This application is related to GB 1013508.5 filed 1 1 August 2010; the contents of which are incorporated herein by reference in their entirety.

The present disclosure relates to certain novel compounds, pharmaceutical compositions comprising same and use of the compounds and compositions for the treatment of microbial infection, particularly Methicillin-resistant Staphylococcus aureus (MRS A) infection. 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 from Actinoplanes garbadinensis, and has 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 disclosed in

WO 2007/0831 12. Deoxyactagardine B is prepared from A. liguriae and has a number of distinguishing features from actagardine, in particular the compounds have differences in the amino acid sequence of the core structure. Additionally actagardine contains an oxidised lanthionine bridge in contrast to deoxyactagardine B, wherein all the lanthionine bridges are present in a reduced form. Obviously different genes and biological machinery is required to make the different compounds. Furthermore, these compounds show different activity when tested against a range of common pathogens. In some instances actagardine and certain compounds derived therefrom exhibit greater activity against a given pathogen than deoxyactagardine B and derivatives thereof. Interestingly, against certain other pathogens deoxyactagardine B and compounds derived therefrom exhibit greater activity than actagardine and derivatives thereof.

Actagardine activity against MRSA when measured by a standard test, such as minimum inhibitory concentrations (MICs), may be as high as about 32 Mg/mL, depending on the strain tested. Thus actagardine has only low to moderate activitity against MRSA because the higher the MIC value the less antimicrobial activity the compound has.

Deoxyactgardine B activity against MRSA when measured by a standard test, such as minimum inhibitory concentrations (MICs), may have an activity as high as about 32 pg/mL, depending on the strain tested. Thus deoxyactagardine also has only low to moderate activity against MRSA.

MRSA is a bacterium responsible for d iff i cu It-to-treat infections in humans and animals. The particular strain(s) of Staphylococcus aureus labeled MRSA is/are resistant to a large group of antibiotics called beta-lactams, which include the penicillins and cephalosporins.

The strain(s) received a significant amount of attention in the media and was branded a "superbug". Patients with open wounds, those who have procedures involving invasive devices, and those with a weakened immune system are most at risk of infection, especially during hospitalization. The infection is highly contagious and if it is identified on a hospital ward, the ward may be closed until it is decontaminated.

Thus antimicrobial compounds with activity against MRSA would be particularly useful. Certain novel compounds have now been identified with activity against MRSA.

Thus in one aspect there is provided a compound of formula (I):

(I)

wherein:

R ¹ together with the carbon to which it is attached and the alpha-nitrogen and

alpha-carbonyl represents an amino acid residue;

R ² together with the carbon to which it is attached and the alpha-nitrogen and

alpha-carbonyl represents an amino acid residue;

X represents a bond or an amino acid residue;

R ³ represents H or C _1-6 alkyl;

R ⁴ represents -R ^A -L-Ar ¹ , or

R ³ together with R ⁴ and the nitrogen to which they are attached form a 5 or 6 membered heterocyclic group optionally including a further heteroatom selected from N, O or S, wherein said heterocyclic group is substituted by -YAr ¹ ;

R ^A represents a bond, -C ₀ . ₉ alkylC ₆ . ₁₀ aryl, -C ₀ . ₉ alkylC ₅ -n heteroaryl,

-C _1-9 heteroalkylC ₅ .iiheteroaryl -C _0-9 alkylC ₃ ^cycloalkyl,

-C _1-9 heteroalkylC _5-1 i heterocyclic or -C _0-9 alkylC _5- n heterocycle;

L represents a straight or branched C ₀ .is alkyl chain wherein optionally one or more carbons are replaced by a heteroatom independently selected from N, O or S, wherein said chain is optionally substituted by one or more (such as 1 or 2), oxo or nitro groups with the proviso that a heteroatom is not bonded directly to the N of the group -NR ³ R ⁴ ;

Y represents a straight or branched C ₀ .is alkyl chain wherein optionally one or more carbons are replaced by a heteroatom independently selected from N, O or S, wherein said chain is optionally substituted by one or more (e.g. 1 or 2), oxo or nitro groups;

Ar ¹ represents a five to ten membered heteroaryl containing one, two or three

heteroatoms independently selected from N, O, S, with the proviso that the heteroaryl does not represent furanyl or imidazolyl, wherein said heteroaryl is optionally substituted on a carbon atom and/or a heteroatom by one, two or three groups independently selected from -(Q) _m (CH ₂ ) _n NR ^B C(0)CCI ₃ , N0 ₂ , halogen, and C

3 haloalkyl, and optionally by one or two -(Q) _m (CH ₂ ) _q NR ⁶ R ⁷ ;

R ^B represents H or -C(0)CCI ₃ ;

R ⁵ together with the carbon to which it is attached and the alpha-nitrogen and alpha- carbonyl represents an amino acid residue;

R ⁶ represents H or alkyl;

R ⁷ represents H or alkyl; or

-NR ⁶ R ⁷ together form a 6 membered heterocyclic ring optionally including a further -NH-

Q represents O, S or NH;

Z represents H, Ci _-6 alkyl, an amino acid residue;

m represents 0 or 1 ; represents 0, 1 , 2, 3 or 4; and

represents 0 or 1 ;

represents 0, 1 , 2, 3 or 4; and

the fragment:

or the E isomer of the latter;

or a pharmaceutically acceptable salt thereof.

The compounds according to the present disclosure generally have activity against a broad range of gram positive bacteria, in particular those associated with soft tissue infections, in particular the gram positive bacteria MRSA and thus are likely to be useful in the treatment of infections of the same.

Alkyl in the context of the present disclosure refers to straight chain or branched chain alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl or t-butyl. Heterocyclic group as employed herein is a saturated or partially unsaturated ring (i.e. a non-aromatic mono or bicyclic ring) comprising one or more heteroatoms selected from O, N and S, for example a 5 or 6 membered heterocycle group such as pyrroline (in particular 1-, 2- or 3-pyrroline), pyrrolidine, tetrahydrofuran, tetrahydrothiophene, pyrazoline (in particular 2- or 3-pyrazoline), 2-imidazoline, pyrazolidine, imidazolidine, 3-dioxolane, thiazolidine, isoxazolidine, pyran (in particular 2H or 4H-pyran), 3,4-dihydro-2H-pyran, piperidine,

1 ,4-oxazine, 1 ,4-dioxine, piperazine, morpholine, 1 ,4-dioxane. It will be understood that in definitions employed herein, such as C ₅ -n heterocycle, that the heteroatom may replace a carbon atom in the ring and therefore C _5-11 heterocycle and a 5 to 11 membered heterocycle are used interchangeably. Other definitions of heterocycles will be construed similarly. The heterocycle may be linked through carbon or nitrogen. Cycloalkyl as employed herein refers to a saturated or partially unsaturated carbocyclic ring, i.e. a non-aromatic carbocyclic ring, for example cyclopropyl, cyclopentyl or cyclohexyl.

Heteroaryl as employed herein refers to an aromatic carbocycle comprising one or more heteroatoms selected from O, N or S including a bicyclic system wherein one or both rings are aromatic and/or one or both rings contain a heteroatom, for example a 5-11 membered heteroaryl, such as pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, triazole, oxadiazole, furazan, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, triazine, 1 H-pyrrolizine, indolizine, indole, isoindole, benzofuran, isobenzofuran, indoline, isoindoline, benzothiophene, indazole, benzimidazole, purine, quinoline, isoquinoline, chromane, isochromane, chromene, cinnoline, quinazoline, quinoxaline, naphthyridine or phthalazine. It will be understood that in definitions employed herein, such as C _5-11 heteroaryl, that the heteroatom may replace a carbon atom in the ring and therefore C _5- n heteroaryl and a 5 to 11 membered heteroaryl are used interchangeably. Other definitions of heteroaryls will be construed similarly. The heteroaryl may be linked through carbon or a nitrogen, as appropriate, in particular carbon. A bicyclic system may be linked to the remainder of the molecule through a ring comprising a heteroatom and/or a ring without a heteroatom, as appropriate. Halogen as employed herein refers to fluoro, chloro or bromo, such as fluoro or chloro, in particular chloro.

Haloalkyl as employed herein refers to alkyl groups having 1 to 6 halogen atoms, for example 1 to 5 halogens , such as per haloalkyl, in particular perfluoroalkyl, more specifically -CCI2CCI3, -CCI3, -CF2CF3 or -CF ₃ .

Heteroalkyl as employed herein represents a straight or branched C ₀ .i ₅ alkyl chain wherein one or more carbons (such as 2 or 3) are replaced by a heteroatom independently selected from N, O or S, wherein said chain is optionally substituted by one or more (for example 1 or 2), oxo or nitro groups.

In relation to a saturated or unsaturated, branched or unbranched alkyl chain, wherein a carbon is replaced by a heteroatom selected from O, N or S, it will be clear to persons skilled in the art that the heteroatom may replace a primary, secondary or tertiary carbon, that is -CH ₃ , -CH ₂ -, a -CH- or a branched carbon group, as technically appropriate. An oxo substituent may be located on a carbon or sulphur atom as desired.

Oxo represents =0. Amino acid as employed herein is a natural or non-naturally occurring amino acid, for example a natural amino acid such as 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 the amino acid employed in the relevant variable is proteinogenic.

Proteinogenic amino acids, as employed herein, is intended to refer to amino acids found in proteins. In one embodiment R ¹ together with the carbon to which it is attached and the alpha- nitrogen and alpha-carbonyl is 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 R ² together with the carbon to which it is attached and the alpha- nitrogen and alpha-carbonyl is 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 R together with the carbon to which it is attached and the alpha- nitrogen and alpha-carbonyl is isoleucine or valine.

In one embodiment R ² together with the carbon to which it is attached and the alpha- nitrogen and alpha-carbonyl leucine or valine.

In one embodiment R ¹ together with the carbon to which it is attached and the alpha- nitrogen and alpha-carbonyl is valine and R ² together with the carbon to which it is attached and the alpha-nitrogen and alpha-carbonyl is leucine. In one embodiment R ¹ together with the carbon to which it is attached and the alpha- nitrogen and alpha-carbonyl is isoleucine and R ² together with the carbon to which it is attached and the alpha-nitrogen and alpha-carbonyl is valine.

In one embodiment X is a bond.

In one embodiment m is 0.

In one embodiment n is 2

In one embodiment p is 0.

In one embodiment p is 1.

In one embodiment q is 2.

In one embodiment R ³ is H. In one embodiment R ⁴ represents R ^A -L-Ar ¹ .

In another alternative embodiment R ³ and R ⁴ together with the nitrogen to which they are attached form a 5 or 6 membered heterocyclic group optionally including a further heteroatom (for example 1 , 2, or 3) selected from N, O, and S, for example pyrrolidine, piperidine, piperazine, such as piperazine or piperidine bearing one -YAr ¹ substitutent, in particular piperazin-4-yl, such as A/-YAr ¹ -piperazin-4-yl.

In one embodiment R ⁵ together with the carbon to which it is attached and the alpha- nitrogen and alpha-carbonyl is a natural amino acid, for example cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine, in particular valine.

In one embodiment R ^A is a bond. When R ^A is a bond then it will be understood that L or Ar ¹ , as appropriate, is directly linked to the nitrogen of -NR ³ R ⁴ .

In one embodiment R ^A is C ₀ . ₉ alkylC ₆ . ₀ aryl, such as Ci alkyl-, C ₂ alkyl-, C ₃ alkyl-, C alkyl-, C ₅ alkyl-, C ₆ alkyl-, C ₇ alkyl- or C ₈ alkyl-phenyl or napthyl, in particular phenyl. When C ₀ is employed then C ₆ . ₁₀ aryl will be linked directly to the nitrogen of -NR ³ R ⁴ .

In an alternative embodiment R ^A is Co _-9 alkylC ₅ .nheteroaryl, C ₀ .g alkylC _3-6 cycloalkyl, or a -Co-9 alkylC ₅ .n heterocyclic group. In an alternative embodiment R ^A is C ₀ -g alkylC _5- nheteroaryl, such as d alkyl-, C ₂ alkyl-, C ₃ alkyl-, C ₄ alkyl-, C ₅ alkyl-, C ₆ alkyl-, C ₇ alkyl- or C ₈ alkyl-heteroaryl, for example selected from pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, triazole, oxadiazole, furazan, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, triazine, 1 H-pyrrolizine, indolizine, indole, isoindole, benzofuran, isobenzofuran, indoline, isoindoline, benzothiophene, indazole, benzimidazole, purine, quinoline, isoquinoline, chromane, isochromane, chromene, cinnoline, quinazoline, quinoxaline, naphthyridine or phthalazine. When C ₀ is employed then C ₅ .naryl will be linked directly to the nitrogen of -NR ³ R ⁴ .

In one embodiment R ^A is C ₀ . ₉ alkylC ₃ . ₆ cycloalkyl, for example C alkyl-, C ₂ alkyl-, C ₃ alkyl-, C ₄ alkyl-, C ₅ alkyl-, C ₆ alkyl-, C ₇ alkyl- or C ₈ alkyl-C _3-6 cycloalkyl selected from cyclopropyl, cyclopentyl or cyclohexyl. When C ₀ is employed then C _3-6 cycloalkyl will be linked directly to the nitrogen of -NR ³ R ⁴ . In one embodiment R ^A is -C ₀ . ₉ alkylC ₅ .n heterocyclic group for example Ci alkyl-, C ₂ alkyl-, C ₃ alkyl-, C ₄ alkyl-, C ₅ alkyl-, C ₆ alkyl-, C ₇ alkyl- or C ₈ alkyl-heterocyclic group for example selected from pyrroline (such as 1 , 2 or 3-pyrroline), pyrrolidine, tetrahydrofuran, tetrahydrothiophene, pyrazoline (such as 2 or 3-pyrazoline), 2-imidazoline, pyrazolidine, imidazolidine, 3-dioxolane, thiazolidine, isoxazolidine, pyran (such as 2H or 4H-pyran), 3,4-dihydro-2H-pyran, piperidine, 1 ,4-oxazine, 1 ,4-dioxine, piperazine, morpholine and 1 ,4-dioxane. When C ₀ is employed then C ₅ -n heterocycle will be linked directly to the nitrogen of -NR ³ R ⁴ . Clearly R ^A is a linking group and thus when it comprises a ring such as a cycloalkyl, heterocycle, heteroaryl or aryl then LAr ¹ may be attached via the ring.

L in one embodiment is C ₀ . When L is C ₀ the Ar ¹ may be linked directly to the nitrogen of -NR ³ R ⁴ . Alternatively when L is C ₀ then Ar ¹ may be linked to R ^A

In an alternative embodiment L is a straight or branched, such as straight, d _-9 alkyl chain wherein optionally one or more, such as one, carbon(s) is/are replaced by a heteroatom selected from O, N and S, such as N, and optionally substituted by oxo (e.g. 1 or 2). For example L is a straight C _1-3 alkyl chain (such as alkyl), such as wherein none of the carbons are replaced by a heteroatom, e.g. wherein the chain does not bear any optional substituents.

In one embodiment L is -(CH ₂ )iNH(CH ₂ )j or -(CH ₂ )kNHC(0)- wherein i is an integer 1 to 12, j is 0 or 1 and k is and integer 1 to 14.

Alternatively, L is a straight C ₆ -9 alkyl chain, wherein one carbon is replaced by a

heteroatom, such as N, and the chain optionally bears one oxo substituent, in particular -CH ₂ CH ₂ CH ₂ NHCH ₂ - or -CH ₂ CH ₂ CH ₂ NHC(0)-. In one embodiment a heteroatom in L is separate from the nitrogen of -NR ³ R ⁴ by at least two carbon atoms.

In one embodiment L is C _1-5 alkyl wherein optionally one carbon atom is replaced by a nitrogen, such as C _1-3 alkyl.

In one embodiment Y represents C ₀ . When Y is C ₀ the Ar ¹ will be linked directly to the relevant heterocycle group. In an alternative embodiment Y is a straight or branched, such as straight, C _1-5 alkyl chain (for example C ₂ , C ₃ or C ₄ chain, such as a C ₄ alkyl chain) wherein optionally one or more, such as one or two carbon(s) is/are replaced by a heteroatom selected from O, N and S, such as N, and optionally substituted by oxo (for example 1 or 2 oxo substituents, in particular on carbon), such as -CH ₂ - or -CH ₂ CH ₂ NHC(0)-.

In one embodiment Ar ¹ bears 1 or 2 chloro or 1 or 2 nitro substituents, in particular 1 or 2 chloros. In one embodiment Ar ¹ bears one group -(Q)m(CH ₂ )qNHC(0)CCI ₃ and optionally one or two groups independently selected from chloro or nitro. In one sub-embodiment m is 0 and q is 1. In one Ar ¹ does not bear any optional substituents.

In one embodiment Ar ¹ is a five or six membered heteroaryi, for example pyrazolyl, pyridinyl, pyrazinyl, for example pyrindin-4-yl.

In one embodiment Ar ¹ is a 5,6 or 6,5 bicyclic heteroaryi system, for example indolyl.

In one embodiment Ar ¹ is a 6,6 bicyclic heteroaryi system, for example quinolinyl or isoquinolinyl. In one or more embodiments -NR ³ R ⁴ represents a substituent selected from Table 1.

TABLE 1

The compounds in the table above are coupled to the lantibiotic moiety through the nitrogen of the terminal -NH ₂ or -NH as appropriate, shown on the left extremity of the fragment. In one embodiment Z represents H, C _1-6 alkyl, for example methyl, ethyl, propyl or butyl, or an amino acid residue, for example 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, such as alanine or serine.

In one embodiment Z is H or alanine, such as H. In one embodiment Z is phenylalanine. In one embodiment the fragment:

In an alternative embodiment the fragment:

In one embodiment the compound is selected from:

Deoxyactagardine B [5-chlorotryptamine] monocarboxamide;

Deoxyactagardine B [4-(4',6'-dichloropyridin-2-yl)piperazine] monocarboxamide;

Deoxyactagardine B [tryptamine] monocarboxamide;

Deoxyactagardine B [7-chloro-4-hydrazinoquinoline] monohydrazide;

Deoxyactagardine B [(2-Chloropyridin-5-yl)methylamine] monocarboxamide;

Deoxyactagardine B [3-(5'-chloro-1 'H-indol-3'yl)methylamino)propylamine]

monocarboxamide;

Deoxyactagardine B [3-(4'6'-dichloro-2'H-benzopyran-3'-yl)methylamino)propylami ne];

monocarboxamide; and

Deoxyactagardine B [4,6-dichlorotryptamine] monocarboxamide. The compounds of 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 a/., J. Pharm. Sci, 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) 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) contains more than one basic group bis salts or tris salts may also be formed and are salts according to 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, glucamate (including N-methyl glucamate and N-ethyl glucamate) glucurinate, alkyl or aryl sulphonates (eg methanesulphonate, ethanesulphonate, benzenesulphonate or p-toluenesulphonate), mesylate 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.

Salts may be employed to optimize the solubility of the compounds of the present disclosure.

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) are within the scope of the disclosure. The salts of the compound of the disclosure may form solvates (e.g. hydrates) and the disclosure also includes all such solvates.

In one embodiment there is provided a process for the preparation of compounds according to the disclosure, for example compounds of formula (I) comprising coupling a compound of formula (II):

with an amine comprising -R ^A -L-Ar ¹ or -YAr, wherein X ¹ represents -OH or an amino acid with a free C-terminal (e.g. where X is an amino acid) and each of the variables are as defined for compounds of formula (I). The amine may be a compound -NHR ³ R ⁴ , where R ³ and R ⁴ are as defined for the compounds of formula (I). In an alternative method, X represents -OH and the amine may be a compound H ₂ NXNHR ³ R ⁴ , where H ₂ N represents the N terminal of the amino acid X (as defined for the compounds of formula (I)).

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) 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). 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 hereinafter to a compound according to the disclosure include both compounds of formula (I) and their pharmaceutically acceptable salts and derivatives. Unless the context specifically indicates otherwise references to compounds of formula (I) includes other compounds within scope of the present invention.

With regard to stereoisomers, the compounds of formula (I) have more than one asymmetric carbon atom. In the general formula (I) 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 formula (I) may also have one or more asymmetric carbon atoms. The compounds of structure (I) may occur as individual enantiomers or diastereomers. All such isomeric forms are included 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) as described herein also extend to tautomeric forms thereof, for example, keto/enol tautomers, where appropriate. The compounds of formula (I) may be in crystalline or amorphous form. Furthermore, some of the crystalline forms of the compounds of structure (I) may exist as polymorphs, all forms which are included in the present disclosure. In another aspect, the invention provides a pharmaceutical composition comprising, as active ingredient, a compound of the disclosure 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 subjects suffering from a condition susceptible to amelioration by an antimicrobial compound.

In another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure and a

pharmaceutically acceptable excipient, diluent and/or carrier (including combinations thereof).

There is further provided by the present disclosure a process of preparing a pharmaceutical composition, which process comprises mixing a compound of the disclosure or a

pharmaceutically acceptable derivative thereof, together with a pharmaceutically acceptable excipient, diluent and/or carrier.

The compounds of 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 pharmaceutical compositions comprising a compound of the disclosure adapted for use in human or veterinary medicine. 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 comprise as - or in addition to - the excipient, diluent and/or carrier any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

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 of the disclosure 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 of the invention may be prepared by processes known in the art, for example see International Patent Application No. 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 compositions of the disclosure include those in a form especially formulated for parenteral, oral, buccal, rectal, topical, implant, ophthalmic, nasal or genito-urinary use. In one aspect of the invention, the agents are delivered orally, hence, the agent is in a form that is suitable for oral delivery.

In some instances it may be possible to deliver the compounds of the disclosure by a topical, parenteral (e. g. by an injectable form) or transdermal route, including mucosal (e. g. as a nasal spray or aerosol for inhalation), nasal, , gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral).

There may be different composition/formulation requirements depending on the different delivery systems. By way of example, the pharmaceutical composition of the present disclosure may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated in an injectable form, for delivery by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes. Where appropriate, the pharmaceutical

compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.

For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.

If a compound of the present disclosure is administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly for example as a bolus fomulation or subcutaneously administering the agent, and/or by using infusion techniques. The compounds of the disclosure can 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 of the disclosure 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 glycollate, 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 as fillers in gelatin or HPMC (hydroxypropyl methylcellulose) capsules. Preferred excipients in this regard 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) and a carrier. Where the capsule is filled with a powder the compounds of formula (I) and/or the carrier may be milled or micronised to provide material with an appropriate particle size.

Compounds of the disclosure 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 a solid dose formulation of a compound of formula (I) for example where the formulation has an enteric coating.

In another aspect the disclosure provides 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.

Having said this it is believed that the compounds of formula (I) are not particularly susceptible to degradation by stomach acid or intestinal enzymes in vivo. The compounds of the disclosure may also be administered orally, in veterinary medicine, in the form of a liquid drench such as a solution, suspension or dispersion of the active ingredient together with a pharmaceutically acceptable carrier or excipient. The compounds of the invention may also, for example, be formulated as suppositories e.g. containing conventional suppository bases for use in human or veterinary medicine or as pessaries e.g. containing conventional pessary bases. In one embodiment the formulation is provided as a formulation for topical administration including inhalation.

Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases. Inhalable powders according to the disclosure containing the active substance may consist solely of the abovementioned active substances or of a mixture of the abovementioned active substances with physiologically acceptable excipient.

These inhalable powders may include monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo- and polysaccharides (e.g.

dextranes), polyalcohols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these with one another. Mono- or disaccharides are preferably used, the use of lactose or glucose, particularly but not exclusively in the form of their hydrates.

Particles for deposition in the lung require a particle size less than 10 microns, such as 1-9 microns suitably from 0.1 to 5 pm, particularly preferably from 1 to 5 pm. The particle size of the active (i.e. the compound according to the disclosure). The propellent gases which can be used to prepare the inhalable aerosols are known from the prior art. Suitable propellent gases are selected from among hydrocarbons such as n- propane, n-butane or isobutane and halohydrocarbons such as chlorinated and/or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The above-mentioned propellent gases may be used on their own or in mixtures thereof.

Particularly suitable propellent gases are halogenated alkane derivatives selected from among TG11 , TG 12, TG 134a and TG227. Of the abovementioned halogenated

hydrocarbons, TG134a (1 ,1 ,1 ,2-tetrafluoroethane) and TG227 (1 ,1 ,1 ,2,3,3,3- heptafluoro propane) and mixtures thereof are suitable for use in formulations of the present invention.

The propellant-gas-containing inhalable aerosols may also contain other ingredients such as co-solvents, stabilisers, surface-active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients are known in the art. The propellant-gas-containing inhalable aerosols according to the invention may contain up to 5 % by weight of active substance. Aerosols according to the disclosure may contain, for example, 0.002 to 5 % by weight, 0.01 to 3 % by weight, 0.015 to 2 % by weight, 0.1 to 2 % by weight, 0.5 to 2 % by weight or 0.5 to 1 % by weight of active.

The compounds of the disclosure may also be used in combination with other therapeutic agents. The disclosure thus provides, in a further aspect, a combination comprising a compound of formula (I) 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) and an antibiotic, such as vancomycin, a beta-lactam (such as a cephalosporin), an aminoglycoside, a macrolide, a tetracyline, a lipopeptide, an

oxazolidinone and/or an anti-inflammatory such as a steriod. The combination may be provided as a co-formulation or simply packaged together as separate formulations, for simultaneous or sequential delivery.

It is to be understood that not all of the compounds of the combination need be administered by the same route. Thus, if the therapy comprises more than one active component, then those components may be administered by different routes.

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 the disclosure or the second therapeutic agent may be administered first. When administration is simultaneous, the combination may be administered either in the same or a different pharmaceutical composition. The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the disclosure. 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 such manner as are known for such compounds in the art. The compositions may contain from 0.01-99 % of the active material. For topical administration, for example, the composition will generally contain from 0.01-10 %, more preferably 0.01-1 % of the active material.

When a compound of the disclosure 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 be the same or differ from that employed when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. It will also 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. 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 and parenteral administration to humans, the daily dosage level of the agent may be in single or divided doses. For systemic administration the daily dose as employed for adult human treatment will range from 2-100mg/Kg body weight, preferably 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.

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

In one embodiment a compound of formula (I) is useful in the treatment of skin infections, in particular bacterial skin and soft tissue infection. In one embodiment a compound as disclosed herein is provided for use in the treatment of gram positive infections, in particular topical or parenteral treatment. In one aspect, the disclosure provides use of a compound of formula (I) in therapy, for example, for treatment of microbial infections such as bacteraemia, pneumonia and microbial infection of soft tissue including surgical wounds, in particular staphylococcal infections including MRSA infection. In one embodiment the compounds of formula (I) are useful for the treatment of enterococcal infections including E. faecalis and E. faecium infection, for example skin and skin structure infections, endocarditis, urinary tract infection and sepsis.

In one embodiment the compounds of formula (I) are useful for the treatment of S.

pyogenes, for example skin infections such as impetigo, erysipelas and cellulitis, throat infections, scarlet fever, and acute glomerulonephritis.

In one embodiment compounds of formula (I) are useful in the treatment of Streptococcus pneumoniae infection, for example pnuemonia, acute sinusitus, otitis media, meningitis, bacteremia, osteomylitis, septic arthritis and endocarditis.

In one aspect the compounds of formula (I) 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.

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

In one aspect there is provided use of a compound of formula (I) for the prophylaxis, treatment or maintenance of IBS (irritable bowel syndrome). See for example Rifaximin Treatment for Symptoms of Irritable Bowel Syndrome. Andrea L. Fumi and Katherine Trexler, The Annals of Pharmacotherap, 2008, 4, 408.

In one embodiment a compound of formula (I) is useful in the treatment of ulcerative colitis including prophylactic treatment to prevent recurrence thereof. The compounds may be particularly suitable for the treatment of steroid refractory ulcerative colitis. See for example steroid-refractory ulcerative colitis treated with corticosteroids, metronidazole and

vancomycin: a case report J. Miner, M. M Gillan, P. Alex, M Centola, BMC Gastroenterology 2005, 5:3. The compounds of the present disclosure may be useful for long term treatment.

In one aspect there is provided a compound of formula (I) or a composition comprising same for use in treatment or prophylaxis for example the treatment or prophylaxis of any one the indications described herein.

In one aspect there is provided a compound of formula (I) or a composition comprising the same for the manufacture of a medicament for one or more of the indications defined above.

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 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 one embodiment the compounds of the present invention have activity against one or more of the following bacteria:

VISA S. aureus V99, S. aureus Ml, S. aureus Mu3, S. aureus 26, S. aureus Mu50, S. aureus 2, S. aureus NJ

MRSA S. aureus R33, S. aureus 12232, S. aureus R36, S. aureus R34, S. aureus R39, S. aureus R37, S. aureus R31 , S. aureus R40, S. aureus W71 , S. aureus W74, S. aureus W82, S. aureus W96, S. aureus W97, S. aureus W98, S. aureus W99, ATCC 33591

MSSA S. aureus G15, S. aureus G20, S. aureus G22, S. aureus G23, S. aureus G28, S. aureus G30, S. aureus G31 , S. aureus G32, S. aureus G33, S. aureus G35, S. aureus G12,

S. aureus G26, S. aureus G29, S. aureus 8325-4, S. aureus SH 1000

MSSE S. epidermidis GRL05001 , S. epidermidis GRL05002, S. epidermidis GRL05003, S. epidermidis GRL05004, S. epidermidis GRL05005, S. epidermidis GRL05006, S.

epidermidis GRL05007, S. epidermidis GRL05008, S. epidermidis GRL05009, S.

epidermidis GRL05010, S. epidermidis 9AF , S. epidermidis C12, S. epidermidis MF 87 , S. epidermidis C 6

MRSE S. epidermidis 7755298, S. epidermidis 7865688, S. epidermidis 7753921 , S.

epidermidis GRL0501 1 , S. epidermidis GRL05012, S. epidermidis GRL05016, S.

ep/ ^' derm/ ^' i//s GRL05017, S. epidermidis GRL05013, S. epidermidis GRL05014, S.

ep/ ^' derm/cf/ ^' s GRLOSOI S, S. epidermidis GRL05019, S. epidermidis GRL05020, S.

epidermidis 7864847, S. epidermidis 7765349 VSE E. faecalis 7754422, E faecium 7865229, E. faecium 19579, E. faecalis GRL05022, E. faecalis GRL05023, E. faecalis GRL05024, E. faecalis GRL05026, E faecalis GRL05027, E faecalis GRL05029, E faecalis GRL05030, E faecalis 7757400, E faecalis 7791220

VRE E faecium 7662769, E faecium 7634337, E. faecium 7865532, E. faecium 9709024, E faecium 9710577, E feeca//s GRL05031 , E faeca// ^' s GRL05032, E faeca//s GRL05033, E aeca//s GRL05034, E faeca// ^' s GRL05035, E faecalis 9758512, E faecium 9704998, E faecium 7860190

S. pyogenes S. pyogenes 7755441 , S. pyogenes 7713283, S. pyogenes 7865253, S.

pyogenes 7757080, S. pyogenes 7755255, S. pyogenes 7865844, S. pyogenes GRL05045, S. pyogenes GRL05046, S. pyogenes 7865289, S. pyogenes GRL05043, S. pyogenes 7755584, S. pyogenes GRL05042, S. pyogenes GRL05041.

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.

Where technically appropriate embodiments may be combined and thus the disclosure extends to all permutations/combinations of the embodiments provided herein.

Preferences given for compounds of formula (I) may equally apply to other compounds of the invention, disclosed herein, as technically appropriate.

EXAMPLES

Example 1

Deoxyactagardine B [5-chlorotryptamine] monocarboxamide

Deoxyactagardine B [DAB] (500 mg), 5-chlorotryptamine (123 mg) and

diisopropylethylamine (157 μΙ_) were dissolved in dry dimethylformamide (3 ml_). A solution of benzotriazole-1 -yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) (278 mg) in dry dimethylformamide (2ml_) was added portionwise. The reaction was followed by analytical hplc (See Table 1) and PyBOP was added until the starting material had been consumed (Figures 1 and 2). Table 1: Analytical HPLC conditions

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 10 min 0% A 100% B

Time 1 1 min 0% A 100% B

Time 1 1.2 min 100% A 0% B

Cycle time 15 min

Injection volume: 10 pL

Detection: 210 nm On completion of the reaction the mixture was poured into a mixture of methanol (8 mL), water (10 mL) and 30%MeCN in 20 mM potassium phosphate buffer pH 7.0 (2 mL) and the resultant solution loaded on to a 10 g C18 silica cartridge. Elution with 40, 50, 60, 70, 80, 90 and 100% aq methanol and evaporation of the appropriate fraction gave the compound as a white solid. Yield 130 mg, 24 %. [M+2H] ⁺² calculated 1025, found 1026.

Table 2: LC/MS conditions

Column: Zorbax 5μ C18(2) 150 x 4.6 mm

Mobile Phase A: 10% acetonitrile, 0.1 % formic acid

Mobile Phase B: 90% acetonitrile, 0.1 % formic acid

Flow rate: 1 mlJmin

Gradient: Time 0 min 100% A 0% B

Time 10 min 0% A 100% B

Time 1 1 min 0% A 100% B

Time 1 1.1 min 100% A 0% B

Cycle time 15 min

Injection volume: 20 ML

Mass Spectrometer parameters

lonisation Electrospray +ve

Mass range 250 - 1500 mu

Capillary voltage 3.10 KV

Cone voltage 40 V

Skimmer lens offset 5 V

Ion energy 1.4 V Example 2

Deoxyactagardine B [4-(4',6'-dichloropyridin-2-yl)piperazine] monocarboxamide

Was prepared from the amide coupling of 50mg deoxyactagardine B with 4-(4',6'- dichloropyridin-2-yl)piperazine according to the procedure described for Example 1.

Yield 6.8 mg, 18 % MH+ calculated 2086, found 2087

Example 3

Deoxyactagardine B [tryptamine] monocarboxamide

Was prepared from the amide coupling of 50mg deoxyactagardine B with tryptamine according to the procedure described for Example 1. Yield 28 mg, 52 % MH+ calculated 2015, found 2016 Example 4

Deoxyactagardine B [(2-Chloropyridin-5-yl)methylamine] monocarboxamide

Was prepared from the amide coupling of 50mg deoxyactagardine B with 2-chloro-5- aminomethylpyridine according to the procedure described for Example 1. Yield 28 mg, 52 %. H+ calculated 1997, found 1998

Example 5

Deoxyactagardine B [3-(5'-chloro-1 'H-indol-3'yl)methylamino)propylamine]

Was prepared from the amide coupling of 50mg deoxyactagardine B with 3-(5'-chloro-1 Ή- indol-3'yl)methylamino)propylamine according to the procedure described for Example 1 . Yield 4.8 mg, 9 %. [M+2H]+2 calculated 1047, found 1047.

Example 6

Deoxyactagardine B [3-(4'6'-dichloro-2'H-benzopyran-3'-yl)methylamino)propylami ne] monocarboxamide

Was prepared from the amide coupling of 50mg deoxyactagardine B with 3-(4'6'-dichloro- 2'H-benzopyran-3'-yl)methylamino)propylamine according to the procedure described for Example 1 . Yield 6.9 mg, 12 %. [M+2H]+2=1071 , found 1072.

Example 7

Deoxyactagardine B [4,6-dichlorotryptamine] monocarboxamide

Was prepared from the amide coupling of 50mg deoxyactagardine B with 4,6- dichlorotryptamine according to the procedure described for Example 1

Yield 43.5 mg, 78 %. MH+ calculated 2083, found 2084

In vitro antibacterial activity of examples 1-7 against Staphylococcus aureus

Susceptibility testing (MIC) was performed by two-fold serial dilutions in Mueller Hinton Broth supplemented with 50pg/ml Ca ²⁺ except that susceptibility testing of S. pneumoniae was performed by two-fold serial dilutions in Brain-Heart-Infusion Broth supplemented with 50pg/ml Ca ²⁺ . Microtitre plates were incubated aerobically for 18-20 hours at 37°C following the recommendations of CLSI. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug that prevented visible growth.

Example Staph, aureus R33 ( RS A) Staph, aureus SH1000 (MSSA)

1 4 4

2 4 - 8 16

3 4 8

4 16 32

5 8 16 Example Staph, aureus R33 (MRS A) Staph, aureus SH1000 (MSSA)

6 8 4 - 8

7 4 4

MIC values in g/mL. Modal average values from up to 8 determinations.

In vitro antibacterial activity of examples 1, 3, 5, 6, 7

MIC values in [iglml. Modal average values from up to 14 determinations.

In vitro antibacterial activity of example i compared with deoxyactagardine B

MRS A Strain Example 1 pg/mL DAB pg/mL

S. aureus R33 4 32

S. aureus 12232 4 16 - 32

S. aureus R36 4 16

S. aureus R34 2 16

S. aureus R39 4 >32

S. aureus R37 4 nt

S. aureus R31 2 nt

S. aureus R40 4 - 8 32 - >32

S. aureus W71 4 >32

S. aureus W74 4 32

S. aureus W82 4 >32

S. aureus W96 4 >32

S. aureus W97 4 >32

S. aureus W98 4 32 - >32

S. aureus W99 4 32 Strain Example 1 DAB

VISA

S. aureus V99 4 32 - >32

S. aureus Ml 16 >32

S. aureus Mu3 4 - 16 32

S. aureus 26 4 >32

S. aureus Mu50 4 32 - >32

S. aureus 2 4 32

S. aureus NJ 4 >32

MSSA

S. aureus G15 2 - 4 >32

S. aureus G20 4 >32

S. aureus G22 4 >32

S. aureus G23 4 >32

S. aureus G28 4 32

S. aureus G30 4 >32

S. aureus G31 4 >32

S. atvrei/s G32 4 >32

S. atvretvs G33 4 >32

S. aureus G35 4 >32

S. aureus GM 4 nt

S. aureus G26 4 nt

S. aureus G29 4 nt

S. aurews 8325-4 4 nt

S. atvrei/s SHI OOO 4 16 - 32

MSSE

S. epidermidis GRL05001 8 >32

S. epidermidis GRL05002 8 >32

S. epidermidis GRL05003 4 32 - >32

S. epidermidis GRL05004 8 32 - >32

S. epidermidis GRL05005 8 32 - >32

S. epidermidis GRL05006 8 >32

S. epidermidis GRL05007 32 >32

S. epidermidis GRL05008 16 >32

S. epidermidis GRL05009 16- 32 >32

S. epidermidis GRL05010 16 - 32 >32

S. epidermidis 9AF 16 - 32 nt

S. epidermidis C12 16 - 32 nt

S. epidermidis F87 4 nt Strain Example 1 DAB

S. epidermidis C16 16-32 nt

MRSE

S. epidermidis 7755298 8-16 32 - >32

S. epidermidis 7865688 16 >32

S. epidermidis 7753921 8-16 >32

S. epidermidis GRL05011 8 32 - >32

S. epidermidis GRL05012 8 nt

S. epidermidis GRL05016 8 nt

S. ep/ctem7/ ^' c(/sGRL05017 16 32 - >32

S. ep/ ^' cte/7n/ci/ ^' sGRL05013 16 32 - >32

S. ep/ ^' ctem7/ ^' (//sGRL05014 16 32 - >32

S. ep/cteAm/(//sGRL05015 8-16 32 - >32

S. epcterm/c// ^' sGRL05019 8-16 32 - >32

S. epidermidis GRL05020 4 32

S. epidermidis 7864847 8-16 nt

S. epidermidis 7765349 16 nt

VSE

E. faecalis 7754422 2 8

E. faecium 7865229 8-16 >32

E. faecium 19579 2-4 32

E. faecalis GRL05022 4 16

E. faecalis GRL05023 2 8

E. faecalis GRL05024 4-8 8

E. faecalis GRL05026 4-8 16

E. faecalis GRL05027 4-8 16

E. faecalis GRL05029 2-4 8-16

E. faecalis GRL05030 2-8 8

E. faecalis 7757400 1 -4 nt

E. faecalis 7791220 4-16 nt

VRE

E. faecium 7662769 8-32 >32

E. faecium 7634337 16-32 >32

E. faecium 7865532 16 >32

E. faecium 9709024 8-16 >32

E. faecium 9710577 16 >32

E. faecalis GRL05031 4 4

E. faecalis GRL05032 8-16 4

E. faecalis GRL05033 8 8 Strain Example 1 DAB

£ faecalis GRL05034 4 >32

£ faecalis GRL05035 8 >32

£ faecalis 9758512 8 nt

£ faecium 9704998 8 nt

£ faecium 7860190 8 nt

S. pyogenes

S. pyogenes 7755441 0.06 - 0.125 <0.5 - 0.5

S. pyogenes 7713283 0.06 1

S. pyogenes 7865253 0.06 <0.5

S. pyogenes 7757080 0.06 nt

S. pyogenes 7755255 0.06 <0.5

S. pyogenes 7865844 8 <0.5

S. pyogenes GRL05045 0.125 - 4 1

S. pyogenes GRL05046 0.06 nt

S. pyogenes 7865289 0.06 1

S. pyogenes GRL05043 2 - 8 nt

S. pyogenes 7755584 0.06 1

S. pyogenes GRL05042 0.06 <0.5

S. pyogenes GRL05041 0.06 - 0.25 1

MIC values in pg/mL. Modal values from up to 8 determinations Pharmacokinetic assessment of Example 1 in male CD-1 mice

A total of 30 male CD-1 mice were divided into 10 groups of 3. Example 1 (5 mg/ml in 5% Tween/45% propylene glycol/50% water) was dosed to each mouse 25 mg/kg /V via the tail vein. Terminal blood samples were collected from groups of 3 mice predose then at 1 min, 5 min, 30 min, 1 h, 2 h, 3 h, 6 h, 8 h and 24 h post dosing. The blood was collected in 1.5 ml polypropylene microcentrifuge tubes pre-prepared with anti-coagulant and centrifuged at 16.100 x g for 5 mins to separate the plasma. The plasma was subsequently transferred to a fresh microcentrifuge tube and stored at -20°C awaiting analysis by LC-MS/MS.

Prior to analysis by LC-MS/MS 200 pL of extraction buffer (acetonitrile with 0.1% formic acid and 500 ng/mL propanolol) were added to 200 pi thawed plasma. The samples were then vortexed for 15 sec and shaken for 10 min on a lab-line 4626-1 CE orbital shaker at a speed of 450 rpm. Following centrifugation for 10 mins in an Eppendorf minispin centrifuge at 16,100 x g the supernatants were analysed by LC-MS/MS and concentrations determined by comparison of the peak areas against a standard calibration curve. Analysis of Example 1 dosed IV 25 mg/kg to male CD1 mice (n=3, mean ± SEM).

Example 1 post IV dosing @ 25mg/kg

Animal ID

Time (hrs) mean SEM

IV10 IV11 IV12

Predose BLOQ BLOQ BLOQ BLOQ ND

-1

E 0.02 546000 480000 412000 479333 22334 o>

c 0.1 445000 475000 41 1000 443667 10674

^■ a 0.5 225000 283000 272000 260000 10269 c

3

o 1 221000 212000 212000 215000 1732

E 2 100000 190000 153000 147667 15079 o

ϋ 3 94000 104000 89000 95667 2546

6 39900 45100 36300 40433 1475

8 15100 15400 22000 17500 1300

24 1 150 1860 1360 1457 122

ND = Not determined, BLOQ = Below limit of quantification.

Summary of LC-MS/MS setup conditions.

Parameter Setting

Autosampler CTC Pal

Injection vol (μί) 20

Mass spectrometer Sciex API 365

Temperature (°C) 400

CAD 5

NEB 10

Capillary (V) 5000

HPLC Agilent 1050

Column Luna C18 5μΜ 150*2.0 mm

Mobile phase A: 0.1 % (v/v) formic acid in water

B: Acetonitrile + 0.1 %(v/v) formic acid

Flow rate (mL/min) 0.3 Parameter Setting

Gradient Time % A % B

(min)

0 95 5

0.6 95 5

1.5 5 95

5 0 100

6 95 5