RICERCA ITALIANA     

Cytotoxic quinones from marine Ascidians:chemical characterization,synthesis and pharmacological activity

Università degli Studi di Roma "La Sapienza"

Abstract

The research program presented here is directed to obtain new derivatives terpene quinones with cytotoxic properties in order to use them as leading molecoles for the development of new drugs in the antitumour therapies. The proposed research takes advantage from the cooperation of two units (Rome-University “La Sapienza” and Naples – University “Federico II”), one devoted to the isolation, structural determination and study of biological properties of new quinones from ascidies, which are marine invertebrates, and the other one devoted to the synthesis of these compounds and structural analogues. All the obtained compounds, from natural sources or through synthesis, will be submitted to biological tests. Regarding the isolation of new compounds, ascidies belonging to the genere Aplidium, a source of quinones, will be investigated. Analysis of the species A.conicum, in order to find minor components, will be continued. Besides of Conicaquinones A and B, the major components of quinone mixture, several other compounds have been isolated: among them it has been isolated the Aplidinone A possessing a benzoquinone structure, condensed with a tiazine ring, as in the case of Coniaquinones. Biological tests have shown that Aplidinone A is able to penetrate cells and to interfere with the membrane redox system, as in the case of capsacidinoides and resiniferatoxin. The substitution pattern of thiazine ring in structure of Aplidinones have been determined by comparison of calculated NMR spectra of semplified models with those obtained from the natural products: the synthetic part of this research, described in the sequel, will be directed towards an unambigous synthesis of Aplidinone A. The program of screening of natural quinones will be carried out in the following way:
1. preparation of extracts from selected and identified materials
2. Preliminary pharmacological screening and fractionation of active extracts with the guide of cytotoxicity tests.
3. Separation of active fractions components.
4. Structural characterization by means of physical measurements.
Since the crucial point in the structural determination of Aplidinones A-C is the regiochemistry of thiazine ring, we have designed an unambiguous synthesis in which the position of the nitrogen was put at the beginning of the synthetic path.
Briefly, the starting material (the commercially available 1,4-dioxy,3-methoxy benzene) can be nitrated in para position in respect to methoxyl after OH protection by acetylation. Subsequent de-protection and exhaustive bromination affords to a dibromoderivative, which is allowed to react with NaHS. The product, after protection of OH and SH, is alkylated. NO2 is then reduced and the product is reacted with 1,2 dibromoethane, after de-protection of SH. Sulfur is oxydized with a peracid and finally the quinone is generated by direct oxidation of protected phenolic OH with Cerium Ammonium Nitrate. The proposed synthesis is characterized by flexibility in order to prepare related compounds, in which, as examples, the lipophilicity of isoprenoid chain is modified and/or functions are introduced in order to “hook” appropriate carriers.
All quinones prepared by synthesis or obtained from natural sources will be submitted to an exhaustive pharmacological characterization, through a study of molecular mechanisms leading to cells apoptosis. The induction of apoptosis process will be evaluated by measuring the amount of cytochrome C in the cythosolic fraction of cells, through analyses western blot, as consequence of exposition to different cytotoxic compounds. Such studies, performed on a wide number of related substances, will enable us to know relationships between structure and activity and the mechanisms of apoptosis. <<<

Principal Investigator

Rosario NICOLETTI Universita' degli Studi di ROMA

Research Objectives

The purpose of this research project is the development of molecules having antitumour activity, starting from the investigations on structures and activity of bio-active marine natural compounds. In particular the quinone derivatives isolated from Ascidia sp, marine invertebrate of Mediterranean see, will be focused. A large number of quinone derivatives, from natural sources or obtained by synthesis, has been tested for their anti- tumour properties, and three groups of quinones, benzo-, nafto-, and anthraquinones have shown interesting cytotoxic properties. Some compounds already in use in therapy, like mytomicine and streptonigrine, possess the benzoquinone moiety whereas some naftoquinones antibiotics, like Lapacol and Lapinon, are cytotoxic towards certain tumour cells. Previous studies have already shown that substances having quinonic structures, as well as hydroquinones, lead to oxidative stress by means of an increase of oxygen reactive species (ROS) and the depletion of reduced glutatione(GSH) amount. It seems likely that such an action is related to the beginning of apoptosis: on the other hand, it is well known that neoplasms are not simply related to the cells proliferation but also to a diminished cells programmed die. There are several examples in the literature, regarding natural compounds having a terpene hydroquinon/quinone structure, isolated from marine or terrestrial sources, mainly from ascidiacei belonging to the order of Aplousobranchiata (family Polyclinidae) as the ascidies of Aplidium genus, which are a rich source of the above mentioned substances with biological activity. The isolated compounds show a wide array of different structures, originated by intra- and inter-molecular cyclizations and/or rearrangements, thus giving macrocyclic or policyclic skeletons. These structures are often linked to amino acids or taurine residues.
This project will be divided in several phases that are, in part, the continuation of already started researches, The collection of substances to be investigated will be done by isolating new substances from natural sources followed by structural and pharmacological characterization as well as by total or partial synthesis. The syntheses of products having structures identical or related to natural substances have the purpose to widen the library of substances to be examined: moreover, it is offered the possibility to obtain easily – once established a synthetic protocol – analogues and compounds having similar structure, differing in functions and physical properties which can be modulated. Those compounds are then suitable for the studies of the above mentioned biological properties.
The final target of this research is to obtain the disposal of a large number of substances having quinone structure, related each others, to submit to biological tests. These investigations have the purposes: i) to understand the apoptosis mechanism ii) to study structure-activity relationships iii) the obtained results will enabled us to use the data collected on significant products as guide to design anti-tumour drug. <<<

First Results

The first year will be devoted to the extraction of biological material and to the isolation and identification of new quinone derivatives. During the same time the unit of Rome will focus his work on the synthesis of Aplidinone A. At the end of this period it will be expected available the synthesis of Aplidinone A and the discovery of new chemical entities.
During the second year the synthesis of structural analogues and/or the synthesis of new identified natural compounds will be carried out. Syntheses will be directed towards those structures which will appear more promising in respect to pharmacological properties. An exhaustive study on pharmacological properties along the lines illustrated in the appropriate parts of this program regarding all the available compounds will be carried out. The synthesis of molecules acting as models to prepare anti cancer drugs is expected. <<<

Timescale

24 months

National and international background

Nowadays it is estimated that approximately 50% of the top-selling drugs in the world are are terrestrial natural products or their derivatives. as many as 25% of the current used anticancer drugs are natural chemicals, with another 25% coming from synthetic derivatives of natural products (1-2) .By comparison, the sea has been scarcely explored although nearly three quarters of the Earth’s surface are covered by water. The marine environments offer very harsh life conditions and the species living within it have been forced to produce a diverse array of chemical compounds. These products, generally called secondary metabolites, are often specie-specific and offer an evolutionary advantage to the producing organisms through the prevention of predation and fouling as well as providing advantages in competition for space. Thousands of new natural products have been reported from marine organisms over the past 20 years and the number of marine bioactive compounds in annually increasing steadily, as well as the number of researches engaged in these tasks. The researches of marine ecology have shown that these metabolites are “chemical weapons” developed as inhibitors of physiological processes to be used against predators or competitors. The interest in these researches is continuously increasing in the last years since investigations on structures and biological activity gave indications that marine organisms will continue to be a significant source of potential drugs (3). Some of these molecules are at the stage of clinical or pre-clinical trials.
Many bioactive molecules from marine sources have been isolated from sessile invertebrates. These organisms are threaded by predators, infections by micro organisms and fouling. Therefore, their chances of surviving are entrusted to their secondary metabolites. Actually, more than 90% of all the new marine metabolites belong to four groups of organisms ( macroalgaes, coelenterates, echinoderms and sponges), but this fact is largely a reflection of the abundance and easy collection of these organisms. Little is known about the metabolism of many other organisms such as Tunicates although in the last 25 years they have shown to be a promising source of biologically active compounds. To figure out the importance of this class of compounds it is enough to say that at present six compounds from marine sources are in clinical phase as antitumors, and three of them – the Didemnina B, the Aplidina and the Ecteinascidina 743 (ET-743) (4-5) – are ascidia metabolites. In particular, the ET-743, isolated from a tunicate – the Ecteinascidia turbinate – is promising for treatments against carcinoma and solid tumors.
Tunicates (or Urocordati) are a sub-phylum of Cordates which include men and other vertebrates. Therefore, from an evolutionistic point of view, they are the most advanced invertebrates. Actually, the sub-phylum comprehends three different classes, but only ascidian species are not only pelagic, but benthonic as well. This brings to an easier collecting, with the result that ascidians are by far the most studied among tunicates.
Nonetheless, the chemistry of Mediterranean ascidiacea has been subjected to a very limited number of investigations, especially if compared with more extensive studies carried out upon other marine invertebrates such as Poriferans and Echinoderms. One of the research units of this group has been committed for long time to the research of new pharmacologically active secondary metabolites from marine resources, and has focused his attention on Mediterranean ascidiacea . Researches have shown ascidiacea secondary metabolites potentially useful in the development of new drugs effective in anti cancer therapies. Analysis of extracts from some species of ascidiacea from the Mediterranean sea have led to the discovery of a number of new molecules provided of antiproliferative activity toward several tumoral cell lines (6-10). Some of this molecules, whose structures are very different from each other, showed a selective behaviour. In particular, from the Sidnyum turbinatum it was isolated the Turbinamide, (11) a new molecule able to inhibit selectively proliferation of nervous system cells (C6, rat glioma), therefore a potential tool in the therapy of gliomas which, as known, are resistant against a large number of chemotherapics . It was demonstrated that cytotoxicity of turbinamide against C6 cells is due to apoptosis, this because the activity of caspases-3 – one of the proteases responsible for apoptosis – is increased in a dose depending way by Turbinamide. This effect is particularly marked in C6 cells (rat glioma) and not observed against rat macrophages (J774). Therefore a new therapy against gliomas could be developed starting from this molecule, also considering its selective effect which would reduce collateral effects such as immunodepression.
Researches upon the Aplidium conicum led to Conaquinones A and B, two new molecule with a tricyclic scaffold containing an inusual 1,1-dioxo-1,4-thiazinic ring condensed with a prenylated naphthoquinone moiety (12). Literature provide many examples of natural compounds with a terpenoid quinone/hydroquinone structure isolated from marine and terrestrial sources. It is possible to quote metabolites with a farnesyl quinone/hydroquinone scaffold such as the Avarol, from the marine sponge Dysidea avara (13) the Cyclozonarone, form a brown seaweed - the Dictyopteris undulata (14)– and a farnesyl hydroquinone isolated from the Wigandia kunthii tree (15). Nonetheless, it is known that marine ascidians belonging to the aplousobranchiata order (family Polyclinadae), like the a. Conicum, are a valuable source of terpenoid quinones and hydroquinones. As a matter of fact, the first biologically active metabolite discovered in tunicates was a prenylated hydroquinone – the geranyl hydroquinone – isolated from a variety of Aplidium (16).
Others biologically active quinones and hydroquinones were isolated from Aplidium ascidia . Namely prenylated hydroquinones from the Aplidium sp (17), the A. Californicum (18), the A. Costellatum (19) and the A. Antillense (20) ; dimeric complexes isolated from the A. Longithorax (21-22) and chromanols from the A. Solidum (23) and the Amarocium Multiplicatum (24). These compounds show a large variety of structures due either to intra and intermolecular cyclizations or rearrangement of the isoprene moieties, that may afford polycyclic or macrocyclic structures. Furthermore, some of these structures can be linked to amino acids or a taurine residue.
Many quinones, both synthetic and naturally occurring, were screened for their antitumor activity. The antitumor activity is exhibited mainly by three groups of naturally occurring quinones, namely benzoquinones, naphtoquinones and anthraquinones. Clinically used mitomycin and streptonigrin possess p-benzoquinone moiety, whereas some naphtoquinone antibiotics such as lapachol and lapinone have also been found to be citotoxic against tumor cells (25). Conicaquinones A and B, isolated from the A. Conicum, exhibited a very strong cytotoxic effect in vitro against C6 (rat glioma).
It is well known that neoplasms are not simply related to the cells proliferation but also to a diminished cells programmed die (26). Moreover, anti-apoptotic processes in tumour cells give a contribution to survival and favour the methastasis.
Previous studies have already shown that substances having quinonic structures, as well as hydroquinones, lead to oxidative stress by means of an increase of oxygen reactive species (ROS) and the depletion of reduced glutatione(GSH) amount. It seems likely that such an action is related to the beginning of apoptosis. The apoptosis is characterized by a cell fragmentation into corpuscles, said apoptotic bodies, covered by plasma membrane, in which the cellular ultra-structures, in the sequel eliminated by phagocytosis, are divided. From a biochemical point of view, apoptosis is characterized by degradation of chromatin, initially in fragments large 50-300 Kbases, and after in fragments smaller than 200 Kbases (27). Then, the DNA fragmentation is a marker of apoptosis. It has been suggested that free radicals modify mitocondrial membranes (28) with a subsequent release in the cytosol factors, like the Citochrome C, activating the Caspasis cascade. In the cytosol, Cytochrome C interact with the apoptotical protease (Apaf-1) giving the aptosome which activates the Caspase 9. Such an activation afford to that of Caspase 3 and the ather Caspases that induce cell die by apoptosis (29).
An investigation on minor quinone components in the same ascidia brought to the isolation of new
compounds, among them the Aplidinone A, all of them characterized by the quinone structure condensed with a thiazine. .
Biological screening showed that Aplidinone A can induce generation of reactive oxygen species (ROS), thus inducing cell death by apoptosis. Furthermore it was possible to demonstrate experimentally an increase in the generation of ROS and a reduction of the mitochondrial transmembrane potential was observed. Therefore, Aplidinone A can penetrate into cells and interfere with the membrane redox system in the same way of capsaicinoids and resiniferatoxin (30). Aplidinones structure was determined by NMR. In particular, the regiochemistry of the thiazine ring (sulphur and nitrogen position) was assigned by comparison of NMR data with those predicted by ab initio calculations.
Considering the pharmacological properties and the interest towards Apilidinone analogues, we decided to undertake the synthesis of Aplidinone A, to define unambiguously its structure and outline a synthetic protocol for the preparation of analogues. Secondly, our aims will be focused on the research of new quinone derivatives from natural sources. Therefore, one of our groups of research will be committed to the synthesis, whereas the other group will carried out researches on new quinones and their pharmacological properties.
Heterocyclic systems like Aplidinones, having a 1,1-dioxo-1,4-thiazinic ring condensed with a benzoquinone ring (I), are not common in literature. This kind of compounds has been synthesised for the first time in 1988 (31) by condensation of hypotaurine with a suitable benzoquinonic derivate.


SCHEME 1

This route shows that Aplidinones can be synthesised in relative few steps. Nonetheless it has the disadvantage of being not regioselective (see scheme 1). The authors managed to solve this problem assigning the structure of the two products by NMR studies carried out on both the regioisomers. Later, Harada worked out the synthesis of Adociaquinones A and B using the same protocol (32). Our researches showed that this is the only procedure used for the synthesis of natural compound similar to Aplidinones. Another possible route to this class of compounds could be through an azido quinone (33)
Being the regiochemistry of the azido moiety one of the crucial point for the determination of the structure of Apilidinones A and C, we have resorted to a synthetic approach where the nitrogen position is defined since the first steps. <<<