WO2022098950A1

WO2022098950A1 - Compounds

Info

Publication number: WO2022098950A1
Application number: PCT/US2021/058182
Authority: WO
Inventors: Pamela Brown; Michael Dawson; Dean Rivers; Roy LESTER; Steven Boakes; Esther DUPERCHY; Scott Coleman; Troy LISTER
Original assignee: Spero Therapeutics, Inc.
Priority date: 2020-11-06
Filing date: 2021-11-05
Publication date: 2022-05-12
Also published as: TW202233649A

Abstract

The invention relates to compounds of formula (I), the use of these compounds in methods of treatment or prophylaxis, including methods for treating a microbial infection, such as a Gram-negative bacterial infection. The compound of formula is (I), wherein ‑R^T is linear C₆ alkyl, and the salts, solvates and protected forms thereof.

Description

COMPOUNDS

RELATED APPLICATION

The present case claims the benefit of, and priority to, US 63/110765 filed on 6 November 2021 (06/11/2021), the contents of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel polymyxin compounds, pharmaceutical compositions comprising the compounds, and the use of the compounds and the pharmaceutical compositions for medical treatment, for example treatment of microbial infections, particularly infections by Gram-negative bacteria.

BACKGROUND

WO 2013/072695 and WO 2014/188178 describe polymyxin derivatives in which the N-terminal fatty acyl moiety and adjacent diaminobutyric acid moiety of Polymyxin B or Colistin are replaced by a terminal group having an amino substituent. Such derivatives have good antibacterial activity whilst having a reduced cytotoxicity.

WO 2015/135976 also describes polymyxin derivatives in which again the N-terminal fatty acyl moiety and adjacent diaminobutyric acid of Polymyxin B or Colistin are replaced by a terminal group having an amino substituent. Here, the specific position of the amino substituent and the placement of other substituents in the N-terminal moiety were shown to be important for strong antimicrobial activity across a range of key pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. The compounds disclosed also retained a low cytotoxicity.

WO 2016/083531 describes polymyxin derivatives in which again the N-terminal fatty acyl moiety and adjacent diaminobutyric acid of Polymyxin B or Colistin are replaced by a terminal group having an amino substituent, such as those groups present in WO 2013/072695, WO 2014/188178 and WO 2015/135976. Additionally, the amino acid residue at positions 6 and/or 7 are substituted with respect to Polymyxin B and Colistin.

Recently, WO 2020/002325 has also described polymyxin derivatives in which again the N-terminal fatty acyl moiety and adjacent diaminobutyric acid of Polymyxin B or Colistin are replaced by a terminal group having an amino substituent. This work shows that certain N terminal groups within the scope of WO 2015/135976 have superior activity. To be more useful for the parenteral therapy of systemic infections than the currently available polymyxins, new polymyxin derivatives must at least match the activity of these known polymyxins whilst having significantly lower renal toxicity in vivo.

SUMMARY OF THE INVENTION

In a general aspect, the invention provides compounds having a deacylated polymyxin core, such as a nonapetide core of Polymyxin B with the amino acid residue at position 3 substituted with Dap, the amino acid at position 6 substituted with D-Leu, the amino acid at position 7 substituted with 2-(S)-aminobutyric acid ((S)-Abu), and having an amino alkyl group, as defined herein, at its N terminal. Such compounds find use in a method of treatment or prophylaxis, optionally in combination with a second active agent. The compounds may be used to treat a microbial infection, such as a Gram-negative bacterial infection.

In a first aspect of the invention, there is provided a compound of formula (I), and pharmaceutically acceptable salts, solvates, protected forms and prodrug forms thereof.

The compound of formula (I) is represented thus:

wherein:

-R^T is linear Ce alkyl (n-Ce alkyl), and salts, solvates and protected forms thereof.

The invention also provides a pharmaceutical composition comprising a compound of formula (I), optionally together with one or more pharmaceutically acceptable carriers.

In a further aspect there is provided a compound of formula (I), or a pharmaceutical composition comprising a compound of formula (I), for use in a method of treatment or prophylaxis. In yet a further aspect there is provided a compound of formula (I), or a pharmaceutical composition comprising a compound of formula (I), for use in a method of treating a microbial infection.

The present invention also provides a method of treatment, the method comprising the step of administering a compound of formula (I), or a pharmaceutical composition comprising a compound of formula (I), to a subject in need thereof. The method may be for the treatment of a microbial infection.

A microbial infection may be a bacterial infection, such as a Gram-negative bacterial infection. The Gram-negative bacterial infection may be selected from the group consisting of Escherichia spp., Klebsiella spp., Enterobacter spp., Salmonella spp., Shigella spp., Citrobacter spp., and other Enterobacteriaceae, Pseudomonas spp., Acinetobacter spp., Stenotrophomonas , and Legionella.

These and other aspects and embodiments of the invention are described in further detail below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds of formula (I), including the compounds of formula (II) and (III) as described in further detail below, for use in medical treatment, optionally together with a second active agent.

To be more useful for the parenteral therapy of systemic infections than the currently known series of polymyxin compounds, new polymyxin derivatives must at least match the antibacterial activity of those known polymyxin compounds whilst having significantly lower renal toxicity in vivo.

It has now been found that it is not sufficient for a polymyxin compound to exhibit lower cytotoxicity, as such is frequently not associated with reduced toxicity in vivo. Thus, the accumulation of the drug in the kidney and its clearance from there must also be favourable. In other words, the combination of cytotoxicity and kidney drug levels after parenteral dosing is what leads to a favourable in vivo toxicity profile.

The present applicant has previously disclosed in WO 2015/135976 polymyxin nonapeptides with an N-terminal y-aminopropyl group substituted at the -position with pentyl or octyl, relative to the -X- group. These are compounds D91 and D105.

WO 2015/135976 also describes a polymyxin nonapeptide with an N-terminal substituted at the a-position with hexyl, relative to the -X- group. This is compound D103. Also disclosed in WO 2015/135976 is a polymyxin nonapeptide with an N-terminal P-aminoethyl group substituted at the a-position with a hexyl group, relative to the -X- group. This is compound DI 02.

Each of the compounds mentioned above are compounds having D-Phe at position 6, and L-Leu at position 7. In contrast, the compounds of the invention have D-Leu at position 6, and L-Abu at position 7.

Whilst these known compounds show promising activity and moderately improved cytotoxicity compared to Polymyxin B, these compounds are inferior to the compounds of the present invention in that they do not display a combination of low cytotoxicity together balanced with acceptable kidney levels after dosage.

Exemplary MIC and cytotoxicity data are shown in Table A below. The structures of the compounds, together with the substituent groups -R¹, are shown beneath the table.

Table A - MIC Data for Known Compounds, Comparative Compounds and Compounds 1 and 2 of the Present Case

* mouse tox score L= lower than PMB, M = similar to PMB, H = higher then PMB ** Cl is the diastereomer of DI 05 and not reported in CAN-21 therefore a comparator compound.

Furthermore, the present inventors have prepared comparator analogues to the compounds of the invention, wherein the only difference is in the length of alkyl substituents at the P-position of the N-terminal group. Exemplary MIC data are set out in Table B below. Unexpectedly, the inventors found that a hexyl chain (n = 5 in Table B) significantly outperforms pentyl (n = 4) or heptyl (n = 6) in terms of cytotoxicity while maintaining reasonable broad spectrum activity. Further increases in the length of the alkyl group increase the cytotoxicity of the resulting compounds, while further decreases in the length of the alkyl group lose efficacy against many of the target microorganisms.

Table B - MIC Data for Known Compounds, Comparative Compounds and Compounds 1 and 2 of the Present Case

^#prepared as a single diasteromer having (R) stereochemistry at the P-branch of the N- terminal group.

When compared with the series of -alkyl-y-aminobutyl N-terminal compounds having a PMB scaffold described above, the compounds of the present invention show a trend different to that which would be expected. The prior art compound DI 05 (having a pentyl -substituent, Table A) and the comparator compound C9 (having a hexyl P- substituent) exhibit equivalent cytotoxicity levels. However an increase in chain length from pentyl to hexyl for compounds having the scaffold of the invention unexpectedly results in a large decrease in cytotoxicity, see compound C6 compared with compounds 1 and 2.

It is difficult to predict the combination of N-terminal group and amino acid residues at positions 6 and 7 which will lead to compounds having favourable properties for activity, cytotoxicity and drug level in kidney. This is in part because changes in lipophilicity generally have opposing effects on cytotoxicity and activity. Furthermore, the levels of drug retention in the kidney do not appear to follow an obvious trend.

Polymyxin Compounds

The compounds of formula (I) are variants and N-terminal derivatives of the polymyxin nonapeptide series of compounds. The core of the compound of formula (I) is a derivative of a nonapeptide, such as the Polymyxin B nonapeptide (PMBN, Polymyxin B 2-10), where the amino acid residue at position 3 is substituted with Dap, the amino acid residue at position 6 substituted with D-Leu, the amino acid residue at position 7 is substituted with (S)-Abu. The compounds of formula (I) have an amino alkyl group at the N terminal of the polymyxin core.

The exemplified compounds of the invention are compounds 1 and 2.

Amino Acid Residue at Position 3

The amino acid residue at position 3 may be Dap (a,P-diaminopropionic acid). This may be L-Dap. Each of compounds 1 and 2 has L-Dap at this 3-position. Alternatively, the amino acid residue at position 3 may be D-Dap.

N Terminal Group

The N terminal group is the group attached to the N terminal of the amino acid residue at position 2 of the polymyxin core.

The terminal may be:

where the asterisk is the point of attachment to the N terminal of the amino acid residue at position 2.

The terminal may be:

The worked examples in the present case include compounds having one or the other of the stereochemistries given above.

Compound (II) and Compound (III)

The compound of formula (I) may be a compound of formula (II) as shown below:

and salts, solvates and protected forms thereof.

The compound of formula (I) may be a compound of formula (III) as shown below:

and salts, solvates and protected forms thereof.

Polymyxin Compounds

The compounds for use in the present case are based on modified forms of known polymyxin compounds, such as Polymyxin B nonapeptide and Colistin nonapeptide.

Polymyxin B nonapeptide has the structure shown below:

H₂N-Thr²-Dab-D ^{4 1}

where positions 2, 4 and 10 are indicated (with reference to the numbering system used for the Polymyxin B decapeptide), and the amino acid residues are in the L-configuration, unless indicated.

The compounds of the invention are derivatives and variants of the polymyxin B nonapeptide, where (i) the N terminal amino group, -NfP, of the residue at position 2 is replaced with the group -NH-C(O)-CH2-CH(R^T)-CH2-NH2, where -R^T is n-hexyl (- (CH₂)SCH3); (ii) the amino acid residue at position 3 is substituted with Dap, (iii) the amino acid residue at position 6 is substituted with D-Leu; and (iv) the amino acid residue at position 7 is substituted with (S)-Abu.

Compounds of the invention, which include the compounds described above, are biologically active. Methods of Synthesis

The preparation of the compounds of the invention will be familiar to those of skill in the art, particularly having knowledge of the methods described in WO 2015/135976 (for the preparation of modified polymyxin nonapeptides, and the methods described in WO 2014/188178 and WO 2016/083531 for the preparation of modified polymyxin nonapeptide variants. The methods described in the art may be readily adapted for use in the preparation of the compounds of the present case, taking into account the N terminal groups employed in the present case, and the amino acid residue substitutions at each of positions 3, 6 and 7 (with respect to the Polymyxin B nonapeptide core).

Generally, a compound of the invention may be prepared by coupling a suitably protected polymyxin nonapeptide intermediate core with a carboxylic acid having the functionality for the N terminal group. The product of this reaction is typically the protected form of the compound of formula (I). Removal of the protecting groups may be undertaken as desired. This is the general strategy known from WO 2015/135976.

As described herein, a compound of the invention may also be prepared by the solid phase synthesis of a suitably protected linear nonapeptide, followed by the addition of a carboxylic acid having the functionality for the N terminal group to the nonapeptide whilst still bound to the resin. Subsequent cleavage of the linear peptide from the resin, deprotection and cyclisation between the amino acid residues at position 4 and 10 leads to a compound of the invention.

A suitably protected nonapeptide intermediate may also be prepared by solid phase synthesis of a linear nonapeptide, followed by cleavage of the linear form from the solid support and then subsequent cyclisation of that linear form between the amino acid residues at positions 4 and 10. The nonapeptide intermediate may be prepared with suitable selections for the amino acid residues at each of the relevant positions.

Compounds of the invention may be made by conventional peptide synthesis, using methods known to those skilled in the art. Suitable methods include solid-phase synthesis such as described by de Visser et al, J. Peptide Res, 61, 2003, 298- 306, Vaara et al, Antimicrob. Agents and Chemotherapy, 52, 2008. 3229-3236, or by Velkov et al. ACS Chem. Biol. 9, 2014, 1172. These methods include a suitable protection strategy, and methods for the cyclisation step.

Where required, the compound of formula (I) may be at least partially purified, for example to separate diastereomeric forms of the product. Protected Forms

Compounds of the invention, such as compounds of formula (I), (II) and (III), may be provided in a protected form. Here, reactive functionality, such as amino functionality, may be masked in order to prevent its reaction during a synthesis step. A protecting group is provided to mask the reactive functionality, and this protecting group may be removed at a later stage of the synthesis to reveal the original reactive functionality.

In one embodiment, the protected form is a compound where amino, and/or hydroxyl functionality is protected (masked) by a protecting group. In one embodiment, the protected form is a compound where the side chain functionality of the amino acids residues within the compound are protected.

In the compound of formula (I), (II) and (III), the amino acid residues at positions 5, 8 and 9 are Dab residues, and the side chain of the Dab residue includes amino functionality. The amino acid functionality of each Dab residue may be protected with an amino protecting group, as described herein. Similarly, the amino acid residue at position 3 is Dap, and the side chain of this amino acid residue includes amino functionality. The amino acid functionality of each Dap residue may be protected with an amino protecting group, as described herein.

Protecting groups, such as those for amino acid residues, are well known and well described in the art.

Amino acids having side group protection, optionally together with amino and carboxy protection, are commercially available. Thus, a protected polymyxin compound may be prepared from appropriately protected amino acid starting materials.

Velkov et al. describe the step-wise preparation of polymyxin compounds on the solid-phase using suitably protected amino acid. The use of protected-forms of threonine and Dab is disclosed (see Supplementary Information).

Where a protecting group is used is it is removable under conditions that do not substantially disrupt the structure of the polymyxin core, for example conditions that do not alter the stereochemistry of the amino acid residues.

In one embodiment, the protecting groups are acid-labile, base labile, or are removable under reducing conditions.

Example protecting groups for amino functionality include Boc (tert-butoxycabonyl), Bn (benzyl, Bzl), CbZ (benzyloxycarbonyl, Z), 2-CL-Z (2-chloro), ivDde (l-[4,4-dimethyl- 2,6-dioxocylcohex-l-ylidene]-3-methylbutyl), Fmoc (fluorenylmethyloxycarbonyl), HSO3- Fmoc (sulfonylated Fmoc, such as 2-sulfo-Fmoc, as described in e.g. Schechter et al, J. Med Chem 2002, 45 (19) 4264), Dde (l-[4,4-dimethyl-2,6-dioxocylcohex-l-ylidene]ethyl), Mmt (4-methoxy trityl), Mtt (4-methyl trityl), Nvoc (6-nitroveratroyloxycarbonyl), Tfa (trifluroacetyl), and Alloc (allyloxycarbonyl).

In one embodiment, the protecting group for amino functionality is selected from Boc, ivDde , CbZ, Bn and Fmoc and HSCh-Fmoc.

In one embodiment, the protecting group for amino functionality is Boc, ivDde, Fmoc or CbZ, such as Boc, ivDde or Cbz.

Boc protection may be provided for the amino functionality present in the side chains of the amino acid residues present at positions 5, 8 and 9, and optionally position 3.

Example protecting groups for hydroxyl functionality include Trt (trityl), Bn (benzyl), and tBu (tert-butyl).

In one embodiment, the protecting group for hydroxyl functionality is tBu.

Further example protecting groups include silyl ether protecting groups, such as TMS, TES, TBS, TIPS, TBDMS, and TBDPS. Such protecting groups are removable with TBAF, for example.

In some embodiments, only some types of functionality are protected. For example, only amino groups may be protected, such as amino groups in the side chain of an amino acid residue.

In one embodiment, amino groups and hydroxyl groups are protected.

Active Agent

The compounds of formula (I), (II) or (III) may each be used together with a second active agent. The inventors have found that such combinations have greater biological activity than would be expected from the individual activity of both compounds. The compounds of formula (I), (II) or (III) can be used to potentiate the activity of the second active agent. In particular, the compounds of formula (I), (II) or (III) may be used together with a second active agent to enhance the antimicrobial activity of that agent, for example against Gram-negative bacteria.

Without wishing to be bound by theory it is believed that the compounds of formula (I), (II) or (III) act on the outer membrane of a cell e.g. a Gram-negative bacterial cell, to facilitate the uptake of the second active agent into that cell. Thus, agents that are otherwise incapable or poor at crossing the outer membrane may be taken up into a target cell by the action of the compounds of formula (I), (II) or (III). In one embodiment, the combination of a compound of formula (I), (II) or (III) with the second active agent is active against Gram-negative bacteria. Here, it is not essential that individually either of the compound of formula (I), (II) or (III) or the second active agent have activity against Gram-negative bacteria.

In one embodiment, the second active agent is an agent having a measured MIC value against a particular microorganism, such as a bacterium, that is less than 10, less than 5, or less than 1 micrograms/mL. The microorganism may be a Gram-negative bacteria, such as a Gram-negative bacteria selected from the group consisting of E. coli, S. enterica, K. pneumoniae, K. oxytoca', E. cloacae, E. aerogenes, E. agglomerans, A. calcoaceticus, A. baumannii; Pseudomonas aeruginosa, and Stenotrophomonas maltophila.

Examples of second active agents that have activity against Gram-negative bacteria include beta-lactams, tetracyclines, aminoglycosides and quinolones.

In one embodiment, the second active agent is an agent having a measured MIC value against a particular microorganism, such as a Gram-negative bacterium, that is more than 4, more than 8, more than 16 or more than 32 micrograms/mL. In this embodiment, the second active agent may be active against Gram-positive bacteria. For example, the second active agent is an agent having a measured MIC value against a particular Gram-positive bacterium that is less than 10, less than 5, or less than 1 micrograms/mL. Here, the compound of formula (I), (II) or (III) acts to facilitate the uptake of the second active agent into the Gramnegative bacterial cell. The second active agent is therefore able to act on a target within the Gram- negative bacterial cell, which target may be the same as the second active agent’s target in a Gram-positive bacterial cell.

The Gram-positive bacteria may be selected from the group consisting of Staphylococcus and Streptococcus bacteria, such as S. aureus (including MRSA), S. epidermis, E. faecalis, and E. faecium.

Examples of second active agents that have activity against Gram-positive bacteria (at the MIC values given above, for example), and moderate activity against Gram-negative bacteria, include rifampicin, novobiocin, macrolides, pleuromutilins. In one embodiment, a compound having moderate activity against Gram-negative bacteria may have a measured MIC value against a Gram-negative bacterium that is less than 32, less than 64, or less than 128 micrograms/mL.

Also suitable for use are agents having activity against Gram-positive bacteria and which are essentially inactive against Gram-negative bacteria. Examples include fusidic acid, oxazolidinines (e.g. linezolid), glycopeptides (e.g. vancomycin), daptomycin and lantibiotics. In one embodiment, a compound having essentially no activity against Gram-negative bacteria may have a measured MIC value against a Gram-negative bacterium that is more than 32, more then 64, more than 128, more than 256 micrograms/mL.

Under normal circumstances such agents are not necessarily suitable for use against Gram-negative bacteria owing to their relatively poor ability to cross the outer membrane of a Gram-negative bacterial cell. As explained above, when used together with a compound of formula (I), (II) or (III), such agents are suitable for use.

In one embodiment, the active agent may be selected from the group consisting of rifampicin (rifampin), rifabutin, rifalazil, rifapentine, rifaximin, aztreonam, oxacillin, novobiocin, fusidic acid, azithromycin, ciprofloxacin, meropenem, tigecycline, minocycline, erythromycin, clarithromycin and mupirocin, and pharmaceutically acceptable salts, solvates and prodrug forms thereof.

The present inventors have found that the polymyxin compounds of formula (I), (II) or (III) may be used together with certain compounds in the rifamycin family to treat microbial infections. The rifamycin family includes isolates rifamycin A, B, C, D, E, S and SV, and synthetically derivatised versions of these compounds, such as rifampicin (rifampin), rifabutin, rifalazil, rifapentine, and rifaximin, and pharmaceutically acceptable salts and solvates thereof.

In one embodiment, the active agent is rifampicin (rifampin) and pharmaceutically acceptable salts, solvates and prodrug forms thereof.

Salts, Solvates and Other Forms

Examples of salts of compound of formula (I), (II) and (III) include all pharmaceutically acceptable salts, such as, without limitation, acid addition salts of strong mineral acids such as HCI and HBr salts and addition salts of strong organic acids such as a methanesulfonic acid salt. Further examples of salts include sulfates and acetates such as acetate itself, trifluoroacetate or trichloroacetate.

In one embodiment the compounds of the present disclosure are provided as a sulfate salt or a trifluoroacetic acid (TFA) salt. In one embodiment the compounds of the present disclosure are provided as acetate salts, such as acetate.

A compound of formula (I), (II) or (III) can also be formulated as a prodrug. Prodrugs can include an antibacterial compound herein described in which one or more amino groups are protected with a group which can be cleaved in vivo, to liberate the biologically active compound. In one embodiment the prodrug is an “amine prodrug”. Examples of amine prodrugs include sulphomethyl, as described in e.g., Bergen et al, Antimicrob. Agents and Chemotherapy, 2006, 50, 1953 or HSO3-FMOC, as described in e.g. Schechter et al, J. Med Chem 2002, 45(19) 4264, and salts thereof. Further examples of amine prodrugs are given by Krise and Oliyai in Biotechnology: Pharmaceutical Aspects, 2007, 5(2), 101-131.

In one embodiment a compound of formula (I), (II) or (III) is provided as a prodrug.

A reference to a compound of formula (I), (II) or (III), or any other compound described herein, is also a reference to a solvate of that compound. Examples of solvates include hydrates.

A compound of formula (I), (II) or (III), or any other compound described herein, includes a compound where an atom is replaced by a naturally occurring or non-naturally occurring isotope. In one embodiment the isotope is a stable isotope. Thus a compound described here includes, for example deuterium containing compounds and the like. For example, H may be in any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Certain compounds of formula (I), (II) or (III), or any other compound described herein, may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exoforms; R-, S-, and meso-forms; D- and E-forms; d- and 1-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and - forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair- forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers,” as used herein, are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, metachlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., Ci-ealkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and paramethoxyphenyl) . Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

One aspect of the present invention pertains to compounds in substantially purified form and/or in a form substantially free from contaminants.

In one embodiment, the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.

Unless specified, the substantially purified form refers to the compound in any stereoisomeric or enantiomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to an equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer.

In one embodiment, the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight.

Unless specified, the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer.

In one embodiment, the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure. Methods of Treatment

The compounds of formula (I), (II) or (III), or pharmaceutical formulations containing these compounds, are suitable for use in methods of treatment and prophylaxis. The compounds may be administered to a subject in need thereof. The compounds are suitable for use together with an active agent (“a second active agent”), for example a second active agent that is an antimicrobial agent.

The compounds of formula (I), (II) or (III) are for use in a method of treatment of the human or animal body by therapy. In some aspects of the invention, a compound of formula (I), (II) or (III) may be administered to a mammalian subject, such as a human, in order to treat a microbial infection.

Another aspect of the present invention pertains to use of a compound of formula (I) or (II) in the manufacture of a medicament for use in treatment. In one embodiment, the medicament comprises a compound of formula (I), (II) or (III). In one embodiment, the medicament is for use in the treatment of a microbial infection.

The term "microbial infection" refers to the invasion of the host animal by pathogenic microbes. This includes the excessive growth of microbes that are normally present in or on the body of an animal. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host animal. Thus, an animal is "suffering" from a microbial infection when excessive numbers of a microbial population are present in or on an animal's body, or when the presence of a microbial population(s) is damaging the cells or other tissue of an animal.

The compounds may be used to treat a subject having a microbial infection, or at risk of infection from a microorganism, such as a bacterium.

The microbial infection may be a bacterial infection such as a Gram-negative bacterial infection.

Examples of Gram-negative bacteria include, but are not limited to, Escherichia spp., Klebsiella spp., Enterobacter spp., Salmonella spp., Citrobacter spp., and other Enterobacteriaceae, Pseudomonas spp., Acinetobacter spp., Stenotrophomonas, and Legionella and numerous others.

Medically relevant Gram-negative bacilli include a multitude of species. Some of them primarily cause respiratory problems (Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary problems (Escherichia coli, Enterobacter cloacae), and primarily gastrointestinal problems (Salmonella enterica). Gram-negative bacteria associated with nosocomial infections include Acinetobacter baumannii, which causes bacteraemia, secondary meningitis, and ventilator-associated pneumonia in intensive-care units of hospital establishments.

In one embodiment the Gram-negative bacterial species is selected from the group consisting of E. coli, S. enterica, K. pneumoniae, K. oxytocw, E. cloacae, E. aerogenes, E. agglomerans, A. calcoaceticus, A. baumannii', Pseudomonas aeruginosa, and Stenotrophomonas maltophila.

In one embodiment the Gram-negative bacterial species is selected from the group consisting of E. coli, K. pneumoniae, Pseudomonas aeruginosa, and A. baumannii.

The compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of skin and soft tissue infections, gastrointestinal infection, urinary tract infection, pneumonia, sepsis, intra-abdominal infection and obstetrical/gynaecological infections. The infections may be Gram-negative bacterial infections.

The compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of Pseudomonas infections including P. aeruginosa infection, for example skin and soft tissue infections, gastrointestinal infection, urinary tract infection, pneumonia and sepsis.

The compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of Acinetobacter infections including A. baumanii infection, for pneumonia, wound infections, urinary tract infection and sepsis.

The compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of Klebsiella infections including K. pneumoniae infection, for pneumonia, intra-abdominal infection, urinary tract infection, meningitis and sepsis.

The compounds of formula (I), (II) or (III) or compositions comprising the same are useful for the treatment of E. coli infection including E. coli infections, for bacteraemia, cholecystitis, cholangitis, intra-abdominal infection, urinary tract infection, neonatal meningitis and pneumonia.

The compounds of formula (I), (II), or (III) or compositions comprising the same may be used together with an active agent in methods of treatment.

The active agent may be an agent that has activity against the microorganism. The active agent may be active against Gram-negative bacteria. The active agent may be active against a microorganism selected from the list given above. In one embodiment, the second active agent has an MIC value of 10 micrograms/mL or less against a microorganism such as E. coli, in the absence of the compound of formula (I), (II) or (III). The microorganism may be a microorganism selected from the group above.

Specific compounds for use as second active agents are described herein and include: rifampicin, rifabutin, rifalazil, rifapentine, and rifaximin; oxacillin, methicillin, ampicillin, cioxacillin, carbenicillin, piperacillin, tricarcillin, flucioxacillin, and nafcillin; azithromycin, clarithromycin, erythromycin, telithromycin, cethromycin, and solithromycin; aztreonam and BAL30072; meropenem, doripenem, imipenem, ertapenem, biapenem, tomopenem, and panipenem; tigecycline, omadacycline, eravacycline, doxycycline, and minocycline; ciprofloxacin, levofloxacin, moxifloxacin, and delafloxacin;

Fusidic acid;

Novobiocin; teichoplanin, telavancin, dalbavancin, and oritavancin; zidovudine (AZT), and pharmaceutically acceptable salts and solvates thereof;

In one embodiment, specific compounds for use as second active agents are described herein and include rifampicin (rifampin), rifabutin, rifalazil, rifapentine, rifaximin, aztreonam, oxacillin, novobiocin, fusidic acid, azithromycin, ciprofloxacin, meropenem, tigecycline, erythromycin, clarithromycin and mupirocin, and pharmaceutically acceptable salts and solvates thereof.

In an alternative aspect, the compounds of formula (I) are suitable for use in the treatment of fungal infections, for example in combination together with an antifungal agent. The antifungal agent may be selected from a polyene antifungal, for example amphotericin B, an imidazole, triazole, or thiazole antifungal, for example miconazole, fluconazole or abafungin, an allylamine, an echinocandin, or another agent, for example ciclopirox.

Treatment

The term “treatment,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term “treatment.”

The term “therapeutically-effective amount,” as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

The term “treatment” includes combination treatments and therapies, as described herein, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.

Combination Therapy

A compound of formula (I), (II) or (III) may be administered in conjunction with an active agent. Administration may be simultaneous, separate or sequential.

The methods and manner of administration will depend on the pharmacokinetics of the compound of formula (I), (II) or (III) and the second active agent.

By “simultaneous” administration, it is meant that a compound of formula (I), (II) or (III) and a second active agent are administered to a subject in a single dose by the same route of administration.

By “separate” administration, it is meant that a compound of formula (I), (II) or (III) and a second active agent are administered to a subject by two different routes of administration which occur at the same time. This may occur for example where one agent is administered by infusion and the other is given orally during the course of the infusion.

By “sequential” it is meant that the two agents are administered at different points in time, provided that the activity of the first administered agent is present and ongoing in the subject at the time the second active agent is administered.

Generally, a sequential dose will occur such that the second of the two agents is administered within 48 hours, such as within 24 hours, such as within 12, 6, 4, 2 or 1 hour(s) of the first agent. Alternatively, the active agent may be administered first, followed by the compound of formula (I), (II) or (III). Ultimately, the order and timing of the administration of the compound and second active agent in the combination treatment will depend upon the pharmacokinetic properties of each.

The amount of the compound of formula (I), (II) or (III) to be administered to a subject will ultimately depend upon the nature of the subject and the disease to be treated. Likewise, the amount of the active agent to be administered to a subject will ultimately depend upon the nature of the subject and the disease to be treated.

Formulations

In one aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I), (II) or (III) together with a pharmaceutically acceptable carrier. The pharmaceutical composition may additionally comprise a second active agent. In an alternative embodiment, where a second active agent is provided for use in therapy, the second active agent may be separately formulated from the compound of formula (I), (II) or (III). The comments below made in relation to the compound of formula (I), (II) or (III) may therefore also apply to the second active agent, as separately formulated.

While it is possible for the compound of formula (I), (II) or (III) to be administered alone or together with the second active agent, it is desirable to present it as a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising at least one compound of formula (I), (II) or (III), as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.

The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.

Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one compound of formula (I), (II) or (III), as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound. The composition optionally further comprises the second active agent in a predetermined amount. The term “pharmaceutically acceptable,” as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005.

The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the compound of formula (I) or (II) with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.

Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, nonaqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in- oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.

Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir.

The compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients. The compound may be presented in a liposome or other microparticulate which is designed to target the compound, for example, to blood components or one or more organs. Where a liposome is used, it is noted that the liposome may contain both the compound of formula (I), (II), (III) and a second active agent. Formulations suitable for oral administration (e.g., by ingestion) include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.

Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Losenges typically comprise the compound in a flavoured basis, usually sucrose and acacia or tragacanth. Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia. Mouthwashes typically comprise the compound in a suitable liquid carrier.

Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills.

Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil- in-water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.

Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid); flavours, flavour enhancing agents, and sweeteners. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach.

Ointments are typically prepared from the compound and a paraffinic or a water- miscible ointment base.

Creams are typically prepared from the compound and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-l,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. A hydrophilic emulsifier may be included together with a lipophilic emulsifier which acts as a stabiliser. It is also possible to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should be a non-greasy, non- staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for intranasal administration, where the carrier is a liquid, include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound. As an alternative method of administration, a dry powder delivery may be used as an alternative to nebulised aerosols.

Formulations suitable for intranasal administration, where the carrier is a solid, include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.

Formulations suitable for pulmonary administration (e.g., by inhalation or insufflation therapy) include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane, carbon dioxide, or other suitable gases. Additionally or alternatively, a formulation for pulmonary administration may be formulated for administration from a nebuliser or a dry powder inhaler. For example, the formulation may be provided with carriers or liposomes to provide a suitable particle size to reach the appropriate parts of the lung, to aid delivery of an appropriate dose to enhance retention in the lung tissue.

Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.

Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration (e.g., for example by injection or infusion, intravenously or subcutaneously), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, sugars, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the compound in the liquid is from about 1 ng/mL to about 500 pg/mL, for example about 1 ng/mL to about 100 pg/mL, for example from about 10 ng/mL to about 10 pg/mL, for example from about 10 ng/mL to about 1 pg/mL. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, 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.

Dosage

Generally, the methods of the invention may comprise administering to a subject an effective amount of a compound of formula (I), (II) or (III) so as to provide an antimicrobial effect. The compound of formula (I), (II) or (III) may be administered at an amount sufficient to potentiate the activity of a second active agent. The second active agent is administered to a subject at an effective amount so as to provide an antimicrobial effect. It will be appreciated by one of skill in the art that appropriate dosages of the compound of formula (I), (II) or (III) or the active agent, and compositions comprising the compound of formula (I), (II) or (III) or the active agent, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound of formula (I) , (II) or (III) or the active agent, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of compound of formula (I), (II) or (III) or the active agent and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

In general, a suitable dose of a compound of formula (I), (II) or (III) or the active agent is in the range of about 10 pg to about 250 mg (more typically about 100 pg to about 25 mg) per kilogram body weight of the subject per day. Where the compound of formula (I), (II) or (III) or the active agent is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Kits

One aspect of the invention pertains to a kit comprising (a) a compound of formula (I), (II) or (III), or a composition comprising a compound as defined in any one of formula (I), (II) or (III), e.g., typically provided in a suitable container and/or with suitable packaging; and (b) instructions for use, e.g., written instructions on how to administer the compound or composition.

The written instructions may also include a list of indications for which the compound of formula (I), (II) or (III) is a suitable treatment.

In one embodiment, the kit further comprises (c) a second active agent, or a composition comprising the second active agent. Here, the written instructions may also include a list of indications for which the second active agent, together with the compound of formula (I), (II) or (III), is suitable for treatment. Routes of Administration

A compound of formula (I), (II) or (III), a second active agent, or a pharmaceutical composition comprising the compound of formula (I), (II) or (III), or the second active agent may be administered to a subject by any convenient route of administration, whether systemically /peripherally or topically (i.e., at the site of desired action).

Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection or infusion, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrastemal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.

The Subject/Patient

The subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orang-utan, gibbon), or a human. Furthermore, the subject/patient may be any of its forms of development, for example, a foetus.

In one embodiment, the subject/patient is a human.

It is also envisaged that the invention may be practised on a non-human animal having a microbial infection. A non-human mammal may be a rodent. Rodents include rats, mice, guinea pigs, chinchillas and other similarly- sized small rodents used in laboratory research. Other Options

Each and every compatible combination of the embodiments described above is explicitly disclosed herein, as if each and every combination was individually and explicitly recited.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. Where technically appropriate embodiments may be combined and thus the disclosure extends to all permutations and combinations of the embodiments provided herein.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein.

Synthesis

All reagents used for chemical synthesis were purchased from commercially available sources and used without further purification. Preparative HPLC was performed on a Gilson preparative HPLC system using a Waters Sunfire C18 OBD 5 pm (19 mm x 150 mm) column eluted with appropriate water/acetonitrile gradients containing 0.15% TFA, with detection at 210 nm. ¹ H NMR spectra were recorded at 400 MHz on a Mercury 400 NMR spectrometer (Agilent Technologies) or Bruker Avance III 400. Chemicals shifts (5) are reported in ppm downfield from TMS. Coupling constants J are recorded in Hertz (Hz).

Mass spectra were recorded on an LCQ DecaXP mass spectrometer with +ve ion electrospray ionisation.

General Method of Solid Phase Synthesis

Synthesis of the protected linear peptide (residues 1-9 and N-terminal group) was carried out on an automated peptide synthesizer using standard Fmoc solid phase peptide chemistry. Specifically, synthesis was undertaken using Fmoc-Thr(tBu)-PEG-PS resin as starting material. Coupling of the Fmoc-amino acids with CBZ protection on the terminal amino groups was performed using 5 molar equivalents (relative to resin loading) of Fmoc amino acid and HATU in DMF with activation in situ, using 10 molar equivalents of DIPEA. Fmoc deprotection was performed using 20% piperidine in dimethylformamide. BOC was used as the orthogonal protecting group on the Dab involved in cyclisation.

The resin-bound linear peptide was treated with TFA/TIS/H2O (96/2/2v/v) for 2 h to reveal the Dab residue involved in cyclisation, and to cleave the peptide from the resin. This material was cyclised using PyBop/HOBt/NMM (4/4/8 molar equivalents relative to the initial loading) in DMF for 3 h. The crude material was partially evaporated, taken up acetonitrile/water and lyophilised overnight. The CBZ groups were then removed using 10% Pd/C in Acetic acid/MeOH/water (5/4/1 v/v).

General Method of Preparation of Acetate Salts

AG1-X2 resin (Bio-Rad Laboratories Ltd) acetate form 200-400 mesh, was regenerated by washing with 10% aqueous acetic followed by 1% aqueous acetic acid, and placed in a fritted cartridge. A solution of the compound as a TFA salt in water was applied to the column, using a loading of 30 g resin to 1 g TFA salt, and the column allowed to drip under gravity, eluting with water. Product-containing fractions were combined and lyophilised to a white solid.

Analytical HPLC conditions for final compounds:

Column: Phenomenex Hyperclone C18 BDS 5 pm x 4.6 mm x 150 mm

Mobile phase: A: water/acetonitrile 90/10, v/v, 0.15% TFA.

B: acetonitrile/water 90/10, v/v, 0.15% TFA

Flow rate: 1 mL/min

Gradient:

Time (mins) % mobile phase A

0 100%

20 40%

21 0%

23 0%

23.5 100

25 100 Detection: 210, 254 nm

Injection volume: 20 |1L

3-({ [(Benzyloxy)carbonyl]amino}methyl)nonanoic acid

(i) Methyl 3-(nitromethyl)nonanoate

To a solution of methyl (E)-non-2-enoate (1.46 g, 6.8 mmol) in nitromethane (10 mL) was added DBU (4.99 mL, 30 mmol) over 15 mins with ice-bath cooling. The reaction mixture was stirred at room temperature over the weekend, concentrated under vacuum and the residue partitioned between 0.5M HC1 (aq) and diethyl ether. The aqueous layer was separated and extracted with additional diethyl ether. The organic extracts were combined, washed with brine, dried with MgSO4, filtered and evaporated to dryness. The residues were purified on silica, eluting with pet ether 40-60 and ethyl acetate (0-50%). The appropriate fractions were combined and evaporated to dryness, producing 5.50g of colourless oil (82%).

^JH NMR (400 MHz, CDC1₃): 0.87 (3H, t, J= 7.0 Hz), 1.17-1.44 (10H, m), 2.44 (2H, d, J = 6.6Hz), 2.55 - 2.69 (1H, m), 3.69 (3H, s), 4.46(2H, dABq, J= 14.7, 6.1 Hz).

(ii) Methyl 3-(benzyloxycarbonylaminomethyl)nonanoate

Zinc dust (14.3 g, 219 mmol) was added portion wise to a solution of methyl 3-(nitromethyl)nonanoate (5.5g, 24 mmol) in acetic acid (65 mL), stirred at 0-5 DC (caution - exothermic) . The mixture was allowed to warm to room temperature, stirring for 17h. The mixture was evaporated to dryness and the residue partitioned between ethyl acetate and saturated NaHCOs (aq.). The mixture was filtered through Celite. The aqueous phase was separated and extracted with additional ethyl acetate. The organic extracts were combined, dried with MgSCL, filtered and evaporated to dryness, producing 2.81g of orange oil (58%). Mass spectrometry gave evidence of the desired product together with the corresponding lactam. m/z 202 [M+H]⁺ (desired product), 170 [M+H]⁺ (lactam). The material was used directly in the next reaction.

A mixture of the crude methyl 3-(aminomethyl)nonanoate (2.81g, 13.96mmol) , water (18ml), sodium bicarbonate (1.76g, 21mmol) , and 1,4-dioxane (9ml) was cooled in an ice bath. A solution of N-benzyloxycarbonyloxy)succinimide (3.83g, 15.35mmol) in 1,4- dioxane (9 ml) was added drop wise. The mixture was stirred at 0-5 DC for 30 minutes and then allowed to warm to room temperature, stirring for 18h. The mixture was evaporated to dryness and the residues partitioned between diethyl ether and 0.5M HCl(aq). The aqueous layer was separated and extracted with additional diethyl ether. The organic extracts were combined, washed with brine, dried with MgSO4, filtered and evaporated to dryness. The residues were purified on silica, eluting with pet ether 40-60 and ethyl acetate. The appropriate fractions were combined and evaporated to dryness, producing 820 mg of colourless oil (2.44 mmol 18% yield); m/z 336 [M+H]⁺ (iii) Title compound

A mixture of methyl 3-(benzyloxycarbonylaminomethyl)nonanoate (0.82 g, 2.44 mmol), lithium hydroxide (0.18 g, 7.33 mmol), water (10 mL) and 1,4-dioxane (10 mL) was stirred at room temperature for 6 h. The mixture was evaporated to dryness, the residues were suspended in water and extracted with ethyl acetate. The organic layer was discarded. The aqueous was acidified with 1 M HCl(aq), then extracted with ethyl acetate (x2). The organic extracts were combined, dried with MgSO4, filtered and evaporated to dryness, producing 419 mg of colourless oil (53%); m/z 322 [M+H]⁺ .

NMR (400MHz, CD₃OD): 0.89 (3H, t, J= 6.6 Hz), 1.21-1.39 (10H, m), 1.94-2.06 (1H, m), 2.24 (2H, dd, 7 = 7.0, 14.7 Hz), 3.11 (2H, dd, 7 = 6.6, 14.0 Hz), 5.04 - 5.11 (2H, m), 7.24 - 7.43 (5H, m).

Alternative Synthesis:

Methyl 3-(nitromethyl)nonanoate was converted to 4-hexylpyrrolidine-2-one as described in the synthesis of 4-pentylpyrrolidine-2-one (Brown et al . ACS Infect. Dis. 2019, 5, 1645). A mixture of 4-hexylpyrrolidine-2-one (250 mg) and 6M HC1 (8.5 mL) was heated to 100DC for 17 h. The mixture was evaporated to dryness, then co-evaporated from dichloromethane to afford the ring-opened amino acid as the hydrochloride salt, as a yellow oil. The material was dissolved in water (3 mL) and 1,4-dioxane (3ml), treated with sodium bicarbonate (522 mg, 2.5 equiv) and cooled to 0DC. A solution of N-(benzyloxycarbonyloxy)succinimide (541 mg, 1.1 equiv) in 1,4-dioxane (1.5 mL) was added dropwise. The mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was evaporated to dryness and the residue partitioned between ethyl acetate and saturated aq. sodium bicarbonate. The aqueous phase was separated and washed with additional ethyl acetate. The organic phase was discarded. The aqueous phase was acidified to pH 4 with citric acid (20% aqueous) and then extracted with ethyl acetate (x3). The organic extracts were combined, dried (MgSO4) and evaporated. The residue was chromatographed in silica eluting with 0-100% ethyl acetate in hexane to afford the title compound as a colourless oil (325 mg, 54%) m/z 322 [M+H]⁺ . (37?)-3-({ [(Benzyloxy)carbonyl]amino}methyl)nonanoic acid

The racemic product was subjected to chiral separation by supercritical fluid chromatography using the preparative separation conditions described below. Enantiomeric purity was confirmed using the chiral analytical chromatography method below.

Preparative chiral supercritical fluid chromatography conditions:

Instrument: Thar 350 preparative SFC (SFC-6)

Column: ChiralPak AY, 300 x 50 mm I.D., 10 pm.

Mobile phase: A: CO2 and B: EtOH

Isocratic method: B 25%

Flow rate: 200 mF /min

Back pressure: 100 bar

Column temperature: 38 DC

Wavelength: 220 nm

Cycle time: 5 min

Work up: After separation, the fractions were dried off via rotary evaporator at bath temperature 40°C to get the desired isomers.

Enantiomer A, faster eluting isomer by SFC on Analytical column 2.6g 97.1% ee Enantiomer B, slower eluting isomer by SFC on Analytical columnl.l7g 99.1% ee Where ee (enantiomeric excess) is defined as the % Enantiomer A - % Enantiomer B Chiral analytical chromatography conditions:

Column: Phenomenex LuxA2, 250 x 4.6 mm I.D., 5 pm

Mobile phase: A: CO2 and Mobile phase B: EtOH (0.2% DEA)

Isocratic conditions: 20% B

Flow rate: 4 mL/min

Back pressure: 125 bar

Column temperature: 40°C

Wavelength: 210 - 400 nm

Fast isomer: retention time on analytical system: 1.90 min Slow isomer: retention time on analytical system: 2.16 min. The slower-eluting enantiomer was used in the preparation of compound CA1338, and shown to correspond to authentic material which had been prepared by separation of the diasteromers of the final product.

Confirmation of Stereochemistry

Ethyl 3-(nitromethyl)nonanoate was suspended in water and treated with Novozyme 435, using the procedure of Tetrahedron Asymmetry 2008, 19, 945-955, which has been demonstrated to hydrolyse only the (S)-enantiomer of the closely related ethyl 3-(nitromethyl)-5-methylhexanoate. The reaction was allowed to proceed to 50% conversion. The unreacted ethyl (R)-3-(nitromethyl)nonanoate was then converted to ( 3/?)-3- ({[(benzyloxy)carbonyl] amino }methyl)nonanoic acid as follows:

To a stirred solution of ethyl (R)-3-(nitromethyl)nonanoate (500 mg) in MeOH (2.5 mL) was charged 10% Pd/C (86.8 mg, dry basis; ca 50% water). The reaction vessel was evacuated for 2 minutes using vacuum, a blanket of hydrogen was introduced, and the evacuation process repeated twice more. The reaction was stirred for 2 days and upon completion of reaction, the reaction was filtered through celite and the celite pad washed with MeOH (5 mL x 2). The filtrate was concentrated under reduced pressure to yield a ~1:1 mixture of ethyl (R)-3-(aminomethyl)nonanoate and (R)-4-hexylpyrrolidin-2-one as a pale orange solid (430.5 mg).

The ~1:1 mixture of ethyl (R)-3-(aminomethyl)nonanoate and (R)-4-hexylpyrrolidin- 2-one (430.5 mg) was put into solution with 6M HC1 (5 mL), the solution was heated to 85°C and stirred for 12 h. Upon completion of reaction, the solution was cooled to ambient temperature and pH of the reaction mixture was adjusted from pH 1-2 to pH 6-7 with 5M NaOH. To the solution was aded DCM (5 mL), K2CO3 (1.3 g) and CbzCl (0.49 mL) and the mixture stirred for 16 h. The pH of reaction mixture was adjusted from pH 10 to pH 3 with 5M HC1 and extracted with DCM (10 mL). The aqueous layer was washed with DCM (10 mL). The combined organic layers were washed with H2O (10 mL) and brine solution (10 mL), dried with Na2SC>4, filtered and concentrated under reduced pressure. The crude material was purified by column chromatography using EtOAc/heptane gradient to provide (3R)-3-({[(benzyloxy)carbonyl]amino}methyl)nonanoic acid as a tan oil (321.4 mg, 73.8% yield). Chiral HPLC on the analytical conditions described above gave a retention time of 2.17 min, corresponding to the slower of the two enantiomers. Enantiomer B (slow running enantiomer) was thus assigned the (R) stereochemistry. [(3R)-3-(aminomethyl)nonanoyl]-Thr-Dap-Cyclo[Dab-Dab-DLeu-Abu-Dab-Dab-Thr] - 1

Solid phase peptide synthesis was carried out as described in the general method using 3-({[(benzyloxy)carbonyl]amino}methyl)nonanoic acid at the N-terminal. The crude deprotected product was then purified by preparative HPLC. Fractions containing the faster- eluting diastereomer were collected and lyophilised to afford the title compound as the TFA salt. The material was converted to the acetate salt as described in the general method, followed by lyophilisation to afford the title compound as the acetate salt as a white solid.

' H NMR (400 MHz, D₂O): 5 (ppm) 0.77 - 0.88 (12H, m), 1.11 - 1.38 (16H, m), 1.50 - 1.68 (4H, m), 1.78 - 2.28 (25H, m, includes 1.85, s, OAc), 2.44 (2H, d, J 6.6 Hz), 2.90-3.13 (9H, m), 3.21-3.33 (2H, m), 3.42 (1 H, dd, J 4.6, 13.4 Hz), 4.14 (1H, d, J 4.6 Hz), 4.16- 4.27(7H, m), 4.35 (1H, d, J 4.1 Hz), 4.47 (1H, dd, J 5.0, 9.3 Hz), m/z (+ve ESI) 1057 [M+H]⁺ , 529 [M+2H]²⁺, 100%.

HPLC retention time (conditions: Analytical method for final compounds) : 7.8 min. This material was assigned the (R) stereochemistry by separate synthesis from optically pure acid (see the alternative synthesis below).

[(3R)-3-(Aminomethyl)nonanoyl]-Thr-Dap-Cyclo[Dab-Dab-DLeu-Abu-Dab-Dab- Thr] - 1 - Alternative synthesis:

The synthetic method was carried out as described above, using the optically pure (3R)-3-({ [(benzyloxy)carbonyl]amino}methyl)nonanoic acid. m/z (+ve ESI) 529 [M+2H]²⁺. NMR and HPLC retention time identical to an authentic sample prepared above.

[(3S)-3-(Aminomethyl)nonanoyl]-Thr-Dap-Cyclo[Dab-Dab-DLeu-Abu-Dab-Dab-Thr] - 2

The synthesis was carried out as for CA1338, using racemic N-terminal acid. After preparative HPLC, fractions containing the slower-eluting diastereomer were collected and lyophilised to afford the title compound as the TFA salt. The material was converted to the acetate salt as described in the general method, followed by lyophilisation to afford the title compound as the acetate salt as a white solid. m/z (+ve ESI) 1057 [M+H]⁺, 529 [M+2H]²⁺, 100%. HPLC retention time: 8.2 min.

Comparator Compounds

The comparator compounds known in the art and described in Tables A, B, C and D, were prepared using the synthetic procedures described in WO 2015/135976 and WO 2020/002325 Furthermore, those comparator compounds not known in the art can be prepared using the same general methods as the compounds of the present invention described under Methods of Synthesis above.

Biological Results

The compounds of the invention were tested, and the results were compared to comparative examples, which includes compounds previously reported in the art. Data is provided in Tables 1 and 2 below.

MIC Determination

The inoculum was prepared by making a direct suspension of isolated colonies (selected from an 18-24 hour Mueller- Hinton agar plate) adjusted to the 0.5 McFarland standard. MIC testing was performed by two-fold serial antibiotic dilutions in cation- adjusted Mueller-Hinton Broth in sterile polypropylene 96-well microtitre plates in a total volume of l70 pL (150 pL broth containing the antimicrobial agent, 20 pL inoculum). The assays were performed in duplicate. Plates were incubated aerobically without shaking for 18-20 hours at 35 °C with the MIC defined as the lowest concentration of drug that prevented visible growth. Several of the compounds were subjected to multiple tests, and where this is the case, the MIC presented is the median value obtained. The MIC values are quoted in pg/mL.

In Vitro Renal Cell Toxicity Assay

The in vitro renal cell toxicity assay was performed according to the following protocol.

HK-2 cells were maintained and assayed in Keratinocyte-SFM media supplemented with 5 ng/mL Epidermal Growth Factor (EGF) and 50 pg/mL Bovine Pituitary Extract (BPE). Cells were seeded at 7,500 cells per well in 96-well plates and allowed to adhere overnight. Polymyxin B (PMB) and test compounds were dissolved in 10% DMSO in water to give a stock solution of 20 and 60 mg/mL, respectively. The test compounds were diluted to give a top concentration of 3,000 or 1,000 pg/mL with semi-log dilutions to give a 9-point concentration range plus vehicle control. PMB was also diluted to give a top concentration of 1,000 pg/ml with semi-log dilutions. Water and DMSO levels were kept constant at 5% and 0.5% respectively. The test compounds were incubated with cells for 24 h at 37DC 5% CO2 in a humidified atmosphere. CellTiter-Blue was diluted in PBS (1:4) and added 20% (v/v) and incubated at 37 DC for 2 h before fluorescent product was detected.

Media only background values were subtracted before the data was analysed using GraphPad Prism. Individual values were normalised to the vehicle control wells for each compound. Compound concentration values were plotted as log values to enable a doseresponse curve to be fitted. The bottom of the curve was constrained to zero and IC50 values were determined.

IC50 values are expressed relative to that for PMB in the same experiment. Where multiple determinations have been made, median values are presented.

Four Hour Kidney Level Measurements Compounds were dosed subcutaneously at 17.2 mg/kg free base to mice (n = 2 or 3). Four hours after dosing the animals were euthanised and kidneys removed, trimmed of fat and connective tissue, weighed and immediately snap-frozen. After thawing at room temperature, pairs of kidneys from each animal were placed in 2 mL conical tubes containing pre-weighed ceria-stabilised zirconium oxide beads. Trifluoroacetic acid, TFA (0.25 mL, 0.15% v/v in water) was added and the tubes were loaded onto a FastPrep-24 homogeniser (MP Biomedicals Europe), and subjected to 3 cycles of 30 seconds each at a speed of 6 m/sec. An aliquot (200 pL) of the homogenate was diluted with a calculated volume of TFA solution (0.15% v/v in water) to give a final concentration of 0.167 grams of kidney /gram of homogenate.

Kidney homogenates (100 pL) were mixed with methanol (190 pL) and TFA (110 pL, 10% v/v in water) and stored overnight at -20 DC for protein precipitation. After 10 min. centrifugation at 13,000 rpm and 6°C, 200pl of the supernatants were transferred into glass inserts and analysed by LC-MS-MS. Table 1

Relative to PMB as reported is in reference to the relative values given in WO 2015/135976. nd = not determined

Table 2

In Vivo Nephrotoxicity

Renal toxicity in male CD-I mice (n = 5) was determined for compound 1 (25, 50, 75 mg/kg/dose) in comparison with PMB (12.5, 25 mg/kg/dose) (Charles River Laboratories

Inc.). Compounds were dosed subcutaneously three times per day (8 hr apart) for either 24 hr (4 doses) or 4 days (12 doses). Immediately after the last dose, animals were transferred to metabolic cages for collection of urine for 24 h after which animals were sacrificed for histopathology. Levels of urinary biomarkers, KIM-1, cystatin C, albumin, P2 microglobulin and NGAL, as well as creatinine, in the urine were measured for compound 1 by Charles River standard analytical methods. Biomarker levels were expressed relative to the urinary creatinine level (supplementary data). Experiments were conducted in accordance with The Guide for the Care and Use of Laboratory Animals and the Current International Council on Harmonisation (ICH) Harmonised Tripartite Guidelines and generally accepted procedures for the testing of pharmaceutical compounds. Results of nephrotoxicity tests are given in tables 3 A and 3B below.

Table 3A - Biomarker data from mouse renal toxicity model and plasma exposure from single dose PK

Doses refer to mg free base/kg Table 3B - Histopathology data from mouse renal toxicity model

^# dose expressed as mg free base/kg

* deceased

Comparison of renal toxicity of compound 2 with comparator compounds CAI 061, Cl and PMB in a mouse renal toxicity assay

Mice (n = 6) were dosed subcutaneously three times per day in two separate experiments with Polymyxin B, CA1061 or Cl at 17.2 mg free base/kg. Starting immediately after the first dose on day 4, mice were transferred to individual metabolic cages and urine was collected over the following 24 hours for determination of biomarker levels (albumin, cystatin C, KIM-1). The geometric mean biomarker levels are presented in Table 3C below for CA1061 and Cl with the data for PMB from the same experiment in parentheses:

Table 3C

Cl was considered to be similarly toxic to PMB, whereas CA1061 appeared more toxic. Mice (n = 6) were dosed subcutaneously with PMB (25 mg free base per kg) or compound 2 (45 mg free base per kg) for four doses at 8 h intervals. After the fourth dose animals were transferred into individual metabolic cages and urine collected for 24 hours for determination of urinary biomarkers. Table 3D below gives biomarker levels, shown as the fold increase compared to the level prior to dosing.

Table 3D

With two of the three biomarkers PMB at 25 mg/kg gave a greater response than compound 2 at 45 mg/kg.

In Vivo Efficacy

Efficacy of compound 1 and compound 2 compared with polymyxin B in a neutropenic murine thigh model infected with E. coli ATCC 25922

After rendering neutropenic (cyclophosphamide 150 mg/kg d-4, 100 mg/kg d-1), CD- 1 mice (n = 5) were inoculated in each thigh with approx. 10⁵ cfu of E. coli ATCC25922. Mice were dosed IV with 0.125, 0.5, and 3 mg/kg of PMB sulphate or test compound (equivalent weight free base) at 1, 3.5 and 6 h post-infection. At 9 h post- infection mice were euthanised and thighs prepared for colony counts. The decrease in colony counts relative to a vehicle control for compound 1 and PMB is shown in Table 4A below.

Table 4A

In a separate experiment compound 2 was compared with PMB, the results are shown in

Table 4B below: Table 4B

Both test compounds performed similarly to PMB.

Efficacy of compound 1 in a neutropenic murine thigh model infected with K. pneumoniae ATCC 43816

After rendering neutropenic (cyclophosphamide 150mg/kg d-4, lOOmg/kg d-1), CD-I mice (n=5) were inoculated in each thigh with approx. 2.5 x 10⁵cfu of K. pneumoniae ATCC43816. Mice were dosed IV with appropriate doses of PMB sulphate or test compound (equivalent weight free base) at 2, 6 and 10 h post-infection. At 16 h post-infection mice were euthanised and thighs prepared for colony counts. The decrease in colony counts relative to a vehicle control is shown in Table 5 below.

Table 5

Compound 1 and PMB performed similarly in this model.

Efficacy of compound 1 in a neutropenic murine thigh model infected with

A. baumannii NCTC 13301

After rendering neutropenic (cyclophosphamide 150 mg/kg d-4, 100 mg/kg d-1), CD- 1 mice (n = 5) were inoculated in each thigh with approx. 3 x 10⁴cfu of A. baumannii NCTC13301. Mice were dosed IV with 0.125, 0.5, 1 and 4 mg/kg of PMB sulphate or test compound (equivalent weight free base) at 2, 6 and 10 h post- infection. At 16 h post- infection mice were euthanised and thighs prepared for colony counts. The decrease in colony counts relative to a vehicle control is shown in Table 6 below.

Table 6

Compound 1 and PMB performed similarly in this model.

Efficacy of compound 1 in a neutropenic murine lung model infected with A. baumannii NCTC 13301

After rendering neutropenic (cyclophosphamide 200 mg/kg d-4, 150 mg/kg d-1), CD- 1 mice (n = 8) were inoculated intranasally with approx. 8 x 10⁶cfu per mouse of A. baumannii NCTC13301. Mice were dosed SC with PMB sulphate (20 mg/kg) or appropriate doses of test compound (equivalent weight free base) at 2, 6 and 10 h post-infection. At 16 h post-infection mice were euthanised and lungs prepared for colony counts. The decrease in colony counts relative to a vehicle control is shown in Table 7 below.

Table 7

nd = not determined PMB was not efficacious in this model at the maximum tolerated dose (20 mg/kg). Compound 1 was more effective at 20 mg/kg and could also be dosed at higher levels to achieve a greater effect due to reduced toxicity. Efficacy of compound 1 in a neutropenic murine lung model infected with P. aeruginosa ATCC 27853

After rendering neutropenic (cyclophosphamide 200 mg/kg d-4, 150 mg/kg d-1), CD- 1 mice (n = 8) were inoculated intranasally with 6 x 10⁴ cfu per mouse of P. aeruginosa ATCC27853. Mice were dosed SC with appropriate doses of PMB sulphate or test compound (equivalent weight free base) at 2, 6 and 10 h post-infection. At 16 h post-infection mice were euthanised and lungs prepared for colony counts. The decrease in colony counts relative to a vehicle control is shown in Table 8 below.

Table 8

Efficacy of compound 1 was superior to PMB in this model.

Claims

Claims:

1. A compound of formula (I):

wherein:

-R^T is linear Ce alkyl, and salts, solvates and protected forms thereof.

2. The compound of claim 1, wherein the terminal is:

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3. The compound of claim 1, wherein the terminal is:

4. The compound of any one of claims 1 to 3, wherein -R^T is a linear alkyl group.

5. The compound of claim 1, wherein the compound of formula (I) is a compound of formula (II):

6. The compound of claim 1, wherein the compound of formula (I) is a compound of formula (III):

7. A pharmaceutical composition comprising a compound of any one of claims 1 to 6 and a biologically acceptable excipient, optionally together with a second active agent.

8. A compound according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7, for use in a method of treatment or prophylaxis.

9. A compound according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7, for use in a method of treating a microbial infection.

10. A compound or pharmaceutical composition according to claim 9, wherein the infection is a bacterial infection.

11. A compound or pharmaceutical composition according to claim 10, wherein the bacterial infection is a Gram-negative bacterial infection.

12. A compound or pharmaceutical composition according to claim 12, wherein the Gram-negative bacterial infection is selected from Escherichia spp., Klebsiella spp., Enterobacter spp., Salmonella spp., Shigella spp., Citrobacter spp., Morganella morganii, Yersinia pseudotuberculosis and other Enterobacteriaceae, Pseudomonas spp., Acinetobacter spp., Moraxella, Helicobacter, Stenotrophomonas , Bdellovibrio, acetic acid bacteria, Legionella and alpha-proteobacteria.

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