WO2010082019A1

WO2010082019A1 - Actagardine derivatives, and pharmaceutical use thereof

Info

Publication number: WO2010082019A1
Application number: PCT/GB2010/000043
Authority: WO
Inventors: Sjoerd Nicolaas Wadman; Michael John Dawson; Jesus Cortes Bargallo
Original assignee: Novacta Biosystems Limited
Priority date: 2009-01-14
Filing date: 2010-01-12
Publication date: 2010-07-22
Also published as: GB0900599D0

Abstract

The present invention pertains generally to the use of certain compounds derived from actagardine for the treatment of C. difficile infection and/or bacterial overgrowth syndrome, for example in the small intestines and/or colon.

Description

ACTAGARDINE DERIVATIVES , AND PHARMACEUTICAL USE THEREOF

The present invention relates to use of certain compounds and use of pharmaceutical compositions comprising same for the treatment of C. difficile infection, in particular C. difficile in the colon and/or lower intestines.

Many antibiotic compounds have been identified from natural sources including microorganisms. Often the antibiotic compounds have a complicated chemical structure and in particular a complicated stereochemical structure.

Actagardine is a natural product prepared by Actinoplanes garbadinensis, which has been identified as having antibiotic properties, see for example EP0195359, in particular against Streptococcus pyogenes, which causes scarlet fever and strep throat infection. Despite the need for new antibiotics in the 22 years since publication of EP0195359 no antibiotics derived from actagardine have been licensed and marketed.

A new family of compounds based on deoxyactagardine B was recently published in WO 2007/083112. Deoxyactagardine B is prepared by A. liguriae and has a number of distiguishing features from actagardine.

It has now been found by the present inventors that certain compounds derived from actagardine have very good activity against C. difficile infection.

In one aspect there is provided use of a compound of formula (I)

wherein

A is -C_1-4 alkyl; B is -C_1-4 alkyl; X is -NH(CH₂)_PNH₂; p is an integer 2 to 12; Z iS -NR¹R²; R¹ is H or C_1-4 alkyl,

R² is H, an amino acid or C_1-4 alkyl, and pharmaceutically acceptable salts, hydrates and solvates thereof, for the treatment of C. difficile infection and/or bacterial overgrowth syndrome.

In another aspect of the invention there is provided a compound of formula (I), and pharmaceutically acceptable salts, hydrates and solvates thereof.

In a further aspect there is provided a compound of formula (I), and pharmaceutically acceptable salts, hydrates and solvates thereof, for use in treatment.

Brief Description of the Figures

Figure 1 shows a HPLC analysis of the starting materials for Example 1.

Figure 2 shows a HPLC analysis after the reaction has gone to completion.

Figure 3 shows a HPLC analysis with more concentrated levels of Example 1. The compounds of the disclosure are advantageous because they have high antibacterial activity against one or more strains of C. difficile, for example when activity is measured by a standard test such as minimum inhibitory concentrations (MICs), generally the compounds employed in the disclosure have an MIC of 4 μg/ml or lower, such as 2 μg/ml.

Additionally, the compounds of the present disclosure may be particularly suited to administration to humans and animals because they have low antibacterial activity against the naturally occurring healthy intestinal flora found in the body. In the case of treatment of diarrhea induced by a microbial infection such as C. difficile it is expected that a reduced recurrence of symptoms will be observed after treatment with the present compounds in comparison to treatment with known antibiotics because of the ability of the natural flora to survive the treatment with the present compounds. In particular the compounds of the disclosure are expected to have very low activity against Bacteroides fragilis, Bacteroides thetaiotaomicron, Bifidobacterium longum, Lactobacillus rhamnosus, and moderately low activity against Peptostreptococcus anaerobius and Bifidobacterium adolescentis.

What is more, when delivered orally the compounds employed in the disclosure are not absorbed systemically, which allows a relatively high concentration of the active to be delivered to the target in the colon/intestines. Thus, because there is no systemic delivery of the compounds when administered orally, then this may minimise any potential exposure to side effects for patients.

C. difficile infection and/or overgrowth is a common problem for patients during hospitalisation. It presents a real burden to the health care system and may be life threatening to vulnerable patients such as elderly patients. Thus treatments specific for the same would be very useful.

It is thought that the present compounds, although comprising peptide components, are not degraded to any significant extent by stomach acid or intestinal enzymes. Thus the compounds employed in the disclosure are particularly suitable for oral delivery. This can be advantageous in that it allows the flexibility to formulate the compounds in a simple and effective formulation enabling delivery of an undegraded active locally to the target tissue.

The physical properties of the compounds of the disclosure such as solubility, stability and the like are adquate for the intended therapeutic use.

Detailed Description

Alkyl in the context of the present disclosure refers to straight chain or branched chain alkyl, for example methyl, ethyl, propyl, isopropyl, n-butyl or t-butyl. In one embodiment A has a structure corresponding to an alkyl side chain of a natural amino acid containing up to 4 carbons.

In one embodiment A is -CH₃.

In one embodiment A is a branched chain alkyl, for example -CH(CH₃)₂, -CH₂CH(CH₃)₂, or -CH(CH₃)CH₂CH₃ SUCh as -CH(CH₃)₂ Or -CH(CH₃)CH₂CH₃, particularly -CH(CH₃)₂.

In one embodiment B has a structure corresponding to an alkyl side chain of a natural amino acid containing up to 4 carbons.

In one embodiment B is -CH₃.

In one embodiment B is is a branched chain alkyl, for example -CH(CH₃)₂, -CH₂CH(CH₃)₂, or -CH(CH₃)CH₂CH₃, such as -CH(CH₃)₂ or -CH(CH₃)CH₂CH₃, or such as -CH(CH₃)₂ or -CH₂CH(CH₃)₂, particularly -CH(CH₃)CH₂CH₃.

In one aspect A is -CH(CH₃J₂ and B is -CH(CH₃)CH₂CH₃.

In one embodiment R¹ is H.

In one embodiment R² is H.

In one embodiment R² is the L or D isomer form of an amino acid residue.

In one embodiment R² is an amino acid residue selected from alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine.

In one embodiment R² is an amino acid residue selected from phenylalanine, tyrosine and alanine (i.e. -C(O)CH(CH₃)NH₂)

In one embodiment Z is -NH₂.

In one aspect A is -CH₂CH(CH₃)₂ and B is -CH(CH₃)₂ and Z is -NH₂.

In one embodiment p is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, such as 2, 3, 7, 9 or 12, in particular 7, 9 or 12.

In one embodiment the p is 3 to 12 or 3 to 8. In one aspect the disclosure provides use of a compound of formula (II)

and pharmaceutically acceptable salts, hydrates and solvates thereof, in the treatment of C. difficile and/or bacterial overgrowth, for example in the small intestines and/or colon.

Methods of preparing compounds similar to compounds of the present disclosure are described in WO 2007/083112.

The compounds employed in the present disclosure may be in the form of and/or may be administered as a pharmaceutically acceptable salt. For a review on suitable salts see Berge et al., J. Pharm. ScL, 1977, 66, 1-19.

Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent, for example, a compound of formula (I) or (II) may be dissolved in a suitable solvent, for example an alcohol such as methanol, and the acid may be added in the same solvent or another suitable solvent. The resulting acid addition salt may then be precipitated directly, or by addition of a less polar solvent such as diisopropyl ether or hexane, and isolated by filtration.

The skilled person will appreciate that where the compound of formula (I) or (II) contains more than one basic group bis salts or tris salts may also be formed and are salts suitable for use in the present disclosure. Suitable addition salts are formed from inorganic or organic acids which form non-toxic salts and examples are lactobionate, mandelate (including (S)-(+)-mandelate, (R)-(-)- mandelate and (R,S)-mandelate), hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, hydrogen phosphate, glutamate, acetate, trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinate, ethyl succinate (4-ethoxy-4- oxo-butanoate), pyruvate, oxalate, oxaloacetate, saccharate, benzoate, glucolate, glucurinate, alkyl or aryl sulphonates (eg methanesulphonate, ethanesulphonate, benzenesulphonate or p-toluenesulphonate) and isethionate.

Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases, including salts of primary, secondary and tertiary amines, such as isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexyl amine and N-methyl-D-glucamine.

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compounds of formula (I) or (II) are within the scope of the disclosure. The salts of the compound of formula (I) or (II) may form solvates (e.g. hydrates) and the disclosure also includes use of all such solvates.

The term "prodrug" as used herein means a compound which is converted within the body, e.g. by hydrolysis in the blood, into its active form that has medical effects. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, "Prodrugs as Novel Delivery Systems", Vol. 14 of the A.C.S. Symposium Series; Edward B. Roche, ed., "Bioreversible Carriers in Drug Design", American Pharmaceutical Association and Pergamon Press, 1987; and in D. Fleisher, S. Ramon and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19(2) 115-130, each of which are incorporated herein by reference.

Prodrugs are any covalently bonded carriers that release a compound of formula (I) or (II) in vivo when such prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound. Prodrugs include, for example, compounds of this disclosure wherein hydroxy, amine or sulfhydryl groups are bonded to any group that, when administered to a patient, cleaves to form the hydroxy, amine or sulfhydryl groups. Thus, representative examples of prodrugs include (but are not limited to) acetate, formate and benzoate derivatives of alcohol, sulfhydryl and amine functional groups of the compounds of formula (I) or (II). Further, in the case of a carboxylic acid (-COOH), esters may be employed, such as methyl esters, ethyl esters, and the like. Esters may be active in their own right and/or be hydrolysable under in vivo conditions in the human body. Suitable pharmaceutically acceptable in vivo hydrolysable ester groups include those which break down readily in the human body to leave the parent acid or its salt.

References herein to a compound employed in the disclosure include both compounds of formula (I) or (II) and their pharmaceutically acceptable salts and derivatives.

With regard to stereoisomers, the compounds of formula (I) or (II) have more than one asymmetric carbon atom. In the general formula (I) or (II) as drawn, the solid wedge shaped bond indicates that the bond is above the plane of the paper. The broken bond indicates that the bond is below the plane of the paper.

It will be appreciated that the substituents in compounds of formulae (I) or (II) may also have one or more asymmetric carbon atoms.

The compounds of structure (I) or (II) may occur as individual enantiomers or diastereomers. All such isomeric forms may be employed within the present disclosure, including mixtures thereof.

Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or HPLC. A stereoisomeric mixture of the agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as by HPLC, of the corresponding mixture using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding mixture with a suitable optically active acid or base, as appropriate.

Compounds of formula (I) or (II) as described herein also extend to tautomeric forms thereof, for example, keto/enol tautomers.

The compounds of formula (I) or (II) may be in crystalline or amorphous form. Furthermore, some of the crystalline forms of the compounds of structure (I) or (II) may exist as polymorphs, all forms of which employed in the present disclosure.

In another aspect, the invention provides a pharmaceutical composition comprising, as active ingredient, a compound of formula (I) or (II) or a pharmaceutically acceptable derivative thereof in association with a pharmaceutically acceptable excipient, diluent and/or carrier for use in therapy, and in particular, in the treatment of human or animal C. difficile infection and/or bacterial overgrowth syndrome.

The compounds employed in the disclosure may be formulated for administration in any convenient way for use in human or veterinary medicine and the disclosure therefore includes within its scope use of pharmaceutical compositions comprising a compound of the invention adapted for use in human or veterinary medicine, as described herein. Such compositions may be presented for use in a conventional manner with the aid of one or more suitable excipients, diluents and/or carriers. Acceptable excipients, diluents and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical excipient, diluent and/or carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may include an excipient selected from a diluent and/or carrier any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s) and mixtures thereof.

Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

For some embodiments, the agents of the present disclosure may also be used in combination with a cyclodextrin. Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of a drug-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule. Drug- cyclodextrin complexes are generally useful for most dosage forms and administration routes. As an alternative to direct complexation with the drug the cyclodextrin may be used as an auxiliary additive, e. g. as a carrier, diluent or solubiliser. Alpha-, beta- and gamma- cyclodextrins are most commonly used and suitable examples are described in WO 91/11172, WO 94/02518 and WO 98/55148.

The compounds may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds may be prepared by processes known in the art, for example see WO 02/00196 (SmithKline Beecham).

The routes for administration (delivery) include, but are not limited to, one or more of: oral (e.g. as a dry powder/ free flowing particulate formulation, tablet, capsule, or as an ingestable solution or suspension) rectal, buccal, and sublingual. The compounds of the disclosure are particularly useful for oral delivery.

The compounds may be administered (e. g. orally or topically) in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications. The compounds may also be presented for human or veterinary use in a form suitable for oral or buccal administration, for example in the form of solutions, gels, syrups, mouth washes or suspensions, or a dry powder for constitution with water or other suitable vehicle before use, optionally with flavouring and colouring agents. Solid compositions such as tablets, capsules, lozenges, pastilles, pills, boluses, powder, pastes, granules, bullets or premix preparations may also be used. Solid and liquid compositions for oral use may be prepared according to methods well known in the art. Such compositions may also contain one or more pharmaceutically acceptable carriers and excipients which may be in solid or liquid form.

The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium sulphate, dibasic calcium phosphate and glycine, mannitol, pregelatinised starch, corn starch, potato starch, disintegrants such as sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.

Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed to fill, for example gelatin or HPMC (hydroxypropyl methylcellulose) capsules. Suitable excipients include microcrystalline cellulose, lactose, calcium carbonate, calcium sulphate, dibasic calcium phosphate and, mannitol, pregelatinised starch, corn starch, potato starch or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

Capsules, may be filled with a powder (of medicament alone or as blend with selected filler(s)) or alternatively a liquid, each comprising one or more compounds of formula (I) or (II) and a carrier. Where the capsule is filled with a powder the compounds of formula (I) or (II) and/or the carrier may be milled or micronised to provide material with an appropriate particle size.

Compounds may be coated, for example with as an enteric coating when administered orally as a tablet or capsule. The tablet or capsule, as appropriate, may, for example be coated by a thin film such as a EUDRAGIT® film available from Rohm Pharma Polymers, which allows controlled dissolution in the gastrointestinal tract. The films are available as cationic polymers such as EUDRAGIT® E 100 (aminoalkyl methacylate copolymers) or as anionic acrylic polymers such as EUDRAGIT® L (methacrylic acid copolymers) and EUDRAGIT S. Permeable acrylic polymers such as EUDRAGIT® RL (amino methacrylate copolymer) and EUDRAGIT® RS are also available.

These coating formulations may be prepared as an aqueous dispersion including optional ingredients such as talc, silicone antifoam emulsion, polyethylene glycol. Alternatively the coating formulation may be prepared as an organic polymer solution.

Alternatively, tablets may be coated using OPADRY® (Surelease®) coating systems, available from Colorcon. Aqueous systems generally comprise up to 15% w/w of OPADRY®. Organic solvent systems generally comprise up to 5% w/w of OPADRY®. The coatings may be prepared by known techniques, for example by;

1. weighing the required quantity of OPADRY® film coating system,

2. weighing the required quantity of water or other solvent(s) into a mixing vessel,

3. with a mixing propeller in the centre of the vessel and as close to the bottom of the vessel as possible, stirring the solvents to form a vortex without drawing air into the liquid, 4. steadily and quickly adding the OPADRY® powder to the vortex, avoiding powder flotation on the liquid surface,

5. increasing the stirrer speed in order to maintain the vortex, if required, and

6. after all the powder ingredients have been added, reducing the mixer speed and continuing mixing for approximately 45 minutes.

Coatings can be applied by known techniques, using tablet coating machines.

The thickness of the coating applied is generally in the range 5 to 35 microns such as 10 to 30 microns, more specifically 10 or 20 microns, depending on the required effect.

Alternatively, the tablet or a capsule, as appropriate, may be filled into another capsule (preferably a HPMC capsule such as Capsugel®) to provide either a tablet in capsule or capsule in capsule configuration, which when administered to a patient yields controlled dissolution in the gastrointestinal tract thereby providing a similar effect to an enteric coating. Thus in one aspect the disclosure provides use of a solid dose formulation of a compound of formula (I) or (II) for example where the formulation has an enteric coating.

In another aspect the disclosure provides use of a solid dose formulation comprising a protective capsule as outer layer, for example as a tablet in a capsule or a capsule in a capsule. The enteric coating may provide an improved stability profile over uncoated formulations.

The compounds employed in the disclosure may also be administered orally in veterinary medicine in the form of a liquid, such as a solution, suspension or dispersion of the active ingredient together with a pharmaceutically acceptable carrier or excipient. The compounds employed in the disclosure may also, for example, be formulated as suppositories e.g. containing conventional suppository bases for use in human or veterinary medicine.

The compounds employed in the disclosure may also be used in combination with other therapeutic agents. The disclosure thus provides, in a further aspect, use of a combination comprising a compound of formula (I) or (II) or a pharmaceutically acceptable derivative thereof together with a further therapeutic agent. The combination may, for example be a combination of a compound of formula (I) or (II) and an antibiotic, such as vancomycin. The combination may be provided as a co-formulation or simply packaged together as separate formulations, for simultaneous or sequential delivery.

When a compound of formula (I) or (II) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone.

Appropriate doses will be readily appreciated by those skilled in the art. It will be appreciated that the amount of a compound of the disclosure required for use in treatment will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian. The compounds of formula (I) or (II) may for example be administered with other active ingredients such as corticosteroids, as appropriate.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus use of pharmaceutical formulations comprising a combination, as defined above, together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the disclosure. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations by any convenient route.

When administration is sequential, either the compound of formula (I) or (II) or the second therapeutic agent may be administered first. When administration is simultaneous, the combination may be administered either in the same or different pharmaceutical composition.

When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, in manner well known in the art.

The compositions may contain from 0.01-99% of the active material. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.

For oral administration to humans, the daily dosage level of the agent may be in single or divided doses. The daily dose as employed for adult human treatment it will range from

2-100mg/kg body weight, for example 5-60mg/kg body weight, which may be administered in 1 to 4 daily doses, for example, depending on the route of administration and the condition of the patient. When the composition comprises dosage units, each unit will preferably contain 100mg to 1g of active ingredient. The duration of treatment will be dictated by the rate of response rather than by arbitrary numbers of days.

In one embodiment the treatment regime is continued for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days.

In one aspect, the disclosure provides use of a compound of formula (I) or (II) in therapy, for example, for treatment of microbial infections such as C. difficile infection, in particular diarrhoea asssociated therewith, particularly by oral delivery of a compound of formula (I) or (II).

In one aspect there is provided use of a compound of formula (I) or (II) for the treatment of cecitis (in an animal subject).

In one aspect the compound of formula (I) or (II) are employed for controlling bacterial overgrowth syndrome. Overgrowth syndrome (BOS) occurs when the normally low bacterial colonization in the upper Gl tract and/or lower intestines significantly increases.

The compounds of the present disclosure may be particularly useful for long term treatment.

As described above the compounds of the present disclosure may be employed in the treatment of humans and/or animals.

In one aspect there is provided a compound of formula (I) or (II) for the manufacture of a medicament for the for treatment of microbial infections such as C. difficile infection, in particular diarrhoea asssociated therewith and/or overgrowth syndrome.

In one aspect there is provided a method of treatment comprising the step of administering a therapeutically effective amount of a compound of formula (I) or (II) or a pharmaceutical composition containing the same to a patient (human or animal) in need thereof, for example for the treatment of an infection/illness or disease as described herein.

In the context of this specification "comprising" is to be interpreted as "including".

Aspects of the invention comprising certain elements are also intended to extend to alternative embodiments "consisting" or "consisting essentially" of the relevant elements.

EXAMPLES

Example 1

Actagardine (7-amino-1-heptylamide monocarboxamide)

Actagardine (25mg), 1 ,7-diaminoheptane (3.4mg) and diisopropylethylamine (5.3μl) were dissolved in dry dimethylformamide (0.25ml). A solution of benzotriazole-1-yl-oxy-tris- pyrrolidino-phosphonium hexafluorophosphate (PyBOP) (10.4mg) in dry dimethylformamide (0.25ml) was added portionwise. The reaction was followed by analytical HPLC (See Table I) and PyBOP was added until the starting material had been consumed (Figures 1 and 2).

Table 1: Analytical HPLC conditions for the separation of lantibiotics (e.g. actagardine) and diaminoalkane derivatised products.

Column: Zorbax 5μ C18(2) 150 x 4.6 mm

Mobile Phase A: 30% Acetonitrile in 20 mM potassium phosphate buffer pH 7.0

Mobile Phase B: 65% Acetonitrile in 20 mM potassium phosphate buffer pH 7.0

Flow rate: 1 ml/min

Gradient: Time 0 min 100% A 0% B Time iO min 0% A 100% B

Time 11 min 0% A 100% B

Time 11.2 min 100% A 0% B Cycle time 15 min Injection volume: 10 μl Detection: 210 nm

The crude reaction mixture was poured into 30% aqueous methanol and the resulting solution was loaded on to a Varian Bond Elut C18 column (500mg). The column was then washed sequentially with 50%, 60%, 70%, 80%, 90% aqueous methanol, with most of the desired material eluting in the 70% fraction (Figure 3) Column chromatography on silica gel (eluent dichloromethane:ethanol:ammonia 10:8:1) gave material of >90% purity by U. V. at 210 nm. Yield 15mg (59%). Samples were analysed by LC-MS. Mass calc (M+2H)⁺² 1001.0, found 1001.02. Example 2 : Actagardine 1,3-diaminopropane monocarboxamide

Was prepared by coupling actagardine with 1 ,3-diaminopropane utilising the procedure described for Example 1. Yield 47%. Mass calc (M+H+Na)⁺² 973.0, found 973.2.

Example 3 : Actagardine 1 ,4-diaminobutane monocarboxamide

Was prepared by coupling actagardine with 1 ,4-diaminobutane utilising the procedure described for Example 1. Yield 50%. Mass calc (M+H+Na)⁺² 990.5, found 989.46.

Example 4 : Antibacterial activity of actagardine and derivatives

The compounds of the invention show antimicrobial activity in vitro and are expected to show similar activity in vivo. They are active against Clostridium difficile and may have improved activity compared to actagardine.

Suceptibility testing for Clostridium difficile strains was performed by two-fold serial antibiotic dilutions in Wilkins-Chalgren Anaerobe agar under anaerobic conditions. Vancomycin was included as a comparator drug. C. dificile cultures were inoculated onto pre-reduced Braziers (CC. E. Y.) agar plates and grown at 37°C for 48 hours under anaerobic conditions. Two to three colonies of the 48 hours cultures were inoculated into 5 ml of pre-reduced Schaedlers Broth and grown at 37°C for 24 hours under anaerobic conditions. This culture was diluted with pre-reduced 0.9% NaCI to achieve the turbidity of the 0.5 McFarland standard and applied to the drug containing plates at a final inoculum of 105 cfu/spot. Drug-free growth control plates were included. The plates were incubated in the anaerobic chamber at 37⁰C for 48 hours and examined for growth. The MIC was the lowest concentration of drug that completely inhibited growth or caused markedly reduction of growth as compared to growth on the drug-free plates.

Table 3: MIC data (μg/ml) for actagardine and derivatives thereof.

(The lower the value of the result the greater the activity of the test compound. )

Claims

1. A compound of formula (I)

wherein

A is -C_1-4 alkyl;

B is -C_1-4 alkyl;

X is -NH(CH₂)_PNH₂; p is an integer 2 to 12;

Z iS -NR¹R²;

R¹ is H or C_1-4 alkyl;

R² is H, an amino acid or C_1-4 alkyl, and pharmaceutically acceptable salts, hydrates and solvates thereof for use in the treatment of C. difficile and/or bacterial overgrowth, for example in the small intestines and/or colon.

2. A compound according to claim 1 , wherein A is selected from -CH(CH₃)₂ and -CH₂CH(CHs)₂.

3. A compound according to claim 2 wherein A is -CH(CH₃J₂.

4 A compound according to any one of claims 1 to 3, wherein B is selected from -CH(CH₃)₂ and -CH(CH₃)CH₂CH₃.

5 A compound according to claim 4, wherein B is -CH(CH₃)CH₂CH₃.

6. A compound according to any one of claims 1 to 5, wherein R² is the L or D isomer form Of -C(O)CH(CH₃)NH₂.

7. A compound according to any one of claims 1 to 5, wherein A is -CH(CH₃)₂and B is -CH(CH₃)CH₂CH₃.

8. A compound according to any one of claims 1 to 7, wherein p is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

9. A compound according to 8, wherein p is 2, 3, 7, 9 or 12.

10 A compound according to claim 9, wherein the p is 7, 9 or 12.

11. A compound according to any one of claims 1 to 10, wherein Z is NH₂.

12. A compound according to claim 1 of formula (II)

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12, and a pharmaceutically acceptable excipient.

14. A composition according to claim 13, for oral administration.

15. Use of a compound as defined in any one of claims 1 to 12 or a composition according to any one of claims 13 to 14, for the manufacture of a medicament for the treatment of

C. difficile infection and/or bacterial overgrowth syndrome.

16. A method of treatment comprising administering a therapeutically effective amount of a compound as defined in any one of claims 1 to 12 or a composition according to any one of claims 13 to 14 to a patient for treatment of C. difficile infection and/or bacterial overgrowth syndrome.

17. A method according to claim 16, for treatment of C. difficile infection.

18. A method according to claim 17, for the treatment of C. difficile infection in the colon and/or lower intestines.

19. A method according to claim 16, for the treatment of bacterial overgrowth in the small intestines.