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of F

8015

-2B

-2I (

9)

Figu

re D

.1.8

HM

BC

spec

trum

(700

MH

z, C

DC

l 3) o

f F80

15-2

B-2

I (9)

Figu

re D

.1.9

1 H N

MR

spec

trum

(700

MH

z, M

eOH

-d4)

of F

8015

-2B

-2B

(10)

Figu

re D

.1.1

0 13

C N

MR

spec

trum

(175

MH

z, M

eOH

-d4)

of F

8015

-2B

-2B

(10)

Figu

re D

.1.1

1 H

SQC

spec

trum

(700

MH

z, M

eOH

-d4)

of F

8015

-2B

-2B

(10)

Figu

re D

.1.1

2 H

MB

C sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f F80

15-2

B-2

B (1

0)

14

4

Figu

re D

.1.1

3 1 H

NM

R sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f F80

15-2

B-2

C (1

1)

Figu

re D

.1.1

4 13

C N

MR

spec

trum

(175

MH

z, M

eOH

-d4)

of F

8015

-2B

-2C

(11)

Figu

re D

.1.1

5 H

SQC

spec

trum

(700

MH

z, M

eOH

-d4)

of F

8015

-2B

-2C

(11)

Figu

re D

.1.1

6 H

MB

C sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f F80

15-2

B-2

C (1

1)

14

5

Figu

re D

.1.1

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f F80

15-2

B-2

E (8

)

14

6

Figu

re D

.1.2

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of F

8015

-2B

-2E

(8)

14

7

Figu

re D

.1.3

HSQ

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of F

8015

-2B

-2E

(8)

14

8

Figu

re D

.1.4

HM

BC

spec

trum

(700

MH

z, C

DC

l 3) o

f F80

15-2

B-2

E (8

)

14

9

Figu

re D

.1.5

1 H N

MR

spec

trum

(700

MH

z, C

DC

l 3) o

f F80

15-2

B-2

I (9)

15

0

Figu

re D

.1.6

13C

NM

R sp

ectru

m (1

75 M

Hz,

CD

Cl 3)

of F

8015

-2B

-2I (

9)

15

1

Figu

re D

.1.7

HSQ

C sp

ectru

m (7

00 M

Hz,

CD

Cl 3)

of F

8015

-2B

-2I (

9)

15

2

Figu

re D

.1.8

HM

BC

spec

trum

(700

MH

z, C

DC

l 3) o

f F80

15-2

B-2

I (9)

15

3

Figu

re D

.1.9

1 H N

MR

spec

trum

(700

MH

z, M

eOH

-d4)

of F

8015

-2B

-2B

(10)

15

4

Figu

re D

.1.1

0 13

C N

MR

spec

trum

(175

MH

z, M

eOH

-d4)

of F

8015

-2B

-2B

(10)

15

5

Figu

re D

.1.1

1 H

SQC

spec

trum

(700

MH

z, M

eOH

-d4)

of F

8015

-2B

-2B

(10)

15

6

Figu

re D

.1.1

2 H

MB

C sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f F80

15-2

B-2

B (1

0)

15

7

Figu

re D

.1.1

3 1 H

NM

R sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f F80

15-2

B-2

C (1

1)

15

8

Figu

re D

.1.1

4 13

C N

MR

spec

trum

(175

MH

z, M

eOH

-d4)

of F

8015

-2B

-2C

(11)

15

9

Figu

re D

.1.1

5 H

SQC

spec

trum

(700

MH

z, M

eOH

-d4)

of F

8015

-2B

-2C

(11)

16

0

Figu

re D

.1.1

6 H

MB

C sp

ectru

m (7

00 M

Hz,

MeO

H-d

4) o

f F80

15-2

B-2

C (1

1)

161

2

.

.

2

A B, A-D 11A020-3A

. 1D, 2D NMR

. A B,

D LPS-

stimulated RAW 264.7

23 μM, 19 μM, 13 μM

, 11A020-3A Bacillus subtilis, Kocuria

rhizophila, Staphylococcus aureus Escherichia coli

0.1 μM, 64 μM, 8 μM, 64 μM .

2

Mycosphaerella nawae F8015-2B-2B, 2C, 2E, 2I 4

. NMR

162

.

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