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Chemical Diversity of Bioactive Marine Natural Products: An Illustrative Case

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Article  in  Current Medicinal Chemistry · August 2004

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 Current Medicinal Chemistry, 2004, 11, 1671-1692 1671

Chemical Diversity of Bioactive Marine Natural Products: An Illustrative

Case Study
Valeria Costantino, Ernesto Fattorusso*, Marialuisa Menna and Orazio Taglialatela-Scafati

Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli “Federico II”, via D. Montesano 49, I-
80131 Napoli, Italy

Abstract: The marine environment contains a number of plants, animals and micro organisms, which, due to
the unique adaptations to their habitat, elaborate a wide diversity of natural products with specific
bioactivities. These products provide a rich source of chemical diversity that can be used to design and
develop new potentially useful therapeutic agents. The huge variety of the structures present in marine
organisms has been illustrated through the case study of the sponge Plakortis simplex, whose chemical
analysis, started in our laboratories about ten years ago, revealed an incredible variety and abundance of
secondary metabolites. The obtained results have been presented with the intention of drawing some
conclusions of general relevance. Particularly, the problem of the limited availability of natural compounds for
both structural and preliminary pharmacological studies has been discussed, this issue becoming a serious
obstacle when the pharmacological research reaches a more advanced stage. Furthermore, the origin of the
chemodiversity in Plakortis simplex and, in general, in marine invertebrates has been discussed; in this
respect, the possible cooperative role of symbiotic micro-organisms in the biosynthesis of the varied
metabolic content typical of these organisms has been considered.
Keywords: Marine, chemical diversity, bioactive natural products, glycolipids, alkaloids, polyketides, Plakortis simplex,
review.

INTRODUCTION examples of valuable natural products widely used in

That nature is a potential important source of useful
drugs has been recognized since ancient times. Its great
diversity developed during several billion years of evolution
results in the coexistence of an enormous number of species
interacting with each other in several ways. In this process
chemistry plays a crucial role; in plain terms, all the
organisms share almost the same biochemistry necessary for
a living cell but, in addition, they produce a wide variety of
so-called “secondary metabolites” which are involved in the
interactions between organisms. Considering the number of
different species and the almost infinite number of possible
interactions, it is not surprising that a wide variety of
secondary metabolites, resulting from evolutionary pressures
to preserve and enhance the life of their producing
organisms, has evolved into structurally and usually
stereochemically complex compounds with biological
potency. Thus, nature has been performing combinatorial
chemistry for thousands of years and represents a real arsenal
of new bioactive compounds.
The study of secondary metabolites has historically
proved of immense benefit in drug discovery, providing a
rich source of structurally novel bioactive molecules, many
of which have become life-saving drugs or biomedical tools.
Approximately one-third of the top-selling drugs in the
world are natural products or their derivatives; well-known
*Address correspondence to this author at the Dipartimento di Chimica
delle Sostanze Naturali, Università di Napoli “Federico II”, via D.
Montesano 49, I-80131 Napoli, Italy; E-mail: fattoru@unina.it
medical and animal health industries include erythromycin
(antibiotic), amphotericin B (fungicidal agent), cyclosporin
A and FK506 (immunosuppressive agents), lovastatin
(anticholesterolemic agent); as many as 25% of the currently
used anticancer drugs are natural chemicals, with another
25% coming from synthetic derivatives of natural products
[1,2]. Therefore, the use of natural products has been up to
now the most successful way to the discovery of new
medicines.
Today, with the continuing need for novel drug-like lead
compounds against an increasing number of ever-more
challenging molecular targets, the availability of rich
libraries of chemically diverse molecules is an essential
requirement. However, drug discovery has entered a more
highly competitive era in which the quality of chemical
collections and the time taken from assay to drug
development are crucial factors. Thus, although the chemical
novelty associated with natural products is higher than that
of any other source, this diversity must be accessed more
efficiently and effectively. Until recently, one major problem
connected with the use of natural products as lead for new
drugs has been the complexity of screening procedures of the
extracts, which were costly and time-consuming; in
addition, they presented the risk that the biological response
could be altered by interfering substances co-occurring in the
extracts.
Currently, most of the new drugs are discovered on the
basis of a molecular approach, which can be performed not
only through the rational drug design aided by computer-
based techniques or through the manipulation of genetic

0929-8673/04 $45.00+.00 © 2004 Bentham Science Publishers Ltd.

1672 Current Medicinal Chemistry, 2004, Vol. 11, No. 13 Fattorusso et al.

targets (antisense approach), but also through the pragmatic
approach of random screening. This last approach to drug
discovery, which is also called combinatorial biology, has
been updated by the recent developments in molecular
biology, instrumentation technology and information, so
that it can now be carried out at high throughputs that could
not have been imagined, even a few years ago [3-6]. The
increasing availability of new molecular targets, the potential
to transform them by genetic engineering, such as the
simplification of the de-replication processes through the use
of clones, make random screening a very promising tool to
the discovery of novel bioactive compounds; a further not
marginal improvement comes from the use of robotics to
conduct the assays. Currently, wide-ranging assays can be
quickly carried out by using micro plates equipped with
numerous wells.
Recent advances in random screenings enabled enormous
increases in throughput to be achieved, but, on the other
hand, they posed some problems in order to be conveniently
applied. A high-throughput screening program requires the
availability of large numbers of compounds for testing
which have to be structurally different in order to increase
the chance of finding activity at the molecular target. This
requirement cannot be supplied by traditional organic
synthesis and so, over the past few years, new approaches,
such as combinatorial chemistry and computer-based
molecular modelling design have become the source of new
levels of chemical diversity. Initially, they seemed to lower
the natural products value in drug discovery; however, as the
use of these techniques has matured, it became clear that
they achieved significant importance mainly in the
generation of focused libraries for specific discovery
programs [5]. Indeed, combinatorial chemistry has failed to
supplant natural products programs as the primary source of
broad chemical diversity. It is now clear that natural
products offer a potentially infinite source of molecular
diversity unmatched by any synthetic chemical collection or
combinatorial chemistry [6]; simple natural organisms,
especially those at lower evolutionary level, are able to create
new structures in a multitude of different ways. The
biosynthesis of secondary metabolites in micro organisms,
plants and invertebrates represents combinatorial chemistry
at its most expansive and versatile level, being guided by

O
OSO3 −
O Cl Cl OH Cl OH OH Cl

OH
1 Cl Cl Cl Cl Cl Cl Cl OH
NC
NCS 5 6 SCN
OH
4
2
NCS
3
Cl Cl O

OHCHN N
OSO2NHM e
NaO3SO OH
Cl 7
OH OH
Cl
5 6
OH OH
Me H
O Me
H
Me O O Me
Me H O
Me O O H H
OH H
Me H H
H H H H HO H O O Me
O O H
O H Me O Me
H
Me OH OH
O O H O
Me Me Me H Me H
H O
HO H
HO O
H
NaO3SO OH
HO Me H OH OH
Me H OH H H H H
OH Me Me H O O O O OH O
H O OH OH
M H H H
e O OH
O H O H O O
OH Me OSO3Na OH
O H H H H H H H O
H H OH OH OH
HO OH HO O
OH H H OH
OH OH
8

Fig. (1). Examples of chemical diversity of compounds isolated from marine organisms.
Bioactive Marine Natural Products
enzymes expressed by a vast gene pool evolved over billions
of years.
The investigation of chemicals produced by natural
sources initially focused on terrestrial organisms. Since
1960s, researchers began to view the oceans as a new and
untouched source of structurally unique compounds –
perhaps not surprisingly considering that more than 60% of
the earth’s biosphere is ocean. The marine environment is a
wealthy source of plants, animals and micro organisms,
which due to their adaptations to this unique habitat,
produce a wide variety of secondary metabolites unlike those
found in terrestrial species. It is well known that most
marine compounds are produced by invertebrates such as
sponges, tunicates and coelenterates [7]. The wealth of their
secondary metabolites has been related to the role of
chemical defence played by these constituents; such
ecological function is believed to be crucial for the survival
of the producer organisms, which are soft-bodied, sessile or
slow-moving animals, lacking, in most cases,
morphological defence structures such as shells or spines [8-
10].
More than 13000 novel marine natural products have
been discovered yet, with a marked increase in the last ten
years; sponges dominate as a source of new compounds
(almost 40%), followed by coelenterates (21%) and micro-
organisms (15%) [11]. All marine organisms have provided
a seemingly endless parade of very unusual novel structures;
biogenetically, they can be included in the major
biosynthetic pathways proposed for terrestrial secondary
metabolites whereas, structurally, they often possess
functional groups appearing uniquely or predominantly
marine. One of the most recurring features is the presence of
halogen atoms, which can be related to the relative
abundance of these elements in the marine environment;
sometimes halogens are present in quite a large number,
such as in compound 1 [12]. Isonitrile, isothiocyanate,
thiocyanate and formamide, represented by compounds 2
[13], 3 [14], 4 [15] and 5 [16] respectively, are further
functionalities which have essentially been found in marine
compounds. Chemical groups as carbonimidic dichloride
present in compound 6 [17] or sulfamate group in
haplosamate A (7) [18] have only been found in spongal
metabolites.
Singularity of marine derived compounds is not limited
to functionalities, but also concerns their carbon skeletons,
for example the polyether structural class, which is the basis
of a majority of marine toxins, well represented by
maitotoxin (8 ), the largest non-proteinaceous toxin
possessing a skeleton composed by 164 carbon atoms [19].
While early studies in marine natural products
constituted a survey of marine secondary metabolism, recent
studies have focused, to a greater extent, on the discovery of
metabolites with biomedically relevant activities [20,21].
Through the combined efforts of marine natural product
chemists and pharmacologists, a number of promising
compounds have been identified and some of them are either
already at advanced stages of clinical trials or have been
selected as promising candidates for extended pre-clinical
evaluation. Most of these products fall within the area of
cancer therapy; the case of the marine alkaloid ecteinascidin
743 (ET-743), an anti-tumour compound especially effective
Current Medicinal Chemistry, 2004, Vol. 11, No. 13 1673
against solid tumours, is an illustrative example [22]. ET-
743 is the most advanced compound among the marine
natural products (or analogues) that are currently under
clinical investigations and expected to enter the drug market
in Europe in the next year. Further examples to be
mentioned are the bryostatins and the dolastatins, isolated
from the bryozoan Bugula neritina and from the sea hare
Dolabella auricularia, respectively, that have also reached
late stages of clinical trials [23-25]. Arabinofuranosyladenine
(ara-A), a derivative of ara-U, isolated from the sponge
Cryptotethya crypta [26], is probably the most prominent
bioactive compound from sponges. It has been found to
display potent anti-herpes virus activity due to the
competitive inhibition of the viral polymerase [27, 28].
Pseudopterosins, unusual diterpene glycosides isolated from
the gorgonian Pseudopterogorgia elisabethae, were shown
to inhibit phospholipase A2; the key enzyme in the
biosynthesis of inflammatory eicosanoid mediators [29]. A
simple analogue of these compounds is currently in phase I
clinical trials as a potential new anti-inflammatory agent. A
further remarkable example is ziconotide, a pain-killing 25-
aminoacid linear peptide that has successfully completed
phase III clinical trials for two therapeutic applications: to
alleviate pain associated with malignant diseases such as
cancer or AIDS, and as analgesic for non-malignant
neuropathic pain. This compound has been found in the
venom of the marine mollusk Conus magusand exerts
analgesic effect due to the blockage of calcium channels [30].
Even if these results illustrate the increasing impact of
this field on the biomedical research, they also demonstrate
that marine organisms have been evaluated for their
biological functionality only at a preliminary level, when
compared to their terrestrial counterparts. The number of
marine derived metabolites, which have entered clinical
trials, is still very small if compared with the ocean rich
chemical diversity of bioactive compounds, which represents
the subject of the present review. Actually, on account of the
large number of bioactive molecules so far isolated from
marine organisms, the extent of the matter is really
remarkable and difficult to be included into a systematic
description. In addition, a comprehensive discussion on
chemical diversity should take into account the producer
organisms belonging to quite different phyla and depending
on their evolutionary level, they may harbour a variety of
unicellular symbionts contributing to their secondary
metabolism. Therefore, we decided to discuss this topic
through the illustrative case of a sponge, Plakortis simplex,
whose chemical analysis, started in our laboratories about
ten years ago, revealed an incredible variety and abundance
of secondary metabolites. The obtained results will be
discussed later with the intention of drawing some
conclusion of general relevance.
BIOACTIVE METABOLITES OF THE SPONGE
PLAKORTIS SIMPLEX
We began to study P. simplex initially focusing our
attention on its glycolipid composition; however, in
addition to a number of new glycolipids, this sponge was
shown to produce also several polyketide and alkaloid
metabolites. Many of these molecules are novel compounds
1674 Current Medicinal Chemistry, 2004, Vol. 11, No. 13 Fattorusso et al.

OH
OH OH
OH O
O R1 OR C 10 H21
O OH
H H NH
HO NH O
OH H
OH HO O C 10 H21
2
O R H O
H H OH
OH

HO OH OH OH
OH
HO OH
OH
O O O
R1 OH
O
OH
O

H H H H
H H H H H H H H
H H H H H H O HO O HO O
O HO O HO O HO O
HO O O O O
HO O
OH H OH OH H OH H OH
H OH H
H
H H H H
H
H H H H H
H H

OH
H
H
HO O
HO H
H OH OH
H O H
H O
HO O
H OH
H HO OH
H O
R1
HO O

O
O COOH
O
O COOCH3 OH
O
O

O O O
O H3 CO
H O COOCH3
O H 3COOC
OH
H3COOC

O COOCH3
H
O COOCH3 Cl OH
H
OH

O O COOH
− H
I OOC OH
O-
O COOCH3
N+ + H
N N O OH
I H CH3

Fig. (2). Representative examples of the molecular diversity found in Plakortis simplex.

endowed with promising bioactivities (immunomodulating, structure of each of the obtained compounds. However, in
antimalarial, cytotoxic). order to highlight the variety of tools developed during last
years to help the work of a natural product chemist, some
In the following pages, we survey the isolation and
general strategies used both for the isolation and for the
structure elucidation of new metabolites discovered from P.
structure elucidation are illustrated. Particular emphasis is
simplex. It is completely outside the intent of the present
devoted to the description of techniques allowing the
review to detail the isolation procedures, as well as the
solution of quite complex stereostructural problems having
spectral and chemical studies, employed to assign the
just little amounts of material.
Bioactive Marine Natural Products Current Medicinal Chemistry, 2004, Vol. 11, No. 13 1675

Scheme 1. Isolation procedure for P. Simplex.

Glycolipids have an amphiphilic behaviour. The isolation procedure we

developed is summarised in Scheme 1.
Many interesting glycolipids are present in sponges in
As an additional advantage, we found that this
small amounts and, very often, as mixture of homologues
methodology put together in well-defined chromatographic
differing in the lipid part of the molecule for the length and
fractions the other different classes of compounds we were
the branching of the alkyl chains. Therefore, to study this
interested in. The pure products could then be easily
kind of molecules we needed to set up simple and efficient
obtained by HPLC.
isolation procedures taking into account that glycolipids

Fig. (3). Effect of acetylation on the 1H NMR resonances of glycolipids.

1676 Current Medicinal Chemistry, 2004, Vol. 11, No. 13
On account of the little amounts generally available for
structural studies, we tried to tackle structure elucidation of
glycolipids taking advantage of the experience we had gained
along the years with NMR techniques rather than using the
degradation methodologies developed by other research
groups [31] that were in use at that time. A remarkable
problem of this approach is the strong overlapping of sugar
protons that resonate in a quite narrow region of the
spectrum. We found that signals overlap can be dramatically
reduced using peracetylated glycolipids because of the better
dispersion of signals in their 1H-NMR spectrum.
Finally, if no further ester functionality is present in the
glycolipid (as it happens for sphingolipids, but not for
glycerolipids) the peracetylated glycolipid can be turned back
to its natural structure, using base-catalysed methanolysis
[32], to perform biological assays. Acetyl groups already
present in natural compounds can be distinguished by
acetylating a small amount of the glycolipid fraction using
trideuteroacetic anhydride instead of acetic anhydride.
A. Glycosphingolipids
Glycosphingolipids (GSLs) are characteristic components
of the membrane of eukaryotic cells, where they serve a
variety of functions through interaction with many
biofactors. At the cell surface, GSLs can interact with
toxins, viruses and bacteria, and interactions with receptors
and enzymes have been described to be of some
physiological significance. A number of GSLs play a role as
tumour associated antigens and in immunotherapy of
individual cancer forms.
A large number of different GSLs have been isolated
until now, mainly from organ tissues of the higher animals.
A significant portion of known GSLs come from marine
organisms [33], most of them being cerebrosides and
gangliosides isolated from echinoderms (starfishes, sea
urchins, holothurians or mollusks).
In the last years, the scientific interest in GSLs is
increasing on account of the role they could play as
therapeutic immunomodulating agents. The first report of
immunomodulating GSLs came from a Japanese group that
in 1993 isolated agelasphins (9) from the sponge Agelas
mauritiana [34].
OH
O
OR
OH
H NH
O C 10H21
H HO 10 R` =
O
R`
H O
H H OH
11 R` =
12 R` =
13 R` =
Fig. (5). Structures of plakosides A-D.
Fattorusso et al.
OH
OH OH
H O R1
H O
H R1 , R2 = alkyl chains
HO NH OH
H OH
H O R2
9 OH
Fig. (4). Structure of agelasphine.
Agelasphins are present in Agelas mauritiana as a
mixture of homologues differing in the composition of the
fatty acid (FA) and LCB chains. Agelasphins showed very
interesting biological activities: they exhibited in vivo
antitumour effects when tested on intraperitoneally
implanted B16 murine melanoma. An additive effect was
observed when these compounds were administrated in
combination with adriamicin, a well-known chemothera-
peutic agent. Agelasphins were then tested for evaluating
their immunomodulating effects using the MLR (mixed
lymphocyte reaction) assay showing a marked
immunostimulating activity at concentrations below 1
µg/ml. In addition agelasphins were shown to be not
cytotoxic, so it is believed that these compounds act as
biological response modifiers (BRMs) [35] and their marked
antitumour activity is due to the activation of the immune
system. The same group synthesised a close analogue of
agelasphin, KRN7000 [36] that is considered as a novel
anticancer agent, acting through stimulation of the immune
system. Further evidence confirming the usefulness of
KRN7000 as a cancer therapy was subsequently obtained. In
particular, the relationship between the antitumour effect and
the activation of natural killer (NK) cells were investigated
and it was suggested that KRN7000 might enhance NK
activity not directly, but via the activation of T cells. In
addition, it has been proposed that KRN-7000 acts through
activation of antigen-presenting cells, such as dendritic cells
[37]. The first clinical trial with this agent, which included
pharmacokinetic studies, has recently been completed [38].
Studying the GSLs composition of the sponge Agelas
[39] and Axinella [40] we found ten more complex α-GSLs
having the first galactose glycosylated by different sugars in
different positions and with a number of sugar units ranging
from 2 to 5.
C 10H21
R= H
C 10H21 R = H
C10 H2 1
R = Glc
C10H21 R = Glc
Bioactive Marine Natural Products
OR
O X
C 10H21
NH
Sugars O C1 0H21
OR
OR
MeO C10 H2 1
X HO
O
1. KMnO4/NalO4
2. CH2N2
O
C1 0H21
MeO X GLC-MS
X, Y = CH2 HC + H2C
CH
CH3 CH3
Scheme 2. Microscale chemical degradation of plakosides.
These compounds have been tested to assess their
immunomodulating activity using a T-cell proliferation
assay, and we found that some, but not all, of them are
immunostimulating agents. Data obtained pointed to the
position 2 of the inner sugar being a crucial point for the
immunostimulating activity: glycosylation at this position
resulted in the loss of the stimulatory effects on the
proliferation of the lymphocytes.
Investigating the glycolipid composition of the sponge
P. simplex we found four unique GSLs having
unprecedented structural features among natural products.
They are β-gactosylceramides, named plakosides A-D (10-
13) [32,41], whose galactose residues are alkylated at O-2 by
a 3,3-dimethyallyl group. Plakosides are very interesting
from a structural point of view, since they are the first
example of GSLs with a prenylated sugar and also the first
natural GSL with a cyclopropane-containing ceramide.
Moreover, the presence of a galactose prenylated at the
very crucial position 2 is of great importance with respect to
their biological activity. In fact, in the T-cell proliferation
assay, they were found to inhibit the proliferation of murine
lymphonode cells activated with concanavalin A. This
immunosuppressive activity is particularly interesting, since,
unlike most immunosuppressors used clinically, plakosides
are not cytotoxic, as shown by the negative response to the
MTT assay.
Due to their unique structure and bioactivity, plakosides
attracted the attention of synthetic chemists. Two
independent syntheses of plakoside A have been reported
[42, 43]. The absolute configuration of the two cyclopropane
rings has been then established by chemical degradation of
the natural plakoside A. Derivatised fragments obtained from
alkyl chains of plakoside A were compared with synthesised
Current Medicinal Chemistry, 2004, Vol. 11, No. 13 1677
OR
O C1 0H2 1
H2/PtO2 NH
AcOH Sugars O C 10H21
Y
OR
1. HCl/M eOH
2. Chromatography
NH2
C 10H21
Y
OR
1. KMnO4/NalO4
2. CH2N2
MeO C10 H2 1
Y
O
+
CH2 H2C
CH3
samples with known absolute configuration [44]. Nicolaou’s
group also evaluated the synthetic compounds by a MLR
and a murine bone marrow cell proliferation assays and
found, in contrast with data reported by us using T-cell
proliferation assay, only a modest immunosuppressive
activity on these cell systems.
A major problem we had in establishing the structures of
plakosides was the determination of the length of the alkyl
chains and the location of the two cyclopropane rings, taking
into account also that we had in our hands just 1-2
milligrams of each one. To solve this problem, we
developed a submilligram scale degradation procedure as
outlined in Scheme 2.
B. Glycoglycerolipids
Glycoglycerolipids (GGLs) are normal constituents of
membranes of photosynthetic organisms, and in the marine
world are present almost exclusively in algae and
cyanobacteria (blue-green algae). Marine GGLs do not show
a great structural variety. Until now the most commonly
found are the compounds in which the glycerol moiety is
linked to one or two galactose residues (monogalactosyl-
diacylglycerol and digalactosyldiacylglycerol) or a unit of
sulfoquinovose (sulfoquinovosyldiacylglycerol).
In the glycolipidic fraction of P. simplex we found
crasserides (1 4 a - 1 4 m ), very close analogues of
glycoglicerolipids, having a five-membered cyclitol, instead
of a sugar moiety, linked to the glycerol molecule through
an ether bond.
Actually, crasserides are widely distributed in marine
sponges. Since our first isolation in the sponge
Pseudoceratina crassa [45], we found them in all Caribbean
species we have studied until now [46], suggesting that
1678 Current Medicinal Chemistry, 2004, Vol. 11, No. 13 Fattorusso et al.

a R1 =
HO OH

b R1 =
HO OH

O c R1 =
O
R1
O d R1 =

O e R1 =

14
HO OH
3` O f R1 =
4` 2` R1
HO 5` O
1` 1``` g R1 =
O
1 h R1 =

2 OH
3 i R1 =
10"
O
1" l R1 =
16
m R1 =

Fig. (6). Structure of crasserides and isocrasserides.

crasserides can play an important ecological role in sponges.
Crasserides are always associated with minor amounts of
isocrasserides (15a-15m), metabolites in which the acyl
chain is linked to one cyclitol hydroxyl group rather than to
the glycerol moiety [46].
Test performed on the fish Carassius auratus showed
that crasserides have antifeedant activity at a concentration of
30 µg/cm 2 of food pellets, pointing to a potential role of
these compounds as natural deterrents from predators.
Almost at the same time, a Japanese group reported the

HO OH HO OH
MeO R1
+
HO OH OH
Et3N/MeOH HO O
O O O
GLC-MS
R1
O OH

O O

HI

AcO AcOAg/AcOH
I
reflux
MeONA
MeOH, reflux

OH CrO3
COOH
AcOH

CH2 N2

COOMe

GLC-MS
Scheme 3. Microscale chemical degradation of crasserides.
Bioactive Marine Natural Products Current Medicinal Chemistry, 2004, Vol. 11, No. 13 1679

H Xyl-II H Xyl-II
H HO H H O
4'
RO O O
RO RO
16
H OR H OR
H H H H
a R=H
b R = CH3CO
c R = CD3CO

H H H H H
H 5 VI H H H H 5 IIIH O H H
4 VI O 4V 5V H O 4I V 5 IVH O 4II I 4 II 5II H O H
RO VI RO V RO 2 IV
RO 2 III
RO 2II
4I
5I
H O
2
RO 2
O O O 111 II I
O II
O 2I O
3VI 1VI 3V 1V 3I V 1 IV 3 1 3 II 1 RO 1
H H H H H 1I
H ORH H ORH H ORH
3I
H ORH H ORH H OR 29
H H
28 24 20 16 12 8
4
17
35 34 33 32 31 30

Fig. (7). Structures of plakopolyprenoside and plaxyloside.

isolation of crasserides [47], from Okinawan marine in the two chair conformations, and the observed chemical
sponges, showing that these compounds also possess shifts depend on the contribution of each of them. Therefore,
stimulation activity on nerve growth factor (NGF). the different conformational behaviour of the sugars in the
Like for plakosides, we needed to perform a chemical peracetylated carbohydrate chain of compound 17b also leads
degradation of crasserides to assess the structure of the acyl to different 1H and 13C chemical shifts in different sugars.
As for the solvent, signals in the 1 H NMR spectrum of
chains and the position of methyl branches in the alkyl
compound 17b showed a remarkably better dispersion when
chains (see Scheme 3).
the spectrum was recorded in C6D6 rather than CDCl3. This
C. Polyisoprenoid Glycolipids result can be explained by the strongly anisotropic shielding
Plakopolyprenoside (16) [48] and plaxyloside (17) [49] effect of the benzene molecule that can more effectively turn
are glycolipids having a C-35 linear polyisoprenoid alcohol differences in conformation into differences in chemical
as aglycon and a sugar chain exclusively composed of β- shifts.
xylopyranose units. H
H H OR
4 5H
Polycyclic terpene glycosides are very common in plants HO O 5 OH 1
2
HO OR H O H
and marine organisms, but plaxyloside and plakopoly- 3 H 1 H H
OH 4 3 2
prenoside, which have an acyclic aglycon, can be considered H H
H OH OH
the first example of a new class of glycolipids, never found
4 1
in other phyla of living organisms that are more similar to C1 C4
fatty-acid-containing glycolipids. Fig. (8). 4C 1 and 1C 4 conformations of a β-xilopyranoside.
Plaxyloside (17) has the same aglycon moiety as
The reason why sugars in a very similar chemical
plakopolyprenoside, but its linear saccharide chain is made
environment show a remarkably different conformational
up of six rather than two, β-xylopyranose residues, making
behaviour is not entirely clear at the moment. This could
the molecule much more polar. Because of its repetitive
originate from the preference of the flexible peracetylated
structure, proton and carbon NMR signals of the
carbohydrate chain for folded conformations, where
carbohydrate chain of compound 17 are strongly overlapped
favourable electrostatic interactions between the partially
to each other, preventing both the assignment of resonances
charged atoms of the acetyl groups of different sugars are
and the measurement of coupling constants. However,
possible.
structure determination of plaxyloside could be successfully
achieved using the peracetylated plaxyloside 17b and C6D6 D. Bacteriohopanoids
as the solvent. In fact, in compound 17b all the six pentose Bacteriohopanoids are typical bacterial metabolites
sugars are in equilibrium between the two possible chair having a mixed-biogenesis origin. They are made up of a
conformations 1C4 and 4C1 (see Fig. 8), but the contribution pentacyclic triterpenoid hopane aglycone linked to a sugar-
of the less stable 1C4 chair proved to be considerably higher derived polyhydroxylated C5 chain. Strictly speaking, they
in the sugars in the middle of the chain than in those at the are not glycolipids, but we found them largely as
end of the chain. constituents of the glycolipid fraction on account of their
This different conformational behaviour was crucial for polarity similar to that of other compounds described until
the structure elucidation because, affecting 1 H and 13 C now. It is well known that bacteriohopanoids play a
chemical shifts of sugars avoided the complete overlapping membrane stabilizer biologic role same as sterols do in
of the sugar signals in both spectra observed for the non- higher organisms.
acetylated compound. Clearly, chemical shifts are different
1680 Current Medicinal Chemistry, 2004, Vol. 11, No. 13
R OH OH
12 OH
OH
18 R=H
19 R=Me
Fig. (9). Structure of hopanoids.
In addition to the simple known tetrol 18, we found the
new 12-methyl derivative 19 [50] and even larger amounts of
a further new bacteriohopanoid, the 32,35-
anydrobacteriohopantetrol 20 [51]. The total amount of the
three metabolites is as high as 50% of sterols in weight.
These data strengthen our hypothesis of a possible structural
role of bacteriohopanoids in the cell membrane of P .
simplex.
It is interesting to note that, although the mechanism by
which hopanes are converted into bacteriohopanoids in
bacteria is not known in detail, it has been demonstrated that
the C5 side chain of most bacteriohopanoids is derived from
ribose, whose stereochemistry is retained in bacterioho-
panetetrol [52]. As shown in the Scheme 4, the C-adenosyl
derivative of 20, which has been isolated from
Rhodopseudomonas acidophila [53] and the C-ribosyl
derivative that is never isolated from a natural source has
been proposed to be a biosynthetic intermediate [54].
Compound 20 fits well into this biogenetic hypothesis,
because it could be produced by cyclisation of
bacteriohopanetetrol 18, through a branch of the above
biogenetic sequence involving the reductive removal of the
adenine, or, alternatively, the reduction of the acetal function
of the intermediate (see Scheme 4).
E. Atypical Glycolipids
The sponge P. simplex is also able to produce atypical
glycolipids that cannot be included in any of the main
classes of glycolipids. We called this metabolite simplexides
(21) [55] because of its very simple structure, that is, an
unusual lipid moiety constituted of a very-long-chain
secondary alcohol composed of 34-37 carbon atoms. The
hydroxyl group, located nearly in the middle of the chain, is
NH2
N N
N N
O OH
hydrolysis
OH OH
?
[H]
?
[H] O
OH OH
Scheme 4. Biogenetic pathway for hopanoids.
Fattorusso et al.
19 35
O
21 30
OH
25 26 17 22
28 OH
29
27
glycosylated by a disaccharide constituted by a β-galactose
linked to an α-glucose through a hydroxyl group at position
4. Simplexides have been isolated as a complex mixture of
homologues having alkyl chains with varied lengths and
branching.
To establish the exact composition of the homologue
alkyl chains and the position of the hydroxyl group on the
chain, we set up a simple microscale (200 µg) degradation
procedure (Scheme 5).
Glycosylated long-chain secondary alcohols have never
been reported as natural compounds, so that simplexides are
the first member of a new class of glycolipids. Tested on T-
cells stimulated with concanavalin A, they showed a potent
inhibitory activity on their proliferation: they caused a 43%
proliferation inhibition at a concentration as low as 0.01
µg/mL, which rose to 79% at 1µg/mL. This immuno-
suppressive activity was not related to a cytotoxic activity,
as shown by the negative response in the MTT assay at all
concentrations tested.
Polyketide Derivatives
Secondary metabolites deriving from the polyketide
pathway [56] and containing a stable cycloperoxide group are
typical components of the organic extracts obtained from
marine sponges belonging to the family Plakinidae. The first
compound of this class to be reported was plakortin (22), a
six-membrered cycloperoxide found in 1978 by Faulkner and
coworkers in Plakortis halicondrioides [57]. After its
isolation, several related polyketides have appeared in the
literature, and most of the isolated compounds maintain
some key features: a 1,2 dioxane ring substituted at position
O
OH
[H]
HO
OH OH
OH OH
Bioactive Marine Natural Products Current Medicinal Chemistry, 2004, Vol. 11, No. 13 1681

OH
H
6'''
α-Hlc
4''' HO
HO 5''' 2''' 1'''
HO H
3'''
H OH 3'
H OHO 2'
6' R 2
6''
4'' H O
H 5'' 2''
1'' O 1 3 R1
HO
3'' H 2 6
β-Gal H OH
H

21

7 7
R1, R2 = 17 11% 18%
18
7 7 19
17 6% 11%

7
5 4%
18

Fig. (10). Structure of simplexides.

3 with an acetate residue and at positions 4,6,6 with alkyl plakortides F-H, isolated from P. halichondrioides [63],
chains, respectively [58]. The nature of the aliphatic chains have been shown to enhance Ca2+ uptake by the cardiac
at position 6 together with the stereochemistry of the sarcoplasmic reticulum through activation of the Ca2 +
asymmetric centers belonging to the cycloperoxide ring ATPase. Although the detailed mechanism of this rich
represents the main sites of structural variation within this variety of bioactivities still needs to be disclosed, it is
class of metabolites. Some analogues possessing a five- commonly believed that the reactive cycloperoxide
membered cycloperoxide ring (1,2-dioxolane) in place of the functionality plays a key role.
more common 1,2 dioxane have also been reported (e.g. These premises encouraged us to begin the chemical
plakortide E, 23) [59]. investigation of the apolar fraction obtained from the organic
Several pharmacological properties have been disclosed extract of P. simplex. This analysis revealed to be
for these cycloperoxide metabolites: plakortin was reported particularly fruitful, resulting in the isolation of more than a
as an antibacterial agent [57], while some of its analogues dozen of new metabolites and in the discovery of interesting
showed antifungal [60], antileishmanial [61], or cytotoxic new bioactivities. As a first result, we found that plakortin
[62] activities. Finally, other members of this family, e.g. constitutes a very major compound of this fraction (about

3' 2 3' 2
6' R 6' R
2' TsCl 2'
21 MeOH/HCl
R1 R1
HO 1 3 TsO 1 3
2 6 2 6

DBU

3' 3' 2
6' R
2 6' R
2' 2'

R1 R1
1 1 3
3 2 6
2 6

KMnO 4/NalO 4

3' 2 3'
6' R 2' 6' R
2
HOOC 2'
1
HOOC R1 COOH 2 R1
1 2 3 HOOC 3
6 6

Scheme 5. Microscale degradation of simplexide.

1682 Current Medicinal Chemistry, 2004, Vol. 11, No. 13 Fattorusso et al.

15
9 5
4
10 6 O COOCH3
O 3 COOCH3 O O
13 O COOCH3
O 1
23 24
22

Fig. (11). Structures of plakortin (22), plakortide E (23), and dihydroplakortin (24).

20% of the entire organic extract, an unusually high
percentage for a secondary metabolite), together with its
novel 9,10-dihydro derivative that we named dihydropla-
kortin (24) [64].
In spite of the wide occurrence of plakortin and of its
possible role in the biogenesis of many analogues (see
below), the absolute configuration of its four stereogenic
centers C-3, C-4, C-6 and C-8 was not known. Indeed, only
the relative configuration around the cycloperoxide ring of
plakortin was determined by Faulkner et al. [57] by a
lanthanide-induced shift study. Therefore, the definition of
the absolute configuration of plakortin and dihydroplakortin
constituted our first task [64]. This was accomplished
through a strategy of chemical degradation followed by
derivatisation with chiral auxiliaries, as shown in the
following Scheme 6.
Two further new metabolites constituted the
cycloperoxide pool of P. simplex, namely plakortide I (25) e
J (26), both characterised by more complex alkyl chains at
C-6. These molecules were isolated together with their
corresponding diol derivatives, e.g. seco-plakortide I (27)
[65], and their stereostructures were determined with the
application of the same strategy described above for
plakortin. This constituted the first report of diol analogues
of plakortin or plakortide cycloperoxides.
These new metabolites were evaluated for cytotoxic
activity against WEHI 164, murine fibrosarcoma cell line,
using the MTT conversion assay to assess the cell viability.
Plakortides I and J inhibited the cell growth at 72 h with
IC 50 2.5 and 4.0 µ g/mL, respectively; while the diol
compounds were much less active than the corresponding
cycloperoxides (IC50 > 15 µg/mL). This result seems to
suggest that the cycloperoxide moiety is largely responsible
for the cytotoxic activity shown by some of these molecules.
However, it should be remarked that a cycloperoxide group
is not enough to provide a significant cytotoxic activity to
the metabolites of this class; indeed, interestingly, plakortin
and above all dihydroplakortin exhibited only a very
marginal cytotoxicity in the same test performed on a large
panel of cell lines [64].
On the other hand, the presence of a cycloperoxide
moiety has been recognised as critical for the anti-malarial
activity of artemisinin (28), a sesquiterpene lactone, first
isolated from the plant Artemisia annua as a potently active
agent against chloroquine-resistant strains of Plasmodium
falciparum. Artemisinin and its oil soluble (e.g. artemether
and arteether) and water soluble (e.g. artesunate) semi-
synthetic derivatives are being increasingly used in the
therapy, especially in combination with traditional
antimalarials (e.g. mefloquine).
Although not yet fully agreed, some evidence suggests
that the endoperoxide group of artemisinin (and of many
semisynthetic derivatives) reacts with the haem iron center
(or with other iron containing cellular structures) giving rise,

O COOCH3 O COOCH3
O O
22 24
KMnO4/NalO4 H2/10% Pd H2/10% Pd
in t=BuOH

HOOC

O COOCH3 HO COOCH3
O HO
(R) or (S)-PGME, (R) or (S) M TPA chloride
PyBoP, HOBT, N-methylmorpholine in dry pyridine
in DM F

H3 COOC HNOC
*
O COOCH3 HO COOCH3
O O O
H3CO *
F3C

Scheme 6. Determination of absolute configurations of plakortin (22) and dihydroplakortin (24).

Bioactive Marine Natural Products Current Medicinal Chemistry, 2004, Vol. 11, No. 13 1683

O COOH O COOH OH COOH

O O HO

25 26 27

Fig. (12). Structures of plakortides I (25) and J (26), and of seco-plakortide I (27).

after structural rearrangements, to C-centered radicals. These falciparum strains; while, interestingly, the structurally
latter species would be the final entities responsible for the similar five-membered cycloperoxide plakortide E (23)
anti-Plasmodium activity, acting as alkylating agents proved practically inactive, suggesting that the size of the
towards the macromolecules within the protozoan [66]. cycloperoxide ring is important for the bioactivity. The
response pattern of plakortin and dihydroplakortin appeared
H to be similar to that of artemisinin although the IC50 s
O differed by almost 2 log.
O
O
H
O
O COOCH3
28 O
O

Fig. (13). Chemical structure of artemisinin (28). 29

Stimulated by the urgent need to discover new drugs
active against malaria, a plague causing at least 2 million
deaths each year, we decided to assess the antimalarial
activity of the sponge-derived cycloperoxides, starting with
the major compounds extracted from P. simplex: plakortin,
dihydroplakortin, and the five-membered cycloperoxide
plakortide E [67].
The three molecules were tested against both D10
(chloroquine-susceptible) and W2 (chloroquine-resistant)
strains of P. falciparum. Parasite growth was determined
spectrophotometrically (OD650) by measuring the activity of
the parasite lactate dehydrogenase (LDH), according to a
modified version of the Makler method in control and drug-
treated cultures. Compounds 22 and 24 showed identical
excellent antimalarial activity (ng/mL range) against both P.
Fig. (14). Chemical structure of plakortide L (29).
Again, the presence of a 1,2-dioxane ring is not enough
to ensure an antimalarial activity. Indeed, plakortide L (29),
a close analogue of plakortin that is isolated from another
Plakortis species, was subsequently shown to be completely
devoid of bioactivity [68].
The mechanism of action of plakortin, as well as of other
simple endoperoxides, is far to be fully elucidated; however,
the above findings suggest that a spatial arrangement of the
cycloperoxide moiety suited to allow the interaction with the
target together with the ability of the molecule to penetrate
within the infected cells are likely to be crucial parameters.
Although plakortin is less active than artemisinin, its
structural simplicity makes it an ideal lead compound for the

9
4
10 6
3 1
O COOCH3
O
22 (C3-O-O-C6)

O O
O COOCH3
H O
H O
OH

H3COOC Plakortones (C3-O-C6 + C1-O-C4)

Furano-esters (C3-O-C6)
Plakortethers (C6-O-C9)

Scheme 7. A schematic view of the relationship of plakortin with three classes of derived metabolites (plakortethers, furano-esters,
and plakortones).
1684 Current Medicinal Chemistry, 2004, Vol. 11, No. 13 Fattorusso et al.

development of a new antimalarial drug alternative to those
now available. In this regard, our efforts are currently
focussed both on the isolation of novel analogues from
marine sources (possibly allowing an extension of the
preliminary structure-activity relationships already
developed) and on the synthesis of the bioactive natural
molecules (plakortin and dihydroplakortin) and of their
analogues in view to better understand the mechanism of
action of simple cycloperoxide derivatives and to design a
drug candidate with stronger antimalarial activity.
15
4 O
O 31
30 2 metabolites of great pharmacological interest: they have
H3COOC
H3COOC
Fig. (15). Chemical structures of furanoesters 30 and 31.
The presence of the highly reactive cycloperoxide group
within the structure of plakortin is not only responsible for
its marked biological activity but, most likely, has a key
role in the production of the rich series of related polyketides
that we have isolated from P. simplex. These molecules
belong to three different structural groups: furanoesters,
plakortones and plakortethers. Scheme 7 schematically
depicts the chemical relationship between these metabolites
and their postulated parent compound plakortin .
Similarly to cycloperoxide derivatives, the gross
structure of these metabolites has been routinely defined
through the application of spectroscopic techniques, while
elucidation of their absolute stereochemistry required
microscale chemical transformation procedures in order to
allow the reaction of the molecule with suitable chiral
auxiliaries or, alternatively, to compare the obtained
products with molecules whose stereochemistry is already
known.
double bond between C-2 and C-3 and of an ether bridge to
connect C-3 and C-6, giving rise to a tetrahydrofuran ring
with a sp2 carbon at C-2 [64].
The absolute stereochemistry of compound 30 has been
deduced with the help of the chemical derivatisation strategy
summarised in Scheme 8 [64]. The absolute configuration
determined for the chiral carbon atoms of 30 is the same of
the corresponding chiral centers of plakortin.
A second class of Plakortis metabolites, plakortones, is
strictly related to the above described furano-esters [59].
Their structures contain a single oxygen atom to connect C-3
and C-6, but, in addition, they show a γ-lactone group
condensed with the tetrahydrofuran ring, originating from
the linkage of C-1 ester oxygen with C-4. Plakortones are
shown potent activation of the sarcoplasmic reticulum Ca2+
ATPase, and, consequently, they have potential application
in correcting relaxation abnormalities related to some forms
of human heart failure. A synthetic approach to their 2,6-
dioxabicyclo[3.3.0]octan-3-one nucleus has been very
recently described [69]. P. simplex afforded two new
members of this class, named plakortone E (32) and F (33),
differing for the alkyl chain at C-6 [70].
6
O O
3
32 H 1 O
O
O
33
H O
Fig. (16). Chemical structures of plakortones E (32) and F (33).

Furanoesters 30 and 31 are closely related to plakortin Furthermore, P. simplex has revealed to be the source of
and dihydroplakortin, respectively, but their chemical plakortethers A-G (34-40) [71], which represents an
structures are highly rearranged, showing the presence of a unprecedented class of non-peroxide plakortin derivatives.

8 OHC
4
6
OsO4/NalO4 O
O

30 O
COOCH3 NaBH4 in MeOH

O
(R) or (S) M TPA chloride
OH2C HOH2 C
in dry pyridine
F 3C
O O
H3CO
O O

LiAIH4 in dry ether

HO OH
OH

Scheme 8. Chemical conversions used to define the absolute stereochemistry of compound 30.
Bioactive Marine Natural Products Current Medicinal Chemistry, 2004, Vol. 11, No. 13 1685

15
11 9
6 1
O COOCH3
H
34
OH O COOCH3
H
35 OH

O COOCH3
H Cl H O
OH COOCH3
36 H
O
37 OH

O COOCH3
HO H H R O COOCH3
OH
38 R = α-OCH3 39 OH
R = β-OCH3 40

Fig. (17). Chemical structures of plakortethers A-G (34-40).

Their structures, characterised by a tetrahydrofuran ring
connecting C-6 and C-9, share with plakortin the carbon
backbone and the absolute stereochemistry of the
corresponding asymmetric centers. The seven plakortethers
basically differ only for the functionalisation at C-10: in
plakortether A (34) C-10 is part of a double bond; in
plakortether B (35) of a single bond; in plakortether D (37);
it is a carbonyl group that in plakortether E (38) is reduced
to an alcohol group. Among the isolated compounds,
plakortether C (36), is particularly interesting because it
represents the first chlorinated compound found in a
Plakortis sponge. Plakortethers F (39) and G (40) are two
further analogues of this class although they consistently
differ from the above described tetrahydrofuran derivatives
because it completely lacks three-carbon fragment C-10/C-
12. In this case, C-9 is an acetal carbon linking directly a
methoxy group, and the two plakortethers F and G are
epimers at C-9.
All the isolated compounds have been evaluated for
cytotoxic activity (using the MTT assay) against two
different tumour cell lines, WEHI 164 (murine fibrosarcoma)
and RAW 264-7 (murine macrophage), proving to be
completely inactive against the first cell line; while,
interestingly, plakortethers A, B, D, and E showed selective
activity against the second cell line. Their IC50, obtained as
a mean of at least three measurements, range between 7 to 11
µ g/mL. Plakortethers C, F, and G were inactive also
towards the second cell line (IC50 > 20 µg/mL). It should be
noted that, among the class of C18 plakortethers, only the
chlorine derivative, plakortether C, is completely inactive.
The gross structure of this new class of metabolites could
be easily determined through the application of 2D NMR
techniques (COSY, HMQC, HMBC), while the relative
geometry around the five-memebered ring was deduced by
inspection of the correlation peaks showed by a 2D NMR
ROESY spectrum. On the contrary, determination of the
absolute stereochemistry of their five (six for plakortethers C
and E) stereogenic carbons proved to be particularly
challenging. Indeed, only one of their chiral carbons (i.e. C-
3) supported a functional group (alcohol) easily amenable to
reaction with chiral auxiliary groups: a procedure generally
required for stereochemistry elucidation of natural products
obtained in small amounts. Therefore, the only remaining
chance was to try to connect the absolute configuration of
one of the novel metabolites with that already determined for
plakortin. To this aim, we elaborated a three-step semi-

Zn/AcOH

O COOCH3 OH
O COOCH3
HO
22
N-iodosuccinimide in CH3CN

O COOCH3
H
35
OH O COOCH3
Ph3SnH/2,2`azobis -(isobutyronitrile) H
+ I OH

O COOCH3
H
OH

Scheme 9. Semisynthesis of plakortether B (35) from plakortin (22).

1686 Current Medicinal Chemistry, 2004, Vol. 11, No. 13 Fattorusso et al.

NaBH4 in MeOH
O O COOCH3
COOCH3 H
H
OH O OH
OH Plakortether D (37)
Plakortether E (38) + HS (CH2 )2 SH/BF3 -Et2 O
its epimer at C-10

Ph3SnH/2,2`azobis -(isobutyronitrile)

O COOCH3
H
OH H
Plakortether B (35) O COOCH3
S S
OH
H2/Pd Ph3SnH/2,2`azobis -(isobutyronitrile)

O COOCH3 O COOCH3
H H
H Cl
OH OH
Plakortether A (34) Plakortether C (36)

Scheme 10. Chemical interconversions among plakortethers.

synthesis of plakortether B framework from plakortin
outlined in Scheme 9.
Plakortethers A (34) and C-E (36-38) were mutually
correlated and/or related to plakortether B (34) through the
series of chemical transformations depicted in scheme 10,
thus accomplishing the unambiguous determination of their
absolute stereochemistry. These conversions indicated that
plakortethers A and C-E actually possess the same absolute
configuration at C-3, C-4, C-6, C-8, and C-9 of plakortether
B.
In order to complete the stereostructure elucidation of
plakortethers C and E, the stereochemistry at C-10 needed to
be connected with those of the other chiral carbons. This
problem was faced up through the use of the J-based
configuration analysis, recently proposed by Murata [72].
Application of this technique to plakortethers C and E,
respectively, allowed the complete stereochemical
elucidation of these molecules (Fig. 18).
In summary, a number of novel polyketides structurally
related to plakortin have been found as minor components of
the apolar fraction of the organic extract of P.simplex and
their stereostructures fully determined using the very limited
material available. It is worthy of note that all these
metabolites lack typical cycloperoxide moiety but share with
plakortin both the carbon skeleton and the stereochemistry of
the corresponding chiral carbon atoms. This observation
strengthens the hypothesis that plakortin could play a crucial
role in the biogenesis of these non-peroxide metabolites. For

H9 H9
H10 C11 H10 OH

C9 C9

C8 O C8 O

Cl C9-C10 axis of plakortether C C11 C9-C10 axis of plakortether E

erithro
3 J (H9-H10) = small 3 J (H9-H10) = small
threo 3 J (H10-C8) = small 3 J (H10-C8) = small
3 J (H9-C11) = small 3 J (H9-C11) = large
2 J (H9-C10) = small 2 J (H9-C10) = large
2 J (C9-H10) = small 2 J (C9-H10) = small

Fig. (18). Application of the Murata method to the C-9/C-10 axis of plakortether C (36) and E (38).
Bioactive Marine Natural Products
O COOCH3
O
H
22
30
H3 COOC
Scheme 11. Proposed biogenetic pathway connecting plakortin (22) and the furano-ester compound 30.
example, a possible biogenetic pathway connecting plakortin
and the tetrahydrofuran-containing compound 30 has
proposed in Scheme 11.
Analogously, plakortethers could biogenetically be
derived from plakortin following the reaction shown in
Scheme 12.
In this case, the reductive opening of the cycloperoxide
ring of plakortin would be driven by the simultaneous
electrophilic cyclisation of the deriving γ,δ unsaturated
alcohol. When the electrophilic group is a proton, this
reaction gives rise to plakortether B (35). All the other
plakortethers could be originated both from a different
electrophilic agent and from subsequent chemical
modifications on plakortether B.
The use of a versatile (easily modifiable by a chemical
point of view) and relatively abundant main structure, as
plakortin certainly is, can be envisaged as a general strategy
used by marine organisms (in this case sponges) to enrich
the chemical (and, consequently, pharmacological) diversity
of their secondary metabolism. In this connection, the above
described role of plakortin in the biosynthesis of many
analogues is the only one of the possible examples. Another
enlightening case is constituted by oroidin [73], the major
pyrrole-imidazole alkaloid of sponges of the genus Agelas.
Oroidin is actually the postulated precursor of many
bioactive alkaloids originating from oxidative/reductive
steps (e.g. the antihistaminic dispacamides [74]),
isomerisations (e.g. the antiserotonergic keramadine [75]),
cyclisations (e.g. the cytotoxic agelastatin [76]),
dimerisations (e.g. the potent antibacterial sceptrin [77]) of
its base structure [78].
O COOCH3
O
H Enz
22
+
E
Scheme 12. Proposed biogenetic pathway connecting plakortin (22) and the plakortethers.
Current Medicinal Chemistry, 2004, Vol. 11, No. 13 1687
-
O COOCH3
O
-B-Enz H-B-Enz
- H2 O
O
OH
H3 COOC
Alkaloids
The alkaloid composition of Plakortis sponges does not
reflect the incredible richness of both polyketide and
glycolipid groups of metabolites: to our knowledge, the
pyrrolidino-tyramine compound plakoridine A [79], the
pyrrolo-acridine plakinidines A-C [80] and the cytotoxic β-
carbolines plakortamines A-D [81] were the only alkaloids
obtained from Plakortis sponges. Our investigation of the
most polar fraction of the organic extract of P. simplex
yielded four new alkaloids; three iodoindoles
plakohypaphorines A-C (41-43) [82], and are pyridinium
derivative simplakidine A (44) [83].
Plakohypaphorines A-C are particularly remarkable
secondary metabolites since they represent the first
iodoindole compounds from either marine and terrestrial
source. Indeed, although some iodine containing alkaloids
have been reported to date from marine organisms (e.g.
geodiamolide A [84] and dakaramine [85]), all of them show
the iodine atom linked to the benzene ring of a tyrosine-
derived unit. In addition, the linkage of an halogen atom at
C-7 of the indole nucleus of plakohypaphorines is worthy of
note since only 5- or 6- halogen (usually bromine)
substituted indole derivatives have been found as spongal
metabolites. On the other hand, 7-haloindoles have been
reported as microbial metabolites [86], and this observation
is suggestive of the microbial origin of plakohypaphorines.
The pyridinium alkaloid simplakidine A (44) possesses a
C17 polyketide moiety, sharing with plakortethers the carbon
backbone and the absolute configuration of the asymmetric
centers but it links a pyridinium ring at C-10. Although
pyridinium alkaloids are not rare in marine sponges, the
O COOCH3
H
E OH
1688 Current Medicinal Chemistry, 2004, Vol. 11, No. 13
O
R
O-
N+
R1 N H
R2 H
41 42 43
R H H I
R1 H I H
R2 I I I
Fig. (19). Chemical structures of plakohypaphorines A-C (41-43) and of simplakidine A (44).
structural diversity within these compounds is somewhat
limited. A first class of pyridinium derivatives comprises
macrocyclic structures with alkyl linear chains linked at
positions C-3 and N-1 of the pyridinium unit (e.g. halitoxin
[87]). Another structural class includes homarine or
trigonelline substituted at C-3 or C-2, respectively, with
simple alkyl chains. Therefore, simplakidine A is a unique
example of pyridinium alkaloid in possessing a trigonelline
nucleus substituted at position 4 with a complex polyketide-
deriving moiety. Simplakidine A could be derived
biogenetically from plakortin as shown in Scheme 13. In
this case, the electrophilic cyclisation would be driven by
simultaneous attack of C-10 at C-4 of a trigonelline unit.
Simplakidine A (44) exhibited only a weak cytotoxicity
towards murine macrophages cell line and, since the only
difference of 44 with the active plakortether B (35) is the
presence of a pyridinium ring at C-10, the high polarity of
this group could have deleterious effects on the ability to
cross the cell membrane.
CONCLUSIVE REMARKS
Both variety and structural innovation are considered
distinctive features of the molecules elaborated by the marine
invertebrates and, undoubtedly, the metabolic content of the
Caribbean sponge P.simplex represents an objective in this
field. Actually, the structural variety and the biological
activity of the metabolites found in this sponge appeared
surprising also for researchers, who have been interested for a
long in the chemistry of marine natural products, providing
the opportunity to make some general remarks.
Microscale Structural Analysis Issue
The first and most evident remark is that an extensive
analysis of the metabolic content of an organism is a
complex, laborious and time-consuming task. One of the
major obstacles to overcome is represented by the limited
availability of the compounds under investigation; in recent
years, this problem is gathering an increasing importance on
account of the difficulties in collecting remarkable amounts
of biological material, which is correctly protected for
reasons of environmental conservation. Sometimes, the
natural product chemist can obtain just a few dozen of grams
of biological material, corresponding to a few grams of
organic extracts, mostly composed by the usual primary
metabolites. So, there are clear difficulties in the isolation
Fattorusso et al.
O COOH
-
OOC OH
+
N 44
CH3
and structure determination of new molecules, as well as in
the investigation of their biological activities having a few
milligrams of pure compounds. Furthermore, this process
has to be managed within the time-frame of modern drug
discovery and speed from prioritising hits to identifying the
chemical structure is imperative.
Fortunately, at present the natural product chemist can
make use of new very informative micro-analytical
techniques; among them, NMR analysis, which is a not-
destructive technique, is fundamental for structural
determination and, thanks to the continuous improvement of
the instrument performances, it requires less sample material
and becomes more informative than a few years ago.
Additionally, NMR spectral analysis affords the invaluable
advantage in recovering the material to be used then for
biological tests. Currently, these techniques allow to assign
structures, including all the stereochemical details, to
molecules also quite complex, such as those of the
plakosides [32], having little more than one milligram of
material.
Access to Pharmaceutical Potential
Chemical identification of the molecules present in
biological extracts is just one step of their full investigation
and, often, it doesn’t represent the first one, a major interest
being their possible use in various applied fields, in
particular as therapeutic agents. The necessary selection of
the employed assays resulted in a consequent limitation to
the detection of the full biological potential of the
metabolites present in the crude extracts. Sometimes,
interesting bioactivities of marine metabolites have been
discovered many years after they were isolated and
chemically investigated. This has happened for plakortin;
this molecule was isolated in 1978 from a Plakortis sp. by
Faulkner [57], who determined its structure, apart from some
stereochemical details, and reported for this compound a
weak antimicrobial activity. After about 25 years, we re-
isolated plakortin from P. simplex and evidenced that it is
potently active against Plasmodium, proposing it as an
interesting lead compound for the antimalarial therapy [67].
Besides plakortin, several parallel cases can be mentioned.
Among them, we can remind the cytotoxic eunicellane
diterpenes from soft corals [88]. Ten years after the report of
sarcodictyns, Fenical et al. revealed that the closely related
eleutherobin and other molecules of the same class are
endowed with a potent anti-tumour activity due to a taxol-
Bioactive Marine Natural Products
like mechanism of action involving tubulin polymerisation
and microtubule stabilisation [89].
From the above considerations it follows that the
discovery of new compounds for pharmaceutical purposes
can be accomplished not only through an accurate analysis of
unexplored extracts, but also through a re-examination of the
extracts from already investigated species, utilising wide
ranging biological screenings. Then, targeted strategies, in
combination with high-throughput screening methods,
ensure that natural chemicals could become novel
pharmacotherapeutic agents.
The Origin of Chemodiversity
Among the multitude of marine organisms, some of
them appear to possess a much more efficient synthetic
potential and this is the case with P. simplex. This capacity
doesn’t represent the attribute of a single species, but it is
shared by a number of other sponges, tunicates, bryozoans or
molluscs. The consequent uncommon chemodiversity of
these invertebrates is a resource to be adequately exploited,
but, at the same time, it represents a matter to be interpreted.
The extremely rich secondary metabolism of marine
invertebrates can be explained in the light of one of the key
issues in modern marine natural products research, namely
the individuation of the real producers of their metabolites.
The question arose with the evidence obtained indicating
that marine invertebrates harbour microorganisms, such as
bacteria, cyanobacteria and fungi, in their tissues, where they
reside in the extra- and intra-cellular spaces. In some cases,
associated micro organisms may constitute up to a 40% of
the biomass, as it has been evidenced for the Mediterranean
sponge Aplysina aerophoba [90-93]. The chemistry of
marine bacteria has received increasing attention over the
past ten years [94-97]. Principal reasons for this interest are
their abundance and phylogenetical diversity, as well as the
capacity of most of them to survive in extreme
environmental conditions due to the unusual enzymatic and
metabolic adaptations. Lately, microorganism-sponge
association has been particularly investigated.
Sponges are primitive metazoans that were probably the
starting point for the metazoan explosion during the
Precambrian. They share many functional features with
unicellular protozoa, such as nutrition, cellular organisation,
gas exchange, reproduction and response to the external
stimuli [98], but, on the other hand, sponges are also true
metazoans having some similarities with higher eukariyotes,
such as genes and proteins that are highly homologous to
vertebrate analogues [99]. Sponges possess amoeboid cells
that phagocytose bacteria and, at the same time, are efficient
filter feeders; as a result of such a continuous functional
activity, a certain amount of transient microorganisms (one
ml of seawater contains an estimated one million microbes)
are trapped within the vascular system or remain attached to
sponge surface. In A. aerophoba, for example, the bacterial
concentration exceeds that of the surrounding seawater by
two or three orders of magnitude.
The relations between sponge and microorganisms living
either permanently or temporarily inside have been currently
understood to a very limited extent. Recent studies based on
feeding experiments with different species of bacteria suggest
Current Medicinal Chemistry, 2004, Vol. 11, No. 13 1689
that the host sponge can differentiate between commensal
bacteria and those permanently associated with the host.
Interestingly, 16S rDNA diversity studies revealed that
sponges belonging to the same species but collected from
different seas and at different depth showed a significantly
uniform microbial community. According to Hentschel,
Author of interesting recent papers in this field, sponges can
be regarded as “microbiological fermenters” containing novel
sponge-specific and evolutionary ancient marine
microorganisms [100].
Several roles are believed to be played by the retained
microorganisms: they serve as food and enrich the diet of
their hosts through nitrogen and carbon fixation. In addition,
quite likely they are involved in the biosynthesis of natural
products recovered from the sponges [101]. Consequently,
the permanent bacterial presence is undoubtedly not
irrelevant for the exceptional chemodiversity of sponges, but
the real contribution of the microorganisms to secondary
metabolism of sponges has not yet been fully understood
and evaluated; this is essentially due to the failure of most
attempts to culturing permanently the sponge-associated
bacteria outside their host.
Sometimes, there are evidences that suggest the
involvement of associated microorganisms in the
biosynthesis of compounds collected from the sponge host.
In this respect, it is worthy of note that the convincing
evidence is present to support the involvement of
cyanobacteria in the biosynthesis of compounds present in
tropical sponge, Dysidea herbacea [102]. Features of the
metabolic content of a sponge could be indicative of
bacterial involvement in the biosynthesis of such
compounds; this happens when one organism is shown to
contain an unusual variety of classes of metabolites, when
the metabolite concentrations are exceedingly low, or when
the structures of the metabolites are reminiscent of bacterial
biogenetic pathways.
In our opinion, P. simplex can be considered a good
example of a sponge with a metabolic content to which
associated microorganisms could have appreciably
contributed. This assumption rests on some pieces of
evidence, such as the unusual structural variety of the
compounds elaborated by the sponge and the presence of
hopanoids, which are generally considered bacterial
metabolites. Actually, P. simplex is the first eukaryote
which was shown to contain them; moreover, one of the
isolated hopanoids has been hypothesised to be a key
intermediate within the accepted hopanoid biogenetic
pathway. If their non-spongal origin is true, the microbial
contribution to the secondary metabolism of P. simplex
should be significant on account of the relatively large
amounts of hopanoid compounds recovered from the sponge
extract.
Further support to the proposed cooperation of bacteria to
the chemodiversity of P. simplex is the presence of
plakohypaphorines, which are iodine-containing molecules
typical of the bacterial metabolism. Finally, biogenetic
hypotheses proposed for plakortin-like molecules envisage a
polyketide pathway with the participation of acyl moieties
generally involved in bacterial metabolisms.
At present, the origin of P. simplex compounds can be
the only object of speculation but we are firmly convinced
1690 Current Medicinal Chemistry, 2004, Vol. 11, No. 13
that the study of the biogenesis of marine invertebrate
metabolites is an intriguing topic, which must be carefully
considered for the future development of the marine natural
products chemistry. A major reason for their unique
molecular diversity likely resides in the marine
environmental conditions, which favour close and permanent
associations between different organisms.
The Problem of Supply
We discussed above the limited availability of most
bioactive compounds elaborated by marine invertebrates,
pointing out that currently it does not represent a serious
obstacle for their isolation and structure determination as
well as for preliminary biological tests. Problems actually
arise when preliminary positive assays refer the product for a
more advanced investigation. They can be easily overcome,
if the research continues using the molecule as a lead; in this
case, preliminary information on the structure-activity
relationships often can be obtained taking advantage from
the frequent co-occurrence of a series of closely related
compounds rather than a single example of a compound
type. The presence of a pool of analogues is probably a
chemical defence strategy, by which the organism is
protected against an array of organisms, since resistance
against a broad range of compounds with similar structures
is less likely to occur. The various compounds may display
synergism in their biological activity and this property may
be of interest for drug development.
The limited availability of a natural compound could
represent a serious obstacle to the research aimed to its direct
use in therapy, which develops according to the usual
procedure, involving first the assays in vivo, followed by
preclinical evaluations and then by clinical trials, where
gram quantities are needed.
The supply of a natural product becomes a large-sized,
very often insoluble, problem, when it is licensed, as drug
and an economically convenient synthesis cannot be carried
out. The industrial use of marine species requires large
amounts of raw material to be collected from natural stocks.
This collection will, in the long run, exert significant
impacts on the benthic community and will be a severe
pressure on the targeted species. Thus, the production of
sponges, tunicates and bryozoans in specially designed mari-
culture plants has been considered. Currently, a number of
multidisciplinary researches are targeting the development of
new technologies for mari-culture plants of marine
invertebrates for the environmentally compatible production
of pharmaceutical relevant species. Even if the obtained
yields of biomass are still far from those that should be
needed for commercial purposes, encouraging progress has
been made in this field and now the bryozoan B. neritina
and the tunicate E. turbinata, the sources of ET-743 and of
bryostatins, respectively, can be successfully cultured [103].
The possible microbial origin of bioactive molecules
recovered from marine invertebrates recently opened new and
interesting perspectives for their synthesis at commercial
level. Isolation and cultivation of the suspected microbial
producers either from the surrounding seawater or from
tissue of invertebrates could provide a more satisfying
answer to the pressing supply problem. If bacteria are
Fattorusso et al.
indeed, the producers of bioactive metabolites of interest,
transfer of the gene clusters responsible for the biosynthesis
of the respective natural products to a vector suitable for
large-scale fermentation could provide an alternative strategy
thereby avoiding the foreseeable difficulties in culturing
symbiotic bacteria.
CONCLUSIONS
Little of the ocean biodiversity has been explored for
pharmaceutical purposes and the continuing demand of new
drugs could be partly satisfied by the large reserves of active
molecules elaborated by marine organisms. Advances in
separation and analytical methods mean that active
compounds can be isolated and identified rapidly from
natural products extracts and remove many of the technical
barriers to using marine derived compounds in high
throughput screening campaigns. To hit this target, the
research needs to be highly interdisciplinary, not being
limited to a strictly chemical investigation. And it is to be
hoped that it could pass the limit of a simple academic
research. Unfortunately, research groups currently interested
in the chemistry of marine products are not very familiar not
only with the molecular trends in drug research, but also
with the issues implied in the drug licensing and marketing.
The chemistry of P. simplex, through which we decided
to illustrate the chemodiversity of bioactive compounds
from the sea, undoubtedly shows the role that marine
metabolites could play as leads for drug discovery. At the
same time, it represents an example of academic research,
which has been extended over several years on a single
organism, and carried out by a group of natural product
chemists episodically interacting with pharmacologists
working in the universities. Thus, it is also illustrative of
the limits of such kind of research, and in part it is also
explicative of the reasons for the modest number of marine
compounds currently in clinical trials. We are firmly
convinced that, within the new era of drug discovery and
development, the opportunities to exploit the molecular
diversity and biofunctionality of marine natural products are
immense, provided that this research will be
multidisciplinary, joining the forces of natural product
chemists, molecular biologists, microbiologists and
scientists of pharmaceutical industries.
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