WO2003081199A2

WO2003081199A2 - Antifouling agent

Info

Publication number: WO2003081199A2
Application number: PCT/EP2003/002912
Authority: WO
Inventors: Lars Bohlin; Martin Sjögren; Per Jonsson; Ulf GÖRANSSON
Original assignee: Innoventus Project Ab; Lars Bohlin; Sjoegren Martin; Per Jonsson; Goeransson Ulf
Priority date: 2002-03-22
Filing date: 2003-03-20
Publication date: 2003-10-02
Also published as: AU2003232189A1; WO2003081199A3; SE0200915D0; AU2003232189A8

Abstract

The present invention relates to novel use of a chemical compound as antifouling agent. The chemical compound can be described as being a halogenated tryptophane residue derivative. Furthermore, the present invention relates to a novel active chemical compound having antifouling effects, such as preventing settlement of marine invertebrates, in particular the settlement of the barnacle Balanus species and the blue mussle Mytilus species, with low toxicity. The chemical compound is used as active component in an antifouling product used for antifouling protection of an underwater structure and vessel hull. The present invention also relates to a method of producing the novel active chemical compound, which method comprises isolating said compound from Geodia barretti or producing it synthetically.

Description

ANTIFOULING AGENT

FIELD OF THE INVENTION The present invention relates to novel use of a chemical compound as antifouling agent. The chemical compound can be described as being a halogenated tryptophane residue derivative. Furthermore, the present invention relates to a novel active chemical compound with low toxicity, having antifouling effects, such as preventing settlement of marine invertebrates, in particular the settlement of the barnacle Balanus species and the blue mussle Mytilus species. The chemical compound is preferrably used as active component in an antifouling product used for antifouling protection of any underwater structure and/or vessel hull. The present invention also relates to a method of producing the novel active chemical compound, which method comprises isolating said compound from Geodia barretti, or producing it synthetically.

BACKGROUND OF THE INVENTION

The growth of marine organisms, i.e. biofouling, on ship hulls and on static constructions, such as off-shore rigs and pipelines, causes substantial economic losses worldwide. For example, biofouling causes exhaustion of materials (corrosion), increased friction and fuel consumption, increased weight and loss of manoeuvrability. Examples of marine organisms colonizing available substrates in water are barnacles, goose-neck barnacles, mussels, blue mussels, sponges, ascidians, green macroalgae, brown macroalgae, red macroalgae, bryozoa, hydroids and biofilm-forming diatoms and bacteria, and shipworms.

Due to an ever-increasing interest for environmental issues, attention has been drawn to the use of toxic anti-fouling agents, e.g. organotin compounds such as (BuO)₃SnO, in the marine transport sector. The need for developing biocompatible substances with low toxicity to replace them with is strong.

Much research has focused on potential chemical defence by secondary metabolites, e.g. peptides, to explain the fouling free body surface of marine sponges. To date, a large number of bioactive components have been isolated from marine sponges, which display widespread activities, i.e. antimicrobial, antiviral and anticancer substances (Holler et al., 1999; McKee et al., 1994; Towle et al., 2001). Some recent reports describe different marine sponges that employ e.g. terpenes, steroids, kalihines and keratinamides with inhibitory effects on barnacle larval settlement (Tsukumoto et al., 1996; 1997, Okino et al; 1995; Hirota et al., 1996). Such compounds have several advantages when used in an antifouling coating over traditional synthetic biocides, since pathways of the degradation of natural compounds are likely to already exist with less risk for bio-accumulation.

1P-2840582-B2 discloses sesquiterpene compounds extracted from Halichondrida axinyssa of Demospongea that exhibit anti-fouling effect and prevents settlement of barnacle.

JP-2862503-B2 discloses phenanthrene derivatives extracted from Octocorallia alcyonacea that can be used as anti-fouling agents. JP-2999398-B2 discloses pyran-substituted naphtalene derivatives extracted from Acanthella cavernosa, useful as anti-fouling agents for periphyton(s), e.g. barnacles.

3P-2999398-B2 discloses decalin-substituted pyran derivatives extracted from Acanthella sponge, useful as anti-fouling agents for mollusks like barnacle.

At the Marine Biofouling Symposium in Plymouth, Great Britain, 7-9 July 1999, M. Sjδgren, U. Gδransson, P. Jonsson, P. Claeson and L. Bohlin reported that isolated fractions from the marine sponge Geodia barrettl inhibited settlement in the barnacle Balanus improvisus. However, they did not disclose any reason, nor any specific compounds responsible for this inhibitory effect.

SUMMARY OF THE INVENTION To provide the means to circumvent the drawbacks associated with the toxic prior art anti-fouling agents, a biocompatible anti-fouling agent that reversibly prevents settlement of marine invertebrates and displays low toxicity towards them is disclosed in the present application.

In this aspect, one object of the invention is to provide novel use of a chemical compound, i.e. for preventing settlement of marine organisms, in particular settlement of the barnacle Balanus species, e.g. Balanus amphitrite and Balanus eburneus, as well as for the blue mussel Mytilus species, such as Mytilus edulis, thereby having low toxic effect on said organisms.

Other examples of marine organisms colonizing available substrates in water, which are also prevented from settlement due to the above described new use of said chemical compound are goose-neck barnacles, mussels, sponges, ascidians, green macroalgae, brown macroalgae, red macroalgae, bryozoa, hydroids and biofilm-forming diatoms and bacteria, and shipworms.

The present invention achieves this object with the novel use of a chemical compound as antifouling agents represented by the general formula:

in either E- and/or Z-configuration, wherein X_!-₄ are independently one or more of hydrogen, halogen (e.g. bromine) and trifluoro methyl, where at least one X^ iε halogen or trifluoromethyl, R is hydrogen or a straight or branched hydrocarbyl moiety containing functionalities, such as carbonyl, keto, aldehyde, carboxyl, hydroxyl, amino, amid, thio, heteroatoms, and saturated or non-saturated, aromatic or allylic substituents, R₂ is hydrogen, lower alkyl or a substituted or non-substituted amino group bound thereto, R_x and R₂ optionally are coupled to each other so as to form a structure containing a ring, and wherein A represents either a single or a double bond.

This chemical compound can in one embodiment of the present invention be exemplified by the following general formula II,

where X^, R_3.-₂ and A are as defined for the compound having the general formula I, and R₃ is hydrogen, lower alkyl, or a substituted or non-substituted amino group bound thereto, the chemical compound with the formulae III-IV,

the chemical compound with the general formula V,

wherein X^ and R^ are as defined for the compound represented by the general formula I, the chemical compound having the formula VI,

or the chemical compound with the general formula VII,

wherein Xi-₄ and R^ are as defined for the compound represented by the general formula I.

Compounds I-VII are represented as the Z-configuration, but the E- configuration is by specific reference also included within the limits of the patent.

Another object of the invention is to provide a novel active chemical compound that prevents settlement of, or repels marine organisms, in particular the barnacle Balanus species such as Balanus improvisus, Balanus amphitrlte and Balanus eburneus, as well as the blue mussel Mytilus species, such as Mytilus edulis without having an essentially toxic effect on them.

Other examples of marine organisms colonizing available substrates in water, which are also prevented from settlement due to the above described use of said new chemical compound are goose-neck barnacles, mussels, sponges, ascidians, green macroalgae, brown macroalgae, red macroalgae, bryozoa, hydroids and biofilm-forming diatoms and bacteria, and shipworms.

The present invention achieves this object with a novel chemical compound having antifouling effect and having the formula VI,

or the general formula VII,

wherein Xl-4 and Rl-2 are as defined for the compound represented by the general formula I.

The above mentioned compounds represented by the formulae I-VII and their derivatives can all be used as antifouling agents in for example an antifouling composition or coating. The compound represented by the formula IV and VI, and derivatives thereof, represented by the general formula V and VII respectively, are currently the most preferred.

In a further aspect, the present invention provides an anti-fouling composition used for anti-fouling protection of an underwater structure and/or vessel hull, which composition comprises one or more of the compounds represented by formula I-VII as the active component(s), at least one binding agent, and optionally a pigment and/or conventional auxiliary.

In a still further aspect, the present invention provides a method of producing a compound represented by formula VI and/or VII, which method comprises isolating said compound from G. barretti and/or any taxonomically related Geodia releasing and/or presenting said compound.

DETAILED DISCLOSURE The present invention is based on the surprising discovery that the marine sponge Geodia barretti has the ability to prevent other organisms to settle on its surface and that it achieves said effect by releasing and/or presenting a specific class of compounds with low toxicity.

The deep sea living (10-300 m) marine sponge Geodia barretti, found in the Kostetfjord at the Swedish west coast, produces a wide spectrum of secondary metabolites, such as histamine, inosine and a previously structurally incorrect defined substance named barrettin (Lidgren et al., 1986), as well as amino acids like taurine and several sterols (Houugaard et al., 1991; Lidgren et al., 1988).

Because of G. barretti's fouling free body, it was of major interest to examine if G. barretti produces defence chemicals that prevent settlement of invertebrate larvae and other biofouling marine organisms, such as barnacles, goose-neck barnacles, mussels, blue mussels, sponges, ascidians, green macroalgae, brown macroalgae, red macroalgae, bryozoa, hydroids and biofilm-forming diatoms and bacteria, and/or shipworms, and in particular, that prevent the settlement of the barnacle Balanus improvisus and the blue mussle Mytilus edulis, which are major fouling organisms in Scandinavian waters.

The inventors have isolated and identified active fractions of substances released from G. barretti. The active compounds comprise brominated cyclodipeptides described by the general formula I and exhibit strong activities as repellants against barnacles, goose-neck barnacles, mussels, blue mussels, sponges, ascidians, green macroalgae, brown macroalgae, red macroalgae, bryozoa, hydroids and biofilm-forming diatoms and bacteria, and/or shipworms, and in particular Balanus species and Mytilus species. In particular, the inventors have successfully isolated and identified two most preferred active fractions of substances released from G. barretti and convincingly shown that specifically one compound in each of these definite fractions indeed has antifouling properties. The two isolated and identified active compounds are cyclo-[(6-Br-8-en-Trp)-Arg] and cyclo-[(6-Br-Trp)-Arg], which herein are referred to as GBBl and GBB2. The structure of GBBl and GBB2 will be described below. The presence of these defence compounds in water, surrounding specimens of G. barretti has also been established. Furthermore, a couple of analogues of the active compounds have been isolated and identified. One of the found active compounds, namely barrettin (herein referred to as GBBl), has previously been structurally determined by a parallel work of Sδlter et al 2002, Tetr. Lett. 2002, 43, 3385-3386, without indication of any biological activity, though.

In the present study, the effect on settlement of cyprid larvae of Balanus improvisus subjected to fractions of water having been inhabited by G. barretti was examined. The settlement rate of laboratory-reared cyprids was studied in Petri dishes, as described in experiment 1. Hydrophiiic fractions from G. barretti obtained through isolation of GBBl and GBB2 from G. barretti, as described in experiment 2, were added to the Petri dishes as well as cyprids. Fractions that were efficient in preventing settlement of B improvisus were further fractionised using reversed-phase high performance liquid chromatography (RP- HPLC). The bioassay-guided fractionation, combined with Mass Spectroscopy (MS) and Nuclear Magnetic Resonance (NMR) analyses, as described in experiment 3, led to the discovery of GBBl and GBB2, both of which comprise of an arginine and a tryptophan residue. The NMR-data of GBBl and GBB2 are shown in table 1 and table 2 below. Table 1 should be studied together with the structural formula of GBBl (IV) presented below the table 1. The structures of GBBl (IV) and GBB2 (VI) are presented in figures 1A and IB.

Table 1. IH and 13C NMR data, HMBC and Roesy correlations for GBBl in DMSO-d6

Position 13C Shift δc IH Shift δh HMBC Roesy correlations correlations

1 11.76 (IH, s) H-2; H-7

2 125.9 7.92 (IH, d) H-l; H-10

3 107.9 H-l; H-2; H-4

3a 125.7 H-4; H-5; H-8

4 119.9 7.58(1H, d) H-5; H-8

5 122.6 7.22(1H, dd) H-4

6 114.6 H-4; H-7

7 114.3 7.61(1H, d) H-l

7a 136.5 H-2

8 107.1 6.95(1H, s) H-4

9 122.9 H-10; H-13

10 9.53(1H, s) H-2

11 166.6 H-10; H-12; H 1-13; r H-15

12 54.7 4.04(1H, t) H-13

13 8.31(1H, d) H-12

14 160.8 H-8; H-10

15 31.1 1.73(2H, q) H-12;H-16;H-17

16 23.9 1.54(2H, qv) H-15; H-17

17 40.4 3.11(2H, q) H-16; H-18

18 7.59(1H, s) H-17

19 156.7 H-17

20 7.15(1H, bs)

21 7.15(2H, bs)

Table 2. IH and 13C NMR data for GBB2

1H-NMR (MeOH-d4) δ: 13C-NMR (MeOH-d4) δ 7.61-7.43 (m, 2H), 169.8, 7.19-7.01(m, 2H) 169.5, 4.39-4.24 (m, IH), 158.6 3.78 (m, IH), 138.7 3.53-3.39 (m, IH), 128.5 3.20-3.02 (m, IH), 127.1 2.77-2.59 (m, 2H), 123.5 2.10-1.96 (m, IH), 122.0 1.07-0.81 (m, IH), 116.1 0.80-0.64 (m, IH), 115.2 0.60-0.44 (m, IH). 110.3

57.5

55.2

41.9

32.2

30.4.

24.6.

The presence of an arginine residue was also shown by quantitative amino acid analysis after acidic hydrolysis of GBBl and GBB2, respectively.

Surprisingly, the structure elucidation showed that both GBBl and GBB2 are made up of at least an arginine and a tryptophan residue, joined N-terminal to C-terminal, forming a cyclic peptid backbone. The structure of the defensive compounds GBBl and GBB2 found in G. barretti are remarkable in that they contain a brominated tryptophan residue. Tryptophan containing substances, e.g. peptides, have been found in organisms such as bryozoans (Kamano et al. 1995) and in gastropods, such as the venomous cone snail Conus imperialis, which contains a heptapeptide in which 6-bromotryptophan is present (Craig et al.1997). In marine sponges, though, the predominating aromatic amino acids in secondary metabolites are tyrosine or phenylalanine (D ^'Auria et al, 1995).

In addition to the structural determination of GBBl and GBB2, it was convincingly determined that GBBl and GBB2 repelled settlement of competent barnacle larvae of B. improvisus. The inhibitory effect, quantified with HPLC, MS and NMR, of a representative of these compounds, purified GBBl, on settlement, and mortality of B. improvisus was determined as described in experiment 5. The result of a settlement inhibition experiment with purified GBBl is shown in fig. 2. The settlement was inhibited in a dose-response manner between 0.1 μM and 1.0 μM with an EC50-value of 5x10-4 mgml-1 (1-factor ANOVA , F= 22.680, p< 0.05).

Further settlement experiments were performed to test and compare the reversible repelling effect on settlement of competent barnacle larvae of β. improvisus of different extracts and fractions of G .barretti, as well as water having been inhabited by G. barretti, purified substances, synthetic L-(+)-arginine and derivates of L-tryptophan. The results and statistical data of the further settlement experiments are summarized in Table 3.

Table 3. The effect of the different fractions, isolated substances from G. barretti and some syntetic derivates of tryptophan and arginine on the cyprid settlement .

Treatment Ef feet on settler. lent Cone μM (EC . F-value P-value

Extract of G. barretti + 55.0 (μg ml-1) 7.2 < 0.05

Fraction 4, 5 of G. barretti ++ 10.0 (μg ml-1) 13.9 < 0.05

Water from G. barrett +++ 2.4 41.9 < 0.05

GBBl +++ 1.2 22.7 < 0.05

GBB2 ++ 10.0 14.3 < 0.05

L-tryptophan - - 3.3 > 0.05

L-arginin - - 1.4 > 0.05

5-bromo-tryptophan - - 3.3 > 0.05

6-fluoro-tryptophan - - 3.3 > 0.05

Tryptophan-arginin-OH - - 3.0 > 0.05

(+++), (++) and (+) denotes the relative efficiency of the individual treatments in the settlement inhibition assay. (-) denotes treatments with no settlement inhibiting activity.

When cyprids were exposed to GBBl, their settlement was inhibited in a dose-dependent manner between 1.2 and 2.4 μM (Fig 3A). The EC₅₀ value of GBBl, i.e. the concentration where the settlement was 50 % of that in the control dishes, was determined to 1.2 μM, and at 2.4 μM the inhibition was complete. When cyprids were exposed to GBB2, settlement was also inhibited in a dose-dependent manner in concentrations between 1.0 μM and 100 μM (Fig 3B). The minimum dose to significantly inhibit settlement was 10 μM, this was also the EC₅₀ value of GBB2.

Since GBBl and GBB2 comprise tryptophan and arginine residues, settlement inhibiting properties of synthetic L-(+)-arginine, L-tryptophan, D-L-5-bromo-tryptophan, 6-fluoro-D-L- tryptophan and tryptophan-arginine-OH were also tested as described in experiment 7, to address the question if the specific molecular structure causes the inhibitory effect, or if a specific part of the molecule in itself could be responsible for the effect.

Interestingly, none of the amino acid residues L-(+)-arginine, L-tryptophan, D-L-5-bromo- tryptophan, 6-fluoro-D-L-tryptophan or tryptophan-arginine-OH had any effect on the settlement of barnacle larvae in concentrations ranging from 0.1 μM to 10 μM (table 3), suggesting that specific molecular structures of GBBl or GBB2 or their derivatives are necessary for the inhibiting effect noticed in the cyprid settlement studies. The about tenfold increase in activity displayed by GBBl in comparison to GBB2 further suggests that the specific chemical structure is of great importance to evoke a non-settlement response, e.g to display an activity in lower concentrations, since the essential difference between the two brominated cyclopeptides is a double-bond in the tryptophan residue. The difference in chemical structure might reflect a difference in the three-dimensional structure of the molecules. In analogy to the above, it can also reasonably be expected that the different configurations, i.e. E and/or Z-configurations of the compounds are important for the bioactivity of the compounds. In one embodiment of the present invention, the different configurations are therefore separated and used separately or combined in the most effective molar ratios with each other as antifouling agents. The mentioned most effective ratios are simply determined by standardised methods known to the skilled artisan.

Furthermore, an experiment using seawater incubated with a living and undamaged G. barretti was also performed, as described in experiment 6. The incubated seawater completely inhibited the settlement of B. improvisus cyprids and indeed this water was shown to contain GBBl and GBB2 by means of LC-MS and by MS fragmentation studies using nanospray infusion (data not shown). The concentration of released GBBl and GBB2 in the tested seawater was determined to 20.5 μM by LC-UV. This relatively highly concentrated solution inhibited 90% of the setttlement of barnacle larvae, even after a ten time dilution (Fig. 4). These results were all in good agreement with the effect mediated by the pure isolated compounds. Many settling larvae and algal-spores have sticky body surfaces or secrete a permanent glue, a process which precedes metamorphosis. Hence, it would be advantageous for the sponge to deter such attempts of attachment already in the ambient water. What is more, the fact that many invertebrate larvae have a speedy metamorphosis (Hadfield, M. G. Cell & Developmental Biology. 2000, 11, 437-443) further points to an additional benefit of early fouling prevention. The observations regarding the release of these compounds into the ambient water show that the concentration in the water surrounding live and undamaged specimens of G. barretti placed in aquaria is sufficient to completely inhibit settlement of cyprid larvae of B. improvisus. Without wanting to limit the present invention to a particular theory, this suggests that the compounds may be released in effective concentrations to reach settling larvae and thereby to form a chemical defence for G. barretti. However, the presence of a chemical defence may have other targets than fouling from invertebrate larvae, e.g act as selective defence against microbial fouling and predation.

Figures 5 and 6 show the release of GBBl from living G. barretti shown by RP-HPLC-MS analysis (mass range m/z 419), after injection of water surrounding an incubated specimen where the spectrum in figure 5 shows both the isomers of GBBl. The main peak in figure 5 monitored at m/z 419 is magnified in figure 6, which clearly shows the typical isotopic distribution of bromine. When injecting the isolated substance as a reference, equivalent chromatograms were obtained. MS-spectra in figures 5 and 6 show a similarity pattern in molecular weight and degradability for GBBl as compared to spectra of purified GBBl indicating a release of the compound into the ambient water of living G. barretti.

A series of experiments was also carried out to establish if the effect of GBBl and GBB2 on the settlement of cyprid larvae was reversible, as described in experiment 8. Cyprids were exposed to GBBl and GBB2 in effective concentrations, as determined by the dose- response experiments described above. The settlement inhibition evoked by GBBl and GBB2 was reversible and the present inventors found that cyprids which had been exposed to GBBl and GBB2 for 24 h, at concentrations of 2.4 μM and 24 μM, respectively, and then were washed and transferred to seawater, metamorphosed to a similar extent as the cyprids in the control dishes. In the dishes where the cyprids remained in GBBl and GBB2 at concentrations of 2.4 μM and 24 μM, respectively, the settlement was completely inhibited (Figs. 7A & B). Thus, it can be concluded that both GBBl and GBB2, due to their low toxicity, are attractive candidates as active ingredients in a marine antifouling paint. Therefore, in two of the most preferred embodiments of the present invention GBBl and/or GBB2 are used as active ingredient(s) in marine antifouling compositions, such as paint.

TBTO (Tributyltin oxide) and other biocides, such as Sea-Nine, which are at present widely used as antifouling substances, are extremely toxic to cyprid larvae so that within 24 h the larvae die. The EC₅₀ value for TBTO and Sea-Nine is 90 ng ml-1 and 330 ng ml-1 respectively (Willemsen et al., 1998). The corresponding values for barretin and GBB2 are 0.5 μg ml-1 (1.2 μM) and 5 μg ml-1 (12 μM), respectively. A comparison of these EC₅₀- values indicates that GBBl and GBB2 are effective in the same range as these biocides and furthermore, when cyprids were exposed to a twentyfold increase in the effective concentration of GBBl (24 μM) (Fig 3A), there is still no significant increase in mortality, further supporting that the compounds comprised in the GBB familly do not act through a toxic mechanism.

Furthermore, the hydrophobic portion of GBBl, GBB2 and their derivatives make them especially suitable for antifouling purposes, since a hydrophobic molecule has the possibility of interacting with important components in the paint formulation, making the active ingredients less prone to leak out into the water. In addition, GBBl, GBB2 and their derivatives possess the attractive feature of being bio-synthesized in the marine environment, i.e. pathways of degradation are likely to already exist.

Field experiments were performed at a raft outside Tjarnδ Marine Biological Laboratory to test the bioactivity of GBBl, GBB2 and the synergistic bioactivity effect of a combination of GBBl and GBB2. Plexiglass® panels were painted with four different non-toxic paints; Spf, TF and H2000 (Loutrec) and Fabi Eco (International) or a combination of one of the four paints and GBBl, or GBB2, or GBBl and GBB2, on settlement of Balanus improvisus and Mytilus edulis. The results of the field experiment on the settlement of Balanus improvisus using the paint Spf are shown in figure 8A, and using the paint Tf in figure 8B. The results of the use of the paints H200 and Fabi Eco on the settlement of Balanus improvisus were similar to the results of the use of Tf and are therefore not shown. The settlement of B. improvisus was significantly inhibited at both 0.1 and 0.01 mg/ml of GBBl used together with Spf, as well as when both 0.1 and 0.01 mg/ml of GBB2 was used together with Spf. 0.1 mg/ml of GBBl in Spf resulted in a 90% reduction of recruitment of B. improvisus. Using Spf, there was also a synergistic inhibitory effect of 0.01 mg/ml GBBl and 0.01 mg/ml GBB2, which inhibitory effect was more efficient than 0.01 mg/ml GBBl or 0.01 mg/ml GBB2 alone.

The results of the field experiment on the settlement of Mytilus edulis using the paint Spf are shown in figure 8C. The settlement of Mytilus edulis was inhibited when both 0.1 and 0.01 mg/ml of GBBl was used together with Spf, as well as when both 0.1 and 0.01 mg/ml of GBB2 was used together with Spf. There was also a synergistic inhibitory effect of 0.01 mg/ml GBBl and 0.01 mg/ml GBB2. A preferred embodiment of the present invention thus relates to the use of a combination of GBBl, GBB2 and/or their derivatives as an even more effective antifouling agent for use in e.g. protecting underwater hulls, structures and/or vessels. Obviously, the synergistic effect of both GBBl and GBB2 compounds will facilitate using less compound to achieve the same or better antifouling effect as described above.

Furthermore, GBBl was synthesized as described in experiment 11 and GBB2 was synthesized according to a conventional method in the art. The bioactivity of synthetic GBBl and GBB2, respectively on settlement of cyprids of B. improvisus was tested as described in experiment 5, but with synthetic GBBl and GBB2 instead of G. barretti fractions, used in the bioassay. The settlement was inhibited by both synthetic GBBl and synthetic GBB2 in a dose-dependent manner. The settlement was inhibited by synthetic GBBl between 0,24μM and 2.4μM and by synthetic GBB2 between 1 and 100 μM (Fig. 9A & 9B). As the dose response of the synthetically produced compounds is essentially similar to the native and isolated compound's, it is thus of course also envisioned to use synthetic GBBl, GBB2 and/or their derivatives for antifouling purposes.

In the present context, when isolating and defining GBBl and GBB2, the inventors also found several analogues to GBBl and GBB2 in the fractions of G. barretti. These analogues can of course also be used for antifouling purposes. Below some particular analogues are shown, which are previously unknown compounds being equally preferred embodiments of all aspects of the present invention.

15

GBBl, GBB2 and their analogues can suitably be used in anti-fouling products for underwater use for inhibition of settlement of barnacles, goose-neck barnacles, mussels, blue mussels, sponges, ascidians, green macroalgae, brown macroalgae, red macroalgae, bryozoa, hydroids and biofilm-forming diatoms and bacteria, and/or shipworms. Such products can be prepared by conventional methods.

In order to form an antifouling composition, GBBl, GBB2 or their analogues can for example be mixed with a binding agent, such as an organopolysiloxane, e.g. a low molecular mass alkoxy-functional silicone resin, a silicone rubber, or an organosilicon copolymer. Such an antifouling composition can additionally comprise inorganic pigments, organic pigments, dyes (which are preferably insoluble in salt water) and/or conventional auxiliaries such as fillers, solvents, plasticizers, catalysts, inhibitors, tackifiers, coating additives and/or common dispersion auxiliaries. It is of course also possible to use other binding agents, pigments and auxiliaries known in the art.

Other examples of antifouling compositions that are meant for use under water and that can be used with anti-fouling agents according to the present invention, are disclosed in US-6 245 784-B1, US-6 217 642-B1, US-6 291 549-B1, US-6 211 172-B1 and US-6 172 132-B1.

The final antifouling products can be used for underwater structures, e.g. in plumbing ports of nuclear power stations, at ocean facilities such as bayshore roads, undersea tunnels, port facilities, and in canals or channels. In a most preferred embodiment, the products can be used for coating marine vessels, such as boats and/or fishing gear (rope, fishing net or the like).

The antifouling compositions can be applied either directly to the surface of vessel hulls and/or underwater structures, or be applied to the surface of vessel hulls and/or underwater structures pre-coated with undercoating material, such as rust preventive and/or a primer.

The antifouling compositions may also be used to repair surfaces of vessel hulls and underwater structures previously coated with a conventional anti-fouling paint and/or anti-fouling coating composition.

The antifouling composition can for example be formulated as a paint, solution or emulsion.

An antifouling paint according to the present invention can comprise of one or more of GBBl, GBB2 and/or their analogues as active ingredient(s), a film-forming ingredient, including a solvent selected according to use, extender pigments, colouring pigments and/or additives. Film-forming ingredients include chlorinated rubber resin, vinyl acetate resin, acrylic resin and natural resin. An antifouling solution is prepared by formulating the active ingredient(s) with a film-forming ingredient as previously described and by dissolving the mixture in a solvent. When the antifouling agent is formulated as an emulsion, an anti-fouling solution can be prepared according to any conventional method in the art, such as by dissolving the active ingredient(s) in solvent and by adding a surfactant to the mixture. Preferred surfactants include those typically used in the art.

Furthermore, the antifouling formulation can also be used to prevent biofouling on surfaces in earlier or later stages of the fouling process, relative to the peak settlement of barnacles, mussels or other macro-organisms. The development of the biofouling layer involves adhesion of macromolecules (within minutes), colonization by bacteria and micro-algae (within hours), settlement of invertebrate larvae and algal spores (within days). Within weeks, the growth of settled propagules will lead to the appearance of macroscopic organisms on the surface, completing the biofouling process. As an example, the antifouling formulation can also be used to control the formation of biofilms or the establishment of mould on facades.

Thus, while there have been shown and described and pointed out fundamental novel features of the invention as applied to embodiments thereof, it will be understood that various omissions and substitutions and changes in details may be made by those skilled in the art without departing from the spirit of the invention. For example, it is expressly intended that all analogues which perform substantially the same function in substantially the same way to achieve the same results are within the scope of the invention.

REFERENCES

Claeson, P.; Gδransson, U.; Johansson, S.; Luijendijk, T.; Bohlin, L. J. Nat. Prod. 1998, 61, 77-81.

Craig, A. G.; Jimenez, E. C; Dykert, J.; Nielsen, D.B.; Gulyas, J.; Abogadie, F. C.jPorter, J.; Rivier, J. E.; Cruz, L. J.; Olivera, B. M.; Mcintosh, J. M. J. Biol. Chem. 1997, 272, 4689- 4698.

D ^'Auria, M. V.; Paloma, L. G.; Minale, L.; Zampella, A.; Debitus, C; Perez, J. J. Nat. Prod. 1995, 58, 121-123.

Hadfield, M. G. Cell & Developmental Biology. 2000, 11, 437-443.

Hirota, H.; Tomono, Y.; Fusitani, N. Tetrahedron. 1996, 52, 2359-2368.

Holler, U.; Konig, G. M.; Wright, A.D. J. Nat Prod. 1999, 62, 114-118.

Hougaard, L.; Christophersen, C; Nielsen, P. H.; Klitgaard, A.; Tendal, O. Biochem. Syst.Ecol .1991, 3, 223-235.

Kamano, Y.; Zhang, H.; Ichihara, Y.; Kizu, H. Tetrahedron. Lett. 1995, 36, 5523-5528.

Lidgren, G.; Bohlin, L.; Bergman, J. Tetrahedron Lett., 1986, 27, 3283.

Lidgren, G.; Bohlin, L; Christophersen Carsten, J. Nat. Prod. 1988, 51, 1277-1280.

McKee, T. C; Cardellina, J. H.; Riccio R. J. Med. Chem. 1994, 37, 793-797.

Okino, T.; Yoshimura, E.; Hirota, H.; Fusitani, N. Tetr. Lett. 1995, 36, 8637-8640.

Schmidt, U.; Lieberknecht, A.; Wild, J. Synth. Commun., 1984, 53.

Schmidt, U.; Wild, J. Liebigs lnn. Chem., 1985, 9, 1882.

Sόlter, S.; Dieckmann, R.; Blumenberg, M.; Francke, W. Tetr. Lett. 2002, 43, 3385-3386.

Towle, M. J.; Salvato, K. A.; Bud row, J. Cancer Res. 2001, 61, 1013-1021.

Tsukumoto, S.; Kato, H.; Hirota, H.; Fusetani, N. Tetrahedron. 1996, 52, 8181-8186.

Tsukumoto, S.; Kato, H.; Hirota, H.; Fusetani, N. Biofouling. 1997, 11, 283-291.

Willelmsen, P. R, Overbeke, K.; Suurmond, A. Biofouling. 1998, 12, 133-147.

Wu, Y.; Matsueda, G.^' R.; Bernatowicz, M.; Synth. Commun., 1993, 23, 3055 FIGURE LEGENDS

Fig. 1 A&B show the chemical configuration of cyclo-[(6-Br-8-en-Trp)-Arg] (GBBl) and cyclo-[(6-Br-Trp)-Arg] (GBB2), isolated and characterized from the marine sponge G. barretti.

Fig. 2 shows the effect of GBBl on the settlement of cyprid larvae of B. improvisus. The response variables are percentage settled and dead cyprids shown as means + SE (n=4).

Fig. 3 A&B show the effect of GBBl and GBB2 on the settlement of cyprid larvae of B. improvisus. The response variables are percentage settled and dead cyprids shown as means ± SE (n=4).

Fig.4 shows the effect of seawater exposed to live G. barretti on the settlement of cyprids of B. improvisus. Data are shown for intact seawater and water diluted 10 times. The respons variables are percentage settled and dead cyprids shown as means ± SE (n=4).

Fig. 5 shows RP-HPLC-MS analysis of GBBl released from living specimens of

Geodia barretti. The presence of both isomers of GBBl (E/Z) is shown. The spectrum clearly shows the typical isotopic distribution pattern of bromine. When injecting the isolated substance as a reference, equivavalent chromatograms were obtained.

Fig. 6 shows RP-HPLC-MS analysis of GBBl released from living specimens of

Geodia barretti wherein the main peak of Fig. 5, monitored at m/z 419, is magnified.

Fig. 7A&B show the reversibility of GBBl and GBB2 respectively after the cyprids are transferred to fresh saltwater. The response variables are percentage settled and dead cyprids shown as means ± SE (n=4).

Fig. 8A-C show the results of a field experiment of the bioactivity of GBBl, GBB2 as well as GBBl and GBB2 in combination with a non-toxic paint. Fig. 8A shows the result of the bioactivity on B. improvisus usin the paint Spf. Fig. 8B shows the result of the bioactivity on B. improvisus using the paint Tf. Fig. 8c shows the result of the bioactivity on Mytilus edulis using the paint Spf.

Fig. 9A&B show the effect of synthetic GBBl and synthetic GBB2 respectively on the settlement of cyprid larvae of B. improvisus. The response variables are percentage settled and dead cyprids shown as means ± SE (n=4). EXPERIMENTS

Experiment 1: Larval bioassav

The brooding stock of adult barnacles (β. improvisus, Darwin 1854) was allowed to settle on Plexiglass® panels in the sea on a raft outside Tjarno Marine Biological Laboratory (58°53'N, 11°8'E). Cleaned from epiphytes they were brought to the laboratory and placed in buckets with running seawater (salinity 32±l%o). When regularly fed, with nauplii of Artemia s.p, B. improvisus will spawn throughout the whole year.

Nauplii were collected and incubated in buckets (20L) with filtered seawater (0.2μm, 32+l%o) and antibiotics (streptomycin 36.5 mg/l, penicillin G 21.9 mg/l) were added. The nauplii larvae were kept at 27-28°C and fed with Thalassiosira pseudomonaε and Isochrysis galbana. After 6-7 days when the nauplii have developed into cyprid larvae they are filtered (320, 230, 160 and lOOμm) and thoroughly washed to remove particles like microalgae and detritus. The inventors have recently found that cyprids of B. improvisus tolerates storage at 13°C and subsequently, that larvae can be used for 4-6 days after hatching.

The experiment for the evaluation of the effect on settlement and mortality was performed using polystyrene Petri dishes (0 48mm, Nunc AS Denmark) to which 10 ml of G. έ>arrett7-fractions dissolved to different concentrations in filtered seawater (0.2μm, 32+l%o) was added. 20±2 competent cyprids were added to each dish in four replicates and dishes with filtered seawater served as controls. Dishes were maintained for 3-4 days in room temperature and after that they were examined under stereomicroscope for attached and metamorphosed individuals and dead cyprids.

Experiment 2: Isolation of GBBl and GBB2 from Geodia barretti

G. barretti, family Geodiidae order Astrophorida (Bowerbank 1858) were collected at 50 to 60 m depth in the Kosterfjord at the Swedish west coast in March 2001. A 1.1 kg specimen was homogenized and freeze-dried. The freeze-dried material was defatted with CH₂CI₂ x 5 for removal of lipophilic compounds and the residue was extracted with EtOH (50 %) x 5, as outlined in a fractionation protocol by Claeson et al (1998). The aqueous ethanolic extract was concentrated and then desalted by reversed phase solid phase extraction (RP-SPE) [Isolute C18(EC), International Sorbent Technologies, Mid Glamorgan, U.K.]. After applying the sample, the RP-SPE column was washed with 0.1% TFA, then 60% AcN, 0.1% TFA to elute captured substances.

AcN was removed from the latter fraction in vacuo, then the extract was subjected to RP- HPLC using an Akta Basic equipped with a 50 ml Superloop (Amersham Pharmacia Biotech, Uppsala, Sweden), enabling unattended repeated injections of 4 ml on a Rainin Dynamax RP18 column (10 x 250 mm, 5 μm, 30θA). A linear gradient elution from 10 to 100% of eluant B [eluant A: 0.1% TFA (v/v), and eluant B: 60 % AcN, 0.1% TFA] over 25 min was used at a flow rate of 4 ml/min. The separation was monitored by UV at 215, 254 and 280 nm, and collected into five fractions that were tested for inhibition of barnacle settlement.

Fractions number 4 and 5 were found to be most active, and were subjected to rechromatography on the same system using more shallow gradients. GBBl was isolated from fraction 5 in this manner and GBB2 from fraction number 4. The purity of the isolated compounds were >95% as determined by analytical HPLC on a GROM ODS-4HE column (2 x 100 mm, 3 μm, 200 A). For GBBl, the ratio of isomers was approximately 87/13 (Z/E) according to peak integration at 280 nm. GBB2 and a mixture of the two isomers from GBBl was used for testing in the "larval bioassay" described above.

Experiment 3:

Structure elucidation

For MS and MS2 a nanospray-ion trap MS [Protana 's NanoES source (MDS Protana A/S, Odense, Denmark) mounted on a LCQ MS (Thermo Finningan, San Jose, USA)] was used. Samples were analysed in the positive ion mode directly after fractionation, or after lyophilising and dissolution in 60% MeOH, 1% HOAc. The spray voltage was set to 0.5 kV and the capillary temperature to 150°C. For MS2, the CID (Collision Induced Disassociation) was set to 35%.

NMR was done on a Bruker DRX-600 spectrometer (Bruker Spectrospin, Milton, ON, Canada) operating at 30°C and equipped with a microprobe. Spectra were recorded with the sample in a 1.7 mm (OD) capillary. IH, COSY, TOCSY, HSQC-DEPT and HMBC spectra were recorded and chemical shifts were determined relative to the internal standards MeOH and DMSO respectively.

The presence of the arginine residue was also shown by quantitative amino acid analysis after acidic hydrolysis of GBBl and GBB2 respectively (24h at 110 C with 6N HCI containing 2 mg/ml phenol, the hydrolysate as analyzed with an LKB Model 4151 Alpha Plus amino acid analyzer using ninhydrin detection).

Experiment 4:

Incubation of water by living G. barretti

A specimen of G. barretti (0,85 kg) was placed in an aquarium containing 5 I filtered seawater for 24 h. Samples of 10 ml was then taken from four different places in the aquarium and were used in the "incubation experiment" (see below). Samples were also taken which were diluted ten times and examined for inhibiting properties on cyprid larval settlement.

LC-MS analysis was done on the LCQ electrospray ion trap MS operated in the positive ion mode, connected to the Akta Basic HPLC. 50 ul incubated water was injected [Grom-Sil ODS-4 HE (2 x 100 mm, 3 μm, 200 A)] and eluted at a flow rate of 0.3 ml/min with a linear gradient from 10 to 100% B over 15 min [A: 0.1% HCOOH (v/v) in H20; B: 60% AcN, 0.1% HCOOH]. The spray voltage was set 4.5 kV and the capillary temperature to 230°C. For determination of the concentration, a calibration curve was created [same HPLC setup as above, eluents (A: 0.1% TFA, B: 60 % AcN, 0.1% TFA), and detection changed (UV 215 nm)], after establishing the molar UV absorbance (e=31205 at 337.5 nm, MeOH) by the quantitative amino acid analysis.

Experiment 5:

Settlement Inhibition Assay The inhibitory effect, quantified with HPLC, MS and NMR, of the purified active substances on settlement, and the mortality rate of β. improvisus was determined using the larval bioassay.

Experiment 6: Incubation experiment

The inhibitory effect of water incubated according to the "incubation of water by living G. barretti" was determined using the "larval bioassay" and the major secondary metabolite (GBBl) was examined with HPLC and MS.

Experiment 7:

Experiments of the effect of synthetic L-C+ . -arginine and derivates of L-tryptophan. The synthetic amino acids L-(+)-arginine, L-tryptophan D-L-5-bromo-tryptophan, 6- fluoro-D-L-tryptophan and the dipeptide tryptophan-arginin-OH were purchased from Sigma-Aldrich (St. Louis, USA). L-(+)-arginine was dissolved in mQ water and added to FSW (10 ml) to give the desired concentration series (0.1, 1.0 and 10 μM). The different tryptophan derivatives were dissolved in di-methylsulfonoxide (DMSO) and prepared as above. Petri dishes of polystyrene (Nunc no 240045) were filled with the different substances in FSW and 20-25 cyprids were added to each dish. Dishes with FSW or DMSO served as controls. Each treatment was replicated four times and the experiment was maintained for 4 days before the dishes were viewed under a stereo microscope to check for attached and metamorphosed individuals, non-attached larvae and also, for dead cyprids.

Experiment 8: Reversible effect of GGB1 and GBB2

A series of experiments was carried out to establish if the effect of GGB1 on the settlement of cyprid larvae was reversible. Cyprids were exposed to GGB1 in the effective concentration as revealed by the dose-response experiment described above. After 24 h the cyprids were washed and then transferred to fresh saltwater. The treatment was replicated 4 times and the experiment was maintained for 4 days. The dishes were then checked in a stereo microscope for attached and metamorphosed individuals, non- attached larvae and also, for dead cyprids.

Experiment 9: Field experiments Field experiments were performed at a raft outside Tjarnδ Marine Biological Laboratory (58°53'N, 11°8'E) to test the bioactivity of GBBl, GBB2 and the synergistic bioactivity effect of a combination of GBBl and GBB2 on Balanus improvisus as well as Mytilus edulis. Plexiglass® panels were painted with four different non-toxic paints; Spf, TF and H2000 (Loutrec) and Fabi Eco (International) or a combination of one of the four paints and GBBl or GBB2 or a combination of GBBl and GBB2. Panels with paint only were used as controls and four replicates were used of the controls and of each combination of one paint and GBBl or GBB2 or a combination of GBBl and GBB2. The concentrations of GBBl tested were 0.01 and 0.1 mg/ml, of GBB2 0.01 and 0.1 mg/ml and the concentration used when combining GBBl and GBB2 was 0.01 mg/ml of each. The plexiglass® panels were held in water during four or eight weeks and the number of individuals of B. improvisus as well as Mytilus edulis were counted on the panels. The field experiment was performed during the period July-September, which is the period when the settling is most intense of B. improvisus.

Experiment 10:

Bioactivity of synthetic GBBl and GBB2

The bioactivity of synthetic GBBl and GBB2 respectively were tested on settlement of cyprids of B. improvisus according to the settlement inhibition assay described above. GBBl was synthesized according to experiment 11 and GBB2 was synthesized according to a method conventional in the art.

Experiment 11:

Synthesis of GBBl NMR spectra were recorded at 300 MHz for IH and 75 MHz for 13C respectively; δ values are given in ppm and coupling constants are reported in Hz. The IR spectra were acquired using a FT-IR instrument. All reagents were purchased from Aldrich or Lancaster and were used as received. All solvents were purified by distillation or were of analytical grade.

Chromatographic separations were performed on silica gel 60 (230-400 mesh). Data for synthetic GBBl (1) agrees completely with the data reported for the natural product

(Lidgren et al., 1986; Sόlter et al., 2002). Compound 2, 4 and 6 was prepared as described elsewhere.2,3,4

3

To a suspension of Cu[Arg^ω'^<0(Boc)₂]₂ (Wu et al., 1993) (2) (2.00 g, 2.48 mmol), ethylenediaminetetra acetic acid (EDTA-4Na.2H₂0) (1.11 g, 2.98 mmol) and NaHC0₃ (834 mg, 9.93 mmol) in H₂0 (15 mL) was dropwise added a solution of di-tert-butyl dicarbonate (Boc₂0) (1.30 g, 5.96 mmol) in acetone (25 mL). The reaction mixture was stirred at room temperature for 12 h and acetone was evaporated. The aqueous mixture was acidified with 5% KHS0₄, until ~ pH 3. The resulting gummy precipitate was extracted with EtOAc (2 x 50 mL). The combined organic phases washed with H₂0 (100 mL), brine (100 mL) and dried over MgS0₄. Evaporation furnished a yellow oil which was subjected to column chromatography using Hexane/EtOAc (50/50) as eluent, yielding 3 as a white solid (1.2 g, 51%).

IR (KBr): 3332, 2980, 1722, 1634, 1616, 1368, 1332, 1158, 1136, 1052 cm-1. 1H-NMR (DMSO-d6): δ 12.53 (s, IH, D₂0 exch.), 11.49 (s, IH, D₂0 exch.), 8.29-8.25 (m, IH, D₂0 exch.), 7.08 (d, J = 8.0, IH, D₂0 exch.), 3.85-3.82 (m, IH), 3.27-3.25 (m, 2H), 1.57- 1.35 (m, 31H) 13C-NMR (CDCI₃) : δ 174.1 (s), 163.1 (s), 155.6 (s), 155.3 (s), 152.1 (s), 82.9 (s), 78.1 (s), 78.0 (s), 53.3 (d), 39.6 (t), 28.2 (q), 28.1 (t), 28.0 (q), 27.6 (q), 25.5 (t).

A solution of compound 4 (Schmidt et al., 1984) (2.23 mg, 5.94 mmol) in EtOH (60 mL) was hydrogenated in the presence of Pd/C (5%; 213 mg) at room temperature for 4.5 h. The reaction mixture was filtered through celite and the filtrate evaporated leaving a clear oil. The free amine was immediately dissolved in CH₂CI₂ (10 mL) and added to an ice-cold mixture of compound 3 (2.56 g, 5.40 mmol), 1-hydroxybenzotriazole hydrate (HOBt) (803 mg, 5.94 mmol), l-ethyl-3-(3 '-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (1.14 mg, 5.94 mmol) and N, N-diisopropylethylamine (DIEA) (1.03 mL, 5.94 mmol) in CH₂CI₂ (15 mL). The reaction mixture was allowed to reach room temperature. After 15 h the solvent was evaporated and the residue was taken up in EtOAc (150 ml) then washed with H₂0 (30 mL) and brine (30 mL). The organic phase was dried ove f MgS0₄ and evaporated. Purification by column chromatography with EtOAc/hexane (70/30) as eluent afforded the title compound 5 as a clear oil. Yield: 2.35 g (64%). IR (KBr): 3332, 2978, 2933, 1752, 1719, 1680, 1639, 1617, 1367, 1330, 1252, 1164, 1134, 1050, 1025 cm-1. 1H-NMR (CDCI₃) : δ 11.45 (s, IH, D20 exch.), 8.34-8.30 (m, IH, D₂0 exch.), 7.27-7.19 (m, IH, D₂0 exch.), 5.42-5.32 (m, IH, D₂0 exch.), 5.19 (d, J = 8.9, IH), 4.22-4.06 (m, 4H), 3.78 (s), 3.43-3.39 (m, 2H), 1.86-1.27 (38 H).

Arginine derivative 5 (2.29 mg, 3.36 mmol) dissolved in CH₂C1₂ (8 mL) was added dropwise to a suspension of potassium tert-butoxide (t-BuOK) (753 mg, 6.73 mmol) in CH₂CI₂ (4 mL) at -17°C under nitrogen atmosphere. After 10 min 6-bromo-3-formyl~ indole-1-carboxylic acid tert-butyl ester (6) (Schmidt, U.; Wild, J. Liebigs Ann. Chem., 1985, 9, 1882) (1.09 mg, 3.36 mmol) in CH₂CI₂ (10 mL) was added. The reaction mixture was allowed to reach room temperature. After 18 h the mixture was evaporated to dryness and the residue dissolved in EtOAc (100 mL), washed with H₂0 (2x200 mL) and brine (100 mL). The organic phase was dried over MgS0₄ and evaporated, leaving a yellow oil which was subjected to column chromatography. Elution with hexane/EtOAc (60/40) afforded 7 as a yellow oil (950 mg, 33%).

IR (KBr) : 3330, 2978, 2933, 1721, 1641, 1619, 1368, 1333, 1251, 1155, 1135, 1051cm-

1.

1H-NMR (CDCI₃) : δ 11.46 (s, IH), 8.52-8.41 (m, IH), 8.31 (s, IH), 8.19 (s, IH), 7.83 (s,

IH), 7.67 (s, IH), 7.53 (d, J = 8.5, IH), 7.39 (dd, J = 8.5; 1.7, IH), 5.72 (d, J = 8.1, IH), 4.51-4.37 (m, IH), 3.81 (s, 3H), 3.60-3.55 (m, IH), 3.49-3.39 (m, IH), 2.02-1.86 (IH), 1.72-1.25 (m, 39H).

13C-NMR (CDCI₃) : δ 171.0 (s), 165.3 (s), 163.4 (s), 156.8 (s), 155.9 (s), 153.4 (s), 148.9 (s), 135.6 (s), 128.6 (d), 128.3 (s), 126.5 (d), 124.6 (d), 123.2 (s), 120.4 (d), 118.8 (s), 118.7 (d), 114.1 (s), 85.2 (s), 83.4 (s), 80.2 (s), 79.6 (s), 54.4 (d), 52.8 (q), 40.0 (t), 29.1 (t), 28.5 (q), 28.3 (q), 28.2 (q), 26.1 (t).

Trifluoroacetic acid (TFA) (0.91 mL) was added a solution of compound 7 (500 mg, 0.59 mmol) in CH2CI2 (10 mL) and stirred at room temperature for 8 h. The solvent was evaporated and the residue dissolved in 1-butanol (lOmL) containing 0.1 M acetic acid. After addition of N-methylmorpholine (NMM) (0.06 mL, 0.59 mmol) the reaction mixture was heated at reflux for 4.5 h. The mixture was allowed to cool and thereafter washed with H20 (2 x 15 mL), brine (10 mL) and dried over MgS0₄. Evaporation of the solvent under reduced pressure afforded GBBl (1) as a dark yellow solid (153 mg, 62%).

Claims

Use of a chemical compound as anti-fouling agent, represented by the general formula I,

in E- and/or Z-configuration, wherein

Xi-₄ are independently one or more of hydrogen, halogen (e.g. bromine) and trifluoromethyl, where at least one Xι-₄ is halogen or trifluoromethyl,

Ri is hydrogen or a straight or branched hydrocarbyl moiety containing functionalities, such as carbonyl, keto, aldehyde, carboxyl, hydroxyl, amino, amid, thio, heteroatoms, and saturated or non-saturated, aromatic or allylic substituents,

R₂ is hydrogen, lower alkyl or a substituted or non-substituted amino group bound thereto,

Ri and R₂ optionally are coupled to each other so as to form a structure containing a ring, and wherein

A represents either a single or a double bond.

2. Use of a chemical compound as anti-fouling agent according to claim 1, wherein the compound is represented by the general formula II,

in E- and/or Z-configuration, where X^, R^ and A are as defined in claim 1 and R₃ is hydrogen, lower alkyl, or a substituted or non-substituted amino group bound thereto.

3. Use of a chemical compound as antifouling agent, represented by the formula III,

in E- and/or Z-configuration.

4. Use of a chemical compound as antifouling agent, represented by the formula IV,

in E- and/or Z-configuration.

5. Use of a chemical compound as antifouling agent, represented by the general formula V,

in E- and/or Z-configuration, where Xι-₄ and Rι-₂ are as defined in claim 1.

6. Chemical compound represented by the formula VI,

in E- and/or Z-configuration.

7. Chemical compound represented by the general formula VII,

in E- and/or Z-configuration, where X^ and Rj-₂ are as defined in claim 1.

8. Use of a chemical compound as antifouling agent, represented by the formula VI,

in E- and/or Z-configuration.

9. Use of a chemical compound as antifouling agent represented by the general formula VII,

in E- and/or Z-configuration, where Xi-₄ and Rι-₂ are as defined in claim 1.

10. A method of producing a compound represented by the formula VI and/or VII, which method comprises isolating said compound from Geodia barretti.

11. Antifouling composition comprising: one or more of the compounds represented by the formulae I-VII, at least one binding agent, pigments and/or conventional auxiliaries.

12. Antifouling composition according to claim 11, wherein the binding agent is an organopolysiloxane, e.g. a low molecular mass alkoxy-functional silicone resin, a silicone rubber and/or an organosilicon copolymer.

13. Antifouling composition according to any of claims 11-12, wherein the pigments are inorganic pigments, organic pigments and/or dyes.

14. Antifouling composition according to any of claims 11-13, wherein the conventional auxiliaries are fillers, solvents, plasticizers, catalysts, inhibitors, tackifiers, coating additives and/or common dispersion auxiliaries.

15. Antifouling composition according to any of claims 11-14 formulated as a paint, solution or emulsion.

16. Coating film formed from the antifouling composition according to any of claims 11- 15.

17. Use of the antifouling composition according to any of claims 11-15 for coating an underwater structure.

18. Method for preventing fouling of underwater structures and/or hulls using the antifouling composition according to any of claims 11-15.

19. Method for preventing settlement of cyprids, barnacles, goose-neck barnacles, mussels, blue mussels, sponges, ascidians, green macroalgae, brown macroalgae, red macroalgae, bryozoa, hydroids and biofilm-forming diatoms and bacteria, and/or shipworms on underwater structures and/or hulls wherein an antifouling composition according to any of claims 11-15 is used.

20. Underwater structure coated with the antifouling composition according to any of claims 11-15.

21. Hull coated with the antifouling composition according to any of claims 11-15.