AU754362B2 - Production of furanones - Google Patents

Description

WO 99/54323 PCT/AU99/00285 1 Production of furanones Technical Field The present invention relates to the side chain functionalisation of fimbrolides (halogenated 3-alkyl-5-methylene-2(5H)-furanones) and their synthetic analogues, that yields fimbrolides substituted with a halogen, an oxygen or a nitrogen functionality in the alkyl chain, especially fimbrolide alcohols, carboxylate and sulfinate and sulfonate esters, ethers, aldehydes, ketones, acids, amides, nitro derivatives, and polymers.

Background Art It is known that a variety of fimbrolides possessing antifungal and antimicrobial properties can be isolated from red marine algae Delisea fimbriata, Delisea elegcas and Delisea pulchra. The very few reported syntheses of functionalised fimbrolides use (E)-P-bromo-p-lithioacrylate or 3formyl-6-methylfuran or allenes as starting materials. These syntheses are unnecessarily long, tedious and give very low yields of the fimbrolides. The present inventors have recently reported the preparation of a range of fimbrolides having different sized chain lengths (Manny et al (1997) Tetrahedron 53: 15813-15826, the disclosure of which is incorporated herein by reference).

Prior to the present invention, it had not been appreciated that the side chains of the fimbrolides could be functionalised directly affording a variety of halogen or oxygen functionalised fimbrolides. We have found that fimbrolides behave like allylic or benzylic compounds in their reactivity and consequently are amenable to free radical functionalisation. The derived halogen compounds can be converted to alcohols or to esters directly from the halogen derivatives or to ketones, esters, amides, alcohols or other halides indirectly from the corresponding esters or alcohols. The fimbrolides substituted with an appropriate group in the alkyl chain are capable of yielding polymers through that group, either directly or via copolymerisation with suitable monomers. It is the preparation of these fimbrolide-based halides, alcohols, esters, ethers, amines, amides, and nitro compounds, ketones, oligomers and polymers that form the major aspect of this invention.

The fimbrolides prepared in accordance with the present invention include not only synthetic versions of the two naturally occurring fimbrolides, but also other functionalised fimbrolides which we believe to be PCT/AU99/00285 Received 21 December 1999 2 novel compounds. The compounds synthesised in accordance with the present invention may be according to formula

Z

R

3 0 O R9

(I)

wherein R, is H, OH, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic;

R

2 and R 3 are independently or both H or halogen; R, is halogen; Z is independently selected from the group halogen, OOH, OC(O)Rg, 0, amine, azide, thiol, R 6 mercaptoalkyl, alkenyloxy, mercaptoalkenyl, aryloxy, mercaptoaryl, arylalkyloxy, mercaptoarylalkyl,

SC(O)R

6 OS(O)R6, OS(O) 2 NHC(O)R, NR 4 or NHR 4 and

R

4 is OH, alkyl, alkoxy, poly(ethylene glycol), alkenyl, aryl or arylalkyl.

Compounds according to Formula apart from those in which R, propyl, R2 Br, R 3

R

9 =Br and Z is OC(O)CH 3 or OH, are believed to be novel and form part of the present invention.

In the structural formulae given herein, a particular geometry is not to be taken as specified unless specifically stated. For example, the formulae are intended to cover the both Z-isomers and E-isomers.

Disclosure of Invention In a first aspect, the present invention provides a method to form a fimbrolide derivative, the method including reacting a fimbrolide with a halogenating agent and/or an oxygenating agent to form compounds with formula (Ia):

K

WO 99/54323 PCT/AU99/00285 3

X

R2 RR2RI 0 0 R9 (la) wherein R1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl, whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; X is a halogen (X F,C1, Br or OH, OOH, OC(0)R 1 or R2 and R3 are independently or both hydrogen or halogen;, and R, is halogen.

The fimbrolide used in the method may be a fimbrolide having the formula: R2 0 0

R

9 wherein R 1

,R

2

R

3 and R, are as defined above.

Preferably the halogenating agent is selected from Nbromosuccinimide, N-chlorosuccinimide, N-iodosuccinimide, bromine, cupric bromide, and phenyltrimethylammonium perbromide. It will be appreciated, however, that other halogenating agents would also be suitable for the present invention.

Preferably the oxygenating agent is selected from lead tetraacetate, Rose Bengal/oxygen gas, hydrogen peroxide/vanadium pentoxide, selenium dioxide, and 3-chloroperoxybenzoic acid. It will be appreciated, however, that other oxygenating agents would also be suitable for the present invention.

The reaction conditions are selected so as to be appropriate to the nature of the reaction being undertaken. Preferably, the reaction conditions WO 99/54323 PCT/AU99/00285 4 when an halogenating agent is used are for example carbon tetrachloride or chloroform or dichloromethane/with or without light/reflux, tetrahydrofuran/room temperature.

Preferably, the reaction conditions when an oxygenating agent is used are acetic acid or acetic acid mixed with a solvent/reflux, pyridine/room temperature, acetone/30°C, dioxane/reflux, and dichloromethane/room temperature.

The present inventors have found that the preferred bromination conditions are N-bromosuccinimide in the presence of catalytic amounts of benzoyl peroxide in carbon tetrachloride and light/reflux. The source of light may be any suitable source for example, the present inventors have found that a 250 W sun lamp is quite suitable.

In a second aspect, the present invention consists in a fimbrolide derivative having formula wherein RI is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; X is a halogen (X Cl, Br or I) or oxygen (X OH, OOH, OC(O)R 1 or R2, R3 are independently or both hydrogen or halogen and; R, is halogen, with the proviso that the following two derivatives are excluded Ri= propyl, X= OH, R2= Br, R3=H; and R1= propyl, X= OC(O)CH 3 R2= Br, R3=H).

In a third aspect, the present invention consists of a method to form a fimbrolide derivative, the method including displacement and/or functionalisation of the halogen or oxygen substituent in the fimbrolide side chain by treating with a nucleophile or an electrophile to form compounds with formula (II): R4 R2 Ri 3R 1 R9

(II)

wherein R1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl, whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; WO 99/54323 PCT/AU99/00285 R2 and R3 are independently or both hydrogen or halogen; R, is halogen; and R4 is selected from the group halogen, amine, azide, hydroxyl, thiol, or any hydrophobic, hydrophilic of fluorophilic alkyl, alkoxy, mercaptoalkyl, alkenyloxy, mercaptoalkenyl, aryloxy, mercaptoaryl, arylalkyloxy, mercaptoarylalkyl, OC(O)R1, SC(O)R 1

OS(O)R

1 OS(0) 2

R

1

NHC(O)R

1

OC(O)NHR

1 or =O.

The fimbrolide treated in the method of the third aspect may be compound of the formula: f7 3R2 R, R9 wherein R 1

,R

2

R

3 and R, are as defined above and R 7 is halogen (F,C1, Br or OH, OOH, OC(O)R, or Preferably the nucleophile is selected from metal halides, water, organic metal carboxylates, organic alcohols, dimethyl sulfoxide, and organonitriles. It will be appreciated, however, that other nucleophiles would also be suitable for the present invention.

Preferably the electrophile is selected from organic acids, isocyanates, carboxylic or sulfonic acid halides or active acylating or sulfinylating agents such as carbonyl imidazoles, carboxylic anhydrides, carbodiimide actived carboxylic acids, sulfonyl halides, and sulfonic anhydrides and diethylaminosulfur trifluoride. It will be appreciated, however, that other electrophiles would also be suitable for the present invention.

The reaction conditions of the method of the third aspect are selected to be appropriate to the nature of the reaction being undertaken.

The reaction conditions suitable when using a nucleophile are acetone or dioxane/room temperature or reflux, water/dioxane or acetone or tetrahydrofuran/reflux, metal acetates/organic acids/neat or high boiling solvents/reflux, organic alcohols/reflux, dimethyl sulfoxide/room temperature, and organonitriles/acid catalyst or silver triflate/reflux.

WO 99/54323 PCT/AU99/00285 6 The reaction conditions suitable when using an electrophile are organic acids/neat and/or solvent/acid catalyst/reflux, organic acid halides or anhydrides or isocyanates /base catalyst/solvent/room temperature, and diethylaminosulfur trifluoride/dichloromethane/low temperature.

In a fourth aspect the present invention consists in a fimbrolide derivative having formula wherein R1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; R2 and R3 are independently or both hydrogen or halogen; R, is halogen, and R4 is selected from the group halogen, amine, azide, hydroxyl, thiol, or any hydrophobic, hydrophilic of fluorophilic alkyl, alkoxy, mercaptoalkyl, alkenyloxy, mercaptoalkenyl, aryloxy, mercaptoaryl, arylalkyloxy, mercaptoarylalkyl, OC(O)R1, SC(O)R1, OS(O)R 1

OS(O)

2

R

1

NHC(O)R

1

OC(O)NHR

1 or with the proviso that the following two derivatives are excluded R1= propyl, X= OH, R2 Br, R3=H; 2. R1 propyl, X= OC(O)CH3, R2= Br, R3=H.

In a fifth aspect, the present invention consists of a method to form a fimbrolide derivative the method including reacting an hydroxyl substituent in the fimbrolide side chain with an oxidising agent to form a compound in accordance with formula (III): 0 R9

(III)

wherein R2 and R3 are independently or both hydrogen or halogen; is OH or the same as R1; R is halogen; and

R

1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic.

WO 99/54323 PCT/AU99/00285 7 The hydroxyl substituted fimbrolide used in the method of the fifth aspect may have the formula:

OH

R2

R

R3 0

R

9 wherein R 5

R

2 and R 3 are as defined above.

Preferably, the oxidising agents are acidic chromium reagents in any form either free or polymer supported Jones reagent, pyridinium chlorochromate, pyridinium dichromate, chromium trioxide etc), manganese dioxide, potassium permanganate, selenium dioxide, ceric ammonium nitrate, ruthenium tetraoxide, and hot nitric acid. It will be appreciated, however, that other oxidation agents may also be used for the present invention.

The reaction condition under which the method of the third aspect is performed may be any suitable conditions. The reaction conditions preferably use Jones reagent/with or without phase transfer catalysts/acetone/room temperature, toluene/reflux, potassium permanganate/buffered solution/room temperature, dioxane/reflux, ceric ammonium nitrate/ aqueous acetic acid/steam bath, carbon tetrachloride/reflux, and acetic acid/steam bath. It will be appreciated, however, that other reaction conditions may also be used for the present invention.

The present inventors have found that the use of Jones reagent in acetone/room temperature is particularly suitable.

In a sixth aspect, the present invention consists in a fimbrolide derivative having the formula (III) wherein R1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; R2 and R 3 are independently or both hydrogen or halogen; R9 is halogen; and is OH or the same as R1.

The present invention also provides a method for forming fimbrolide oximes, imines, hydrazones and amines.

WO 99/54323 PCT/AU99/00285 8 Accordingly in a seventh aspect, the present invention consists of a method to form a fimbrolide analogue derived from a compound of formula (III), the method including reacting an aldehyde or ketone substituent in the fimbrolide side chain of the compound with an amine derivative to form a compound with formula (IV) or N R 8 N-R8 R2 R R2

RI

RR

1 3R 1 R9 R9 (IV) (V) wherein R1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; R2 and R3 are independently or both hydrogen or halogen; R, is halogen and R8 is OH, NHRI, NHC(X)NH 2

NHC(X)NHR

1 S, NRi) or any R 1 Preferably, the amine derivatives used are hydroxyl amine hydrochloride, alkyl and aryl hydrazines, alkyl or aryl amine in the presence or absence of a reducing agent. It will be appreciated, however, that other amine derivatives may also be used for the present invention.

The reaction conditions used in the method of the seventh aspect may be any conditions suitable for the nature of the reaction carried out. For example when using an amine derivative suitable conditions are ethanol or methanol/room temperature or reflux, toluene in the presence of a catalyst/room temperature or reflux and ethanol or methanol in the presence of sodium borohydride or sodium cyanoborohydride/room temperature or reflux.

In a eighth aspect, the present invention consists in a fimbrolide derivative having the formula (IV) and wherein R1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or WO 99/54323 PCT/AU99/00285 9 fluorophilic; R2 and R3 are independently or both hydrogen or halogen, R, is halogen and R8 is OH, NHR,, NHC(X)NH 2 NHC(X)NHRi S, NR 1 or any R 1 In a ninth aspect the present invention provides an oligomer or polymer formed by oligomerising or polymerising a compound in accordance with the present invention directly or with one or more other monomers.

The one or more other monomer may be any suitable polymerisable copolymer eg acrylate ester such as alkyl, hydroxyalkyl, aminoalkyl, or substituted aryl, acrylates or methacrylates, crotonates, substituted or unsubstituted acrylonitriles, vinyl alcohols or acetates, and styrene.

In a tenth aspect, the present invention consists in incorporation of fimbrolides according to the first, third, fifth or seventh aspects of the present invention either in surface coatings or polymers through the newly introduced functionality on the alkyl chain or the alkyl chain itself via direct polymerisation or copolymerisation with suitable monomers.

In a eleventh aspect, the present invention consists in a fimbrolide derivative produced by the method according to the first, third, fifth or seventh aspects of the present invention.

In an twelfth aspect, the present invention consists in the use of a fimbrolide derivative according to the present invention. The present inventors have found that many of the fimbrolide derivatives having the formula have antimicrobial, antiseptic, microbacterial static and/or antifouling properties. Accordingly, the fimbrolide derivatives are suitable for use as antimicrobial and/or antifouling agents.

In a thirteenth aspect the present invention provides a compound of formula (VI): 13 0 0 R2

(VI)

wherein R 2

R

3

R

9 and R 1 (except where R, is hydrogen) are as defined above.

4 4 WO 99/54323 PCT/AU99/00285 An example of a compound in accordance with this form of the invention is 4-Bromo-5-(bromomethylene)-3-(l-butenyl)-2(5H)-furanone.

The compound of the thirteenth aspect may be formed by dehydrating hydroxyl substituent in the fimbrolide side chain. The dehydration may be catalysed by H2S04 in the presence of toluene.

As used herein and in the claims: The term "halogen" means F, Cl, Br, or I.

The term "alkyl" is taken to mean straight chain, branched chain and cyclic alkyl or cycloalkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like. Preferably the alkyl group is of 1-25 carbon atoms. The alkyl group may be optionally substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C1-C4-alkoxycarbonyl, hydroxyl, carbonyl and aryl groups.

The term "aryl" is taken to include substituted and unsubstituted phenyl, napthyl or other benzenoid aromatic or any aromatic heterocyclic nucleus containing N, O, S, P or chalcogen heteroatom such as pyridyl, pyrimidyl, indolyl or furanyl.

The term "alkoxy" as used herein and in the claims denotes straight chain or branched alkoxy, preferably containing 1 to 25 carbon atoms and like functional groups, such as polyethylene glycol (PEG) and cyclic ethers.

The term "alkenyl" is taken to mean a straight chain, a branched chain or cycloalkyl group having one or more double bonds. Preferably the alkyl group is 1-25 carbon atoms. The alkyl group may optionally be substituted by one or more halogen atoms, carbonyl, hydroxyl, carboxyl, C1-C4alkoxycarbonyl groups.

The term "amine" as used herein and in the claim means any basic primary, secondary or tertiary nitrogen containing group or molecule, aromatic or non-aromatic.

WO 99/54323 PCT/AU99/00285 11 Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or step, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples and accompanying drawings.

Brief Description of Drawings Figure 1 shows the structure of Delisea pulchra furanones and synthetic analogues and derivatives tested in barnacle settlement assay.

Figure 2 shows the effect of furanones 2, 281, 2223, 2425, 26, 27, and 28 on the settlement of barnacle cyprid larvae as measured by settlement expressed as a percent of the control.

Figure 3 shows growth curves of Staphylococcus aureus against different furanones.

Figure 4 shows growth curves of Staphylococcus aureus against compounds 33/34 and Figures 5 -51 show the structural formulae for other specific examples of compounds in accordance with the present invention.

Modes for Carrying Out the Invention EXPERIMENTAL DETAILS Fimbrolide Production General. Melting points are uncorrected. Microanalyses were performed by Dr H.P. Pham of The University of New South Wales Microanalytical Laboratory. 1H NMR spectra were obtained in CDC13 on a Bruker AC300F (300 MHz) or a Bruker DMX500 (500 MHz) spectrometer.

1 3 C NMR were obtained in the same solvent on a Bruker AC300F (75.5 MHz) or a Bruker DMX500 (125.8 MHz) spectrometer. Chemical shifts were measured on the d scale internally referenced to the solvent peaks: CDC1 3 (d 7.26, d 77.04). Ultraviolet spectra were measured on an Hitachi U-3200 spectrophotometer and refer to solutions in absolute MeOH. Infrared spectra were recorded on a Perkin-Elmer 298 or a Perkin-Elmer 580B spectrophotometer and refer to paraffin mulls. The electron impact mass spectra were recorded on a VG Quattro mass spectrometer at 70eV ionisation WO 99/54323 PCT/AU99/00285 12 voltage and 200 0 C ion source temperature. FAB spectra were recorded on an AutoSpecQ mass spectrometer. Column chromatography was carried out using Merck silica gel 60H (Art. 7736), whilst preparative thin layer chromatography was performed on 2 mm plates using Merck silica gel 60GF2 5 4 (Art. 7730).

RESULTS

Fimbrolide Production Examples of a number of fimbrolides produced are provided below.

EXAMPLE 1 and 5-(dibromomethylene)-3-(1- N-bromosuccinimide (17.3 g, 0.097 mol) was added to a solution of 4and/or 5-(dibromomethylene)-3-ethyl-2(3H)furanone (22.6 g, 0.08 mol) in carbon tetrachloride (500 ml) containing benzoyl peroxide (0.25 The mixture was irradiated with a 250 W lamp and refluxed in an oil bath for 18h. After cooling the mixture to room temperature it was filtered and the precipitate washed with carbon tetrachloride (50 ml). The filtrate was evaporated under reduced pressure and the crude product was purified by silica gel chromatography using dichloromethane light petroleum (2 3) as the eluent to yield the bromo compounds (22.0 g, 76%) as a 4 1 mixture.

4-Bromo-5-(bromomethylene)-3-( 1-bromoethyl)-2(5H)-furanone A pale yellow solid, m.p. 79 0 C. vmax 2850, 1750, 1630, 1580, 1440, 1360, 1270, 1180, 1065, 1000, 970, 940, 1080, 755 cm- 1 Xmax 306 nm (e 10826). 1 H n.m.r. 8 (CDCI 3 2.06, d, J 7.2 Hz, 5.00, q, J 7.2 Hz, Hi'; 6.45, s, 5-CHBr. 1 3 C n.m.r. 5 (CDC13): 22.3, C2'; 35.7, Cl'; 94.3, 5-CHBr; 130.5, C4; 133.7, C; 149.5, C5; 165.8, C2. Mass spectrum: m/z 364 (M 8 1Br3), 362 (M 8 1 Br2, 7 9 Br), 360 (M 8 1 Br 7 9 Br2), 358 (M 7 9 Br3), 283 281 (100); 279 202 200 173 158 156 145 143 133 121 (26).

5-(Dibromomethylene)-3-(1-bromoethyl)-2(5H)-furanone A white solid m.p. 119°C. vmax 2900, 1720, 1590, 1450, 1370, 1250, 1170, 1080, 1060, 1000, 960, 840, 770, 720 cm- 1 .max 319 nm (e 12225). 1

H

n.m.r. 5 (CDC13): 1.99, t, J 7.2Hz, 4.87, q, J 7.2 Hz, Hi'; 7.56, s, H4. 13C n.m.r. 5 (CDC13): 23.9, C2'; 36.0, Cl'; 82.8, (5-CBr2); 134.7, C4; 138.2, C3; 149., C5; 165.5, C2. Mass spectrum: m/z 364 (M 8 1 Br3), 362 (M 8 1 Br2, WO 99/54323 WO 9954323PCT/AU99/00285' 13 79 Br), 18); 360 (M 8 1 Br 7 9 Br2), 18); 358 (M 7 9 Br3), 283 281 (100); 279 227 225 223 202 200 174 172 146 145 144 143 EXAMPLE 2 3-(l-Bromobutyl).5-(dibromometbylene)-2(5H)-furanone The procedure described for 4-bromo-5-(bromoinethylene)- and (dibromomethylene)-3-(1-bromomethyl)-2(5H)-furaiione was used to treat 3butyl-5-(dibromomethylene)-2(5H)-furanone (4.95g, 16 mmol) with Nbromosuccinimide (3.83g, 22 mmol) in carbon tetrachloride (70 Ml) to give after chromatography the bromobutyl furanone as a yellow solid (5.48g, 88%) m.p. 55'C. vmax 3087, 2924, 2854, 1778, 1463, 1377, 967, 832 cm-i. XM' 314.2 nm (e 28115). 1 H NMvR d: 0.99 t, 3H, 1.50 m, 2H, 2.10 m, 2H, 4.72 t, 1H, 7.54 s, 1H, H4. Mass spectrum: m/z 392 (M+1( 8 lBr3); 389(M+1( 8 lBr, 7 9 Br2); 386(M+1( 7 9 Br3); 311; 309; 307; 269; 267 265.

EXAMPLE 3 and 5-(dibromomethylene)- 1acetoxybutyl) -2 (5H)-fura none A solution of 4-bromo-5-(bromomethylene)- and/or (dibromomethylene)- 1-bromobutyl)-2 (5H)-furanone (3.00 g, 7.7 mmnol) in glacial acetic acid (160 ml) containing sodium acetate (1.20 g, 15 mmol) was refluxed for 18h. The mixture was concentrated to approximately 20 ml and neutralised with excess saturated sodium carbonate solution. The residual oil was extracted with ether (3 x 100 ml), washed with brine, dried over sodium sulfate and evaporated. The crude product was chromatographed on silica gel using dichioromethane light petroleum (1 1) as eluent to yield the ace toxybutylfuranones (0.96 g, 34%) as a 4 1 mixture.

4-Bromo-5-(bromometbylene)-3-( 1-acetoxybutyl)-z A pale yellow oil vmax 2940, 1775, 1740, 1640, 1600, 1450, 1420, 1370, 1220, 1100, 1020, 985, 760, 730 cm- 1 %max 295 nm (e 6265). 1 H n.m.r.

8 (CDC13) 0.93, t, J 7.2 Hz, (H4') 3 1.35, mn, 1.84, m, 2.07, s,

GOGH

3 5.50, bt, J 7.2 Hz, Hl': 6.37, s, 5-CHBr. 1 3 C n.m.r. 8 (GDCl3): 13.5, C4'; 18.5, GOGH 3 20.6, CT'; 33.7, C2'; 68.2, Cl'; 93.5, 5-CHBr; 130.2, C4; 131.4, C3; 149.7, C5; 164.2, C2; 170.2, CO. Mass spectrum: rn/z 370, (M 8 1 Br2), 368 (M 8 1 Br, 7 9 Br), 366, (M 7 9 Br2), 327 325 WO 99/54323 WO 99/54323PCT/A U99/0 0285 14 323 289 287 285 283 281 247 245 229 227 149 (28).

5-(Dibromomethylene)-3-( 1-acetoxybutyl)-2(5H)-furanone A pale yellow solid nip 76 0 C. vmax 2880, 1760, 1735, 1445, 1370, 1225, 1170, 1100. 1030, 950, 840, 765, 7320 cm- 1 kmax 314 nm (e 8900). 1 H n.m.r.

6 (CDCl 3 0.94, t, J 7.2 Hz, 1.36, m, 1.84, m, 2.12, s,

COCH

3 5.59, bt, J 6.2 Hz, Hi'; 7.39, bs, H4. 1 3 C n.m.r. 6 (CDCl3): 13.6, C4'; 18.3, COCH3; 20.9, C3S; 34.8, C2'; 68.3, Cl'; 81.6, 5-CBr2; 135.0, C4; 136.1, C3; 149.3, C5; 166.1. C2; 169.9, CO. Mass spectrum: rn/z 370, (M 8 1 Br2), 28%); 368 (M 8 1 Br, 79 Br), 54); 366, (M 7 9 Br2), 28); 328 327 326 (36); 325 324 323 289 287 247 245 229 (12); 227 198 EXAMPWLE 4 5-(Dibromomethylene)-3-( 1-acetoxyethyl)-2(5H)-furanone The procedure described for 4-bromo-5-(bromomethylene)-3-(1acetoxybutyl) -2 (5H)-furanone was used to treat 5 -(dibromomethylene)-3-( 1- (2.80 g, 7.7 mmol) with sodium acetate (1.20 g, mmol) in glacial acetic acid (160 ml) to give after chromatogrphy the acetoxyethyl furanone as a white solid (0.88 g, 34%) m.p. 124'C. Vmax 2880, 1750, 1610, 1445, 1365, 1230, 1170, 1080, 1030, 990, 960, 930, 835, 760, 715 cm- 1 Xmax 313 fnm (e 31296). 1H n.m.r. 8 (CDCl3) 1.53, d, J 6.2 Hz, (H2') 3 2.13, s. COCH 3 5.66, m, 1H, Hl'; 7.43, bs, H4. 1 3 C n.m.r. 5 (CDCl 3 18.9, CH3; 20.9, C2'; 53.4, Cl'; 81.7, 5-CHBr; 134.6, C4; 136.7, C3; 149.2, C5; 166.0, C2; 169.6, CO. Mass spectrum: m/z 342, (M 8 1 Br2), 340 (M 8 1 Br, 7 9 Br), 338, (M 7 9 Br2), 300 299 298 297 296 (32); 295 281 279 (18);261 259 219 217 201 200 199 174 172 170 157 145 143 (24).

EXAMPLE 5-(Dibromomethylene)-3-(1-thioacetoxyetbyl)-2(5H)-furanone A solution of 5-(dibromomethylene)-3-( 1-bromoethyl)-2 (3.00 g, 7.7 mmol) in glacial acetic acid (160 ml) containing potassium thioacetate (1.20 g, 15 mmol) was refluxed for 12h. The mixture was concentrated to approximately 20 ml and neutralised with excess saturated sodium carbonate solution. The residual oil was extracted with ether (3 x 100 ml), washed with brine, dried over sodium sulfate and evaporated. The crude product was chromatographed on silica gel using dichloromethane WO 99/54323 PCT/AU99/00285 light petroleum (1 1) as eluent to yield the thioacetoxyethvlfuranones (0.96 g, 34%) as a yellow oil vmax 3200, 2910, 2850, 1780, 1730, 1690, 1600, 1450, 1420, 1380, 1350, 1270, 1170, 1105, 1010, 960, 880, 850, 810, 770 cm- 1 Xmax 297 nm (e 6664). 1H NMR d: 1.61 3H, J 7.2 Hz, 2.52 3H,

SCOCH

3 4.49 1H, J 7.2 Hz. 7.44 1H, H4). Mass spectrum: m/z 358 (M 8 1 Br2)); 356 (M( 8 1 Br, 7 9 354 (M 79 Br2)); 316, 314, 312, 283, 281,279, 277, 275, 235, 233, 200, 172, 153, 143.

EXAMPLE 6 4-Bromo-5-(bromomethylene)-3-( Trimethylsilyl trifluoromethanesulfonate (0.1 ml) was added with stirring to a cooled solution of 4-bromo-5-(bromomethylene)-3-(1- (0.12 g, 0.37 mmol) in acetonitrile (10 ml) at After stirring the reaction mixture at room temperature for 1h, it was quenched with water (20 ml) and extracted with ether (3 x 40 ml). The combined ether extracts was washed with brine, dried over sodium sulfate and evaporated to yield the amide as a light tan oil (0.lg, 74%).

Recrystallisation of the crude product from dichloromethane/light petroleum gave the pure amide as yellow powder, m.p. 153-55 0 C 1H n.m.r. 6 (CDC13) 0.93, t, J 7.2 Hz, 1.24-1.40, m, 1.66-1.77, m, 1,98, s, NHCOCH3; 5.02, q, 7.9 Hz, Hi'; 6.25, bd, J 8.7 Hz, NH; 6.38, s, 5-CHBr. 13C n.m.r. 6 (CDC13): 13.5, C4'; 19.0, C3'; 23.1, NHCOCH3; 35.2, C2'; 45.7, C1'; 93.6, 5-CHBr; 130.9, C4; 131.9, C3; 149.6, C5; 165.3, C2; 169.6, NHCO. Mass spectrum: m/z 369, (M 8 1 Br2), 367 (M 8 1 Br, 7 9 Br), 365, (M 79 Br2), 362 364 326 324 322 284 282 (53); 280 EXAMPLE 7 5-(Dibromomethylene)-3-(1-hydroxyethyl)-2(5H)-furanone A solution of 5-(dibromomethylene)-3-(1-bromoethyl)-2(5H)-furanone (18.0 g, 0.05 mol) in a mixture of dioxane (200 ml) and sulfuric acid (3M, ml) was refluxed in an oil bath for 3h. After cooling the mixture to room temperature, it was diluted with water (300 ml) and extracted with dichloromethane (3 x 200 ml). The combined dichloromethane extracts were washed with water, dried and evaporated. The crude product was purified by silica gel chromatography using dichloromethane light petroleum (1 1) as an eluent to yield the hydroxyethyl furanone (9.6 g, 62%) as a white solid.

m.p. 100 0 C. vmax 3300, 2870, 1750, 1595, 1440, 1370, 1250, 1170, 1030, 985, 4 WO 99/54323 PCT/AU99/00285 16 955, 835, 770, 720 cnV 1 Xmax 311 nm (e 5832). 1 1H ii.m.r. 5 (CDG1 3 1.50, d, J 7.2 Hz, 4.72, in. 1H, HI'; 7.49, bs, H4. 1 3 C n.m.r. 5 (CDCl 3 21.8, C2'; 63.4, CV'; 81.3, 5-CHBr; 133.7, C4; 140.3, C3; 149.5, C5; 167.3, C2. Mass spectrum: nl/z 300, 1 8 1 Br2), 298 1 8 1 Br, 79 Br), 36); 296, 1 7 9 Br2). 18); 285 283 281 25 7 255 (100); 253 (78); 219 217 201 200 199 174 172 170 (18); 147 145 (28);:119 117 (38).

EXAMPLE 8 5-(Dibromomethylene)-3-(l-hydroxybutyl)-2(5H)..furanone The procedure described for 5-(dibromome thylene)-3-( 1-hydroxyethyl)- 2 (5H)-furanone was used to treat 3 (1 -ace toxyb utyl) -5 ibromomethyle ne) (0.70g, 1.9 mmol) with sulfuric acid (3 M, 5 ml) in dioxane ml) to give after chromatography the hydroxybutyl furanone as a yellow oil (0.42g, 68%) vmax 3441, 2960, 2931, 2873, 1779, 1615, 1267, 1174, 1020, 965, 848 cm-i. Xmax 303.6 nmn (e 1161). 1 H NMR d: 0.95 3H, 1.43 (in, 2H, 1.78 (in, 2H, 3.22 1H, OH); 4.58 1H, 7.52 (s, 1H, H4). Mass spectrum: rn/z 328 (M 8 1 Br2)); 326 (M( 8 1Br, 7 9 324

(M(

7 9 Br2)); 299; 297; 285; 283 281; 257; 255; 253; 247; 245; 203; 205; 175; 173.

EXAMPLE 9 5-(Dibromomethylene)-3-(1-fluoroethyl)-2(5H)..furanone A cooled solution of 5-(dibromomethylene)-3-(1-hydroxyethyl)-2(5H).

furanone (0.47g, 1.6 mmol) in analytical grade dichloromethane (2 ml) was added dropwise with stirring to a solution of (die thylani no) sulfur trifluoride (1 ml) in dichloromethane (2 ml) held in a dry ice/acetone cooling bath. The progress of the recation was monitored by thin layer chromatography. Upon completion of the reaction, the mixture was added dropwise to a conical flask containing water (100 ml). The product was extracted with dichloromethane (3 x 50 ml) and the organic layer was dried over anhydrous sodium sulfate.

The crude product was chromatographed on a silica column using dichloromethane as the eluent. The fraction with Rf of 0.90 in dichioromethane was collected and evaporated to yield the fluoro compound (0.47g, 97%) as a yellow solid m.p. 41'C. Vmax 3096, 2924, 2854, 1790, 1754, 1609, 1463, 1376, 1264, 1192, 1092, 990, 847, 771 cm- 1 Xmax 306.4 nrn (e 4269). IH NIMR d: 1. 62 3H, 5.34, 5.5 2(m, 1H, 7.5 8(s, 1H, 5-CHBr), 1 3 C n.m.r. 5 (CDCl3): 19.7 and 19.8, C2'; 82.3, CBr2; 83.4 and WO 99/54323PCAU99028 PCT/AU99/00285 17 85.6, Cf: 134.4, G4; 136.5 and 138.5, C3: 149.2, C5; 165.7, CO. Mass spectrum: rnlxz 302 (M 8 1 Br2); 300 (M( 8 1 Br, 7 9 Br); 298 (M( 7 9 Br2); 202; 200; 198: 175 172; 170.

EXAMPLE 5-(Dibromomethylene)-3-( 1-fluorobutyl)-2(5H)-furainone The procedure described for 5-(dibromomethylene-3-( 1-fluoroethyl)- 2 (5H)-furanone was used to treat S -(dibromomethylene)-3-(1-hydroxybutyl).

2 (5H)-furailone 24g, 0.74 mmol) with (diethylamino)sulfur trifluoride ml) in dichioromethane (3 ml) to give after chromatography the fluorobutyl furanone as a pale yellow oil 23g? 9 V~a 3084, 2961, 28 74, 1780, 1614.1465, 1379, 1266, 1180, 1026, 966, 847, 784, 680 cm-I. kmax 308.6nm (e 24923). 1 H NlvMR d: 0.95 3H, 1.52 (in, 2H, 1.88 (in, 2H, H- 5.2, 5.4 (in, 1H, H-1'(CHIF)); 7.56 1H, 5-CHBr). 1 3 C n.m.r. 6 (CDCI 3 13.5, C4'; 17.8, CT'; 35.6 and 35.9, C2'; 82.1, 5-CBr 2 86.5 and 88.8, C1,; 134.8, C4; 135.7 and 135.7 and 136.0, C3; 149.3, C5; 165.7 and 165.8, CO. Mass spectrum: m/z 330 (M 8 1 Br2); 328 (M( 8 1 Br, 7 9 Br); 326 (M( 7 9 Br2); 288; 286; 284; 247 207; 205.

EXAMPLE 11 4 -Bromo-5-(bromomethylene)-3-(l-butanoyloxybutyl))..2(5H)-furanone 4-Bromo-5-(bromomethylene)-3-( 1-hydroxybutyl))-2(5H)-furanone (4.75 g, 0.015 mnol) and butanoyl chloride (7.8 ml, 0.075 mol) were refluxed together for 7h then cooled and poured into water (50 ml) and extracted with ether (3 x 30 ml). The combined ether extracts were washed sequentially with saturated sodium bicarbonate (2 x 50 ml) and brine (50 ml), dried over sodium sulfate, and evaporated. The crude product was purified by silica gel chromatography using ether light petroleum (1 9) as the eluent to yield the butanoyloxybutyl furanone as a pale yellow oil (3.60 g, VMax 2950, 1780, 1730, 1635, 1600, 1450, 1380, 1280, 1240, 1165, 1060, 980, 840, 770 cm- 1. km 289 nin (e 14900). 1 H n.m.r. 8 (CDCl 3 0.91, t, J 7.4 Hz, OCOCH2CH2CH 3 0.93, t, J 7.2 Hz, (H4') 3 1.35, m, 1.66, q, J 7.4 Hz, OCOCH2CH 2 CH3; 1.80-1.95, m, 2.32, t, J 7.4 Hz, OCOCH2CH2CH 3 5.50, dd, J 6.4 Hz 8.0 Hz, Hi'; 6.36, s, 5-CH-Mr. 1 3 C n.m.r. 5 (CDCl 3 13.4, OCOCH2CH2CH3; 13.5, C4'; 18.2, OCOCH2CH 2

CH

3 18.4, C3T; 33.5, C2'; 35.7, OGOCH2CH2CH3; 68.0, Cl'; 93.2. 5-CI~r; 130.6, C4; 132.4, C3; 149.6, CS: 165.9, C2; 172.7, CO. Mass spectrum: m/Vz 399, (M+1 8 1 Br2), 397 (M+1 8 1 Br, 7 9 Br), 395, (M+1 7 9 Br2), 327 325 323 (18); WO 99/54323 WO 9954323PCT/AU99/00285 18 317 315 311 309 307 283 281 279 267 265 247 245 223 221 (44).

EXAMPLE 12 4-Bromo-5-(bromomethylene)-3.( 4-Bromo-5-(bromomethylene)-3-( 1-hydroxybutyl))-2 (5H)-furanone (0.24 g, 0.7 nunol) and octadecanoyl chloride (0.3 ml, prepared from octadecanoic acid and thionyl chloride) were stirred in an oil bath at 110'C for 24 h. The reaction mixture was diluted with ether (50 ml) and washed with water (3 x ml) followed by brine (30 ml). The organic phase was dried over sodium sulfate and evaporated to yield a brown oil. The crude product was purified by silica gel chromatography using dichloromethane as the eluent to yield the octadecanoyloxybutyl furanone as dark tan oil (0.14 g, 1 H n.m.r. (CDCl 3 0.87, t, J 7.2 Hz, OCO(CH2) 1 6CH 3 0.95, t, J 7.2 Hz, (H4') 3 1.28, m, OCOCH2(CH2115CH 3 1. 35-1.45, mn, 1.58-1.60, mn, OCOCH2CH 2 1.75- 2.05, m, 2.34, t, J 7.2 Hz, OCGCH2(CH2)lSGH 3 5.43, dd, J 6.2 Hz 7.7 Hz, HI'; 6.37, s, 5-CHBr. 1 3 C n.m.r. 6 (CDCl 3 13.5, OCO(CH2) 1 6

CH

3 14.1, C4'; 18.6, 22.7, 24.8, 29.2, 29.3, 29.4, 29.6, 29.7, 31.9, 33.8, 33.9, CH2; 68.0, Cl'; 93.3, 5-CHBr; 130.7, C4; 131.3, C3; 149.8, C5; 163.7, C2; 173.1, CO.

EXAMvPLE 13 Method A 4-Bromo-5-(bromomethylene)-3-( 1-acryloyloxybutyl)-2(SH)-furanone The procedure described for 4-bromo-5-(bromomethylene)-3-( 1butanoyloxybutyl))-2 (5H) -furanone was used to treat 4-bromo-5 (bromoi-ethylene)-3-( 1-hydroxybutyl))-2 (5H)-furanone (4.75 g, 0.015 mol) with acryloyl chloride (6.0 ml, 0.073 mol). The crude product was purified by silica gel chromatography using ether light petroleum (1 as the eluent to yield the acryloyloxybutyl furanone as a pale yellow oil (3.60 g, VMax 3060, 2940, 2850, 1770, 1710, 1620, 1590, 1430, 1390, 1385, 1280, 1250, 1160, 1095, 1030, 970, 835, 795, 760, 700 cni- 1 Xmax 293 nm (e 18170). 1

H

n.m.r. 6 (CDCl3) 0.91, t, J 7.4 Hz, ester CH3; 0.9-7, t, J 7.4 Hz, (H4') 3 1.38, m, 1.84-2.04, m, dd, J 6.7 Hz 8.2 Hz, Hi'; 5.88, d, J 10.7 Hz, CH=CH2; 6.14, dd, J 10.7 Hz 16.3 Hz, CH=CH 2 6.39, s, 5-CHBr; 6.46, d, J 16.3 Hz, CH=CH 2 1 3 C n.rn.r. 6 (CDCl3): 13.5, C4'; 18.5, C3T; 33.7, C2'; 68.2, C1'; 93.5, 5-CHBr; 127.5, CH=CH2; 130.4, C4; 131.5, CH=CH2; 132.1, C3; 149.8, C5; 163.7, C2; 165.2, CO. Mass spectrum: m/z 382, (M 8 1 Br2), 380 (M 8 1 Br, 7 9 Br), 378, (M 7 9 Br2), 327 325 323 (14); WO 99/54323 PCT/AU99/00285 19 301 299 283 281 279 269 267 265 229 227 225 223 203 201 175 173 147 145 143 (48).

Method B Concentrated sulfuric acid (1 drop) was added to a solution of 4-bromo- 5-(bromoinethylene)-3-(1-hydroxybutyl))-2(5H)-furanone (0.94 g, 3.0 mmol) and acrylic acid (2 ml) in benzene (5 ml). The mixture was refluxed for 4 h, and after cooling to room temperature, poured into water (50 ml). The crude product was extracted with ether (2 x 50 ml), and the combined ether extract washed with sodium carbonate solution. The extract was dried over anhydrous sodium sulfate, evaporated and chromatographed over silica column using dichloromethane/light petroleum as the eluent to yield the pure acryloyloxybutyl furanone as a tan oil (0.48 g, 42%).

EXAMPLE 14 4-Bromo-5-(bromomethylene)-3-(1-butanoyl)-2(5H)-furanone To an ice cooled solution of 4-bromo-5-(bromomethylene)-3-(l- (2.77 g, 8.5 mmol) in acetone (75 ml) was added dropwise with stirring Jones reagent (12 ml, prepared by dissolving chromium trioxide (13.36 g) in sulfuric acid (11.2 ml) and water (38.5 ml).

The mixture was stirred at room temperature for Ih and the progress of the reaction monitored by thin layer chromatography. After the completion of the reaction, the mixture was poured into water (200 ml) and extracted with ether (3 x 100 ml). The combined ether extracts were washed with brine (100 ml), dried over sodium sulfate and evaporated to yield the crude ketone (2.23g, 81%) as a yellow solid. Recrystallisation of the crude ketone from dichloromethane/hexane gave the pure ketone as yellow plates, m.p. 83-84°C vmax 1700, 1680, 1630, 1540, 1310, 1000 cm- 1 1 H n.m.r. 6 (CDC13) 0.97, t, J 7.2 Hz, (H4') 3 1.70, m, 2.93, t, J 7.2 Hz, 6.74, s, 5-CHBr. 13C n.m.r. 6 (CDC13): 13.6, C4'; 16.7, C3'; 44.4, C2'; 99.3, 5-CHBr; 125.7, C4; 138.1, C3; 150.4, C5; 163.5, CO; 194.1, Cl'. Mass spectrum: m/z 326, (M 8 1 Br2), 324 (M (81Br, 7 9 Br), 320, (M 79 Br2), 298 296 281 279 225 131 77 71 43 (100).

Covalently Bound Furanone Polymer Synthesis EXAMPLE Preparation of furanone acrylate homopolymer

J

WO 99/54323 PCT/AU99/00285 A mixture of 4-bromo-5-(bromomethylene)- and 3 -(1-acryloyloxybutyl)-2(5H)-furanone (0.36 AIBN (0.003 g) and toluene (0.75 ml) was degassed and then heated at 60 0 C for 24h. Hexane was added to the mixture and the precipitated polymer was washed once with methanol.

The final product was collected and dried to yield the polymer (0.04 g, 11% conversion) of average mass 14284.

EXAMPLE 16 Preparation of furanone acrylate-polymethyl methacrylate copolymer A mixture of methyl methacrylate (3.0 (bromomethylene)- and 5-(dibromomethylene)- 3-(1-acryloyloxybutyl)-2(5H)furanone (0.74 g) and AIBN (0.006 g) was degassed for 1/2h by purging with nitrogen gas and then heated at 60 0 C for 24h. Hexane (50 ml) was added to the mixture and the precipitated polymer was washed once with methanol.

The polymer was further purified by reprecipitation from chloroform and excess methanol. The final product was collected and dried to yield the polymer (1.74 g, 47% conversion) of average mass 7578.

EXAMPLE 17 Preparation of furanone acrylate-polystyrene copolymer A mixture of styrene (15 4-bromo-5-(bromomethylene)- and (dibromomethylene)-3-(l-bromoethyl)-2(5H)-furanone (0.16 g) and AIBN (0.023 g) was degassed for 1/2h by purging with nitrogen gas and then heated at 60°C for 3h. After the completion of polymerisation, the mixture was poured into hexane and the precipitated polymer was washed twice with ether and dried in vacuo (0.1 mm Hg) at 40 0 C for 24h to yield the polymer (12.9 g, 85% conversion). An XPS analysis of the powdered polymer in aluminium foil confirmed the presence of bromine.

EXAMPLE 18 Preparation of furanone acrylate-poly(Styrene/MEMA/MMA) polymer To a solution of styrene (5 MMA (5 g) and HEMA (5 g) in toluene (8 ml) was added 4-Bromo-5-(bromomethylene)- and 5-(dibromomethylene)-3- (1-bromoethyl)-2(5H)-furanone (0.15 g) followed by dodecanethiol (2 ml) and AIBN (0.4 The mixture was degassed by two freeze-thaw cycles and then heated at 700C for 24h. After the completion of polymerisation, the mixture was treated with hexane and the precipitated polymer was washed with hexane and dried in vacuo (0.1mm Hg) at room temperature for 24h to yield the polymer (22.2 g, 87% conversion).

WO 99/54323 PCT/AU99/00285 21 EXAMPLE 19 4-Bromo-5-(bromomethylene)-3-(l-butenyl)-2(5H)-furanone Concentrated sulfuric acid (2 drops) was added to a solution of (bromomethylene)-3-(1-hydroxybutyl)-2(5H)-furanone (2.0 g) in toluene The mixture was refluxed for 4h, and after cooling to room temperature, poured into water (50 ml). The crude product was extracted with ether (2 x ml), and combined ether extract washed with sodium carbonate solution.

The extract was dried over sodium sulfate, evaporated and chromatographed over silica column using light petroleum as the eluent to yield the pure 1-butenyl)-furanone as a light yellow oil (0.40 'Hnmr d (CC1 3 1.10, t, J 7.2

H

z (H4') 3 2.26, q, J 7.2 (H3') 2 6.20, d, CH=CH; 7.20, d, CH=CH, 6.24, s, Fimbrolide Biological Activitiy MATERIALS AND METHODS Inhibition of Cyprid Settlement The effects of synthetic furanones on the settlement of barnacle larvae were tested using cyprids of the cosmopolitan fouling barnacle Balanus amphitrite Darwin. The naturally occurring furanone 2, and the synthetically prepared compounds 281 (a 1:1:1 mixture of synthesised 2 8 2223 (a 1:1 mixture of synthesised 22 23), 2425 (a 1:1 mixture of synthesised 24 26, 27, and 28 (Figure 1) were compared for their efficacy in deterring barnacle cyprid settlement. Compounds were dissolved in ethanol (99.7% purity) at a concentration of 180 /g.ml-1 to 1.8 jpg.ml- 1 A 0.5 ml aliquot of each compound to be tested was added to treatment petri dishes (surface area 9 cm 2 and 0.5 ml of ethanol only was added to ethanol control dishes.

Dishes were dried on a shaker resulting in a coating of extract on treatment dishes with a concentration range of 10 gg.cm 2 to 100 ng.cm 2 for each compound.

Cypris larvae were obtained from laboratory cultures of adult brood stock of Balanus amphitrite. Nauplii of B. amphitrite were collected and reared on Skeletonema costatum until reaching cyprid stage. Cypris larvae were filtered and maintained in filtered seawater at 5 0 C for five days prior to use in settlement assays (Rittschof et al., 1992).

WO 99/54323 PCT/AU99/00285 22 Settlement tests were conducted by adding 25-35 cyprids to either treatment dishes, ethanol control dishes, or untreated dishes, each containing 4 ml of sterilised filtered seawater (0.22 All the treatments and controls were tested in triplicate. Test dishes were incubated for 24 h at 28 0 C in a 15:9 h light-dark cycle (Rittschof et al., 1992). After 24 h, the test was terminated by the addition of three drops of 40% formaldehyde, and nonsettled larvae filtered from the dish. The percent settlement of cyprids was then determined by counting settled and non-settled larvae.

Statistical Analyses The data from the bioassays were analysed by analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Data were analysed as percentages after arcsin Ap transformations.

RESULTS

Inhibition of Cyprid Settlement The settlement of Balanus amphitrite Cypris larvae was significantly inhibited by the compounds tested (Figure 2; two-factor ANOVA [metabolite x concentration] followed by Tukey's test). All treatments completely inhibited settlement at the highest concentration (10 Ag.cm- 2 Ethanol controls were used in the analysis as ethanol had no significant effect on settlement (single factor ANOVA, P=0.17). The synthetic furanone 2223 (Figure 1) was the most active metabolites (Figure At a concentration of 1 tg.cm- 2 2223 completely inhibited settlement and inhibited settlement by compared to the control at 500 ng.cm- 2 The next most inhibitory compound was the furanone 28 (Figure 1) which inhibited settlement completely at 5 Ag.cm 2 and inhibited settlement by 90% at 1 Mg.cm 2

A

group of furanones, 2425, 26 and 27 completely inhibited settlement at Ag.cm- 2 but had no effect at 1 btg.cm- 2 The furanone 2 and the synthetic analogue 281, a 1:1:1 mixture of 2, 8 and 1 (Figure 1) were the least effective compounds completely inhibiting settlement at 10 /g.cm 2 Inhibition of Staphylococcus aureus Staphylococcus aureus is a facultatively anaerobic, nonmotile, grampositive coccus and is normally associated with the skin, skin glands, and mucous membranes of humans. S. aureus is the most important human staphylococcal pathogen and causes, for example, boils, abscesses and wound infections.

WO 99/54323 PCT/AU99/00285 23 A screening experiment of the different furanones against the growth of S. aureus was performed in a BioRad 3550 Microplate reader. The growth was measured as absorbance at 610 nm up to 9 h. A complex growth media, Nutrient Broth, was used and the cells were grown at 37 0 C. Both natural furanones (compounds 2, 3 and 4) and synthesised furanones (compounds 33/34 and 45) were used in the experiment at the concentration 10 ug/ml.

The results showed that the synthesised furanones (33/34 and inhibited growth of S. aureus more effectively than the natural furanones (Figure The growth of the cells inoculated with 33/34 and 45 was completely inhibited for 9 h compared to 2 h for those inoculated with the natural compounds. All furanones, however, inhibited the growth ofS.

aureus compared to the control.

Further experiments were performed with the synthesised furanones and 33/34 at the concentrations 10 ug/ml and 5 pg/ml. The cells were grown in side arm flasks in NB media at 37C. The growth of the cells were measured at 610 nm for up to 48 h.

The results showed that compound 33/34 was more effective at inhibiting growth of S. aureus compared to compound 45 (Figure 4), however, both compounds at both concentrations inhibited the growth completely for 9 h. Growth of the cells occurred after 9h with compound at the concentration 5g/ml and after 15 h at the concentration 10 ig/ml.

Compound 33/34 at 54g/ml inhibited the growth for 15 h and at the concentration 10 ug/ml the growth of S aureus was completely inhibited for 34 h.

DISCUSSION

The derivatisation of naturally occurring furanones resulted in an increase in the deterrence of barnacle settlement. For example, manipulation of the length of the acyl side chain and the functionality on the 1' position of the acyl side chain of the furanone resulted in a significant increase in activity. This is clearly demonstrated in a comparison of the activity of furanones 2 and 2425. In 2425 a bromine has been added in the 1' position of acyl chain resulting in a five fold increase in activity in the settlement bioassay (Figure All of the synthesised furanones are either novel compounds not being previously reported in the literature or are racemic mixtures of a naturally occurring furanone. The racemic analogues of the naturally occurring compounds have the same activity as the naturally WO 99/54323 PCT/AU99/00285 24 occurring optically pure form. Therefore, the synthetic furanones, both analogues of naturally occurring compounds and novel compounds, have activity comparable to or better than the compounds from which their structure was derived, e.g. furanone 2 vs 2425.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims (20)

1. A compound according to formula R6 R 3 R9 (I) wherein R 6 is H, OH, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; R 2 and R 3 are independently or both H or halogen; Rs is halogen; Z is independently selected from the group R 6 halogen, OOH, OC(O)R 6 O, amine, azide, thiol, mercaptoalkyl, alkenyloxy, mercaptoalkenyl, aryloxy, mercaptoaryl, arylalkyloxy, mercaptoarylalkyl, SC(O)R 6 OS(O)R 6 OS(O) 2 R 6 NHC(O)R 6 NR 4 r NHR 4 and R 4 is OH, alkyl, alkoxy, poly(ethylene glycol), alkenyl, aryl or arylalkyl. provided that: when R 6 is propyl, R 2 is Br, R 3 is H or Br and R 9 is Br, then Z is other than H, OC(O)CH 3 or OH; when R 6 is propyl, R 2 is Br, R 3 is H and R 9 is I, then Z is other than OC(O)CH 3 or OH; when R 6 is propyl, R 2 is Br, R 3 is H and R 9 is CI, then.Z is other than OH; when R 6 is propyl, R 2 is H, R 3 and R9 are Br, then Z is other than H; when R 6 is propyl, R 2 is Br, R 9 is Cl and Z is H, then R 3 is other than CI; and when Re is methyl, R 2 is H, R 3 and Rg are Br, then Z is other than Br

2. A compound according to claim 1 of formula (la): PCT/AU99/00285 Received 21 December 1999 26 X R2 R 1 R3 (la) wherein R1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; X is a halogen, OH, OOH, OC(O)Rj or =O; R 2 and R 3 are independently or both hydrogen or halogen;, and R, is halogen, provided that: when R 1 is propyl, R 2 is Br, R 3 is H or Br and R, is Br, then X is other than OC(O)CH 3 or OH; when R 1 is propyl, R 2 is Br, R 3 is H and R, is I, then X is other than OC(O)CH 3 or OH; when RI is propyl, R 2 is Br, R 3 is H, R, is Cl, then X is other than OH.

3. A compound according to claim 1 of formula (II): R 4 R2_ RI R3 R9 (II) wherein R 1 is hydrogen, unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl; R 2 and R 3 are independently or both hydrogen or halogen; R, is halogen; and R 4 is selected from the group halogen, amine, azide, hydroxyl, thiol, or any hydrophobic, hydrophilic of fluorophilic alkyl, alkoxy, mercaptoalkyl, PCT/AU99/00285 Received 21 December 1999 27 alkenyloxy, mercaptoalkenyl, aryloxy, mercaptoaryl, arylalkyloxy, mercaptoarylalkyl, OC(O)R1, SC(O)R1, OS(O)R1, OS(0) 2 R1, NHC(O)R 1 OC(O)NHR1, or provided that: when R 4 is propyl, R 2 is Br, R 3 is H or Br, and R, is Br, then R 1 is other than H, OC(O)CH 3 or OH; when R 4 is propyl, R 2 is Br, R 3 is H, R 9 is I, then R, is other than OC(O)CH 3 or OH; when R 4 is propyl, R 2 is Br, R 3 is H, R, is Cl, then R, is other that OH; when R 4 is propyl, R 2 is H, R 3 and R 9 are Br, then R, is other than H; when R 4 is propyl, R 2 is Br, R 3 and R, are Cl, then R 1 is other than H.

4. A compound according to claim 1 of formula (III): R2 R 5 o 0 R9 (III) wherein R 2 and R 3 are independently or both hydrogen or halogen; R 5 is OH or the same as R 1 Rg is halogen; and R 1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic. A compound according to claim 1 of formula (IV) or PCT/AU99/00285 Received 21 December 1999 28 N/R /R8 N RWR R2 R, R2 R, R3 R3 0 0 0 0 R R9 (IV) (V) wherein R, is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; R2 and R 3 are independently or both hydrogen or halogen; R, is halogen and R8 is OH, NHR 1 NHC(X)NH 2 NHC(X)NHRi S, NR 1 or any R 1

6. A method for forming a fimbrolide derivative, the method including reacting a fimbrolide with a halogenating agent and/or an oxygenating agent to form compounds with formula (Ia): X R2\ RI (Ia) wherein R, is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; X is a halogen (X F,C1, Br or OH, OOH, OC(O)R 1 or R 2 and R 3 are independently or both hydrogen or halogen;, and R 9 is halogen. A 'i PCT/AU99/00285 Received 21 December 1999 29

7. A method according to claim 6 wherein the halogenating agent is selected from the group N-bromosuccinimide, N-chlorosuccinimide, N- iodosuccinimide, bromine, cupric bromide, and phenyltrimethylammonium perbromide.

8. A method according to claim 6 wherein the oxygenating agent is selected from lead tetraacetate, Rose Bengal/oxygen gas, hydrogen peroxide/vanadium pentoxide, selenium dioxide, and 3-chloroperoxybenzoic acid.

9. A method for forming a fimbrolide derivative, the method including displacement and/or functionalisation of the halogen or oxygen substituent in the fimbrolide side chain by treating with a nucleophile or an electrophile to form compounds with formula (II): R2R R2 RI R3 R9 (II) wherein R 1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; R 2 and-R 3 are independently or both hydrogen or halogen; R is halogen; and R 4 is selected from the group halogen, amine, azide, hydroxyl, thiol, or any hydrophobic, hydrophilic of fluorophilic alkyl, alkoxy, mercaptoalkyl, alkenyloxy, mercaptoalkenyl, aryloxy, mercaptoaryl, arylalkyloxy, mercaptoarylalkyl, OC(O)R1, SC(O)R1, OS(O)R 1 OS(0) 2 R 1 NHC(O)R1, OC(0)NHR 1 or =0 provided that when R4 is propyl, R 2 is Br, R 3 and R, are Cl, then R, is other than H. PCT/AU99/00285 Received 21 December 1999 A method according to claim 9 wherein the nucleophile is selected from metal halides, water, organic metal carboxylate, organic alcohols, dimethyl sulfoxide, and organonitrile/acid catalyst, and silver triflate.

11. A method according to claim 9 wherein the electrophile is selected from organic acids, isocyanates, acid halides or active acylating agents such as carbonyl imidazoles or anhydrides (including activated hydrophilic PEG acids, PEG acid chlorides, PEG-oxycarbonylimidazoles and PEG-isocyanates) organic sulfonyl chlorides, and diethylaminosulfur trifluoride.

12. A method for forming a fimbrolide derivative the method including reacting an hydroxyl substituent in the fimbrolide side chain with an oxidising agent to form a compound in accordance with formula (III): R3 2 Rs ^Y0 0 R9 (III) wherein R 2 and R 3 are independently or both hydrogen or halogen; R 5 is OH or the same as R1; R, is halogen; and R1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic.

13. A method according to claim 12 wherein the oxidising agents is selected from the group consisting of acid dichromate reagents in any form which may be free or polymer supported, chromium trioxide, manganese dioxide, potassium permanganate, selenium dioxide, ceric ammonium nitrate, ruthenium tetraoxide, and hot nitric acid. -vAm PCT/AU99/00285 Received 21 December 1999 31

14. A method according to claim 13 wherein the acid dichromate agent is selected from the group consisting of Jones reagent, pyridinium chlorochromate, pyridinium dichromate. A method for forming a fimbrolide analogue derived from a compound of formula (III) O R2 R R3 0 0 (III) wherein R2 and R3 are independently or both hydrogen or halogen; is OH or the same as R1; R 9 is halogen; and R 1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic, the method including reacting an aldehyde or ketone substituent in the fimbrolide side chain of the compound with an amine derivative to form a compound with formula (IV) or Rs I Rg N FN R2R, R2R R3 I R3 0y o y 0 0 R9 R9 (IV) (V) PCT/AU99/00285 Received 21 December 1999 32 wherein R1 is hydrogen, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; R2 and R3 are independently or both hydrogen or halogen; R, is halogen and R8 is OH, NHR 1 NHC(X)NH 2 NHC(X)NHR, S, NR 1 or any R 1

16. A method according to claim 15 wherein the amine derivative is selected from the group hydroxyl amine hydrochloride, alkyl and aryl hydrazines, alkyl or aryl amine optionally in the presence of a reducing agent.

17. A fimbrolide derivative produced by a method in accordance with any one of claims 6 to 16.

18. An oligomer or polymer formed by oligomerisation or polymerisation of a fimbrolide compound of the formula: Z R2 2 Re 0 0O R 9 wherein Ro is H, OH, alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl whether unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; R 2 and R 3 are independently or both H or halogen; R, is halogen; Z is independently selected from the group R, halogen, OOH, OC(O)R,, O, amine, azide, thiol, mercaptoalkyl, alkenyloxy, mercaptoalkenyl, aryloxy, mercaptoaryl, arylalkyloxy, mercaptoarylalkyl, SC(O)R, OS(O)R 6 OS(O) 2 R 6 NHC(O)R 6 NR 4 or NHR4; and R 4 is OH, alkyl, alkoxy, poly(ethylene glycol), alkenyl, aryl or arylalkyl, n zIfV-, PCT/AU99/00285 Received 21 December 1999 33 optionally with at least one other monomer.

19. A polymer according to claim 18 wherein the polymer is a homopolymer of the fimbrolide compound of claim 18. A polymer according to claim 18 wherein the polymer is a copolymer of at least one fimbrolide compound in accordance with claim 18 and at least one other polymerisable monomers. 21 Use of a compound in accordance with any one of claims 1 to 5 or 17 as antimicrobial, antiseptic, microbacterial static and/or antifouling agent.

22. An antimicrobial, antiseptic and/or microbacterial static composition including at least one compound in accordance with claims 1 to 5 or 17, or an oligomer or polymer according to any one of claims 18 to

23. An antifouling composition including at least one compound in accordance with claims 1 to 5 or 17, or an oligomer or polymer according to any one of claims 18 to

24. A surface coating composition incorporating at least one compound according to any one of claims 1 to 5 or 17 or an oligomer or polymer according to any one of claims 18 to A compound of formula (VI): (VI) wherein Rlis alkyl, alkoxy, oxoalkyl, alkenyl, aryl or arylalkyl unsubstituted or substituted, straight chain or branched chain, hydrophobic, hydrophilic or fluorophilic; PCT/AU99/00285 Received 21 December 1999 34 R2 and R3 are independently or both hydrogen or halogen; and is halogen.

26. A compound according to claim 25 which is (broimomethylenie)-3-( 1-butenyl)-2(5H)-furanone. 7 EC~f~J