WO2014183164A1 - Dihydropyrrolones and their use as antimicrobial agents - Google Patents

Abstract

The present invention relates generally to antimicrobial dihydropyrrolone compounds, methods for their use and methods for preparing surfaces to which the dihydropyrrolone compounds are attached.

Description

Dihydropyrrolones and their use as antimicrobial agents

Field of the invention

The present invention relates generally to antimicrobial dihydropyrrolone compounds, methods tor their use and methods for preparing surfaces to which the dihydropyrrolone compounds are attached.

Background of the Invention

The use of biomaterial implants and medical devices has increased dramatically over the last decade. Whilst providing an improvement in the quality of life of patients, device- related infections remain a significant concern. Infection is the most common reason for failure of implanted devices and infections associated with implanted devices account for over 50% of all nosocomial infections.

The number of device-related infections in the United States is approximately one million per year, with the economic cost of device-related infec tions est imated to be in the order of about $US3 billion. With ever increasing life expectancies and advances in medical technology an increase in the number of implant operations is expected in the future together with a concomitant increase in device-related infections. There is therefore a clear need to develop medical devices having improved antimicrobial properties.

To this end there is increasing interest in the development of antimicrobial surface coatings. Current strategies tor combating infections include the incorporation of antibiotics, silver or silver-based nanoparticles and quaternary ammonium compounds into biomaterials. These types of coatings have however met with limited success and suffer from a number of disadvantages. For example, the effectiveness of an antibiotic-releasing coating is strongly dependent on the rate and manner in which the drug is released, and therefore presents a major challenge for the administration of an accurate antibiotic dose. Development of microbial resistance to the antibiotic also presents a significant problem. Th antibacterial properties of silver coatings are strongly dependent on the rate of silver release, and at certain concentrations silver has been shown to exhibit undesirable cytotoxic effects on fibroblasts and endothelial cells. Moreover, silver-resistant bacteria have been reported. Quaternary' ammonium compounds have been shown to be toxic to human cells thereby rendering such compounds undesirable as surface coatings for biomaterials. Against this background, the present inventors have developed a class of dihydropyrrolone (DHP) compounds which possess remarkable antimicrobial activity when attached to surfaces utilising Click chemistry.

Summary of the Invention

In a first aspect the present invention provides a compound of the formula ( I), or a salt thereof:

wherein,

Ri is selected from the group consisting of: hydrogen, halogen, heteroaryl, aryl, Ci-C_o alkyl, CN-Cao alkenyl and C Cy, alkynyl, wherein the heteroaryl, aryl, CJ-CJG alkyl.. the Cj- C20 alkenyl and the C2-C20 alkynyl groups are optionally substituted with one or more of the following substituents: hydroxy, halogen or OCi-C_f, alkyl;

i is selected from the group consisting of: hydrogen, halogen, alkyl, heteroaryl, and aryl, wherein the alkyl, heteroaryl, aryl groups are optionally substituted with one or more of the following substituents: halogen, hydroxy, OCi-Q. alkyl, amino, alkenyl or alkynyl;

and R₄ are independently selected from the group consisting of: hydrogen and halogen;

R5 is selected from the group consisting of: C1-C20 alkylene, C2-C20 alkenylene, C2-C20 alkynylene -(CH₂}_w~Pb-(CH;)_y- -(CH₂) _v-Ph-J-(CH₂)y- or -(CH₂)_W-Ph-(CH₂)_V-J- wherein w is 0, 1 , 2, 3, 4, 5 or 6, y is 0, 1 , 2, 3, 4, 5 or 6, J is O, S or NH; and

X is a functional group suitable for attachment to a solid surface.

In an embodiment X may lie a functional group suitable for covalent attachment to a solid surface.

In another embodiment X may be a functional group that is capable of undergoing a click reaction with a functional group attached to a solid surface to which it is desired to attach the compound of formula (I).

In another embodiment the solid surface may be a functionalised solid surface. In an embodiment X may be selected from the group consisting of azido. ethvnyl, epoxide,

» ₌©_₀Θ

aziridme, amino, thiol or ?

Accordingly in an embodiment the compound of formula (I), or salt thereof, may be represented by:

In one embodiment, Ri is selected from the group consisting of: hydrogen, halogen, Cj-Go alky! and G- « alkenyl, wherein the G«G.> atkyl and the C₂-G« alkenyl groups ar optionally substituted with one or more of the following substituents: hydroxy or halogen;

In another embodiment, | is selected from the group consisting of: hydrogen and G-G alkyl, wherein the G-G. alky) group is optionally substituted with one or two of the following substituents: hydroxy or halogen.

In a further embodiment. R\ is selected from the group consisting of: hydrogen and Ci-Go alkyl.

In a further embodiment, R| is selected from the group consisting of: hydrogen and G-G alkyl.

In one embodiment, R₂ is selected from the group consisting of: hydrogen, halogen and phenyl, wherein the phenyl group is optionally substituted with one or more of the following substituents: halogen, hydroxy or OG-G, alkyl.

In another embodiment, R₂ is selected from the group consisting of: hydrogen, halogen and phenyl, wherein the phenyl ('Ph') group is optionally substituted with between one and three halogens.

In a further embodiment, R₂ is selected from the group consisting of: hydrogen, halogen and phenyl wherein the phenyl group is optionally substituted with one or two fluoro groups.

In yet another embodiment, R₂ is selected from the group consistiug of: hydrogen, bromo and phenyl, wherein the phenyl group is optionally substituted with a single fluoro group.

In one embodiment, R3 and R are independently selected from the group consisting of: hydrogen, iodo, chloro and bromo.

In another embodiment, Rj and R4 are independently selected from the group consisting of: hydrogen and bromo.

In one embodiment, R₅ is G-G, alkylene, -<C¾ Ph-(CH2)y or -<CH₂)_w-Ph-0-(CH₂)_v~ wherein w is 0, I or 2 and y is 0, 1 or 2.

In another embodiment, Rj is G-G, alkylene or -(CH₂)_w-Ph-(CH2)_y or -(CH₂)_w-Ph-0- (CH₂)y-, wherein w is 0 or I and y is 0 or 1.

In yet another embodiment, R5 is G-G alkylene, ~Ph- or ~Ph-OCH₂- or -Ph~OCH₂CH₂~ In still a frirther emjbod^ent, Rs is G -G* aJkyle»e¾ -Ph- or -Plv-QCHr-_* In a second aspect the present invention provides an antimicrobial composition comprising a compound according to the first, aspect.

The composition may further comprise one or more additional antimicrobial agents.

In a third aspect the present invention provides a method for eliminating or inhibiting the growth of one or more microorganisms, or the colonisation of an environment by the microorganisms, the method comprising contacting the one or more microorganisms, or an environment inhabited by the microorganisms, with an effective amount of a compound of the first aspect, or a composition of the second aspect.

In a fourth aspect the present invention provides a method for inhibiting the adherence of one or more microorganisms to a surface, the method comprising attaching to the surface at least one compound according to the first aspect.

The surface may be a solid surface. The solid surface may be colonised by, or be capable of being colonised by, the microorganisms.

The one or more microorganisms may be selected from bacteria, fungi, yeast and protozoa.

The bacteria may be Gram-negative or Gram-positive bacteria. In particular embodiments the bacteria are Staphylococcus spp., such as S. aureus, or Pseudomonas spp., such as P. aeruginosa.

In a fifth aspect the present invention provides a method for preventing the occurrence of microbial infection on or around the sur face of a medical device inserted into a patient, or at or near the site of insert ion of the medical device., the method comprising attaching to a surface of the device, or coating a surface of the device with, at least one compound of the first aspect.

In a sixth aspect the present invention provides a method for preparing a device having at least one surface, the method comprising reacting the at least one surface with at least one compound as defined in the first aspect.

The device may be a medical device.

In a seventh aspect the present invention provides a method for modifying a surface, the method comprising reacting the surface with at least one compound as defined in the first aspect.

The surface may be a solid surface.

In an eighth aspect the present invention provides a surface, wherei at least one compound of the first aspect is attached to the surface.

In an ninth aspect the present invention provides a device, wherein at least one compound of the first aspect is attached to a surface of the device.

In a tenth aspect the present invention provides a compound array comprising:

a) a functional ised solid surface; and

b) at least one compound of formula (la),

wherein Ri„ R;, R5, R,s and R5 are as defined in the first aspect, and Y is a residue of a bivalent functional group attached to the functionaltsed solid surface.

Brief Description of the Drawings

Embodiments of the invention are described herein, by way of non-limiting example only, with reference to the following drawings.

Figure 1 shows XPS high resolution N 1 s spectra of A) CVD AzPTS and B) dip-coat AzPTS functionalised surfaces. An azide characteristic double-peak at 404 eV and 401 eV was observed for both samples.

Figure 2 shows XPS high resolution N Is spectra of alkyne-F8-phenyl-DHP attachment on A) CVD AzPTS and B) dip-coat AzPTS surfaces- Disappearance or reduction of the characteristic azide peak at 404 eV was observed, giving rise to an amide peak at 401 eV and a 1,2,3-triazole peak at 400 eV.

Figure 3 shows XPS high resolution C 1 s spectra of A) CVD alkyne-PTS functionalised surface and B) the subsecjuent azide-F8-phenyl-DHP immobilised surface. Appearance of C-Br peak at 288 eV indicated successful attachment of the DHP.

Figure 4 shows Two-dimensional (2-D) fluorescence microscopy images of/', aeruginosa adhered on A) untreated control (blank); B) Azide-terminated surface; C) Azide surface coupled with alkyne-F8-DHP D) Azide surface coupled with alkyne-DHPl ; E) Alkyne- terminated surface; and F) Azide-F8-DHP coupled to alkyne surface. Live bacteria stain green and bacteria with damaged membranes stain red. Images were taken under x200 magnification. Scale bar = 100 u Figure 5 shows Two-dimensional (2-D) fluorescence microscopy images of S. aureus adhered on A) untreated control (blank); B) Azide-terminated surface; C) Azide surface coupled with alkyne-FS-DHP: D) Azide surface coupled with alkyne-DMPl ; E) Alkyne- terminated surface; and F) Azide-F8-DHP coupled to alkyne surface. Live bacteria stain green and bacteria with damaged membranes stain red. Images were taken under x200 magnification. Scale bar = 100 μηι.

Figure 6 shows image analysis of the percentage of surfaces co%¾red by (A) P. aeruginosa and (B) $. aureus stained using live/dead fluorescent stain. Areas covered by live (green- staining) bacteria are proportionately represented by green and the areas covered by dead (red-staining) bacteria are proportionately represented by the red area in the bars: the combination of green and red bars represents the total areas. Error bars = standard derivations; ""indicates p < 0.001 compared to untreated control; Δ indicates p < 0.05 when compared between DHPs.

Detailed Description

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

In the context of this specification, the term "about" is understood to refer to a range of numbers or values that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but. not the exclusion of any other integer or step or group of integers or steps.

In the context of this specificat ion, the term "microorganism" is used in its broadest sense and is therefore not limited in scope to prokaryotic organisms. Rather, the term "microorganism" includes within its scope bacteria, archaea, yeast, fungi, protozoa and algae.

In the context of this specification, the term "antimicrobial composition" is understood to mean a composition that is capable of eliminating, preventing, inhibiting or retarding the growth of at least one microorganism, the colonisation of an environment by the microorganism, or the adherence to a surface by the microorganism. In the context of this specification, the term "environment inhabited by" in the context of microorganisms encompasses any environment (solid, fluid or gaseous, including surfaces of cells, tissues, organs or inanimate objects) that is inhabited or colonised by, or is capable of being inhabited or colonised by, microorganisms*

As used herein the term "antimicrobial agent" refers to any agent that, alone or in combination with another agent such as an antibiotic, is capable of killing or inhibiting the growth of one or more species of microorganisms.

in the context of this specification, the term "effective amount" refers to an amount of a compound which is sufficient to cause a Log reduction in the number of microorganisms of at least 1.0, which means that less than 1 microorganism in 10 remains. The compounds of the present invention may provide Log reductions in the number of microorganisms of at least about 2.0, or at least about 3.0, or at least about 4.0, or at least about 5.0, or at least about 6.0, or at least about 7.0.

In the context of this specification, the term "medical device" refers to any device that is designed for use within, or in contact with cells, tissue or organs of a human or animal body.

In the context of this specification, the term "C1 -C20 alkyl" is taken to include straight chain and branched chain monovalent saturated hydrocarbon groups having 1 to 20 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyJ, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like.

In the context of this specification, the term "Ci -Ci alkyl" is taken to include straight chain and branched chain monovalent saturated hydrocarbon groups having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyl, hexyl, heptyl, octyl and the like.

in the context of this specification, the term "G-C<_> alkyl" is taken to include straight chain and branched chain monovalent saturated hydrocarbon groups having I to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl and the like.

In the context of this specification, the term "C.-C20 alkenyl" is taken to include straight chain and branched chain monovalent hydrocarbon radicals having 2 to 20 carbon atoms and at least one carbon-carbon double bond, such as vinyl, propenyl, 2-methyl-2-propenyl, butenyl, pentenyl, hexenyl, hepteny , undecenyl and the like.

In the context of this specification, the term "C2-C10 alkenyl" is taken to include straight chain and branched chain monovalent hydrocarbon radicals having 2 to 10 carbon atoms and at least one carbon-carbon double bond, such as vinyl, propenyl, 2-methyl-2-propenyl, butenyl. pentenyl, bexenyl, heptenyl and the like.

In the context of this specification, the term "C2-C20 alkynyl" is taken to include straight chain and branched chain monovalent hydrocarbon radicals having 2 to 20 carbon atoms and at least one carbon-carbon triple bond, such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, imdecynyl and the like.

in the context of this specification, the term "aryl" is taken to include monovalent aromatic radicals having between 6 and 30, or between 6 and 20, or between 6 and 15. or between 6 and 10, carbon atoms, for example phenyl, biphen l, naphthyl, anthracenyl, phenanthrenyl, pyrenyl and the like.

In the context of this specification, the term "heteroarylene" is taken to include bivalent aromatic radicals having between 4 and 25 atoms, wherein at least one atom is a beteroatom selected from nitrogen, oxygeu and sulfur, for example furanylene. quinazolinylene, quinoiinylene, isoquinolhiylene, indolylene, isomdolylene, benzopyranylene, benzooxazolylene, benzimidazolylene. pyrazolylene, tetrazolylene, oxazolylene, oxadiazolylene, isoxazolylene, thiadiazolylene, quinolizinylene, pyranylene, isothiazolylene, thiazolylene, thienylene, imidazolylene, pyrazinylene, pyridazinylene. pyrimidinylene, isothiazolylene, pyridylene, triazolylene, benzothienylene, pyrrolyiene, benzothiazolylene, quinoxalinylene, naphthyridinyJene, pteridinylene, ca bazolylene, azepinylene, acridinylene, benzisothiazolylene, benzoxazolylene, benzisoxazolylene, benzorurylene, purinylene.. benzimidazolylene, triazinylene and the like.

In the context of this specification, the term "Ci-Csu alkylene" is taken to include straight chain and branched chain bivalent saturated hydrocarbon groups having I to 20 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, bexylene, heptylene, oclylene, dodecylene and the like.

In the context of this specification, the term "Ct-C<> alkylene" is taken to include straight chain and branched chain bivalent saturated hydrocarbon groups having I to 6 carbon atoms, such as methylene, ethylene, propylene, butylene, pentylene and the like.

In the context of this specification, the term "C1-C4 alkylene" is taken to include straight chain and branched chain bivalent saturated hydrocarbon groups having I to 4 carbon atoms, such as methylene, ethylene, propylene and the like.

In the context of this specification, the term "C2^C¾> alkenylme" is taken to include straight chain and branched chain bivalent hydrocarbon groups having 2 to 20 carbon atoms and at least one carbon-carbon double bond, such as propeirylene, 2-methyl-2-propenylene, butenylene, pentenylene, hexenylene, heptenylene, undecenylene and the like.

In the context of this specification, the term "C Cao alkynylene" is taken to include straight chain and branched chain bivalent hydrocarbon groups having 2 to 20 carbon atoms and at least one carbon-carbon triple bond, such as ethynylene, propynylene, butynylene, pentynylene, hexynylene, undecynylene and the like.

in the context of this specification, the terms "halo" and "halogen" may be used interchangeably and are taken to include fhioro, chloro, bromo and iodo.

in one aspect, the present invention provides a compound of the formula (I), or a salt thereof:

wherein

Rt is selected from the group consisting of: hydrogen, halogen, heteroaryl, aryl, C1-C2 alkyl, C20 alkenyl and C2-C20 alkynyl, wherein the heteroaryl, aryl, CVC20 alkyl, the C2- C20 alkenyl and the C2-C20 alkynyl groups are optionally substituted with one or more of the following substituents: hydroxy, halogen or OCi-C<_> alkyl:

R2 is selected from the group consisting of: hydrogen, halogen, heteroaryl, and aryl, wherein the heteroaryl, aryl group is optionally substituted with one or more of the following substituents: halogen, hydroxy, OC_rO, alkyl or amino;

and R4 are independently selected from the group consisting of: hydrogen and halogen;

R5 is selected from the group consisting of: G-C20 alkylene, C2-C20 alkenylene, Ci-Cio alkynylene, -(CH2)_¾-Ph-(CH₂)y-, -iCH₂)w-Ph-J-(CH₂) or -(CH₂)_W-Ph-(CH₂)_V-J- vvherein. w is 0, 1 , 2, 3, 4, 5 or 6, y is 0, 1 , 2, 3, , 5 or 6, J is O, S or NR; and

X is a functional group suitable for attachment to a solid surface.

In an embodiment X may be a functional group suitable for covalent attachment to a solid surface.

In another embodiment the solid surface may be a functionalised solid surface. In an embodiment X may be, for example, azido, ethyn l or *

In one embodiment, Ri is selected from the group consisting of: hydrogen, halogen, G-Go alkyl and C₂-G0 alkenyl, wherein the G-Go alkyl and the G-Go atkenyl groups are optionally substituted with one or more of the following substituents: hydroxy or halogen;

In another embodiment, Ri is selected from the group consisting of: hydrogen and G-G alkyl, wherein the G-G; alkyl group is optionally substituted with one or two of the following substituents: hydroxy or halogen.

In a further embodiment, R_t is selected from the group consisting of: hydrogen and G-G<> alkyl.

Tn a further embodiment, Ri is selected from the group consisting of: hydrogen and G-G, alkyl.

in one embodiment, 2 is selected from the group consisting of: hydrogen, halogen and phenyl, wherein the phenyl group is optionally substituted with one or more of the following substituents: halogen, hydroxy or OG-G. alkyl.

In another embodiment. R₂ is selected from the group consisting of: hydrogen, halogen and phenyl, wherein the phenyl group is optionally substituted with between on and three halogens.

in a further embodiment, is selected from the group consisting of: hydrogen, halogen and phenyl, wherein the phenyl group is optionally substituted with one or two fluoro groups.

In yet another embodiment, R2 is selected from the group consisting of: hydrogen, bromo and phenyl, wherein the phenyl group is optionally substituted with a single fluoro group.

In one embodiment, R3 and R4 are independently selected from the group consisting of: hydrogen, iodo, chloro and bromo.

In another embodiment, R3 and are independently selected from the group consisting of: hydrogen and bromo.

In one embodiment, Rs is Ci-G, alkylene, -(CHJ V-PMCHJ), or H H2)«-Ph-0-(CH₂)_>-. wherein w is 0, 1 or 2 and y is 0, I or 2.

In another embodiment, R₃ is G-G alkylene or -(CH₂)„-Ph-(CH2>y or -(CH₂)«-Ph-0- (CHaiv- wherein w is 0 or 1 and y is 0 or 1. In yet another embodiment, R5 is CrC₄ alkylene, -Ph- or -Ph-OCHa- or -Ph-OCH₂CH₂-

In still a further embodiment, Rj is C1-C4 alkylene, -Ph- or -Ph-OCH -.

In one embodiment, Ri is selected from the group consisting of: hydrogen, halogen, Cj-Cio alkyl and C2-C10 alkenyl, wherein the C1-C10 alkyl and the C Cto alkenyl groups are optionally substituted with one or more of the following substituents: hydroxy or halogen, R.2 is selected from the group consisting of: hydrogen, halogen and phenyl, wherein the phenyl group is optionally substituted with one or more of the following substituents: halogen, hydroxy or OCi- ft alkyl, R and R4 are independently selected from the group consisting of: hydrogen, iodo, chloro and bromo, R5 is Ci-CV, alkylene, -(CHs v-Ph-iCrtyv or -(CHj - h-CKCHi -, wherein w is 0, 1 or 2 and y is 0, 1 or 2, and X is a functional group suitable for covalent attachment to a solid surface, for example azido, ethynvl or

In another embodiment, R i is selected from the group consisting of: hydrogen and O-C* alkyl, wherein the Cj -Cr_> alkyl group is optionally substituted with one or two of the following substituents: hydroxy or halogen, R2 is selected from the group consisting of: hydrogen, halogen and phenyl, wherein the phenyl group is optionally substituted with between one and three halogens, R¾ and R4 are independently selected from the group consisting of: hydrogen and bromo, R5 is C1-C4 alkylene or -(CHiXv-PIMO^K- or -

(CH2)_w- h-0- CH2>_v- wherein w is 0 or I and y is 0 or I , and X is a functional group suitable tor covalent attachment to a solid sur ace, for example azido, ethvnvl or

In yet another embodiment, Ri is selected from the group consisting of: hydrogen and C|- C10 alkyl, Ra is selected from the group consisting of: hydrogen, halogen and phenyl, wherein the phenyl group is optionally substituted with one or two fluoro groups, 3 and R₄ are independently selected from the group consisting of: hydrogen and bromo, R5 is Ci- C₄ alkylene, -Ph- -Ph-OCHj- or -Ph-OCH₂CH₂- and X is a functional aroup suitable for covalent attachment to a solid surface, for example azido, ethynyl or

In a further embodiment, Ri is selected from the group consisting of: hydrogen and C)- , alkyl, Rj is selected from the group consisting of: hydrogen, bromo and phenyl, wherein the phenyl group is optionally substituted with a single fluoro group, R? and R₄ are independently selected from the group consisting of: hydrogen and bromo. R5 is C₁-C4 alkvlene, -Ph-_t -Ph-OCHr- or -PIV-OCH2CH2-. and X is a functional group suitable for covalent attachment to a solid surface, for example azido, ethynyl or <

In one embodiment, the compound of formula (I) is selected from the group consisting of:

The present invention also relates to intermediate compound of the formula (II)

wherein Rj, R_, R_¾ R*, Rj and X are as defined in the first aspect.

The compounds of formula (I) and (Π) may have one or more chiral centres. The present invention includes all enantiomers and diastereo isomers, as well as mixtures thereof in any proportions. The invention also extends to isolated enantiomers or pairs of enantiomers.

Also within the scope of the compounds of formula (I) and (U) are salts, including pharmaceutically acceptable salts. Salts of the compounds of formula (I) and (11) may be prepared by conventional methods known to those skilled in the art. For example, acid addition salts may be prepared by reacting the compounds of formula (Γ) and (H) with organic or inorganic acids. Examples of such salts include HC1, HBr and HI salts, salts of other mineral acids such as sulfate, nitrate, phosphate and the like, alkyl and monoary Sulfonates such as ethanesulfonatev toluenesulfonste and benzene sulfonate;, and salts of other organic acids, such as acetate, trifluoroacetate, tartrate, maleate, citrate, benzoate, ascorbate and the like. Compounds of the formula (I) and (II) may also be quaternised by reaction with compounds such as (Ci~C_i)alkyl halides, tor example, methyl, ethyl, isopropyl and butyl halides.

Compounds of the formula (l) may be prepared from known foranone compounds according to Scheme I .

)

Scheme 1: Preparation of compounds of the formula (I) from the corresponding turanone derivatives.

The reaction depicted in Scheme 1 may involve direct conversion to the compound of formula (I), or alternatively the reaction may proceed via the intermediate shown below from which water is eliminated to provide the compound of formula (1).

Where X is an azide functional group, compounds of the formula (I) may be prepared according to Scheme 2 or Scheme 3.

Scheme 2: Preparation of compounds of the formula (I) from the corresponding furanone compounds wherein X is azide.

Scheme 3: Alternative preparation of compounds of the formula (i) from the corresponding furanone compounds wherein X is azide.

Where X is an ethynyl functional group, compounds of the formula (1) may be prepared according to Scheme 4 or Scheme 5.

Scheme 4: Preparation of compounds of the formula (1) from the corresponding furanone compounds wherein X is ethynyl.

Scheme 5: Alternative preparation of compounds of the formula (I) from the corresponding furanone compounds wherein X is ethynyl.

Scheme 6: Alternative preparation of compounds of the formula (I) from the corresponding furanone compounds wherein X is

The present invention also relates to antimicrobial compositions comprising one or more compounds according to the first aspect, optionally together with one or more acceptable carriers, excipients or diluents. The carriers, excipients and diluents may be pharmaceutically acceptable. The compositions may be used to eliminate, reduce or inhibit microbial growth or the colonisation of an environment by microorganisms by allowing the composition to contact the microorganisms to be eliminated, reduced or inhibited and/or the environment of the microorganisms. The compositions may be used in a range of different environments that are inhabited or colonised by, or susceptible to being inhabited or colonised by. unwanted microorganisms.

The compositions may take any suitable form depending on the intended use thereof. For example, the compositions may take the form of a solution which can be applied to a surface or area by spraying. Alternatively, the compositions may take the form of a solution into which articles may be immersed. In another embodiment the compositions may take the form of a wipe comprising the compounds which can be used to apply the compounds to a surface or article.

The compounds and compositions of the present invention find application in a range of industrial and domestic applications, including but not limited to water supply reservoirs and teed pipes, drain pipes (domestic or industrial scale), process equipment of, for example, cooling towers, water treatment plants, dairy processing plants, food processing plants, chemical manufacturing plants, pharmaceutical or biopharmaceutical manufacturing plants, oil pipelines and oil refinery equipment, and pulp and paper mills. Other amenable environments and settings include, for example, as marine anti-fouJing paints or coatings, for example in treating ship hulls, aquaculture equipment, fishing nets or other in-water structures. Additional environments include domestic and commercial dishwashers and domestic or industrial clothes washing machines as well as point of use filters and water purification membranes.

I n particular embodiments the compounds of the first aspect are attached to a solid surface, for example the surface of a device, such as a medical device. The medical device may be. for example, a contact lens, a corneal only or inlay device, fluid collection bag, sensor, medical dressing, hydrogel bandage, tubing, stent, shunt, drain, surgical equipment, pulmonary device, laparoscopic device, ear plugs, heart valve, an implant, such as a hearing implant, a knee implant, a hip implant, an intraocular lens implant, an implantable electrode, an implantable neuroprosthetic electrode array (such as those manufactured by Cochlear), a catheter (such as an indwelling catheter) or carrier for artf biotiCi diagnostic or therapeutic agents. Those skilled in the art will appreciate that the compounds of the first aspect may be attached to the surface of other medical devices which are susceptible to contamination by microorganisms.

The surface of the device may comprise a polymer, for example a hydrogel, a silicon hydrogel, a polymer or copolymer of 2-hydroxyethylmethacrylate, silicone rubber, polyurethane, polypropylene, polyethylene, polyacrylamide, polytetrafluoroethylene (Teflon), polyimide, or biodegradable polymer, such as poly-lactide.

The solid surface may be a glass or metal surface or a metal-containing surface, for example a transition metal surface or a transition metal-containing surface. In one embodiment, the transition metal is titanium.

The compounds may also be attached to surfaces intended tor use in fields other than the medical field, wherein the surfaces are susceptible to being inhabited or colonised by, unwanted microorganisms. In other embodiments the compounds may be attached to surfaces located in food preparation areas, for example kitchens, water and oil pipes, packaging, chinking water systems, filters, marine surfaces, for example ships, and in- water structures.

In one aspect the present invention provides a surface, wherein at least one compound of the first aspect is attached to the surface.

In a further aspect the present invention provides a device, wherein at least one compound of the first aspect is attached to the surface of the device. n particular embodiments the device is a medical device as defined above.

The compound of formula (I) may be directly covalemly attached or indirectly covalently attached to the surface. In one embodiment, the compound of formula (I) is indirectly covalently attached to the surface via a divalent group comprising or consisting of W, wherein W is a heteroarylene group.

The heteroarylene group may have between 4 and 10 atoms, wherein at least one atom is a heteroatom selected from nitrogen, oxygen and sulfur.

In another embodiment, the heteroarylene group has between 4 and 6 atoms, wherein at least one atom is a heteroatom selected from nitrogen, oxygen and sulfur.

In a further embodiment, the heteroarylene group may have 5 or 6 atoms, wherein at least one atom is a heteroatom selected from nitrogen and oxygen. In still a further embodiment, the heteroarylene group may have 5 or 6 atoms, wherein between 1 and 4 atoms are heteroatoms selected from nitrogen and oxygen.

In one embodiment, the heteroarylene group is a triazolylene or an isoxazolylene group.

In another embodiment, the compound of formula (I) is indirectly covalemly attached to the surface via a divalent group comprising or consisting of W and a linker, wherein the linker is located between the surface and the group W.

The presence of a linker allows modulation of the distance between the surface and the compound of formula (I). Suitable linkers will be well known to those skilled in the art. In one embodiment, the linker is C1-C20 alkylene, C2-C20 alkenylene or C2-C20 alkenylene. each of which may optionally be interrupted by one or more of the following groups: -0-, - -C(0)- and -NH-.

in an alternative embodiment the linker may be O-C20 alkylene which may optionally be interrupted by one or more of the following groups: -0-, -C(0)- and -NH-.

In a further embodiment the linker may be Ci-C o alkylene or O-Cut alkylene- NHC(0)0(CH₂)_p, wherein p is 0, I, 2, 3, 4 or 5.

In still a further embodiment the linker may be CrOo alkylene or C Cw alkylene- NHC(0)0(CH₂)_P> wherein p is 0, L 2 or 3.

In yet another embodiment the linker may be O-G, alkylene or Ci-C« alkylene- NHC(0)0(CH2)_P, wherein p is 0, 1 or 2.

In a further embodiment the linker may be Q-C4 alkylene or C1-C4 alkyiene- NHC(0)0(CH₂)_P> wherein p is 1 or 2.

In a further embodiment, the linker may be polyethylene glycol, phenylene or cycloalkylene.

The mode of attachment of the linker to the surface will be dependent on the functional groups present on the surface of the device prior to attachment of the compound of the formula (I). For example, where the functional groups present on the surface prior to attachment of a compound of the formula (I) are hydroxy groups, the linker may be attached to the surface via an -Si-(O)s- group as follows: surface-0-Si(0)₂-linker. Accordingly, in one embodiment, the compound of formula (I) is indirectly covalemly attached to the surface of the device as follows:

wherein the linker, W and compound of formula (I) are defined above.

Alternative modes of attachment:

- immobilisation of amino substituted DHPs onto epoxidated or aziridinated surfaces.

- coupling of epoxide substituted DHPs to amino-functionalised surfaces, in which the amine surface can be generated by either silanisation or plasma polymerisation.

- immobilisation of amino substituted DHPs to carboxyl-ftmctionaUsed surfaces via carbodiimide chemistry.

- thiol substituted DHPs attach via reaction with maleimide-functionalised surfaces.

- direct attachment of azido substituted DHPs under photochemical conditions mediated with or without benzophenones.

In another aspect, the present invention provides a compound array comprising:

a) a functionalised solid surface; and

b) at feast one compound of formula (la),

wherein R Ra, R¾ R4 and R are as defined Iri the first aspect and Y is a divalent functional group attached to the functionalised solid surface.

In one embodiment, Y is a heteroarylene group which is defined as per variable W above.

In another embodiment, Y and the functioualised solid surface taken together have the following structures:

wherein the linker is as defined above.

The array may comprise at least 2, at least 10, at least 50, at least 100, at least 500, at least 1000, or at least 10,000 compounds of formula ( la). The compounds may be the same or they may be different.

The compound array may be prepared utilising the methodology described below.

In another aspect the present invention provides a method for inhibiting the adherence of one or more microorganisms to a sur&ce, the method comprising attaching to the surface at least one compound of the formula (I). The surface may be a solid surface as described above.

In a further aspect the present invention also provides a method for preventing the occurrence of microbial infection on or around the surface of a medical device inserted into a patient, or at or near the site of insertion of the medical device, the method comprising attaching to a surface of the device, or coating a surface of the device with, at least one compound of the formula (i).

In yet another aspect the present invention provides a method for preparing a device having at least one surface, the method comprising reacting the at least one surface with at least one compound of the formula (1).

In still a further aspect the present invention provides a method for modifying a surface, the method comprising reacting the surface with at least one compound of the formula (I).

In accordance with the above aspects, attaching to a surface, coating a surface, or reactin a surface with a compound of formula (I) may include reaction of a functional group Z of the solid surface with functional group X of the compound of formula (Ϊ). Functional group Z may be capable of undergoing a Cl ick reaction wit h functional group X.

"Click chemistry" is a term that describes reactions that are high yielding, simple to perform, stereospecific, solvent insensitive, create only easily removable byproducts, and can be easily purified without the use of chromatography.

In one embodiment, X is azido. ethvnyl or 4

, and the functional group of the solid surface is azido, ethynyl or

, wherein when X is azido or

, Z is ethynyl, and when X ethynyl, Z is azido or

In other embodiments, Z is azido and X is ethynyl or X is azido and Z is ethynyl.

Alternatively, attaching to a surface, coating a surface, or reacting a surface with a compound of the formula (I) may include the step of functional isation of the surface so as to provide a functional group which is capable of reacting with functional group X of the compound of formula (I). Functionalisation of the solid surface will be necessary where the solid surface does not possess functional groups which are capable of reacting with functional group X of the compound of formula (1). Such functional groups may be incorporated by conventional synthetic methods known to those skilled in the art.

In embodiments of the fourth to seventh aspects attaching to a surface, coating a surface, or reacting a surface with a compound of the formula (I) may comprise the following steps:

(i) functionalisation of the surface so as to provide a functional group Z which is capable of reacting with functional group X of the compound of formula (I);

(ii) reaction of the junctional group Z with the functional group X.

Z may be a terminal functional group.

The reaction in step (it) may be a click reaction.

X may be azido, ethynyl or

. and the functional group of the solid surface may be azido. ethynyl or

. wherein when X is azido or

, Z is ethynyl, and when X ethynyl. Z is azido or

In particular embodiments, Z is azido and X is ethynyl or X is azido and Z is ethynyl.

Step (i) may comprise reaction of a functional group of the surface with a compound of the formula M-T-Z, wherein Z is as defined above, T is a linker and M is a functional group which is capable of reacting with the functional group of the surface. The reaction in step (i) may be carried out by methods known to those skilled in the art, for example by chemical vapour deposition or by dip-coating. The linker may be as defined above.

Those skilled in the art will appreciate that the identity of M will be dependent on the type of functional group or groups on the surface which are intended to be functionalised. For example, where the timet ional groups on the surface are hydroxy groups, M may be, for example, -Si(OCi-C<;alkyl).¾.

Surfaces can be functionalised with amine or epoxide groups using plasma polymerisation. M may be allylamine (or polyaJlylamine) for amine surfaces. For epoxide surfaces, may be polymer of glyc idyl methacrylates or allyl glycidyl ether. Plasma polymerisation can tie performed on a variety of surfaces such as Teflon, silicone rubber, titanium, aluminium and glass surfaces.

Another example, noble metal such as gold and platinum surfaces can react with terminal thiols. Therefore M may be -S-, T may be alkyl chain, and Z may be azido, ethynyl, or amine.

Scheme 7 shows an example of reaction of a functional group of a surface (-OH) with a compound of the formula -M-T-Z. wherein M is (EtOVSi-, T is or -

(CHj).,NHC(O)OCH₂-₍ and Z is azido or ethynyl.

Scheme 7: Reaction of a surface so as to provide a functional group of the formula Z which is capable of reacting with the functional group X of the compound of the formula

(I).

Reaction of the functional group Z with the functional group X may be achieved by a click reaction as shown below in Scheme 8.

Scheme 8: Schematic representation of the immobilisation of alkyne/azide-functionalised DHP onto azide/alkyne-tenmnated surfaces via a click reaction (copper(I)-catalysed azide- alkyne cycloaddition or CuAAC).

In Scheme 8, a surface of a substrate (tor example a surface of a medical device) having hydroxy groups is functionalised with a triethoxysilane derivative which includes a terminal azide functional group (AzPTS) or a terminal alkyne functional group (Alkyne- PTS). Utilising a click reaction, the azide-functionalised surface is reacted with a compound of formula (I) wherein X is^≡ (denoted in the scheme as "DHP-- '), and the alkyne-functionalised surface is reacted with a compound of formula (I) wherein X is azido, (denoted in the scheme as 'ΌΗΡ-Ν/), to provide surfaces on which compounds of the formula (Γ) are indirectly attached. The click reaction results in the formation of a

1 ,2,3-triazolyl ring. In the above noted scheme an * group may be substituted for the azido group which, following the click reaction, results in the formation of an isoxazolyl ring rather than a J ,2,3-triazolyl ring. in embodiments of the fourth to seventh aspects attaching to a surface, coating a surface, or reacting a surface with a compound of the formula (I) may include the following steps:

(a) reaction of a functional group of the surface with a compound of the formula M- T-Z, wherein M and T are as defined above; and

(b) reaction of the functional group Z with the functional group X of the compound of the formula (I),

wherein Z is azido and X is ethynyl, or X is azido and Z is ethynyl.

Step (a) may be carried out by chemical vapour deposition. In a particular embodiment, Z is azido and X is ethynyl and Step (a) is carried out by chemical vapour deposition.

The compound array of the tenth aspect may be prepared utilising the methods described above. For example, a solid surface may be functionalised so as to provide functional groups Z which are capable of reacting with functional groups X of compounds of formula (I). Reaction of functional groups Z with functional groups X utilising a click reaction generates bivalent group Y thereby forming the array in which compounds of the formula (T) are indirectly attached to the solid surface.

The compounds, compositions, devices and methods of the invention find use against a range of microorganisms, for example, bacteria, fungi (including mould and yeast), and protozoa.

The bacteria may be Gram-negative or Gram-positive bacteria, such as for example Staphylococcus spp., Streptococcus spp., Enterococcus spp., Acinetobacter spp., Pseudomonas spp., Haemophilus spp., Proteus spp., Senatia spp., Escherichia spp., Salmonella spp.. Klebsiella spp.. Bacillus spp. and Listeria spp. Exemplary species include but are not limited to Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus ( RSA), Staphylococcus epidermidis. coagulase negative staphylococci, Enterococcus faecalis, Enterococcus faecium including vancomycin-resistant Enterococcus faecium. Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli. Salmonella enterica. Salmonella typhi, Bacillus cerern, and Listeria monocytogenes.

Those skilled in the art will appreciate that the compounds and compositions may be useful in the control of other bacterial species in addition to those specifically recited above.

As demonstrated in the Examples below, surfaces to which are attached compounds of the formula (1) via the click reaction shown Scheme 1 have, surpr -singly, been found to possess remarkable antimicrobial activity in reducing bacterial adhesion and biofilm formation.

Those skilled in the art will appreciate that the embodiments described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications which fall within the spirit and scope. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the present application. Further, the reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Certain embodiments will now be described with reference to the following examples which are intended tor the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

Examples

Example 1: Synthesis of compounds

Synthesis of azide-DHP derivative

Method ./.·

I-(4-(MunopheMyl)-3-biriy!-5-(di romomeih l)-5-hydrox

3-Butyl-5-(dibromomethylene)furan-2(5H)-one (1.0 g; 3.23

mmol) was first dissolved in dry acetonitrile (10 mL), followed

by addition of/>-phenyIenediamuie (0.854 g; 7.90 mmol) in dry

acetonitrile (10 mL). The reaction was stirred and heated to

reflux for 48 h. The reactant mixture was chromatographed on

silica gel using 50 % ethylacetate/«-hexane to yield the title compound as yellow solid (0.46 g; 34 %). Ή NMR (300 MHz, CDCl?): δ 0.95 it, 3H, CHj), 1.44 Cm, 2H, CH₂), 1.62 (m, 2R CH₂), 2.34 (m, 2H, CH₂), 3.78 (s, 2H, NH₂), 4.57 (s, 1H, OH), 5.48 (s, CHBr₂), 6.62-6.65 (m, 2H, 2 x H aryl), 6.76 (s, IH, CH), 7.15-7.18 (m, 2H, H aryl);^,3C NMR: ό 13.7 (CH;). 22.3 (CH₂), 25.1 (CH₂), 29.3 (CH₂), 47.0 (CHBr₂)_; 91.9 (C), 1 15.6 (2 x CH aryl), 124.5 (C), 128.7 (2 x CH aryl), 136.0 (CH), 144.1 (C), 145.4 (C), 169.4 (CO). l-(4-amhmphenyl)-3-bwyl-5-(l bramometh lene^

A mixture of the above compound (0.46 g; 1.10 mmol) and

thionyl chloride (0.16 tnL; 2.20 mmol) in dry toluene (10 mL) was

heated to 80 °C with stirring for 2 h. Solvent and thionyl chloride

were removed by vacuum, and the product was purified by flash

column chromatography with silica gel and ethylacetate/»-hexane

to yield to title compound as yellow solid (0.15 g; 33 %). Ή NMR

(300 MHz, CDCh). δ 0.95 (t, 3H, CHj), 1.44 (m, 2H, CH₂), 1.60 (m, 2H, CH₂), 2.41 (m, 2H, CH₂), 3.78 (s, 2H. N.H₂>, 6.70-6.73 (m, 2H, 2 x H aryl), 6.99-7.02 (m, 2R H aryl), 7.13 (s, 1H, CH);^,3C NMR: δ 13.7 (CH_S), 22.3 (CH;), 25.2 (CH₂), 29.5 (CH₂), 76.4 (CBr₂), 1 15.61 (2 x CH aryl), 125.3 (C), 130.4 (2 x CH aryl), 131.6 (CH), 138.8 (C), 140.3 <C), 146.7 (C), 172.1 (C=0). l-<4Hizi^>phenyl)-3-biHyl-5-{d^romomelhylene (azkk- 8-phenyl- D P)

The mixture of the above compound (0.1 1 g; 0.28 mmol) in HCl

(2M; 8 mL) was stirred in an ice bath at 0 °C followed by the

addition of NaN<¾ (55 mg; 0.78 mmol). The mixture was left to

stir for 1.5 h, followed by addition of NaN* (60 mg; 0.92 mmol)

and stirred for a further 45 miu. The product was extracted into

CH2 I2, dried with sodium sulphate and the solvent removed by vacuum to yield the title compound as white solid (0.104 g; 89 %). M.p. 59-60 °C; Ή NMR (300 MHz. CDClj): δ 0.95 (t, 3H, CH-,), 1.39 (m, 2H, CH₂), 1.58 (m, 2H, CH₂). 2.37 (t, 2H, CH₂), 7.06-7.09 (m, 2H, H aryl), 7.16 (t, I H, CH)_S 7.19-7.22 (m, 2H, H aryl); ¹³C NMR: δ 13.7 (CH,), 22.3 (CH₂), 25.2 (C¾), 29.5 (CH₂), 76.4 (CBr₂), 119.4 (2 x CH aryl), 130.7 (2 x CH aryl), 13.1.6 (C), 132.2 (CH), 138.9 (C), 140.1 (C), 140.2 (C), 171.7 (00). IR ( Br, v^, cm^'1): 3395, 2957, 2929, 2870, 2430, 2130, 2101 , 1701 , 1590, 1505, 1467, 1375, 1308, 1279, 1210, 1 191 , 1 169, 1 1 16, 1095, 836, 655; HRMS (ESI) m/z Calcd. for CisHwBrjN^Na (M + Na)^! 446.9432. Found 446.9437. Method 2:

4-Azidoamlitie

4-Bromoaniline (2.02 g; 1 1.7 mmol), NaN_* (1.57 g; 24.1 mmol), sodium ascorbate (0.22 g; 1.1 mmol), Cul (0.33 g; 1.7 mmol), NJf- dimethyiethylenediamine (200 pL; 1.8 mmol), and 7 mL ethanol/water (7:3) were stirred and heated to reflux for 45 min under an argon atmosphere. The

crude mixture was then washed with water and saturated sodium chloride solution and extracted into ethylacetate. The organic layer was collected, dried with sodium sulphate and concentrated in vacuo. The residue was purified by filtration through a short column of silica gel, giving the desired 4-azidoaniline as yellow crystal (0.96 g; 40 ¾).^lH NMR (300 MHz, CDC ): δ 3.63 (s, 2H, NH₂), 6.66-6.69 (m, 2H, H aryl), 6.82-6.85 (m, 2H, H aryl); C NMR: δ 1 16.3 (2 x CH aryl), 120.0 (2 x CH aryl), 1 0.2 (C), 143.7 (C); [R (neat, \ , cin ' ): 3397, 3321, 3222, 2928, 2853, 2105, 2067, 1724, 1631 , 1599, 1505, 1302, 1267, 1 126, 1080, 832, 81 , 783, 696, 686, 678, 666.

I-(4-a∑Ul0phenyl)-3-bitty!-5-(dibwm0mefh)fy

A mixture of 3-butyl-5-(dibromometliylene)iuran-2(5H)-one

(0.43 g; 1.39 mmol) and 4-azidoaniline (0.46 g: 2.22 mmol)

were healed together at 120 °C. The residue was redissolved in

CH2CI2 and successively washed with water. The organic

phase was separated and dried with sodium sulphate and the

solvent evaporated in vacuo. The reactant residual solid was

purified by chromatography on silica gel using ethylacetate w-hexane to yield the title compound as brown solid (0.35 g; 57 %). Ή NMR (300 MHz, CDClj): δ 0.95 (t, 3H, CHj), 1.39 (m, 2H, CH₂). 1.58 (m, 2H, CH₂). 2.37 (m, 2H, CH₂), 4.29 (s. Hi OH), 5.47 (s, CH.Br₂), 6.80 (s, IH, CH), 6.96-6.99 (m, 2H, 2 x H aryl), 7.44-7.46 (m, 2H, H aryl). ^,3C NMR: 6 13.7 (CHj), 22.3 (CH₂), 25.1 <CH₂). 29.3 (CH₂), 46.3 (CHBr₂), 92.3 (C), 1 19.4 (2 x CH aryl), 127.9 (2 x CH aryl), 130.6 (C), 136.6 (CH), 138.9 (C), 143.8 (C), 169.4 (CO).

I-(4- zidopbenyt)-3-biAtvl-5-{dibrami ietin^!len { zide-F8-phem'l- DHP

The above lactam intermediate was mixed with Ρ₂θ (0.56 g;

3.96 mmol) in CH>C1 (10 mL) and refluxed for 2 h. The solvent

was evaporated under vacuum and the residue was purified by

silica gel chromatography (ethylacetate/ CH₂CI₂) to afford the

title compound as white solid (0.26 g; 77 %).

Sy thesis of a!kyne derivatives of PHP

p-aminotrimethyhilam

A mixture of /.'-aminophenol ( 1.0 g; 9. 16 mmol), hexamet yldisilazane

(0.89; 5.52 mmol) and a few drops of chlorotrimethylsilane were heated

together with stirring at 140-150 "C for 2 h. The mixture was cooled and

distilled under vacuum at 120 °C to yield a colourless mobile liquid (1.92

g; 82 %). *H NMR (300 MHz, CDC ): δ 0.22 (s, 9H, CH._¾), 3.46 (s, 2H, NH>), 6.55-6.58 (m, 2H, H aryl), 6.65-6.68 (m, 2H, H aryl); C NMR: δ 0.16 (3 x CH,), 1 16.4 (2 x CH aryl), 120.7 (2 x CH aryl), 140.5 (C), 147.7 (C).

3-buiyI-5-(cfihr m<meihylene)-!-(4- ydrox^

3-butyl-5-(dibromomethylene)ruran-2(5H)-one (0.34 g; 1.10

mmol) and />-aminophenyItrimethylsilane (1.5 g; 5.90 mmol)

were heated together with stirring at 120°C for 24 h. The

mixture was cooled to room temperature, washed with HQ

(2M) and extracted into QfcCfc. The solution was then dried

with sodium sulphate and passed through a flash column with CH2CI2 to remove any unreacted 3-butyl-5-(dibromomethylene)furan-2(5H)-one. Finally, the product was eluted with 50 % CH Cyethylacetate, and solvent removed by vacuum to afford the intermediate product 3-butyl-5-(dibromomethyl)-5-bydroxy- 1 -(4-((trimethylsily I )oxy)phenyl)- 1 H- pyrrol-2f 5H)-one as white solid (0.40 g; 74 %).

The intermediate product (0.40 g; 0.95 mmol) was dehydrated with borontrifluoride diethyl etherate (BFa.CXCjl fc: 300 μΐ; 1.12 mmol) in dry C¾Cl; (10 ltiL), with stirring and refluxed for 2 h. Purification of the residue by silica gel chromatography (20 % ethylacetate/CHiClj) afforded the title compound as colourless solid needles (0.32 g; 83

%).

3~butyl-5-(dibr<momei ykme)-J-(4-(pnw~2'yn~l'ylo^

(alkyne-l^'8-phenyl-DHP)

The compound above (0.19 g; 0.47 tmnol) was first

dissolved in dry acetone (5 mL)₅ followed by the addition of

K.2CO* (0.1 g; 1.09 mmol) and propargyl bromide (80 %wt

in toluene; 200 μΐ.; 1.80 mmol). The solution was stirred

and refluxed for 4 h, and the product was purified by

column chromatography (50 % CH₂Cl /«-hexane) to yield the desired product as white solid (0.21 g; 99%). M.p. 86-87 °C; Ή NMR (300 MHz, CDCI,): δ 0.95 (I, 3H, CH₃), 1 .35 (m, 2H, CHj), 1.59 (m, 2H, CHj), 2.3? (t, 2H, CH₂), 2.53 (t, I H, CCH), 4.70 (d, 2H, OCH2), 6.99-7.02 (m_? 2H, 2 x CH aryl), 7. ! 2-7. J 5 (m, 3H, 2 x CH acryl, CH). C NMR: δ 13.8 (CH._¾), 22.4 (CH₂), 27.3 (CH₂), 31.4 (CH₂), 56.1 (OCH₂), 74.2 (CBr₂), 76.2 (≡CH), 77.5 (CCH), 1 19.4 (2 x CH aryi), 128.4 (C), 130.6 (2 x CH aryl), 131.8 (C), 138.9 (C), 140.2 (C), 157.5 (C), 171.9 (C=0). IR ( Br, v_ft¾aX( cm ¹ ): 3256, 2958, 2921 , 2848, 2126, 1895, 1701 , 1606, 1588, 1509, 1463, 1449, 1380, 1293, 1241 , 1 193, 1 175, 1 122, 1031 , 844, 825, 723, 682, 661 , 533; HRMS (ESI) m z Calcd. for QeHnBfc OzNa (M + Na)* 459.9529. Found 459.9527.

3- bui f-5-(dibwmome(hyk e)-J-(pjvp-2-m-l-y -lH^ym>l-2(5H)-one ( lkyne-FS-DHP) A solution of 3-butyi-5-(dibromomethylene)furan-2(5H)-one (0.41

g; 1.21 mmol) in CH₂CI₂ (5 mL) was stirred in an ice bath at 0 °C

followed by dropwise addition of propargyiamine (0.38 mL; 5.93

mmol) in CH₂C1₂ (10 mL). The solution was then heated to 40 °C

and allowed to react for 2 h. The resulting brown mixture was concentrated under reduced pressure. The lactam intermediate was then mixed with P2O5 (0.72 g; 5.07 mmol) in CH2CI2 ( 15 mL) and refluxed tor 2 h. The solvent was evaporated under vacuum and the residue was purified by silica gel chromatography (10 % ethyl acetate/ CH₂Cl ) to afford the title compound as light brown solid (0.32 g; 70 %). M.p. 36 °C; Ή NMR δ (CDClj): 0.93 (m, 3H, CH*), 1.36 (m, 2H, CH₂), 1.56 (m,2H,CH₂),2.24 (t, I H,≡CH\ 2.33 (m, 2H ,CH₂),4.78 (d, 2H, -CH₂-), 7.04 (s, I H, H4); C NMR: δ 13.8 (CH. , 22.4 (CH_:), 25.3 (CH₂), 29.6 (Ci½), 1.2 (NCH₂), 72.1 (CBr₂), 75.3 (=CH), 79.1 (C), 132,7 (CH), 138.8 (C). 139.7 (C), 171.3 (C=0); IR (neat, v*«, cm^"1): 3232, 2957, 2925, 2853, 1695, 1590, 1461, 1439, 1343, 1304, 1 191, 1 146, 1094, 935, 852, 833, 735; HRMS (ESI) m/z Calcd. for C,₂H,₄Br₂NO (M + H)^† 345.9442. Found 345.9437.

4- bromo-5^ro omethylem)-l^roi>-2-y^i-i-yl)-IH-pyrrol-2(5 )- iie (alkyne-FSO-DhfP)

A solution of 4-bromo-5-(bromomethylene)furan-2(5H)-one (0.80 g; 3.15

mmol) in (10 mL) was stirred in an ice bath at 0 °C followed by dropwise

addition of propargyiamine (0.96 mL; 14.99 mmol) in (15 mL). The

stirred mixture was allowed to react for 3 h at 0 °C and the solution was

concentrated under vacuum. A mixture of the resulting lactam intermediate and P₂0< (0.43 g; 3.03 mmol) in dry CH₂CI₂ ( 10 mL) was refluxed for 2 h. The solvent was evaporated under reduced pressure and the residue was purified by silica gel chromatography (20 % ethylacetate/ CH₂CI₂) to yield the product as yellow solid (0.58 g; 63 %); m.p. 114-1 16 °C, ^JH NMR (300 MHz, CDCb): 56.40 (s, I H, H3), 6.3? (s, I H, <Ή)Α 78-4.79 (ro, 2H, NCH₂),2.24-2.26 (m, IH,≡CU). ⁿC NMR: 167.8 (C=0), 138.9 (C), 1 1.9 (C), 123.7 (CH),93.6 (CHBr),78.5 (C),72.2 (≡CH),30.5 CNCH2). IR (mijol, v, cm⁰): 2923, 2854, 1716, 1633. 1563, 1462, 1377, 1342, 1266. UV (C¾OH): J 293.0 nm (22960 M ¹); HRMS (ESI) m/z Calcd. for QHsBraNONa (M + Na)' 31 1.8630. Found 31 1.8649. Anal. Cakt. for C»H₅Br₂NO: C, 33.03; H, 1.73; N, 4.81. Found: C, 33.20; H, 2.02; N, 4.78.

Other alkyne-DHP analogues were synthesised using the same method from the corresponding furanone starting material.

5-methylene-4-p enyl-i-ipwp-2-yit-l-}i)-lH-pyrrol-2(SH)-one (alkyne-DHP 1)

67 % yield; M.p. 77-78 °C;^!H NMR (300 MHz, CDCI₃): δ 7.48 (s, 5H,

ArH), 6.26 (s, ΓΗ, H3), 5.30-5.31 (m, I H, =CH), 5.12-5.13 (m, I H,

=CH), 4.53 (d, 2H, NC¾), 2.27-2.29 (t, IH,≡C ); "C NMR: 151.5

(CO), 143.8 (C), 131.3 (C), 129.9 (C), 128.9 (4 x CH_ary]), 128.7 (2 x

CH_a„), 120.0 (C), 101.1 (CH2), 72.5 (=CH), 28.9 (NCH2); 1R (neat,

v , cm^''): 3238, 3202, 3075, 2956, 2921 , 1695, 1635, 1487, 1445_r. 1427,

1393, 1352, 1338, 1298, 1262, 1169, 1 129, 1072, 1029, 916, 872, 848, 830, 806, 775, 688, 656; HRMS (ESI) m z Calcd. for CuHjiNONa (M + Naf 232.0738. Found 232.0733.

4-(2-j (orophmyl)-5-methylem-l-ipr p-2-yn-1-yi)- (alkyne-DHP2) 64 % yield; M.p. 78-80 °C;^lH NMR (300 MHz, CDCb): δ 7.20-7.43 (m.

4H, ArH), 6.33 (s, IH, H3), 5.27-5.28 (m, IH, =CH), 4.97-4.98 (m, IH,

=CH), 4.51 (d, 2H, NCH₂), 2.27-2.28 (t, I H,≡CH); C NMR: 167.9

(CO), 161.4 fCF , 158.1(C), 144.1 (C), 131.1 (CH^ 130.9 (CH^),

131.1 (CH^ , 124.2 (CH_aryl), 123.5 (CH^,), 1 9.5 (C% 1 16.5 (CH₂), 97.9

(C), 72.0 (=CH), 28.6 (NCH2); IR (neat, \ , cm-'): 3258, 2925, 1697,

1635, 1619, 1488, 1447, 1433, 1401, 1348, 1225, 1 104, 941, 876, 852, 820, 787, 760, 715, 684; HRMS (ESI) m/z Calcd. for C_l4Hi₀FNONa (M + Na)¹ 250.0644. Found 250.0639.

3-Azk propyl(rielhoxysikme (AzP^'J^'S)

A mixture of 3-chloropropyltriefhoxysilane (2.0 niL; 7.89 mmol)

and sodium azide ( 1.60 g; 24.6 mmo ) in dimethvlformamide (2

mL) was stirred and heated to 120 °C for 24 h. The solution was

washed with distilled water and saturated NaCL and extracted into CH₂CI₂. The solvent was removed under vacuum to yield a pale yellow liquid (1.8 inL; 92 %). Ή NM (300 MHz, CDCI. : δ 0.69-0.63 (t, 2H, CHfeSi), 1.24-1.19 (t, 9H, CH ),1.73-1.67 (q, 2H, CCH₂C),3.27-3.23 (t, 2H, CH₂N3), 3.84-3.76 (q, 6H, OCH₂); '¾ NMR: δ 7.6 <SiCH₂), 18.3 (CH₃),22.7 (CH₂),53.8 (CH₂N.458.4 (OCH₂); I (neat. \ , cm⁰): 2975, 2928, 2886, 2094, 1443, 1390, 1344, 1276, 1242, 1166, 1100, 1070, 954, 775, 680; HRMS (ESI) m/z Calcd. for C^N^SiNa ( + Na/ 270.1250. Found 270.1244.

Preparation of prop-2-yv- l-ylO-(triethoxyu!yl)propyl)carhamate (A Iky/ie-PT )

A solution of propargyl alcohol (1.2 mL; 20.8 mniol) and

triethylarnine (2.4 mL; 17.2 nimol) in CH₂C1₂00 mL)

was stirred and cooled in an ice bath at 0 °C, followed by

dropwise addition of 3-isocyanatopropyltriethoxysilane (4.5 mL; 18.1 mmol) in CH₂Ci₂ (5 mL). The mixture was stirred at room temperature tor 24 h, washed with distilled water and extracted into CH₂Cl2. The solution was then concentrated under reduced pressure to yield 3-Azidopropyltriethoxysilane as orange oil (4.6 mL; 83 %). Ή NMR (300 MHz, CDCU): δ 0.54-0.48 (t, 2H, CH₂Si), 1.13- .1.08 (t. 9H, CH. , 1.56- 1.46 (m, 2H, CCH₂C),2.39-2.38 (t, 1 H, CCH),3.10-3.03 (q, 2H, CH₂N),3.73-3.66 (q, 6H, OCH₂),4.55 (d, 2H, CH₂CC),5.32 (s, 1 H, NH); ^,SC NMR: δ 7.4 (SiCH₂), 18.0 (CH₃),23.0 (CCH₂C),43.3 (NCH₂),52.0 (OCH₂C),58.2 (OCH₂),74.3 (CH),78.3 (C),155.4 (NCOO); IR (neat, v_m¾x, cm^'1): 3312, 2975, 2929, 2886, 1707, 1532, 1443, 1391, 1244, 1195, 1166, 1 100. 1072, 954, 770, 672; HRMS (ESI) m/z Calcd. for Cr,H₂₅N0₅SiNa (M + Na)^: 326.1400. Found 326.1394.

Example 2: Attachment of a DHP to a surface using Click Chemistry

General procedure for azide/alkvne si fan linker auachmeitl to glass

Glass coverslips (No. I , diameter 13mm D 263 M glass, ProSciTech, Australia) were washed with freshly prepared piranha solution (3: 1 v/v sulfuric acid to 30% hydrogen peroxide) for 30 min, followed by sonication (Unisonics, FXP 10M, Australia) in distilled

H₂0 for 3 x 15 min. The cleaned glass coverslips were stored in absolute ethanol before use.

The attachment of the azide- and alkyne-tenninated silanes (termed AzPTS and alkyne- PTS respectively) was carried out utilising chemical vapour deposition (CVD) and dip- coating. The CVD method involved placing cleaned glass coverslips on steel mesh within a glass vessel. A solution containing the appropriate linker (10% v/v in dry toluene) was transferred into the glass vessel underneath the coverslips. The glass vessel was sealed and placed in an oven at 120*C tor 18 hours. After this time the glass coverslips were rinsed twice with dry toluene and once in absolute ethanol. They were then air-dried and stored in die dark prior to use. For dip-coating, cleaned glass coverslips were immersed in a solution of a silane linker (5 % v/v in dry toluene) for 10 minutes. The substrates were then rinsed twice with toluene to remove excess silane reagent and cured at 100 °C tor 30 minutes. The samples were then stored in the dark prior to use.

General procedure for attachment of PHP via a click reaction

A solution of 50 % ethanol/water comprising of azide- or alkyne-DHPs (6.8 mM), copper sulfate ( 7.5 mM) and sodium ascorbate (34.2 mM) was prepared. 1 mL of this solution was added to a well of a high density polyethylene case each containing an alkyne- or azide-functionalised glass coverslip. The reaction vessels were sealed and agitated overnight following by rinsing with 50 % ethanol/water (2x) and absolute ethanol (Ix). The resulting samples were air-dried and stored in sterile dust-f ee glass containers.

Optimisation of PHP attachment efficiency via Azide- arid Aikyne-lerminated coupling agents

In order to identify the best attachment strategy for DHPs, two complimentary approaches were investigated. One based on the coupling of alkyne- functionaJized DHP onto azide- terminated surface, and the other involved the reaction between azide-functionalized DHP and alkyne-terminated surface (see Scheme 7). In addition to that, two deposition methods (chemical vapour deposition (CVD) and dip-coating) for the preparation of azide- alkyne- terminated surfaces were explored. The azide-Zalkyne-terminated surfaces were subsequently coupled with the appropriate alkvne-/azide-F8-phenyl-DHP using click chemistry (CuAAC). The attachment of DHP was analysed using X-ray photoelectron spectroscopy (XPS; ESCALAB220-iXL, VG Scientific, West Sussex, England). The X- ray source was monochromated Al Ka and the photoenergy was 1486.6 eV with a source power of 120 W. Vacuum pressure was < 10^"8 mbar. The elemental compositions of the surfaces are shown in Table 1. Table I: Elemental analysis of azide- and alkyne-term ated surfaces, and the subsequently attached FS-phenyl-DHP surfaces.

Successful glass surface modification by AzPTS and alkyne-PTS via the two deposition methods is revealed by the changes in carbon and nitrogen compositions on the surfaces. Both AzPTS and alkyne-PTS samples prepared by either CVD or dip-coating methods exhibited significant but varied increases in both carbon and nitrogen contents compared to the blank glass surface. For AzPTS treated surfaces, the CVD surface exhibited considerable increases of 5.9 % C and 4.3 % N in comparison to the blank surface, while the dip-coated surface gave marked increases of 17.7 % C and 12.2 % N. The dip-coated AzPTS surface had close to three times as much carbon and nitrogen content compared to the CVD AzPTS surface indicating that a greater amount of azide-functionality was deposited when samples were prepared by the dip-coating method. A similar trend was observed for alkyne-PTS treated surfaces, wherein the CVD sample had increases of 18.1 % C and 2.3 % N respectively, while the dip-coated sample exhibited 42.1 % C and 6.2 % N increase. These results suggest that the dip-coating deposition method gives rise to surfaces with approximately three times more silaue linkers than the CVD deposition method.

The subsequent addition of DHPs to the azide-Zalkyne-functionalised surfaces gave further changes in carbon composition (Table 1 ). For the AzPTS surfaces coupled with alkyne- F8-phenyl-DHP, a more than 35 % increase in carbon content was observed for both samples coupled via the CVD and dip-coat AzPTS linkers indicating successful attachment of DHP to the surfaces. Similarly, for the complimentary immobilisation approach azide- F8-phenyl-DHP attachment onto alkyne-PTS functionalized surfaces gave a further increase in carbon content of 6.2 % and 4.1 % for CVD and dip-coated samples respectively.

Furthermore, the detection of bromine further confirmed the successful coupling of DHP, as well as illustrated the relative amounts of attached DHP on the surfaces generated via the two different approaches (Table 1 ). DHP attachment via the AzPTS linker led to more detectable bromine compared to attachment via the alkyne-PTS linker, with bromine compositions of 2.8 and 2.3 Br for DHP surfaces coupled by CVD and dip-coat AzPTS respectively, and 1.3 and 1.7 %Br respectively for CVD and dip-coat alkyne-PTS surfaces.

High resolution XPS measurements revealed the progression of the click chemistry reactions, particularly at the N 1s region. Representative XPS N Is narrow scan spectra of AzPTS modification via CVD and dip-coat deposition are shown in Figure 1 and the N Is spectra of the subsequent attachment of DHP are shown in Figure 2. The C Is and N I s binding energies with respective assignments to surface functionalities are shown in Tables 2 and 3.

The CVD and dip-coat AzPTS N Is spectra showed similar signals which were deconvoluted and fitted to three peaks (Figure 1 ). The two peaks at 400.7 and 404.3 eV with a ratio of approximately 2:1 displayed the characteristic azide double-peak, in which the higher binding energy component is attributed to the relatively electron-poor middle nitrogen atom of the azide group. The third peak at 399.3 eV can. be assigned to degradation products of azide, such as N2O and NO which arise due to prolonged scanning.

Changes in intensity of peaks in the N Is narrow scan illustrated the completeness of the click reaction. Upon coupling the azide-terminated surfaces with alkyne-FS-phenyl-DHP, the intensity of the characteristic azide double-peak markedly reduced (Figure 2). For the DHP sample coupled via the CVD AzPTS linker, the azide peak (404.3 eV) was no longer observable, whilst two new peaks at 400.5 eV and 401.7 eV arise representing characteristic peaks for 1,2,3-triazole and amide (from the DHP) respectively. This indicated that all of the azide functionality had reacted and been convened into the triazole group, evidence of a complete click reaction and successful attachment of DHP. Similarly for the DHP sample coupled via the dip-coat AzPTS linker, a 1,2,3-triazole peak was formed at 400.5 eV and an amide peak at 401.8 eV, however a small peak at 404.5 eV representing the characteristic azide peak remained. In addition, by calculating the percentage of area reduction of the 404 eV peak it was estimated that approximately 85% of the azide on the dip-coat Az^'PTS surface was reacted to give the triazole moiety.

Table 2: The C is binding energies and proposed assignments.

Table 3: The Λ' Is binding energies and proposed assignments.

For DHP attachment on the alkyne-terminated surlaces, a less distinctive difference was observed between the CVD and dip-coat alkyne- PTS surfaces due to lack of unique peak for the alkyne functionality. The resolved C Is spectrum revealed three peaks at 285.0, 286.5 and 289.3 eV which correspond to aliphatic carbon (C-C and C=C), carbon-nitrogen and carbon-oxyget (C-N and C~0), and c^bamaie (N-CO-O) xe^peetively (see Figure and Table 2). No distinctive difference apart from overall peak intensity was observed between the samples prepared by CVD and dip-coat method. Upon coupling with azide- F8-phenyl-DH^'P, an extra peak arises at 286.4 eV, corresponding to the carbon-bromine bond (C-Br) from the DHP (see Figure 3B) indicating successful surface modification via this approach.

Attachment of Other Alkyne-DHP Derivatives

From the results shown above the coupling of alkyne-mnetionalised DHP with a gas-phase deposited azide-tenmnated surface was found to give the greatest DHP attachment efficiency. This approach was selected for attachment of other DHP derivatives. The carbon, nitrogen and halogen contents of the surfaces coupled with a range of alkyne-DHP derivatives are shown in Table 4.

Table 4: Elemental analysis of alkyrie-DHPs coupled surfaces.

Significant increases in carbon and nitrogen percentages were observed tor all alkyne-DH P treated samples in comparison to the CVD azide surface indicating successful coupling of DHPs. Moreover, detection of halogen for alkyne-F8-DHPs, alkyne-F30-DHP and alkyne- DHP2 coupled surfaces indicated that considerable amounts of DHPs were immobilized.

Contact angle measurement

Changes in surface hydrophobicity provide an alternative indication of successful suriace modifications. Contact angles were measured using a contact angle goniometer ( ame- Hart, inc. NRL USA, Model no. 100-00). Multiple drops of deionised water were placed on each surface using a micro-syringe. The angle between the water droplet and surface was measured with a 50 mm Cosmicar Television Lens (Japan), and the contact angle was calculated by Rame-Hart Imaging software. A minimum of 5 measurements were made on 5 different samples. Larger contact angles correspond to increased hydrophobicity. Static water contact angles were measured for all samples and are shown in Table 5.

Table 5: Contact angle measurements

A significant increase in hydrophobicity was observed after the modification of AzPTS. and no significant difference was observed between the CVD and dip-coat deposition methods. The contact angles were 80° and 76° tor CVD and dip-coat AzPTS respectively. Similarly, for alkyne-PTS treated surfaces contact angle increased markedly with no significant difference between the two deposition methods (66° and 71 ° tor CVD and dip- coat respectively). Both increases in surface hydrophobicity indicate successful modification of surface with the silane coupling agents.

The subsequent coupling of all DHPs resulted in further increases in hydrophobicity (81^c to 90°), which is consistent with the addition of hydrophobic carbon components from the DHPs.

DL ion

It was found that the coupling of alkyne-DHP with azide-tenninated surfaces yielded greater attachment efficiency compared to the coupling of azide-DHP with alkyne- terminated surfaces. The results above suggest thai the optimum strategy for the attachment of DHP via CuAAC is through the coupling of an alkyne-functionalised DHP with a gas-phase deposited azide surface.

The attachment of DHP to a surface via a Click reaction is significantly more efficient than attachment of DHP to a surface via Michael addition (Ho el al, Biofouling. vol. 26, No. 8, 2012, 13-921). The DHP coating produced through CuAAC consists of approximately twice as much DHP attached to the surface compared to a surface produced using the Michael addition approach. This is evidenced by the fluorine detection of samples treated with a compound that has a similar structure (0.99 %F detection for DHP2 surface vs. 1 .9 %F for alkyne-DHP2 surface: DHP2 has the same structure as the alkyne-DHP2 but with an acrylate group in place of the ethynyl group).

Example 2 - In vitro antimicrobial efficacy of surfaces to which are attached DHP

In this Example, the antimicrobial efficacy of the optimised DHP surfaces shown below was assessed against two clinically relevant pathogens. These coatings were prepared by the general method depicted in Scheme 8.

Antibacterial activity

The strains of bacteria used for this study were Staphylococcus aureus strain 38 and P idomonas aeruginosa PAO I . These strains were streaked onto LB agar and incubated overnight at 37°C. A single colony was cultured overnight in 10 ml of tryptone soya broth (TSB; Oxoid, UK) medium at 37~C The resulting bacteria were collected by centrifugation and re-suspended in the same volume of TSB twice. Optical density of the resulting culture was adjusted to OD^ = 0.1 in TSB supplemented with 0.25% w/v glucose. The glass surfaces to be tested were placed individually in 6-well plates followed by addition of 4 ml of the bacterial suspension. The plates were incubated at 37°C with shaking at 120 rpm for 24 h, then the media was replaced with fresh TSB containing 2.5% w v glucose (4 ml) and plates incubated as before for a farther 24 h. The surfaces were then gently rinsed twice with phosphate buffered saline (PBS) to remove non-adherent bacteria before examination by fluorescent microscopy or scanning electron microscopy (SEM).

Analysis of Bacterial Adhesion

The glass samples with adhered bacteria prepared as described above were stained with Live/Dead BacLight Bacterial Viability Kits L-7007 (Molecular Probes, Inc, Eugene, OR) according to the manufacturers' procedure. Briefly, 2 μΙ_ of the two components were mixed thoroughly in 1 L of PBS. 10 μL· of the solution were then trapped between the sample and the glass microscopy slide and allowed to incubate at room temperature in the dark for .15 min. The samples were observed and imaged with an Olympus FV1000 Confocal Inverted Microscope. For bacterial adhesion, images from 15 representative areas on each of triplicate samples tor each surface were taken. Cells that were stained green were considered to be viable, those that stained red were considered to be dead as were those that stained both green and red.

A11 confocal images were analysed using ImageJ software, which measured the area fraction covered by green (live) or red (dead) cells in the field of view. The image analysis results were reported as the average percentage coverage of live cells and dead cells in the fields of view.

Statistical Analysis

Data were analysed by the one-way analysis of variance (ANOVA) using IBM SPSS Statistics 20 software (version 20.0.0). Differences between the groups were analysed using post hoc Games-Howell correction and results with p < 0.05 were considered significant.

Cytotoxic assay

A direct contact material toxicity assay was conducted to determine the effect of the DHP- coated material on mammalian cells and ISO 10993-5 procedures were followed |iSO 10993 Part 5 Biological evaluation of medical devices - Part 5: tests for cytotoxicity: in vitro methods. (1995E)]. Earle's L, NCTC clone 929 (Murine) cells were grown in minimum essential media with non-essential amino acids (MEM/NA) supplemented with 10% fetal bovine serum (FBS), and were grown to near confluency in plastic petri dishes. The medium was aspirated and replaced with a small volume of fresh medium and materials to be tested were placed directly on the cell monolayer for 24 h. During this time any cytotoxic components emanating from the test materials will disrupt the normal functions of cells beneath and perhaps adjacent to the samples. After the incubation period cells were stained with a vital stain (Trypan Blue) and cytotoxicity was assessed using bright field and phase-contrast microscopy. Silastic medical grade tubing (Dow Corning Corporation, USA) was used as negative control and surgical latex glove (Ansell Medical Victoria, Australia) was used as positive control. Cytotoxic responses were graded according to a standard key, which quantifies the zonal extent of cell damage (0 to 4 maximum). A reactivity grade >1 is indicative of a significant cytotoxic response under the conditions of this assay.

Antimicrobial Activity by Fluorescence Microscopy

The click-DHP coatings described above were assessed for their antimicrobial activity against . aeruginosa and S. aureus. Fluorescence microscopy with the aid of live/dead staining was used to investigate the bacterial adhesion and biofilm formation on the modified samples. Representative images for untreated (blank), process controls (azide- and alkyne-treated), and three selected DHP coated samples are shown in Figure 4 and Figure 5 for P. aeruginosa and S. aureus respectively. The areas of the surfaces covered by bacteria and the relative proportions of live and dead bacteria (stained green and red, respectively) for each surface were evaluated by image analysis and the results are shown in Figure 6.

Extensive bacterial colonization and biofilm formation were observed on the untreated control by both P. aeruginosa and '. aureus, as indicated by the high density of live (green-stained) bacterial coverage (Figure 4 and Figure 5). Samples treated with azide and alkyne coupling agents exhibited similar patterns of bacterial colonisation but with reduction in bacterial adhesion for both strains of bacteria. Further reductions in bacterial cell adhesion were observed for all DHP-treated samples for both bacterial strains compared to the controls, with no signs of biofilm formation.

To compare the level of bacterial adhesion between samples quantitative image analysis was performed on the confocal micrographs. Results of image analysis showed high percentage coverage by both/', aeruginosa and S. aureus on the blank control, with 13.3 ± 1.6% and 15.9 ± 2.1 % of total adherent bacteria respectively (Figure 6).

For aeruginosa, the azide- and alkyne-terminated surfaces exhibited significant reductions in the surface area covered by adherent bacteria of 51.5 ± 10.6% and 39.5 ± 12.6 % respectively compared to control (p < 0.05), with no significant difference between the two process control samples (Figure 6). More pronounced reductions in bacterial coverage were observed for all DHP-treated samples, with reduction of 90.6 - 97.3 % compared to control (p < 0.001). Of these coatings, surfaces coupled with alkyne-F30- DHP and alkyne-DHPl performed significantly better than other DHP samples against P. aeruginosa (p < 0.05), with 94.3 ± 6.2 % and 97.3 ± 1.6 % reduction respectively. There was no significant difference between the samples coupled with aikyne-F8-DHP, alkyne- FSs-DHP, and alkyne-DHP2_? whereas azide-F8-DHP coated sample was significantly less active compared to all DHPs (p < 0.05). There was no significant increase in the proportion of dead (red-staining) cells for all modified samples.

For 8. aureus, similarly to P. aeruginosa, significant reductions in the surface area covered by adherent bacteria were observed for the azide and alkyne process controls with reductions of 60.9 ± 6.3 % and 68.6 ± 4.9 % respectively, and they were not significantly different from each other (Figure 6). Markedly further reductions in bacterial coverage were observed for all DHP-treated samples compared to control (p < 0.001 ). Alkyne- DHPl and alkyne-DHP2 showed significantly fewer adherent bacteria than other DHP samples (p < 0.05), with reductions of 96.8 ± 1.4 % and 96.3 ± 1.4 % respectively compared to untreated control. There were no significant differences between the alkyne- F8-DHP, alkyne-FSs-DHP and alkyne-F30-DHP samples, whilst azide-F8-DHP was significantly different from all DHPs (p < 0.05). The proportion of red staining cells did not increase for all modified samples.

Cytotoxicity of dick-D P Samples

The direct contact material toxicity assay showed no negative effect on the L929 cells (no cell damage) when in contact with untreated, azide-terminated and alkyne-F8-DHP samples (grade 0). The negative and positive controls behaved as expected with the negative control showing slight physical cell damage (grade 1) and positive control exhibiting all dead cells (grade 4). The FS-DHP glass was thus not toxic within the parameters of the assay. Discussion

The covalently bound DHP analogues were found to be able to significantly reduce adhesion of both P. aeruginosa and . aureus bacteria at up to 97 % reduction in bacterial adhesion compared to the untreated control. Of these compounds, covalently bound aikyne-F30-DHP and alkyne-DH.Pl were found to lie the most effective in reducing the bacterial adhesion of P. aeruginosa. While tor S. aureus the. most effective compounds were alkyne-DHPl and alkyne-DHP2. Together, the results indicate that while all the DHP compounds tested are effective in reducing bacterial adhesion, alkyne-DHPl gave the best broad spectrum activity.

Substrates with higher concentration of surface-bound DHP demonstrated greater effectiveness in resisting bacterial colonisation. The substrate generated by coupling alkytie-F8-DHP to an azide-temunated surface was found to give approximately double the amount of surface-attached DHP when compared to the substrate prepared via the complimentary approach (azide-F8-DHP coupled to alkyne-terminated surface). The increased surface concentration of DHP endowed the former sample with significantly greater reduction in adhesion of both /·'. aeruginosa and .V. aureus. Although the difference between the two complimentary approaches was not substantial, it indicates that the antimicrobial efficacy of a DHP coating is dependent on the surface concentration of DHP.

The DHP coatings produced using a click, reaction showed a vast improvement in antimicrobial efficacy compared to coatings produced using Michael addition chemistry (Ho et £?/, Biofouling, vol. 26, No. S, 2012, 913-921). Coatings prepared using Michael addition chemistry exhibited reductions of 65.8 % and 79.3 % for P. aeruginosa and S. aureus respectively. In contrast, the alkyne-DHPl coating demonstrated reductions of 97.3 and 96.8 % respectively for . aeruginosa and S. aureus, representing a remarkable improvement in antimicrobial performance of over 31 % for P. aeruginosa and 17 % for S. aureus. Hence, optimisation of surface attachment of DHP using a Click reaction dramatically increases the antimicrobial efficacy of the coating.

Claims

Claims

1. A compound of formula (I), or a salt thereof:

wherein,

R.j is selected from the group consisting of: hydrogen, halogen, heteroar l, aryl, O-C20 alkyl, C2-C20 alken l and C2-C20 alkynyl, wherein the heteroaryl. aryl, Ci-Cio alkyl. the C2- C20 alkenyl and the C2-C20 alkynyt groups are optionally substituted with one or more of the following substituents: hydroxy, halogen or OC1-C5 alkyl;

R2 is selected from the group consisting of: hydrogen, halogen., alkyl, heteroaryl, and aryl, wherein the alkyl, heteroaryl, aryl groups are optionally substituted with one or more of the following substituents: halogen, hydroxy, OC1-C0 alkyl. amino, alkenyl or alkynyl;

R j and R are independently selected from the group consisting of: hydrogen and halogen;

R; is selected from the group consisting of: C1-C20 alkylene, C2-C20 alkenylene, C -C2 alkynylene, -<CH2)_w-Ph-(CH₂) -, -(CH^Ph-HCHz - or -^CHjk-Ph-CCH.y-J- wherein w is 0, I , 2, 3, 4, 5 or 6, y is 0, 1 , 2, 3, 4, 5 or 6, J is 0, S or NH; and

X is a functional group suitable for attachment to a solid surface.

2. A compound according to claim 1 , or salt thereof, represented by:

3. A compound according to claim 1 or claim 2, or a salt thereof, wherein Ri is selected from the group consisting of: hydrogen and C\-C(, alkyl, wherein the Cj-Gs a!kyl group is optionally substituted with one or two of the following substituems: hydroxy or halogen.

4. A compound according to any one of claim 1 to 3, or a salt thereof, wherein is selected from the group consisting of: hydrogen, halogen and phenyl, wherein the phenyl group is optionally substituted with one or more of the following substituenls: halogen, hydroxy or OCi-C« alkyl.

5. A compound according to any one of claim 1 to 4, or a salt thereof, wherein Ra and 4 are independently selected from the group consisting of: hydrogen, iodo. chloro and bromo.

6. A compound according to any one of claim 1 to 5, or a salt thereof, wherein R? is Cj-C« alkytene, -(CHi -P HCH^ or -(CH₂ P ~0« CH₂)^ wherein w is 0, I or 2 and y is 0. 1 or 2.

7. A compound according to claim 1 selected from the group consisting of:

azide-FS-benzene-DHP alkyne-F8- enzene-DHP alkyne-F8-DHP

Br

alkyne-F30-DHP

alkyne-DHP1 alkyne-DHP2

8. A compound of formula (II), or a salt thereof:

wherein Rt, Ra, R.*, R , R5 and X are as defined in claim l .

9. An antimicrobial composition comprising a compound according any one of claims 1 to 8.

10. An antimicrobial composition according to claim 9, wherein the composition further comprises one or more additional antimicrobial agents.

1 1. A method tor eliminating or inhibiting the growth of one or more microorganisms, or the colonisation of an environment by one or more microorganisms, the method comprising contacting the one or more microorganisms, or an environment inhabited by the microorganisms, with an effective amount of a compound according to any one of claims Ϊ to 8, or a composition according to claim 9 or claim 10.

12. A method for inhibiting the adherence of one or more microorganisms to a surface, the method comprising attaching to the surface at least one compound according to anyone of claims 1 to 8,

13. A method for preventing the occurrence of microbial infection on or around the surface of a medical device inserted into a patient, or at or near the site of insertion of the medical device, the method comprising attaching to a surface of the device, or coating a surface of the device ith, at least one compound according to any one of claims 1 to 8.

14. A method for preparing a device having at least one surface, the method comprising reacting the at least one surface with at least one compound according to any one of claims 1 to 8.

15. A method according to claim 14, wherein the device is a medical device.

16. A method tor modifying a surface, the method comprising reacting the surface with at least one compound according to any one of claims I to 8.

17. A compound array comprising:

a) a functionalised solid surface; and

b) at least one compound of formula (la),

(la)

wherein i, R>, R*. Rt and R$ are as defined in claim 1 and Y is a divalent functional group attached to the functionalised solid surface.