WO2012051663A1 - Antimicrobial compounds - Google Patents

Abstract

The present invention relates to antimicrobial compounds and their uses, and in particular to peptide antibiotics which may be used in the treatment of bacterial infections such as Gram-negative bacterial infections, particularly those caused by multidrug-resistant (MDR) pathogens.

Description

Antimicrobial compounds

Field of the invention The present invention relates to antimicrobial compounds and their uses, and in particular to peptide antibiotics which may be used in the treatment of bacterial infections such as Gram-negative bacterial infections, particularly those caused by multidrug-resistant (MDR) pathogens.

Background of the invention

The world is facing an enormous and growing threat from the emergence of bacteria that are resistant to almost all available antibiotics. Whilst a small number of new antibiotics targeting MDR Gram-positive bacteria have been approved in the past two decades, there has been a marked decline in the discovery of novel antibiotics for the treatment of Gram- negative bacteria.

Representative genera of Gram-negative bacteria are: Acinetobacter; Actinobacillus; Bartonella; Bordetella; Brucella; Burkholderia; Campylobacter; Cyanobacteria; Enterobacter; Erwinia; Escherichia; Francisella; Helicobacter; Hemophilus; Klebsiella; Legionella; Moraxella; Morganella; Neisseria; Pasteurella; Proteus; Providencia; Pseudomonas; Salmonella; Serratia; Shigella; Stenotrophomonas; Treponema; Vibrio; and Yersinia.

Representative genera of Gram-positive bacteria are: Actinobacteria; Bacillus; Clostridium; Corynebacterium; Enterococcus; Listeria; Nocardia; Staphylococcus; and Streptococcus.

While the recently approved tigecycline is active against a range of clinically important Gram-negative pathogens, including Acinetobacter baumannii, it is reported to not be effective against Pseudomonas aeruginosa. Numerous hospitals worldwide have experienced outbreaks of infections caused by P. aeruginosa, A. baumannii or Klebsiella pneumoniae that are resistant to all commercially available antibiotics, including the last- line therapies colistin (polymyxin E) and polymyxin B. The Infectious Diseases Society of America (IDSA) has placed P. aeruginosa, A. baumannii and K. pneumoniae on a 'hit list* of the six top-priority dangerous MDR microorganisms, the so-called 'superbugs', in its recent ' Bad Bugs Need Drugs' campaign.

Polymyxins belong to a class of peptides which was discovered more than 50 years ago. The structures of representative polymyxins are shown below:

Polymyxin B1 & Colistin A =

6-methyloctanoic acid Polymyxin B2 & Colistin B R*i = 6-methylheptanoic acid

Polymyxins are produced by nonribosomal biosynthetic enzymes from the secondary metabolic pathways of Bacillus polymyxa. There are two polymyxins clinically available, colistin (polymyxin E) and polymyxin B, and cross resistance exists between these two polymyxins. The efficacy of polymyxins in treating certain Gram-negative bacterial infections has been demonstrated by recent clinical studies. Parenteral administration of colistin and polymyxin B can be potentially nephrotoxic and/or neurotoxic. Polymyxins are now being used as last-line antibiotics in patients where all other available antibiotics are inactive. Although the incidence of resistance to polymyxins is currently relatively low, it has been demonstrated that resistance can emerge in vitro in P. aeruginosa, A. baumannii and K. pneumoniae, and polymyxin resistance in hospitalised patients has been increasingly reported. There is only one amino acid difference between colistin and polymyxin B and, not surprisingly, cross resistance has been found to exist. In essence, resistance to polymyxins implies a total lack of antibiotics for treatment of life-threatening infections caused by these MDR Gram-negative 'superbugs'. Accordingly there exists an urgent need to discover new antibiotics which are effective against Gram-negative bacteria, particularly those resistant to all currently available antibiotics, including the last-line therapy polymyxins.

Summary of the invention It has now been found that certain polymyxin analogs are effective against Gram-negative bacteria, including both polymyxin-susceptible and -resistant MDR Gram-negative bacteria. Some of these polymyxin analogs may also have activity against Gram-positive bacteria, such as methicillin-resistant and vancomycin-resistant Gram-positive bacteria. Accord

wherein:

Ri is a lipophilic group comprising five or more carbon atoms and optionally comprising one or more O, N or S atoms, wherein the or each carbon atom in R\ that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more O, N or S atoms in Ri, where present, are each independently conjugated to the or each distal carbon atom in Ri by a moiety comprising three or more sequential carbon atoms;

Y is selected from -C(0)-; -C(S)-; -C(NH)-; and -S(0)₂-; m, n and p are each independently 0 or 1 ;

R₃, R4 and R₅ where present and R₂, Re, R9, Rio and Rj 1 are each independently selected from the side chain of an amino acid selected from α,γ-diaminobutyric acid, arginine, histidine, lysine, leucine, ornithine,- glutamic acid, aspartic acid, glutamine, asparagine, homoserine, serine, threonine and cysteine;

X is a residue of the side chain of an amino acid selected from α,γ-diaminobutyric acid, arginine, histidine, lysine, ornithine, glutamic acid, aspartic acid, glutamine, asparagine, homoserine, serine, threonine and cysteine;

R₇ is a lipophilic group comprising five or more carbon atoms and optionally comprising one or more O, N or S atoms, wherein the or each carbon atom in R₇ that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more O, N or S atoms in R₇, where present, are each independently conjugated to the or each carbon atom in R₇ that is distal to the peptide backbone by a moiety comprising four or more sequential carbon atoms;

Re is a lipophilic group comprising two or more carbon atoms and optionally comprising one or more O, N or S atoms, wherein the one or more O, N or S atoms in Re, where present, are each independently conjugated to the or each carbon atom in Kg that is distal to the peptide backbone by a moiety comprising four or more sequential carbon atoms; or a pharmaceutically acceptable salt thereof.

In some embodiments the total number of carbon, nitrogen, oxygen and sulfur atoms in Ri is 30 or less.

In some embodiments the total number of carbon, nitrogen, oxygen and sulfur atoms in R₇ is 30 or less.

In some embodiments the total number of carbon, nitrogen, oxygen and sulfur atoms in Rg is 30 or less.

In one aspect the present invention provides a peptide of formula (I):

wherein:

Ri is a lipophilic group comprising from 5 to 22 carbon atoms selected from Cs-2₂alkyl, C₅. 22alkenyl, C^ i l, C₅.i₂cycloalkyl, C6-i6arylCi.₆alkyl, C_6-i6arylC₂-6alkenyl, cyclopropylC2- ₆alkyl, C₄.₁2cycloalkylCi-6alkyl and Cs.^cycloalky^-ealkenyl wherein, where present, any one or more CH₂ groups in Ri is independently optionally replaced with O, C(O), N(Ri2) or S, wherein the or each carbon atom in Ri that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more O, N or S atoms in Rj, where present, are each independently conjugated to the or each distal carbon atom in Ri by a moiety comprising three or more sequential carbon atoms;

Y is selected from -C(O)-; -C(S)-; -C(NH)-; and -S(0)₂-; m, n and p are each independently 0 or 1 ; R3, R4 and R5 where present and R₂, R^, R9, Rio and Ru are each independently selected from the side chain of an amino acid selected from α,γ-diaminobutyric acid, arginine, histidine, lysine, leucine, ornithine, glutamic acid, aspartic acid, glutamine, asparagine, homoserine, serine, threonine and cysteine;

X is a residue of the side chain of an amino acid selected from α,γ-diaminobutyric acid, arginine, histidine, lysine, ornithine, glutamic acid, aspartic acid, glutamine, asparagine, homoserine, serine, threonine and cysteine;

R.7 is a lipophilic group comprising from 5 to 22 carbon atoms and is selected from C5. ₂₂alkyl, C_5-22alkenyl, C6-i6aryl, C5.12cycloa.kyl, C6-i6arylCi^alkyl, C6-i6arylC₂-6alkenyl, cyclopropylC_2-6alkyl, C4-i₂cycloalkylCi.6alkyl and C3_-12cycloalkylC2-6a-kenyl wherein, where present, any one or more CH₂ groups in R₇ is independently optionally replaced with O, C(O), N(Ri₂) or S; wherein the or each carbon atom in R7 that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more O, N or S atoms in R₇, where present, are each independently conjugated to the or each distal carbon atom in R₇ by a moiety comprising four or more sequential carbon atoms;

Rg is a lipophilic group comprising from 2 to 22 carbon atoms and is selected from C_{2- 22}alkyl, C_2-22alkenyl, C6-i6aryl, C3-i2cycloalkyl, C₆-i6arylCi^alkyl, C6-i6arylC₂^alkenyl, C₃. i₂cycloalkylCi.6alkyl and C₃.i2cycloalkylC₂^alkenyl wherein, where present, any one or more CH₂ groups in Rg is independently optionally replaced with O, C(O), N(Ri2) or S; wherein the one or more O, N or S atoms in Rg, where present, are each independently conjugated to the or each carbon atom in Rg that is distal to the peptide backbone by a moiety comprising four or more sequential carbon atoms; wherein, where present, Ri₂ is independently selected from hydrogen, Ci^alkyl, C₂. ₂₂alkenyl, C6-i6aryl, C3.12cycloa.kyl,

C6-i6arylC_2-6alkenyl, C₃. i₂cycloalkylCi.6alkyl and C₃.i₂cycloalkylC2-6alkenyl; wherein any alkyl, alkenyl, aryl and cycloalkyl group within the peptide is optionally substituted with one or more groups selected from halo, C1.22a.kyl, C2-22alkenyl, C6-i6aryl, C_3-12cycloalkyI, hydroxy,

amino, aminoCi_-22alkyl, Ci-2₂alkyloxy, Cj.

22alkylamino, (C].22alk lXCi.₂₂alkyI)amino, Ci_-22alkylcarbonyloxy, C2-

22alkenylcarbonyloxy, C6-i6arylcarbonyloxy, C₃.₁₂cycloalkylcarbonyloxy, Cj.

22alkylcarbonylamino, C₂-22alkenylcarbonylamino, C6.i6arylacylamino and C₃. i₂cycloalkylcarbonylamino; or a pharmaceutically acceptable salt thereof.

It has now been found that the peptides of the present invention are active against Gram- negative bacteria, and surprisingly the peptides are active against not only polymyxin- susceptible but also polymyxin-resistant MDR Gram-negative bacteria. Without wishing to be limited by theory, it is believed that the combination of:

a) the absolute stereochemistry of the amino acid residue comprising the R₇ group shown in Formula (I);

b) the or each carbon atom in R₇ that is distal to the peptide backbone being conjugated to the peptide backbone by five or more sequential atoms, and the one or more O, N or S atoms in R₇, where present, are each independently conjugated to the or each distal carbon atom in R₇ by a moiety comprising four or more sequential carbon atoms; and

c) the or each carbon atom in R₈ that is distal to the peptide backbone being conjugated to the peptide backbone by one or more sequential atoms, and the one or more O, N or S atoms in Rg, where present, are each independently conjugated to the or each distal carbon atom in R₈ by a moiety comprising four or more sequential carbon atoms;

provides the peptides with sufficient localized lipophilicity to render the peptide effective against Gram-negative bacteria, particularly both polymyxin-susceptible and -resistant Gram-negative bacteria. Some of the compounds may also be active against Gram- positive bacteria, which suggests that the compounds may have secondary cellular target(s) because Gram-positive bacteria do not possess the same primary target for polymyxins as possessed by Gram-negative bacteria. The number of sequential atoms that conjugate the peptide backbone to the or each distal carbon atom in R₇ may be greater than 5, greater than 6 or greater than 7. The number of sequential carbon ^"atoms that conjugate the peptide backbone to the or each distal carbon atom in Rg may be greater than 1 or greater than 2.

In some embodiments the number of sequential carbon atoms that conjugate the peptide backbone to the or each distal carbon atom in Rg may be 9 or less atoms, 8 or less atoms, or 7 or less atoms.

In some embodiments the Y group is -C(O)-. In some embodiments the R₇ group does not comprise O, C(O), N(Ri₂) or S.

In some embodiments the Re group does not comprise O, C(O), N(R[₂) or S.

In another aspect the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a peptide as hereinbefore defined, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.

In another aspect the invention provides a method of preventing or treating a bacterial infection comprising the step of administering a therapeutically effective amount of a peptide as hereinbefore described, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

In another aspect the invention provides a use of a peptide as hereinbefore described, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the prevention or treatment of a bacterial infection.

In another aspect the invention provides a peptide as hereinbefore described, or a pharmaceutically acceptable salt thereof, for the prevention or treatment of a bacterial infection. Brief description of the Figures

Figure 1 shows static time-kill of compound 1 and colistin against a polymyxin-resistant MDR clinical P. aeruginosa isolate D.

Figure 2 shows static time-kill of compound 1 and colistin against P. aeruginosa ATCC 27853.

Detailed description of the invention

The initial cellular target of polymyxins in Gram-negative bacteria is the lipopoly saccharide (LPS) component of the outer membrane (OM). It is believed that the LPS target is conserved across most, if not all, Gram-negative bacteria. LPS is composed of three domains, a conserved inner core 2-keto-3-deoxyoctanoic acid bound to lipid A and a variable O-antigen composed of repeating units of various polysaccharides. The consensus structure of lipid A consists of a β- -6-linked D- glucosamine disaccharide that is phosphorylated at the 1- and 4'-positions and is shown below:

Lipid A usually contains six acyl chains. Four β-hydroxy acyl chains (usually Cio and Q₂ in length) are attached directly to the glucosamine sugars, while a secondary acyl chain is often attached to the β-hydroxy group on each of two of the chains. Lipid A acts as a hydrophobic anchor with the tight packing of the fatty acyl chains helping to stabilise the overall membrane structure.

It is believed that there is an initial polar interaction between the cationic polymyxin peptide (particularly the charged α,γ-diaminobutyric acid (Dab) residues) and the lipid A component of LPS in the OM, thereby displacing divalent cations (Ca²⁺ and Mg²⁺) from the negatively charged phosphate groups of lipid A. This initial interaction is followed by uptake across the OM and interaction with the cytoplasmic membrane. It has now been found that the binding of a naturally occurring polymyxin with lipid A may be enhanced by substitution with one or more lipophilic groups at particular locations within the polymyxin peptide structure.

As noted above, it is believed that a critical first step in the action of polymyxins on Gram- negative bacteria is the polar interaction between the positively charged Dab residues of polymyxins and the negatively charged phosphate groups on lipid A. It has been discovered that many of the bacterial mechanisms of resistance to polymyxins are based on modifications to lipid A which reduce or abolish this initial polar interaction. In Escherichia coli, Salmonella enterica serovar Typhimurium, K. pneumoniae and P. aeruginosa, modification of one or both of the phosphates of lipid A with moieties, such as 4-amino-4-deoxy-L-arabinose (L-Ara4N) and/or phosphoethanolamine (PEtn), reduces the net negative charge of lipid A thereby reducing the initial interaction of a polymyxin with lipid A leading to polymyxin resistance. One example of such a modification is shown below:

In addition, in K. pneumoniae the presence of a capsule can also cause polymyxin resistance.

The issue of polymyxin resistance is complicated by the recent discovery from population analysis profiles of 42 P. aeruginosa, 46 A. baumannii and 30 K. pneumoniae polymyxin- susceptible (minimum inhibitory concentrations (MICs, the lowest concentration of an antibiotic that inhibits visible growth of a microorganism) < 2 mg/L) MDR clinical strains from various regions in the world that contained a small sub-population that was able to grow in the presence of 4 mg/L, or higher, of colistin - a phenomenon known as heteroresistance which potentially explains how apparently susceptible Gram-negative bacteria can rapidly develop polymyxin resistance. Consistent with such findings of heteroresistance to colistin, recent studies have revealed very high ratios of colistin minimum bactericidal concentration (MBC, the lowest concentration of an antibiotic that results in the killing of more than 99.9% of the bacteria being tested) to MIC in MDR strains of all three species (MBC/MIC >32). In other words, whilst visible bacterial growth was inhibited at MICs of colistin, 100% of bacterial cells were not killed. It has also been recently demonstrated in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model which mimics clinical dosing regimens that polymyxin-resistant sub-populations of A. baumannii have the potential to rapidly amplify upon exposure to colistin.

It has now been surprisingly found that the peptides of the present invention are not only effective against polymyxin-susceptible but also polymyxin-resistant MDR Gram-negative bacteria. Without wishing to be bound by theory it is believed that sufficient binding between a peptide and lipid A may be achieved by overcoming, or at least ameliorating, the adverse polar interactions between a modified moiety on the phosphate group of lipid A and the positively charged residues on polymyxins through enhancement of the interactions between the lipophilic groups on lipid A and certain residues on the peptide.

It is believed that, for the polymyxin analogs in this invention, the lipophilic groups Ri, R₇ and R8 interact with the acyl chains in lipid A, whereas the X, R₂, ¾, R9, Rio and Ru groups and the R3, R4 and R5, where present, which are generally hydrophilic, interact with the polar portions of lipid A. If lipid A has undergone glycosylation or modification with phosphoethanolamine as part of the resistance mechanism in the bacteria, it is believed that the lipophilic groups and the generally hydrophilic groups remain able to maintain these same interactions. Without wishing to be bound by theory it is believed that being able to maintain these interactions results in the peptides of the present invention being effective against not only polymyxin-susceptible but also polymyxin-resistant MDR Gram-negative bacteria.

As used herein the expression "peptide backbone" takes its standard meaning and typically refers to the regular structure of alternating alpha-carbon units and peptide (amide) bonds. With reference to the peptides of the present invention, the peptide backbone is the sequence of carbon and nitrogen atoms extending between, and including, the N-terminus nitrogen atom proximal to R] and the C-terminus (in acyclic peptide) carbonyl carbon atom proximal to Ru- As used herein "conjugated" refers to a bonding of two or more elements of the peptide. Typically such conjugation will be by covalent bonding of the two or more elements of the peptide. It has now been discovered that the interaction between the lipophilic groups Ri, R₇ and Rg and the acyl chains in lipid A can be enhanced by using Ri, R₇ and Rg groups that extend a sufficient distance, or reach, from the peptide backbone. For example, in narurally- occurring polymyxin B the group corresponding to the R₇ position is the benzyl side chain of phenylalanine. It will be appreciated that the carbon atom in the side chain of phenylalanine that is distal to the peptide backbone is conjugated to the peptide backbone by 4 sequential atoms as shown below:

carbon atom

It has now been found that providing the R group with a distal carbon atom that is further from the peptide backbone than is found in phenylalanine, in combination with the other features of the invention described herein, provides the peptides of the present invention with advantageous properties.

For the avoidance of any doubt, a reference to the or each carbon atom that is "distal" to the peptide backbone refers to the or each carbon atom that is structurally furthest from the peptide backbone, rather than referring to a carbon atom that may conformationally be furthest from the peptide backbone at any particular point in time. The following structure shows the carbon atom in a hexyl group that is distal to the peptide backbone, as distinguished from a different carbon atom that may be more remote conformationally at any particular point in time due to folding of the chain, etc: n atom

The hexyl group shown above is one example of an R₇ side chain of the present invention where the carbon atom in the side chain that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms.

In some groups two or more carbon atoms may be equally distal to the peptide backbone. Examples of such groups are cyclopentylbutyl and 2-butyl-2-phenylethyl:

In such embodiments it will be understood that each carbon atom that is equally distal to the peptide backbone must be conjugated to the peptide backbone by 5 or more sequential atoms.

As used herein, the expression "sequential atoms" refers to atoms that are in sequence such that the atoms may be thought of as being part of a linear chain of atoms, although the skilled person will recognise that typically the bonds between atoms are disposed at angles to each other, such as the tetrahedral angle. In some embodiments, sequential atoms may form part of a cyclic structure. With reference to the following structure showing a biphenylmethyl side chain, the numbered atoms can be seen to be in sequence:

distal carbon atom

It will be understood that the atoms marked A, B, C and D are not in sequence with all of the other atoms. Such atoms that are not in sequence may be thought of as being in parallel or as part of branches of the side chain. It has also been found that where the R₇ and/or Rg group comprise a O, N or S atom, such atoms must not be located too close to the or each carbon atom in each group that is distal to the peptide backbone. In particular it has been found that such a O, N or S atom must be separated from the or each carbon atom in each group that is distal to the peptide backbone by a moiety comprising four or more sequential carbon atoms. As can be seen in the following example of an R₇ and/or Rg group, the O atom is conjugated to the distal carbon atom by a moiety comprising four or more sequential carbon atoms:

In some examples, where present, any one or more CH₂ groups in the Ri, R₇ and/or Rg groups is independently optionally replaced with O, C(O), N(Ri₂) or S. Examples of such groups are ethers, ketones, amines and thioethers, such as octyloxymethyl, octanylcarbanylethyl, (4-ethylphenyl)(methyl)amino and (4-phenylethylthio)but-l-yl. It will be understood that the Ri, R₇ and/or Rg groups may contain a plurality of O, C(O), N(Rj₂) and/or S groups. In some embodiments the Rj, R₇ and or Rg groups may contain two such groups adjacent to eachother. For example, where a CH₂ group in R₇ is replaced with O and an adjacent CH₂ group in R₇ is replaced with C(O) the skilled person will recognise that the combined functionality is an ester. By way of further example, where a CH₂ group in Ri is replaced by N(Rj₂) and an adjacent CH₂ group in Ri is replaced by C(O) the skilled person will recognise that the combined functionality is an amide. Other functionality combinations are contemplated including carbamate (-0-C(0)-N(R|₂)-), carbonate (-O-C(O)-O-) and uryl (-N(Ri₂)-C(0)-N(R₎₂)-). Peroxy (-0-0-) and hydrazine (-N-N-) groups are typically not found in living organisms, in part because of their relative instability. To this end the person skilled in the art would not typically use such peroxy and hydrazine groups in the R_\, R₇ and/or Rg groups of the invention. Surprisingly it has also been found that some compounds of the invention exhibit activity against a number of Gram-positive bacteria such as Staphylococcus aureus and Enterococcus faecium strains, particularly multidrug-resistant strains S. aureus ATCC 43300 (methicillin resistant), S. aureus ATCC 43300 (vancomycin intermediate resistant), S. aureus ATCC 43300 (vancomycin resistant) and E. faecium ATCC 700221 (vancomycin resistant). These results suggest that the compounds may have secondary cellular target(s) because Gram-positive bacteria do not possess lipopolysaccharide, a well- accepted primary target of polymyxins in Gram-negative bacteria.

In some embodiments, the side chain residue X is the residue of the side chain of α,γ- diami

In some embodiments, the linear peptide portion of the molecule comprises three amino acids, as do the naturally occurring polymyxin structures. In these cases, m and p may be equal to 1 and n may be equal to 0.

In some embodiments the X, R₃, Re, R9, Rio and Rn groups in the peptide of formula (I):

are the same as the corresponding groups that are generally common to the naturally occurring polymyxin structures, m and p equals 1 and n equals 0, and the peptide may accordingly be depicted as formula (III):

wherein R|, R₇ and Rg are lipophilic groups as hereinbefore defined;

R₂ is the side chain of α,γ-diaminobutyric acid or threonine;

R5 is the side chain of α,γ-diaminobutyric acid, serine or threonine.

In some embodiments the X, R₂, R3, Ri, R9, Rio and Rn groups in the peptide of formula (I):

are the same as the corresponding groups that are common to the naturally occurring polymyxin structures, m and p equals 1 and n equals 0, and the peptide may accordingly be depicted as formula (IV):

wherein Ri, R₇ and Re are lipophilic groups as hereinbefore defined;

R5 is the side chain of α,γ-diaminobutyric acid or serine. Except for the amino acid residue bearing the R₇ substituent, for which the stereochemistry is as depicted in Formula (I), each amino acid residue within the peptide may have any possible stereochemical configuration such as an L- or D-form. In some embodiments the absolute stereochemistry at each of the stereocentres in the compound of formula (V) shown below is the same as the corresponding stereochemistry in the naturally occurring polymyxin

In the compound of formula (V) (as well as compounds of formulae (I) to (IV)) shown above, the absolute stereochemistry of the stereocentre adjacent to the Re group has not been defined. It will be understood that where the amino acid(s) providing the Rg group is used as a mixture of enantiomers, such as a racemate, the peptide so formed that includes the Rg group will, in the absence of purification of an intermediate peptide, generally be prepared as a mixture of diastereomers. In some embodiments, the peptide may be used as a mixture of diastereomers, such as in the treatment of a bacterial infection. In some embodiments the peptide may be used as a single diastereomer, which may be isolated from a mixture of diastereomers or may be prepared using an enantiopure, or enantioenriched, amino acid that provides the Rg group. It is believed that preferred peptides are those in which the stereocentre adjacent to the Rg group has the stereochemistry of naturally occurring amino acids such as L-leucine. Where the peptide is used as a single diastereomer, it is typically preferable that the peptide is made using an enantiopure, or enantioenriched, amino acid containing the Rg group rather than being purified following synthesis using a racemic mixture of the amino acid.

Preferred examples of Rj lipophilic groups are C5.i6a.kyl, C5.gcycl0a.kyl,

6alkyl, C₆-ioaryl,

C₆.ioarylCi.6alkoxyC₆- 10arylC1.6a.kyl, C6-io rylCi.6alkoxyCi.6alkyl, C6-i2alkoxyCi.₆alkyl, C₆-i2alkylthioCi.6alkyl and Ci-i₂alkylC6-ioarylCi-6alkyl wherein, where present, any one or more CH₂ groups in Ri is independently optionally replaced with O, C(O), N(Rj₂) or S, wherein the or each carbon atom in Ri that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more O, N or S atoms in Ri, where present, are each independently conjugated to the or each distal carbon atom in Ri by a moiety comprising three or more sequential carbon atoms, wherein any alkyl, cycloalkyl or aryl group within Ri lipophilic group is optionally substituted with one or more groups selected from halo, d.₂₂alkyl, C₂.₂2alkenyl, Ce-ioaryl, C3-i₂cycloalkyl, hydroxy, hydroxy Ci.₂₂alkyl, amino, aminoCi_-22alkyl, Ci.₂₂alkyloxy, Ci.22alkylamino, (Ci.₂₂alkylXCi-₂₂alkyl)amino, Ci. ₂₂alkylcarbonyloxy, C2-22alkenylcarbonyloxy, C6.i₆arylcarbonyloxy, C3. i2cycloalkylcarbonyloxy, Ci^alkylcarbonylarnino, C₂.₂₂alkenylcarbonylamino, C6-i6arylacylamino and C3.i2cycloalkylcarbonylamino. For example Ri may be 2- chlorophenylamino, phenyl, biphenyl, biphenylmethyl, C4-9alkyl (such as butyl, heptyl or nonyl),

(such as trans-4-propylcyclohexanyl)..

Preferred examples of R₇ lipophilic groups are C₆-i6alkyl (such as Cg-^alkyl), C3- gcycloalkyl, C3^cycloalkylCi-6alkyl, Ce-ioarylCi-ealkyl (such as phenylethyl), C_6-ioarylC6-

^alkoxyCi^alkyl, C6-i₂alkylthioC|-6alkyl and Ci.i₂alkylC6arylCi_-6alkyl wherein, where present, any one or more CH₂ groups in R₇ is independently optionally replaced with O, C(O), N(Ri₂) or S; wherein the or each carbon atom in R₇ that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more 0, N or S atoms in R₇, where present, are each independently conjugated to the or each distal carbon atom in R₇ by a moiety comprising four or more sequential carbon atoms, wherein any alkyl, cycloalkyl or aryl group within R₇ lipophilic group is optionally substituted with one or more groups selected from halo, Ci-2₂alkyl, C₂.₂₂alkenyl, C6-i6ar l, C3-i₂cycloalkyl, hydroxy, hydroxyCi.22alkyl, amino, aminoCi.^alkyl, Ci. 22alkyloxy, Ci^alkylamino, (Ci-22alkyIXCi.22alkyl)amino, Ci.₂2alkylcarbonyloxy, C₂. 22alkenylcarbonyloxy, C6-i6arylcarbonyloxy, C3-i2cycloalkylcarbonyloxy, C|. 22alkylcarbonylamino, C₂.22alkenylcarbonylamino, Ce-iearylacylamino and C₃. i2cycloalkylcarbonylamino. For example R₇ may be octyl, biphenylmethyl, (4- phenylmethoxy)phenylmethyl, phenylmethoxymethyl, hexyloxymethyl, hexylthiomethyl, 4-methylphenylmethyl and 4-trifluoromethylphenylmethyl. It will be appreciated that a number of these amino acids may be derived from naturally occurring and/or commercially available amino acids such as tyrosine, serine and cysteine through 0-, O- and S- alkylation reactions respectively.

Preferred examples of Rg lipophilic groups are C2-i6alkyl (such as C_2-7alkyl), C₃. gcycloalkyl, C3-gcycloalkylCi.6alkyl, Ce-ioaryl, Ce-ioarylCi^alkyl Ce-io rylCe-ioarylCi. ₆alkyl, C₆-ioarylCi_-6alkoxyC6-ioarylCi.6alkyl,

C_6-i2alkoxyCi. ₆alkyl, C6.i₂alkylthioCi_-6alkyl and Ci.^alkylCe-ioarylCi^alkyl wherein, where present, any one or more CH₂ groups in R₈ is independently optionally replaced with 0, C(O), N(Ri₂) or S; wherein the one or more O, N or S atoms in Rg, where present, are each independently conjugated to the or each carbon atom in Rg that is distal to the peptide backbone by a moiety comprising four or more sequential carbon atoms, wherein any alkyl, cycloalkyl or aryl group within Rg lipophilic group is optionally substituted with one or more groups selected from halo, Ci^alkyl, C₂.₂₂alkenyl, C6-i6aryl, C3.i₂cycloalkyl, hydroxy, hydroxyCi_-22alkyl, amino, aminoCi_-22alkyl, Ci-₂₂alkyloxy, Ci-₂₂alkylamino, (Ci- ₂₂alkyl)(Ci.2₂alkyl)amino, Ci^alkylcarbonyloxy, C₂.22alkenylcarbonyloxy,

C6.₁₆arylcarbonyloxy, C_3-i2cycloalkylcarbonyloxy, Ci.₂2alkylcarbonylamino, C₂. 22alkenylcarbonylamino, C6-i6arylacylamino and Cs-^cycloalkylcarbonylamino. For example R» may be butyl (such as isobutyl), octyl, phenyl, bi-phenyl, cyclohexylbutyl, trans-4-propylcyclohexanyl, cyclododecanyl and cyclopentylphenyl. The skilled person will recognise that the Y group may be the residue of functionality that is useful in conjugating an P group to the nitrogen atom of the N-terminus of the peptide. A preferred example of the Y group is -C(O)-.

As used herein, the term "alkyl", used either alone or in compound words denotes straight chain or branched alkyl. Preferably the alkyl group is a straight chain alkyl group. Prefixes such as "Ci-2₂" are used to denote the number of carbon atoms within the alkyl group (from 1 to 22 in this case). Examples of straight chain and branched alkyl include methyl, ethyl, /j-propyl, isopropyl, H-butyl, sec-butyl, /-butyl, n-pentyl, hexyl, heptyl, 5-methylheptyl, 5- methylhexyl, octyl, nonyl, decyl, undecyl, dodecyl and docosyl (C22).

As used herein, the term "cycloalkyl", used either alone or in compound words denotes a cyclic alkyl group. Prefixes such as "C3.12" are used to denote the number of carbon atoms within the cyclic portion of the alkyl group (from 3 to 12 in this case). Examples of cyclic alkyl include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and cyclododecyl.

As used herein, the term "alkenyl", used either alone or in compound words denotes straight chain or branched hydrocarbon residues containing at least one carbon to carbon double bond including ethylenically mono-, di- or polyunsaturated alkyl groups as previously defined. Preferably the alkenyl group is a straight chain alkenyl group. Prefixes such as "€2-22" are used to denote the number of carbon atoms within the alkenyl group (from 2 to 22 in this case). Examples of alkenyl include vinyl, allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 1-hexenyl, 3-hexenyl, 1-heptenyl, 3- heptenyl, 1-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1 -decenyl, 3-decenyl, 1,3- butadienyl, 1 ,4-pentadienyl, 1 ,3-hexadienyl, 1 ,4-hexadienyl and 5-docosenyl (C22). As used herein, the term "aryl" denotes any single- or polynuclear, conjugated or fused residues of aromatic hydrocarbon ring systems. Prefixes such as "C^ie" are used to denote the number of carbon atoms within the cyclic portion of the aryl group (from 6 to 16 in this case). Examples of aryl include phenyl (single nuclear), naphthyl (fused polynuclear), biphenyl (conjugated polynuclear) and tetrahydronaphthyl (fused polynuclear).

As used herein, the term "halo" refers to halogen atoms such as F, CI, Br, I. In some embodiments halo is CI. In some embodiments halo is F. It will be understood that a fluorine atom may function as an isostere for hydrogen, and accordingly the skilled person may substitute one or more hydrogen atoms in an alkyl, alkenyl, aryl and/or cycloalkyl group, for example, for fluorine atom(s).

As used herein, the term "lipophilic" refers to a property of a chemical compound, or part thereof, wherein the chemical compound or part thereof more readily associates with members of a large and diverse group of oils, fats and fat like substances (that occur, for example, in living organisms) than with water. As referred to herein, "lipophilic groups" are groups which, of themselves, display a preference to be solubilised by lipidic solvents rather than aqueous solvents.

As used herein, reference to an amino acid "side chain" takes its standard meaning in the art. Examples of side chains of amino acids are shown below:

side

of α,γ-diaminobutyric acid arginine histidine threonine cysteine

side chain of side chain of side chain of side chain of side chain of l sine ornithine glutamatic acid glutamate glutamine

side chain of side chain of side chain of side chain of side chain of aspartic acid aspartate asparagine serine leucine

side chain of

phenylalanine

In addition to the negatively charged side chains shown above, it will be appreciated that a number of the side chains may also be protonated and so become positively charged, such as the side chain of lysine. The present invention contemplates within its scope these protonated side chains as well.

As discussed above, it is understood that the peptides of the present invention may exist in one or more stereoisomeric forms (eg enantiomers, diastereomers). Other than the stereochemistry indicated for the carbon atom adjacent to the R₇ group in compound (I), the present invention includes within its scope all of these stereoisomeric forms either isolated (in for example enantiomeric isolation), or in combination (including racemic mixtures and diastereomic mixtures). The present invention contemplates the use of amino acids in both L and D forms, including the use of amino acids independently selected from L and D forms. For example, where the peptide comprises two Dab residues, each Dab residue may have the same, or opposite, absolute stereochemistry. It is understood that the R7 and Rg groups in the peptide of formula (I):

are appended to a 2-aminoethanoic acid subunit and accordingly are amenable to incorporation into the peptide backbone through standard peptide chemistry. The actual synthesis of the R₇ and R_% appended 2-aminoethanoic acid reagents may be accomplished using known techniques - for example: Wimmer, N., et al. (2002) Syntheses of polycationic dendrimers on lipophilic peptide core for complexation and transport of oligonucleotides, Bioorg. Med Chem. Lett. 12, 2635-2637; and Gibbons, W. A., et al. (1990) Synthesis, Resolution and Structural Elucidation of Lipidic Amino Acids and Their Homo- and Hetero-Oligomers, Liebigs Ann. Chem. 1 175. The entire contents of these documents are incorporated herein by reference.

Lipidic amino acids such as:

may be prepared for use as the amino acid bearing the Rg group in the synthesis of the polymyxin peptide analogs of the present invention.

Lipidic amino acids such as:

may be prepared for use as the amino acid bearing the R₇ group in the synthesis of the polymyxin peptide analogs of the present invention. As noted previously, fluorine may be used as an isostere of hydrogen.

It will be appreciated that a number of these amino acids may be derived from naturally occurring and/or commercially available amino acids such as tyrosine, serine and cysteine through 0-, O- and S- alkylation reactions respectively. The R.7 and Rg appended amino acids ("lipidic" amino acids) may be prepared as racemates or, alternatively, the amino acids or synthetic intermediates may be resolved to provide enantiopure, or at least enantioenriched, products. The resolution may take place chemically or enzymatically for example. The amino acids bearing the R₇ and Rg side chains may be synthesised in enantiopure, or at least enantioenriched, form through stereoselective synthesis and/or purification techniques such as recrystallisation. A number of amino acids bearing lipophilic groups defined by R₇ and/or Rg in enantiopure, or enantioenriched, form are commercially available. Examples of R₇ appended amino acids that may be used in enantiopure, or at least enantioenriched, form are shown below:

Examples of Rg appended amino acids that may be used in enantiopure, or at least enantioenriched, form are shown below:

In general, techniques for preparing the peptides of the invention are well known in the art for example see:

a) Alewood, P.; Alewood, D.; Miranda, L.; Love, S.; Meutermans, W.; Wilson, D., "Rapid in situ neutralisation protocols for Boc and Fmoc solid-phase chemistries" Meth. Enzymol. 1997, 289, 14-28;

b) Merrifield, J. Am. Chem. Soc. 1964, 85, 2149;

c) Bodanzsky, "Principles of Peptide Synthesis", 2nd Ed., Springer- Verlag (1993); and

d) Houghten, Proc. Natl. Acad. Sci. USA, 1985, 82, 5131.

Of particular relevance to polymyxin peptide synthesis are: Kline, T., Holub, D., Therrien, J. et al. (2001), Synthesis and characterization of the colistin peptide polymyxin El and related antimicrobial peptides, J. Pept. Res. 57, 175-187; and Vaara, M., Fox, J., Loidl, G., Siikanen, O. et al. (2008), Novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents, Antimicrob. Agents Chemother. 52(9), 3229- 3236. The entire contents of these documents are incorporated herein by reference. Known solid or solution phase techniques may be used in the synthesis of the peptides of the present invention, such as coupling of the N- or C-terminus to a solid support (typically a resin) followed by step-wise synthesis of the linear peptide. An orthogonal protecting group strategy may be used to facilitate selective deprotection and cyclization to form the cyclic heptapeptide core of the peptide. Protecting group chemistries for the protection of amino acid residues, including side chains, are well known in the art and may be found for example in: Theodora W. Greene and Peter G. M. Wuts, Protecting Groups in Organic Synthesis, (Third Edition, John Wiley & Sons, Inc, 1999) - the entire contents of which is incorporated herein by reference.

As a general strategy, the synthesis of the peptides of the present invention may be performed in four stages. In the first stage, amino acids may be protected for incorporation into the peptide, such as the protection of -biphenylglycine as Fmoc-biphenylglycine. Second, a partially protected linear peptide which selectively exposes only the functional groups required for cyclization may be synthesised using solid phase techniques. Third the cyclization reaction may be performed in solution to produce the protected cyclic lipopeptide. Fourth the remaining side chain protecting groups may be deprotected to furnish the peptide.

Where the compounds of the present invention require purification, chromatographic techniques such as high-performance liquid chromatography (HPLC) and reversed-phase HPLC may be used. The peptides may be characterised by mass spectrometry and/or other appropriate methods.

Where the peptide comprises one or more functional groups that may be protonated or deprotonated (for example at physiological pH) the peptide may be prepared and/or isolated as a pharmaceutically acceptable salt. It will be appreciated that the peptide may be zwitterionic at a given pH. As used herein the expression "pharmaceutically acceptable salt" refers to the salt of a given compound, wherein the salt is suitable for administration as a pharmaceutical. For example, such salts may be formed by the reaction of an acid or a base with an amine or a carboxylic acid group respectively.

Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Examples of organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

Pharmaceutically acceptable base addition salts may be prepared from inorganic and organic bases. Corresponding counterions derived from inorganic bases include the sodium, potassium, lithium, ammonium, calcium and magnesium salts. Organic bases include primary, secondary and tertiary amines, substituted amines including naturally- occurring substituted amines, and cyclic amines, including isopropylamine, trimethyl amine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2- dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, and N-ethylpiperidine.

Acid/base addition salts tend to be more soluble in aqueous solvents than the corresponding free acid/base forms.

The compounds of the invention may be in crystalline form or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. The term "solvate" is a complex of variable stoichiometry formed by a solute (in this invention, a peptide of the invention) and a solvent. Such solvents should not interfere with the biological activity of the solute. Solvents may be, by way of example, water, ethanol or acetic acid. Methods of solvation are generally known within the art.

The compounds of the invention may be in the form of a "pro-drug". The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the peptides of the invention. Such derivatives would readily occur to those skilled in the art and include, for example, compounds where a free hydroxy group is converted into an ester derivative or a ring nitrogen atom is converted to an N-oxide. Examples of ester derivatives include alkyl esters (for example acetates, lactates and glutamines), phosphate esters and those formed from amino acids (for example valine). Any compoimd that is a prodrug of a peptide of the invention is within the scope and spirit of the invention. Conventional procedures for the preparation of suitable prodrugs according to the invention are described in text books, such as "Design of Prodrugs" Ed. H. Bundgaard, Elsevier, 1985 - the entire contents of which is incorporated herein by reference.

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a peptide as hereinbefore defined, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.

The term "composition" is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.

While the peptide as hereinbefore described, or pharmaceutically acceptable salt thereof, may be the sole active ingredient administered to the subject, the administration of other active ingredient(s) with the compound is within the scope of the invention. For example, the compound could be administered with one or more therapeutic agents in combination. The combination may allow for separate, sequential or simultaneous administration of the peptide as hereinbefore described with the other active ingredient(s). The combination may be provided in the form of a pharmaceutical composition.

As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the peptide actives care should be taken to ensure that the activity of the peptide is not destroyed in the process and that the peptide is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the peptide by means known in the art, such as, for example, micro encapsulation. Similarly the route of administration chosen should be such that the peptide reaches its site of action.

Those skilled in the art may readily determine appropriate formulations for the peptides of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or sulphite, and metal chelators such as EDTA.

The peptide as hereinbefore described, or pharmaceutically acceptable salt thereof, may be prepared in parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery. The pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage and may be preserved against reduction or oxidation and the contaminating action of microorganisms such as bacteria or fungi. The solvent or dispersion medium for the injectable solution or dispersion may contain any of the conventional solvent or carrier systems for peptide actives, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include agents to adjust osmolality, for example, sugars or sodium chloride. Preferably, the formulation for injection will be isotonic with blood. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Pharmaceutical forms suitable for injectable use may be delivered by any appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.

Sterile injectable solutions are prepared by incorporating the active peptide in the required amount in the appropriate solvent with various of the other ingredients such as those enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying or freeze-drying of a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.

Other pharmaceutical forms include oral and enteral formulations of the present invention, in which the active peptide may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active peptide may be incorporated with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active peptide in such therapeutically useful compositions is such that a suitable dosage will be obtained. The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active peptide, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active peptide(s) may be incorporated into sustained-release preparations and formulations, including those that allow specific delivery of the active peptide to specific regions of the gut.

Liquid formulations may also be administered enterally via a stomach or oesophageal tube.

Enteral formulations may be prepared in the form of suppositories by mixing with appropriate bases, such as emulsifying bases or water-soluble bases. It is also possible, but not necessary, for the peptides of the present invention to be administered topically, intranasally, intravaginally, intraocularly and the like.

The present invention also extends to any other forms suitable for administration, for example topical application such as creams, lotions and gels, or compositions suitable for inhalation or intranasal delivery, for example solutions, dry powders, suspensions or emulsions.

The peptides of the present invention may be administered by inhalation in the form of an aerosol spray from a pressurised dispenser or container, which contains a propellent such as carbon dioxide gas, dichlorodifluoromethane, nitrogen, propane or other suitable gas or combination of gases. The peptides may also be administered using a nebuliser.

Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

As mentioned above the principal active ingredient may be compounded for convenient and effective administration in therapeutically effective amounts with a suitable pharmaceutically acceptable vehicle in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.25 μg to about 2000 mg. Expressed in proportions, the active compound may be present in from about 0.25 μg to about 2000 mg/mL of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. The term "therapeutically effective amount" refers to that amount which is sufficient to effect treatment, as defined below, when administered to an animal, preferably a mammal, more preferably a human in need of such treatment. The therapeutically effective amount will vary depending on the subject and nature of bacterial infection being treated, the severity of the infection and the manner of administration, and may be determined routinely by one of ordinary skill in the art. The terms "treatment" and "treating" as used herein cover any treatment of a condition or disease in an animal, preferably a mammal, more preferably a human, and includes: (i) inhibiting the bacterial infection, eg arresting its proliferation; (ii) relieving the infection, eg causing a reduction in the severity of the infection; or (iii) relieving the conditions caused by the infection, eg symptoms of the infection. The terms "prevention" and preventing" as used herein cover the prevention or prophylaxis of a condition or disease in an animal, preferably a mammal, more preferably a human and includes preventing the bacterial infection from occurring in a subject which may be predisposed to infection but has not yet been diagnosed as being infected.

In some embodiments the bacterial infection is a Gram-negative bacterial infection. The bacterial infection may be caused by one or more species selected from one or more of the genera: Acinetobacter; Actinobacillus; Bartonella; Bordetella; Brucella; Burkholderia; Campylobacter; Cyanobacteria; Enterobacter; Erwinia; Escherichia; Francisella; Helicobacter; Hemophilus; Klebsiella; Legionella; Moraxella; Morganella; Neisseria; Pasteurella; Proteus; Providencia; Pseudomonas; Salmonella; Serratia; Shigella; Stenotrophomonas; Treponema; Vibrio; and Yersinia. Specific examples of species are Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Stenotrophomonas maltophilia; Escherichia coli and Salmonella enterica.

In some embodiments the bacterial infection is a Gram-positive bacterial infection. The bacterial infection may be caused by one or more species selected from one or more of the genera: Actinobacteria; Bacillus; Clostridium; Corynebacterium; Enterococcus; Listeria; Nocardia; Staphylococcus; and Streptococcus. Specific examples of species are Staphylococcus aureus; and Enterococcus faecium.

The invention will now be described with reference to the following non-limiting examples: Example 1: Synthesis of compounds of the invention

(a)

2-Aminodecanoic acid:

is prepared according to the procedure described in Gibbons, W. A., et al. (1990) Synthesis, Resolution and Structural Elucidation of Lipidic Amino Acids and Their Homo- and Hetero-Oligomers, Liebigs Ann. Chem. 1175.

L-2-aminodecanoic acid is available commercially from a number of sources including: www.chemicalbook.com; www.anaspec.com; www.chemexper.com; and www.icis.com.

L-2-aminooctanoic acid is available commercially from a number of sources including: www.chemicalbook.com.

was prepared from Fmoc-D-Cys-OH by reaction with Bromohexane (2eq) in a (1 :1) mixture of dimethylformamide, 10% sodium carbonate solution overnight. The reaction solution was diluted with water and acidified with concentrated hydrochloric acid whilst cooling in an ice/water bath. Acetonitrile was then added to the reaction mixture, which was lyophilised overnight. The solid material obtained was extracted into diethylether which was then concentrated under nitrogen. The crude Fmoc-D-Cys(Hexyl)-OH was isolated by lyophilization from a mixture of acetonitrile and water and used without further purification. The purity was >90% as estimated by reversed-phase HPLC. The compound was confirmed as having the correct molecular weight by ESI-MS analysis: m/z (monoisotopic) 428.32 [M+H]⁺. (c)

A representative synthesis used to make compounds of the invention including compound 1:

by Fmoc-based solid phase peptide synthesis is detailed below:

Peptides where synthesized using standard Fmoc solid phase peptide synthesis methodology. The Dab γ-amino groups were protected by t-butoxycarbonyl (tBoc), and threonine was protected as the t-butyl ether (tBu). The γ-amino group of the Dab residue involved in cyclization was protected by ivDde, which is a hydrazine labile group that can be removed prior to the cleavage step. Synthesis of the linear sequence was carried out in a CEM Liberty automated microwave synthesizer using super acid labile 2-chlorotrityl or trityl resin preloaded with Fmoc- Thr(tBu)-OH. Fmoc deprotection was performed using the default instrument protocol: 20% piperidine in DMF (1 x 30 s, 1 x 3 min) at 75 °C (35 W microwave power). Couplings were performed using the default instrument protocol: 5 eq of Fmoc amino acid and HCTU with activation using diisopropylethylamine (10 eq) in dimethylformamide over 2 min at room temperature then at 50 °C (25 W microwave power) over 4 min. Coupling of the N-terminal octanoyl group was performed twice using the default instrument protocol: 5 eq of octanoic acid and HCTU with activation using diisopropylethylamine (10 eq) in dimethylformamide over 2 min at room temperature then at 50 °C (25 W microwave power) over 4 min. Removal of the ivDde group was achieved using 2% hydrazine in DMF.

Cleavage of the peptide from the resin was achieved by treating the resin with 1% TFA in dichloromethane. The crude partially protected linear peptide was isolated by lyophilization from a mixture of acetonitrile and water.

Cyclization of the linear sequence was performed using (DPP A) diphenylphosphorylazide (3 eq) and (DIPEA) diisopropylethylamine (6 eq) in DMF overnight. The crude protected cyclic peptide was isolated by lyophilization from a mixture of acetonitrile and water. The residual protecting groups were removed by treating the peptide with 97.5% TFA: 2.5% triisopropylsilane.

The crude deprotected and cyclized peptide was first purified by ion exchange chromatography using a VariPure™ IPE column followed by further purification by reversed-phase chromatography using conventional gradients of acetonitrile:water:trifluoroacetic acid. Compound 1 was dried by lyophilization. The purity was >95% as estimated by reversed-phase HPLC. The compound was confirmed as having the correct molecular weight by ESI-MS analysis: /n/z(monoisotopic) 1211.95 [M+H]⁺, 606.80 [M+2H]²⁺. It will be understood that this representative synthesis may be applied to the synthesis of a range of compounds described herein. For example, the representative synthesis may be ap lied to the synthesis of the following compounds 2 to 8:

ESI-MS analysis: /n z(monoisotopic) 1268.00 [M+H]⁺, 634.85 [M+2H]²⁺;

NH₂

ESI- ²⁺;

ESI-MS analysis: mz(monoisotopic) 1240.00 [M+H]⁺, 620.80 [M+2H]²⁺;

NH₂

ESI-MS analysis: mz(monoisotopic) 1229.95 [M+H]⁺, 615.80 [M+2H]²⁺;

NH₂

ESI-MS analysis: z(monoisotopic) 1283.90 [M+H]⁺, 642.80 [M+2H]²⁺.

Example 2: Measurements of minimum inhibitory concentrations (MICs) MICs of the lipopeptides (trifluoroacetic acid salt, TFA) were determined by broth microdilution in cation-adjusted Mueller-Hinton broth (CAMHB) (Oxoid Australia, Thebarton, SA, Australia) according to Clinical and Laboratory Standards Institute standards (Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; eighteenth informational supplement M100-S18. Wayne, PA, 2008). Colistin (sulfate) was employed as control. Both Gram-negative and Gram-positive bacteria were examined for 1 and 2: (1) for Pseudomonas aeruginosa, ATCC 27853 and 4 polymyxin-resistant multidrug-resistant clinical isolates; (2) for Acinetobacter baumannii, ATCC 19606, one laboratory-derived polymyxin-resistant strain and one polymyxin-resistant multidrug-resistant clinical isolate; (3) for Klebsiella pneumoniae, ATCC 13883 and one polymyxin-resistant clinical isolate; (4) for Stenotrophomonas maltophilia, 3 polymyxin-resistant multidrug-resistant clinical isolates; (5) for Gram-positive bacteria, Staphylococcus aureus ATCC 43300 (methicillin resistant), ATCC 700698 (vancomycin intermediate resistant) and ATCC 700699 (vancomycin resistant), and Enterococcus faecium ATCC 700221 (vancomycin resistant). Compounds 3 to 8 were examined against P. aeruginosa ATCC 27853 and one polymyxin-resistant multidrug-resistant clinical isolate (strain D). The results for compounds 1 - 8 are shown in Table 1 :

Table 1: MICs (mg/L) for compounds and colistin (colistin resistance: colistin MIC > 2 mg/L); ND = not determined; * = lab-derived strain that has no LPS.

Strain Test molecule

Colistin 1 2 3 4 5 6 7 8

Gram-negative bacteria

P. aeruginosa

ATCC 27853 1 1 2 4 32 2 2 2 4

A >128 2 2 ND ND ND ND ND ND

D >128 3 2 8 4 2 2 8 4

U >128 4 4 ND ND ND ND ND ND

V >128 8 4 ND ND ND ND ND ND

A. baumannii

ATCC 19606 1 2 2 ND ND ND ND ND ND

J* 128 16 8 ND ND ND ND ND ND

16 4 4 ND ND ND ND ND ND

I. pneumoniae

ATCC 13883 1 4 4 ND ND ND ND ND ND

P >128 >32 >32 ND ND ND ND ND ND

Ste. mat tophilia

W 128 4 2 ND ND ND ND ND ND

X 128 2 4 ND ND ND ND ND ND

Y 64 2 2 ND ND ND ND ND ND

Gram-positive bacteria

S. aureus

ATCC 43300 >128 8 4 ND ND ND ND ND ND

ATCC 700698 >128 16 8 ND ND ND ND ND ND

ATCC 700699 >128 8 8 ND ND ND ND ND ND

E.faecium

ATCC 700221 >128 4 4 ND ND ND ND ND ND Interestingly, unlike polymyxins which are only active against Gram-negative bacteria, the peptides of the present invention showed activity against Gram-positive pathogens S. aureus and E. faecium with MICs 4 - 16 mg/L. This implies that the peptides of the present invention may have secondary cellular target(s) because Gram-positive bacteria do not possess lipopolysaccharide, a well-accepted primary target of polymyxins in Gram- negative bacteria.

Example 3: Static time-kill studies The time-killing kinetics of compound 1 (TFA) and colistin (sulfate) were examined against a polymyxin-resistant multidrug-resistant clinical P. aeruginosa isolate (strain D) and P. aeruginosa ATCC 27853 (polymyxin susceptible), and compound 1 (TFA) against a vancomycin-resistant S. aureus ATCC 700699 (intrinsically polymyxin resistant). Compound 1 or colistin was added to a logarithmic-phase broth culture of approximately 10⁶ CFU/mL to yield concentrations of 0, 0.5, 1 and 4> MIC of the isolate (32 mg/L for colistin against the polymyxin-resistant P. aeruginosa isolate). Viable counting was performed on samples collected at 0, 0.5, 1, 2, 3, 4 and 24 h (plus 5 and 6 h for 5. aureus ATCC 700699) after antibiotic addition. After appropriate dilutions with saline, samples of bacterial cell suspension (50 μί) were spirally plated on nutrient agar plates (Medium Preparation Unit, University of Melbourne) using a Whitley automatic spiral plater (WASP, Don Whitley Scientific, West Yorkshire, UK). Colonies were counted by a ProtoCOL automated colony counter (Synbiosis, Cambridge, UK) after 24 h incubation of subcultures at 35 °C. The lower limit of counting was 20 CFU/mL. The results of the antibacterial killing kinetics of compound 1 against P. aeruginosa, with one polymyxin-resistant (colistin MIC > 128 mg/L) multidrug-resistant clinical isolate and ATCC 27853 (reference strain), and against S. aureus ATCC 700699 (intrinsically polymyxin resistant with a colistin MIC > 128 mg/L) are shown in Figures 1, 2 and 3, respectively. Against the polymyxin-resistant P. aeruginosa isolate, compound 1 at 4* MIC achieved ~6 logio kill with no viable cells detected over the 2- to 6-h interval; while no substantial killing was observed with colistin even at 32 mg/L. Figure 2 shows that compound 1 had comparable antibacterial killing to colistin against polymyxin-susceptible P. aeruginosa ATCC 27853. Compound 1 at 4x MIC achieved >5 log kill at 24 h against vancomycin-resistant S. aureus ATCC 700699, showing its antibacterial activity against Gram-positive S. aureus which is intrinsically resistant to polymyxin B and colistin (Figure 3). The time-kill data are consistent with the MIC results above.

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

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.

Claims

Claims:

1. Peptide of formula (I) :

wherein:

Ri is a lipophilic group comprising five or more carbon atoms and optionally comprising one or more O, N or S atoms, wherein the or each carbon atom in Ri that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more 0, N or S atoms in Ri, where present, are each independently conjugated to the or each distal carbon atom in Ri by a moiety comprising three or more sequential carbon atoms;

Y is selected from -C(O)-; -C(S)-; -C(NH)-; and -S(0)₂-; m, n and p are each independently 0 or 1 ;

R₃, R» and R₅ where present and R₂, Re, R9, Rio and Rn are each independently selected from the side chain of an amino acid selected from α,γ-diaminobutyric acid, arginine, histidine, lysine, leucine, ornithine, glutamic acid, aspartic acid, glutamine, asparagine, homoserine, serine, threonine and cysteine;

X is a residue of the side chain of an amino acid selected from α,γ-diaminobutyric acid, arginine, histidine, lysine, ornithine, glutamic acid, aspartic acid, glutamine, asparagine, homoserine, serine, threonine and cysteine;

R₇ is a lipophilic group comprising five or more carbon atoms and optionally comprising one or more O, N or S atoms, wherein the or each carbon atom in R₇ that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more O, N or S atoms in R₇, where present, are each independently conjugated to the or each carbon atom in R₇ that is distal to the peptide backbone by a moiety comprising four or more sequential carbon atoms;

Rg is a lipophilic group comprising two or more carbon atoms and optionally comprising one or more O, N or S atoms, wherein the one or more O, N or S atoms in Rg, where present, are each independently conjugated to the or each carbon atom in Rg that is distal to the peptide backbone by a moiety comprising four or more sequential carbon atoms; or a pharmaceutically acceptable salt thereof.

2. A peptide according to claim 1 of formula (I):

wherein:

Ri is a lipophilic group comprising from 5 to 22 carbon atoms selected from Cs-₂₂alkyl, C_5- 22alkenyl, C_6-i6aryl, C₅-i₂cycloalkyl, C6-i6arylCi.₆alkyl, C₆.i6arylC₂-6alkenyl, cyclopropylC₂. ealkyl, C4-i₂cycloalkylCi.6alkyl and C3.i₂cycloalkylC₂-6alkenyl wherein, where present, any one or more CH₂ groups in Ri is independently optionally replaced with O, C(O), N(R₁₂) or S, wherein the or each carbon atom in Rj that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more O, OC(O), N(Rj₂), N(R|₂)C(0) or S in R|, where present, are each independently conjugated to the or each distal carbon atom in Ri by a moiety comprising three or more sequential carbon atoms; Y is selected from -C(O)-; -C(S)-; -C(NH)-; and -S(0)₂-; m, n and p are each independently 0 or 1 ;

R₃, R4 and R5 where present and R₂, Re, R9, Rio and Ri 1 are each independently selected from the side chain of an amino acid selected from α,γ-diaminobutyric acid, arginine, histidine, lysine, leucine, ornithine, glutamic acid, aspartic acid, glutamine, asparagine, homoserine, serine, threonine and cysteine;

X is a residue of the side chain of an amino acid selected from α,γ-diaminobutyric acid, arginine, histidine, lysine, ornithine, glutamic acid, aspartic acid, glutamine, asparagine, homoserine, serine, threonine and cysteine;

R₇ is a lipophilic group comprising from 5 to 22 carbon atoms and is selected from C5. ₂₂alkyl, C5_-22alkenyl, Ce-iearyl, Cs-ucycloalkyl, C₆-i6arylCi-6alkyl, C6-i6arylC_2- alken l, cyclopropylC_2-6alkyl, C4.i₂cycloalkylCi.6alkyl and C₃.i₂cycloalkylC₂-₆alkenyl wherein, where present, any one or more CH₂ groups in R₇ is independently optionally replaced with O, C(O), N(R]₂) or S, wherein the or each carbon atom in R₇ that is distal to the peptide backbone is conjugated to the peptide backbone by five or more sequential atoms, and the one or more 0, N or S atoms in R₇, where present, are each independently conjugated to the or each distal carbon atom in R₇ by a moiety comprising four or more sequential carbon atoms;

Rs is a lipophilic group comprising from 2 to 22 carbon atoms and is selected from C₂. ₂₂alkyl, C₂.₂₂alkenyl, C₆-i6aryl, C3-i₂cycloalkyl, Ce-iearylQ-ealkyl, Ce-iearylC^alkenyl, C₃. i₂cycloalkylCi-6alkyl and C_3-i₂cycloalkylC₂-6alkenyl wherein, where present, any one or more CH₂ groups in Rg is independently optionally replaced with O, C(O), N(R₁₂) or S, wherein the one or more O, N or S atoms in Re, where present, are each independently conjugated to the or each carbon atom in Rs that is distal to the peptide backbone by a moiety comprising four or more sequential carbon atoms; wherein, where present, R]₂ is independently selected from hydrogen, Ci.^alkyl, C₂. ₂₂alkenyl, C₆-i₆aryl, C₃.i₂cycloalkyl, C₆-i₆arylCi-6alk l, C₆-i₆arylC₂^alkenyl, C₃.

and C3.i₂cycloalkylC₂.₆alkenyl; wherein any alkyl, alkenyl, aryl and cycloalkyl group within the peptide is optionally substituted with one or more groups selected from halo, Q.^alkyl, C₂.₂₂alkenyl, Ce-iearyl, C₃-i₂cycloalkyl, hydroxy, hydroxyCi_-22alkyl, amino, aminoCi.₂₂alkyl, Ci_-22alkyloxy, C_\. ₂₂alkylamino, (Ci_-22alkylXCi,₂₂alkyl)amino, Ci.₂₂alkylcarbonyloxy, C₂. ₂₂alkenylcarbonyloxy, C6.i₆arylcarbonyloxy, C₃.i₂cycloalkylcarbonyloxy, Cj. ₂₂alkylcarbonylamino, C₂.₂₂alkenylcarbonylamino, Ce-iearylacylamino and C₃. ncycloalkylcarbonylamino; or a pharmaceutically acceptable salt thereof.

3. Peptide according to claim 1 or claim 2 wherein the number of sequential atoms that conjugate the peptide backbone to the or each distal carbon atom in R₇ is greater than 5.

4. Peptide according to claim 1 or claim 2 wherein the number of sequential atoms that conjugate the peptide backbone to the or each distal carbon atom in R₇ is greater than 6.

5. Peptide according to claim 1 or claim 2 wherein the number of sequential atoms that conjugate the peptide backbone to the or each distal carbon atom in R₇ is greater than 7.

6. Peptide according to claim 1 or claim 2 wherein the number of sequential atoms that conjugate the peptide backbone to the or each distal carbon atom in Rg is greater than 1.

7. Peptide according to claim 1 or claim 2 wherein the number of sequential atoms that conjugate the peptide backbone to the or each distal carbon atom in Rg is greater than 2.

8. Peptide according to claim 1 or claim 2 wherein the number of sequential carbon atoms that conjugate the peptide backbone to the or each distal carbon atom in Rg is 9 or less atoms.

9. Peptide according to claim 1 or claim 2 wherein the number of sequential carbon atoms that conjugate the peptide backbone to the or each distal carbon atom in Rg is 8 or less atoms.

10. Peptide according to claim 1 or claim 2 wherein the number of sequential carbon atoms that conjugate the peptide backbone to the or each distal carbon atom in Rg is 7 or less atoms.

11. Pharmaceutical composition comprising a therapeutically effective amount of a peptide according to claim 1 , or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.

12. Method of preventing or treating a bacterial infection comprising the step of administering a therapeutically effective amount of a peptide according to claim 1, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

13. Use of a peptide according to claim 1 , or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the prevention or treatment of a bacterial infection.

14. Peptide according to claim 1, or a pharmaceutically acceptable salt thereof, for the prevention or treatment of a bacterial infection.