WO2016166103A1

WO2016166103A1 - Polymyxin derivatives

Info

Publication number: WO2016166103A1
Application number: PCT/EP2016/058019
Authority: WO
Inventors: Ingrid Malene APELAND; Vidar BJØRNSTAD; Adnan BUDNJO; Sølvi GUNNES; Robert Fletcher Kenyon; Carl Åke Rikard LARSSON
Original assignee: Xellia Pharmaceuticals Aps
Priority date: 2015-04-13
Filing date: 2016-04-12
Publication date: 2016-10-20

Abstract

The present invention relates to polymyxin derivatives and their use as antimicrobial compounds, especially for use in the treatment of bacterial infections caused by gram-negative bacteria, including infections caused by multi drug resistant gram-negative bacteria.

Description

POLYMYXIN DERIVATIVES

FIELD OF THE INVENTION

The present invention relates to polymyxin derivatives with antibiotic activity.

BACKGROUND OF THE INVENTION

Polymyxins were discovered in 1947 as antibiotics produced by Paenibacillus polymyxa. Naturally occurring polymyxins are antibiotic decapeptides containing a heptapeptide ring and a N-terminal amide coupled fatty acid. Today, two commercial polymyxin mixtures are in clinical use; Polymyxin B (PMB) and Polymyxin E/Colistin (PME). Both mixtures comprise a variety of closely related peptides obtained by fermentation as described by Goevaerts et al 2002 and Van den Bossche et al 201 1.

Polymyxins are highly bactericidal to Gram-negative bacteria and considered one of the most efficient cell-permeabilizing compounds. However, the therapeutic applications of polymyxins are very limited because of their relatively high toxicity, and they are therefore used as a last resort in patients with infections that are resistant to other available types of antibiotics. Currently recommended dosage regimens of polymyxins are suboptimal and higher doses are required in order to minimize potential emergence of resistance.

Unfortunately, polymyxin-induced nephrotoxicity is the major dose-limiting factor as it occurs in up to 60% of patients when the polymyxins are administered intravenously. The toxicity of polymyxins is considered to be effected by accumulation of polymyxin in the epithelial cells in the kidneys leading to cell death triggered by apoptosis. Accordingly, there exists a need to develop polymyxin derivatives that have a similar or better efficacy as the currently used polymyxin, and at the same time a lower toxicity.

SUMMARY OF THE INVENTION

The present inventors have identified polymyxin derivatives having reduced toxicity relative to colistin sulfate while at the same time retaining the efficacy against gram- negative bacteria. The present invention relates to compounds having the structure and absolute configuration of Formula I:

wherein

R may be selected from C₆-Ci₄ aryl substituted by one or more electron withdrawing groups, or C1-C12 heteroaryl, optionally substituted by one or more electron withdrawing groups;

n may be 0, 1 , 2, 3 or 4;

R₃ may be selected from -CH₂OH, -CH₂NH₂ or -CH2CH2N H2;

R₆ may be selected from -CH₂Phenyl or -CH₂CH(CH₃)2;

R₇ may be selected from -CH₂CH(CH₃)₂, -CH(OH)CH₃, -CH(CH₃)₂, -CH₂CH₂CH₃ or -CH(CH₃)CH₂CH₃;

or a pharmaceutically acceptable salt thereof.

One embodiment of the invention relates to pharmaceutical compositions comprising compounds or pharmaceutically acceptable salts thereof as described herein and at least one pharmaceutically acceptable carrier and/or diluent. One embodiment relates to compounds or pharmaceutically acceptable salts thereof as described herein for use as a medicament. One embodiment relates to compounds or pharmaceutically acceptable salts thereof as described herein, for use in the treatment or prevention of gram-negative bacterial infections, including infections by multi drug resistant gram-negative bacteria. One embodiment relates to a method of treating or preventing a bacterial infection in a mammal, comprising administering a therapeutically effective amount of

compounds or pharmaceutically acceptable salts thereof as described herein to the mammal. One embodiment relates to compounds or pharmaceutically acceptable salts thereof as described herein, in the manufacture of a medicament for the prevention or treatment of a bacterial infection.

One embodiment relates to a kit comprising a therapeutically effective amount of a compound as described herein or a pharmaceutically acceptable salt thereof and instructions for use thereof.

DETAILED DISCLOSURE OF THE INVENTION

Polymyxins are cationic molecules which bind to anionic lipopolysaccharide (LPS) molecules within the outer membranes of Gram-negative bacteria. The initial association of polymyxins with the bacterial membrane occurs through electrostatic interactions between the cationic polypeptide and anionic LPS molecules in the outer membrane of the Gram-negative bacteria. Polymyxins displace magnesium (Mg²⁺) and calcium (Ca²⁺), from the negatively charged LPS, leading to a local disturbance of the outer membrane. The result of this process causes an increase in the permeability of the cell envelope consisting of the cell wall and the cytoplasmic membrane, leakage of cell contents, and subsequently cell death.

It is well-known that polymyxin nonapeptides, that may be derived from enzymatic removal of the polymyxin fatty acyl-Dab 1 , retains the ability of binding to bacterial LPS and attenuates the toxicity expressed by the parent polymyxin. The latter can probably be explained by lower absorption into the cells of the patient due to the decreased lipophilic character of the nonapeptides. However, the antimicrobial activity of such polymyxin nonapeptides is very low.

The present inventors have surprisingly found that removing the L-Dab at position in a naturally occurring polymyxin and instead attaching an amino acid having a D- configuration to the N-terminal of the polymyxin nonapeptide, provides compounds having a lower toxicity and a MIC in the same range or even better than Polymyxin E and B. Every amino acid besides glycine can occur in at least two stereoisomeric forms because of the stereogenic alpha carbon atom. Most naturally occurring amino acids (with the exception of threonine and isoleucine) have only one stereogenic center, i.e. they may occur as two different enantiomers. These forms are denoted D- and L-forms or configurations based on the direction in which they are able to rotate plane polarized light.

Most naturally occurring amino acids are found in the L-configuration and naturally occurring polymyxins contain L-Dab in position 1.

In one embodiment, the present invention concerns polymyxin derivatives wherein the o amino acid residue at osition 1 has a D-configuration.

The D-configuration corresponds to the ^-configuration when the four substituents attached to the a carbon consist of an amino functional group, a hydrogen atom, a carbonyl group and a side chain with lower priority than the carbonyl group, whereas it corresponds to the S-configuration when the four substituents attached to the a carbon consist of an amino functional group, a hydrogen atom, a carbonyl group and a side chain with higher priority than the carbonyl group. As mentioned above, naturally occurring polymyxins contain L-Dab in position 1 ; thus the ocarbon of this amino acid in naturally occurring polymyxin has S-configuration. The polymyxin derivatives according to the invention comprise an oamino acid residue at position 1 with D-configuration.

Definitions

The terms "naturally produced polymyxins" or "naturally occurring polymyxins" are intended to mean polymyxins produced by Paenibacillus polymyxa. A naturally produced polymyxin is a branched cyclic decapeptide containing a heptapeptide ring and an N- terminal amide coupled fatty acid. The N-terminal amino acid residue is referred to as amino acid position 1. The amino acid residue in position 10 forms a lariate structure with the amino acid residue in position 4. Accordingly, the N-terminal fatty acyl group is amide- coupled to L-Dab residue in position 1 and the threonine residue in position 10 is amide coupled to L-Dab residue in position 4 in naturally occurring polymyxins. The naturally produced polymyxins encompass Polymyxin A1 , Polymyxin A2, Polymyxin B1 , Polymyxin lle-B1 , Polymyxin B2, Polymyxin B3, Polymyxin B4, Polymyxin B5, Polymyxin B6, Polymyxin C, Polymyxin D1 , Polymyxin D2, Polymyxin E1 , Polymyxin E2, Polymyxin E3, Polymyxin E4, Polymyxin E7, Polymyxin lle-E1 , Polymyxin lle-E2, Polymyxin lle-E8, Polymyxin Nval-E1 , Polymyxin Val-E1 , Polymyxin Val- E2, Polymyxin M, Polymyxin P1 and Polymyxin P2.

The positions of the amino acids residues are depicted as (n) above or below the residues:

7 - MB, 7 - NH₂ Table A below shows the variations of R-i , R₆ and R₇ in the major components of polymyxin B and E

Table A

By the term "absolute configuration" is intended to mean the spatial arrangement of atoms of a chiral molecular entity. When the term "absolute configuration" is used in the present claims it refers to the configuration as depicted in Formula I.

An amino acid residue is defined as the unit in a peptide which comprises

-NH-CHR-COOH when it is a C-terminal residue

NH₂-CHR-CO- when it is an N-terminal residue

-NH-CHR-CO- when it is an internal residue

wherein R is the side chain of the amino acid residue.

By the term "oamino acid" is meant an amino acid containing an amino group attached directly to the alpha carbon, i.e. the backbone carbon atom neighboring the carboxylic group. The "side chain" of an oamino acid residue is -H for glycine, -CH₂OH for serine, -CH₃ for alanine etc. Accordingly, the "side chain" of an oamino acid residue is the moiety attached to the a carbon.

By the term "oamino acid residue at position 1 " is meant the N-terminal amino acid residue. By the term "L-configuration" is meant the configuration of an amino acid which is similar to L-glyceraldehyde as a standard. The term is used interchangeable with L-enatiomer, L- isomer and L-form. By the term "D-configuration" is meant the configuration of an amino acid which is similar to D-glyceraldehyde as a standard. The term is used interchangeable with D-enatiomer, D-isomer and D-form

The term "alkyl" as used herein encompasses linear aliphatic hydrocarbons, such as e.g. methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl.

By the term "aryl" is meant any aromatic hydrocarbon ring structure comprising only carbon atoms in the ring structure. By the term "C₆ - Ci₄ aryl" is meant an aryl comprising from 6 to 14 carbon atoms in an aromatic ring structure.

By the term "heteroaryl" is meant any aryl, including mono- and bicyclic heteroaryls, comprising at least one nitrogen, oxygen or sulphur atom in the ring system, and wherein at least one ring is an aromatic ring.

The term C₁-C₁₂ heteroaryl means a heteroaryl, including mono- and bicyclic heteroaryl, comprising between 1 and 12 carbon atoms in addition to at least one nitrogen, oxygen or sulfur atom. Examples of monocyclic heteroaryls include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxidiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl and triazinyl. Bicyclic heteroaryls may consist of a monocyclic heteroaryl fused to a phenyl or two monocyclic heteroaryls fused together. Examples of bicyclic heteroaryls includes benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzoxazolyl,

benzothiazolyl, furopyridinyl, indolyl, isoquinolinyl, naphthyridinyl, phthalazinyl,

pyrrolopyridinyl, quinazolinyl, quinolinyl, quinoxalinyl and thienopyridinyl.

By the term "electron withdrawing group" is meant an atom or functional group that draws electron density from neighboring atoms towards itself by resonance or inductive electron withdrawal. I.e. in this case it means a group that reduces the electron density of the aryl or heteroaryl by resonance or inductive electron withdrawal.

By the term "Dab" is meant the compound 2,4-diaminobutyric acid, but when present in a peptide sequence it represents the 2,4-diaminobutyric acid residue.

By "Thr" is meant threonine, but when present in a peptide sequence it represents the threonine residue. By "Phe" is meant phenylalanine, but when present in a peptide sequence it represents the phenylalanine residue.

By "Leu" is meant leucine, but when present in a peptide sequence it represents the leucine residue.

By "He" is meant isoleucine, but when present in a peptide sequence it represents the isoleucine residue.

The terms "optional" or "optionally" as used herein means that a subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, "optional bond" means that the bond may or may not be present, and that the description includes single, double, or triple bonds. The term "salts" or "salt thereof" as described herein, refers to a compound

comprising a cation and an anion, which can be prepared by any process known to one of ordinary skill, e.g., by the protonation of a proton-accepting moiety and/or

deprotonation of a proton-donating moiety. Alternatively, the salt can be prepared by either a cation/anion metathesis or a cation/anion exchange reaction.

The term "more than X% of a compound as measured by a validated HPLC-method " as described herein is to be understood as the relative integrated area of the pertaining peak in the chromatogram resulting from such HPLC method. An example of a suitable HPLC method is described in WO2014/195405. A "composition" is any mixture of more than one type of compound. E.g., a mixture of a polymyxin and an excipient, a mixture of a polymyxin and another active ingredient or a mixture of two polymyxins.

A "pharmaceutical composition" is any composition suitable for use in vivo either directly or after reconstitution or after dilution.

The term "effective amount" as used herein means an amount of the compound or composition required to provide the desired effect in a patient, i.e. reduce symptoms of a bacterial infection, decrease the number of bacteria in the affected tissue, and/or prevent bacteria in the affected tissue from increasing in number. The dose will be adjusted to the individual requirements in each particular case. That dosage can vary within wide limits depending upon numerous factors such as the severity of the disease to be treated, the age and general health condition of the patient, other medicaments with which the patient is being treated, the route and form of administration and the preferences and experience of the medical practitioner involved.

The term "patient" or "subject" refers to a warm-blooded animal such as, e.g. a human, livestock, a dog, a cat, a rabbit or a monkey who is receiving a compound or composition according to the invention.

In one embodiment the invention relates to a compound having the structure and absolute configuration of Formula I:

wherein

R is selected from C₆-Ci₄ aryl substituted by one or more electron withdrawing groups, or C1-C12 heteroaryl, optionally substituted by one or more electron withdrawing groups; n is 0, 1 , 2, 3 or 4;

R₃ is selected from -CH₂OH, -CH₂NH₂ or -CH₂CH₂NH₂;

R₆ is selected from -CH₂Phenyl or -CH₂CH(CH₃)₂;

R₇ is selected from -CH₂CH(CH₃)₂, -CH(OH)CH₃, -CH(CH₃)₂, -CH₂CH₂CH₃ or -CH(CH₃)CH₂CH₃;

or a pharmaceutically acceptable salt thereof.

In another embodiment the invention relates to a compound having the structure and configuration of Formula II:

wherein the alpha amino acid residue 1 has a D-configuration

and wherein

R is selected from C₆-C₁₄ aryl substituted by one or more electron withdrawing groups, or C-I-C-I2 heteroaryl, optionally substituted by one or more electron withdrawing groups; n is 0, 1 , 2, 3 or 4;

R₃ is selected from -CH₂OH, -CH₂NH₂ or -CH₂CH₂NH₂;

R₆ is selected from -CH₂Phenyl or -CH₂CH(CH₃)₂;

or a pharmaceutically acceptable salt thereof.

Accordingly, the invention relates to decapeptides comprising a nonapeptide structure obtained from polymyxin B or E, but wherein the N-terminal fatty acid tail and the L-Dab amino acid at position 1 has been removed and exchanged with an amino acid having a D-configuration.

The inventors have surprisingly found specific derivatives of polymyxin, in which the fatty acid tail and L-Dab at position 1 have been removed and exchanged with an amino acid moiety having a D-configuration, which are less toxic than colistin sulfate while retaining their efficacy. The inventors have further found that certain side chains of the amino acid at position 1 are providing compounds having a good efficacy and at the same time a low toxicity.

Accordingly, in one embodiment the invention relates to compounds as described herein, wherein R is a C₆-Ci₄ aryl substituted by one or more electron withdrawing groups.

In one embodiment R is a phenyl group substituted by one or more electron withdrawing groups. In one embodiment the invention relates to compounds as described herein, wherein R is a C -C heteroaryl, optionally substituted by one or more electron withdrawing groups.

In one embodiment, R is a pyridyl, pyrimidinyl or thienyl group, optionally substituted by one or more electron withdrawing groups.

In one embodiment, R is a pyridyl, pyrimidinyl or thienyl group, substituted by one or more electron withdrawing groups.

The one or more electron withdrawing group may be selected from halides and haloalkyls, including trihalides, such as, e.g. trihalomethyl, formyl, alkanoyl, carboxyl, alkanoyloxy, carbamoyl, cyano, triflate, triflyl, sulfonyl, quaternary ammonium and nitro groups.

In one embodiment the invention relates to the compounds as described herein, wherein the one or more electron withdrawing groups are one or more halides.

In one embodiment the invention relates to the compounds as described herein, wherein the one or more halides are selected from CI, Br, F and I.

In one embodiment the invention relates to the compounds as described herein, wherein the one or more electron withdrawing groups are one or more trihalomethyl groups.

In one embodiment the invention relates to the compounds as described herein, wherein the one or more trihalomethyl groups are selected from CF₃ and CCI₃. In one embodiment the invention relates to the compounds as described herein, wherein the one or more trihalomethyl groups are CF₃.

In one embodiment the invention relates to compounds as described herein, wherein R is a phenyl, pyridyl, pyrimidinyl or thienyl group substituted by one or more halides.

In one embodiment the invention relates to compounds as described herein, wherein R is phenyl, pyridyl, pyrimidinyl or thienyl group substituted by one or more haloakyl groups. In one embodiment the invention relates to compounds as described herein, wherein R is phenyl, pyridyl, pyrimidinyl or thienyl group, substituted by one or more trihalomethyl groups.

In one embodiment, the invention relates to compounds as described herein, wherein R is a phenyl group substituted by one or more halides.

In one embodiment, the invention relates to compounds as described herein, wherein R is a phenyl group substituted by one or more trihaloalkyl groups. In one embodiment, the invention relates to compounds as described herein, wherein R is a phenyl group substituted by one or more CF₃ groups.

In one embodiment, the invention relates to compounds as described herein, wherein R is a phenyl group substituted by a halide or haloalkyl in the para-position.

In one embodiment, the invention relates to compounds as described herein, wherein R is a phenyl group substituted by a CF₃ group in the para-position.

The R-group may be directly attached to the alpha carbon atom. Accordingly, in one embodiment, the invention relates to a compound as described herein, wherein n is 0.

The R group may also be attached to the alpha carbon atom via one or more

methylenegroups. In one embodiment, the invention relates to a compound according to the invention, wherein the R group is attached to the alpha carbon atom via one methylene group, i.e. wherein n is 1 .

The compounds according to the invention may comprise different amino acid residues at position 3, 6 and 7, selected from serine, 2,3-diaminopropionic acid and 2,4-dibutyric acid for position 3, phenylalanine and leucine for position 6 and leucine, threonine, valine and norvaline and isoleucine for position 7.

In one embodiment, the compounds according to the invention comprises a 2,4- diaminobutyric acid residue at position 3, i.e. R₃ may be -CH₂CH₂NH₂ as in the naturally occurring polymyxins E and B.

In one embodiment, the compounds according to the invention may comprise a phenylalanine residue at position 6, i.e. R₆ may be -CH₂Phenyl as in the naturally occurring polymyxin B, such as, e.g. polymyxin B1 , B2, B1-i and B3.

In one embodiment, the compounds according to the invention may comprise a leucine residue at position 6, i.e. R₆ may be -CH₂CH(CH₃)₂ as in the naturally occurring polymyxin E, such as, e.g. polymyxin E1 , E2, E1-i, E3 and E1-7.

In one embodiment, the compounds according to the invention may comprise a leucine residue at position 7, i.e. R₇ may be -CH₂CH(CH₃)₂ as in the naturally occurring polymyxin B1 , B2, B3, E1 , E2, E3 and E1 -7. In one embodiment, the compounds according to the invention may comprise a isoleucine residue at position 7, i.e. R7 -CH(CH₃)CH₂CH₃ as in the naturally occurring polymyxin E1-I and B1-i.

In one embodiment, the compounds according to the invention are based on polymixin E and comprise a leucine at position 6 and a leucine or isoleucine at position 7.

In one embodiment the compounds according to the invention comprise a leucine at position 6 and a leucine at position 7. In one embodiment, the compounds according to the invention are based on polymyxin B and comprise a phenylalanine at position 6 and a leucine or isoleucine at position 7. In one embodiment, the compounds according to the invention are based on polymyxin B and comprise a phenylalanine at position 6 and a leucine at position 7.

In one embodiment, the compounds according to the invention are selected from the following:

O

Thr-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr Thr-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr

or a pharmaceutically acceptable salt thereof.

one embodiment the compounds are selected from the following

"Thr-Dab-Dab-Dab- D-Leu-Leu-Dab-Dab-Thr

NH

^■-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr

Thr-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr

or a pharmaceutically acceptable salt thereof.

In one embodiment the compounds are selected from the following:

Thr-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr

NH₂

or a pharmaceutically acceptable salt thereof. Preparation

Polymyxin B and Polymyxin E are commercially available as the sulfate salts from a variety of sources, including Xellia Pharmaceuticals and Sigma-Aldrich.

Polymyxin nonapeptides from Polymyxin E or Polymyxin B can be achieved by enzymatic digestion of the polymyxin by cysteine endopeptidases such as ficin and papain, where the fatty acyl-Dab 1 fragment is selectively cleaved off the parent molecule (see e.g. Agr. Biol. Chem, vol 37 (1 1 ) 1973 Chihara et al and Antimicrobial Agents and Chemotherapy, vol 30 no. 2 1986 by Duwe et al.) followed by attachment of a suitable amino acid to the N-terminal part of the nonappetide. The compounds according to the invention may also be produced by solid phase synthesis using commonly known techniques.

If the compounds according to the invention require purification, HPLC or reversed-phase HPLC may be used e.g. as described in WO2014/195405.

One embodiment is directed to compositions comprising 70-100% of the compounds described herein as measured by validated HPLC methods. In one embodiment the compositions may comprise more than 75% of the compounds described herein as measured by validated HPLC methods.

In one embodiment, the compositions may comprise more than 80% of the compounds described herein as measured by validated HPLC methods.

In one embodiment, the compositions may comprise more than 85% of the compounds described herein as measured by validated HPLC methods.

In one embodiment the compositions may comprise more than 90% of the compounds described herein as measured by validated HPLC methods.

In one embodiment the compositions may comprise more than 95% of the compounds described herein as measured by validated HPLC methods. In one embodiment the compositions may comprise more than 97% of the compounds described herein as measured by validated HPLC methods.

In one embodiment the compositions may comprise more than 98% of the compounds described herein as measured by validated HPLC methods.

In one embodiment the compositions may comprise more than 99% of the compounds described herein as measured by validated HPLC methods. The charge of the compounds of the present invention will vary depending on the environment in which they appear. E.g. in aqueous solution, the pH will affect the degree of protonation of the amino and carboxylic acid groups. Thus the structures representing the compounds are simplified illustrations of the actual compounds. The present invention will cover any solid or dissolved basic and acidic salt forms , especially pharmaceutically acceptable salts of the claimed compounds.

Pharmaceutically acceptable base additional salts may be prepared from inorganic and organic bases. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium and magnesium slats. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally-occurring substituted amines, and cyclic amines, including isopropylamine, trimethyl amine, diethylamine, trimethylamine, tripropylamine, ethanolamine, 2-demethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N- alkylglucamine, theobromine, purines, piperazine, piperidine and N-ethylpiperidine.

Pharmaceutically acceptable acid additional salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloride acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Salts derived from organics 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.

In one embodiment the pharmaceutically acceptable salt is prepared from sulphuric acid, hydrochloride or acetic acid.

Further covered is any ionic species of the claimed compounds to be found in any solid or liquid state. Even further covered is any solid state of the compounds including amorphous material, crystals, co-crystals and solvates.

Pharmaceutical compositions In one embodiment, the compounds and salts as described herein may be used as a medicament.

The compounds or compositions may be formulated for administration by any route known in the art, such as subdermal, inhalation, oral, topical or parenteral. The compounds and compositions may be in any form known in the art including but not limited to liquid preparations, powders, granules, tablets, capsules, lozenges or creams.

The pharmaceutical composition according to the invention may comprise one or more pharmaceutically acceptable excipients and/or carriers and/or adjuvants and/or diluents.

The pharmaceutically acceptable excipient may further include a preservative, a buffer and/or an antioxidant. Examples of carriers and excipients include corn starch, gelatin, lactose, surose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acic. Suitable diluents include, but are not limited to, water for injection, 0.9% NaCI, 5% dextrose in 0.9% NaCI, 5% dextrose in water, 5% dextrose in 0.45% NaCI, 5% dextrose in 0.225% NaCI and Lactated Ringer's solution.

The pharmaceutical composition may be a lyophilized product capable of being reconstituted using a suitable diluent for topical (e.g. by inhalation using for example a nebulizer or other such inhalation device) or parenteral administration (e.g. intravenous administration). For parenteral administration fluid unit dosage forms are prepared utilizing the compound or composition and a sterile vehicle. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle or other suitable solvent. Examples of suitable solvents and vehicles include water, ethanol, benzyl alcohol, polyols (such as, e.g. glycerol, propylene glycol and polyethylene glycol) and suitable mixtures thereof.

Pro -drug

In one aspect of the invention the compound according to the invention may be formulated as a prodrug. One example of a prodrug is a compound according to the invention wherein one or more amino groups have been protected by a group which can be cleaved off in vivo. An example of a group which can be used to protect one or more amino group is sulfomethyl, (-CH₂S0₃), providing a compound according to the invention having between 1 to 8 sulfomethyl groups attached to the γ-amino groups on the DAB residues.

Kits

An embodiment of the invention relates to a kit comprising a compound according to the invention or a composition comprising a compound according to the invention and instructions for use, e.g. written instructions of how to administer the compound or composition according to the invention. The compound or composition and the instructions may be provided in a suitable container or package.

Use

The compounds, salts and compositions described herein may be used for the treatment and/or prevention of bacterial infections, such as, e.g. gram-negative bacterial infections. The infections may include those caused by susceptible and multi-drug resistant gram- negative bacteria, including, but not limited to, infections caused by Acinetobacter spp., Acinetobacter baumannii, Aeromonas spp., Bordetella spp., Borrelia spp., Campylobacter spp., Citrobacter spp., Enterobacter spp., Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Francisella tularensis, Fusobacterium spp., Helicobacter pylori,

Hemphilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella

pneumophila, Moraxella catarrhalis, Morganella morganii, Neisseria gonorrhoeae, Neisseria meningitides, Proteus vulgaris, Porphyromonas spp., Prevotella soo.,

Pasteurella spp., Proteus mirabilis., Pseudomonas aeruginosa, Salmonella spp., Shigella app., Vibrio spp. and Yersinia spp. In one embodiment the gram-negative bacteria are selected from the group consisting of Acinetobacter baumannii, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.

In one embodiment the the gram-negative bacteria is selected from Acinetobacter baumannii, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, wherein the bacterial strain is resistant to at least one antibacterial drug. In one embodiment the the gram-negative bacteria is selected from Acinetobacter baumannii, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae and

Pseudomonas aeruginosa, wherein the bacterial strain is multi-drug resistant. Examples of infections that may be treated with the compounds and compositions according to the invention include urinary tract infections, systemic infections,

gastrointestinal infections, lung infections, such as pneumonia (including hospital acquired pneumonia), and chronic lung infections in patients with cystic fibrosis or non-CF bronchiectasis.

Dosage

The invention also encompasses administering to a subject in need thereof a

therapeutically effective dose of a compound or a pharmaceutically acceptable salt thereof according to the invention. The selected dosage level will depend on the activity of the compound, the route of administration, the severity of the condition being treated, the age, body weight, gender and health of the subject, the tolerance of the subject to the compound, rate of excretion of the compound and the type of microorganism involved in the infection. In one embodiment, the pharmaceutical composition may be administered once per day. However, if desired, the daily dosage may be divided into multiple doses, e.g. two to six separate doses per day. The treatment regime may require administration for several day or even for weeks. Administration route

Any route typically used to treat bacterial infections may be used to administer the compounds or compositions according to the invention, including oral, parenteral, topical, intestinal, rectal and transmucosal administration. Examples of parenteral administration routes encompass intravenous, intramuscular, intradermal, intranasal, intraventricular and subcutaneous injections or infusion

techniques. Transmucosal administration encompasses nasal aerosol or inhalation applications. The present invention will now be described in further details in the following Examples. It is to be understood that the examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention in any way. EXAMPLES

Example 1 : Exemplified syntheses

Example 1 a: Preparation of Polymyxin E (Colistin) nonapeptide

Chemical Formula: C₄oH₇₆N₁₄Oi -|

Molecular Weight: 929.14

Polymyxin E1 (13.61 g, 1 1 .63 mmol) (isolated from colistin originating from Xellia

Pharmaceuticals, e.g. as described in WO201 1/051070) was suspended in a 0.1 M sodium phosphate buffer (900 ml_, pH 5.1 ). Potassium chloride (2.14 g, 28.71 mmol), disodium ethylenediaminetetraacetate dihydrate (3.55 g, 9.54 mmol) and L-cysteine (0.12 g, 0.99 mmol) were added and the pH was adjusted to 5.5 using 10% aqueous phosphoric acid. Finally, papain (4.08 g) was added, and the reaction was stirred at 37 °C. After 3 days, more papain (0.43 g) was added and the mixture was stirred for 3 more days. The pH was adjusted to 8 and the solution was lyophilized to give a white solid (46.88 g). An HPLC standard curve was used to estimate the amount of polymyxin E nonapeptide in the crude product (9.51 g, 88%). m/z: [M + Na]⁺ calculated for C4oH₇₆N₁₄OiiNa 951.6; found 951.5 Example 1 b: Preparation of tetra-Boc polymyxin E (Colistin) nonapeptide

Molecular Weight: 1329.60 Polymyxin E nonapeptide (9.51 g, 10.24 mmol) obtained from Example l awas suspended in water (160 mL), triethylamine (160 mL) and 1 ,4-dioxane (160 mL). The mixture was cooled to 0 °C, followed by addition of 2-(tert-butoxycarbonyloxyimino)-2- phenylacetonitrile (10.09 g, 40.97 mmol, 4 mol equiv). After 4 hours, the reaction was quenched by the addition of 7 M ammonia in methanol (160 mL) and left stirring overnight. The mixture was concentrated on a rotary evaporator and transferred to a separation funnel using dichloromethane (500 mL) and water (500 mL). The two phases were separated and the aqueous phase was extracted with dichloromethane (3 x 300 mL). The combined organic phases were dried (MgS0₄), filtered and concentrated on a rotary evaporator. The product was purified by multiple flash chromatography using silica gel and heptane/ethyl acetate (1 :1 ) followed by dichloromethane/methanol (9:1 ) to give the title compounds as a white solid (3.34 g, 25%). m/z: [M + Na]⁺ calculated for C₆oHio8Ni₄Oi₉Na 1351 .8; found 1351 .3 Example 1 c: Attachment of the N-terminal g-amino acid.

General procedure:

\) Coupling: The α-amino acid was dissolved in dimethylformamide followed by the addition of N,N- diisoproylethylamine, 2-(1 H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyl uranium

hexafluorophosphate (HBTU) and tetra-Boc polymyxin E nonapeptide. The reaction mixture was stirred at room temperature for 2-16 hours. The reaction mixture was diluted with ethyl acetate, and washed with 1 M aqueous hydrochloric acid, saturated aqueous sodium bicarbonate and brine. The organic phase was filtered through a phase separator for removal of water and concentrated on a rotary evaporator. ii) Boc deprotection:

To the above crude coupled intermediate was added a TFA/TIS/H₂0 (95:2.5:2.5, v/v/v) mixture. The resulting solution was stirred at room temperature for 2 hours, after which the volatiles were evaporated, and the residual oil was crystallized from diethyl ether, washed repeatedly with diethyl ether and dried under vacuum. iii) Purification:

All crude peptides were purified by reverse phase semi preparative HPLC utilizing a Ci₈- column (XSelect CSH™ Prep C18, 5 μηι, OBD, 19x150 mm) in combination with UV- detection at 215 nm. Elution was achieved with a gradient mixture consisting of water and acetonitrile, both of which contained 0.1 % trifluoroacetic acid. The purified peptides were lyophilized and analyzed by HPLC using a Cie-column (ACE III C18, 3 μηη, 4.6x150 mm) at 210 nm, eluting with a ramping mixture of 0.03 M sodium sulfate buffer (pH 2.3) and acetonitrile.

In Table 1 are shown examples of compounds prepared by the methods as described above. Compounds C1 , C2, C13, C14, C19 and C20 are for comparison reasons only and are not encompassed by the present invention: Table 1

Example 2: Minimal Inhibitory Concentration (MIC) testing

The purified compounds were tested for in vitro activity against a panel of four strains of Gram negative pathogens, i.e., Pseudomonas aeruginosa, Klebsiella pneumonia,

Acinetobacter baumannii, and Proteus mirabilis. The testing was performed based on EUCASTguidelines.

The test organisms were cultured in shake flasks in Mueller Hinton Broth (BD 275730) for approximately 16 hours at 250 rpm. They were recultured in Mueller Hinton Broth to a turbidity of 0.5 McFarland and further diluted in Mueller Hinton Broth to 1x10^s CFU/mL and frozen with glycerol at -75 °C. The test samples were prepared by dissolving the purified compounds in Mueller Hinton Broth in concentrations of 5 mg/mL. Two-fold serial dilutions were made in sterile 96-well plates using Mueller Hinton Broth. An equal part of bacterial suspension was added, giving the first dilution a concentration of 256 Mg/mL and the last one 0.125 Mg/mL. Plates with Pseudomonas aeruginosa were incubated at 32-34 °C overnight, whereas plates with Klebsiella pneumoniae, Acinetobacter baumannii and Proteus mirabilis were incubated at 37 °C with shaking overnight. The results are provided in Table 2.

Compounds C1 , C2, C13, C14, C19 and C20 are for comparison reasons only and are not encompassed by the present invention.

Table 2

From the results presented in Table 2, it can be concluded that the stereogenic center in o position of the a-amino acid residue 1 is highly important for the overall biological activity of the compound. The D-isomers (1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 22, 24, 26 and 28) are in each case more potent than their L-counterparts (2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 23, 25, 27 and 29, respectively). Furthermore, the results also indicate that electron-withdrawing groups in the para-position of the aromatic ring in a-amino acid residue 1 increases the biological activity of the compounds.

Example 3: In vitro renal cell toxicity assay

A selection of compounds (3, 5, 6, 15, 16, 21 , 24, 25, 26, 28 and 30) were monitored and analyzed for cytotoxic effects in a human kidney transformed cell line (HK-2: human papillomavirus 16 (HPV-16) transformed cell line from the proximal tube of kidney nephron).

HK-2 cells were seeded on 96 wells plates and cultured for 24 hours. They were then treated with the different conditions in the presence of a fluorescent DNA intercalating agent for the following 72 hours. Time-lapsing imaging was performed with a sampling rate of 1 image every 2 hours. The number of fluorescent/cytolytic cells was counted and reported.

Compounds 3, 5, 6, 15, 16, 24, 25, 26 and 28 were transformed to the HCI salt by dissolving them in 0.1 M aq. HCI followed by lyophilization prior to submitting them for testing.

Compounds 21 and 30 were obtained as acetate salts. They were suspended in complete culture medium to prepare 10 mg/mL stock solutions. The doses tested were 0, 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1.0 and 1.2 mg/mL (the highest tested concentration for compound 3 was 1.0 mg/mL). The different concentrations were prepared as a 2 times concentrated solution in the culture medium. The treatment was performed by adding 100 pL of the 2 times concentrated solutions to the 100 pL of culture medium already present in the well.

The cellular viability post-treatment was measured by analyzing the plasma membrane integrity based on the incorporation of a non-permeant and fluorescent DNA intercalating agent that selectively stained cytolytic cells with compromised plasma membranes. The cytotoxic effect of the different tested compounds was analyzed by reporting the kinetic of the cytolysis (cytolyzed cells numeration), the dose dependent cytotoxic effect (cytolyzed cells numeration) of the compound at different time point of the treatment, by quantifying the dose dependent effect of the compound (% of cytolyzed cells) at the end of the treatment and by counting the total number of seeded cells at the end of the treatment.

In order to compare the cytotoxic effect of the different compounds to that of colistin, they were ranked on the basis of the EC50 of cytolysis induction (percentage) at the end of each treatment (Table 3). All the compounds exhibited a dose dependent cytotoxic effect after 48 hours of treatment.

Table 3

Claims

1. A compound having the structure and absolute configuration of Formula I:

wherein

R is selected from C₆-Ci₄ aryl substituted by one or more electron withdrawing groups, or Ci- Ci₂ heteroaryl, optionally substituted by one or more electron withdrawing groups;

n is 0, 1 , 2, 3 or 4;

R₃ is selected from -CH₂OH, -CH₂NH₂ or -CH₂CH₂NH₂;

R₆ is selected from -CH₂Phenyl or -CH₂CH(CH₃)₂;

R₇ is selected from -CH₂CH(CH₃)₂, -CH(OH)CH₃, -CH(CH₃)₂, -CH₂CH₂CH₃ or

-CH(CH₃)CH₂CH₃;

or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 , wherein R is a C₆-Ci₄ aryl substituted by one or more electron withdrawing substituents, or Ci-Ci₂ heteroaryl, optionally substituted by one or more electron withdrawing groups, and wherein the one or more electron withdrawing groups are selected from halides, haloalkyi, formyl, alkanoyi, carboxyl, alkanoyloxy, carbamoyl, cyano, triflate, triflyl, sulfonyl, ammonium and nitro groups.

3. A compound according to claim 1 or 2, wherein the one or more electron withdrawing groups are one or more halides.

4. A compound according to claim 3, wherein the one or more halides are selected from CI, F and I.

5. A compound according to any of the preceding claims, wherein the one or more electron withdrawing groups are one or more trihalomethyl groups.

6. A compound according to claim 5, wherein the one or more trihalomethyl groups are selected from CF₃ and CCI₃.

7. A compound according to claim 6, wherein the one or more trihalomethyl groups are CF₃.

8. A compound according to any of the preceding claims, wherein R is selected from phenyl, pyridyl, pyrimidinyl and thienyl.

9. A compound according to claim 8, wherein R is a phenyl.

10. A compound according to claim 9, wherein R is a phenyl substituted by one or more halides.

1 1. A compound according to claim 9, wherein R is a phenyl substituted by one or more trihaloalkyl groups.

12. A compound according to claim 1 1 , wherein R is a phenyl group substituted by one or more CF₃ groups.

13. A compound according to claim 10-12, wherein R is a phenyl group substituted by a halide or haloalkyl in the para-position.

14. A compound according to claim 13, wherein R is a phenyl group substituted by CF₃ in the para-position.

15. A compound according to any of the preceding claims wherein n is 1.

16. A compound according to any of claims 1-14, wherein n is 0.

17. A compound according to any of the preceding claims wherein R₃ is -CH₂CH₂NH₂.

18. A compound according to any of the preceding claims wherein R₆ is -CH₂Phenyl.

19. A compound according to any of claims 1-17, wherein R₆ is -CH₂CH(CH₃)₂.

20. A compound according to any of the preceding claims wherein R₇ is -CH₂CH(CH₃)₂.

21 . A compound according to any of claims 1 -19, wherein R7 -CH(CH₃)CH₂CH₃.

22. A compound selected from the following

O

Thr-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr Thr-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr

or a pharmaceutically acceptable salt thereof.

23. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to any of claims 1 to 22, and at least one

pharmaceutically acceptable carrier and/or diluent.

24. A compound or a pharmaceutically acceptable salt thereof according to any of claims 1 to 22 for use as a medicament.

25. A compound or a pharmaceutically acceptable salt thereof according to claim 24, for use in the treatment or prevention of gram-negative bacterial infections, including infections by multidrug resistant gram-negative bacteria.

26. A compound or a pharmaceutically acceptable salt thereof according to claim 25, for use in the treatment or prevention of a bacterial infection caused by sensitive or multidrug resistant Enterobacteriacae, Pseudomonas aeruginosa and/or Acinetobacter baumannii.

27. A compound according to claim 25 or 26, wherein the infection is caused by a bacterial strain which is resistant to at least one antibacterial drug.

28. A method of treating or preventing a bacterial infection in a mammal, comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any of claims 1 to 23 to the mammal.

29. A method according to claim 28, wherein the infection is caused by sensitive or multi-drug resistant Enterobacteriacae, Pseudomonas aeruginosa and/or Acinetobacter baumannii.

30. Use of a compound or a pharmaceutically acceptable salt thereof according to any of claims 1 to 23, in the manufacture of a medicament for the prevention or treatment of a bacterial infection.

31 . A kit comprising a therapeutically effective amount of a compound according to any of claims 1 to 23 or a pharmaceutically acceptable salt thereof and instructions for use thereof.