ACTAUNIVERSITATISUPSALIENSISUPPSALA2007

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 63

Bioactive Compounds in theChemical Defence of MarineSponges

Structure-Activity Relationships and PharmacologicalTargets

ERIK HEDNER

ISSN 1651-6192ISBN 978-91-554-6971-9urn:nbn:se:uu:diva-8218

Dissertation presented at Uppsala University to be publicly examined in B7:113a, BMC,BMC, Uppsala, Friday, October 19, 2007 at 13:15 for the degree of Doctor of Philosophy(Faculty of Pharmacy). The examination will be conducted in English.

Abstract

Hedner, E. 2007. Bioactive Compounds in the Chemical Defence of Marine Sponges.Structure-Activity Relationships and Pharmacological Targets. Acta Universitatis Upsaliensis.Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy63. 54 pp. Uppsala. ISBN 978-91-554-6971-9.

Marine invertebrates, in particular sponges, represent a source of a wide range of secondarymetabolites, many of which have been attributed various defensive capabilities againstenvironmental stress factors. In this thesis sponge-derived low-molecular peptide-likecompounds and associated analogs are investigated for bioactivity and pharmacologicaltargets.The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol

-3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge Geodia barrettiand its ability to inhibit larval settlement of the barnacle Balanus improvisus was determined.With an EC

50value of 15 nM, this compound’s antifouling effect was higher than those of the

previously reported brominated dipeptides from Geodia barretti, i.e., barettin and8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible.However, the compound lacked affinity for 5-HT

1-7receptors, whereas barettin possessed

specific affinity to 5-HT2A, 5-HT

2Cand 5-HT

4, while 8,9-dihydrobarettin interacted with

5-HT4. In an attempt to evaluate structure-activity relationships synthesized analogs with

barettin and dipodazine scaffolds were investigated for antifouling activity. The analogbenso[g]dipodazine, with an EC

50value of 34 nM, displayed the highest settlement inhibition.

The studies of the structure-activity relationships of sponge-derived compounds wereextended to cover analogs of agelasines and agelasimines originally isolated from sponges ofthe genus Agelas. Synthesized (+)-agelasine D and two structurally close analogs wereinvestigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broadspectrum antibacterial activity found prompted a further investigation of structure-activityrelationships in 42 agelasine and agelasimine analogs and several characteristics thatincreased bioactivity were identified.In conclusion this work has produced new results regarding the potent bioactivity of

compounds derived from the sponges Geodia barretti and Agelas spp. and increased SARknowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of thesecompounds.

Keywords: 5-hydroxytryptamine, Agelas, agelasine, agelasimine, antibacterial, antifouling,barettin, bromobenzisoxazolone barettin, cytotoxic, Geodia barretti, marine, secondarymetabolite, sponge

Erik Hedner, Department of Medicinal Chemistry, Division of Pharmacognosy, Box 574,Uppsala University, SE-75123 Uppsala, Sweden

© Erik Hedner 2007

ISSN 1651-6192ISBN 978-91-554-6971-9urn:nbn:se:uu:diva-8218 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8218)

Distributor: Uppsala University Library, Box 510, SE-751 20 Uppsala

www.uu.se, acta@ub.uu.se

“Haraka haraka haina baraka”

There is no benefit in hurrying (Swahili saying)

List of Papers

This doctoral thesis is based on the following papers, referred to in the text by their Roman numerals (I�V):

I. Hedner, E., Sjögren, M., Andersson, R., Göransson, U., Hodzic, S.,

Jonsson, P. R. and Bohlin, L. Antifouling activity of a novel dibro-minated cyclopeptide from the sponge Geodia barretti. In manu-script.

II. Sjögren, M., Johnson, A-L., Hedner, E., Dahlström, M., Shirani, H.,

Göransson, U., Bergman, J., Jonsson, P. R. and Bohlin, L. (2006) Antifouling activity of synthesized peptide analogs of the sponge metabolite barettin. Peptides 27: 2058�2064.

III. Hedner, E., Sjögren, M., Frändberg, P.-A., Johansson, T., Görans-

son, U., Dahlström, M., Jonsson, P. R., Nyberg, F. and Bohlin, L. (2006) Brominated cyclodipeptides from the marine sponge Geodia barretti as selective serotonin 5-HT2 ligands. Journal of Natural Products 69: 1421�1424.

IV. Vik, A., Hedner, E., Charnock, C., Samuelsen, Ø., Larsson, R.,

Gundersen, L.-L. and Bohlin, L. (2006) (+)-Agelasine D: improved synthesis and evaluation of antibacterial and cytotoxic activities. Journal of Natural Products 69: 381�386.

V. Vik, A., Hedner, E., Charnock, C., Tangen, L., Samuelsen, Ø., Lars-

son, R., Bohlin, L. and Gundersen, L.-L. (2007) Antimicrobial and cytotoxic activity of agelasine and agelasimine analogs. Bioorganic and Medicinal Chemistry 15: 4016�4037.

All papers were written by the first author with comments and suggestions given by the co-authors. In I and III EH was responsible for all analyses and the major part of the laboratory work. Settlement inhibition in II was planned by MS. Synthesis and NMR analysis in IV-V were performed by AV. Antimicrobial activity in IV-V was measured by ØS. Cytotoxicity in IV-V was measured by EH.

Table of contents

1. Introduction.................................................................................................9 1.1 Natural product chemistry ....................................................................9 1.2 Marine natural products......................................................................11 1.3 Marine bioprospecting........................................................................12 1.4 Secondary metabolites and host defence............................................12 1.5 Biofouling...........................................................................................14 1.6 Sponge physiology and biology .........................................................15 1.7 Geodia barretti ...................................................................................15 1.8 Agelas species ....................................................................................17

2. Aims of the study ......................................................................................19

3. Brominated peptides from Geodia barretti (I�III)....................................20 3.1 Isolation and structure characterization..............................................20 3.2 Barettin and dipodazine analogs.........................................................22 3.3 Larval settlement assay ......................................................................23 3.4 Serotonin receptor affinity..................................................................26

4. Agelasine and agelasimine analogs (IV�V)..............................................28 4.1 Analog structures................................................................................28 4.2 Cytotoxicity in the FMCA assay ........................................................29 4.3 Antimicrobial activity ........................................................................32

5. Discussion .................................................................................................34 5.1 Antifouling activity of bromobenzisoxazolone barettin (I)................34 5.2 Antifouling and barettin analogs (II)..................................................35 5.3 Geodia barretti metabolites as 5-HT receptor ligands (III) ...............35 5.4 Biological activity of agelasine D analogs (IV) .................................37 5.5 SAR studies of agelasine and agelasimine analogs (V) .....................37 5.6 Chemical defence compounds – potential applications......................39

6. Conclusion and future perspectives ..........................................................40

7. Populärvetenskaplig sammanfattning .......................................................42

8. Acknowledgements...................................................................................44

9. References.................................................................................................47

Abbreviations and conventions

5-HT 5-hydroxytryptamine/serotonin ACHN renal adenocarcinoma cell line AcN acetonitrile Arg arginine Br bromine CEM/s leukemia cell line DKP diketopiperazine DMSO dimethylsulphoxide EC50 effective concentration (50%) ESI-MS electrospray ionization mass spectrometry FDA fluorescein diacetate FMCA fluorometric microculture cytotoxicity assay FSW filtered seawater GPCR G-protein coupled receptor HEK293 human embryonic kidney cells HPLC high performance liquid chromatography IC50 inhibitory concentration (50%) MDR multidrug resistance MIC minimal inhibitory concentration MS mass spectrometry NCE new chemical entity NCI National Cancer Institute NMR nuclear magnetic resonance PBS phosphate buffered saline Pro proline RP reversed phase RPMI 8226/s myeloma cell line SAR structure�activity relationship SI survival index sp./spp. species (singular/plural) TFA trifluoroacetic acid Trp tryptophan U-937 GTB lymphoma cell line

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1. Introduction

The term “pharmacognosie” was used for the first time, in 1811, in Lehrbuch der Materia Medica by Johann Adam Schmidt (Sandberg and Corrigan 2001). The word is derived from the two Greek words pharmakon and gno-sis, which mean “drug” and “knowledge”, respectively (Samuelsson 2004). The common and original definition of pharmacognosy is the scientific study of drugs derived from nature. However, the focus of pharmacognosy has shifted numerous times: the classical botanical aspects have successively been deemphasized in favour of a more modern pharmacognosy, dealing more with natural product chemistry and pharmacology (Bruhn and Bohlin 1997; Bohlin et al. 2007). An expanded model of pharmacognosy that incor-porates a broad range of areas has recently been proposed (Larsson 2007).

Figure 1. The model depicts the multidisciplinary aspects of modern pharmacognosy and the connections between biology, pharmacology and chemistry (Larsson 2007).

1.1 Natural product chemistry The rich structural diversity and complexity of natural products have re-sulted in numerous new drugs and inspired chemists to produce synthetic

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analogs with enhanced bioactivity (Faulkner 2000a). Although most of the drugs currently in development are results of either semi- or complete syn-thesis, natural products continue to play a significant role in the discovery and development of new pharmaceuticals, as was recently highlighted (Faulkner 2000a; Newman and Cragg 2007). Scientists have only scratched the surface of many unconventional natural product sources (Tulp and Boh-lin 2004). Of all drugs developed between 1981 and 2006, 28% were either natural products or derived from them. Another 20% can be categorized as natural product mimics (NM). This label is somewhat controversial, as it can be interpreted as exaggerating the roles natural products play in NM devel-opment. However, one can argue that nature played an inspirational role in the initial development stage. Immunosuppression, antiinfection, oncology and metabolic diseases are regarded as the predominant therapeutic areas of natural product-derived drugs (Butler 2004).

Figure 2. New chemical entities by source (01/1981–06/2006). B (biological), N (natural product), ND (derived from natural products, usually semisynthetic), S (synthetic), S* (synthetic, but with pharmacophore from natural products), V (vac-cine), S/NM (synthetic/natural product mimic) S*/NM (synthetic, but with pharma-cophore from natural products/natural product mimic) (Newman and Cragg 2007).

In the 1990s the pharmaceutical industry in practice selected lead com-pounds exclusively on the basis of Lipinski’s rule of five, although the rule is not directly applicable to natural products (Lipinski et al. 1997; Macarron 2006). As a consequence of the rigorous application of this rule, natural products were deprioritized or even eliminated from the drug discovery process. The concept of the discovery process has since then gradually changed and a renewed interest in natural products has resurged. Natural sources offer excellent opportunities for finding leads for novel targets (Tulp and Bohlin 2002). Today there is a growing awareness that natural products

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tend to occupy a wider chemical space than do synthetic compounds – a fact the scientific community is starting to take into account in the early drug discovery process (Feher and Schmidt 2003; Larsson et al. 2007).

1.2 Marine natural products The ocean represents a unique resource providing a diverse array of natural products. The greatest biodiversity is found in ecosystems, such as rocky coasts, kelp beds and coral reefs, where species diversity and population density are exceedingly high (Haefner 2003). In fact, 34 of the 36 phyla of life are represented in the marine environment, implying chemical evolution along many separate lineages. Only a fraction of marine organisms have so far been investigated, but even so over 12,000 novel compounds have been discovered (Donia and Hamann 2003), with hundreds of additional com-pounds being discovered annually (Faulkner 2002). Properties in the marine environment force aquatic organisms to produce molecules that can differ substantially in structural terms from terrestrial substances. Physical condi-tions, such as lack of light, low temperature, high salinity or extreme pres-sure force marine organisms to produce secondary metabolites to overcome these obstacles.

Despite four decades of intense research, marine pharmacognosy is still considered a relatively young field compared to terrestrial pharmacognosy. Originally, pharmacognosy dealt exclusively with the study of drugs derived from terrestrial plants and animals. However, in the 1950s, marine organisms were identified as an excellent source of new biologically active compounds (Blunt et al. 2007). It was not until 20 years later, however, that systematic investigation of the marine environment was initiated. There are several profound differences between the marine and terrestrial environments for which scientists must account, such as the sources of the natural products in question. Marine invertebrates and microorganisms have yielded substan-tially more bioactive natural products than seaweeds have, unlike the terres-trial environments, where plants are considerably richer in secondary me-tabolites (Proksch 1994; Faulkner 2002).

Several sessile invertebrates have intimate, symbiotic relationships with microorganisms and several natural products isolated from marine inverte-brates are suspected to be of microbial origin; however scientific evidence for this is often scarce and incomplete (Engel et al. 2002; Proksch et al. 2002). The last decade of marine natural product research has witnessed a gradual shift of focus from invertebrates and other macroorganisms to mi-croorganisms.

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1.3 Marine bioprospecting Marine bioprospecting is a rapidly expanding enterprise that entails the in-vestigation of the marine environment in search of novel biomolecules (Newman et al. 2003; Mayer et al. 2007). There is enormous potential for industrial development of marine resources, not only as pharmaceuticals, but also as nutritional supplements, molecular probes, cosmetics, fine chemicals, agrichemicals and paint ingredients. Researchers must also pay attention to the growing awareness of sustainable use of marine resources and to over-coming challenges, such as supplying sufficient biomass without disrupting ecosystems and modernizing coarse collection methods (Brümmer and Nickel 2003).

The first commercialized marine biomolecule of medical interest was cephalosporin, isolated from the marine fungus Cephalosporium acremo-nium in 1948, which functioned as a template for the development of the early antibiotic agents (Newton and Abraham 1955). Since then, only a handful of drugs of marine origin has reached the market; however, there is currently an influx of promising drug candidates in phase II/III trials, such as the anticancerogenic compounds Ecteinascidin-743, Aplidine and Kahalalide F (Schwartsmann et al. 2001; Haefner 2003; Fayette et al. 2006). Moreover, marine natural products that fail in clinical trials may still be introduced into the market as tools for biomedical research (Folmer et al. 2007).

Table 1. Commercially available marine-derived products.

Product Class Application Natural Source

Ara-A (Acyclovir) Nucleoside Antiviral Sponge, Cryptotethya crypta

Ara-C (Cytarabine) Nucleoside Antitumour Sponge, Cryptotethya crypta Manoalide Terpenoid Molecular probe Sponge, Luffariella variabilis Cephalosporin ß-laktam Antibiotic Fungus, Cephalosporium acremonium Omega-3 Lipids Prevent heart disease Fish Resilience® Extract Additive in creams Gorgonian, Pseudopterogorgia elisabethae

Prialt® (Ziconitide) Polypeptide Chronic pain Cone snail, Conus magus

1.4 Secondary metabolites and host defence Secondary metabolites are assumed to have evolved from primary metabo-lites. Their biological roles have been debated, though the prevailing view is that they offer evolutionary advantages to the host organisms (Firn and Jones 2000). Secondary metabolites with adaptive characteristics would contribute to the survival of new strains (Faulkner 2000b). A widely accepted definition of secondary metabolites is “substances that are formed in organisms but that do not participate in those metabolic processes which are necessary for the

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life and development of the organism” (Samuelsson 2004). While secondary metabolism has different functions than primary metabolism, they cant be sharply distinguished from one another. The genes coding for enzymes in-volved in secondary metabolisms are different, but the precursors are the same (Cavalier-Smith 1992).

Secondary metabolites in marine organisms, especially in invertebrates, are often attributed a defensive function, but reports of how they function in an ecological context is scarce (Hay and Fenical 1988; Pawlik 1993; Engel et al. 2002). The composition and type of compounds involved in the chemi-cal defence can vary dramatically among geographic regions, habitats and between individuals in a local habitat, and even within a single individual (Harvell et al. 1993; Hay 1996). It is currently assumed that tropical plants and invertebrates have more dynamic chemical defences than do their tem-perate counterparts, as the intense competition and predation in these spe-cies-rich tropical areas have led to the evolution of a wider range of secon-dary metabolites (Bolser and Hay 1996). In the marine environment, secon-dary metabolites appear to be most common and most ecologically important in tropical benthic organisms subject to high rates of attack by consumers on coral reefs (Bolser and Hay 1996; Faulkner 2002). However, secondary me-tabolites also play important roles in temperate and polar benthic communi-ties (Toth and Pavia 2007). Chemically defended organisms often produce multiple secondary metabolites, which opens up the possibility of synergistic or additive effects among various metabolites (Hay 1996).

Marine secondary metabolites, such as terpenes, alkaloids and polypheno-lics, can differ fundamentally from terrestrial secondary metabolites. Incor-poration of halogen is a very characteristic feature, and many marine organ-isms possess chemical skeletons and structures not found in terrestrial organ-isms (Rinehart 1992; Faulkner 2002). Secondary metabolites can have a wide range of activity, more commonly functioning as antifeedants, antifoul-ers, microbial pathogens, gamete attractants and trail markers. Marine sec-ondary metabolites involved in the host defence are often active at minute concentrations; the compounds dilute rapidly when released into the sur-rounding water and must be extremely potent if they are to have the intended effect.

Over the last forty years, sponges (phylum Porifera) have been identified as an excellent source of unique marine natural products, having a higher incidence of biologically active compounds than any other single marine phylum (Harper et al. 2001). The high incidence of bioactive compounds in these primitive filter feeders is likely related to their chemical defence against environmental stress factors. There is a strong correlation in sponges between the absence of physical defence mechanisms and the presence of unconventional biomolecules (Pawlik 1993).

Sponges in particular, and sessile marine invertebrates in general, are evolving as economically important sources of compounds. The past decade

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has seen a dramatic increase in the number of lead compounds from diverse marine life entering preclinical and clinical trials (Simmons et al. 2005). The largest group of new chemical entities of natural product origin has antican-cer indications, followed by NCEs with antibacterial indications (Newman and Cragg 2007). Hitherto, NCI has conducted the most extensive screening of marine organisms for anticancer activity and, when investigated for an-tileukemic activity the sponges proved to be the largest source of NCEs with a hit rate of 8.7% (Cragg et al. 2006). However there is a rapidly evolving recognition that a significant number of marine natural products are actually produced by symbiont microbes (Hoffmann et al. 2005).

Table 2. Examples of sponge-derived compounds for treating cancer and inflamma-tion in preclinical/clinical trials.

Metabolite Disease area Source sponge

Halichondrin B Cancer Halichondria okadai KRN7000 Cancer Agelas mauritianus LAF389 Cancer Jaspis digonoxea Mycaperoxide B Cancer Mycale sp. Laulimalide Cancer Cacospongia mycofijiensis IPL 512.602 Inflammation Petrosia contignata IPL 576.092 Inflammation Petrosia contignata Manoalide Inflammation Luffariella variablis

1.5 Biofouling Control of fouling on ship hulls and piping systems are estimated to cost the industry several billion dollars annually (Townsin 2003). The problem has so far been handled by using coatings that include high concentrations of heavy metals (Wahl 1989). Some coatings, such as TBT have been banned and other face global bans due to their unwanted effects on the environment and new alternatives must be developed (Clare 1996; IMO 1999). Practically all surfaces in the sea are subject to fouling, but somehow certain marine organ-isms are able to remain completely fouling free. Several such organisms have been studied in antifouling assays, and it has been concluded that many leak specific substances into the surrounding water, substances that prevent foulers from attaching (DeNys et al. 1995; Sjögren et al. 2004a). Conse-quently, there have been increased efforts to find natural compounds with potent antifouling activities, to develop eco-friendly antifouling compounds to replace the toxic biocides currently used in marine coatings. Sponges gen-erally have a fouling free body surface, which have led researchers to inves-tigate sponge physiology and chemistry in search for antifouling compounds.

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1.6 Sponge physiology and biology Sponges belong to the phylum Porifera, are regarded as the most primitive of the multicellular animals and arose approximately 1.6 billion years ago. Sponges are subdivided into three classes based on their spicule structure: Calcarea (calcium carbonate skeleton), Demospongiae (sponging and/or siliceous skeleton), and Hexactenellida (silica glass skeleton) (Bergquist 1978). All currently known sponge taxa are aquatic and there are an esti-mated 15,000 species (Leys et al. 2005). Sponges can be found in every type of marine habitat, from deep polar oceans to tropical reefs. They are sessile, soft-bodied marine invertebrates and most lack obvious physical defences, although calcareous and siliceous spicules may provide some physical de-fences. Adult sponges are sedentary and are anchored to a stationary sub-strate, such as a rock or another organism. Sponges are suspension feeders and feed by inhaling and filtering seawater through numerous pores (ostia) located in the external body wall. The diet available may differ markedly between sponges in different habitats, but the lower particle size limit is approximately 0.2 �m (Ribes et al. 1999). Sponges lack true tissue, such as muscles, conventional nerves and internal organs. Their anatomy is made up of different specialized cell types, organized in a relatively simple manner. The outer surfaces consist of pinacocytes (epithelial cells), while the meso-phyle forms most of the sponge biomass and consists of non-differentiated cells that form a gelatinous matrix interlaced with spicules (calcium carbon-ate spikes for structure and defence), porocytes (tubular cells that form pores in the sponge body), choanocytes with flagellum (that drive the water cur-rent) and pseudopodium collars (that act as sieves for particles) (Thakur and Müller 2004). Sponges are hermaphroditic and their reproduction patterns can vary greatly, ranging from asexual reproduction by budding of body parts, to sexual reproduction primarily by fertilizing eggs. After fertilization, larvae are released to find substrata suitable for settlement (Bergquist 1978).

Sponges harbour diverse and complex microbial communities, which are phylogenetically distinct from the microorganisms found in the surrounding water and sediment. The roles of these microorganisms are not fully under-stood, but there appear to be symbiotic relationships between the host sponge and many such microbes (Pietra et al. 1997).

1.7 Geodia barretti Geodia barretti (class Demospongiae, order Choristida, family Geodiidae) is a sponge characteristic of deep, rocky beds in the North Atlantic and is also found in Koster Fjord off the northwest Swedish coast. Sponges of the fam-ily Geodiidae are generally massive with a smooth, unfouled and solid cor-

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tex. Geodia barretti is a coldwater sponge that grows at depths below 50 m; it can reach 50 cm in diameter and weigh up to 24 kg.

Figure 3. (A) Specimen of the sponge Geodia barretti at a depth of 50 m in Koster Fjord, Sweden; its unfouled body is evident. (B) A field of Geodia barretti at a depth of 123 m. Photos: Tomas Lundälv, Tjärnö Marine Biological Laboratory.

In 1983, the sponge Geodia barretti was included in a collection of Swedish marine organisms that were screened for biological activity. Extracts of G. barretti produced a strong contractile response in a guinea pig ileum assay (Andersson et al. 1983). The novel compound barettin was isolated using bioassay-guided isolation (Lidgren et al. 1986). The structure of this bromi-nated cyclodipeptide has been under some controversy. The structure was initially determined to be cyclo[(6-bromo-8-en-tryptophan)proline] by Lidgren et al. (1986; Figure 4a); this proposed structure was later shown to be invalid, first by Lieberknecht and Griesser (1987) and then by Sölter et al. (2002), eventually being finalized as (cyclo-[(6-bromo-8-en-tryptophan)-arginine]) (Figure 4b).

Figure 4. (A) Proposed structure of barettin: cyclo[(6-bromo-8-en-tryptophan)proline]. (B) Finalized structure of barettin: (cyclo-[(6-bromo-8-en-tryptophan)-arginine]).

The chemical composition of Geodia barretti has been investigated and found to include metabolites such as histamine, adenosine, inosine, the free amino acids taurine and glycine and several sterols (Lidgren et al. 1988; Hougaard et al. 1991). Sponges from other Geodia spp. have yielded several

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bioactive compounds, such as the cyclodepsipeptides geodiamolides and the triterpenoid stelliferin riboside (Tinto et al. 1998; Tabudravu and Jaspars 2001). G. barretti is also a rich source of halogenated compounds and Sjögren et al. (2004b) examined G. barretti and isolated 8,9-dihydrobarettin, which is identical in structure to barettin apart from the double bond in posi-tion C-8. Barettin and 8,9-dihydrobarettin are potent antifoulers that inhibit the settlement of barnacle cyprids (Balanus improvisus), having EC50 values of 0.9 �M and 7.9 �M, respectively � in the range of antifouling agents used today (Sjögren et al. 2004b). This activity was retained when the cyclodipep-tides were incorporated into non-toxic coatings and investigated in the field (Sjögren et al. 2004a). Both barettin and 8,9-dihydrobarettin have been suc-cessfully synthesized (Johnson et al. 2004).

1.8 Agelas species Tropical marine sponges of the genus Agelas (class Demospongiae, order Poecilosclerida, family Agelasidae) are rich sources of alkaloids. Agelasines are methyladeninium salts carrying a diterpenoid side chain (Figure 5) and associated with bioactivities such as antimicrobial and cytotoxic effects, smooth muscle contractive responses and Na and K-ATPase inhibition (Ko-bayashi et al. 1987; Mangalindan et al. 2000). More than ten agelasines are currently known, and agelasine A�F have been synthesized (Asao et al. 1989; Piers et al. 1992; Piers et al. 1995; Utenova and Gundersen 2004; Bakkestuen et al. 2005; Marcos et al. 2005). Agelasimines, another class of secondary metabolites isolated from Agelas sp., are neutral 3,7-dialkylated purines (Figure 5); agelasimines A and B have been reported to inhibit the proliferation of leukemia cells (Fathi-Afshar et al. 1989).

Figure 5. Structures of agelasine D�F and agelasimine A�B.

There are reports that some compounds isolated from Agelas sponges have antifouling activities. The diterpene-alkaloid epi-agelasine C from Agelas

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mauritiana has been reported to inhibit settlement of the macroalga Ulva conglobata (Hattori et al. 1997).

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2. Aims of the study

The work presented here is part of the Marine Pharmacognosy research pro-gramme, at the Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, implemented in collaboration with the Tjärnö Marine Biological Laboratory, Göteborg University. The overall ob-jective of this programme is to identify bioactive compounds of marine ori-gin and evaluate their pharmacological effects and mechanisms of action at a molecular level. The specific objectives of this thesis were:

� To explore novel bioactive secondary metabolites found in Geodia barretti.

� To investigate settlement inhibition effect of secondary metabolites

from G. barretti.

� To investigate and evaluate structure�activity relationships of the settlement inhibition exerted by barettin analogs on cyprids of Balanus improvisus.

� To identify receptor targets associated with the pharmacological ef-

fects of cyclopeptides obtained from G. barretti, with an emphasis on human serotonin receptor subtypes.

� To investigate bioactivity of agelasine and agelasimine analogs,

with an emphasis on cytotoxicity.

� To investigate and evaluate structure�activity relationships of age-lasine and agelasimine analogs, with an emphasis on cytotoxicity.

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3. Brominated peptides from Geodia barretti (I�III)

Many secondary metabolites produced in sponges are likely involved in the chemical defence that target foulers and predators. Barettin and 8,9-dihydrobarettin isolated from Geodia barretti have been confirmed to inhibit the settlement of cyprids (Sjögren et al. 2004b). In I the chemical profile of Geodia barretti is further investigated, resulting in the isolation of the novel dibrominated compound bromobenzisoxazolone barettin. In II analogs with barettin and dipodazine scaffolds are investigated for antifouling struc-ture�activity relationships. In III barettin and 8,9-dihydrobarettin are inves-tigated for serotonin receptor affinity to provide clues of their pharmacologi-cal targets.

3.1 Isolation and structure characterization A specimen of the sponge Geodia barretti Bowerbank was collected in Kos-ter Fjord, off the west coast of Sweden at a depth of 60 m. The sponge was fractionated according to the procedure presented in Figure 6.

Figure 6. Protocol used for isolating bromobenzisoxazolone barettin from Geodia barretti.

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The sponge was defrosted, homogenized and extracted repeatedly using AcN:H2O (15:85). The concentrated extract was fractionated using RP-HPLC and screened using ESI-MS for traces of brominated compounds. Fraction 36 was found to contain a minor constituent with a 1:2:1 dibromi-nated isotope pattern (Figure 8B) and, after purification with analytical RP-HPLC, yielded bromobenzisoxazolone barettin (Figures 7�8). The structure was elucidated using high-resolution MS and 1D- and 2D-NMR (Figure 9).

Figure 7. Chemical structures of (1) barettin, (2) 8,9-dihydrobarettin and (3) bromo-benzisoxazolone barettin.

Figure 8. (A) HPLC chromatogram of (1) barettin, (2) 8,9-dihydrobarettin and (3) bromobenzisoxazolone barettin. The gradient (0�100% [B]) is represented by the red solid line. (B) Mass spectrum of bromobenzisoxazolone barettin.

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Bromobenzisoxazolone barettin is structurally related to 8,9-dihydrobarettin; an arginine joined with a tryptophan residue forming a diketopiperazine-type cyclic peptide backbone. However, bromobenzisoxazolone barettin is further substituated at position C-8. 1H NMR revealed a broad signal at �H 8.41 consistent with a hydroxy-group with NOESY correlations to H-2 and H-9. HMBC correlation 4J C-22,H-2 indicated that position C-8 is further substitu-ated (Figure 9). 1H NMR displayed two aromatic doublets (�H 6.78 and 7.36; J 8.6 Hz) having correlations in HMBC 3J C-22,H-24, 3J C-25a,H-24, 3J C-26,H-

25, 4J C-27a,H-22 indicating a heterocycle at C-25a/C-27a and a C-X substituent in position C-23 (Figure 9). HRFAB-MS and ESI-MS confirmed the second bromine assigned to C-23 through an isotope cluster. N-H bonds were con-firmed with 323 K 1H NMR; all N-H bonds shifted upfield.

Figure 9. HMBC and 1H spectra of bromobenzisoxazolone barettin (recorded in DMSO-d6, 600 MHz).

3.2 Barettin and dipodazine analogs

In II, barettin and dipodazine were used as the basis for synthesizing 14 ana-logs. Barettin and 8,9-dihydrobarettin consist of a joined arginine and tryp-tophan residue that form a cyclic peptide backbone. Cyclic dipeptides are often referred to as diketopiperazines and these compounds have attracted attention for their significant bioactivity (Prasad 1995; De Rosa et al. 2003). Dipodazine, originally isolated and elucidated from the fungi Penicillium dipodomyis (Sörensen et al. 1999; Johnson et al. 2002), was chosen for its

23

structural similarities to barettin; instead of arginine, however, a glycine is connected to the tryptophan residue. The use of dipodazine and its analogs was motivated by our desire to focus on the tryptophan residue and the im-portance of modifying substituents in it for monitoring accompanying changes in bioactivity. Dipodazine analogs are considerably easier to synthe-size than barettin analogs are. Barettin was used as a starting point for the design of the analogs 5-bromobarettin and debromobarettin, and dipodazine for the remaining 12 analogs (Figure 10).

Figure 10. Chemical structures of barettin (1), 8,9-dihydrobarettin (2), 5-bromobarettin (3), debromobarettin (4), dipodazine (5), 5-bromodipodazine (6), 5-methoxydipodazine (7), 5-nitrodipodazine (8), 6-chlorodipodazine (9), 5-methyldipodazine (10), benzo(e)dipodazine (11), benzothiophenedipodazine (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]pyrazine-1,4-dione (14), 6-bromo-1H-indole-3-carboxalde-hyde (15), benzo(g)dipodazine (16) and 5-hydroxytryptamine (17).

3.3 Larval settlement assay Bromobenzisoxazolone barettin was investigated for its effect on the settle-ment of competent larvae of the barnacle Balanus improvisus, and was found

24

to inhibit cyprid larvae in a dose-dependent manner (I; Figures 11-12); its EC50 value was determined to be 15 nM, which is approximately 60 times more potent than the settlement inhibition of barettin. The settlement inhibi-tion of bromobenzisoxazolone barettin was found to be reversible. To inves-tigate SARs for barettin-like compounds exerting settlement inhibition, sev-eral barettin and dipodazine analogs were investigated using the larval set-tlement assay (II).

Figure 11. Three stages in the development process of Balanus improvisus. (A) Free-swimming nauplius larvae. (B) Cyprid larvae searching and attaching to sub-stratum. (C) Newly metamorphosed juvenile barnacle. Photos: Tjärnö Marine Bio-logical Laboratory.

Larval rearing and cyprid preparation was conducted using the system de-veloped at Tjärnö Marine Biological Laboratory (Berntsson et al. 2000). Nauplius larvae were fed plankton in rearing containers and moulted into cyprid larvae in 6�7 days (Figure 11); cyprids were stored at 4°C. In short, the compounds were dissolved in 10 mL of filtered seawater (0.2 �m) in the following concentrations: 10 �M, 1 �M, 0.1 �M, 10 nM, 1 nM and 0.1 nM (I), and 100�M, 10 �M and 1 �M (II). Competent cyprids (20 � 2 individu-als) were added to each dish. Petri dishes containing filtered seawater served as controls. The experimental dishes were kept for 3�5 days at room tem-perature, after which they were investigated under a stereo microscope for attached/metamorphosed individuals and non-attached individuals. Reversi-bility was investigated by exposing 40 cyprids to 10 �M of the compound. After 48 hours, 20 cyprids were washed and transferred to new saltwater; the experiment was maintained for 5 days. The dishes were investigated for at-tached/metamorphosed individuals and non-attached individuals.

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Figure 12. Effect of bromobenzisoxazolone barettin on the settlement of cyprid larvae of Balanus improvisus.

The 14 analogs tested can be divided into two main groups, namely, analogs with a barettin scaffold and analogs with a dipodazine scaffold (II). In the barettin group, 5-bromobarettin significantly stimulated settlement while debromobarettin did not produce any significant effect. In the dipodazine group, 12 different compounds were examined in the settlement assay. Four of the dipodazine analogs (6, 11, 13, 15) proved to significantly inhibit the settlement of B. improvisus cyprids (Figure 10; Table 3). Analog 15 dis-played moderate activity, with an EC50 value of 6.7 μM (Figure 10); analogs 13, 6 and 11 displayed EC50 values of 2.4 μM, 5.8 μM and 1.5 μM, respec-tively (Figure 10). The most effective compound found in these experiments was benzo[g]dipodazine with an EC50 value 34 nM (Table 3). The remaining dipodazine analogs (5, 7�10, 12) did not significantly affect the settlement of B. improvisus cyprids (Figure 10; Table 3).

Table 3. EC50 values of barettin and dipodazine analogs for the settlement inhibition (I) and stimulation (S) of Balanus improvisus cyprids.

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3.4 Serotonin receptor affinity In order to further establish the mode of action, the barettins were investi-gated for their affinity for human serotonin receptors (III). Serotonin recep-tors were chosen based on several observations and previous reports; like serotonin, the barettins have an indole nucleus in the tryptophan residue. Serotonin is hydroxylated in position C-5, whereas barettin and 8,9-dihydrobarettin are substituted with a bromine in position C-6. A similar brominated tryptophan residue can be found as a 6-Br-Trp moiety in the 41-amino acid peptide �-conotoxin, a polypeptide that selectively inhibits the 5-HT3 receptor through competitive antagonism (England et al. 1998). Addi-tionally, sponge-derived indole alkaloids, with 6-Br-tryptamine moieties, have displayed antiserotoninergic activity (Bifulco et al. 1995) and, barettin and 8,9-dihydrobarettin have structural similarities to high-affinity 5-HT receptor ligands, like the drug tegaserod (Beattie et al. 2004). These findings prompted us to investigate a possible interaction with the serotonergic sys-tem.

Membranes were prepared from human embryonic kidney cells (HEK293) transfected with human serotonin receptors. The following sero-tonin receptor subtypes were prepared from a glycerol stock: 5-HT1A, 5-HT1D, 5-HT2A, 5-HT2C, 5-HT3A, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7A. �N-methyl-3HLSD, �9-methyl-3HBRL-43694, �N-methyl-3HGR113808 and �1,2-3H5-carboxamidotryptamine were used as radioligands. Saturation studies were conducted on transfected HEK293 cell membranes expressing 5-HT2A, 5-HT2C or 5-HT4 receptors.

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Table 4. Affinity of barettin, 8,9-dihydrobarettin, 5-HT, methysergide and tegaserod for serotonin subreceptors expressed in HEK293 cell membranes.

Of the panel of receptors tested, covering all the major 5-HT subfamilies, the HEK293 cell membranes expressing 5-HT2A, 5-HT2C and 5-HT4 receptors displayed receptor�ligand affinity for barettin below a concentration of 10 �M. Barettin displaced the radioligands with dose-responsive kinetics. The affinity constant Ki for barettin at the 5-HT2A receptor was determined to be 1.93 �M (Table 4). Barettin does not have as high an affinity for 5-HT2A as does the more selective ligand methysergide, but is nearly as selective as endogenous 5-HT (0.69 �M). In the case of 5-HT2C, both barettin and 8,9-dihydrobarettin were able to displace �N-methyl-3HLSD, producing Ki val-ues of 0.34 �M and 4.63 �M, respectively (Table 4). Barettin displayed a Ki of 1.91 �M at the 5-HT4 receptor, while 8,9-dihydrobarettin was not able to displace the radioligand. Tegaserod was introduced as a 5-HT4 receptor-selective ligand and produced a Ki of 31 nM. Saturation experiments were performed to confirm that the HEK293 cells expressing the desired receptor were robust.

Bromobenzisoxazolone barettin failed to interact with the serotonin recep-tors 5-HT2A, 5-HT2C or 5-HT4 (data not shown). This was somewhat unex-pected, since barettin has affinity for 5-HT2A, 5-HT2C or 5-HT4 and 8,9-dihydrobarettin for 5-HT4 at concentrations below 10 �M. The brominated benzisoxazolone side chain is a relatively large substituent and it is plausible that it obstructs the binding site of the serotonin receptor, preventing the molecule from docking at the receptor.

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4. Agelasine and agelasimine analogs (IV�V)

Agelas is a common sponge genus and is abundant in a variety of habitats, predominantly tropical. The chemical compositions of species such as A. clathrodes, A. conifera, A. dispar, A. inaequalis, A. mauritiana, A. sceptrum and A. wiedenmayeri have been investigated (Assmann et al. 2000) and sev-eral constituents have been attributed defensive capabilities, for example the brominated pyrrole alkaloids Nagelamides A-H with their antibacterial activ-ity (Endo et al. 2004). Antimicrobial alkaloids and peptides are crucial build-ing blocks in the innate host defence for many invertebrates against bacteria and fungus (Dimarcq et al. 1998).

There are a number of bioactive compounds in the Agelas sp. but this the-sis focus on the biological activity of agelasines and agelasimines (IV�V).

4.1 Analog structures Previously, (+)-agelasine D has been synthesized from commercially avail-able (+)-manool (Utenova and Gundersen 2004); however, in IV we report an improved conversion of (+)-manool to (+)-agelasine D along with anti-bacterial and cytotoxic data for this natural product and its two synthetic intermediates (Figure 13).

Figure 13. Chemical structures of (+)-agelasine D (10) and analogs 9a and 9b.

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In V, the range of compounds is expanded with both agelasine and age-lasimine analogs, to obtain information on structure�activity relationships (SARs) for antibacterial and cytotoxic activities (Figure 14; Tables 5 and 7). Agelasine and agelasimine are purine derivatives bearing a diterpenoid chain; both the purine ring structure and the terpenoid chain were modified.

Figure 14. Chemical structures of some agelasine analogs (3l, 5d, 6a, 7, 9b, 13b) and agelasimine analogs (15b, 16�18) in V.

4.2 Cytotoxicity in the FMCA assay Cytotoxicity was measured using the fluorometric microculture cytotoxicity assay (FMCA), an in vitro assay based on measurements of fluorescence generated by the hydrolysis of FDA to fluorescein by cells with intact plasma membranes (Larsson and Nygren 1989; Dhar et al. 1996). Com-pounds were dissolved in 1% DMSO and dispensed into 96-well microtiter plates together with cancer cells suspended in cell growth medium. After incubation for 72 hours at 37°C and 5% CO2, the cells were washed with PBS and FDA was added. The cells were incubated for 40 minutes and the generated fluorescence was measured.

(+)-Agelasine D and two N6-alkoxy derivatives (IV; Figure 13) were screened against four human tumour cell lines: lymphoma (U-937 GTB), myeloma (RPMI 8226/s), leukemia (CEM/s), and renal adenocarcinoma

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(ACHN). The last is a multi-drug resistant cell line, whereas the others are regarded as drug sensitive cell lines. (+)-Agelasine D and one analog exhib-ited cytotoxicity against all cell lines, particularly to RPMI 8226/s (Figure 15). Both compounds displayed dose-dependent cytotoxicity with activity in the range of clinical drugs used today. Their activities were comparable to that reported for agelasine G against another lymphoma cell line (L1210; IC50 3.1 �g/mL � 4.8 �M) (Ishida et al. 1992).

Figure 15. Survival index (SI) curves of (+)-agelasine D and analog 9a for four cancer cell lines.

Cytotoxicity against the same four cancer cell lines was examined for the agelasine analogs 3, 5�7, 9b, 11 and 13 and for agelasimine analogs 15�18 (V; Figure 14; Table 5). Compounds 1a�f, 8, 10a�b, 12 and 14 were starting reagents, compounds 2a�j were synthetic intermediates and compounds 3b�d, 5a and 11a�b were weak antimycobacterials and compound 4 was only isolated in minor amounts (V; Figure 14). Related structures were only weak inhibitors of Mycobacterium tuberculosis, so the bioactivities of these compounds were not determined.

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Table 5. Cytotoxicity of agelasine and agelasimine analogs examined in V against the cancer cell lines U-937 GTB, RPMI 8226/s, CEM/s and ACHN. See Figure 14 and V for chemical structures.

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4.3 Antimicrobial activity (+)-Agelasine D and two N6-alkoxy derivatives (IV; Figure 13) were screened against the following bacteria: Staphylococcus aureus, Streptococ-cus pyogenes, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Bacteroides fragilis, Bacteroides thetaiotaomicron and Myco-bacterium tuberculosis. MIC was determined using a standard broth mi-crodilution technique (Vorland et al. 1998), and the three compounds were found to exhibit relatively broad antibacterial spectra (Table 6). The highest activities were found against the gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes. Bacteroides fragilis is the most com-monly isolated group of anaerobes in clinical settings. Members of this group have displayed increased resistance to most of the antimicrobial agents traditionally used for treating anaerobic infections. The MIC value of (+)-agelasine D was comparable to that obtained using rifampicin, gentamy-cin or metronidazole. Metronidazole has been the drug of choice for prevent-ing and treating B. fragilis infections for 40 years.

Table 6. Antibacterial activity of compounds 9a, 9b and (+)-agelasine D and clini-cally used drugs against Mycobacterium tuberculosis, Staphylococcus aureus, Strep-tococcus pyogenes, Enterococcus faecalis, Escherichia coli, Pseudomonas aerugi-nosa, Bacteroides fragilis and Bacteroides thetaiotaomicron.

Antibacterial activities were examined for agelasine analogs 3, 5–7, 9b, 11, and 13, and agelasimine analogs 15–18 against the following bacteria: Staphylococcus aureus, Escherichia coli, Bacteroides fragilis and Bacter-oides thetaiotaomicron as well as the mycobacteria Mycobacterium tubercu-losis (V; Figure 14; Table 6).

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Table 7. Antimicrobial activity of agelasine and agelasimines analogs examined in V against Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli, Bac-teroides fragilis and Bacteroides thetaiotaomicron. See Figure 14 and V for chemical structures.

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5. Discussion

5.1 Antifouling activity of bromobenzisoxazolone barettin (I) Several brominated natural products are of marine origin and brominated amino acid derivatives are present in complex structures, such as cyclic pep-tides and indole alkaloids (Bittner et al. 2007). The barettins isolated from Geodia barretti have 6-BrTrp moieties (Figure 7). Many other pharmaco-logically active marine compounds, such as the styelins, are brominated in position C-6 (Taylor et al. 2000).

Bromobenzisoxazolone barettin inhibited the settlement of cyprids of Balanus improvisus with an EC50 value of 15 nM (Figure 12); this effect was reversible. Compared to 8,9-dihydrobarettin, the EC50 value differs approxi-mately 500-fold while the difference in chemical structure is limited to a hydroxy- and a bromobenzisoxazolone substituent in C-8 (Figure 7). The bromobenzisoxazolone substituent greatly enhances the settlement inhibi-tion. Benzisoxazolone, i.e., [benzisoxazol-3(1H)-one] has been synthesized and has moderate antimicrobial and antileukemic activity (Wierenga et al. 1984). Based on the strong antifouling activity, it is plausible to assume that bromobenzisoxazolone barettin plays a prominent role in the chemical de-fence of G. barretti. Bromobenzisoxazolone barettin may even have other or additional targets than just the cyprid larvae.

Since the structurally related barettin and 8,9-dihydrobarettin selectively target specific serotonin subreceptors, bromobenzisoxazolone barettin was investigated for affinity for 5-HT2A, 5-HT2C and 5-HT4 receptors transfected into HEK293 cells. Bromobenzisoxazolone barettin failed to interact with these serotonin subreceptors. In III we hypothesized that serotonin receptors may be the targets of barettin and 8,9-dihydrobarettin, however our findings suggests that 5-HT receptors may not be the primary molecular target of bromobenzisoxazolone barettin in cyprids of B. improvisus.

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5.2 Antifouling and barettin analogs (II) Fourteen synthetic barettin analogs were designed (Figure 10) to investi-

gate the structure�activity basis of settlement inhibition, in order to develop compounds with increased antifouling effects while preserving reversibility. The analogs were synthesized from either a barettin scaffold or from a dipo-dazine scaffold (Johnson et al. 2004). The dipodazine scaffold allowed easier additions of substituents to the indole moiety and additionally allowed us to evaluate the effect of removing the arginine residue.

Both the presence and location of the bromine are crucial for bioactivity in barettin and dipodazine analogs. When Br was substituted for Cl, NO2, CH3 or MeO in positions 5 or 6, the bioactivity was either decreased or lost. These results suggest that the size and electronegativity in these positions directly influence antifouling activity. It was interesting that Br in position 5, as in 5-bromobarettin (analog 3), stimulated settling; 5-bromobarettin has structural similarities to serotonin and we have previously demonstrated that barettin interacts with 5-HT subreceptors. The affinity of 5-bromobarettin for the 5-HT2C receptor is 0.12 μM (unpublished data), which is lower than that of barettin. However, Br substituated in position 5 with the dipodazine scaffold, as in 5-bromodipodazine (analog 6), significantly reduced the set-tlement. Isomerization seems to play an important role in antifouling activity as well, when comparing isomers 13 and 14 (Figure 10), the particular stereochemistry of the compounds influenced the settlement inhibition of B. improvisus.

Benzo[g]dipodazine (analog 16) was the most effective analog of the 14 synthesized compounds, with an EC50 value of 34 nM. Benzo[g]dipodazine is not brominated, but the phenyl substituent in position 6�7 could act in a way similiar to that of the electronegative bromine in position 6. The in-creased hydrophobicity may explain the increased settlement inhibition of benzo[g]dipodazine since penetration of cyprid tissues and cellmembranes is likely facilitated.

When summarizing the results, it is clear that minor alterations in chemi-cal configuration radically alter the settlement inhibitory effect on B. impro-visus larvae. Similar changes in bioactivity recur in compounds when func-tional groups are modified, which may be explained through a change in affinity for the molecular targets (Pla et al. 2006; Bittner et al. 2007).

5.3 Geodia barretti metabolites as 5-HT receptor ligands (III) The two brominated compounds barettin and 8,9-dihydrobarettin isolated from the sponge Geodia barretti were investigated for affinity for serotonin receptors. The receptor studies were based on previous reports that bromi-

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nated tryptophan residues interact with serotonin receptors. It has been re-ported that �-conotoxin from the cone snail Conus inactivates 5-HT3 recep-tors (England et al. 1998) and that sponge-derived compounds with BrTrp moieties interact with serotonin receptors in displacement studies (Hu et al. 2002; Segraves and Crews 2005).

Barettin interacted specifically with 5-HT2A, 5-HT2C and 5-HT4 receptors, while 8,9-dihydrobarettin only interacted with the 5-HT2C receptor (Table 4). The 5-HT2A receptor is associated with several actions in the PNS, such as smooth muscle contraction and vasoconstriction (Rang et al. 1995). Both the 5-HT2A and 5-HT2C receptors are associated with neuronal excitation in the CNS and are implicated in treating depression and anxiety disorders (Barnes and Sharp 1999). The 5-HT4 receptor is mainly linked to gastrointestinal motility (Read and Gwee 1994; Barnes and Sharp 1999). All serotonin re-ceptors, except the ligand-gated ion channel 5-HT3, are G protein-coupled transmembrane receptors and it is estimated that approximately 60% of drugs today act through GPCRs (Wilson et al. 1998). GPCRs belong to the rhodopsin-like receptor family and are highly conserved in their protein structure (Meeusen et al. 2003); GPCR representatives have been identified in simple eukaryotes (Peroutka and Howell 1994).

The small difference in chemical backbone between barettin and 8,9-dihydrobarettin, i.e., the double bond in the tryptophan residue, resulted in vastly different affinities. The double bond may give barettin a better fit in the receptor binding pocket through a more rigid steric orientation of the bromo-tryptophan residue. The bromine is another structural constituent that likely influences interaction with the receptor. Halogenation has been high-lighted in structure�activity studies and resulted in drastic improvements in affinity and in changes in selectivity (Bózsing et al. 2002). The struc-ture�activity relationships of barettin and 8,9-dihydrobarettin in the recep-tor�ligand binding assay were reflected in the settlement inhibition of B. improvisus cyprids (Sjögren et al. 2004b).

It has been established that a G protein-coupled 5-HT receptor with DNA sequence homologies to mammalian 5-HT1 receptors is present in the barna-cle Balanus amphitrite (Kawahara et al. 1997). In a crustacean, the spiny lobster Panulirus interuptus, a 5-HT2 receptor has been functionally charac-terized (Clark et al. 2004). The 5-HT2 receptor may be the primary molecular target of barettin and 8,9-dihydrobarettin in cyprids of B. improvisus. Sponges lack a nervous system so the barettins are likely involved in the arsenal of chemical defences that the sponge G. barretti uses to deter preda-tion and fouling by means of serotonergic ligand-binding actions in the tar-get organism. The role of the 5-HT4 receptor remains to be elucidated, as this receptor subclass has been neither cloned nor functionally characterized in invertebrates.

Future research to further define the functional roles of 5-HT receptors in invertebrates is necessary if we are to understand the roles of barettin and

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8,9-dihydrobarettin. Additional structure�activity studies are likely to give clues as to the relationships between the compounds and the 5-HT receptors.

5.4 Biological activity of agelasine D analogs (IV) In IV an improved synthesis of (+)-agelasine D is presented along with anti-bacterial and cytotoxic data. Compounds derived from Agelas spp. have previously displayed activity against microorganisms (Mangalindan et al. 2000; Bakkestuen et al. 2005). (+)-Agelasine D and two structurally close analogs (Figure 13) were investigated for inhibition of M. tuberculosis, S. aureus, S. pyogenes, E. faecalis, E. coli, P. aeruginosa, B. fragilis and B. thetaiotaomicron. All three compounds exhibited relatively broad antibacte-rial spectra with moderate inhibition of the majority of the bacterial strains. The highest activity was recorded against the S. aureus and S. pyogenes strains; in the case of S. pyogenes, the MIC was equivalent to that of the clinically used drug gentamycin.

The compounds were screened for cytotoxicity against a panel of four human cancer cell lines: U-937 GTB (lymphoma), RPMI 8226/s (myeloma), CEM/s (leukemia) and ACHN (renal adenocarcinoma) (Figure 15). The tert-butoxy derivative displayed no activity against any of the cell lines, while agelasine D and the methoxy derivative had activities in range of clinically used drugs against all tested cancer cell lines, including the multidrug-resistant cell line ACHN. Our results indicate that the alkoxy substituent strongly influences the cytotoxicity, though no significant differences were found in terms of antibacterial activity. Agelasine analogs with enhanced antibacterial activities may be candidates for drug development for treating microbial infections. The tert-butoxy derivative is a potential antibacterial candidate due to its low cytotoxicity.

Armed with the cytotoxic and antibacterial results from IV, it is plausible to consider the involvement of agelasine D as a chemical deterrent in the host defence of sponges from Agelas spp. The early structure�activity rela-tionships examined in IV prompted more detailed SAR studies, which re-sulted in V and Proszenyák et al. (accepted in Archiv der Pharmazie).

5.5 SAR studies of agelasine and agelasimine analogs (V) Several agelasine and agelasimine analogs (V; Figure 14) were synthesized to obtain SAR knowledge of antimicrobial and cytotoxic activities (V; Ta-bles 5 and 7).

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The length of the terpenoid side chain in position N-7 was the single most important substituent for both the antibacterial and cytotoxic activities. Un-saturations in the terpenoid chain increased antibacterial activity, which was unaffected by altered geometry. Larger alkoxy substituents in the N6-position generally decreased cytotoxicity, while causing no significant variation in antimicrobial activity. Removal of the N6-alkoxy group to produce adenini-um salts resulted in substantially reduced activities. Variations in the N-9 position had no significant influence on either cytotoxicity or antimycobacte-rial activity, while antibacterial activity decreased with larger N-9 substitu-ents.

Mono-alkylated neutral purines displayed decreased cytotoxicity compa-red to that of cationic 7,9-dialkylated purinium and no activity was recorded for S. aureus or E. coli. However, 7-geranylgeranyladenine (analog 7) (Figu-re 14) was one the most active inhibitors of M. tuberculosis and was substan-tially more active than the N6-alkoxy derivatives 6a�b were (V; Figure 14). It is possible that a cationic charge is required for antimycobacterial activity and that analog 7, but not the less basic analogs 6a-b, is protonated in the cell culture. In analogs 11c�d (V) the intact purine ring was removed and repla-ced with either protons to form imidazolium salts or a benzene ring to form benzimidazolium salts; however, doing so resulted in decreased cytotoxic and antibacterial activities. In the agelasimine analogs 15-18 (V; Figure 14) a long terpenoid side chain in N-7 was necessary for inhibition of M. tuber-culosis, which resulted in low MIC against M. tuberculosis, E. coli and S. aureus. Cytotoxicity was moderate to high for the agelasimine analogs 15b and 18 (Figure 14).

Based on the structure�activity data generated in this study, we hypothe-size that their amphiphilic characteristics permit the agelasine and age-lasimine analogs to interact with cell membranes. Many naturally occurring amphiphilic peptides exert their effects through cell membrane interaction (Hancock and Scott 2000). A long hydrophobic terpenoid chain and a hy-drophilic purine ring were found to increase the bioactivities greatly. From a drug development perspective this is favourable, since cell membrane inter-actions avoid many drug resistance mechanisms that are associated with cell membrane penetration (Mor 2000). Additional SAR information about selec-tivity and cell membrane interaction may lead to the development of low cytotoxic agelasine and agelasimine analogs that specifically target bacterial strains. In V, the analog 7-geranylgeranyladenine was found to have an in-teresting biological profile with low MIC against M. tuberculosis; the anti-bacterial activity could theoretically be enhanced and/or the cytotoxicity reduced with further structural modifications.

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5.6 Chemical defence compounds – potential applications

Upon commencing this work, the primary objective was to investigate the structural and biological characteristics of cyclic peptides obtained from cold-water invertebrates. The focus rapidly shifted to the sponge Geodia barretti to further characterize the chemical profile and to investigate the pharmacological targets and the mechanism of action of the barettins in or-der better understand their function in the chemical defence of the sponge. The role of barettin, 8,9-dihydrobarettin and bromobenzisoxalone barettin in the sponge host defence appear to be settlement inhibition, possible mediated through a receptor-ligand interaction. Its unlikely that they are cytotoxic, since barettin and 8,9-dihydrobarettin have only displayed weak signs of cytotoxicity against cancer cell lines (U-937 GTB, RPMI 8226/s, CEM/s and ACHN) in concentrations up to 100 �M (unpublished data). The potential commercial value of the barettins is primarily as coating additives. Bromo-benzisoxazolone barettin is an attractive candidate to be an active ingredient in marine paints, since it inhibits settlement at very low concentrations and its effect is reversible. Field investigations would be needed to determine whether it can be incorporated into antifouling paints as successfully as ba-rettin and 8,9-dihydrobarettin can (Sjögren et al. 2004a). Complete synthesis must be accomplished to solve the supply and to evaluate the final cost as an additive.

My initial focus was purely on the barettins, but was expanded to include two additional groups of compounds from sponges of the genus Agelas, i.e., agelasines and agelasimines. The antifouling activity of (+)-agelasine D and 29 agelasine analogs have been investigated for settlement inhibition in a preliminary study and found to have weak to moderate antifouling activity (unpublished data). Instead, their role in the sponge host defence appears to be fundamentally different from that of the barettins; they are primarily cyto-toxic and antimicrobial. The results from IV�V indicate that SAR studies of agelasine and agelasimine analogs can generate data and clues for new an-timicrobial pharmaceuticals. Enhancing the antimycobacterial activity, while reducing the general cytotoxicity could result in agelasine analogs with at-tractive characteristics for early development of drugs that target M. tubercu-losis.

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6. Conclusion and future perspectives

The conclusions of the studies included in this thesis are as follows:

� The novel secondary metabolite bromobenzisoxazolone barettin isolated from Geodia barretti inhibits the settlement of Balanus improvisus cyprids, with an EC50 value of 15 nM.

� Characteristics of the settlement inhibition of B. improvisus

cyprids were investigated. Altering substitutions in positions C-5 and C-6 of analogs with barettin and dipodazine scaffolds greatly affects their activity. The dipodazine analog benso[g]dipodazine was identified as having an EC50 value of 34 nM.

� Receptor-ligand affinity was discovered for two brominated

dipeptides from G. barretti. Barettin interact with the receptor subtypes 5-HT2A, 5-HT2C and 5-HT4 and 8,9-dihydrobarettin in-teract with 5-HT2C.

� (+)-Agelasine D and an N6-alkoxy derivative are cytotoxic in the

micromolar range against the following human cancer cell lines: U-937 GTB, RPMI 8226/s, CEM/s and ACHN. Both compounds and an additional N6-alkoxy derivative have broad antibacterial spectra.

� The structure�activity characteristics for cytotoxicity and antim-

icrobial activity were identified for 42 agelasine and agelasimine analogs. The length of the N-7 terpenoid side chain was identified as the single most critical substitution influencing bioactivity.

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Future Perspectives

Proposals for future studies:

� The potential for incorporating bromobenzisoxazolone barettin in antifouling coatings applied in the field.

� Complete synthesis of bromobenzisoxazolone barettin.

� Investigating the microorganism population in G. barretti to de-

termine whether the barettins examined in I�III are of bacterial origin.

� Resolving the functionality of the receptor�ligand interaction of

barettin and 8,9-dihydrobarettin with regard to their selective 5-HT subreceptors to provide more clues as to how interaction at the GPCRs takes place (work in progress at the Division of Pharmacognosy, Uppsala University).

� Investigating the structure�activity relationship of serotonin re-

ceptor affinity for analogs with barettin scaffolds (work in pro-gress at the Division of Pharmacognosy, Uppsala University).

� Investigating whether barettin, 8,9-dihydrobarettin and bromo-

benzisoxazolone barettin have other targets than 5-HT receptors.

� The results presented in IV and V indicate that agelasines are very interesting antimicrobial agents, and that less cytotoxic ana-logs may have potential as antibiotics. Determining the antibacte-rial mechanisms of action may provide clues in how to synthesize compounds with higher activity, less cytotoxicity and/or that tar-get specific bacterial strains.

� Further investigating the substituents in agelasine and age-

lasimine analogs from a SAR perspective, with an emphasis on the terpenoid side chain (work in progress with an article, Proszenyák et al., accepted for publication in Archiv der Phar-mazie).

� Investigating the settlement inhibition of agelasine and age-

lasimine analogs (work in progress at the Division of Pharmacog-nosy, Uppsala University).

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7. Populärvetenskaplig sammanfattning

I den marina miljön finns organismer som inte förekommer på land. Svampdjuren (Porifera) är en sådan grupp. Dessa organismer är evertebrater som lever fastsittande på ett substrat. Eftersom de saknar möjlighet att för-flytta sig så kan de inte fly från predatorer eller andra hot. Svampdjuren har vid sidan om fysiska försvarsmekanismer utvecklat ett kemiskt försvar där de avger bioaktiva substanser. Dessa ämnen måste vara potenta, eftersom de omedelbart späds ut i det omgivande vattenskiktet. I synnerhet utgör påväxt av alger och marina bakterier ett allvarligt hot, eftersom svampdjur livnär sig via filtrering av plankton och liknande mikroorganismer. Påväxt är ett fenomen som drabbar alla ytor i havet. Oönskad påväxt på båtskrov och konstruktioner ger upphov till problem som ökad bränsleförbrukning och ökade förslitningsskador. Flera komponenter i befintliga skyddsfärger är skadliga för ett stort antal marina livsformer. På grund av långsam nedbryt-ning av komponenterna i skyddsfärgerna är det vanligt att de ackumuleras i bottensediment. Förbud och restriktioner, bland annat förbudet av TBT 2003, har påskyndat utvecklingen av alternativ med bättre miljöprofil. Ett huvudsyfte med denna avhandling har varit att identifiera och isolera natur-ligt förekommande substanser från marina livsformer samt att undersöka deras och syntetiserade analogers förmåga att inhibera tillväxt av havstul-panslarver.

Svampdjuret Geodia barretti innehåller flera bromerade substanser och två av dessa, de cykliska dipeptiderna barettin och 8,9-dihydrobarettin, har i studier visat sig inhibera påväxt av havstulpanslarver (Balanus improvisus) vid mycket låga koncentrationer. I ett försök att förstärka denna effekt samt för att få en inblick i verkningsmekanismen syntetiserades 14 strukturlika analoger. Variationer av substituenter i position 5 och 6 i tryptofandelen resulterade i dramatiska förändringar i effekt. Analogen benzo[g]dipodazine, där brom ersattes med en bensenring, var avsevärt potentare. En nackdel är att synteskostnaden för dessa substanser är för hög för framgångsrik kom-mersialisering.

Nyligen isolerade och strukturbestämde vi ytterligare en cyklisk dipeptid, bromobenzisoaxolon barettin, vilken precis som barettin är uppbyggd av aminosyrorna tryptofan och arginin, men är ytterligare substituerad med en hydroxygrupp samt en ovanlig bromerad cyklisk struktur. Denna substans inhiberar påväxt av havstulpaner vid mycket låga koncentrationer, med ett

43

EC50-värde på 15 nM. Ytterligare syntes- och fältstudier är nödvändiga för att kunna avgöra dess kommersiella potential.

För att bättre förstå hur dessa bromerade substanser utövar sin effekt på molekylär nivå genomfördes receptorstudier där affinitet till serotoninrecep-torer undersöktes. Hos människa är transmittorsubstansen serotonin viktig för flera funktioner, såsom humörreglering, gastrointesitinal mobilitet och reglering av kärltonus. Hos evertebrater är däremot dess biologiska roll ofullkomligt kartlagd. Det finns dock studier som pekar på att havstulpan-slarver kan innehålla serotoninreceptorer och isåfall skulle hämning av påväxt kunna ske via en interaktion med dessa receptorer. Vi konstaterade att barettin och 8,9-dihydrobarettin binder in specifikt till subreceptorerna 5-HT2A, 5-HT2C och 5-HT4 när de uttrycks i ett humant cellsystem. Dessa re-sultat kan utnyttjas dels för att hitta mer hållbara antifoulinglösningar och dels för att utveckla farmakologiska verktyg i jakten på effektivare läke-medel som interagerar med serotoninreceptorer.

En annan typ av svampdjur, från släktet Agelas, producerar indolalka-loiderna agelasiner och agelasiminer. Dessa substansklasser uppvisar ett flertal biologiska effekter och är sannolikt involverade i svampdjurets kemi-ska försvar mot bakterier och andra mikrober. Tillsammans med en grupp forskare i Oslo har vi undersökt strukturaktivitetssamband för dessa substan-ser, med tyngdpunkt på cytotoxitet och antibakteriell aktivitet. Framförallt var längden på terpensidokedjan kritisk för effekten, men även flera andra strukturer i molekylen som direkt påverkar effekten identifierades. Genom att ytterligare modifiera strukturer i molekylen skulle man kunna erhålla ledtrådar för utvecklingen av ny antibiotika mot humana sjukdomar som exempelvis tuberkulos, där resistensutveckling idag är ett allvarligt hot för att framgångsrikt kunna bekämpa sjukdomen.

Sammanfattningsvis visar denna avhandling att detaljerad kunskap om bioaktiva naturliga substanser involverade i det kemiska försvaret hos ma-rina svampdjur, kan utgöra en viktig ledtråd för identifiering av analoger som kan ligga till grund för utveckling av nya läkemedelskandidater eller biologiskt verksamma substanser för industriell applikation.

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8. Acknowledgements

This research was carried out at the Division of Pharmacognosy, Department of Medicinal Chemistry, Faculty of Pharmacy, Uppsala University. I have meet many inspiring people during my Ph.D. years and I would like to ex-press my sincere gratitude to all the people that contributed to this thesis. I would especially like to acknowledge the following: My supervisor Professor Lars Bohlin – thank you for introducing me to the fascinating field of marine science. Your guidance kept me on track and is vital for the work presented in this thesis. My co-supervisor Professor Per Jonsson – for giving me access to the fa-cilities at Tjärnö Biological Marine Laboratory and for helping me out set-ting up the settlement assay. Dr Ulf Göransson – for always fixing the HPLC after we wrecked it and for all the knowledge you passed down to the laboratory grunts. Dr Martin Sjögren and Dr Mia Dahlström – for taking good care of me at Tjärnö and for giving me insights into the marine world while taking Tilda for a stroll. Professor Rolf Larsson, at the Division of Clinical Pharmacology, Uppsala Academic Hospital – for giving me access to an excellent laboratory and a superior coffee-machine. And to Dr Sumeer Dhar and Dr Joachim Gullbo – for helpful advice and a lot of laughs, while drinking all that great coffee.

The NMR guru, Rolf Andersson – for all those days in the NMR-bunker puzzling out the spectras.

Dr Hesham El-Seedi, – for setting the standards as a scientist and for always cheering people up, although you did tend to blame me when something broke in the lab ….. All the great people in the pharmacognosy-gang; Dr Erika Svangård – for showing me around the lab and teaching me advanced soxhlet, Dr Ulrika Huss – for revealing the fundamental scientific saying: det spelar ingen roll

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hur länge du jobbar, du får ändå inte gjort mer än 15 minuters effektivt ar-bete per dag, Docent Anders Backlund – for all the mushroom field trips, I will try to avoid lömsk flugsvamp and toppig giftspindling in the future, Dr Petra Lindholm – for good company while being my room-mate for a few short weeks until you threw me out, Dr Sofia Ortlepp – for good company while being my new room-mate until you fled to Germany, Dr Sonny Lars-son – for being a living dictionary and for teaching a hopeless case like me some basic botany, Catarina Ekenäs – for nostalgic reflections when we were young and promising pharmacy students, Jenny Pettersson – for cheer-ing me on while I tried to chop up a champagne-bottle with a sword and for good times hanging out in Helsingfors, Josefin Rosén – for dragging me along to Stång Intensiv, Thanks you helped shape my bod!, Anders Herrmann – dispite your habit of putting your coffee mugs all over the lab you’re a really cool guy with tons of great advice, especially regarding train-ing, dating and other important issues. Thank you for all the laughs (I´ll never forget bananen at P3 Fredagsflörten), Robert Burman – thanks for great company while lunching, all the booze at Midsummer and for teaching me how to shoot the bow (I hit the beerbottles at last!), and the divisions Ethiopian team Teshome “Back-floor” Aboye and Mariamawit Yonathan Yeshak. Kerstin Ståhlberg, “the real boss at the division” – for all your enthusiasm and the fun stuff you organized. It´s great to be able to crosscountryski, skate, bowl and do kayak during working hours! And thanks to the depart-ment administrators, Maj Blad and Siv Berggren –for helping out every time I needed a course-certificate and for always bringing the newspaper on time. My students; Said Hodzic – for being a great bud and for all the effort and interest you shown in the marine project, Menaz Kermali – for expanding my Swahili vocabular with useful phrases such as Akili ni nywele kila mtu ana yake (Brains are like hair, everyone has there own), to Hélène Harne-mark – for turning me into a sudoku maniac, Eva Gerold – for all those looong days at the HPLC, Catherine Duarte-Martins – for all the laughs while running the cancerassay (doesn´t matter that the experiments botched), Anette Jansson and Emilia Dessle – for being so fascinated by invertebrate chemistry. “Alla goa gubbar” at the Division of Analytical Pharmaceutical Chemistry; Henrik, Jakob, Sheila, Ylva, Annika, Matilda, Anita, Victoria and Ingela – for all the relaxing time at the coffee breaks. One never knew where the con-versations would end up. Mikael, Maria, Snitting, Ante, Hallberg and Mats – for all the laughs and cursing while watching soccer and for just hanging out over the years.

46

Petter – for being a good friend and for keeping me company in the gym, so I could vent my frustrations over all my failed experiments. Alma – for your support and friendship and for all the fond memories. My grandparents, Sten – thanks for the whisky and the philosophical talks, Ingrid – thanks for the dinners and the practical talks and Dagny – thanks for the housing and some good games of “Hjärter”. My “extra-family” Anders – for letting me visit “västkusten för att tråla makrill”, to Nina – for giving me down-to-earth advice in life and Benjamin – for inspiring me to head east. My brother Per – for being my best friend and for your relaxed and positive outlook-on-life, his girlfriend Anna – welcome to the family and their new-born son / my godson Ingemar – for showing me the joy of life. To my sisters, Margareta – for being a great confidant and for taking the time to hang out with your big bro and Kristina – for being your crazy self and for looking-up to your orderly and proper big bro. My father Thomas – for encouraging me to be the best I can be. My mother Marie-Louise – for always believing in me. Thanks for all the help and for just being around!

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 63

Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofPharmacy. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy”.)

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