AU2003257229A1 - Furanone derivatives and methods of making same - Google Patents

Description

WO 2004/016588 PCT/AU2003/001053 "Furanone derivatives and methods of making same" technical Field The present invention relates to novel synthesis methods, to the 5 products of such novel methods, and to uses of these products. In particular, the present invention provides methods for the reactions of furanones, in particular fimbrolides, with amines. The invention has particular application in the synthesis of halogenated 1,5-dihydro-pyrrol-2-one, 5-halormethylene substituted 1,5-dihydropyrrol-2-ones (lactam analogues of fimbrolides), 5-amino 10 substituted furanones and 5-aminomethylens-2(5H)-furanones and their synthetic analogues. The invention also relates to novel compounds and uses thereat Background 15 Fimbrolides (halogenated 5-methylene-2(5H)-furanones) possess a wide range of important biological properties including antifungal and antimicrobial properties (see WO 96/29392 and WO 99153915, the disclosures of which are incorporated herein by cross-reference). These metabolites can be isolated from red marine algae Delisea fimbriata, Delisea elegans and Delisee pulchra. 20 Despite their biological activity very few hetero atom containing analogues of these molecules have been reported in the literature. The majority of the published syntheses of fimbrolides focus on the preparation of naturally occurring fimbrolides themselves. Recently we have developed methods that yield both the natural and non-natural fimbrolides in good yields (see WO 25 99/54323 and WO 0200639 the disclosure of which is incorporated herein by cross-reference). We have now found that, surprisingly, fimbrolides undergo reactions with amines under mild conditions. We have found this discovery to be particularly useful in the synthesis of 5-hydroxy-5-alkyl substituted 1,5-dihydro-pyrrol-2 30 one, 5-amino-5-alkyl substituted 2(SH)-furanones and 5-aminomethylene substituted 2(5H)-furanones. Furthermore 5-hydroxy-5-halomethyl substituted 1,5-dihydro-pyrrol-2-one generated under these conditions can be dehydrated to yield 5-halomethylene substituted 1,5-dihydropyrrol-2-ones (lactam analogues of fimbrolides), and the 5-amino-5-bromomethyl substituted 2(5H) 35 furanones can be dehydrobrominated to yield a range of 5-aminomethylene WO 2004/016588 PCT/AU2003/001053 2 substituted 2(5H)-furanones. These furanones can be further functionalised to yield a range of novel analogues. Summary of the Invention in a first aspect, the present invention provides a method for the 5 preparation of compound of formula 11 O R4 Rr5 wherein R 1 and R 2 are independently selected from the group H, 10 halogen, substituted or unsubstituted alky. substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic; R$ and R4 are independently selected from the group H. halogen, 15 substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl; R6 is selected from the group consisting of H, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or 20 unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic, or forms part of an amino acid, or is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a 25 surface; the method comprising reacting a compound of formula I WO 2004/016588 PCT/AU2003/001053 3 Ri R2 0 o R

R

4 wherein R 1 and R 2 are independently H, halogen, alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or 5 unsubstituted alkanyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic;

R

3 and R4 are independently H, halogen, alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or arylalkyl; and R is 10 hydroxy, halogen; and "---" represents a single bond, in which case R is absent, or a double bond, provided that at least one of R1, R 2 , R 3 and R 4 Is halogen, with a compound of formula R 5

NH

2 wherein Rs is selected from the group consisting of H, substituted or 15 unsubstituted alkyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic, or 20 forms part of an amino acid, or is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a surface. The reaction may optionally be carried out in the presence of solvent. Preferably, in the compound of formula 1I, at feast one of R 1 , R 2 , R 3 and 25 R 4 is halogen. In the structural formulae described herein, a particular geometry is not to be taken as specified. For example, the formulae covers both Z- and E isomers. The reaction may be performed in the presence or absence of a solvent. 30 The solvent may be any suitable solvent. Preferable solvents in the present invention include alkyl acetates, aromatic hydrocarbons, chlorinated alkanes, cyclic or open chain ethers such as tetrahydrofuran, diethyl ether, dioxane, and WO 2004/016588 PCT/AU2003/001053 4 Cl-C3 acids. More preferably, the solvents are aromatic hydrocarbons and chlorinated alkanes. Most preferably, the solvent is dichloromethane, as well as dichioroethane and trichloroethane. The reaction is preferably carried out at mild temperatures. Preferably 5 the cyclisation reaction is performed at a temperature in the range of 20-150 0 C. Where a solvent is present, the cyclisation may be performed at reflux temperature, for example, at the reflux temperature of dichloromethane. Optionally the reaction may be carried out below reflux temperature under pressure. 10 The reaction time may vary from about 2 hours to 12 hours or more and is typically about 2 hours or more, It will be appreciated that reaction conditions may be varied depending upon the individual nature of the substrate and the desired rate of the reaction. Non-limiting examples of compounds of formula 11, which may be 15 described as 5-alkyl-5-hydroxy substituted 1,5-dihydro-pyrrol-2-ones, that can be synthesised by the method of the Invention Include: r Sr rO rr - r OH 5r 6 WO 2004/016588 PCT/AU2003/001053 5 In a second aspect, the present invention provides a compound of formula II: OR1 Rs 5 wherein R 1 and R 2 are independently H, halogen, alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, 10 straight chain or branched chain, hydrophilic or fluorophilic;

R

3 and R 4 are independently H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or arylalkyl;

R

5 is selected from the group consisting of H, hydroxy, substituted or 15 unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic, or 20 forms part of an amino acid, or is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a surface, Particularly preferred are compounds of formula I in which at least one of R 1 , R 2 , R 3 and R 4 is halogen. 25 The inventors have found the 5-alkyl-5-hydroxy substituted 1,5-dihydro pyrrol-2-one of formula il can be dehydrated to yield a range of 5 (halomethylene)- 1,5-dlhydro-pyrrol-2-one, 5-(dihalomomethylene)-1,5-dihydro pyrrol-2-one. Accordingly in a third aspect, the present invention provides a method for 30 the dehydration of a compound of formula 1I above, to prepare a compound of formula 1i1; WO 2004/016588 PCT/AU2003/001053 6 OR R3 wherein R1 and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, 5 substituted or unsubstituted oxoalkyi, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic;

R

3 and R 4 are independently selected from H, halogen, substituted or 10 unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl; and R5 is a defined above, the method comprising contacting a compound of formula 11 with a dehydrating agent 15 Preferably at (east one of R 1 , R 2 , R 3 and R 4 in formula IlIl is halogen; Examples of suitable dehydrating agents include phosphorus pentoxide, silica gel, molecular sieves, alumina, acidic resins and polymers, phosphorus oxychloride, acetic anhydride, N,N'-dicyclohexylcarbodiimide (DCC), trifluoroacetic acid, sulfuric acid, trifluoroacetic anhydride, trifluorosulfonic acid 20 anhydride (triflic anhydride). Preferably dehydration is carried out using phosphorus pentoxide in the presence of a solvent. The solvent may be any suitable solvent. Preferable solvents in the present invention include alkyl acetates, aromatic hydrocarbons, chlorinated alkanes, tetrahydrofuran, diethyl ether, dioxane and Cl-G3 acids. 25 More preferably, the solvents are aromatic hydrocarbons and chlorinated alkanes. Most preferably, the solvent is dichloromethane, as well as dichloroethane and trichloroethane. The reaction is preferably carried out at mild temperatures. Preferably the dehydration reaction is performed at a temperature in the range of from 30 about 20-15000.

WO 2004/016588 PCT/AU2003/001053 7 Where a solvent is present, the cyclisation may be performed at reflux temperature of the solvent, for example, at the reflux temperature of dichloromethane. The reaction time may range from about 2 hours to 12 hours or more and 5 is typically about 2 hours or more. It will be appreciated that reaction conditions may be varied depending on the individual nature of the substrate and the desired rate of the reaction. Non-limiting examples of furanones (111) that can be synthesized by this procedure are listed below. 10 OH Or Br BBr Or N0 N U EBr Hr er N NN Or Or" We believe that the 1.5-dihydro-pyrrol-2-ones prepared of formula Ill are novel compounds. 15 Thus, in a fourth aspect the present invention provides a compound of formula Ill: WO 2004/016588 PCT/AU2003/001053 8 .-. 0 R3 R95 R4 wherein R 1 and R2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, 5 substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atorns, straight-chain or branched chain, hydrophilic or fluorophilic;

R

3 and R 4 are independently selected from H, halogen, substituted or 10 unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or arylalkyl; and Rt is as defined above. Preferably at least one of R 1 , R 2 , Ra and R 4 is halogen, Furthermore the present inventors have also found that furanones of 15 formula (I) when treated with certain amines can yield 5-amino substituted or 5 aminomethylene substituted furanones. Alternatively, the compounds of formula I can be treated with an alcohol to yield 5' alkoxy substituted furanones. For example when 4-bromo-5-bromomethylene-2(5H)-furanone was treated with aniline it gave 4-bromo-5-phonylaminomethylene-2(SH)-furanone in good 20 yields. In contrast, the reaction of 4-bromo-5-bromomethylene-2(5H)-furanone with benzyl amine, gave the corresponding 5-benzylamino-4-bromo-5 bromomethyl-2(6H)-furanone. Accordingly, in a fifth aspect, the present invention provides a method for the preparation of a compound of formula IV 25

R

1 R2 Rl 3 a H X IV R5 WO 2004/016588 PCT/AU2003/001053 9 wherein R 1 and R2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, 5 substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic;

R

3 and R 4 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or 10 unsubstituted aryl or substituted or unsubstituted arylalky; Rs is as defined above, X Is 0 or NR 0 , where R 6 may be R 1 , the method comprising reacting a compound of formula I wherein R 3 is a hydrogen and represents a double bond. 15 Preferably at least one of R 1 , R 2 , R 3 and R 4 is halogen. Preferably Re is H. Representative examples of furanones (IV) that can be synthesised by this procedure are listed below. Br Br Br Br Br Br o 0' NH0 NH0 N Ph HH o NH 0 NH 20 In yet a sixth aspect, the present invention provides a compound of formula IV WO 2004/016588 PCT/AU2003/001053 10 R4 O H 0 H x IV IRS wherein R 1 and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, 5 substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or urisubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic;

R

3 and R 4 are independently selected from H, halogen, substituted or 10 unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl; and

R

6 and X are as defined above, Preferably, at least one of R 1 , R 2 , Rs and R 4 is halogen. Accordingly a seventh aspect, the present invention provides for a 15 method for preparation of a compound of formula V. R, R2 O R3 0)7 V X-RS wherein R 1 and R 2 are independently selected from H, halogen, 20 substituted or unsubstitutod alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxcalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally Interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic; 25 R 3 is selected from H, halogen, substituted or unsubstituted alkyl, substituted or. unsubstituted alkoxy, substituted or unsubstituted aryl or WO 2004/016588 PCT/AU2003/001053 11 arylalkyl; wherein R5 is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, 5 straight chain or branched chain, hydrophilic or fluorophilic; X is 0 or NR 6 , where R is as defined above; and Rs is as defined above. Non-limiting examples of furanones of formula (V) that can be synthesised by this procedure are listed below. 10 Br 'Br Br H H H HN HNH6 In an eighth aspect, the present invention provides a compound of formula V: 15 R1 R2 O R3 wherein R 1 and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, 20 substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluormphilic;

R

3 is selected from H, halogen, substituted or unsubstituted alkyl, 25 substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or arylalkyl; WO 2004/016588 PCT/AU2003/001053 12 X is 0 or NR 6 , where R is as defined above; and Rs is as defined above. In yet a ninth aspect the present invention provides a compound of formula (VI): z R1 .. ON 5 VI wherein R 1 and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylaikyl, 10 optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic;

R

3 and R 4 are independently selected from H, halogen, alkyl, substituted or unsubstituted aryl or arylalkyl; wherein R 5 is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, 15 substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic:

R

5 is defined as above; and Z is selected from the group R 2 , halogen, OC(O)R. =0, amine azide, 20 thiol, R 2 , mercaptoaryl, arylalkoxy, mercaptoarylakyl, SC(O)R2, OS(O) 2

R

2 , NHC(0)R 2 , =NR 2 or NHR 2 . The compounds of formula Vi may be prepared by functionalizing a fimbrolide of formula (IlIl) wherein, R 1 , R 2 , R$ and R 4 are as defined above, with a reagent described in WO 99154323, (the disclosure of which is incorporated 25 herein by cross-reference). Reagents for introduction and manipulation of the Z group include halogenating and oxidising agents (N-halosuccinimide, lead tetraacetate, selenium dioxide, Jones reagent), nucleophiles (including organic metal carboxylates, organic alcohols, dimethyl sulfoxide and organonitriles) and 30 electrophiles including (organic acids, isocyanates, carboxylic or sulfonic acid halides and diethylaminosulfur trifluoride).

WO 2004/016588 PCT/AU2003/001053 13 Non-limiting examples of furanones of formula (VI) that can be synthesised by this procedure are listed below. Br Br Br -Br ~B' Br re Br Br Br S B N B r Br br OH OH Br N Brr O'>2H 3 0 0 SSr N ~ Br In a tenth aspect, the present invention provides an oligomer or polymer 10 formed by oligomerising or polymerising a compound of formula II - VI, described herein directly or with one or more other monomers, The one or more other monomer may be any suitable polymerisable copolyrmer e.g. acrylate ester such as alkyl, hydroxyalkyl, aminoalkyl, or substituted substituted or unsubstituted aryl acrylates or methacrylates; WO 2004/016588 PCT/AU2003/001053 14 crotonates, substituted or unsubstituted acrylonitriles, vinyl alcohols or acetates, styrene and siloxanes.

R

5 may be a residue of a natural or synthetic compound. Rs may be a biological or non-biological compound. For example, R 5 may be a coenzyme or 5 cofactor. R 5 may be an oligomer or a polymer, which may be biological or synthetic. For example, the oligomer or polymer may be a peptide or polyamide. The polymer may be a protein, for example, an enzyme or a receptor, Re may be an oligomer or polymer comprising nucleic acid residues. The polymer may be a polynucleotide, for example, DNA or RNA. R 5 may form 10 part of or be bonded to a nucleoside. The nucleoside may be a D- or L nucleoside. R5 may be linked to a sugar moiety of the nucleoside.

R

6 may be a surface or substrate with which the nitrogen atom of is associated, The association may be chemical bonding, for example covalent bonding. The surface or substrate may be biological or synthetic. Alternatively, 15 the association may be by means of adsorption. Methods for forming such associations are described in more detail below. Re may also be a dendrimer. A review of dendrimers Is provided in Klajnert, B. and Bryszewska, M. (2001) Dendrimers:properties and applications, Acta Biochemica Polonica Vol. 48 No.1/2001, the disclosure of 20 which is incorporated herein by reference. A plurality of compounds in accordance with the invention may be carried by the dendrimer. The compound may be immobilised directly onto at least part of the surface of the material of the substrate or via one or more intermediate layers interposed between the substrate material and the immobilised layer. The 25 intermediate layer (s) may be bonding layer (s). The substrate may be shaped or non-shaped. The substrate may be solid, semi-solid or flexible. The substrate may be a woven or non-woven film or sheet. The substrate may be a natural or synthetic filament or fibre. The substrate may be a natural material, for example, a plant seed. The material 30 from which the substrate is formed may be selected to suit the particular application. For example, In the case of a shaped biomedical device the material may meet other specifications of the application, such as mechanical and optical properties, The substrate may be a shaped article including, but are not limited, 35 medical devices, for example, implantable biomedical devices such as urinary WO 2004/016588 PCT/AU2003/001053 15 catheters, percutaneous access catheters, stents, as well as non-implantable devices such as contact lenses, contact lens storage cases, and the like. The material from which the article is formed can be a metal, a ceramic, a solid synthetic polymer, or a solid natural polymer, for example a solid 5 biopolymer. Examples of useful materials for this invention are titanium, hydroxyapatite, polyethylene (which are useful materials for orthopaedic implants), polyurethanes, organosiloxane polymers, perfluorinated polymers (which are useful materials for instance for catheters, soft tissue augmentation, and blood contacting devices such as heart valves), acrylic hydrogel polymers 10 and siloxane hydrogel polymers (for Instance for contact lens and intraocular lens applications), and the like, and any combination thereof, The surfaces of these materials can be chemically inert or contain reactive functional groups. Further examples of substrates include archival documents, antiques and art, rare and valuable seeds intended for storage (e. g. seed banks of 15 conservation groups), etc in which case the substrate may be paper, material or other natural or synthetic material. The substrate may be a shell fish or aquaculture apparatus, for example, that described in PCT/AU98/00508, the disclosure of which is incorporated herein by reference. 20 As mentioned above, R5 may be associated with a surface of substrate. If necessary, the surface of the substrate may be optionally treated at least in part to activate the surface, to which the compounds of the present invention may be reacted to immobilise the compound. Reference to at least part of the surface of the substrate includes a 25 surface of one or more intermediate layers applied to the substrate. The compounds may be immobilised on the substrate surface by any suitable technique, Immobilization may be by covalent or non covalent means. Preferably, the compounds are immobilized on the substrate surface by means of covalent bonds. 30 The immobilization of furanone compounds on to the substrate prevents their loss from the surface, thus ensuring long-lasting antimicrobial action. The association between the compounds of the invention and the substrate may be characterised by the formula: X-Y-Z where X is a substrate. Y Is an optional chemical linking moiety and Z is a compound In accordance 35 with the present invention. The linking moiety, if present, may be a homobifunctional or heterobifunctional linking moiety. Y may be a simple WO 2004/016588 PCT/AU2003/001053 16 component (eg a short molecule) or it may comprise a plurality of units or components that may be the same of different. Y may comprise a number of components or units that may be "built up"in a stepwise fashion. The formation of a covalent interfacial linkage is much preferable to an 5 ionic bond since in biological media where the salt content is such that ionic bonds are interfered with and ironically attached molecules can be displaced from a surface. In the context of substrates that are medical devices, covalent anchoring of the compound(s) also serves to eliminate concerns regarding possible 10 deleterious effects that compounds might cause at sites distant from the device, such as in the liver, brain, or kidney tissues of a living human organism. In medical applications it is important to anchor the furanone compound (s) via an interfacial covalent-bond that is not subject to cleavage in the host environment that the biomedical device is to be placed in. 15 Methods for the covalent immobilization of organic molecules onto solid surfaces are well known to those skilled in the art. Interfacial reactions leading to the formation of covalent interfacial bonds are derived from well known organic-synthetic reactions. The choice of immobilization reaction depends on both the nature of the substrate material and the chemical composition of the 20 furanone derivative (s) that are desired for a particular application. For example, a compound that contains a hydroxyl group in a side chain distal to the ring system, can be linked covalently onto surfaces using epoxide chemistry analogous to the reaction pathway described for the immobilization of polysaccharides onto epoxidated surfaces in Li et at, Surface Modification of 25 Polymeric Biornaterials (OD Ratner and DG Castner, Eds), Plenum Press, NY, 1996 pages 165-173 (the disclosure of which is incorporated herein in its entirety), through isocyanate groups attached to the surface to produce stable urethane linkages through thermal processes, or through carboxylic acid groups or their equivalents, such as acid chlorides, on the surface to produce 30 ester linkages. A compound that contains an aldehyde group can be linked onto surface amine groups using a reductive animation reaction. A compound that contains a carboxylic acid group can be linked onto surface amine groups using carbodlimide chemistry. Interfacial coupling reactions must of course be selected not only for their 35 ability to achieve the desired covalent linkage but also for avoidance of adverse effects on the furanone compound (s) to be attached. Particularly, the furanone WO 2004/016588 PCT/AU2003/001053 17 ring system tends to be labile to alkaline conditions. Such limitations are well known to those skilled in the art. Among the many possible interfacial coupling reactions known in the art, there is sufficient scope for selection of reactions that proceed in a suitable pH range and with furanones substituted with various 5 functional groups in various positions. Some solid substrate materials possess reactive surface chemical groups that can undergo chemical reactions with a partner group on a compound and thereby form a covalent interfacial linkage directly. Alternatively, in situ covalent linkage can be made directly through the 10 addition of a doubly functionalised linker molecule to the active surface in the presence of an appropriate compound, or stepwise by sequential addition of doubly functionalised linker molecules and then an appropriate compound. It is not always possible to immobilize furanone compounds directly onto solid substrate materials; in these cases surface activation or one or more interfacial 15 bonding layer (s) is used to effect covalent immobilization of the compounds. Such surface activation is essential when immobilizing compounds onto polymeric materials such as fluoropolymers and polyolefins. Surface activation of solid substrate materials can be achieved in a number of ways. Examples are corona discharge treatment or low pressure 20 plasma treatment of polymers. These methods are well known to introduce a variety of functional groups onto polymeric surfaces. An alternative approach is to provide an interracial bonding layer interspersed between the solid substrate material or medical device and the compound layer. The application of a thin interfacial bonding layer can be done 25 using methods such as dip coating, spin coating, or plasma polymerization. The chemistry of the bonding layer is selected such that appropriate reactive - chemical groups are provided on the surface of this layer, groups that then are accessible for reaction with compound of the invention. Particularly versatile is the subsequent application of multiple thin 30 interfacial bonding layers; this method can provide a very wide range of desired chemical groups on the surface for the immobilization of a wide range of functionalized furanones and enables usage of compounds optimized for their biological efficacy. By providing a thin, surface-coated layer of compounds, the optical 35 quality of antibacterial devices of this invention is not reduced, which makes the WO 2004/016588 PCT/AU2003/001053 18 invention applicable to transparent ophthalmic devices such as contact lenses and intraocular lenses. The present invention provides thin surface coatings that provide antimicrobial properties and/or antifungal properties to solid materials onto 5 which the coatings have been applied. More particularly, the coatings may be designed to reduce or prevent colonization of biomedical devices by bacteria that cause adverse effects on the health of human users of biomedical devices when such devices are colonized by bacteria. The active antibacterial layer comprises one or a plurality of furanone 10 compounds selected for both their antibacterial activity and absence of cytotoxicity as well as any other adverse biomedical effect on the host environment that the coated device contacts. In an eleventh aspect, the present invention provides incorporation of compounds produced by the methods according to the first, third, fifth, seventh, 15 ninth, or tenth aspects either in surface coatings or polymers through any part of the molecule, for example, newly introduced functionality on the alkyl chain or the alkyl chain or the halornethylene functionality itself via direct polymerisation or copolymerisation with suitable monomers. In an twelfth aspect, the present invention provides a compound 20 produced by the method according to the first, third, fifth, seventh, ninth, or eleventh aspects of the present invention. In a thirteenth aspect, the present invention provides the use of a compound produced according to the present invention. The present inventors have found that many of the 1,5-dihydro-pyrrol-2-one derivatives and furanones 25 having the formula (11), (11), (IV), (V) and (VI) have antimicrobial and/or antifouling properties, Accordingly, the fimbrolide derivatives are suitable for use as antimicrobial and/or antifouling agents. Thus in a fourteenth aspect, the present invention provides methods of use of compounds of formula (11), (1i1), (IV), (V) and (VI) in medical, scientific 30 and/or biological applications. For these and other applications, the compounds of the present invention may be formulated as a composition. In a fifteenth aspect, the present invention provides a composition comprising at least one compound of formula (Ii), (ll), (IV), (V) or (VI). 35 The compositions of the third aspect of the invention may be in any suitable form. The composition may include a carrier or diluent. The carrier may WO 2004/016588 PCT/AU2003/001053 19 be liquid or solid. For example, the compositions may be in the form of a solution or suspension of at least one of the compounds in a liquid. The liquid may be an aqueous solvent or a non-aqueous solvent. The liquid may consist of or comprise a one or more organic solvents, The liquid may be an ionic 5 liquid. Particular examples of carrier or diluents include, but are not limited to, water, polyethylene glycol, propylene glycol, cyclodextrin and derivatives thereof. The composition may be formulated for delivery in an aerosol or powder form. 10 The composition may include organic or inorganic polymeric substances. For example, the compound of the invention may be admixed with a polymer or bound to, or adsorbed on to, a polymer. When the composition is to be formulated as a disinfectant or cleaning formulation, the composition may include conventional additives used in such 15 formulations. Non-limiting examples of the physical form of the formulations include powders, solutions, suspensions, dispersions, emulsions and gels. Formulations for pharmaceutical uses may incorporate pharmaceutically acceptable carriers, diluents and excipients known to those skilled in the art. The compositions make be formulated for parenteral or non-parenteral 20 administration. The composition of the invention may be formulated for methods of introduction including, but not limited to, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, and oral routes. It may be formulated for administration by any convenient route, for example by infusion or bolus injection, by absorption 25 through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration may be localized or systemic. The composition may be formulated for intraventricular and intrathecal injection. Pulmonary administration can also be employed, e.g., by use of an inhaler or 30 nebulizer, and formulation with an aerosolizing agent. In certain preferred embodiments the composition further comprises other active agents such as antibiotics and cleaning agents. In a sixteenth aspect, the present invention provides a method of treating an infection in a human or animal subject the method comprising administration 35 to the subject of an effective amount of the compound of the invention. The treatment may therapeutic and/or prophylactic.

WO 2004/016588 PCT/AU2003/001053 20 The compounds of the present invention can act as quorum sensing inhibitors and therefore find use in any application where such as effect is desired. For example, the compounds of the present invention may have use in preventing the establishment and expression of virulence by microorganisms 5 through the inhibition of quorum sensing systems and/or other extracellular systems (eg see, International patent application No. PCTIAUO1/01621, the disclosure of which is incorporated herein in its entirety). The present invention is suitable for biofilms originating from a single type of organism and for mixed biofil ms. By "mixed biofilms" is meant biofi Ems 10 .created by more than one type of microorganism. Most preferably, it is envisioned that biofilms will be created by at least two organisms from the group consisting of bacteria, algae, fungi, and protozoa. The effects of treating biofilms with homoserine factones have been demonstrated with Pseudomonas aeruginosa. The HSLs have generally been 15 isolated from a wide range of bacteria known to produce biofilms. Among these are the enterobacteria. The presence of the HSLs in a wide range of bacteria indicates that the compounds of the present invention can be used to effectively treat not only Pseudomonas sp. biofilms but also mixed biofilms containing Pseudomonas sp. and biofilms composed of bacteria other than 20 Pseudomonas aeruginosa. The following is a list of groups of Gram-Negative bacteria that have members which use homoserine lactones for cell-cell communication: anaerobic Gram Negative Straight, Curved and Helical Rods; Bacteroidaceae; The Rickettslas and Chlamydias; Dissimilatory Sulfate-or Sulfur-Reducing 25 Bacteria; the Mycoplasmas; The mycobacteria; Budding and/or Appendaged Bacteria; Sheathed Bacteria; Nocardioforms and Actinomycetes, for example. See Bergey's Manual of Systematic Bacteriology, First Ed., John G. Holt, Editor in Chief (1984), incorporated herein by reference. The method of the sixteenth aspect may be used to treat an infection or 30 condition in a subject that is characterized by biofilm formation. Non-limiting examples of human infections involving biofilms include dental caries, periodontitis, otitis media, muscular skeletal infections, necrotising fascitis, biliary tract infection, osteomyelitis, bacterial prostatitis , native valve endocarditis, cystic fibrosis pneumonia, meloidosis, and nosocomial infections 36 such as ICU pneumonia, sutures, exit sites, arterlovenous sites, soleral buckles, contact lenses, urinary catheter cystitis, peritoneal dialysis (CAPD) WO 2004/016588 PCT/AU2003/001053 21 peritonitis, IUDs, endotracheal tubes, Hickman catheters, central venous catheters, mechanical heart valves, vascular grafts, biliary stent blockage, and orthopedic devices, penile prostheses. Further applications are described in Costerton J et al, (1999) Vol. 284, Science pp1318-1322 and Costerton J and 5 Steward, (2001) Battling Biofilms, Scientific American pp 75-81, the disclosures of which are incorporated herein by reference. Other locations in which biofilms may form included drinking water pipes, which may lead to corrosion or disease, household drains, dental plaque which may lead to gum disease and cavities, which may lead to gun disease or 10 cavities, contact lenses which may lead to eye infections, ears which may lead to chronic infection and lungs which may lead to pneumonia. The condition may be cystic fibrosis. The infection may be that resulting from a skin infection, burn infection and/or wound infection. The method and composition of the invention may be particularly suitable for the treatment of 15 infection in immuno compromised individuals. In yet a seventeenth aspect, the present invention provides a method for treating biofilm formation on a surface by contacting the surface with a compound in accordance with the present invention. The term "surface" as used herein relates to any surface which may be 20 covered by a biofilm layer. The surface may be a biological (eg tissue, membrane, skin etc) or non-biological surface. The surface may be that of a natural surface, for example, plant seed, wood, fibre etc. The surface or substrate may be any hard surface such. as metal, 25 organic and inorganic polymer surface, natural and synthetic elastomers, board, glass, wood, paper, concrete, rock, marble, gypsum and ceramic materials which optionally are coated, eg with paint, enamel etc; or any soft surface such as fibres of any kind (yams, textiles, vegetable fibres, rock wool, hair etc.); or porous surfaces; skin (human or animal); keratinous materials 30 (nails etc.). The hard surface can be present in process equipment or components of cooling equipment, for example, a cooling tower, a water treatment plant, a dairy, a food processing plant, a chemical or pharmaceutical process plant. The porous surface can be present in a filter, eg. a membrane filter. 35 Particular examples of surfaces that may be treated in accordance with the invention include, but are not limited to, toilet bowls, bathtubs, drains, WO 2004/016588 PCT/AU2003/001053 22 highchairs, counter tops, vegetables, meat processing rooms, butcher shops, food preparation areas, air ducts, air-conditioners, carpets, paper or woven product treatment, nappies (diapers), personal hygiene products (eg sanitary napkins) and washing machines. The cleaning composition may be in the form 5 of a toilet drop-in or spray-on devices for prevention and removal of soil and under rim cleaner for toilets. The compositions and methods of the present invention also have applications in cleaning of Industrial surfaces such as floors, benches, walls and the like and these and other surfaces in medical establishments such as hospitals (eg surfaces in operating theatres), veterinary 10 hospitals, and in mortuaries and funeral parlours. A compound of the invention may be incorporated into epidermal bandages and lotions. Alternatively, the compounds of the invention may be incorporated into cosmetic formulations, for example, aftershave lotions. Compositions of the present invention may be in the form of an aqueous 15 solution or suspension containing a cleaning-effective amount of the active compound described above. The cleaning composition may be in the form of a spray, a dispensable liquid, or a toilet tank drop-in, under-rim product for prevention, removal and cleaning of toilets and other wet or intermittently wet surfaces in domestic or industrial environments. 20 The compositions of the present invention may additionally comprise a surfactant selected from the group consisting of anionic, non-ionic, amphoteric, biological surfactants and mixtures thereof. Most preferably, the surfactant is sodium dodecyl sulfate. One or more adjuvant compounds may be added to the cleaning solution 25 of the present invention. They may be selected from one or more of biocides, fungicides, antibiotics, and mixtures thereof to affect planktonics. pH regulators, perfumes, dyes or colorants may also be added. By "cleaning-effective" amount of active compound, it is meant an amount of the compound which is necessary to remove at least 10% of 30 bacteria from a biofilm as determined by a reduction in numbers of bacteria within the biofilm when compared with a biofilm not exposed to the active compound. The cleaning methods of the present invention are suitable for cleaning surfaces. They may be used to treat hard, rigid surfaces such as drain pipes, 35 glazed ceramic, porcelain, glass, metal, wood, chrome, plastic, vinyl and formica or soft flexible surfaces such as shower curtains, upholstery, laundry WO 2004/016588 PCT/AU2003/001053 23 and carpeting. It is also envisioned that both woven and non woven and porous and non-porous surfaces would be suitable. In other embodiments of the present invention, the composition of the invention may be formulated as a dentifrice, a mouthwash or a composition for 5 the treatment of dental caries. The composition may be formulated for acne treatment or cleaning and disinfecting contact lenses (eg as a saline solution). The method of the invention'may be used to treat medical devices, In yet a further aspect, the present invention extend to a medical device having a least one surface associated with a compound(s) in accordance with 10 the present invention. The method of the invention may be used to treat Implanted devices that are permanent such as an artificial heart valve or hip joint, and those that are not permanent such as indwelling catheters, pacemakers, surgical pins etc. The method may further be used in situations involving 15 bacterial infection of a host, either human or animal, for example in a topical dressing for burn patients. An example of such a situation would be the infection by P. aeruginosa of superficial wounds such as are found in burn patients or in the lung of a cystic fibrosis patient. In other forms, the present invention can be used to treat integrated 20 circuits, circuit boards or other electronic or microelectronic devices. In yet another aspect, the present invention provides a method for the inhibition of a biological pathway is a cell, the method comprising administering to the cell a compound in accordance with the present invention. 25 Terminology The term "alkyl" is taken to mean both straight chain alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, and the like. Preferably the alkyl group is a lower alkyl of 1 to 6 carbon atoms. The alkyl group may optionally be substituted by one or more groups selected from 30 alkyl, cycloalkyl, alkeny, alkynyl, halo, carboxyl, haloalkyl, haloalkynyl, hydroxy, substituted or unsubstituted alkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocyclyl, heterocyoloxy, 35 heterocyclamino, haloheterocyclyl, alkylsulfenyl, alkylcarbonyloxy, alkylthio, acylthio, phosphorus-containing groups such as phosphono and phosphinyl, WO 2004/016588 PCT/AU2003/001053 24 The term "alkoxy" denotes straight chain or branched alkyloxy, preferably C1.10 alkoxy. Examples include methoxy, ethoxy, n-propoxy, isopropoxy and the different butoxy isomers. The term "alkenyl" includes groups formed from straight chain, branched 5 or mono- or polycyclic alkenes and polyene. Substituents include mono- or poly-unsaturated alkyl or cycloalkyl groups as previously defined, preferably C2 10 alkenylt Examples of alkenyl include vinyl, allyl, 1-methylvinyl, butenyl, iso butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, 10 cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3 butadienyl, 1,4-pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4 hexadieny, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, or 1,3,5,7-cyclooctatetraenyl. The term "halogen" includes fluorine, chlorine, bromine or iodine, 15 preferably bromine or fluorine. The term "heteroatoms" denotes 0, N, S or Si. The term "acyl" used either alone or in compound words such. as "acyloxy", "acylthio", "acylamino" or diacylanino" denotes an alkanoyl, aroyl, heteroyl, carbamoyl, alkoxycarbonyl, alkanesulfonyl, arysulfonyl, and is 20 preferably a C 1

.

10 alkanoyl. Examples of acyl include carbamoyl; straight chain or branched alkanoyl, such as formyl, acetyl, propanoyl, butanoyl, 2 methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptancyl, octanoyl, nonanoyl, decanoyl; alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, t-pentyloxycarbonyl or heptyloxycarbonyl; 25 cycloalkanecarbonyl such as cyclopropanecarbonyl cyclobutanecarbonyl, cyclopentanecarbonyl or cyclohexanecarbonyl; alkanesulfony, such as methanesulfonyl or ethanesulfbnyl; alkoxysulfonyl, such as methoxysulfony or ethoxysulfonyl; heterocycloalkanecarbonyl; heterocyclyoalkanoyl, such as pyrrolidinylacetyl, pyrrolidinylpropanoyl, pyrrolinylacetyl, pyrrolylacetyl, 30 pyrrolidinylbutanoyl, pyrrolidinylpentanoyl, pyrrolldinylhexanoyl or thiazolidinylacetyl; heterocyclylalkenoyl, such as heterocyclylpropenoyl, heterocyclylbutenoyl, heterocyclylpentenoyl or heterocyclylhexenoyl; or heterocyclylglyoxyloyl, such as, thiazolidinylglyoxyloyl or pyrrolidinylglyoxyloyl. The term "aryr' refers to atyl groups having 6 through 10 carbon atoms 35 and includes, for example, phenyl, naphthyl, indenyl. Typically the aryl group WO 2004/016588 PCT/AU2003/001053 25 will be phenyl or naphthyl as compounds having such groups are more readily available commercially than other aryl compounds, The term "substituted aryl" refers to aryl groups having 1 through 3 substituents independently selected from the group of lower alkyl, lower 5 substituted or unsubstituted alkoxy, halonitro, or haloalkyl having I through 3 carbon atoms and I through 3 halo atoms. Typical substituted aryl groups include, for example, 2-fluorophenyl, 2-chlorophenyl, 2,6-dimethylphenyl, 4 fluorophenyl, 2-methylphenyl, 2-chloro, 3-chloromethylphenyl, 2-nitro, 5 methylphenyl, 2,6-dichlorophenyl, 3-trifluoromethylphenyl, 2-methoxyphenyl, 2 10 bromonaphth-1-y, 3-methoxyinden-1-y, and the like. Carboxyaryl eg carboxy phenyl, aminoaryl eg amlnophenyl The term "fluorophilic" is used to indicate the highly attractive interactions between certain groups, such as highly fluorinated alkyl groups of C4-C10 chain length, towards perfluoroalkanes and perfluoroalkane polymers. 15 The term "amino acid" as used herein includes any compound having at least one amino group and at least one carboxyl group. The amino acid may be a naturally occurring amino acid or it may be a non-naturally occurring amino acid. The amines used in this invention may be soluble in the reaction medium 20 or insoluble in the reaction medium. Examples of soluble amines include ammonia, alkyl-, aryl-, arylalkyl-, and heterocyclic amines. Examples of insoluble amines include basic amine resins and amine containing biological and synthetic polymers. The term "optionally substituted" includes, but is not limited to such 25 groups as halogen; hydroxy; hydroxy substituted alkyl; substituted or unsubstituted S(O)m alkyl or S(O)m aryl wherein m is 0, 1 or 2. such as methyl thio, methylsulfinyl or methyl sulfonyl; amino, mono and di-substituted amino; alkyl, cycloalkyl, or cycloalkyl alkyl group; halosubstituted alkyl, such as CF 3 ; an optionally substituted aryl, optionally substituted arylalkyl, such as benzyl or 30 phenethyl, wherein these aryl moieties may also be substituted one to two times by halogen; hydroxy; hydroxy substituted alkyl; alkoxy; S(O)m alkyl; amino, mono and di- alkyl substituted amino, substituted or unsubstituted alkylsiO for example (CH 3

)

3 SiO-. The term "Medical devices" as used herein includes disposable or 35 permanent catheters, (e.g., central venous catheters, dialysis catheters, long term tunneled central venous catheters, short-term central venous catheters, WO 2004/016588 PCT/AU2003/001053 25 peripherally inserted central catheters, peripheral venous catheters, pulmonary artery Swan-Ganz catheters, urinary catheters, and peritoneal catheters), long term urinary devices, tissue bonding urinary devices, vascular grafts, vascular catheter ports, wound drain tubes, ventricular catheters, hydrocephalus shunts 5 heart valves, heart assist devices (e.g., left ventricular assist devices), pacemaker capsules, Incontinence devices, penile implants, small or temporary joint replacements, urinary dilator, cannulas, elastomers, hydrogels, surgical instruments, dental instruments, tubings, such as intravenous tubes, breathing tubes, dental water lines, dental drain tubes, and feeding tubes, fabrics, paper, 10 indicator strips (e.g., paper indicator strips or plastic Indicator strips), adhesives (e.g., hydrogel adhesives, hot-melt adhesives, or solvent-based adhesives), bandages, orthopedic implants, and any other device used in the medical field. "Medical devices" also include any device which may be inserted or implanted into a human being or other animal, or placed at the insertion or implantation 15 site such as the skin near the insertion or implantation site, and which include at least one surface which is susceptible to colonization by biofilm embedded microorganisms. Medical devices also include any other surface which may be desired or necessary to prevent biofilm embedded microorganisms from growing or proliferating on at least one surface of the medical device, or to 20 remove or clean biofilm embedded microorganisms from the at least one surface of the medical device, such as the surfaces of equipment in operating rooms, emergency rooms, hospital rooms, clinics, and bathrooms. In one specific embodiment, the biofilm penetrating composition is integrated into an adhesive, such as tape, thereby providing an adhesive which may prevent 25 growth or proliferation of biofilm embedded microorganisms on at least one surface of the adhesive. Implantable medical devices include orthopedic implants. Insertable medical devices include catheters and shunts which. The medical devices may be formed of any suitable metallic materials or non-metallic materials known to 30 persons skilled in the art. Examples of metallic materials include, but are not limited to, tivanium, titanium, and stainless steel, and derivatives or combinations thereof. Examples of non-metallic materials include, but are not limited to, thermoplastic or polymeric materials such as rubber, plastic, polyesters, polyethylene, polyurethane, silicone, Gortex 35 (polytetrafluoroethylene), Dacron 'm (polyethylene tetraphthalate), Teflon WO 2004/016588 PCT/AU2003/001053 27 (polytetrafluoroethylene), latex, elastomers and DacronTM sealed with gelatin, collagen or albumin, and derivatives or combinations thereof. The present invention also extends to a method of regulating a cells characterised by AHL-mediated quorum sensing or an AI-2 pathway comprising 5 contacting the cells with a compound in accordance with the present invention. Throughout this specification 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 steps, but not the exclusion of any other element, integer or step, or group of elements, 10 integers or steps. Reference is made to patent applications PCTAU01/01621, PCT/AUO2/00797, PCT/AU99/00284, PCT/AU99/00285, PCT/AUO0/01553, PCT/AUO1/00296, PCT/AUDI/00295, PCT/AU01/00407, PCT/AU89/00508 15 and PCT/AU01/00781 which relate to furanones and analogues and to uses of these compounds and the entire disclosures of which are incorporated herein by reference.

WO 2004/016588 PCT/AU2003/001053 28 Modes for Carrying Out the Invention The'invention is further described in and illustrated by the following examples. The examples are not to be construed as limiting the invention In 5 any way. EXPERIMENTAL DETAILS General. Melting points are uncorrected. Microanalyses were performed by Dr H.P, Pham of The University of New South Wales Microanalytical 10 Laboratory. 1H NMR spectra were obtained in CDC1 3 on a Bruker AC300F (300 MHz) or a Bruker DMX500 (500 MHz) spectrometer. 13 C NMR were obtained in the same solvent on a Bruker AC300F (75.5 MHz) or a Bruker DMX50O (125,8 MHz) spectrometer. Chemical shifts were measured on the 8 scale internally referenced to the solvent peaks: CDC1 3 (8 7.26, 8 77.04). 15 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 an VG Quattro mass spectrometer at 70eV lonisation voltage and 200*C ion source 20 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 60GF 254 (Art. 7730). 25 3-Butyl-5-dibromomethyl-S.hydroxy-i-phenyl-1,5-dihydropyrrol-2-one A solution of 3-butyl-5-dibromomethylene-2(SH)furanone (0.20 g; 0.66 mmol) in aniline (5 ml) was allowed to stand at room temperature for 24 h. The mixture was diluted with dichloromethane (25 ml) and washed with aqueous hydrochloric acid (2M, 20 ml). The organic phase was dried over sodium 30 sulfate and evaporated to yield a yellow viscous oil (0.30 g). The crude product was chromatographed on silica using dichloromethane/ethylacetate (19:1; v:v) as the eluent. The major product, a pale yellow band, was collected and recrystallised from light petroleum to yield 3-butyl-5-dibromomethyl-5-hydroxy 1-phenyl-1,5-dihydropyrrol-2-one as colourless prisms (0.24 g, 92%), m.p. 96 35 98"C vma 3211, 2957, 1679, 1597, 1500, 1417, 1117, 1058, 760, 698 cm- 1 . Xm,: 263nm (am 2,955), 202 (2,464), 1H n.m.r. 8 (CDC 3 ): 7.54-7.37, m, Ph; WO 2004/016588 PCT/AU2003/001053 29 6.82, 1H, s, C4-H; 6.56, 1H, s, -CH1r2; 3.42, s, CS-OH; 2.43-2.41, m, 2H, CH 2 ; 1.64-0.97, m, C3-chain. "C n.m.r. 8 (CDC1s): 13.7, 14.0, 22.3, 40.6, 92.2, 126.7, 127.4, 129.0, 134.0,135.5,9136.0, 144.3, 169.0. 5 5-Dibromomethyl-3-hexyl-5-hydroxy-1-phenyl-1,5-dihydropyrrol-2-one A mixture of 3-hexy-5-dibromomethylene-2(5H)furanone (0.40g, 1.18 mmol) and aniline (1 ml) in ethanol (6 ml) was refluxed for 3h. The solvent was evaporated off and the residue extracted with diohloromethane (25 ml). The organic phase was washed with aqueous hydrochloric acid (2M, 2 x 20 ml), 10 dried over sodium sulfate and evaporated to yield a semi-solid (0.39 g). The crude product was chromatographed on silica using dichloromethane/ethylacetate (19:1; v;v) as the eluent. The major product, a pale yellow band, was collected and recrystallised from light petroleum to yield 5-dibromomethyl-3-hexyl-5-hydroxy-l-phenyl-1,5-dihydropyrrol-2-one 15 as a semi-crystalline-solid (23%), msp. 43-45* (Found (HRESMS) 451,962217.

C

17

H

2 ,Br 2

NO

2 Na* (?"Br) requires 451.953106). vmax: 3186, 2926, 1680,1659, 1492, 1372, 1095, 1059, 897, 850, 766, 747, 699, 671 cm. Ams: 261nm (smax 4051), 206 (26.550). 1 H n.m.r. 5 (CDCs): 7.5-7.25, m, 5H, Ph; 6.8, s, 1H, C4-H; 5.5, s, 1H, -CHBr,; 3.77, brs, 1H, C5-OH; 2.44-2.34, m, 2H, CH 2 ; 2.03-0.91, 20 11H, C3-chain. 13C n.m.r. S ( CDCia): 14.2, 25.3, 29.0, 31.0, 46.6, 92.0, 104.8, 126.7, 127.0, 136.0, 144.0,169.0, 172.0. I-Benzyl-3-butyl-5-dibromomethyl-5-hydroxy-1,5-dihydropyrrol-2-one A solution of 3-butyl-5-dibromomethylene-2(5H)furanone (1.03 g: 3.32 mmol) in 25 benzyl amine (2ml) was allowed to stand at room temperature for 1 h during which time the reaction mixture solidified. The solid was dissolved in dichloromethane (25. ml) and washed with aqueous hydrochloric acid (2M, 20 ml). The organic phase was dried over sodium sulfate and evaporated to yield a yellow viscous oil. The crude product was triturated with light petroleum to 30 yield a white solid (1.0 g; 74%) which was recrystallised from light petroleum to yield 1-benzyl-3-butyl-5-dlbromomethyl-5-hydroxy-1,5-dihydropyrrol-2-one as colourless needles, mp. 92-93*C (Found (HRESMS) m/z 479,974243.

C

1 sH 21 2r 2

NO

3 Na* ( T9 Br) requires 479.978123). vmm: 2987, 2953, 2920, 1677, 1650, 1449, 1424, 1069 cm-'. kmax: 207 (88,250). 'Hn.m.r. 8 (CDC13): 7.4-7.29, 35 m, 5H, Ph; 6.72, s, 1 H, C4-H; 5.56, s, 1 H, -CHBr 2 ; 4.54, bs, 2H, CH 2 Ph; 3.0, WO 2004/016588 PCT/AU2003/001053 30 1 H, C-6 OH; 1.54-0.97, m, C-3 chain. 13 C n.m.r.6 (CDCI 3 ): 13,7, 22.3, 29.3, 42.6, 46.8, 91.5, 127.6,128.3, 128.7, 136.9.137.0,160.8, 170.6. 1-Benzyl-5-dibromomethyl-3-hexyl-5-hydroxy-4,5-dihydropyrrol-2-one 5 Method A A solution of 3-hexyl-5-dibromomethylene-2(5H)furanone (1.03 g: 3.32 mmol) in benzyl amine (2 ml) was stirred at room temperature for 0.5h. Dichloromethane (16 ml) was added to the reaction mixture and the 10 precipitated solid was filtered off. The filtrate was washed with aqueous hydrochloric acid (2M, 20 ml), dried over sodium sulfate and evaporated to yield a yellow viscous oil (0.36g). The crude product was chromatographed on silica using dichloromethanelethyl acetate (1:19) as the eluent and recrystallised from light petroleum to yield 1-benzyl-5-dibromomethyl-3-hegyl-5 15 hydroxy-1,6-dihydropyrrol-2-one (0.11g) as colourless needles m.p.105-10800. (Found (HRESMS) m/z 465.994011. CGH 23 Br 2

NO

2 Na* ( 79 Br) requires 465.998758. vmax. 3195, 2987, 2924, 2858, 1676, 1649, 1425, 1153, 1068, 968, 845, 730, 599 cf 1 . ?.ma: 205 (em 7740) nm. 1H n.m.r.6(CDCI): 7.39 7.26, m, 5H, Ph; 6.7, s, IH, C4-H; 5.6, s, IH, -CHBr 2 ; 4.54, d, J 15 Hz, 2H, 20 CH 2 Ph; 2.89-2.35, m, 2H, CH 2 ; 1.60-0.87, m, 13H, C3-chain. '3C n.m.r. a (CODCa): 14, 22.45, 25, 27, 28.8, 31.4, 42,5, 46.7, 91.5, 127.6, 128.5, 128.6, 136.6, 136.7, 144.0, 170.0. Method B 25 A mixture of 3-hexyl-5-dibromomethylene-2(5H)furanone (1.03 g: 3.32 mmol) and benzyl amine (2ml) in ethanol (5 ml) was stirred at room temperature for 2.5h, The crude product was isolated and purified as described above to yield 1-benzyl-5-dibromomethyl-3-hexyl-5-hydroxy-1,5-dihydropyrrol-2-one in (72%) yield. 30 1-Butyl-5-dibromomethyl-3-hexyl-5-hydroxy-1,5-dihydropyrrol-2-one n-Butylamine (0.272 g; 3.72 mmol) was added dropwise to a solution of 5 dibromomethylene-3-hexy-2(5H)furanone (0.314 g; 0.93 mmol) in CH 2 Cl 2 (10 ml). The mixture was stirred at room temperature for 5 hrs. Column 35 chromatography on silica with CH2C2 followed by CH 2 CI2/EtOAc (19:1) afforded the major product -as a colourless oil (0.20 g) which upon WO 2004/016588 PCT/AU2003/001053 31 recrystallisation from petrol gave 1-butyl-5-dibromomethyl-3-hexyl-5-hydroxy 1,5-dihydropyrrol-2-one (52%) as colourless needles, m.p. 85-86". (Found(HRESMS) m/z 432.013664. C 1

H

25 Br 2

NO

2 Na* (7Br) requires 432.014407). vmx 3230, 2957, 2859, 1672, 1650, 1458, 1422, 1375, 1270, 5 1233, 1139, 1079, 1023, 728, 666, 612 cm-1. Xma 259 (smax 945), 206 (9658) nm. 'H n.m.r. 8 (CDCil): 6.68, s, 1H, C4-H; 5.8, s, IH, CHBr 2 ; 3.45, m, 1H, N CHz; 3.10, m, IH, N-CH 2 ; 3.15, bs, OH; 3.20, m, 2H, -CH 2 ; 2.33-2.31, m, -CH 2 chain; 1.65-0.88, m, 14H, alkyl chain. 'C n.m.r. S (CDCIs): 13.8, 20, 22, 25, 27, 29, 30.75, 31, 39, 46.6, 91.4, 136,144.5,170. 10. N-(2-Hydroxyethyl)-3.butyl-5-dibrommethyl-5-hydroxy-2(5H)pyrolinone A solution of ethanolamine (1.13g; 18.5 mmol) in CH2Cl2 (5 ml) was added dropwise to an ice-cooled solution of 3-butyl-5-dibromomethylen-2(5H) furanone (1.0g; 9.25 mmol) in dichloromethane. The mixture was stirred at this 15 temperature for 1 hr and then at room temperature for further lh. The mixture was washed with waster (3 x 50 ml), dried over sodium sulfate and evaporated to yield a viscous oil (0.63g). The cuude product was chromatographed on silica using EtOAc as an eluent to yield N-(2-hydroxyethyl)-3-butyl-5 dibmmmethyl-5-hydroxy--2(5H)pyrolinone as an oil which solidified on keeping, 20 Crystallisation from (CH 2 Cl2/petrol) afforded the title compound as colurless needles, m.p. 68-70". vm: 3439, 3105, 3065, 2957, 2927, 1701, 1593, 1496, 1465, 1370, 1189, 1139, 1095, 1069, 1037, 945, B35, 763 cm- 1 . X max. 203 nm 'H nmr 8 (CDCIS): 0.93, t, 3H, CH 3 ; 1.25-1.45, m, 4H, CH 2 ; 2.35, m, 2H, CH 2 ; 3.10, rn, IH, NCH 2

CH

2 OH; 3.84, m, 2H, NCH 2

CH

2 OH; 4.10, m, 1H, 25 NCH 2

CH

2 OH; 5.43, bs, 1 H, OH:, 5.84, a, I H, CHBr 2 ; 6.78, a, 1 H, H4. 1C n.m.r. S (CDC13): 13.73, 22.2, 24.9, 29.3 41.3, 46.7, 61.3, 90.3, 137.5, 142.8, 171.1. 5-Dibromomethyl-3-hexyl-5-hydroxy-1,5-dihydropyrrol-2-one Liquid ammonia (5ml) was added to 5-dibromomethylene-3-hexyl 30 2(5H)furanone (0.50 g; 1,48 mmol) in a sealed tube held in a acetone/liquid nitrogen'bath. The reaction mixture was allowed to warm up gradually and kept at room temperature overnight. After gradual evaporation of ammonia the product was extracted with EtOAc (20 ml), washed with water, dried over Na 2

SO

4 , and evaporated to yield a solid (0.30g). The crude product was 35 purified on a silica column using first CH 2

CI

2 as the eluent followed by EtOAc/MeOH (4:1). The yellow band upon solvent removal and crystallisation WO 2004/016588 PCT/AU2003/001053 32 from petrol afforded a yellow crystalline solid (0.07g ) of 5-dibromomethyl-3 hexyl-5-hydroxy-1,5-dihydropyrrol-2-one, mp. 106-109'C. 1 H n.m.r. 6(CDC 3 ): 6.61; s, iH, C4-H; 0.26, s, 1H, -NH; 5.68, s, 1H, CHBr 2 ; 3.2, s, C5-OH; 2.28 2.23, m, -CH 2 , chain; 1.55-0.91, m, 11 H, chain, 13C n.m.r. 5 (CDCI 0 ): 13.9, 22, 5 25.5, 27, 29, 31.4, 129, 140, 142, 170.5. 4-bromo-5-hydroxy-5-hydroxyrnethyl-1,5-dihydropyrrol-2-one A suspension of 4-bromo-5-bromomethylene-2(5H)-furanone (1.30g, 5.16 mmol) in aqueous ammonia solution (20% wlw) was stirred at room 10 temperature for 1/2 h. During this time a complete dissolution of furanone was observed. The solution was evaporated to dryness in vacuo at ca 35-40 CC, and finally under high vacuum at room temperature. The resulting solid (1.70 g) was recrystallised from ethanol to yield 4-bromo-5-hydroxy-5 -hydroxymethyl 1,5-dihydropyrrol-2-one as colourless granules (1.0 g). m.p. 140-1426C 15 (decomp); vfl: 3259, 3100, 2949, 1667, 1592, 1419, 1370, 1152, 1076, 981, 872, 56:3 cm-1. Xm 220 (a m, 6077). 1 H n.m.r. 8 (CDC1): 8.09, s, -NH; 6.22, d, 2 Hz, H3; 4.97, t, 2 Hz, -CH 2 OH; 3.37, q, J 2 Hz, OH; 2.48, d, 2Hz, -CH 2 OH. 3C n,m.r. 6 (CDC3): 69.6, 84.3,132,8,152,3,174.1. 20 4-Bromo-3-hexyl-5-hydroxy-5-hydroxymethyl-1,5-dihydropyrrol-2-one A suspension of 4-bromo-3-hexyl-5-bromomethylene-2(5H)-furanone (0.50 g; 1.48 mmol) in aqueous ammonia solution (30 mIs; 28%) was stirred at room temperature for 2h, during. which time the solid completely dissolved. The solution was evaporated to dryness, and the residue extracted with 25 dichloromethane (26 ml). The organic phase was dried over anhydrous sodium sulfate and evaporated to yield a red viscous oil. Chromatography on silica using ethyl acetate followed by ethyl acetate/methanol (4:1). gave a solid which upon recrystallisation from light petroleum yielded 4-bromo-3-hexyl-5-hydroxy 5-hydroxymethyl-1,5-dihydropyrrol-2-one as colourless granules (0.16g: 36%), 30 m.p. 134-135 0. vma: 3304, 3256, 3185, 2961, 1670, 1589, 1441, 1350, 1136, 1069, 983 cm-1; am: 221 (Cma 6.678), 196 (3,415) nm. 5-Ethyl-5-hydroxy-4-methyl-1,S-dihydropyrrol-2-one A mixture of 5-ethylidene-4-methyl-2(5H)furanone (0,02 g; 0.162 mmol) in 35 aqueous ammonia solution ( 5 ml; 28% wiw) was stirred at room temperature for 1.5 h during which time all of the furanone dissolved. The solution was WO 2004/016588 PCT/AU2003/001053 33 evaporated in vacuo to dryness leaving 5-othyl-5-hydroxy-4-methyl-1,5 dihydropyrrol-2-one as a white solid (0.015g; 65%), m.p. 182-186*C. (Found(HRESMS) m/z 1(54.067448, C 6

H

1 lNO 2 Na' requires 160.06815). vmax: 3204, 2980, 1698, 1664, 1633.5, 1445, 1157, 1080, 1016, 983, 852, 769, 578. 5 Amax 207 (emax23,180 ) nm. 1 H n.m.r. 5(DMSO)-de) 7.97, s, 1H, -NH; 5.55, s, 1H, C3-H; 3.18, a, 1H, C5-OH; 1.79, s, 3H, C4-Me; 1.69-1,52, m, 2H, C5-2ja Me; 0.34, t, 3-1, Me. I3C n.m.r. 5 (DMSO-de): 7.9, 11.9, 29.2, 90.2, 121,7, 162, 171.6. 10 1-Benzyl-5-ethyl-5-hydroxy-4-methyl-1,5-dihydropyrrol-2-one A solution of 5-ethylidene-4-methyl-2(5H)furanone (0.124 g; 10 mmol) in benzylamine (0.128g; 12 mmol) was the left to stand at room temperature for 72 hrs, during which time a solid precipitated from the reaction. The reaction mixture was triturated with CH 2

CI

2 /petrol (1:3) and the precipitated solid was 15 filtered and recrystallised from EtOAc/petrol to yieldl-benzyl-5-ethyl-5-hydroxy 4-methyl-1,5-dihydropyrrol-2-one as colourless crystals m.p. 129-132" (70%). Vmax: 3247, 3082, 2964, 1669, 1638. 1496, 1353, 1101, 1053, 902, 708 cm 1 . max: 276 (smax 2,101), 237 (16,321), 243 (39,646) nm.iH n.m.r. 5 (CDCla): 7.4 7.24, m, SH, Ph; 5.79, s, 1H, C3-H; 4.46, 2 d, J 15 Hz, -CH 2 Ph; 3.81, bs, C6 20 OH; 1.92, s, C4-Me; 1.83-168, m, C5-CH 2 Me; 0.34, t, J 7.51 Hz, C5-CH2Me. 13C n.m.r. 5(CDC 3 ): 6.8, 11.85, 26, 41.9, 94, 122, 127, 128, 128.5, 138, 159, 170. 5-Aminomethyl-4-hoptyl-5-hydroxy-1,5-dihydropyrrol-2-one 25 5-Bromo-5-bromomethyl-4-heptyl-2(SH)furanone (0.50 g; 1.47 mmol) was dissolved in liquid ammonia in a sealed tube, and left to stand at room temperature for 72 h. Ammonia was allowed to gradually evaporated leaving behind a yellow crystalline solid. The solid was dissolved in hot ethylacetate (ca 25) ml to remove ammonium bromide and the clear filtrate was concentrated to 30 a small volume (ca 7 ml), to yield 5-aminomethyl-4-heptyl-5-hydroxy-1,5dihydropyrrol-2-one as a crystalline solid, (0.1 g; 34%); m.p. 176'C. vmax: 3370, 3248, 2956, 2926, 2855, 1674, 1627, 1469, 1350, 1227,1095, 1082, 954, 855 cmt. Anax: 208 (smax 6846), 291 (2754) nm. 1H n.m.r. 8 (CDCl): 7.53, s, -NH; 5.49, d, C5-CH 2 N]; 3.35, 3H, m, -C5-OH and -CH2NH 2 ; 2.23-2.0, m, CH2; 35 1.52-0,85, m, 13H, alkyl chain. 13C (CDCl 3 ): 14, 22.4, 26, 26.5, 28.9,29, 31.6, 66, 73, 78, 120.5, 167,5, 171.6.

WO 2004/016588 PCT/AU2003/001053 34 5-Bromomethyl-4-heptyl-5-hydroxy-1-phenyl-1,5-dihydropyrrol-2-one 5-Bromo-5-bromomethyl-4-heptyl-2(H)furanone (0.51g; 1.5 mmol) was dissolved in dry aniline (5 ml). The mixture soon solidified; it was allowed to 5 stand at room temperature for 24 h. Dichloromethane (25 mIs) was added to the mixture and the organic phase was washed with aqueous hydrochloric acid (2M) and brine. The dried (Na 2

SO

4 ) organic phase was evaporated to yield a yellow solid (0,50 g; 91%). Recrystallisation from light petroleum gave 5 bromomethyl-4-heptyl-5-hydroxy-1-phenyl-1,5-dihydropyrrol-2-one as 10 colourless needles, m.p. 152-154"C. vma: 3194, 2956, 1930, 2854, 1676, 1626, 1589, 1502, 1494, 1393, 1246, 1141, 836, 758, 692 cm. X,: 257( smn 3947), 202 (27,313) nm. IH n.m.r. 5 (CDCIs): 7.55-7.26, m, SH, Ph; 5.79, a, C3 H; 4.52, 1H, C5-OH; 3.39, d, 2H, C5-CH 2 Br 2.27-2.12, m, 2H, chain; 1.6-0.91, m, 13H, chain. 1 3 C n.m.r. 5(CDC 3 ): 14, 22.5, 25.6, 25.8, 20, 29.2, 30.4, 31.6, 15 121.6, 126, 126.7,130,134.6, 163, 170.5. 1-Benzyl-5-bromomethyl-4-heptyl-5-hydroxy-1,5-dihydropyrrol-2-one A mixture of 5-bromo-5-bromomethyl-4-heptyl-2(5H)furanone (0.51g, 1.5 mmol) in benzylamine (0.30g; 2.82 mmol) in ethanol (6 ml) was stirred at room 20 temperature for I h. Dichloromethane (25 ml) was added to the reaction mixture and the organic phase was washed with aqueous hydrochloric acid (2M) followed by brine. After drying over-sodium sulfate the solvent was evaporated in vacuo to yield 1-benzyl-5-bromomethyl-4-heptyl-5-hydroxy-1,5-dihydropyrrol 2-one as a viscous oil (0.52 g; 97%) which solidified on standing in the fridge. 25 Colourless needles from light petroleum; m.p. 9 4 -96 0 .vm,: 3270, 3062-, 3033, 2957, 2854, 1667, 1637, 1607, 1496, 1416, 1335, 1297, 1257, 1190, 1161, 1140, 1109, 1030, 950, 884, 865,769 oa'. ,a: 251 (esm 2391), 206 (18,974) nm. 1H n.m.r. S(CDCIs): 7.36-7.28, m,. 5H, Ph; 5.85, s, C3-H; 4.54 and 3,42, 2d, 2H each, C5-CH 2 Br and CH 2 Ph; 3.42, bs, IH, CS-OH, 2,31-2.15, m, 2H, ClH; 30 1.62-0.88, m, 13H, alkyl chain.'C, n.m~r. 5(CDCs): 14, 22.5, 25.5, 26, 29, 29.2, 30.87, 41.9, 122, 127, 128.3, 137.5, 163, 171. Synthesis of 3-alkyl-6-holomethylene-1,5-dihydropyrrol-2-one 35 3-Butyl-5-dibromomethylene-I-phenyl-1,5-dihydropyrrol-2-one WO 2004/016588 PCT/AU2003/001053 35 Phosphorus pentoxide was added to a solution of 3-butyl-5-dibromomethyl-5 hydroxy-1-phenyl-1,5-dihydropyrrol-2-one in chloroform. The resulting mixture .was stirred overnight at room temperature and passed through a pad of Celite. The crude product was chromatographed on silica and recrystallised from light 5 petroleum to yield 3-butyl-5-dibromomethylene-1-phonylA,5-dihydropyrrol-2 one as orange needles (78%), orange crystals from petrol. (Found(HRESMS) m/z 419.054622. C 16

H

17 Br 2 NONa+ ( 7 9Br) requIres 419.955896). Xma 202 (6max 8137), 195 (3850) nm. 1 H n.m.r. B(CDC 3 ): 7.22-7.17, m, 5H, Ph; 7.17, s, C4-H; 2.38-2.36, m, 2H, CH 2 ; 1.65-0.96, m, C3-chain. 1*C nm.r. 8 (CDC]): 13.6, 22.3, 10 25,2, 29,5, 128.3, 128.8,132.1, 139, 140,.171.8. 3-Hexyl-5-dibromomethylene-1-phenyl-1,5-dihydropyrrol-2-one 3-Hexyl-5-dibromomethylene-1-pheriyl-1,5-dihydropyrrol-2-one was prepared from 3-hexyl-5-dibromomethyl-5-hydroxy-l-phenyl-1,5dihydropyrrol-2-one as 15 described above. Y6Ilow granules from petrol. vmax: 3378, 2957, 2925, 2854, 1692, 1598, 1501, 1492, 1445, 1122, 1081, 743, 677 cm-1. Xmax: 309 (Smax 19,681) nm. 'H n.m.r. & (CDC)$): 7.4-7.17, n, 6H, Ph and H4; 2.37-2.34, m, 2H,'

CH

2 ; 1.57-0.89, m, 11 H, 03-chain. 20 1-Benzyl-3-butyl-5-dibromomothylone-1,5-dihydropyrrol-2-one 1 -Benzyl-3-butyl-6-dibromomethyl-5-hydroxy-1,5-dihydropyrrol-2-one was dehydrated with P 2 0 5 in CHCs at room temperature for 72 hrs. The mixture was filtered through celite and the solvent evaporated in vacuo to yield a viscous oil, which solidified on keeping in a refrigerator. The solid was 25 recrystallised from methanol/water to yield 1 -benzyl-3-butyl-5 dibromomethylene-1,5-dihydropyrrol-2-one as colourless plates, m.p. 56-58*C (91%). vmax: 2954, 1706, 1626, 1495, 1453, 1494, 1435, 1386, 1352, 1269, 1235, 1095, 765 cm-1. Xmx: 324 (emax 5285), 283 (16,201), 206 (10,972) nm.'H n.m.r. S (CODCs): 7.3-7.07, m, OH, Ph and H4; 5.26, s, CH 2 Ph, 2.4-2.36, m, 2H, 30 CH 2 ; 1.6-0.95, m,.C3-chain. 13 C n.m.r. 8 (CDCl 3 ): 13.7, 22, 25, 29.6, 44.2, 74.7, 89.25, 126, 127, 128, 132, 137.8, 138.8, 140, 172.1 1 -Benzyl-5-dibromomethylene-3-hexyl-1,5-dihydropyrrol-2-one This compound was prepared according to the procedure described for 1 35 benzyl-3-butyl-5-dibromomethylene-1,5-dlhydropyrrol-2-one. vmax: 2960, 2848, 2923, 2854, 1696, 1592, 1496, 1453, 1354, 1316, 977, 830, 738, 630 cm-. 1

H

WO 2004/016588 PCT/AU2003/001053 36 n.m.r. 8CDCia): 7.3-7.08, m, SH, Ph; 7.26, s, 1H, H4; 5,26, 2H, -C.HPh; 2.4 2.36, m, 2H, CH 2 ; 1.56-1.32, m, C3-chain. 1-Butyl-5-dibromomethylene-3-hexyl-1,5-dlhydropyrrol-2-one 5 This compound was prepared according to the procedure described for 1 benzyl-3-butyl-5-dibromomethylene-1,5-dilhydropyrrol-2-one. Yield (30%). vmr: 2956, 2928, 2858, 1705, 1586, 1452, 1360, 1335, 1194, 1135, 1058, 846, 829 741 cm'- ; Xmax: 290 (sa& 18,927), 20$ (9,409) nm. 1H n.m.r. 8(CDCl): 7.0, a, 1H, C4-H,; 3.99-3.93, t, 2H, -CH 2 N-; 2.3, t, -CH 2 - chain; 1.56-0.88, m, 16H, 10 chain. 12C n.m.r. 5 (CDC[$); 13.7, 14, 19.7, 22.4, 25, 27, 29, 31.4, 32.1, 40.6, 132,137,139,140.6,172.0. 5-Dibromomethylene-3-hexyl-1,5-dihydropyrrol-2-one This product was prepared by the dehydration of 5-dibromomethyl-3-hoxyl-5 15 hydroxy-1,5-dihydropyrrol-2-one as described above, rn.p. 103-105". 5-Ethylidene-4-methyl-1,5-dihydropyrrol-2-one 5-Ethyl-5-hydroxy-4-methyl-2(5H)pyrrolinone was dehydrated to 5-ethylidene-4 methyl-1,5-dihydropyrrol-2-one with P 2 0 6 in dichloromethane. vmax: 3158, 3093, 20 3036, 1670, 1495, 1434, 1397, 1381, 1356, 1279, 956, 867, 796, 639. Xma: 173 (gm. 33,010) nm. 'H n.m.r. S(CDC 3 ): 8.94, s, IH, -NH; 5.85, 1H, s, C3-H; 5.33, q, J 7.53 Hz, -=CHCH 3 ; 2.1, s, 3H, C4-Me; 1,92, d, J 7.53, 05-Me-CH=. 13 n.m.r. 5 (CDC13): 11.7, 12.9, 107, 120.5, 140, 148, 172.0. 25 1-Berzyl-5-ethylidene-4-methyl-1,5-dihydropyrrol-2-one I-Benzyl-5-ethylidene-4-methyl-1,5-dihydropyrrol-2-one was prepared by the dehydration of 1--benzyl-5-ethyl-5-hydroxy-4-methyl-1,5-dihydropyrrol-2-one as described before. Xmax: 206 (cmax213 2 ) nm. 30 5-Bromomethylene-4-hoptyl-1-phenyl-1,5-dihydropyrrol-2-one p-Toluenesulfonic acid (0.05g) was added to a solution of 5-bromomethyl-5 hydroxy-4-heptyl-1 -phenyl-1,5-dihydropyrrol-2-one in toluene. The mixture was refluxed for 1/2h and after cooling, washed with set. NaHCO 3 . The organic phase was dried over Na 2

SO

4 , and evaporated to yield an E,Z mixture of 5 35 bromomethylene-4-hoptyl-1-phonyl-1,5-dihydropyrrol-2-one as a colourless oil which solidified on Standing, m.p. 63-66', vmax: 3414, 3080, 2952, 2853, 1695, WO 2004/016588 PCT/AU2003/001053 37 1627, 1597, 1499, 1446, 1382, 1269, 1074, 907, 831 cm' Xma: 317 (smEx 22,834), 278 (43,910), 204 (46,925) nm; 1 H n.m.r. S(CDCIa): 7.4-7.24, m,,5H, Ph, 6.04 and 5.94, 2 s, 1H each, =CHBr and C3-H; 2.45, m, 2H, 0H 2 ; 1.65-0.9, m, IH, alkyl chain. 5 1 -Benzyl-5-bromomethylene-4-heptyl-1,5-dihydropyrrol-2-one 1 -Benzyl-5-bromomethyl-4-hepyl-5-hydroxy-I1,5-dihydropyrrol-2-one dehydrated smoothly to an E and Z mixture of 1-benzyl-5-bromomethylene-4 heptyl-1,5-dihydropyrrol-2-one upon heating a solution of 1-benzyl-5 10 bromomethyl-4-heptyl-5-hydroxy-1,5-dihydropyrrol-2-one with p-toluenesulfonic acid in toluene; m.p. 52-55*; vmar: 3096, 2927, 2857, 1704, 1630, 1387, 1357, 954, 856, 843 cm";mx: 319 (smq 10,220), 276 (19,433), 206 (17,040) nm; "H n.m,r. 8 (CDCIs): 7.29-7.15, m, 5H, Ph; 6.15 and 5.98, 2s, each 1H, =CHBr and C3-H; 2.39, m, 2H, CHz; 1.7-0.89, m, 13H, alkyl chain, 15 Reaction of N-(2-Hydroxyethyl)-3-butyl-2(5H)pyrolinone with acetic anhydride and Triethylamine N-(2-.Acetoxyethyl)-3-butyl-5-(dibromomethylene)-2(5H)pyrolinone 20 A mixture of N-(2-hydroxyethyl)-3-butyl-5-dibromomethyl-5-hydroxy 2(5H)pyrrolinone (0.2g, 0.54 mmol), acetic anhdride (0.44g; 4.4 mmol) and triethylamine (0.44g; 4.4 mmol) in dry dichloromethane (10 ml) was refluxed for 2 hr. After cooling to room temperature, the mixture was washed with aqueous sodium bicarbonate and brine. The organic phase was dried over anhydrous 25 sodium sulfate and evaporated to yield a viscous oil. 1 H n.m.r showed it to be a mixture of the mono- (88%) and di-acetate (12%) derivatives. Chromatography on silica using EtOAc/CH2C2 (5:1) as an fluent yielded 5-acetoxy-N-(2 acetoxyethyl)-3-butyl-5-dibromomethyl-2(5H)pyrrolinone (12%) as an oil. vmax: 2957, 2931, 2875, 1766, 1720, 1433, 1369, 1236, 1044, 1013, 855, 707 cm 1 . 30 kmax 217 (emax 1692), 268 (738) nm. 1H n.mr. 6(CDCla) 0.91 (t, 3H, CH 3 ); 1.38 (m, 2H, CH 2 ); 1.55 (m, 2H, CH 2 ); 2.05 and 2.10 (each s, 3H, OH 3 ); 2.34 (m, 2H,

CH

2 ); 3.61 (m, 1H, NCH 2

CH

2 ); 3.64- (m, I H, NCH 2

CH

2 ); 4.27 (m, 2H,

NCH

2 CHg); 6.26 (s, I-H, CHBrz); 6.S (s, 1H, H4). ' 3 C nm.r. 8(CDC 3 ) 13.7, 20.8, 21.2, 22.1, 24.9, 38.7, 44.1, 61.5, 94.1, 134.2,144.3,168.4, 170.6,171.0. 35 N-(2-acetoxyethyl)-3-butyl-5-dibromomethyl-5-hydroxy-2(5H)pyrrolinone (88%) H n.m.r. 8(0DCl 3 ) 0.93 (t, 3H, CH3); 1.38 (m, 2H, CH 2 ); 1.55 (m, 2H, CH 2

):

WO 2004/016588 PCT/AU2003/001053 38 2.21 (s, 3H, CHs), 2.34 (m, 2H, CH 2 ); 3.27 (M, 1H, NCH 2

CH

2 ); 4.04 (in, 21-1,

NCH

2

CH

2 ); 4.30 (, 1H, OH); 4.62 (m, IH, NCH 2

CH

2 ); 5.85 (s, IH, CHBr 2 ); 6.73 (s, 1H, H4). 13C n.m.r. 6(CDCIa) 13.7, 20.9, 22.2, 24.9, 29.3, 38.1. 45.9, 62.5, 91.0, 137.4,143.3, 170.4,171.9. 5 Dehydration of N-(2-acetoxyethyl)-3-butyl-5-dibromomethyl-5-hydroxy 2(5H)pyrrolinone with p-toluenesulfonic acid in toluene gave quantitatively N-(2 acetoxyethyl)-3-butyl-5-(dibromomethylene)-2(5H)pyrolinone. vma. 2957, 2929, 2870, 1744, 1705, 1441, 1368, 1229, 1177, 1161, 1130, 1035, 830. 764 cm". 10 'H n.m.r. a(CDC 3 ) 0.93 (t, 3H, CH 3 ); 1.36 (m, 2H, CH 2 ); 1.55 (m, 2H, C-12); 2.02 (s, 3H, CH 3 ); 2.32 (m, 2H, CH 2 ); 4.25-4.31 (m, 4H, NCH 2 CH2); 7.05 (s, 1H, 14). "C n.m.r. 5(CDCis) 13.7, 20.7, 22.3, 25.1, 29.5, 39.5. 62.3, 73.8, 132.4, 138.6, 140.3, 170.6, 172.0. 15 Hydrolysis of N-(2-acetoxyethyl)-3-butyl-2(H)pyrolinone N-(2-hydroxyethyl)-3-butyl-5-(dibromomethylene)2(5H)pymlinone A solution of potassium carbonate (19) in water (3 ml) was added dropwise to a solution of N-(2-acetoxyethyl)-3-butyl-5-(dibromomethylene)-2(5H)pyrolinone (0.2g, 0.51 mmol) in methanol (7ml). After stirring the mixture at room 20 temperature for 20 mins, methanol was removed in vacuo and the product extracted with ethylacotate (2 x 40 ml). The resulting extracts were combined, washed with brine, dried (Na 2

SO

4 ), and evaporated to yield an oil (0.18 g; 94.5%), which solidified upon standing in the fridge. Crystallisation from light petroleum gave N-(2-hydroxyethyl)-3-butyl-5-dibromomothylene 25 2(5H)pyrolinone as colurless granules, m.p. 48-50' max 3404, 2957, 2930, 2880, 1720, 1651, 1465, 1348, 1207, 1081, 1054, 1018, 936, 850, 716 cm-'. Vmax 206 (smc 25,389), 239 (6,758), 288 (2,186) nm. 'H n.m.r. 5(CDCh3) 0.91 (t, 3H, CH 3 ); 1,36 (m, 2H, CH 2 ); 1.54 (m, 2H, CH 2 ); 2.29 (m, 2H, CH 2 ); 3.83 (M, 2H, NCHzCH 2 ); 4.20 (m, 2H, NCH2CH 2 ); 7.02 (s, IH, H4). '3C n.m.r 8 (CDCI 3 ) 30 13.7, 22.3, 24.9, 29.5. 43.3, 46.8, 61.9, 74.5, 132.3, 138.6, 140.5, 173.2 Synthesis of 5-phenylaminomethylene-2(5H)furafnone 4-Bromo-5-phenylaminomethylene-2(H)furanone 35 A solution of 4-bromo-5-bromomethylene-2(5H)furanone (0.30 g: 0,79 mmol) was dissolved in aniline (5 mi), and left to stand at room temperature for 3 hrs, WO 2004/016588 PCT/AU2003/001053 39 during which time the mixture solidified. The solid was triturated with CH2C2/petml (1:1; v/v. 20 ml) and filtered. The resulting solid was dried and recrystallised from ethanol to yield 4-bromo-5-phenylaminomethylene 2(5H)furanone (0,24g, 49%) as yellow needles, m.p. 200-202*0 (decomp). 5 (Found (HRESMS) m/z 287.963053. CilHaBrNO2Na* ( 9 Br) requires m/z 287.963840). v mx 3233, 3127, 1730, 1697, 1595, 1498, 1276, 1195,932, 798, 756 cm~'. X., 397 nm (smx 50,686); 246 (12,769), 202 (15,961), 1 H n.m.r. S (CDCIa): 9.99, d, J 10.44 Hz, IH, -NHPh; 7.31-6.99, m, Ph; 7.07, d, J 10.44 Hz, IH, =CHNHPh; 6.16, s, 03-H. "3C n.m.r. 8 (CDCIa): 109.0, 116.2, 117.9; 129.9, 10 129.8, 133.9, 167.5. 5-Phenylaminomethylene-4-bromo-$-butyl-2(5H)-furanone A solution of 4-bromo-3-butyl-5-bromomethylene-2(5H)-furanone (0.25 g; 0.81 mmol) in aniline (0.082 g; 0.88 mmol) was left to stand at room temperature for 15 72 h. The mixture was diluted with CH 2 C1 2 (50 ml), washed with aqueous hydrochloric acid (2M) and dried over anhydrous sodium sulfate. The solvent was removed in vacuo leaving behind a brown viscous oil (0.29 g). The crude product was chromatographed on silica using dichloromethane to yield 5 phenylaminomethylene-4-bromo-3-butyl-2(5H)-furanone as a yellow solid. 'H 20 n.m.r S(CDCIs): 7.40-6.80, m, 5H, Ph; 6.70, d J 12.5 Hz, CH(NH)Ph; 2.42 2.40, m, 2H, CH 2 -chain; 1.7-1.2, m, 4H, CH2-chain; 0.95, t J 7.3 Hz, CH3. (Found (HRESMS) m/z 344.021931. CjsHuBrNO 2 Na* ('Br) requires m/z 344.021891). 25 4-Bromo-5-phenylaminomethylene-3-hexyl-2(5H)furanone A mixture of 4-bromo-3-hexyl-5-bromomethylene-2(5H)-furanone (0.50g; 1.48 mmol) and aniline (Iml) in ethanol (10 ml) was heated at reflux for 2 h. After cooling to room temperature, the mixture was evaporated to dryness and the residue extracted with dichloromethane (20 ml). The organic phase was 30 washed with aqueous hydrochloric acid (2M) and dried over anhydrous sodium sulfate- Removal of the solvent and recrystallisation of the solid from light petroleum gave 3-bromo-5-phenyaminomethylene-3-hxyl-2(5H)furanone (0.50g; 100%) as yellow needles; rn.p. 147-148 0 C. vmax: 3242, 3161, 3109, 29212, 2842, 1728, 1683, 1600, 1581, 1500, 1350, 1236, 1055, 960, 750, 673 36 cm-1. Xmax: 394 (ema 26,287) , 247 (8002) nm. 'H n.m.r.6(CDCl): 7.32-6.97, m, 5H, Ph: 6.98, s, -NHPh; 8.73, s, C5 =CH-NHPh; 2.4, t, -CH2-chain; 1.61- WO 2004/016588 PCT/AU2003/001053 40 0.88, m, 11H, chain. ' 3 C n.mr.S(CDCla): 14.0, 22.0, 24.8, 27.6, 29, 31.0, 103.0, 113,0, 115.0, 122.5, 124.0, 129.5, 129.5, 131.0. 139.9, 167.0. 5-Phenylaminomethylene-4-hepty-2(H)furanone 5 5-Phenylaminomethyl-4-heptyl-5-hydroxy-2(5H)pyrrolinone 5-Bromomethylene-4-heptyl-2(5H)furanone (0.44g; 1.61 m~nmol) was dissolved in dry aniline (2 ml) and left to stand at room temperature for 24 h. Dichloromethane (10 ml) was added to the reaction mixture and the organic phase was washed with aqueous hydrochloric (2M) followed by water. After 10 drying over sodium sulfate, the solvent was evaporated off to yield a pale yellow solid. The crude product was chromatographed on silica column using dichloromethane followed by CH 2 Cl 2 /EtOAc (2:1; v:v) as the eluents to yield 5 phenylaminomethyl-4-hepty-5-hydroxy-2(5H)furanone (0.43g; 88%) as a pale yellow solid, m.p, 172-174"C. vmax: 3192, 3037, 2957, 2931, 2953, 1676, 1643, 15 1598, 1502, 1493, 1336, 1250, 1160, 923, 757 cm-f. Xmax 278 (smax 7188), 203 (8609) nm. "H n.m.r, 6 (CDCla): 7.53-7.25, 6H, Ph and -NHPh; 5.73, s, 1 H,C3 H; 5.11,s. 1H, C5-OH; 3.37, d, 2H, -CH 2 NHPh; 22-2.0, m, 2H, -CH 2 -chain; 1.25-0.91, m, 13H, chain. '3C n.m.r.8 (CDCl): 14.0, 22.6, 25.5, 25.3, 25,8, 29.0, 29.2, 30.4, 31.6, 93.4, 121.8,126.0, 126.7, 129.0,134.6, 163.0,170.4. 20 5-Phonylaminomethylene-4-heptyl-2(5H)furanone A sample of 5-phenylaminomethyi-4-hoptyl-5-hydroxy-2(5H)furanone was dehydrated using p-toluenesulfonio acid in toluene to yield an E and Z mixture of 5-phenylaminomethylene-4-heptyl-2(5H)furanone as a colourless oil which 25 solidified on standing in the fridge. vma: 3088(-NH), 3052, 2927, 2856, 1712, 1626, 1598, 1499, 1454, 1264, 1195, 759, 699 cm'1 X m 292 (ernax 7623), 204 (4728) nm. 'H n.m.r. 8(CDCla): 7.4-7.25, SH, Ph and -NHPh; 6.19-6.1, d, 1H, C3-H; 5.93-6.0, 1H, d, 05- =Cj!NHPh; 1.68-0.90, 15H, chain. 13 C n.m.r.8(CDCIs): 14, 22.5, 26.4, 28.1, 28.9, 29.0, 29,2, 30.0, 31.6, 31.7, 88.7 30 93.0, 118.5, 122.6, 127.8, 128.2, 128.4, 128.6, 128.7, 129.3, 129.5, 134.0, 135.0, 142.0,143.0, 152,0,153.2,168.0. 4-Methyl-5-(1-phenylamino-ethylidene)-H-furan-2-on A solution of 5-ethylidene-4-methyl-2(5H)furanone (0,31 g; 2.5 mmol) in aniline 35 (0.26 g; 2.75 mmol) was left to stand at r.t. for 3 hrs, during which time a solid precipitated from the reaction. The reaction mixture was triturated with WO 2004/016588 PCT/AU2003/001053 41

CH

2 C1 2 /petrol (1:1) and the solid filtered and recrystallised from EtOAc/petrol to yield 5-ethyl-5-hydroxy-4-methyl-1-phenyl-1,5-dihydropyrrol-2-one as colorless crystals (70% ); m.p. 97-100*. vmax: 3287, 1884, 1704, 1530, 1496, 1353, 1101, 1053, 971, 897, 790, 756, 688, 638 cm". Xmex: 273 (Ema 15,256), 5 226 (16,382), 243 (39,646) nm. 1 H n.m.r. 8 (DMSO-de) 10.11, s, 1H, -NH; 7.57, d, 2H, ArH; 7.30, t, 3H, ArH; 6.08, s, 1H, 03-H; 3.27, s, 3H, CH 3 ; 1.96, s, 3H,

OH

3 . 1C n.m.r. 5 (CDICl):. 20.9, 119.6, 123.7, 123.8, 129.1, 139.3, 142.9, 163.1, 170.4. 10 Synthesis of 5-arylamino and arylalkylamino-2(5H)furanones 4-bromo-5-benzylamino-5-bromomethyl-2(SH)furanone Benzyl amine (0.10 g; 0.95 mmol) was added with stirring to an Ice-cooled solution of the 4-bromo-5-(bromomethylene)-2(5H)furanone (0.16 g; 0.84 mmol) in dichloromethane (10 ml). The mixture was stirred at room temperature 15 for 2.5 h, washed with aqueous hydrochloric acid solution (1 M, 10 ml), dried (Na 2 SO4), and evaporated to yield a brown oil. The crude product was chromatographed on silica using dichloromethane/ethyl acetate (1:4; v:v) as the eluent and recrystallised from dichloromethanellight petroleum to yield 4 bromo-5-benzylamino-6-bromomethyl-2(5H)furanone as orange flakes. m.p. 20 137-139 * (Found (HRESMS) m/z 381.901032. C 1 2 H11Br 2

NO

2 Na* (91Br) requires 381.904812). vmax 3256, 1674, 1655, 1431, 1413, 1352, 1072, 1054, 699 cm 1 . Xmar 257 (Em* 2879) nm. 1H n.m.r. 8.(CDC): 7.38,d, J 11 Hz, IH,

NHCH

2 -; 7,37-7.29, m, Ph; 6.38, s, 03-H, 4.65, d, J 15 Hz, 1H, -CH 2 Br; 4.44, d, J 15 Hz, 1 H, -CH 2 Br and 3.58-3.44, dd, J 15 Hz, CH 2 Ph. ' C n.m.r.5 (CDCI): 25 30.6, 42.8, 53.0, 92.2, 128,0, 128.2,128.9,137.0, 142.0, 168.0. 4-Bromo-5-benzylamino-5-bromomethyl-3-hexyl-2(5H)furanone Benzylamine (0.32g; 2.96 mmol) was added with stirring to a solution of 4 bromo-3-hexyl-5-bromomethylene-2(5H)-furanone (0.50 g; 1.48 mmol) in 30 ethanol (6 ml). The mixture was stirred at room temperature for 1 h and evaporated to dryness. The residue was extracted with dichloromethane (20 ml) and the dichloromethane extract washed with aqueous hydrochloric acid (2M). After drying over anhydrous sodium sulfate, removal of the solvent gave a thick viscous oil, Column chromatography on silica gel using dichloromethane 35 followed by dichloromethane/ethyl acetate (19:1) as the eluents afforded 4 bromo-5-benzylamino-5-bromomethyl-3-hexyl-2(5H)furanone (0.36g; 56%) as a WO 2004/016588 PCT/AU2003/001053 42 viscous oil; m.p. 7 2

-

7 5".vma 32 77, 3065, 3032, 2954, 2928, 2857,1681, 1496, 1411, 1355, 1151, 1064, 1104, 1030, 988, 907, 726, 698. Xmax: 277 (cmax: 39,542), 205 (38,034) nm. 'H n.m.r. 6(CDC 3 ): 7.4-7.26, m, Ph; 4.8-4.74, d, and 4.4, d, C5-CHZBr; 3.6 and 3.53, d, C5-NHCHPh; 2.42-2.33. m. -CH 2 , chain; 5 1.56-0,85, n, IIH, chain. 1 3 C n.m.r. 8(CDCI 3 ): 22.0, 25.0. 27,0, 28.8, 31.4, 42.9, 46.7, 49.5, 90.6, 91.6, 127.0, 128.0, 129.0, 136.0, 136.7, 136.9, 138.0, 140.0, 144.0, 168.0, 170.6. 5-Phenylamino-3,5-dimethyl-2(5H)-furanone 10 Method A A solution of 3,5-dimethyl-5-hydroxy-2(5H)-furanone (0.13g; 1.02 mmol) in dry aniline (2 mIs) was stirred at room temperature for 1 hr. A thin layer chromatography analysis of the mixture (developing solvent; CH 2

C

2 ) indicated completion of the reaction as indicated by the disappearance of the starting 15 material. Dichloromethane (25 mIs) were added to the mixture and the solution washed with aqueous hydrochloric acid solution (1 M; 3 x 20 mIs). The organic layer was dried over anhydrous sodium sulfate and evaporated to yield 5 phenylamino-3,5-dimethyl-2(5H)-furanone as a viscous oil which solidified on keeping (0.013 g), A sample was recrystallised from dichloromethane/light 20 petroleum to yield the furanone as colourless needles v max 3360, 3088, 2965, 1770, 1601, 1570, 1536, 1294, 1246, 1132, 1040, 999, 867, 756, 697 crf'. AmZ 236 nim. 'H n.m.r. S (CDCla): 7.18, t, 2H Ph; 6.90, t, IH, ArH, 6.89, s, 1H, H4, 6.83, d, 2H, ArI; 4.24, bs, 1h, OH; 1,91, s, 3H, C3-Me;1.75, s, 3H, -Me. ' 3 C n.m.r. S (CDCl): 10.4, 26.2, 95.5, 121.3, 122.7, 128.9, 132.4, 133.5, 141.9, 25 148.8, 156.5,171.9. Method B A mixture of 3,5-dimethyl-5-hydroxy-2(5H)-furanone (0.13g; 1,02 mmol) and aniline (2 ml) in dry toluene (10 ml) was refluxed for 5h. The mixture was cooled and evaporated. The residue was dissolved in dichloromethane (25 ml) 30 and the solution washed with aqueous hydrochloric acid solution (1M; 3 x 20 ml). The organic layer was dried over anhydrous sodium sulfate and evaporated to yield 5-phenylamino-3,5-dimethyl-2(5H)-furanone as a viscous oil. The crude product was chromatographed on silica using dichloromethane/ethyl acetate (19:1) as the eluent (Yield 58.0%). 35 5-Phenylamino-6-mthyl-4-phen~yl-2(5H)-furanlone WO 2004/016588 PCT/AU2003/001053 43 A mixture of 5-hydroxy-5-methyl-4-phenyl-2(5H)-furanone (0.13g; 1.02 mmol) and aniline (2 ml) in dry toluene (10 ml) was refluxed for Sh. The mixture was cooled and washed with aqueous hydrochloric acid solution (2M; 3 x 2Q| mis). The organic layer was dried over anhydrous sodium sulfate arid evaporated to 5 yield a viscous oil, The crude product was chromatographed on silica using dichloromethane/ethyl acetate (19:1) as the eluent and recrystallised; from dichloromethane/light petroleum to yield 5-phenylamino-5-methyl-4-phenyl 2(5H)-furanone (0.10 g; 72%) as colourless flakes. m.p. 158-16 0 "Cvmax: 3356, 1724, 1608, 1534, 1501, 1320, 1291, 1376, 1030, 943, 846. 770, 7568 691, 10 639. %ma, 276(amt. 7056), 238 (5615)nm. 'IH n.m.r. 8(CDCI): 7,94-7.44, m, 5H, Ph; 7.14-6.82, m, 5H,Ph; 6.4, s, 1H, CS-H; 4,53, bs, 1H, -NE-Ph; 1.9, s, C5-Me, "C n.mr.5 (CDC 3 ): 117.3,120,122,5,128, 129,5,131, 142,159,160,170. 5-Benzylaminomethyl-3-methyl-2(SH)furanone 15 Phosphorus pentoxide (2g) was added to a solution of 3,5-dimethyl-5-hydroxy 2(5H)-furanone (0.50g; 2.15 mmol) in dichloromethane (25 ml). The mixture was refluxed for 2h -and the cooled solution was filtered through celite and evaporated in vacuo to yield 3-methyl-5-methylene-2(5H)-furanone as a colourless oil (0.37 g; 82%). The methylene product was dissolved in 20 dichloromethane (5 ml) and benzylamine (1.15 g; 10.8 mmol) was added at room temperature. The mixture was stirred at room temperature for 1 h. After evaporation of the solvent the crude product was chromatographed on silica using dichloromethanellight petroleum as the elent to yield 5 benzylaminomethyl-3-methyl2(5H)furanone as a colourless oil (0.12 g; 26%). 25 vmk: 2929, 2854, 1788, 1747, 1715, 1618, 1456, 1388, 1373, 845, 712 crrF'. ma: 308 nm (sm. 1462), 260 (5243).'H n.m.r.8 (CDCla): 7.29-7.21, m, 6H, Ph and -NHCH2Ph; 6.65, s, 1H, C 4 rH; 4.82,s, 2H, -CH2Ph; 4.70, d, -CHNHPh; 2.02, s, C 3 -Me. 13C n.m.r. 5 (CDCIs): 10.8, 25.9, 42.9, 95.0, 105.3, 126.9,127.1, 128.5, 131.2, 134.2, 137.2, 148.4. 30 Side-chain functionalization 3.(1'-Bromohexyl)-1-butyl-5-dibromomethylene-1,5-dihydropyrrol-2-one N-Bromosuccinimide (02g; 1.79 mmol) was added to a solution of 1-butyl-5 dibromomethyl-3-hexyl-1,5-dihydropyrrol-2-one (0.64 g; 1.63 mmol) containing 35 few crystals of benzoyl peroxide in CC14 (25 ml). The mixture was heated at reflux under a 100 watt fluorescent lamp for 24 h. The reaction mixture was WO 2004/016588 PCT/AU2003/001053 44 cooled and passed through a pad of Celite. The filtrate was evaporated to dryness to yield a brown oil which was chromatographed on a silica column using CH 2 Cl 2 petrol (1:) as the eluent to yield 3-(1'-Bromohexyl)-1-Butyl-5 dibromomethylene-1,5-dihydropyrrol-2-one (0.46 g; 59.8%) as a pale yellow oil. 5 (Found): HRESMS): m/z 483.849575. CjsH14NB40a) requires 483.851758. vmax: 3017,2950, 1709, 1598, 1593,1480, 1215, 1194, 845, 695, 668 cm'. Amax: 326 (Emax 4070) nm. 'H n.m.r. S(CDCIa): 7.28, s, IH, H4; 4.78, t, -CHBr-chain; 219 2.11,m, -CHrchain; 1.53-0.98, m, alkyl chain. 1 3 C n.m.r. S(CDC): 13.12, 20.95, 26.8, 39, 43.9, 79.5. 95, 128.6, 128.9, 129.4, 133.8, 134.5, 138.2, 139.6, 10 168.7 3-(1'-Bromobutyl)-1-butyl-5-dibromomethylene-1,5-dihydropyrrol-2-one N-Bromosuccinimide (0.32g; 1.79 mmol) was added to a solution of N-butyl-5 dlbromomethyl-3-hexyl-2(5H)pyrrolinone (0.64 g; 1.63 mmof) containing few 15 crystals of benzoyl peroxide (0.01g) in CC1 4 (25 ml). The mixture was heated at reflux under a 100 watt fluorescent lamp for 24 h. The reaction mixture was cooled and passed through a pad of Celite. The filtrate was evaporated to dryness to yield a semi-solid (0.69g) which was chromatographed on a silica column using CH 2 Cl 2 /petrol (1:1) as the eluent to yield 3-(1'-bromobutyl)-1 20 butyl-5-dibromomethylene-1,5-dihydropyrrol-2-one (0.46g; 60%) as a pale yellow oil. Xmax: 2930, 2957, 2871, 1705, 1584, 1357, 1192, 1055, 902, 769, 651 cm'; %ma: 325 (Smax 11,669), 202 (9,879) nm. 1iH n,rm,r, 5(CDC 3 ): 7.29, d, 1H, C4-H; 4.78, t, IH, C3-CHBr- chain; 3.98,t, 2H, >NC..H-; 2.10, m, -CH chain; 1.58-0.93, m, 12H, chain; 13C n.m.r. 6(CDCl$): 13.78, 13.96, 19,8, 22.4, 25 27.4, 31, 32.2, 37, 41, 43.9, 98.7, 132.5, 138.2, 140, 169.0, 3.(1 '-Bromobutyl)-5-dibromomethylene- N-phonyl-1,5-dihydropyrrol-2-one N-Bromosuccinimide (0.056g; 0.316 mmol) was added to a solution of 5 dibromomethyl-3-butyl-1-phenyl-1,5-dihydropyrrol-2-one (0.64 g; 1.63 mmol) 30 containing few crystals of benzoyl peroxide (0.01g) in 0C1 4 (10 ml). The mixture was heated at reflux under a 100 watt fluorescent lamp for 24 h. The reaction mixture was cooled and passed through a pad of Celite. The filtrate was evaporated to dryness to yield a brown oil (0.17g) which was chromatographed on a silica column using CH 2 C1 2 /petrol (1:1) as the eluent to 35 yield 3-(l'-bromobutyl)-5-dibromomethylene-1-phenyl-1,5-dihydropyrmi-2-one as a pale viscous oil (0.10g). (Found:HRESMS) m/z: 483.849575, WO 2004/016588 PCT/AU2003/001053 45

C

15 H1 4 BrNONa* (Br") requires 483.851758. vm.: 3017, 2950, 1709, 1598, 1593, 1480, 1215, 1194, 1122, 845, 756, 695, 668 Grrdf.Amc: 326 (sma 3,896), 202 (5,566) nm. 1H n.m.r. SCDC 3 ): 7.45, m, 6H, Ph and C3-H; 4.86, t, 1H, C3 CHBr- chain; 2.16, m, -CH 2 chain; 1.53-0.98, m, 5H, alkyl chain. 13 C 5 n.m.r.$(CDC 3 ): 13, 21, 26.8, 39, 43, 79.5, 95, 107, 128.6, 129.4, 134, 134.5, 138, 139.6,169. N-Phenyl-3-(1'hydroxybutyl)-5-dibromomethylene-2(5H)pyrrolinonS A solution of N-Phenyl-3-(1-bromobutyl)-5-dibromomethylene-2(5H)pyrolinone 10 (0.0194 mol) in DMSO (60 ml) containing few drops of water was left to stand aside at room temperature for 6 days. The mixture was diluted with dichloromethane (100 ml) and the resulting solution washed with brine (3 x 120 ml). The organic phase was dried over anhydrous sodium sulfate and evaporated to yield a pale yellow oil.(9.08g). The crude product was purified on 15 a silica column using initially dichloromethane followed by dichloromethane/ethyl acetate to afford N-phenyl-5-dibrommethylene-3(1' hydroxybutyl)-2(5H)pyrolinone (6.83g; 88%) as pale yellow needles (dichloromethane/light petroleum), m.p. 93-95*. vmax: 3439, 3065, 2957, 2927, 2871, 1701, 1496, 1455, 1370, 1189, 1139, 1095, 1069, 1038, 945, 835, 763, 20 697 cm'. Xmax 203 (sm. 11,968), 313 (10,707) nm. 1H n.m.r. 6 (CDCla) 0.97 (t, 3H, CH); 1.39 (m, 2H, CH 2 ); 1.79 (m, 2H, CH 2 ); 4.61, m, 1H, HI'; 2.71, bs, IH, OH; 7.23 (s, IH, H4); 7.21-7.44 (m, 5H, ArH). 13 C n.m.r. 6(CDCIs) 13.7, 14, 18.5, 37.8, 67.4, 128.6, 128.9,128.3,129.5, 131.5, 134.5, 139.8, 170.8. 25 N-Phenyl-3-(1'-acetoxybutyl)--dibromomethylne-2(5H)pyrrolinone A solution of acetyl chloride (0.25 ml, 3.2 mmol) in dichloromethane (3 ml) was added dropwise to an ice-cooled solution of N-Phenyl-3-(1-hydroxybutyl)-5 dibromomethylene-2(5H)pyrolinone (0.1g, 0.25mmol) in dichloromethane (10 ml) containing triethylamine (0.25 ml, 2.47 mmol). The mixture was stirred in ice 30 for 1h and then at room temperature overnight. The mixture was poured Into saturated sodium bicarbonate solution (20 ml) and extracted with dichloromethane (3 x 30 ml). The organic phase washed with water (3 x 20 ml), dried over anhydrous sodium sulfate -and evaporated to yield a pale yellow oil (0.11g). The crude product was purified on a silica column using 35 dichloromethane/ethyl acetate (15:1) to afford N-phenyl-5-dibrommethylene 3(1 '-acetoxybutyl)-2(5H)pyrolinone (0.1g) as a viscous oil vn. 2960, 2931, WO 2004/016588 PCT/AU2003/001053 46 2873, 1753,1712, 1592, 1494, 1454, 1362, 1262, 1232, 1193, 1148, 1096, 1060, 836, 753, 698 cm- . %m 281, 321 nm. 'H n.m.r. 8 (CDC3) 0.96 (t, 3H, Cl-3); 1.38 (m, 2H, CH 2 ); 1.91 (m, 2H, CH 2 ); 5.72, m, 1H, HI'; 7.21 (s, 1H, H4); 7.21-7-40 (m, 5H, ArH). 5 Malonic acid mono-[1-(5-dibromomethylene-2-oxo-1-phenyl-2,5-dihydro I H-pyrrol-3-yI)-butyl] ester A solution of N-Phenyl-3-(1-hydroxybutyl)-54dibromomethylene-2(5H)pyrolinone (0.5g, 1.25 mmol) in dichloromethane (15 ml) containing triethylamine (0.25 ml, 10 2.47 mmol) was added drop wise over a period of 9h to an Ice-cooled solution of malonyl dichloride (0.36g, 25 mmol) in dichloromethane (10 ml). The mixture was allowed to stand at room temperature overnight, washed with brine (3 x 20 ml), dried over anhydrous sodium sulfate and evaporated to yield a brown viscous oil. The crude product was purified on a silica column using ethyl 15 acetate/methanol (1:4) to afford malonic acid mono-[1-(5-dibromomethylene-2 oxo-1 -phenyl-2,5-dihydro-1 H-pyrrol-3-yl)-butyl] ester (0.48g) as a viscous oil vm: 3470, 2959, 2873,1709, 1594, 1494, 1454, 1362, 1245, 1194, 1148, 1096, 1060, 835, 753, 698 cm". 'rm 271, 303 nm, 1H n.m.r. 8 (CDCls) 0.96 (t, 3H CH 3 ); 1.46 (m, 2H, CH 2 ); 1.88 (m, 2H, CH 2 ); 3.01, m, 2H, 20 COCH 2 CO; 5.67, m, 1H, HI'; 7.23, m, 2H, ArH; 7.43 (m. 3H, ArH); 7,59, S, 1 H, H4. Preparation of 2(5H)pyrrolinone-polystyrene copolymer A mixture of styrene (7.13g), 3-(1'-Bromobutyl)-5-dibromomethylene- N-phenyl 25 1,5-dihydropyrrol-2-one (0.37g) and AIBN (0.026g) was degassed for 112h bby purging with argon gas and then heated at 65"C for 3h, After completion of polymerisation, the mixture was poured into methanol and precipitated polymer was filtered, washed extensively with methanol and dried to yield the copolymer (2.38g, 32%). 30 Surface attachment of 2(5H)pyrrolinone A layer of malonic acid mono-[l-(5-dibromomethylene-2-oxo-1-phenyl-2,5 dihydro-1H-pyrrof-3-yl)-butyl] ester was covalently attached to a surface containing amino groups by immersing the surface in a solution of 35 2(5H)pyrrolinone (2mg/ml) in acetonitrilelwater containing NHS, N-hydroxy succinimide. The mixture was shaken for 10 minutes and EDC, N-(3- WO 2004/016588 PCT/AU2003/001053 47 dimethylaminopropyl)-N'-ethylacrbodiimide hydrochloride, was added to the solution to give a final concentration of (2mg/mI). After shaking the solution for 24h, the surface was taken out of the solution and washed thoroughly with water and dried. The surface analysis was performed using XPS and 5 %bromine was used a s a marker for determining the extent of covalent attachment. Biological activity of furanones 10 Effect of furanones as inhibitor of AHL-mediated quorum sensing, Al-2 pathway and growth of S. aureus Methods Gfp assay Briefly, the Gfp assay determines the relative effectiveness of a compound as 15 an inhibitor of AHL mediated quorum sensing. The assay is dependent on a bacterial strain that carries a reporter plasmid. This plasmid expresses the green fluorescent protein (Gfp) in the presence of AHLs (2). The presence of a competitor will prevent AHL mediated Gfp expression of the reporter. The assay can be used to generate an index of inhibition for each compound. The 20 results here, presented as good, moderate, or poor, are based on the index of each of the compounds as an inhibitor of AHL mediated quorum sensing using this bioassay. Attachment/Biofilm formation 25 The ability of furanones to inhibit biofilm formation or attachment has been determined using a modification of the 96 well microtitre method described by Christensen et al. ((1)). The furanones are added to the wells of the microplate and the solvent is allowed to evaporate, leaving the furanones adsorbed onto the plate. Then a suspension of the monitor bacterium, Pseudomonas 30 aeruginosa, is added to each well and incubated for 24h. Following incubation, the wells are rinsed to remove unattached or loosely adhered cells. The attached wells are fixed with formaldehyde and subsequently stained with cyrstal violet. Following extensive washing to remove the crystal violet, the wells are read at 600 nm. The attachment/biofilm formation in the presence of 35 the furanones is calculated as the percentage of the controls, which are not exposed to the furanones.

WO 2004/016588 PCT/AU2003/001053 48 Two-Component signal transduction Assays Taz-I Assay The Taz-assay carried out according to the method of Jin and Inouye 5 (1993) with the following alterations. E coli RU1012 (pYTO301) were grown overnight In M9 medium at 37"C supplemented with 100 ug/mI ampicillin and 50 ug/ml kanamycin. This overnight culture was then used to inoculate 50 ml M9 medium in side-arm flasks which were then incubated at 37C and shaken at 180 rpm. The OD10 of the growing cultures was monitored regularly and 10 when the OD 61 m- 0.2 the cultures were placed on ice. Aspartate was added to side-arm -flasks to give a final concentration of 3 mM (aspartate stock solution made up in M9 salts). The test compound or mixtures of compounds were dissolved in ethanol 15 and added to cultures to give the required final concentrations. Negative controls were prepared with equal volumes of ethanol. Cultures were then placed in a 37"C incubator and shaken for 4 hours (ODe1 approximately 0.7) before being removed and put on ice. Samples were then removed for eta galactosidase assays carried out according to the method of Miller (1972). 20 V. harveyi bioassay for the detection of AI-2 activity The V. harveyi bioassay was performed as described previously (Surette and Bassler, 1998). The V. harveyi reporter strain BB170 was grown for 16 hours at 30*C with shaking in AR medium. Cells were diluted 1:5,000 into 30*C 25 prewarmed AB medium and 90 ul of the diluted suspension was added to wells containing supernatant. Furanones were added to the wells to achieve the desired final concentrations and the final volume in each well was adjusted with sterile medium to 100 ul. Ten ul of V. harveyi BB152 (Al-1-, Al-2+) supernatant was used as a positive control and 10 ul of E coli DH5c supernatant or sterile 30 media was used as a'negative control. This strain of E coi has previously been shown to harbor a mutation in the AI-2. synthase gene, ygaG, which results in a truncated protein with no Al-2 activity (Surette et al. 1998). The microtiter plates were incubated at 30*C with shaking at 175 rpm. Hourly determinations of the total luminescence were quantified using the 35 chemiluminescent setting on a Wallac (Gaithersburg, MD) model 1450 Microbeta Plus liquid scintillation counter. The V. harveyi cell density was WO 2004/016588 PCT/AU2003/001053 49 monitored by the use of a microplate reader (Bio-Rad, Hercules, CA). Activity is reported as the percentage of activity obtained from V. harveyi BB152 cell free supernatant. While the absolute values of luminescence varied considerably between experiments, the pattern of results obtained was 5 reproducible. Growth of Staphylococcus aureus Material and methods The growth of Staphylococcus aureus against furanones was tested in sidearm 10 flasks. One percent of an overnight culture was added to the growth media, Nutrient Broth, containing furanones at the concentrations 1-50 pg/ml. The bacteria were incubated at 37C and growth was measured at 610 nm. The results of these experiments are summarised in the table 1. 15 Table 1. Summary of activity for lactam and other N containing analogues as inhibitor of AHL-mediated- quorum sensing, AI-2 pathway and growth of S. aureus. Compound AHL Al-2 S. aureus (%.of control) (% of control) 26%, 50 ug/ml NE at 50 ug/ml 57%, 1Oug/ml 80%, Sug/mI +++ 21 %, 50 ug/ml NE at 50 ug/mI NE at 50 ug/mi 0% growth at ug/ml for 10hrs D h H_________________

______________________

WO 2004/016588 PCT/AU2003/001053 50 39% 102% O (100 ugfml) (25 pg/ml) Br .

2% 104% (50 pg/ml) (60 pg/mI) 61 % No effect (20 pg/mI) SOpg/ml +-+ -50% No effect (100 pg/ml) 50pg/ml ++ No effect sopg/ml CH 4+++ +++r Noeffect (50pg/mI) CF, - 4+No effe ct o 4NBr (b0pgfmI) Rr 0 WO 2004/016588 PCT/AU2003/001053 51 ++ No effect (50 pg/mi) P2 Other si gnal regulated phenotypes 40-50% reduction 30% 40% reduction in in swimming reduction In cholera toxin motility in V. attachment production by V. cholerae and V. by V cho/erae vulnificus vulnificus 40-50% reduction 50-80% reduction 30% 40% reduction In in swimming in protease reduction in cholera toxin motility in V. production by V. attachment production by V cholerae and V. vtlnificus by V. cholerae vulnificus vulnificus 40-50% reduction 30% 40% reduction in in swimming reduction in cholera toxin motility in V. attachment production by V. cholerae and V. by V. cholerae vuinifious vulnflcus Christensen, G. D., W. A. Simpson, J. J. Younger, L. M. Baddour, F. F. Barrett, D. M. Melton, and E. H. Beaohey. 1985. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J. Clin. Microbiol. 22(6):996 10 1006. Andersen, J. B., C. Sternberg, L. K. Poulsen, S. P. Bjom, M. Givskov, and S. Molin. 1998. New unstable variants of green fluorescent protein for studies of WO 2004/016588 PCT/AU2003/001053 52 transient gene expression in bacteria. Appi. Environ. Microbiol. 64(6):2240 2246. Jin, T,, and M. Inouye. 1993. Ligand binding to the receptor domain regulates 5 the ratio of kinase to phosphatase activities of the signalling domain of the hybrid Escherichia coli transmembrane receptor, Tazi. J, Mol. Biol. 232: 484 49 Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor 10 Laboratory, Cold Spring Harbor,. N.Y. Surette, M. G., and B. L. Bassler. 1998. Quorum sensing in Escherichia coli and Salmonella typhimurium. Proc. Nati. Acad. Soi., USA 95:7046-7050. 15 Surette, M. G., M. B. Miller, and B. L. Bassler. 1999. Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harvey: a new family of genes responsible for autoinducer production. Proc, Nati, Acad. Sci., USA 96:1639-1644. Any description of prior art documents herein is not to be taken as an 20 admission that the documents form part of the common general knowledge of the relevant art. 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 25 as broadly described, The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Any description of prior art documents herein is not to be taken as an admission that the documents form part of the common general knowledge of 30 the relevant art.

Claims (117)

1. A method for the preparation of compound of formula I OR4 I OH 5 lR wherein R 1 and R.

2 are independently selected from the group H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted substituted or unsubstituted oxoalkyl, substituted or 10 unsubstituted substituted or unsubstituted alkenyl, substituted or unsubstituted substituted or unsubstituted aryl or arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydophilic or fluorophilic R 3 and R 4 are independently selected from the group H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, 15 substituted or unsubstituted substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl; Rs is selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylsily, substituted or unsubstituted substituted or unsubstituted 20 oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic, or forms part of an amino acid, or 25 is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a surface, the method comprising reacting a compound of formula I WO 2004/016588 PCT/AU2003/001053 .54 R1 R2 0 o R wherein R1 and R 2 are independently H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or 5 unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic; R 3 and P4 are independently H, halogen, substituted or unsubstituted 10 alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or arylalkyl; and R Is hydroxy, halogen; and "----" represents a single bond, in which case R is absent, or a double bond, provided that at least one of R1, R 2 , R 3 and R 4 is halogen, with a compound of formula R 5 NH 2 15 wherein RS is selected from the group consisting of H, substituted or unsubstituted alkyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, 20 hydrophilic or fluorophilic, or forms part of an amino acid, or is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a surface. 25 2. A method according to claim 1, wherein, at least one of Ri, R2, R3 and R4 is halogen,

3. A method according to claim 1 or 2, wherein R is a residue of a naturally occurring compound. 30

4. A method according to claim 1 or claim 2, wherein R5 is a biomolecule.

5. A method according to claim 4, where Re is a coenzyme or cofactor. WO 2004/016588 PCT/AU2003/001053 55

6. A method according to any one of claims 1 to 5, wherein R 5 is an oligomer or a polymer

7. A method according to claim 6, wherein the oligomer or polymer is a 5 biomolecule.

8. A method according to claim 7, wherein Rs is a peptide or polyamide.

9. A method according to any one of claims I to 6, wherein R 8 is a protein 10 residue.

10.A method according to claim 9, where R8 is an enzyme or a receptor.

11.A method according to any one of claims I to 7, wherein R. is an oligomer 15 or polymer comprising nucleic acid residues.

12.A method according to claim 11, wherein R is a polynucleotide.

13.A method according to claim 12, wherein the polynucleotide is DNA or RNA. 20

14. A method according to claim 1, wherein R6 is a surface or a substrate with which the nitrogen atom of compound 11 is associated.

15.A method according to claim 14, wherein the association is chemical 25 bonding.

16.A method according to claim 15, wherein the association is covalent bonding. 30

17. A method according to any one of claim I or claim 14, wherein the surface or substrate may be biological or synthetic.

18. A compound of formula ll: 0 R4 RS 35 I wherein R 1 and R 2 are independently H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or WO 2004/016588 PCT/AU2003/001053 56 unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic; R 3 and R4 are independently H, halogen, substituted or unsubstituted 5 alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl; R 5 is selected from the group consisting of H, hydroxy, substituted or .unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or 10 unsubstituted aryl, substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic, or forms part of an amino acid, or is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a 15 surface.

19. A compound according to claim 18, wherein Rs is a D- or L- nucleoside.

20. A compound according to claim 18, wherein R5 is an oligomer or a polymer. 20

21,A compound according to claim 18, wherein R5 is dendrimer.

22. A compound according to claim 18, wherein R5 is a substrate. 25

23.A compound, according to claim 18, wherein R. is a surface.

24.A compound according to claim 18, wherein at least one of R 1 , R 2 , R 3 and R 4 is halogen. 30

25.A method for preparing a compound of formula IlI, the method comprising dehydration a compound of formula II according to claim 18 or 19: R5 Ill WO 2004/016588 PCT/AU2003/001053 57 wherein R 1 and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, 5 substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic; R 3 and R 4 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or 10 unsubstituted aryl or arylalkyl; and R is selected from the group consisting of H, substituted or unsubstituted alkyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or 15 branched chain, hydrophilic or fluorophilic, or forms part of an amino acid, or is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a surface. 20 25. A method according to claim 25, wherein at least one of R 1 , R 2 , R 3 and R4 in formula IlIl is halogen.

26.A method according to claim 24 or 25, wherein the dehydration is carried out in the presence of a dehydrating agent. 25

27.A method according to claim 26, wherein the dehydrating agent is selected from the group consisting of phosphorus pentoxide, silica gel, molecular sieves, alumina, acidic resins and polymers, phosphorus oxychloride, acetic anhydride, N,N'-dicyclohexylcarbodiimide (DCC), trifluoroacetic acid, 30 sulfuric acid, trifluoroacetic anhydride, and trifiuorosulfonic acid anhydride (triflic anhydride).

28.A compound of formula Ill: WO 2004/016588 PCT/AU2003/001053 58 R 111 wherein R 1 and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, 5 substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic; R 3 and R4 are independently selected from H, halogen, substituted or 10 unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or arylalkyl; and R 5 is selected from the group consisting of H, substituted or unsubstituted alkyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted oxcalkyl, substituted or unsubstituted alkenyt, substituted or 15 unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic, or forms part of an amino acid, or is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a 20 surface.

29.A compound according to claim 28, wherein at least one of R 1 , R 2 , R 3 and R 4 is halogen. 25

30.A compound according to claim 28 or 29, selected from the group consisting of: WO 2004/016588 PCT/AU2003/001053 59 Er Br .,7 Br H OH B5 Br Sr r Br Br Sr NN rr Br Br -Br ~ HH NN N Sr of~ Br

31 .A method for the preparation of a compound of formula IV Ri R2 R4 O H 5 IV wherein R, and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted arkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, 10 substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic; WO 2004/016588 PCT/AU2003/001053 60 R 3 and R 4 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or arylalkyl; and Ra is selected from the group consisting of H, substituted or 5 unsubstituted alkyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic, or 10 forms part of an amino acid, or is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a surface, X is 0 or NR 0 , where R 5 be independently selected from R 1 , the method comprising reacting a compound of formula I as defined in 15 claim 1, wherein R 3 is a hydrogen and "---" represents a double bond.

32. A method according to claim 32, wherein at least one of R 1 , R 2 , R 3 and R 4 is halogen. 20

33.A method according to claim 31 or 32, wherein Re is H.

34.A compound of formula IV R4 R2 R3 R4 0 C3 H 0 0 x IVR5 25 wherein R 1 and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, WO 2004/016588 PCT/AU2003/001053 61 optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic; R 3 and R 4 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or 5 unsubstituted aryl or arylalkyl; R 5 is selected from the group consisting of H, substituted or unsubstituted alkyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally 10 interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic, or forms part of an amino acid, or is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a surface; and 15 X is O or NRe, where R6 be independently selected from R 1 .

35.A compound according to claim 30, wherein at least one of RI, R 2 , Rs and R 1 is halogen. 20

36.A compound according to claim 34 or 35, wherein X is NR 0 .

37.A compound according to claim 36, wherein R 6 is an optionally substituted arylalkyl. 25

38.A compound according to claim 34 selected from the group consisting of: WO 2004/016588 PCT/AU2003/001053 62 Br Br Br 7 Br Br Br o N H 0 N H 0 N H Ph H3CH OH 3 0% \OHCH o NH "NH NH

39.A compound of formula V: R1 R2 O R3 0 )7 5 X'R wherein R 1 and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, 10 substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilio; R 3 is selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl or 15 arylalkyl; X is 0 or NR 8 , where R 6 Is as defined above; and R 5 is selected from the group consisting of H, substituted or unsubstituted alkyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstitutod alkenyl, substituted or WO 2004/016588 PCT/AU2003/001053 63 unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilic, or forms part of an amino acid, or 5 is a nucleoside, an oligomer, a polymer, a dendrimer, a substrate or a surface.

40.A compound of formula (VI): z N I -R4 VI 10 wherein R 1 and R 2 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched 15 chain, hydrophillc or fluorophllc; R 3 and R4 are independently selected from H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or arylalkyl; Rs is selected from the group consisting of H, substituted or unsubstituted alkyl, hydroxy, -substituted or unsubstituted alkoxy, substituted or 20 unsubstituted oxoalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl or substituted or unsubstituted arylalkyl, optionally interrupted by one or more hetero atoms, straight chain or branched chain, hydrophilic or fluorophilio, or forms part of an amino acid, or 26 is a nucleoside,. an oligomer, a polymer, a dendrimer, a substrate or a surface; and Z is selected from the group R 2 , halogen, OC(O)Ra, =O, amine azide, thlol, R2, mercaptoaryl, arylalkoxy, mercaptoarylalkyl, SC(O)R 2 , -OS(O)AR 2 , NHC(O)R 2 , =NR 2 or NHR 2 . 30 WO 2004/016588 PCT/AU2003/001053 64

41.An oligomer or a polymer formed by oligomerising or ,polymerising a compound of formula II - VI directly or with one or more other monomers.

42.An oligomer or a polymer according to claim 41, wherein the one or more 5 other monomer is selected from the group acrylate ester such as substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, or substituted substituted or unsubstituted aryl acrylates or methacrylates, crotonates, substituted or unsubstituted acrylonitriles, vinyl alcohols or acetates, styrene and siloxanes. 10

43.A surface coating or polymer having incorporated therein a compound according to any one of the preceding claims.

44. Use of a compound according to any one of claims as antimicrobial and/or 15 antifouling agent.

45. Use of a compound according to any one of claims 18, 28, 34, 38, 39 or 40 in a medical, scientific and/or biological application(s). 20

46. A composition comprising at least one compound according to any one of claims 18, 28, 34, 38, 39 or 40 and a carrier or diluent.

47. A composition according to claim 46, where the carrier or diluent is a liquid 25

48.A composition according to claim 46, where the composition is in the form of a solution or suspension of at least one of the compounds

49. A composition according to claim 47 or 48, wherein the liquid is an aqueous solvent or a non-aqueous solvent. 30

50.A composition of claim 46, wherein the solvent is a one or more organic solvent(s).

51.A composition according to claim 47, wherein the liquid is an ionic liquid. 35 WO 2004/016588 PCT/AU2003/001053 65

52.A composition according to any one of claims 46 to 51, in an aerosol or powder formulation.

53.A composition according to any one of claims 46 to 52, including organic or 5 inorganic polymeric substances.

54.A composition according to claim 53, wherein the compound is admixed with a polymer or bound to, or adsorbed on to, a polymer. 10

55.A composition according to any one of claims 46 to 54 formulated as a disinfectant or cleaning formulation.

56.A composition according to any one of claim 46 to 55 in the form of a powder, solutions, suspension, dispersion, emulsion or gel. 15

57.A composition according to any one of claims 46 to 54 in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent andlor excipient. 20

58.A composition according to claim 57, wherein the composition is a form suitable for parenteral or non-parenteral administration.

59. A composition according to claim 58 formulated for topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, 25 epidural, ophthalmic, or oral administration.

60. A composition according to claim 57 formulated for administration by infusion or bolus injection, absorption through epithelial or mucocutaneous linings and may be administered together with other biologically active 30 agents.

61.A composition according to claim 57 formulated for use in an inhaler or nebulizer. WO 2004/016588 PCT/AU2003/001053 66

62.A method of treating an infection in a human or animal subject the method comprising administration to the subject of an effective amount a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40. 5

63.A method according to claim 62 wherein treatment is therapeutic or prophylactic,

64.A method or treating an Infection or condition in a subject that is characterised by biofilm formation comprising administering a compound 10 according to any one of claims 18, 28, 34, 38, 39 or 40.

65.A method according to claim 64, wherein the condition is cystic fibrosis.

66.A method according to claim 64, wherein the .condition is dental caries, 15 periodontitis, otitis media, muscular skeletal infections, necrotizing fascitis, biliary tract infection, osteomyelitis, bacterial prostatitis , native valve endocarditis, cystic fibrosis pneumonia, meloidosis.

67. A method according to claim 64, wherein the condition is nosocomial 20 infection.

68.A method according to claim 67, wherein the infection is IOU pneumonia-or an infection associated with sutures, exit sites, arteriovenous sites, scleral buckles, contact lenses, urinary catheter cystitis, peritoneal dialysis (CAPD) 25 peritonitis, IUDs, endotracheal tubes, Hickman catheters, central venous catheters, mechanical heart valves, vascular grafts, biliary stent blockage, and orthopaedic devices, penile prostheses.

69.A method according to claim 64, wherein the infection is selected from the 30 group a skin infection, burn infection and wound infection.

70.A method according to any one to claims 64 to 69, wherein the Is an immunocompromised individuals- WO 2004/016588 PCT/AU2003/001053 67

71.A method for treating or preventing biofilm formation on a surface, the method comprising contacting the surface with a compound according to any one of claims 18, 28, 34, 38, 39 or 40. 5

72.A method according to claim 71, wherein the surface is a non-blological surface.

73.A method according to claim 71, wherein the surface is a natural surface. 10

74.A method according to claim 71, wherein the surface is a surface of a plant, seed, wood, fibre or hair.

75.A method according to claim 71, wherein the surface is a biological surface. 15

76.A method according to claim 75, wherein the surface is a surface of a tissue, membrane or skin.

77.A method according to claim 71, wherein the surface is a hard surface. 20

78.A method according to claim 77, wherein the surface is formed of a metal, an organic and inorganic polymer, a natural or synthetic elastomer, board, glass, wood, paper, concrete, rock, marble, gypsum and ceramic materials which optionally are coated. 25

79.A method according to claim 71, wherein the surface is a coating.

80.A method according to claim 79, where in the coating is an enamel, vanish or paint. 30

81.A method according to claim 71, wherein the surface is a soft surface.

82.A method according to claim 81, wherein the surface is a surface of a fibre.

83.A method according to claim 82, wherein the fibre is in the form of a yam, a 35 textile, a vegetable fibre, rock wool. WO 2004/016588 PCT/AU2003/001053 68

84. A method according to claim 71, wherein the surface is a porous surfaces.

85.A method according to claim 71, wherein the surface Is a surface of process equipment or components of cooling equipment. 5.

86.A method according to claim 85, wherein the process equipment is-for a cooling tower, a water treatment plant, a dairy processing plant, food processing plant, a chemical process plant or a pharmaceutical process plant or a component thereof. 10

87.A method according to claim 86, wherein the surface is that of a filter.

S8, A Imethod according to claim 87, wherein the filter is a membrane filter. 15

89.A method according to claim 71, wherein the surface is a surface of toilet bowls, bathtubs, drains, highchairs, counter tops, vegetables, meat processing rooms, butcher shops, food preparation areas, air ducts, air conditioners, carpets, paper or woven product treatment, nappies(diapers), personal hygiene products, and washing machines. 20

90.'A method according to claim 71, wherein the surface is an industrial surface.

91.A method according to claim 71, wherein the surface is a medical surface. 25

92.A method according to claim 71, wherein the surface is a hospital, veterinary hospital surface, mortuary surface and funeral parlour surface.

93.A dentifrice, a mouthwash or a composition for the treatment of dental 30 caries comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40.

94.A composition for treatment of acne comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40. 36

95.A composition for cleaning and disinfecting contact lenses comprising a WO 2004/016588 PCT/AU2003/001053 69 compound in accordance with anyone of claims 18, 28, 34,38,39 or 40.

96.A medical device incorporating a compound of any one of claims comprising a compound in accordance with any one of claims 18, 28, 34, 5 38, 39 or 40 on at least one surface thereof.

97.An implant device having at least one surface associated with compound according to any one of claims comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40. 10

98.An implant device according to claim 97, wherein the device is an artificial heart valve or hip joint, an indwelling catheter, pacemaker, surgical pin and the like. 15

99.An antifouling composition comprising an effective amount of a compound of any one of claims comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40.

100. An antifouling coating composition, the composition comprising an 20 effective amount of a compound according to any one of claims comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40.

101. An shellfish or aquaculture apparatus having at least one surface associated with a compound of any one of claims comprising a compound 25 in accordance with any one of claims 18, 28, 34, 38, 39 or 40.

102. A biofilm removing or inhibiting composition comprising an amount of a of a compound according to any one of comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40 and a vehicle or 30 carrier, wherein the amount of the mixture is effective to remove or disrupt, a bacterial biofilm or inhibit normal biofilm formation.

103. A composition according to claim 102, additionally comprising a surfactant selected from group consisting of anionic, nonionic, amphoteric, 35 biological surfactants and mixtures thereof. WO 2004/016588 PCT/AU2003/001053 70

104. A composition of claim 103 further comprising a compound selected from the group consisting oftbiocides, fungicides, antibiotics, and mixtures thereof. 5

105. A method of removing biofilm from a surface comprising the step of administering a cleaning-effective amount of a compound of claim comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40 to a biofilm-containing surface. 10

106. A method of preventing bofilm formation on a surface comprising the step of administering an effective amount of the compound according any one of claims comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40 to a surface, wherein the amount is effective to prevent biofilm formation. 15

107, A method of claim 106, wherein the surface is. a hard, rigid surface.

108. A method of clairn106, wherein the surface is selected from the group consisting of a drainpipe, glaze ceramic, porcelain, glass, metal, wood, 20 chrome, plastic, vinyl, and formica.

109. A method of claim 106, wherein the surface is a soft, flexible surface.

110. A method of claim106, wherein the surface is selected from the group 25 consisting of shower curtains or liners, upholstery, laundry, and carpeting.

111. A method of claim 106, wherein the biofilm is produced by a bacteria of the class Pseudomonas. 30

112. The method of claim106, wherein the bacteria is of the species Pseudomonas aeuroginosa.

113. A method of claim1O, wherein the biofilm is produced by an organism selected from the group consisting of bacteria, algae, fungi and protozoa. 35

114. A dentifrice comprising an effective amount of a compound of any one of WO 2004/016588 PCT/AU2003/001053 71 claims comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40, wherein the amount is effective to either prevent or remove biofilm formation. 5

115. A mouthwash comprising an effective amount of a compound of any one of claims comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40, wherein the amount is effective to either prevent or remove biofilm formation. 10

116. An optical lens, wherein at least a part of a surface of the lens is associated with a compound according to any one of claims comprising a compound in accordance with any one of claims 18, 28, 34, 38, 39 or 40.

117. An optical lens according to claim 116, wherein the lens in a contact 15 lens.