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WO2024256834A1 - Analogues des teixobactines sous la forme d'agents anti-biofilm - Google Patents

Analogues des teixobactines sous la forme d'agents anti-biofilm Download PDF

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WO2024256834A1
WO2024256834A1 PCT/GB2024/051527 GB2024051527W WO2024256834A1 WO 2024256834 A1 WO2024256834 A1 WO 2024256834A1 GB 2024051527 W GB2024051527 W GB 2024051527W WO 2024256834 A1 WO2024256834 A1 WO 2024256834A1
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compound
side chain
group
use according
biofilm
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Ishwar Singh
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University of Liverpool
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/15—Depsipeptides; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid

Definitions

  • the present invention relates to new uses of teixobactin analogues and, in particular, the use of such compounds in the killing, inhibition or prevention of microbial biofilms.
  • Biofilm treatment or prevention is primarily useful in a clinical setting, particularly with patients that are suffering from biofilm infections, though the invention includes the treatment or prevention of biofilms in non-living materials, such as in medical devices.
  • Biofilms occur on both inert and living supports, in natural environments and in industrial installations.
  • a biofilm is a structured community of microorganisms encapsulated within a selfdeveloped polymeric matrix and adherent to a living or inert surface. Biofilms are also often characterized by surface attachment, structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances.
  • Single-celled organisms generally exhibit two distinct modes of behavior. The first is the familiar free floating, or planktonic, form in which single ceils float or swim independently in some liquid medium. The second is an attached state in which cells are closely packed and firmly attached to each other and usually form a solid surface. A change in behavior is triggered by many factors, including quorum sensing, as well as other mechanisms that vary between species. When a cell switches modes, it undergoes a phenotypic shift in behavior in which large suites of genes are up- and down-regulated.
  • Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists adhere to the surface initially through weak, reversible van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili. The first colonists facilitate the arrival of other cells by providing more diverse adhesion sites and beginning to build the matrix that holds the biofilm together. Some species are not able to attach to a surface on their own but are often able to anchor themselves to the matrix or directly to earlier colonists. It is during this colonization that the cells are able to communicate via quorum sensing. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment.
  • Biofilms are usually found on solid substrates submerged in or exposed to some aqueous solution, although they can form as floating mats on liquid surfaces and also on the surface of leaves, particularly in high humidity climates. Given sufficient resources for growth, a biofilm will quickly grow to be macroscopic.
  • Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae; each group performing specialized metabolic functions. However, some organisms will form monospecies films under certain conditions. Biofilms appear able to defend themselves against disinfectants and antibiotics, phagocytes and the human immune system. Extracellular matrix The biofilm is held together and protected by a matrix of excreted polymeric compounds called EPS. EPS is an abbreviation for either extracellular polymeric substance or exopolysaccharide. This matrix protects the cells within it and facilitates communication among them through biochemical signals. Some biofilms have been found to contain water channels that help distribute nutrients and signaling molecules.
  • biofilms can become fossilized. Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways.
  • One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased 1000 fold (see Stewart P, Costerton J, 2001, Lancet 358 (9276):135–8).
  • the tolerance of biofilms for antibiotics can be attributed to limited drug diffusion through the extra-cellular polymeric substances (EPS) and cell conversion from the planktonic to dormant state, with altered metabolic activities resulting in phenotypic antimicrobial tolerance.
  • EPS extra-cellular polymeric substances
  • the release of EPS and formation of the biofilm matrix is a fundamental step in the process of biofilm development, as it protects the cells from threats in the surrounding environment (e.g. immune cells and antibiotics) and provides mechanical and structural support.
  • the EPS mostly constitutes of proteins, polysaccharides, extracellular DNA and lipids, which have a wide range of functions including adhesion, water retention, structural integrity and enzymatic activity.
  • the biofilm matrix plays a role in antibiotic decomposition by enzymatic action, low pH and high concentration of metals. Bacterial cells inside the biofilm have been shown to be 1000 times more tolerant to antibiotics than planktonic cells in some cases.
  • Biofilms and infectious diseases Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one estimate 80% of all infections (see “Research on microbial biofilms (PA-03- 047)", NIH, National Heart, Lung, and Blood Institute, 2002-12-20). Infectious processes in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque, gingivitis, coating contact lenses, and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and heart valves.
  • biofilms are present on the removed tissue of 80% of patients undergoing surgery for chronic sinusitis.
  • the patients with biofilms were shown to have been denuded of cilia and goblet cells, unlike the controls without biofilms who had normal cilia and goblet cell morphology.
  • Biofilms were also found on samples from two of 10 healthy controls mentioned.
  • the species of bacteria from interoperative cultures did not correspond to the bacteria species in the biofilm on the respective patient's tissue. In other words, the cultures were negative though the bacteria were present.
  • Mortality relating to antimicrobial resistance (AMR) in 2019 was estimated as 1.27 million deaths based on data gathered from 204 countries.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Teixobactin is a recently-discovered depsipeptide antibiotic that acts through a novel mechanism of action (Ling L.L et al., Nature, 2015, 517, 455-459). Teixobactin inhibits bacterial cell wall synthesis by binding to precursors of essential cell wall components. As such, it is likely to induce resistance at a considerably slower rate than antibacterials that act at intracellular protein targets. Teixobactin’s unusual structure comprises D-amino acid residues, and an L-allo-enduracididine residue. The manufacture of Teixobactin in a commercial scale is difficult and expensive, in part due to the presence of the L-allo-enduracididine residue.
  • a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or clathrate thereof, for use in killing, inhibiting or preventing the growth of a microbial biofilm wherein: R 1 represents H, C 1-6 alkyl, C 1-6 acyl, benzyl or benzoyl; AA 1 represents any hydrophobic proteinogenic or hydrophobic non-proteinogenic amino acid; AA 2 , AA 5 and AA 6 each independently represents an isoleucine or an allo-isoleucine residue; AA 3 and AA 4 each independently represents a proteinogenic or non-proteinogenic amino acid; AA 7 represents a serine residue; R 8 represents hydrogen or C1-4 alkyl; R 9 represents a proteinogenic or non-proteinogenic amino acid side chain; R 10 represents, a hydrophobic proteinogenic or hydrophobic non-proteinogenic amino acid side chain; R 11 represents a proteinogenic or non-proteinogenic amino acid side chain; and Z
  • L-Chg10-teixobactin may be described as having the following structure:
  • the invention relates to a compound of formula (I), or a pharmaceutically-acceptable salt, solvate or clathrate thereof, as defined above, provided that the compound of formula (I), or the pharmaceutically-acceptable salt, solvate or clathrate thereof, is not: (R)-N1-((2R,3S)-1-(((2S,3S)-1-(((S)-1-(((3S,6S,9S,12R,13S)-3-((S)-sec-butyl)-6- cyclohexyl-9,13-dimethyl-2,5,8,11-tetraoxo-1-oxa-4,7,10
  • salts Compounds, salts, solvates, and clathrates of formula (I) are referred to hereinafter as the “compounds of the invention”.
  • pharmaceutically-acceptable salt we mean an acid addition or base addition salt suitable for use in pharmaceuticals.
  • Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of the invention with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration).
  • Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
  • suitable ion exchange resin examples include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic and arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.
  • mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids
  • organic acids such as tartaric, acetic, trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succin
  • solvate we mean a solid form wherein the relevant compound (e.g. a compound of formula (I)) is associated with one or more solvent molecules.
  • the term solvate includes hydrates and other solvates of pharmaceutically acceptable solvents.
  • a preferred solvent for solvate formation is DMSO.
  • clathrate we mean a solid form wherein the relevant compound (e.g. a compound of formula (I)) forms a lattice that contains a guest molecule (e.g. a pharmaceutically- acceptable solvent) within the lattice structure.
  • biofilm we include microbial (e.g.
  • amino acid and “residue” (for example D-phenylalanine “residue”) we mean the dehydrated portion of an amino acid present in polypeptide chains and represented by the following formula S. C. wherein S. C. represents an amino acid side chain.
  • amino acid includes non-proteinogenic amino acids unless otherwise specified.
  • amino acid side chain or “side chain of an amino acid” we mean the group attached WR ⁇ WKH ⁇ SRVLWLRQ ⁇ ⁇ WR ⁇ WKH ⁇ FDUER[ ⁇ O ⁇ DQG ⁇ DPLQR ⁇ JURXSV ⁇ LQ ⁇ ⁇ -amino acids, including non- SURWHLQRJHQLF ⁇ -amino acids and particularly proteinogenic amino acids.
  • proteogenic amino acids are the 22 amino acids that may be naturally encoded or naturally found in the genetic code of organisms.
  • Non-proteinogenic amino acids are those not naturally encoded or found in the genetic code of any organism.
  • the set of non- proteinogenic amino acids is generally considered to include all organic compounds with an amine (-NH2) and a carboxylic acid (-COOH) functional group linked via a single additional carbon atom, as well as a side chain and a hydrogen bound to that single additional carbon atom, but excluding selenocysteine, pyrrolysine and the 20 standard amino acids that are incorporated into proteins during translation.
  • Non-proteinogenic amino acids include those amino acids that are intermediates in biosynthesis, those that are post-translationally formed in proteins, and those that possess a physiological role (e.g. components of bacterial cell walls, neurotransmitters, and toxins).
  • references to hydrophobic non-proteinogenic amino acid side chains are references to hydrophobic side chains (particularly those formed primarily of alkyl and/or aryl groups in the absence of polar groups) which are capable of being bound to an amino acid backbone.
  • references to polar non-proteinogenic amino acid side chains are references to polar side chains (particularly those comprising a hydroxyl group or an amide functional group (e.g. wherein one or more of said groups is bound to the amino acid via a linear, branched, cyclic or part cyclic C1-8 alkylene group)) which are capable of being bound to an amino acid backbone.
  • the amino acid residues may be provided in their naturally occurring stereochemical configuration (e.g. the l-configuration), or the alternative stereochemical configuration.
  • L 2 represent linking groups which form a bridge between two separate portions of the molecule.
  • such linking groups include -OC(O)-.
  • the left-hand hyphen in such linking groups represents the point of attachment to L 1
  • the right-hand hyphen in such linking groups represents the point of attachment to L 3 .
  • alkyl groups as defined herein may be straight-chain or, when there is a sufficient number (i.e. a minimum of three) of carbon atoms, be branched-chain and/or cyclic. Further, when there is a sufficient number (i.e.
  • alkyl groups may also be part cyclic/acyclic. Such alkyl groups may also be saturated or, when there is a sufficient number (i.e. a minimum of two) of carbon atoms, be unsaturated. Unless otherwise specified, alkyl groups may also be substituted with one or more halo, and especially fluoro, atoms. Unless otherwise specified, alkylene groups as defined herein may be straight-chain or, when there is a sufficient number (i.e. a minimum of two) of carbon atoms, be branched- chain. Such alkylene chains may also be saturated or, when there is a sufficient number (i.e. a minimum of two) of carbon atoms, be unsaturated.
  • alkylene groups may also be substituted with one or more halo atoms.
  • heterocyclyl group refers to a 3- to 6- membered aromatic, saturated or part saturated heterocyclic ring containing one or two heteroatoms selected from O and N.
  • acyl refers to alkyl groups having a carbonyl group attached to the carbon which forms the point of attachment to the rest of the molecule.
  • R 1 represents H, C 1-6 alkyl, C 1-6 acyl, benzyl or benzoyl.
  • R 1 may represent H, C 1-4 alkyl, C 1-4 acyl, benzyl or benzoyl.
  • R 1 represents H, methyl, or acetyl; preferably methyl.
  • AA 1 represents any hydrophobic proteinogenic amino acid, such as alanine, valine, leucine, isoleucine, methionine, tyrosine, tryptophan or, particularly, phenylalanine.
  • AA 1 represents an L- or D- phenylalanine residue; preferably a D-phenylalanine residue.
  • AA 2 , AA 5 and AA 6 each individually represents an L-, L-allo-, D- or D-allo-isoleucine residue.
  • AA 2 and AA 6 each independently represents an L- or D-isoleucine residue, preferably an L-isoleucine residue.
  • AA 5 represents a D-allo-isoleucine or a D- isoleucine residue.
  • AA 7 represents an L- or D-serine residue, preferably an L-serine residue.
  • Adjacent AA groups may be linked together via an amide bond between the C1 (carbon number one) of one amino acid with the nitrogen attached to the alpha carbon in the adjacent amino acid, as in a so-called eupeptide bond.
  • any adjacent AA groups may be linked via an isopeptide bond; that is, the side chain of at least one of these amino acids may form part of the backbone of the polypeptide chain.
  • a serine residue e.g.
  • isopeptide bonds may be advantageous for one or more properties of the compound of the invention as, for example, it may improve the solubility of the compound. Isopeptide bonds are also capable of being converted into eupeptide bonds under suitable conditions.
  • the AA 6 and AA 7 groups are linked via a eupeptide or O-acyl isopeptide bond, preferably via a eupeptide bond.
  • the AA 2 and AA 3 groups are linked via a eupeptide or O-acyl isopeptide bond, preferably via a eupeptide bond.
  • the AA 6 and AA 7 groups are linked via an O- acyl isopeptide bond, and/or the AA 2 and AA 3 groups are linked via an O-acyl isopeptide bond.
  • O-acyl isopeptide bonds are formed via the oxygen atom in the HO-CH 2 - side chain of a serine residue in the AA 7 or AA 3 position.
  • AA 2 represents an L-isoleucine residue
  • AA 5 represents a D-allo-isoleucine or D-isoleucine residue
  • AA 6 represents an L-isoleucine residue
  • AA 7 represents an L-serine residue
  • AA 1 , AA 3 and AA 4 may be varied as described herein.
  • the serine and glutamine residues normally present at these positions in native teixobactin may be more readily substituted.
  • the solubility of the compound can be improved by using a polar amino acid at one or both of these positions (and also at R 9 ).
  • Uncharged polar amino acids such as serine, threonine, asparagine and glutamine may therefore be used.
  • Particular amino acids that may be mentioned in this respect are positively charged amino acids, such as arginine, histidine, lysine, ornithine, 2,3-diaminopropanoic acid, and 2,4-diaminobutyric acid.
  • Other similar structures that are described herein may also be used at these positions.
  • the side chain of AA 3 represents a moiety selected from the group consisting of a positively charged amino acid side chain, -CH 2 -NH 2 , -(CH 2 ) 2 - NH 2 , -(CH 2 ) 3 -NH 2 , and a fragment of formula -L 1 -L 2 -L 3 -X 1 ; wherein L 1 represents a linear or branched C1-12 alkylene linker; L 2 is selected from the group consisting of -O-, -N(X a )-, -[N(X a ) 2 ] + -, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)N(X a )-, -OC(O)N(X a )-, -NHC(O)O-, or -NHC(O)N(X a )-;
  • X 1 represents a
  • the side chain of AA 3 represents a side chain of an amino acid selected from the group consisting of arginine, lysine, histidine, ornithine, 2,3-diaminopropanoic acid, and 2,4-diaminobutyric acid; preferably arginine, even more preferably L-arginine.
  • AA 3 represents an L- or D-serine residue, preferably an L-serine residue.
  • the side chain of AA 4 may represent a moiety selected from the group consisting of a positively charged amino acid side chain, -CH 2 -NH 2 , -(CH 2 ) 2 -NH 2 , -(CH 2 ) 3 -NH 2 , and a fragment of formula -L 1 -L 2 -L 3 -X 1 as defined hereinabove.
  • the side chain of AA 4 represents a side chain of an amino acid selected from the group consisting of arginine, lysine, histidine, ornithine, 2,3-diaminopropanoic acid, and 2,4- diaminobutyric acid; preferably arginine, even more preferably D-arginine.
  • AA 4 represents an L- or D-glutamine residue, preferably a D- glutamine residue. While it may be advantageous for at least one of the side chains of AA 3 and AA 4 to be a moiety selected from the group consisting of positively charged amino acid side chains, - CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, and a fragment of formula -L 1 -L 2 -L 3 -X 1 as defined hereinabove, in one embodiment the side chains of both AA 3 and AA 4 are (independently) moieties selected from this group. Compounds of the invention have hydrophobic groups elsewhere on the molecule, e.g.
  • AA 1 may represent an L- or D-phenylalanine residue
  • AA 2 may represent an L- or D-isoleucine residue
  • AA 5 may represent an L-, L-allo-, D- or D-allo-isoleucine residue
  • AA 6 may represent an L- or D-isoleucine residue
  • AA 7 may represent an L- or D-serine residue.
  • R 8 represents hydrogen or C1-4 alkyl, preferably hydrogen or a methyl group, even more preferably a methyl group. Irrespective of the group that R 8 represents, it is preferred that the carbon to which this group is attached is in the D-configuration, i.e.
  • R 9 represents a proteinogenic or non-proteinogenic amino acid side chain.
  • R 9 represents -CH 2 -NH 2 , -(CH 2 ) 2 -NH 2 , -(CH 2 ) 3 - NH 2 , a hydroxy group, an amide functional group (which latter two groups are bound to the remainder of the molecule via a linear, branched, cyclic or part cyclic C 1-8 alkylene group), the side chain of an amino acid selected from the group consisting of histidine, lysine, arginine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, norvaline, cyclohexylglycine, cyclohexylalanine, phenylglycine, biphen
  • R 9 represents a moiety selected from the group consisting of a positively charged amino acid side chain, -CH 2 -NH 2 , -(CH 2 ) 2 -NH 2 , -(CH 2 ) 3 - NH 2 , and a fragment of formula -L 1 -L 2 -L 3 -X 1 as defined hereinabove.
  • R 9 represents a side chain of an amino acid selected from the group consisting of arginine, lysine, and histidine; preferably arginine. Alternatively, R 9 may represent methyl, in line with the structure of teixobactin.
  • R 10 represents a hydrophobic proteinogenic or hydrophobic non-proteinogenic amino acid side chain. Preferably R 10 is a hydrophobic side chain bound to the rest of the molecule in the L-configuration. Particular hydrophobic side chains that may be mentioned include C 1-6 alkyl groups (e.g.
  • hydrophobic side chains that may be mentioned include -C1-6 alkylene-NH(R 10a ) and -C1-6 alkylene-NHCH2(R 10a ), wherein R 10a represents a C1-6 alkyl group (e.g. methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopentyl or cyclohexyl) or phenyl.
  • R 10 represents a linear or branched C1-6 alkyl, optionally substituted by -NH(R 10a ) or -NHCH2(R 10a ).
  • R 10 represents a linear or branched C1-6 alkyl. It has surprisingly been found that compounds having a non-cyclic alkyl group at the R 10 position have better anti-biofilm properties in certain circumstances (e.g. against S. epidermidis biofilms) compared to compounds with a cyclic group at this position. Therefore, particularly preferred R 10 groups include branched and linear propyl and butyl groups (e.g. n-propyl, n-butyl, sec-butyl, iso- butyl).
  • R 10 groups include branched and linear propyl and butyl groups substituted by -NH(R 10a ) or -NHCH 2 (R 10a ). Most preferably, R 10 groups include linear propyl and butyl groups. In one embodiment, R 10 is butyl. In a particular embodiment of the invention, R 10 represents a cyclic C1-6 alkyl, preferably cyclohexyl. It has surprisingly been found that compounds with a cyclic alkyl group at this position have better anti-biofilm properties in certain circumstances (e.g. against S. aureus biofilms) compared to compounds with a non-cyclic alkyl group at this position.
  • R 11 represents a hydrophobic proteinogenic or hydrophobic non- proteinogenic amino acid side chain (such as that of alanine (i.e. a methyl group), valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, norvaline, cyclohexylglycine, cyclohexylalanine, phenylglycine, biphenylglycine, biphenylalanine, naphthylglycine or naphthylalanine).
  • the group at R 11 may be bound in either the D- or L- configuration, though it is preferred that it is present in the L-configuration.
  • R 11 represents a butyl group (e.g. the side chain of isoleucine)
  • the chiral carbon to which R 11 is bound is preferably in the S-configuration (thus corresponding to the L-isoleucine that is present at this position for Teixobactin).
  • R 11 represents an L- or D-isoleucine side chain, preferably an L- isoleucine side chain.
  • the carbon to which each group is attached is in the L-configuration, i.e. the configuration most commonly seen in naturally occurring amino acids.
  • Z is -O- or -NH-, preferably -O-.
  • AA 1 represents a D-phenylalanine residue
  • AA 2 represents an L-isoleucine residue
  • AA 5 represents a D-allo-isoleucine or D-isoleucine residue
  • AA 6 represents an L-isoleucine residue
  • AA 7 represents an L-serine residue
  • R 8 represents a methyl group
  • R 11 represents an L-isoleucine side chain
  • Z is -O-.
  • one or more of R 9 , the side chain of AA 3 and the side chain of AA 4 represents a moiety selected from the group consisting of a positively charged amino acid side chain, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, and a fragment of formula -L 1 -L 2 -L 3 -X 1 as defined hereinabove.
  • one or more of R 9 , the side chain of AA 3 and the side chain of AA 4 represents a side chain of an amino acid selected from the group consisting of arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, and 2,4-diaminobutyric acid. More preferably, one or more of R 9 , the side chain of AA 3 and the side chain of AA 4 , represents an arginine side chain. In yet another embodiment, AA 4 represents an arginine residue. All of these compounds are particularly suited to combating S. epidermidis biofilms. Further increasing the number of positively charged groups at these positions (i.e.
  • two or more of R 9 , the side chain of AA 3 and the side chain of AA 4 represent a side chain of an amino acid selected from the group consisting of arginine, lysine, histidine, ornithine, 2,3-diaminopropanoic acid, and 2,4-diaminobutyric acid. More preferably, two or more of R 9 , the side chain of AA 3 and the side chain of AA 4 , represent an arginine side chain. In yet another embodiment, AA 4 and AA 3 represent an arginine residue.
  • R 9 , the side chain of AA 3 and the side chain of AA 4 each independently represent a moiety selected from the group consisting of a positively charged amino acid side chain, -CH 2 -NH 2 , -(CH 2 ) 2 -NH 2 , -(CH 2 ) 3 -NH 2 , and a fragment of formula -L 1 -L 2 -L 3 -X 1 as defined hereinabove (e.g. an amino acid selected from the group consisting of arginine, lysine, histidine, ornithine, 2,3-diaminopropanoic acid, and 2,4- diaminobutyric acid).
  • R 9 , the side chain of AA 3 and the side chain of AA 4 are all arginine side chains. These compounds are particularly suited to combating S. epidermidis biofilms.
  • the combination of positively charged groups at one or more of R 9 , AA 3 and AA 4 together with a hydrophobic group at R 10 is found to be particularly effective, and it is believed that this arises from an improved overall solubility for the compound due to the balance of charges.
  • R 9 represents a side chain of an amino acid selected from the group consisting of arginine, lysine, histidine, ornithine, 2,3-diaminopropanoic acid, and 2,4-diaminobutyric acid; and R 10 represents a linear or branched C 1-6 alkyl optionally substituted by -NH(R 10a ) or -NHCH 2 (R 10a ), preferably a linear or branched propyl or butyl group, even more preferably a linear propyl or butyl group.
  • At least two of R 9 , the side chain of AA 3 and the side chain of AA 4 represents a side chain of an amino acid selected from the group consisting of arginine, lysine, histidine, ornithine, 2,3-diaminopropanoic acid, and 2,4-diaminobutyric acid; and R 10 represents a linear or branched C1-6 alkyl or a cyclic C3-6 alkyl, preferably a linear or branched propyl or butyl group, even more preferably a linear propyl or butyl group.
  • R 9 , the side chain of AA 3 and the side chain of AA 4 represents an arginine side chain; and R 10 represents a linear or branched C 1-6 alkyl, preferably a linear or branched propyl or butyl group, even more preferably a linear propyl or butyl group.
  • R 9 represents an alanine side chain
  • AA 3 represents a serine residue
  • AA 4 represents a glutamine residue
  • R 10 represents a linear or branched C1-6 alkyl, preferably a linear or branched propyl or butyl group, even more preferably a linear propyl or butyl group.
  • R 9 represents an alanine side chain
  • AA 3 represents a serine residue
  • AA 4 represents a glutamine residue
  • R 10 represents a cyclic C1-6 alkyl, preferably a cyclohexyl group.
  • Chg 10 -Teixobactin or a pharmaceutically-acceptable salt, solvate or clathrate thereof
  • the compound of formula (I), or a pharmaceutically- acceptable salt, solvate or clathrate thereof is selected from the group consisting of the compounds in the following table:
  • the compound of formula (I), or a pharmaceutically-acceptable salt, solvate or clathrate thereof is selected from the group consisting of: (D-Arg 4 -Leu 10 -teixobactin);
  • the compounds of the invention may be prepared in accordance with techniques known to those skilled in the art, for example as described in WO 2018/162922.
  • the novel compounds of the invention including for example Arg4-Nle10-teixobactin and Nle10-teixobactin, can be synthesised using this method.
  • Nle10- teixobactin compound 16
  • the amino acid at position 10 is replaced with norleucine during the synthesis of the compound (e.g. during step (a) in Example 2 of WO 2018/162922).
  • the amino acid at position 10 may be replaced with norleucine and the amino acid at position 4 is replaced with arginine.
  • compounds of the invention that contain one or more isopeptide bonds (e.g.
  • a compound of the invention i.e. a compound of formula (I) or a pharmaceutically-acceptable salt, solvate or clathrate thereof as defined above, is used for killing, inhibiting or preventing the growth of a microbial biofilm.
  • the compounds of the invention are useful because they possess pharmacological activity. They are therefore indicated as pharmaceuticals.
  • compositions comprising a compound of the invention in combination with a pharmaceutically-acceptable adjuvant diluent or carrier.
  • formulations are referred to hereinafter as the “formulations of the invention”.
  • the use of compounds or formulations of the invention in medicine includes their use as pharmaceuticals (both for human and veterinary use).
  • the compositions of the present invention may also be useful in other fields of industry. For example, the compositions may be useful in hygiene and sterilisation procedures (e.g. in scientific laboratories).
  • the compounds of the invention may be formulated at various concentrations, depending on the efficacy/toxicity of the compound being used and the purpose for which it is being used.
  • the compound when used in medicine, the compound will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19 th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA). Suitable routes of administration are discussed below, and include topical, intravenous, oral, pulmonary, nasal, aural, ocular, bladder and CNS delivery. It will be appreciated that the compounds for use according to the invention may be employed for killing a number of types of biofilm-forming microorganisms, including bacteria, archaea, protozoa, fungi and algae.
  • microorganisms may be resistant to one or more conventional antibiotics, such as methicillin (e.g. MRSA).
  • the microorganisms are bacteria.
  • bacteria and derivatives thereof, such as “bacterial infection” includes references to organisms (or infections due to organisms) of the following classes and specific types: Gram-positive cocci, such as Staphylococci (e.g. Staph. aureus, Staph. epidermidis, Staph. saprophyticus, Staph. auricularis, Staph. capitis capitis, Staph. c. ureolyticus, Staph. caprae, Staph.
  • Staphylococci e.g. Staph. aureus, Staph. epidermidis, Staph. saprophyticus, Staph. auricularis, Staph. capitis capitis, Staph. c. ureolyticus, Staph. caprae, Staph.
  • Streptococci e.g. beta-haemolytic, pyogenic streptococci (such as Strept. agalactiae, Strept. canis, Strept. dysgalactiae dysgalactiae, Strept. dysgalactiae equisimilis, Strept. equi equi, Strept. equi zooepidemicus, Strept. iniae, Strept. porcinus and Strept. pyogenes), microaerophilic, pyogenic streptococci (Streptococcus “milleri”, such as Strept. anginosus, Strept. constellatus constellatus, Strept.
  • streptococci of the “mitis” alpha-haemolytic - Streptococcus “viridans”, such as Strept. mitis, Strept. oralis, Strept. sanguinis, Strept. cristatus, Strept. gordonii and Strept. parasanguinis
  • mitis alpha-haemolytic - Streptococcus “viridans”, such as Strept. mitis, Strept. oralis, Strept. sanguinis, Strept. cristatus, Strept. gordonii and Strept. parasanguinis
  • salivarius non-haemolytic, such as Strept. salivarius and Strept. vestibularis
  • mutans teeth-surface streptococci, such as Strept. criceti, Strept. mutans, Strept. ratti and Strept. sobrinus
  • Strept. acidominimus Strept.
  • Gram-negative cocci such as Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria sicca, Neisseria subflava and Neisseria weaveri; Bacillaceae, such as Bacillus anthracis, Bacillus subtilis, Bacillus thuringiensis, Bacillus stearothermophilus and Bacillus cereus; Enterobacteriaceae, such as Escherichia coli, Enterobacter (e.g.
  • Enterobacter aerogenes Enterobacter aerogenes, Enterobacter agglomerans and Enterobacter cloacae
  • Citrobacter such as Citrob. freundii and Citrob. divernis
  • Hafnia e.g. Hafnia alvei
  • Erwinia e.g. Erwinia persicinus
  • Morganella morganii Salmonella (Salmonella enterica and Salmonella typhi), Shigella (e.g. Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Klebsiella (e.g. Klebs. pneumoniae, Klebs. oxytoca, Klebs.
  • Shigella dysenteriae Shigella flexneri
  • Klebsiella e.g. Klebs. pneumoniae, Klebs. oxytoca, Klebs.
  • Enterococci e.g. Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus and Enterococcus solitarius); Helicobacter (e.g.
  • Helicobacter pylori Helicobacter cinaedi and Helicobacter fennelliae
  • Acinetobacter e.g. A. baumanii, A. calcoaceticus, A. haemolyticus, A. johnsonii, A. junii, A. lwoffi and A. radioresistens
  • Pseudomonas e.g. Ps. aeruginosa, Ps. maltophilia (Stenotrophomonas maltophilia), Ps. alcaligenes, Ps. chlororaphis, Ps. fluorescens, Ps. luteola. Ps. mendocina, Ps.
  • clostridioforme C. cochlearium, C. cocleatum, C. fallax, C. ghonii, C. glycolicum, C. haemolyticum, C. hastiforme, C. histolyticum, C. indolis, C. innocuum, C. irregulare, C. leptum, C. limosum, C. malenominatum, C. novyi, C. oroticum, C. paraputrificum, C. piliforme, C. putrefasciens, C. ramosum, C. septicum, C. sordelii, C. sphenoides, C. sporogenes, C.
  • Mycoplasma e.g. M. pneumoniae, M. hominis, M. genitalium and M. urealyticum
  • Mycobacteria e.g.
  • Mycobacterium tuberculosis Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium leprae, Mycobacterium smegmitis, Mycobacterium africanum, Mycobacterium alvei, Mycobacterium asiaticum, Mycobacterium aurum, Mycobacterium bohemicum, Mycobacterium bovis, Mycobacterium branderi, Mycobacterium brumae, Mycobacterium celatum, Mycobacterium chubense, Mycobacterium confluentis, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium flavescens, Mycobacterium gadium, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium goodii, Mycobacterium haemophilum
  • Brucella abortus Brucella canis, Brucella melintensis and Brucella suis
  • Campylobacter e.g. Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus
  • Listeria monocytogenes Vibrio (e.g.
  • Vibrio cholerae and Vibrio parahaemolyticus Vibrio alginolyticus, Vibrio carchariae, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus and Vibrio vulnificus); Erysipelothrix rhusopathiae; Corynebacteriaceae (e.g. Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium urealyticum); Spirochaetaceae, such as Borrelia (e.g.
  • Pasteurella e.g. Pasteurella aerogenes, Pasteurella bettyae, Pasteurella canis, Pasteurella dagmatis, Pasteurella gallinarum, Pasteurella haemolytica, Pasteurella multocida multocida, Pasteurella multocida gallicida, Pasteurella multocida septica, Pasteurella pneumotropica and Pasteurella stomatis
  • Bordetella e.g.
  • Nocardiaceae such as Nocardia (e.g. Nocardia asteroides and Nocardia brasiliensis); Rickettsia (e.g. Ricksettsii or Coxiella burnetii); Legionella (e.g.
  • Capnocytophaga e.g. Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Capnocytophaga gingivalis, Capnocytophaga granulosa, Capnocytophaga haemolytica, Capnocytophaga ochracea and Capnocytophaga sputtona
  • Bartonella Bartonella bacilliformis, Bartonella clarridgeiae, Bartonella elizabethae, Bartonella henselae, Bartonella quintana and Bartonella vinsonii arupensis
  • Leptospira e.g.
  • Fusobacterium e.g. F. gonadiaformans, F. mortiferum, F. naviforme, F. necrogenes, F. necrophorum necrophorum, F.
  • Chlamydia e.g. Chlamydia trachomatis
  • Chlamydophila e.g.
  • Chlamydophila abortus Chlamydia psittaci
  • Chlamydophila pneumoniae Chlamydia pneumoniae
  • Chlamydophila psittaci Chlamydophila psittaci
  • Leuconostoc e.g. Leuconostoc citreum, Leuconostoc cremoris, Leuconostoc dextranicum, Leuconostoc lactis, Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides
  • Gemella e.g.
  • compounds of the invention may be used to kill any of the above-mentioned bacterial organisms. It will be further appreciated by skilled persons that the compounds may be used to prevent and/or treat infection with such microorganisms, i.e. the compounds are suitable for prophylactic and/or therapeutic treatment. For example, the compounds may be used to prevent or reduce the spread or transfer of a pathogen to other subjects, e.g. patients, healthcare workers, etc.
  • the bacteria are Gram-positive bacteria, such as those selected from the group consisting of Staphylococci and Enterococci.
  • the bacteria may be Staphylococci, such as Staphylococcus aureus, (e.g. methicillin-resistant Staphylococcus aureus, MRSA).
  • the bacteria are Staphylococcus epidermidis.
  • Other Staphylococci include Staph. aureus, Staph. epidermidis, Staph. saprophyticus, Staph. auricularis, Staph. capitis capitis, Staph. c. ureolyticus, Staph. caprae, Staph.
  • Enterococci include Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus and Enterococcus solitarius.
  • the bacteria are Gram-negative bacteria, such as those selected from the group consisting of Acinetobacter (e.g. A.
  • Enterobacteriaceae such as Escherichia coli, Klebsiella (e.g. Klebs. pneumoniae and Klebs. oxytoca) and Proteus (e.g. Pr. mirabilis, Pr. rettgeri and Pr. vulgaris); and Pseudomonas (e.g. Ps. aeruginosa, Ps. maltophilia (Stenotrophomonas maltophilia), Ps. alcaligenes, Ps.
  • Enterobacteriaceae such as Escherichia coli, Klebsiella (e.g. Klebs. pneumoniae and Klebs. oxytoca) and Proteus (e.g. Pr. mirabilis, Pr. rettgeri and Pr. vulgaris)
  • Pseudomonas e.g. Ps. aeruginosa, Ps. maltophilia (Stenotrophomonas maltophilia
  • Dosages of the compound for use according to the invention will depend on several factors; including the particular compound used, the formulation, route of administration and the indication for which the compound is used. Typically, however, dosages will range from 0.01 to 20 mg of compound per kilogram of body weight, preferably from 0.1 to 15 mg/kg, for example from 1 to 10 mg/kg of body weight.
  • biofilm may be associated with either an inert support or a living support.
  • the biofilm is associated with a living support.
  • the biofilm may grow or be susceptible to growth on a surface within the human or animal body.
  • the invention provides a compound as defined above for use in the treatment or prevention of a condition or disorder associated with the presence or growth of a biofilm on or in the body of a living mammal.
  • a further aspect of the invention provides a method for treating or preventing a condition or disorder associated with the presence or growth of a biofilm on or in the body of a living mammal, said method comprising administering a compound as described herein to a patient suffering from or susceptible to a disease or condition associated with or caused by a biofilm.
  • a yet further aspect of the invention provides the use of a compound of formula (I) as described herein in the manufacture of a medicament for treating or preventing a condition or disorder associated with the presence or growth of a biofilm on or in the body of a living mammal.
  • the invention provides a compound for use in killing, inhibiting, or preventing the growth of a microbial biofilm as hereinbefore defined, wherein the biofilm is on or in the body of a living mammal.
  • the mammal is human.
  • the compounds described herein may be used to treat or prevent a disorder or condition associated with the growth of a biofilm (e.g. a microbial biofilm) at one of the following sites within the body: (a) The oral cavity, including the surfaces of the teeth and gums (for example, dental plaque, gingivitis, endodontic infections, oral candidiasis, oral aspergillosis, periodontitis). (b) The urinary tract (for example, cystitis).
  • the sinuses for example, chronic sinusitis.
  • the ear for example, middle ear infections).
  • the heart for example, endocarditis).
  • the prostate for example, chronic bacterial prostatitis.
  • the bone for example, osteomyelitis
  • the lungs for example, infections in cystic fibrosis such as pneumonia
  • the kidneys for example, infectious kidney stones and in peritoneal dialysis.
  • the skin is associated with an inert support, e.g. an inert support within the body.
  • the biofilm may grow or be susceptible to growth on the surface of a device implanted or otherwise inserted within the human or animal body.
  • the compounds described herein may be used to treat or prevent an infection associated with the growth of a microbial biofilm on one of the following inert surfaces within the body:
  • a catheter for example, for intravascular or urinary tract use.
  • a stent for example, a coronary stent.
  • a shunt for example, a cerebrospinal shunt).
  • An ophthalmic device for example, contact lenses, scleral buckles and intraocular lenses).
  • a joint prosthesis i.e.
  • an implantable medical device which is impregnated, coated or otherwise treated with a compound as described herein.
  • the implantable medical device may be selected from the group consisting of intravascular devices, catheters, shunts, intubating and tracheotomy tubes, opthalmic devices, joint prostheses, artificial heart valves and breast implants.
  • implantable device we include devices attached to surface of body, e.g. contact lenses.
  • the implantable medical device is packaged in a sealed and sterile container prior to use.
  • the compound when used in medicine, the compound will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA).
  • suitable routes of administration are discussed below, and include topical, intravenous, oral, pulmonary, nasal, aural, ocular, bladder and CNS delivery.
  • a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA).
  • Suitable routes of administration are discussed below, and include topical, intravenous, oral, pulmonary, nasal, aural, ocular, bladder and CNS delivery.
  • for application topically e.g.
  • the compounds can be administered in the form of a lotion, solution, cream, gel, ointment or dusting powder (for example, see Remington, supra, pages 1586 to 1597).
  • the compounds can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • a suitable lotion or cream suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, e-lauryl sulphate, an alcohol (e.g. ethanol, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol) and water.
  • mineral oil sorbitan monostearate
  • a polyethylene glycol liquid paraffin
  • polysorbate 60 e.g. sorbate 60
  • cetyl esters wax e-lauryl sulphate
  • an alcohol e.g. ethanol, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol
  • water e.g. ethanol, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol
  • Formulations suitable for topical administration in the mouth further include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • the medicament for use may also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray, or nebuliser may contain a solution or suspension of the active compound, e.g.
  • a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch. Aerosol or dry powder formulations are preferably arranged so that each metered dose or “puff” contains at least 1 mg of a compound for delivery to the patient. It will be appreciated that the overall dose with an aerosol will vary from patient to patient and from indication to indication, and may be administered in a single dose or, more usually, in divided doses throughout the day.
  • the medicament for use according to the invention may be delivered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
  • the medicament may also be administered intra-ocularly (see below), intra-aurally or via intracavernosal injection.
  • the medicament may also be administered parenterally, for example, intravenously, intra- arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously (including via an array of fine needles or using needle- free Powderject® technology), or they may be administered by infusion techniques.
  • parenterally for example, intravenously, intra- arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously (including via an array of fine needles or using needle- free Powderject® technology), or they may be administered by infusion techniques.
  • They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the medicament may also be administered by the ocular route, particularly for treating diseases of the eye.
  • the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
  • a compound is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
  • the medicaments may be stored in any suitable container or vessel known in the art. It will be appreciated by persons skilled in the art that the container or vessel should preferably be airtight and/or sterilised.
  • the container or vessel is made of a plastics material, such as polyethylene.
  • the compounds for use according to the invention may also be employed to kill, inhibit or prevent the growth of microbial biofilms in vitro.
  • the compounds may also be used in the form of a sterilising solution or wash to prevent the growth of microbial biofilms on a surface or substrate, such as in a domestic environment (e.g. kitchen work surfaces, showers, pipes, floors, etc.) or a commercial or industrial environment (e.g. within cooling systems, pipes, floor surfaces, etc.) environment.
  • such a medicament comprises the antimicrobial compound in solution at a concentration of 1 to 100 ⁇ g/ml.
  • the solution further comprises a surface-active agent or surfactant.
  • Suitable surfactants include anionic surfactants (e.g. an aliphatic sulphonate), amphoteric and/or zwitterionic surfactants (e.g. derivatives of aliphatic quaternary ammonium, phosphonium and sulfonium compounds) and nonionic surfactants (e.g. aliphatic alcohols, acids, amides or alkyl phenols with alkylene oxides)
  • the surface-active agent is present at a concentration of 0.5 to 5 weight percent.
  • the compounds for use in the first and second aspects of the invention are preferably exposed to the target surface for at least five minutes.
  • the exposure time may be at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3, hours, 5 hours, 12 hours and 24 hours.
  • step a Synthesis of D-Arg 4 -Chg 10 -teixobactin (3) and D-Arg 4 -Leu 10 -teixobactin (17)
  • step b The Fmoc protecting group was deprotected using 20% piperidine in DMF by shaking for 3 min, followed by draining and shaking again with 20% piperidine in DMF for 10 min. AllocHN-D-Thr-OH was then coupled to the resin by adding 3 eq. of the AA, 3 eq. HATU and 6 eq. DIPEA in DMF and shaking for 1.5h at room temperature.
  • step c Esterification was performed using 10 eq. of Fmoc- Ile-OH, 10 eq. DIC and 5 mol% DMAP in DCM and shaking the reaction for 2h.
  • step d Fmoc- Chg-OH was coupled using 4 eq. of AA, 4 eq. HATU and 8 eq. DIPEA in DMF and shaking for 1h followed by Fmoc deprotection using 20% piperidine in DMF as described earlier.
  • step e The N terminus of Chg was protected using 10 eq. Trt-Cl and 15% Et 3 N in DCM and shaking for 1h. The protection was verified by the Ninhydrin colour test.
  • step f The Alloc protecting group of D-Thr was removed using 0.2 eq.
  • step g All amino acids were coupled using 4 eq. Amino Acid, 4 eq. DIC/Oxyma using a microwave peptide synthesizer. Coupling time was 10 min. Deprotection cycles were performed as described earlier.
  • step i The solvent was evaporated and the peptide was redissolved in DMF to which 1 eq. HATU and 10 eq. DIPEA were added and the reaction was stirred for 30 min to perform the cyclization.
  • the peptide was precipitated using cold Et 2 O (-20°C) and centrifuging at 7000 rpm to obtain a white solid. This solid was further purified using RP-HPLC using the protocols described below.
  • Fmoc-D-allo-Ile-OH and oxyma pure were purchased from Merck Millipore.
  • the side chain protecting groups for the amino acids are tBu for Ser, Pbf for Arg and Trt for Gln and Thr unless specified otherwise.
  • Diisopropylethylamine (DIPEA) supplied as extra dry, redistilled, 99.5 % pure, Acetic anhydride, allyl chloroformate, CDCl3 and polysorbate 80 and were purchased from Sigma Aldrich.
  • Tritylchloride and 4-(Dimethylamino)pyridine were purchased from Alfa Aesar. Dimmethylformamide (DMF) peptide synthesis grade was purchased from Rathburn chemicals.
  • Triethylamine, Diethyl ether (Et 2 O), Dimethylsulfoxide (DMSO), Dichloromethane (DCM), Tetrahydrofuran (extra dry with molecular sieves), Formic acid 98-100% purity and Acetonitrile (HPLC grade) were purchased from Fisher Scientific. Water with the Milli-Q grade standard was obtained in-house from an ELGA Purelab Flex system. 2-Chlorotritylchloride resin (manufacturer’s loading: 1.20 mmol/g) was purchased from Fluorochem. All chemicals were used without further purification.
  • MHB Meuller-Hinton broth
  • MIC values were determined in the media containing polysorbate 80 (0.002%, v/v) to prevent non-specific adsorption of the peptides to plastic surfaces.
  • the final peptides concentrations ranged from 0.0625 – 32 ⁇ g/ml (for lower range 0.031 – 16 ⁇ g/ml was used).
  • Positive and negative controls contained 200 ⁇ l of inoculum without any peptide dissolved in broth, respectively.
  • the 96 well plates were then incubated at 37 °C for 24 h.
  • Arginine substitutions in case of Chg 10 -teixobactin had the lowest effect on the GM-MIC values compared to Leu 10 - and Nva10-teixobactin analogues, making it easier to balance the amphipathic nature of Chg10-teixobactin. Most of the analogues showed potent antibacterial activity (MIC) against MRSA/S.
  • aureus strains (0.125- ⁇ NjJ ⁇ P/ ⁇ 7R ⁇ GHWHUPLQH ⁇ WKH ⁇ HIIHFW ⁇ RI ⁇ FDWLRQLFLW ⁇ RQ ⁇ EDFWHULFLGDO ⁇ SURSHUWLHV ⁇ we determined the minimum bactericidal concentration (MBC) of teixobactin analogues against tested MRSA/ S. aureus strains.
  • MBC minimum bactericidal concentration
  • Example 4 in vitro Biofilm Studies Study
  • Commensal biofilm former pathogens such as S. aureus and S. epidermidis are known to colonize implants at the insertion sites and they are challenging to treat due to their inherent resistance to antimicrobial therapies and the host immune responses.
  • To test the potency of teixobactin peptides we determined their anti-biofilm properties against prolific biofilm forming strains S. aureus 15981 and S.
  • the acquired images were processed using IMARIS version 9 software (Bitplane AG, Zurich, Switzerland) to obtain the biovolumes of the live and dead cell populations and the dead-to-live biovolume ratio graphs were plotted using GraphPad Prism 7 software (United States).
  • Results The teixobactin analogues displayed potent antibiofilm properties against both S. aureus and S. epidermidis biofilms when compared to comparator antibiotics (vancomycin at 10 ⁇ g/mL). In the case of S. epidermidis biofilm, the dead/live biovolume ratio remained similar for untreated and vancomycin (p>0.05)-treated groups (Fig. 1a and Fig. 1c).
  • the preformed biofilms were treated with a teixobactin analogue (3, 5 and 16) for 5 h, and the teixobactin analogues displayed potent antibiofilm properties against both S. aureus and S. epidermidis biofilms when compared to comparator clinical antibiotic (vancomycin at 10 ⁇ g/mL).
  • vancomycin vancomycin at 10 ⁇ g/mL
  • the dead/live biovolume ratio remained similar for untreated and vancomycin (p>0.05)-treated groups (Fig. 1a and Fig. 1c).
  • Example 5 in vivo Biofilm Studies Study: In vivo efficacy of teixobactin analogue in a murine non-neutropenic subcutaneous foreign body implant model (Teflon catheter) of Staphylococcus aureus ATCC 43300 infection.
  • the teixobactin analogue 17 (as obtained in Example 2) was evaluated for prevention of biofilm formation in subcutaneous implanted Teflon catheters infected with S aureus ATCC 43300 in a 7 day murine model.
  • the male CD 1 mice were implanted with a single Teflon catheter. 100 ⁇ L of bacterial inoculum was administered into each catheter lumen (1.7 x 10 6 CFU/catheter implant).
  • Teixobactin analogue 17 administered SC Q8h 25 and 50 mg/kg starting at 2h post infection showed robust efficacy and resulted in a statistically significant burden reduction of 5.25 and 5.11 Log 10 CFU/catheter compared to vehicle, corresponding to 5.92 and 5.78 Log 10 CFU/catheter below the level of stasis, respectively.
  • 6 out of 8 implants from each treatment group had bacterial burden below the limit of detection.
  • Rifampicin administered PO Q24h at 10 mg/kg reduced bacterial burden by 5.33 Log 10 CFU/catheter compared to vehicle, corresponding to 6.00 Log 10 CFU/catheter below the level of stasis. 5 out of 7 implants had burden below the limit of detection. Results are shown in Fig. 3.
  • Example 6 Synthesis of Further Teixobactin Analogues (Compounds 31-34) The following compounds may be made by methods analogous to those in Examples 1 and 2: Table 5: Compound number and name for compounds 31-33. The following compound has been made by a method analogous to that in Example 1: Table 6: Compound number, name, chemical formula, exact mass and mass found for compound 34.
  • Example 7 Synthesis of Further Teixobactin Analogues (Compounds 35-38) The following compounds have been made using methods analogous to those in Examples 1 and 2: Table 7: Compound number, name, chemical formula, exact mass and mass found for compounds 35-38.
  • Example 8 – MIC Testing MIC testing for compounds 34 to 38 was performed in accordance with the methods disclosed in Example 3. The results are shown below.
  • Example 9 MBEC testing MBEC testing was performed for compounds 35 to 38 using the following assay: I. Innoculum Preparation and Plating 1. The selected bacterial strain was streaked on Mueller-Hinton Agar (MHA) and incubated at 37 °C overnight. 2. Prepare MHA and pour 50 mL each to square Petri dishes. Set aside and keep in the fridge. (This will be used for biofilm stamping) 3.
  • MHA Mueller-Hinton Agar
  • TSA Tryptic soy broth
  • TSG D-glucose sterile filtered solution 1% w/v
  • 4. 2-3 colonies of bacterial strain are emulsified in 3 mL of phosphate buffered saline to match the turbidity of 0.5 McFarland standard solution. At least 2 triplicates of inoculum were prepared for each bacterial strain tested. 5. Dilute each inoculum PBS by 1:100 into TSG (3 separate tubes) to achieve a colony count of about 1 x 10 6 CFU/mL. (i.e. A1 B, A2 B) 6.
  • REU relative fluorescence units

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Abstract

L'invention concerne un composé de formule (I), ou un sel, solvate ou clathrate pharmaceutiquement acceptable de celui-ci, pour une utilisation dans la destruction, l'inhibition ou la prévention de la croissance d'un biofilm microbien, formule (I) dans laquelle R1, AA1, AA2, AA3, AA4, AA5, AA6, AA7, R8, R9, R10, R11 ont les significations données dans la description. De préférence, les biofilms sont produits par Staphylococcus aureus ou Staphylococcus epidermidis.
PCT/GB2024/051527 2023-06-14 2024-06-14 Analogues des teixobactines sous la forme d'agents anti-biofilm Pending WO2024256834A1 (fr)

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