Patent Description:
There is currently an urgent need for new antibiotics and strategies for treating drugresistant bacterial infections. Bacterial resistance has developed against many, if not all, of current antibiotics. In the meantime, few antibiotics have being developed over the last number of decades. A primary cause of drug resistance is the over-use of antibiotics that can result in alteration of microbial permeability, alteration of drug target binding sites, induction of enzymes that destroy antibiotics (such as β-lactamases) and induction of efflux mechanisms.

The need for new antibiotics is even more urgent in the case of gram-negative bacteria such as Pseudomonas, Klebsiella and other bacteria such as Escherichia and Acinetobacter.

Many of the currently known antibiotics are structurally related to one another, and therefore are targets for drug resistance in bacteria. The inventors have unexpectedly identified a new class of organometallic compounds which have been found to have antibacterial properties.

<NPL>) describes the testing of a variety of different ruthenium II complexes against Gram positive and Gram negative bacteria. The compounds include a number of complex pyridine-containing multi ring structures, but no indole-containing structures. <NPL> describes the production (<NUM>-amido-<NUM>-imine) metallacycles. There is no suggestion of any anti-bacterial activity for such compounds. <NPL> was published after the priority date of the invention and discloses indole based half-sandwich complexes.

The invention provides a compound of formula I for use in the prophylaxis or treatment of a bacterial infection as defined in the claims.

Components A and B are bound to the transition metal M through the nitrogen atom of the indole or pyridine and are additionally bound to one another by a bond, such as a carbon-carbon bond. That bond is typically between the carbons of each of A and B immediately adjacent to the nitrogen atom of the indole or pyridine. That is, carbon <NUM> or <NUM> of the pyridine or carbon number <NUM> of the indole group.

The indoles or pyridines may be functionalised with one or more substituents which will influence the lipophilicity of the resulting organometallic compound (e.g. sulfonic acid, benzene), have an effect on the electronic properties of the complexes (e.g. electrondeficient ligands such as carboranes), or bio-active ligands (e.g. sugars).

L may comprise one or two aromatic rings. The rings may be linked via a carbon-carbon bond for example, or may share a common bond. L may be a substituted or non-substituted cyclopentadienyl or a substituted or non-substituted benzene ring. Functionalisation of the ligand L may be performed to modify the chemical and physical properties of the resulting complexes, for example by introducing one or more side groups selected from a sugar, hydroxyl, halogen, carboxylate, amide, triazole or a C1 - C4 straight or branched chain alkyl group. The halogen may be selected from, for example, chlorine, fluorine, bromine or iodine.

One or more active targeting moieties against bacteria may be connected to the aromatic ring via, for example, a carboxylate acid, amide or triazole group. The substitutions may be used to adjust the activity of the compound against the bacteria, alternatively, for example, adjust the solubility of the compound to allow the mode of administration to a subject to be optimised. One, two, three, four, five or all of the carbon atoms of the aromatic ring may be substituted.

L, A or B may be substituted by, for example, one or more methyl or -CH (CH<NUM>)<NUM>.

L may comprise a C5 or C<NUM> aromatic ring.

L may be selected from para-cymene, pentamethylcyclopentadiene, hexamethyl benzene, biphenyl and cyclopentadienyl.

M is typically selected from RuII, RhIII, IrIII and OsII.

Z is typically selected from a halogen or coordinating ligand compound wherein Z is preferably selected from Cl, Br, I, water, methanol, dimethyl sulfoxide, acetonitrile and dimethylformamide.

More preferably, the compound is selected from FKB3, FKB10 and FKB11.

The chlorine atom of the compound may be replaced by one or more alternative halogen or coordinating ligands as defined above.

The compounds and pharmaceutically acceptable salts may be used in combination with the pharmaceutically acceptable carrier. The carrier may, for example, be a solvent, such as an aqueous solvent, or alternatively a filler or bulking agent such as talc or carboxymethylcellulose.

The compound of pharmaceutically acceptable salt may be in a form suitable to be administered to a subject. These include, for example, oral tablets, oral suspensions, oral solutions, injectable formulations, such as intra-venously, intra-muscularly or intraperitonealy, as eye drops or topical lotions or creams. The formulation may also be in a form suitable for administration through the nose or lungs, such as in the form of an aerosol.

Oral administration of compounds can be enclosed in hard or soft shell gelatine capsules, compressed into tablets or incorporated directly into the food of a patient's diet. Compounds can also be combined with one or more excipients and be used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Such compounds and preparations will typically contain at least <NUM>% wt of active compound. The percentage of these compositions and preparations may vary depending on a given dosage form. Binders such as gum, tragacanth, acacia, corn starch or gelatine may be used. Excipients such as dicalcium phosphate, a disintegrating agent such as corn starch or potato starch, or a lubricant such as magnesium stearate may be used. Sweetening agents such as sugars or aspartame, or flavouring agents such as peppermint oil may be used. Additionally, when the dosage form is in the form of a capsule, a liquid carrier such as a vegetable oil or a polyethylene glycol may be used. Preservatives such as methyl and propyl parabens may be used together with one or more dyes or coatings, such as gelatine, wax, shellac and sugar or the like.

The subject to be treated may be a mammal, such as a human, but may also include cats, dogs, pigs, sheep, cows or rodents such as rats or mice.

A bacterial infection may be caused by a Mycobacterium, a Gram-positive bacterium or a Gram-negative bacterium, most typically a Gram-negative bacterium. The bacterium may be antibiotic resistant, for example by producing a beta-lactamase or carbapenumase.

Bacteria may be selected from the genus Mycobacterium, Escherichia, Salmonella, Clostridium, Streptococcus, Staphylococcus, Pseudomonas, Klebsiella, Acinetobacter, and Enterobacter, most preferably the bacterium is selected from Mycobacterium abcsessus, Escherichia coli, Pseudomonas aeruginosa or Klebsiella pneumonia.

The invention will now be described by way of example only with reference to the following figures:.

FKB3: p-cymene ruthenium dimer (<NUM>, <NUM> mmol) and <NUM>-(<NUM>-pyrindyl)-<NUM>-Indole (<NUM>, <NUM> mmol) were placed in a <NUM> <NUM> neck round bottom flask and dissolved in <NUM> dichloromethane. Triethylamine (<NUM>µl, <NUM>) was added to the reaction mixture. The dark orange solution was then left stirring overnight at room temperature under nitrogen. The solvent was removed under vacuum. The orange crude was dissolved in ethyl acetate and washed with <NUM> HCl (<NUM> x <NUM>) and brine (<NUM> x <NUM>). The combined organic layer was dried over MgSO<NUM>, filtered and dried over vacuum to obtain an orange powder. The product was purified by chromatography (acetone/dichloromethane <NUM>:<NUM> v/v). Yield: <NUM> (<NUM> %). HRMS-ESI+: calculated (M-Cl)+ <NUM>/z, found <NUM>/z. <NUM> NMR (<NUM>, CDCl<NUM>): δH <NUM> ppm (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, t, J = <NUM>), <NUM> (<NUM>, m), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, s), <NUM> (<NUM>, sept, J = <NUM>), <NUM>. 31v(<NUM>, s), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>).

FKB10: Rh dimer [(Cp*)RhCl<NUM>]<NUM> (<NUM>, <NUM> mmol) and <NUM>-(<NUM>-pyrindyl)-<NUM>-Indole (<NUM>, <NUM> mmol) were placed in a <NUM> neck round bottom flask and dissolved in <NUM> of dichloromethane to obtain an orange solution. Triethylamine (<NUM>µL, <NUM> mmol) was added and the reaction mixture left to react for <NUM> at room temperature. The solvent of the reaction mixture was removed under vacuum and the orange solid dissolved in dichloromethane (<NUM>) and extracted with <NUM> HCl solution (<NUM> x <NUM>). The combined organic layer was dried over magnesium sulfate, filtered and dried over vacuum to obtain an orange powder. The product was purified by column chromatography in silica, acetone / dichloromethane (<NUM>:<NUM> v/v) and recrystallised in dichloromethane to obtain crystalline orange needles. Yield: <NUM> (<NUM> %). HRMS-ESI+: calculated (M-Cl)+ <NUM>/z, found <NUM>/z. <NUM> NMR (<NUM>, CDCl<NUM>): δH <NUM> ppm (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, t, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, m), <NUM> (<NUM>, s), <NUM> (<NUM>, t, J = <NUM>), <NUM> (<NUM>, s).

FKB11: FKB11 was synthesised following the preparation described for compound FKB10 with the following modifications. Ir dimmer [(Cp*)IrCl<NUM>]<NUM> (<NUM>, <NUM> mmol), <NUM>-(<NUM>-pyrindyl)-<NUM>-Indole (<NUM>, <NUM> mmol). The product was obtained as a yellow powder. Yield: <NUM> (<NUM> %). HRMS-ESI+: calculated (M-Cl)+ <NUM>/z, found <NUM>/z. <NUM> NMR (<NUM>, CDCl<NUM>): δH <NUM> ppm (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, t, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, m), <NUM> (<NUM>, m), <NUM> (<NUM>, s).

Stability of FKB3, FKB10 and FKB11: Each complex was dissolved in MeOD (<NUM>) and diluted to a final concentration of <NUM> with either MeOD or D<NUM>O. <NUM>H NMR spectra was recorded at t = <<NUM>, <NUM> and <NUM>.

Each complex was also dissolved in MeOD (<NUM>) and diluted to a final concentration of <NUM> with D<NUM>O. The sample was then reacted with <NUM> mol. of silver nitrate to obtain the fully hydrolyzed specie.

From the NMR spectra of <FIG> it can be observed that FKB10 is stable over <NUM> both in methanol and in aqueous solution. Furthermore, addition of silver nitrate does not produce any shift of the <NUM>H NMR resonances, suggesting that ligand substitution occurs replacing the chloride ligand by either methanol or water.

The iridium compound FKB11 shows no changes in the <NUM>H NMR spectra, which indicates stability; as a side note, the compound is quite insoluble, and it precipitates over time. The ruthenium complex FKB3 is fully stable. Interestingly, when the compound is dissolved in <NUM> % MeOD, two species can be observed, corresponding to the chloride and the methoxide adducts.

<FIG>: Stability of FKB10 in MeOD (A) and MeOD/D<NUM>O (B), FKB11 in MeOD (C) and MeOD/D<NUM>O (D) and FKB3 in MeOD (E) and MeOD/D<NUM>O (F).

In vitro chemosensitivity tests were performed against HCT116 (colorectal cancer cell line), A2780 and A2780cisR (cisplatin sensitive and cisplatin resistant ovarian cancer cell lines, respectively). Cancer cell lines were routinely maintained as monolayer cultures in RPMI medium supplemented with <NUM>% foetal calf serum, penicillin (<NUM> I. /ml) and streptomycin (<NUM>µg/ml), sodium pyruvate (<NUM>) and L-glutamine (<NUM>). For chemosensitivity studies, cells were incubated in <NUM>-well plates at a concentration of <NUM> × <NUM><NUM> cells per well and the plates were incubated for <NUM> hours at <NUM> and a <NUM>% CO2 humidified atmosphere prior to drug exposure.

Complexes were dissolved in dimethylsulfoxide (DMSO) to provide stock solutions which were further diluted with media to provide a range of final concentrations. Drug-media solutions were added to cells (the final concentration of DMSO was less than <NUM>% (v/v) in all cases) and incubated for <NUM> hours at <NUM> and <NUM>% CO<NUM> humidified atmosphere. The drug-media was removed from the wells and the cells were washed with PBS (<NUM>µL, twice), and <NUM>µL of complete fresh media were added to each well. The plates were further incubated for <NUM> hours at <NUM> in a humidified atmosphere of <NUM>% CO2 to allow for a period of recovery. <NUM>-(<NUM>,<NUM>-dimethylthiazol-<NUM>-yl)-<NUM>,<NUM>-diphenyltetrazolium bromide (MTT) (<NUM>µL, <NUM>/mL) was added to each well and incubated for <NUM> hours at <NUM> and <NUM>% CO2 humidified atmosphere. All solutions were then removed and <NUM>µL of DMSO was added to each well in order to dissolve the purple formazan crystals. A Thermo Scientific Multiskan EX microplate photometer was used to measure the absorbance in each well at <NUM>. Cell survival was determined as the absorbance of treated cells divided by the absorbance of controls and expressed as a percentage. The IC<NUM> values were determined from plots of % survival against drug concentration. Each experiment was repeated in duplicate of triplicates and a mean value was obtained and stated as IC<NUM> (µM) ± SD. Cisplatin was used as a positive control. As a side note, the higher the IC<NUM> values are, the less cytotoxic the compounds are.

All Mycobacterium abscessus (M. abs) cultures were grown in Middlebrook 7H9 media, supplemented with glycerol (<NUM>% v/v), ADC (acid-dextrose-glucose) (<NUM>% v/v) and tween80 (<NUM>% w/v), at <NUM> with shaking at <NUM> rpm for <NUM>. The optical density (OD<NUM>) was adjusted to <NUM> prior to microtiter plate set-up.

The gram negative organisms selected were Escherichia coli (E. coli ATCC, J53 2138E, J53 2140E and I496 ESBL), Pseudomonas aeruginosa ATCC and Klebsiella pneumoniae H467. All organisms were grown in nutrient broth at <NUM> with <NUM> rpm shaking, for <NUM>. The optical density (OD<NUM>) was adjusted to <NUM> prior to microtiter plate set-up.

abscessus is an example of a mycobacterium capable of causing lung infections. J53 <NUM> E, J532140 E and I496ESBL are β-lactamase producing strains. H467 produces a carbapenumase.

Each compound was made in DMSO (dimethyl sulfoxide) to a stock concentration of <NUM>. A master plate was used to generate the desired concentrations for testing. This was achieved by serial <NUM>-fold dilution (repeated <NUM> times, the final row was DMSO only (<NUM>)).

For each compound, <NUM>µl of culture (OD <NUM>) was added to the microtiter plate and <NUM>µl of serially diluted compound added. The concentrations tested were: <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. All plates were read in the plate reader at <NUM> (T=<NUM>). abs the plates were incubated at <NUM> for <NUM> with reads being taken every <NUM>. For gram negative organisms the plates were incubated at <NUM> and read at <NUM> and <NUM>. After the final read for all organisms the experiment was plated out to observe bactericidal activity. abs was plated onto Middlebrook 7H11 agar supplemented with glycerol (<NUM>%) and incubated at <NUM> for <NUM>-<NUM>. The gram negative organisms were plated onto nutrient agar and incubated at <NUM> for <NUM>. Colony forming units were counted for all organisms.

All OD readings were transferred to excel and adjusted to account for OD of the broth and compounds. The data was then transferred to GraphPad Prism <NUM> for data analysis.

Out of the compounds tested, activity was observed for a total of <NUM> compounds. Three of the compounds, FKB3, FKB10 and FKB11, exhibited varying levels of growth inhibition against all organisms tested.

Reduction in optical density reading was observed for all compounds tested against M. abs at <NUM> (<FIG>). Uniquely, FKB10 showed a further reduction in optical density at both <NUM> and <NUM>, suggesting the highest potency against M. abs (<FIG>).

All compounds tested against E. coli ATCC showed activity down to <NUM> (<FIG>). Both FKB10 and FKB11 were the most potent, showing the least change in optical density over <NUM> at <NUM> concentration (<FIG>). FKB3 and FKB11 both have minimum bactericidal concentrations (MBC) of <NUM>.

All of compounds tested against E. coli J53 2138E showed a reduction in optical density. FKB3 and FKB11 were the only compounds with an observable MBC of <NUM> respectively.

All of the compounds tested show a reduction of optical density over <NUM>, with the largest reduction in change of optical density seen for FKB11 at <NUM> (<FIG>). FKB3 and FKB11 were the only compounds with an observable MBC of <NUM> respectively.

All of the compounds tested show a reduction of optical density over <NUM>, with the largest reduction in change of optical density seen for FKB3 at <NUM>, as well as FKB10 at <NUM> (<FIG>). FKB3 and FKB11 were the only compounds with an observable MBC of <NUM> respectively.

All of the compounds tested caused a reduction in change of optical density at <NUM> (<FIG>). The largest of these reductions occurred when P. aeruginosa was treated with FKB11 (<FIG>). However, despite this, there was no observable MBC for any compound against P. aeruginosa, suggesting these compounds are bacteriostatic, rather than bactericidal against this organism.

Each compound tested against K. pneumoniae H467 showed a reduction in optical density over <NUM>. The largest reduction occurred with the addition of FKB11 at <NUM> (<FIG>). Both FKB3 and FKB11 gave an MBC of <NUM>.

Claim 1:
A compound of Formula I for use in the treatment or in the prophylaxis of a bacterial infection:
<CHM>
wherein L is a substituted or non-substituted aromatic ligand; Z is a halogen or coordinating ligand; M is a group <NUM> or group <NUM> transition metal selected from; Fe, Ru, Co, Rh and Os; wherein one of A or B is a substituted or non-substituted, especially non-substituted, indole; and the second of B or A is a substituted or non-substituted, especially a non-substituted, pyridine,
and A and B coordinate to the transition metal M through the nitrogen atom of the indole or pyridine.