Field of the Invention

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.

Background of the Invention

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. In the case of 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. Similarly, 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.

It is therefore clear that the inhibition or dispersion of biofilms is a key strategy in controlling and reducing microbial infectious diseases.

Against this background, the present inventors have developed 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.

Summary of the Invention

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

wherein L is a linker and Y is a nitric oxide (NO) donor.

In a second aspect, the invention provides a compound of formula (II), or a salt thereof:

wherein L _\ is a linker, Yi is an NO donor and A is O or NR', and wherein 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.

In a third aspect, the invention provides a compound of formula (III), or a salt thereof

wherein R4 is selected from H, OC1-C alkyl and Ci-C ₆ alkyl, and 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 and phenyl.

In a fourth aspect, 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.

In a fifth aspect, 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.

In a sixth aspect, the invention provides a method for inhibiting biofilm formation and/or development, the method comprising exposing biofilm-forming microorganisms to an 201

4

effective amount of a compound of the first, second or third aspects or a composition of the fourth aspect.

In accordance with the fifth and sixth 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.

In particular embodiments of the fifth and sixth 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,

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. Alternatively, or in addition, methods may comprise increasing the sensitivity of a microorganism to antimicrobial agents.

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.

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 spp., Serratia spp., Porphyromonas spp., Fusobacterium spp. and Vibrio spp. In particular embodiments 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. In particular embodiments, 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.

Accordingly, in a seventh aspect, there is provided 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, the method comprising administering to the subject an effective amount of a compound of the first or second aspects or a composition of the third aspect.

In an eighth 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.

In a ninth aspect, 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.

In a tenth aspect, 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.

In an eleventh aspect, 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.

In a twelth aspect, 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.

In a thirteenth aspect, 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. In a fourteenth aspect, 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.

Brief Description of the Drawings

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

Figure 1: 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. 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 3: Biological screening assay for QS inhibition by AHL derivative 97a. 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 YlasB::gfp (ASV) fusion plasmid was employed.

Figure 4: Nitrite standard curve obtained using the Griess assay.

Figure 5: Nitrite concentration (μΜ) 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 (A and B): Results from biofilm dispersion study of IND-2.

Detailed Description

The articles "a" and "an" are used herein to refer to one or to more than one {i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

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

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.

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.

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

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.

As used herein, 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.

As used herein, the term "exposing" means generally bringing into contact with. Typically 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. Typically 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. Thus, a microorganism or biofilm may be exposed to 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 defined herein also includes the administration of the compound or composition to a subject in or on which the biofilm or microorganisms reside. Thus, in the present disclosure the terms "exposing", "administering" and "delivering" and variations thereof may, in some contexts, be used interchangeably. )

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 effect the expression or activity of molecules which in turn regulate biofilm formation or development.

Similarly, the term "promoting" 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.

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.

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.

As used herein, 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. Thus, the association between the infection, disease or condition and the biofilm may be direct or indirect and may be temporally separated.

In the context of this specification, the term "NO donor" is understood to mean a functional group which is capable of releasing one or more NO groups.

In the context of this specification, the term "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.

In the context of this specification, the term "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.

In the context of this specification, the term "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.

In the context of this specification, the term "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.

In the context of this specification, the term "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.

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

In the context of this specification, the term "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.

In the context of this specification, the term "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, benzothienyl, pyrrolyl, benzothiazolyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, acridinyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, benzofuryl, purinyl, benzimidazolyl, triazinyl and the like.

In the context of this specification, the term "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.

In the context of this specification, the term "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. In the context of this specification, the term "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.

In the context of this specification, the term "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.

In the context of this specification, the term "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.

In the context of this specification, the term "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.

In the context of this specification, the term "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.

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

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. Typically, 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.

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

wherein 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. ,

In another embodiment, 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.

In another embodiment, 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.

In another embodiment, 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. In another embodiment, 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.

In another embodiment, 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.

In another embodiment, 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.

In yet another embodiment, 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.

In yet another embodiment, 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. Those skilled in the art will readily appreciate that additional NO donor functional groups may also be utilised.

In some embodiments, Y is -SNO, ON0 ₂ or diazeniumdiolate. Typically, 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. For example, in one embodiment, 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:

wherein Ri is an organic functional group, T is a linker and 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.

In an alternative embodiment, 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.

In another embodiment, 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.

In still a further embodiment, 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.

In yet another embodiment, 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.

In another embodiment, 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.

In another embodiment, Ri is phenyl, methyl, ethyl, propyl or isopropyl, wherein the phenyl group may optionally be substituted with one or two nitro groups.

In still a further embodiment, 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.

In another embodiment, 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.

In still a further embodiment, 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.

In a particular embodiment, the diazeniumdiolate has the structure:

wherein A and Ri are as defined above.

In some embodiments, T may be a divalent hydrocarbon group having between 1 and 40, or between 1 and 30, or between 1 and 20 carbon atoms.

In another embodiment, 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.

In a further embodiment, 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.

In a further embodiment, 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-.

In a further embodiment, T is branched or straight chain C1-C6 alkylene.

In still a further embodiment, T is -NHC1-C6 alkylene-, wherein the -NH group is bonded to variable L.

Exemplary compounds of formula (I) include:

In a second aspect, the present invention provides compounds of formula (II), or a salt thereof:

wherein L is a linker, Yi is an NO donor and A is O or NR ¹ , and wherein 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.

In one embodiment, 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.

In another embodiment, 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.

In a further embodiment, 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.

In a further embodiment, 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.

In another

wherein the lower squiggly line of each structure denotes attachment of h _\ to the ring system, and wherein 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. In an alternative embodiment, s is an integer between 1 and 6, t is an integer between 1 and 10, and 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. Those skilled in the art will readily appreciate that additional NO donor functional groups may also be utilised.

In one embodiment, A is O.

In another embodiment, when A is O, Y _\ is not -ON0 ₂ .

In one embodiment, A is O, Y\ is a diazeniumdiolate and Lj is selected from:

wherein u and t are as defined above.

In a further embodiment, A is NR.'.

In yet another embodiment, A is NR' and Yj is -ON0 ₂ .

In yet another embodiment, A is NR', and L| is:

wherein 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.

In one embodiment, 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. In another embodiment, 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.

In a further embodiment, 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.

In yet another embodiment, R' is H, butyl, (CH ₂ )-phenyl or phenyl, wherein the phenyl may be substituted with 1 or 2 halo substituents.

In yet another embodiment, R' is H, (CH ₂ )-phenyl or phenyl, wherein the phenyl may be substituted with 1 or 2 halo substituents.

In still a further embodiment, R' is H, butyl^ (CH ₂ )-phenyl, phenyl or bromophenyl,

In some embodiments, Yi is a diazeniumdiolate. Typically, 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. Alternatively, the N-diazeniumdiolate may be attached to variable Li via one of the oxygen atoms of the N-diazeniumdiolate.

In one embodiment, 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 _\ :

wherein R\ is an organic functional group, T is a linker and 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-, -ΝΗ- 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.

In an alternative embodiment, 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.

In another embodiment, 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.

In still a further embodiment, 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

In yet another embodiment, 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.

In another embodiment, 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.

In another embodiment, Ri is branched or straight chain C1-C6 alkyl.

In one embodiment, 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.

In another embodiment, 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.

In a further embodiment, 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.

In one particular embodiment, the diazeniumdiolate has the structure:

N-O-Ri

wherein A and Rj are as defined above.

In some embodiments, T may be a divalent hydrocarbon group having between 1 and 40, or between 1 and 30, or between 1 and 20 carbon atoms.

In another embodiment, 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: - Ο-, -NH-, -S- and carbonyl.

In a further embodiment, 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.

In a further embodiment, 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-.

In a further embodiment, T is branched or straight chain Ci-C ₆ alkylene.

In still a further embodiment, T is -NHQ-Ce alkylene-, wherein the -NH group is bonded to variable Li.

In an alternative embodiment, the diazeniumdiolate has the following structure, wherein the squiggly line denotes the point of attachment of the diazeniumdiolate moiety to the variable

wherein R ₂ and R3 are organic functional groups.

In one embodiment, 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.

In another embodiment, 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.

In yet another embodiment, 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.

In still a further embodiment, 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.

In yet another embodiment, 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.

In yet another embodiment, R ₂ and R ₃ are independently selected from C C6 alkyl.

In yet another embodiment, R ₂ and R3 are independently selected from the group consisting of: methyl, ethyl, propyl and isopropyl.

In particular embodiments, 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:

Further exemplary compounds of formula (II) include:

In a third aspect, the invention provides a compound of formula (III), or a salt thereof

(HI)

wherein R4 is selected from H, OC ₍ -C6 alkyl and Ci-Ce alkyl, and 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 ₆ alkyl and phenyl.

In one embodiment, Rg is H.

In one embodiment, R4 is selected from H and OCi-C ₆ alkyl.

In another embodiment, R4 is selected from H, methoxy, ethoxy, propoxy and isopropoxy. In a further embodiment, R4 is selected from H and methoxy.

In another embodiment, R4 is located at the 5-position.

In a further embodiment, 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.

In still a further embodiment, 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.

In an emtodiment,' the protecting groups on the glycoside moiety, if present, are selected from trimethylsilyl, tertiarybutyldimethylsilyl, acetyl, or benzyl.

In a further embodiment, 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.

In yet another embodiment, R ₅ is selected from the group consisting of: an alkali metal cation, -CH ₂ OMe, -C¾OEt, CH ₂ OC(0)Me, CH ₂ C(0)OMe, CH ₂ C(0)OEt, -CH ₂ -Ph or - CH CH ₂ -Ph.

of formula (HI) is

301 , wherein P is selected from H, trimethylsilyl, acetyl, or benzyl.

Exemplary compounds of formula (III) include:

wherein 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.

Also within the scope of the compounds of formula (I), (II) and (III) are 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. For example, base-addition salts may be prepared by reacting the compounds of formula (I), (II) or (III) 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), (II) or (III) with organic or inorganic acids. Examples of such salts include HC1, HBr and HI salts, salts of other mineral acids such as sulfate, nitrate, phosphate and the like, alkyl and 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 Ζ,-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.

Scheme 1: Preparation of nitrate compounds of formula (I)

Alternatively, 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.

Scheme 2: Preparation of nitrate compounds of formula (I) having a 3-oxo functionality

Compounds having an NH functionality at the 3 -position may be prepared as depicted in Scheme 3. 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.

9

O

DIPEA, PyBOP X,.ON0 ₂

HO Z

2a

wherein Z is as defined for X

Scheme 3: Preparation of nitrate compounds of formula (I) having an NH functionality at the 3-position Exemplary diazeniumdiolate compounds of formula (I) may be prepared as depicted below in Scheme 4.

11 13

Scheme 4: Preparation of diazeniumdiolate compounds of formula (I)

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.

11 17

Scheme 5: Preparation of intermediate compounds 11

In scheme 5, anhydrides 15 are reacted with benzyl alcohol to provide carboxylic acids 16, which are coupled with -homoserine lactone hydrobromide 1 to provide the protected acids 17, which are deprotected under standard conditions to provide intermediate compounds 11.

An exemplary S-nitroso compound of formula (I) may be prepared according to Scheme 6.

22

Scheme 6: Preparation of an S-nitroso compound of formula (I)

In Scheme 6, penicillamine 18 is reacted with acetic anhydride in the presence of base to provide the thietanone derivative 19, which is then reacted with -homoserine lactone 1 furnishing the thiol 21. The thiol functional group is then converted to an S-nitroso (S- NO) functional group via reaction with t-butyl nitrite to afford the S-NO compound 22.

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.

wherein J is O or S

Scheme 7: Preparation of fimbrolide-TV-diazeniumdiolate compounds of formula (II)

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.

Scheme 8: Preparation of fimbrolide-iV-diazeniumdiolate compounds of formula (II) l,5-dihydropyrrol-2-one nitrate compounds of formula (II) may be prepared according to Scheme 9. Fimbrolide derivatives 28 are treated with amines, which after dehydration afford l,5-dihydropyrrol-2-one derivatives 29. Bromination followed by reaction with

Scheme 9: Preparation of l,5-dihydropyrrol-2-one nitrate compounds of formula (II)

Further compounds of formula II analagous to compound 183 may be synthesised using the reaction scheme set out in scheme 10. Treatment of thio compound 188 with formic acid in the presence of acetic anhydride and sodium acetate provides amide 191, which is then reacted with bromofimbrolide 156a to provide thiazolidine fimbrolide derivative 192. Ring opening followed by reaction with f-butyl nitrate afforded compound 183.

A person skilled in the art could readily adapt the reactions set out in any of the schemes described herein using methods and reactions known in the art to arrive at further compounds of the invention.

Scheme 10: Preparation of a dibromo (nitrosothio)butanoate compound of formula (H)

Compounds of formula (II) analagous to compound 300 may also be prepared using the

Scheme 11: Preparation of a further compound of formula (II)

Indole diazeniumdiolate compounds 33 of formula (ΠΙ) 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.

Scheme 12: Preparation of indole diazeniumdiolate compounds of formula (III)

Scheme 13: Preparation of indole diazeniumdiolate compounds of formula (III)

Representative diazeniumdiolate compounds 34 to 36 and 38 may be prepared from compound 33 as depicted in Scheme 13.

Those skilled in the art will appreciate that embodiments of the present invention are applicable to single species or mixed species biofilms. 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. coli, Shigella spp., Mycobacterium spp., Enterococcus spp., Salmonella spp., Legionella spp., Haemophilus spp., Bacillus spp., Desulfovibrio spp., Shewanella spp., Geobacter spp., Klebsiella spp. such as AT. pneumoniae, Proteus spp. such as P. mirabilis, Serratia spp. such as S. marcescens, Porhyromonas spp., Fusobacterium spp., Proteus spp., Aeromonas spp., Arthrobacter spp., Micrococcus spp., and Burkholderia spp.. Alternatively those skilled in the art will appreciate that in some applications of the present invention, the 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.

In accordance with embodiments disclosed herein, 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. The concentration may be in the nanomolar, micromolar, or millimolar range. In particular embodiments, 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 μΜ, between about 500 nM and 100 μΜ, or between about 1 μΜ and about 50 μΜ. 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 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.

Compounds, compositions, uses and methods of the present invention may be employed in combination with at least one additional antibiotic or antimicrobial agent. Alternatively, or in addition, compounds of the present invention may be administered or delivered in conjunction with one or more antibiotics or antimicrobial agents, either simultaneously or sequentially. For sequential application 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. alginate lyases) and various other proteolytic enzymes and nucleases. The 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.

The compounds, compositions uses and methods 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.

Compounds, compositions, uses and methods 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. 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. Accordingly, compositions of the invention may be formulated as pharmaceutical compositions or form components of, for example, surgical dressings, mouthwash, toothpaste or saline solutions.

Compounds and 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. The term "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. 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).

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). Further, 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. 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.

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. 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 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.

Typically for anti-fouling and other industrial applications, 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 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.

Typically, 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. Those skilled in art will appreciate that a number of controlled release systems are applicable to the delivery of agents according to the present invention. By way of example only, release may be diffusion controlled, chemically controlled or solvent activated.

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 fibres. 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.

In typical chemically controlled systems 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. 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.

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.

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. When 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.

In 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 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.

Those skilled in the art will readily appreciate that the delivery systems and methods described above are merely examples of suitable methods and systems that may be employed in the present invention. 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 invention.

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, 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. Typically, the carrier or carriers will form from 1% to 99.9% by weight of the compositions.

For pharmaceutical applications, compositions may be formulated for delivery by any route, for example oral, topical, intracavitary, intravesical, intramuscular, intraarterial, intravenous or subcutaneous.

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. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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

The present invention is further described by reference to the following non-limiting examples.

Examples

[Example 1: Synthesis of compounds

2-(Nitrooxy)acetic acid

A mixture of 2-bromoacetic acid (1.00 g, 7.19 mmol) and silver nitrate (1.83 g, 10.8 mmol) in anhydrous acetonitrile (30 mL) was stirred at 70 °C for 18 h. The resulting product was filtered through a column of Celite and silica to remove the silver salt formed. The filtrate was evaporated to dryness and DCM (50 mL) was added. After standing at room temperature for 2 h the mixture was filtered to remove any leftover silver salt precipitate. The removal of DCM gave the title compound as a yellow oil (0.74 g, 86%). 1H NMR (300 MHz, CDCb): δ 4.96 (s, 2H, CH2), 9.53 (bs, 1H, OH); ¹³ C NMR (75.6 MHz, CDCb): δ 66.3 (CH2), 171.6 (C=0).

3- (Nitrooxy)propanoic acid

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%). Ή NMR (300 MHz, CDCb): δ 2.82 (t, J = 6.0 Hz, 2H, CH2COO), 4.72 (t, J = 6.0 Hz, 2H, CH2ONO2), 11.56 (bs, 1H, OH); ¹³ C NMR (75.6 MHz, CDCb): δ 30.0 (CH2COO), 66.2 (CH2ONO2), 173.9 (C=0).

8-(Nitrooxy)octcmoic acid

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%). ^l U NMR (300 MHz, CDCb): δ 1.35-1.41'(m, 6H, 3 ^χ Cl ), 1.60-1.72 (m, 4H, 2 * CH2), 2.34 (t, J= 7.3 Hz, 2H, CH2COO), 4.42 (t, J= 6.6 Hz, 2H, CH2ONO2), 11.37 (bs, 1H, OH). ll-(Nitrooxy)undecanoic acid

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. 40-41 °C; Ή NMR (300 MHz, CDCb): δ 1.28-1.43 (m, 12H, 6 * CH2), 1.62-1.73 (m, 4H, 2 ^χ CHa), 2.34 (t, J = 6.7 Hz, 2H, CH2COO), 4.43 (t, J= 6.6 Hz, 2H, CH2ONO2), 10.23 (bs, 1H, OH); ¹³ C NMR (75.6 MHz, CDCb): δ 24.5 (CH2), 25.5 (Ctb), 26.6 (CH2), 28.9 (CH2), 29.0 (CH2), 29.1 (CH2), 33.99 (CH2COO), 73.3 (CH2ONO2), 180.3 (C=0).

4- (Nitrooxymethyl)benzoic acid

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. 165 °C; Ή NMR (300 MHz, DMSO-<fc): S 5.63 (CH2ONO2), 7.54 (d, J= 9.0 Hz, 2H, H2, H6), 7.95 (d, J= 9.0 Hz, 2H, H3, H5); ¹³ C NMR (75.6 MHz, CDCb): δ 74.5 (CHa), 129.3 (ArCH), 129.9 (ArCH), 131.7 (ArC), 137.6 (ArC), 167.2 (C=0).

6-(Nitrooxy)hexanoic acid

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%). 1H NMR (300 MHz, CDCb): δ 1.41-1.49 (m, 2H, CH2), 1.63- 1.75 (m, 4H, 2 ^χ CY ), 2.36 (t, J = 7.1 Hz, 2H, CH2COO), 4.43 (t, J = 6.4 Hz, 2H, CH2GNO2), 11.72 (bs, 1H, OH); ¹³ C NMR (75.6 MHz, CDCb): δ 22.3 (CH_), 23.4 (CH2), 24.7 (CH2), 32.0 (CH2COO), 71.3 (CH2ONO2), 178.4 (C=0).

2-Oxo-2-(2-oxotetrahydrqfuran-3-ylamino)ethyl nitrate

To a stirred solution of L-homoserine lactone hydrobromide (0.36 g, 3.56 mmol) in water (4.00 mL), triemylamine (0.54 mL, 3.92 mmol) was added followed by the addition of acid 2-(Nitrooxy)acetic acid (0.64 g, 5.34 mmol) and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) (1.09 g, 5.70 mmol). The mixture was stirred at room temperature for 40 h and then evaporated in vacuo to dryness. The residue was partitioned between water (20 mL) and ethyl acetate (50 mL) and the organic layer successively washed with 5% sodium bicarbonate (NaHCCb) solution (2 ^χ 20 mL), 1M potassium hydrogen sulfate (KHSO4) solution (2 * 20 mL) and brine. The organic layer was dried over sodium sulfate (NaSO) and evaporated to dryness to yield the title compound as a white solid (0.21 g, 54%). M.p. 96-98 °C; 1H NMR (300 MHz, CDCb) δ 2.16-2.28 (m, 1H, H4a), 2.82-2.91 (m, 1H, H4b), δ 4.27-4.36 (m, 1H, H5a) 4.51 (t, J = 9.1 Hz, 1H, H5b), 4.56-4.65 (m, 1H, H3), 4.95 (s, 2H, CH2ONO2), 6.57 (bs, 1H, NH); ¹³ C NMR (75.6 MHz, CDCb) δ 30.1(CH2) , 49.1 (CH), 66.1 (CH2), 69.0 (CH2), 165.4 (C=0), 174.4 (C=0); IR (neat): v™* 3351, 3000, 2948, 2922, 1764, 1688, 1646, 1546, 1421, 1380, 1288, 1247, 1224, 1195, 1181, 1144, 1046, 1010, 982, 953, 938, 848, 815, 768, 739, 720 cm ^"1 ; HRMS (ESI) m/z Calcd. for CeHs^OeNa (M + Na)+ 227.0280. Found 227.0271. 3-Oxo-3-(2-oxotetrahydrqfuran-3-ylamino)propyl nitrate

The title compound synthesis was carried out as described for the preparation of compound 2-Oxo-2-(2-oxotetrahydrofuran-3-ylamino)ethyl nitrate using 3-(nitrooxy)propanoic acid (0.72 g, 5.34 mmol) to give the title compound as an off white solid (0.21 g, 49%). M.p. 74-76 °C; Ή NMR (300 MHz, CDCb) δ 2.13-2.28 (m, 1H, H4a), 2.67-2.71 (dt, J= 3 and 9, 2H, COCH2), 2.73-2.82 (m, 1H, H4b), 4.25-4.34 (m, 1H, H5a), 4.44-4.51 (dt, J = 9.0, 3.0 Hz, 1H, H5b), 4.56-4.65 (m, 1H, H3), 4.77 (t, J= 6 Hz, 2H, CH2ONO2), 6.85 (bd, J= 6 Hz, NH); ^,3 C NMR (75.6 MHz, CDCb) 29.5 (CH2), 33.0 (CH2), 49.1 (CH), 66.1 (CH2), 68.4 (CH2), 169.2 (C=0), 175.5 (C=0); IR (neat): vmax 3307, 3098, 2938, 1774, 1649, 1614, 1558, 1456, 1408, 1376, 1355, 1268, 1212, 1170, 1007, 951, 895, 862, 792, 760, 688 cm ^"1 ; HRMS (ESI) m/z Calcd. for C?HioN206Na (M + Na)+ 241.0437. Found 241.0426.

4 Oxo-4-(2-oxotetrahydrofuran-3-ylamino)hexyl nitrate

The title compound synthesis was carried out as described for the preparation of compound 2-oxo-2-(2-oxotetrahydrofuran-3-ylamino)ethyl nitrate using 8-(nitrooxy)octanoic acid (0.94 g, 5.34 mmol) to give the title compound as an off white solid (0.24 g, 48%). M.p. 50 °C; 1H NMR (300 MHz, CDCb) δ 1.42-1.50 (m, 2H, CH2), 1.62-1.80 (m, 4H, CH2), 2.06- · 2.21 (m, 1H, H4a), 2.27 (t, J= 6, 2H, COCH2), 2.79-2.88 (m, 1H, H4b), 4.43-4.50 (m, 3H, CH2ONO2 and H5b merge), 4.52-4.59 (m, 1H, H3), 6.06 (bd, J = 3 Hz, NH); ¹³ C NMR (75.6 MHz, CDCb) δ 24.6 (Ctb), 25.1 (CH2), 26.4 (CH2), 30.43 (CH2), 35.5 (CH2), 49.2 (CH), 66.0 (CH2), 72.8 (CH2), 172.9 (C=0), 175.3 (C=0); IR (neat): vmax 3309, 3074, 2954, 2865, 1775, 1639, 1622, 1543, 1493, 1451, 1382, 1360, 1279, 1225, 1167, 1107, 1014, 998, 976, 947, 899, 865, 759, 700, 684, 657 cm ¹ ; HRMS (ESI) m/z Calcd. for CioHieNiOeNa (M + Na)+ 283.0906. Found 283.0900.

11-Oxo-l l-(2-oxotetrahydrofuran-3-ylamino)undecyl nitrate

l l-(Nitrooxy)undecanoic acid (0.67 g, 2.67 mmol) was coupled to L-homoserine lactone hydrobromide (0.18 g, 1.78 mmol) using the method described for compound 2-oxo-2-(2- oxotetrahydrofuran-3-ylamino)ethyl nitrate to give the title compound as a white solid (0.11 g, 36%). M.p. 62 °C; Ή NMR (300 MHz, CDCb) δ 1.28-1.43 (m, 12H, 6 * CH2), 1.58-1.75 (m, 4H, 2 CH2), 2.05-2.20 (m, 1H, H4a), 2.24 (t, J = 6.0, 2H, COCH2), 2.80- 2.89 (m, 1H, H4b), 4.23-4.32 (m, 1H, H5a), 4.43 (t, J= 6.0 Hz, 2H, CH2ONO2), 4.46 (dt, J = 12.0, 3.0 Hz, 1H, H5b), 4.52-4.58 (m, 1H, H3), 6.04 (bs, 1H, NH); ^I3 C NMR (75.6 MHz, , .„

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CDCb) δ 25.2 (CH2), 25.5 (Ofc), 26.6 (CH2), 28.9 (CH2), 29.0 (CH2), 29.12 (CH2), 29.13 (CH2), 29.16 (CH2), 30.5 (CH2), 36.0 (CH2), 49.1 (CH), 66.0 (CH2), 73.3 (CH2), 173.6 (C=0), 175.4 (C=0); IR (neat): vmax 3317, 3073, 2924, 2852, 1775, 1642, 1622, 1545, 1468, 1381, 1361, 1278, 1225, 1170, 1107, 1012, 999, 946, 865, 803, 758, 722 cm ^'1 ; HRMS (ESI) m/z Calcd. for 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.

4-(2-Oxotetrahydrofuran-3-ylcarbamoyl)benzyl nitrate

4-(Nitrooxymethyl)benzoic acid (0.73 g, 3.71 mmol) was coupled to L-homoserine lactone hydrobromide (0.25 g, 2.47 mmol) using the method described above for 2-oxo-2-(2- oxotetrahydrofuran-3-ylarnino)ethyl nitrate to give the title compound as a white solid (0.17 g, 46%). M.p. 152-154 °C; 1H NMR (300 MHz, CDCb) δ 2.15-2.27 (m, 1H, H4a), 2.87-2.96 (m, 1H, H4b), δ 4.25-4.34 (m, 1H, H5a) 4.50 (dt, J = 9.0, 1.2 Hz, H5b), 4.62- 4.70 (m, 1H, H3), 5.40 (s, 2H, CH2ONO2), 6.67 (bs, 1H, NH), 7.41 (d, J = 10.2 Hz, 2H, 2 x ArH), 7.82 (d, J= 10.2 Hz, 2H, 2 χ ArH); ¹³ C NMR (75.6 MHz, CDCb) δ 30.1 (CH2), 49.8 (CH), 66.3 (CH2), 73.6 (CH2), 127.7 (ArCH), 128.9 (ArCH), 133.8 (ArC), 136.4 (ArC), 167.0 (C=0), 175.4 (CO); IR (neat): Vmax 3299, 2925, 1775, 1614, 1577, 1533, 1506, 1449, 1384, 1342, 1281, 1221, 1184, 1167, 1115, 1024, 992, 975, 955, 882, 850, 756, 732, 696 cm ^'1 ; UV (MeOH): ^228 nm (ε 12933 M-icm-i); HRMS (ESI) m/z Calcd. for Ci2Hi2N206Na (M + Na)+ 303.0593. Found 303.0587.

11, 13-Dioxo-13-(2-oxotetrahydrofuran-3-ylamino)tridecyl nitrate

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) and 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 resulting residue was dissolved in DMF (10 mL) and L- homoserine lactone hydrobromide (0.30 g, 1.64 mmol) was added. The mixture was stirred at room temperature for 1 h and at 60 °C for an additional 4 h. The resulting solution was diluted with ethyl acetate 50 mL, and washed with saturated sodium carbonate solution, 1 M sodium hydrogen sulfate solution and brine. The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo. Further purification by vacuum chromatography using ethyl acetate/ methanol (9.5:0.5) gave the title compound as a yellow solid (0.32 g, 54%). M.p. 58 °C; 1H NMR (300 MHz, CDCb) δ 1.28-1.43 (m, 12H, 6 x CH2), 1.53-1.58 (m, 2H, CH2), 1.65-1.74 (m, 2H, CH2), 2.19-2.28 (m, 1H, H4a), 2.49 (t, J = 6.0, 2H, COCH2), 2.66-2.76 (m, 1H, H4b), 3.45 (s, 2H, COCH2CO) 4.21-4.28 (m, 1H, H5a), 4.40 (t, J= 6.0 Hz, 2H, CH2ONO2), 4.45 (dt, J= 12.0, 3.0 Hz, 1H, H5b), 4.56- 4.62 (m, 1H, H3), 7.65 (bs, 1H, NH); ¹³ C NMR (75.6 MHz, CDCb) δ 23.2 (CHa), 25.5 (CH2), 26.7 (CH2), 28.9 (CH2), 29.0 (CH2), 29.21 (CH2), 29.25 (CH2), 29.3 (CH2), 29.6 (CH2), 43.7 (CH2), 48.3 (CH2), 49.0 (CH), 65.9 (CH2), 73.4 (CH2), 166.4 (C=0), 174.9 (C=0), 206.4 (CO). IR (neat): vmax 3265, 2923, 2853, 1783, 1722, 1647, 1642, 1548, 1467, 1421, 1383, 1348, 1280, 1167, 1033, 999, 976, 866, 721, 702 cm ^"1 ; UV (MeOH): max 249 nm (ε 12943 M-icm-i); HRMS (ESI) m/z Calcd. for C17H29N2O7 (M + H)+ 373.1975. Found 373.1959.

12, 14-Dioxo-14-(2-oxotetrahydrofuran-3-ylamino)tetradecyl nitrate

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. 62 °C; 1H NMR (300 MHz, CDC13): 1.25-1.39 (m, 14H, 7 ^χ CFh), 1.53-1.58 (m, 2H, CH2), 1.67-1.72 (m, 2H, CH2), 2.18-2.27 (m, 1H, H4a), 2.51 (t, J = 7.3 Hz, 2H, COCH2), 2.67-2.76 (m, 1H, H4b), 3.45 (s, 2H, COCH2CO), 4.21-4.30 (m, 1H, H5a), 4.42 (t, J= 6.6 Hz, 2H, CH2ONO2), 4.46 (dt, J= 9.0, 1.4 Hz, 1H, H5b), 4.53-4.62 (m, 1H, H3), 7.65 (bd, J= 6.5 Hz, 1H); ^l3 C NMR (75.6 MHz, CDCb) δ 23.3 (CH2), 25.6 (CH2), 26.7 (CH2), 28.9 (CH ₂ ), 29.0 (CH2), 29.30 (CH2), 29.36 (CH2), 29.4 (CH2), 29.7 (CH2), 43.8 (CH2), 48.2 (CH2), 49.0 (CH), 65.9 (CH2), 73.4 (CH2), 166.4 (C=0), 174.9 (C=0), 206.5 (C=0). IR (neat): vmax 3290, 2919, 2850, 1776, 1715, 1642, 1625, 1543, 1468, 1418, 1381, 1342, 1278, 1225, 1173, 1016, 1000, 949, 865, 720, 699 cm ^"1 ; UV (MeOH): 251 nm (ε 11285 M-icm-i); HRMS (ESI) m/z Calcd. for Ci8H30N2O7Na (M + Na)+409.1951. Found 409.1931. ll-Oxo-Jl-(2-oxo-2-(2-oxotetrahydrofiiran-3-ylamino)ethylami no)undecyl nitrate

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. The residue was partitioned between water (20 mL) and ethyl acetate (50 mL) and the organic layer successively washed with 5% sodium bicarbonate (NaHCCb) solution (2 x 20 mL), 1M potassium hydrogen sulfate (KHSO4) solution (1 x 20 mL) and brine. The organic layer was dried over sodium sulfate (Na2S04) and evaporated to dryness to yield the title compound as a white solid. 1H NMR (300 MHz, CDCb) δ 1.40 (s, 9H, 3 x CH3), 2.20-2.28 (m, 1H, H4a), 2.59-2.68 (m, 1H, H4b), 3.81-3.83 (m, 2H, COCH2NH) 4.20-4.29 (m, 1H, H5a) 4.41 (dt, J = 8.9, 1.1 Hz, H5b), 4.56-4.64 (m, 1H, H3), 5.59 (bs, 1H, NH), 7.35 (bs, 1H, NH); ^,3 C NMR (75.6 MHz, CDCb) δ 28.2 (3 ^χ C¾) , 29.3 (CH2), 48.8 (CH), 66.0 (CH2), 156.2 (C=0), 170.6 (C=0), 175.5 (C=0). The product was treated with trifluoroacetic acid (5 mL) to remove the Boc group to give the desired product 104 as yellow oil. 1H NMR (300 MHz, DMSO-efe) δ 2.13-2.24 (m, 1H, H4a), 2.40-2.46 (m, 1H, H4b), 3.63 (s, 2H, COCH2NH) 4.20-4.28 (m, 1H, H5a) 4.37 (dt, J= 1.5 and 8.7 Hz, H5b) , 4.67-4.73 (m, 1H, H3), 8.11 (bs, 1H, NH), 8.94 (bd, J = 7.8, 1H, NH); ¹³ C NMR (75.6) MHz, DMSO-rfi) δ 28.7 (Ofe), 40.5 (CH2), 48.6 (CH), 65.8 (CH2), 166.6 (C=0), 175.2 (C=0).

Sodium l-(4-Ethoxyc rbonylpiperazin-l-yl)diazen-l-ium-l, 2-diolate

A solution of l-ethoxycarbonylpiperazine (5.42 g, 34.0 mmol) in 60 mL of anhydrous ether and methanol (1:1) was placed in a Parr bottle. The solution was treated with sodium methoxide (NaOMe) (2.03 g, 37.0 mmol) and the Parr apparatus was clamped. The apparatus was purged and evacuated with nitrogen (3x) followed by charging with 5 atm. nitric oxide (NO) at 25 °C for 48 h. The excess NO was then vented by purging with nitrogen followed by addition of anhydrous ether. The white precipitate was collected by filtration and washed with cold methanol as well as with copious amounts of anhydrous ether. The product was dried under vacuum to afford the title compound as a white solid (3.90 g, 47%). M.p. 184 °C (dec), lit. m.p. 184-185 °C; 1H NMR (300 MHz, 0.1 M NaOD in D2O) δ 1.69 (t, J = 7.1 Hz, 3H, CHs), 3.02 (m, 4H, 2 CH2), 3.59 (m, 4H, 2 CH2), 4.05 (q, J= 7.1 Hz, 2H, OCH2); ¹³ C NMR (75.6 MHz, 0.1M NaOD in D2O) δ 13.7 (CHs), 42.5 (CH2), 44.1 (CH2), 51.2 (CH2), 62.7 (CH2), 156.8 (C=0).

Sodium l-(4-tert-Butoxycarbonylpiperazin-l-yl)diazen-l-ium-l,2-diol ate The title compound reaction was carried out as described for the preparation of Sodium 1- (4-ethoxycarbonylpiperazin-l-yl)diazen-l-ium-l,2-diolate. A solution of tert- butoxycarbonylpiperazine (5.00 g; 2.68 mmol) and sodium methoxide (1.59 g, 2.95 mmol) in 50 mL of ether and methanol was exposed to nitric oxide to give the title compound as a free flowing solid (1.76 g, 24%). M.p. 260 °C, lit. m.p. 262-264 °C; 1H NMR (300 MHz, 0.1 M NaOD in D2O) δ 1.38 (s, 9 H, 3 ^χ CHs), 3.01 (m, 4H, 2 ^χ CHa), 3.56 (m, 4H, 2 x CH2); ¹³ C NMR (75.6 MHz, 0.1 M NaOD in D2O) δ 30.44 (CHa), 45.44 (CH2), 54.19 (CH2), 85.05 (C), 159.08 (CO).

02-Methyl-l-(4-ethoxycarbonylpiper(^in-l-yl)diazen-l-i m-l,2-diolate

To a solution of sodium l-(4-emoxycarbonylpiperazin-l-yl)diazen-l-ium-l,2-diolate (1.63 g, 6.80 mmol) in 30 mL of methanol was added 1.70 g of anhydrous potassium carbonate, and the resulting slurry was cooled in an ice bath. Dimethyl sulfate (0.80 mL, 8.16 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 1 h and then allowed to warm gradually to room temperature. After an additional hour, the solution was concentrated on a rotary evaporator; the residue was extracted with dichloromethane, washed with water, dried over sodium sulfate, and filtered through a layer of sodium sulfate. The solvent was evaporated under reduced pressure to give the title compound as a light yellow solid (0.84 g, 54%). M.p. 42-44 °C, lit. m.p. 46 °C; 1H NMR (300 MHz, CDCb) δ 1.26 (t, J= 5.5 Hz, 3H, CH3), 3.35 (m, 4H, 2 x CH2), 3.63 (m, 4H, 2 ^χ Cth), 4.00 (s, 3H, OCHa), 4.15 (q, J= 5.5 Hz, 2H, OCH2).

02-Methyl l-(piperazin-l-yl)diazen-l-ium-l,2-diolate

A mixture of 02-methyl-l-(4-ethoxycarbonylpiperazin-l-yl)diazen-l-ium-l,2 -diolate (0.65 g) in 20 mL of 10% sodium hydroxide in ethanol, and 1 mL of water was heated at reflux. After 45 min, no starting material remained in the mixture, as assessed from thin-layer chromatography. The solution was allowed to cool to room temperature and evaporated to a viscous residue, which was extracted with dichloromethane, dried over sodium sulfate, filtered, and evaporated to give the title compound as a yellow oil (0.32 g, 71%). 1H NMR (300 MHz, CDCb) δ 3.02 (m, 4H, 2 x CH2), 3.36 (m, 4H, 2 x CH2), 4.00 (s, 3H, OCHs); ¹³ C NMR (75.6 MHz, CDCb) δ 44.8 (CHz), 52.5 (CH2), 60.9 (CH2).

02-(2 -DMtrophenyl)-l-[4-(tert-butoxycarbonyl)piperazin-l-yl]dia^ To a solution of sodium l-[4-(/e^butoxycarrx)nyl)piperazin-l-yl]diazen-l-ium-l,2-dio late (1.00 g, 3.72 mmol) in 5% sodium bicarbonate (15 mL) at 0 °C under a steady stream of nitrogen gas was added 2,4-dinitrofluorobenzene (0.69 g, 3.72) in tert-butyl alcohol (10 mL). The reaction was warmed to room temperature and stirred for 24 h. Precipitation occurred at such a rapid rate that stirring was impeded throughout the course of the reaction. The solvent was evaporated off and the residue extracted with dichloromethane. The dichloromethane was removed in vacuo and the yellow mixture

was recrystallised in EtOH to give the title compound as yellow crystals (0.41 g, 26%). M.p. 118 °C, lit m.p. 115-117 °C; 1H NMR (300 MHz, CDCb) δ 1.41 (s, 9H), 3.53-3.62 (m, 8H), 7.58 (d, J = 9.2 Hz, 1H), 8.37-8.41 (dd, J= 9.2 and 2.7 Hz, 1H), 8.80 (d, J= 2.7 Hz, 1H); ^,3 C NMR (75.6 MHz, CDCB) δ 28.2 (CHs), 42.0 (CH2), 50.5 (CH2), 80.9 (CH2), 117.6 (ArH), 122.1 (ArH), 129.0 (ArH), 137.3 (ArC), 142.4 (ArC), 153.7 (ArC), 154.1 (ArC).

02-(2,4-Dinitrophenyl) 1 -(piper azin-l-yl)diazen-l-ium-l,2-diolate, hydrochloride salt To a solution of 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. The mixture was filtered and washed with ether to yield the title compound as a white solid (0.26 g, 98% yield). M.p. 182-184 °C, lit. m.p. 184-185 °C; Ή NMR (300 MHz, DMSO-cfe) δ 3.38 (m, 4H, 2 x CH2), 3.89 (m, 4H, 2 ^χ CH2), 7.95 (d, J = 9.3 Hz, 1H, ArH), 8.56-8.60 (dd, J= 9.3 and 2.7 Hz, 1H, ArH), 8.89 (d, J= 2.6 Hz, 1H, ArH), 9.35 (bs, 1H, NH); ¹³ C NMR (75.6 MHz, DMSO-afe) 541.3 (CH2), 47.3 (CH2), 118.7 (ArCH), 122.2 (ArCH), 130.1 (ArCH), 137.4 (ArC), 142.8 (ArC), 152.9 (ArC).

4-(Benzyloxy)-4-oxobutanoic acid

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 ^χ 75 mL). The aqueous extracts were carefully acidified to pH 2 with concentrated HC1 and then extracted with diethyl ether (2 ^χ 100 mL). The organic layer was dried (Na2S04), filtered and concentrated to give the title compound as a white solid (8.84 g, 85 %). M.p. 52-54 °C, lit. 56-57 °C; 1H NMR (300 MHz, acetone-tfc): δ 2.68-2.71 (m, 4H, 2 ^χ CH2), 5.14 (s, 2H, CHiAr), 7.34-7.36 (m, 5H, ArH).

6-(Benzyloxy)-6-oxohexanoic acid

The title compound was synthesized following the procedure described for compound 4- (benzyloxy)-4-oxobutanoic acid using adipic anhydride and benzyl alcohol, the resulting crude mixture was purified by washing to afford the title compound as a white waxy oil (7.45, 81%).^ NMR (300 MHz, CDCb): δ 1.65-1.70 (m, 4H, 2 x CH2) 2.32-2.39 (m, 4H, 2 x CH2), 5.11 (s, 2H, CH2Ar), 7.31-7.36 (m, 5H, ArH); ^,3 C NMR (75.6 MHz, CDCb) δ 24.0 (CH2), 24.3 (CHi), 33.1 (CH2), 33.8 (CH2), 66.2 (CH2), 128.2 (ArH), 128.5 (ArH), 173.1 (CO), 178.7 (C=0).

4-Oxo-4-(2-oxotetrahydrofuran-3-ylamino)butanoic acid

To a stirred solution of L-homoserine lactone hydrobromide (0.5 g, 2.73 mmol) in DCM (10 mL) triethylamine (0.42 mL, 3.00 mmol) was added followed by the addition of 4- (benzyloxy)-4-oxobutanoic acid (0.62 g, 3.00 mmol) and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) (0.78 g, 4.10 mmol). The mixture was stirred at room temperature for 30h and then evaporated in vacuo to dryness. The residue was partitioned between water (20 mL) and ethyl acetate (50 mL) and the organic layer successively washed with 5% sodium bicarbonate (NaHC03) solution (2 x 20 mL), 1M potassium hydrogen sulfate (KHSO4) solution (1 x 20 mL) and brine. The organic layer was dried over sodium sulfate and evaporated to dryness to yield the benzyl protected compound as a colourless oil (0.53 g, 66%). 1H NMR (300 MHz, CDCb): δ 2.05-2.20 (m, 1H, H4a), 2.53-2.58 (m, 2H, Ctfe), 2.68-2.73 (m, 3H, CH2 and H4b), 4.18- 4.27 (m, 1H, H5a), 4.40 (dt, J= 9.0, 1.3 Hz, 1H, H5b), 4.49-4.53 (m, 1H, H3), 5.11 (s, 2H, ClbAr), 6.68 (bd, J = 6.3 Hz, 1H, NH), 7.32 (m, 5H, ArH). 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. 112-114 °C; 1H NMR (300 MHz, DMSO- de): δ 2.07-2.18 (m, 1H, H4a), 2.33-2.51 (m, 5H, 2 CH2 and H4b), 4.15-4.24 (m, 1H, H5a), 4.33 (dt, J= 8.7, 1.8 Hz, 1H, H5b), 4.49-4.58 (m, 1H, H3), 6.68 (bd, J= 7.9 Hz, 1H, NH), 12.09 (bs, COOH); ¹³ C NMR (75.6 MHz, DMSO-<fc) S 28.7 (CH2), 29.3 (CH2), 30.1 (CHi), 48.3 (CH), 65.7 (CH2), 171.5 (C=0), 174.1 (C=0), 175.7 (CO); IR (neat): vmax 3352, 2920, 2519, 1771, 1715, 1611, 1556, 1442, 1427, 1384, 1353, 1277, 1238, 1179, 1131, 1103, 1022, 999, 952, 919, 845, 754, 699, 676 cm ¹ ; HRMS (ESI) m/z Calcd. for CsHnNOsNa (M + Na 224.0535. Found 224.0524; Anal. Calcd. for CsHnNOs: C, 47.76; H, 5.51; N, 6.96. Found: C, 47.87; H, 5.49; N, 6.91.

6-Oxo-6-(2-oxotetrahydrofuran-3-ylamino)hexanoic acid

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%). ^l H NMR (300 MHz, CDCb): S 1.67 (m, 4H, 2 ^χ CHi), 2.05-2.20 (m, 1H, H4a), 2.23-2.38 (m, 4H, 2 * CH2), 2.75-2.78 (m, 1H, H4b), 4.18-4.31 (m, 1H, H5a), 4.43 (dt, J= 9.0, 1.1 Hz, 1H, H5b), 4.49-4.53 (m, 1H, H3), 5.10 (s, 2H, CifcAr), 6.26 (bd, J = 5.2 Hz, 1H, NH), 7.32 (m, 5H, ArH). Deprotection gave the title compound as a white solid (0.27 g, 78%). M.p. 142-144 °C; 1H NMR (300 MHz, OMSO-de): δ 1.48-1.52 (m, 4H, 2 ^χ CH.), 2.05-2.19 (m, 5H, H4a and 2 CH2), 2.33-2.42 (m, 1H, H4b), 4.15-4.23 (m, 1H, H5a), 4.33 (dt, J = 8.8, 1.8 Hz, 1H, H5b), 4.47-4.56 (m, 1H, H3), 8.32 (bd, J = 7.9 Hz, 1H, NH). ¹³ C NMR (75.6 MHz, DMSO-<fc) δ 24.5 (CH2), 25.1 (Ctfc), 28.7 (CH2), 33.9 (CH2), 35.2 (CH2), 48.2 (CH), 65.6 (CH2), 172.4 (C=0), 174.8 (C=0), 175.8 (C=0); IR (neat): vmax 3342, 3291, 2955, 2934, 2872, 1763, 1692, 1640, 1536, 1467, 1429, 1407, 1359, 1282, 1251, 1223, 1190, 1171, 1103, 1080, 1015, 999, 945, 920, 803, 734, 716 cm ^-1 ; HRMS (ESI) m/z Calcd. for 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)but anoy yl)diazen-l-ium-l,2-diol te

To a solution of 4-oxo^-(2-oxotetrahydrofuran-3-ylamino)butanoic acid (0.15 g, 0.75 nunol) and Ch-methyl l-(piperazin-l-yl)diazen-l-ium-l,2-diolate (0.14 g, 0.90 mmol) in water/acetonitrile (5:1) (6 mL) EDC (0.2 lg, 1.12 mmol) was added. The reaction mixture was allowed to stir at r.t. for 40 h. The solvent was concentrated under vacuo, and extracted with EtOAc (2 ^χ 50 mL). The organic layer was washed with brine (2 25 mL), dried over anhydrous Na2S04 and evaporated to give a crude residue. Purification by vacuum chromatography using ethyl acetate/methanol (9.5:0.5) gave the title compound as a yellow oil (0.11 g, 43%). 1H NMR (300 MHz, CDCb): δ 2.12-2.26 (m, 1H, H4a), 2.57- 2.62 (m, 2H, CH2), 2.66-2.73 (m, 3H, CH2 and H4b), 3.36-3.44 (m, 4H, 2 ^χ CH.), 3.65- 3.78 (m, 4H, 2 x CHz), 4.00 (s, 3H, OCHa), 4.20-4.29 (m, 1H, H5a), 4.43 (dt, J= 9.0, 1.3 Hz, 1H, H5b), 4.49-4.54 (m, 1H, H3), 6.86 (bd, J = 6.6 Hz, 1H, NH); ¹³ C NMR (75.6 MHz, CDCh) δ 28.1 (CH2), 29.8 (CH2), 30.7 (CH2), 40.4 (CH2), 43.9 (CH2), 49.0 (CH),

51.1 (CH2), 61.2 (OCH3), 65.9 (CH2), 170.3 (CO), 172.7 (C=0), 175.2 (C=0); IR (neat): vmax 3309, 2919, 2866, 1775, 1637, 1534, 1494, 1442, 1380, 1281, 1213, 1170, 1 1 12, 1028, 946, 914, 813, 727 cm ^-1 ; UV (MeOH): λα« 244 nm (ε 5012 M-icm-i); HRMS (ESI) m/z Calcd. for CnH2iN506Na (M + Na 366.1390. Found 366.1379.

02-Methyl-l-(4-(4-oxo-4-(2-oxotetrahydrofuran-3-ylamino)h exam

yl)diazen-l-ium-l,2-diolate

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): δ 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, 4H, 2 CHa), 3.60-3.77 (m, 4H, 2 CH2), 4.00 (s, 3H, OCHa), 4.20-4.29 (m, 1H, H5a), 4.43 (dt, J= 8.9, 1.4 Hz, 1H, H5b), 4.53-4.62 (m, 1H, H3), 6.94 (bd, J = 6.7 Hz, 1H, NH); ^,3 C NMR (75.6 MHz, CDCb) δ

24.2 (CH2), 24.8 (Ok), 29.7 (CH2), 32.5 (CH2), 35.5 (CH2), 40.1 (CH2), 43.9 (CH2), 48.9 (CH), 51.2 (CH2), 61.2 (OCHa), 65.9 (CH2), 171.3 (C=0), 173.3 (C=0), 175.4 (C=0); IR (neat): vmax 3299, 2943, 2869, 2247, 1774, 1629, 1534, 1494, 1439, 1380, 1280, 1218, 1175, 1111, 1064, 1031, 947, 915, 727 cm ^'1 ; UV (MeOH): m« 242 nm (ε 5420 M-icm-i); HRMS (ESI) m/z Calcd. for CtsIfeNsOeNa (M + Na)+ 394.1703. Found 394.1693. 02-(2 -Dinitrophenyl)'l-(4-(4-oxo-4-(2-oxotetrahydrofuran-3- ylamino)butanoyl)piperazin-l-yl)diazen-l-ium-l,2-diolate

The title compound was synthesized following the procedure for 02-methyl-l-(4-(4-oxo-4- (2-oxotetrahydrofuran-3 -ylamino)butanoyl)piperazin- 1 -yl)diazen- 1 -ium- 1 ,2-diolate using 4-oxo-4-(2-oxotetrahydrofuran-3-ylarnino)butanoic acid (0.1 g; 0.50 mmol) and 02-(2,4- dinitrophenyl) l-(piperazin-l-yl)diazen-l-ium-l,2-diolate, hydrochloride salt (0.19 g, 0. 55 mmol) to give the title compound as a yellow solid (0.17 g, 79%). M.p. 112-114 °C; 1H NMR (300 MHz, CDCb): δ 2.17-2.29 (m, IH, H4a), 2.63-2.66 (m, 2H, CH2), 2.69-2.76 (m, 3H, CH2 and H4b), 3.36-3.87 (m, 8H, 4 * CH2), 4.22-4.31 (m, IH, H5a), 4.46 (dt, J = 9.0 and 1.2 Hz, IH, H5b), 4.49-4.57 (m, IH, H3), 6.61 (bd, J= 6.5 Hz, IH, NH), 7.92 (d, J = 9.0 Hz, ArCH), 8.54 (dd, J = 9.0 and 1.2 Hz, ArCH), 8.87 (d, J = 1.2 Hz, ArCH); ¹³ C NMR (75.6 MHz, CDCb) S 28.1 (CH2), 29.8 (CH2), 30.8 (CH2), 40.2 (CH2), 43.8 (CH2), 49.1 (CH), 50.5 (CH2), 65.9 (CH2), 117.8 (ArCH), 122.1 (ArCH), 129.1 (ArCH), 170.3 (C=0), 172.6 (CO), 175.1 (C=0); IR (neat): vma* 3313, 3053, 2916, 2857, 1780, 1674, 1605, 1527, 1472, 1447, 1343, 1264, 1239, 1209, 1158, 1114, 1068, 1012, 959, 909, 850, 835, 731, 699 cm ^"1 ; UV (MeOH): λπ««297 nm (ε 58484 M-icm-i); HRMS (ESI) m/z Calcd. for Ci8H2iN?OioNa (M + Na)+ 518.1248. Found 518.1240.

02-(2,4-Dinitrophenyl)-l-(4-(4-oxo-4-(2-oxotetrahydrofura n-3- ylamino)hexanoyl)piperazin-l-yl)diazen-l-ium-l,2-diolate

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%). M.p. 108-110 °C, δ 2.16-2.21 (m, IH, H4a), 2.25-2.34 (m, 4H, 2 CH2), 2.64-2.73 (m, IH, H4b), 3.57-3.64 (m, 4H, 2 x CH2), 3.73-3.79 (m, 4H, 2 Cttz), 4.20-4.29 (m, IH, H5a), 4.43 (dt, J = 1.4 and 8.9 Hz, IH, H5b), 4.53-4.62 (m, IH, H3), 6.94 (bd, J= 6.7 Hz, IH, NH), 7.68 (d, J = 9.0 Hz, ArCH), 8.47 (dd, J = 9.0 and 1.2 Hz, ArCH), 8.89 (d, J = 1.2 Hz, ArCH); ¹³ C NMR (75.6 MHz, CDCb) S 24.2 (CH2), 24.8 (CH2), 29.7 (CH2), 32.5 (CH2), 35.5 (CH2), 40.1 (CH2), 43.9 (CH2), 48.9 (CH), 51.2 (CH2), 61.2 (OCHs), 65.9 (CH2), 118.3 (ArCH), 122.3 (ArCH), 129.6 (ArCH), 171.3 (C=0), 173.3 (C=0), 175.4 (CO); IR (neat): vmax 3313, 3053, 2916, 2857, 1779, 1664, 1605, 1527, 1472, 1447, 1342, 1264, 1239, 1209, 1158, 1114, 1068, 1012, 959, 909, 848, 832, 731 , 667 cm ¹ ; HRMS (ESI) m/z Calcd. for C2oH2sN70ioNa (M + Na 546.1561. Found 546.1554.

3-Acetamido-4,4-dimethylthietan-2-one

Acetic anhydride was added dropwise to an ice-cooled mixture of D-penicillamine and potassium carbonate and stirred overnight. To the reaction mixture ice-cold water was added and the precipitate filtered to yield the title compound as a white solid. M.p. 164- 166 °C; ^l H NMR (300 MHz, CDCb): δ 1.62 (s, 3H, CHa), 1.84 (s, 3H, CHa), 2.04 (s, 3H, NHCOCH3), 5.64 (d, J = 8.0 Hz, 1H, CH), 6.46 (bs, 1H, NH); ^,3 C NMR (75.6 MHz, CDCb) δ 22.5 (CHJ), 26.2 (CH3), 30.2 (OCH3), 51.2 (C) 76.3 (CH), 169.5 (C=0), 192.2 (CO).

2-Acetamido-3-mercapto-3-methyl-N-(2-oxotetrahydrofuran-3 -yl)butanamide

A solution of L-homoserine lactone (0.3 g, 1.64 mmol), triethylamine (0.25 mL, 1.80 mmol) and 3*acetamido-4,4-dimemylthietan-2-one (0.34 g, 1.96 mmol) in dimethylformamide was stirred overnight. The solvent was evaporated to dryness and purified by column chromatography using ethyl acetate/methanol (95:5) as mobile phase to yield the title compound. 1H NMR (300 MHz, CDCb) δ 2.16-2.28 (m, 1H, H4a), 2.82-2.91 (m, 1H, H4b), δ 4.27-4.36 (m, 1H, H5a) 4.51 (t, J= 9.1 Hz, H5b ), 4.56-4.65 (m, 1H, H3), 4.95 (s, 2H, CH2ONO2), 6.57 (bs, 1H, NH); ¹³ C NMR (75.6 MHz, CDCb) δ 30.1(CH2) , 49.1 (CH), 66.1 (CH2), 69.0 (CH2), 165.4 (C=0), 170.2 (C=0), 174.4 (C=0); IR (neat): vmax 3313, 3261, 3083, 2968, 1784, 1635, 1563, 1538, 1462, 1376, 1278, 1171, 1143, 1022, 949, 812, 735, 699 cm ^"1 ; HRMS (ESI) m/z Calcd. for CnHisNaCfcSNa (M + Na)+ 297.0885. Found 297.0872.

(S)-3-Acetamido-2-met^l-4-oxo-4-(2-oxotetrahydrofuran-3-y lamino)butan-2-yl nitrothioite

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. 1H NMR (300 MHz, CDCb) δ 1.35 (s, )2.00 (s, Ctfc), 2.02 (s,CH3) 2.16-2.24 (m, 1H, H4a), 2.56-2.60 (m, 1H, H4b), δ 4.27-4.36 (m, 1H, H5a) 4.51 (t, J = 9.1 Hz, H5b ), 4.56-4.68 (m, 1H, H3), 5.25 (d, H, CH), 6.46 (bs, 1H, NH). ¹³ C NMR (75.6 MHz, CDCb) δ 30.1(CH2), 49.1 (CH), 66.1 (CH2), 69.0 (CH2), 165.4 (CO), 171.1 (CO), 174.4 (CO); HRMS (ESI) m/z Calcd. for CiiHnNsOsSNa (M + Na> 326.0787. Found 326.0951.

3-(l -Bromobutyl)-5-(dibromomethylene)furan-2(5H)-one

N-bromosuccinimide (NBS) (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. 76 °C; 1H NMR (300 MHz, CDCb): δ 0.98 (t, J= 7.1 Hz, 3H, CHa), 1.47 (m, 2H, CH2), 2.08-2.15 (m, 2H, CH2), 4.73 (m, 1H, CHBr), 7.55 (s, 1H, CH); ^,3 C NMR (75.6 MHz, CDCb): δ 13.1 (CH3), 20.9 (CH2), 38.6 (CHa), 42.1 (CHBr), 82.7 (CBn), 135.4 (CH), 137.4 (C), 149.1 (C), 165.7 (CO); IR (neat): Vmax3086, 2964, 2872, 1779, 1612, 1597, 1463, 1376, 1321, 1267, 1185, 1165, 1108, 1061, 1037, 968, 917, 898, 845, 777, 735, 665 cm ^'1 ; HRMS (ESI) m/z Calcd. for CoHioBnCfe (M + H 386.8231. Found 386.8224

3-(l-Hydroxybutyl)-5-(dibromomethylene)furan-2(5H)-one

3-(l-Bromobutyl)-5-(d^bromomemylene)furan-2(5H)-one (7 g, 18.00 mmol) was dissolved in DMSO (10 mL) and few drops of water was added. The mixture was stirred at room temperature for 72 h. To the mixture water (60 mL) was added and extracted with DCM (2 x 20 mL). The organic phase was washed with brine solution (2 x 20 mL), then dried and evaporated. Vacuum chromatography of the residue through silica gel gave the title compound as an off white solid (3.5 g, 59%). M.p. 56 °C; 1H NMR (300 MHz, CDCb): δ 0.97 (t, J= 7.1 Hz, 3H, CH3), 1.46-1.52 (m, 2H, CHa), 1.68-1.81 (m, 2H, CH2), 2.25 (d, J = 6.1 Hz, 1H, OH) 4.54-4.60 (m, 1H, CHOH), 7.50 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.7 (CH ₃ ), 18.4 (CH2), 37.6 (CH2), 67.0 (CHOH), 81.1 (CBn), 134.1 (C),

139.5 (CH), 149.5 (C), 165.7 (CO); IR (neat): vmax 3262, 3092, 2959, 2931, 2871, 1755, 1606, 1464, 1378, 1340, 1265, 1235, 1180, 1128, 1064, 1019, 962, 950, 917, 889, 802, 784, 745, 682 cm- ¹ ; UV (MeOH): 305 nm (ε 207289 M-icm-i); HRMS (ESI) m/z Calcd. for C ^g HioBnOsNa (M + Na)+ 346.8894. Found 346.8891 l-(5-(Dibromomethylene)-2-oxo-2,5-dihydrofuran-3-yl)butyl 2-bromoacetate A solution of the 2-bromoacetylbromide (0.08 mL, 0.92 mmol) was slowly added to a stirred solution of 3-(l-hydroxybutyl)-5-(dibromomethylene)furan-2(5H)-one (0.20 g, 0.61 mmol) and dry pyridine (0.99 mL, 1.22 mmol) in anhydrous CH2CI2 (10 mL) at 0 °C under argon. The reaction was allowed to reach room temperature and then stirred for 2.5 h. The mixture was washed with 2 M HC1 (3 * 10 mL) and brine (lx 20 mL). The combined organic layers were dried, filtered, and concentrated under reduced pressure. The crude product thus obtained was purified by vacuum chromatography to yield, the title compound as a white solid (0.24 g, 87%). M.p. 78 °C; Ή NMR (300 MHz, CDCb): δ 0.95 (t, J= 7.3 Hz, 3H, CH3), 1.37-1.54 (m, 2H, CH2), 1.85-1.92 (q, J = 7.4 Hz, 2H, CH2), 3.87 (s, 2H, CH2Br), 5.65 (t, J = 7.1 Hz, 1H, CHOCO), 7.50 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.5 (CKs), 18.2 (CH2), 25.2 (CH2), 34.6 (CH2), 70.2 (CH), 82.3 (CBn), 135.0 (C=CH), 135.2 (CH), 149.2 (C=CBn), 165.9 (C=0), 166.3 (C=0); IR (neat): vmax 3089, 2962, 2872, 1762, 1734, 1608, 1463, 1405, 1381, 1273, 1216, 1186, 1156, 1107, 1033, 966, 917, 885, 842, 770, 683 cm ^"1 ; UV (MeOH): λαη 308 nm (ε 261761 M-icm-i); HRMS (ESI) m/z Calcd. for CnHiiBnOtNa (M + Na)+ 466.8105. Found 466.8100; Anal. Calcd. for C11H11BDO4: C, 29.56; H, 2.48. Found: C, 29.78; H, 2.49.

Sodium l-(N,N-diethylamino)diazen-l-ium-l,2-diolate

N^V-Diethylamine (7.00 g, 96.6 mmol) in 60 mL ether: methanol (1:1) and sodium methoxide (6.26 g, 116 mmol) was exposed to NO as described for the preparation of Sodium l-(4-ethoxycarbonylpiperazin-l-yl)diazen-l-ium-l,2-diolate to give the title compound as a white solid (5.60 g, 37%). M.p. 240 °C (dec); 1H NMR (300 MHz, 0.1 M NaOD in D2O) δ 0.88 (t, J= 7.1 Hz, 3H, CH3), 2.88 (q, J = 7.1 Hz, 2H, CH2); ^,3 C NMR (75.6 MHz, 0.1 M NaOD in D2O) δ 10.7 (CFb), 48.2 (CH2). Z) -(2-(l-(5-(Dibromomet ylene)-2-oxo-2,5-dxlty

3,3-diethyltriaz-l-ene 2-oxide

l-(5-(Dibromomemylene)-2-oxo-2,5-dihydrofuran-3-yl)butyl 2-bromoacetate (0.1 g, 0.22 mmol) was dissolved in DMSO containing NaHC03 (0.018 g, 0.22 mmol) and the resulting mixture was stirred at 0 °C. Sodium l-(N _> N-diethylamino)diazen-l-ium-l,2-diolate (0.042 g, 0.26 mmol) was added before the reaction mixture solidified and the reaction was kept at 0 °C for 20 min. DCM (20 mL) was added and the organic layer was washed with water (2 x 25 mL) and brine (25 mL). The organic layer was dried over Na2S0 and evaporated under vacuo. The crude product was purified by silica gel preparative TLC using ethyl acetate/n-hexane (4:6) to yield the title compound as an off white solid (0.051 g, 46%). M.p. 60-62 °C; 1H NMR (300 MHz, CDCb): δ 0.92 (t, J= 7.4 Hz, 3H, CHa), 1.10 (t, J = 7.1 Hz, 6H, 2 CH3 ), 1.33-1.42 (m, 2H, CH2), 1.82-1.90 (m, 2H, CH2), 3.12 (q, J = 7.1 Hz, 4H, 2 x CH2), 4.82 (s, 2H, CH ₂ ON=NO), 5.70 (t, J - 6.8 Hz, 1H, CHOC=0), 7.44 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 11.4 (Clfc), 13.4 (CH3), 18.1 (CH2), 34.5 (CH2), 48.6 (CH2), 68.9 (CH2), 69.4 (CH), 82.2 (CBrc), 134.9 (C=CH), 135.3 (CH), 149.0 (C=CBn), 165.7 (C=0), 166.8 (CO); IR (neat): Vmax 3108, 2957, 2931, 2875, 1775, 1752, 1604, 1513, 1464, 1450, 1416, 1383, 1282, 1261, 1230, 1204, 1177, 1141, 1098, 1033, 1005, 964, 951, 905, 843, 776, 725, 660 cm ^-1 ; UV (MeOH): 307 nm (ε 314073 M-icm- 1); HRMS (ESI) m/z Calcd. for CisHziBrcNjOeNa (M + Na)+ 519.9695. Found 519.9692.

(Z)-2-(2-(l-(5-(Dibromomethylene)-2-oxo-2,5-dihydro^

(4-(ethoxycarbonyl)piperazin-l-yl)diazene oxide

The title compound was synthesized following the procedure described for (Z)-l-(2-(l-(5- (dibromomemylene)-2-oxo-2,5-dihydrofuran-3-yI)butoxy)-2-oxoe moxy)-3,3-diemyltriaz- 1-ene 2-oxide using l-(5-(dibromomemylene)-2-oxo-2,5-dihydrofuran-3-yl)butyl 2- bromoacetate (0.2 g, 0.44 mmol) and sodium l-(4-Ethoxycarbonylpiperazin-l-yl)diazen-l- ium-l,2-diolate (0.12 g, 0.53 mmol) to give the title compound as a white solid (0.15 g, 58%). M.p. 84-86 °C; 1H NMR (300 MHz, CDCb): δ 0.93 (t, J = 7.4 Hz, 3H, CH3), 1.26 (t, J= 7.1 Hz, 3H, CH3 ), 1.30-1.38 (m, 2H, CH2), 1.83-1.91 (m, 2H, CH2), 3.40 (t, J= 4.9 Hz, 4H, 2 CH2), 3.64 (t, J= 4.9 Hz, 4H, 2 ^χ CH.), 4.13 (q, J= 7.1 Hz, 2H, CH2), 4.78 (s, 2H, CH20N=NO), 5.69 (t, J = 6.7 Hz, 1H, CHOC=0), 7.43 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.4 (Ctb), 14.5 (CH3), 18.1 (CH2), 34.5 (CH2), 42.2 (CH2), 50.8 (CH2), 61.8 (CH2), 69.0 (CH2), 69.3 (CH), 82.3 (CBn), 134.8 (C=CH), 135.4 (CH), 149.0 (C=CBn), 154.9 (CO), 165.7 (C=0), 166.9 (C=0); IR (neat): vmax 3099, 2957, 2932, 2862, 1773, 1697, 1607, 1504, 1473, 1421, 1386, 1283, 1268, 1248, 1215, 1196, 1183, 1135, 1096, 1057, 1041, 1010, 968, 947, 921, 894, 854, 835, 780, 767, 726, 682 cm ¹ ; UV (MeOH): 308 nm (ε 311580 M-icm-i); HRMS (ESI) m/z Calcd. for CisHwBn fOeNa (M + Na)+ 604.9859. Found 604.9855.

Sodium l-(morpholin-l-yl)diazen-l-ium-l,2-diolate

The title compound synthesis was carried out as described for the preparation of sodium 1- (4-ethoxycarbonylpiperazin-l-yl)diazen-l-ium-l,2-diolate. A solution of morpholine (10.0 g; 114.7 mmol) and sodium methoxide (6.82 g, 126.2 mmol) in 120 mL of ether and methanol (1:1) was exposed to nitric oxide to give the title compound as a white solid (8.90 g, 45%). M.p. 184-186 °C (dec) Ή NMR (300 MHz, 0.1 M NaOD in D2O) <5 3.16 (t, J = 4.6 Hz, 4H, 2 CH2), 3.90 (t, J =4.8 Hz, 4H, 2 x CH2); ¹³ C NMR (75.6 MHz, 0.1 M NaOD in D2O) δ 51.6 (Ctb), 65.6 (CH2).

(Z)-2-(2-(l-(5-(Dibromomethylene)-2-oxo-2,5-dihydrofiiran -3-yl)buto

morpholinodiazene oxide

The title compound was synthesized following the procedure described for (Z)-l-(2-(l-(5- (dibromomemylene)-2-oxo-2,5-dmydrofuran-3-yl)butoxy)-2-oxoem oxy)-3,3-diemyltriaz- 1-ene 2-oxide using l-(5-(cUbromomemylene)-2-oxo-2,5-dihy kofuran-3-yl)butyl 2- bromoacetate (0.25 g, 0.55 mmol) and sodium l-(morpholin-l-yl)diazen-l-ium-l,2-diolate (0.11 g, 0.67 mmol) to give the title compound as a sticky oil (0.11 g, 40%). 1H NMR (300 MHz, CDCb): δ 0.93 (t, J= 7.4 Hz, 3H, CHs), 1.27-1.31 (m, 2H, CH2), 1.77-1.85 (m, 2H, CH2), 3.42-3.46 (m, 4H, 2 ^χ CHa), 3.82-3.85 (m, 4H, 2 ^χ CHa), 4.78 (s, 2H, CH20N=NO), 5.71 (t, J = 6.7 Hz, 1H, CHOC=0), 7.45 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.5 (CH3), 18.2 (CH2), 34.6 (CH2), 51.4 (CHa), 65.6 (CH2), 69.1 (CH2), 69.4 (CH), 82.4 (CBn), 134.9 (C=CH), 135.5 (CH), 149.1 (C=CBr2), 165.8 (C=0), 167.0 (C=0); IR (neat): vmax2962, 2929, 2865, 1767, 1611, 1500, 1456, 1380, 1267, 1228, 1194, 1099, 1064, 1036, 963, 927, 874, 848, 774, 728, 681 cm ^-1 ; UV (MeOH): max 308 nm (ε 270071 M-icm-i); HRMS (ESI) m/z Calcd. for CisHaiBnNsOyNa (M + Na)+ 533.9487 Found 533.9487.

4-(l-(5-(Dibromomethylem)-2-oxo-2,5-dihydrof ran-3-yl)butoxy)-4-oxobutano

To a solution of succinyl dichloride (0.42 mL, 3.83 mmol) at 0 °C, a solution of 3-(l- hydroxybutyl)-5-(dibromomethylene)furan-2(5H)-one (0.5 g, 1.53 mmol) and pyridine (0.36 mL, 4.60 mmol) in DCM was added slowly over a period of 45 min. The reaction mixture was stirred further for 10 min at 0 °C and then at room temperature for 2h. The organic layer was washed with 1M HC1 (2 20 mL), water (2 20 mL) and brine (20 mL). The organic layer was dried, evaporated and the crude product was purified by vacuum chromatography using ethyl acetate/n-hexane (4:6) to give the title compound

177a as a white solid (0.56 g, 86%). M.p. 130-132 °C; 1H NMR (300 MHz, CDCb): δ 0.93 (t, J= 7.3 Hz, 3H, CH3), 1.33-1.41 (m, 2H, CH2), 1.81-1.89 (m, 2H, CH2), 2.68-2.73 (m, 4H, 2 CH2C=0), 5.61 (t, J= 6.7 Hz, 1H, CHOC=0), 7.42 (s, 1H, CH); ^,3 C NMR (75.6 MHz, CDCb): δ 13.5 (CHs), 18.2 (CHa), 28.6 (CH2), 28.7 (CH2), 34.6 (CH2), 68.9 (CH), 81.8 (CBn), 135.1 (CH), 135.7 (C=CH), 149.2 (C=CBn), 166.0 (C=0), 171.1 (C=0), 177.2 (C=0); IR (neat): Vmax 3095, 2974, 1768, 1731 , 1712, 1409, 1234, 1204, 1169, 1035, 968, 905, 843, 771, 678 cm ^"1 ; UV (MeOH): max 307 nm (ε 217372 M-i cm ^'1 ); HRMS (ESI) m/z Calcd. for CnHuBnOeNa (M + Na> 446.9055. Found 446.9051 ; Anal. Calcd. for Ci3Hi4Br206: C, 36.65; H, 3.31. Found: C, 36.90; H, 3.28.

3-(l-Bromohexyl)-5-(dibromomethylene)fiiran-2(5H)-one

The title compound synthesis was carried out as described for the preparation of compound 3-(l-bromobutyl)-5-(dibromomethylene)furan-2(5H)-one using the fimbrolide derivative, 3-hexyl-5-(dibromome lene)-2(5H)-furanone (2.0 g, 5.91 mmol) and NBS (6.10 mmol) to give the title compound as an off white solid (2.39 g, 97%). M.p. 76 °C; Ή NMR (300 MHz, CDCb): δ 0.91 (t, J= 7.1 Hz, 3H, CHs), 1.29-1.56 (m, 6H, 3 * CH2), 2.05-2.15 (m, 2H, CH2), 4.68-4.73 (m, 1H, CHBr), 7.54 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ

13.9 (CH3), 22.3 (CHa), 27.3 (CHz), 30.8 (CH2), 36.7 (CH2), 42.4 (CHBr), 82.6 (CBn), 135.7 (CH), 137.4 (C=CH), 149.1 (C=CBn), 165.7 (CO); IR (neat): Vma* 3091 , 2951, 2857, 1775, 1614, 1597, 1466, 1375, 1269, 1215, 1177, 1115, 1052, 999, 966, 912, 848, 833, 777, 669 cm ^"1 ; HRMS (ESI) m/z Calcd. for CnHi3Bo02Na (M + Na)+ 436.8363. Found 436.8358

5-(Dibromomethylene)-3-(l-hydroxyhexyl)furan-2(5H)-one

The title compound was synthesized following the procedure for compound 3-(l- hydroxybutyl)-5-(dibromomethylene)furan-2(5H)-one using 3-( 1 -bromohexyl)-5- (dibromomemylene)furan-2(5H)-one (2 g; 4.79 mmol) and DMSO H2O to give the title compound as a yellow solid (1.05 g, 62%). M.p. 40 °C; 1H NMR (300 MHz, CDCb): δ 0.97 (t, J = 7.1 Hz, 3H, Ctb), 1.36-1.48 (m, 4H, 2 CH2), 1.63-1.93 (m, 4H, 2 * CH2), 4.53-4.58 (m, 1H, CHOH), 7.48 (s, 1H, CH); ^,3 C NMR (75.6 MHz, CDCb): δ 13.9 (CHa), 22.5 (Ctb), 24.8 (CH2), 31.4 (CH2), 35.5 (CH2), 67.3 (CHOH), 81.1 (CBn), 134.1 (C), 139.5 (CH), 149.5 (C), 167.2 (CO); IR (neat): vmax 3262, 3092, 2959, 2931, 2871 , 1755, 1606, 1464, 1378, 1340, 1265, 1235, 1 180, 1 128, 1064, 1019, 962, 950, 917, 889, 802, 784, 745, 682 cm ^"1 ; UV (MeOH): nu 305 nm (ε 120858 M-icm-i); HRMS (ESI) m/z Calcd. for CiiHuBnOaNa (M + Na)+ 374.9207. Found 374.9200.

3-(l-(5-(Dibromomethylem)-2-oxo-2,5-dihydrofuran-3-yl)hex yloxy)-3-oxopropa^ The desired compound was synthesized using malonyl dichloride (0.35 mL, 3.53 mmol), 5-(dibromomemylene)-3-(l-hydroxyhexyl)ruran-2(5H)-one (0.5 g, 1.41 mmol) and pyridine (0.34 mL, 4.23 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 (0.46 g, 75%). M.p. 74-76 °C; 1H NMR (300 MHz, CDCb): δ 0.88 (t, J= 7.3 Hz, 3H, CH3), 1.30-1.34 (m, 6H, 3 * Cth), 1.84-1.95 (m, 2H, CH2), 3.52 (d, 2H, CH2), 5.61-5.66 (m, 1H, CHOC=0), 7.55 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.8 (CHs), 22.2 (CH2), 24.4 (CH2), 31.1 (CH2), 32.4 (CH2), 40.7 (CH2), 70.2 (CH), 81.8 (CBn), 135.0 (C=CH), 135.3 (CH), 149.1 (C=CBn), 165.1 (C=0), 165.9 (C=0), 171.2 (C=0); IR (neat): v _mf * 3104, 2955, 2926, 2859, 1750, 1698, 1609, 1550, 1467, 1421, 1405, 1335, 1264, 1208, 1184, 1147, 1066, 1042, 1027, 988, 967, 892, 857, 844, 775, 751, 725, 686, 670 cm ^'1 ; UV (MeOH): ax 306 nm (ε 266715 M-i cm ^"1 ); HRMS (ESI) m/z Calcd. for CuHieBrcCteNa (M + Na)+ 460.9211. Found 460.9212; Anal. Calcd. for CwHi BnOe: C, 38.21; H, 3.66. Found: C, 38.40; H, 3.55.

3-(l-Bromododecyl)-5-(dibromomethylene)furan-2(5H)-one

The fimbrolide derivative, 3-dodecyl-5-(dibromomethylene)-2(5H)-furanone (2.0 g, 4.73 mmol) was brominated using NBS (1.01 g, 5.68 mmol) by the method described for 3-(l- bromobutyl)-5-(dibromomethylene)furan-2(5H)-one to give the title compound as an off white solid (2.23 g, 94%). M.p. 52 °C; 1H NMR (300 MHz, CDCb): δ 0.87 (t, J= 6.9 Hz, 3H, CH3), 1.26 (m, 18H, 9 x CH2), 2.08-2.13 (m, 2H, CH2), 4.71 (m, 1H, CHBr), 7.54 (s, 1H, CH); ^,3 C NMR (75.6 MHz, CDCb): δ 14.1 (CHs), 22.6 (CH2), 27.6 (CH2), 28.6 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 31.9 (CH2), 36.7 (CH2), 42.4 (CHBr), 82.6 (CBn), 135.3 (CH), 137.4 (C=C), 149.1 (C=CBn), 165.7 (C=0); IR (neat): vmax 3091, 2921, 2852, 1777, 1699, 1613, 1597, 1464, 1265, 1198, 1177, 1014, 966, 907, 849, 832, 778, 670 cm ^"1 ; HRMS (ESI) m/z Calcd. for CnHasBnCfeNa (M + Na 520.9302. Found 520.9290.

5-(Dibromomethylene)-3-(l-hydrox dodecyl)furan-2(5H)-one

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. 40 °C; 1H NMR (300 MHz, CDCb): δ 0.85 (t, J= 6.9 Hz, 3H, CH3), 1.17-1.43 (m, 18H, 9 C\ ), 1.87-1.95 (m, 2H, CH2), 4.53-4.58 (m, 1H, CHOH), 7.48 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 14.1 (CH3), 22.6 (CH2), 25.1 (CH2), 29.2 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 31.9 (CH2), 35.6 (CH2), 67.3 (CH2OH), 81.0 (CBn>), 135.0 (CH), 139.5 (C), 149.0 (C), 165.7 (C=0); IR (neat): vmax 3394, 3086, 2917, 2850, 1755, 1617, 1601, 1466, 1377, 1264, 1181, 1044, 965, 911, 845, 778, 720, 708, 681 cm- ¹ ; UV (MeOH): 305 nm (ε 114642 M-i cm ^'1 ); HRMS (ESI) m/z Calcd. for CnmeBnOsNa (M + Na)+ 459.0146. Found 459.0136.

3- (l-(5-(Dibromometf^lem)-2-oxo-2,5-dihydrofiiran-3-yl)dodecyl oxy)-3-oxopropanoic acid

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. 46-48 °C; 1H NMR (300 MHz, CDCb): δ 0.88 (t, J= 7.3 Hz, 3H, CH3), 1.23-1.30 (m, 18H, 9 * CH2), 1.84-1.89 (m, 2H, CH2), 3.45 (d, 2H, CH2), 5.61-5.66 (m, 1H, CHOCO), 7.55 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 14.1 (CHa), 22.6 (CH2), 24.9 (CH2), 29.0 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6 (CH2), 31.9 (CH2), 32.5 (CH2), 40.8 (CH2), 70.3 (CH), 77. 2 (CH), 82.2 (CBrc), 135.1 (C=CH), 135.5 (CH), 149.2 (C=CBn), 165.3 (CO), 166.0 (CO), 171.1 (CO); IR (neat): Vn«* 3092, 2919, 2850, 1759, 1699, 1608, 1466, 1423, 1334, 1266, 1236, 1208, 1183, 1152, 1068, 1040, 1026, 989, 966, 892, 850, 774, 750, 723, 684, 670 cm ^"1 ; UV (MeOH): 307 nm (ε 290145 M- lcm-i); HRMS (ESI) m/z Calcd. for C2oH28Bn0 Na (M + Na 545.0150. Found 545.0146; Anal. Calcd. for C2oH2sBr206: C, 45.82; H, 5.38. Found: C, 45.52; H, 5.30.

4- (l-(5-(Dibromomethylene)-2-oxo-2,5-dihydrof ran-3-yl)dodecylo

The desired compound was synthesized using succinyl dichloride (0.31 mL, 2.85 mmol),

5- (dibromomemylene)-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 (0.50 g, 83%). M.p. 58-60 °C; 1H NMR (300 MHz, CDCb): δ 0.88 (t, J= 6.9 Hz, 3H, CH3), 1.23-1.30 (m, 18H, 9 * CH2), 1.83-1.88 (m, 2H, CH2), 2.69-2.73 (m, 4H, 2 x CH2), 5.59 (t, J = 5.3 Hz, 1H, CHOCO), 7.55 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 14.1 (CH3), 22.6 (CH2), 24.9 (CTfc), 28.5 (CH2), 28.7 (CH2), 29.1 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6 (CH2), 31.9 (CH2), 32.6 (CH2), 69.2 (CH), 77. 2 (CH), 81.7 (CBn), 135.1 (CH), 135.7 (COH), 149.2 (C=CBn), 166.0 (CO), 171.1 (C=0), 176.3 (CO); IR (neat): vmax 3086, 2924, 2852, 1759, 1740, 1709, 1608, 1466, 1441, 1417, 1370, 1322, 1279, 1262, 1162, 1086, 1045, 1020, 965, 913, 847, 800, 774, 720, 684 cm ^"1 ; UV (MeOH): λ™χ 307 nm (ε 223431 M-icm-i); HR S (ESI) m/z Calcd. for 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.

(Z)-l-(4-(4-(l-(5-(Dibromomethy!ene)-2-oxo-2,5-dihydrofit ran-3-yl)buto

oxobutanoyl)piperazin-l-yl)-2-methoxydiazene oxide

To the solution of 4-(l-(5-(dibromomemylene)-2-oxo-2,5-dihydrofuran-3-yl)butoxy )-4- oxobutanoic acid (0.2 g, 0.47 mmol) and 02-methyl l-(piperazin-l-yl)diazen-l-ium-l,2- diolate (0.09 g, 0.56 mmol) in DCM was added EDC (0.13 g, 0.70 mmol) and the mixture was stirred for 7h at room temperature. The organic layer was successively washed with water (2 ^χ 20 mL), brine, dried over sodium sulfate and evaporated to dryness followed by vacuum chromatography using ethyl acetate/n-hexane (50:50) to yield the title compound as sticky brown oil (0.15 g, 57%). ^l H NMR (300 MHz, CDCb): δ 0.92 (t, J= 7.4 Hz, 3H, CH ₃ ), 1.36-1.41 (m, 2H, CH2), 1.78-1.85 (m, 2H, CH2), 2.60-2.75 (m, 4H, 2 x CH2CO), 3.35-3.43 (m, 4H, 2 ^χ CHa), 3.65 (t, J = 5.2 Hz, 2H, CH2), 3.76 (t, J = 5.2 Hz, 2H, CH2), 4.00 (s, 3H, OCH3), 5.61 (m, 1H, CHOCO), 7.61 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.6 (CHs), 18.3 (CH2), 27.9 (CH2), 29.0 (CH2), 29.6 (CH2), 34.6 (CH2), 40.3 (CH2), 43.7 (CH2), 51.0 (CH2), 51.2 (CH2), 61.2 (OCH3), 69.0 (CH), 81.3 (CBn), 135.3 (CH), 135.9 (C=CH), 149.5 (C=CBn»), 166.2 (CO), 169.5 (CO), 172.0 (CO); IR (neat): VOMX 2961, 2933, 2871, 1769, 1736, 1645, 1496, 1440, 1371, 1281, 1214, 1156, 1109, 1066, 1030, 965, 912, 847, 774, 725, 683 cm ^*1 ; UV (MeOH): max 307 nm (ε 174472 M-i cm ^"1 ); HRMS (ESI) m/z Calcd. for CisHwBnNtCbNa (M + Na 588.9909. Found 588.991 1.

(Z)-l-(4-(3-(J-(5-(Dibromomethylene)-2-oxo-2 -dihydrofuran-3-yI)hexyloxy)-3- oxopropanoyl)piperazin-l-yl)-2-methoxydiazene oxide

The title compound was synthesized following the procedure described for (Z)-l-(4-(4-(l- (5-(dibromomemylene)-2-oxo-2,5-dihyarofu^

yl)-2-methoxydiazene oxide using 3-(l-(5-(dibromomethylene)-2-oxo-2,5-dihydrofuran-3- yl)hexyloxy)-3-oxopropanoic acid (0.2 g, 0.454 mmol), <¾-Methyl l-(piperazin-l- yl)diazen-l-ium-l,2-diolate (0.08 g, 0.496 mmol) and EDC (0.11 g, 0.59 mmol) to give the title compound as a yellow sticky oil (0.21 g, 79%). ^l H NMR (300 MHz, CDCb): 60.88 (t, J= 7.3 Hz, 3H, CH3), 1.28-1.33 (m, 6H, 3 χ CH2), 1.85-1.89 (m, 2H, CH2), 3.40-3.48 (m, 4H, 2 x CH2), 3.53 (d, J= 2.8 Hz, 2H, CH2), 3.63 (t, J= 5.4 Hz, 2H, CH2), 3.82 (t, J= 5.7 Hz, 2H, CH2), 4.02 (s, OCH3), 5.58-5.62 (m, 1H, CHOC=0), 7.62 (s, 1H, CH); ^,3 C NMR (75.6 MHz, CDCb): δ 13.9 (CHa), 22.3 (CH2), 24.6 (CH2), 31.2 (CH2), 32.5 (CH2), 40.5 (CH2) _> 40.79 (CH2), 44.88 (CH2), 51.03 (CH2), 61.2 (OCHs), 69.9 (CH), 82.2 (CBn), 135.0 (C=CH), 135.7 (CH), 1492 (C=CBn), 164.0 (C=0), 166.1 (C=0), 166.3 (C=0); IR (neat): vmax 2930, 2860, 1769, 1744, 1648, 1497, 1442, 1380, 1316, 1266, 1213, 1150, 1111, 1069, 1035, 1003, 962, 914, 848, 776, 728, 684 cm ^-1 ; UV (MeOH): 308 nm (ε 218887 M-i cm ^"1 ); HRMS (ESI) m/z Calcd. for Ci«H24BnN407 a (M + Na)+ 603.0066. Found 603.0059.

(Z)-l-(4-(3-( ^] -(5-(Dibromomethylene)-2-oxo-2,5-dihydrofiiran-3-yl)do decyloxy)-3- oxopropanoyl)piperazin-l-yl)-2-methoxydiazene oxide

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%). 1H NMR (300 MHz, CDCb): «5 0.86 (t, J= 7.3 Hz, 3H, CH3), 1.23-1.30 (m, 18H, 9 x CH2), 1.84-1.89 (m, 2H, CH2), 3.39-3.46 (m, 4H, CH2), 3.52 (d, J= 2.8 Hz, 2H, CH2), 3.62 (t, J= 5.4 Hz, 2H, CH2), 3.81 (t, J= 5.4 Hz, 2H, CH2), 4.01 (s, 3H, OCH3), 5.57-5.62 (m, 1H, CHOCO), 7.62 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 14.1 (Ctts), 22.6 (CH2), 24.9 (CH2), 29.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 31.9 (CH2), 32.5 (CH2), 40.4 (CH2), 40.7 (CH2), 44.8 (CH2), 51.0 (CH2), 61.2 (OCH3), 69.9 (CH), 82.2 (CBn), 135.0 (C=CH), 135.7 (CH), 149.2 (C=CBn), 164.0 (C=0), 166.1 (C=0), 166.3 (C=0); IR (neat): Vmax 2923, 2853, 1773, 1748, 1650, 1498, 1442, 1316, 1266, 1212, 1150, 1111, 1069, 1035, 1005, 962, 848, 774, 729, 684 cm ^" UV (MeOH): Xmax 308 nm (ε 279253 M-icm-i); HRMS (ESI) m/z Calcd. for Ci8H24BnN40?Na (M + Na)+ 687.1005. Found 687.1003.

(Z)-l-(4-(4-(l-(5-(Dibromomethylene)-2-oxo-2,5-dihydrofur an-3-yl)dodecylo^

oxobutanoyl)piperazin-l-yl)-2-methoxydiazene oxide

The title compound was synthesized following the procedure described for (Z)-l-(4-(4-(l- (5-(dibromomemylene)-2-oxo-2,5-dmydrofuran-3-yl)butoxy)-4-ox obutanoyl)piperazin-l- yl)-2-methoxydiazene oxide using 4-(l-(5-(dibromomethylene)-2-oxo-2,5-dmydrofuran-3- yl)dodecyloxy)-4-oxobutanoic acid (0.2 g; 0.38 mmol), 02-methyl l-(piperazin-l- yl)diazen-l-ium-l,2-diolate (0.08 g, 0.45 mmol) and EDC (0.11 g, 0.57 mmol) to give the tide compound as a yellow sticky oil (0.18 g, 71%). Ή NMR (300 MHz, CDCb): δ 0.86 (t, J= 6.9 Hz, 3H, CH3), 1.23-1.28 (m, 18H, 9 * CH2), 1.81-1.87 (m, 2H, CH2), 2.60-2.76 (m, 4H, 2 x CH2), 3.35-3.44 (m, 4H, CH2), 3.65 (t, J= 5.2 Hz, 2H, CH2), 3.78 (t, J = 5.1 Hz, 2H, CH2), 4.01 (s, 3H, OCH3), 5.49-5.54 (t, J= 5.3 Hz, 1H, CHOC=0), 7.61 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 14.1 (CHs), 22.6 (CH2), 24.9 (CH2), 27.9 (CH2), 29.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 31.9 (CH2), 32.6 (CH2), 40.3 (CH2), 43.7 (CH2), 51.0 (CH2), 51.1 (CH2), 61.2 (OCH3), 69.2 (CH), 77. 2 (CH), 81.3 (CBn), 135.3 (CH), 135.8 (C=CH), 149.5 (C=CBr2), 166.2 (C=0), 169.5 (C=0), 172.1 (C=0); IR (neat): vmax 2923, 2853, 1774, 1738, 1648, 1498, 1440, 1372, 1281, 1214, 1155, 1112, 1068, 1031, 1004, 967, 848, 775, 730, 683 cm ^"1 ; UV (MeOH): 307 nm (ε 295837 M-icm-i); HRMS (ESI) m/z Calcd. for CisH24BnN407Na (M + Na)+ 701.1161. Found 701.1163. l,3-Dibutyl-5-(dibromomethylene)-lH-pyrrol-2(5H)-one

A solution of 3-butyl-5-(dibromomethylene)furan-2(5H)-one (1.00 g, 3.22 mmol) in DCM (20 mL) was stirred in an ice bath at 0 °C followed by dropwise addition of «-butylamine (0.70 g, 9.68 mmol) in DCM (2 mL). The mixture was allowed to stir for 1 h at 0 °C and, then the mixture was successively washed with HC1 (2M, 10 mL), water (10 mL) and brine solution (10 mL). The organic phase was separated, dried (Na2S04) and the solvent evaporated in vacuo. A mixture of the residual oil and >-TsOH (0.02 g, 0.1 1 mmol) in toluene (10 mL) was refluxed for 1 h. The solvent was evaporated in vacuo and the residue purified by silica gel chromatography (2:1 dichloromethane light petroleum) to afford the title compound as a yellow oil (0.66 g, 56%). 1H NMR (300 MHz, CDCb): δ 0.92 (m, 6H, 2 x CHJ), 1.25-1.43 (m, 4H, 2 x CH2), 1.51-1.63 (m, 4H, 2 x CH2), 2.28-2.33 (m, 2H, CH2), 3.96 (t, J = 7.6 Hz, 2H, CH2), 6.99 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 14.2 (2 x CH3), 20.2 (CH2), 22.8 (CH2), 25.5 (CH2), 30.0 (CH2), 32.5 (CH2), 41.1 (CH2), 73.9 (C), 132.2 (CH), 139.2 (C), 141.0 (C), 172.3 (C=0); IR (neat): vmax2957, 2871, 1701, 1586, 1455, 1361, 1194, 1168, 1134, 1047, 829; UV (CH3OH): ax 205.0 nm (9270 M-i cm ^'1 ), 284.0 nm (20630 M-i cm ^"1 ).

3-Butyl-5-(dibromomethylene)-lH-pyrrol-2(5H)-one A solution of 3-butyl-5-(dibromomemylene)furan-2(5H)-one (1.00 g, 3.22 mmol) in ether (30 mL) was stirred in a dry-ice bath at -78 °C followed by slow bubbling of ammonia gas for approximately 15 min. The stirred mixture was slowly brought to room temperature and allowed to react for 18 h. The crude reaction mixture was successively washed with water (20 mL), HC1 (2M, 20 mL) and brine solution (20 mL). The organic phase was separated, dried (Na2S04) and the solvent evaporated in vacuo. The residual solid and p- TsOH (0.02 g, 0.11 mmol) in toluene (10 mL) was refluxed for 1 h. The solvent was evaporated in vacuo and the residue purified by silica gel chromatography (2:1 ethyl acetate/n-hexane) to afford the title compound as a white solid (0.66 g, 36%). M.p. 104- 106 °C Ή NMR (300 MHz, DMSO-ifc): δ 0.86 (t, J = 7.2, 3H, CHs), 1.24-1.31 (m, 2H, CH2), 1.44-1.49 (m, 2H, CH2), 2.15-2.26 (m, 2H, CH2), 6.95 (s, 1H, CH), 10.37 (bs, 1H, NH); ¹³ C NMR (75.6 MHz, DMSO-ifc): δ 14.0 (CHa), 22.1 (CH2), 24.9 (CH2), 29.6 (CH2), 74.6 (C), 129.6 (CH), 141.6 (C), 141.7 (C), 171.6 (C=0); IR (neat): Vmax 3142, 3093, 3034, 2946, 2925, 2869, 2762, 1705, 1618, 1465, 1431, 1386, 1314, 1298, 1201, 1 131, 1097, 1005, 929, 862, 845, 793, 743, 712 cm ¹ ; UV (MeOH): 311 nm (ε 130984 M-icm-i); HRMS (ESI) m/z Calcd. for CoHnBnNONa (M + Na)+ 329.9105. Found 329.9096. l-Bemyl-3-butyl-5-(dibrornomethylene)-lH-pyrrol-2(5H)-one

A solution of 3-buryl-5-(dibromomemylene)furan-2(5H)-one (1.03 g, 3.32 mmol) in DCM (20 mL) was stirred in an ice bath at 0 °C followed by dropwise addition of benzylamine (0.98 g, 9.1 mmol) in DCM (2 mL). The stirred mixture was brought to room temperature and allowed to react for 2 h and then successively washed with water (10 mL), HC1 (2M, 10 mL) and brine solution (10 mL). The organic phase was separated, dried (Na2S04) and the solvent evaporated in vacuo. A mixture of the residual solid and p-TsOH (0.02 g, 0.1 1 mmol) in toluene (10 mL) was refluxed for 2 h. The solvent was evaporated in vacuo and the residue purified by silica gel chromatography (2:1 dicMoromethane/light petroleum) to afford the title compound 212c as a yellow solid (0.78 g, 60%). M.p. 112-114 °C; ^l H NMR (300 MHz, CDCh): δ 0.98 (t, J = 7.2 Hz, 3H, CHa), 1.30-1.47 (m, 2H, CH2), 1.51-1.58 (m, 2H, CH2), 2.28-2.34 (m, 2H, CHa), 5.18 (s, 2H, NCH2), 6.98-7.22 (m, 6H, CH and Haryl); ¹³ C NMR (75.6 MHz, CDCb): ά 14.7 (CHs), 22.8 (CH2), 25.7 (CH2), 30.0 (CH2), 44.6 (NCH2), 75.1 (C), 126.4 (2 x ArCH), 127.4 (ArCH), 128.9 (2 x ArCH), 132.7 (CH), 138.2 (C), 139.2 (C), 140.8 (C), 172.5 (CO); IR (KBr disc): Vmax 2953, 1706, 1626, 1494, 1435, 1352, 1268, 1235, 1094, 747, 721; UV (CHJOH): 206.0 nm (10970 M ^' ), 283.0 nm (16200 M-i cm ^'1 ). 3-Butyl-5-(dibromomethylene)-l -phenyl- lH-pyrrol-2(5H)-one

A mixture of 3-butyl-5-(dibromomethylene)furan-2(5H)-one (1 g, 3.22 mmol) and aniline (0.94 g, 0.96 mmol) was refluxed for 3 h. The residue was dissolve in DCM (30 mL) and successively washed with water (20 mL), HC1 (2M, 20 mL) and brine solution (20 mL). The organic phase was separated, dried (Na2S04) and the solvent evaporated in vacuo. The residual solid was purified by flash chromatography (dichloromethane). A mixture of the residual solid and p-TsOH (0.02 g, 0.11 mmol) in toluene (10 mL) was refluxed for 1 h. The solvent was evaporated in vacuo and the residue purified by silica

gel chromatography (dichloromethane) to afford the title compound as a yellow solid (0.53 g, 43%). M.p. 48 °C; Ή NMR (300 MHz, CDCb): δ 0.95 (t, J = 7.2, 3H, CH ₃ ), 1.37-1.45 (m, 2H, CH2), 1.58-1.64 (m, 2H, CH2), 2.35-2.40 (m, 2H, CH2), 7.16-7.37 (m, 6H, CH and ArH); ^,3 C NMR (75.6 MHz, CDCb): δ 13.7 (CHa), 22.3 (CH2), 25.2 (CH2), 29.5 (CH2), 76.1 (C), 128.3 (ArCH), 128.8 (2 x ArCH), 129.3 (2 x ArCH), 132.0 (CH), 135.0 (C), 138.9 (C), 140.2 (C), 171.7 (C=0); IR (neat): vmax 3107, 3047, 2962, 2919, 2853, 1692, 1592, 1498, 1460, 1356, 1186, 1 172, 1124, 1083, 1067, 1038, 857, 833, 797, 778, 755, 740, 687 cm ^"1 ; UV (MeOH): L™* 308.0 nm (72090 M^cm ^"1 ). HRMS (ESI) m/z Calcd. for CisHieBrcNO (M + H)+ 383.9599. Found 383.9590. l-(4-Bromophenyl)-3-butyl-5-(dibromomethylene)-lH-pyrrol-2(5 H)-one

A mixture of 3-butyl-5-(dibromomethylene)furan-2(5H)-one (1.00 g, 3.22 mmol) and 4- bromoaniline (1.6 g, 0.96 mmol) were heated at 80 °C for 18h without any solvent. The residue was redissolve in DCM (15 mL) and successively washed with water (10 mL), HC1 (2M, 10 mL) and saturated NaCl solution (10 mL). The organic phase was separated, dried (Na2S04) and the solvent evaporated in vacuo. The residual solid was purified by flash chromatography (dichloromethane). A mixture of the residual solid and P2O5 (0.02 g, 0.1 1 mmol) in toluene (10 mL) was refluxed for 1 h. The solvent was evaporated in vacuo and the residue purified by silica gel chromatography (dichloromethane/n-hexane) to afford the title compound 212e as an off white solid (0.95 g, 64%). M.p. 88 °C; 1H NMR (300 MHz, CDCb): δ 0.95 (t, J =7.4, 3H, CH3), 1.37-1.44 (m, 2H, CH2), 1.57-1.63 (m, 2H, CH2), 2.34-2.40 (m, 2H, CH2), 7.09-7.12 (m, 2H, ArH), 7.17 (s, 1H, CH), 7.53 _T 7.56 (m, 2H, ArH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.7 (CHa), 22.3 (CH2), 25.2 (CH2), 29.5 (CH2), 53.3 (C), 122.3 (ArC), 130.8 (2 x ArH), 132.0 (2 x ArH), 132.3 (CH), 134.1 (C), 138.8 (C), 139.9 (C), 171.4 (C=0); IR (neat): v _max 3091, 2955, 2930, 2863, 1698, 1585, 1485, 1464, 1397, 1360, 1196, 1123, 1091, 1066, 1010, 935, 864, 838, 825, 737, 716, 658 cm ¹ ; UV (MeOH): 308 nm (ε 84538 M ^{^} 'cm ^"1 ); HRMS (ESI) m/z Calcd. for Ci5Hi4Br3NONa (M + Na)+483.8523. pound 483.8508.

3-(l-Bromobutyl)-l-butyl-5-(dibromomethylene)-lH-pyrrol-2 (5H)-orte

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%). 1H NMR (300 MHz, CDCh): δ 0.92 (m, 6H, 2 x CH3), 1.24-1.63 (m, 8H, 4 x CH2), 1.99-2.12 (m, 2H, CH2), 3.97 (t, J= 7.6 Hz, 2H, CH2), 4.76-4.81 (m, 2H, CH2), 7.28 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCh): 6 13.1 (CH3), 13.6 (CH3), 19.7 (CH2), 20.9 (CH2), 32.0 (CH2), 32.0 (CH2), 38.8 (CH2), 40.8 (CH2), 43.4 (CH), 73.9 (C), 133.3 (CH), 138.0 (C), 139.9 (C), 169.0 (C=0); IR (neat): vmax 3094, 2953, 2931, 2871, 1688, 1605, 1581, 1464, 1454, 1440, 1378, 1337, 1254, 1194, 1169, 1132, 1052, 1009, 864, 833, 776, 743, 732, 669; UV (CFbOH): λπ«« 280 nm (ε 53916 M-'cm ^"1 ); 325 nm (ε 57589 M^cm ^"1 ); HRMS (ESI) m/z Calcd. for CnHisBrsNONa (M + Na)+ 463.8836. Found 463.8824.

3~(l-Brpmobutyl)-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. A solution of 3-butyl-5- (dibromomethylene)-lH-pyrrol-2(5H)-one (0.32 g; 1.03 mmol), benzoyl peroxide (0.03 g) and NBS (0.20 g, 1.13 mmol) in 15 mL carbon tetrachloride was reacted to give the title compound as a light yellow solid (0.34 g, 85%). M.p. 108 °C; 1H NMR (300 MHz, CDCb): δ 0.96 (t, J = 7.3, 3H, CH3), 1.40-1.60 (m, 2H, CH2), 1.85-1.93 (m, 2H, CH2), 5.72-5.77 (m, 1H, CH), 7.12 (s, 1H, CH), 7.95 (bs, lH, NH); ¹³ C NMR (75.6 MHz, CDCh): δ 13.6 (CHs), 18.3 (CH2), 34.1 (CH2), 77.4 (CH), 79.7 (C), 130.4 (CH), 137.6 (C), 139.5 (C), 168.0 (C=0); IR (neat): Vmax 3091, 2961, 2931, 2870, 1698, 1613, 1583, 1492, 1454, 1373, 1195, 1174, 1136, 1086, 1040, 1006, 892, 869, 833, 782, 738, 692 cm ^"1 ; UV (MeOH): 323 nm (ε 92630 M ^{' 1} ); HRMS (ESI) m/z Calcd. for C HioBrsNONa (M + Na)+407.8210. Found 407.8205. l-Benzyl-3-(l-bromobutyl)-5-(dibromomethylene)-lH-pyrrol-2(5 )-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%). M.p.76 °C; ^J H NMR (300 MHz, CDCh): δ 0.98 (t, J= 7.3 Hz, 3H, CHa), 1.32-1.59 (m, 2H, CH2), 2.00-2.11 (m, 2H, CH2), 4.77-4.82 (m, 1H, CHBr), 5.21 (s, 2H, NCHa), 6.98-7.24 (m, 5H, ArH), 7.32 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.2 (CHa), 21.0 (CH2), 38.9 (CH2), 43.5 (CHBr), 44.4 (NCH2), 78.3 (C), 125.9 (2 x ArCH), 127.1 (ArCH), 128.6 (2 x ArCH), 133.8 (CH), 137.3 (C), 138.1 (C), 139.7 (C), 169.3 (C=0); IR (neat): vmax 3376, 2955, 2933, 2871, 1688, 1604, 1581, 1493, 1450, 1431, 1369, 1347, 1328, 1291, 1190, 1166, 1144, 1085, 1072, 1025, 956, 920, 887, 856, 826, 778, 743, 709, 695, 660; UV (CHaOH): λίι» 325 nm (ε 49141 M ^" 'cm ^"1 ); HRMS (ESI) m/z Calcd. for CieHieBraNONa (M + Na)+ 497.8680. Found 497.8668.

3-(l-Bromobutyl)-5-(dibromomethylene)-l^henyl-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. A solution of 3-butyl-5- (dibromomethylene)-l -phenyl- lH-pyrrol-2(5H)-one (0.3 g; 0.77 mmol), benzoyl peroxide (0.03 g) and NBS (0.15 g, 0.85 mmol) in 15 mL carbon tetrachloride was reacted to give the title compound as a light brown solid (0.47 g, 80%). M.p. 52-54 °C; 1H NMR (300 MHz, CDCb): δ 1.00 (t, J = 7.3, 3H, CHa), 1.46-1.70 (m, 2H, CH2), 2.10-2.20 (m, 2H, CH2), 4.85-4.90 (m, 1H, CH2), 7.21-7.41 (m, 5H, ArH), 7.49 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.2 (CHa), 21.0 (CH2), 39.0 (CH2), 43.5 (CHaBr), 79.7 (CBn), 128.7 (ArCH), 129.0 (2 x ArCH), 129.4 (2 x ArCH), 133.8 (CH), 134.5 (C), 138.2 (C), 139.6 (C), 168.8 (CO); IR (neat): Vmax 3388, 3092, 2962, 2930, 2869, 1698, 1583, 1491, 1454, 1371, 1193, 1169, 1135, 1085, 1040, 887, 832, 782, 763, 740, 691, 661 cm ¹ ; UV (MeOH): λπιβχ 325 nm (ε 78315 M ^{' 1} ); HRMS (ESI) m/z Calcd. for CisHisBraNO (M + H)+ 461.8704. Found 461.8695.

3-(l-Bromobutyl)-l-(4-bromophenyl)-5-(dibromomethylem)-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)-on e (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%). M.p. 88 °C; Ή NMR (300 MHz, CDCb): δ 0.98 (t, J = 7.4, 3H, CHa), 1.46-1.65 (m, 2H, CFb), 2.07-2.18 (m, 2H, CH2), 4.81-4.86 (m, 1H, CHBr), 7.09-7.15 (m, 2H, ArH), 7.47 (s, 1H, CH), 7.55-7.58 (m, 2H, ArH); ^,3 C NMR (75.6 MHz, CDCb): δ 13.2 (CHa), 21.0 (CH2), 39.0 (CH2), 43.2 (CH), 79.9 (C), 122.7 (ArC), 130.9 (ArH), 132.2 (ArH), 133.6 (C), 133.9 (2 χ ArH), 138.2 (C), 139.3 (C), 168.6 (C=0); IR (neat): Vmax 3093, 2964, 2868, 1698, 1606, 1580, 1486, 1462, 1375, 1255, 1189, 1131, 1089, 1070, 1012, 938, 898, 867, 835, 824, 776, 744, 714, 687, 667 cm ^'1 ; UV (MeOH): 321 nm (ε 89517 M ^' W); HRMS (ESI) m/z Calcd. for CisHiaBwNONa (M + Na 561.7628. Found 561.7613. l-(l-Butyl-5-(dibromomethylene)-2-oxo-2,5-dihydro-lH-pyrrol- 3-yl)butyl nitrate

A solution of 3-(l-bromobutyl)-l-butyl-5-(dibromomethylene)-lH-pynOl-2(5H) -one (0.5 g, 1.12 mmol) and silver nitrate (0.22 g, 1.35 mmol) in anhydrous acetonitrile (20 mL) was heated at 50 °C for 5h. The crude product was filtered through a pad of Celite and silica to remove the silver salt formed. The filtrate was evaporated to dryness and the residue purified by vacuum chromatography using dichloromethane/n-hexane to give the title compound as a light yellow solid (0.34 g, 71%). M.p. 36-38 °C; Ή NMR (300 MHz, CDCb): δ 0.93 (m, 6H, 2 x CHa), 1.22-1.61 (m, 8H, 4 x CH2), 1.99-2.16 (m, 2H, CH2), 3.98 (t, J = 7.6 Hz, 2H, CH2), 4.72-4.81 (m, 2H, CH2), 7.2 (s, 1H, CH); ^l3 C NMR (75.6 MHz, CDCb): δ 13.6 (CHa), 13.7 (CHa), 18.3 (CH2), 19.7 (CH2), 32.1 (CH2), 34.2 (CH2), 40.9 (CH2), 77.5 (CH), 73.9 (C), 133.1 (CH), 134.4 (C), 139.9 (C), 169.1 (C=0); IR (neat): vmax 3153, 2957, 2930, 2871, 1684, 1633, 1581, 1465, 1453, 1437, 1353, 1334, 1296, 1272, 1196, 1144, 1103, 1046, 967, 915, 873, 833, 771, 746, 691, 677; UV (CHaOH): max 281 nm (ε 53936 M ^{' "1} ); 331 nm (ε 66072 M-'cm ^'1 ); HRMS (ESI) m/z Calcd. for Ci3Hi8Br2N204Na (M + Na)+ 446.9531. Found 446.9521.

1

l-(5-(Dibromomethylene)-2-oxo-2,5-dihydro-lH-pyrrol-3-yl) butyl nitrate

The title compound was synthesized following the procedure for compound l-(l-butyl-5- (dibromomethylene)-2-oxo-2,5-dihydro-lH-pyrrol-3ryl)butyl nitrate using 3-(l- bromobutyl)-5-(dibromomethylene)-lH-pyrrol-2(5H)-one (0.13 g; 0.35 mmol) and silver nitrate (0.75 g, 0.43 mmol) to give the title compound as a yellow solid (0.09 g, 78%). M.p. 90-92 °C; Ή NMR (300 MHz, CDCb): δ 0.96 (t, J = 7.2, 3H, CHa), 1.39-1.64 (m, 2H, CH2), 2.01-2.17 (m, 2H, CH2), 4.75-4.80 (m, 1H, CH), 7.14 (s, 1H, CH), 7.75 (bs, 1H, NH). ^I3 C NMR (75.6 MHz, CDCb): δ 14.0 (CH3), 22.1 (Cth), 24.9 (CH2), 29.6 (CH2), 74.6 (C), 129.6 (CH), 141.6 (C), 141.7 (C), 171.6 (C=0); IR (neat): Vmax 3116, 3040, 2966, 2931, 2872, 1692, 1651, 1625, 1464, 1455, 1379, 1327, 1298, 1273, 1201, 1160, 1114, 1102, 1051, 1019, 966, 881, 871, 833, 775, 751, 700, 669 cm ^"1 ; UV (MeOH): 322 nm (ε 99037 M-'cm ^"1 ); HRMS (ESI) m/z Calcd. for C HioBnN204Na (M + Na)+ 390.8905. Found 390.8896. l-(l-Benzyl-5-(dibromomethylem)-2-oxo-2,5-dihydro-lH-pyrrol- 3-yl)butyl nitrate

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. 76 °C; Ή NMR (300 MHz, CDC1 ₃ ): δ 0.98 (t, J = 7.1 Hz, 3H, CHs), 1.43-1.53 (m, 2H, CH ₂ ), 1.89-1.97 (m, 2H, CH ₂ ), 5.28 (s, 2H, NCH ₂ ), 5.81-6.86 (m, 1H, CHON0 ₂ ), 7.04-7.07 (m, 2H, ArH), 7.25-7.35 (m, 3H, ArH), 7.37 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDCb): δ 13.6 (CHs), 18.4 (CH2), 34.3 (CH2), 44.4 (NCHa), 77.5 (CH), 79.2 (C), 125.9 (2 x ArCH), 127.2 (ArCH), 128.7 (2 x ArCH), 133.5 (CH), 134.4 (C), 137.2 (C), 139.6 (C), 169.4 (C=0); IR (neat): vmax 2962, 2929, 1687, 1633, 1585, 1496, 1454, 1434, 1382, 1332, 1319, 1294, 1266, 1191, 1153, 1133, 1107, 1074, 993, 946, 923, 884, 863, 831, 754, 738, 701, 693, 677; UV (CH ₃ OH): ^ 281 nm (ε 49297 M ^{' 1} ); 327 nm (ε 62742 M' ¹ ); HRMS (ESI) m/z Calcd. for Ci6Hi ₆ Br ₂ N ₂ 0 ₄ Na (M + Na 480.9375. Found 480.9366. l-(5-(Dibromomethylene)-2-oxo-l-phenyl-2,5-dihydro-lH^yw

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)-on e (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 %). ^l H NMR (300 MHz, CDC1 ₃ ): δ 0.97 (t, J = 7.2, 3H, CH ₃ ), 1.43-1.54 (m, 2H, CH ₂ ), 1.89-1.97 (m, 2H, CH ₂ ), 5.80-5.85 (m, 2H, CH ₂ ON0 ₂ ), 7.22-7.44 (m, 6H, CH and ArH); ¹³ C NMR (75.6 MHz, CDC1 ₃ ): δ 13.6 (CH ₃ ), 18.4 (CH ₂ ), 34.2 (CH ₂ ), 77.6 (CH ₂ ), 80.6 (C), 128.8 (ArCH), 129.0 (ArCH), 129.1 (ArCH), 129.3 (ArCH), 129.4 (ArCH), 133.3 (CH), 134.2 (C), 134.4 (C), 139.4 (C), 168.9 (C=0); IR (neat): vmax 2959, 2918, 2849, 1708, 1634, 1595, 1497, 1455, 1368, 1270, 1192, 1096, 1071, 963, 887, 753, 740, 694 cm ^"1 ; HRMS (ESI) m/z Calcd. for C ₁₅ H, ₄ Br2N ₂ 0 ₄ Na (M + Na)+ 466.9218. Found 466.9205. l-(l-(4-Bromophenyl)^5-(dibromomethyIene)-2-oxo-2,5-dihydro- JH-pyrrol-3-yl)butyl nitrate

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-p yrrol-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. 88 °C; 1H NMR (300 MHz, CDC1 ₃ ): δ 0.98 (t, J= 7.4, 3H, CH ₃ ), 1.43-1.54 (m, 2H, CH ₂ ), 1.89-1.97 (m, 2H, CH ₂ ), 5.78-5.83 (m, 1H, CHBr), 7.11-7.13 (m, 2H, ArH), 7.43 (s, 1H, CH), 7.56-7.59 (m, 2H, ArH); ¹³ C NMR (75.6 MHz, CDC1 ₃ ): δ 13.6 (CH ₃ ), 18.4 (CH ₂ ), 34.2 (CH ₂ ), 77.4 (CH), 80.9 (C), 122.9 (ArC), 130.9 (2 x ArH), 132.1 (2 x ArH), 133.6 (CH), 134.5 (C), 139.2 (C), 168.6 (C=0); IR (neat): vmax 3121, 2962, 1695, 1625, 1583, 1485, 1464, 1359, 1296, 1270, 1192, 1169, 1097, 1068, 1011, 953, 882, 860, 841, 823, 772, 754, 716, 675, 655 cm ^-1 ; UV (MeOH): 325 nm (ε 85764 M-! cm ^-1 ); HRMS (ESI) m/z Calcd. for Ci ₅ H ₁₃ Br ₃ N ₂ 0 ₄ Na (M + Na)+ 544.8323. Found 544.8310.

Sodium l-(lH-indol-3-yl)-diazen-l-ium-l,2-diolate

A solution of indole (4.00 g, 34.14 mmol) in anhydrous ether and methanol (1:1) was placed in a Parr bottle. The solution was treated with sodium methoxide (NaOMe) (3.68 g, 68.28 mmol) and the Parr apparatus was clamped. The apparatus was purged with nitrogen and evacuated (3x) followed by charging with 5 atm. nitric oxide (NO) at 25 °C for 72h. The excess NO was vented by purging with nitrogen followed by addition of anhydrous ether. The resulting precipitate was collected by filtration and washed with copious amounts of anhydrous ether. The product was dried under vacuum to afford the title compound as a yellow solid (3.91 g, 57%). M.p. 220 °C (dec); 1H NMR (300 MHz, DMSO-ifo): δ 7.05 (t, J= , 1H, H5), 7.14 (t, J= , 1H, H6), 7.38 (d, J= 7.3, 1H, H7), 7.56 (s, 1H, H2), 8.00 (d, J= 7.3, 1H, H4), 11.28 (bs, 1H, NH); ¹³ C NMR (75.6 MHz, DMSO- de): δ 112.2 (C7), 116.1 (C4), 119.3 (C3), 119.9 (C5), 121.8 (C6), 122.1 (C2), 125.1 (C3a), 135.8 (C7a); IR (neat): vmax 3403, 3144, 2831, 1594, 1454, 1437, 1354, 1329, 1302, 1252, 1229, 1159, 1112, 1098, 995, 892, 851, 820, 766, 740; UV (10 mM aqueous NaOH ): max 273 nm (ε 20924 M-'cm ^"1 ), 299 nm (ε 22367 M ^' W ¹ ); HRMS (ESI) m/z Calcd. for CsHeNsNazCh (M + Na 222.0255. Found 222.0248.

Sodium l-(5-methoxy-lH-indol-3-yl)-diazen-l-ium-l,2-diolate

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. 180 °C (dec); 1H NMR (300 MHz, 0.1M NaOD in D2O): δ 3.80 (s, 3H, OCH3), 6.88 (m, 1H, H6), 7.36 (m, 2H, H4 and H7), 7.62 (s, 1H, H2); ¹³ C NMR (75.6 MHz, 0.1M NaOD in D2O): δ 57.3 (OCH3), 103.2 (C4), 114.4 (C7), 114.7 (C6), 120.5 (C3), 120.6 (C2), 123.1 (C3a), 131.8 (C7a), 155.4 (C5); IR (neat): vmax 3330, 3119, 2831, 2702, 1579, 1435, 1359, 1300, 1280, 1252, 1222, 1143, 1023, 1008, 919, 899, 879, 854, 799, 767; UV (10 mM aqueous NaOH ): 273 nm (ε 27499 M ^' W), 299 nm (ε 21770 M ^' W ¹ ); HRMS (ESI) m/z Calcd. for CgHeNsNazCb (M + Na 252.0361. Found 252.0354.

(Z)-l-(lH-Indol-3-yl)-2-methoxydiazene oxide

To a solution of sodium l-(lH-indol-3-yl)-diazen-l-ium-l,2-diolate (1.00 g, 5;02 mmol) in 30 mL of methanol at 0 °C under argon was added 1 g of anhydrous potassium carbonate, and the resulting slurry was cooled in an ice bath. Dimethyl sulfate (0.52 mL, 5.50 mmol) was added dropwise and the reaction mixture was allowed to warm gradually to room temperature. After an additional two hour, the solution was concentrated on a rotary evaporator, the residue was extracted with dichloromethane, washed with water, dried over sodium sulfate, and filtered through a pad of sodium sulfate. The solvent was evaporated under reduced pressure and the residue chromatographed on silica gel. Elution with ethyl acetate/n-hexane (1:1) gave the title compound as a light brown crystalline solid (0.58 g, 61%). M.p. 216-218 °C (dec); 1H NMR (300 MHz, CDCb): S 4.29 (OCHa), 7.27-7.34 (m, 2H, H5 and H6), 7.41-7.46 (m, 1H, H7), 7.97 (d, J = 3.3 Hz, 1H, H2), 8.06-8.09 (m, 1H, H4), 8.86 (bs, 1H, NH); ¹³ C NMR (75.6 MHz, CDCb): 6 61.6 (OCHs), 112.0 (C7), 118.4 (C3), 120.7 (C4), 121.8 (C5), 122.3 (C6), 123.9 (C2), 125.1 (C3a), 135.8 (C7a); IR (neat): vmax 3187, 2946, 2834, 1641, 1586, 1530, 1466, 1440, 1400, 1369, 1330, 1273, 1239, 1211, 1198, 1128, 1061, 1007, 937, 882, 839, 774, 731, 659; UV (MeOH): 253 nm (ε 72648 M-'cm ^"1 ), 318 nm (ε 65001 M ^' W ¹ ); HRMS (ESI) m/z Calcd. for C-rtfeNaCfeNa (M + Na)+ 214.0592. Found 214.0584. (Z)-l-(lH-Indol-3-yI)-2-(methoxymethoxy)diazene oxide

To the solution of sodium l-(lH-indol-3-yl)-diazen-l-ium-l,2-diolate (0.50 g, 2.51 mmol) in THF (10 mL) at 0 °C under argon was added chloromethyl methyl ether (0.2 mL, 2.51 mmol). The reaction mixture was brought to room temperature and stirred for 16h. The solution was concentrated on a rotary evaporator and the residue was extracted with dichloromethane. The organic layer was washed with water, dried over sodium sulfate, and filtered through a pad of sodium sulfate. The solvent was evaporated under reduced pressure and the residue was purified by silica gel vacuum chromatography. Elution with ethyl acetate/n-hexane (1 :1) gave the title compound as a brown crystalline solid (0.31 g, 56%). M.p. 116 °C (dec); 1H NMR (300 MHz, CDCb): S 3.57 (s, 3H, OCHs), 5.49 (s, 2H, OCH2), 7.28-7.33 (m, 2H, H5 and H6), 7.43-7.46 (m, 1H, H7), 8.00 (d, J = 3.0 Hz, 1H, H2), 8.09-8.11 (m, 1H, H4), 9.13 (bs, 1H, NH); ¹³ C NMR (75.6 MHz, CDCb): δ 57.3 (OCH3), 98.4 (OCH2), 112.0 (C7), 118.5 (C3), 120.7 (C4), 122.2 (C5), 122.4 (C6), 123.9 (C3a), 124.0 (C2), 135.8 (C7a); IR (neat): vmax 3218, 2992, 2943, 1587, 1526, 1445, 1404, 1367, 1330, 1273, 1237, 1208, 1158, 1107, 1054, 998, 947, 920, 837, 729, 670; UV (MeOH): max 251 nm (ε 72619 M ^{' 1} ), 318 nra (ε 61447 M ^"1 cm ^"1 ); HRMS (ESI) m/z Calcd. for CioHiiNsOaNa (M + Na)+ 244.0698. F,ound 244.0690.

(Z)-2-(2-Ethoxy-2-oxoethoxy)-l-(lH-indol-3-yl)diazene oxide

Sodium l-(lH-indol-3-yl)-diazen-l-ium-l,2-diolate (0.50 g, 2.51 mmol), was alkylated using ethyl-2-bromoacetate (0.28 mL, 2.51 mmol) by the method described for compound (2)-l-(lH-indol-3-yl)-2-(methoxymethoxy)diazene oxide to give the title compound as a brown solid (0.25 g, 39%). M.p. 134-136 °C (dec); 1H NMR (300 MHz, CDCb): S 1.30 (t, J= 7.1, 3H, CHJ), 4.29 (q, J= 7.1, 2H, OCHa), 4.98 (s, 2H, OCH2CO), 7.24-7.29 (m, 2H, H5 and H6), 7.41-7.44 (m, 1H, H7), 7.94-7.96 (m, 2H, H2 and H4), 9.02 (bs, 1H, NH); ¹³ C NMR (75.6 MHz, CDCb): δ 14.1 (CHa), 61.7 (OCH2), 69.9 (OCH2CO), 112.1 (C7), 118.4 (C3), 120.5 (C4), 122.4 (C5 and C6), 123.3 (C3a), 123.9 (C2), 135.0 (C7a), 168.1 (C=0); IR (neat): vmax 3247, 3167, 2999, 2935, 1758, 1587, 1537, 1446, 1436, 1421, 1382, 1325, 1246, 1226, 1214, 1153, 1086, 1054, 1023, 1015, 949, 848, 773, 750, 723, 669; UV (MeOH): Xmax 251 nm (ε 84612 M 'cm ^"1 ), 320 nm (ε 71450 M ¹ ); HRMS (ESI) m/z Calcd. for CnHoNaOiNa (M + Na)+ 286.0804. Found 286.0795.

(Z)-2-(Benzyloxy)-l-(lH-indol-3-yl)diazene oxide Sodium l-(lH-indol-3-yl)-diazen-l-ium-l,2-diolate (0.5 g, 2.51 mmol), was alkylated using benzyl bromide (0.33 mL, 2.76 mmol) by the method described for (Z)-l-(lH-indol- 3-yl)-2-(methoxymethoxy)diazene oxide to give the title compound as a brown solid (0.24 g, 36%). M.p. 174-176 °C (dec); 1H NMR (300 MHz, CDCb): δ 4.29 (OCHa), 7.27-7.34 (m, 2H, H5 and H6), 7.36-7.41 (m, 4H, H7, 3 x ArH), 7.49-7.53 (m, 2H, 2 ^χ ArH), 7.90- 7.94 (m, 2H, H2 and H4), 8.65 (bs, 1H, NH); ^,3 C NMR (75.6 MHz, CDCb): S 111.8 (C7), 118.4 (C3), 120.7 (C4), 121.7 (C5), 122.3 (C6), 123.9 (C2), 125.1 (C3a), 128.6 (ArH), 128.7 (ArH), 128.8 (ArH), 135.8 (C7a); IR (neat): vmax 3298, 3132, 2956, 1732, 1675, 1587, 1529, 1451, 1416, 1372, 1343, 1328, 1245, 1205, 1194, 1094, 1065, 1032, 981, 835, 771, 750, 696; UV (MeOH): max 254 nm (ε 68676 M ^' W), 319 nm (ε 61251 M ^' W ¹ ); HRMS (ESI) m/z Calcd. for C15H14N3O2 (M + H)+ 268.1086. Found 268.1075.

3-Formyl-2, 2, 5, 5-tetramethylthiazolidine-4-carboxylic acid

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%). M.p. 178 °C; Ή NMR (300 MHz, CDC1 ₃ ): δ 1.50 (s, 3H, CH ₃ ), 1.65 (s, 3H, CH ₃ ), 1.93 (s, 6H, 2 ^χ CH ₃ ), 4.53 (s, 1H, CH), 8.36 (s, 1H, CHO); ¹³ C NMR (75.6 MHz, acetone-*/*): δ 25.9 (CH ₃ ), 32.9 (CH ₃ ), 33.7 (CH ₃ ), 34.5 (CH ₃ ), 50.2 (>C-S), 70.2 (>C-S), 72.0 (CH), 161.4 (C=0), 170.2 (C=0); IR (neat): 2976, 2944, 2507, 1738, 1613, 1466, 1364, 1318, 1194, 1163, 1137, 1109, 953, 917, 733, 717, 691 cm ^"1 ; HRMS (ESI) m/z Calcd. for C ₉ Hi ₅ N0 ₃ SNa (M + Na) ⁺ 240.0607. Found 240.0659. l-(5-(Dibromomethylene)-2-oxo-2, 5-dihydrofuran-3-yl)butyl 3-formyl-2, 2, 5, 5- tetramethylthiazolidine-4-carboxylate

To a solution of bromofimbrolide (2 g, 5.14 mmol) and powdered potassium hydroxide (0.4 g, 7.19 mmol) in DMF (7 mL), was added 3-formyl-2,2,5,5-tetramethylthiazolidine-4- carboxylic acid (1.3 g, 6.68 mmol) and stirred for 72h at room temperature. Water was added to the reaction mixture and the resulting mixture was extracted with DCM. The organic layer was further washed with water, brine and evaporated to dryness. The crude product was purified by vacuum chromatography using ethyl acetate/n-hexane to yield two diasteromeric products. Isomer 1 (0.74 g, 27%): M.p. 134-136 °C; 1H NMR (300 MHz, CDC1 ₃ ): δ 0.95 (t, J= 7.4 Hz, 3H, CH ₃ ), 1.37-1.43 (m, 2H, CH ₂ ), 1.46 (s, 3H, CH ₃ ), 1.69 (s, 3H, CH ₃ ), 1.81-1.88 (m, 2H, CH ₂ ), 1.93 (s, 2H, CH ₃ ), 4.81 (s, 1H, CH), 5.54-5.58 (m, 1H, CHOC=0), 7.80 (s, 1H, CH), 8.34 (s, 1H, CHO); ^,3 C NMR (75.6 MHz, CDCI3): δ

13.4 (CH ₃ ), 18.5 (CH ₂ ), 25.7 (CH ₃ ), 33.0 (CH ₃ ), 33.5 (CH ₃ ), 34.3 (CH ₂ ), 34.5 (CH ₃ ), 50.1 (>C-S), 70.3 (>C-S), 70.5 (CH), 72.4 (CH), 81.8 (CBr ₂ ), 135.3 (C), 135.5 (CH), 149.0 (C), 160.2 (CHO), 166.1 (CO), 168.3(C=0); IR (neat): 2960, 2928, 1768, 1748, 1671, 1462, 1372, 1325, 1177, 1136, 1113, 1062, 1026, 964, 881, 842, 772, 680 cm ^"1 ; HRMS (ESI) m/z Calcd. for Ci ₈ H ₂₄ Br ₂ N0 ₅ S (M + H) ⁺ 523.9742. Found 523.9730. Isomer 2 (0.52 g, 19%): M.p. 98 °C; Ή NMR (300 MHz, CDC1 ₃ ): δ 0.92 (t, J= 7.4 Hz, 3H, CH ₃ ), 1.37- 1.50 (m, 2H, CH ₂ ), 1.40 (s, 3H, CH ₃ ), 1.66 (s, 3H, CH ₃ ), 1.86-1.88 (m, 2H, CH ₂ ), 1.93 (s, 3H, CH ₃ ), 4.73 (s, 1H, CH), 5.58-5.63 (m, 1H, CHOC=0), 7.57 (s, 1H, CH), 8.38 (s, 1H, CHO); ¹³ C NMR (75.6 MHz, CDC1 ₃ ): δ 13.4 (CH ₃ ), 18.5 (CH ₂ ), 25.7 (CH ₃ ), 33.0 (CH ₃ ),

33.5 (C¾), 34.3 (CH ₂ ), 34.5 (CH ₃ ), 50.1 (>C-S), 70.3 (>C-S), 70.5 (CH), 72.4 (CH), 81.8 (CBr ₂ ), 135.3 (C), 135.5 (CH), 149.0 (C), 160.2 (CHO), 166.1 (C=0), 168.3(C=0); IR (neat): 3096, 2967, 2934, 2872, 1767, 1749, 1669, 1468, 1386, 1370, 1326, 1267, 1175, 1142, 1118, 1058, 1026, 965, 915, 890, 844, 774, 735, 714 cm ¹ ; HRMS (ESI) m/z Calcd. for Ci ₈ H ₂ Br ₂ N0 ₅ S (M + H) ⁺ 523.9742. Found 523.9728. l-(5-(Dibromomethylene)-2-oxo 2,5-dihydrojw-an-S-yl)butyl-2-acetam^

methylbutanoate

The 1 -(5-(dibromome lene)-2-oxo-2,5-dihydrofuran-3-yl)butyl 3-formyl-2,2,5,5- tetr emylthiazolidine-4-carboxylate (0.4 g, 0.81 mmol) was dissolved in a mixture of 1M methanolic hydrochloric acid (1.5 mL) and methanol (2 mL) by gentle warming. The reaction mixture was kept at 50 °C for 18h and the solvent evaporated to dryness. Aqueous sodium bicarbonate solution (7 mL, 0.81 mmol) was added to the residue and the reaction mixture was cooled in an ice bath. Acetic anhydride (2 mL) was added dropwise to the mixture followed by stirring for 18h at room temperature. The reaction mixture was concentrated to dryness and the residue purified by vacuum chromatography using ethyl acetate/n-hexane to give title compound as an off white solid (0.16 g, 42%). M.p. 134-136 °C; ^l H NMR (300 MHz, CDC1 ₃ ): δ 0.93 (t, J= 7.4 Hz, 3H, CH ₃ ), 1.31-1.43 (m, 2H, CH ₂ ), 1.37 (s, 3H, CH ₃ ), 1.52 (s, 3H, CH ₃ ), 1.81-1.89 (m, 2H, CH ₂ ), 2.04 (s, 3H, COCH ₃ ), 2.12 (s, 1H, SH), 4.54 (d, J= 8.7 Hz, 1H, CH), 5:53-5.58 (m, 1H, CHOC=0), 6.43 (bd, J= 8.6 Hz, 1H, NH) 7.61 (s, 1H, CH); ¹³ C NMR (75.6 MHz, CDC1 ₃ ): δ 13.5 (CH ₃ ), 18.4 (CH ₂ ), 23.0 (CH ₃ ), 29.3, (CH ₃ ), 31.3 (CH ₃ ), 34.7 (CH ₂ ), 45.6 (>C-SH), 60.7 (CH), 70.1 (CH), 82.1 (CBr ₂ ), 135.2 (C), 135.6 (CH), 149.3 (C), 166.1 (C=0), 170.1 (C=0), 170.3(C=O); IR (neat): 2964, 2919, 2857, 1769, 1748, 1667, 1463, 1372, 1325, 1262, 1175, 1136, 1113, 1062, 1026, 964, 912, 849, 772, 755, 714 cm ^"1 ; HRMS (ESI) m/z Calcd. for C ₁₆ H ₂ iBr ₂ N0 ₅ SNa (M + Na) ⁺ 519.9405. Found 519.9412. l-(5-(Dibromomethylene)-2-oxO'2,5-dih drofuran-3-yl)butyl-2-a^

(nitrosothio)butanoate

To a solution of l-(5-(Dibromomemylene)-2-oxo-2,5-dihydroiuran-3-yl)butyl-2- acetamido-3-mercapto-3-methylbutanoate (1.0 mmol) in DCM (15 mL) was added /-butyl nitrite (2.0 mmol) and the solution was stirred at 0 °C for 20 min. The crude reaction mixture was evaporated to dryness under vacuo and the residue purified by vacuum chromatography to yield the title compound as a green sticky oil. *H NMR (400 MHz, CDC1 ₃ ): δ 0.89 (t, J = 7.4 Hz, 3H, CH ₃ ), 1.31-1.37 (m, 2H, CH ₂ ), 1.78-1.84 (m, 2H, CH ₂ ), 1.97 (s, 3H, CH ₃ ), 2.02 (s, 3H, CH ₃ ), 2.07~(s, 3H, COCH ₃ ), 5.35 (d, J =9.0 Hz, 1H, CH), 5.53 (t, J= 6.0 Hz, 1H, CHOC=0), 6.41 (bd, J= 8.1 Hz, 1H, NH) 7.51 (s, lH, CH).

Example 2 - Quorum sensing inhibition assay

Assay method

Selected compounds were analysed using the QS inhibitory screening system developed by Hentzer et a/. ¹ The system uses the QS monitor strain P. aeruginosa MH602 which carries a plasmid containing a reporter gene (Gfp (ASV)) 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.

When 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 μΜ to 1.3 μΜ, 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).

In order to compare the relative strength of the inhibitors, 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. Thus, the potency of a compound as a QS inhibitor is directly correlated with its calculated percentage of QS inhibition.

Initially, the assay was performed on two standard QS inhibitors, (Z)-4-bromo-5- (bromomethylene)-3-butylfuran-2(5H)-one (compound 34a) and (Z)-4-bromo-5- (bromomethylene)-furan-2(5H)-one (compound 37). (Z)-4-bromo-5-(bromomethylene)-3- b tylfuran-2(5H)-one is a natural fimbrolide derivative from Delisea pulchra which is known to be a potent QS inhibitor. (Z)-4-bromo-5-(bromomethylene)-furan-2(5H)-one is a synthetic QS inhibitor and is considered as one of the "gold standards" for QS inhibition studies.

The data is presented graphically below as the Relative Fluorescence Units (RFU) and OD values observed during the course of the assay (Figures 1 and 2). The RFU/OD ratio is also calculated to correct for differences in growth observed at given time points. The percentage QS inhibition (based on RFU) values of the two compounds at various concentrations measured at the highest QS activity time point (8 h) in control is tabulated in Table 1

Table 1: Percentage quorum sensing inhibition at different concentrations for standard compounds. Concentration (uM)

Compound

266 88.8 29.6 9.9 3.3 1.0

34a 75.8 39.9 5.9 NA NA NA

37 GI GI 61.9 29.8 3.7 NA

NA- No activity (No reduction in RFU compared to control), GI- Growth inhibition of test species MH602 (greater than 15%)

From the results, it is evident that of the two standard compounds, compound 37 has a higher QS inhibition potency. At 37 μΜ, 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 μΜ, whereas compound 34a did not affect the growth of the bacterial host even at 333 μΜ.

Screening results

AHL-based NO donors

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.

i

Table 2: Percentage quorum sensing inhibition at different concentrations of AHL derivatives.

Concentration (μΜ)

Compound

266 88.8 29.6 9.9 3.3 1.0

95a 26.42 5.56 NA NA NA NA

95d 70.07 25.20 NA NA NA NA

95e 62.18 14.16 NA ^" NA NA NA

97a 74.45 47.07 29.90 8.68 1.41 2.86

97b 59.99 20.08 1.08 NA NA NA

105 62.26 20.36 4.08 NA NA NA

121a 43.07 13.63 2.93 NA NA NA 121b 41.66 13.85 2.49 2.07 NA NA

122a 57.85 25.43 11.77 3.32 0.34 0.41

NA- Not active (No reduction in RFU compared to control)

The results show that bulkier hydrophobic substitution on the acylated homoserine lactone scaffold is more conducive for inhibitory activity, possibly due to better complementarities with the hydrophobic pocket of the LasR receptor protein, leading to superior inhibition of AHL-mediated QS pathway.

Fimbrolide-based NO donors

Selected fimbrolide-based NO derivatives were screened for biological activity using the same methodology as described above. The results of this assay are presented in Table 3.

Table 3: Percentage quorum sensing inhibition at different concentrations of the fimbroiide derivatives.

Concentration (μΜ)

Compound

266 88.8 29.6 9.9 3.3 1.0

172 74.45 53.31 25.34 3.12 NA NA

174 72.71 48.83 25.81 6.57 NA NA

175 75.38 51.79 18.01 NA NA NA

178a 70.69 40.38 15.89 1.26 NA NA

178b 67.25 47.22 28.33 12.55 3.77 NA

178c 44.57 2.53 NA NA NA NA

178d 44.32 3.58 NA NA NA . NA

NA- No activity (no reduction in RFU compared to control), GI- Growth inhibition of MH602 (greater than 15%)

In general, appending of NO donors to the fimbroiide derivative led to an increase in QS inhibitory activity which was evident from the higher QS inhibitory activity of fimbroiide NO hybrids. 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.

Dihydropyrrolone-based NO donors

Selected dihydropyrrolone-based NO hybrid derivatives were screened for biological activity using the same methodology as described above. The results of this assay are presented in Table 4.

Table 4: Percentage quorum sensing inhibition at different concentrations of dihydropyrrolones derivatives.

Concentration (μΜ)

Compound

266 88.8 29.6 9.9

210a 29.57 12.65 6.11 NA '

210b 33.36 8.67 NA NA

210c 68.92 29.09 10.56 NA

210d 70.09 27.81 4.76 NA

210e 40.95 9.05 NA NA

NA- No activity (no reduction in RFU compared to control)

[Example 3 - Nitric oxide release studies

A wide range of detection techniques have been developed for nitric oxide. 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.

Quantification of nitrate/nitrite by the Griess Reaction

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).

Reagent B Reagent A Azo dye

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.

R-NO ₃ — - R-OH + N0 ₂ - NO

2NO + 0 ₂ — - 2N0 ₂

NO + N0 ₂ — - N ₂ 0 ₃ and ₍

N ₂ 0 ₃ + H ₂ 0 - 2N0 ₂ - + 2H ⁺

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. However, the enzymatic approach requires NADPH (nicotinamide adenine dinucleotide phosphate-oxidase) as a cofactor, and this compound has been shown to interfere with the Griess assay. Organic nitrate can also be reduced to NO using xanthine oxidase in presence of xanthine and cysteine.

The use of xanthine oxidase along with xanthine and cysteine was found to be most suitable and was thus utilized for the study of the selected compounds.

Assay Procedure In a 96- well plate containing a solution of the test compound (7.5 or 15 μΐ from 5 mM stock solution in 0.1 M phosphate buffer, pH 7.4) was added a freshly prepared solution of L-cysteine (15 μΐ of a 5 mM solution in 0.1 M phosphate buffer, pH 7.4), xanthine (3 μΐ of a 1 mM solution in 0.1 M phosphate buffer, pH 7.4) and xanthine oxidase (6 μΐ of a 2 mg mL solution in 0.1 M phosphate buffer, pH 7.4). PBS was added to the mixture to a total volume to 150 μΐ, and the mixture was incubated at 37 °C for 1 h at 180 rpm. The plate was brought to room temperature, an aliquot of the Griess reagent (20 μΐ), 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 μΐ 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).

Solutions of 0 to 100 μΜ sodium nitrite were used to prepare a standard curve of nitrite absorbance versus concentration under the same experimental conditions. The concentration of nitric oxide released (quantitated as nitrite ions) was calc lated from the standard curve.

Nitric oxide quantitation results by Griess assay

As shown in Figure 4, the standard curve gave a good linear fit between 0 and 120 μΜ (R = 0.995). 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.

Nitric oxide measurement by Apollo

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. For NO release measurements, 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. Aliquots of the indole diazeniumdiolate derivatives IDN-1 (compound 237) and IND-2 (compound 243d) in aqueous medium were injected in the solution through the septum and the change in current was monitored. Two set of experiments, one with gentle nitrogen (N ₂ ) purging and another without mtrogen purging was studied. When the NO scavenger PTIO [2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-l-oxyl-3 -oxide] was added to the solution, an immediate drop in the measured current was observed, which confirmed that the change in current was due to the production of NO.

The results indicated that the indole diazeniumdiolate derivatives 237 (IND-1) and 243d (IND-2) can release NO spontaneously in aqueous solution (Figures 6 and 7).

Example 4 - Biofilm dispersion assay

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.

1) Biofilm dispersion study of IND-1 in combination with 0.1% SDS

Combinatorial treatments of IND-1 with the antimicrobial surfactant SDS were assessed on P. aeruginosa biofilms. Biofilms were grown for 6 h in the absence of any treatment, then the NO donor IND-1 was added at various concentrations for 15 min, after which 0.1% SDS was added to the wells and the plates were incubated for a final 45 min. For quantification in microtitre plates, a method similar to that reported by O'Toole et al? was used. Wells containing biofilms were washed twice with phosphate buffered saline (PBS) and stained for 20 min with 1 mL of crystal violet (0.2 %). The wells were washed again three times with PBS, and the remaining crystal violet was dissolved in 1 mL of absolute ethanol. Biofilm biomass was quantified by measurement at OD550. The results are shown in Table 5.

removed vs

The data suggested that IND-1 exhibits potent biofilm dispersion activity with and without the addition of SDS.

2) Biofilm dispersion study of IND-2

The dispersal of P. aeruginosa biofilms was assayed by treating grown biofilms (6 h) with NO donors for 10 min. For quantification in microtitre plates, a method similar to that reported by O'Toole et al? was used. Wells containing biofilms were washed twice with PBS and stained for 20 min with 1 mL of crystal violet (0.2 %). The wells were washed again three times with PBS, and the remaining crystal violet was dissolved in 1 mL of absolute ethanol. Dispersal was quantified by measurement at OD550. MAHMA NONOate (NOC-9) was used as standard. The results are shown in Table 6 and Figure 8 (A and B).

GI- Growth inhibition; BD- Biofilm dispersion

The PAOl biofilm dispersion assay indicated that compound 243d at 100 μΜ 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.

References

1. Hentzer, M; Riedel, K.; Rasmussen, T. B.; Heydorn, A.; Andersen, J. B.; Parsek, M. R.; Rice, S. A.; Eberl, L.; Molin, S.; H iby, N.; Kjelleberg, S.; Givskov, M. Microbiology 2002, 148. O'Toole, G. A.; Kolter, R. Mol. Microbiol. 1998, 28, 449-461.

Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Biochem. J. 2001, 357, 593-615.