ANTIMICROBIAL AGENTS TO TREAT BACTERIAL INFECTIONS

Field of the Invention

The present invention relates to compounds suitable for use against pathogens. In particular, the compounds can be used as antimicrobial agents to treat bacterial infections.

Background of the Invention

There is increasing concern at the rapid emergence of multi-drug-resistant (MDR) pathogens, which is exacerbated by a lack of new antibiotics in development. In particular, the MDR problem is greater for gram-negative 'superbugs' because there are few therapeutic options against them currently available or on the horizon.

Gram-negative pathogens of the genera Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae are responsible for many infections in hospitals (intensive care and/or immunocompromised patients). In addition, they are becoming increasingly prevalent outside the clinic, as shown by increases in seemingly trivial community-acquired infections such as bacterial otitis externa (swimmer's ear).

The outer membrane of gram-negative bacteria is comprised of a lipopolysaccharide (LPS). In addition to the effects of infection, this can cause more acute symptoms: the human immune system recognises LPS endotoxin and triggers immune responses that can initiate septic shock, leading to multiple organ failure and death.

Agents that bind LPS and thereby reduce the blood concentrations of endotoxin can mitigate the onset of sepsis.

One available antibiotic that binds LPS is the naturally occurring antimicrobial colistin, which shows excellent activity against many clinical isolates of P aeruginosa, A baumannii and K pneumoniae in vitro.

Colistin, or polymyxin E, is a member of the polymyxin family (polymyxin A, B, C, D and E), originally isolated in the 1940s. Unfortunately, colistin's side-effects - in particular, its levels of nephrotoxicity and neurotoxicity - limits its use in therapy. In addition, it must be administered intravenously, limiting its use outside the clinic. Simple modifications to colistin have not been successful in producing a more effective LPS binding agent. Other alternatives that have been investigated include simple amphiphilic species, including polyamines, acylpolyamines, bis-guanidines, tercyclopentanes, and dendrimers. Whilst some antibacterial activity has been observed, it is not comparable to that of colistin.

There is thus a need for new agents that are effective against pathogens, in particular gram-negative microorganisms, but with lower toxicity than colistin and which are suitable for administration by alternative routes.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was, in Australia, known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Summary of the Invention

The invention provides a new class of compounds which can be directed against the cell membrane of a microorganism. In particular, they are believed to target lipid A in the LPS of the cell membrane. The compounds are conformationally organised to present one or more binding portions that recognise and bind the anionic groups on the membrane, and a hydrophobic portion able to penetrate the membrane.

In one embodiment, the invention provides a compound including: a core having a first face and a second face; a binding portion attached to the first face of the core, wherein the binding portion is capable of binding to an anionic group present in a cell membrane of a microorganism; and a hydrophobic portion attached to the second face of the core, wherein the hydrophobic portion is capable of interacting with the cell membrane of the microorganism.

In some embodiments, the core includes a bicyclic moiety. In some embodiments, the bicyclic moiety has a structure of Formula (I) wherein

X is selected from the group consisting of O, N and (CH ₂ ) _m , where m is 1 or 2.

In some embodiments, the compound has a structure of Formula (IA)

wherein each R _A is independently an optional substituent or a binding portion, and wherein at least one R _A is a binding portion; and

wherein each R _B is independently an optional substituent or a hydrophobic portion, and wherein at least one R _B is a hydrophobic portion, or two Re taken together form a hydrophobic portion.

In some embodiments, the core includes norbornane or norbornene.

In some embodiments, the compound includes two binding portions.

In some embodiments, the compound is of Formula (II):

(II) or a salt or ion thereof, wherein R ¹ is a moiety forming part of a hydrophobic portion; R ² is a first binding portion; and R ³ is a second binding portion.

In some embodiments, the salt is at least one selected from the group consisting of chloride, sulfate, bromide, mesylate, maleate, citrate, phosphate and trifluoroacetate salts.

In some embodiments, each binding portion is adapted to bind to a phosphate group present in a cell membrane of a microorganism. In some embodiments, the binding portions are separated by a distance of between about 12 to 14A. In some embodiments, the binding portions are joined to the core in a structural arrangement of any combination of exo and endo.

In some embodiments, each binding portion independently has a structure of Formula (III):

S ^{V / rΛ} ' P (in)

or a salt or ion thereof, wherein

D at each occurrence is independently selected from the group consisting of a bond or a functional group;

L at each occurrence is independently an optionally substituted spacer group; A at each occurrence is independently a binding moiety capable of binding with an anionic group present in a cell membrane of a microorganism;

Z at each occurrence is independently a substituent group selected from the group consisting of hydrogen, optionally substituted Ci to C- ₁ 4 alkyl, optionally substituted 5 to 8-membered aryl and optionally substituted 3 to 8-membered cycloalkyl, wherein said alkyl, aryl and cycloalkyl may be optionally interrupted by one or more heteroatoms selected from the group consisting of O, N and S; or other heterocycle;

n is an integer selected from the group consisting of 1 , 2, 3, 4, 5 and 6; and p is an integer selected from the group consisting of 1 , 2, 3, 4, 5 and 6.

In some embodiments, n = 1. In some embodiments, p = 1.

In some embodiments, D is a functional group. Amide functional groups, for example - CONH-, may be suitable. In some embodiments, D is selected from the group consisting Of -CH ₂ NH-, -CH ₂ O-, NH ₂ and O.

In some embodiments, L includes a hydrogen bonding moiety adapted to participate in hydrogen bonding interactions with the cell membrane of a microorganism. In some embodiments, L is selected from the group consisting of a Ci to Ci ₀ alkyl or a C ₂ to C-io alkenyl chain and an amino acid residue, where the amino acid may include glycine, arginine, asparagine, glutamine and lysine or a non-natural amino acid. In some embodiments, L is a C ₂ to Ce alkyl or C ₂ to C _& alkenyl chain. In some embodiments, L may include amide, urea, thiourea, guanidine, ammonium, imidazolinium and combinations of these moieties. In some embodiments, L is -CH ₂ CH ₂ - _.

As n is an integer and may vary, and as D and L may vary, the total length of the group (D-L) _n may vary. For ease of reference, the total length is herein designated q. In some embodiments A is capable of binding to LPS using hydrogen bonding, ionic bonding and/or electrostatic forces.

In some embodiments A may be amide, urea or guanidine. In some embodiments, A may include a primary, secondary, tertiary or quaternary amine or ammonium moiety; or guanidine, guanidinium, amidine, amidinium, imidazolium, thiouronium, amide, urea, thiourea or pyrrole.

In some embodiments, A may include additional functionality directly bonded to the groups listed above in order to further enhance their binding properties. In some embodiments, a carbonyl or an amino group sits adjacent to a urea. For example an additional amino group bonded to a urea forms a hydrazinecarboxamide functional group. A carbonyl adjacent to a guanidine forms a guanidinycarbonyl. In some embodiments, each group -A-Z is independently selected from the group consisting of: or a salt or ion thereof.

In some embodiments, Z is selected from the group consisting of H, Ci to Cu alkyl, 5 to 8-membered aryl and 3 to 8-membered cycloalkyl, heterocycloalkyl or heteroaryl. In some embodiments, Z is an optionally substituted aryl, an optionally substituted alkyl, an optionally substituted cycloalkyl or an optionally substituted heterocycle. In some embodiments, Z may contain an optional substituent selected from the group consisting of alkyl esters (for example Ci to C ₄ alkyl esters), halogens, nitrite (CN) and nitro (NO ₂ ) groups. In some embodiments Z contains one or more optional substituents independently selected from the group consisting of F, Cl, Br, I, CH ₃ , CH ₂ CH ₃ , OH, OCH ₃ , CO ₂ CH ₃ , CO ₂ Et, CF ₃ , OCF ₃ , NO ₂ , NH ₂ , and CN. In some embodiments, Z = H.

In some embodiments, the optional substituent may be an electron withdrawing group. Electron withdrawing groups may activate the binding moiety A to enhance its interaction with amine groups present in the cell membrane. In some embodiments, the substituent may be F. In some embodiments, Z is a 5 to 8-membered aryl in which one or more of the carbons has been interrupted by a heteroatom which may be selected from the group consisting of nitrogen, oxygen and sulphur or combinations thereof such that Z is a heteroaryl group. In some embodiments, Z is an optionally substituted cycloalkyl in which one or more of the carbons has been interrupted by a heteroatom which may be selected from the group consisting of nitrogen, oxygen and sulphur or combinations thereof such that Z is a heterocycloalkyl group.

In some embodiments, the hydrophobic portion or the moiety forming part of the hydrophobic portion includes 4 to 20 carbon atoms. In some embodiments, the hydrophobic portion or the moiety forming part of the hydrophobic portion is selected from the group consisting of optionally substituted C ₄ to C2 ₀ alkyl, optionally substituted C ₄ to C ₂ o alkenyl, optionally substituted C ₄ to C ₂ o alkynyl, optionally substituted C ₄ to C ₂ o cycloalkyl and optionally substituted C ₆ to C2 ₀ aryl.

In some embodiments, the hydrophobic portion or the moiety forming part of the hydrophobic portion is selected from the group consisting of optionally substituted C ₆ to C ₈ alkyl and optionally substituted C ₆ to C1 ₀ aryl.

In some embodiments the hydrophobic portion includes a linking moiety linking the moiety forming part of the hydrophobic portion to the core. In some embodiments the hydrophobic portion includes an acetal moiety linking the moiety forming part of the hydrophobic portion to the core

In some embodiments, the compounds are selected from the group consisting of (where, for example, the designation "(1.2HCI)" refers to the HCI salt of Compound 1 )

(1.2HCI)

D

(6)

D D

(10.2HCI)

In respect of compound 6, the optionally substituted fluorine on Z acts as an electron- withdrawing group, which further activates the thiourea groups for stronger hydrogen bonding.

In some embodiments, the compounds of the invention may have multiple binding portions. In some embodiments, these may be different, having different A-Z groups.

In some embodiments, the invention provides compositions including at least one compound of the invention and a carrier, excipient or diluent. In some embodiments, the compositions are formulated for oral administration to a subject. In some embodiments, the compositions are formulated for topical application to a surface.

In some embodiments, the invention provides a method for the treatment or prophylaxis of infection of a mammal by a microorganism, the method comprising administering to the mammal in need thereof an effective amount of a compound of the invention. In some embodiments, the microorganism is gram-negative bacteria. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human animal.

In some embodiments, the invention provides a method for treating or preventing contamination of a substrate by a microorganism, the method comprising applying an effective amount of a compound of the invention. In some embodiments, the antimicrobial compound is applied to a surface of the substrate. In some embodiments, the microorganism is bacteria.

In some embodiments, there is provided use of the compounds of the invention in the treatment or prophylaxis of infection of a mammal by a microorganism. In some embodiments, there is provided use of the composition of the invention in the treatment or prophylaxis of infection of a mammal by a microorganism.

In some embodiments, there is provided use of the compounds of the invention in treating or preventing contamination of a substrate by a microorganism. In some embodiments, there is provided use of the compositions of the invention in treating or preventing contamination of a substrate by a microorganism.

In some embodiments, there is provided a compound of the invention for use in the treatment or prophylaxis of infection in a mammal. In some embodiments, there is provided a composition according to the invention for use in the treatment or prophylaxis of infection of a mammal by a microorganism.

Detailed Description of the Invention In this specification a number of terms are used which are well known to a skilled addressee. Nevertheless for the purposes of clarity a number of terms will be defined.

The term "optionally substituted" as used throughout the specification denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more non-hydrogen substituent groups. In certain embodiments the substituent groups are one or more groups independently selected from the group consisting of halogen, =O, =S, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxycycloalkyl, alkyloxyheterocycloalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkyloxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, -C(=O)OH, -C(=O)R ^a , -C(=O)OR ^a , C(=O)NR ^a R ^b , C(=NOH)R ^a , C(=NR ^a )NR ^b R ^c , NR ^a R ^b , NR ^a C(=O)R ^b , NR ^a C(=O)OR ^b , NR ^a C(=O)NR ^b R ^c , NR ^a C(=NR ^b )NR ^c R ^d , NR ^a SO ₂ R ^b , -SR ^a , SO ₂ NR ³ R ⁶ , -OR ^a _, OC(=O)NR ^a R ^b , OC(=O)R ^a and acyl, wherein R ^a , R ^b , R ^c and R ^d are each independently selected from the group consisting of H, C _r C ₁₂ alkyl, CrCi ₂ haloalkyl, C ₂ -Ci ₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₂ -C ₁₀ heteroalkyl, C ₃ -Ci ₂ cycloalkyl, C ₃ -Ci ₂ cycloalkenyl, C ₂ -Ci ₂ heterocycloalkyl, C ₂ -Ci ₂ heterocycloalkenyl, C ₆ -Ci ₈ aryl, Ci-Ci ₈ heteroaryl, and acyl, or any two or more of R ^a , R ^b , R ^c and R ^d , when taken together with the atoms to which they are attached form a heterocyclic ring system with 3 to 12 ring atoms.

In some embodiments each optional substituent is independently selected from the group consisting of: halogen, =O, =S, -CN ₁ -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, -COOH, -SH, and acyl.

Examples of particularly suitable optional substituents include F, Cl, Br, I, CH ₃ , CH ₂ CH ₃ , OH, OCH ₃ , CF ₃ , OCF ₃ , NO ₂ , NH ₂ , and CN.

As used herein the term "amino acid" refers to a molecule which contains both an amine and a carboxyl function. The amino acid may be a natural or a non-natural amino acid (that is, an amino acid not found in nature).

"Alkenyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain. The group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl. The group may be a terminal group or a bridging group.

"Alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C1-C ₁ 2 alkyl, more preferably a C ₁ -C ₁₀ alkyl, most preferably CrC ₆ unless otherwise noted. Examples of suitable straight and branched CrC ₆ alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t- butyl, hexyl, and the like. The group may be a terminal group or a bridging group.

"Alkynyl" as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-12 carbon atoms, more preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain. Exemplary structures include, but are not limited to, ethynyl and propynyl. The group may be a terminal group or a bridging group.

"Aryl" as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring. Examples of aryl groups include phenyl, naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C _5- C ₇ cycloalkyl or C _5- C ₇ cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl. The group may be a terminal group or a bridging group. Typically an aryl group is a C6-C18 aryl group.

"Cycloalkyl" refers to a saturated monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane. A cycloalkyl group typically is a C ₃ -C ₉ cycloalkyl group. The group may be a terminal group or a bridging group.

"Halogen" represents chlorine, fluorine, bromine or iodine. "Heteroalkyl" refers to a straight- or branched-chain alkyl group preferably having from 2 to 12 carbons, more preferably 2 to 6 carbons in the chain, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced by a heteroatomic group selected from S, O, P and NR' where R' is selected from the group consisting of H, optionally substituted d-C^alkyl, optionally substituted C- ₃ -Ci ₂ cycloalkyl, optionally substituted C ₆ -C ₁₈ aryl, and optionally substituted Ci-Ci ₈ heteroaryl. Exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, amides, alkyl sulfides, and the like. Examples of heteroalkyl also include hydroxyC _r C ₆ alkyl, Ci-C ₆ alkyloxyC-ι-C ₆ alkyl, aminoCi-C ₆ alkyl, Ci-C _θ alkylaminoCrCealkyl, and di(Ci-C ₆ alkyl)aminoCrC ₆ alkyl. The group may be a terminal group or a bridging group.

"Heteroaryl" either alone or part of a group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, 1 H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-, 3- or 4-pyridyl, 2-, 3-, A-, 5-, or 8-quinolyl, 1-, 3-, 4-, or 5-isoquinolinyl 1-, 2-, or 3- indolyl, and 2-, or 3-thienyl. A heteroaryl group is typically a CrCi ₈ heteroaryl group. The group may be a terminal group or a bridging group.

In some embodiments, the invention provides a compound including: a core having a first face and a second face; a binding portion attached to the first face of the core, wherein the binding portion is capable of binding to an anionic group present in a cell membrane of a microorganism; and a hydrophobic portion attached to the second face of the core, wherein the hydrophobic portion is capable of interacting with the cell membrane of the microorganism.

The compounds of the invention are directed to the LPS of the outer membrane of gram-negative bacteria. This represents an ideal target as: (i) the cellular membrane of vertebrates does not contain a large number of charged moieties and thus the compound is less likely to induce lysis of mammalian cells; and (ii) the LPS component is a highly conserved feature of gram-negative bacteria, and therefore agents targeting lipid A/LPS have a wide spectrum of activity.

The LPS outer membranes of gram-negative bacteria vary slightly between species, yet all contain a central core of lipid A. Lipid A contains two anionic phosphate groups on each side and in the intact LPS layer the phosphate groups of individual lipid A units are linked by Ca ²⁺ and/or Mg ²⁺ ions.

Without wishing to be bound by theory, it is thought that the mode of action of the new compounds involves disruption of this bacterial outer membrane using molecular recognition, in particular anion recognition. The one or more binding portions (or "arms") recognise the anionic groups, and bind electrostatically to them, while the hydrophobic portion (the "tail") penetrates and disrupts the membrane. The binding portions bind electrostatically to the negatively charged phosphate groups of lipid A. Following the initial electrostatic binding, the hydrophobic portion penetrates the LPS layer and disruption of the outer membrane ensues. The combined effect leads to lysis and destruction of the microorganism.

Compared to colistin, the new compounds are of relatively low molecular weight (MW 400-700) and non-peptidic. Thus they are more drug-like, and have potential for oral bioavailability (poor oral availability is still a major limitation of peptide drugs).

The activity is enhanced where the compounds have an amphiphilic structure i.e. the anion binding groups on one side are complemented by hydrophobic groups on the other. the compounds may have a facially amphiphilic structure - that is, the one or more binding portions on one face of the core and the hydrophobic portion on the second face - the well spaced positively charged groups on one face able to bind with the negatively charged anionic groups of lipid A of LPS in the bacterial outer membrane. The strong binding results in disruption of the LPS membrane and lysis leading to rapid bacterial death.

Thus conformational organisation enhances activity.

The desired conformational organisation, to ensure the binding portions and the hydrophobic portion are structurally positioned to interact with the membrane, results from the features of the core structure. The anion targeting groups are optimally positioned to facilitate the hydrophobic interactions between the compounds and LPS, for enhanced antimicrobial activity.

Preferably, the core structure has a degree of rigidity to maintain the binding and hydrophobic portions in their desired positions. Cyclic moieties give good rigidity, as do bicyclic moieties.

In one embodiment, the core includes a bicyclic moiety, for example a bicyclic moiety of the structure of Formula (I)

wherein „,

X is selected from the group consisting of O, N and (CH ₂ ) _m , where m is 1 or 2.

In some embodiments, the compound has a structure of Formula (IA) (IA) wherein each R _A is independently an optional substituent or a binding portion, and wherein at least one RA is a binding portion; and

wherein each R _B is independently an optional substituent or a hydrophobic portion, and wherein at least one R ₈ is a hydrophobic portion, or two R ₈ taken together form a hydrophobic portion.

Suitable molecules for the core include norbornane and norbornene.

In some embodiments, the compound is of Formula (II):

or a salt or ion thereof, wherein

R ¹ is a moiety forming part of a hydrophobic portion; R ² is a first binding portion; and R ³ is a second binding portion.

In some embodiments, the salt is selected from the group consisting of chloride, sulfate, bromide, mesylate, maleate, citrate, phosphate and trifluoroacetate salts.

In some embodiments, each binding portion is adapted to bind to an anionic group, such as a phosphate group present in a cell membrane of a microorganism, in particular, gram-negative bacteria. The compounds of the invention include at least one binding portion, but it has been found that those including two binding portions show particularly good activity against target microorganisms. Where there is more than one binding portion, as in Formula II, the core acts to maintain a distance between plural binding portions such that each of the binding portions may be able to bind to separate anionic groups on the microorganism membrane. For example, it has been observed that an efficacious distance is about 12 to 14A between the binding portions to achieve this. The angle in relation to the C1-C2 bond of the bicyclic coremay range between -90° and +90°. In some embodiments, the binding portions are joined to the core in a structural arrangement of any combination of exo and endo.

For example, a compound of the invention may employ the structurally rigid endo/exo norbomane to orient two binding portions or 'arms' at an angle of approximately 110° from each other and thus optimise their ability to act independently.

An exemplary structure for a binding portion is shown in Formula (III):

S ^{V / nV} ' P (Hi) or a salt or ion form thereof, wherein D at each occurrence is independently selected from the group consisting of a bond or a functional group; L at each occurrence is independently an optionally substituted spacer group;

A at each occurrence is independently a binding moiety capable of binding with an anionic group present in a cell membrane of a microorganism;

Z at each occurrence is independently a substituent group selected from the group consisting of hydrogen, optionally substituted Ci to C ₁₄ alkyl, optionally substituted 5 to 8-membered aryl and optionally substituted 3 to 8-membered cycloalkyl, wherein said alkyl, aryl and cycloalkyl may be optionally interrupted by one or more heteroatoms selected from the group consisting of O, N and S; or other heterocycle; n is an integer selected from the group consisting of 1 , 2, 3, 4, 5 and 6; and p is an integer selected from the group consisting of 1 , 2, 3, 4, 5 and 6.

In some embodiments, n = 1. In some embodiments, p = 1. In some embodiments, D is a functional group. Amide functional groups, for example -CONH-, may be suitable. In some embodiments, D is selected from the group consisting Of -CH ₂ NH-, -CH ₂ O-, NH ₂ and O.

In some embodiments, L includes a hydrogen bonding moiety adapted to participate in hydrogen bonding interactions with the cell membrane of the microorganism. In some embodiments, L is selected from the group consisting of a C ₁ to Cio alkyl or a C ₂ to do alkenyl chain and an amino acid residue, where the amino acid may include glycine, arginine, asparagine, glutamine and lysine or a non-natural amino acid. In some embodiments, L is a C ₂ to Ce alkyl or C ₂ to Ce alkenyl chain. In some embodiments, L may include amide, urea, thiourea, guanidine, ammonium, imidazolinium and combinations of these moieties. In some embodiments, L is - CH ₂ CH ₂ - _.

As n is an integer and may vary, and as D and L may vary, the total length of the group (D-L) _n may vary. For ease of reference, the total length is herein designated q. In some embodiments, A may be amide, urea or guanidine. In some embodiments, A may include a primary, secondary, tertiary or quaternary amine or ammonium moiety; or guanidine, guanidinium, amidine, amidinium, imidazolium, thiouronium, amide, urea, thiourea or pyrrole.

In some embodiments, A may include additional functionality directly bonded to the groups listed above in order to further enhance their binding properties. In some embodiments, a carbonyl or an amino group sits adjacent to a urea. For example an additional amino group bonded to a urea forms a hydrazinecarboxamide functional group. A carbonyl adjacent to a guanidine forms a guanidinycarbonyl.

In some embodiments, each group -A-Z is independently selected from the group consisting of:

or a salt or ion thereof.

In some embodiments, Z is selected from the group consisting of H, Ci to C ₁₄ alkyl, 5 to 8-membered aryl and 3 to 8-membered cycloalkyl, heterocycloalkyl or heteroaryl. In some embodiments, Z is an optionally substituted aryl, an optionally substituted alkyl, an optionally substituted cycloalkyl or an optionally substituted heterocycle. In some embodiments, Z may contain an optional substituent selected from the group consisting of alkyl esters (for example Ci to C ₄ alkyl esters), halogens, nitrite (CN) and nitro (NO ₂ ) groups. In some embodiments, Z = H.

In some embodiments, the optional substituent may be an electron withdrawing group. Electron withdrawing groups may activate the binding moiety A to enhance its interaction with amine groups present in the cell membrane. In some embodiments, the substituent may be F.

In some embodiments, Z is a 5 to 8-membered aryl in which one or more of the carbons has been interrupted by a heteroatom which may selected from the group consisting of nitrogen, oxygen and sulphur or combinations thereof such that Z is a heteroaryl group. In some embodiments, Z is an optionally substituted cycloalkyl in which one or more of the carbons has been interrupted by a heteroatom which may be selected from the group consisting of nitrogen, oxygen and sulphur or combinations thereof such that Z is a heterocycloalkyl group.

In addition to the binding portions which include the anion recognition groups targeting the phosphate moieties of LPS, the compounds include a hydrophobic portion (for example, an alkyl or aryl chain) for efficient penetration of the LPS layer. In some embodiments, the core and hydrophobic portion are of Formula IV:

where n2 is an integer. In some embodiments, n2 may be selected from the group consisting of 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 and20.

In some embodiments, the core and hydrophobic portion are of Formula IVA:

where R _E is chosen from the group consisting of optionally substituted alkyl or optionally substituted aryl.

The compounds of the invention can be customised for strong binding:

(i) The actual binding group that will interact with the phosphate anionic groups of LPS can be varied at A and some or all of ionic, electrostatic and/or hydrogen bonding forces can be used to maximise attractive forces.

(ii) the distance between the binding groups on the binding portions can be customised by altering the total spacer length q, (iii) the size of the hydrophobic alkyl chain can be varied by changing n2.

Variations to the features n2, q and Z can all be readily incorporated during synthesis.

i. Hydrophobic portion n2: A hydrophobic "tail" of six to eight carbons gives good activity. A range of alkyl lengths have been investigated, and can conveniently be attached to the core using an acetal linkage. In one embodiment, the norbornene alkene (which can be prepared by a Diels-Alder reaction) is typically first oxidised to a c/s-diol (such as by the use of OsO4/NMO), which can form the desired acetal with a range of suitable aldehydes to furnish the required hydrophobic tail. This approach allows for a wide variety of commercially available or 'tailor made' aldehydes to be attached with ease. It has been found that compounds possessing a phenyl group in the hydrophobic portion show good antibacterial activity, as do those with an octyl group.

In some embodiments, the hydrophobic portion or the moiety forming part of the hydrophobic portion includes 4 to 20 carbon atoms. In some embodiments, the hydrophobic portion or the moiety forming part of the hydrophobic portion is selected from the group consisting of optionally substituted C ₄ to C20 alkyl, optionally substituted C ₄ to C ₂₀ alkenyl, optionally substituted C ₄ to C ₂ o alkynyl, optionally substituted C ₄ to C ₂₀ cycloalkyl and optionally substituted C ₆ to C ₂ o aryl.

In some embodiments, the hydrophobic portion or the moiety forming part of the hydrophobic portion is selected from the group consisting of optionally substituted Cβ to C ₈ alkyl and optionally substituted C ₆ to C10 aryl.

In some embodiments the hydrophobic portion includes a linking moiety linking the moiety forming part of the hydrophobic portion to the core. In some embodiments the hydrophobic portion includes an acetal moiety linking the moiety forming part of the hydrophobic portion to the core

In order that the optimal distance between the two anion recognition groups can be achieved, the spacer lengths can be varied. The distance between the two phosphate groups in lipid A has been experimentally determined as between 12.5 and 14A. Therefore, in some embodiments the binding groups in the new compounds also span this distance. For example, a two carbon ethylene spacer is sufficient for the terminal nitrogen atoms (in the case of the primary ammonium group) to span up to 13.2A and thus this spacer is suitable. These alkyl chains are readily incorporated by heating an alkyl diamine with the commercially available (or readily synthesised) endolexo diester. For good antibacterial activity, total spacer lengths of q=2 to about q=6 are preferred.

In addition to alkyl chains, short amino acid chains may also be used in the binding portions. The chains still span the required distance however the amide bond may afford additional interactions (such as hydrogen bonding interactions) with the LPS phosphate groups and/or disaccharide unit core of lipid A and may aid binding to the hydrocarbon portion of LPS.

ii. Binding group A: Colistin and others of the polymyxin family employ a primary ammonium group to interact with the phosphate groups of lipid A. In the compounds of the invention, the binding group A may be a range of groups including amide, urea, thiourea, imidazolinium, guanidine and guinidinylcarbonyl, which also interact with phosphate anions, or a primary, secondary, tertiary or quaternary ammonium moiety. In some embodiments, compounds of the invention include guanidines, which have been found to give good binding. Many of these groups can employ dipole-dipole interactions, H-bonding, ionic bonding, van der Waals forces and/or electrostatic forces to bind the target anion. As such, a range of phosphate binding functional groups can be synthesised by modifying Z and/or A. The synthesis of substituted guanidines can be easily achieved by treating primary amines with aminonitriles (RNHCN).

Multiple binding groups can be incorporated (for example an arginine spacer between the core and the binding group will add an additional guanidine binding unit) in order to achieve even more potent binding of the lipid A phosphate groups.

Suitable compounds and salts include

(1.2HCI)

(6)

(10.2HCI) In respect of compound 6, the optionally substituted fluorine on Z acts as an electron- withdrawing group, which further activates the thiourea groups for stronger hydrogen bonding.

In some embodiments, the compounds of the invention may have multiple binding portions. In some embodiments, these may be different, having different A-Z groups.

The compounds may be racemates, or in the form of single enantiomers.

The compounds of the invention may be included in therapeutic compositions, or formulated for topical or oral administration. They are particularly effective against bacteria, in particular gram-negative bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae.

In some embodiments, the invention provides a method for the treatment or prophylaxis of infection of a mammal by a microorganism, the method comprising administering to a subject in need thereof an effective amount of a compound of the invention. The mammal may be a human or a non-human animal.

In some embodiments, the invention provides a method for treating or preventing contamination of a substrate by a microorganism, the method comprising applying an effective amount of a compound of the invention to the substrate or its surface.

In some embodiments, there is provided use of the compounds of the invention in the treatment or prophylaxis of infection of a mammal by a microorganism. In some embodiments, there is provided use of the composition of the invention in the treatment or prophylaxis of infection of a mammal by a microorganism.

In some embodiments, there is provided use of the compounds of the invention in treating or preventing contamination of a substrate by a microorganism. In some embodiments, there is provided use of the compositions of the invention in treating or preventing contamination of a substrate by a microorganism. in some embodiments, there is provided a compound according as described for use in the treatment or prophylaxis of infection in a mammal. In some embodiments, there is provided a composition according as described for use in the treatment or prophylaxis of infection of a mammal by a microorganism.

Administration of the compounds of the invention to humans or non-human animals may be by any of the accepted modes for enteral administration such as oral or rectal, or by parenteral administration such as subcutaneous, intramuscular, intravenous and intradermal routes. Injection can be bolus or via constant or intermittent infusion. The active compound is typically included in a pharmaceutically acceptable carrier or diluent and in an amount sufficient to deliver to the patient a therapeutically effective dose.

In using the compounds they can be administered in any form or mode which makes the compound bioavailable. One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the compound selected, the condition to be treated, the stage of the condition to be treated and other relevant circumstances. We refer the reader to Remingtons Pharmaceutical Sciences, 19 ^th edition, Mack Publishing Co. (1995) for further information.

The compounds can be administered alone or in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier, diluent or excipient. The compounds, while effective themselves, are typically formulated and administered in the form of their pharmaceutically acceptable salts as these forms are typically more stable, more easily crystallised and have increased solubility.

The compounds are, however, typically used in the form of pharmaceutical compositions which are formulated depending on the desired mode of administration. As such in some embodiments there is provided a pharmaceutical composition including a compound of the invention and a pharmaceutically acceptable carrier, diluent or excipient. The compositions are prepared in manners well known in the art. The present teachings in other embodiments provide a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions. In such a pack or kit can be found a container having a unit dosage of the agent(s). The kits can include a composition comprising an effective agent either as concentrates (including lyophilized compositions), which can be diluted further prior to use or they can be provided at the concentration of use, where the vials may include one or more dosages. Conveniently, in the kits, single dosages can be provided in sterile vials so that the physician can employ the vials directly, where the vials will have the desired amount and concentration of agent(s). Associated with such containers) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The compounds may be used or administered in combination with one or more additional drug(s) for the treatment of the disorder/diseases mentioned. The components can be administered in the same formulation or in separate formulations. If administered in separate formulations the compounds may be administered sequentially or simultaneously with the other drug(s).

In addition to being able to be administered in combination with one or more additional drugs, the compounds may be used in a combination therapy. When this is done the compounds are typically administered in combination with each other. Thus one or more of the compounds may be administered either simultaneously (as a combined preparation) or sequentially in order to achieve a desired effect. This is especially desirable where the therapeutic profile of each compound is different such that the combined effect of the two drugs provides an improved therapeutic result.

Pharmaceutical compositions of the present teaching for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin.

If desired, and for more effective distribution, the compounds can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

The active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. Dosage forms for topical administration of a compound of the invention include powders, patches, sprays, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required.

The amount of compound administered will preferably treat and reduce or alleviate the condition. A therapeutically effective amount can be readily determined by an attending diagnostician by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the therapeutically effective amount a number of factors are to be considered including but not limited to, the species of animal, its size, age and general health, the specific condition involved, the severity of the condition, the response of the patient to treatment, the particular compound administered, the mode of administration, the bioavailability of the preparation administered, the dose regime selected, the use of other medications and other relevant circumstances.

A preferred dosage will be a range from about 0.01 to 300 mg per kilogram of body weight per day. A more preferred dosage will be in the range from 0.1 to 100 mg per kilogram of body weight per day, more preferably from 0.2 to 80 mg per kilogram of body weight per day, even more preferably 0.2 to 50 mg per kilogram of body weight per day. A suitable dose can be administered in multiple sub-doses per day.

Examples

In some embodiments, the compounds of the invention may be synthesised in accordance with the Scheme below. Example 1. Synthesis of Compounds 1. 4 and 8

11 1.1 Synthesis of dimethyl ester 11

Trifluoroacetic acid (5 drops) and MgSO ₄ .anhydrous (2 g) was added to a solution of dimethyl δ.β-dihydroxy-bicyclo^^.ilheptane^.S-dicarboxylate (420 mg, 1.74 mmol) and octanal (244 mg, 1.91 mmol) in CHCb (5 ml_) and the solution stirred at room temperature for 16 hrs. The suspension filtered, the filtrate collected and reduced to dryness in vacuo to give a pale yellow oil. Analysis by ¹ H NMR spectroscopy indicated the presence of the desired compound and excess octanal. Purification by silica gel chromatography (CH ₂ CI ₂ ) gave diester 11 as a colourless oil (470 mg, 76%). u _max (thin film, cm ^'1 ) 2952.6, 2926.1 , 2856.9, 1734.6, 1436.6, 1299.6, 1242.8, 1195.2, 1028.5; δ _H (270 MHz, CDCI ₃ ) 4.66 (1 H, t, J 4.9 Hz), 4.01 (1 H, d, J 5.5 Hz), 3.89 (1 H, d, J 5.5 Hz), 3.69 (6H, br s), 3.21 (1 H, t, J 5.1 Hz), 2.70 - 2.64 (3H, m), 1.77 (1 H, d, J 10.6 Hz), 1.60 (2H, m), 1.40 - 1.20 (11 H, m), 0.85 (3H, m); δ _c (CDCI ₃ , 67.5 MHz) 174.0, 172.8, 104.3, 81.3, 78.9, 52.4, 52.2, 45.4, 45.1 , 43.7, 43.3, 32.8, 31.8, 31.7, 29.6, 29.2, 24.3, 22.7, 14.1 ; HRMS (+ ve) calcd for Ci ₉ H ₃₁ O ₆ 355.2115 [M+H]\ found 355.2118.

THF (1 ml_) was added to a solution of diester 11 (248 mg, 7.0 mmol) in sodium hydroxide (2 M, 15 ml_) and the solution stirred at room temperature for 4 hrs. Water (30 ml_) was added and the aqueous phase extracted with ethyl acetate (3 x 25 ml_), the aqueous phase was then acidified (HCI, 2 M) to pH 1 followed by extraction with ethyl acetate (3 x 30 ml_). The combined organic phases (from the acidic aqueous wash) were dried (MgSO ₄ ) and the solvent removed to give diacid 12 as white plates (193 mg, 84%). Analysis by ¹ H NMR spectroscopy showed the desired compound in > 95% purity, m.p. 153 - 154 ⁰ C; u _max (KBr/cm ^"1 ) 3158.1 , 3153.9, 2945.1 , 2860.3, 1733.1 , 1710.4, 1421.9, 1295.3, 1200.4, 1 1 18.5, 1042.8, 710.3; δ _H (270 MHz, d _e -DMSO) 12.42 (2H, br s, COOH), 4.63 (1 H, t, J 4.7 Hz),

3.97 (1 H, d, J 5.6 Hz), 3.90 (1 H, d, J 5.6 Hz), 2.99 (1 H, dd, J 5.2, 5.2 Hz), 2.35 - 2.50 (3H ₁ m), 1.54 (3H, m), 1.24 (11 H, m), 0.85 (3H, m); δ _c (67.5 MHz, CDCI ₃ ) 175.1 , 173.8, 103.8, 81.2, 78.7, 45.6, 45.0, 43.8, 43.1 , 32.8, 31.7, 29.5, 29.5, 24.2, 22.6, 21.6, 14.5; HRMS (+ve) calcd Ci ₇ H ₂₇ O ₆ Na 349.1621 [M+Na]\ found 327.1620.

13

EDCI (260 mg, 1.35 mmol), HOBt (8 mg, 61 μmol) were added to a solution of diacid 12 (200 mg, 0.613 mmol) in CHCI ₃ (8 ml_) and the solution stirred for 5 minutes at room temperature. A solution of benzyl (2-aminoethyl)carbamate (262 mg, 1 .35 mmol) in CHCI ₃ (2 ml_) was added and the solution stirred at room temperature overnight. Water (25 ml_) was added and the aqueous phase extracted with EtOAc (3 * 30 ml_), the combined organic phases were washed with brine (1 * 30 ml_), dried (MgSO ₄ ) and the solvent removed to give a white resin. Analysis of the crude resin by ¹ H NMR spectroscopy indicated the presence of the desired compound, . purification by silica gel chromatography (EtOAc/Petrol 1 :1 -> EtOAc/MeOH 9.5:0.5) gave diamide 13 (261 mg, 63%) as a white gum. u _max (thin film/cm ^'1 ) 3341.8, 3067.4, 2926.3, 2855.1 , 1703.8, 1644.3, 1542.5, 1455.3, 1263.5, 1 145.6, 1034.5; δ _H (270 MHz, CDCI ₃ ) 7.56 (2H, br s, NH), 7.40 (1 H, br s, NH), 7.20 - 7.35 (10H, m), 6.00 (2H, br s, NH), 5.02 (4H, br s), 4.59 (1 H, t, J 4.7 Hz), 4.03 (1 H, d, J 4.8 Hz), 3.97 (1 H, d, J 4.8 Hz), 3.10 - 3.42 (8H, m), 2.45 - 2.60 (3H, m), 1.73 (1 H, d, J 10.0 Hz), 1.50 - 1.60 (2H, m, OCH ₂ ) 1.49 (1 H, d, J 10 Hz), 1.23 (10H, m), 0.85 (3H, m); δ _c (67.5 MHz, CDCI ₃ ) 174.7, 172.6, 157.2 (x 2), 136.5, 128.6 (x 5), 128.2 (x 2), 128.1 (x 5), 104.0, 81 .6. 78.8, 66.9, 66.8, 46.6, 46.3, 44.0 (x 2), 40.7, 39.9, 39.7, 33.0, 32.1 , 31.9, 29.6, 29.3, 24.3, 22.7, 14.2; HRMS (+ve) Calcd for C ₃₇ H _5I N ₄ O ₈ 679.3701 [M+H]\ found 679.3724.

Pd/C (5% Pd loading on Carbon) (67 mg, 20% w/w) was added to a solution of protected amide 13 (366 mg, 0.89 mmol) in methanol (20 ml_). The system was flushed with hydrogen gas (balloon) and the solution stirred for 16 hours at room temperature. The system was then flushed with nitrogen gas three times, celite added to the reaction mixture and the resulting slurry filtered through a pad of celite. The filtrate was collected and the solvent removed in vacuo to give a white gum which was confirmed as diamine 1 by ¹ H NMR spectroscopy (266 mg, 96%). U _max (thin film/ cm ^"1 ) 3400.5, 2955.3, 2929.0, 2859.0, 2522.4, 2236.2, 2077.8, 1646.4 (broad), 1455.3, 1212.9, 1116.4, 103635, 971.9; δ _H (270 MHz, d ₄ - methanol) 4.65 (1 H, t, J 4.6 Hz), 3.20 - 3.60 (8H, m), 3.21 (1 H, t, J 4.7 Hz), 2.40 - 2.65 (3H, m), 1.73 (1 H, d, J 10 Hz), 1.58 (3H m), 1.46 (1 H, d, J 10 Hz), 1.28 (10H ₁ m), 0.88 (3H, m) (amine and amide hydrogens not observed due to deuterium exchange); δ _c (67.5 MHz, d ₄ -methanol) 175.5, 173.6, 103.7, 81.5, 78.7, 46.0, 45.9, 43.8, 43.7, 39.7 (x 2), 37.9 (x 2), 32.5, 31.6, 31.4, 29.4, 29.3, 23.9, 22.3, 11.5; HRMS (+ve) calculated C _2I H ₃₈ N ₄ O ₄ 411.2971 [M+H]\ found 411.2944. *Note: Diamine 1 was characterised as the free amine and converted to the corresponding hydrochloride salt (1.2HCI) via treatment with anhydrous HCI in methanol prior to biological evaluation.

4-Fluorophenyl isothiocyanate (69 mg, 0.44 mmol) was added to a solution of amine 1 (186 mg, 0.44 mmol) in CHCI ₃ (4 ml_) and the solution stirred at room temperature for 30 minutes then warmed to 50 ⁰ C for 16 hours. The solvent was removed in vacuo to give an orange solid, recrystalisation from CH ₂ Cl2/Petrol gave thiourea 4 as a white powder (173 mg, 55%). m.p. 243 ⁰ C (dec); u _max (thin film/cm- ¹ ) 3430.1 , 2939.5, 2929.0, 2858.2, 1644.8 (broad), 1535.9, 1509.2, 1216.6; δ _H (270 MHz, d ₆ -DMSO/d ₄ -MeOH) 7.20 - 7.27 (4H, m), 6.97 (4H, m), 3.00 (2H, m), 3.56 (1 H, m). 3.00 - 3.34 (8H, m), 2.13 - 2.51 (3H, m), 2.13 (1 H, d, J 1.6 Hz) ₁ 1.57 (1 H, d, J 9.6 Hz) ₁ 1.47 (2H. m), 1.18 (11 H, m), 0.79 (3H, t, J 7.0 Hz); δ _c (67.5 MHz ₁ d ₄ -MeOH) 182.3, 181.6, 174.8, 173.0, 160.8 (d J ₁ ^F 242 Hz) ₁ 160.6 (d, J ₁ ^F 241 Hz), 136.8, 134.1 , 128.7 (d, J ₂ ^F 8.3 Hz) ₁ 127.0 (br s), 115.6 (d, J ₃ ^F 22.7 Hz), 114.5 (d, J ₃ ^F 22.7 Hz) ₁ 103.7, 81.5, 78.7, 52.2, 46.2, 46.1 , 45.9, 43.9, 43.7, 38.9, 37.3 (x 3), 32.6, 31.6, 31.4, 29.3 29.0, 23.9, 22.3, 13.1 ; HRMS (-ve) calculated for C ₃₅ H ₄₇ F ₂ N ₆ O ₄ S ₂ 715.2912 [M-HJ ^" , found 715.2900.

Diamine 1 (226 mg, 0.55 mmol) in CHCI ₃ (3 mL) was added to a solution phenylcyanamide (143 mg, 1.2 mmol) in CHCI ₃ (5 mL) and the sealed vessel heated at 90 ⁰ C for 48 hours. The solvent was then removed in vacuo to give a pale yellow solid. Recrystallisation of the solid (CH ₂ CI ₂ /Petrol) gave a white precipitate which was collected by vacuum filtration and air dried to give guanidine 8 as white blocks (264 mg, 73%). Analysis of the solid by ¹ H NMR spectroscopy indicated the presence of the desired compound, m.p. 158 - 160 ⁰ C; v _max (thin film) 3311.4, 3088.4, 2932.4, 1711.6, 1641.1 , 1593.7, 1453.0, 1263.8, 1073.5; δ _H (270 MHz, d ₄ -MeOH) 6.79 - 7.07 (10H, m), 4.62 (1 H, m), 4.0 (2H, m), 3.00 - 3.50 (9H, m), 2.42 -2.71 (4H, m), 1.71 (1 H, d, J 10.9 Hz), 1.57 (2H, m), 1.26 (11 H ₁ m), 0.88 (3H, t, J 5.9 Hz); δ _c (100 MHz, de-DMSO) 172.1 , 170.2, 154.8, 151.5, 129.1 (2 x C) , 128.1 (2 x C), 128.0 (2 x C), 127.5 (2 x C), 127.2, 121.2, 119.2, 117.2, 112.9, 102.1 , 80.3, 77.4, 34.8, 31.6, 30.4, 28.1 , 27.8, 22.9, 21.3, 13.2; HRMS predicted for C35H51N5O/ [M +H] ⁺ 647.40274, found 647.4020. Guanidine 8 was converted to the corresponding hydrochloride salt (8.2HCI) by treatment with anhydrous HCI in methanol prior to biological evaluation.

Among the compounds synthesised are those shown in Table 1.

Table 1

Example 2. Activity testing

Preliminary antibacterial activity screen using a disc diffusion method (at 30 μg/disc) demonstrated that sample compounds are active against Acinetobacter baumannii ATCC19606, Pseudomonas aeruginosa ATCC27853 and/or Staphylococcus aureus ATCC43300. The results are shown in Table 2. The salt of Compound 8 showed excellent activity against all strains. The salts of the compounds tested are shown below.

Based on the inhibition zones and that of colistin, the minimum inhibitory concentration (MIC) of compound 8 is comparable to that of colistin which is approximately 1 μg/mL

Compound or Acinetobacter Pseudomonas Staphylococcus salt of baumannii aeruginosa aureus compound ATCC19606 ATCC27853 ATCC43300

1.2HCI Weak Moderate Moderate

2.2HCI Weak Moderate Weak

7.2HCI Moderate Weak Moderate

8.2HCI Strong Strong Strong

9.4HCI Weak Weak Strong

10.2HCI Moderate Weak Weak

Table 2 - Preliminary antibacterial activity screen, disc diffusion method

Finally, it will be appreciated that various modifications and variations of the compounds, methods and compositions of the invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the art are intended to be within the scope of the present invention.