Antitumor potential of natural products from Mediterranean ascidians

Abstract

Ascidians, invertebrates belonging to the subphylum Urochordata (Tunicata), are renowned for their great chemical diversity,
and during the last 25years, they have been shown to produce an array of cytotoxic molecules. Among the first six marine-derived
compounds that have reached clinical trials as antitumor agents, three are derived from ascidians, as evidence of the high
potential of these organisms as a new source of antitumor compounds. Reported in this communication are some recent results
on the chemistry of Mediterranean ascidians; a number of new molecules with different structural features but all endowed
with antiproliferative or cytotoxic activity are discussed. These results strongly evidence the highly significant role that
Mediterranean ascidians natural products could play in anticancer drug discovery and development process.

Full text

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Antitumor potential of natural products
from Mediterranean ascidians
Marialuisa Menna
Received: 30 December 2008/Accepted: 27 March 2009/Published online: 22 April 2009
? Springer Science+Business Media B.V. 2009
Abstract
subphylum Urochordata (Tunicata), are renowned for
their great chemical diversity, and during the last
25 years, they have been shown to produce an array
of cytotoxic molecules. Among the first six marine-
derived compounds that have reached clinical trials
as antitumor agents, three are derived from ascidians,
as evidence of the high potential of these organisms as
a new source of antitumor compounds. Reported in
this communication are some recent results on the
chemistry of Mediterranean ascidians; a number of
new molecules with different structural features but
all endowed with antiproliferative or cytotoxic acti-
vity are discussed. These results strongly evidence
the highly significant role that Mediterranean ascidi-
ans natural products could play in anticancer drug
discovery and development process.
Ascidians, invertebrates belonging to the
Keywords
Tunicates ? Ascidians ? Cytotoxicity ?
Anticancer activity
Marine natural products ?
Introduction
Approximately one-third of today’s best selling drugs
are either natural products or have been developed
based on lead structures provided by nature. How-
ever, it is surprising that up to now almost all
medicinally used natural products or derivatives
thereof were obtained from terrestrial organisms
rather than from those inhabiting the sea, considering
that the oceans cover more than 70% of the earth’
surface. With regard to drug discovery and develop-
ment, natural products marine sources started to
attract interest from pharmaceutical companies and
research institutions only 40 years ago, with the
advent of high performance liquid chromatography
(HPLC), and the new NMR and mass techniques.
Even if the systematic study has involved only a
small percentage of the organisms of marine habitat,
since then several thousand of new compounds have
been isolated so far, many of them being structurally
unique and absent in terrestrial organisms (Blunt
et al. 2008). As with plants, researchers have
recognized the potential use of this chemical arsenal
to kill bacteria or cancer cells.
Incidence of biological activity in marine-derived
compounds is high, especially with regard to cytoto-
xicity where marine-derived extracts surpass those of
terrestrial origin. It is no surprise, therefore, that
marine natural products have their stronghold in
the area of anticancer chemotherapy, as indicated
by the list of compounds currently under clinical
M. Menna (&)
Dipartimento di Chimica delle Sostanze Naturali,
Universita ` degli Studi di Napoli ‘‘Federico II’’,
Via D. Montesano 49, 80131 Napoli, Italy
e-mail: marialuisa.menna@unina.it
123
Phytochem Rev (2009) 8:461–472
DOI 10.1007/s11101-009-9131-y

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investigation(Amadoretal.2003;NewmanandCragg
2004a). The first marine-derived cancer drugs can be
considered the unusual nucleosides spongothymidine
and spongouridine discovered by Bergman and his co-
workers in 1950’s from the Caribbean sea Cryptotet-
hya crypta, (Bergmann and Feeney 1951) which
served as lead structures for the development of
nowadays commercially important anticancer drug for
leukemia, ara-C, currently sold under the brand name
Cytosar-URand which presently remains the only
marine-derived anticancer agent in continuousclinical
use. In recent years, researchers have discovered
dozens of ocean-derived chemicals that appear to be
powerful cancer cell killers and, thus, potential
anticancer new drugs. Majority of bioactive molecules
from the sea has been isolated from invertebrates
(Blunt et al. 2008); the wealth of their secondary
metabolites is related to the role of chemical defense
played by these constituents and this 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 defense structures such as shells or
spines. Among marine invertebrates, tunicates have
received more attention. More commonly known as
sea squirts, tunicates are a group of marine organisms
that spend most of their lives attached to docks, rocks,
or the undersides of boats; they belong to the phylum
Chordata, which encompasses all vertebrate animals,
including mammals. Therefore, they represent the
most highly evolved group of the animals commonly
investigated by marine natural products chemists and
are evolutionarily more closely related to vertebrates
like ourselves than to most other invertebrate animals.
Members of the class Ascidiacea (Ascidians) are the
mostly investigated tunicates, since they present a
benthonic stage in their life, making their collection
easier. The chemistry of ascidians has become one of
the mostactive fields ofmarine natural products;it has
been amply demonstrated that these sea creatures are
prolific producers of unusual structures with signifi-
cant bioactivities. Most of these products fall within
the area of cancer therapy (Rinehart 2000) and a
significant number of ascidian-derived compounds
have entered into preclinical (Fig. 1) and clinical trials
as antitumor agents (Newman and Cragg 2004a, b).
Surprisingly, the chemistry of ascidians species of the
Mediterranean sea has been until now subjected to a
very limited number of investigations and this appears
to be in striking contrast to the extensive chemical
analysis carried out in the last 30 years of other
Mediterranean invertebrates, like Porifera and Echi-
noderms. The focus of this review is to highlight the
highly significant role that ascidians metabolites could
play in anticancer drug discovery and development
process, with particular regard to the still not quite
explored potential of Mediterranean species. Some of
the recent results obtained within a research, aimed to
unearth the chemistry of marine ascidians coming
from the Mediterranean area and to discover new
chemical entities with useful biological activities, are
here reported. They are preceded by a brief outline
about the progress in the development and acquisition
ofanticancer agentsfrommarineascidians,notonlyto
underline the scientific background of this research
activity but also in order to give an idea of the rough
road that a natural compound must do before beco-
ming a promising drug. A number of new bioactive
molecules, all endowed with antiproliferative or
cytotoxic activity (see Table 1), will be discussed,
drivinghomealsothehugechemicaldiversitywhichis
characteristic of ascidians natural products.
Significant progress in the development
and acquisition of anticancer agents
from marine ascidians
Didemnin B (1), the first marine natural product
which entered anticancer clinical trials, was first
isolated from the Caribbean tunicate Trididemnum
solidum in 1981 (Rinehart et al. 1981). Early
investigation into the bioactivity of this compound
revealed its strong antiproliferative effects in vitro
against a variety of human tumor cell lines. Thus, it
proceeded through Phase I and entered into Phase II
clinical trials but, although it possessed promising
antitumor, antiviral and immunosuppressive activity,
it showed high toxicity, poor solubility, and a short
bioactivelifespanand
(Newman and Cragg 2004a). Although didemnin B
was never carried into Phase III trials, activity
focused on developing the compound as a potential
cancer treatment helped pave the way for the rest of
the marine-derived products following it into the
development pipeline. Aplidine (dehydrodidemnin B,
2), a second-generation didemnin isolated from
Aplidum albicans, a different tunicate species, is
trialswereterminated
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currently in clinical trials for a variety of cancers. In
preclinical animal tests, Aplidine exhibited marked
anticancer properties, outperforming the related com-
pound didemnin B by a factor of six (Urdiales et al.
1996); the molecule has been described as a multi-
factor apoptosis inducer, and it has low toxicity and a
high specificity for tumor cells. The varied inferred
mechanisms of action thus far reported include rapid
and persistent activation of apoptosis and interruption
of the tumor cell cycle at the G1–G2. The compound
also inhibits the activity of key enzymes, including
elongation factor 1-alpha, ornithine decarboxylase,
and palmitoyl protein thioesterase. In addition, it was
shown to inhibit flt-1 gene expression encoding
vascular
involved in growth and vascularization of human
leukemia cells (Faivre et al. 2005). Synthetically
derived Aplidine, under the PharmaMar trade name
Aplidin?, is currently in Phase II trials for a variety of
cancers, including multiple myeloma, renal cancer
and aggressive lymphomas (http://www.pharmamar.
com/aplidin.aspx).
Vitilevuamide (3) is a bioactive cyclic peptide
which has been isolated from the ascidians Didem-
num cuculiferum and Polysyncraton lithostrotum
(Ireland and Fernandez 1998); it is one of several
novel tubulin interactive agents recently discovered
from marine invertebrates (Edler et al. 2002).
endothelialgrowthfactor receptor-1
N
CH3
O
O
CH3
O
NH
OCH3
O
N
O
NH
O
O
O
O
H3C
N
H
O
N
CH3
O
N
O
HO
OH
N
N
OH
OCH3
OCOCH3
OH
O
OS
O
NH
O
H3CO
HO
N
CH3
O
O
CH3
O
NH
OCH3
O
N
O
NH
O
O
O
O
H3C
N
H
O
N
CH3
O
N
O
O
OH
HO
H
N
O
HN
O
O
N
H
NH
Cl
O
N
O
N
Cl
HOOC
H
N
O
O
NH
O
HN
O
H
N
O
O
NH
N
H
OO
OH
HN
O
N
OH
OCH3
O
N
H
O
H
N
O
N
H
O
NH
O
S
NH
O
N
O
1
2
3
4
5
Fig. 1 Ascidians-derived
compounds currently in
clinical or advanced
preclinical trials
Phytochem Rev (2009) 8:461–472463
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Research on the mechanism of action of this two-
ringed marine peptide reveal that vitilevuamide
inhibit tubulin polymerization and can arrest the cell
cycle of target cells in the G2/M phase. The molecule
exhibits activity in vivo against the P388 lymphocytic
leukemia line. An intriguing finding is that tubulin
binding and inhibition by this compound occurs at a
site on the tubulin molecule distinct from the
interaction sites of dolastatin 10, colchicine, and the
vinca alkaloids. Vitilevuamide is currently in the pre-
clinical evaluation phase as a potential anticancer
agent (Kijjoa and Sawangwong 2004; Newman and
Cragg 2004a).
Diazonamide (4) is a further tubulin interactive
agent, isolated by Fenical’s group from the ascidian
Diazona angulata (Lindquist et al. 1991); this
compound is currently subjected to anticancer pre-
clinical studies. In preliminary bioactivity screens,
this compound killed lab-cultured colon cancer cells
but, unfortunately, a more detailed characterization
and biomedical evaluation was hindered for a long
period due to supply problems. The structural com-
plexity of the molecule made laboratory synthesis
difficult, but funding from organizations like the
American Cancer Society and the efforts of several
organic chemistry groups working towards the goal
Table 1 Effects of different compounds isolated from Mediterranean ascidians on cell viability
CompoundModel Biological effect Reference
6
IGR-1 (human melanoma) AntiproliferativeAiello et al. (2001b)
J774 (murine monocyte/macrophage)
WEHI 164 (murine fibrosarcoma)
P388 (murine leukemia)
7–9, 15
WEHI 164 (murine fibrosarcoma)Antiproliferative Aiello et al. (2001b)
10, 11
WEHI 164 (murine fibrosarcoma)CytotoxicAiello et al. (2000)
C6 (rat glioma)
12
WEHI 164 (murine fibrosarcoma)CytotoxicAiello et al. (2000)
13
WEHI 164 (murine fibrosarcoma)AntiproliferativeAiello et al. (1997a, b)
J774-murine monocyte/macrophage
P388 (murine leukemia)
GM-7373 (bovine endothelial)
14
WEHI 164 (murine fibrosarcoma)Antiproliferative Aiello et al. (1997a, b)
J774-murine monocyte/macrophage
P388 (murine leukemia)
IGR-1 (human melanoma)
16
C6 (rat glioma) Cytotoxic, cell membrane
lipoperoxidation inducer,
Pro-apoptotic
Aiello et al. (2001a)
SH-SY5Y (human neuroblastoma)
17, 18
C6 (rat glioma)CytotoxicAiello et al. (2003)
RBL-2H3 (rat basophilic leukemia)
19, 20
Jurkat (human T lymphoma) Cytotoxic, Pro-apoptotic,
influence on intracellular
ROS level
Aiello et al. (2005a)
25, 26
AGS (gastric carcinoma) Cytotoxic, pro-apoptoticAiello et al. (2007)
T47D (breast carcinoma)
A549 (lung carcinoma)
Jurkat (human T lymphoma)
32a
AGS (gastric carcinoma) CytotoxicAiello et al. (2009)
36a
AGS (gastric carcinoma)Cytotoxic Aiello et al. (2009)
A549 (lung carcinoma)
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ultimately proved fruitful. In the process, synthetic
chemists discovered that the original structure
reported for the natural product was incorrect; an
analog was also produced also possessing potent
microtubulin interactive activity (http://www.marine
biotech.org/diazonamidea.html). Diazonamide A is
an inhibitor of microtubule assembly, arresting the
process of cell division in cultures exposed to treat-
ment. Examination of treated cells reveals a loss of
spindle microtubule assemblies and also microtubules
associated with the interphase stage of the cell cycle
(Cruz-Monserrate et al. 2003).
One tunicate living in the crystal waters of West
Indies coral reefs and mangrove swamps, Ecteina-
scidia turbinata, turned out to be the source of an
interesting anticancer drug called Ecteinascidin 743
(ET-743; Etrabectedin, 5) first isolated in 1990
(Rinehart et al. 1990; Wright et al. 1990). Preclinical
trials showed ET-743 was active against a range of
tumor types in standard animal models. Subsequent
human trials showed efficacy against advanced soft
tissue sarcoma, osteosarcoma and metastatic breast
cancers. Research into the mode of action of ET-743
has revealed that binding of the drug to target cell
DNA inhibits cell division and leads to apoptosis of
cancer cells. Setting the drug apart from others that
trigger ‘cell suicide’ mechanisms is the fact that
ET-743 induces apoptosis only during active gene
transcription. This makes actively dividing cancer
cells more vulnerable to drug toxicity than normal
cells because they exhibit greatly accelerated tran-
scription and translation rates. Ecteinascidin 743 also
may hold promise in keeping tumors from becoming
resistant to chemotherapy; it interferes with the gene
that produces P-glycoprotein, a membrane protein
that confers drug resistance on cancer cells by
actively transporting toxic compounds (like drug
therapies) out of the cells. Such activity suggests that
ET-743 may become important as a key ingredient in
multi-drug ‘cocktails’ if it can prevent target cells
from developing resistance to the other drug therapies
(Manzanares et al. 2001; Bonetta 2001; Takebayashi
et al. 2001; Kijjoa and Sawangwong 2004). ET-743
has been co-developed under the trade name Yond-
elis?by the Spanish marine pharmaceutical com-
pany. The drug is well along in Phase II clinical trials
in the United States and four other countries; the
company reports that Yondelis is the first new drug in
20 years that is effective against soft tissue sarcoma
(STS) a rare but pernicious form of cancer (http://
www.pharmamar.com/yondelis.aspx).
Sulfated alkanes and alkenes from Mediterranean
ascidians
Sulfated metabolites, although relatively unusual,
occur in marine organisms. The sulfate group is a
common feature of echinoderm metabolites, like
sulfated saponins with triterpenoid or steroidal agly-
cones (Riccio et al. 1987) and has also been
encountered in sponges metabolites, in the form of
sulfated sterols and phenol sulfates (Aiello et al.
1999). Only a few examples of sulfated alkanes/
alkenes have been reported from marine sources
(Findlay et al. 1990, 1991; Nakao et al. 1993;
Roccatagliata et al. 1997); although the isolation of
some alkanes/alkenes sulfate esters from two Japa-
nese ascidians has been reported (Tsukamoto et al.
1994; Fujita et al. 2002), recently they have been
shown to be commonly present in remarkable
amounts in Mediterranean tunicates. Most of these
compounds, often having quite simple structures, are
cytotoxic or antimicrobial. Solitary ascidians of the
family Ascidiidae and Pyuridae (Ascidia mentula,
Microcosmus vulgaris, Halocynthia papillosa) as
well as colonial Polyclinidae species (Sidnyum tur-
binatum) have been the sources of compounds 6–15
(Crispino et al. 1994; Aiello et al. 1997a, b; De Rosa
et al. 1997; Aiello et al. 2000, 2001b). Compounds
6–11 are of polyketide derivation, but carbon ske-
letons of compounds 12–15 are clearly referable to
terpenoid structures; all compounds were endowed
with cytotoxic and/or antiproliferative activity. More
complex polysubstituted alkyl sulfates have also been
isolated from ascidians (Lievens and Molinski 2005).
An intriguing metabolite, turbinamide (16), has been
isolated from S. turbinatum, a rather uncommon
colonial ascidian collected in the Bay of Naples
(Aiello et al. 2001a). Turbinamide showed a strong
and selective cytotoxic effect against neuronal rather
than immune system cells; C6 rat glioma cells were
shown to be very sensible to this metabolite. This
result was of particular interest, since gliomas are
also considered among the most malignant forms of
cancer, whose therapeutic treatment is up to now only
palliative for. Studies on the cellular effects of
turbinamide have been performed and it has been
Phytochem Rev (2009) 8:461–472465
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demonstrated that turbinamide-induced cytotoxicity
in C6 cells is due to apoptosis. The activity of
caspase-3 is enhanced in a concentration-dependent
manner by this compound, having no effect on J774
cells (Esposito et al. 2002). Thus, turbinamide may
represent a promising novelty in the therapy of
gliomas avoiding the toxic side-effects such as
immunosuppression, usually observed with conven-
tional antiblastic drugs (Fig. 2).
Pro-apoptotic meroterpenes from marine
ascidians belonging to Aplidium genus
Quinones represent a clinically important category of
chemotherapeutic agents with a wide range of appli-
cations in both antitumor and antimicrobial therapy
(Smith 1985). A large number of quinones, both
synthetic and naturally occurring, have been screened
for their antitumor activity; the antitumor activity is
OSO3-Na+
OSO3-Na+
OSO3-Na+
OSO3-Na+
OSO3-Na+
OSO3-Na+
OSO3-Na+
OSO3-Na+
OSO3-Na+
OH
OH
OH
OH OSO3-Na+
OH
H2N
O
OH
OH
OSO3-Na+
OSO3-Na+
6
7
8
9
1011
12
13
14
16
OSO3-Na+
OSO3-Na+
15
Fig. 2 Sulfated alkanes/alkenes from Mediterranean ascidians. Sources: A. mentula (6, 14); M. sulcatus (13); H. papillosa (10–12);
P. adriaticus (12); A. conicum (12); P. mammillata (12); P. fumigata (12); A. virginea (12); S. turbinatum (7–9, 14–16)
466 Phytochem Rev (2009) 8:461–472
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exhibited predominantly by three main groups of
naturally occurring quinones such as benzoquinones,
naphtoquinones and anthraquinones. Many natural
products with structures featuring both terpene and
quinone/hydroquinone moieties have been reported
from natural source. Representative compounds based
on the farnesyl quinone/hydroquinone skeleton within
marine environment include avarone and avarol from
the marine sponge Dysidea avara (Minale et al. 1974)
and cyclozonarone from the marine brown alga
Dictyopteris undulata (Kurata et al. 1996). Above
all, marine ascidians of the order Aplousobranchiata,
family Polyclinidae have been reported to synthesise
prenyl quinones and hydroquinones, either linear or
cyclic; these metabolites of mixed biogenesis are
generally known as meroterpenes and originated from
intra- and inter-molecular cyclizations and/or rear-
rangements, thus giving macrocyclic or policyclic
skeletons, often linked to amino acids or taurine
residues (Zubı `a et al. 2005).
Indeed, the first biologically active tunicate meta-
bolite, geranylhydroquinone, was isolated from an
Aplidiumsp.andwasshowntoofferprotectionagainst
leukemia and tumor development in test animals
(Fenical 1976). Further examples include prenylated
quinones from Aplidium sp. (Fig. 3), A. californicum,
O
N
H
S
O
O
OH
OO
O
N
H
S
O
O
OH
OO
S
H
N
O
O
OO
S
H
N
O
OO
O
H2N
S
H
N
O
O
OO
HN
SO3
S
H
N
O
O
OO
H3CO
N
H
S
O
O
OO
17
18
19
20
21
22
23
Fig. 3 Prenyl quinones from A. conicum (Sardinia)
Phytochem Rev (2009) 8:461–472 467
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A. costellatum, and A. antillense, dimeric prenylated
quinones from A. longithorax, and chromenols from
A. solidum and Amaroucium multiplicatum (Zubı `a
et al. 2005). The Mediterranean ascidian A. conicum
has been extensively studied. The sulfated normono-
terpene 12 was initially reported as the major consti-
tuent of this ascidian (De Rosa et al. 1997); later, a
specimen of A. conicum from Tarifa Island afforded
four meroterpenoids but their pharmacological pro-
perties have not been evaluated (Garrido et al. 2002).
An extensive study of another sample of the same
ascidian, collected along Sardinia coasts, gave rise to
the isolation of a large group of unique new mero-
terpenes, compounds 17–23, whose quite different
structures have in common the presence of an unusual
dioxo-thiazine ring and for their interesting cytotoxic
and pro-apoptotic properties in vitro (Aiello et al.
2003, 2005a, b).
Based on the reported anticancer and cancer-
protective properties of prenylated quinones, coni-
caquinones A and B (17 and 18) were tested in vitro
against two different cultured cell lines; cytotoxicity
was evaluated on rat glioma (C6) and rat basophilic
leukemia (RBL-2H3) cell lines. Both showed a
marked and selective effect on rat glioma cells
(Aiello et al. 2003).
Thiaplidiaquinones A and B (19 and 20), posses-
sing an unprecedented tetracyclic structure (Aiello
et al. 2005a), were strongly cytotoxic against Jurkat
cell line, derived from a human T lymphoma, with a
IC50around 3 lM, which is in the range of other
antitumor quinones such as doxorubicin (de Tinguy-
Moreaud et al. 1994). It has also been demonstrated
that they induce cell death by apoptosis and that the
pro-apoptotic mechanism involve the induction of a
strong production of intracellular reactive oxygen
species (ROS), which mediate the collapse of
the mitochondrial transmembrane potential (DWm).
The increase of ROS generation is likely due to the
inhibition of the plasma membrane NADH-oxidore-
ductase (PMOR) system, through interference with
the coenzyme-Q binding site leading to the redirec-
tion of normal electron flow in the complex and
generating an excess of intracellular ROS (Ly and
Lawen 2003; Lawen et al. 1994). The PMOR may be
an important target for anticancer drugs, since its up-
regulation could be a mechanism by which cancer
cells can tolerate the high levels of ROS formed due
to their increased metabolic rate. Moreover, cancer
cells also have a low superoxide dismutase activity
and are thus especially sensitive to drugs that
interfere with the regulation of intracellular ROS
(Inbaraj et al. 1999).
Tests on leukemia Jurkat cells showed that apli-
dinone C (21) is completely inactive while aplidinone
A (22) is moderately cytotoxic and, like thiaplidiaqui-
nones, induces apoptosis by overproduction of intra-
cellular ROS; it was impossible to test aplidinone B
(23) due to supply problems. Actually, spectroscopic
data obtained for aplidinones did not allow to assign
the regiochemistry (sulfur and nitrogen position) of
heterocyclic ring, which was determined for Aplidi-
none A by comparison of experimental
chemical shifts with those predicted by quantum
mechanical calculations (Aiello et al. 2005b). In order
to validate the structural assignment made for apli-
dinones by theoretical means a synthetic approach has
beenundertakenwhichyieldedsomesyntheticanalogs
of Aplidinone A in which the geranyl chain isreplaced
by other alkyl chains. The spectroscopic analysis
carried out on synthetic compounds confirmed the
proposed regiochemistry for aplidinone. Moreover,
the availability of these compounds allowed simple
structure-activity relationships study (SARs) which
evidencedthatcytotoxicactivitydependsonthenature
andthelengthofsidechainlinkedtothebenzoquinone
ring and, mainly, on its position respect to the
dioxothiazine ring. Pharmacological studies per-
formed on the synthetic analogs of Aplidinone A
evidenced that some of them are endowed with potent
cytotoxic and pro-apoptotic activities in several tumor
cell lines and also inhibit TNFa-induced NF-jB
activation (Menna et al. unpublished results).
13C NMR
Long-chain amino alcohols
In 1999, researchers of PharmaMar reported at the
AACR-NCI-EORTC International Conference on
Molecular Targets and Cancer Therapeutics on the
initial studies with spisulosine (24), a molecule
isolated from the marine clam Spisula polynyma
(Jimeno et al. 1999). This initial report was followed
by paper of Cuadros et al. (2000) reporting that this
compound causes a loss of actin stress fibers,
probably due to its resemblance to lysophosphatidic
acid (LPA) and hence an interaction with the LPA
receptor, which is known to modulate the levels of
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the Rho proteins. The compound demonstrated a
wide in vitro therapeutic index when tumor cells were
compared to normal cell lines, with a 50–100 fold
difference in IC50values and appears to interact with
the endothelial cell differentiation gene (EDG)
receptors. Spisulosine is currently in Phase I trials
against solid tumors in Europe (Sa ´nchez et al. 2008).
Several spisulosine-related amino alcohols have
been isolated from the Mediterranean ascidia Clave-
lina phlegraea (Clavaminols A-N, compounds 25–36,
Fig. 4) with cytotoxic and pro-apoptotic activities
(Aiello et al. 2007; Aiello et al. 2009). The effects of
clavaminolsA-Nontheviabilityofdifferentcelllines,
T47D (breast cancer), A549 (lung carcinoma), and
NH2
OH
NH2
OH
NH2
OCOCH3
NHCOCH3
OH
NHCOCH3
OH
NHCOCH3
OH
25 26
27
28
29
30
7
24
NH2
OH
31 R=COCH3
31a R=H
36 R=COCH3
36a R=H
32 R1=COCH3; R2=H
32a R1=R2=H
33
35
34
NH-R
OH
NH-R1
O-R2
O-R2
NHCOCH3
OCOCH3
NHCHO
OCOCH3
NHCOCH3
OH
NH-R
OH
Fig. 4 Spisulosine (24) from the marine clam S. polynyma and clavaminols A–F (25–30) from the Mediterranean ascidia
C. phlaegraea
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AGS (gastric carcinoma), were studied using the
sensitive calcein–AM assay; the hydrolysis products
of clavaminols G, H, and N (compounds 31a, 32a, and
36a) were also tested. Pharmacological profiles of
these compounds allowed to draw some consideration
on the structural features required for the cytotoxic
activity and to define SAR. Clavaminol A (25) is the
most potent cytotoxic compound of the series, with an
IC50close to 5 lg/ml for AGS cells, however, it was
less active than the related spisulosine possessing the
opposite configuration at carbons 2 and 3 (see Fig. 4).
The cytotoxic activity of clavaminol A was not
restricted to the gastric tumoral cell line and similar
antitumoralactivitywasfoundagainstA549andT47D
cell lines. The effect of clavaminol A on cellular
apoptosiswasinvestigatedinleukemiaJurkatcellline.
This study revealed that the molecule induces a strong
increase in the percentage of hyplodiploid cells, thus
indicating that it causes cell death through activation
of the apoptotic process.Moreover, analysisof the cell
cycle indicated that most of DNA fragmentation, the
hallmark fortheapoptoticpathway,occursinthe G1/S
phase (Aiello et al. 2007). Remarkably, clavaminol B
(26) was less potent than clavaminol A, indicating that
additional unsaturation was detrimental for the activ-
ity. Compounds with acetylated amino or hydroxyl
groups (27–36) actually resulted inactive. This sug-
gested that the presence of free hydroxyl and amino
groups is critical for the cytotoxic activity, which on
the other hand is not affected by branching of the alkyl
chain since compound 36a exerts a cytotoxic effect
comparable to that of clavaminol A in both cell lines.
Interestingly, compound 32a, which is related to
clavaminol A by the presence of an extra hydroxyl
group, showed significant cytotoxic activity in AGS
cells,eveniflowerthanthatofclavaminolAanditwas
inactive against lung cancer cell line A549. This
indicated that additional hydroxylation admittedly
decreases the activity but may confer cell selectivity
to clavaminols.
Conclusions
The continuing and overwhelming contribution of
ascidians metabolites to the expansion of the chemo-
therapeutic armamentarium is clearly evident and
much remains to be explored. Still, cancer is the main
cause of death in developed countries besides
cardiovascular pathologies and much attention is
devoted to research programs aimed to the discovery
of new anticancer drugs. Although many approaches
can be used in the discovery and development of new
anticancer drugs, one of the main strategies is still the
use of natural products as leads for the production of
new drugs. Discovery of natural bioactive molecules
is of course only the first step. The hardest challenge
is turning these products, which are often found in
very small quantities, into useful medicines. In
addition, isolation and chemical characterization is
often a laborious and, therefore, time-consuming
task. By definition, there is a biological source
involved in the production of any natural product,
and this poses also the problem of the environmental
impact. The role of natural products was improved by
the recent findings in the genomic field; there is even
suspicion that some of the biologically active com-
pounds isolated from the larger organisms may be
products of symbiotic microorganisms. The increa-
sing genomic information and progress in molecular
biology in general, combined with biochemical
studies leading to a comprehensive understanding of
complex natural product biosynthesis at the molec-
ular level, as well as technologies exploiting this
knowledge are expected to give natural products
research a promising future.
References
Aiello A, Fattorusso E, Menna M, Carnucccio R, D’Acquisto F
(1997a) Novel antiproliferative alkyl sulfates from the
Mediterranean tunicate Ascidia mentula. Tetrahedron 53:
5877–5882
Aiello A, Fattorusso E, Menna M, Carnucccio R, Iuvone T
(1997b) A new antiproliferative sulfated alkene from the
Mediterranean tunicate Microcosmus vulgaris. Tetrahe-
dron 53:11489–11492
Aiello A, Fattorusso E, Menna M (1999) Steroids from spon-
ges: recent reports. Steroids 64:687–714
Aiello A, Carbonelli S, Esposito G, Fattorusso E, Iuvone T,
Menna M (2000) Novel bioactive sulfated alkene and
alkanes from the Mediterranean ascidian Halocynthia
papillosa. J Nat Prod 63:1590–1592
Aiello A, Carbonelli S, Esposito G, Fattorusso E, Iuvone T,
Menna M (2001a) Turbinamide, a new selective cytotoxic
agent from the Mediterranean tunicate Sidnyum turbina-
tum. Org Lett 3(19):2941–2944
Aiello A, Carbonelli S, Fattorusso E, Iuvone T, Menna M
(2001b) New bioactive sulfated metabolites from the
Mediterranean tunicate Sidnyum turbinatum. J Nat Prod
64:219–221
470Phytochem Rev (2009) 8:461–472
123

Page 11

AielloA,FattorussoE,LucianoP,MennaM,EspositoG,Iuvone
T, Pala D (2003) Conicaquinones A and B, two novel
cytotoxic terpene quinones from the Mediterranean ascid-
ian Aplidium conicum. Eur J Org Chem 2003(5):898–900
AielloA,FattorussoE,LucianoP,MachoA,MennaM,Mun ˜ozE
(2005a) Antitumor effects of two novel naturally occurring
terpene quinones isolated from the Mediterranean ascidian
Aplidium conicum. J Med Lett 48:3410–3416
Aiello A, Fattorusso E, Luciano P, Mangoni A, Menna M
(2005b) Isolation and structure determination of Aplidi-
nones A-C from the Mediterranean ascidian Aplidium
conicum: a successful regiochemistry assignment by
quantum mechanical
tions. Eur J Org Chem 2005(23):5024–5030
Aiello A, Fattorusso E, Giordano A, Menna M, Navarrete C,
Mun ˜oz E (2007) Clavaminols A–F, novel cytotoxic
2-amino-3-alkanols from the ascidian Clavelina phleg-
raea. Bioorg Med Chem 15(8):2920–2926
Aiello A, Fattorusso E, Giordano A, Menna M, Navarrete C,
Mun ˜oz E (2009) Clavaminols G-N, six new marine
sphingoids from the Mediterranean ascidian Clavelina
phlegraea. Tetrahedron. doi:10.1016/j.tet.2009.03.056
Amador ML, Jimeno J, Paz-Ares L, Cortes-Funes H, Hidalgo
M (2003) Progress in the development and acquisition of
anticancer agents from marine sources. Ann Oncol
14:1607–1615
Bergmann W, Feeney R (1951) Contribution to the study of
marine products. XXXII. The nucleosides of sponges.
J Org Chem 16:981–987
Blunt JW, Copp BR, Hu W et al (2008) Marine natural pro-
ducts. Nat Prod Rep 25:35–94
Bonetta L (2001) Anticancer squirt. Nat Med 7:891
Crispino A, De Giulio A, De Rosa S, De Stefano S, Milone A,
Zavodnik N (1994) A sulfated normonoterpenoid from the
Ascidian Polycitor adriaticus. J Nat Prod 57:1575–1577
Cruz-Monserrate Z, Vervoort HC, Bai R, Newman DJ, Howell
SB, Los G, Mullaney JT, Williams MD, Pettit GR, Fenical
W, Hamel E (2003) Diazonamide A and a synthetic
structural analog: disruptive effects on mitosis and cellu-
lar microtubules and analysis of their interactions with
tubulin. Mol Pharmacol 63:1273–1280
Cuadros R, Montejo de Garcini E, Wandosell F, Faircloth G,
Fernandez-Sousa J, Avila J (2000) The marine compound
spisulosine, an inhibitor of cell proliferation promotes the
disassembly of actin stress fibers. Cancer Lett 152:23–29
De Rosa S, Milone A, Crispino A, Jaklin A, De Giulio A (1997)
Absolute configuration of2,6-dimethylheptylsulfate and its
distribution in Ascidiacea. J Nat Prod 60:462–463
de Tinguy-Moreaud E, Pourquier P, Montaudon D, Robert J
(1994) Relationships between DNA damage and growth
inhibition induced by topoisomerase II-interfering drugs
in doxorubicin-sensitive and -resistant rat glioblastoma
cells. Anticancer Res 14:99–103
Edler MC, Fernandez AM, Lassota P, Ireland CM, Barrows LR
(2002) Inhibition of tubulin polymerization by vitilevua-
mide, a bicyclic marine peptide, at a site distinct from
colchicine, the vinca alkaloids, and dolastatin 10. Bio-
chem Pharm 63(4):707–715
Esposito G, Aiello A, Carbonelli S, Menna M, Fattorusso E,
Iuvone T (2002) Mechanism of cytotoxicity of turbina-
mide in vitro. Anticancer Res 22:2827–2832
13C NMR chemical shift calcula-
Faivre S, Chie `ze S, Delbaldo C, Ady-vago N, Guzman C,
Lopez-Lazaro L, Lozahic S, Jimeno J, Pico F, Armand JP,
Lopez Martin JA, Raymond E (2005) Phase I and phar-
macokinetic study of Aplidine, a new marine cyclodep-
sipeptide in patients with advanced malignancies. J Clin
Oncol 23:7871–7880
Fenical W (1976) Geranyl hydroquinone, a cancer-protective
agent from the tunicate Aplidium species. Food-Drugs
Sea Proc 4:388–394
Findlay JA, He Z, Calhoun LA (1990) Forbesin: a novel sul-
fated glycolipid from the starfish Asterias forbesi. J Nat
Prod 53:1015–1018
Findlay JA, Yayli N, Calhoun LA (1991) Novel sulfated
hydrocarbons from the sea cucumber Cucumaria frond-
osa. J Nat Prod 54:302–304
Fujita M, Nakao Y, Matsunaga S, Nishikawa T, Fusetani N
(2002) Sodium 1-(12-hydroxy) octadecanyl sulfate, an
MMP2 inhibitor, isolated from a tunicate of the family
Polyclinidae. J Nat Prod 65:1936–1938
Garrido L, Zubia E, Ortega MJ, Salva ` J (2002) New mero-
terpenoids from the ascidian Aplidium conicum. J Nat
Prod 65:1328–1331
Inbaraj JJ, Gandhisan R, Murugesan R (1999) Cytotoxicity and
superoxide anion generation by some naturally occurring
quinones. Free Radic Biol Med 26:1072–1078
Ireland CM, Fernandez A (1998) Cyclic peptide antitumor
agent from an ascidian. PCT Int. Appl. 02.04.1998
Jimeno J, Garcı ´a-Gra ´valos D, A´vila J, Smith B, Grant W,
Faircloth GT (1999) ES-285, a marine natural product
with activity against solid tumors. AACR-NCI-EORTC
international conference on molecular targets and cancer
therapeutics. Washington DC, 16–19 Nov 1999, Abstract
no. 314
Kijjoa A, Sawangwong P (2004) Drugs and cosmetics from the
sea. Mar Drugs 2004:73–82
Kurata K, Taniguchi K, Suzuki M (1996) Cyclozonarone, a
sesquiterpene-substituted benzoquinone derivative from
the brown alga Dyctiopteris undulate. Phytochemistry
47:749–752
Lawen A, Martinus RD, Mcmullen GL, Nagley P, Vaillant F,
Wolvetang EJ, Linnane AW (1994) The universality of
bioenergetic disease: the role of mitochondrial mutation
and the putative inter-relationship between mitochondria
and plasma membrane NADH oxidoreductase. Mol
Aspects Med 15(Suppl):13–27
Lievens SC, Molinski TF (2005) Sagittamides A and B.
Polyacetoxy long-chain acyl amino acids from a Didem-
nid Ascidian. Org Lett 7(11):2281–2284
LindquistN,FenicalW,VanDuyneGD,ClardyJ(1991)Isolation
and structure determination of diazonamides A and B, unu-
sualcytotoxicmetabolitesfromthemarineascidianDiazona
chinensis. J Am Chem Soc 113(6):2303–2304
Ly JD, Lawen A (2003) Transplasma membrane electron
transport: enzymes involved and biological function.
Redox Rep 8:3–21
Manzanares I, Cuevas C, Garcia-Nieto R, Marco E, Gago F
(2001) Advances in the chemistry and pharmacology of
ecteinascidins, a promising new class of anticancer agents.
Curr Med Chem-Anti-Cancer Agents 1:257–276
Minale L, Riccio R, Sodano G (1974) Avarol, a novel ses-
quiterpenoid hydroquinone with a rearranged drimane
Phytochem Rev (2009) 8:461–472 471
123

Page 12

skeleton from the sponge Dysidea avara. Tetrahedron Lett
38:3401–3404
Nakao Y, Matsunaga S, Fusetani N (1993) Toxadocial A: a
novel thrombin inhibitor from the marine sponge tox-
adocia cylindrical. Tetrahedron Lett 34(9):1511–1514
Newman DJ, Cragg GM (2004a) Advanced preclinical and
clinical trials of natural products and related compounds
from marine sources. Curr Med Chem 11:1693–1713
Newman DJ, Cragg GM (2004b) Marine natural products and
related compounds in clinical and advanced preclinical
trials. J Nat Prod 67(8):1216–1238
Riccio R, Iorizzi M, Minale L (1987) Recent advances in the
chemistry of Echinoderms. In: Hostettmann K, Lea PJ
(eds) Biologically active natural products. Clarendon
Press, Oxford
Rinehart LK (2000) Antitumor compounds from tunicates.
Med Res Rev 20(1):1–27
Rinehart LK, Gloer JB, Carter Cook J, Mizsak S, Scahill TA
(1981) Structure of the didemnins, antiviral and cytotoxic
depsipeptides from a Caribbean tunicate. J Am Chem Soc
103:1857–1859
Rinehart K, Holt TG, Fregeau NL, Stroh JG, Kiefer PA, Sun F,
Li LH, Martin DG (1990) Ecyeinascidin 729, 743, 759A,
and 770: potent antutumor agents from the Caribbean
tunicate Ecteinascidia turbinata. J Org Chem 55:
4512–4515
Roccatagliata AJ, Maier MS, Seldes AM, Zea S, Duque C
(1997) A new sulfated alkene from the ophiuroid Ophio-
coma echinata. J Nat Prod 60:285–286
Sa ´nchez AM, Malagarie-Cazenave S, Olea N, Vara D, Cuevas
C, Dı ´az-Laviada I (2008) Spisulosine (ES-285) induces
prostate tumor PC-3 and LNCaP cell death by de novo
synthesis of ceramide and PKCf activation. Eur J Phar-
macol 584:237–245
Smith MT (1985) Quinones as mutagens, carcinogens and
anticancer agents: introduction and overview. J Toxicol
Environ Health 16:665–672
Takebayashi Y, Pourquier P, Zimonjic DB, Nakayama K,
Emmert S, Ueda T, Urasaki Y, Kanzaki A, Akiyama S,
Popescu N, Kraemer KH, Pommier Y (2001) Antiprolif-
erative activity of ecteinascidin 743 is dependent upon
transcription-coupled nucleotide-excision repair. Nat Med
7:961–966
Tsukamoto S, Kato H, Hirota H, Fusetani N (1994) Antibac-
terial and antifungal sulfated alkane and alkenes from the
Hepatopancreas of the ascidian Halocynthia Roretzi. J Nat
Prod 57:1606–1609
Urdiales JL, Morata P, Nunez de Castro I, Sanchez-Jimenez F
(1996) Antiproliferative effect of dehydrodidemin B
(DDB), a depsipeptide isolated from Mediterraneam
tunicates. Cancer Lett 102:31–37
Wright AE, Forleo DA, Gunawardana GP, Gunasekera SP,
Koehn FE, McConnell OJ (1990) Antitumor tetrahydro-
isoquinoline alkaloids from the colonial ascidian Ectein-
ascidia turbinata. J Org Chem 55:4508–4512
Zubı `a E, Ortega M, Salva ` J (2005) Natural products chemistry
in marine ascidians of the genus Aplidium. Mini-Rev Org
Chem 2:546–564
472 Phytochem Rev (2009) 8:461–472
123