WO2021159176A1 - Antimicrobial compounds - Google Patents

Abstract

Provided herein are compounds of formula (I) which comprise a cephalosporin antibiotic (present moiety X), bonded to a piperidine diazeniumdiolate (NONOate) or a salt thereof. Also provided are compositions that include a compound described herein and methods for promoting the dispersal of microorganisms from biofilms, inhibiting biofilm formation or development, treating and preventing biofilm-associated diseases and disorders or a Pseudomonas aeruginosa infection in a subject suffering from cystic fibrosis, and killing a biofilm-forming microorganism. (I)

Description

Antimicrobial compounds

Field of the disclosure

[0001] The present disclosure relates generally to antimicrobial compounds and uses thereof.

Background of the disclosure

[0002] Any discussion of the prior art throughout this specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Biofilms are high-density populations of bacterial cells encapsulated within a self- produced polysaccharide matrix. They protect bacteria from a range of stressors such as antibiotics, a hosts immune system and hydrodynamic shear forces. Biofilm-encased bacteria are estimated to be up to 1000-fold more resistant to antibiotics than their planktonic counterparts. In addition to affording protection from antibiotics, biofilms protect pathogens in vivo from host immune antibodies and phagocytes.

[0004] More than 80% of all infections are estimated to be biofilm-related, with many of these being chronic, such as infections on artificial heart valves and other medical indwelling devices, as well as urinary tract infections, diabetic wounds and respiratory infections in cystic fibrosis sufferers. Currently there are few options for treating biofilm infections. In this context, there is a need for new antimicrobial compounds, particularly compounds that disrupt or disperse biofilms or inhibit biofilm formation or development.

Summary of the disclosure

[0005] In a first aspect there is provided a compound of the formula (I), or a salt thereof

wherein X is:

R₁ is a substituent that corresponds to a substituent attached to the 7-NHC(0)- group of a cephalosporin antibiotic, and

T is a straight-chain or branched-chain alkanediyl group having between 1 and 8 carbon atoms.

[0006] In one embodiment, T is a straight-chain or branched-chain alkanediyl group having between 1 and 4 carbon atoms.

[0007] In another embodiment, T is -CH₂- or -CH₂CH₂-.

[0008] In a second aspect there is provided a composition for promoting the dispersal of microorganisms from a biofilm or inhibiting the formation and/or development of a biofilm, the composition comprising a compound according to the first aspect.

[0009] In a third aspect there is provided a method for killing a biofilm-forming microorganism, the method comprising exposing the microorganism to an effective amount of a compound of the first aspect.

[0010] The microorganism may have been dispersed from a biofilm.

[0011] The microorganism may be part of a biofilm.

[0012] In a fourth aspect there is provided a method for promoting dispersal of microorganisms from a biofilm, the method comprising exposing the biofilm to an effective amount of a compound of the first aspect or a composition of the second aspect.

[0013] In a fifth aspect there is provided a method for inhibiting biofilm formation and/or development, the method comprising exposing biofilm-forming microorganisms to an effective amount of a compound of the first aspect or a composition of the second aspect.

[0014] In accordance with the fourth and fifth aspects, the compound or composition comprising the same may be coated, impregnated or otherwise contacted with a surface or interface susceptible to biofilm formation. In one embodiment the surface may be a surface of an implantable medical device, prosthesis or medical or surgical equipment.

[0015] In some embodiments of the fourth and fifth aspects the biofilm may be on a bodily surface of a subject, internal or external to the subject, and exposure of the biofilm or biofilm- forming microorganisms to the compound or composition may be via administration of the compound or composition to the subject Administration may be via any suitable route depending on the nature and location of the biofilm or biofilm-forming microorganisms.

[0016] In accordance with the above aspects and embodiments, the biofilm may be surface- associated or suspended. The suspended biofilm may be in the form of floes or granules. Typically in accordance with the above aspects and embodiments the biofilm or biofilm- forming microorganisms are exposed to an effective amount of a compound or composition as defined herein such that the concentration of the nitric oxide donor or nitric oxide released and thus exposed to the biofilm or microorganisms is non-toxic to the environment or to the subject in which the biofilm or microrganisms are found. For example, the concentration of nitric oxide may be in the nanomolar, micromolar or millimolar range. The nitric oxide concentration may be, for example, from about 1 nM to about 500 μΜ.

[0017] The microorganisms present in the biofilm may be of a single species or of multiple species. The microorganisms present within the biofilm or capable of forming a biofilm may be bacteria. The microorganisms within the biofilm or capable of forming a biofilm may comprise one or more species selected from, for example, Acinetobacterspp.,Pseudomonas spp.,Pseudoalieromonas spp.,Staphylococcus spp,.Streptococcus spp.,Shigella spp., Mycobacterium spp.,Enterococcusspp.,Escherichiaspp.,Salmonellaspp.,Legionellaspp., Haemophilus spp.,Bacillus spp.,Desulfovibrio spp.,Shewanella spp.,Geobacter spp., Klebsiellaspp.,Neisseriaspp.,Proteusspp Aeromonasspp.,Arthrobacterspp.,Micrococcus spp.,Burkholderia spp.,Serratiaspp.,Porphyromonasspp.,Fusobacterium spp.and Vibrio spp.Inparticularembodimentsthemicroorganism isPseudomonasaeruginosa.

[0018] In a sixth aspect there is provided a method for treating or preventing a biofilm- associated infection, disease or disorder in a subject wherein the infection is caused by a microorganism capable of forming a biofilm, the method comprising administering to the subject an effective amount of a compound of the first aspect or a composition of the second aspect.

[0019] The disease or disorder may be, for example, chronic obstructive pulmonary disease (CORD), bacterial endocarditis, gonorrhoea, otitis media, Legionnaire's disease, tuberculosis, kidney stones, urinary tract infections, pulmonary infections, dental plaque, dental caries and infections associated with surgical procedures or bums.

[0020] The compound or composition may be administered with a β-lactamase inhibitor.

[0021] The β-lactamase inhibitor may be clavulanic acid, tazobactam, sulbactam, avibactam, vaborbactam or relebactam.

[0022] The compound or composition may be administered contemporaneously, simultaneously or sequentially with the β-lactamase inhibitor.

[0023] In a seventh aspect there is provided a method for treating or preventing a Pseudomonas aeruginosa infection in a subject suffering from cystic fibrosis, the method comprising administering to the subject an effective amount of a compound of the first aspect or a composition of the second aspect.

[0024] The Pseudomonas aeruginosa may be present in the form of, or as part of, a biofilm.

[0025] The compound or composition may be administered to the subject by inhalation.

[0026] The compound or composition may be administered with a polymyxin or tobramycin.

[0027] The compound or composition may be administered contemporaneously, simultaneously or sequentially with the polymyxin or tobramycin.

[0028] The polymyxin may be colistin.

[0029] In an eight aspect there is provided use of a composition of the first aspect for inhibiting biofilm formation and/or development.

[0030] In a ninth aspect there is provided use of a compound of the first aspect for promoting dispersal of microorganisms from a biofilm.

[0031] In a tenth aspect there is provided use of a compound of the first aspect in the manufacture of a medicament for treating or preventing a biofilm-associated infection, disease or disorder in a subject wherein the infection is caused by a microorganism capable of forming a biofilm.

[0032] In an eleventh aspect there is provided use of a compound of the first aspect in the manufacture of a medicament for treating or preventing a Pseudomonas aeruginosa infection in a subject suffering from cystic fibrosis. [0033] In a twelfth aspect there is provided use of a compound of the first aspect for killing a biofilm-forming microorganism.

Brief description of the drawings

[0034] Figure 1. Stability of compounds 17 to 22 in the presence of (i) B. cereus and (ii) P. aeruginosa β-lactamases. Arrows indicate addition of the following to a reaction vessel containing 20 mL PBS (pH 7.4) at 25 °C: a. test compound at final concentration 50 μΜ, b. 100 μL of 40 U mL^-1 B. cereus β-lactamase or 25 μL of 40 U mL^-1 P. aeruginosa β-lactamase.

[0035] Figure 2. Dispersal responses of P. aeruginosa PA30 (left) and PA68 (right) clinical isolate ex-vivo biofilms following treatment with varying concentrations of the spontaneous NO donor sodium nitroprusside (SNP). The means of six technical replicates and standard deviations are shown. Dashed lines indicate the mean of untreated controls. Experiments were repeated three times with similar results. One-way ANOVA statistical testing with Dunnett’s multiple comparison test was used to compare treated biofilms with untreated controls.

[0036] Figure 3. Activity of compound 22 and ceftazidime (CAZ) against PA30 ((a) and (b)) and PA68 ((c) and (d)) biofilms. Biofilms were grown for 24 hours and treated with each compound (128 μg/mL, 3 h) before staining remaining cells with SYT09 (green; alive) and propidium iodide (red; dead). COMSTAT analysis was performed with image quantification of total biomass, thickness distribution, maximum thickness and total surface area coverage. Representative 3D CLSM images obtained after compound treatments are shown for: (a) PA30 and (c) PA68 biofilms. One-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis, with compound treatment groups compared to controls and to each other, ns = not significant.

[0037] Figure 4. Compound 22 concentration versus time profiles in mouse: (a) plasma and (b) lung tissue homogenate after SC dosing (75 mg/kg). Data represent the mean ± SEM.

[0038] Figure 5. Effects of CAZ and compound 22 in an acute P. aeruginosa (FDA-CDC AR- BANK#0264) lung infection model in neutropenic mice. Animals were inoculated intranasally (0.02 mL/lung) with 9.1 x 10⁵ CFU per mouse. Ceftazidime and compound 22 (50 and 120 mg/kg; n = 5 for each cohort) were administered SC every 4 hours starting 2 hours after infection. At 26 hours after inoculation, lung tissues were harvested and weighed and the lung bacterial burden measured (CFU/g). ‘Significant difference (p < 0.05) compared to the vehicle control was determined by one-way ANOVA followed by Dunnett’s test. Definitions

[0039] 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.

[0040] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely".

[0041] The term "about" is understood to refer to a range of +/- 10%, preferably +/- 5% or +/- 1% or, more preferably, +/- 0.1%.

[0042] 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.

[0043] As used herein the term "biofilm" refers to any three-dimensional, matrix-encased microbial community displaying multicellular characteristics. Accordingly, as used herein, the term biofilm includes surface-associated biofilms as well as biofilms in suspension, such as floes and granules. Biofilms may comprise a single microbial species or may be mixed species complexes, and may include bacteria as well as fungi, algae, protozoa, or other microorganisms.

[0044] The term "biofilm-forming microorganism" refers to any microorganism that is capable of forming biofilms, either single species or mixed species biofilms.

[0045] As used herein the term "dispersal" as it relates to a biofilm and microorganisms making up a biofilm means the process of detachment and separation of cells and a return to a planktonic phenotype or behaviour of the dispersing cells.

[0046] As used herein the term "effective amount" includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired effect. The exact amount required will vary from subject to subject depending on factors such as the species of microorganisms being treated, the extent, severity and/or age of the biofilm being treated, whether the biofilm is surface-associated or suspended, the particular agent(s) or compound(s) being administered, the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

[0047] As used herein the term "exposing" means generally bringing into contact with. Typically, direct exposure refers to administration of the agent or compound to the microorganism or biofilm to be treated or otherwise bringing the microorganism or biofilm into contact with the agent itself. Typically, indirect exposure refers to the administration of a precursor of the active agent or compound, or a compound or molecule capable of generating, either solely or in reaction with other compounds or molecules, the active agent or compound to the microorganism or biofilm, or otherwise bringing the microorganism or biofilm into contact therewith. Thus, a microorganism or biofilm may be exposed to a compound or composition as defined herein directly or indirectly. Further, a microorganism or biofilm may be exposed to nitric oxide released from a compound directly or indirectly. In the context of the present disclosure, indirectly "exposing" a biofilm or microorganisms to a compound or composition as described herein also includes the administration of the compound or composition to a subject in or on which the biofilm or microorganisms reside. As such, in the present disclosure the terms "exposing", "administering" and "delivering" and variations thereof may, in some contexts, be used interchangeably.

[0048] The term "inhibiting" and variations thereof such as "inhibition" and "inhibits" as used herein in relation to biofilms means complete or partial inhibition of biofilm formation and/or development, and also includes within its scope the reversal of biofilm development or processes associated with biofilm formation and/or development. Further, inhibition may be permanent or temporary. The inhibition may be to an extent (in magnitude and/or spatially), and/or for a time, sufficient to produce the desired effect. Inhibition may be prevention, retardation, reduction or otherwise hindrance of biofilm formation or development. Such inhibition may be in magnitude and/or be temporal or spatial in nature. Further, such inhibition may be direct or indirect. By indirect inhibition is meant that the agent may affect the expression or activity of molecules which in turn regulate biofilm formation or development.

[0049] The term "pharmaceutically acceptable" as used herein refers to substances that do not cause substantial adverse allergic or immunological reactions when administered to a subject. A "pharmaceutically acceptable carrier'' includes, but is not limited to, solvents, coatings, dispersion agents, wetting agents, isotonic and absorption delaying agents and disintegrants.

[0050] As used herein the term "surface" includes both biological surfaces and non-biological surfaces. Biological surfaces typically include surfaces both internal (such as organs, tissues, cells, bones and membranes) and external (such as skin, hair, epidermal appendages, seeds, plant foliage) to an organism. Biological surfaces also include other natural surfaces, such as wood or fibre. A non-biological surface may be any artificial surface of any composition that supports the establishment and development of a biofilm. Such surfaces may be present in industrial plants and equipment, and include medical and surgical equipment and medical devices, both implantable and non-implantable. Further, for the purposes of the present disclosure, a surface may be porous (such as a membrane) or non-porous, and may be rigid or flexible.

[0051] As used herein the terms "treating", "treatment", "preventing" and "prevention" refer to any and all uses which remedy a condition or symptoms, prevent the establishment of a condition or disease, or otherwise prevent, hinder, retard, or reverse the progression of a condition or disease or other undesirable symptoms in any way whatsoever. Thus the terms "treating" and "preventing" and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery.

[0052] In the context of this specification the term "alkanediyl" is understood to refer to a bivalent saturated branched-chain or straight-chain hydrocarbon group conforming to the formula C_nH_2n.

Detailed description

[0053] Low concentrations of nitric oxide (NO) have been found to modulate bacterial signalling and metabolic processes, rendering biofilm bacteria more susceptible to antibiotics. To reduce potential safety risks associated with systemic NO exposure, the present inventors have developed NO donor compounds that release NO upon reaction with bacterial enzymes, thereby localising NO exposure to sites of infection.

[0054] In particular, the present inventors have developed cephalosporin-3'- diazeniumdiolates as a class of β-lactams that carry a chemically stable, O²linked diazeniumdiolate NO donor at the cephalosporin 3'-position. The compounds may undergo β-lactam ring cleavage in the presence of transpeptidases/penicillin-binding proteins (PBPs), thereby triggering ejection of an unstable diazeniumdiolate anion (NONOate) which, in turn, spontaneously emits NO leading to biofilm dispersal. The reactivity towards PBPs is such that as well as promoting biofilm dispersal, the compounds may also kill planktonic cells thereby negating the need to co-administer antibiotics. In direct contrast to known cephalosporin-3'- diazeniumdiolates, the present class of compounds also possess β-lactamase stability, thereby optimising their reactivity with PBPs. [0055] In one aspect there is provided a compound of the formula (I), or a salt thereof:

wherein X is

R₁ is a substituent that corresponds to a substituent attached to the 7-NHC(0)- group of a cephalosporin antibiotic, and,

T is a straight-chain or branched-chain alkanediyl group having between 1 and 8 carbon atoms.

[0056] The compounds of formula (I) may have two chiral centres. The present disclosure includes all enantiomers and diastereomers, as well as mixtures thereof in any proportions. The disclosure also extends to isolated enantiomers or pairs of enantiomers. In embodiments of the disclosure X has the following structure:

[0057] Also within the scope of the compounds of formula (I) are salts, including pharmaceutically acceptable salts. Salts of the compounds of formula (I) may be prepared by conventional methods known to those skilled in the art. For example, base-addition salts may be prepared by reacting the compounds of formula (I) with a suitable base. Examples of such salts include alkali metal salts, such as lithium, potassium and sodium, and alkali earth metal salts, such as calcium, magnesium and barium. Additional basic salts include, but are not limited to, ammonium, copper, iron, manganese and zinc salts. Acid addition salts may be prepared by reacting the compounds of formula (I) with organic or inorganic acids. Examples of such salts include HCI, HBr and HI salts, salts of other mineral acids such as sulfate, nitrate, phosphate and the like, alkyl and monoarylsulfonates such as ethanesulfonate, toluenesulfonate 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) may also be quatemised by reaction with compounds such as (C₁-C₄)alkyl halides, for example, methyl, ethyl, isopropyl and butyl halides.

[0058] T may be a straight-chain or branched-chain alkanediyl group having between 1 and 6 carbon atoms, or between 1 and 5 carbon atoms, or between 1 and 4 carbon atoms, or between 1 and 3 carbon atoms.

[0059] In some embodiments, T is-CH₂-or-CH₂CH₂-.

[0060] R₁ may be a substituent corresponding to a substituent attached to the 7-NHC(0)- group of any one or more of the following: ceftolozane, cefmatilen, cefaloram, cefazolin, cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazedone, cefazaflur, cefradine, cefroxidine, ceftezole, cefaclor, cefamandole, cefminox, cefonicid, ceforanide, cefotiam, cefprozil, cefbuperazone, cefuroxime, cefuzonam, , cefotetan, cefmetazole, flomoxef, cefixime, ceftriaxone, ceftazidime, cefoperazone, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, cefteram, ceftibuten, ceftiolene, ceftizoxime, latamoxef, cefepime, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline fosamil, an oxacephem, cefclidin, cefluprenam, cefoselis, cefozopran, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide, or ceftiofur.

[0061] In some embodiments, R₁ is a substituent corresponding to a substituent attached to the 7-NHC(0)- group of a cephalosporin antiobiotic, and wherein the substituent includes the following functional group:

[0062] In alternative embodiments, R₁ is a substituent corresponding to a substituent attached to the 7-NHC(0)- group of cefaloram, cefotaxime or ceftazidime.

[0063] In alternative embodiments, R₁ is a substituent corresponding to a substituent attached to the 7-NHC(0)- group of cefotaxime or ceftazidime.

[0064] In alternative embodiments, R₁ is a substituent corresponding to a substituent attached to the 7-NHC(0)- group of ceftolozane, cefotaxime or ceftazidime.

[0065] In further embodiments, R₁ is a substituent corresponding to a substituent attached to the 7-NHC(0)- group of ceftolozane, cefepime, cefuroxime, ceftazidime, cefotaxime, ceftriaxone or cefozopran.

[0066] In alternative embodiments, R₁ is a substituent corresponding to a substituent attached to the 7-NHC(0)- group of ceftolozane or ceftazidime.

[0067] In a further embodiment, R₁ is a substituent corresponding to a substituent attached to the 7-NHC(0)- group of cefepime, ceftolozane or ceftazidime.

[0068] In one embodiment, the compound of formula (I) has a structure selected from the group consisting of:

[0069]Inanotherembodiment,thecompoundofformula(I)hasastructureselectedfrom the groupconsistingof:

[0070] The compounds of formula (I) may be prepared according to Scheme 1 below:

Scheme 1 - Preparation of compounds of formula (I)

[0071] Synthesis of the sodium diazeniumdiolate reagent 24 may be achieved by dissolving the N-Boc-4-alkylamino piperidine 23 in a MeOH/Et₂O mixture containing sodium methoxide in a Paar-Knorr vessel and treating with nitric oxide gas under pressure at 50 psi. Shaking at room temperature for 48 h delivers 24 in high yield. In situ Finkelstein conversion of the orthogonally-protected 3-chloro-cephalosporin intermediate 25 with Nal/acetone, followed by reaction with sodium diazeniumdiolate reagent 24 afforded the penultimate intermediate 26. Subsequent deprotection with TFA (between about 20 and about 50 equivalents) in molten phenol provides compounds of formula (I).

[0072] Intermediate 25 may be prepared according to Scheme 2, in which commercially available 7-amino-p-methoxybenzyl-protected 3-chloro-cephalosporin ester. HCI salt 8 is coupled with appropriately substituted carboxylic acids using EDCI and DMAP. The Ri group of the acid may be chosen based on the desired Ri group to be included in the compounds of formula (I).

Scheme 2 - Preparation of intermediate 25

[0073] Compositions according to the present disclosure may be in any suitable form. Typically, the form will depend on that which is most suitable for application or delivery to the required site and thus will vary with different medical, industrial and domestic applications. For example, a composition may be formulated for in vivo administration, such as in the form of a liquid, suspension, nasal spray, eyedrops, powder, tablet, capsule, cream, paste, gel or lotion. For industrial and domestic applications the composition may be formulated as a paint, wax, other coating, emulsion, solution, gel, suspension, beads, powder, granules, pellets, flakes or spray. The skilled addressee will also recognise that the appropriate formulation will depend on the particular application and the proposed route of delivery. Suitable routes of administration for in vivo applications include, for example, oral, nasal, parenteral (e.g. intravenous, topical, intraarterial, intramuscular, intraocular), transdermal and subcutaneous administration.

[0074] Compositions of the present disclosure may also include carriers, diluents or excipients. Suitable carriers, diluents and excipients are known to those skilled in the art. The diluents, adjuvants and excipients must be "acceptable" in terms of being compatible with the other ingredients of the composition, and in the case of pharmaceutical compositions, not deleterious to the recipient thereof. Carriers may be liquid or solid. In the case of liquid carriers, the liquid may be an aqueous or non-aqueous solvent

[0075] Examples of pharmaceutically acceptable diluents may include demineralised or distilled water; saline solution; vegetable-based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3- butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly.

[0076] In some examples, a further level of controlled release may be desirable. For pharmaceutical applications, a number of suitable controlled release systems are known in the art. For example, polymeric colloidal particles or microencapsulates (microparticles, microspheres or nanoparticles) in the form of reservoir and matrix devices may be employed, or the compound may be contained by a polymer containing a hydrophilic and/or teachable additive eg, a second polymer, surfactant or plasticiser, etc. to give a porous device, or a device in which the drug release may be osmotically 'controlled' (both reservoir and matrix devices). Large cage-like molecules such as the C₆₀ Buckminster-fullerenes ('Buckyballs') or hyperbranched (starburst) dendrimers may also be used.

[0077] Typically, for anti-fouling and other industrial applications, the composition, for example in the form of a paint or other surface coating, may employ a carrier enabling the controlled release of the compound temporally and/or spatially. A variety of methods to achieve controlled release of bioactive agents are known to those skilled in the art and may include, for example, encapsulation of the active agent in a suitable polymer or polymer-based product. The polymer may be an organic or inorganic polymer, for example a polyolefin, polyether, polyester, polyamide, polyurethane or polypeptide. Suitable polymers for providing controlled release are known to those skilled in the art, for example as disclosed in United States Patent No. 6,610,282.

[0078] Typically, the rate of release of the substance is influenced by the properties of the polymer itself as well as environmental factors (such as pH, temperature etc). Controlled release systems are capable of delivering substances slowly and continuously for up to several years. By way of example only, release may be diffusion controlled, chemically controlled or solvent activated. [0079] In diffusion controlled systems, diffusion of the agent trapped within a polymer matrix is the rate-determining factor for the overall release rate. One type of diffusion controlled system employs a reservoir device in which the agent forms a core surrounded by an inert diffusion barrier. These systems include membranes, capsules, microcapsules, liposomes, and hollow fibers. Alternatively, the device may be a monolithic device in which the active agent is dispersed or dissolved in an inert polymer. Diffusion through the polymer matrix is the rate-limiting step, and release rates are determined in part by the choice of polymer and its consequent effect on the diffusion and partition coefficient of the agent to be released.

[0080] In typical chemically controlled systems a polymer degrades overtime and releases an agent in an amount proportional to the gradual erosion. Chemical control can be achieved using bioerodible or pendant chains. In a bioerodible system the agent is ideally distributed uniformly throughout a polymer in the same way as in monolithic diffusion systems. As the polymer surrounding the agent is eroded, the agent escapes. In a pendant chain system, the agent is covalently bound to the polymer and is released by bond scission owing to water or enzymes.

[0081] In typical solvent-activated controlled systems, the active agent is dissolved or dispersed within a polymeric matrix and is not able to diffuse through that matrix. Osmotic pressure is used as the driving force for release of the agent. In one type of solvent-controlled system, as the environmental fluid (e.g., water) penetrates the matrix, the polymer (e.g a hydrogel) swells and its glass transition temperature is lowered below the environmental (host) temperature. Thus, the swollen polymer is in a rubbery state and allows the drug contained within to diffuse through the encapsulant

[0082] With regard to anti-fouling applications, self-polishing antifouling coatings are known in the art. Such coatings are typically based on polymers of tributyltin methacrylate, methyl methacrylate and film softening monomers, such as 2-ethylhexyl acrylate. An organotin polymer typically acts as the paint binder. Such paints may also contain a toxicant additive, such as cuprous oxide or a triorganotin compound. In addition, the usual paint additives such as pigments, thixotropic agents may also be present. In normally alkaline seawater, the polymeric organotin binder is gradually hydrolyzed, and the tributyltin is liberated in a form that is an active antifoulant The hydrolyzed polymer formed is water-soluble or water-swellable and is easily eroded off the surface by moving seawater, exposing a fresh surface of paint.

[0083] Compounds and compositions of the present disclosure may be included in pharmaceutical, cosmetic, dermatological or topical delivery compositions as preservatives to inhibit or prevent the growth and/or colonisation of unwanted microorganisms. The compositions of the disclosure are therefore useful for preventing spoilage and hence increasing the usable shelf life of any type of pharmaceutical, cosmetic, dermatological or topical delivery compositions to which they are added. The compounds or compositions of the disclosure may be conveniently included in any solid or liquid pharmaceutical, cosmetic, dermatological or topical delivery composition during the manufacture thereof, or alternatively after manufacture. The term "cosmetic composition" is understood to mean a composition intended for placement in contact with any external part of an animal body, including the mucous membranes of the oral cavity, the teeth, the hair and the nails, for the purpose of, for example: protecting, perfuming, cleansing, maintaining (i.e. moisturising or exfoliating), beautifying, altering the appearance of, or altering the odour of, the body. Examples of cosmetic compositions include but are not limited to: nail care products, make up, products intended for application to the lips, face masks and scrubs, hair tints, dyes and bleaches, products for waving, straightening and fixing hair, cleansing products such as lotions, powders and shampoos, conditioning products such as. lotions, creams, oils, hairdressing products such as lotions and lacquers, products for care of the teeth and the mouth, including toothpastes, mouthwashes, tongue cleaners, dental bleaches/whiteners and denture cleansers, perfumes, toilet waters, Eau de colognes, feminine hygiene products, deodorants, antiperspirants, cleansers such as toilet soap, deodorant soap, astringent and skin washes, shaving products such as creams, foams and lotions, bath and shower preparations such as salts, foams, oils, gels, etc., depilatories, after-bath powders, hygienic powders, moisturising products such as creams, lotions, gels and foams, sunbathing products (without SPF or SPF <4), anti-wrinkle products (without SPF) and anti-ageing products (without SPF).

[0084] Those skilled in the art will readily appreciate that the delivery systems, compositions and methods described above are merely examples of suitable methods and systems that may be employed. Any other suitable carriers and delivery systems may be employed to achieve the desired means of application of agents according to embodiments of the present disclosure.

[0085] The compounds and compositions disclosed herein find application in a wide range of environments and circumstances. The following is a brief discussion of some general areas of application. However, those skilled in the art will readily appreciate that any environment or situation in which biofilm development is a problem, or in which it is desirable to inhibit microbial growth, will be potentially suitable.

[0086] The present disclosure provides methods of treating a biofilm-associated infection, disease or disorder in a subject the method comprising administering to the subject an effective amount of a compound or a composition of the disclosure. The infection may, for example, be an oral cavity infection, a lung infection, a urinary tract infection, a digestive tract infection, a respiratory tract infection, a cervical infection, an ear infection, an eye infection, a wound (e.g., diabetic, bum, cut, abrasion) infection or a heart infection. The compounds of the present disclosure may be used to treat a range of biofilm-associated diseases or disorders, including chronic obstructive pulmonary disease (CORD), bacterial endocarditis, gonorrhoea, otitis media, Legionnaire's disease, tuberculosis, kidney stones, urinary tract infections, pulmonary infections, dental plaque, dental caries and infections associated with surgical procedures or bums.

[0087] The compounds of the present disclosure preferably disrupt biofilms, inhibit the formation or development of biofilms or kill biofilm-forming microorganisms. The biofilm may be a bacterial biofilm comprising Gram-positive or Gram-negative bacteria. The biofilm may comprise one or more bacterial species selected from Pseudomonas spp., Pseudoalteromonas spp., Staphylococcus spp., Streptococcus spp., Shigella spp., Mycobacterium spp., Enterococcus spp., Escherichia spp., Salmonella spp., Legionella spp., Haemophilus spp., Acinetobacter spp., Neisseria spp., Bacillus spp., Desulfovibrio spp., Shewanella spp., Geobacter spp., Klebsiella spp., Proteus spp., Aeromonas spp., Arthrobacter spp., Micrococcus spp., Burkholderia spp., Serratia spp., Porphyromonas spp., Fusobacterium spp. and Vibrio spp. For example, the bacteria may be Pseudomonas aeruginosa, Staphylococcus epidennidis, Staphylococcus aureus, Mycobacterium tuberculosis, Escherichia coli, Bacillus licheniformis, Bacillus cereus, Burkholderia ce nocepacia, Serratia marcescens, Fusobacterium nucleatum, Streptococcus pneumoniae, Klebsiella pneumoniae, Acinetobacter baumannii, Neisseria gonorrhoeae, Enterococcus faeca!is, Haemophilus influenzae, Proteus mirabilis, Vibrio cholera or any combination thereof.

[0088] By virtue of their preferential reactivity with PBPs, the compounds of the present disclosure find use in killing biofilm-forming microorganisms. The microorganisms may be as defined in the previous paragraph. In some embodiments, the microorganisms may be microorganisms that have been dispersed from a biofilm (such as, for example, planktonic cells). In alternative embodiments, the microorganisms may be part of a biofilm.

[0089] Cystic fibrosis (CF) is the most frequently passed genetic disorder in Western Europe. CF patients suffer from chronic Pseudomonas aeruginosa infections. When infecting the lung of a CF patient, Pseudomonas aeruginosa undergoes a characteristic transition from an acute virulent pathogen to a CF-adapted pathogen allowing it to persist in the lung for many years, even decades. This is due to the overproduction of the matrix polysaccharide alginate, leading to the formation of a mucoid biofilm that tolerates antibiotics, components of both the innate and adaptive immune response and resists phagocytosis. As described further below, compounds of the present disclosure possess activity against a number of clinical isolates obtained from CF patients. As such, the present disclosure further provides a method for treating or preventing a Pseudomonas aeruginosa infection in a subject suffering from cystic fibrosis comprising administering to the subject an effective amount of a compound or composition of the disclosure.

[0090] In some examples, compounds of the present disclosure inhibit Pseudomonas aeruginosa growth with a minimum inhibitory concentration (MIC) of between about 0.05 μg/mL and 70 μg/mL, such as between about 1 μg/mL and 70 μg/mL, or between about 1 μg/mL and 60 μg/mL, or between about 1 μg/mL and 50 μg/mL, or between about 1 μg/mL and 40 μg/mL, or between about 1 μg/mL and 35 μg/mL, or between about 1 μg/mL and 20 μg/mL, or between about 1 μg/mL and 10 μg/mL, or between about 1 μg/mL and 5 pg/mL

[0091] The compounds of the present disclosure may, if desired, be administered to a subject together with a further antibiotic or antimicrobial agent either contemporaneously, simultaneously or sequentially. The further antibiotic or antimicrobial agent may be, for example, a polymyxin including colistin or an octapeptin, a β-lactam, a monopenem, a carboxypenem, an aminoglycoside, a quinolone, a macrolide, a lincozamide, a tetracycline, a streptogramin, a glycopeptide, a rifamicin, a sulfonamide, chloramphenicol, nalidixic acid, an azole-containing compound, a peptide antibiotic, ceftazidime, azithromycin, tobramycin, tetracycline, a detergent, a surfactant, an agent that induces oxidative stress, a bacteriocin, an antimicrobial enzyme such as a lipase, a pronase, a nuclease, a lyase (e.g. alginate lyases) and various other proteolytic enzymes, a peptide or a phage. The antibiotics and antimicrobial agents may be natural or synthetic.

[0092] In some embodiments, the compounds of the present disclosure may be administered to a subject together with a β-lactamase inhibitor either contemporaneously, simultaneously or sequentially. Non-limiting examples of β-lactamase inhibitors include clavulanic acid, tazobactam, sulbactam, avibactam, vaborbactam and relebactam. Those skilled in the art will recognise that many new β-lactamase inhibitors are currently in development and that all such inhibitors that are developed for clinical use, as well as further inhibitors that may be developed for clinical use in the future, are within the scope of the present disclosure.

[0093] The compounds of the present disclosure may be administered to a subject by any suitable route, for example inhalation, oral, rectal, transmucosal, topical, intestinal, parenteral, intramuscular, transcutaneous, intradermal, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, intraocular, intracavitary, intravesical, intramuscular, intraarterial, intravenous or subcutaneous. [0094] The compounds described herein are preferably used in amounts such that a low, non- toxic concentration of NO is released in the vicinity of the target microorganism. The concentration may be in the nanomolar, micromolar, or millimolar range. In some examples, the concentration is between about 1 nM and about 100 mM, between about 10 nM and about 50 mM, between about 25 nM and about 50 mM, between about 50 nM and about 25 mM, between about 100 nM and about 10 mM, between about 200 nM and about 1 mM, between about 500 nM and 500 μΜ, between about 500 nM and 100 μΜ, or between about 1 pM and about 50 pM. In one embodiment, the concentration is about 500 nM. The most suitable concentration to achieve the desired effect will depend on a number of factors and may be determined by those skilled in the art using routine experimentation. Such factors include, but are not limited to, the particular compound used for NO release, the means or route of administration of the compound, the nature, structure and age of the biofilm, the species of microorganism to be treated and so on.

[0095] The compounds of the present disclosure may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the infection or condition being treated, whether a recurrence is considered likely, etc. The administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, e.g., once per day over a period of days, once per hour over a period of hours, or any other such schedule as deemed suitable.

[0096] Compounds, compositions and methods of the present disclosure may also be used in coating medical devices, including medical and surgical equipment and implantable medical devices, including but not limited to venous catheters, drainage catheters (e.g. urinary catheters), dialysis catheters, stents, pacemakers, contact lenses, hearing-aids, percutaneous glucose sensors, dialysis equipment, drug-pump related delivery cannula, prostheses such as artificial joints, hearts, heart valves or other organs and medical fixation devices (e.g. rods, screws, pins, plates and the like). Further, embodiments of the present disclosure may find application in wound repair, as for example, compounds and compositions comprising the same may be impregnated or coated onto sutures and wound dressings such as bandages.

[0097] Compounds, compositions and methods of the present disclosure may also find application in a range of industrial and domestic applications, including but not limited to water supply reservoirs and feed 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 suitable applications may include, for example, marine anti-fouling paints or coatings, for example in treating ship hulls, aquaculture equipment, fishing nets or other in-water structures.

[0098] Those skilled in the art will appreciate that the aspects and 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.

[0099] The present disclosure is further described by reference to the following non-limiting examples.

Examples

Example 1 - Synthesis of compounds

[00100] Piperidines 1 and 2 were purchased from Combi-Blocks, San Diego CA. Nitric oxide gas was purchased from Asia Pacific Gas Enterprise Company Ltd. Anhydrous acetone was prepared by heating analytical reagent (AR) grade solvent at reflux overnight with Drierite granules (anhydrous CaSO*) before distilling onto 4A molecular sieves (pre-dried overnight at 400 °C) under N₂. Anhydrous methanol was prepared by distilling AR grade solvent onto 3A molecular sieves (pre-dried overnight at 400 °C) under N2. Anhydrous ether was prepared by distillation from sodium/benzophenone ketyl. The term "petroleum spirit" or "pet. spirit" refers to petroleum spirit within the boiling range 40-60 ⁰ C. Analytical thin layer chromatography (TLC) was performed using SiliaPlate Aluminium Backed F254, 200 μm plates and SiliaPlate

Aluminium Backed C18 F254, 150 μm plates from Silicycle. Normal phase column chromatography was performed using SiliaFlash P60, 40-63 μm , 230-400 mesh silica gel. Reverse phase column chromatography was performed using SiliaBond C18 (carbon 17 %), 60 A, 40-63 μm , 230-400 mesh silica gel from Silicycle. Centrifugation was performed using a REMI R-8C BL centrifuge. ¹H and ¹³C NMR spectra were recorded on Broker 400 and 500 MHz spectrometers. Chemical shifts (δ) are relative to tetramethylsilane (TMS, 0 ppm). High resolution electrospray mass spectra were recorded on a Waters XEVO Ultima spectrometer. The final target compounds were lyophilised using a CHRIST Alpha 1-2 LD freeze dryer at - 53 °C and 0.15 mbar.

Compounds 3 and 4

[00101] Compounds 3 and 4 were prepared according to Scheme 3 below:

[00102] /V-Boc-4-alkylamino piperidines 1 and 2 (2.5 g) were dissolved in a mixture containing 10 mL of anhydrous 25% sodium methoxide/methanol and 40 mL of anhydrous diethyl ether in a Paar-Knorr reaction vessel. The mixture was purged with nitrogen (bubbling for 15 mins) before the reaction vessel was sealed and charged with nitric oxide gas at 50 psi. After shaking for 48 h the white precipitate was collected by vacuum filtration, washed with copious dry diethyl ether under argon and dried under vacuum.

[00103] Compound 3 was prepared using the general procedure above from compound 1 (2.50g,11.7mmol)togive3asawhitesolid(3.11g,90%).¹H NMR (DMSO-d₆,500MHz)δ 6.86(bs,1H),2.94(d,2H,J= 10.7Hz),2.88-2.81(m,4H),1.68(d,2H,J= 11.5Hz),1.37(bs, 9H),1.36-1.39(m,1H,overlapped),1.26 (qd,2H,J=11.8 Hz,J= 3.5 Hz).¹³C NMR (DMSO- d6,125MHz)d155.8,77.4,51.9,45.1,35.3,28.9,28.3.

[00104]Compound 4 was prepared using the generalprocedure above from compound 2 (2.50g,10.9 mmol)togive4 asawhite solid (3.16 g,93 %).¹H NMR (DMSO-d₆,500 Hz)δ 6.78 (t,1H,J= 5.4 Hz),2.97-2.84 (m,6H),2.88-2.81(m,4H),1.76-1.69 (m,2H),1.37 (bs, 9H),1.34-1.24 (m,5H).¹³C NMR (DMSO-d₆,125 MHz)d 155.6,77.3,51.8,37.5,35.9,32.0,

31.1,28.3.

Compounds 5 and 6

[00105] Compounds 5 and 6 were prepared according to Scheme 4 below:

[00106] Protected compounds 5 and 6 were prepared from the salt 8 as follows. To a stirred solution of the acid (1 eq.), 8 (1 eq.) and 4-N , N-dimethylaminopyridine (DMAP) (0.5 eq.) in CH₂CI2 (15 mL) at 0 °C was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (1.1 eq.) in small portions and the resulting mixture was stirred for 1 h. The reaction mixture was diluted with CH₂Cl₂(150 mL) and washed with water (100 mL) and brine (50 mL). The organic fraction was dried over anhydrous MgSO₄, filtered and evaporated to give a yellow residue that was purified by silica gel column chromatography to afford the acylated products.

[00107] Compound 5 was prepared using the above general procedure from 9 (2.00 g, 4.51 mmol), 8 (1.83 g, 4.51 mmol), EDCI (0.770 g, 4.96 mmol) and DMAP (0.275 g, 2.25 mmol) to give 5 as a pale yellow solid (3.01 g, 84%). ¹H NMR (CDCl₃, 400 MHz) δ 7.34-7.27 (m, 17H), 7.04 (s, 1H), 6.91-6.87 (m, 3H), 6.67 (s, 1 H), 5.91 (dd, 1H, J = 8.9 Hz, J = 4.9 Hz), 5.23 (d, 1H, J= 11.8 Hz), 5.19 (d, 1H, J= 11.8 Hz), 5.02 (d, 1 H, J= 5.0 Hz), 4.54 (d, 1H, J= 11.8 Hz), 4.42 (d, 1 H J = 11.8 Hz), 4.06 (s, 3H), 3.80 (s, 3H), 3.64 (d, 1H, J= 18.4 Hz), 3.46 (d, 1H, J = 18.3 Hz). ¹³C NMR (CDCl₃, 100 MHz) δ 168.6, 163.9, 162.3, 161.1 , 160.1, 147.2, 143.2, 140.1, 130.8, 129.3, 128.3, 127.7, 126.7, 126.2, 125.8, 114.1, 113.1, 71.9, 68.4, 63.4, 59.1 , 57.7, 55.4, 43.3, 27.4.

[00108] Compound 6 was prepared using the above general procedure from 10 (2.00 g, 3.50 mmol), 8 (1.42 g, 3.50 mmol), EDCI (0.597 g, 3.84 mmol) and DMAP (0.214 g, 1.75 mmol) to give 6 as a pale yellow solid (2.52 g, 78%). ¹H NMR (CDCI₃, 500 MHz) δ 8.21 (d, 1H, J = 9.0 Hz), 7.26-7.35 (m, 18H), 6.89 (d, 2H, J = 8.5 Hz), 6.72 (s, 1 H), 5.98 (dd, 1 H, J = 8.8 Hz, 5.0 Hz), 5.26 (d, 1 H, J = 12.0 Hz), 5.19 (d, 1 H, J= 12.0 Hz), 5.02 (d, 1 H, J= 5.0 Hz), 4.53 (d, 1 H, J = 11.5Hz),4.44 (d,1H,J= 11.5 Hz),3.81(s,3H),3.61(d,1H,J= 18.0 Hz),3.44 (d,1H,J = 18.0Hz),1.63(s,3H),1.59(s,3H),1.41(s,9H).¹³C NMR (CDCl₃,125MHz)δ174.2,168.2, 164.2,163.4,161.3,160.0,149.3,143.3,141.5,130.8,129.4,128.3,127.7,126.8,126.3, 125.9,114.1,112.6,83.1,82.3,71.9,68.3,59.3,57.9,55.4,43.5,28.1,27.5,24.4,23.8.

Compounds11to16

[00109]Compounds11to16werepreparedaccordingtoScheme5below:

[00110]Compounds11to16werepreparedbyreactionofcompounds5to7withcompounds 3 and 4 as follows. Sodium iodide (1 eq.)was added to a suspension ofPMB-protected cephalosporinesters5to7(1eq.)inanhydrousacetone(10mL)underargonandthemixture was stirred in the dark at room temperature for 1 h. Freshly prepared sodium diazeniumdiolates3and4(1eq.)wereaddedinoneshotandthemixturewasstirredatroom temperatureforafurther1.5hrs.Thesolventwasremovedunderreducedpressureandthe residuepurifiedbysilicagelcolumnchromatographytogivethedesiredadducts.

[00111]Compound11waspreparedusingtheabovegeneralprocedurefrom 7(500mg,1.03 mmol),Nal(154 mg,1.03 mmol)and 3(304 mg,1.03 mmol)togive 11asanoffwhitesolid (342mg,46%).¹H NMR (CDCl₃,500MHz)d7.38-7.26(m,7H),6.88(d,2H,J=8.7Hz),6.19 (d,1H,J=8.7 Hz),5.82 (dd,1H,J=9.0 Hz,J=4.9 Hz),5.21-5.15(m,3H),4.96 (d,1H,J= 14.0 Hz),4.91(d,1H,J=4.9 Hz),4.62 (bs,1H),3.86 (d,2H,J= 9.6 Hz),3.80(s,3H),3.67 (d,1H,J= 16.0Hz),3.62(d,1H,J= 16.0Hz),3.49(d,1H,J= 18.5Hz),3.41(d,1H,J= 18.5 Hz),3.01(t,2H,J=6.2Hz),2.84(t,2H,J= 12.0Hz),1.78(d,2H,J= 12.5Hz),1.65-1.55(m, 1H,overlappedbyH₂0),1.44(s,9H),1.36(t,2H,J= 12.8 Hz).¹³C NMR (CDCl₃,125MHz)d 171.3,164.8,161.4,160.1,156.2,133.8,130.8,129.6,129.3,127.9,126.8,126.4,125.8, 114.1,79.6,71.8,68.3,59.4,57.7,55.4,51.4,45.6,43.4,35.9,28.6,28.3,26.2.ESI-HRMS calcd.for747.2783[M+Na]⁺ C₃₅H₄₄O₉N₆SNa,found747.2791.

[00112]Compound12waspreparedusingtheabovegeneralprocedurefrom 7(500mg,1.03 mmol),Nal(154 mg,1.03 mmol)and4 (319 mg,1.03 mmol)togive 12asanoffwhitesolid (326mg,43%).1H NMR (CDCl₃,500MHz)δ7.36-7.25(m,7H),6.89(d,2H,J=8.7Hz),6.13 (d,1H,J=9.1Hz),5.81(dd,1H,J=9.1Hz,J=4.9 Hz),5.24-5.10(m,3H),4.93(d,1H,J= 14.1Hz),4.90(d,1H,J=4.9Hz),4.52(bs,1H),3.84-3.76(m,5H),3.66(d,1H,J=16.1Hz), 3.61(d,1H,J= 16.1Hz),3.50(d,1H,J= 18.6 Hz),3.40(d,1H,J= 18.6 Hz),3.15(d,2H,J= 5.9 Hz),2.82 (t,2H,J= 11.6 Hz),1.82 (d,2H,J= 10.8 Hz),1.46-1.33 (m,14H).¹³C NMR (CDCl₃,125 MHz)δ 171.1,164.7,161.3,159.9,156.0,133.7,130.7,129.5,129.2,127.8,

126.7,126.4,125.5,114.0,79.5,71.7,68.1,59.2,57.5,55.3,51.6,43.3,38.1,36.1,32.5,

30.7,28.4,26.1.ESI-HRMS calcd.for761.2939[M+Na]⁺ C₃₆H₄₆O₉N₆SNa,found761.2957.

[00113]Compound 13 was prepared using the above generalprocedure from 5 (500 mg, 0.629 mmol),Nal(94 mg,0.629 mmol)and 3 (187 mg,0.629 mmol)to give 13 asayellow solid(182 mg,28%).¹H NMR (CDCl₃,500MHz)d7.33-7.24 (m,18H),7.04 (bs,1H),6.89(d, 2H,J=8.7Hz),6.68(s,1H),5.90(dd,1H,J=8.8Hz,J=4.9),5.25-5.13(m,3H),5.03-4.98 (m,2H),4.65 (bs,1H),4.06 (s,3H),3.87(d,2H,J= 11.3 Hz),3.81(s,3H),3.54 (d,1H,J= 18.5Hz),3.46(d,1H,J= 18.4 Hz),3.01(t,2H,J=6.4 Hz),2.85(t,2H,J= 12.0Hz),1.78(d, 2H,J= 10.5 Hz),1.56 (bs,1H),1.41(s,9H),1.40-1.32 (m,2H).¹³C NMR (CDCl₃,125 MHz) d 168.7,163.9,162.2,161.4,160.1,156.1,147.3,143.2,141.0,130.8,129.4,128.4,127.8, 126.8,126.5,125.8,114.2,113.2,79.6,71.9,71.7,68.3,63.4,59.1,57.6,55.4,51.4,45.5, 35.9,28.5,28.3,26.4.ESI-HRMS calcd.for 1032.3743 [M+H]⁺ C₅₂H₅₈O₉N₆S₂Na ,found 1032.3762. [00114]Compound 14 was prepared using the above generalprocedure from 5 (500 mg, 0.629 mmol),Nal(94 mg,0.629mmol)and4 (195 mg,0.629mmol)togive 148asayellow solid (151mg,23%).¹H NMR (CDCl₃,400 MHz)d7.34-7.28 (m,17H),7.03 (s,1H),6.89 (d, 2H,J= 8.7 Hz),6.84 (d,1H,J=8.9 Hz),6.70 (s,1H),5.91(dd,1H,J=9.0 Hz,J=4.9 Hz), 5.27(d,1H,J= 14.0Hz),5.22(d,1H,J= 11.8Hz),5.17(d,1H,J= 11.8Hz),5.01(d,1H,J= 5.0Hz),4.97(d,1H,J= 14.0Hz),4.50(bs,1H),4.06 (s,3H),3.84-3.75(m,5H),3.55(d,1H, J= 18.5Hz),3.46(d,1H,J= 18.6Hz),3.15(d,2H,J=5.9Hz),2.83(t,2H,J= 11.1Hz),1.82 (d,2H,J= 10.2 Hz),1.44-1.40 (m,14H).¹³C NMR (CDCl₃,100MHz)d 168.7,163.9,162.2,

161.4,160.1,156.1,147.2,143.2,141.0,130.8,129.4,128.4,127.8,126.8,126.6,125.7, 114.2,113.2,79.4,71.9,71.8,68.3,63.5,59.1,57.5,55.4,51.8,38.2,36.3,32.6,30.8,28.5,

26.4.ESI-HRMS calcd.for1046.3899[M+H]⁺ C₅₃H₆₀O₁₀N₉S₂,found 1046.3905.

[00115]Compound 15 was prepared using the above generalprocedure from 6 (500 mg, 0.542 mmol),Nal(81mg,0.542 mmol)and 3 (161mg,0.542 mmol)to give 15 asayellow solid(195mg,31%).¹H NMR (CDCl₃,500MHz)d8.12(d,1H,J=8.7Hz),7.34-7.26(m,17H), 6.91-6.88 (m,3H),6.72 (s,1H),5.99 (dd,1H,J= 8.8 Hz,J= 5.0 Hz),5.29 (d,1H,J= 14.0 Hz),5.24(d,1H,J= 11.8Hz),5.17(d,1H,J= 11.8Hz),5.00(d,1H,J=5.0Hz),4.96(d,1H, J= 14.1Hz),4.63(bs,1H),3.86-3.81(m,5H),3.52(d,1H,J= 18.5Hz),3.43(d,1H,J= 18.5 Hz),3.03(t,2H,J=6.3 Hz),2.84 (tt,2H,J= 12.0 Hz,J=2.6 Hz),1.81(d,2H,J= 13.0 Hz), 1.62(s,3H),1.58(s,3H),1.60-1.54(m,1H,overlapped),1.44(s,9H),1.41(s,9H).1.41-1.36 (m,2H,overlapped).¹³C NMR (CDCl₃,125MHz)d174.0,168.2,164.3,163.4,161.6,160.0,

156.1,149.3,143.3,141.5,130.8,129.5,128.4,127.7,126.9,126.8,125.9,114.2,112.6,

83.1,82.2,79.6,72.0,68.2,59.3,57.8,55.4,51.5,45.5,35.9,28.5,28.4,28.2,26.5,24.4, 23.9.ESI-HRMS calcd.for1160.4580[M+H]⁺ C₅₉H₇₀O₁₂N₉S₂,found 1160.4546.

[00116]Compound 16 was prepared using the above generalprocedure from 6 (500 mg, 0.542 mmol),Nal(81mg,0.542 mmol)and 4 (168 mg,0.542 mmol)to give 16 asayellow solid(236mg,37%).¹H NMR (CDCl₃,500MHz)d8.11(d,1H,J=8.8Hz),7.34-7.26(m,17H), 6.91-6.88 (m,3H),6.72 (s,1H),5.99 (dd,1H,J= 8.8 Hz,J= 5.0 Hz),5.30 (d,1H,J= 14.1 Hz),5.24(d,1H,J= 11.8Hz),5.17(d,1H,J= 11.8Hz),5.00(d,1H,J=5.0Hz),4.95(d,1H, J= 14.1Hz),4.49(bs,1H),3.83-3.78(m,5H),3.52(d,1H,J= 18.4 Hz),3.43(d,1H,J= 18.5 Hz),3.16 (d,2H,J=5.9Hz),2.83(tt,2H,J= 11.6 Hz,J=2.7 Hz),1.82(d,2H,J= 10.0 Hz), 1.62 (s,3H),1.58(s,3H),1.48-1.44 (m,2H,overlapped,observedingHSQC),1.45-1.41(m, 1H,overlapped,observed in gHSQC),1.44-1.38 (m,2H,overlapped,observed in gHSQC), 1.44 (s,9H),1.41(s,9H).¹³C NMR (CDCl₃,125 MHz)d 174.1,168.3,164.3,163.4,161.7,

160.1,156.1,149.3,143.4,141.5,130.9,129.5,128.4,127.7,127.0,126.8,125.8,114.2, 112.6,83.1,82.2,79.4,72.0,68.2,59.3,57.8,55.4,51.8,38.2,36.3,32.6,30.8,28.6,28.2, 26.5,24.4,23.9.ESI-HRMS calcd.for1174.4742[M+H]⁺ C₆₀H₇₂O₁₂N₉S₂,found 1174.4760.

Compounds17-22

[00117]Compounds17to22werepreparedbydeprotectionofthecorrespondingcompounds 11to16asfollows.

[00118]Mixturescontainingcompounds11to16(1eq.)andphenol(3-4g)wereheatedto45 °C in a dry flask under an atmosphere ofargon. Upon complete melting of phenol, trifluoroaceticacid(0-50eq.)wasaddedandthemixturesstirredat45°C for2.5hrs.Addition of50% pet.spirit/ether(100 mL)tothe cooled reaction mixtures(room temperature)caused precipitationofawhitesolidthatwascollectedbycentrifugation(3000rpm,5min).Thepellets werewashedwithicecold anhydrousdiethyletherandthecrudesolidwaspurified byd e- reverse phase silicagelcolumnchromatography. Fractionscontainingthe pure product(as indicatedbyC18-reversephasesilicagelTLC)werecombinedandlyophilised.

[00119]Compound 17 was prepared using the above generalprocedure from 11 (200 mg,0.276 mmol)andTFA (422 μL,5.5mmol)togive 17asa fluffywhitesolid(102 mg,73%).¹H NMR (DMSO-

d₆,400MHz)d9.03(d,1H,J=8.4Hz),8.48(bs,2H),7.31-7.20(m,5H),5.54(dd,1H,J=8.4 Hz,J=4.8 Hz),5.25(d,1H,J= 11.7Hz),4.98(d,1H,J=4.9 Hz),4.77(d,1H,J= 11.7 Hz), 3.76(t,2H,J= 12.2 Hz),3.58(d,1H,J= 13.9Hz),3.50(d,1H,J= 14.0Hz),3.45(d,1H,J= 17.4Hz),3.30(d,1H,J= 17.5Hz),2.88-2.78(m,2H),2.76-2.67(m,2H),1.84(d,2H,J= 13.2 Hz),1.80-1.70 (m,1H),1.40-1.24 (m,2H).¹³C NMR (DMSO-d₆,100 MHz)d 171.0,164.3, 163.3,135.9,134.7,129.0,128.2,126.4,112.2(observedinHMBC),73.0,58.6,57.4,50.25, 50.17,42.9,41.6,32.8,27.4,27.325.5.ESI-HRMS calcd.for505.1864[M+H]⁺ C₂₂H₂₉N₆O₆S, found505.1886.

[00120]Compound 18 was prepared using the abovegeneralprocedurefrom 12(200mg,0.271 mmol)andTFA (414μL,5.4mmol)togive18as a fluffy white solid (114 mg,81%).¹H NMR

(DMSO-d₆,500MHz)d9.02(d,1H,J=8.6 Hz),8.24(bs,2H),7.30-7.20(m,5H),5.53(d,1H, J=9.2Hz),5.23(d,1H,J= 10.1Hz),4.97(s,1H),4.80(d,1H,J= 10.2Hz).¹³C NMR (DMSO- d₆,126 MHz)d 171.0,164.0,163.3,136.0,134.6 (observed ingHMBC)129.0,128.2,126.4, 112.5 (observed in gHMBC)73.0,58.6,57.4,50.9,41.6,36.4,32.9,31.5,29.9,25.4.ESI- HRMS calcd.for519.2020[M+H]⁺ C₂₃H₃₁N₆O₆S,found519.2051. [00121]Compound 19 was prepared using the above generalprocedure from 13 (200 mg, 0.194mmol)andTFA (593μL,7.8mmol)togive 19asa fluffywhitesolid(57mg,52%).¹H NMR (DMSO-d₆,400MHz)d9.54(d,1H,J=8.2 Hz),

8.45(bs,2H),7.22(s,2H),6.73(s,1H),5.65(dd,1H,J=4.8Hz,J=7.9Hz),5.24(d,1H,J= 11.7Hz),5.06(d,1H,J=4.9Hz),4.77(d,1H,J= 11.8Hz),3.83(s,3H),3.76(t,2H,J= 11.6 Hz),3.47(d,1H,J= 17.4Hz),3.30(d,1H,J= 17.5Hz),2.83(t,2H,J= 10.9Hz),2.72(d,2H, J=6.0Hz),1.83(d,2H,J=12.0Hz),1.75(m,1H),1.32(t,2H,J=11.1Hz).¹³CNMR(DMSO- d₆,100 MHz)d 168.4,164.3,163.0,162.4,149.1,142.6,134.6 (observedingHMBC),113.4 (observedingHMBC),108.9,73.0,61.9,58.2,57.5,50.3,50.2,43.0,32.8,27.44,27.38,25.7. ESI-HRMS calcd.for570.1548[M+H]⁺ C₂₀H₂₈O₇N₉S₂,found570.1561.

[00122]Compound20waspreparedusingthe above generalprocedure from 14 (200 mg,

0.191 mmol)and TFA (585 μL,7.6 mmol)to give20asafluffywhitesolid(51mg,46%).¹H NMR (DMSO-d₆,400 MHz)d9.54 (d,1H,J=

9.54 Hz),8.27(bs,2H),7.22 (s,2H),6.73(s,1H),5.65(dd,1H,J=7.9 Hz,J=4.8 Hz),5.22

(d,1H,J=11.7Hz),5.05(d,1H,J=4.8Hz),4.79(d,1H,J= 11.6Hz),3.83(s,3H),3.77-3.70 (m,2H),3.46(d,1H,J=17.5Hz),3.35-3.25(m,1H,overlappedbyH₂O,observedingHSQC), 2.85-2.77(m,4H),1.76 (d,2H,J= 12.6 Hz),1.52-1.40(m,3H),1.28-1.17(m,2H).¹³C NMR (DMSO-d₆,100MHz)d168.4,164.1,163.0,162.3,149.1,142.6,135.3(observedingHMBC),

113.1(observedingHMBC),108.9,73.0,61.8,58.2,57.5,50.9,50.8,36.4,32.9,31.5,30.0, 29.9.ESI-HRMS calcd.for584.1704[M+H]⁺ C₂₁H₃₀O₇N₉S₂,found584.1724.

[00123]Compound 21was prepared using the above generalprocedure from 15 (200 mg, 0.172mmol)andTFA (659μL,8.6mmol)togive 21asa fluffywhitesolid(43mg,39%).¹H NMR (DMSO-d₆,500MHz)d10.17(bs,1H),8.38(bs, 2H),7.25(bs,2H),6.73(s,1H),5.77(dd,1H,J

=7.7Hz,J=5.0Hz),5.11(d,1H,J=5.0Hz),5.04(d,1H,J= 12.0Hz),4.97(d,1H,J= 12.0 Hz),3.82 (d,2H,J= 11.2 Hz),3.50 (d,1H,J= 17.7 Hz),3.37 (d,1H,J= 17.9 Hz),2.92 (d, 1H,J= 11.4 Hz),2.87(d,1H,J= 11.4 Hz),2.68(d,2H,J=6.1Hz),1.82-1.68(m,3H),1.46 (s,3H),1.43(s,3H),1.38-1.17(m,2H).¹³C NMR (DMSO-d₆,125MHz)d176.3(observedin gHMBC),168.4,1637,163.3,163.2,149.7,142.9,132.0 (observed in gHMBC),117.2 (observed in gHMBC), 109.8, 82.3, 72.0, 58.4, 57.7, 49.9, 43.0, 32.8, 26.7, 25.5, 24.4, 23.9. ESI-HRMS calcd.for642.1759[M+H]⁺ C23H32N9O9S2,found642.1789.

[00124]Compound 22 was prepared using the above generalprocedure from 16 (200 mg,0.170mmol)andTFA (652μL,8.5mmol) togive22asafluffywhitesolid(37mg,33%). ¹H NMR(DMSO-d₆,500MHz)d9.96(bs,1H), 8.27(bs,2H),7.26(bs,2H),6.74(s,1H),5.75

(dd,1H,J= 7.7 Hz,J= 5.0 Hz),5.13 (d,1H,J= 11.9 Hz),5.09 (d,1H,J= 5.0 Hz),4.85(d, 1H,J= 11.9Hz),3.79-3/73(m,2H),3.49(d,1H,J= 17.5Hz),3.33(d,1H,J= 17.5Hz),2.83- 2.76 (m,4H),1.72 (d,2H,J = 11.6 Hz),1.46 (s,3H),1.43 (s,3H),1.49-1.40 (m,2H, overlapped,observedingHSQC),1.49-1.40(m,1H,overlapped,observedingHSQC),1.25- 1.16(m,2H).¹³C NMR (DMSO-d₆,125MHz)d176.0,168.4,164.0,163.2,163.1,149.6,142.8,

133.8(observedingHMBC),115.1(observedingHMBC),109.8,82.2,72.5,58.4,57.6,50.8, 50.7,36.4,32.9,31.5,29.7,25.7,24.4,23.9.ESI-HRMS calcd.for678.1735 [M+Na]⁺ C₂₄H₃₃N₉0₉S₂Na,found678.1727.

Example 2 - Antibacterial activity

[00125] Compounds 17 to 22 were screened for antibacterial activity against the following human pathogens: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae,

Acinetobacter baumannii, Enterococcus faecalis, Mycobacterium tuberculosis, Neisseria

Gonorrhoeae, Streptococcus pneumoniae and Pseudomonas aeruginosa. MIC measurements were obtained for a variety of strains from multiple laboratories using standard broth microdilution methods performed in accordance with the Clinical Laboratory Standards Institute (CLSI) guidelines. MICs of the corresponding cephalosporin antibiotics were measured for comparison, where available.

[00126] Briefly, overnight cultures were grown in LB broth. Antibiotic and compound stocks were prepared from powder fresh each day and filtered sterilised (except cephaloram and its associated compounds which were dissolved in DMSO). Antibiotics and compounds were plated as a 2-fold dose response from 128 μg/mL to 0.125 μg/mL with a maximum of 0.5% DMSO, final in assay concentration. Different 96 well plates were used for each strain to eliminate any chance of cross contamination. Growth inhibition of all bacteria was determined by measuring absorbance at 600 nm (OD₆₀₀) using a Tecan M1000 Pro monochromator plate reader. The percentage of growth inhibition was calculated for each well using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references.TheMIC foreachantibioticandcompoundwasrepeatedtwicemoreondifferent daysto providethree biologicalreplicates,andthefinalMIC valuewasthe modevaluefrom thethreerepeats.Table 1listsbacterialstrainsthatarerelevanttothisexample.

Table 1:Bacterialstrains

[00127]Withregardtothe Nanyang MIC measurements,MIC weredeterminedaccordingto standardprotocols(Wiegandetal.Nat.Protocol.(2008)3(2):163-175).Briefly,bacteriawere growninMullerHintonBroth(MHB)at37°C overnightandsubsequentlyinoculatedinto5mL offreshMHB at37°C untiltheyreachedmid-logphase(4-5h).Mid-logcellsweredilutedinto fresh MHB to afinaldensityof5x10⁵ cellspermL. MHB (50 uL)wasadded into a 96well plate and the compounds of interest were serially diluted across the plate. The bacterial suspension (50 μL) was added to each well to achieve a final cell density of 2.5 x10⁵ cells per mL. The plates were incubated at 37 "C with shaking (200 rpm) for 16-18 h at which time the MIC was determined by visual inspection as the highest dilution showing no visible growth.

[00128] The results are presented in Table 2.

T d C

A C I

Example3-AntibacterialactivityagainstapanelofP.aeruginosa CF clinicalisolates [00129]MICsofcompounds19to22andtheircorrespondingcephalosporinantibioticcontrols weremeasuredagainstP.aeruginosalaboratorystrainPA01andtenclinicalisolatesobtained from theexpectoratedsputum ofCF patients.The minimum inhibitoryconcentrations(MIC) ofcompounds 19 to 22 againstP.aeruginosa PA01 and clinicalisolateswere determined using the broth microdilution method in accordance with ClinicalLaboratory Standards Instituteguidelines.¹ Two-foldserialdilutionsoftestcompoundswere prepared ina96well plate. Compound dilutionswere transferredto a new 96 wellplate intriplicate,followed by addition ofinoculated cation-adjusted MuellerHinton broth (CAMHB)broth,prepared by diluting an overnightculture 1in 500 to achieve approximately 1x 10⁵to 10⁶ cells. A CFU countwascarriedouttoconfirm theexactcellnumberoftheinoculum.Blanksampleswere preparedusingcompounddilutionsanduninoculatedCAMHB broth.The96wellplateswere incubatedovernightat37 °C andtheOD valuesreadat584 nm usingthe FLUOstarOmega Microplate Reader(BMG Labtech).The MIC wasrecordedasthe lowestconcentrationthat inhibitedbacterialgrowth,asdetermined byvisibleturbidity.Theresultsareshownbelow in Table3.

[00130]Itwas observed thatcompounds 21 and 22 eithermaintained orimproved activity againstten ofthe eleven strains (MICs 0.5-16 μg/mL) relative to the parentantibiotic ceftazidime.Comparatorcompounds30to33showedpooractivityagainstmostoftheclinical isolates.

Example4-β-lactamasestability

[00131]The stability ofcompounds 17 to 22 in the presence oftwo representative β- lactamasesfrom BacilluscereusandPseudomonasaeruginosawasstudiedamperometrically using an Apollo 4000 free radical analyser (World Precision Instruments) fitted with an NO- specific probe (ISO-NOP). A saturated solution of NO (1.91mM at 20 °C) was prepared by bubbling NO into 10 mL of degassed distilled water for 10 min according to the manufacturer’s instructions. A 10-fold dilution was performed giving a 191 μΜ stock solution from which subsequent aliquots (10, 20, 40, 60, 80 and 100 μL) were diluted into 20 mL of PBS buffer (pH = 7.4,2.7 mM KCI,135 mM NaCI,1.75 mM KH₂P0₄,10 mM Na₂HPO₄)to calibrate the instrument response. Baseline NO levels were monitored for 5 min in 20 mL PBS buffer solution (pH = 7.4 at 25 °C) followed by injection of compounds 17 to 22 (50 mM). NO levels were monitored for 15 min. Cleavage of the compounds was promoted by addition of 25 μL 40 U mL¹ Pseudomonas aeruginosa β-lactamase (Sigma, L6170) or 100 μL40 U mL^-1 Bacillus cereus β-lactamase (Sigma, 61305). After steady state concentrations were reached, another aliquot of β-lactamase was added. Multiple trials of each compound were performed and a representative curve of each is presented in Figure 1.

[00132] Compound 18 underwent hydrolysis in the presence of the B. cereus enzyme, while compounds 17, 19 and 20 were more stable. Both of the ceftazidime-based analogues 21 and 22 were stable in the presence of this enzyme. Compounds 17 and 18 were highly susceptible to the P. aeruginosa β-lactamase while all four oxyimino ether analogues 19 to 22 showed enhanced stability. As this technique directly measures NO concentrations in solution, these assays also served to confirm that NO is released from the compounds following enzyme-mediated β-lactam ring opening.

Example 5 - NO-mediated dispersion of P. aeruginosa biofilms

[00133] P. aeruginosa clinical isolates PA30 and PA68 were confirmed to form reproducible ex-vivo biofilms using a crystal violet (CV) assay in 96 well microtitre plates. Biofilms were cultivated in M9 minimalmedia (pH 7;Formedium)containing 48 mM Na₂HP0₄,22 mM KH₂PO₄,9 mM NaCI,19 mM NH₄CI,and supplementedwith2 mM MgSO₄ (SigmaAldrich), 100 uM CaCI₂ (SigmaAldrich)and 20 mM glucose (Sigma Aldrich).Non-selective tryptone soya agar (TSA) plates (Oxoid) and LB agar plates (Formedium) were used for colony forming unit (CFU) counting. Overnight cultures were diluted 1 in 100 into freshly prepared M9 minimal media and added into a flat-bottom, tissue culture-treated 96 well microtitre plate (Thermo Scientific) with a minimum of six replicate wells per strain. The location of wells was altered such that for each group, 3 wells were located near the edge of the plate and 3 near the centre to control for the effects of well location on biofilm growth. Uninoculated M9 minimal media served as the negative control. Four replicate 96 well microtitre plates were set up for each strain for sacrificial analysis on days 1, 2, 3 and 5, following incubation at 37 "C. Daily media changes with fresh M9 minimal media were carried out for biofilms grown for more than 1 day. After incubation, the planktonic suspension was removed and discarded and biofilms were washed twice with PBS to remove any non-adherent cells. Biofilms were then stained with 0.1 % CV (Sigma Aldrich) for 15 minutes. The CV solution was removed and the biofilms rinsed with water 3 to 4 times, until clear, and left to air dry overnight. CV stained biofilms were resolubilised in 30% acetic acid (Sigma Aldrich) for 20 minutes and transferred to a new 96 well microtitre plate, where the absorbance was measured at 584 nm (LUOstar Omega Microplate Reader, BMG Labtech). Consistent and high absorbance readings were obtained with PA30 and PA68 on all days, confirming reproducible biofilm growth by both strains.

[00134] The dispersal responses of biofilms grown from P. aeruginosa CF clinical strains PA30 and PA68 to the spontaneous nitric oxide donor sodium nitroprusside (SNP) were examined using the crystal violet microtitre plate assay described above, with minor modifications. Briefly, overnight cultures were diluted 1 in 100 into freshly prepared M9 minimal media and added into a flat-bottom tissue culture-treated 96 well microtitre plates (Thermo Scientific) and incubated at 37 "C for 24 hours. Uninoculated M9 minimal media served as the negative control. After incubation, the planktonic suspension was removed and discarded and the biofilms were washed once with M9 minimal media to remove non-adherent cells. Sodium nitroprusside solution prepared fresh in M9 minimal media was serially diluted and added to wells containing biofilms. A minimum of 6 replicate wells were used for each treatment group, with the location of wells altered such that in each treatment group 3 wells were located near the edge of the plate and 3 near the centre. Biofilms were washed twice with PBS, stained with 0.1% CV for 15 mins, resolubilised in 30% acetic acid and the absorbance was measured at 584 nm. The dispersal responses of PA30 and PA68 ex-vivo biofilms to varying concentrations of SNP are shown in Figure 2.

Example 6 - Activity of compound 22 against P. aeruginosa ex-vivo biofilms grown from cystic fibrosis clinical isolates PA30 and PA68

[00135] Confocal laser scanning microscopy (CLSM) experiments were used to compare the effects of compound 22 and its parent antibiotic ceftazidime on PA30 and PA68 biofilms. Overnight cultures of the strains were diluted 1 in 100 in M9 minimal media, and 2.5 mL volumes were used to inoculate 35 mm glass-bottom microwell plates (MatTek Corporation). The plates were incubated at 37 "C with gentle shaking (50 rpm), with media changes at 24- hour intervals if grown for more than 24 hours. Biofilms were then treated with test compounds solubilised in M9 minimal media and re-incubated. Following treatment, biofilms were washed twice with Hank’s balanced salt solution (HBSS) to remove non-attached cells and biofilm viability was assessed using a LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies), containing dyes SYT09 and Propidium Iodide (PI). SYT09 stains bacterial cells with intact membranes fluorescent green, whereas PI stains cells with compromised membranes fluorescent red. Stock solutions of each dye were diluted to 2 μg/mL, added to the glass- bottom microwell plates and incubated in the dark at room temperature for 20 minutes. Biofilms were then examined under an inverted Leica SP8 confocal laser scanning microscope using the x63 oil immersion lens, with sequential scanning at 1 pm intervals. Argon and DPSS laser lines at 488 nm and 561 nm were used to excite the dyes, and the FITC filter set cube was used to acquire fluorescence images. A total of 5 replicate images were taken from each biofilm grown in the microwell plates. The locations within each well from where the images were taken were kept constant for each plate to avoid bias. The first image was taken from the centre of the well and four subsequent images were taken from the centre of each theoretical quadrant of the well. Images were obtained and analysed using the LAS AF software (Leica Microsystems GmbH). COMSTAT 2.0 software was used for quantitative analysis of images. Representative 3D CLSM images obtained after compound treatments are shown for PASO and PA68 biofilms in Figure 3 (a) and Figure 3 (c) respectively.

[00136] Treatment of PA30 biofilms with ceftazidime (128 μg/mL) showed an unexpected effect, where statistically significant increases were observed for three of the four biofilm parameters relative to untreated controls (Figure 3 (b)). No significant decreases were seen in any of the parameters relative to controls with 22 (128 μg/mL), however, all four showed reductions relative to ceftazidime. Treatment of PA68 biofilms with compound 22 (Figure 3(d)) produced a greater dispersion effect, where statistically significant reductions were observed for all four parameters relative to controls, whereas no differences were observed with ceftazidime. Significant reductions were observed for all parameters with compound 22 relative to ceftazidime, except maximum thickness.

Example 7 - Activity of compound 22 in an acuta P. aeruginosa lung infection model [00137] The efficacy of compound 22 was examined alongside ceftazidime in an acute P. aeruginosa lung infection model in neutropenic mice. In preparing for the model, a preliminary dose-escalation study was carried out (in mice) with compound 22 to investigate its tolerability. Male Institute of Cancer Research (ICR) mice weighing 25 ± 5 g were provided by BioLasco Taiwan (under Charles River Laboratories License). Animals were acclimated for 3 days prior to use and were confirmed to be in good health. All animals were maintained in a hygienic environment with controlled temperature (20 - 24 °C), humidity (30% - 70%) and 12-hour light/dark cycles. Free access to sterilized standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water were made available. All aspects of this work, including housing, experimentation, treatments and disposal of animals, were performed in accordance with the Guide for the Care and Use of Laboratory Animals: Eighth Edition (National Academy Press, Washington, D. C., 2011) in an AAALAC-accredited animal laboratory facility. The animal care and use protocol was reviewed and approved by the Institutional Animals Care and Use Committee (IACUC) at Pharmacology Discovery Services Taiwan, Ltd.

[00138] Compound 22 was administered via subcutaneous injection (SC) to groups of three male ICR mice. The compound was formulated in water for injection (WFI) at 10, 15, 20 and 60 mg/mL and administered at 5 mL/kg. Animals received an initial dose of 50 mg/kg. If the animals survived for 72 hours, the dose was increased for the next cohort to 100 mg/kg. If the animals survived for 72 hours after 100 mg/kg, the dose was increased to the highest tested dose 300 mg/kg for the next cohort Full clinical examinations and body weight changes were assessed. At each dose level, animals were observed for the presence of acute toxic symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhoea, salivation, lacrimation, vasodilation, piloerection, etc.) during the first 30 minutes, and again at 1 hour. Body weights were recorded pre-dose and at 72 hours. The animals were observed and mortality noted daily.

[00139] Compound 22 did not produce any adverse effects 30 minutes or 1 hour after SC injection of 50, 100 and 300 mg/kg (the maximum dose tested, Table 4).

Table 4: Acute toxicity symptoms in mice 1-hour post-dosing SC with compound 22. - No effects, ± Slight to moderate effects, + Severe effects, Inc. Increased , Dec. Decreased, Spont Spontaneous, C. Chronic , T. Tonic, C-T Chronic-Tonic, F. Fluid, V. Viscosity, Voc. Vocalization.

[00140]Alltreated animals survived (Table 5)with no significantbody weightchanges observedovera72-hourobservationperiod(Table6).

Table5:MousedeathsafterSC dosingwithcompound22.

Table6:Micebodyweightpre-andpost-dosingSC withcompound22.

Example8-Plasmaandlungpharmacokineticsofcompound22inmiceaftersubcutaneous dosing

[00141]The pharmacokinetics of compound 22 in mice were examined by monitoring concentrationsofthedruginplasmaandlungtissuehomogenatesovertimefollowingasingle SC bolus injection of 75 mg/kg. Compound 22 was dissolved in water for injection and administered via SC injection at 75 mg/kg (5 mL/kg) to male ICR mice weighing 20-30 g (n = 3). Drug-free mice (n = 3) were included as controls. Blood aliquots (300-400 μL ) were collected via cardiac puncture from individual anesthetized mice 5, 15, 30, 45, 60, 180, 360 minutes after injection in tubes coated with lithium heparin, mixed gently, then kept on ice and centrifuged at 2,500 xg for 15 minutes at 4°C, within 1 hour of collection. For control animals, blood was collected by cardiac puncture. The plasma was then harvested and kept frozen at -70°C until further processing. Immediately after the blood sampling, the whole lungs were quickly removed, rinsed with cold saline (0.9 % NaCI, g/mL), blotted with dry gauze, weighed, and kept at -70 °C until further processing. Lungs were homogenized in an appropriate volume of cold PBS (pH 7.4) for 10 seconds on ice. The lung homogenate was then stored at -70 °C until further processing.

[00142] The plasma samples were processed using acetonitrile precipitation and analyzed by LC-MS/MS. A plasma calibration curve was generated from aliquots of drug-free plasma spiked with compound 22 at the specified concentrations. The spiked plasma samples were processed together with the unknown plasma samples using the same procedure. The processed plasma samples were stored at -70 °C until LC-MS/MS analysis, at which time peak areas were recorded and the concentrations of compound 22 in the unknown plasma samples were determined using the appropriate calibration curve. The reportable linear range of the assay was determined, along with the lower limit of quantitation (LLQ).

[00143] Each lung homogenate was centrifuged at 5,400 xg for 15 minutes at 4 °C. Supernatants were subsequently processed using acetonitrile precipitation and analyzed by LC-MS/MS. A lung calibration curve was generated from aliquots of drug-free lung homogenate spiked with compound 22 at the specified concentration levels. The spiked lung homogenate samples were processed together with the unknown lung homogenate samples using the same procedure. The processed lung samples were stored at -70°C until the LC- MS/MS analysis, at which time peak areas were recorded and the concentrations of compound 22 in the unknown lung samples were determined using the appropriate calibration curve. The reportable linear range of the assay was determined, along with the LLQ. Plots of plasma and lung concentration of compound 22 versus time were constructed and lung:plasma ratios were calculated.

[00144] A mean peak plasma concentration of 104 μg/mLwas observed after 15 minutes and the levels reduced to 8 μg/mL after 1 hour (Figure 4). The compound was still detectable in plasma (66 ng/mL) after 6 hours. A peak lung concentration of 35 μg/g was observed 15 minutes post-injection, and after 1 hour, the concentration was 4 μg/g. At the 6-hour mark the lung concentration had reduced to 236 ng/g. The lung: plasma ratio remained steady (0.26- 0.33) until 45 minutes after injection but rose dramatically after 1 hour (0.49) and increased further with time (Table 7), suggesting compound 22 is cleared more rapidly from the plasma than from the lungs.

Example 9 - In vivo testing of compound 22

[00145] Pseudomonas aeruginosa strain FDA-CDC AR-BANK#0264 was received from the FDA-CDC AR Bank and cryopreserved as single-use frozen working stock cultures, stored at -80 °C. The stock was thawed at room temperature prior to use. A 0.2 mL aliquot was inoculated into 20 mL Tryptic soy broth (TSB) and incubated at 35-37 °C with shaking (250 rpm) for 6 h. One mL of the 6 h culture was used to seed 99 mL TSB and incubated at 35-37 °C with shaking at 250 rpm for 16 h. Cells in 20 mL culture were pelleted by centrifugation (3,500 x g) for 15 minutes and re-suspended in 10 mL cold PBS >1 x 10⁹ CFU/mL, ODKO 1.2- 1.6. The culture was diluted in PBS to a target inoculum of 5 ^χ 10⁷ CFU/mL. The actual colony counts were determined by plating dilutions onto nutrient agar (NA) plates followed by incubation for 20-24 h. The CFU counts were 4.55 x 10⁶ CFU/mL.

[00146] Groups of 5 female Bltw:CD1 (ICR) mice weighing 22 ± 2 g were used. Animals were immunosuppressed by two intraperitoneal injections of cyclophosphamide, the first 150 mg/kg 4 days before infection (Day -4) and the second 100 mg/kg 1 day before infection (Day -1). On day 0, animals were anesthetized with etomidate-lipuro emulsion (20 mg/10 mL; 20 mg/kg IV) and inoculated intranasally with P. aeruginosa FDA-CDC AR-BANK#0264 suspension (0.02 mL/mouse). The target inoculation density was 1.0 x 10⁶ CFU/mouse and the actual count was 9.1 ^χ 10^s CFU/mouse. Vehicle (5% DMSO in 0.9% NaCI), compound 22 and ceftazidime (both at two doses; 50 and 120 mg/kg in 5% DMSO in 0.9% NaCI) were administered SC six times at 4-hr intervals (q4h) starting 2 hr after infection for a total dose of 300 and 720 mg/kg over 24 h. Animals were sacrificed 26 h after inoculation by CO₂ asphyxiation and the lung tissues were harvested and weighed for each of the test animals. The removed lung tissue was homogenized in 1 mL sterile PBS (pH 7.4) and stored on ice for up to 2 h. Serial 10-fold dilutions in PBS were generated. Aliquots of the dilutions (100 μL) were separately plated onto MacConkey II agar plates for bacterial enumeration. The bacterial counts (CFU/g) in lung tissue homogenates were calculated and the percentage decrease in counts compared to the corresponding vehicle control was calculated using the following formula:

Decrease (%) = [(CFU/g of vehicle -CFU/g of treatment)/ (CFU/g of vehicle)] x 100%

One-way ANOVA with Tukey’s multiple comparisons test was performed to assess the statistical difference in the lung bacterial counts for compound 22 and ceftazidime-treated mice compared to vehicle control, and to each other (p < 0.05). The MICs for ceftazidime and compound 22 against Pseudomonas aeruginosa were measured at 1 μg/mL and 4 μg/mL, respectively, immediately prior to the experiment.

[00147] Two hours after inoculation the lung burden was 5.54 ± 0.12 log CFU/g in the vehicle control group, which increased to 9.50 ± 0.17 log CFU/g at the end of the 26-hour period (Figure 5). Mice treated with 50 mg/kg ceftazidime showed a statistically significant reduction in lung bacteria relative to controls (7.57 ± 0.39 log CFU/g) and the effect was increased at 120 mg/kg (6.40 ± 0.24 log CFU/g). Treatment with compound 22 also significantly reduced bacteria at 50 mg/kg (8.06 ± 0.21 log CFU/g) and 120 mg/kg (7.22 ± 0.23). No statistically significant differences were evident in the ceftazidime versus compound 22 treated mice at 50 mg/kg or 120 mg/kg, indicating that both compounds show equivalent effects in this model.

References

1. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; CLSI document M07-A9 (ISBN1-56238-784-7). Vol 32 No. 2. Clinical and Laboratory Standards Institute, Wayne, PA 19087 USA.

Claims

Claims

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

wherein X is:

Ri is a substituent that corresponds to a substituent attached to the 7-NHC(0)- group of a cephalosporin antibiotic, and

T is a straight-chain or branched-chain alkanediyl group having between 1 and 8 carbon atoms.

2. The compound according to claim 1, wherein T is a straight-chain or branched-chain alkanediyl group having between 1 and 4 carbon atoms.

3. The compound according to claim 2, wherein T is -CH₂-or-CH₂CH₂-.

4. The compound according to any one of claims 1 to 3, wherein R₁ is a substituent that corresponds to a substituent attached to the 7-NHC(0)- group of a cephalosporin antibiotic selected from the group consisting of: ceftolozane, cefmatilen, cefaloram, cefazolin, cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazedone, cefazaflur, cefradine, cefroxidine, ceftezole, cefaclor, cefamandole, cefminox, cefonicid, ceforanide, cefotiam, cefprozil, cefbuperazone, cefuroxime, cefuzonam, cefotetan, cefmetazole, flomoxef, cefixime, ceftriaxone, ceftazidime, cefoperazone, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, cefteram, ceftibuten, ceftiolene, ceftizoxime, latamoxef, cefepime, cefozopran, cefpirome, cefquinome, ceftobiprole,ceftaroiinefosamil,anoxacephem,cefdidin,cefluprenam,cefoselis,cefozopran, cefaparole,cefcanel,cefedrolor,cefempidone,cefetrizole,cefivitril,cefmepidium,cefoxazole, cefrotii,cefsumide,ceftioxideandceftiofur.

5. The compound according to any one ofclaims 1 to 3,wherein R₁ is a substituent correspondingtoasubstituentattachedtothe7-NHC(O)-groupofacephalosporinantibiotic, andwhereinthesubstituentincludesthefollowingfunctionalgroup:

6. The compound according to any one ofclaims 1 to 3,wherein R₁ is a substituent corresponding to a substituentattached to the 7-NHG(O)-group ofceftolozane,cefepime, cefuroxime,ceftazidime,cefotaxime,orcefozopran.

7. The compound according to any one ofclaims 1 to 3,wherein R₁ is a substituent correspondingtoasubstituentattachedtothe7-NHC(O)-groupofceftolozaneorceftazidime.

8. The compound according to any one ofclaims 1 to 3,wherein R₁ is a substituent correspondingtoasubstituentattachedtothe7-NHC(O)-groupofceftolozane.

9. Thecompoundaccordingtoanyoneofclaims1to8,whereinX is:

10. A compound of formla(I) asdefinedinclaim 1selectedfrom thegroupconsistingof:

11. A compound of formula (I) as defined in claim 1 selected from the group consisting of:

12. A compound of formula (I) as defined in claim 1 having the following structure:

13. A composition for promoting the dispersal of microorganisms from a biofilm or inhibiting the formation and/or development of a biofilm, the composition comprising a compound according to any one of claims 1 to 12.

14. A method for promoting dispersal of microorganisms from a biofilm, the method comprising exposing the biofilm to an effective amount of a compound of any one of claims 1 to 12, or a composition of claim 13.

15. A method for inhibiting biofilm formation and/or development, the method comprising exposing biofilm-forming microorganisms to an effective amount of a compound of any one of claims 1 to 12, or a composition of claim 13.

16. The method of claim 15, wherein the compound or composition is coated, impregnated or otherwise contacted with a surface or interface susceptible to biofilm formation.

17. The method of claim 16, wherein the surface is a surface of an implantable medical device, prosthesis or medical or surgical equipment.

18. The method of any one of claims 14 to 17, wherein the biofilm is formed on a bodily surface of a subject, internal or external to the subject, and exposure of the biofilm or biofilm- forming microorganisms to the compound or composition is via administration of the compound or composition to the subject.

19. The method of any one of claims 14 to 18, wherein the microorganisms within the biofilm or capable of forming a biofilm comprises one or more species selected from: Acinetobacter spp.,Pseudomonas spp., Pseudoalieromonas spp., Staphylococcus spp,. Streptococcusspp.,Shigellaspp.,Mycobacterium spp.,Enterococcusspp.,Escherichiaspp,, Salmonella spp., Legionella spp., Haemophilus spp., Bacillus spp., Desulfovibrio spp,, Shewanella spp.,Geobacterspp.,Klebsiella spp.,Neisseria spp.,Proteusspp.,Aeromonas spp.,Arthrobacterspp.,Micrococcusspp.,Burkholderia spp.,Serratiaspp.,Porphyromonas spp.,Fusobacterium spp.and Vibriospp.

20. The method of claim 19, wherein the microorganism is Pseudomonas aeruginosa.

21. A method for treating or preventing a biofilm-associated infection, disease or disorder in a subject wherein the infection is caused by a microorganism capable of forming a biofilm, the method comprising administering to the subject an effective amount of a compound of any one of claims 1 to 12, or a composition of claim 13.

22. The method of claim 21, wherein the disease or disorder is chronic obstructive pulmonary disease (CORD), bacterial endocarditis, gonorrhoea, otitis media, Legionnaire's disease, tuberculosis, kidney stones, urinary tract infections, pulmonary infections, dental plaque, dental caries and infections associated with surgical procedures or bums.

23. The method of claim 21 or claim 22, wherein the compound or composition is administered with a β-lactamase inhibitor.

24. The method of claim 23, wherein the β-lactamase inhibitor is selected from the group consisting of: clavulanic acid, tazobactam, sulbactam, avibactam, vaborbactam and relebactam.

25. The method of claim 23 or claim 24, wherein the compound or composition is administered contemporaneously, simultaneously or sequentially with the β-lactamase inhibitor.

26. A method for treating or preventing a Pseudomonas aeruginosa infection in a subject suffering from cystic fibrosis, the method comprising administering to the subject an effective amount of a compound of any one of claims 1 to 12, or a composition of claim 13.

27. The method of claim 26, wherein the Pseudomonas aeruginosa is present in the form of, or as part of, a biofilm.

28. The method of claim 26 or claim 27, wherein the compound or composition is administered to the subject by inhalation.

29. The method of any one of claims 26 to 28, wherein the compound or composition is administered with a polymyxin or tobramycin.

30. The method of claim 29, wherein the compound or composition is administered contemporaneously, simultaneously or sequentially with the polymyxin or tobramycin.

31. The method of claim 29 or claim 30, wherein the polymyxin is colistin.

32. A method for killing a biofilm-forming microorganism, the method comprising exposing the microorganism to an effective amount of a compound of any one of claims 1 to 12.

33. The method of claim 32, wherein the microorganism is selected from the group consisting of: Acinetobacter spp., Pseudomonas spp., Pseudoalieromonas spp., Staphylococcus spp,. Streptococcus spp., Shigella spp., Mycobacterium spp., Enterococcus spp., Escherichia spp., Salmonella spp., Legionella spp., Haemophilus spp., Bacillus spp., Desulfovibrio spp., Shewanella spp., Geobacter spp., Klebsiella spp., Neisseria spp., Proteus spp., Aeromonas spp., Arthrobacter spp., Micrococcus spp., Burkholderia spp., Serratia spp., Porphyromonas spp., Fusobacterium spp. and Vibrio spp.

34. The method of claim 33, wherein the microorganism is Pseudomonas aeruginosa.