I

Chemical and Bioactivity Studies on metabolites

from Three Fijian Marine Sponges

A thesis submitted to the University of the South Pacific in partial

fulfillment of the requirements for the degree of

Master of Science in Chemistry

By

Sachin Singh (BSc)

University of the South Pacific

February 2005

II

Declaration

I declare that this submission is a result of my own investigation and that, to the best of

my knowledge, it contains no material written by another author or previously reported,

nor any material that has been submitted as a diploma or any form of another degree at

any university or institute of higher learning, except where due acknowledgement is

credited in the text.

Candidate: Sachin Singh

Supervisors: Prof. Subramanium Sotheeswaran

University of the South Pacific

Prof. Robert J. Capon

University of Melbourne

Associate Prof. Sadaquat Ali

University of the South Pacific

Date submitted

III

Dedication

To my parents Arjun Singh and Sushila Wati who have been very supportive

throughout my research and to my sister Roshila, without whom this thesis would not

have been possible.

IV

Acknowledgements

I would like to thank my Research Supervisors, Professor Subramanium Sotheeswaran

and Professor Robert J. Capon for their guidance and support during my research. I am

also indebted to Associate Professor Sadaquat Ali for critically reviewing my manuscript.

I wish to thank Ernest Lacey and Jennifer H. Gill, from Microbial Screening

Technologies Pty Ltd, for conducting all bioassays.

I am indebted to Dr Colin Skene for his guidance during my fellowship at University of

Melbourne, Australia.

I am thankful to the Department of Chemistry at the University of Melbourne especially

to the Marine Natural Products Research Group (Big Ben and little Ben, Shirley, Mike,

Alison, Ed and Michelle) for teaching me how to use instruments like the NMR and Mass

Spectrometer. I would also like to thank the Staff and students at Ormond College

(Victoria, Australia) for making my visit enjoyable.

I would like to thank Alison Drechsler for helping me obtain journal articles.

I would like to thank Francis Mani for allowing me the use of his office during the

Christmas break.

V

I would like to acknowledge my friends Kirti, Anand, Modi, David and Artika for their

help in printing, locating journal articles and for being a general nuisance.

VI

Table of Contents

Declaration

Dedication

Acknowledgements

Table of Contents

List of Abbreviations

Abstract

Chapter 1

1.0 Introduction

1.1 Introduction

1.1.1 Historical uses of Natural Products

1.1.2 The Marine environment

1.1.3 Agrochemicals

1.1.4 Drug Discovery strategies

1.1.5 Limitations

1.2 Present study

1.2.1 The Project: Nematocides from marine sources

1.2.2 Phylum Porifera

1.2.3 Bioactive metabolites from Fijian sponges

1.2.4 Genus Spongosorites

ii

iii

iv

vi

x

xii

1

1

2

4

6

7

8

8

10

11

12

VII

1.2.4.1 Alkaloids

1.2.5 Genus Stelleta

1.2.5.1 Terpenes

1.2.5.2 Alkaloids

1.2.6 Genus Stylotella

1.2.6.1 Cyclopeptides

1.2.6.2 Terpenes

1.2.6.3 Alkaloids

Chapter 2

2.0 Experimental

2.1 General Experimental Procedures

2.2 General Methodology

2.2.1 Collection and Identification

2.2.2 Extraction and Isolation

2.2.2.1 Spongosorites sp

2.2.2.1.1 Spongosoritin A

2.2.2.2 Stelleta splendens

2.2.2.2.1 Marfey’s analysis

2.2.2.2.2 Jaspamide/jasplakinolide

2.2.2.3 Stylissa massa

2.2.2.3.1 Oroidin

2.2.2.3.2 Sceptrin

12

16

16

19

22

22

23

24

26

26

27

27

28

29

29

32

32

33

35

36

36

VIII

2.2.2.3.3 Trigonelline

2.2.2.3.4 Zooanemonin

2.2.2.3.5 Hexabromodiphenyl ether

2.2.2.3.6 Taurine

Chapter 3

3.0 Discussion

3.1 Polyketide

3.1.1 Spongosoritin A

3.2 Depsipeptide

3.2.1 Jaspamide/Jasplakinolide

3.3 Bromopyrrole-Imidazole alkaloids

3.3.1 Oroidin

3.3.2 Sceptrin

3.4 Polybrominated diphenyl ether

3.4.1 hexabromodiphenyl ether

3.5 Aminosulfonic Acid

3.5.1 Taurine

3.6 Nicotinic Acid

3.6.1 Trigonelline

3.7 Pyridinium salt

3.7.1 Zooanemonin

37

37

38

38

39

39

39

47

47

51

51

56

59

60

61

61

65

65

67

67

IX

Chapter 4

4.0 Conclusion

Chapter 5

5.0 References

6.0 Appendix

Spectroscopic data for Spongosoritin A

69

70

70

90

90

X

List of abbreviations

13C-NMR Carbon 13 nuclear magnetic resonance

1H-1H COSY 1H-1H Correlation spectroscopy

1H-NMR Proton nuclear magnetic resonance

2D-NMR Two dimensional nuclear magnetic resonance spectroscopy

bs Broad singlet

CyLD99 Lethal dose required to kill 99% of cells

CyT Titre volume for cytotoxicity test.

(Increase in titre volume= decrease in concentration)

d Doublet

dd Doublet of doublets

DEPT-135 Distortionless enhancement by polarisation transfer-135

dt Doublet of triplets

ESIMS Electrospray ionisation mass spectroscopy

FT-ICR MS Fourier transform ion cyclotron resonance mass spectroscopy

FTIR Fourier transform infrared spectroscopy

HMBC Heteronuclear multiple bond correlation

HMQC Heteronuclear multiple-quantum coherence

HREIMS High resolution electron ionisation mass spectroscopy

m Multiplet

mult Multiplicity

NeLD99 Lethal dose required to kill 99% of nematode population

NeT Titre volume required for nematode toxicity test.

XI

nOe Nuclear overhauser effect

ppm Parts per million

q Quartet

s Singlet

SPE Solid phase extraction

t Triplet

UV Ultraviolet radiation

�C 13-C signal in parts per million

�H 1-H signal in parts per million

XII

Abstract

Eight metabolites have been isolated from three Fijian marine sponges (Spongosorites sp,

Stelleta splendens and Stylissa massa).

A novel polyketide, Spongosoritin A, was isolated from Spongosorites sp. This

compound was biologically inactive against all bioassays conducted.

From Stelleta splendens the known cyclodepsipeptide jaspamide/jasplakinolide, was

isolated. Jaspamide exhibited excellent antihelminthic properties against the parasitic

nematode Haemonchus contortus (NeT=64, NeLD99=2.6�g/mL) and cytotoxicity (

CyT=1024, CyLD99= 0.16�g/mL).

Stylissa massa yielded six known metabolites: Bromopyrrole-imidazoles oroidin and

sceptrin, Hexabromodiphenyl ether, taurine, zooanemonin and trigonelline. Moderate

antihelminthic properties were shown by Oroidin (NeT=8, NeLD99=15�g/mL), taurine

(NeT=4, NeLD99=42�g/mL), zooanemonin (NeT=4, NeLD99=50�g/mL).

Hexabromodipehnyl ether (NeT=16, NeLD99=8.9�g/mL) and trigonelline (NeT=16,

NeLD99=3.4�g/mL) showed good antihelmintihic activity. Sceptrin was inactive against

bioassays conducted.

Structural elucidation was made by spectroscopic methods (1H-NMR, 13C-NMR, 1H-1H

COSY NMR, HMBC, HMQC, ESIMS, FTIR) and using MarinLit database.

1

1.0 Introduction

1.1 Introduction

1.1.1 Historical uses of Natural Products

Man has harnessed drugs from nature for thousands of years. Extracts from animal or

plant sources have been used as medicines, poisons and for recreational purposes. The

benefits of these have been enormous. Natural products have been the basis of ancient

practices of the Ayuverda in India and in Chinese medicine for centuries. The plant and

animal extracts contained healing properties that cured various ailments. Monks of the

Benedictine order applied Papaver somniferum to treat pain and anaesthetize patients.

The active ingredient was discovered to be morphine and was first isolated in 1806

(Grabley and Thiericke 2000). In the 17th century the bark of the Peruvian Cinchona tree

was imported to Europe, where its extract was used to treat malaria. Its active ingredient,

quinine was first isolated in1820. This was later replaced by synthetic derivatives.

Salicin, the active principle in willow-tree bark was used by the Greeks and Romans

since 400 BC for its antipyretic and analgesic properties. The acetyl derivative of

salicylic acid (more active degradation product of salicin), aspirin, is now one of the

most common drugs used in modern day medicine (Grabley and Thiericke 2000).

The discovery of penicillin by Alexander Fleming in 1928 added a new dimension to

Natural Product Chemistry. While plants had been the main source of Natural Product

drugs, microorganisms were now being looked at with growing interest. Unlike plants,

2

microbes could be cultured to provide almost unlimited supply of new raw material for

drugs (at moderate costs).

1.1.2 The Marine Environment

The Natural Products Chemist began to look towards the ocean depths in search for

useful, natural compounds. The harsh, competitive Marine environment was home to

many species, which employed biochemical warfare frequently as a means of survival.

Consequently, a whole new range of exciting chemistry and potential biologically active

compounds was available. An added bonus was that many invertebrates had symbiotic

relationships with microorganisms, which was sometimes responsible for the production

of chemical compound of interest. Most of the chemicals found have been highly toxic, a

direct result of trying to survive in the highly competitive marine environment.

Brevetoxin B is a neurotoxin produced by the dinoflagellate Ptychodiscus brevis

(Grabley and Thiericke 2000) and is associated with the Red Tide catastrophe’s that

occurs along coastlines causing death of marine life and human poisoning. Other marine

natural products exhibiting toxicity are being tested clinically for anticancer treatment.

O

O

O

O

O

O

O

O

O

O

O

CHO

O

CH3

CH2

OH

H

HH

CH3

H

H

HH

HHHH

CH3

H

H H

HCH3

CH3CH3

Brevetoxin

3

Didemnin B, a cyclic peptide was first isolated from the ascidian Trididemnum solidum

(Rinehart et. al. 1981) and became the first marine metabolite to reach human clinical

trials. Didemnin B had anticancer, antiviral as well as immunosuppressant abilities. It

was withdrawn from clinical trials after the compound proved to be too toxic.

Dehydrodidemnin B, isolated from another ascidian Aplidium albicans, exhibited

superior anticancer activity to didemnin B (Sakai et. al. 1996). This compound can be

prepared by oxidation of didemnin B or by total synthesis and is undergoing clinical

trials.

CH3N

O

OH

NO

CH3

NH

CH3

CH3

ONHO

N

OMe

CH3

O

N

O

NH

CH3

CH3O

CH3

OCH3

CH3

O

CH3

CH3

CH3OH

Didemnin B

4

Bryostatin 1, a macrocyclic metabolite was first isolated from the bryozoan Bugula

neritina (Pettit et. al. 1982). It was until recently undergoing clinical trials for anticancer

treatment. Wender et. al. (1998) synthesized a simplified analog that retains the

bioactivity of bryostatin 1.

1.1.3 Agrochemicals

Both agricultural and pharmaceutical chemistry are vital tools, on the basis of which

modern society exists. One gives rise to an abundant food supply while the other provides

a healthy mind and body. On the agricultural scene, industrial companies focussed on

O O

CH3

OH

CH3 CH3

O

COOMe

OCH3

OOH

HCH3

CH3OH

OO

OH

O

COOMe

H

CH3

Bryostatin 1

5

producing synthetic agrochemicals. The success of these synthetic chemicals (particularly

pesticides) accelerated developments in synthetic chemistry. In the pharmaceutical

industry synthetic compounds began to quickly replace natural product therapy. Soon

problems with the use of synthetic pesticides surfaced. Chlorinated hydrocarbons, such as

DDT, began causing problems in the food chain. DDT had been directly linked to the

production of thin eggshells in wild birds. Methyl bromide, a popular fumigant and soil

sterilant was discovered in groundwater. Methyl bromide causes sterility in human males

exposed to it and contributes to the ozone hole at the polar icecaps. The shift to

biologically active natural product remedies had major advantages. They were target

specific, had high specific activity and were biodegradable (Cutler and Cutler 1999).

Insecticides developed from the natural product template pyrethrin became commercially

available in the late 1980s. Cyclopenol, a benzodiazepine from the fungus Penicillium

cyclopium (Cutler and Cutler 1999) is active against Phytophthora infestans, which is

responsible for the potato late blight.

NH

NH

O

O

OH

O

Cyclopenol

6

Ivermectin , a broad-spectrum antihelminthic is a modified version of avermectins, a

group of compounds produced naturally by Streptomyces avermitilis (Grabley and

Thiericke 2000; Cutler and Cutler 1999).

1.1.4 Drug discovery Strategies

Due to the amount of investment involved in researching natural products, it is no longer

viable to research “blindly”. Today a chemical screening strategy is in place at most

institutions where natural product research is undertaken. Crude extracts taken from test

samples are screened for potential bioactivity. Once a “hit” (Crude showing positive

O

OCH3

OH

OMe

O

OMe

CH3

O

H

CH3

OCH3

OMe

OH

OO

O

O

CH3

H

R

CH3

H

Ivermectin

Mixture of B1a: R= CH(CH3)CH2CH3

and B1b: R= CH(CH3)2

7

bioactivity) is obtained further research is conducted to identify and isolate the bioactive

metabolite (Koch et. al. 2000). This reduces the guesswork involved and increases the

chances of finding a biologically active compound.

Bioactivity studies are conducted either at each stage of fractionation or only on the

isolated pure metabolite. Bioassay guided fractionation tends to take much longer and is a

little more costly but the more active “hits” usually yield the metabolites of interest to the

Natural Products Chemist (Faulkner 2000).

1.1.5 Limitations

The biggest drawback of marine natural product chemistry is the insufficient supply of

material. To obtain grams of a pure metabolite, hundreds of kilograms of animal material

are required. This cannot be obtained without severely affecting the marine ecosystem.

Clinical trials are limited due to this reason.

Other problems include getting permission to collect biological specimens and access to

collection sites.

8

1.2 Present Study

The main objectives of this project were to:

� Isolate biologically active secondary metabolites from Marine sources, primarily

targeting metabolites with antihelminthic properties. Secondary targets were

cytotoxic, antifungal and antibacterial metabolites.

� Isolate a novel compound.

� Isolate a compound previously undiscovered in the particular species of sponges

being investigated.

1.2.1 The Project: Nematocides from marine sources

Nematodes or Roundworms (Phylum Nematoda) are the most abundant of metazoan

animals. They inhabit almost every substrate, geographical area and multicellular

organism. Over 12,000 species have been described, however, biologists believe more

unidentified species exist. Although some are free-living freshwater, marine and

terrestrial species most are parasitic (Nybakken 1996). The parasitic forms are best

known and are of great medical and agricultural significance. Unlike microbial

pathogens, nematodes are larger, multicellular organisms. As a result their biochemical

processes are different from microorganisms. Therefore most antihelminthic agents

(Agosta 1996) tend to be chemically different from drugs used to combat bacteria and

fungi.

9

Marine Natural Products group (MNP) at Melbourne University primarily targets the

parasitic nematode Haemonchus contortus. This particular nematode is a parasite of

ruminants and is a major pest to livestock. The MNP group is dedicated to finding

antihelminthic agents from marine sources to combat nematodes affecting the agricultural

industry. Some success has been obtained in the past with the discovery of phoriospongin

A (Capon et. al. 2002) obtained from the sponges Phoriospongia sp and Callyspongia

bilamellata. A nematocidal depsipeptide (LD99 �������� ��������������������������

2001) and its methyl ester (LD99 ������� ���99 �������������� �������!"���#��

sponge Trachycladus laevispirulifer, and thiocyanatin A (a dithiocyanate LD99 1.3

�����!"���#���$�����Oceanapia sp (Capon et. al. 2001).

NHNH

NH

OO

O

O

CH3

NH

O

NH

CH3 O

CH3

CH3

OH

NH

O

CH3

N

O

CH3

NH

O

CH3

O

NH

CH3

Cl

CH3

O NH

NH2

O

CH3

CH3

OH

O

Phoriospongin A

10

1.2.2 Phylum Porifera

Sponges (Phylum Porifera) have been a rich source of biologically active, structurally

novel natural products. The most primitive of multicellular organisms, they specialise in

O NHCH3

CH3

CH2

O

OHOMe

OMe

OCH3

OMe

OH

OH O

Onnamide F

NCSSCN

OH

Thiocyanatin A

11

being sessile. This characteristic, in conjunction with their relative abundance in shallow

waters, make sponges an easy target for natural product chemists.

Sponges exhibit a wide variety of colours depending on the species. This makes them

rather conspicuous to predators. Other problems arise from harmful microorganisms

ingested while filter feeding and from fungal growth. To combat these problems sponges

have resorted to chemical warfare as a defence mechanism.

Chemical warfare is not the only reason that chemicals are produced. Reproduction,

growth and signalling all involve chemistry.

These chemicals are sought after by Natural Products Chemists in the hope of finding a

miracle drug to cure the many ailments that afflicts human health and quality of life.

1.2.3 Bioactive Metabolites from Fijian Sponges

Fiji boasts the third largest reef system in the world. Most of this is undocumented in

terms of fauna. Natural products chemists have shown some interest in sponges from Fiji.

One of the more interesting classes of bioactive metabolites reported from Fijian sponges

are bengamides (Quinoa et. al. 1986). Bengamides have exhibited a variety of biological

activity and are currently undergoing clinical trials.

Three Fijian marine sponges: Spongosorites sp, Stylissa massa and Stelleta splendens

were chemically investigated in an effort to discover new bioactive metabolites.

12

1.2.4 Genus Spongosorites

Sponges of the genus Spongosorites (Demospongiae, Halichondriidae) are generally

found in deep marine waters in the Indo-Pacific region. These sponges have received

somewhat limited attention from Marine natural products chemists, with only one major

structural class of bioactive metabolites, the bis-indole alkaloids having been discovered.

1.2.4.1 Alkaloids

Topsentin and its analogues are a series of bis-indole alkaloids that were isolated from

Topsentia genitrix (Bartik et. al. 1987), Spongosorites sp. (Murray et. al. 1995; Sakemi

and Sun 1991; Wright et. al. 1992) and Hexadella sp. (Morris and Andersen 1988)

sponges.

Topsentin, bromotopsentin and a related dihydro compound were isolated from a

Caribbean sponge of the genus Spongosorites and were active as antiviral and cytotoxic

(P388 leukemia cell line) agents (Tsujii and Rinehart 1988). Nortopsentins A, B and C

were isolated from the deep-sea sponge, Spongosorites ruetzleri and exhibited cytotoxic

and antifungal properties (Sakemi and Sun 1991).

13

NH

N

NH

O

NH

Br

4,5-Dihydro-6”- deoxybromotopsentin

NH

NH

NR1

NH

R2

Nortopsentins

A R1 =R2= BrB R1= Br, R2=HC R1= H, R2=Br

NH

N

NH

O

NH

R1

R2

1. R1= H, R2 = OH (Topsentin)2. R1=Br, R2 = OH (Bromotopsentin)3. R1= OH, R2 = H (Isotopsentin)4. R1= R2 = OH (Hydroxytopsentin)5. R1= R2 = H (Deoxytopsentin)

Topsentin and analogues

14

Researchers at the Harbor Branch Oceanographic Institution, along with Dr Robert

Jacobs at UC Santa Barbara, demonstrated that topsentins are potent mediators of both

immunogenic and neurogenic inflammation (Faulkner 2000). At the 9th International

Symposium on Marine Natural Products (Townsville, July 1998), Dr Jacobs and

associates reported synthesising a simple bis-indole derivative possessing excellent anti-

inflammatory activity.

Dragmacidins

The dragmacidin series of bisindolyl piperazines are a new class of marine natural

products. The first Dragmacidin was isolated in 1988 from the deepwater marine sponge,

Dragmacidon sp. It was the first bisindolyl marine alkaloid to contain an unoxidized

piperazine ring, and exhibited a wide range of biological activity. (Kohmoto et. al.1988).

All members of the series contain a central piperazine ring and two indole units.

Wright et.al. reported dragmacidin D in 1992, from a Spongosorites sp of sponge.

Dragmacidin D was found to inhibit growth of feline leukemia virus and P388 and A549

tumor cell lines. It also exhibited activity against fungal pathogens Cryptococcus

neoformans and Candida albicans. In 1998 Capon et. al. isolated dragmacidin D and E

from a southern Australian sponge of the same genus. Dragmacidin E was found to be a

potent inhibitor of serine-threonine protein phosphatases.

15

Methylated Purine Base

Methylated purine bases have been isolated from both marine and terrestrial sources.

Many of these have exhibited a wide range of biological activity.

An example of this structure class, 1, 9-dimethylhypoxanthine was isolated from a

Spongosorites sp obtained from Southern Australia. This was found to be biologically

inactive (Capon et. al. 2000).

Br

NH

NH

NH

O

NH

OH

NH

+

NH

NH2

CH3

Dragmacidin E

NH NH

N

O

NH

NH

N+

NH2H

Br

OH

Dragmacidin D

CH3

N

NN

N

O

CH3

1,9-dimethylhypoxanthine

16

1.2.5 Genus Stelleta

The major natural metabolites reported from different species of the genus Stelleta belong

to the classes of terpenes, terpenoids and alkaloids.

1.2.5.1 Terpenes

All Triterpenoids reported from genus Stelletta, had isomalbaricane skeletons. In 1982,

McCabe et. al. reported a biolocally inactive triterpenoid pigment. Su et. al. (1994)

reported stelletin A, a biologically active triterpenoid pigment. Subsequent researchers

reported stelletins C, D, E, F and G (McCormick et. al. 1996). All these stelletins were

found to be cytotoxic.

OCH3

CH3

O

CH3

OCH3

CH3R2

CH3

HR1

CH3

H

A R1= R2= OC R1= OAc, R2= H Stelletins

17

Globostellatic Acids A-D were isolated from S. Globostelleta (Ryu et. al. 1996). These

exhibited antitumour properties.

CH3

NaO 2C

AcO

CH3

O

CH3

CH3 CH3 CH3

OH

O

CH3

HH

Globostellatic Acid A

CH3

NaO2C

AcO

CH3

CH3 CH3

CH3

CH3

OH

OCH3

CH3

O

H

H

Globostellatic acid B

CH3

CH3 CH3

CH3

CH3

OH

OCH3

RO

NaO2C

CH3

CH3

H

O

H

Globostellatic acid C, R = Ac

Globostellatic acid D, R = H

18

Stelletadine A, an acylated bisguanidine sesquiterpene alkaloid was reported (Tsukamoto

et. al. 1996) to induce metamorphosis of ascidian larvae.

Tsukamoto et. al. (1999b) reported bistelletadines A and B, dimers of the sesquiterpene

stelletadines.

CH3

CH3

O

NH NH

NH2+

NH NH2

NH2+

NH NH

CH3

O NH2+

NH NH2

NH2+

R

A R=HB R= (CH3)2C=CHCH2-

Bistellettadines

CH3 NH NHNH NH2

CH3CH3

CH3

CH2 NH2+

NH2+

Stellettadine A

19

1.2.5.2 Alkaloids

A large number of alkaloids have been reported from the genus Stelletta.

Fused ring alkaloids isolated from sponges of genus Stelletta were cytotoxic acridine

alkaloids, nordercitin, dercitamine and dercitamide[Gunawardana et. al. 1989].

Also reported were indolizidine alkaloids, stellettamide A (Hirota et. al. 1990) and B

(Shin et. al. 1997).

S

N NH

N

R

Nordercitin R= N(CH3)2Dercitamine R= NHCH3Dercitamide R= NHCOC2H5

CH3 NH

CH3 CH3 CH3

O

N+

CH3

H

Stelletamide A

20

Fusetani et. al. (1994) reported a family of pyridine alkaloids, cyclostellettamines A-F

from the sponge S. maxima. Cyclostellettamines were found to inhibit the muscarinic

acetylcholine binding receptors, which play a role in memory and learning.

N+ N

+

)m

) n(

(

A. m= 1, n=1B. m= 1, n=2C. m= 2, n=2D. m= 1, n=3E. m= 2, n=3F. m= 3, n=3

Cyclostellettamines

21

Stellettazole A, B and C were found to be antibacterial. Stelletazole A (Tsukamoto et. al.

1999a), B and C have all been classified as alkaloids (Matsunaga et. al. 1999).

CH3 NH

N+

NH NH

CH3 CH3 CH3 CH3 O N

CH3

NH2

CH3 NH

N+

NH2

CH3 CH3 CH3 CH3 O NCH3

CH3

CH3 CH3

NH

N+

NH

O N

CH3

NH2+

NH2CH3

Stellettazole A

Stellettazole B

Stellettazole C

22

1.2.6 Genus Stylotella

Sponges of the genus Stylotella (Demospongiae, Dictyonellidae) have been found to

contain cyclopeptides, as well as terpenes and alkaloids.

1.2.6.1 Cyclopeptides

Two cycloheptapeptides were isolated from sponges of the genus Stylotella. Stylostatin 1

(Pettit et. al. 1993) has antitumour properties whereas stylopeptide 1 is biologically

inactive (Pettit et. al. 1995).

NH

NH

O

NH

NH2O

ONHO

OH

CH3 CH3

NH

O

NH

CH3

O

N

OCH3

O

CH3

CH3

Stylostatin 1

23

1.2.6.2 Terpenes

Stylotelline, belonging to a rare class of sesquiterpene isocyanides (Pais et. al. 1987) and

stylotellanes, a group of dichloroimines (Simpson et. al. 1997) were isolated from two

different sponges of the genus Stylotella. The former was found to have antibiotic and

antitumour activity while the latter was inactive.

NH

NH

OO

CH3

CH3

OCH3CH3

NH

NH

O

NH

O

CH3

CH3

NH

OH

ON

O

H

H

Stylopeptide 1

CH3 N Cl

ClCH3 CH3 CH3

CH3

CH3 CH3 CH2

N Cl

ClCl

CH3

CH3

CH3

CN

CH3

Stylotelline

Stylotellane A

Stylotellane B

24

1.2.6.3 Alkaloids

An important family of C17N9 bioactive bisguanidine alkaloids was isolated from

palau’an sponges, S. aurantium and S. agminata. Palau’amine (Kinnel et. al. 1998) is

composed of six contiguous rings with an unbroken chain of eight chiral centres. In 1995

Kato et. al. reported styloguanidines (isopalau’amines) which also belong to this family

and were isolated from the same genus of sponge. Palau’amines and its congeners exhibit

a wide range of biological activity which includes antibiotic, immunosuppressive and

antitumour activity.

N

N

O

NH

N

NH2

NH

NNH2

OH Cl

R2

R1

CH2NHR3

Palau’amines

NH

N

O

NH

N

NH2

NH

NNH2

OH

CH2NH2

Cl

R2

R1

Styloguanidines

5. R1 = R2 = H, 6. R1 = H, R2 =Br7. R1 = R2 =Br

1. R1 = R2 =R3=H2. R1 = R2 =H, R3=Ac3. R1 =H, R2 =Br, R3=H4. R1 = R2 =Br , R3=H

25

Also found were a range of known ‘nuisance’ compounds: sceptrin, oroidin,

dibromophakellin, hymenin, hymenidin, hymenialdisine, and debromohymenialdisine

(Patil et al. 1997; Williams and Faulkner 1996). These compounds are found in a large

number of sponges of the class Demospongiae. Of these, debromohymenialdisine or

DBH has been patented as a Protein Kinase C inhibitor and more recently as a treatment

for osteoarthritis (Faulkner 2000). There is still considerable interest in commercial

development of DBH as it can easily be synthesized (Xu et. al. 1997).

N

NHNH

O

NHO

NH2

Debromohymenialdisine

26

2.0 Experimental

2.1 General Experimental Procedures (Capon et. al. 2003; Capon per comm.

2002)

High performance liquid chromatography (HPLC) was performed using either a Waters

600 solvent delivery system equipped with a Waters 2700 sample manager and Waters

996 photodiode array detector, or a Waters 2790 separations module equipped with

Waters 996 photodiode array detector, Alltech 500 evaporative light scattering detector

with low temperature adapter, and Waters Fraction Collector II. Both systems operated

under PC control running Waters Millenium software operating through Microsoft NT.

Size exclusion chromatography was performed using sealed columns packed with

Sephadex LH-20 or Sephadex G-10. Fractions generated on the open columns were

collected on an ISCO Retriever IV fraction collector. Sealed columns were all connected

to an ISCO Tris peristaltic pump fitted with an ISCO UA-60 UV detector and an ISCO

UA-500 fraction collector through a column switch.

1H and 13C as well as the 2-D NMR experiments were performed on either a Varian Unity

400 MHz or a Varian Inova 400 MHz spectrometer in the solvents indicated. All spectra

were referenced to residual 1H signals in deuterated solvents. ESIMS were acquired

using a Waters 2790 separations module equipped with a Micromass ZMD mass

spectrometer at the cone voltage indicated and recorded using Masslynx 3.5 operating

through Microsoft NT. High resolution ESI measurement were recorded on a Bruker

27

BioApex 47e FT-ICR MS (FTMS) fitted with an analytical electrospray at a capillary

voltage of 100-150 eV.

%#�"����$��&���"������������"�������'()D) were recorded at room temperature on a

Jasco Dip-1000 digital polarimeter in a 100 mm x 2 mm cell. Ultraviolet (UV) absorption

spectra were obtained using a Hitachi Model 150-20 double beam spectrophotometer,

while infrared (IR) spectra were acquired using a Bio-rad FTS 165 FT-IR spectrometer

under PC control running Bio-rad Win-IR software. Solvents indicated were

spectroscopic grade solvents.

Bioassays

Bioassays were performed by Microbial Screening Technologies Pty Ltd., using

published procedures (Gill et al. 1995). Screening was done for nematocides, cytotoxic

agents, Protox (procaryotes) and Eutox (Eucaryotes).

2.2 General Methodology

2.2.1 Collection and Identification

Spongosorites sp (USP ID 55-087) was collected on 10th March, 2001 at a depth of 5-

15m(18’20.788S; 177’59.751E) from Filis Delight. This sponge was identified by Lisa

Goudie (Museum of Victoria, Nov 2002). This has been registered at Museum of Victoria

(Registration No.MVF83540).

28

Stylissa massa (USP ID 55-033) was collected on 9th November, 2000 at a depth of

1.5m(18’20.788S; 177’59.751E) from Suva Barrier Reef. Dr John Hooper (Queensland

Museum, Apr 2003) identified this sponge. This has been registered at Queensland

Museum (Registration No.QMG319998). Stylissa massa was previously known as

Stylotella aurantium.

Stelletta splendens (USP ID 55-029) was collected on 9th November, 2000 at a depth of

5-15m(18’20.788S; 177’59.751E) from Denise Patch. Dr John Hooper (Queensland

Museum, Apr 2003) identified this sponge. This has been registered at Queensland

Museum (Registration No.QMG20000). Stelletta splendens was previously known as

Dorypleres splendens.

A voucher specimen of all three sponges is being kept at Cold Room 6, Department of

Chemistry, University of the South Pacific.

2.2.2 Extraction and Isolation

The samples of Spongororites sp, Stelleta splendens and Stylissa massa were collected

and stored in 95% Ethanol. For each of the samples stored in EtOH, one third of the

solvent was decanted, the bottle refilled with fresh EtOH and stored for future use. The

EtOH extracts were dried in vacuo. Thus Spongosorites gave 65.4mg of crude extract.

The masses of Stelleta splendens (524.4mg) and Stylissa massa (1621.8mg) were

obtained and recorded.

29

The crude extracts were extracted with DCM (3 x 10mL) followed by BuOH (30mL) and

H2O (30mL). The BuOH and H2O were placed in a separating funnel, shaken and left to

stand until two distinct layers formed. The layers were collected separately. All extracts

were dried in vacuo and the mass obtained.

2.2.2.1 Spongosorites sp

Three fractions were generated: DCM soluble (24.1 mg, 36.3%), BuOH soluble (6.2 mg,

9.5%) and H2O soluble (35.1 mg, 57.5%). The DCM solubles (CyT =256, CyLD99 =0.65)

and BuOH solubles (CyT =128, CyLD99 =1.70) exhibited bioactivity. The bioactive

solubles (BuOH and DCM) displayed similar HNMR and HPLC characteristics and were

thus combined. The combined fraction was fractionated further on silica SPE. Material

eluting from 20-40% EtOAc in Petroleum spirit was subjected to semipreparative HPLC

(gradient 80-100% MeCN in H2O @ 2mL/min), to yield a biologically inactive pure

compound (2.2mg, 3.4%) which was later identified as Spongosoritin A.

2.2.2.1.1 Spongosoritin A

Numbering shown on structure (Page 43-44).

'()D -148.10 (c 1.54, MeOH); -111.00(c 1.11, CH2Cl2)

30

IR (CHCl3) cm-1: 1699, 1674, 1625, 1461, and 1434.

���������max : 272.5 nm

1H-NMR (400MHz,CDCl3� �� = 0.75(t, H-17); 0.93(t, H-12); 1.12(t, H-14); 1.16 (m, H-

16a);1.36(m, H-16b);1.40(s, H-15); 1.76(m, H-7a); 1.76(m, H-8); 1.96(m, H-7 b); 1.96(m,

H-11); 2.10(q, H-13); 3.70(s, OMe); 4.80(bs, H-4); 5.02(dd, H-9); 5.23(dt, H-10); 6.21(s,

H-5).

13C-NMR (100MHz, CDCl3� �� = 11.3(C-17); 11.7(C-14); 13.9(C-12); 18.5(C-13);

25.6(C-11); 26.3(C-15); 29.4(C-16); 40.1(C-8); 45.0(C-7); 50.5(OMe); 83.9(C-4);

95.1(C-6); 132.2(C-10); 133.8(C-9); 138.2(C-3); 141.7(C-5); 166.9(C-1); 171.5(C-2);

13C-NMR (DEPT 135, CDCl3� ��� 11.3(C-17, CH3); 11.7(C-14, CH3); 13.9(C-12, CH3);

18.5(C-13, CH2); 25.6(C-11, CH2); 26.3(C-15, CH3); 29.4(C-16, CH2); 40.1(C-8, CH);

45.0(C-7, CH2); 83.9(C-4, CH); 50.5(OMe, CH3); 132.2(C-10, CH); 133.8(C-9, CH);

141.7(C-5, CH).

1H-1H COSY ���6.21: 4.80, 2.10(H5: H4, H13); 5.23: 5.02, 1.96 (H-10: H-9, H-11);

5.02: 1.76, 1.76, 1.96, 1.96, 5.23(H-7a, H-8, H-7b, H-11, H-10); 4.80: 6.21(H-4:H-5);

2.10: 6.21,1.12(H-13: H-5, H-14); 1.96: 1.76, 1.76, 5.02(H-7b:H-7a, H-8, H-9); 1.96:

5.23, 0.93(H-11: H-10, H-12); 1.76: ,1.76, 1.96 (H-7a: H-8, H-7b); 1.76: 1.76, 1.96, 5.02,

31

1.16, 1.36 (H-8: H-7a, H-7b, H-9, H-16a, H-16b ); 1.36: 1.76, 1.16, 0.75 (H-16b: H-8, H-

16a, H-17); 1.16: 1.76, 0.75, 1.36(H-16a: H-8, H-17, H-16b); 1.12: 2.10 (H-14: H-13);

0.93: 1.96 (H-12: H-11); 0.75: 1.16, 1.36(H-17: H-16a, H-16b).

HMQC � = 11.3: 0.75(C-17); 11.7:1.12(C-14); 13.9: 0.93(C-12); 18.5:2.10(C-13); 25.6:

1.96 (C-11); 26.3: 1.40(C-15); 29.4: 1.16, 1.36(C-16); 40.1: 1.76(C-8); 45.0: 1.76,

1.96(C-7); 50.5: 3.70(OMe); 83.9: 4.80(C-4); 132.2: 5.23(C-10); 133.8: 5.02(C-9); 141.9:

6.21(C-5).

HMBC � = 4.80: 171.5, 138.2 (H-4: C-2, C-3); 6.21: 171.5, 138.2, 95.1, 18.5 (H-4: C-2,

C-3, C-6, C-13); 1.76: 40.1, 133.8 (H-7a: C-8, C-9); 1.96: 141.7, 95.1, 40.1, 29.4 (H-7b:

C-5, C-6, C-8, C-16); 1.76: 133.8 (H-8: C-9); 5.02: 40.1, 132.2, 25.6 (H-9: C-8, C-10, C-

11); 5.23: 40.1, 133.8, 25.6, 13.9 (H-10: C-8, C-9, C-11, C-12); 1.96: 132.2, 13.9 (H-11:

C-10, C-12); 0.93: 132.2, 25.6 (H-12: C-10, C-11); 2.10: 138.2, 141.7, 11.7 (H-13: C-3,

C-5, C-14); 1.12: 138.2, 18.5(H-14: C-3, C-13); 1.40: 141.7, 95.1, 45.0 (H-15: C-5, C-6,

C-7); 1.16: 40.1 (H-16a: C-8); 1.36: 40.1 (H-16b: C-8); 0.75: 40.1, 29.4 (H-17:C-8, C-16);

3.70: 166.9(OMe: C-1).

nOe: 4.80: 2.10, 3.70(H-4: H-13,OMe); 6.21: 4.80, 2.10, 1.12(H-5: H-4, H-13, H-14);

3.70: 4.80, 1.12 (OMe: H-4, H-14).

ESI (+) ms: 293 [M+H]+, 315 [M+Na]+

ESI (+) HRMS: m/z 315.19124 with an error of -3.8ppm, for C18H28O3Na.

32

[Calculated for C19H34S2N2ONa: 393.2010]

Molecular Formula: C18H28O3

2.2.2.2 Stelleta splendens

The crude was partitioned into DCM solubles (16.5mg, 3.3%), BuOH solubles (70.9mg,

13.5%) and H2O solubles (437mg, 83.3%). The bioactive DCM and BuOH solubles

revealed identical HNMR and HPLC characteristics and were combined.

Solvent trituration of the combined fraction was conducted using the following solvents

(in order): hexane, EtOAc, DCM and MeOH (30 mL each). EtOAc fraction yielded

Jaspamide (13.6mg, 2.6%) as colourless oil.

Marfey’s Analysis was carried out to confirm stereochemistry as that published in

literature (Fujii et. al. 1997).

2.2.2.2.1 Marfey's Analysis

Used to determine D and L isomers of common amino acids.

Acid Hydrolysis

Samples to be analysed (100 �g) were dissolved in 6N HCl (0.5 mL) and stirred for 24

hrs at 110�C. The resulting solution was dried in vacuo and redissolved in H2O (50 �L).

33

Marfey's Reaction

50�L of the acid hydrolystate (or authentic amino acid standard) was added to 20�L of

1M sodium bicarbonate and 100�L of 1% 1-Fluoro-2,4-dinitrophenyl-5-L-

alaninamide(1-FDAA) in acetone. The resulting mixture was stirred at 37� C for 60

minutes. The solution was then neutralized with 1N HCl and diluted with 810 �L of

acetonitrile.

HPLC analysis

Column: Phenomex Luna C18(2) 4.6mm x 150mm

Flow rate: 1 mL/min

Gradient: linear 45 min [15% B to 45% B] in A.

A: 0.1M aqueous NH4Ac adjusted to pH 3 with Trifluoroacetic acid (TFA)

B: MeCN

Detection: PDA with selective monitoring at 340 nm

2.2.2.2.2 Jaspamide/Jasplakinolide

Numbering shown on structure (Page 48).

'()D +30.4 (0.93, CH2Cl2)

'()D +20.7 (0.97, CH3OH)

34

1HNMR (CDCl3������� �� =0.72(3H, d, H-39); 0.80(3H, d, H-20); 1.02(3H, d, H-

21); 1.09(3H, d, H-18); 1.27(2H, m, H-7); 1.53(3H, s, H-19); 2.13(1H, m, H-6); 2.60(2H,

ddd, H-2, H-10); 2.95(3H, s, H-38); 3.32(2H, m, H-28); 4.70(3H, m, H-5, H-8, H-16);

5.22(1H, m, H-11); 5.75(1H, dd, H-14); 6.60(NH, d, H-17); 6.65(2H, d, H-24, H-26);

6.90(2H, d, H-23, H-27); 7.15(1H, m, H-35); 7.20(1H, s, H-36); 7.50(1H, m, H-33); 8.50

(NH, s, H-29).

13CNMR (CDCl3������� ��� 17.7(C-39); 18.5(C-19); 19.0(C-21); 20.3(C-18);

21.9(C-20); 23.3(C-28); 29.2(C-6); 30.8(C-38); 39.7(C-10); 40.1(C-2); 40.7(C-3);

43.3(C-7); 45.9(C-16); 48.9(C-11); 55.5(C-14); 70.7(C-8); 109.0(C-30); 110.6(C-36);

115.5(C-24, C-26, C-31); 118.1(C-33); 120.1(C-35); 122.4(C-34); 127.2(C-23, C-27);

127.8(C-5); 131.3(C-4, C32); 133.6(C-22); 136.1(C-37); 155.6(C-25); 168.9(C-15);

170.8(C-13); 174.3 (C-9); 175.1(C-1).

HPLC retention times for Marfey's Analysis

Sample Retention Time (min)

L-Alanine 13.10

D-Alanine 18.11

Jaspamide (Marfey's deriv.) 13.60, 37.38, 39.83

ESI (+) ms = 709.26 [M+H]+

ESI (-) ms = 707.26 [M-H]+

Molecular Formula: C36H45N4O6Br

35

This was determined by using Mass Spec data to conduct a search on Marinlit database.

A match was made with known compounds. Spectroscopic data matched well with that of

known compound, Jaspamide (Zabriskie et al 1986; Crews et al 1986).

2.2.2.3 Stylissa massa

The Crude (1621.8mg) was partitioned into DCM solubles (95mg, 5.9%), BuOH solubles

(208.6mg, 15.2%) and H2O solubles (1298.4, 80.1%).

BuOH solubles were fractionated further by C18 Solid Phase Extraction to obtain 11

fractions. The fractions (each 10mL) were eluted in order with the following solvent

mixture (H2O: MeOH): 1 (Insoluble residue in 80:20), 2-3 (80:20), 4-5(60:40), 6-7

(40:60), 8-9 (20:80), 10-11 (100% MeOH).

Based on HNMR and ESI (±) MS data, a hexabromodiphenyl ether (Fr 1-2, 58.3mg)

eluted early from the SPE, followed by known bromo sponge metabolites oroidin (Fr 4,

3.3mg) and Sceptrin (Fr 11, 2.6mg). Fraction 3 was subjected to semipreparative C18

HPLC (gradient) to yield Trigonelline (2.0mg).

Part of the H2O solubles (256mg) were fractionated further on a G10 Sephadex column

using H2O as the eluent. 60 fractions were collected at a flowrate of 2.7mL/min with each

fraction volume being 8mL respectively. Zooanemonin (24.1mg) eluted early (Fr 10-13),

followed by taurine (35.7mg, Fr 20-25) and additional trigonelline (58.2mg, Fr 32-43).

36

2.2.2.3.1 Oroidin

1H-NMR (400MHz,CDCl3): � = 4.02(H-8: d, 2H), 6.03(H-9: dt, 1H), 6.24(H-10: d, 1H),

6.74(H-12: s, 1H,), 6.80(H-4: s, 1H).

ESI(+)MS = 390 [M+H]

Molecular Formula: C11H13N5OBr2 (MarinLit)

A search on marinlit database using Mass Spectrometry data found a match with the

known compound, Oroidin. Spectroscopic data (experimental) matched that of literature

(Garcia et al 1973; Lindel et al 2000).

2.2.2.3.2 Sceptrin (Dimer of Debromooroidin/Hymenidin)

1H-NMR (400MHz,CDCl3� �� = 2.29(H-8:s, 1H), 3.43(H-9:s, 2H), 6.40(H-4:s, 1H),

6.70(H-14/16:s, 1H), 6.90(H-16/14:s, 1H)

EESI(+)MS = 619 [M+H], 621[M+2+H], 623[M+4+H]

Molecular Formula: C22H26N10O2Br2 (Marinlit)

A search on marinlit database using Mass Spectrometry data found a match with the

known compound, Sceptrin. Spectroscopic data (experimental) matched with that of

literature (Walker et al 1981).

37

2.2.2.3.3 Trigonelline

1H-NMR (400MHz,CDCl3� �� = 4.28(H8: s, 3H), 7.82(t), 8.70(d), 9.01(s) [4H].

ESI(+)MS = 138 [M+H]

Molecular Formula:

A search on marinlit database using Mass Spectrometry data found a match with the

known compound, Trigonelline.

2.2.2.3.4 Zooanemonin

1H-NMR (400MHz,CDCl3� �� = 3.46(H-6/7: s, 3H), 3.58(H-6/7: s, 3H), 3.70(H-8: s,

2H), 7.06(H-5: s, 1H), 8.40(H-2: s, 1H).

ESI(+)MS = 155 [M+H]+

Molecular Formula: C7H10N2O2 (Marinlit)

A search on marinlit database using Mass Spectrometry data found a match with the

known compound, Zooanemonin. Spectroscopic data (experimental) matched that of

literature (Hattori et al 2001).

38

2.2.2.3.5 Hexabromodiphenyl ether

1H-NMR (400MHz,CDCl3� �� = 6.50(H-4’:s), 7.37(H-5’:s)

.

ESI(+)MS = 670 [M]-, 672[M+2]-, 674[M+4]-

Molecular Formula: C12H4O3Br6 (Marinlit)

A search on marinlit database using Mass Spectrometry data found a match with the

known compound, Hexabromodiphenyl ether. Mass spectrum detected the presence of six

Br atoms. Spectroscopic data (experimental) matched that of literature (Carte & Faulkner

1981).

2.2.2.3.6 Taurine

1H-NMR (400MHz,CDCl3� ����3.11(t), 3.30(t)

Molecular Formula: C2H7NO3S

A search on marinlit database using Mass Spectrometry data found a match with the

known compound, Taurine. Comparison of experimentally obtained data was made with

spectroscopic data obtained from an authentic standard of Taurine, with results matching

perfectly.

39

3.0 Discussion

3.1 Polyketide

The structure elucidation of a novel polyketide, Spongosoritin A, from the sponge

Spongosorites sp is described below.

3.1.1 Spongosoritin A

This compound was isolated as a clear oil.

It was assigned the Molecular formula C18H28O3 by High Resolution Mass Spectrometry

( (M+Na, m/z= 315.1924). Five double bond equivalents were calculated from the

molecular formula.

H to C assignments was made on the basis of DEPT 135 and HMQC experiments (Refer

Table 1).

From the 1HNMR and 13CNMR the following were deduced:

1. *�+����!���&�&�" ������"��$"�����������#�"����#���,��-��&�$"��������C=132.2, 133.8,

.��/ �/0/�1 �/1/�� �/����2��H=5.23, 5.02, 6.21)

2. 3#"���$"��"-���#-�����C=//�� //�1 /��.�2���H=0.75, 1.12, 0.93)

3. �������#���������"���"-���#-���"��$���C=�.�0 ��H=1.24)

4. ������#�+-���"��$���C =�4�� ��H =3.70)

5. 5�����#������ ��������� -������+-�������C=���� ��H=4.80).

40

6. 5�������"�&�" ��-����C= 166.9).

The C-C connectivity was deduced using 1H-1H COSY and HMBC (see Table 1).

The following moiety was deduced from 1H-1H COSY:

Fig 1.1H-1H COSY correlations

From HMBC (Refer Table1) this moiety was further elucidated and two possible partial

structures were deduced:

78

910

11

16

CH3 17

CH3 12R

32

56

78

910

11

16

13

CH3 17

CH3 15

CH3 12

CH314

R1

R2

41

Fig. 2 HMBC Correlations (H to C)

Comparing the molecular formula of the tentative partial structures (Fig 2) with the

molecular formula for the compound showed that C3H4O3 were still unassigned. This

consisted of a methine ��C=���� ��H=0��4� ���#�+-���"��$���C =�4�� ��H =3.70), and an

����"�&�" ��-����C= 166.9); deduced previously from NMR data. Drawing possible

structures showed that one of the oxygen atoms on the methine was from the methoxyl

group while the other was part of an ester. The carbonyl present is actually an ester as no

other C (apart from the ones mentioned) shows a downfield shift associated with having

oxygen attached. This formed the moiety:

Fig.3

35

26

78

910

11

16

13

CH3 17

CH3 15

CH3 12

CH314

R2

R1

O O

R1

OCH3

R2

42

When combining the two moieties deduced (Fig 1 and Fig 2), comparison of the

molecular unsaturation number to the count of unsaturation sites indicated that one

additional ring was present which could contain the ester. This ring was either 6-

membered or 5-membered.

Combining this data gave four possible structures:

R1 = -CH2CH(C2H5)CH=CHC2H5

R2 = -CH=C(CH3)CH2CH(C2H5)CH=CHC2H5

Fig. 4

O

O

OCH3

CH3

R1

CH3

O

CH3

R1

O

OCH3

CH3

OO OCH3

R2CH3

O O

R2CH3

H3CO

(i) (ii)

(iii) (iv)

43

6�&���"�5,�"#����"�7!!�&����5����+$�"�������#�������#��&���������80���H=4.80),

8/����H=��/4� �8/0���H=/�/����#���8����H=���/�������""������� �59����H=3.70), H13

��H=��/4���#���80���H=0��4�������""������������80���H=4.80), H14 (1.12) when OMe

��H=3.70) was irradiated. This meant that C4 was attached to C3 (Thus enhancing H13

and OMe but not affecting H15).

As H15 was unaffected by any of the above nOe experiments the number of possible

structures were now limited to two (Structures: ii and iii).

Relooking at HMBC correlations (H to C) for H15 showed that neighbouring carbons

were C5, C6 and C7, which supported structure (iii).

Hence the structure was tentatively determined as 1a and was assigned the trivial name,

Spongosoritin A.

4

32

56

78

910

11

16 1

OO

13

OMe

CH3 17

CH3 15

CH3 12

CH314

1a

44

While this structure matched with most of the spectroscopic data there were still

irregularities:

1) Olefinic sp2 carbons do not usually occur at 95 ppm (C6), especially in a

trisubstituted double bond as proposed in 1a.

2) Acetal carbons (C4) don’t usually occur at 84 ppm.

An alternative to 1a, is a lower homologue of a polyketide sponge metabolite 2, reported

by Faulkner et al in 1980. The spectroscopic data for 2 agree closely with those of

Spongosoritin A.

In Spongosoritin A (1b) the C6 ethyl substituent in 2 is replaced by a methyl substituent.

The IR spectrum of ester 2 contained a complex group of bands at 1710, 1690

and1640cm-1. This compared relatively well to Spongosoritin A, which had IR bands at

1699, 1674 and 1625cm-1.

Faulkner deduced the structure of ester 2 by 1H-NMR, 13C-NMR and ozonolysis of the

ester 2.

6

5

O

4

3

13 CH314

2

COOMe1

78

CH3 15

910

16CH3 17

11CH3 12

O

CH3

COOMe

CH3

CH3

CH3

21b

45

In comparing the two possible structures for Spongosoritin A: 1a and 1b; C7 to C12 and

C16, C17 moieties were the same in both structures.

The notable differences between 1a and 1b were the carbon appearing at 95 ppm (C6),

was not an sp2 hybridized carbon but a tertiary sp3 carbon attached to an oxygen atom and

the acetal carbon (C4) in 1a was an sp2 hybridized carbon forming an alkene bond (C2) in

1b. Even with this rearrangement, the 2D-NMR data matches with the new structure

proposed.

Again there are some doubts, as tertiary sp3 hybridised carbons even those with a single

oxygen substituent, do not occur easily at 95 ppm and olefinic carbons very rarely occur

at 84 ppm. Both structures: 1a and 1b correspond to most of the spectroscopic data

including the 2D-NMR data. Both structures are tentative at this stage as neither

possibility can be ruled.

Unfortunately spectroscopic data available is insufficient to conclusively determine the

final structure. Further analysis is required to discard one of the possible structures.

Although the crude fraction showed cytotoxic activity (CyT= 256, LD99= 0.65)

Spongosoritin A was completely inactive against all bioassays. As this was a bioassay-

guided fractionation, this compound has either degraded or needs to work in tandem with

another compound to be biologically active.

46

Table 1: NMR data for Spongosoritin A

Atom #

13C NMR(ppm)

1HNMR(ppm)

Type of C(HMQC, DEPT-135)

1H-1H COSY HMBC(H to C Correlations)

1 166.9 C2 171.5 C3 138.2 C4 83.9 4.80 (bs) CH H5 C2, C35 141.7 6.21 (s) CH H4, H13 C2, C3, C6, C136 95.1 C7a7b

45.0 1.76 (m)1.96 (m)

CH2 H8H8, H9

C8, C9C5, C6, C8, C16

8 40.1 1.76 (m) CH H7a, H7b, H9, H11, H16a,H16b

C9

9 133.8 5.02 (dd) CH H7b, H8, H10, H11

C8, C10, C11

10 132.2 5.23 (dt) CH H9, H11 C8,C9,C11, C1211 25.6 1.96 (m) CH2 H8, H9, H10,

H12C10, C12

12 13.9 0.93 (t) CH3 H11 C10, C1113 18.5 2.10 (q) CH2 H5, H14 C3, C5, C1414 11.7 1.12 (t) CH3 H13 C3, C1315 26.3 1.40 (s) CH3 C5,C6,C716a16b

29.4 1.16(m, 1H)1.36(m, 1H)

CH2 H8, H16b, H17H8, H16a, H17

C8C8

17 11.3 0.75 (t, 3H) CH3 H16a, H16b C8, C16OMe 50.5 3.70 (s, 3H) CH3 C1

Assignments supported by HMQC and Dept 135 experiments.Numbering follows Structure 1aChanges to numbering 1a=1b: C2=C3, C3=C4, C4=C2. All other assignments are identical.

47

3.2 Depsipeptide

A known depsipeptide was isolated from the sponge Stelleta splendens. The structure of

this depsipeptide was elucidated by the spectroscopic methods described below.

3.2.1 Jaspamide/Jasplakinolide

The molecular formula was determined as C36H45O6N4Br ([M]+ =709) by its ESIMS, 1H-

NMR and 13C- NMR. The 13C-NMR showed signals for four carbonyl groups including

��������"���C= 175.1, 174.4, 170.5, 168.9, 70.7). 1H-NMR showed signals for a p-

�� ��������� ��:����"����'�H=6.90(d), 6.65(d), 6.65(d), 6.90(d)], o-substituted benzene

"����'�H= 7.50(m), 7.15(m), 7.20(s)] and a tri-�� �����������;�����H=4.70).

A search on Marinlit Database using the mass number ([M]+ 709) found a match with the

known marine compound, Jaspamide/ Jasplakinolide (Zabriskie et al 1986; Crews et al

1986). Spectroscopic data were compared and a near perfect match was obtained (refer to

Table 2).

As an additional confirmation test Marfey’s analysis was carried out. Jaspamide is

composed of three amino acids: L-alanine, N-methyltryptophan (2- "��� "���������<-

tyrosine. Acid hydrolysis and subsequent HPLC analysis showed that there were three

amino acids present. One of these was confirmed to be L-alanine by using authentic

standards of L and D-alanine. The other two amino acids are very rare and authentic

standards of these were unavailable for comparison.

48

Hence the structure was determined to be jaspamide/Jasplakinolide (3).

3NH17

5

4

O

8

910

11

7

6O15

16

13

O

NH12

MeN38

14

O

1 2

CH319

O CH318

CH320

CH3 21

CH3 39

22

24

23

2526

27

OH

2831

32

37

33

36

34

35

NH29

30

Br

3

49

Table 2: Comparison of NMR data for jaspamide (jasplakinolide)

Jaspamide(Zabriskie et. al. 1986)

Jasplakinolide(Crews et. al. 1986)

Experimental

13C (ppm) 1H (mult) 13C (ppm) 1H(mult) 13C (ppm) 1H(mult)1 175.1 175.3 175.12 40.1 2.50(m) 40 2.50(m) 40.1 2.60(ddd)3 40.7 2.38(dd)

1.89(d)41.1 2.39

1.8940.7 2.40(m)

1.90(d)4 131.1 133.8 131.35 127.8 4.75(d) 128.3 4.74(m) 127.8 4.70(m)6 29.2 2.23(m) 29.3 2.28(m) 29.2 2.13(s)7 43.3 1.32(m) 43.7 1.30(m) 43.3 1.27(m)8 70.8 4.62(m) 70.7 4.62(m) 70.7 4.70(m)9 174.4 170.8 174.310 39.7 2.65(dd) 2.65(dd) 40.4 2.65

2.6339.7 2.60(ddd)

11 49 5.26(dd) 49.2 5.27(m) 48.9 5.22(m)12 7.65(d) 7.67(d)13 170.5 169.1 170.814 55.5 5.85(dd) 55.7 5.85(dd) 55.5 5.75(dd)15 168.9 174.7 168.916 45.8 4.75(m) 46.1 4.74(m) 45.9 4.70(m)17 6.63(bs) 6.70(d) 6.60(d)18 20.3 1.12(d) 20.4 1.11(d) 20.3 1.09(d)19 18.5 1.56(s) 18.5 1.55(s) 18.5 1.53(s)20 21.9 0.81(d) 21.9 0.80(d) 21.9 0.80(d)21 19 1.05(d) 19.2 1.05(d) 19 1.02(d)22 133.6 131.7 133.623 127.1 6.94(d) 127.4 6.93(d) 127.2 6.90(d)24 115.6 6.66(d) 115.7 6.70(d) 115.5 6.65(d)25 155.7 155.9 155.626 115.6 6.66(d) 115.7 6.70(d) 115.5 6.65(d)27 127.1 6.94(d) 127.4 6.93(d) 127.2 6.90(d)28 23.2 3.38(dd)

3.24(dd)23.4 3.37

3.2423.3 3.32(m)

29 8.70(br s) 9.20(s) 8.50(s)30 109 109.2 10931 111.1 110.432 131.3 127.5 131.333 118.1 7.24(d) 118.3 7.53(d) 118.1 7.50(m)34 122.3 7.13(dd) 120.4 7.09(t) 122.435 120.9 7.10(dd) 122.5 7.11(t) 120.1 7.15(m)36 110.6 7.56(br d) 110.6 7.21(d) 110.6 7.20(s)37 136.1 136.5 136.138 30.8 2.98(s) 30.9 2.95(s) 30.8 2.95(s)39 17.8 0.70(d) 17.9 0.66(d) 17.7 0.72(d)

50

Jaspamide exhibited antihelminthic properties against the parasitic nematode, H.

contortus, (NeT=64, LD99=2.6), as well as cytotoxicity (CyT=1024, LD99=0.16).

This is a known compound and its biological activity has been well documented in

literature.

Jaspamide/ jasplakinolide is a cyclic depsipeptide that has been isolated exclusively from

marine sponges. It was discovered independently by two different research groups.

Zabriskie and associates (1986) reported Jaspamide, derived from the Jaspis sp collected

from Suva harbour in Fiji and from a marine lake in Palau. They found that the

compound was a potent insecticide, with activity against Heliothis virescens (LC50

4ppm).

Crews et. al. (1986) reported the cyclic depsipeptide as Jasplakinolide. This compound

was derived from a Jaspis sp of sponge from Beqa lagoon in Fiji. This research group

found that jaspamide possessed antihelminthic properties (in vitro 7��4=�/�������������

the nematode Nippostrongylus braziliensis) as well as cytotoxicity against larynx

�$��#������&�"&������4���������������#����� "-���&������&����������4�4/������

Both groups found that jaspamide had potent activity against the fungus Candida

albicans but was inactive against a variety of gram positive and gram negative bacteria.

Clinical trials on jaspamide were discontinued after the compound proved to be too toxic

(Faulkner 2000). However, further research on jaspamide continued. Studies showed that

the drug possessed in vitro cytotoxicity to HT-29 cells (Crews et. al. 1994) as well as

51

antiproliferative activity in the NCI-60 cell line screen. It was most effective against a

number of tumour derived cell lines, human prostrate carcinoma (Crews et. al. 1994) and

myeloid leukemia (Fabian et. al. 1995). Odaka et. al. (2000) attempted to explain the

mode of action regarding the antiproliferative activity of jaspamide. This group found

that Jaspamide induces cell death via apoptosis. They also reported that transformed cells

were more susceptible to jaspamide induced apoptosis than normal non-transformed cell.

In summary, jaspamide possessed excellent anticancer properties.

Jaspamide’s biosynthetic origin is as follows. It is a mixed polyketide consisting of three

amino acids: (R)-2-bromoabrine (N-methyltryptophan), (R)-<-tyrosine and (S)-Alanine

and a propionate unit (Zabriskie et. al. 1986; Crews et. al. 1986). The former two amino

acids are exceptionally rare.

3.3 Bromopyrrole-imidazole alkaloids

The following known bromopyrroles were isolated from the marine sponge Stylissa

massa. Structures of these were elucidated by spectroscopic methods outlined below.

3.3.1 Oroidin

Molecular mass of this compound was found to be 389 ([M+]). The

[M+H]+: [M+2+H]+: [M+4+H]+ peak height ratios were 1:2:1, showing presence of 2 Br

atoms. The 1H-NMR showed presence of 2 vinylic pro�����'�H= 6.03(1H, dt), 6.24(1H,s)]

���������-��&�$"������'�H= 4.02(2H,d)]. From this the following moiety was deduced: -

CH2CH=CH-.

52

A search on Marinlit database found a close match to the known marine natural product

Oroidin. The 1H-NMR data of the literature (Lindel et. al. 2000) matched (as shown in

table 3) that of the experimental obtained data. Thus the structure was concluded to be

that of oroidin(4).

Table 3: comparison of literature and experimental 1H-NMR of Oroidin

Literature (Lindel et. al. 2000)18�69>�� �%�%�3)

Experimental 18�69>�� �CDCl3) {ppm}

E-isomer (ppm) Z-isomer (ppm)4.04 (dd, 2H) 4.07 (dd, 2H) 4.02 (dd, 2H)6.01 (dt, 1H) 5.75 (dt, 1H) 6.03 (dt, 1H)6.30 (d, 1H) 6.20 (d, 1H) 6.24 (d, 1H)6.74 (s, 1H) 6.81 (s, 1H) 6.74 (s, 1H)6.82 (s, 1H) 6.82 (s, 1H) 6.80 (s, 1H)

Oroidin showed no biological activity against the bioassays conducted.

54

2

3

6NH7

89

1011

14N15

NH13

12

NH216

O

Br

Br

NH1

4

53

Oroidin is a major metabolite of several species of the Genus Agelas. It was first isolated

from Agelas oroide in 1971 (Forenza et. al.) but an error was made in deducing its

structure. Garcia et al proposed a revised structure in 1973. It functions as a secondary

defense metabolite acting as a feeding deterrent against several predatory fish (Chanas et.

al. 1996). Van Soest and Richelle-Maurer (2000) found that the concentration of oroidin

(as well as sceptrin) increased in sponge cells after damage to sponges and during

confrontation (stony corals placed near sponge substrate). Oroidin has displayed

antimuscarinic inhibition activity, inhibiting acetylcholine receptors (Rosa et. al. 1992).

Oroidin is the precursor molecule for over 50 bioactive pyrrole-imidazole alkaloids. The

C11N5 skeleton of oroidin forms part of many derivatives through cyclization,

dimerisation, isomerisation of the double bond and/or oxidation/reduction. This includes

compounds like the Palau’amines, isophakellin, sceptrin, hymenidin and

hymenidialdisine.

In nature, six modes of oroidin cyclization have been identified (Lindel 1999; Fattorusso

and Taglialatela-Scafati 2000). These have been classified according to their linkage

formation (see Table 4).

54

Table 4: Cyclization modes of the Oroidin skeleton.

Linkage formation Examples

2 C4/C10 Hymenialdisine (Kitagawa et al 1983)

3 N1/C12 + N7/C12 Dibromoagelaspongine (Fedoreyev et. al. 1989)

4 N1/C12 + N7/C11 Dibromophakellin (Sharma and Magdoff-Fairchild 1977)

5 C4/C12 + N7/C11 Dibromoisophakellin (Fedoreyev et. al. 1986)

6 N1/C9 + C8/C12 Agelastatin (D’Ambrosio et. al. 1993)

7 N1/C9 Cyclooroidin (Fattorusso and Taglialatela-Scafati 2000)

55

Fig 5: Six modes of cyclization of the Oroidin skeleton.

NH

NH

O

NH O

NNH2

3

45

2 NH1

Br

Br6

NH7

O

8

910

1112

NH15

N13

14

NH216

N

Br

Br

N

O

NH

OH

NNH2N

Br

Br

N

O NH

NH

NH

NHBr

Br

O

NNH

NNH2

NBr

NH

O

NHNCH3

O

OH

H

H

H

NBr

Br

NH

O

NH

NH2

N

1

23

4

5

6

7

56

3.3.2 Sceptrin

Molecular mass of this compound was found to be 622. The [M]+: [M+2]+: [M+4]+ peak

height ratios were 1:2:1, showing the presence of 2 Br atoms. The 1H-NMR revealed the

following:

a) ���"����&�$"�������������'�H= 6.90(s), 6.46(s), 6.77(s)].

b) CH2-CH-CH- !��&��������-�'�H= 3.43(d), 2.30(dt), 2.94(d)].

The lack of proton signals suggested that the compound was symmetrical. A search on

MarinLit database came up with a close match with the known bromopyrrole-imidazole,

sceptrin (5).

Spectroscopic data matched well with that published in literature (Walker et. al. 1981).

12

16 NH13

1415

11

NH10

98

7

5

2

N+

3NH1 4

NH2 6

H

O

NH

NHN

+

NH NH2

HOBr

Br

5

57

Table 5: Comparison of Literature and Experimental 1H-NMR of Sceptrin

Literature1H NMR[Walker et. al. 1981]

�� 9�2SO-d6)

Experimental/8�69>�� %�%�3)

2.29 (br s, 1H) 2.29 (s, 1H)3.10 (d, 1H) *

3.42 (br s, 2H) 3.43 (s, 2H)6.66 (s, 1H) 6.40 (s,1H)6.97 (s, 1H) 6.70 (s, 1H)6.99 (s, 1H) 6.90 (s, 1H)

7.33 (br s, 2H) *8.59 (br t, 1H) *

*H corresponding to N-H bonds

Sceptrin showed no biological activity against the bioassays conducted.

Walker et. al. (1981) reported sceptrin from the sponge Agelas sceptrum. It functions as a

chemical defense metabolite, acting as a feeding deterrent against against predatory fish

(Chanas et. al. 1996; Assmann et. al. 2000). Concentrations of sceptrin and oroidin

increase in sponge cells after damage and during confrontation with another species (Van

Soest and Richelle-Maurer 2000).

Sceptrin and its analogues have potent antibacterial/antifungal activity (Bernan et. al.

1993), anti-muscarinic (Rosa et. al. 1992) and antihistaminic activity (Cafieri et. al.

1997).

Sceptrin is formed via a head-to-head [2+2] cycloaddition of debromooroidin. The

reaction was believed to be photochemical in nature. Hao and associates (2001) refuted

58

this, arguing that if the reaction was photochemically driven, then oroidin (which is

achiral) should yield sceptrin as a racemic mixture. Sceptrin is a chiral compound

suggesting that the reaction is in fact an enzyme catalyzed one. If this were the case, then

Sceptrin would be the first example of a biological [2+2] cycloaddition or pericyclic

reaction.

One of the possibilities proposed by Hao et. al. (2001) involves a polar conjugate addition

and not a pericyclic reaction.

Fig 6: Possible polar Mechanism. Two Sequential conjugate additions catalyzed by protonation of one debromo-oroidin molecule.

NH

NH

N

NH

NH3+

O

Br

NH

NH

N

NH

NH2OBr

N

NH

NH

OBr

NH

NH2+

NH

NH

O

Br

C-

N

NH

NH3+

NH

NH N+N

H

NH2

HO

NH NH

N+

NH

NH2

H

OBr

Br

59

3.4 Polybrominated diphenyl ether

Polybrominated Diphenyl ethers (PBDEs) are a class of chemicals that are commercially

used as flame-retardants. Products based on penta-, octa- and deca- BDEs are added to

plastics used in electrical appliances as well as building materials and transport. Hexa-

BDEs are present in small amounts in both penta- (4-8%) and octa- BDEs (10-12%)

based flame-retardants.

PBDEs are ecologically of concern as pollutants. Similar to Polychlorinated biphenyls,

PBDEs are a threat to wildlife and humans. They are persistent, lipophilic and are able to

bioaccumulate. Many PBDEs can travel long distances from its origin in the

environment. When combusted many PBDEs form Polybrominated dibenzodioxins

(PBDDs) and Polybrominated dibenzofurans (PBDFs) which have similar toxicity and

environmental impact as well known pollutants, Polychlorinated dibenzofurans (PCDFs)

and Polychlorinated dibenzodioxins (PCDFs). PBDEs are known to disrupt human

endocrine systems.

(van Esch, International Programme on Chemical Safety)

A known polybrominated diphenyl ether (PBDE) was isolated from the marine sponge

Stylissa massa. The structure of this was elucidated from the spectroscopic methods

mentioned below.

60

3.4.1 Hexabromodiphenyl ether

Hexabromodiphenyl ether was assigned the molecular formula C12H4O3Br6 on the basis

of ESI (-) MS ([M]+ =670) and 1H NMR. The [M]+: [M+2]+: [M+4]+ :[M+6]+ :[M+8]+

:[M+10]+ :[M+12]+peaks had peak height ratios of 1:6:15:20:15:6:1. This showed

presence of 6 bromine atoms. There were only two signals in the 1H-NMR. Both were

!"���"����&�$"�������H 7.37(s) and 6.50(s). DBE was calculated to be 8. Therefore

structure was composed of two benzene rings substituted with 6 Br atoms and 2 protons.

The two rings were either linked directly or by an oxygen atom. The unassigned oxygen

and two protons were in the form of two hydroxyl groups. Therefore the aromatic rings

were linked via the oxygen atom as an ether link.

Substitution pattern of Br atoms on the aromatic rings was deduced by comparing

spectroscopic data with that of known hexabromodiphenyl ether (Faulkner and Carte

1981, see Table 6).

Table 6: Comparison of 1H-NMR of Hexabromodiphenyl ether

Literature (Carté and Faulkner 1981){ppm}

Experimental(ppm)

7.37 7.376.50 6.50

The structure was found to be 2-(3’, 5’-dibromo-2’-hydroxyphenoxy)-3,4,5,6-

tetrabromophenol.

61

Hexabromodiphenyl ether exhibited antihelminthic properties against the parasitic

nematode, H.contortus, (NeT=16, LD99=21).

Faulkner and Carte first reported 2- (3’ -5 ‘-dibromo-2’ -hydroxyphenoxy)-3,4,5,6-

tetrabromophenol in 1981, from the sponge Phyllospongia foliascens. It was isolated as

an inseparable mixture with 2-(3’ -5 ‘-dibromo-2’ -hydroxyphenoxy)-3,5,6-

tribromophenol. There was no mention of biological activity.

3.5 Aminosulfonic acid

The isolation and structure elucidation of the known aminosulfonic acid, taurine, from the

marine sponge Stylissa massa by spectroscopic methods are outlined below.

3.5.1 Taurine

This compound was obtained as a clear gum. The molecular mass was established by

6

3

4

2

5

1

6

OH

5'

3'

OH

O Br

BrBr

Br

Br

Br

62

ESI (-) MS ([M]+ = 125). The 1H NMR revealed only two proton signals (3.10[t],

3.29[t]), that corresponded to the moiety: -CH2-CH2-. A search on Marinlit database

found a match with the common marine metabolite, taurine (C2H7NO3S). Comparison

was made with spectroscopic data obtained from an authentic sample of taurine. This data

matched perfectly with experimental data.

Taurine exhibited antihelminthic activity against the parasitic nematode, H. contortus

(NeT=4, LD99= 42).

Taurine or 2-aminoethanesulfonic acid was first discovered in ox bile in 1827. It was not

until 1975 that its significance in human nutrition was realized (Birdsall 1998). Taurine is

a semi-essential amino acid. It is not utilized in protein synthesis and is found in either

free form or as simple peptides. Unlike other amino acids, taurine contains a sulfonic acid

moiety instead of a carboxylic acid. It is one of the most abundant free amino acids found

in human tissue including skeletal and cardiac muscle, and the brain (Huxtable 1992).

Taurine is synthesized from methionine and cysteine. Jacobsen and Smith (1968)

demonstrated five pathways of taurine synthesis from methionine. The most common

pathway is methionine – cysteine – cystein - sulfuric acid - hypotaurine- taurine. In the

human body there are three known pathways of taurine synthesis from cysteine. All three

NH2 1 2

3

SO3H4

7

63

pathways require the active coenzyme form (pyridoxal-5’-phosphate (P5P) of vitamin B6

(Shin and Linkswiler 1974).

Taurine plays an important role in several human physiological functions. Two major

roles are:

Conjugation of bile acids (Birdsall 1998)

To solubilize at the pH levels inside the human body bile acids need to be conjugated

through peptide linkages with either glycine or taurine to form bile salts. Taurine-

conjugated acids increase the excretion of cholesterol. Bile acids help to absorb lipids and

fat-soluble vitamins.

Detoxification

Taurine acts as an antioxidant by neutralizing hypochlorous acid (a potent oxidising

agent). Taurine has a sulfonic acid group in place of a carboxylic acid. Instead of forming

an aldehyde from hypochlorous acid, a relatively stable chloroamine compound is

produced, thus protecting the body from toxic effects of aldehyde release that can cause

DNA damage (Kozumbo et. al. 1992).

Other functions include membrane stabilization, osmoregulation and modulation of

cellular calcium levels (Birdsall 1998).

64

Taurine has been used clinically to treat several conditions including cardiovascular

diseases, epilepsy and other seizure disorders (Fariello et. al. 1985), Alzheimer’s disease

(Tomaszewski et. al. 1982; Csernansky et. al. 1996), hepatic disorders (Matsuyama et. al.

1983) and cystic fibrosis (Smith et. al. 1991; Carrasco et. al. 1990). Acamprosate (an

analogue of taurine) is used in treatment of alcoholism (Wilde and Wagstaff 1997; Sass

et. al. 1996; Whitworth et. al. 1996; Paille et. al. 1995; Bara et. al. 1995; Guiet-Bara

1995).

Most sponges contain hypotaurine. Taurine is also present in high quantities. Ackermann

and List (1959) reported trimethyltaurine from Geodia sp while Bergquist and Hartman

(1969) reported taurocyamine in many species of Hadromerida, Spirophorida and

Choristida. More recently Fattorusso and Taglialatela-Scafati (2000) reported

taurodispacamide A, an antihistaminic bromo-pyrrole alkaloid containing a taurine

moiety, from Agelas oroides sponge.

NH

Br

Br NH

O

NH

+

NNH

NH2

SO3-

Taurodispacamide A

65

3.6 Nicotinic acid

The isolation and structure elucidation of the known nicotinic acid analogue, trigonelline

from the marine sponge, Stylissa massa, by spectroscopic methods are outlined below.

3.6.1 Trigonelline (N-methyl nicotinic acid)

This compound was obtained as a yellow gum. The molecular formula, C7H7NO2, was

established by ESI (+) MS and 1H-NMR. The 1H-NMR revealed the following

functionalities:

a) /�$"��"-���#-��'�H = 4.21(s)] unattached to any neighbouring protons and

b) A 6-membered m-substituted aromatic rin��'�H = 7.92(t), 8.68(d), 8.98(s): Integrating

for 4H]. This ring was deduced to be a pyridine ring (from the molecular formula and

1H-NMR).

A search on Marinlit database showed a close match to the natural product, trigonelline

(8).

2

1

3

N+

6

4

5

CH38

COO-7

8

66

Trigonelline exhibited antihelminthic properties against the nematode, H. contortus

(NeT= 16, LD99= 3.4).

Trigonelline’s presence has been detected in various terrestrial plants (mainly legumes

and coffee beans) and in several marine organisms.

In plants, trigonelline (TRG) acts as a natural hormone, controlling plant growth by

inducing G2 arrest in root and shoot meristems (Evans et. al. 1979; Evans and

Tramontano 1981). This was proposed after Evans and Tramontano (1981) found that by

adding TRG to plants, it replaced cotyledons (which contained natural TRG) in

promoting G2 arrest. The proportion of G2 arrested cells were indirectly related to TRG

levels in plants. TRG induces defense metabolism in plants and accumulation of

secondary defense metabolites (Berglund 1994). It also serves as an osmoregulator

(Tramontano and Jouve 1997), achieving this feat by increasing the in vitro thermal and

salt stability of pyruvate kinase (Shomerilan et. al. 1991).

TRG acts as a reusable storage form of the vitamin, niacin (nicotinic acid) and can re-

enter the nicotinamide metabolic pathway by demethylation (Minorsky 2002). In coffee

beans, the roasting process degrades TRG into volatile flavoured compounds that give

coffee its distinct smell (Saldana 1997). TRG is thought to be of nutritional importance

to humans. It enhances the performance of the Central nervous system, secretion of bile

and the intestine (Saldana 1997). Fenugreek (Trigonella foenungreca) is an herb that is

used widely throughout Asia and Southern Europe. It is used as a natural cure for

67

diabetes, migraines, allergies, elevated cholesterol and constipation (Shapiro and Gong

2002). TRG is a major metabolite of this plant and has a hypoglycemic effect on humans

(Mishkinsky et. al. 1967). In Fiji the seeds are known as methi, and are used as a spice.

Fenugreek is also a vital component of several commercially available breast

enlargement products.

Presence of TRG has been detected in several marine organisms including shellfish.

Trigonelline has antifouling activity against cyprids of the barnacle, Balanus amphitrite

(Miki et. al. 1996).

3.7 Pyridinium salt

The isolation and structure elucidation of the known pyridinium salt, zooanemonin, from

the marine sponge Stylissa massa by spectroscopic methods are outlined below.

3.7.1 Zooanemonin

This compound was isolated as a white powder. The molecular formula, C7H10N2O2, was

established from the ESI (+) MS and 1H-NMR. The 1H-NMR revealed the following

functionalities:

a) ����$"��"-���#-�����H= 3.46[s], 3.58[s])

b) ?��������"����&�$"��������H= 8.40[s], 7.06[s]).

c) 5�����#��������&#��������&�" �+-���"��$���H= 3.70[s])

68

The lack of carbons meant the ring was heterocyclic. A search on MarinLit found a close

match to the known alkaloid, zooanemonin (9).

Zooanemonin showed moderate antihelmintic activity against the nematode H.contortus

(NeT= 4, LD99=50).

Zooanemonin has been isolated from the marine sponge Protophlitaspongia aga as an

antifouling substance against the barnacle Balanus amphitrite (Hattori et. al. 2001) and as

a constituent of the Chilean marine invertebrate Antholoba achates (Gonzalez et. al.

1984). Fouling organisms cause serious problems by settling on ships' hulls, and other

marine infrastructures. Organotin compounds are currently used as antifoulants but cause

too many environmental problems. Many sessile marine organisms produce antifouling

compounds as a secondary defense metabolites. Natural compounds tend to exhibit a

lower toxicity than substances in current use or are more environmentally friendly.

2

N+

3

N1

5

4

CH3 78

CH3 6

COO-9

9

69

4.0 Conclusion

Eight natural products have been isolated from three different species of Fijian marine

sponges.

A novel polyketide, Spongosoritin A, was isolated from Spongosorites sp.

Spongosoritin A exhibited no bioactivity in all bioassays conducted.

Stelleta splendens yielded the known cyclodepsipeptide, jaspamide/jasplakinolide.

Jasplamide proved to be both cytotoxic and antihelminthic.

Stylissa massa yielded six known metabolites: hexabromodipheny ether, oroidin,

sceptrin, trigonelline, taurine and zooanemonin. All these compounds with the exception

of sceptrin showed, moderated to good, antihelminthic activity.

Unfortunately a new potential agrochemical was not discovered in this project. However

a new compound, although biologically inactive, has been discovered and its structure

determined. Marine invertebrates are still an excellent source for potential agrochemicals

and pharmaceuticals and continued research may uncover a veritable goldmine in terms

of biologically active compounds and interesting chemistry.

70

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Appendix

Spectroscopic data for Spongoritin A, a novel polyketide from the Fijian marine sponge

Spongosorites sp.

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