WO2014071457A1 - Donneurs d'oxyde nitrique à double action et leur utilisation comme agents antimicrobiens - Google Patents

Donneurs d'oxyde nitrique à double action et leur utilisation comme agents antimicrobiens Download PDF

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WO2014071457A1
WO2014071457A1 PCT/AU2013/001292 AU2013001292W WO2014071457A1 WO 2014071457 A1 WO2014071457 A1 WO 2014071457A1 AU 2013001292 W AU2013001292 W AU 2013001292W WO 2014071457 A1 WO2014071457 A1 WO 2014071457A1
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compound
alkyl
optionally
group
branched
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PCT/AU2013/001292
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Nicolas Barraud
Naresh Kumar
Samuel Kunjunju KUTTY
Scott Rice
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Newsouth Innovations Pty Limited
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Publication of WO2014071457A1 publication Critical patent/WO2014071457A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/26—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D307/30—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/32—Oxygen atoms
    • C07D307/33—Oxygen atoms in position 2, the oxygen atom being in its keto or unsubstituted enol form
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/40—Nitrogen atoms, not forming part of a nitro radical, e.g. isatin semicarbazone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/58—One oxygen atom, e.g. butenolide

Definitions

  • the present invention relates generally to conjugates comprising a nitric oxide donor and an acyl homoserine lactone, fimbrolide, fimbrolide derivative, dihydropyrrolone or indole, and to the use of such conjugates as antimicrobial agents.
  • Biofilms are three dimensional microbial growth forms comprising microbial communities and the extracellular matrix they produce. Biofilms are ubiquitous in nature, forming on any surface or at any interface where water or suitable fluid is available, or in suspension, for example as floes or granules.
  • Biofilms are etiologic agents of a number of diseases and are associated with a variety of chronic infections in humans, forming on a variety of surfaces within the body, for example on surfaces in the respiratory tract and lungs (associated with cystic fibrosis and Legionnaire's disease), on surfaces of the ear (associated with otitis media), and on surfaces of the heart and heart valves (associated with bacterial endocarditis).
  • Biofilms offer increased protection to the microorganism inhabitants, for example in the form of substantially increased resistance to antibiotics compared to planktonie cells and resistance to phagocytosis, which render biofilms very difficult to eradicate and explains the severity and high level of persistence of biofilms and the morbidity associated with infections produced by biofilms.
  • cystic fibrosis for example, a principal cause of respiratory infections is Pseudomonas aeruginosa, and P. aeruginosa biofilms on the surface of the lungs in cystic fibrosis sufferers imparts a greater degree of antibiotic resistance and resistance to host immune defences. Consequently the major cause of chronic lung infections, and in turn of morbidity and mortality, in cystic fibrosis sufferers is biofilm-associated P. aeruginosa.
  • Biofilms also readily form on medical equipment such as catheters and cannulas, and on implantable medical devices including stents and contact lenses. Indeed many long term catheterization patients acquire infections caused by biofilm-forming bacteria, and more generally biofilms are responsible for a range of hospital acquired infections, adding considerable cost to health systems. From a public health perspective, biofilms are important reservoirs of pathogens in water systems such as drinking water, reservoirs, pipes and air-conditioning ducts. Biofilms also cause significant industrial damage, causing, for example, fouling and corrosion in fluid processes such as water distribution and treatment systems, pulp and paper manufacturing systems, heat exchange systems and cooling towers, and contributing to the souring of oil in pipelines and reservoirs.
  • Biofilms are essentially multicellular microbial communities, the formation and development of which are dependent on various multicellular traits of the member organisms, such as cell-cell signalling.
  • Extracellular signalling systems such as quorum sensing are used by bacteria to assess cell density and initiate changes in gene expression and phenotypes when sufficient concentrations of signalling molecules are reached.
  • intracellular signals such as nitric oxide (NO) are also used by bacteria to control biofilm development. Both intracellular and extracellular signals are associated with differential gene expression, leading to the induction of, for example, virulence factors and/or defence mechanisms, and with cell differentiation such that biofilm- associated cells become highly differentiated from planktonic cells.
  • antimicrobial compounds comprising an NO donor and either an acyl homoserine lactone, fimbrolide, fimbrolide derivative or indole.
  • the compounds exploit the differing modes of action of NO and acyl homoserine lactone/fimbrolide/dihydropyrrolone/indole compounds against bacteria so as to provide enhanced efficacy in inhibiting biofilm formation and/or development.
  • the invention provides a compound of formula (I), or a salt thereof:
  • L is a linker and Y is a nitric oxide (NO) donor.
  • the invention provides a compound of formula (II), or a salt thereof:
  • L ⁇ is a linker
  • Yi is an NO donor and A is O or NR'
  • R' is H, Ci-Ce alkyl, C2-C6 alkenyl, (CH 2 )r-phenyi phenyl, wherein the phenyl may be substituted with one or more substituents selected from: halo, C1-C alkyl, hydroxy, amino, nitro and O Ci-C 6 alkyl, r is an integer between 1 and 6, and R6 and R 7 are independently selected from H and Br.
  • the invention provides a compound of formula (III), or a salt thereof
  • R4 is selected from H, OC1-C alkyl and Ci-C 6 alkyl
  • R 5 is selected from the group consisting of: an alkali metal cation (for example Na + or K + ), branched or straight chain C1-C10 alkyl, wherein one or more methylene groups of the alkyl chain may optionally be replaced by a group selected from: -0-, -NH-, -S- and carbonyl, or R5 is - (CH 2 ) z -phenyl or (CH2) z -pyridyl, wherein the phenyl and pyridyl groups may optionally be substituted with one or more substituents selected from the group consisting of: trihalomethyl and nitro, z is an integer between 0 and 8, or R5 is a glycoside moiety which is optionally protected with one or more protecting groups, and R $ is selected from the group consisting of: H, Ci-C 6 alkyl, COO Q-C6 alkyl
  • the invention provides an antimicrobial composition comprising a compound according to the first, second or third aspects.
  • the composition may further comprise one or more additional antibiotics or antimicrobial agents.
  • the invention provides 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, second or third aspects, or a composition of the fourth aspect.
  • the invention provides a method for inhibiting biofilm formation and/or development, the method comprising exposing biofilm-forming microorganisms to an 201
  • the compound or composition comprising the same may be coated, impregnated or otherwise contacted with a surface or interface susceptible to biofilm formation.
  • the surface may be a surface of an implantable medical device, prosthesis or medical or surgical equipment.
  • 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,
  • Methods for promoting dispersal of microorganisms from a biofilm and methods for inhibiting biofilm formation and/or development may comprise inducing differentiation events in microorganisms within biofilms which lead to dispersal, or may comprise preventing induction of differentiation events in microorganisms which lead to biofilm formation.
  • methods may comprise increasing the sensitivity of a microorganism to antimicrobial agents.
  • the biofilm may be surface- associated or suspended.
  • the suspended biofilm may be in the form of floes or granules.
  • the microorganisms present in the biofilm may be of a single species or of multiple species.
  • the microorganisms within the biofilm or capable of forming a biofilm may comprise one or more species selected from, for example, Pseudomonas spp., Pseudoalteromonas 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., Proteus spp., Aeromonas spp., Arthrobacter spp., Micrococcus spp., Burkholderia s
  • the microorganism may be Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus, Mycobacterium tuberculosis, Escherichia coli, Bacillus licheniformis, Burkholderia cenocepacia, Serratia marcescens, Fusobacterium nucleatum, or Vibrio cholerae.
  • the biofilm is on or within the body of a subject and may be associated with a disease or condition suffered by the subject.
  • the disease or condition may be, for example, cystic fibrosis, bacterial endocarditis, otitis media, Legionnaire's disease, tuberculosis.or kidney stones.
  • a method for treating or preventing a biofilm-associated infection, disease or condition in a subject wherein the infection, disease or condition is caused by, or associated with, a microorganism capable of forming a biofilm comprising administering to the subject an effective amount of a compound of the first or second aspects or a composition of the third aspect.
  • the invention provides a method for inhibiting quorum sensing- mediated activity in microorganisms, the method comprising exposing the microorganisms to a compound of the first, second or third aspects, or a composition of the fourth aspect.
  • the microorganism may be gram-negative bacteria.
  • the invention provides a use of a compound of the first, second or third aspects for the manufacture of a composition for use in promoting dispersal of microorganisms from a biofilm or for inhibiting the formation or development of a biofilm.
  • the invention provides a use of a compound of the first, second or third aspects for the manufacture of a medicament for treating or preventing a biofilm- associated infection, disease or condition in a subject wherein the infection is caused by a microorganism capable of forming a biofilm.
  • the invention provides a use of a compound of the first, second or third aspects for the manufacture of a composition for use in inhibiting quorum sensing- mediated activity in microorganisms.
  • the invention provides a compound of the first, second or third aspects, or a composition of the fourth aspect, for promoting dispersal of microorganisms from a biofilm or for inhibiting the formation or development of a biofilm.
  • the invention provides a compound of the first, second or third aspects, or a composition of the fourth aspect, for treating or preventing a biofilm- associated infection, disease or condition in a subject wherein the infection is caused by a microorganism capable of forming a biofilm.
  • the invention provides a compound of the first, second or third aspects, or a composition of the fourth aspect, for inhibiting quorum sensing-mediated activity in microorganisms.
  • Figure 1 Biological screening assay for QS inhibition by known natural fimbrolide 34a Upper: Relative Fluorescence Units (RFU), centre: optical density (OD) and lower: RFU/OD as a function of time.
  • the monitor strain P. aeruginosa PAOl harbouring the VlasB::gfp (ASV) fusion plasmid was employed.
  • Figure 2 Biological screening assay for QS inhibition by known synthetic furanone 37.
  • the monitor strain P. aeruginosa PAOl harbouring the VlasB::gfp (ASV) fusion plasmid was employed.
  • FIG. 3 Biological screening assay for QS inhibition by AHL derivative 97a.
  • the monitor strain P. aeruginosa PAOl harbouring the YlasB::gfp (ASV) fusion plasmid was employed.
  • Figure 4 Nitrite standard curve obtained using the Griess assay.
  • FIG. 5 Nitrite concentration ( ⁇ ) obtained for different test compounds using the Griess assay.
  • Figure 6 NO release study of 237 (IND-1) using Apollo 4000 NO analyser.
  • the arrow heads 1 and 2 indicates the addition of compound 237 and PTIO respectively for each set of experiment.
  • Figure 7 NO release study of 243d (IND-2) using Apollo 4000 NO analyser.
  • Figure 8 Results from biofilm dispersion study of IND-2.
  • 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.
  • 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.
  • biofilm-forming microorganism refers to any microorganism that is capable of forming biofilms, either single species or mixed species biofilms.
  • 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.
  • the term "effective amount” includes within its meaning a non-toxic but sufficient amount of an agent 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) being administered and 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.
  • the term "exposing" means generally bringing into contact with.
  • direct exposure refers to administration of the agent to the microorganism or biofilm to be treated or otherwise bringing the microorganism or biofilm into contact with the agent.
  • indirect exposure refers to the administration of a precursor of the active agent or a compound or molecule capable of generating, either solely or in reaction with other compounds or molecules, the active agent to the microorganism or biofilm or otherwise bringing the microorganism or biofilm into contact therewith.
  • a microorganism or biofilm may be exposed to compound or composition as defined herein directly or indirectly.
  • a microorganism or biofilm may be exposed to nitric oxide released from a compound directly or indirectly.
  • indirectly “exposing” a biofilm or microorganisms to a compound or composition as defined herein also includes the administration of the compound or composition to a subject in or on which the biofilm or microorganisms reside.
  • the terms “exposing”, “administering” and “delivering” and variations thereof may, in some contexts, be used interchangeably.
  • inhibitors 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 effect the expression or activity of molecules which in turn regulate biofilm formation or development.
  • promotion and variations thereof, such as “promotion” and “promotes”, as used herein in the context of promoting the dispersal of microorganisms from a biofilm also complete or partial promotion of dispersal, which may be permanent or temporary, to an extent (in magnitude and/or spatially), and or for a time, sufficient to produce the desired effect. Such promotion may be direct or indirect.
  • 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.
  • treating refers 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.
  • treating does not necessarily imply that a patient is treated until total recovery.
  • the term "associated with” when used in the context of an infection, disease or condition “associated with” a biofilm means that the disease or condition, or a symptom thereof, may result from, result in, be characterised by, or otherwise associated with the formation and/or presence of a biofilm.
  • the association between the infection, disease or condition and the biofilm may be direct or indirect and may be temporally separated.
  • NO donor is understood to mean a functional group which is capable of releasing one or more NO groups.
  • C 1 -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, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like.
  • Ci-Cto 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.
  • Q-Ce alkyl is taken to include straight chain and branched chain monovalent saturated hydrocarbon groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl and the like.
  • C 2 -C 2 o 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, heptenyl, undecenyl and the like.
  • C2-C6 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 and the like.
  • 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, undecynyl and the like.
  • aryl is taken to include monovalent aromatic radicals having between 6 and 30 carbon atoms, for example phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl and the like.
  • heteroaryl is taken to include monovalent aromatic radicals having between 4 and 25 atoms, wherein 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 or 2 atoms are heteroatoms selected from nitrogen, oxygen and sulfur, for example furanyl, quinazolinyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, benzopyranyl, benzooxazolyl, benzimidazolyl, pyrazolyl, tetrazolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, quinolizinyl, pyranyl, isothiazolyl, thiazolyl, thienyl, imidazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, isothiazolyl, pyridyl, triazolyl
  • C1-C20 alkylene is taken to include straight chain and branched chain bivalent saturated hydrocarbon groups having 1 to 20 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene, octylene, dodecylene and the like.
  • C1-Q5 alkylene is taken to include straight chain and branched chain bivalent saturated hydrocarbon groups having 1 to 15 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene, octylene, dodecylene and the like.
  • C1-C12 alkylene is taken to include straight chain and branched chain bivalent saturated hydrocarbon groups having 1 to 12 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene, octylene, dodecylene and the like.
  • C1-Q0 alkylene is taken to include straight chain and branched chain bivalent saturated hydrocarbon groups having 1 to 10 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene, octylene and the like.
  • C1-C6 alkylene is taken to include straight chain and branched chain bivalent saturated hydrocarbon groups having 1 to 6 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene and the like.
  • C2-C20 alkenylene is taken to include straight chain and branched chain bivalent hydrocarbon radicals having 2 to 20 carbon atoms and at least one carbon-carbon double bond, such as vinyl, propenylene, 2-methyl-2- propenylene, butenylene, pentenylene, hexenylene, heptenylene, undecenylene and the like.
  • C2-C20 alkynylene is taken to include straight chain and branched chain bivalent hydrocarbon radicals 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.
  • NO is involved in cell death and the dispersal of biofilms.
  • the molecular mechanism involved in biofilm dispersal at low NO concentrations involves the secondary messenger c-di-GMP.
  • Exogenous NO, at sub-lethal concentrations is able to induce the transition of bacteria in biofilms from a sessile mode of growth to a free-swimming, planktonic mode.
  • NO at non-lethal concentrations has also been shown to increase the sensitivity of various biofilms to antimicrobial treatments.
  • the compounds of the present invention are conjugates comprising an NO donor and an acyl homoserine lactone, fimbrolide, fimbrolide derivative, dihydropyrrolone or indole.
  • the compounds exploit the differing modes of action of NO and acyl homoserine lactone/fimbrolide/dihydropyy-Tolone/indole compounds against bacteria so as to provide enhanced efficacy in inhibiting biofilm formation and/or development.
  • the acyl homoserine lactones, fimbrolides, fimbrolide derivatives and dihydropyrrolones exert their effect by inhibiting the quorum sensing phenomenon that is used by bacteria to communicate.
  • the invention provides compounds of formula (I), or a salt thereof:
  • L is a linker and Y is a nitric oxide (NO) donor.
  • L may be a divalent organic group having between 1 and 40, or between 1 and 30, or between 1 and 20 carbon atoms.
  • L may be a divalent organic group having between 1 and 40, or between 1 and 30, or between 1 and 20 carbon atoms, and at least one carbonyl group.
  • L may be -C(0)-X-, wherein X is selected from the group consisting of: branched or straight chain Ci-C 2 o alkylene, branched or straight chain C 2 -C 2 o alkenylene and branched or straight chain C2-C20 alkynylene, wherein one or more methylene groups of the alkylene, alkenylene and alkynylene chains may optionally be replaced by a group selected from: -0-, -NH-, -S-, -phenylene- and carbonyl, and wherein the alkylene, alkenylene and alkynylene chains may optionally be substituted with one or more -NHC ⁇ Ci-Ci alkyl groups.
  • L is -C(0)-X-, wherein X is branched or straight chain Q-Cao alkylene wherein one or more methylene groups of the alkylene chain may optionally be replaced by a group selected from: -0-, -NH-, -S-, -phenylene- and carbonyl, and wherein the alkylene chain may optionally be substituted with one or more -NHC(0)Cj-C6 alkyl groups.
  • L is -C(0)-X-, wherein X is branched or straight chain Q-ds alkylene wherein between 1 and 3 methylene groups of the alkylene chain may optionally be replaced by a group selected from: -0-, -NH-, -phenylene- and carbonyl, and wherein the alkylene chain may optionally be substituted with one or more -NHC(0)Ci-C6 alkyl groups.
  • L is -C(0)-X-, wherein X is branched or straight chain Ci-C ⁇ alkylene wherein 1 or 2 methylene groups of the alkylene chain may optionally be replaced by a group selected from: -NH-, -phenylene- and carbonyl, and wherein the alkylene chain may optionally be substituted with one or more -NHC(0)Ci-C 6 alkyl groups.
  • L is -C(0)-X- wherein X is branched or straight chain C1-C15 alkylene wherein 1 or 2 methylene groups of the alkylene chain may optionally be replaced by a group selected from: -NH-, -phenylene- and carbonyl.
  • L is -C(0)-X- wherein X is branched or straight chain C1-C15 alkylene wherein 1 or 2 methylene groups of the alkylene chain may optionally be replaced by a group selected from: -NH-, -phenylene- and carbonyl.
  • X is selected from the group consisting of: - CH 2 ) n -, -Ph- (CHz , -CH 2 C(0)(CH 2 )p-, -CH 2 HC(0)(CH 2 ) p - or -(CH 2 ) q C(0)-, wherein n is 1 to 12, m is 1 to 4, p is 7 to 12 and q is 1 to 6.
  • X is selected from the group consisting of: -(CH 2 )n- > -Ph- (CH 2 ) m -, -CH 2 C(0)(CH 2 ) p -, -CH 2 NHC(0)(CH 2 ) p - or -(CH 2 ) q C(0)-, wherein n is 1 to 9, m is 1 or 2, p is 9 to 12 and q is 2 to 4.
  • Non-limiting examples of NO donors include: nitrates, nitrites, diazeniumdiolates, N- nitrosoamines, C-nitrosamines, S-nitrosamines, furoxans, benzofuroxans, sydnonimines, oximes, hydroxylamines, S-nitrosothiols (S-NO) and N-hydroxyguanidines.
  • NO donors include: nitrates, nitrites, diazeniumdiolates, N- nitrosoamines, C-nitrosamines, S-nitrosamines, furoxans, benzofuroxans, sydnonimines, oximes, hydroxylamines, S-nitrosothiols (S-NO) and N-hydroxyguanidines.
  • NO donors include: nitrates, nitrites, diazeniumdiolates, N- nitrosoamines, C-nitrosamines, S-nitrosamine
  • Y is -SNO, ON0 2 or diazeniumdiolate.
  • the diazeniumdiolate is an N-diazeniumdiolate.
  • the N-diazeniumdiolate may be attached to variable L via a nitrogen atom of the N-diazeniumdiolate that does not bear an oxygen atom.
  • the nitrogen atom of the N-diazeniumdiolate that does not bear an oxygen atom may be directly or indirectly attached to the variable L.
  • the diazeniumdiolate has one of the following structures, wherein the squiggly line denotes the point of attachment of the N-diazeniumdiolate moiety to variable L:
  • Ri is an organic functional group
  • T is a linker
  • ring A is a 4-, 5-, 6- or 7- membered saturated or unsaturated ring which may optionally contain an additional nitrogen atom, and may optionally be substituted with C 1 -C alkyl.
  • Ri may be aryl or heteroaryl, branched or straight chain C 1 -C20 alkyl, branched or straight chain C 2 -C 2 o alkenyl, branched or straight chain C 2 -C 2 o alkynyl, wherein the alkyl, alkenyl and alkynyl groups may optionally be interrupted by one or more of the following: -0-, - NH- or -S-, and wherein the aryl, heteroaryl, alkyl, alkenyl and alkynyl groups may Optionally be substituted with one or more substituents selected from the group consisting of: halo, nitro, hydroxy, SH, OC(0)Ct-C 6 alkyl, OQ-Cealkyl, -NHCi-C 6 alkyl and Ci- C 6 alkyl.
  • Rj is aryl, branched or straight chain Ci-C 2 o alkyl, or branched or straight chain C2-C20 alkenyl, wherein the alkyl or alkenyl groups may optionally be interrupted by one or more of the following: -0-, -NH- or -S-, and wherein the aryl, alkyl and alkenyl groups may optionally be substituted with one or more substituents selected from the group consisting of: halo, nitro, hydroxy, amino, OCi- C 6 alkyl, -NHCi-C 6 alkyl and Ci-C 6 alkyl.
  • Ri is aryl, branched or straight chain C 1 -C 20 alkyl, wherein the aryl group may optionally be substituted with one or more substituents selected from the group consisting of: halo, nitro, hydroxy, OCi-C6alkyl, -NHCi-C 6 alkyl and Ci-Cealkyl.
  • Ri is phenyl or branched or straight chain C1-C20 alkyl, wherein the phenyl group may optionally be substituted with between one and three substituents selected from the group consisting of: halo, nitro, hydroxy, OCi-C6alkyl, and d-Cealkyl.
  • Ri is phenyl or branched or straight chain Ci-Cto alkyl, wherein the phenyl group may optionally be substituted with between one and three substituents selected from the group consisting of: nitro, hydroxy, methyl or ethyl.
  • R ⁇ is phenyl or branched or straight chain d-Ce alkyl, wherein the phenyl group may optionally be substituted with one or two nitro groups.
  • Ri is phenyl, methyl, ethyl, propyl or isopropyl, wherein the phenyl group may optionally be substituted with one or two nitro groups.
  • ring A is a 5-, 6- or 7-membered saturated or unsaturated ring which may optionally contain an additional nitrogen atom, and may optionally contain 1 or 2 oxygen or sulfur atoms.
  • ring A is a 5- or 6-membered saturated ring which contains an additional nitrogen atom, and may optionally contain 1 or 2 oxygen or sulfur atoms, and wherein the ring is connected to variable L via the additional nitrogen atom in the ring.
  • ring A is a 5- or 6-membered saturated ring which contains an additional nitrogen atom, and wherein the ring is connected to variable L via the additional nitrogen atom in the ring.
  • the diazeniumdiolate has the structure:
  • T may be a divalent hydrocarbon group having between 1 and 40, or between 1 and 30, or between 1 and 20 carbon atoms.
  • T is selected from the group consisting of: branched or straight chain Ci-C 2 o alkylene, branched or straight chain C 2 -C 2 o alkenylene and branched or straight chain C2-C20 alkynylene, wherein one or more methylene groups of the alkylene, alkenylene and alkynylene chains may optionally be replaced by a group selected from: - 0-, -NH-, -S- and carbonyl.
  • T is branched or straight chain C1-C20 alkylene, wherein one or more methylene groups of the alkylene chain may optionally be replaced by a group selected from: -0-, -NH-, -S- and carbonyl.
  • T is branched or straight chain C1-C20 alkylene wherein one or more methylene groups of the alkylene chain may optionally be replaced by -NH-.
  • T is branched or straight chain C1-C6 alkylene.
  • T is -NHC1-C6 alkylene-, wherein the -NH group is bonded to variable L.
  • Exemplary compounds of formula (I) include:
  • the present invention provides compounds of formula (II), or a salt thereof:
  • L is a linker
  • Yi is an NO donor and A is O or NR 1
  • R is H, Ci-C 6 alkyl, C 2 -C6 alkenyl, (CltVphenyl and phenyl, wherein the phenyl may be substituted with one or more substituents selected from: halo, C 1 -C6 alkyl, hydroxy, amino, nitro and O C1-C6 alkyl, r is an integer between 1 and 6, and R $ and R 7 are independently selected from H and Br.
  • R$ may be H and R 7 may be Br.
  • Li may be a divalent hydrocarbon chain having between 1 and 40, or between 1 and 30, or between 1 and 20 carbon atoms.
  • Li is a divalent hydrocarbon chain having between 1 and 40, or between 1 and 30, or between 1 and 20 carbon atoms, wherein the hydrocarbon chain may optionally be interrupted by one or more of the following groups: -C(0)0-, -C(S)0-, - C(0)S- and carbonyl.
  • L ⁇ is branched or straight chain C ! -C 2 o alkylene, branched or straight chain C 2 -C 2 o alkenylene or branched or straight chain C 2 -C 2 o alkynylene, wherein one or more methylene groups of the alkylene, alkenylene and alkynylene chains may optionally be replaced by a group selected from: -0-, -NH-, -S- and carbonyl.
  • Li is branched or straight chain C1-C 2 0 alkylene wherein one or more methylene groups of the alkylene chain may optionally be replaced by a group selected from: -0-, -NH-, -S- and carbonyl.
  • h ⁇ is branched or straight chain Ci-C 2 o alkylene wherein between one and three methylene groups of the alkylene chain may optionally be replaced by a group selected from: -0-, -S- and carbonyl.
  • squiggly line of each structure denotes attachment of h ⁇ to the ring system
  • the upper squiggly line denotes attachment of Li to Y l5 and wherein s is an integer between 0 and 12, t is an integer between 0 and 15, and u is an integer between 1 and 15.
  • s is an integer between 1 and 6
  • t is an integer between 1 and 10
  • u is an integer between 1 and 5.
  • Non-limiting examples of NO donors include: nitrates, nitrites, diazeniumdiolates, N- nitrosoarnines, C-nitrosamines, S-riitrosarriines, furoxans, benzofuroxans, sydnonimines, oximes, hydroxylamines, 5-nitrosothiols and N-hydroxyguanidines.
  • NO donors include: nitrates, nitrites, diazeniumdiolates, N- nitrosoarnines, C-nitrosamines, S-riitrosarriines, furoxans, benzofuroxans, sydnonimines, oximes, hydroxylamines, 5-nitrosothiols and N-hydroxyguanidines.
  • A is O.
  • A is O
  • Y ⁇ is a diazeniumdiolate
  • Lj is selected from:
  • A is NR.'.
  • A is NR' and Yj is -ON0 2 .
  • A is NR', and L
  • s is an integer between 0 and 12, or between 1 and 10, or between 1 and 8, or between 1 and 4, or between 1 and 3.
  • R' is H, Ci-C 6 alkyl, (CH 2 ) r -phenyl or phenyl, wherein the phenyl may be substituted with one or more substituents selected from: halo, Ci-C 6 alkyl, hydroxy and OCi-C 6 alkyl, and wherein r is an integer between 1 and 6.
  • R' is H, Q-C6 alkyl, (CH 2 )r-phenyl or phenyl, wherein the phenyl may be substituted with one or more substituents selected from: halo, methyl, ethyl, propyl, isopropyl and butyl, and wherein r is an integer between 1 and 3.
  • R' is H, methyl, ethyl, propyl, isopropyl, butyl, (CH 2 ) r -phenyl or phenyl, wherein the phenyl may be substituted with one or more substituents selected from: halo and methyl, and wherein r is an integer between 1 and 3.
  • R' is H, butyl, (CH 2 )-phenyl or phenyl, wherein the phenyl may be substituted with 1 or 2 halo substituents.
  • R' is H, (CH 2 )-phenyl or phenyl, wherein the phenyl may be substituted with 1 or 2 halo substituents.
  • R' is H, butyl ⁇ (CH 2 )-phenyl, phenyl or bromophenyl,
  • Yi is a diazeniumdiolate.
  • the diazeniumdiolate is an N- diazeniumdiolate.
  • the N-diazeniumdiolate may be attached to variable Li via a nitrogen atom of the N-diazeniumdiolate that does not bear an oxygen atom.
  • the nitrogen atom of the N-diazeniumdiolate that does not bear an oxygen atom may be directly or indirectly attached to the variable Li.
  • the N-diazeniumdiolate may be attached to variable Li via one of the oxygen atoms of the N-diazeniumdiolate.
  • the diazeniumdiolate has one of the following structures, wherein the squiggly line denotes the point of attachment of the N-diazeniumdiolate moiety to the variable L ⁇ :
  • R ⁇ is an organic functional group
  • T is a linker
  • ring A is a 4-, 5-, 6- or 7- membered saturated or unsaturated ring which may optionally contain an additional nitrogen atom, and may optionally be substituted with CrC 6 alkyl.
  • Ri may be aryl or heteroaryl, branched or straight chain C1-C20 alkyl, branched or straight chain C 2 -C 2 o alkenyl, branched or straight chain C -C 2 o alkynyl, wherein the alkyl, alkenyl and alkynyl chains may optionally be interrupted by one or more of the following groups: - 0-, - ⁇ - or -S-, and wherein the aryl, heteroaryl, alkyl, alkenyl and alkynyl groups may optionally be substituted with one or more substituents selected from the group consisting of: halo, nitro, hydroxy, SH, OC(0)C 1 -C 6 alkyl, Od-Qalkyl, -NHCj-Cealkyl and C r C 6 alkyl.
  • Rj is aryl, branched or straight chain C1-C20 alkyl or branched or straight chain C2-C20 alkenyl, wherein the alkyl and alkenyl chains may optionally be interrupted by one or more of the following: -0-, -NH- or -S-, and wherein the aryl, alkyl and alkenyl groups may optionally be substituted with one or more substituents selected from the group consisting of: halo, nitro, hydroxy, OCi-Cealkyl, - NHCi-C 6 alkyl and d-Cealkyl.
  • R ⁇ is aryl, branched or straight chain C1-C20 alkyl, wherein the alkyl group may optionally be interrupted by one, two or three of the following: -0-, -NH- or -S-, and wherein the aryl group may optionally be substituted with one or more substituents selected from the group consisting of: halo, nitro, hydroxy, OCi-Cgalkyl, - NHd-Cealkyl and Ci-C 6 alkyl.
  • Ri is phenyl or branched or straight chain C1-C20 alkyl, wherein the phenyl group may optionally be substituted with between one and three substituents selected from the group consisting of: halo, nitro, hydroxyl
  • Ri is phenyl or branched or straight chain Ct-Cio alkyl, wherein the phenyl group may optionally be substituted with between one and three substituents selected from the group consisting of: halo, nitro, methyl and ethyl.
  • Ri is phenyl or branched or straight chain C C 6 alkyl, wherein the phenyl group may optionally be substituted with between one and two substituents selected from the group consisting of: nitro, methyl and ethyl.
  • Ri is branched or straight chain C1-C6 alkyl.
  • ring A is a 5-, 6- or 7-membered saturated or unsaturated ring which may optionally contain an additional nitrogen atom and may optionally contain 1 or 2 oxygen or sulfur atoms.
  • ring A is a 5- or 6-membered saturated ring which contains an additional nitrogen atom, and may optionally contain 1 or 2 oxygen or sulfur atoms, and wherein the ring is connected to variable Lj via the additional nitrogen atom in the ring.
  • ring A is a 5- or 6-membered saturated ring which contains an additional nitrogen atom, and wherein the ring may optionally be substituted with Ci-C 6 alkyl, and wherein the ring is connected to variable L via the additional nitrogen atom in the ring.
  • the diazeniumdiolate has the structure:
  • T may be a divalent hydrocarbon group having between 1 and 40, or between 1 and 30, or between 1 and 20 carbon atoms.
  • T is selected from the group consisting of: branched or straight chain C1-C20 alkylene, branched or straight chain C 2 -C 2 o alkenylene and branched or straight chain C2-C20 alkynylene, wherein one or more methylene groups of the alkylene, alkenylene and alkynylene chains may optionally be replaced by a group selected from: - ⁇ -, -NH-, -S- and carbonyl.
  • T is branched or straight chain C1-C20 alkylene, wherein one or more methylene groups of the alkylene chain may optionally be replaced by a group selected from: -0-, -NH-, -S- and carbonyl.
  • T is branched or straight chain C1-C20 alkylene wherein one or more methylene groups of the alkylene chain may optionally be replaced by -NH-.
  • T is branched or straight chain Ci-C 6 alkylene.
  • T is -NHQ-Ce alkylene-, wherein the -NH group is bonded to variable Li.
  • the diazeniumdiolate has the following structure, wherein the squiggly line denotes the point of attachment of the diazeniumdiolate moiety to the variable
  • R 2 and R3 are organic functional groups.
  • R2 and R 3 are independently selected from CrQo alkyl, or alternatively R2 and R3, together with the nitrogen to which they are attached, form a 5- or 6-membered ring which may optionally contain between 1 and 3 additional nitrogen atoms, and may optionally contain an oxygen atom, wherein the ring may optionally be substituted with one or more of the following: d-C6 alkyl and C(0)OCi-C6 alkyl.
  • R 2 and R3 are independently selected from Ci-C 10 alkyl, or alternatively R 2 and R3, together with the nitrogen to which they are attached, form a 5- or 6-membered ring which may optionally contain an additional nitrogen atom and may optionally contain an oxygen atom, wherein the ring may optionally be substituted with one or more of the following: Q-Q alkyl and C(0)OCi-C 6 alkyl.
  • R 2 and R3 are independently selected from Cj-Ce alkyl, or alternatively R 2 and R3, together with the nitrogen to which they are attached, form a saturated 5- or 6-membered ring which may optionally contain an additional nitrogen atom and may optionally contain an oxygen atom, wherein the ring may optionally be substituted with one or two of the following: methyl, ethyl, C(0)OMe and C(0)OEt.
  • R 2 and R3 are independently methyl, ethyl or propyl, or alternatively R 2 and R3, together with the nitrogen to which they are attached, form a saturated 6-membered ring which contains an additional nitrogen atom and/or an oxygen atom, wherein the ring may optionally be substituted with one of the following: C(0)OMe and C(0)OEt.
  • R 2 and R3 are independently methyl or ethyl, or alternatively R 2 and R3, together with the nitrogen to which they are attached, form a 6-membered ring which contains an additional nitrogen atom and or an oxygen atom, and wherein the ring may optionally be substituted with C(0)OMe.
  • R 2 and R 3 are independently selected from C C6 alkyl.
  • R 2 and R3 are independently selected from the group consisting of: methyl, ethyl, propyl and isopropyl.
  • R 2 and R3, together with the nitrogen to which they are attached form the following structures:
  • part i cularly i n which the squiggly line denotes the point of attachment to the variable Li.
  • Exemplary compounds of formula (II) include:
  • the invention provides a compound of formula (III), or a salt thereof
  • R4 is selected from H, OC ( -C6 alkyl and Ci-Ce alkyl
  • R 5 is selected from the group consisting of: an alkali metal cation (for example Na + or K + ), branched or straight chain C1-C10 alkyl, wherein one or more methylene groups of the alkyl chain may optionally be replaced by a group selected from: -0-, -NH-, -S- and carbonyl, or R 5 is - (CH 2 )z-phenyl or (CH 2 ) z -pyridyl, wherein the phenyl and pyridyl groups may optionally be substituted with one or more substituents selected from the group consisting of: trihalomethyl and nitro, z is an integer between 0 and 8, or R5 is a glycoside moiety which is optionally protected with one or more protecting groups, and Re is selected from the group consisting of: H, C ⁇ -C 6 alkyl, COO Ci-C
  • Rg is H.
  • R4 is selected from H and OCi-C 6 alkyl.
  • R4 is selected from H, methoxy, ethoxy, propoxy and isopropoxy. In a further embodiment, R4 is selected from H and methoxy.
  • R4 is located at the 5-position.
  • R 5 is selected from the group consisting of: an alkali metal cation, branched or straight chain Ct-Cio alkyl, wherein one or more methylene groups of the alkyl chain may optionally be replaced by a group selected from: -O- and carbonyl, a - (CH2) Z -Ph, wherein z is an integer between 1 and 4, and a glycoside moiety which is optionally protected with one or more protecting groups.
  • R 5 is selected from the group consisting of: an alkali metal cation, branched or straight chain Ci-Cio alkyl, wherein between one and three methylene groups of the alkyl chain may optionally be replaced by a group selected from: -O- and carbonyl,a -(CH 2 ) Z -Ph, wherein z is an integer between 1 and 4, and a glycoside moiety which is optionally protected with one or more protecting groups.
  • the protecting groups on the glycoside moiety are selected from trimethylsilyl, tertiarybutyldimethylsilyl, acetyl, or benzyl.
  • R 5 is selected from the group consisting of: an alkali metal cation, branched or straight chain Ci-C 6 alkyl, wherein one or two methylene groups of the alkyl chain may optionally be replaced by a group selected from: -O- and carbonyl, or R 5 is -(CH 2 ) Z -Ph, wherein z is 1 or 2.
  • R 5 is selected from the group consisting of: an alkali metal cation, -CH 2 OMe, -C3 ⁇ 4OEt, CH 2 OC(0)Me, CH 2 C(0)OMe, CH 2 C(0)OEt, -CH 2 -Ph or - CH CH 2 -Ph.
  • P is selected from H, trimethylsilyl, acetyl, or benzyl.
  • Exemplary compounds of formula (III) include:
  • P is selected from H, trimethylsilyl, acetyl, or benzyl.
  • the compounds of formula (I), (II) and (III) may have one or more chiral centres.
  • the present invention includes all enantiomers and diastereoisomers, as well as mixtures thereof in any proportions.
  • the invention also extends to isolated enantiomers or pairs of enantiomers.
  • salts including pharmaceutically acceptable salts.
  • Salts of the compounds of formula (I), (II) and (III) may be prepared by conventional methods known to those skilled in the art.
  • base-addition salts may be prepared by reacting the compounds of formula (I), (II) or (III) with a suitable base.
  • alkali metal salts such as lithium, potassium and sodium
  • 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), (II) or (III) with organic or inorganic acids.
  • organic or inorganic acids examples include HC1, 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 formula (I), (II) or (III) may also be quaternised by reaction with compounds such as (Ci-G alkyl halides, for example, methyl, ethyl, isopropyl and butyl halides.
  • Nitrate compounds of formula (I) may be prepared as depicted in Scheme 1 by coupling of ⁇ ,-homoserine lactone hydrobromide (a-amino-y-butyrolactone) 1 with appropriately functionalised nitrate carboxylic acid derivatives 2 using a peptide coupling agent to provide the series of compounds of formula 3.
  • the nitrate carboxylic acid derivatives 2 may be nitrate.
  • nitrate compounds of formula (I) having a 3-oxo functionality may be prepared as depicted in Scheme 2.
  • Appropriately functionalised nitrate carboxylic acid derivatives 4 are reacted with Meldrum's acid 5 to provide acylated intermediates 6, which are subsequently reacted with -homoserine lactone hydrobromide 1 to provide the 3-oxo series of compounds 7.
  • Boc-glycine 8 is coupled to Z-homoserine lactone hydrobromide 1 via EDC coupling followed by deprotection of the Boc-group.
  • the homoserine lactone-glycine derivative 9 is then coupled with appropriately functionalised nitrate carboxylic acid derivatives 2a to provide the series of compounds 10 having an NH functionality at the 3- position.
  • the carboxylic acids 11 are coupled with appropriately functionalised diazeniumdiolates 12 under standard conditions to provide diazeniumdiolate compounds of formula 13.
  • the intermediate carboxylic acids of formula 11 may be prepared according to Scheme 5.
  • Fimbrolide-JV-diazeniumdiolate compounds 25 of formula (II) wherein the diazeniumdiolate is linked via one of its oxygen atoms may be prepared as shown in Scheme 7, by reaction of the fimbrolide derivatives 23 with diazeniumdiolates 24.
  • Fimbrolide-N-diazeniumdiolate compounds 27 of formula (II) wherein the diazeniumdiolate is linked via the nitrogen not attached to an oxygen atom may be prepared as shown in Scheme 8, by reaction of the fimbrolide derivatives 26 with diazeniumdiolates 12.
  • Indole diazeniumdiolate compounds 33 of formula ( ⁇ ) may be prepared by reaction of indoles of formula 32 with sodium methoxide and NO under high pressure (about 80 psi) as shown in scheme 12.
  • Representative diazeniumdiolate compounds 34 to 36 and 38 may be prepared from compound 33 as depicted in Scheme 13.
  • Species may include, but are not limited to, Pseudomonas spp. such as P. aeruginosa, Pseudoalteromonas spp. such as P. tunicata, Staphylococcus spp. such as S. aureus and S. epidermidis, Streptococcus spp., Escherichia spp. such as E.
  • marcescens Porhyromonas spp., Fusobacterium spp., Proteus spp., Aeromonas spp., Arthrobacter spp., Micrococcus spp., and Burkholderia spp..
  • identities of the particular species in the mixed communities of the biofilm to be treated are undetermined and are not critical to the applicability of the invention.
  • compounds of formula (I), (II) and (III) are typically used in amounts such that a low, non-toxic concentration of nitric oxide is released in the vicinity of the biofilm or biofilm-forming microorganisms.
  • concentration may be in the nanomolar, micromolar, or millimolar range.
  • the concentration may be 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 ⁇ , between about 500 nM and 100 ⁇ , or between about 1 ⁇ and about 50 ⁇ .
  • 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 employed, 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.
  • antibiotics or antimicrobial agents may be employed in combination with at least one additional antibiotic or antimicrobial agent.
  • compounds of the present invention may be administered or delivered in conjunction with one or more antibiotics or antimicrobial agents, either simultaneously or sequentially.
  • the antibiotics or antimicrobial agents may be formulated into the same composition as the compounds of the present disclosure.
  • Suitable antimicrobial agents include, but are not limited to, detergents, surfactants, agents that induce oxidative stress, bacteriocins and antimicrobial enzymes, peptides and phage.
  • Antimicrobial enzymes include but are not limited to lipases, pronases, lyases (e.g.
  • antibiotics and antimicrobial agents may be natural or synthetic.
  • the antibiotic or antimicrobial agent employed may be selected for the particular application of the invention on a case-by-case basis, and those skilled in the art will appreciate that the scope of the present invention is not limited by the nature or identity of the particular antimicrobial agent.
  • compositions of the present disclosure find particular application in the treatment, prevention and ongoing management of infectious diseases and of diseases and conditions associated with, characterised by, or caused by biofilms and biofilm-forming microorganisms.
  • infectious diseases and of diseases and conditions associated with, characterised by, or caused by biofilms and biofilm-forming microorganisms For example, a variety of bacterial infections associated with biofilm formation may be treated with methods and compositions of the invention, such as cystic fibrosis, otitis media, bacterial endocarditis, kidney stones, legionnaire's disease, urinary tract infections, pulmonary infections, dental plaque, dental caries and infections associated with surgical procedures or burns.
  • compositions of the invention may be formulated as pharmaceutical compositions or form components of, for example, surgical dressings, mouthwash, toothpaste or saline solutions.
  • compositions of the present invention 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 invention are therefore useful for preventing spoilage and hence increasing the usable life of any type of pharmaceutical, cosmetic, dermatological or topical delivery compositions to which they are added.
  • the compounds or compositions of the invention may be conveniently included in any solid or liquid pharmaceutical, cosmetic, dermatological or topical delivery composition during the manufacture thereof, or alternatively after manufacture.
  • cosmetic composition is understood to mean a composition intended for placement in contact with any external part of a human or 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, mamtaining (i.e. moisturising or exfoliating), beautifying, altering the appearance of, or altering the odour of, the body.
  • 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).
  • Compounds, compositions, methods and uses of the present invention 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), 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, medical fixation devices (e.g. rods, screws, pins, plates and the like).
  • embodiments of the present invention 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.
  • Compounds, compositions, uses and methods of the present invention 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 amenable environments and settings include, for example, as marine anti-fouling 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.
  • compositions according to the present invention 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.
  • 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.
  • the composition may be formulated as a paint, wax, other coating, emulsion, solution, gel, suspension, beads, powder, granules, pellets, flakes or spray.
  • 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.
  • compositions of the invention typically 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.
  • Controlled release of the compounds of the invention may be desirable and may be imparted by the formulation of compounds into compositions.
  • a number of suitable controlled release systems are known in the art.
  • polymeric colloidal particles or microencapsulates in the form of reservoir and matrix devices may be employed, or the agent may be contained by a polymer containing a hydrophilic and/or leachable additive e.g., 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 C6o Buckminster-fullerenes ('Buckyballs') or hyperbranched (starburst) dendrimers may also be used.
  • the composition for example in the form of a paint or other surface coating, employs a carrier enabling the controlled release of the active agent temporally and/or spatially.
  • a carrier enabling the controlled release of the active agent 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, the disclosure of which is incorporated herein by reference.
  • the rate of release of the substance is determined 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.
  • release may be diffusion controlled, chemically controlled or solvent activated.
  • 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 fibres.
  • 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.
  • a polymer degrades over time and releases an agent in an amount proportional to the gradual erosion.
  • Chemical control can be achieved using bioerodible or pendant chains.
  • the agent 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.
  • the agent In a pendant chain system, the agent is covalently bound to the polymer and is released by bond scission owing to water or enzymes.
  • 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.
  • the environmental fluid e.g., water
  • the polymer e.g. a hydrogel
  • the swollen polymer is in a rubbery state and allows the drug contained within to diffuse through the encapsulant.
  • Chemical bonding of a bioactive agent to a polymer can be accomplished in several general ways based on different methods of synthesis well known to those skilled in the art including: reaction on preformed polymers; reactions on naturally-occurring polymers; polymerization of vinyl monomers containing the active ingredient; and step growth polymerizations.
  • the bioactive agent is chemically bonded to a polymer, the bond has to be cleaved by a chemical reaction- typically enzymatic, hydrolytic, thermal, or photochemical.
  • a variety of chemical and physical variables can affect the rate of bond cleavage and subsequent release of chemically attached materials from polymers including the nature of the labile bone, length of the spacer group, molecular weight, hydrophilicity, neighbouring group effects, environmental factors and physical form and dimensions.
  • 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.
  • paints may also contain a toxicant additive such as cuprous oxide or a triorganotin compound.
  • the usual paint additives such as pigments, thixotropic agents may also be present.
  • the polymeric organotin binder is gradually hydrolysed, and the tributyltin is liberated in a form that is an active antifoulant.
  • the hydrolysed polymer formed is water-soluble or water-swellable and is easily eroded off the surface by moving seawater, exposing a fresh surface of paint.
  • Examples of pharmaceutically acceptable diluents are 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 iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol,
  • compositions may be formulated for delivery by any route, for example oral, topical, intracavitary, intravesical, intramuscular, intraarterial, intravenous or subcutaneous.
  • the compound was prepared as described above for 2-(nitrooxy)acetic acid using 3- bromopropanoic acid (1.00 g, 6.53 mmol) and silver nitrate (1.66 g, 9.80 mmol) in acetonitnle (30 mL) at 70 °C for 18 h to yield the title compound as a yellow oil (0.82 g, 94%).
  • the compound was prepared as described above for compound 2-(nitrooxy)acetic acid using 8-bromooctanoic acid (0.75 g, 3.36 mmol) and silver nitrate (1.19 g, 7.05 mmol) in acetonitrile (20 mL) to give the title compound as a yellow oil (0.64 g, 94%).
  • the compound was prepared as described for compound 2-(nitrooxy)acetic acid by using 11-bromoundecanoic acid (3.00 g, 11.3 mmol) and silver nitrate (2.40 g, 14.7 mmol) in acetonitrile (60 mL) at 70 °C for 18 h followed by workup to yield the title compound as a yellow solid (2.68 g, 96%). M.p. 38-40 °C, lit.
  • the compound was prepared as described for compound 2-(nitrooxy)acetic acid by using 4-(bromomethyl)benzoic acid (2.00 g, 9.30 mmol) and silver nitrate (2.05 g, 12.1 mmol) in acetonitrile (50 mL) at 70 °C for 18 h followed by workup to yield the title compound as a white solid (1.72 g, 94%). M.p. 162-164 °C, lit.
  • the compound was prepared as described above for 2-(nitrooxy)acetic acid, using 6- bromohexanoic acid (2.00 g, 10.2 mmol) and silver nitrate (2.26 g, 13.3 mmol) in acetonitrile (50 mL) at 70 °C for 18 h followed by workup to yield the title compound as a yellow oil (1.68 g, 93%).
  • CisIfcetoOeNa M + Na 353.1689. Found 353.1681; Anal. Calcd. for C15H26N2O6: C, 54.53; H, 7.93; N, 8.48. Found: C, 54.98; H, 8.15; N, 8.31.
  • N,N-(dimethylamino)pyridine (DMAP) (0.22 g, 1.96 mmol
  • N,N- dicyclohexylcarbodiimide (DCC) (0.40 g, 1.18 mmol
  • l l-(nitrooxy)undecanoic acid (0.40 g, 1.64 mmol)
  • Meldrum's acid (0.23 g, 1.64 mmol) were dissolved in 20 mL of dichloromethane. The resulting solution was stirred overnight, cooled to r.t. and then filtered to remove N.JV-dicyclohexyl urea formed in the reaction. The filtrate was concentrated in vacuo.
  • the title compound was synthesized as described above for l l,13-dioxo-13-(2- oxotetrahydrofuran-3-ylamino)tridecyl nitrate by reacting 6-(nitrooxy)hexanoic acid (0.42 g, 1.64 mmol) with Meldrum's acid followed by L-homoserine lactone hydrobromide, the resulting crude mixture was purified by vacuum chromatography to afford the title compound as a light yellow solid (0.36 g, 57%). M.p.
  • the preparation of the title compound involved the synthesis of 2-amino-N-(2- oxotetrahydrofuran-3-yl)acetamide by coupling L-homoserine lactone hydrobromide (0.5 g, 2.73 mmol) with N-Boc-glycine 8 (0.57 g, 3.28 mmol) in the presence of EDC (0.783 g, 4.10 mmol) and triethylamine (0.41 mL, 3.07 mmol) in DCM. The mixture was stirred at room temperature for 24h and then evaporated in vacuo to dryness.
  • 02-(2,4-dinitrophenyl)-l-[4-(tert-butoxycarbonyl)piperazin-l-yl]diazen-l- ium-l,2-diolate (0.35 g) in 22 mL of ethyl acetate was added 1.5 mL of concentrated hydrochloric acid.
  • the resulting yellow solution was stirred at room temperature for 2 h, during which time a white precipitate was evident.
  • Method A A mixture of succinic anhydride (5 g, 50.0 mmol), benzyl alcohol (5.69 mL, 55.0 mmol) and catalytic amount DMAP in anhydrous THF (20 mL) was heated at reflux for 12 h. The solution was made acidic with IN HC1 and THF was evaporated. The residue was taken up in ethyl acetate and washed with IN HC1. The organic layer was made basic with 5% NaHC03 and washed with ethyl acetate, and organic layer was discarded. The basic layer was acidified with IN HC1 and extracted with ethyl acetate, dried (Na2S04) and filtered and the solvent was removed under vacuum to provide the title compound as a white solid (8.63 g, 83 %).
  • Method B Succinic anhydride (5 g, 50.0 mmol) was dissolved in anhydrous DCM (40 mL). Benzyl alcohol (5.69 mL, 55.0 mmol), triethylamine (7.50 mL, 55.0 mmol), and a catalytic amount of DMAP were added to this solution. The resulting clear solution was stirred at room temperature for 18h, after which the, all the volatiles were removed under vacuum. The crude residue was taken up in diethyl ether (200 mL) and was extracted with 2N NaOH (2 ⁇ 75 mL). The aqueous extracts were carefully acidified to pH 2 with concentrated HC1 and then extracted with diethyl ether (2 ⁇ 100 mL).
  • the benzyl group was removed by hydrogenation using 10% Pd/C (0.20 g) and H2 gas at atmospheric pressure in THF at reflux for 30h.
  • the crude reaction mixture was filtered through a column of Celite and silica to remove Pd C.
  • the filtrate was evaporated to dryness to give the title compound as a white solid (0.30 g, 84%). M.p.
  • the title compound was synthesized following the procedure for 4-oxo-4-(2- oxoteti ⁇ ydroiuran-3-ylamino)butanoic acid using acid 6-(Benzyloxy)-6-oxohexanoic acid (0.64 g; 3.00 mmol) first to synthesize the protected acid (0.5 g, 58%).
  • CioHisNOsNa (M + Na)+ 252.0848. Found 252.0840; Anal. Calcd. for C10H15NO5: C, 52.40; H, 6.60; N, 6.11. Found: C, 52.42; H, 6.73; N, 6.17. 02-Methyl-l-(4-(4-oxo-4-(2-oxotetrahydrofiiran-3-ylamino)butanoy yl)diazen-l-ium-l,2-diol te
  • 6-Oxo-6-(2-oxotetrahydrofuran-3-ylamino)hexanoic acid (0.2 g, 0.87 mmol) was coupled with C -methyl l-(piperazin-l-yl)diazen-l-ium-l,2-diolate (0.15 g, 0.95 mmol) as described for 02-memyl-l-(4-(4-oxo-4-(2-oxotetrahydrofuran-3- ylamino)butanoyl)piperazin-l-yl)diazen-l-ium-l,2-diolate to give the title compound as a yellow oil (0.11 g, 35%).1H NMR (300 MHz, CDCb): ⁇ 2.16-2.21 (m, 1H, H4a), 2.23-2.35 (m, 4H, 2 x CH2), 2.64-2.73 (m, 1H, H4b), 3.34-3.40 (m, 4
  • the title compound was synthesized following the procedure for 02-methyl-l-(4-(4-oxo-4- (2-oxotetrahydrofuran-3-ylammo)butanoyl)piperazin- 1 -yl)diazen- 1 -ium- 1 ,2-diolate using 6-oxo-6-(2-oxotetrahydrofuran-3-ylamino)hexanoic acid (0.1 g, 0.43 mmol) and 02-(2,4- dinitrophenyl) l-(piperazin-l-yl)diazen-l-ium-l,2-diolate, hydrochloride salt (0.16 g, 0.47 mmol) to give the title compound as a yellow solid (0.16 g, 72%).
  • the title compound was obtained by reacting 2-acetamido-3-mercapto-3-methyl-N-(2- oxotetrahydrofuran-3-yl)butanamide (0.02g, 0.072 mmol) with i-butyl nitrite (0.02 mL, 0.14 mmol) in chloroform at r.t. for 45 minutes. Evaporation of the solvent yielded the title compound as yellow green material.
  • N-bromosuccinimide (3.15 g, 17.74 mmol) was added to a solution of 3-butyl-5- (dibromomemylene)-2(5H)-furanone (5 g, 16.13 mmol) and catalytic amount benzoyl peroxide in carbon tetrachloride (CCk) (60 mL). The mixture was irradiated with 250W lamp and was refluxed for 18 h. The crude product was filtered through a pad of silica, filtrate evaporated and purified by vacuum chromatography using dichloromethane/n- hexane to afford the title compound as a light yellow solid (6.27 g, 100%). M.p.
  • the title compound was synthesized following the procedure for compound 3-(l- hydroxybutyl)-5-(dibromomemylene)furan-2(5H)-one using the bromofimbrolide 3-(l- bromododecyl)-5-(dibromomethylene)furan-2(5H)-one (2 g, 4.73 mmol) and DMSO/H2O to give the title compound as a yellow solid (1.20 g, 58%). M.p.
  • the desired compound was synthesized using malonyl dichloride (0.40 g, 2.85 mmol), 5- (dibromomethylene)-3-(l-hydroxydodecyl)furan-2(5H)-one (0.5 g, 1.14 mmol) and pyridine (0.27 mL, 3.42 mmol) as described for 4-(l-(5-(dibromomethylene)-2-oxo-2,5- dmydrofuran-3-yl)butoxy)-4-oxobutanoic acid to yield the title compound as an off white solid (0.47 g, 79%). M.p.
  • the desired compound was synthesized using succinyl dichloride (0.31 mL, 2.85 mmol),
  • CiiIiboBnOeNa (M + Na)+ 559.0307. Found 559.0304; Anal. Calcd. for CioHasBnOe: C, 46.86; H, 5.62. Found: C, 47.12; H, 5.48.
  • the desired compound was synthesized using 3-(l-(5-(dibromomethylene)-2-oxo-2,5- dihydrofiiran-3-yl)dodecyloxy)-3-oxopropanoic acid (0.2 g, 0.37 mmol), 02-methyl 1- (piperazin-l-yl)diazen-l-ium-l,2-diolate (0.07 g, 0.44 mmol) and EDC (0.10 g, 0.55 mmol) as described for (Z)-l-(4-(4-(l-(5-(dibromomethylene)-2-oxo-2,5-dihydrofuran-3- yl)butoxy)-4-oxobutanoyl)piperazin-l-yl)-2-methoxydiazene oxide to yield the title compound as a white solid (0.20 g, 80%).
  • N-Bromosuccinimide (0.22 g, 1.24 mmol) was added to a solution of l,3-dibutyl-5- (dibromomethylene)-lH-pyrrol-2(5H)-one (0.412 g, 1.12 mmol) and benzoyl peroxide (0.03 g) in carbon tetrachloride (CCU) (30 mL). The mixture was irradiated with 250W lamp and was refluxed for 24 h. The crude product was filtered through a pad of silica- evaporated and purified by vacuum chromatography using dichloromethane/n-hexane to afford the title compound as a yellow oil (0.4 g, 79%).
  • the title compound was prepared as described for the compound 3-(l-bromobutyl)-l- butyl-5-(dibromomethylene)-lH-pyrrol-2(5H)-one.
  • the title compound was prepared as described for the compound 3-(l-bromobutyl)-l- butyl-5-(dibromomethylene)-lH-pyrrol-2(5H)-one.
  • the fimbrolide derivative, l-benzyl-3- butyl-5-(dibromomethylene)-lH-pyrrol-2(5H)-one (0.4 g, 1.00 mmol) was brominated using NBS (0.21 g, 1.20 mmol) in presence of benzoyl peroxide (0.04 g) to give the title compound as an off white solid (0.48 g, 100%).
  • the title compound was prepared as described for the compound 3-(l-bromobutyl)-l- butyl-5-(dibromomethylene)-lH-pyrrol-2(5H)-one.
  • the title compound was prepared as described for the compound 3-(l-bromobutyl)-l- butyl-5-(dibromomethylene)-lH-pyrrol-2(5H)-one.
  • the fimbrolide derivative, l-(4- bromophenyl)-3-butyl-5-(dibromomethylene)-lH-pyiTol-2(5H)-one (0.50 g, 1.07 mmol) was brominated using NBS (0.23 g, 1.29 mmol) and benzoyl peroxide (0.03 g) to give the title compound as an off white solid (0.57 g, 97%).
  • the title compound was synthesized following the procedure for l-(l-butyl-5- (dibromomethylene)-2-oxo-2,5-dihydro-lH-pyrrol-3-yl)butyl nitrate using l-benzyl-3-(l- bromobutyl)-5-(dibromomethylene)-lH-pyrrol-2(5H)-one (0.3 g, 0.62 mmol) and silver nitrate (0.13 g, 0.81 mmol) to give the title compound as a yellow solid (0.20 g, 69%). M.p.
  • the title compound was synthesized following the procedure for l-(l-butyl-5- (dibromomethylene)-2-oxo-2,5-dihydro-lH-pyrrol-3-yl)butyl nitrate using 3-(l- bromobutyl)-5-(dibromomethylene)-l-phenyl-lH-pyrrol-2(5H)-one (0.22 g; 0.47 mmol) and silver nitrate (0.10 g, 0.61 mmol) to give the title compound as a light brown solid (0.15 g, 72 %).
  • the title compound was synthesized following the procedure for l-(l-butyl-5- (dibromomemylene)-2-oxo-2,5-dihydro-lH-pyrrol-3-yl)butyl nitrate using the bromo- derivative 3-(l-bromobutyl)-l-(4-bromophenyl)-5-(dibromomethylene)-lH-pyrrol-2(5H)- one (0.30 g, 0.55 mmol) and silver nitrate (0.12 g, 0.71 mmol) to give the title compound as a white solid (0.18 g, 74%). M.p.
  • the desired compound was prepared from 5-methoxyindole (3.00 g, 20.38 mmol), NaOMe (2.20 g, 40.76 mmol), and nitric oxide as described above for sodium l-(lH-indol-3-yl)- diazen-l-ium-l,2-diolate to give the title compound as a yellow solid (3.4 g, 72%). M.p.
  • Acetic anhydride (10 mL, 15.84 mmol) was added dropwise over a period of 30 min to a stirring solution of formic acid (25 mL, 15.84 mmol) containing the thiazolidine derivative (3 g, 15.84 mmol) and sodium formate (1.18 g, 17.43 mmol) at 0 °C.
  • the reaction mixture was further stirred at 0 °C for lh, followed by room temperature for 6h. At the end of this period, ice water was added and the resulting precipitate collected by filtration to give the title compound as a white solid (2.1 g, 63%).
  • Gfp a reporter gene fused to the promoter of the quorum sensing regulated gene lasB from P. aeruginosa.
  • the half life of Gfp (ASV) is approximately 110 minutes allowing online monitoring of changes in gene expression over a time span of a few hours.
  • the gene encoding the modified Gfp (ASV) fused to a promoter is positively regulated by quorum sensing (such as the lasB promoter)
  • the elevated expression of the quorum sensing-controlled gene will result in an increase in fluorescence.
  • the reporter plasmid is carried in the wild-type strain P. aeruginosa PAOl, which produces its own AHLs.
  • the reporter construct, VlasBr.gfp is therefore induced in this strain and an increase in Gfp production can be observed during the normal growth of the strain.
  • the addition of a quorum sensing inhibitor to this bioreporter will result in a lowered expression of Gfp (ASV) to an extent that correlates with the efficiency of the inhibitor.
  • the assay is performed by measuring the Gfp output in fluorescence units in the presence of the inhibitor compounds at various concentrations, and the degree of inhibition is determined by comparison with Gfp expression in control cells in the absence of inhibitor.
  • the final concentration of the inhibitors for this assay ranged from 1000 ⁇ to 1.3 ⁇ , and the plates were incubated in a microtitre plate reader, Wallac Victor 2 (Perkin Elmer). No inhibitor was added to the last row of the plate and hence was used as a control. The measurements were made every 30 min over 15 h for the cell growth (OD600) and the expression of Gfp (fluorescence, excitation 485 nm, emission 535 am).
  • the percentage quorum sensing (QS) inhibition values were calculated and compared to the control without inhibitor, at concentrations exhibiting less than 15% growth inhibition on the bacterial host. This latter point is important as it excludes QS inhibitory effects that are due to inhibition on bacterial growth or viability, and therefore identifies those compounds that have specific QS inhibition activities.
  • the untreated control value was considered as 0% QS inhibition and the percentage inhibition was calculated at the time point of about 8 to 9 h corresponding to the maximum QS activity in the untreated control.
  • the potency of a compound as a QS inhibitor is directly correlated with its calculated percentage of QS inhibition.
  • compound 37 has a higher QS inhibition potency. At 37 ⁇ , compound 37 inhibited QS activity by 62%, compared to only 6% for compound 34a. It was also interesting to note that compound 37 was more toxic to the test species with growth inhibition (> 40%) at 111 ⁇ , whereas compound 34a did not affect the growth of the bacterial host even at 333 ⁇ .
  • the QS inhibitory assay was performed on selected AHL-based compounds and the results of this assay are shown below.
  • the representative RFU, OD and RFU/OD data for compound 97a is presented in Figure 3.
  • Table 2 Percentage quorum sensing inhibition at different concentrations of AHL derivatives.
  • Table 3 Percentage quorum sensing inhibition at different concentrations of the fimbroiide derivatives.
  • Fimbroiide derivatives with shorter alkyl chains and with substitution at the C-a-position (CI') were the most potent inhibitors. Furthermore, longer alkyl chain lengths result in weaker growth inhibition. Thus, a compromise between QS inhibitory activity and growth inhibition may be maintained by controlling the chain length in order to achieve a desired biological effect. In case of the C-a-substituted derivatives, smaller hydrophobic or hydrophilic substituents resulted in stronger QS inhibition compared to larger hydrophobic or hydrophilic substituents.
  • Table 4 Percentage quorum sensing inhibition at different concentrations of dihydropyrrolones derivatives.
  • the spectroscopic methods for NO detection include absorbance spectroscopy using the Griess reagent, fluorescence-based detection involving ' diaminofluoresceins (DAFs) and chemiluminescence methods that employ the reaction of NO with ozone.
  • Other techniques include electron paramagnetic resonance (EPR) and electrochemical methods using an amperometric NO sensor.
  • the NO detection method employed in this study is based on the quantification of nitrite using Griess reagent and an amperometric NO sensor instrument Apollo 4000.
  • the most common approach for the detection of nitrite is the Griess assay.
  • the Griess reagent assay typically relies on the diazotization of a suitable aromatic amine sulfanilamide by acidified nitrite (N0 2 " , followed by a subsequent coupling reaction with N-l-naphthylethylenediamine dihydrochloride (NED), producing a highly coloured azo chromophore (see scheme below).
  • the absorption maximum for the azo product is at 540 nm and can be detected using conventional UV-visible absorption spectroscopy, from which the concentration of nitrite can be assessed.
  • the Griess assay can also be utilized for the measurement of organic nitrate (R-NO 3 ) after reduction of nitrate to nitrite (N0 2 ⁇ ). Nitrate can also be converted via N0 2 ⁇ to NO which in turn due to its reactive nature converts back to nitrite (N0 2 -) in oxygenated aqueous media as shown below.
  • Methods for nitrate reduction to nitrite include treatment with thiols (cysteine) or metal reductants such as cadmium, zinc, and vanadium chlorides.
  • Enzymatic reduction may also be employed using nitrate reductase obtained from bacteria.
  • NADPH nicotinamide adenine dinucleotide phosphate-oxidase
  • Organic nitrate can also be reduced to NO using xanthine oxidase in presence of xanthine and cysteine.
  • xanthine oxidase along with xanthine and cysteine was found to be most suitable and was thus utilized for the study of the selected compounds.
  • the plate was brought to room temperature, an aliquot of the Griess reagent (20 ⁇ ), freshly prepared by mixing equal volumes of 1.0% sulfanilamide (reagent B) and 0.1% N- naphthylethylenediamine dihydrochloride (reagent A) was added, followed by the addition of 130 ⁇ of 0.1 M phosphate buffer, pH 7.4. After 30 min, the absorbance was measured at 540 nm using a plate reader (Wallac Victor 2 , Perkin-Elmer).
  • the Griess assay results indicate that the nitrate derivatives can be converted into NO, and can be quantified as nitrite ions using the Griess reagent.
  • Compound 95a had a better total nitrite release compared to the standard ISDN 43 (see Figure 5) which has two nitroester (ON0 2 ) group in the structure, compared to one nitroester group in case of the synthesized derivatives tested.
  • An Apollo 4000 free radical analyzer (World Precision Instruments (WPI), Sarasota, USA) was used as an alternative technique to measure NO concentrations and is particularly useful for spontaneous NO releasing compounds.
  • An ISO-NOP electrode (2 mm in diameter) was calibrated daily using SNAP and Cu(II) as a catalyst according to the manufacturer's instructions. The calibration curve obtained was in the range of 1 to 2 pA for 1 nM NO.
  • the electrode was plunged through the hole of a silicone septum of a glass vial containing 10 ml of PBS buffer (pH 7.4) and gently agitated with a magnetic stirrer.
  • Biofilm dispersal assay is carried out on established biofilms and is utilized to study the effect of NO on dispersion of established biofilms. This assay is particularly useful for spontaneous NO releasing compounds, thus compound 237 (IND-1) and 243d (IND-2) which can release NO spontaneously were studied via this assay.
  • the PAOl biofilm dispersion assay indicated that compound 243d at 100 ⁇ could induce more than 60% dispersion of the biofilm within 10 min, with better potency than the standard NO donor NOC 9 and exhibited no bacterial growth inhibition.

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Abstract

L'invention concerne de manière générale des conjugués comprenant un donneur d'oxyde nitrique et un acyl homosérine lactone, un fimbrolide, un dérivé de fimbrolide, un dihydropyrrolone ou un indole, ainsi que l'utilisation de ces conjugués en qualité d'agents antimicrobiens.
PCT/AU2013/001292 2012-11-08 2013-11-08 Donneurs d'oxyde nitrique à double action et leur utilisation comme agents antimicrobiens WO2014071457A1 (fr)

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WO2017034482A3 (fr) * 2015-08-24 2017-04-06 Agency For Science, Technology And Research Conjugués
WO2021159176A1 (fr) * 2020-02-12 2021-08-19 The University Of Wollongong Composés antimicrobiens

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