Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 32

Bioactive Compounds from the Marine Sponge Geodia barretti

Characterization, Antifouling Activity and Molecular Targets

MARTIN SJÖGREN

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 UPPSALA ISBN 91-554-6534-X 2006 urn:nbn:se:uu:diva-6797                       !"    #$ % &  " ' &    & ("  " )*   & ("  +, !"    -      .-",

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This thesis is based on the following papers, which will be referred to by their Roman numerals:

I Antifouling activity of brominated cyclopeptides from the marine sponge Geodia barretti Sjögren, M., Göransson, U., Johnsson, AL., Dahlström, M., Andersson, R., Bergman, J., Bohlin, L. Journal of Natural Products 2004, 67, 368-372.

II Synthesis of barettin Johnson, AL., Bergman, J., Sjögren, M., Bohlin, L. Tetrahedron 2004, 60, 961-965

III Recruitment in the field of Balanus improvisus and Mytilus edulis in response to the antifouling cyclopeptides barettin and 8,9- dihydrobarettin from the marine sponge Geodia barretti Sjögren, M., Dahlström, M., Göransson, U., Jonsson, PR., Bohlin, L. Biofouling 2004, 6, 291-297

IV Antifouling activity of synthesized structure analogs of the sponge metabolite barettin Sjögren, M., Johnson. AL., Hedner, E., Dahlström, M., Göransson, U., Shirani, H., Bergman, J., Bohlin, L. Accepted for publication in Peptides

V Brominated cyclodipeptides from the marine sponge Geodia barrette as selective 5-HT ligands Sjögren, M., Hedner, E., Frändberg, PA., Johansson, T., Göransson, U., Dahlström, M., Jonsson, PR., Nyberg, F., Bohlin, L. In manuscript

Contents

1. Introduction...... 11 1. 1 Secondary metabolites...... 11 1.2 Host defence interactions as a basis for finding new bioactive compounds ...... 13 1.3 Bioactive compounds of marine origin with the potential of serving as drug leads ...... 14 1.4. Bioactivity of Swedish marine organisms – focus on the sponge Geodia barretti...... 15 1.5. Marine secondary metabolites as antifoulants...... 16 1.5.1. Study organism: biology of sponges...... 16 1.5.2 Marine biofouling and antifouling compounds of marine origin17 1.6. History of barettin ...... 18 1.7. Synthesis of natural products ...... 19 1.8. Bioactivity of diketopiperazines...... 20 1.9. Analogs of bioactive natural products...... 20 1.10. Molecular target; highlighting G protein-coupled receptors ...... 21 1.11. Aims of the present study...... 23 2. Methodological considerations ...... 24 2.1. Bioassay-guided fractionation and isolation ...... 24 2.2. Structure elucidation ...... 25 2.3. Rearing of barnacle cypris larvae...... 26 2.4. Experimental design...... 27 2.5. Synthesis of barettin and 8,9-dihydrobarettin ...... 28 2.6. Methods in field experiments...... 29 2.7. Analogs of the dipeptides barettin and dipodazine...... 29 2.7.1 Peptide synthesis...... 29 2.8. Receptor ligand binding assay...... 31 3. Results and discussion ...... 32 3.1. Settlement inhibition displayed by barettin and 8,9-dihydrobarettin 32 3.2. Synthesised barettins as antifouling compounds...... 33 3.3. Field experiment to evaluate antifouling activity...... 33 3.4. Analogs as antifouling compounds ...... 36 3.5. Barettins as selective serotonin ligands...... 39 Concluding remarks...... 42 Populärvetenskaplig sammanfattning ...... 45 Acknowledgement ...... 47 References...... 49 Abbreviations

aa amino acid Ala Alanine Arg Arginine AcN Acetonitrile Br Bromine DKP:s Diketopiperazines DMSO Dimethylsulphoxide EC50 Effective Concentration (50%) EtOH Ethanol FSW Filtered Seawater GPCR G protein-coupled receptors His Histidine HPLC High Performance Liquid Chromatography LC Liquid Chromatography Leu Leucine MS Mass Spectrometry MW Molecular weight NMR Nuclear Magnetic Resonance Phe Phenylalanine Pro Proline RP Reversed Phase RP-SPE Reversed Phase-Solid Phase Extraction SPC Self Polishing Copolymer SPE Solid Phase Extraction TBTO Tributyltinoxide Tyr Tyrosine Trp Tryptophan Val Valine

1. Introduction

1. 1 Secondary metabolites Fossil records indicate that humans have used plants for medicinal purposes since at least the Mid Paleolithic age (app. 60 000 years ago) (Solecki and Shanidar, 1975). Apart from archeological records, there are written records dating back at least 5000 years that report on plants for medicinal use (Raskin et al., 2002). The rich variety of bioactive compounds originating from natural sources such as terrestrial plants, fungi and bacteria has been a very valuable source both for compounds used as drugs and as leads in drug discovery (Fabricant and Farnsworth, 2001). Well-known examples with a direct use as drugs are antibiotics from actinomycetes, the antitumor agent paclitaxel from the yew tree Taxus brevifolia and morphine, vinblastine, vincristine and reserpine from different plant sources. It is estimated that 122 compounds from 94 species of plants are used globally as drugs. The number of described higher plants is estimated at 250 000 (but may be much higher) and only 6% of these have been screened for biological activity (Fabricant and Farnsworth, 2001). The bioactive natural compounds stem from the development in terrestrial as well as aquatic organisms of means to produce sophisticated chemical compounds that partake in various interactions among species. These compounds are not directly involved in the maintenance of cells and cell primary metabolism, but are instead believed to have evolved to contribute to the overall fitness of the organism (Qi et al., 2004). The production of such compounds is often referred to as the secondary chemistry of the organism and the compounds themselves are termed secondary metabolites. The role and adaptive significance of secondary metabolites have been under debate for some time (Williams et al., 1989; Stone and Williams, 1992). Traditionally, secondary metabolites have been regarded as evolutionary neutral or waste products. However, the presently most accepted view is that many secondary metabolites are adaptive and play key roles in the host defence against pathogens, parasites, predators, competitors and epibiota, reviewed by (Harper et al., 2001). Although a striking number of secondary metabolites have been discovered from a wide array of organisms, molecules possessing a potent biological activity, i.e. displaying activity in the submicromolar range, are still quite rare (Firn and Jones, 2000). Firn and Jones (2000) recently

11 proposed a model for the evolution of secondary metabolism, which reconciles both secondary metabolites, which display a lack of biological activity and molecules with very potent activity. The model holds that if enzymes involved in the production of secondary metabolites would have a high substrate specificity, the chemical diversity would most likely not be favoured nor would the evolution of a putative biological potency of a molecule. Therefore, to ensure chemical diversity the enzymes involved must tolerate a range of substrates and thus, a range of new compounds can be produced, one of which may have the desired potent biological activity. In parallel to this, evidence has been put forward that points to a large discrepancy between chemical diversity and functional diversity, e.g., the chemical diversity is much greater than the functional diversity (Tulp and Bohlin, 2002). In addition to the lack of potency of many isolated secondary metabolites, there is also the problem of lack reproducibility of bioactivity (Cordell, 2000). More than 40% of different plant extracts have displayed bioactivity in screening assays but when plants are resampled or fractions have been purified, the activity is lost. This is attributed to changes in the ecological context of the organism, i.e., plants are harvested at different times and in different locations, the microenvironment may change, there may be temporal and spatial differences in physical and chemical stimuli. Also, differences in stress pressure from consumers and pathogens may alter the content of bioactive secondary metabolites (Raskin et al., 2002). In addition, plant bioactive secondary metabolites may act in concert, i.e., synergistically (or additively), and during purification procedures, in attempts to obtain one molecule, bioactivity is lost because several metabolites that act in synergy (or additively) produce the bioactivity. Another explanation for loss of bioactivity could be that the metabolites degrade or oxidize during the fractionation procedures. Many enzymatic pathways responsible for the production of secondary metabolites have also proven to be highly inducible, particularly the production of some alkaloids (Facchini, 2001). Inducible defences against herbivory have also been shown with brown algal secondary metabolites in the marine environment (Toth and Pavia, 2000). Thus, the ecological basis for the production of bioactive secondary metabolites cannot be overlooked and a more thorough consideration in the future of the role of ecology in the production of secondary metabolites may prove very useful for an understandable search for new leads in drug discovery.

12 1.2 Host defence interactions as a basis for finding new bioactive compounds Classically, in pharmacognosy, the isolation and structural characterisation of bioactive compounds have been guided by their effect in mammalian systems, often based on their traditional use in ethnopharmacology as medicinal plants. However, the failure of the pharmaceutical industries to find novel leads for drug development from natural products in their large screening programs conducted during the twentieth century, may be attributed to the general disregard in such schemes for the impact of ecological interactions on secondary metabolite production. As mentioned above, ecological interactions, habitat conditions and the intricate mechanisms at the molecular level, that regulate secondary chemistry may render the production highly variable. However, increased knowledge in chemical ecology has for example shown that neuropharmacological compounds with a human medicinal history, e.g. cocaine, are indeed secondary metabolites synthesised by plants as natural insecticides (Nathanson et al., 1993). The sources commonly explored for the discovery of compounds of marine origin are sessile invertebrate organisms like sponges, tunicates, bryozoans and mollusks. The production of secondary metabolites by these organisms is a possible consequence of their inability to hide or flee from predators or fouling organisms, i.e. the compounds synthesised are used in various ecological interactions, e.g., host defence interactions. These organisms not only carry the largest number of secondary metabolites, they also have the ability to synthesise a diverse range of organic compounds, i.e. polyketides, alkaloids, peptides and terpenes (Proksch et al., 2002). Growing knowledge of chemical interactions in the marine environment suggests that secondary metabolites produced by sponges, bryozoans and tunicates serve as a defence against predators such as fish (Proksch, 1994; Paul and Puglisi, 2004). Evidence has also emerged that secondary metabolites can serve as a protection against settlement and growth of fouling organisms e.g. barnacles, bryozoans and tubeworms (de Nys et al., 1995; Assmann et al., 2000; Engel and Pawlik, 2000). An illustrative example of the ecological role of marine secondary metabolites is the field experiment, conducted by (Bakus, 1981). In this study it was found that 70% of the associated fauna of coral reefs, including invertebrates such as sponges, tunicates and corals, that lived exposed to predators such as fish and herbivores, were ichthyotoxic, whereas the less exposed, cave-living fauna were non-toxic to fish predators (Bakus, 1981). Marine sponges are pre-eminent producers of secondary metabolites and are also one of the richest sources of marine alkaloids. Alkaloids have long been recognised as one of the most potent groups of compounds and several toxins and bioactive compounds belong to this group, e.g. nicotine, cocaine,

13 the bungarotoxins and the conotoxins. In the marine environment, several chemical interactions have shown to be mediated by bioactive alkaloids (Assmann et al., 2000; Engel and Pawlik, 2000). For example, in the red- coloured tropical sponge Oceanapia sp., the pyridoacrine alkaloids kuanoniamine C and D seem to be responsible for the red colour and also serve as a protection against predatory fish (Schupp et al., 1999). In the Aplysina family, e.g. A. aerophoba, the brominated isoxazoline alkaloid aeroplysisin, serves as a defence of the sponge against predators or fouling organisms (Teeyapant and Proksch, 1993; Ebel et al., 1997).

1.3 Bioactive compounds of marine origin with the potential of serving as drug leads The first compounds isolated from marine organisms and used as tools in drug development were the unusual nucleosides spongouridin and spongothymidin from the sponge Cryptotethya crypta (Bergmann and Feeney, 1951). These two compounds made it possible to develop the antiviral drugs, vidarabin and cytarabin. Also, an important milestone in the discovery of potential leads from marine natural sources was the isolation and structural elucidation of the prostaglandins from the gorgonian Plexaura homomalla (Wienheimer and Spraggins, 1969). To date, about 10.000 different natural compounds of marine origin have been discovered (Faulkner, 2002). Several substances of natural origin have successfully been characterized and assayed for bioactivity with potential pharmaceutical use as well as for use in pest management. For example, the marine sponge Theonella swinhoei, a marine sponge found in the sea outside Papua New Guinea, produces many different secondary metabolites, e.g. swinhoeiamide with insecticidal properties, which is effective against the pest insect Spodoptera littoralis (Edrada et al., 2002). Furthermore, the potent protein phosphatase inhibitors calyculinamides and clavosines have been isolated from the marine sponges Discodermia calyx (Kato et al., 1986) and Myriastra clavosa (Fu et al., 1998). Moreover, a recent review (Newman and Cragg, 2004) has highlighted the status of bioactive marine natural products in different states of preclinical trials, i.e., compounds with antitumor, anti-viral and anti- inflammatory activities. A large number of these compounds derive from marine sponges. For example the imidizole, giroline, from the sponge Pseudaxinyssa cantharella (Tsukamoto et al., 2004), affects protein synthesis in eukaryotes. The compound passed phase 1 trials in humans but failed later in drug development because of its hypertensive side effects in patients. Another more promising attempt is the compound KRN-7000, an

14 analog to agelaphines synthesized from Agelas mauritianus (Natori et al., 1994), with anti-tumor activity especially on murine melanoma. This compound has now reached phase II clinical trials for cancer immunotherapy.

1.4. Bioactivity of Swedish marine organisms – focus on the sponge Geodia barretti A screening survey was conducted in the beginning of the 1980s, in which Swedish marine organisms were assayed for their potential production of bioactive secondary metabolites. Of 25 different species sampled from the Koster Fjord area, it was found that extracts from Geodia barretti displayed unusual bioactivity. G. barretti is known to produce a wide spectrum of secondary metabolites such as histamine, inosine, the amino acid taurine and several sterols (Hougaard et al., 1991). Fractions from G. barretti caused contraction of isolated guinea pig ileum (Andersson et al., 1983). Later it was found that the bioactivity could be traced to the compound barettin (Lidgren and Bohlin, 1986). However, the chemical structure of barettin was then debated for the following two decades (Lieberknecht and Griesser, 1987). The story of the barettin molecule is told below (1.6). In their screening study, Andersson et al., 1983 also found that chloroform extracts of the red alga Ceramium rubrum significantly inhibited (>99%) stimulated guinea pig ileum. The compound responsible for this effect has not been isolated. Furthermore, fractions from the sponge Halichondria panicea, the bryozoan Flustra foliacea and the echinoderm Asterias rubens displayed unusual bioactivity. At least F. foliacea and H. panicea have been much studied by others as to their production of bioactive compounds. For example, it has been shown that H. panicea produces sesquiterpenes (Ye et al., 1996), glycosphingolipids (Nagle et al., 1992), and glycoglycerolipids (Wicke et al., 2000). The latter have been shown to be produced by symbiotic bacteria (Wicke et al., 2000).The bryozoan F. foliacea, sampled from locations in the North Sea, is rich in secondary metabolites and produces bioactive brominated alkaloids and monoterpenes (Peters et al., 2002; Peters et al., 2004). Among the alkaloids are the compounds called flustramine A-E (Holst et al., 1994) of which at least flustramine C and E have been successfully synthesized (Morales-Rios et al., 2002). In the study by Andersson et al. (1983), petroleum ether extracts of F. foliacea caused >99% inhibition of stimulated guinea pig ileum.

15 1.5. Marine secondary metabolites as antifoulants In 1998, G. barretti was re-examined for its content of bioactive compounds. Following the observation that the sponge possessed an almost entirely fouling-free body surface, the emphasis was now on a possible defence against on-growth mediated by chemical metabolites. Chemical compounds that might be effective against larval settlement, could serve as the sponge’s outside shield. In addition to answering questions regarding defence mechanisms in coldwater marine organisms, such molecules could also present a new means to combat fouling on manmade structures.

1.5.1. Study organism: biology of sponges Marine sponges (Porifera, Metozoa) produce a wide range of secondary metabolites with bioactivity against settling marine invertebrate larvae (Okino et al., 1995; Hirota et al., 1996; Tsukamoto et al., 1996). Sponges are the most primitive of all multicellular animals and are subdivided into three major classes, the Hexactinellida, the Demospongiae, and the Calcarea (Müller and Müller, 2003). They are entirely aquatic and most of them are marine and live deep in the oceans as well as in more shallow waters. They are sessile and live their adult life attached to the substratum. Upon fertilization, the larvae that are released swim or creep off to find new sites for settlement (Müller and Müller, 2003). Sponges are filter feeders and filter water through many small pores, i.e., ostia (Müller and Müller, 2003) forming extensive water canals. These pores and canal systems in sponges allow them to filter water of their own mass every 5 seconds. They also have excreting pores, oscules that are less in number than the filtering ostia (Müller and Müller, 2003). Sponges do not possess true tissues but are instead equipped with different varieties of cells, ranging from simple asconoids to the more intricate leuconoids. The totipotent archaeocytes, which can transform into reproductive, skeletal or epidermis cells, have attracted research interest because of their similarity to stem cells. Sponges are often equipped with spicules to form a robust skeleton and their constitution is in general based on silica oxide (SiO/H2O) as in Hexactinellida and in Demospongiae, or calcium carbonate (CaCO3) as in Calcarea (Müller and Müller, 2003). It has recently been highlighted that marine sponges harbor diverse and complex microbial communities, phylogenetically distinct from those found in the marine plankton or marine sediment (Fieseler et al., 2004). Still, the role of microbial communities that live associated to sponges is poorly understood. However, it has been shown that some sponges live in symbiosis with cyanobacteria, which supply the sponge with nutrients from photosynthesis (Hoffmann et al., 2005). The marine sponge G. barretti Bowerbank (family Geodiidae, class Demospongiae, order Astrophorida), the study organism in the present

16 thesis, lives on the Atlantic continental shelf at depths between 10 and 500 m. It is much less studied than its congener G. cyonidum.

Figure 1. The marine sponge G. barretti living at 50 to 250m depth in the Koster Fjord, Sweden. The left-hand picture was taken at 70m and the right-hand picture was taken at 123m depth. The picture also serves to illustrate the fouling-free body surface of the sponge. Photos by courtesy of: Tomas Lundälv, Tjärnö Marine Biological Laboratory.

1.5.2 Marine biofouling and antifouling compounds of marine origin The unwanted growth of sessile organisms, e.g. algae, hydroids, blue mussels, and barnacles, on manmade surfaces in the sea, so-called biofouling, causes severe and far-reaching technical and economic problems for the shipping and aquaculture industries (Wahl, 1989). Traditionally, mankind has tried to solve this problem by using heavy metal-based marine coatings, e.g., over the past decades using TBTO and copper as active ingredients. However, due to the undesirable side effects of primarily TBTO on non-target organisms (Alzieu et al., 1986; Alzieu et al., 1989; Evans et al., 1996) the IMO., (1999) has passed a resolution to control the use of organotin substances, both on national levels as well as on a global level (IMO, 1999). These restrictions call for a development of new non-toxic, biodegradable alternatives with a target-specific mode of action (Clare et al., 1992). Fouling avoidance can involve physical, mechanical or chemical processes, all of which have been the subject of research studies. Berntsson et al., (2000a) report promising results in which avoidance of the unwanted growth of the barnacle B. improvisus is achieved by microtextures. The microgeometry of the surfaces successfully counteracts with the settlement behaviour of cypris larvae and inhibits both attachment and metamorphosis. Also, Dahlström et al., (2000) have reported the antifouling effect of nanomolar concentrations of receptor-specific synthetic chemical compounds. Even if all surfaces, both artificial and natural, in the marine environment are targets for colonising attempts, several organisms still remain un-fouled.

17 Therefore, marine organisms and putative host defence interactions have been the subject of research efforts to find antifouling substances of natural origin. In addition to the antifouling compounds isolated from marine sponges, a number of secondary metabolites isolated from different marine organisms have been described to date, which display inhibitory effects on larval settlement. For example, substances with antifouling activity have been isolated from the whip coral (Rittschof et al., 1985), from marine bryozoans (Konya et al., 1994a; Konya et al., 1994b) and from red algae (de Nys et al., 1995).

1.6. History of barettin

The history of barettin begins in the mid 1980s. When it was isolated (Lidgren et al., 1986), the structure was confirmed and determined as cyclo[(6-bromo-8-en-tryptophan)proline] (Fig. 2 A). Lidgren et al., suggested that barettin consisted of a proline connected through a cyclic formation with tryptophan. The structure suggested was disproved by Lieberknecht and Griesser in 1987 (Lieberknecht and Griesser, 1987) when this group attempted to perform a full synthesis of the molecule. Lieberknecht and Griesser instead proposed a structure with double molecular weight of cyclo[(6-bromo-8-en-tryptophan)proline] (Fig 2 B). However, this attempt also failed to produce the correct structure of barettin. In the late 1990s, our group restarted the marine branch of Swedish pharmacognosy. Antifouling research was at this time (and still is) a very hot topic because of the recent bans of toxic ingredients in marine paints. The Marine Pharmacognosy project commenced a bioassay-guided fractionation of G. barretti based on the observation of G. barretti’s fouling-free body surface in the search for natural compounds with antifouling properties. Coincidently, the bioactivity in the settlement assay using barnacle larvae was traced to barettin, because we obtained NMR spectra identical to those presented by Lidgren et al., with the compound purified by aid of the antifouling assay. However, after chemical analysis with HPLC, MS and NMR the structure instead could be assigned to (cyclo-[(6-bromo-8-en- tryptophan)-arginine]). Thus we realized that proline should be replaced with arginine for the correct structure of barettin (Fig. 2 C). Because the inhibiting effect of barettin on barnacle larvae was promising, we decided to patent the molecule for antifouling applications. What we didn’t know was that a German group led by Professor Francke also had an interest in resolving the structure of barettin. Our manuscript was ready to be submitted but we needed a go-ahead from the patent bureau that it was all right to disclose the chemical structure of barettin. Because of patent problems Sölter

18 et al. (2002) published the correct structure of barettin, before us and identical to the one we had found.

A) B) O O H H N N N O N¨ Br Br HN O Br N N N N H H H O O C) O

H N NH2 Br N H O NH

Figure 2 A-C. Chemical proposed structures of barettin

1.7. Synthesis of natural products It has been suggested that nearly 50% of the drugs in use today are of natural origin (Newman et al., 2003). In several cases, the drugs represent the synthetic derivative of the native substance but also, a large number of drugs represent a biomimetic of the natural substance. These compounds are used mainly for therapeutic purposes, and isolation and structural elucidation of the native compounds are often guided by their bioactivities in pharmacological assays, for example, antitumor, antiviral and antibacterial screening assays (Napolitano et al., 2001; Nakagawa et al., 2003; Turan- Zitouni et al., 2004). Where the isolation of compounds, based on chemically mediated ecological interactions (to study for example roles of chemical defence) is concerned, the main purposes are often to gain insights into organism biology and evolutionary strategies (Hay, 1996; Cruz-Rivera and Hay, 2003). Hence, the main purpose is not to isolate compounds for human applications and therefore, a complementary synthesis of the isolated compounds is not always performed. Complementary synthesis is important for confirming the originally characterized compound, preserving the analyzed organisms from extinction (for example, 17 tons of the bryozan Bugula neritinia were collected by divers over a two-year period to obtain sufficient amounts of the cytotoxic compound bryostatin 1) (Haefner, 2003) and also to lower the production costs of the molecules.

19 1.8. Bioactivity of diketopiperazines The dipeptides barettins belong to a group of compounds called diketopiperazines (DKPs). These molecules consist of dipeptides forming a cyclic ring structure. The natural sources of these compounds are most commonly organisms from the protist and plant kingdom. These molecules have attracted recent research interest because of their potent biological activity (De Rosa et al., 2003). For example, they have been shown to be involved in cell-to-cell signaling in bacteria, the so-called quorum-sensing (Holden et al., 1999; Degrassi et al., 2002). Examples of other DKPs activities are growth inhibitors of cancer cells of cyclo(Phe-Pro) (Brauns et al., 2004) and the cyclo(His-Pro) is endogenous to mammals (Prasad, 1995). The DKP cyclo(Leu-Gly) have been reported to interact with a dopaminergic target (Walter et al., 1975). Recent findings stated that 13 DKPs were found in roasted cocoa nibs (Theobroma cacao) and also were responsible for the bitter taste in the cocoa (Stark and Hofmann, 2005), However, little is known of the mechanism of action of these group of compounds in mammalians.

1.9. Analogs of bioactive natural products The advantages of synthesizing analogs of bioactive natural products are apparent. Often, the native compound has chemical entities that can be further explored and modified so that structure-activity relationships may be unraveled through changes in bioactivity. The aim of modifications in the chemical entities of the native molecule through the synthesis of analogs is often to obtain stronger biological activity. Concomitantly, information on where the biological activity rests is often gained. A further reason for designing and synthesizing analogs may be, as in the case with the barettins, the cost associated with the synthesis of the natural compounds. The cost for synthesizing the barettins (in full scale) was estimated to 60 000 € kg-1, which is far too expensive for use as an antifouling additive. Thus, the work of synthesizing analogs was undertaken to find more cost-efficient molecules. Several successful analogs of natural compounds have been synthesized, primarily for use in pharmacological applications. Examples are compounds that interact with mammalian receptors, like analogs to a pyridazine compound targeting GABA receptors (Carling et al., 2005; Wellendorph et al., 2005) or the analogs to a beta-tetralonohydantoins compound targeting specific 5-HT receptors, i.e., 5-HT1A and 5-HT2A receptors (Byrtus et al., 2005).

20 1.10. Molecular target; highlighting G protein-coupled receptors Marine natural products have been investigated primarily for cytotoxic effects, e.g., bryostatins, but also for activity against several other disease targets, such as antiviral, anti-parasitic and anti-inflammatory activity (Faulkner, 2002; Gul and Hamann, 2005). Recently, there is growing research interest in these compounds’ ability to interact with molecular targets to evaluate possible roles as neuropharmacological agents in disease states such as psychiatric disorders that are mediated for example by serotonin receptors and enzymes involved in monoamine transport and metabolism. The conotoxins produced by the gastropod snail Conus sp are prominent examples of naturally derived marine peptides that have been used as tools in pharmacological research to discriminate between different ion-channel receptor subtypes (Terlau and Olivera, 2004). The approximately 500 different Conus species produce some 100 peptides each with little or no overlap in peptide profile between species. It is hypothesized that a majority of these peptides target a unique ion channel receptor. Bioactive compounds regularly interact to affect physiological functions on enzymes, ion- channels, carrier proteins and receptors, all of which are proteins. It is estimated that approximately 60% of the drugs prescribed today act through G protein-coupled receptors (Wilson et al., 1998). The G protein-coupled receptor family includes for example muscarinic acetylcholine receptors (mAch), adrenoceptors, dopamine receptors, 5-HT receptors (except 5-HT3 which is an ion-channel receptor) and many neurotransmitters and neurohormones act through interaction to G protein-coupled receptors (Brody and Cravchik, 2000). The G protein-coupled receptor family is very much conserved in its protein structure (Hamm, 1998; Meeusen et al., 2003), and representatives have been found in simple eukaryotes like yeasts and molds (Peroutka and Howell, 1994). It was only recently that the crystal structure of a G-protein was revealed (Palczewski et al., 2000). The chemical entity in form of the indole nucleus of the neurotransmitter serotonin has encouraged some researchers to examine marine-derived indole alkaloids for their ability to interact with serotonin receptors (Bifulco et al., 1994; Bifulco et al., 1995; England et al., 1998; Hu et al., 2002). Of the biogenic amines, the serotonin receptor family consists of the largest number of receptor subtypes (Peroutka, 1994). Six of these receptor subtypes are G protein-coupled receptors, namely, 5-HT1,2,4-7 where 5-HT1 and 5-HT2 are further subdivided. The 5-HT3 receptor is as mentioned above a ligand- gated cation-channel receptor. It has been suggested that catecholaminergic receptors (dopamine, noradrenaline and adrenaline) evolved from the serotonin 5-HT1 receptor. It is further believed that serotonin evolved more

21 than 750 million years ago (Peroutka and Howell, 1994; Walker et al., 1996). The gelliusines A and B are brominated tris-indole alkaloids isolated from the New Caledonian sponge Gellius sp. (Bifulco et al., 1994). (r) gelliusine A (Fig. 3 A) caused serotonin-like contraction on guinea pig ileum in a concentration range between 5 and 70 µg ml-1 whereas at lower concentrations the compound antagonized serotonin-induced contraction (Bifulco et al., 1994). However, no further reports on (r) gelliusine A have been presented in which the specific serotonin receptor affinity has been investigated. Because of the indoleamine pharmacophore of one pyrroloiminoquinone and six aplysinopsins isolated from the Jamaican sponge Smenospongia aurea, these substances were screened for their ability to displace the binding of radio-labeled ligands of 5-HT2A and 5-HT2C receptors (Hu et al., 2002). Of the compounds from S. aurea, the 6-bromoaplysinopsin (Fig 3 B), the 6-bromo-2’-de-N-methylaplysinopsin (Fig 3 C) and N-3’- ethylaplysinopsin, displaced high-affinity radio-labeled ligands from cloned human serotonin 5-HT2A and 5-HT2C receptor subtypes. The other compounds from S. aurea showed no such activity (Hu et al., 2002). Interesting to note is that these substances displaying serotonin-like activity (Bifulco et al., 1994) or specific serotonin receptor affinity (Hu et al., 2002) have an indole residue, which is brominated in the 6th position. The 6-bromo-tryptophan residue of V-conotoxin also prompted by England et al., to investigate the activity of this specific conotoxin at serotonin receptors. They showed that V-conotoxin exhibited specific binding properties to the only ion channel receptor in the serotonin receptor family, namely, the 5-HT3 receptor.

A) B) C)

Br NH2

H3C NH H NH HO NH N N 2 H H N N HN CH N 3 CH3 H O O

NH2 N H Br Br Br

Figure 3 A-C. The chemical structures of natural products from marine sponges with specific serotonin-like activity or serotonin receptor affinity. A) (r) gelliusine with serotonin-like activity on guinea pig ileum. B) 6-bromoaplysinopsin with high affinity for 5-HT2A and 5-HT2C receptors (Ki µM close to that of endogenous serotonin which is 0.32 and 0.13 µM for 5-HT2A and 5-HT2C receptors respectively). 6-bromoaplysinopsin displayed higher affinity for the 5-HT2C receptor than for 5- HT2A receptors. C) 6-bromo-2'-de-N-methylaplysinopsin with a 40-fold higher affinity for the 5-HT2C receptor than for the 5-HT2A receptor.

22 1.11. Aims of the present study The work presented in this thesis is part of an ongoing project, Marine Pharmacognosy, at the Division of Pharmacognosy, Uppsala University and at Tjärnö Marine Biological Laboratory, Göteborg University, investigating the presence and potential applications of bioactive compounds from marine organisms. The overall objective of this project is to identify bioactive compounds from marine coldwater organisms with emphasis on the compounds’ antifouling activities and their mechanism of action at the molecular level.

The specific objectives of the present thesis were:

• to perform a bioassay-guided isolation and consequent structural elucidation of natural compounds from Geodia barretti with specific activity against the macrofouling organism Balanus improvisus • to investigate and evaluate the effects of the isolated compounds, i.e., dipeptides, on colonising attempts of Balanus improvisus and Mytilus edulis in situ • to perform a full synthesis of the cyclopeptides from G. barretti, i.e., the barettins, with a subsequent evaluation of their bioactivity in the settlement assay, and to confirm the chemical structure • to synthesize analogs of the barettins as well as analogs of the naturally occurring barettin congener dipodazine, and to evaluate their antifouling activity • to establish the specific molecular target/s of the barettins in a receptor affinity assay, with emphasis on mammalian 5-HT (serotonin) receptor subtypes

23 2. Methodological considerations

The aim of this section is to present a summary and some general considerations of the different methods used in the different papers comprising this thesis.

2.1. Bioassay-guided fractionation and isolation

Geodia barretti tissue; homogenize and freeze dry

Pre-extraction with Removal of lipohilic dichloromethane contituents

G. barretti Dichloromethane residue extract Extraction with ethanol (50%) Extraction of peptides

Ethanol extract Size-exclusion Removal of low-molecular weight chromatography compounds on Sepahdex G 10

High-molecular Low-molecular weight fraction weight fraction

Solid-phase extraction Removal of salts and on RP-18 material polysaccharides

Peptide Salts and poly- fraction saccharides

Figure 4. The protocol for the extraction of polypeptides as described by Claeson et al. (1998). Slightly modified, this extraction procedure was used to obtain peptide fractions from G. barretti (paper I).

24 Specimens of G. barretti were collected in the Koster Fjord outside Tjärnö Marine Biological Laboratory on the Swedish west coast at a depth of 60-90 m. The sponges were immediately homogenized and freeze-dried. The freeze-dried material was defatted with dichloromethane for removal of lipophilic substances. The dichloromethane extract was evaporated and saved for future analysis. The remaining sponge material was extracted in 50% ethanol (Claeson et al., 1998) (Fig. 4). The aqueous ethanol extract was concentrated and then desalted by reversed phase solid phase extraction (RP- SPE). The sample was applied on the RP-SPE column and then washed with 0.1% trifluoro acetic acid (TFA) and then with 60% acetonitrile (AcN with 0.1% TFA) to elute captured substances. Upon in vacuo removal of AcN the extract was subjected to RP-HPLC with an Äkta Basic equipped with a 50 mL superloop. Five fractions were collected and tested for inhibitory effects in the barnacle larvae settlement assay. Fractions denominated 4 and 5 were found to be the most active. Barettin was present in fraction 5 and 8,9- dihydrobarettin in fraction 4. These fractions were further purified by fractionation on an analytical column (Amersham C18, 4.6x150mm) using the HPLC system described above. The purity of each of the compounds were >95% as determined by analytical HPLC.

2.2. Structure elucidation For structure elucidation the MS and NMR analyses are imperative steps for the characterisation of the active molecules. MS and MS-MS can give information regarding features such as the molecular weight, the molecular backbone, and the degradation profile of the molecule, while the chemical structure can be determined by using NMR. Our work on the isolation of barettin was performed independently and in parallel to the work by Sölter et al., (2002) as discussed in the introduction section (1.6). Our analysis revealed that the spectroscopic properties of the compound with the molecular weight of m/z 421, 423 [M+H]+ isolated from fraction 4 were very similar to those of barettin. The combined NMR analysis, including 2D experiments, revealed that the compound was a cyclic dipeptide consisting of tryptophan and arginine, brominated in the 6-position of the indole nucleus. Similar features had previously been determined for barettin. The differences in molecular weight (i.e. the molecular weight of barettin + 2) indicated that the structure was 8,9-dihydrobarettin. This was further confirmed by 1H1H-COSY sequence 8-H; 9-H; 10-H and via 3 3 HMBC-correlations J C-3, H-9 ; J C-11, H-9. All proton signals were assigned to their respective carbon by HMQC. 1H and 13C-NMR data as well as correlations from 1H1H-COSY and HMBC experiments are listed in table 1.

25 1 13 Table 1. H-NMR (Bruker DPX 300, 300 MHz, DMSO-d6) and C-NMR (Bruker DPX 300, 75 MHz, DMSO-d6) data of 8,9-dihydrobarettin.

Carbon G Proton G (ppm) J (Hz) COSYa HMBCa DMSO-d6 (ppm) DMSO-

d6 1-H 11.07 S 2-H; 7-H C-3, C-3a, C-7a C-2 125.7 2-H 7.09 S 1-H; 8-H C-3, C-3a, C-7a C-3 108.7 C-3a 126.6 C-4 120.7 4-H 7.54 d, J = 8.3b 5-H C-3, C-6, C-7a C-5 121.3 5-H 7.107.06 dd, J = 8.5; 4-H; 7-H C-3a, C-7 1.7b C-6 113.6 C-7 113.7 7-H 7.51 s 1-H; 5-H C-3a, C-5 C-7a 136.7

C-8 28.7 8-H2 3.282.97 2-H; 9-H C-2, C-3, C-3a, C-9 C-9 55.3 9-H 4.124.05 m 8-H; 10-H C-3, C-11 10-H 8.15 s 9-H C-12, C-14 C-11 167.3 C-12 52.9 12-H 3.032.97 m 13-H; 15-H C-14, C-16 13-H 7.92 s 12-H C-9, C-11 C-14 167.9

C-15 29.2 15-H2 1.551.48 m 12-H; 16-H

C-16 23.4 16-H2 1.471.24 m 15-H; 17-H

C-17 40.2 17-H2 3.032.97 m 18-H 18-H 7.45 t, J = 5.4 17-H C-19, C-15 C-19 156.6

20-/21- H3 7.30-6.75 br a b Correlations of protons in COSY and HMBC spectrum (300 MHz, DMSO-d6). Matching J values could not be obtained because of slightly overlapping signals.

2.3. Rearing of barnacle cypris larvae The rearing of cypris larvae was performed as described by (Berntsson et al., 2000a). Nauplius larvae of B. improvisus were collected from spawning adults and kept in 20L buckets with aerated FSW (0.2 Pm, salinity 30.0‰) and antibiotics (streptomycin 36.5 g L-1 and penicillin G 21.9 mg L-1). The nauplius larvae were fed with the diatom Thalassiosira pseudonana and the prymnesiophyte Isocrysis galbana and the water (FSW) in the rearing containers was changed every third day. When rearing containers are kept at 27-28qC, the development into cypris larvae takes 6-7 days. The newly

26 moulted cyprid larvae were filtered through a stack of sieves with mesh sizes of 320 Pm, 230 Pm, 160 Pm and 100 Pm. Cyprids were washed thoroughly to remove algae and detritus before being used in settlement experiments. The barnacle rearing at Tjärnö Marine Biological Laboratory is shown in Figure 5.

Figure 5. Picture of the laboratory rearing of the barnacle Balanus improvisus.

2.4. Experimental design Barettin and 8,9-dihydrobarettin were tested for their ability to inhibit settlement of B. improvisus cyprids. The two compounds were dissolved in FSW to give the desired concentration series (0.1-100 µM). The different concentrations of the compounds were added to Petri dishes (10 mL in FSW) in four replicates. Dishes containing FSW (10 mL) served as controls. Competent cyprids (20r2 individuals) were added to each dish and the dishes were maintained for 3-4 days at room temperature after which they were examined under a stereo-microscope for 1) attached and metamorphosed, 2) living cyprids and 3) dead cyprids. Figure 6 shows the experimental set-up of the barnacle settlement assay and also, the different stages in the settlement process from cyprid to juvenile.

Figure 6. The experimental set-up for the cyprid assay and also, pictures showing the sequential events taking place in the dishes: from cyprid to a newly metamorphosed juvenile barnacle of B. improvisus. The settlement assay is typically concluded after 3-4 days and effects of fractions or purified compounds is evaluated.

27 2.5. Synthesis of barettin and 8,9-dihydrobarettin To the best of our knowledge and based on extensive literature searches, barettin and 8,9-dihydrobarettin are probably the first reported naturally- derived compounds with antifouling properties to have been synthesized (Johnson et al., 2004). They consist of the two amino-acids tryptophan and arginine and form a cyclic dipeptide. The main characteristics of the aa:s are the indole unit in the tryptophan and the guanidine unit in the arginine. These chemical units are exclusive for these aa and provide a unique chemistry to the molecules, enabling them to interact with physiological targets such as enzymes and receptors. However, the indole and the guanidine may pose problems when resolving the synthesis of compounds containing these units (Rzeszotarska and Masiukiewicz, 1988). This is particularly true for the guanidine group in arginine. Because the guanidine group in the arginine has highly nucleophilic properties it must be carefully protected during synthesis. There are few options of groups to be used for protecting arginine (although several are found for indole). Commonly, arginine is protected with a nitro group or by protonation (Gish and Carpenter, 1953) but these protection methods have proven not fully reliable (Rzeszotarska and Masiukiewicz, 1988). For example, problems with deprotection and solubility arise upon using these methods. An emerging avenue for arginine protection is instead the guanylation of the G-amino groups of ornithine-containing precursors used when synthesizing arginine- containing peptides. This approach eliminates many problems of the standard protocols (Wu et al., 1993). Thus, for the synthesis of barettin and 8,9-dihydrobarettin a protected arginine derivative, ND-(tert-butoxycarbonyl)-NZ, NZc-bis(tert-butoxycarbon- yl)-L-arginine was prepared (paper II). When synthesizing the unsaturated analog of barettin, 8,9-dihydrobarettin, this protected arginine could be used in standard protocols with 6-bromotryptophan methyl ester. It was however, not possible to dehydrogenate 8,9-dihydrobarettin into barettin and hence, a different scheme had to be used for the synthesis of barettin (paper II). To introduce the double bond in barettin we decided toprepare phosphonoglycinate. Phosphoglycinate was prepared by hydrogenolysis of the known methyl 2-benzyloxycarbonyl-amino-2-(diethoxyphosphinyl)- acetate coupled to the protected arginine mentioned above. A subsequent Horner-Wadsworth-Emmons (HWE) reaction with 6-bromo-1-(tert- butoxycarbonyl)-indole-3-carboxaldehyde yielded compound (1) in Figure 8. The Boc-protecting groups were removed with TFA, and cyclisation was performed according to standard protocols to yield barettin, compound (2) in Figure 7.

28 (1) (2)

O O

OMe NH H HN O HN N NH2 Br N Br N Boc H H N NHBoc O NH BocHN NBoc Figure 7. The final step in the synthesis of barettin. The Boc-protecting groups of compound (1) were removed by treatment with TFA. After that cyclisation was performed in refluxing 1-butanol with 0.1 M acetic acid to yield barettin (2).

2.6. Methods in field experiments Generally, there is a lack of data describing the substance of the marine coatings e.g. solvents, pigments and additives, that are available on the market today. This problem makes it more difficult to evaluate the results obtained in field experiments. In the field experiment in paper III, barettin and 8,9-dihydrobarettin were included in different concentrations in four different, commercially available non-toxic marine paints. The concentrations tested as well as the technology of the different coatings, as stated by the manufacturers, are listed in table 2. Panels were painted in replicates (n=4) with the different treatments. Panels coated with the respective paint without the addition of barettin or 8,9- dihydrobarettin served as controls. The panels were randomly placed on racks and deployed in a bay outside Tjärnö Biological Laboratory (58q53’N, 11q8’E) at depths between 0.3 and 1.1 m, so as to avoid a possible interaction with depth. The experiment was maintained for an 8-week period from the beginning of July to the end of August, representing a period with intense settlement of the barnacle B. improvisus and the blue mussel Mytilus edulis (Protobranchia, Lamellibranchia) in Swedish waters. When the experiment was terminated the panels were brought to the laboratory and the recruitment of B. improvisus and M. edulis was evaluated.

2.7. Analogs of the dipeptides barettin and dipodazine

2.7.1 Peptide synthesis The synthesis of barettin is both time-consuming and highly expensive (60 000 € kg-1, in large scale) (Jonsson, 2005) In order for the molecule to find use as a commercial antifouling additive, a more cost effective synthesis needs to be developed. Because such a synthesis is not within reach in the very near future, two sets of analogs to barettin were designed and tested for their antifouling effect. In the first analog series, the barettin skeleton was

29 modified in the tryptophan residue and resulted in 5-bromobarettin (3) and debromobarettin (4) (Figure 8). Barettin has a structural resemblance to a natural product named dipodazine isolated from the fungi Penicillium dipodomys (Sörensen, 1999) and recently synthesized (Johnson et al., 2002). Thus, the other series of analogs made use of this compound as a starting- point. These were dipodazine (5), 5-bromodipodazine (6), 5- methoxydipodazine (7), 5-nitrodipodazine (8), 6-chlorodipodazine (9), 5- methyldipodazine (10), benzo[e]dipodazine (11), 3-[1-benzothiophen-2-yl- methylidene]-piperazine-2,5-dione (12), 3-[1-(6-bromo-1H-indol-3-yl)- meth-(E)-ylidene]-hexahydro-pyrrolo-[1,2-a]pyrazine-1,4-dione (13), 3-[1- (6-bromo-1H-indol-3-yl)-meth-(Z)-ylidene]-hexahydro-pyrrolo[1,2-a]pyr- azine-1,4-dione (14), 6-bromo-1H-indole-3-carboxaldehyde (15) and benzo[g]dipodazine (16) (Figure 8). For characterization of the analogs NMR, IR, melting points and MS were employed. The NMR data were obtained at 300 MHz for 1H and at 75 MHz for 13C. The IR spectra were obtained with FT-IR equipment. The melting point was established by capillary method. The MS data were acquired on a Thermo-Finnigan and the MW was determined with one decimal (paper IV). The cyprid settlement assay was performed as outlined in paper I.

O O X NH NH H H HN N NH2 HN N NH Y N Br N 2 H H O NH O NH 1 X = H, Y = Br 2 3 X = Br, Y = H 4 X = H, Y = H

O O O X NH NH HN HN HN NH Y N N S H H O O O

5 X = H, Y = H 11 12 6 X = Br, Y = H O O 7 X = MeO, Y = H 8 X = NO2, Y = H N H 9 X = H, Y = Cl HN 10 X = Me, Y = H Br N Br N H H O O 13 E isomer 15 14 Z isomer NH HN N H O

16 Figure 8. Chemical structures of the different analogs of barettin and dipodazine, where barettin is (1) and dipodazine is (5).

30 2.8. Receptor ligand binding assay As mentioned above, the work by England et al (1998) describing the interaction of the V-conotoxin containing 6-bromo-tryptophan residues with the serotonin ion channel receptor, i.e., 5-HT3, prompted us to investigate the possible ligand binding properties of the barettins to 5-HT receptors. The natural ligand to these receptors namely serotonin has an indole amine pharmacophore but is substituted in the 5th position, i.e. 5-hydroxy- tryptamine. Thus, the barettins were isolated and their chemical structures were confirmed as outlined as in paper I. The purified compounds were tested for their ability to displace radiolabeled high-affinity ligands to ten different 5-HT receptor subtypes. Membranes were prepared from human embryonic kidney-293 (HEK-293) cells, which were transfected with the selected subtypes of human serotonin receptors. The following serotonin receptor subtypes were prepared from a glycerol stock: 5-HT1A, 5-HT1D, 5- HT1F, 5-HT2A, 5-HT2C, 5-HT3A, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7A as outlined in paper V. The following radioligands were used in the binding assay; >N-methyl-3H@ LSD, >9-methyl-3H@ BRL-43694, >N-methyl-3H@ GR113808 and >1,2-3H@ 5-carboxamidotryptamine. Non-specific radioligand binding was defined by 10 PM 5-hydroxytryptamine. All experiments were performed in triplicates. After the first activity screening, it was found that the barettins specifically interacted with 5-HT2A, 5-HT2C and 5-HT4. Therefore, saturation studies were conducted on HEK-293 cells expressing 5-HT2A, 5-HT2C and 5- 3 HT4 receptors with increasing concentrations of radioligand (>N-methyl- H@ 3 LSD for 5-HT2A, 2C; >N-methyl- H@ GR113808 for 5-HT4) and a fixed concentration of 400 PM 5-hydroxytryptamine. 10 Pl 5-hydroxytryptamine or 10 Pl buffer were added to each sample to determine non-specific and total binding (paper V).

31 3. Results and discussion

3.1. Settlement inhibition displayed by barettin and 8,9- dihydrobarettin Marine natural sources are beginning to be explored for environmentally non-hazardous antifouling compounds (Nagata et al., 2001; Rittschof et al., 2003; Stupak et al., 2003; Kanagasabhapathy et al., 2004). These research efforts are guided by ecological interactions in an attempt to “to let nature itself solve the problem”. Indeed, the two cyclopeptides barettin and 8,9- dihydrobarettin, from G. barretti, were isolated and structurally elucidated by their ability to inhibit settlement of barnacle larvae of B. improvisus (paper I).

A) B) O O

H H N NH 2 N NH 2 Br N Br N H H O NH O NH Figure 9 A and B. The chemical structures of the two congeneric cyclopeptides: A) barettin and B) 8,9-dihydrobarettin.

Barettin and 8,9-dihydrobarettin (Fig. 9 A and B) inhibit settlement of B. improvisus in a dose-response manner between 0.1 and 100 µM (Figure 10 A and B). The EC50 value, i.e., the concentration at which the settlement was 50% of that in the control dishes, of barettin was determined to 0.9 µM and at 1.9 µM the settlement inhibition was complete (Fig. 10 A). The potency of 8,9-dihydrobarettin was approximately one order of magnitude lower (Fig. 10 B) and the corresponding EC50 was determined to 7.9 µM. A study, that fortifies that G. barretti may be a key organism in the search for new antifouling compounds, examined several marine sponges for their associated free-living and sessile fauna (Klitgaard, 1995). This study showed that G. barretti was by far the least fouled sponge both in the number of organisms and in the area fouled.

32 Settlement % Living (swimming) larvae % Dead larvae % A) barettin B) 8,9-dihydrobarettin 100 100 80 80 60 60 40 40

Response % 20 20 0 0 Control 0.12 0.24 0.48 0.95 1.9 19 Control 0.79 7.9 79 Concentration [µM] Concentration [µM]

Figure 10. The effect of A) barettin and B) 8,9-dihydrobarettin on the settlement of cypris larvae of B. improvisus as reflected by percentages for settled, living, not settled and dead cyprids presented as means r SE (n=4).

Our results thus indicate a possible role for these compounds as defence agents against fouling growth. There might be other effects of these substances, for example, they may act as feeding deterrents in a defence against predators. However, no such studies have as yet been performed.

3.2. Synthesised barettins as antifouling compounds When testing the synthesized barettins, the effects in the cyprid settlement assay of these substances were identical to the effects of the native barettin and 8,9-dihydrobarettin (Johnson et al., 2004). The EC50-values of synthesized barettin and 8,9-dihydrobarettin were 0.9 µM and 7.9 µM, respectively. Data acquired with NMR and mass spectrometry demonstrated identical molecular weights and MSn fragmentations for the synthesised compounds as compared to the native compounds. Both native and synthesized compounds displayed a reversible effect when tested in the cyprid settlement assay. After the 48h exposure to native or synthesized barettin or 8,9-dihydrobarettin, cyprids were washed and transferred to fresh FSW. The settlement of the cyprids pre-exposed to native or synthetic barettin or 8,9-dihydrobarettin did not significantly differ from the settlement in the control dishes. This indicates that the compounds do not exert their action through an irreversible mechanism (paper I and Paper II).

3.3. Field experiment to evaluate antifouling activity Deploying artificial panels in the sea is an often-used method for studies of the recruitment and succession of benthic organisms in the field (Chalmer, 1982; Henrikson and Pawlik, 1995; Hills and Thomason, 1998; Berntsson

33 and Jonsson, 2003). It is also common to use artificial static panels for studying the effects of various factors on fouling organisms for the purpose of developing antifouling methods (Roberts et al., 1991; Swain and Schultz, 1996; Berntsson et al., 2000b; Dahlström et al., 2000). Even if the artificial panels are not natural substrates for settling marine organisms, they make it possible to vary for example, concentrations of bioactive ingredients, surface chemistry, surface geometry as well as coatings types. Previously, we have shown in in vivo laboratory experiments that there is a strong effect on the settlement and metamorphosis of cypris larvae of B. improvisus of the brominated cyclopeptides barettin and 8,9-dihydrobarettin, isolated from the marine sponge G. barretti (Paper I). However, laboratory experiments are very different from field conditions. Thus, in Paper III, we wanted to examine whether barettin and 8,9-dihydrobarettin displayed preserved antifouling activity when subjected to field conditions. Because the synthetic compounds display identical settlement inhibitory effects as the native compounds (papers II and III), these were used in four different marine paints subjected to field conditions. The results obtained in paper III provide initial information on the applicability of the brominated cyclic peptide barettin and its structural congener 8,9-dihydrobarettin as antifouling agents in marine paints. We show that these compounds display a significant antifouling effect over an eight-week period when subjected to field conditions.

Table 2. The reduction in recruitment of the barnacle B. improvisus when adding barettin or 8,9-dihydrobarettin in four different commercially available paints. The results are presented as percent reduction compared to control panels over 8 weeks of field exposure.

Paint Type barettin 8,9-dihydrobarettin

0.1% 0.01% 0.1% 0.01%

SPF SPC 89*** 67** 61* 43

FabiEco SPC 49 45 69*** 59*

TF Solid paint/weak SPC 17 25 -1 -10

H2000 Solid paint/weak SPC 23 28 20 46

*** = p<0.001, ** = p=0.001-0.01, * = p=0.01-0.05. SPC=Self-Polishing Copolymer.

The results presented in paper III show that there is a strong effect of the paint used in combination with barettin and 8,9-dihydrobarettin. Of the four different coatings used in paper III, the self-polishing coating SPF by far displayed the most promising results. When barettin or 8,9-dihydrobarettin was incorporated in the SPF-paint, the recruitment of B. improvisus was

34 strongly reduced in response to both of the compounds as compared to control panels (table 2). The blue mussel M. edulis is also a major fouling organism in Swedish waters (Berntsson and Jonsson, 2003), and therfore, we also checked the test panels with barettin and 8,9-dihydrobarettin for their ability to affect the recruitment of M. edulis. The results obtained for the recruitment of M. edulis in response to barettin and 8,9-dihydrobarettin included in the SPF-paint, show results similar to those for the recruitment of B. improvisus. Both 0.1 and 0.01% barettin as well as 0.1% 8,9- dihydrobarettin strongly reduced recruitment of M. edulis when included in the SPF-paint (table 3). However, the concentration of 0.01% 8,9- dihydrobarettin did not significantly affect the B. improvisus or the M. edulis recruitment in the SPF-paint as compared to the controls.

Table 3. The reduction in recruitment of the blue mussel M. edulis when adding barettin or 8,9-dihydrobarettin in four different commercially available paints. The results are presented as percent reduction compared to control panels over 8 weeks of field exposure.

Paint Type barettin 8,9-dihydrobarettin

0.1% 0.01% 0.1% 0.01%

SPF SPC 81*** 83*** 72** 52

FabiEco SPC 47 51 67** 50*

TF Solid paint/weak SPC 6 51* 4 -28

H2000 Solid paint/weak SPC 12 26 39 53*

*** = p<0.001, ** = p=0.001-0.01, * = p=0.01-0.05. SPC=Self-Polishing Copolymer. In conclusion, the results presented in paper III show that of the four different coatings used, the self-polishing coating SPF displayed the most promising results. When barettin and 8,9-dihydrobarettin were incorporated in this paint, there was a significant reduction of recruitment of both B. improvisus and M. edulis compared to the controls. However, the recruitment of B. improvisus to control panels was one order of magnitude higher than the recruitment of M. edulis. This may reflect the relative abundance of larvae of the respective species in the water. Still, the results of the field test, presented in paper III indicate that barettin and 8,9- dihydrobarettin are promising candidates as non-toxic antifouling agents against B. improvisus and M. edulis when these substances are used in a self- polishing coating.

35 3.4. Analogs as antifouling compounds Because the synthesis of the barettins, in particular barettin, proved to be highly costly on a large scale, a set of analogs were designed and synthesized in an attempt to find more cost-efficient molecules with preserved or improved bioactivity. In addition, the synthesis of analogs enabled an investigation of structure-activity relationships through the modification of the barettin pharmacophore. As mentioned above, we included the natural product dipodazine and analogs thereof in this study, due to the strong structural resemblance of dipodazine to the barettins.

Table 4. Summary of the EC50 values of settlement inhibition and stimulation on the barnacle Balanus improvisus of analogs of barettin and dipoazine including F-values and p-values.

Substance MW EC50 p-value F-value *(I)/(S) (µM barettin (1) 421 0.9 0.0001 22.7 I 8,9-dihydrobarettin (2) 423 7.9 0.03 7.3 I 5-bromobarettin (3) 421 4.1 0.0001 21.5 S debromobarettin (4) 342 *--- 0.34 1.7 --- dipodazine (5) 241 --- 0.87 0.03 --- 5-bromodipodazine (6) 320 5.8 0.008 9.4 I 5-methoxydipodazine (7) 271 --- 0.24 1.5 --- 5-nitrodipodazine (8) 286 --- 0.1 2.9 --- 6-chlorodipodazine (9) 276 --- 0.24 1.5 --- 5-methyldipodazine (10) 255 --- 0.53 0.4 --- benzo(e)dipodazine (11) 291 1.5 0.0001 31.2 I 3-[1-benzothiophen-2-yl-methylidene]- 258 --- 0.4 0.6 --- piperazine-2,5-dione (12) 3-[1-(6-bromo-1H-indol-3-yl)-meth- 361 2.4 0.043 8.5 I (E)-ylidene]-hexahydro-pyrrolo[1,2- a]pyrazine-1,4-dione (13) 3-[1-(6-bromo-1H-indol-3-yl)-meth- 361 --- 0.5 0.5 --- (Z)-ylidene]-hexahydro-pyrrolo[1,2- a]pyrazine-1,4-dione (14) 6-bromo 1H-indole-3-carboxaldehyde 225 6.7 0.0004 20.1 I (15) benzo(g)dipodazine (16) 291 0.034 0.0001 11.8 I

--- = no significant effect on settlement of B. improvisus larvae. “I” denotes inhibition and “S” stimulation. The F-values and the p-values were obtained in a 1-factor ANOVA and in Means Comparison Contrast (MCC) tests to detect significant settlement differences between the treatments of different analogs.

36 The analogs can be divided in two main groups: the barettin group and the dipodazine group. In Table 4, the effects of the different analogs on the settlement of B. improvisus larvae are summarized. In the barettin group two analogs were synthesized namely 5-bromobarettin (3), which significantly stimulated settlement (EC50: 4.1 µM), and debromobarettin (4), which did not display any significant effect, either stimulatory nor inhibitory, on the settlement of cypris larvae. In the dipodazine group, comprising the remaining 12 compounds, five of the analogs displayed significant settlement inhibition (6, 11, 13, 15, and 16) ranging from 1.5 to 6.7 µM (Table 4). The most promising candidate of the analogs developed was benzo[g]dipodazine (16) with an EC50 value of 0.034 µM (Fig. 11 A). This compound also displayed a reversible mechanism. When cyprids pre- exposed for 24h to 34 µM benzo[g]dipodazine were washed and transferred to FSW, there was a significant recovery of settlement (50% settlement) as compared to larvae that remained in 34 µM benzo[g]dipodazine (Fig. 11 B).

A) B) 100 100 O

80 NH 80 HN N H 60 O 60

40 40

20 Settlement (%)

Settlement (%) 20

0 0 Control, Control 0.034 0.34 3.4 34 Control, Control, 34 µM 34 µM, FSW DMSO FSW DMSO transferred (0.1%) Concentration [µM] (0.1%) to FSW Treatment Figure 11 A and B. Effect of benzo(g)dipodazine on the settlement of B. improvisus cyprids (A). The lethal effect of benzo(g)dipodazine has not yet been established. The response variables are percentage settlement shown as means r SE (n=4). B) The reversible effect of benzo(g)dipodazine. Dishes in which the cyprids were maintained in the effective concentration of benzo(g)dipodazine (34 µM) throughout the experiment are denominated 34 µM. Dishes in which cyprids were exposed to the effective concentration (see above) of benzo(g)dipodazine for 24 h and after 24 h they were washed and transferred to seawater are denominated 34 µM, transferred to FSW. The response variables are percentage settled, non-settled and dead cyprids shown as means r SE (n=4).

The remaining dipodazine compounds (5, 7, 9, 10, 12, and 14) did not significantly affect settlement of B. improvisus cyprids (Table 1) (Paper IV). A great number of natural products, from both terrestrial and marine sources, have been isolated and characterized. In several cases, compounds with promising bioactivities have been used as the framework for synthesis of active analogs in a bioassay-guided approach (Cichewicz et al., 2005; Oberthur et al., 2005). In paper IV, we employed a bioassay-guided

37 approach to find structure-activity relationships of barettin and dipodazine analogs. We focused mainly on how changes in the substitution in the indole nucleus would affect bioactivity. When evaluating the effects of the 14 analogs in the cyprid assay we found that five of the analogs had moderate but significant settlement inhibitory effects (6, 11, 13, 15). In addition, we found that the barettin analog 5-bromobarettin (3) had a stimulatory effect on larval settlement (Paper IV). One compound was singularly effective in inhibiting barnacle larval settlement, namely benzo[g]dipodazine (16). This compound displayed an EC50 value of 0.034 µM, which represents a 20-fold increase in effective concentration as compared to barettin. In our strategy to synthesize analogs with preserved or improved bioactivity we first focused on the significance of the 6-position bromine in the indole residue of tryptophan in barettin (1). The assumption that substitution in this particular position may alter bioactivity was based on the results presented by England et al. (1998) on peptides from the gastropod Conus sp. In this study, V-conotoxin consisting of 6-bromotryptophan and an arginine residue, specifically interacted with the ion channel receptor 5-HT3. Also, directing bromine to the 6th position of the indole residue in the synthesis is difficult and expensive, whereas bromination in the 5th position is easily achieved and consequently less expensive. Altering the position of bromine to the 5th position instead of the 6th position (5-bromobarettin (3)) resulted in a significant stimulation of settlement (Table 4). Removing the bromine atom entirely from the barettin scaffold (debromobarettin, (4)) resulted in a total loss of the inhibitory effect on larval settlement. In a second step analogs were synthesized from the dipodazine scaffold (5) (Fig. 8), a diketopiperazine isolated from Penicillium dipodomyis (Sörensen et al., 1999) and synthesized by Johnson et al., (2002). The aim of using the dipodazine molecule as a starting point for analog synthesis was partly to test the effect of removing the arginine residue and partly, because this molecule allows easier additions of substituents to the indole moiety. The most promising dipodazine analog, benzo[g]dipodazine, has an increased bioactivity compared to barettin. This compound is probably more hydrophobic than barettin and thus, is more readily to penetrate the hydrophobic chitinous shell of the cyprid. A more hydrophobic compound also has better tissue-penetration when the cyprid outer shell has been passed, enabling the molecule to interact with its molecular target. It is hypothesized that the phenyl in the 6-7-position in benzo[g]dipodazine could act similar to the bromine in the 6-position in barettin (Paper IV). On the basis of the results in paper V (discussed below) we have begun to examine the affinity of the analogs, which displayed bioactivity in the cyprid settlement assay, for the subtypes of serotonin receptors described in paper V. The effects of these compounds in the settlement assay using B. improvisus larvae (paper IV), i.e., settlement EC50 values, are in good

38 agreement with results obtained in the serotonin receptor assay (Hedner and Sjögren et al, manuscript in prep).

3.5. Barettins as selective serotonin ligands The dose-dependent effect of barettin and 8,9-dihydrobarettin in the barnacle cyprid settlement assay as well as their reversible mechanism of action on settlement, raise questions as to these molecules’ endogenous targets. The dose-dependent effect indicates that the target may be a receptor protein. Studies were therefore undertaken to investigate the possible interactions of barettin and 8,9-dihydrobarettin with mammalian receptors (5-HT-receptors have not yet been found in B. improvisus), primarily ion- channel receptors and G- protein-coupled receptors. Based on the finding by England et al., (1998) that the 6-bromotryptophan containing V-conotoxin GVIIIA purified from the venomous cone snail Conus geographus produced a complete inactivation of the excitatory serotonin 5-HT3 receptor (ion channel receptor) and also, because of the indole pharmacophore of the natural ligand to 5-HT receptors, serotonin [5-hydroxy tryptamine], we commenced the search for the molecular target of the barettins by investigating their effect on serotonin receptors. Serotonin receptors can be divided into seven major subtypes and these are 5-HT1, 5-HT2, 5-HT3, 5- HT4, 5-HT5, 5-HT6, 5-HT7, where 5-HT1 and 5-HT2 are further divided into 5-HT1A-F, and 5-HT2A-C. All serotonin receptors except 5-HT3 are G protein- coupled receptors. The G protein-coupled receptors (GPCR:s) belong to the rhodopsin-like receptor superfamily. GPCR:s are involved in chemosensation, in neuromodulation and in the transmission of the action of neurohormones. Ligands interacting to GPCRs can be both physical, i.e. photons, and chemical (Brody and Cravchik, 2000). These receptors are a highly important group as targets for drug action. It is currently estimated that approximately 60% of the drugs prescribed today act through G protein- coupled receptors (Wilson et al., 1998). Natural ligands to the GPCR family include all the major biogenic amines, i.e., serotonin, dopamine, noradrenaline, adrenaline, histamine and gamma-butyric acid (GABA). Approximately a thousand GPCRs have been found to date and more are continuously being discovered. The GPCRs family is highly conserved in its protein structure (Hamm, 1998; Meeusen et al., 2003), and representatives have been found in simple eukaryotes like yeasts and molds (Peroutka and Howell, 1994).

39 The marine sponge G. barretti produces the two dipeptides, barettin and 8,9-dihydrobarettin (paper I). There is approximately one order of magnitude difference in the inhibitory effect on settlement of B. improvisus larvae between the two compounds (0.9 and 7.9 µM, respectively). This difference in bioactivity is reflected in their ability to displace high-affinity ligands to 5-HT receptors. Barettin selectively interacted with the serotonin receptors 5-HT2A, 5-HT2C and 5-HT4 with the corresponding Ki values being 1.93 PM, 0.34 PM and 1.91 PM, respectively (Figs 3 and 4). Barettin interacted with these receptors at concentrations close to that of endogenous serotonin. Previously reported affinity values for serotonin are: Ki 0.32 µM at the 5- HT2A receptor and Ki 0.13 µM at the 5-HT2C receptor (Almaula et al., 1996). When investigating 8,9-dihydrobarettin, it was found that 8,9- dihydrobarettin only interacted with the 5-HT2C receptor, the Ki value for this interaction being 4.63 PM (Fig 3) (Paper V). In addition, 8,9-dihydrobarettin did not show affinity to either 5-HT2A nor 5-HT4, which indicates that the double bond between the tryptophan and arginine residue plays an important role in the interaction with the receptor proteins. Serotonin, the endogenous ligand to 5-HT receptors is biosynthesised from tryptophan and is substituted in the 5-position (hydroxyl) in the indole nucleus. Thus, for substances interacting with 5-HT receptors, like barettin and 8,9-dihydrobarettin, the indole nucleus and the possibly the substitution in the 6th position are expected to be of importance. 5-HT receptors are ancient receptors and serotonin is present in yeasts and molds (Peroutka and Howell, 1994) as well as in most metazoan species, except possibly the Porifera (Walker et al., 1996). It has also been established that a G protein-coupled 5-HT receptor with DNA sequence homologies to mammalian 5-HT1 receptors is present in the barnacle B. amphitrite (Kawahara et al., 1997), closely related to B. improvisus. However, this barnacle serotonergic GPCR has not been functionally cloned, nor has its pharmacology, e.g., affinity of different ligands and activation of a subsequent intracellular signal pathway, been established. However, it is highly likely that 5-HT receptors are present also in B. improvisus, and we suggest that the effect of barettin (1) and 8,9-dihydrobarettin (2) is exerted through specific ligand-binding mechanisms to serotonin receptors, possibly 5-HT2 receptors. Furthermore, studies have been performed that illustrate the importance of serotonin in the settlement and subsequent metamorphosis of B. amphitrite (Yamamoto et al., 1996; 1998; reviewed by Dahlström and Elwing, in press). In pharmacological assays using cypris larvae of B. amphitrite, different serotonin receptor agonists and antagonists were used to screen for effects on both attachment and metamorphosis. The pharmacological interpretation, as suggested by Clare and Matsumura (2000) was employed, in which the settlement process of barnacle larvae can be separated pharmacologically into attachment and metamorphosis. The substance most effective in enhancing attachment was the 5-HT3 agonist 5-

40 HTQ, which increased attachment in the submicromolar concentrations varying from 0.1-10 µM. Yamamoto et al., (1999) also tested the effect of 5- HT2 antagonists where the 5-HT2 antagonists cyproheptadine (Fig. 12) and LY-53 857 were the most efficient in reducing attachment and metamorphosis of cyprids (100 nM). When using cyproheptadine at 10 µM, a significant increase in metamorphosis without prior attachment was noted. This was only observed at this concentration.

Figure 12. The chemical structure of the non- specific 5-HT2 antagonist cyproheptadine. Reported to inhibit settlement of cypris larvae of the barnacle B. amphitrite at 100 nM. Cyproheptadine is also a migraine N prophylactic

CH3

Based on these results, Yamamoto et al., (1999) suggested that 5-HT2 receptors might be particularly important in the linkage between attachment and metamorphosis. 5-HT2 receptors in mammals are coupled to an intracellular signal pathway, which increases cellular levels of diacylglyceride (DAG) and inositol-tri-phosphate (IP3). To date, no attempts have been made to characterise the intracellular signal pathway involved in the settlement inhibition displayed by the 5-HT2 antagonists tested on B. amphitrite. Yamamoto et al. (1999) also established the presence of serotonin in cypris larvae by analyzing larval tissue with high-performance liquid chromatography (HPLC) with electrochemical detection. The concentration of serotonin per larvae was quite high, approximately 0.5 ng per cyprid. The available evidence supports the involvement of serotonin in the settlement process of the barnacle B. amphitrite and studies are on under way to establish the role of serotonin also in B. improvisus (Zega, Dahlström et al., submitted manuscript) It is desirable to gain more knowledge of the pharmacology of the serotonin receptors involved in the settlement process, primarly to functionally clone the receptor(s) involved and to establish its pharmacological profile, as well as the intracellular signal pathways involved.

41 Concluding remarks

The aim when commencing this project was to acquire a deeper understanding of the mechanisms involved in the fouling-free body surface of the marine sponge G. barretti. That aim implicated chemical as well as ecological and molecular tasks. The hypothesis was that the fouling-free body surface of G. barretti was the result of a chemical defence directed against foulers and other competitors including predators. A chemical defence that successfully combats fouling attempts and predator attacks necessarily must rely on 1) potent molecules continuously produced by the animal in such amounts that hydrodynamic mixing and dissolution play minor roles in the molecule’s effective interface, 2) molecules that are lipophilic or amphiphilic by their nature and thus do not easily dissolve in the surrounding water but may remain close to the body surface of the producing organism where their action is exerted, 3) production of the defensive compound that has a high degree of plasticity and may be induced upon attacks, and 4) defence constituted of several compounds acting in concert to achieve the desired effect at lower concentrations than each compound alone could achieve, i.e., a synergistic action. These are all complex issues and need to be approached with consideration. Within the work comprising this thesis, a broad knowledge platform has been created that will constitute a valuable basis for the continuing search for answers regarding mechanisms underlying defences mediated by chemical compounds in the marine environment and their underlying mechanisms. This knowledge platform has been built up gradually, in a hierarchical manner, including bioassay-guided isolation of bioactive compounds, resolving the synthesis of complex diketopiperazine backbones, approaching the hard work of designing, developing and testing synthetic analogs with the aim of preserving or improving bioactivity until finally, resolving the molecular target of the compounds isolated. I present here some concluding remarks on the work conducted so far. During the continuing work with this thesis, from the initial anti- settlement tests of fractions from G. barretti to the isolation and structural elucidation of the two compounds responsible for the anti-settlement activity, the barettins, barettin and 8,9-dihydrobarettin, we did not fully understand the size and measure this project was to take. One of the major breakthroughs along the way was when the syntheses of the barettins were resolved. For barettin these efforts involved great intellectual challenges, the

42 result of which was a highly complex organic synthesis comprising 14 steps for obtaining the final product. The other barettin compound, lacking the double bond in the tryptophan residue, namely 8,9-dihydrobarettin, was a challenge as well but still, much easier to synthesize. Another important issue was to establish whether bioactivity could be preserved when the barettins were included in commercially available paints particularly designed for the harsh marine environment and also, the degree of bioactivity of the barettins under field conditions, with particular emphasis on their effects on two of the most aggressive fouling organisms in Swedish waters, the barnacle B. improvisus and the blue mussel M. edulis. In this particular study (paper III) an approximate 70-90% reduction of recruitment of both of the tested organisms was obtained. Again, barettin was the most effective substance, reflecting the results obtained in the static laboratory assays (papers I and II). We now, to a certain extent, had a proof-of-concept and an incitement for the development of a marine coating using barettin as a non-toxic and presumably biodegradable additive in non-hazardous antifouling coatings. Hence, it was disappointing when we realized that the synthesis of barettin was by far too expensive for this purpose. The cost for synthesizing barettin was estimated to 60 000 € kg-1 (in large scale). To achieve a 90% reduction of recruitment 1 g of barettin would need to be added to 1 L of paint. The mere cost for barettin in such a paint would be 60 €. The paint manufacturers set the limit for the cost of biocide/additive in the paint to approximately 2 €. But when learning about science one realizes that when one door closes another doors opens. Hence, because of the set back of the costly synthesis of barettin, we were encouraged to design and develop barettin analogs. In addition to the applied interest, the work with analog synthesis was scientifically challenging and held great promise to afford insights into structure-activity relationships of the barettin chemical scaffold. The results obtained also showed that small changes in for example the position of the bromine atom radically changed the bioactivity. When bromine was placed in the 5th position (5-bromobarettin) instead of the 6th (barettin) in the indole moiety of tryptophan, this gave rise to a significant stimulatory effect in the cyprid settlement assay. Studies are now under-way to determine the effect of 5-bromobarettin in the serotonin receptor assay. During the work of developing and testing analogs of barettin and dipodazine, we found one compound (benzo[g]dipodazine) that was even more effective than barettin in inhibiting settlement of B. improvisus. In addition, this compound demonstrated the same reversible mechanism of action as barettin and also had the good fortune of being much less expensive to synthesize. In parallel to the work with the analogs, we initiated studies to determine the molecular target of the barettins in order to take one step closer to an understanding of their mechanism of action in the cyprid settlement assay.

43 Based on the indole pharmacophore of the barettins, we chose to start to investigate a possible serotonin receptor affinity. The results we obtained clearly point to a specific affinity of barettin for 5-HT2A, 5HT2C, and 5-HT4 receptors. 8,9-dihydrobarettin only displayed affinity for the 5-HT2C receptor. There is to date limited information about the role of 5-HT2 receptors in invertebrates. It has been suggested that a 5-HT2-like receptor in the fruitfly Drosophila melanogaster is highly important in embryological development. The 5-HT2A receptor is widely distributed in peripheral and central tissues in mammals, i.e., rats, mice and humans. These receptors mediate contractile responses in a series of vascular smooth muscle preparations. In central tissues 5-HT2A receptors are present in the cortex, claustrum and basal ganglia. The activation of 5-HT2A receptors results in a stimulation of hormone secretion, e.g., corticosterone, oxytocin, renin and prolactin. The 5-HT2C receptor, on the other hand is much more difficult to characterize. This is largely due to the lack of selective ligands for this receptor, because ligands interacting with 5-HT2C receptors very often also display selectivity for 5-HT2A receptors, like barettin in our study (paper V). Here, 8,9-dihydrobarettin might prove highly valuable as a pharmacological tool because it only interacts with 5-HT2C receptors. Barettin also interacted with the 5-HT4 receptor which is involved in regulating neuronal excitation and gastrointestinal motility. Recently, the 5-HT4 antagonist tegaserod was approved for clinical treatment of irritable bowel syndrome. Tegaserod displays a structural resemblance to the barettins (paper V). We are currently evaluating the barettin and dipodazine analogs for their ability to displace high-affinity ligands to serotonin receptor subtypes. With the knowledge of a specific molecular target of both the barettins and the analogs described herein, highly useful information will be gained in the development of new molecules to serve as pharmacological tools or be tested for different disease targets, i.e., as antipsychotic drugs (5-HT2A/5-HT2C) or for diseases related to the gut (5-HT4). There is also great promise in developing applied environmental methods, such as non-hazardous antifouling solutions, from the knowledge platform developed within this project. The research concerning natural products derived from marine organisms is still in its infancy but results obtained so far point to the ocean being a great source of unusual and intriguing molecules, quite different in their structure from their terrestrial counterparts. Marine natural products may find use in a wide range of areas and thus, maintaining marine biodiversity is pivotal for maintaining the unexplored chemical diversity in the world’s oceans. From a long-term perspective, this would benefit mankind and hold promise to provide sophisticated solutions to old as well as new problems.

44 Populärvetenskaplig sammanfattning

I den marina miljön är påväxt ett naturligt fenomen. Sporer av mindre alger sätter sig och tillväxer på större alger och larver av ryggradslösa fastsittande djur behöver ett ledigt substrat för att kunna sätta sig och genomgå metamorfos. Ytor som är fria från organismer, d.v.s är ”lediga”, är ofta en begränsande faktor i havet. En oönskad påväxt är den som uppstår på konstruktioner som människan introducerat i havet, såsom båtskrov, redskap i akvakultur, på och i pipelines och på oljeriggars nedsänkta delar. Påväxt skapar en rad problem för mänsklig aktivitet i marin miljö som t.ex ökad bränsleförbrukning och ökande förslitningsskador för fartygsindustrin Länge har vi människor skyddat våra marina konstruktioner genom att belägga skrovet med färger som innehåller starkt giftiga komponenter (fr.a. tenn och kopparföreningar). Båda dessa biocider är dessutom ackumulerbara i sediment och bryts ned mycket långsamt eller inte alls. Genom en resolution i FN-organet IMO (International Maritime Organisation) 1999 förbjöds användningen av den organiska tennföreningen tributyltenn (TBT). Det hade då larmats sedan 1980-talet om TBT:s mycket skadliga effekter på icke-målorganismer i den marina miljön, såsom sedimentlevande snäckor och musslor. Fartyg får sedan 2003 inte nymåla sina skrov med färger som innehåller TBT och användningen av koppar har starkt begränsats i flera EU- länder. Dessa förbud har skyndat på en utveckling mot nya påväxtskydd och många nya och nygamla biocider har dykt upp i de kommersiella produkterna. Tyvärr har flera forskningsrapporter visat att många av dessa ämnen är mycket skadliga för den marina miljön. Restriktioner eller förbud mot deras användning har således införts bl. a i Sverige, Storbritannien och Danmark. Två bioaktiva cyclodipeptider från det marina svampdjuret Geodia barretti, s.k. diketopiperaziner, (DKP:s) med inhiberande effekt på havstulpanlarvers settlingsförmåga (Balanus improvisus) har isolerats, strukturbestämts och syntetiserats. Dipeptiderna, barettin och 8,9- dihydrobarettin, består av de två aminosyrorna tryptofan och arginin som bildar en cyklisk ringstruktur. Ringstrukturen gör så att molekylen troligen får ökad stabilitet och därmed ej bryts ned så snabbt i naturen vilket är en viktig egenskap för att bibehålla molekylens effekt längre inblandad en marin färg.

45 Syntes av barettinerna (och för alla bioaktiva naturligt utvunna molekyler) är av största vikt då man inte kan skövla havet på den producerande organismen för att erhålla tillräcklig mängd av dessa ämnen. En annan del av projektet var att försöka bredda kunskapen om hur barettinerna fungerar d.v.s deras verkningsmekanism. Detta gjorde vi genom att syntetisera analoger till barettin. Analoger görs genom att förändra molekylen som t.ex.att byta ut eller flytta vissa delar i molekylen. I barettins fall t.ex ändra positionen av bromatomen och studera om egenskaperna som effekt på settlingsbeteendet hos havstulpanlarverna förändras. Vi syntetiserade 14 olika nya molekyler och kunde konstatera att små förändringar av molekylen fick dramatiska förändringar av effekten på settling hos B. improvisus larver. Vissa analoger hade ingen effekt alls medan en visade högre effektivitet än barettin hos larverna. Med dessa intressanta analogdata ville vi försöka nå en djupare förståelse för hur barettinerna utövar sin effekt på molekylär nivå. Eftersom barettin har stora likheter med serotonin eller 5-HT som denna signalsubstans också kallas. Serotonin finns hos alla djurgrupper med ett nervsystem, från enkla ryggradslösa djur till människor. Serotonin utövar sin funktion genom att binda in till receptorproteiner. Det finns idag 14 olika humana subtyper av serotoninreceptorer beskrivna. Således testades om barettinerna kunde binda in till olika typer av serotoninreceptorer. Serotonin är inblandade i många funktioner hos människor, som t.ex. depression, ångest och migrän, men hos ryggradslösa djur vet man väldigt lite om dess funktion. De få data som finns, tyder dock på att B. improvisus innehåller serotonin, och en molekyl som har förmågan att binda in till och inhibera en serotoninreceptor kan i så fall påverka settlingsförmågan. I vår studie med barettinerna fann vi en specific aktivitet på tre olika typer av 5-HT receptorer. Ur antifoulingsynpunkt är dessa data viktiga då man kanske kan lösa frågorna ur ett mer hållbart och miljövänligt perspektiv. Ett exempel är att designa molekyler som inte är skadliga för målorganismen utan fungerar repellerande (larverna kan simma därifrån och istället hitta en naturlig yta att sätta sig på). En annan aspekt är att det också är möjligt att designa molekyler som bryts ned naturligt och inte lagras upp i sediment och ackumuleras i närningskedjor under lång tid. Avslutningsvis och med det nya kunnandet om barettinernas specifika aktivitet kan dessa data appliceras även inom t.ex.medicin där de kan fungera som farmakologiska verkyg för att hitta nya och mer effektiva läkemedel.

46 Acknowledgement

This work was carried out at the Division of Pharmacognosy, Department of Medicinal Chemistry, Faculty of Pharmacy, Uppsala University. The field experiments were conducted Tjärnö Marine Biological Laboratory. I wish to express my sincere gratitude to: My supervisor Lars Bohlin for skillully guiding and supporting me through this interesting research field My assistant supervisor Per Johnson: for your great knowledge, for inspiring discussions, and for never-failing enthusiasm. Mia Dahlström:, your inspiration and your importance when it comes to solving difficulties are priceless. The late Lars Afezelius, with thanks for introducing me to this exciting field of research. Ulf Göransson:, for your valuable ideas and for your skillful help with HPLC and MS. I really enjoy working with you. Kent Berntsson, for all your help with barnacle issues. Tomas Lundälv:, for excellent Geodia barretti pictures Karl Anders Hagskiöld:, for technical support concerning my research. My co-authors and collaborators for their invaluable help: Jan Bergman, Rolf Andersson, Ann-Louise Johnsson, Erik Hedner, Hamid Shirani, Fred Nyberg and Per-Anders Frändberg My generous Pharmacognosy colleagues, present and former: Erika Svangård, Petra Lindholm, Sofia Lindgren, Ulrika Huss, Therese Lindholm, Anders Backlund, Erik Hedner, Anders Herrmann, Sonny Larsson, Jenny Pettersson, Josefin Larsson, Kerstin Ståhlberg and Maj Blad. All the generous and lovely personnel at TMBL:, especially, Eva Marie Rödström, Hans G Hansson, Martin Ogemark and all the PhD-students. The Chemical Ecology group at TMBL, especially Gunilla Toth, Henrik Pavia, Göran Nylund, Lena Granhag, Ann Larsson, Gunnar Cervin, Erik Selander and Fredrik Lindgren. Finn Sandberg, Wenche Rolfson, Jan Bruhn and the late Premila Perera, for interesting discussions concerning Pharmacognosy. Alla “goa gubbar och gummor” jag har fått lära känna under min tid på Tjärnö; Kent Berntsson, Gunilla Toth, Susanne Svensson, Lena Granhag, Anette Ungfors, Niklas Samuelsson, Patrik Bågenholm, Solveig van Nes Karin Bergström och alla andra.

47 Alla i innebandygänget; “vinna är inte en fråga om liv och död, det är mycket viktigare än så”. Min familj i Bovallstrand; min mor, far; min syster Annika med familjen Wilgot, Mauritz och Freja och min lillasyster Torun med sin dotter Isa. Vår labrador Tilda som under alla promenader inspirerat till många ideer Mia Dahlström, min livskamrat och bästa vän.

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57 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 32

Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy”.)

ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-6797 2006