chemical and bioactivity studies on...
TRANSCRIPT
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
5.0 References
Ackermann, D. and List, P.H. (1959) Ube das vorkommen von Taurobetain. Taurin und
Inosit in Riesenkieselschwamm. Hoppe-Seylers Z Physiological Chemistry 317, 78-81
cited in Bergquist, P. R. (1978) Sponges. Hutchinson & Co (Publishers) Ltd., 209.
Agosta, William (1996) Bombardier Beetles and Fever Trees: A close-up look at
Chemical Warfare and Signals in Animals and Plants Addison-Wesley Publishing
Company, Inc.
Assmann, M.; Lichte, E.; Pawlik, J.R. and Köck, M (2000) Chemical defenses of the
Caribbean sponges Agelas wiedenmayeri and Agelas conifera. Marine Ecology Progress
Series Vol. 207: 255-262.
Bara, M.; Guiet-Bara, A.; Durlach, J. and Pechery, C. (1995) Comparative studies of Ca
N-acetylhomotaurinate and Ca N-acetyltaurinate. I. Effects on the ionic transfer through
the isolated human amnion. Experimental Clinical Pharmacology 17:233-240 cited in
Birdsall, T. C. (1998) Therapeutic application of taurine. Alternative Medicine Review
3(2):128-136.
Bartik, K; Braekman, J.C.; Daloze, D.; Stoller, C.; Huysecom, J.; Vandevyver, G. and
Ottinger, P (1987) Topsentins, new toxic bis- indole alkaloids from the marine sponge
Topsentia genitrix. Canadian Journal of Chemistry 65, 2118–2121.
71
Berglund, T (1994) Nicotinamide, a missing link in the early stress-response in
eukaryotic cells. A hypothesis with special reference to oxidative stress in plants FEBS
Lett 351: 145-149 in Minorsky, P.V. (2002) The Hot and the Classic. TRIGONELLINE:
A DIVERSE REGULATOR IN PLANTS. Plant Physiology, January 2002, Vol. 128, 7-8
Bergquist, P. R. and Hartman, w. D. (1969) Free amino acid patterns and the
classification of the Demospongiae. Marine Biology 3, 247-268.
Bernan, V.S; Roll, D.M.; Ireland, C.M.; Greenstein, M; Maiese, W.M. and DA Steinberg
(1993) A study on the mechanism of action of sceptrin, an antimicrobial agent isolated
from the South Pacific sponge Agelas mauritiana. Journal of Antimicrobial
Chemotherapy, Vol 32, 539-550.
Birdsall, T. C. (1998) Therapeutic application of taurine. Alternative Medicine Review
3(2): 128-136.
Cafieri, F.; Carnuccio, R.; Fattorusso, E.; Tagilialatela-Scafati, O. and Vallelfuoco, T
(1997) Chemical Letters, 7, 2283 cited in Hao, E.; Fromont, J.; Jardine, D. and Karuso, P.
(2001) Natural products from sponges of the Genus Agelas- a trail of the [2+2]
photoaddition enzyme. Molecules 6, 130-141.
Capon, R.J.; Skene, C.; Stewart, M.; Ford, J.; O’Hair, R.A.J.; Williams, L.; Lacey, E.;
Gill, J.H.; Heiland, K. and Friedel, T (2003) Aspergillicins A-E: 5 novel depsipeptides
72
from the marine-derived fungus Aspergillus carreus. Organic Biomolecular Chemistry 1,
1856-1862.
Capon, R.J. (2002) personal communication.
Capon, R.J.; Ford, J.; Lacey, E.; Gill, J.H.; Heiland, K. and Friedel, T. (2002)
Phoriospongin A and B: Two new nematocidal depsipeptides from the Australian marine
sponges Phoriospongia sp. and Callyspongia bilamellata. Journal of Natural Products
65(3), 358-363.
Capon, R. J.; Skene, C.; Liu, E. H.; Lacey, E.; Gill, J. H.; Heiland, K.; Friedel, T. (2001)
The Isolation and Synthesis of Novel Nematocidal Dithiocyanates from an Australian
Marine Sponge, Oceanapia sp. Journal of Organic Chemistry 66(23), 7765-7769.
Capon, R.J.; Rooney, F. and Murray, L.M. (2000) 1, 9-Dimethylhypoxanthine from a
southern Australian marine sponge Spongosorites sp. Journal of Natural Products 63
261-262.
Capon, R.J.; Rooney, F.; Murray, L.M.; Collins, E.; Sim, A.T.R.; Rostas, J.A.P.; Butler,
M.S. and Carroll, A.R. (1998) Dragmacidins: new protein phosphatase inhibitors from a
Southern Australian deep-water marine sponge, Spongosorites sp. Journal of Natural
Products 61, 660-662.
73
Carrasco S, Codoceo R, Prieto G, et al. (1990) Effect of taurine supplements on growth,
fat absorption and bile acid on cystic fibrosis. Acta Univ Carol 36:152-156 cited in
Birdsall, T. C. (1998) Therapeutic application of taurine. Alternative Medicine Review
3(2): 128-136
Carte, B. and Faulkner J. (1981) Polybrominated diphenyl ethers from Dysidea herbacea
and Phyllospongia foliascens. Tetrahedron 37, 2335-2339.
Chanas, B.; Pawli, J.R.; Lindel, T. and Fenical, W (1996) Chemical defense of the
Carribean sponge Agelas clathrodes (Schmidt) Journal of Experimental Marine Biology
and Ecology 208, 185-196.
Crews, P., J. J. Farias, R. Emrich, and P. A. Keifer. 1994. Milnamide A, an unusual
cytotoxic tripeptide from the marine sponge Auletta cf. constricta. J. Org. Chem.
59:2932-2936 in Odaka,C.; Sanders, M.L.; and Crews, P. (2000)Jasplakinolide Induces
Apoptosis in VariousTransformed Cell Lines by a Caspase-3-LikeProtease-Dependent
Pathway. Clinical and Diagnostic Laboratory Immunology, November 2000, p. 947-952,
Vol. 7, No. 6.
Crews, P.; Manes, L.V.; and Boehler, M. (1986) Jasplakinolide, a cyclodepsipeptide from
the marine sponge, Jaspis sp. Tetrahedron Letters, Vol. 27(No.25) 2797-2800.
Csernansky, J.G.; Bardgett, M.E.; Sheline, Y.I.; et al. (1996) CSF excitatory amino acids
and severity of illness in Alzheimer's disease. Neurology 46:1715-1720 cited in Birdsall,
74
T. C. (1998) Therapeutic application of taurine. Alternative Medicine Review 3(2): 128-
136 .
Cutler, Horace. G.; Cutler, Stephen. J. (Eds) (1999) Biologically Active Natural
Products: Agrochemicals CRC Press. 4-10.
D’Ambrosio, M.; Guerriero, A.; Debitus, C.; Ribes, O.; Pusset, J.; Leroy, S. and Pietra, F.
(1993) Journal of the Chemical Society., Chemical Communications 1305-1306.
Evans, L.S. and Tramontano, W.A. (1981) Is trigonelline a plant hormone pea seedlings?
Am J Bot 68: 1282-1289 in Minorsky, P.V. (2002) The Hot and the Classic.
TRIGONELLINE: A DIVERSE REGULATOR IN PLANTS. Plant Physiology, January
2002, Vol. 128, 7-8.
Evans, L.S.; Almeida, M.S.; Lynn, D.G. and Nakanishi, N (1979) Chemical
characterization of a hormone that promotes cell arrest in G2 in complete tissues.
Science 203: 1122-1123 in Minorsky, P.V. (2002) The Hot and the Classic.
TRIGONELLINE: A DIVERSE REGULATOR IN PLANTS. Plant Physiology, January
2002, Vol. 128, 7-8.
Fabian; Shur, I.; Bleiberg I.; Rudi, A.; Kashman, Y. and Lishner, M.(1995) Growth
modulation and differentiation of acute myeloid leukemia cells by jaspamide.
Experimental. Hematology. 23:583-587 in Odaka,C.; Sanders, M.L.; and Crews, P.
75
(2000) Jasplakinolide Induces Apoptosis in VariousTransformed Cell Lines by a
Caspase-3-LikeProtease-Dependent Pathway. Clinical and Diagnostic Laboratory
Immunology, November 2000, p. 947-952, Vol. 7, No. 6.
Fariello, R.G; Golden, G.T and McNeal, R.B Jr (1985) Taurine and related amino acids
in seizure disorders -current controversies. Prog Clin Biol Res 179:413-424 cited in
Birdsall, T. C. (1998) Therapeutic application of taurine. Alternative Medicine Review
3(2): 128-136.
Fattorusso, E; Taglialatela-Scafati, O (2000) Two novel pyrrole-imidazole alkaloids from
the Mediterranean sponge Agelas oroides Tetrahedron Letters 41, 9917-9922.
Fatturusso, E. and Taglialatela-Scafati, O. (2000) Two novel pyrrole-imidazole alkaloids
from the Mediterranean sponge Agelas oroides. Tetrahedron Letters 41, 9917-9922.
Faulkner, J. D. Antonie van Leeuwenhoek 77:: 135–145, 2000. 135 Marine pharmacology
© 2000 Kluwer Academic Publishers.
http://www.reefcheck.org/headlines/june/pdf/marine_pharmacology.pdf
Faulkner, J.D. and Stierle, D.B (1980) Metabolites of three marine sponges of the Genus
Plakortis Journal of Organic Chemistry 45, 3396-3401.
Fedoreyev, S.A.; Ilyin, S.G.; Utkina, N.K.; Maximov, O.B.; Reshetnyak, M.V.; Antipin,
M.Y.; Struchkorf, Y.T. (1989) The structure of debromoagelaspongin- A novel bromine
76
containing Guanidine derivative from the marine sponge Agelas sp. Tetrahedron 45,
3487-3492.
Fedoreyev, S.A.; Utkina, N.K.; Ilyin, S.G.; Reshetnyak, M.V. and Maximov, O.B. (1986)
The structure of dibromophakellin from the marine sponge Acanthella carteri.
Tetrahedron Letters 27, 3177-3180.
Forenza, S; Minale, L.; Riccio, R. and Fattorusso, E. (1971) New bromo-pyrrole
derivatives from the sponge Agelas oroides Journal of the Chemical Society. Chemical
Communications 0, 1129-1130.
Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M. and Harada, K.I. (1997) A
nonempirical method using LC/MS for the determination of the absolute configuration of
constituent amino acids in a peptide: Elucidation of limitations of Marfey’s method and
of its separation mechanism. Analytical Chemistry 69, 3346-3352.
Fusetani, N.; Asai, N.; Matsunaga, S.; Honda, K. and Yasumuro, K. (1994)
Cyclostellettamines A-F pyridine alkaloids inhibit binding of methyl quinuclidinyl
benzilate (QNB) muscarinic acetylcholine receptors from sponge Stelletta maxima
Tetrahedron Letters 35, 3967-3970.
77
Garcia, E.; Benjamin, L.E. and Fryer, R. I. (1973) Reinvestigation into the structure of
oroidin, a bromopyrrole derivative from the marine sponge, Agelas oroides Journal of the
Chemical Society., Chemical Communications , 78-79.
Gill, J.H.; Redwin, J.M.; Van Wyk, J.A. and Lacey, E. (1995) International Journal of
Parasitology 25, 463-470.
Gonzalez, F.; Silva, M.and Bittner, M. (1984) Some constituents of the Chilean marine
invertebrate Antholoba achates Coutony 1846. Revista Latinoamericana de Quimica.
Vol.15 (2), 87-88.
Grabley, S. and Thiericke, R. (Eds) (2000) Drug Discovery from Nature Springer edition
Series 5-9, 27-30.
Guiet-Bara, A.; Bara, M.; Durlach J. and Pechery, C. (1995) Comparative studies of Ca
N-acetylhomotaurinate and Ca N-acetyltaurinate. II. Preventive and opposing actions of
the acute ethanol depletive effect on the ionic transfer through the isolated human
amnion. Methods Find Exp Clin Pharmacol 17:361-368 cited in Birdsall, T. C. (1998)
Therapeutic application of taurine. Alternative Medicine Review 3(2): 128-136
Gunawardana, G.P.; Kohmoto, S. and Burres, N.S. (1989) New cytotoxic acridine
alkaloids from two deepwater marine sponges of the family Pachastrellidae. Tetrahedron
Letters 30, 4359-4362.
78
Hao, E.; Fromont, J.; Jardine, D. and Karuso, P. (2001) Natural products from sponges of
the Genus Agelas- a trail of the [2+2] photoaddition enzyme. Molecules 6, 130-141.
Hattori, T.; Matsuo, S.; Adachi, K.and Shizuri, Y. (2001) Isolation of antifouling
substances from the Palauan sponge Protophlitaspongia aga. Fisheries Science Vol.
67(4), 690-693.
Hirota, H.; Matsunaga, S. and Fusetani, N. (1990) Stellettamide A, an antifungal alkaloid
from a marine sponge of the genus Stelletta. Tetrahedron Letters. 31 4163-4164.
Huxtable, R. J. (1992) Physiological actions of taurine. Physiological Review 72:101-
163.
van Esch, Dr G.J. INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 162 BROMINATED DIPHENYL ETHERS.
Bilthoven, Netherlands www.inchem.org/documents/ehc/ehc/ehc162.html.
Jacobsen, J. G. and Smith, L. H. (1968) Biochemistry and Physiology of taurine and
taurine derivatives. Physiological Review 48, 424-511.
79
Kato, T.; Shizuri, Y.; Izumida, H.; Yokoyama. A. and Endo. M. (1995)
Styloguanidines,new chitinase inhibitors from the marine sponge Stylotella aurantium.
Tetrahedron Letters. 36 2133-2136.
Kinnel, R.B.; Gehrken, H.; Swali, R.;Skoropowski, G. and Scheuer, P.J. (1998)
Palau'amine and its congeners: a family of bioactive bisguanidines from the marine
sponge Stylotella aurantium Journal of Organic Chemistry 63, 3281-3286.
Kitagawa, I.; Kobayashi, M.; Kitanaka, K.; Kido, M. and Kyogoku, Y. (1983) Chemical
Pharmaceutical Bulletin 31, 2321-2328.
Koch, C.; Neumann, T., Thiericke, R. and Grabley, S. (2000) A central Natural Product
pool-New approach in Drug Discovery Strategies in Grabley, S. and Thiericke, R. (Eds)
(2000) Drug Discovery from Nature Springer edition Series.
Kohmoto, S.; Kashman, Y.; McConnell, O. J.; Rinehart, K. L.; Wright, A.; Koehn, F.
(1988) Dragmacidin, a new cytotoxic Bis(indole) Alkaloid from a deep-water marine
sponge, Dragmacidon sp. Journal of Organic Chemistry 53, 3116.
Kozumbo, W.J.; Agarwal, S. and Koren, H.S. (1992) Breakage and binding of DNA by
reaction products of hypochlorous acid with aniline, l-naphthylamine or l-naphthol.
Toxicol Appl Pharmacol 115:107-115 cited in Birdsall, T. C. (1998) Therapeutic
application of taurine. Alternative Medicine Review 3(2): 128-136.
80
Lindel, T. and Hochgurtel, M. (2000) Synthesis of the marine natural product oroidin
and it’s Z-isomer Journal of Organic Chemistry 65, 2806-2809.
Matsunaga, S.; Yamashita, T.; Tsukamoto, S. and Fusetani, N. (1999) Three new
antibacterial alkaloids from a marine sponge Stelletta species Journal of Natural
Products 62, 1202-1204.
Matsuyama, Y.; Morita, T.; Higuchi, M.; and Tsujii, T. (1983) The effect of taurine
administration on patients with acute hepatitis. Prog Clin Biol Res 125:461-468 cited in
Birdsall, T. C. (1998) Therapeutic application of taurine. Alternative Medicine Review
3(2):128-136 http://www.thorne.com/altmedrev/fulltext/taurine3-2.html.
McCabe, T.; Clardy, J.; Minale, L.; Pizza, C.; Zollo, F. and Riccio, R. (1982) A
triterpenoid pigment with the isomalabaricane skeleton from the marine sponge Stelletta
sp. (Choristida) Tetrahedron Letters 23, 3307-3310.
McCormick, J.L.; McKee, T.C.; Cardellina, J.H.; Leid, M. and Boyd, M.R.(1996)
Cytotoxic triterpenes from a marine sponge, Stelletta sp . Journal of Natural Products, 59
1047-1050.
81
Miki, W.; Kon-ya, K.; Mizobuchi, S. (1996) Biofouling and marine biotechnology: New
antifoulants from marine invertebrates. Journal of Marine Biotechnology Vol. 4, Iss. 2;
117-120.
Minorsky, P.V. (2002) The Hot and the Classic. TRIGONELLINE: A DIVERSE
REGULATOR IN PLANTS. Plant Physiology, January 2002, Vol. 128, 7-8.
http://www.plantphysiol.org/cgi/content/full/128/1/7.
Mishkinsky, J.; Joseph, B.; Sulman, F.G. (1967) Hypoglycaemic effect of trigonelline.
Lancet Vol. 2 (7521): 1311-1312 cited in Shapiro, K and Gong W.C (2002) Natural
products used for diabetes. Journal of the American Pharmaceutical Association. Vol. 42,
No. 2, 216-226.
Morris, S.A. and Andersen, R.J. (1988) Nitrogenous metabolites from the deep water
sponge Hexadella sp. Canadian Journal of Chemistry 67, 677–681.
Murray, L.M.; Lim, T.K.; Hooper, J.N.A. and Capon, R.J (1995) Isobromotopsentin: a
new bis(indole) alkaloid from a deep-water marine sponge Spongosorites sp Australian
Journal of Chemistry 48, 2053-2058.
Nybakken, James. W. (1996) Diversity of Invertebrates Wm. C. Brown Publishing 107-
109.
82
Odaka,C.; Sanders, M.L.; and Crews, P. (2000) Jasplakinolide Induces Apoptosis in
VariousTransformed Cell Lines by a Caspase-3-LikeProtease-Dependent Pathway.
Clinical and Diagnostic Laboratory Immunology, November 2000, p. 947-952, Vol. 7,
No. 6.
Paille, F.M.; Guelfi, J.D.; Perkins, A.C.; et al. (1995) Double-blind randomized
multicentre trial of acamprosate in maintaining abstinence from alcohol. Alcohol
30:239-247 cited in Birdsall, T. C. (1998) Therapeutic application of taurine. Alternative
Medicine Review 3(2): 128-136.
Pais, M.; Fontaine, C.; Laurent, D.; La Barre, S. and Guittet, E (1987) Stylotelline,a new
sesquiterpene isocyanide from the sponge Stylotella sp. Use of 2D NMR in structure
determination. Tetrahedron Letters 28, 1409-1412.
Patil, A.D.; Freyer, A.J.; Killmer, L.; Hofmann, G.; Johnson, R.K. (1997) Z-
axinohydantoin and debromo-Z-axinohydantoin from the sponge Stylotella aurantium.
Inhibitors of protein kinase C Natural Products Letters 9, 201-207 CA127: 147240c.
Pettit, G.R.; Srirangam, J.K.; Herald, D.L.; Erickson, K.L.; Doubek, D.L.; Schmidt, J.M.;
Tackett, L.P. and Bakus, G.J. (1993) Antineoplastic agents. 251.Isolation and structure of
stylostatin 1 from Papua New Guinea sponge Stylotella sp. Journal of Organic Chemistry
58, 3222.
83
Pettit, G.R.; Srirangam, J.K.; Herald, D.L.; Xu, J.; Boyd, M.R.; Cichacz, Z.; Kamano, Y.;
Schmidt, J.M. and Erickson, K.L. (1995) Isolation and crystal structure of stylopeptide 1,
a new marine Porifera cycloheptapeptide Journal of Organic Chemistry 60, 8257-8261.
Pettit, GR.; Herald, C.L.; Doubek, D.L.; Herald, D.L.; Arnold, E. and Clardy, J. (1982)
Isolation and structure of bryostatin 1. Journal of the American Chemical Society 104:
6846–6848.
Quinoa, E.; Adamczeski, M.; Crews, P. and Bakus, G. (1986) Bengamides, heterocyclic
antihelminthics froma Jaspidae marine sponge. Journal of Organic Chemistry 51, 4494-
4497.
Rinehart, K.L. Jr.; Gloer, J.B.; Hughes, R.G. Jr; Renis, H.E.; McGovren, J.P.;
Swynenberg, E.B.; Stringfellow, D.A.; Kuentzel, S.L. and Li, L.H. (1981) Didemnins:
antiviral and antitumor depsipeptides from a Caribbean tunicate. Science 212: 933–935.
Rosa, R; Silva, W.; Escalona de Motta, G.; Rodriguez, A.D; Morales, J.J and Ortiz, M
(1992) Antimuscarinic activity of a family of C11N5 compounds isolated from Agelas
sponge Experimentia 48, 885-887.
Ryu, G.; Matsunaga, S.; and Fusetani, N. (1996) Globostellatic acids A-D, new cytotoxic
isomalabaricane triterpenes from the marine sponge Stelletta globostellata. Journal of
Natural Products 59, 512-514.
84
Sakai, R.; Rinehart, K.L.; Kishore, V.; Kundu, B.; Faircloth, G.; Gloer, J.B.; Carney, J.R.;
Namikoshi, M.; Sun, F.; Hughes, R.G. Jr; Grávalos, D.G.; de Quesada, T.G.; Wilson,
G.R. and Heid, R.M. (1996) Structure–activity relationships of the didemnins. Journal of
Medicinal Chemistry 39: 2819–2934.
Sakemi, S and Sun, H.H (1991) Nortopsentins A, B,and C. Cytotoxic and antifungal
imidazolediylbis[indoles] from the sponge Spongosorites ruetzleri. Journal of Organic
Chemistry 56, 4304-4307.
Saldana, M. D. A.; Mazzafera, P. E and Mohamed, R. S. (1997) EXTRAÇÃO DOS
ALCALÓIDES: CAFEÍNA E TRIGONELINA DOS GRÃOS DE CAFÉ COM C
SUPERCRÍTICO. Ciênc. Tecnol. Aliment., Dez. 1997, vol.17, no.4, p.371-376..
Sass, H.; Soyka, M.; Mann, K. and Zieglgansberger,W. (1996)Relapse prevention by
acamprosate. Results from a placebo-controlled study on alcohol dependence. Arch Gen
Psychiatry 53:673-680 cited in Birdsall, T. C. (1998) Therapeutic application of taurine.
Alternative Medicine Review 3(2): 128-136
Shapiro, K and Gong W.C (2002) Natural products used for diabetes. Journal of the
American Pharmaceutical Association. Vol. 42, No. 2, 216-226.
85
Sharma, G. and Magdoff-Fairchild, B. (1977) Natural Products of marine sponges 7. The
constitution of weakly basic guanidine compounds, dibromophakellin and
monobromophakellin. Journal of Organic Chemistry 42, 4118-4124.
Shin, J.; Seo, Y.; Cho, K.W.; Rho, J.R. and Sim, C.J. (1997) Stellettamide B, a new
indolizidine alkaloid from a sponge of the genus Stelletta. Journal of Natural Products 60
611-613.
Shin, H.K. and Linkswiler, H.M. (1974) Tryptophan and methionine metabolism of adult
females as affected by vitamin B6 deficiency. Journal of Nutrition 104:1348-1355.
Shomerilan, A; Jones, G.P. and Paleg, L.G. (1991) In vitro thermal and salt stability of
pyruvate-kinase are increased by proline analogs and trigonelline. Aust J Plant Physiol
18: 279-286 in Minorsky, P.V. (2002) The Hot and the Classic .TRIGONELLINE: A
DIVERSE REGULATOR IN PLANTS. Plant Physiol, January 2002, Vol. 128, 7-8.
Simpson, J.S.; Raniga, P.; Garson, M.J. (1997) Biosynthesis of dichloroimines in the
tropical marine sponge Stylotella aurantium Tetrahedron Letters 38, 7947-7950.
Smith, U.; Lacaille, F.; Lepage, G.; et al. (1991)Taurine decreases fecal fatty acid and
sterol excretion in cysticfibrosis. A randomized double-blind study. Am J Dis Child
145:1401-1404 cited in Birdsall, T. C. (1998) Therapeutic application of taurine.
Alternative Medicine Review 3(2):128-136
86
Su, J.Y.; Meng, Y.H.; Zeng LM; Fu X; Schmitz FJ (1994) Stellettin A, a new triterpenoid
pigment from the marine sponge Stelletta tenuis Journal of Natural Products 57 1450-
1451.
Tomaszewski, A.; Kleinrok, A.; Zackiewicz, A.; et al. (1982) Effect of various amino
acids on acetylcholine metabolism in brain tissue. Ann Univ Mariae Curie Sklodowska
37:61-70 cited in Birdsall, T. C. (1998) Therapeutic application of taurine. Alternative
Medicine Review 3(2): 128-136.
Tramontano, W.A. and Jouve D. (1997) Trigonelline accumulation in salt-stressed
legumes and the role of other osmoregulators as cell cycle control agents.
Phytochemistry 44: 1037-1040in Minorsky, P.V. (2002) The Hot and the Classic.
TRIGONELLINE: A DIVERSE REGULATOR IN PLANTS. Plant Physiol, January 2002,
Vol. 128, 7-8.
Tsujii, S.; Rinehart, K.L.; Gunasekera, S.P.; Kashman, Y.; Cross, S.S.; Lui, M.S.;
Pomponi, S.A. and Diaz, M.C. (1988) Topsentin, bromotopsentin, and
dihydroxybromotopsentin: antiviral and antitumor bis(indolyl)imidazoles from
Caribbean deep-sea sponges of the family Journal of Organic Chemistry 53, 5446-5453.
Tsukamoto, S.; Kato, H.; Hirota, H. and Fusetani, N. (1996) Stellettadine A: A new
acylated bisguanidinium alkaloid which induces larval metamorphosis in ascidians from
a marine sponge Stelletta sp Tetrahedron Letters 37, 5555-5556.
87
Tsukamoto, S.; Yamashita, T.; Matsunaga, S. and Fusetani, N. (1999b) Bistellettadines A
and B: two bioactive dimeric stellettadines from a marine sponge Stelletta sp. Journal of
Organic Chemistry 64, 3794-3795.
Tsukamoto, S.; Yamashita, T.; Matsunaga, S.and Fusetani, N. (1999a) Bioactive marine
metabolites - Part 89 - Stellettazole A: An antibacterial guanidinoimidazole alkaloid
from a marine sponge Stelletta sp. Tetrahedron Letters. 40 737-738.
Van Soest and Richelle-Maurer (2000) Ecological Significance of compounds
http://wwwzma.bio.uva.nl/departments/coel/symbiosponge/web/rep&pub/protocols/Chap
ter%204.pdf
Vuong, Dat; Capon, Robert J.; Lacey, Ernest; Gill, Jennifer H.; Heiland, Kirstin; Friedel,
Thomas. (2001) Onnamide F: A new nematocide from a Southern Australian marine
sponge, Trachycladus laevispirulifer. Journal of Natural Products 64(5), 640-642.
Walker, R.P.; Faulkner, D.J.; van Engen, D. and Clardy, J. (1981) Sceptrin, an
antimicrobial agent from the sponge Agelas sceptrum. Journal of American Chemical
Society 103: 6772-6773.
Wender PA, De Brabander J, Harran PG, Jiminez J-M, Koehler MFT, Lippa B, Park C-M
& Shiozaki (1998) Synthesis of the first members of a new class of biologically active
bryostatin analogues. Journal of the American Chemical Society 120: 4534–4535.
88
Whitworth, A.B; Fischer, F; Lesch, O.M, et al. (1996) Comparison of acamprosate and
placebo in long-term treatment of alcohol dependence. Lancet 347:1438-1442 cited in
Birdsall, T. C. (1998) Therapeutic application of taurine. Alternative Medicine Review
3(2): 128-136
Wilde, M.I. and Wagstaff, A.J.(1997) Acamprosate. A review of its pharmacology and
clinical potential in the management of alcohol dependence after detoxification. Drugs
53:1038-1053 cited in Birdsall, T. C. (1998) Therapeutic application of taurine.
Alternative Medicine Review 3(2): 128-136
Williams, D.H. and Faulkner, D.J. (1996) Isomers and tautomers of hymenialdisine and
debromohymenialdisine Natural Products Letters 9, 57-64.
Wright, A.E.; Pomponi, S.A.; Cross, S.S. and McCarthy, P (1992) A new bis-(indole)
alkaloid from a deep-water marine sponge of the genus Spongosorites Journal of Organic
Chemistry 57, 4772-4775.
Xu, Y.; Yakushijin, K. and Horne, D.A (1997) Synthesis of C11N5 marine sponge
alkaloids (±) hymenin, Stevensine, hymendialdisine and debromohymendiadisine. Journal
of Organic Chemistry 62, 456-464.
89
Zabriskie, M.; Klocke, J.A.; Ireland, C.M.; Marcus, A.H.; Molinski, T.F.; Faulkner, J.;
Xu, C. and Clardy, J.C. (1986) Jaspamide, a modified peptide from a Jaspis sponge, with
insecticidal and antifungal activity. Journal American Chemical Society, 108, 3123-
3124.
90
Appendix
Spectroscopic data for Spongoritin A, a novel polyketide from the Fijian marine sponge
Spongosorites sp.
91
92
93
94
95
96
97
98
99
100