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이학석사 학위논문

Identification of Secondary Metabolites

from the Marine Plant and Bacteria

- 해양식물과 해양미생물의 2차 대사산물 규명-

Hyunji Kim

August 2014

Laboratory of Marine Drug

School of Earth and Environment Sciences

Seoul National University

i

Abstract

The marine environment has established to be a very rich source of particularly

potent compounds that have demonstrated significant activities in antitumor, anti-

inflammatory, analgesia, immunomodulation, allergy and anti-viral assays.

The chemical inquiry of several marine plant and bacteria with the isolation and

structure elucidation of secondary metabolites with biological activities are

described in this thesis.

Actinobacteria from Rayong beach, Thailand, Streptomycetes sp., let us the

isolation of new compound with naphthalene moiety. The structure was elucidated

by extensive spectroscopic data (IR, MS, 1H and

13C NMR, COSY, HSQC,

HMBC) analysis.

Continuous research on marine derived bacteria led to collections of a number of

actinomycetes. Actinobacteria from the mud flat, streptomyces sodiiphilus, let us

the isolation of butanamide. 1D and 2D NMR study revealed the moiety, which is

common to natural products. It is reported alkaliphilic actinomycetes that its

secondary metabolites able of quenching quorum sensing controlled behaviors in

gram-negative reporter strains.

A collection of the marine plant Peucedanum japonicum (Umbelliferae) from the

south of Korea beach, led to the isolation of the five novel khellactone.

ii

Key words

Marine natural products, Secondary metabolite, Peucedanum japonicum,

Streptomyces sodiiphilus

Student Number: 2012-23078

iii

Contents

Abstract ................................................................................................................ ⅰ

Contents................................................................................................................ ⅲ

List of Figures ...................................................................................................... ⅴ

List of Tables ........................................................................................................ ⅵ

Chapter 1 General Introduction of Marine Natural Products

1. Marine Natural Products in General Introduction .............................................. 1

Chapter 2 Naphthalene derivative from Streptomycete sp.

2. 1 Introduction ..................................................................................................... 5

2. 2 Result and Discussion ..................................................................................... 6

2.2.1 Structural Elucidation of coumarines ...................................................... 6

2.2.1.1 13A001-1 .......................................................................................... 6

2.3 Experimental Section ....................................................................................... 9

2.3.1 Instruments and Data Collection ............................................................. 9

2.3.2 Collection, Extraction and Isolation ...................................................... 10

Chapter 3 Butanamide from Streptomyces sodiiphilus

3. 1 Introduction ................................................................................................... 11

3. 2 Result and Discussion ................................................................................... 12

3.2.1 Structural Elucidation of Butanamide ................................................... 12

3.2.1.1 Butanamide..................................................................................... 12

3.3 Experimental Section ..................................................................................... 15

3.3.1 Instruments and Data Collection ........................................................... 15

3.3.2 Isolation of the Secondary Metabolites ................................................. 16

Chapter 4 Khellactone from the marine plant, Peucedanum japonicum

4. 1 Introduction ................................................................................................... 17


4.2.1 Structural Elucidation of Khellactone ................................................... 22

4.2.1.1 cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-dihydroseselin ... 22

4.2.1.2 Compound 1-4 ................................................................................ 25

4.3 Experimental Section ............................................................................... 37

4.3.1 Instruments and Data Collection ....................................................... 37

iv

4.3.2 Collection, Extraction and Isolation .................................................. 38

4.3.3 Plant material..................................................................................... 39

Reference .............................................................................................................. 40

Appendix .............................................................................................................. 42

한글초록 .............................................................................................................. 66

v

List of Figures

Figure 1.1 Chemical structures of marine-derived drugs ............................ 2

Figure 1.2 Experimental sample places in marine environment ................. 4

Figure 2 COSY and HMBC correlations of 13A001-1 ............................... 7

Figure 3 COSY and HMBC correlations of Butanamide .......................... 13

Figure 4.1 Chemical structures of the marine plant, Peucedanum japonicum

................................................................................................................... 18

Figure 4.2 COSY and HMBC correlations of cis-3’-Acetoxy-4’-(2-

methylbutyroyloxy)-3’,4’-dihydroseselin ................................................. 22

Figure 4.3 Structure of cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’,4’-

dihydroseselin ............................................................................................ 23

Figure 4.4 Structures of compounds 1-4 ................................................... 26

Figure 4.5 Spectroscopic data of compounds 1-4...................................... 27

vi

List of Tables

Table 1 A number of novel/new metabolites produced by marine

actinomycetes during the period 2005- 2010 (Subramani et al., 2012) .... 3

Table 2 Physical and spectral properties of 13A001-1 ................................ 8

Table 3 Physical and spectral properties of butanamide ............................ 14

Table 4.1 Physical and spectral properties of cis-3’-Acetoxy-4’-(2-

methylbutyroyloxy)-3’, 4’-dihydroseselin ................................................ 24

Table 4.2 Spectral properties of compound 1 ............................................ 27




1

Chapter 1

General Introduction of Marine Natural Products

1. Marine Natural Products in General Introduction

The largely uninvestigated marine world that apparently harbors the most

biodiversity may be the greatest resource to discover novel ‘validated’ structures

that considered with biologically relevant chemical space. Nature is an ancient

pharmacy that made use of the solitary source of therapeutics for the early times.

Natural products are a major part of the modern pharmaceuticals that we use to

cure human disease and have been the mainstay of disease treatment for most of

human history. The diversity of organisms in the marine environment has

motivated researchers to indentify novel marine natural products for many years

that could finally be developed into drugs. The first FDA-approved marine-derived

drugs, Cytarabine (Ara-C) and vidarabine (Ara-A) (Figure 1.1), are synthetic

pyrimidine and purine nucleosides, respectively, developed from naturally existing

nucleosides originally isolated from the sponge Tethya crypta in Carinnean.

Cytarabine was approved by the FDA in 1969 as an anticancer drug, while

vidarabine was approved in 1976 as an antiviral agent. It has taken over 30 years

for another marine-derived natural product to get approval. Ziconotide (Prialt® ) for

the treatment of ordinary to chronic pain gained FDA approval in 2004. It is a

naturally occurring peptide isolated from the venom of the cone snail Conus magus.

Travectedin (Yondelis® ) has received European approval for the treatment of soft

tissue sarcoma in 2007, and for ovarian carcinoma in 2009. It is a marine alkaloid

isolated from the marine tunicate Ecteinascidia turbinate. Eribulin mesylate

(Halaven™) is the latest drug to the market from the marine sponge Halichondria

okadai in 1986. Incidentally numerous other marine natural products or derivatives

thereof are in different phases of clinical trials.

2

Cytarabine, Ara-C Vidarabine, Ara-A

Prialt®

Yondelis® Eribulin Mesylate(Halaven™)

Figure 1.1 Chemical structures of marine-derived drugs

3

Table 1 demonstrated that some examples of new secondary metabolites isolated

from marine actinomycetes from 2005 to 2010. Among them, a few compounds are

of particular interest due to their rarity and potent and diverse bioactivity such as

staurosporinone, salinosporanide A, lodopyridone, arenimycin, marinomycins and

proximicins from Table 1.

Table 1 A number of novel/new metabolites produced by marine actinomycetes

during the period 2005- 2010 (Subramani et al., 2012)

Compound Source Biological activity

Chinikomycins Streptomyces sp. Anticancer

Chloro-dihydroquinones Novel actonomycete Antibacterial; anticancer

Glaciapyrroles Streptomyces sp. Antibacterial

Frigocyclinone Streptomyces griseus Antibacterial

Lajollamycin Streptomyces nodosus Antibacterial

Mechercharmycins Thermoactinomyces sp. Anticancer

Salinosporamide A Salinispora tropica Anticancer; Antibacterial

Sporolide A Salinispora tropica Unknown

Salinosporamides B & C Salinispora tropica Cytotoxicity

2-Allyloxyphenol Streptomyces sp. Antibacterial; food preservative; oral

disinfectant

Saliniketal Salinisporta arenicola Cancer chemoprevention

Marinomycins A-D Marinispora Antibacterial; anticancer

Cyanosporaside A Salinispora pacifica Unknown

Lodopyrideone Saccharomonospora sp. Anticancer

Arenimycin Salinispora arenicola Antibacterial; anticancer

Salinispyrone Salinispora pacifica Unknown

Salinopyrones A & B Salinispora pacifica Mild cytotoxicity

Pacificanones A & B Salinispora pacifica Antibacterial

Arenicolides A-C Salinispora arenicola Mild cytotoxicity

1-hycroxy-1-norresistomycin Streptomyces chinaensis Antibacterial; anticancer

Resistoflavin methyl ether Streptomyces sp. Antibacterial; anti-oxidative

saturosporinone Streptomyces sp. Antitumor; phycotoxicity

4

The high degree of innovation in the field of marine natural products will lead to

successful marine drug discovery and development, and provides grounds for our

optimism that marine natural products will form a new wave of drugs that flow into

the market and pharmacies in the future.

We have isolated actinobacteria from sediment, beach, and plant in shore. We

elucidated structures of a new one and six previously reported ones. (Figure 1.2)

Figure 1.2 Experimental sample places in marine environment

5

Chapter 2

Naphthalene derivative from Streptomycete sp.

2.1. Introduction

The sea, covering more than 70% of the surface of planet Earth, contains an

exceptional biological diversity, accounting for more than 95% of the whole

biosphere. The sea, covering more than 70% of the surface of planet Earth,

contains an exceptional biological diversity, accounting for more than 95% of the

whole biosphere. Of the total sea surface, only 7–8% is coastal area and the deep

sea, of which 60% is covered by water more than 2000 m deep. The deep sea is a

unique and extreme environment characterized by high pressure, low temperature,

lack of light, and oxygen concentration.

As a part of our continuous research for secondary metabolites discovery from

Rayong beach, Thailand, Streptomycete sp. was isolated from a sediment sample.

Chemical investigation of this strain gave new compound.

6

2.2 Result and Discussion

2.2.1 Structural Elucidation of naphthalene

2.2.1.1 13A001-1

13A001-1 had the molecular formula C20H14O4, as determined by LFABMS with

14 degrees of unsaturation. The UV peak near 400 nm shows us that there are

conjugated bonds.

The four contiguous aromatic proton signals at δ 6.6, 6.9, 7.43, 8.39 and HMBC

correlation, respectively, were indicative of a 1, 2-disubstituted naphthalene. The

HMBC spectrum from H-5’ to C-4’ is suggested that there’s a methyl ester in the

naphthalene. The assignments of the signals of the hydroxyl proton are

straightforward on the basis of their chemical shift. C-2’ exhibits two ling range

coupling interactions of unequal magnitude with H-1’ and H-7. In particular, in

CDCl3, one could observe the HMBC correlations of the exchageable OH carbon to

the sp proton of the naphthalene ring.

The 1H NMR spectrum showed two-proton doublets at δ 7.46 (d, J = 8.0 Hz) and

δ 7.8 (d, J = 8.0 Hz), and four-proton triplets at δ 7.42, respectively, suggested

mono-substituted phenyl ring. The HMBC correlation of H-6’’ to the carbon

resonating at δ 151.19 allowed the assignment of the presence of a C-1’-substituted

naphtho[1,8-b,c]pyran (Figure 2).

7

Figure 2 COSY and HMBC correlations of 13A001-1

8

Table 2 Physical and spectral properties of 13A001-1

NMR Dataa

No. δC, mb δH, m, J (Hz) COSY HMBC

1 158.8, C

2 99.7, CH 6.6 (s) 1, 3, 4, 8a

3 167.4, C

4 98.8, C

4a 133.9, C

5 122.8, CH 8.39 (d, 8.7) 6 4, 7, 8a

6 130.1, CH 7.43 (t, 15, 7.8) 7 4a, 8

7 116.3, CH 6.9 (d, 7.5)

8 130.7, C

8a 118.1, C

1’ 151.1, C

2’ 103.6, CH 6.65 (s) 7, 8a, 1’, 1’’

1’’ 132.2, C

2’’ 124.7, CH 7.8 (d, 7.5) 3’’

3’’ 128.7, CH 7.46 (t, 15, 7.8) 4’’ 1’’, 5’’

4’’ 129.5, CH 7.42 (t, 7, 3.5) 5’’ 2’’

5’’ 128.7, CH 7.46 (t, 15, 7.8) 6’’

6’’ 124.7, CH 7.8 (d, 8) 1’, 2’’, 4’’

1’’’ 172.6, C

2’’’ 52.1, CH3 4.06 (s)

OH 12.57

a In CDCl3, at 700 MHz for

1H and

13C NMR.

bThe numbers of attached protons

were determined from 1H,

13C and HSQC NMR spectroscopic data.

Yellow amorphous solid

Molecular fomula: C20H14O4

LREIMS: m/z 318

IR(film) vmax: 2917, 2849 cm-1

UV(CDCl3)λmax: 240, 280, 400 nm

9

2.3 Experimental Section

2.3.1 Instruments and Data Collection

UV spectra were obtained with a Waters UV spectrophotometer. IR spectra were

acquired on a JASCO FT/IR 4200 spectrophotometer. All MNR spectra were

recorded on a Bruker Ascend 700 spectrometers using chloroform-d1 as the solvent.

The optical rotations were measured in MeOH using a 1.0 cm cell on a Rudolph

Research Autopol 3.

NMR solvents were obtained from Cambridge Isotope Laboratories (CIL) Inc.

Chemical shifts were reported with reference to the respective solvent peaks [δ

7.26 and δ 77.0 for CDCl3]. Electrospray ionization source (ESI) low resolution

mass spectra were recorded on an Agilent Technologies 6120 Quadrupole mass

spectrometer coupled with an Agilent Technologies 1260 series HPLC. Separation

of extracts by HPLC WATERS 1525 binary HPLC pump, WATERS 2489

UV/visible detector was carried out using Shiseido CAPCELL C18 (250 × 10 mm,

5 μm) reversed-phase semipreparative column.

10

2.3.2 Isolation of the Secondary Metabolites

The extract was separated by silica normal phase column using step-gradient

elution of MeOH in dichloromethane (0%, 1%, 2%, 5%, 10%, 20%, 50%, 90% and

100%). Fraction 1 was further purified using reversed-phase HPLC (Capcell C18

250 × 10 mm, 5 μm, 2.0 mL/min, UV = 210 nm and 280 nm; CH3CN:H2O= 70:30).

11

Chapter 3

Butanamide from Streptomyces sodiiphilus

3.1. Introduction

Alkaliphilic actinomycetes that thrive in alkaline environments have typical

nutrient requirements, cultural conditions and physiological properties. The deep

sea is a unique and extreme environment characterized by high pressure, low

temperature. In deep sea sediments, marine micro-organisms occupy the main

ecological niche, which carries out an important role in the recycling of carbon and

nitrogen sources on the sea floor.

It is known that actinomycetes grow well in neutral and slightly alkaline media.

Alkaliphilic actinomycetes were first isolated from various soils by Taber. These

alkaliphilic streptomycetes showed maximal radial rates of colony growth at pH 8.

It is obligately Na+-dependent, and showed sensitivity to K

+.

Taxonomically diverse marine bacteria have proven to be a rich resource for the

discovery of structurally unique and bioactive secondary metabolites. Given the

intense microbial competition for resources such as space and nutrients, it is

probable that many excreted metabolites help mediate microbe-microbe

interactions. Various antibiotics have been implicated as chemical defenses for

marine bacteria, thus suggesting a role for the biosynthesis of toxic metabolites.

Though not yet widely studied, the secretion of nontoxic molecules could also

play important roles in antagonistic marine microbial interactions. Quorum sensing

pathways of competing bacteria are potential targets for such nontoxic chemical

defenses. Bacterial communication is facilitated by the production and subsequent

recognition of small signaling molecules (autoinducers) and can regulate important

phenotype, including bioluminescence, biofilm formation, swarming motility,

antibiotic biosynthesis, and virulence factor production.

12


3.2.1 Structural Elucidation of Butanamide

3.2.1.1 Butanamide

The molecular formula of butanamide was determined to be C13H19NO by

LRFABMS of the molecular ion at m/z 205. It is 5 degrees of unsaturation and the

five contiguous aromatic proton signals at δ 7.22(d, J = 4.5 Hz), 7.24(t, J = 15, 7.5

Hz), 7.32(t, J = 15, 7.5 Hz), respectively, were indicative of mono-substituted

phenyl ring. The IR spectrum showed bands at 3275 and 1648cm-1

, pointing to the

presence of an amide group. The 1H and

13C NMR data confirmed the presence of

an amide carbonyl and indicated three methylenes and two methyl.

The 1H NMR spectrum of butanamide showed a broad D2O exchangeable signal

at δ 5.45(1H) for NH and/or OH groups. The signals at δ 5.12 (1H) and δ 4.78 (1H)

were attributed to olefinic protons. A triplet of doublet at δ 3.96 for a methine

proton connected either with a nitrogen or an oxygen atom, a 1H multiplet at δ 1.80,

a doublet of triplet (2H) at δ 1.56 and a doublet for six protons at δ 0.86 (isopropyl

group) were observed at the aliphatic region. The 13

C NMR and APT spectrum of

this compound showed eight signals. The signals at δ 166.4 and δ 158.2 were

interpreted as carbonyl signal of carboxylic acids, amides or esters. The signals at δ

134.6 and δ 98.9 represent a C=CH2 fragment in conjugation with a carbonyl group,

while the signals at δ 22.1 were accounted for the two methyl group of an isopropyl

residue. Finally the structure of the compound was assigned by COSY, HMQC and

HMBC couplings as butanamide.

13

Figure 3 COSY and HMBC correlations of Butanamide

14

Table 3 Physical and spectral properties of butanamide

NMR Dataa


1 172.5, C

2 46.3, CH2 1.99 (d) 3 1

3 26.2, CH 2.15 (m) 4,5 1

4 22.6, CH3 0.9 (d, 7.0)

5 22.6, CH3 0.9 (d, 7.0)

1’ 40.6, CH2 3.56 (t, 7.0) 2’

2’ 35.9, CH2 2.85 (t, 7.0) 1’’, 2’’

1’’ 139, C 3’’

2’’ 128.6, CH 7.2 (d, 7.5) 4’’

2’’ 128.6, CH 7.2 (d, 7.5)

3’’ 128.7, CH 7.31 (t, 15, 7.5)

3’’ 128.7, CH 7.31 (t, 15, 7.5)

4’’ 126.6, CH 7.24 (t, 15, 7.5)

NH 5.4 (br s)


1H and

13C NMR.





Molecular fomula: C13H19NO

LRFABMS: m/z 205.30

IR(film) vmax: 3301, 1637, 1541 cm-1

UV (CDCl3) λmax: 200, 250 nm

15



Streptomyces sodiiphilus was obtained from marine sediment from JangBong-

Island, Gyeong Gi-do, South Korea in 2013. Sampled mud sediments were dried

by air for 24 hours in a clean bench and given a heat shock at 55℃ for 9 minutes in

a low-temperature incubator. Aggregated clumps were lightly mortared by glass

rod and stamped by sponge-plug onto various prepared solid agar media. Some of

the dried samples were suspended in sterilized sea water and diluted plastic rod.

These crude plates were placed in 27℃ chamber and monitored for 1 to 3 months

to obtain a unique actinomycete like colonies. Strain 13B033 was peaked from 1/5

ISP 1 media agar plate showing white filamentous colony. The 16S r RNA gene

was cloned using universal primers 27F and 1492R and showed 99.5% similarity to

Streptomuces sodiiphilus strain YIM 80305.

The bacterial strain 13B033 was cultured at 25℃ with shaking at 150 rpm in 30

L Pyrex flask each containing 1L of the medium SYP (10 g of starch soluble, 4 g of

yeast extract, 2 g of peptone, dissolved in 75% of 1 L filtered-seawater). After 10

days, the broth was extracted two times with ethyl acetate and evaporated to yield

2.37 g of crude organic extract.

16



elution of MeOH in dichloromethane(0%, 1%, 2%, 5%, 10%, 20%, 50%, 90% and



17

Chapter 4

Khellactone Derivatives from the Marine Plant, Peucedanum

japonicum

4.1 Introduction

Peucedanum japonicum, a medicinal plant belonging to the family of

Umbelliferae, grows on the cliffs of Island, Korea. Peucedanum japonicum leaves

are traditionally consumed on Island as a medicinal herb for the treatment of cough.

The root of this plant has been used as a folk medicine for cold and neuralgic

diseases in Taiwan (Chen et al., 1996). A number of studies gave reported on the

physiological activities of PJT including antioxidant activity (Hisamoto et al.,

2003), tyrosinase inhibitory effect (Hisamoto et al., 2004), and anti-platelet

aggregation activity (Chen et al., 1996) in vitro. Furthermore, several study have

demonstrated the instance of hypolipidaemic compounds in the leaves of

Peucedanum japonicum (Hsu and Yen, 2007; Li et al., 2006).

It has been reported that some coumarins, glucosides, and chromones.(Figure

4.1)

19

norisoprenoid glucoside

phenylpropanoid glucoside

Praeruptorin A

psoralen xanthotoxin

20

divaricataesters A divaricataesters B

divaricataesters C

21

Panaxynol

Panaxydol

Panaxytriol


22


4.2.1 Structural Elucidation of Khellactone

4.2.1.1 cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-dihydroseselin

Cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-dihydroseselin had the [M]+

peak at m/z 389 in the low resolution electron ionization mass spectrum which

indicated the molecular formula was C24H28O7. It requires ten degrees of

unsaturation. The absorption bands of a carbonyl (1722 cm-1

) and an aromatic

system (1612 cm-1

, 1492 cm-1

) in the IR spectrum are characteristic of a coumarin

skeleton.

The 1H NMR spectrum in the aromatic proton region of it contained two pairs of

doublets at δ 6.23 (1H, d, J = 9.5 Hz), 7.61 (1H, d, J = 9.5 Hz) and at δ 7.37 (1H, d,

J = 8.6 Hz), 6.82 (1H, d, J = 8.6 Hz), which are in agreement with the H-3 and H-4

signals of the α-pyrone ring system and signals of H-5 and H-6 of the benzene ring,

indication that it is a coumarin substituted at the C-7 and C-8 positions. A pair of

doublets at δ 5.38 (1H, d, J = 3.5 Hz) and δ 6.27(1H, d, J = 3.5 Hz) was assigned to

two ester groups, showing C-7 and C-8 formed a dihydropyran ring. The signals at

δ 2.42 (m), 1.5 (m), 1.75 (m), 1.23 (d, 7.2), 0.96 (t) are due to a methylbutyryl

group and at δ 5.34 (d, 5.0), 6.57 (d, 5.0) are due to an acetoxy group.


methylbutyroyloxy)-3’, 4’-dihydroseselin

23

The relative cis-configuration of compound was assigned on the basis of the

coupling constant J3’, 4’, which was between 4.1 and 5.0 Hz. The optical rotations

for compound [α]D25

= + 0.96° indicates the configuration 3’R, 4’R.

Figure 4.3 Structure of cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-

dihydroseselin

24



NMR Dataa


2 159.7, C

3 113.2, CH 6.25 (d, 9.6) 4 2, 9

4 143.1, CH 7.61 (d, 9.6) 5, 7, 9, 10

5 129.1, CH 7.36 (d, 8.6) 6 9, 10

6 114.3, CH 6.81 (d, 8.6) 7, 8

7 156.6, C

8 107.5, C

9 154.3, C

10 112.4, C

2’ 77.7, C

3’ 70.1, CH 5.34 (d, 5.0) 4’ 1’’

4’ 60.9, CH 6.57 (d, 5.0) 7, 8, 2’, 2’-Me

1’’ 166.4, C

2’’ 41.2, CH 2.42 (m), 4’’, 3’’

3’’ 26.3, CH2 1.5 (m), 1.75 (m)

4’’ 16.3, CH3 1.23 (d, 7.2) 2’’, 3’’

5’’ 11.6, CH3 0.96 (t 1’’

1’’’ 166.2, C 2’’’

2’’’ 20.6, CH3 2.12(s)

2’-Me 23.7, CH3 1.42 (s) 2’, 3’

2’-Me 22.4, CH3 1.46 (s)


1H and

13C NMR.




Colorless amorphous solid

Molecular formula: C21H24O7

LRFABMS: m/z 388.15

IR(film) vmax: 2925, 1666, 1461 cm-1

UV(CDCl3) λmax: 210, 250, 325 nm

25

4.2.1.2 Compounds 1-4

In addition to the cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-dihydroseselin,

four previously reported khellactone, Anomalin (1), Isosamidin (2), (3’S, 4’S)-3’-

isovaleryl-4’-(2-methylbutyryl)-khellactone (3), cis-3’, 4’-disenecioylkhellatone

(4) were also isolated from Peucedanum japonicum. Anomalin (1) and isosamadin

(2) were previously isolated from Saseli resinosum (Alev et al., 2006), S. unicaule

(Barrero et al., 1990), and Peucedanum japonicum (Ikeshiro et al., 1992; Fan et al.,

2000). While cis-3’, 4’-disenecioylkhellatone (4) reported from Peucedanum

japonicum (Ting-Ting Jong et al., 1992), (3’S, 4’S)-3’-isovaleryl-4’-(2-

methylbutyryl)-khellactone (3) was obtained from the Peucedanum praeruptorum

(Huawei Lv et al., 2013). The structures were elucidated by extensive spectroscopic data (IR, MS,

1H and

13C NMR, COSY, HSQC, and HMBC) analysis.

26

1 2

3 4

Figure 4.4 Structures of compounds 1-4

27

Figure 4.5 Spectroscopic data of compounds 1-4

Compound 1



LREIMS: m/z 426

IR (film) vmax: 1731, 1604 cm-1

UV (CDCl3) λmax: 209, 255, 300 nm

[α]D25

= + 9.3° (c 0.0015, MeOH)

Compound 3



LREIMS: m/z 430

IR (film) vmax: 2925, 1666, 1461 cm-1

UV (CDCl3) λmax: 250 nm

[α]D25

= + 29.3° (c 0.00075, MeOH)

Compound 2



LRFABMS: m/z 386.1

IR (KBr) vmax: 2965, 1742, 1609 cm-1

UV (CDCl3) λmax: 225, 250, 322nm

[α]D25

= - 67.31° (c 0. 0083, MeOH)

Compound 4



LRFABMS: m/z 426

IR (KBr) vmax: 2965, 1742, 1609 cm-1

UV (CDCl3) λmax: 248, 300, 325 nm

[α]D25

= + 12.3° (c 0.083, MeOH)

28

Table 4.2 Spectral properties of Compound 1

NMR Dataa


2 159.8, C

3 113.3, CH 6.21 (d, 9.57) 4 2, 9

4 143.2, CH 7.61 (d, 9.57) 5, 7, 8, 9, 10

5 129.4, CH 7.38 (d, 8.58) 6 9, 10

6 114.4, CH 6.82 (d, 8.58) 7, 8

7 156.7, C

8 107.6, C

9 154.1, C

10 112.5, C

2’ 77.5, C

3’ 70.2, CH 5.44 (d, 5.0) 1’’

4’ 60.2, CH 6.70 (d, 5.0) 7, 8, 2’, 2’-Me

1’’ 166.4, C

2’’ 139.8, C

3’’ 127.4, CH 6.13(1H, m) 5’’ 2’’, 3’’

4’’ 20.4, CH3 1.97(3H, m)

5’’ 15.8, CH3 1.84(3H, m) 2’’

1’’’ 166.3, C

2’’’ 138.4, C

3’’’ 127,CH 5.98(1H, m) 5’’’ 2’’’, 3’’’

4’’’ 20.3, CH3 1.97(3H, m)

5’’’ 15.6, CH3 1.84(3H, m) 2’’’

2’-Me 25.4, CH3 1.46 (s) 2’, 3’

2’-Me 22.5, CH3 1.50 (s)


1H and

13C NMR.




29


NMR Dataa


2 159.5, C

3 112.8, CH 6.23 (d, 9.5) 4 2, 9

4 143, CH 7.61 (d, 9.5) 5, 7, 8, 9, 10

5 128.7, CH 7.36 (d, 8.5) 6 9, 10

6 114.1, CH 6.81 (d, 8.5) 7, 8

7 156.4, C

8 135.9, C

9 153.6, C

10 112.3, C

2’ 77.7, C

3’ 68.7, CH 5.35 (d, 5.0) 4’ 1’’

4’ 61.1, CH 6.57 (d, 5.0) 7, 8, 2’, 2’-Me

1’’ 169.5, C 2’’

2’’ 20.6, CH3 2.11 (s)

1’’’ 164.9, C

2’’’ 114.8, CH 5.69 (s) 4’’, 5’’

3’’’ 158.4, C

4’’’ 27.4, CH3 2.17 (s) 2’’’, 3’’’

5’’’ 20.3, CH3 1.9 (s) 1’’’

2’-Me 23.1, CH3 1.41 (s) 2’, 3’

2’-Me 24.5, CH3 1.45 (s)


1H and

13C NMR.




30


NMR Dataa


2 159.6, C

3 113.5, CH 6.26 (d, 9.5) 4 2, 9

4 143.2, CH 7.63 (d, 9.5) 5, 7, 8, 9, 10

5 129, CH 7.39 (d, 8.5) 6 9, 10

6 114.5, CH 6.83 (d, 8.5) 7, 8

7 156.2, C

8 107, C

9 153.2, C

10 112.7, C

2’ 77.5, C 3’

3’ 69.8, CH 5.35 (d, 5.0) 4’, 5’ 2’, 1’’, 2’-Me

4’ 60.3, CH 6.58 (d, 5.0) 2’, 1’’’

1’’ 171.9, C

2’’ 43.1, C 2.25(2H,m)

3’’ 25.3, CH 2.15(1H, m)

4’’ 22.5, CH3 1.00(3H, d, 7.0)

5’’ 22.5, CH3 1.01(3H, d, 7.0)

1’’’ 175.4, C

2’’’ 41, C 2.44(m) 3’’’, 4’’’

3’’’ 26.5,CH 1.5, 1.75(2H, m)

4’’’ 16.1, CH3 1.25(3H, t, 7.5) 5’’’

5’’’ 11.5, CH3 0.9(3H, d, 7.0)

2’-Me 25.5, CH3 1.45 (s) 1’, 2’, 3’

2’-Me 22.4, CH3 1.49 (s)


1H and

13C NMR.




31


NMR Dataa


2 159.9, C

3 113.2, CH 6.21 (d, 9.5) 4 2, 9

4 143.2, CH 7.57 (d, 9.5) 5, 7, 8, 9, 10

5 129.0, CH 7.34 (d, 8.6) 6 9, 10

6 114.3, CH 6.79 (d, 8.6) 7, 8

7 156.6, C

8 107.62, C

9 154.1, C

10 112.5, C

2’ 77.7, C

3’ 69.4, CH 5.36 (d, 4.9) 4’ 1’’

4’ 59.8, CH 6.62 (d, 54.9) 7, 8, 2’, 2’-Me

1’’ 165.2, C

2’’ 115.2, CH 5.62 (br s) 4’’, 5’’

3’’ 158.3, C

4’’ 27.4, CH3 2.15 (d, 1.1) 2’’, 3’’

5’’ 20.3, CH3 1.87 (s) 1’’

1’’’ 165.1, C 2’’’

2’’’ 115.2, CH 5.66 (br s)

3’’’ 157.6, C

4’’’ 27.4, CH3 2.19 (d, 1.1)

5’’’ 20.3, CH3 1.88 (s)

2’-Me 25.1, CH3 1.42 (s) 2’, 3’

2’-Me 22.6, CH3 1.46 (s)


1H and

13C NMR.




32







Research Autopol 3.

NMR solvents were obtained from Cambridge Isotope Laboratories (CIL), Inc.








33

4.3.2 Collection, Extraction and Isolation

The root of Peucedanum japonicum (dry wt. 1000 g) were extracted three times

with 50% MeOH in CH2Cl2 at room temperature (rt). These extracts were

combined and partitioned three times between MeOH and n-Haxane. The MeOH

layer was further partitioned between H2O and Ethyl acetate to afford an H2O-

soluble fraction and an Ethyl acetate-soluble fraction (8.41 g). The Ethyl acetate

fraction was subjected to reversed-phase silica gel flash column chromatography

(YMC Gel ODS-A, 120 Å, 40-60 μm), eluting with a step gradient solvent system

of 100% to 0% CH2Cl2/MeOH and washing with acetone, to afford 11 fractions (1-

11). They were separated on a reversed-phase preparative HPLC column (Shiseido

CAPCELL C18 5 μm, 250 × 10 mm, 2.0 mL/min, UV detection at 210 nm) eluting

with 70% CH3CN with a retention time of 23 min. from fraction 2.

34

4.3.3 Plant material

Cultivated Korean Peucedani Radix (Peucedanum japonicum, Umvelliferae) was

purchased from a market in Sunchoen, Korea in July, 2013. A voucher specimen

was deposited at the Center for Marine Natural Products and Drug Discovery,

Seoul National University, Korea.

35

Reference

Alejandro M. S. M.; Keith B. G.; Carmen C.; Robert S. J.; William K.; R.Daniel L.;

J. Michael M.; David J. N.; Barbara C. P.; Dale E. S. Trends in Pharmacological

Sciences 2010 31 255-265

Alev T.; Masaki B.; Ozlem B.; Toru O. Pharmaceutical Biology 2006 44 528-533

Antonio G. G.; Juan T. B.; Hermelo L. D.; Juan R. L.; Francisco R. L.

Phytochemistry 1979 18 1021-1023

Chuen-neu W.; Young-ji S.; Yueh-hsiung K.; Chien-chih C.; Yun-lian L. Planta

Med. 2000 65 644-647

Dang T. L. H.; Hee cheol C.; Tae cheol R.; Hyun sun L.; Myung koo L’; Young ho

K. Arch. Pharm. Res. 1999 22 324-326

Gwang L.; Hyoung-gun P.; Mi-lim C.; Young ho K.; Young bok P.; Kyoung-sik S.;

Chaejoon C.; Young-seuk B. J. Microbiol. Biotechnol. 2000 10 394-398

Huawer Lv. Jianguang Luo; Xiaobing W. Chromatographia 2013 76 141-148

Ih-sheng C.; Chin-teng C.; Wine-show S.; Che-ming T.; Ian-lih T.; Chang-yih D.;

Feng-nien Ko Phytochemistry 1996 41 525-530

Jie K.; Lei Z.; Jing-hao S.; Min Ye; Jian H.; Bao-rong W.; De-an Guo Journal of

Asian National Products Research 2008 10 971-976

Jimmy Y. C. W.; Wang-fun F.; Jin-xia Z.; Chung-hang L.; Hoi-lung K.; Meng-su Y.;

Ding Li, Hon-Yeung C. European Journal of Pharmacology 2003 473 9-17

Ling-yi K.; Fei Zhi Journal of Asian Natural Products Research 2003 5 183-187

Ling-yi K.; Yi Li; Zhi-da M.; Xian Li; Ting-ru Z. Phytochemistry 1996 41 1423-

1426

Margaret E. T.; Jiayuan L.; Joselynn W.; Fatemeh A.; David C. R. Applied and

Environmental Microbiology 2009 75 567-572

Masashi H.; Hiroe K.; Hajime O.; Nobuji N. J. Agric. Food Chem. 2003 51 5255-

5261

Masashi H.; Hiroe K.; Nobuji N. J. Agric. Food Chem. 2004 52 445-450

Naoki T.; Toshio K.; Yoco A.; Junji O.; Izumi M.; Kiyoshi W.; Seisho T. Chem.

Pharm. Bull. 1991 39 1415-1421

Natthanan N.; Takafumi O.; Takayoshi T.; Masashi I.; Hironori I.; Hirosuke O.

36

International Journal of Life Science and Medical Research 2012 2 28-34

Sung ok L.; Sang zin C.; Jong hwa L.; Sung hyun C.; Sang hyun P.; Hee chol K.;

Eun young Y.; Hijae C.; Kang ro L. Arch Pharm Res. 2004 27 1207-1210

Taro K.; Masahiko T.; Makio S.; Nian-he W.; Kimiye B. J.Nat. Med. 2008 62 87-90

Thomas R. H.; Christopher S. J.; Feng S. Narure protocols 2007 2 2451-2458

Ting-ting J.; Hwey-ching H.; Meei-yueh J.; Journal of Natural Products 1992 55

1396-1401

Wen-jun Li; Yong-guang Z.; Yu-qin Z.; Shu-kun T.; Ping Xu; Li-hua Xu; Cheng-lin

J. International Journal of Systematic and Evolutionary Microbiology 2005 55

1329-1333

Yasumasa I.; Izumi M.; Yutaka T. Phytochemistry 1992 31 4303-4306

Yasumasa I.; Izumi M.; Yutaka T. Phytochemistry 1994 35 1339-1341

37

Appendix

Figure S1 1H NMR spectrum of 13A001-1 in CDCl3 ............................... 39

Figure S2 13

C NMR spectrum of 13A001-1 in CDCl3 .............................. 40

Figure S3 COSY NMR spectrum of 13A001-1 in CDCl3 ......................... 41

Figure S4 HSQC NMR spectrum of 13A001-1 in CDCl3 ......................... 42

Figure S5 HMBC NMR spectrum of 13A001-1 in CDCl3 ........................ 43

Figure S6 1H NMR spectrum of Butanamide in CDCl3 ............................ 44

Figure S7 COSY NMR spectrum of Butanamide in CDCl3 ...................... 45

Figure S8 HSQC NMR spectrum of Butanamide in CDCl3 ...................... 46

Figure S9 HMBC NMR spectrum of Butanamide in CDCl3 .................... 47

Figure S10 1H NMR spectrum of cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-

3’, 4’-dihydroseselin in CDCl3 .................................................................. 48

Figure S11 COSY NMR spectrum of cis-3’-Acetoxy-4’-(2-

methylbutyroyloxy)-3’, 4’-dihydroseselin in CDCl3 ................................. 49

Figure S12 HSQC NMR spectrum of cis-3’-Acetoxy-4’-(2-


Figure S13 HMBC NMR spectrum of cis-3’-Acetoxy-4’-(2-


Figure S14 1H NMR spectrum of Compound 1 in CDCl3 ......................... 52

Figure S15 COSY NMR spectrum of Compound 1 in CDCl3 .................. 53



Figure S18 HSQC NMR spectrum of Compound 2 in CDCl3 .................. 56

Figure S19 HMBC NMR spectrum of Compound 2 in CDCl3 ................. 57



Figure S22 HSQC NMR spectrum of Compound 3 n CDCl3 ................... 60


38



Figure S26 HSQC NMR spectrum of Compound 4 in CDCl3 .................. 64


39

Figure S1 1H NMR spectrum of Compound 13A001-1 in CDCl3

40

Figure S2 13

C NMR spectrum of Compound 13A001-1 in CDCl3

41

Figure S3 COSY NMR spectrum of Compound 13A001-1 in CDCl3

42

Figure S4 HSQC NMR spectrum of Compound 13A001-1 in CDCl3

43

Figure S5 HMBC NMR spectrum of Compound 13A001-1 in CDCl3

44

Figure S6 1H NMR spectrum of Butanamide in CDCl3

45

Figure S7 COSY NMR spectrum of Butanamide in CDCl3

46

Figure S8 HSQC NMR spectrum of Butanamide in CDCl3

47

Figure S9 HMBC NMR spectrum of Butanamide in CDCl3

48

Figure S10 1H NMR spectrum of Compound cis-3’-Acetoxy-4’-(2-

methylbutyroyloxy)-3’, 4’-dihydroseselin in CDCl3

49

Figure S11 COSY NMR spectrum of cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’,

4’-dihydroseselin in CDCl3

50

Figure S12 HSQC NMR spectrum of cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’,


51

Figure S13 HMBC NMR spectrum of cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’,


52

Figure S14 1H NMR spectrum of Compound 1 in CDCl3

53

Figure S15 COSY NMR spectrum of Compound 1 in CDCl3

54


55


56

Figure S18 HSQC NMR spectrum of Compound 2 in CDCl3

57

Figure S19 HMBC NMR spectrum of Compound 2 CDCl3

58


59


60


61

Figure S23 HMBC NMR spectrum of Compound 3 in CDCl3

62


63


64


65

Figure S27 HMBC NMR spectrum of Compound 4 in CDCl3

66

한글초록

해양환경은 신약 소재의 잠재적 가치를 가지고 있는 매우 다양한

물질이 존재하는 것으로 알려져 있다. 이러한 물질들은 항암, 항염, 무통,

면역제어, 알레르기 그리고 항바이러스 분석을 통해 특이한 활성으로

증명되었다.

본 석사학위 논문은 다양한 해양 식물과 해양미생물의 화학적인

조성을 연구하고 그들의 이차대사산물의 분리, 정제 및 구조 규명에

관한 것이다.

태국 Rayong 해변에서 분리한 악티노박테리아인

스트렙토마이세테스에서 나프탈렌 계열의 새로운 물질을 분리하였다.

구조는 여러가지 분광학 자료(IR, MS, 1H and 13C NMR, COSY, HSQC,

HMBC) 분석을 통하여 구조를 규명하였다.

해양에서 유래된 박테리아의 지속적인 연구 결과 여러 개의

악티노마이세테스를 분리하였다. 해양 퇴적물에서 분리한 박테리아인

스트렙토마이세스 소디필러스에서 1D, 2D NMR을 통해 밝혀진 구조를

규명하였다. 이 물질은 알칼리필릭한 악티노마이세테스로 이차

대사산물이 그람 음성균의 쿼럼센싱의 조절자로 알려져 있다.

한국 남해안 근처에서 자라는 해양 식물인 식방풍에서 다섯개의

켈락톤 구조의 물질을 규명하였다.

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이학석사 학위논문

Identification of Secondary Metabolites

from the Marine Plant and Bacteria

- 해양식물과 해양미생물의 2차 대사산물 규명-

Hyunji Kim

August 2014

Laboratory of Marine Drug

School of Earth and Environment Sciences

Seoul National University

i

Abstract

The marine environment has established to be a very rich source of particularly

potent compounds that have demonstrated significant activities in antitumor, anti-

inflammatory, analgesia, immunomodulation, allergy and anti-viral assays.

The chemical inquiry of several marine plant and bacteria with the isolation and

structure elucidation of secondary metabolites with biological activities are

described in this thesis.

Actinobacteria from Rayong beach, Thailand, Streptomycetes sp., let us the

isolation of new compound with naphthalene moiety. The structure was elucidated

by extensive spectroscopic data (IR, MS, 1H and

13C NMR, COSY, HSQC,

HMBC) analysis.

Continuous research on marine derived bacteria led to collections of a number of

actinomycetes. Actinobacteria from the mud flat, streptomyces sodiiphilus, let us

the isolation of butanamide. 1D and 2D NMR study revealed the moiety, which is

common to natural products. It is reported alkaliphilic actinomycetes that its

secondary metabolites able of quenching quorum sensing controlled behaviors in

gram-negative reporter strains.

A collection of the marine plant Peucedanum japonicum (Umbelliferae) from the

south of Korea beach, led to the isolation of the five novel khellactone.

ii

Key words

Marine natural products, Secondary metabolite, Peucedanum japonicum,

Streptomyces sodiiphilus

Student Number: 2012-23078

iii

Contents

Abstract ................................................................................................................ ⅰ

Contents................................................................................................................ ⅲ

List of Figures ...................................................................................................... ⅴ

List of Tables ........................................................................................................ ⅵ

Chapter 1 General Introduction of Marine Natural Products

1. Marine Natural Products in General Introduction .............................................. 1

Chapter 2 Naphthalene derivative from Streptomycete sp.

2. 1 Introduction ..................................................................................................... 5

2. 2 Result and Discussion ..................................................................................... 6

2.2.1 Structural Elucidation of coumarines ...................................................... 6

2.2.1.1 13A001-1 .......................................................................................... 6

2.3 Experimental Section ....................................................................................... 9

2.3.1 Instruments and Data Collection ............................................................. 9

2.3.2 Collection, Extraction and Isolation ...................................................... 10

Chapter 3 Butanamide from Streptomyces sodiiphilus

3. 1 Introduction ................................................................................................... 11


3.2.1 Structural Elucidation of Butanamide ................................................... 12

3.2.1.1 Butanamide..................................................................................... 12

3.3 Experimental Section ..................................................................................... 15

3.3.1 Instruments and Data Collection ........................................................... 15

3.3.2 Isolation of the Secondary Metabolites ................................................. 16

Chapter 4 Khellactone from the marine plant, Peucedanum japonicum

4. 1 Introduction ................................................................................................... 17


4.2.1 Structural Elucidation of Khellactone ................................................... 22

4.2.1.1 cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-dihydroseselin ... 22

4.2.1.2 Compound 1-4 ................................................................................ 25

4.3 Experimental Section ............................................................................... 37

4.3.1 Instruments and Data Collection ....................................................... 37

iv

4.3.2 Collection, Extraction and Isolation .................................................. 38

4.3.3 Plant material..................................................................................... 39

Reference .............................................................................................................. 40

Appendix .............................................................................................................. 42

한글초록 .............................................................................................................. 66

v

List of Figures

Figure 1.1 Chemical structures of marine-derived drugs ............................ 2

Figure 1.2 Experimental sample places in marine environment ................. 4

Figure 2 COSY and HMBC correlations of 13A001-1 ............................... 7

Figure 3 COSY and HMBC correlations of Butanamide .......................... 13


................................................................................................................... 18


methylbutyroyloxy)-3’,4’-dihydroseselin ................................................. 22

Figure 4.3 Structure of cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’,4’-

dihydroseselin ............................................................................................ 23

Figure 4.4 Structures of compounds 1-4 ................................................... 26

Figure 4.5 Spectroscopic data of compounds 1-4...................................... 27

vi

List of Tables

Table 1 A number of novel/new metabolites produced by marine

actinomycetes during the period 2005- 2010 (Subramani et al., 2012) .... 3

Table 2 Physical and spectral properties of 13A001-1 ................................ 8

Table 3 Physical and spectral properties of butanamide ............................ 14


methylbutyroyloxy)-3’, 4’-dihydroseselin ................................................ 24





1

Chapter 1

General Introduction of Marine Natural Products

1. Marine Natural Products in General Introduction

The largely uninvestigated marine world that apparently harbors the most

biodiversity may be the greatest resource to discover novel ‘validated’ structures

that considered with biologically relevant chemical space. Nature is an ancient

pharmacy that made use of the solitary source of therapeutics for the early times.

Natural products are a major part of the modern pharmaceuticals that we use to

cure human disease and have been the mainstay of disease treatment for most of

human history. The diversity of organisms in the marine environment has

motivated researchers to indentify novel marine natural products for many years

that could finally be developed into drugs. The first FDA-approved marine-derived

drugs, Cytarabine (Ara-C) and vidarabine (Ara-A) (Figure 1.1), are synthetic

pyrimidine and purine nucleosides, respectively, developed from naturally existing

nucleosides originally isolated from the sponge Tethya crypta in Carinnean.

Cytarabine was approved by the FDA in 1969 as an anticancer drug, while

vidarabine was approved in 1976 as an antiviral agent. It has taken over 30 years

for another marine-derived natural product to get approval. Ziconotide (Prialt® ) for

the treatment of ordinary to chronic pain gained FDA approval in 2004. It is a

naturally occurring peptide isolated from the venom of the cone snail Conus magus.

Travectedin (Yondelis® ) has received European approval for the treatment of soft

tissue sarcoma in 2007, and for ovarian carcinoma in 2009. It is a marine alkaloid

isolated from the marine tunicate Ecteinascidia turbinate. Eribulin mesylate

(Halaven™) is the latest drug to the market from the marine sponge Halichondria

okadai in 1986. Incidentally numerous other marine natural products or derivatives

thereof are in different phases of clinical trials.

2

Cytarabine, Ara-C Vidarabine, Ara-A

Prialt®

Yondelis® Eribulin Mesylate(Halaven™)

Figure 1.1 Chemical structures of marine-derived drugs

3

Table 1 demonstrated that some examples of new secondary metabolites isolated

from marine actinomycetes from 2005 to 2010. Among them, a few compounds are

of particular interest due to their rarity and potent and diverse bioactivity such as

staurosporinone, salinosporanide A, lodopyridone, arenimycin, marinomycins and

proximicins from Table 1.

Table 1 A number of novel/new metabolites produced by marine actinomycetes

during the period 2005- 2010 (Subramani et al., 2012)

Compound Source Biological activity

Chinikomycins Streptomyces sp. Anticancer

Chloro-dihydroquinones Novel actonomycete Antibacterial; anticancer

Glaciapyrroles Streptomyces sp. Antibacterial

Frigocyclinone Streptomyces griseus Antibacterial

Lajollamycin Streptomyces nodosus Antibacterial

Mechercharmycins Thermoactinomyces sp. Anticancer

Salinosporamide A Salinispora tropica Anticancer; Antibacterial

Sporolide A Salinispora tropica Unknown

Salinosporamides B & C Salinispora tropica Cytotoxicity

2-Allyloxyphenol Streptomyces sp. Antibacterial; food preservative; oral

disinfectant

Saliniketal Salinisporta arenicola Cancer chemoprevention

Marinomycins A-D Marinispora Antibacterial; anticancer

Cyanosporaside A Salinispora pacifica Unknown

Lodopyrideone Saccharomonospora sp. Anticancer

Arenimycin Salinispora arenicola Antibacterial; anticancer

Salinispyrone Salinispora pacifica Unknown

Salinopyrones A & B Salinispora pacifica Mild cytotoxicity

Pacificanones A & B Salinispora pacifica Antibacterial

Arenicolides A-C Salinispora arenicola Mild cytotoxicity

1-hycroxy-1-norresistomycin Streptomyces chinaensis Antibacterial; anticancer

Resistoflavin methyl ether Streptomyces sp. Antibacterial; anti-oxidative

saturosporinone Streptomyces sp. Antitumor; phycotoxicity

4

The high degree of innovation in the field of marine natural products will lead to

successful marine drug discovery and development, and provides grounds for our

optimism that marine natural products will form a new wave of drugs that flow into

the market and pharmacies in the future.

We have isolated actinobacteria from sediment, beach, and plant in shore. We

elucidated structures of a new one and six previously reported ones. (Figure 1.2)

Figure 1.2 Experimental sample places in marine environment

5

Chapter 2

Naphthalene derivative from Streptomycete sp.

2.1. Introduction

The sea, covering more than 70% of the surface of planet Earth, contains an

exceptional biological diversity, accounting for more than 95% of the whole

biosphere. The sea, covering more than 70% of the surface of planet Earth,

contains an exceptional biological diversity, accounting for more than 95% of the

whole biosphere. Of the total sea surface, only 7–8% is coastal area and the deep

sea, of which 60% is covered by water more than 2000 m deep. The deep sea is a

unique and extreme environment characterized by high pressure, low temperature,

lack of light, and oxygen concentration.

As a part of our continuous research for secondary metabolites discovery from

Rayong beach, Thailand, Streptomycete sp. was isolated from a sediment sample.

Chemical investigation of this strain gave new compound.

6


2.2.1 Structural Elucidation of naphthalene

2.2.1.1 13A001-1

13A001-1 had the molecular formula C20H14O4, as determined by LFABMS with

14 degrees of unsaturation. The UV peak near 400 nm shows us that there are

conjugated bonds.

The four contiguous aromatic proton signals at δ 6.6, 6.9, 7.43, 8.39 and HMBC

correlation, respectively, were indicative of a 1, 2-disubstituted naphthalene. The

HMBC spectrum from H-5’ to C-4’ is suggested that there’s a methyl ester in the

naphthalene. The assignments of the signals of the hydroxyl proton are

straightforward on the basis of their chemical shift. C-2’ exhibits two ling range

coupling interactions of unequal magnitude with H-1’ and H-7. In particular, in

CDCl3, one could observe the HMBC correlations of the exchageable OH carbon to

the sp proton of the naphthalene ring.

The 1H NMR spectrum showed two-proton doublets at δ 7.46 (d, J = 8.0 Hz) and

δ 7.8 (d, J = 8.0 Hz), and four-proton triplets at δ 7.42, respectively, suggested

mono-substituted phenyl ring. The HMBC correlation of H-6’’ to the carbon

resonating at δ 151.19 allowed the assignment of the presence of a C-1’-substituted

naphtho[1,8-b,c]pyran (Figure 2).

7

Figure 2 COSY and HMBC correlations of 13A001-1

8

Table 2 Physical and spectral properties of 13A001-1

NMR Dataa


1 158.8, C

2 99.7, CH 6.6 (s) 1, 3, 4, 8a

3 167.4, C

4 98.8, C

4a 133.9, C

5 122.8, CH 8.39 (d, 8.7) 6 4, 7, 8a

6 130.1, CH 7.43 (t, 15, 7.8) 7 4a, 8

7 116.3, CH 6.9 (d, 7.5)

8 130.7, C

8a 118.1, C

1’ 151.1, C

2’ 103.6, CH 6.65 (s) 7, 8a, 1’, 1’’

1’’ 132.2, C

2’’ 124.7, CH 7.8 (d, 7.5) 3’’

3’’ 128.7, CH 7.46 (t, 15, 7.8) 4’’ 1’’, 5’’

4’’ 129.5, CH 7.42 (t, 7, 3.5) 5’’ 2’’

5’’ 128.7, CH 7.46 (t, 15, 7.8) 6’’

6’’ 124.7, CH 7.8 (d, 8) 1’, 2’’, 4’’

1’’’ 172.6, C

2’’’ 52.1, CH3 4.06 (s)

OH 12.57


1H and

13C NMR.





Molecular fomula: C20H14O4

LREIMS: m/z 318

IR(film) vmax: 2917, 2849 cm-1

UV(CDCl3)λmax: 240, 280, 400 nm

9







Research Autopol 3.

NMR solvents were obtained from Cambridge Isotope Laboratories (CIL) Inc.








10



elution of MeOH in dichloromethane (0%, 1%, 2%, 5%, 10%, 20%, 50%, 90% and



11

Chapter 3

Butanamide from Streptomyces sodiiphilus

3.1. Introduction

Alkaliphilic actinomycetes that thrive in alkaline environments have typical

nutrient requirements, cultural conditions and physiological properties. The deep

sea is a unique and extreme environment characterized by high pressure, low

temperature. In deep sea sediments, marine micro-organisms occupy the main

ecological niche, which carries out an important role in the recycling of carbon and

nitrogen sources on the sea floor.

It is known that actinomycetes grow well in neutral and slightly alkaline media.

Alkaliphilic actinomycetes were first isolated from various soils by Taber. These

alkaliphilic streptomycetes showed maximal radial rates of colony growth at pH 8.

It is obligately Na+-dependent, and showed sensitivity to K

+.

Taxonomically diverse marine bacteria have proven to be a rich resource for the

discovery of structurally unique and bioactive secondary metabolites. Given the

intense microbial competition for resources such as space and nutrients, it is

probable that many excreted metabolites help mediate microbe-microbe

interactions. Various antibiotics have been implicated as chemical defenses for

marine bacteria, thus suggesting a role for the biosynthesis of toxic metabolites.

Though not yet widely studied, the secretion of nontoxic molecules could also

play important roles in antagonistic marine microbial interactions. Quorum sensing

pathways of competing bacteria are potential targets for such nontoxic chemical

defenses. Bacterial communication is facilitated by the production and subsequent

recognition of small signaling molecules (autoinducers) and can regulate important

phenotype, including bioluminescence, biofilm formation, swarming motility,

antibiotic biosynthesis, and virulence factor production.

12


3.2.1 Structural Elucidation of Butanamide

3.2.1.1 Butanamide

The molecular formula of butanamide was determined to be C13H19NO by

LRFABMS of the molecular ion at m/z 205. It is 5 degrees of unsaturation and the

five contiguous aromatic proton signals at δ 7.22(d, J = 4.5 Hz), 7.24(t, J = 15, 7.5

Hz), 7.32(t, J = 15, 7.5 Hz), respectively, were indicative of mono-substituted

phenyl ring. The IR spectrum showed bands at 3275 and 1648cm-1

, pointing to the

presence of an amide group. The 1H and

13C NMR data confirmed the presence of

an amide carbonyl and indicated three methylenes and two methyl.

The 1H NMR spectrum of butanamide showed a broad D2O exchangeable signal

at δ 5.45(1H) for NH and/or OH groups. The signals at δ 5.12 (1H) and δ 4.78 (1H)

were attributed to olefinic protons. A triplet of doublet at δ 3.96 for a methine

proton connected either with a nitrogen or an oxygen atom, a 1H multiplet at δ 1.80,

a doublet of triplet (2H) at δ 1.56 and a doublet for six protons at δ 0.86 (isopropyl

group) were observed at the aliphatic region. The 13

C NMR and APT spectrum of

this compound showed eight signals. The signals at δ 166.4 and δ 158.2 were

interpreted as carbonyl signal of carboxylic acids, amides or esters. The signals at δ

134.6 and δ 98.9 represent a C=CH2 fragment in conjugation with a carbonyl group,

while the signals at δ 22.1 were accounted for the two methyl group of an isopropyl

residue. Finally the structure of the compound was assigned by COSY, HMQC and

HMBC couplings as butanamide.

13

Figure 3 COSY and HMBC correlations of Butanamide

14

Table 3 Physical and spectral properties of butanamide

NMR Dataa


1 172.5, C

2 46.3, CH2 1.99 (d) 3 1

3 26.2, CH 2.15 (m) 4,5 1

4 22.6, CH3 0.9 (d, 7.0)

5 22.6, CH3 0.9 (d, 7.0)

1’ 40.6, CH2 3.56 (t, 7.0) 2’

2’ 35.9, CH2 2.85 (t, 7.0) 1’’, 2’’

1’’ 139, C 3’’

2’’ 128.6, CH 7.2 (d, 7.5) 4’’

2’’ 128.6, CH 7.2 (d, 7.5)

3’’ 128.7, CH 7.31 (t, 15, 7.5)

3’’ 128.7, CH 7.31 (t, 15, 7.5)

4’’ 126.6, CH 7.24 (t, 15, 7.5)

NH 5.4 (br s)


1H and

13C NMR.





Molecular fomula: C13H19NO

LRFABMS: m/z 205.30

IR(film) vmax: 3301, 1637, 1541 cm-1

UV (CDCl3) λmax: 200, 250 nm

15



Streptomyces sodiiphilus was obtained from marine sediment from JangBong-

Island, Gyeong Gi-do, South Korea in 2013. Sampled mud sediments were dried

by air for 24 hours in a clean bench and given a heat shock at 55℃ for 9 minutes in

a low-temperature incubator. Aggregated clumps were lightly mortared by glass

rod and stamped by sponge-plug onto various prepared solid agar media. Some of

the dried samples were suspended in sterilized sea water and diluted plastic rod.

These crude plates were placed in 27℃ chamber and monitored for 1 to 3 months

to obtain a unique actinomycete like colonies. Strain 13B033 was peaked from 1/5

ISP 1 media agar plate showing white filamentous colony. The 16S r RNA gene

was cloned using universal primers 27F and 1492R and showed 99.5% similarity to

Streptomuces sodiiphilus strain YIM 80305.

The bacterial strain 13B033 was cultured at 25℃ with shaking at 150 rpm in 30

L Pyrex flask each containing 1L of the medium SYP (10 g of starch soluble, 4 g of

yeast extract, 2 g of peptone, dissolved in 75% of 1 L filtered-seawater). After 10

days, the broth was extracted two times with ethyl acetate and evaporated to yield

2.37 g of crude organic extract.

16



elution of MeOH in dichloromethane(0%, 1%, 2%, 5%, 10%, 20%, 50%, 90% and



17

Chapter 4

Khellactone Derivatives from the Marine Plant, Peucedanum

japonicum

4.1 Introduction

Peucedanum japonicum, a medicinal plant belonging to the family of

Umbelliferae, grows on the cliffs of Island, Korea. Peucedanum japonicum leaves

are traditionally consumed on Island as a medicinal herb for the treatment of cough.

The root of this plant has been used as a folk medicine for cold and neuralgic

diseases in Taiwan (Chen et al., 1996). A number of studies gave reported on the

physiological activities of PJT including antioxidant activity (Hisamoto et al.,

2003), tyrosinase inhibitory effect (Hisamoto et al., 2004), and anti-platelet

aggregation activity (Chen et al., 1996) in vitro. Furthermore, several study have

demonstrated the instance of hypolipidaemic compounds in the leaves of

Peucedanum japonicum (Hsu and Yen, 2007; Li et al., 2006).

It has been reported that some coumarins, glucosides, and chromones.(Figure

4.1)

19

norisoprenoid glucoside

phenylpropanoid glucoside

Praeruptorin A

psoralen xanthotoxin

20

divaricataesters A divaricataesters B

divaricataesters C

21

Panaxynol

Panaxydol

Panaxytriol


22


4.2.1 Structural Elucidation of Khellactone

4.2.1.1 cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-dihydroseselin

Cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-dihydroseselin had the [M]+

peak at m/z 389 in the low resolution electron ionization mass spectrum which

indicated the molecular formula was C24H28O7. It requires ten degrees of

unsaturation. The absorption bands of a carbonyl (1722 cm-1

) and an aromatic

system (1612 cm-1

, 1492 cm-1

) in the IR spectrum are characteristic of a coumarin

skeleton.

The 1H NMR spectrum in the aromatic proton region of it contained two pairs of

doublets at δ 6.23 (1H, d, J = 9.5 Hz), 7.61 (1H, d, J = 9.5 Hz) and at δ 7.37 (1H, d,

J = 8.6 Hz), 6.82 (1H, d, J = 8.6 Hz), which are in agreement with the H-3 and H-4

signals of the α-pyrone ring system and signals of H-5 and H-6 of the benzene ring,

indication that it is a coumarin substituted at the C-7 and C-8 positions. A pair of

doublets at δ 5.38 (1H, d, J = 3.5 Hz) and δ 6.27(1H, d, J = 3.5 Hz) was assigned to

two ester groups, showing C-7 and C-8 formed a dihydropyran ring. The signals at

δ 2.42 (m), 1.5 (m), 1.75 (m), 1.23 (d, 7.2), 0.96 (t) are due to a methylbutyryl

group and at δ 5.34 (d, 5.0), 6.57 (d, 5.0) are due to an acetoxy group.



23

The relative cis-configuration of compound was assigned on the basis of the

coupling constant J3’, 4’, which was between 4.1 and 5.0 Hz. The optical rotations

for compound [α]D25

= + 0.96° indicates the configuration 3’R, 4’R.

Figure 4.3 Structure of cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-

dihydroseselin

24



NMR Dataa


2 159.7, C

3 113.2, CH 6.25 (d, 9.6) 4 2, 9

4 143.1, CH 7.61 (d, 9.6) 5, 7, 9, 10

5 129.1, CH 7.36 (d, 8.6) 6 9, 10

6 114.3, CH 6.81 (d, 8.6) 7, 8

7 156.6, C

8 107.5, C

9 154.3, C

10 112.4, C

2’ 77.7, C

3’ 70.1, CH 5.34 (d, 5.0) 4’ 1’’

4’ 60.9, CH 6.57 (d, 5.0) 7, 8, 2’, 2’-Me

1’’ 166.4, C

2’’ 41.2, CH 2.42 (m), 4’’, 3’’

3’’ 26.3, CH2 1.5 (m), 1.75 (m)

4’’ 16.3, CH3 1.23 (d, 7.2) 2’’, 3’’

5’’ 11.6, CH3 0.96 (t 1’’

1’’’ 166.2, C 2’’’

2’’’ 20.6, CH3 2.12(s)

2’-Me 23.7, CH3 1.42 (s) 2’, 3’

2’-Me 22.4, CH3 1.46 (s)


1H and

13C NMR.






LRFABMS: m/z 388.15

IR(film) vmax: 2925, 1666, 1461 cm-1

UV(CDCl3) λmax: 210, 250, 325 nm

25

4.2.1.2 Compounds 1-4

In addition to the cis-3’-Acetoxy-4’-(2-methylbutyroyloxy)-3’, 4’-dihydroseselin,

four previously reported khellactone, Anomalin (1), Isosamidin (2), (3’S, 4’S)-3’-

isovaleryl-4’-(2-methylbutyryl)-khellactone (3), cis-3’, 4’-disenecioylkhellatone

(4) were also isolated from Peucedanum japonicum. Anomalin (1) and isosamadin

(2) were previously isolated from Saseli resinosum (Alev et al., 2006), S. unicaule

(Barrero et al., 1990), and Peucedanum japonicum (Ikeshiro et al., 1992; Fan et al.,

2000). While cis-3’, 4’-disenecioylkhellatone (4) reported from Peucedanum

japonicum (Ting-Ting Jong et al., 1992), (3’S, 4’S)-3’-isovaleryl-4’-(2-

methylbutyryl)-khellactone (3) was obtained from the Peucedanum praeruptorum

(Huawei Lv et al., 2013). The structures were elucidated by extensive spectroscopic data (IR, MS,

1H and

13C NMR, COSY, HSQC, and HMBC) analysis.

26

1 2

3 4

Figure 4.4 Structures of compounds 1-4

27

Figure 4.5 Spectroscopic data of compounds 1-4

Compound 1



LREIMS: m/z 426

IR (film) vmax: 1731, 1604 cm-1

UV (CDCl3) λmax: 209, 255, 300 nm

[α]D25

= + 9.3° (c 0.0015, MeOH)

Compound 3



LREIMS: m/z 430

IR (film) vmax: 2925, 1666, 1461 cm-1

UV (CDCl3) λmax: 250 nm

[α]D25

= + 29.3° (c 0.00075, MeOH)

Compound 2



LRFABMS: m/z 386.1

IR (KBr) vmax: 2965, 1742, 1609 cm-1

UV (CDCl3) λmax: 225, 250, 322nm

[α]D25

= - 67.31° (c 0. 0083, MeOH)

Compound 4



LRFABMS: m/z 426

IR (KBr) vmax: 2965, 1742, 1609 cm-1

UV (CDCl3) λmax: 248, 300, 325 nm

[α]D25

= + 12.3° (c 0.083, MeOH)

28


NMR Dataa


2 159.8, C

3 113.3, CH 6.21 (d, 9.57) 4 2, 9

4 143.2, CH 7.61 (d, 9.57) 5, 7, 8, 9, 10

5 129.4, CH 7.38 (d, 8.58) 6 9, 10

6 114.4, CH 6.82 (d, 8.58) 7, 8

7 156.7, C

8 107.6, C

9 154.1, C

10 112.5, C

2’ 77.5, C

3’ 70.2, CH 5.44 (d, 5.0) 1’’

4’ 60.2, CH 6.70 (d, 5.0) 7, 8, 2’, 2’-Me

1’’ 166.4, C

2’’ 139.8, C

3’’ 127.4, CH 6.13(1H, m) 5’’ 2’’, 3’’

4’’ 20.4, CH3 1.97(3H, m)

5’’ 15.8, CH3 1.84(3H, m) 2’’

1’’’ 166.3, C

2’’’ 138.4, C

3’’’ 127,CH 5.98(1H, m) 5’’’ 2’’’, 3’’’

4’’’ 20.3, CH3 1.97(3H, m)

5’’’ 15.6, CH3 1.84(3H, m) 2’’’

2’-Me 25.4, CH3 1.46 (s) 2’, 3’

2’-Me 22.5, CH3 1.50 (s)


1H and

13C NMR.




29


NMR Dataa


2 159.5, C

3 112.8, CH 6.23 (d, 9.5) 4 2, 9

4 143, CH 7.61 (d, 9.5) 5, 7, 8, 9, 10

5 128.7, CH 7.36 (d, 8.5) 6 9, 10

6 114.1, CH 6.81 (d, 8.5) 7, 8

7 156.4, C

8 135.9, C

9 153.6, C

10 112.3, C

2’ 77.7, C

3’ 68.7, CH 5.35 (d, 5.0) 4’ 1’’

4’ 61.1, CH 6.57 (d, 5.0) 7, 8, 2’, 2’-Me

1’’ 169.5, C 2’’

2’’ 20.6, CH3 2.11 (s)

1’’’ 164.9, C

2’’’ 114.8, CH 5.69 (s) 4’’, 5’’

3’’’ 158.4, C

4’’’ 27.4, CH3 2.17 (s) 2’’’, 3’’’

5’’’ 20.3, CH3 1.9 (s) 1’’’

2’-Me 23.1, CH3 1.41 (s) 2’, 3’

2’-Me 24.5, CH3 1.45 (s)


1H and

13C NMR.




30


NMR Dataa


2 159.6, C

3 113.5, CH 6.26 (d, 9.5) 4 2, 9

4 143.2, CH 7.63 (d, 9.5) 5, 7, 8, 9, 10

5 129, CH 7.39 (d, 8.5) 6 9, 10

6 114.5, CH 6.83 (d, 8.5) 7, 8

7 156.2, C

8 107, C

9 153.2, C

10 112.7, C

2’ 77.5, C 3’

3’ 69.8, CH 5.35 (d, 5.0) 4’, 5’ 2’, 1’’, 2’-Me

4’ 60.3, CH 6.58 (d, 5.0) 2’, 1’’’

1’’ 171.9, C

2’’ 43.1, C 2.25(2H,m)

3’’ 25.3, CH 2.15(1H, m)

4’’ 22.5, CH3 1.00(3H, d, 7.0)

5’’ 22.5, CH3 1.01(3H, d, 7.0)

1’’’ 175.4, C

2’’’ 41, C 2.44(m) 3’’’, 4’’’

3’’’ 26.5,CH 1.5, 1.75(2H, m)

4’’’ 16.1, CH3 1.25(3H, t, 7.5) 5’’’

5’’’ 11.5, CH3 0.9(3H, d, 7.0)

2’-Me 25.5, CH3 1.45 (s) 1’, 2’, 3’

2’-Me 22.4, CH3 1.49 (s)


1H and

13C NMR.




31


NMR Dataa


2 159.9, C

3 113.2, CH 6.21 (d, 9.5) 4 2, 9

4 143.2, CH 7.57 (d, 9.5) 5, 7, 8, 9, 10

5 129.0, CH 7.34 (d, 8.6) 6 9, 10

6 114.3, CH 6.79 (d, 8.6) 7, 8

7 156.6, C

8 107.62, C

9 154.1, C

10 112.5, C

2’ 77.7, C

3’ 69.4, CH 5.36 (d, 4.9) 4’ 1’’

4’ 59.8, CH 6.62 (d, 54.9) 7, 8, 2’, 2’-Me

1’’ 165.2, C

2’’ 115.2, CH 5.62 (br s) 4’’, 5’’

3’’ 158.3, C

4’’ 27.4, CH3 2.15 (d, 1.1) 2’’, 3’’

5’’ 20.3, CH3 1.87 (s) 1’’

1’’’ 165.1, C 2’’’

2’’’ 115.2, CH 5.66 (br s)

3’’’ 157.6, C

4’’’ 27.4, CH3 2.19 (d, 1.1)

5’’’ 20.3, CH3 1.88 (s)

2’-Me 25.1, CH3 1.42 (s) 2’, 3’

2’-Me 22.6, CH3 1.46 (s)


1H and

13C NMR.




32







Research Autopol 3.

NMR solvents were obtained from Cambridge Isotope Laboratories (CIL), Inc.








33

4.3.2 Collection, Extraction and Isolation

The root of Peucedanum japonicum (dry wt. 1000 g) were extracted three times

with 50% MeOH in CH2Cl2 at room temperature (rt). These extracts were

combined and partitioned three times between MeOH and n-Haxane. The MeOH

layer was further partitioned between H2O and Ethyl acetate to afford an H2O-

soluble fraction and an Ethyl acetate-soluble fraction (8.41 g). The Ethyl acetate

fraction was subjected to reversed-ph

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