SUCCINATE DERIVATIVES AND THEIR THERAPEUTIC USE

Field of the Invention

The present invention relates to succinate derivatives, to pharmaceutical compositions comprising them and to their use in the treatment of inflammatory skin conditions which are characterised by colonisation with Staphylococcus aureus. Background of the Invention

Atopic dermatitis (AD), sometimes referred to as eczema, is a chronic, relapsing condition which is characterised by pruritus, erythema, dry skin and inflammation. The pathogenesis of AD is still not fully understood, although excessive T-cell activation in response to antigen stimulation and hyperstimulation of T-cells by atopic Langerhans cells are said to be important factors. Levels of IgE production correlate well to the severity of the disease, and although allergen specific IgE may be observed in many patients, it is not clear that this finding indicates sensitisation to a specific allergen.

Prevalence of the disorder varies widely, but has been estimated to be as high as 20% among children in some western countries. AD is frequently seen in families with a history of atopic diseases (asthma, allergic rhinitis and AD).

Staphylococcus aureus has been found to colonise the skin lesions of more than 90% of AD patients (Leyden et al, Br. J. Dermatol. 90:525-530, 1974), while being present in only 5% of normal subjects. The bacteria have been shown to be important in the exacerbation and chronicity of AD through the release of toxins (e.g. enterotoxins A, B, C and D; toxic shock syndrome toxin), many of which are highly antigenic in nature, thus exacerbating the inflammatory responses in the skin (Leung et al, J. Clin. Invest. 92 1374-80, 1993). One particular study in children found that 81% of patients had Staphylococcus aureus colonisation (compared to 4% of the control group) showing that disease severity could be correlated with colonisation by toxigenic strains (Bunikowski et al, J. Allergy Clin Immunol. 105(4):814-819, 2000). Current treatments typically involve a number of approaches, such as (i) skin hydration, including batching and use of moisturiser; (ii) the use of medicaments to reduce or modulate the immune response, such as steroids (glucocorticoids) and immunosuppressants (e.g. cyclosporine A, tacrolimus and pimecrolimus); and (iii) elimination of contributory factors such as irritants,

allergens, emotional stress factors and infectious agents. Although the current treatments can effectively deal with acute phases of the disorder, there are questions over their long-term use due to the potentially severe side-effects associated with extended use of steroids and immunosuppressants. Oral antibiotics are often used to treat superinfections, although the general use of antibiotics, especially topical antibiotics, is generally discouraged due to the risk of the development of antibiotic-resistant bacterial strains.

Aureolysin (EC 3.4.24.29) is a metalloprotease which is secreted by Staphylococcus aureus (Dubin, Biol. Chem. 383:1075-1086, 2002). Aureolysin is a member of the thermolysin protein family, being dependent upon zinc and calcium for its activity, and has a low substrate specificity.

Investigation of the proteolytic activity of a range of Staphylococcus aureus strains from patients with AD found that of those strains showing moderate to high proteolytic activity, aureolysin contributed between 25-100% of the proteolytic activity (Miedzobrodzki et al, Eur. J. CHn. Microbiol Infect. Dis. 21:269-276, 2002).

Matrix metalloproteases (MMPs) have long been of interest, as drug targets in cancer and inflammatory diseases. MMPs (M10) and the adamalysin (M12) family of metalloproteases are metzincins (characterised by the 'metzincin' fold near the zinc binding motif (Bode et al, FEBS Lett, 331 , 134-40, 1993), and are distinct from the gluzincins (characterised by the presence of a GIu residue in the zinc binding domain) of which aureolysin (M4) is a member. Thus aureolysin is not categorised as an MMP.

WO2007/025999 (published after the priority date claimed herein) discloses the treatment or prevention of an inflammatory skin condition which is characterised by colonisation with Staphylococcus aureus, comprising the topical administration of an aureolysin inhibitor. WO2007/025999 also discloses succinate derivatives as aureolysin inhibitors. Summary of the invention According to a first aspect of the present invention, novel compounds are of formula (I):

wherein R ¹ is C1-4 alkyl, allyl or propargyl; R ² is C _1-6 alkyl, -(CH ₂ ) _m -aryl or -(CH ₂ ) _m -heteroaryl; R ³ is H or C _1-4 alkyl;

R ⁴ is C _1-4 alkyl, -(CH ₂ ) _n -aryl or -(CH ₂ ) _n -heteroaryl; R ⁵ is H ₁ OH or C _1-4 alkoxy; and m and n independently represent 0 or an integer of up to 3; or a pharmaceutically acceptable salt or solvate thereof. In a second aspect of the present invention is a method for the treatment or prevention of an inflammatory skin condition in a mammal which is characterised by colonisation with Staphylococcus aureus, comprising the topical administration of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof. A further aspect of the present invention is the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a topical medicament for the treatment or prevention of an inflammatory skin condition in a mammal which is characterised by colonisation with Staphylococcus aureus. Another aspect is a topical pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier or excipient, for use in the treatment of an inflammatory skin condition which is characterised by colonisation with Staphylococcus aureus. The methods, uses and compositions are expected to be useful in veterinary applications (i.e. wherein the mammal is a domestic or livestock mammal e.g. cat, dog, horse, pig etc.). However, the principal expected use or method is in pharmaceutical applications (i.e. wherein the mammal is a human).

A potential advantage provided by the λ/-alkylated succinate derivatives of formula (I) with respect to previously known succinate derivatives is their resistance to nucleophilic attack. For example, analogues of compounds of formula (I) in which R ¹ represents hydrogen may undergo an undesirable cyclisation reaction during synthesis and therefore synthetic strategies must be tailored to prevent, or at least to minimise, the formation of thermodynamically favoured by-products. Furthermore, such reactivity may, to some extent, be retained by the resultant product and may therefore compromise stability. The λ/-alkylated succinate derivatives of the present invention are more resistant to such nucleophilic attack. The resultant compounds may therefore be synthesised more easily (i.e. require fewer steps) and may confer enhanced stability upon the product. Detailed Description of the Invention

The term 'C _1-4 alkyl' as used herein as a group or a part of the group refers to a linear or branched saturated hydrocarbon group containing from 1 to 4 carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, terf-butyl, and the like.

The term 'C _1-6 alkyl' as used herein as a group or a part of the group refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or hexyl and the like. The terms 'Ci- ₆ alkoxy' and 'C1. ₆ fluoroalkyl' may be interpreted in an analogous fashion.

The term 'aryl' as used herein refers to a C ₆ -i2 monocyclic or bicyclic hydrocarbon ring wherein at least one ring is aromatic and which may optionally be substituted. Examples of such groups include phenyl, naphthyl or tetrahydronaphthalenyl and the like, particularly phenyl.

The term 'heteroaryl' as used herein refers to a 5-7 membered monocyclic aromatic ring or a fused 8-10 membered bicyclic aromatic ring containing 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur and which may optionally be substituted.

Examples of such monocyclic aromatic rings include thienyl, furyl, furazanyl, pyrrolyl, triazolyl, tetrazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, isothiazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazolyl, pyrimidyl, pyridazinyl,

pyrazinyl, pyridyl, triazinyl, tetrazinyl and the like. Pyridyl (e.g. 2-, 3- or 4-pyridyl) is a very typical example.

Examples of such fused aromatic rings include quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pteridinyl, cinnolinyl, phthalazinyl, naphthyridinyl, indolyl, isoindolyl, azaindolyl, indolizinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, benzofuranyl, isobenzofuranyl, benzothienyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoxadiazolyl, benzothiadiazolyl and the like.

Exemplary optional substituents (e.g. 1 , 2 or 3 substituents) for the aryl or heteroaryl groups include C _1-6 alkyl (e.g. methyl), halogen (e.g. Cl, Br, F),

Ci _-6 alkoxy (e.g. methoxy), hydroxyl, C _1-6 fluoroalkyl (e.g. trifluorom ethyl), cyano and -SO ₂ Ci _-4 alkyl, particularly Ci _-6 alkyl (e.g. methyl), halogen (e.g. Cl, Br, F) and

Ci _-6 alkoxy (e.g. methoxy).

Examples of an -(CH ₂ ) _n -aryl or an -(CH ₂ )m-aryl group include -CH ₂ phenyl and -CH ₂ CH ₂ phenyl.

Examples of an -(CH ₂ ) _n -heteroaryl or an -(CH ₂ ) _m -heteroaryl group include -CH ₂ -(2-pyridyl) and -CH ₂ CH ₂ -(2-pyridyl).

Preferably, R ¹ is -Ci _-4 alkyl, such as methyl or ethyl, particularly methyl.

Preferably, R ² is Ci _-6 alkyl (e.g. tert-butyl), -(CH ₂ ) _m -aryl (e.g.-(CH ₂ ) ₂ - phenyl) or -(CH ₂ ) _m -heteroaryl (e.g. -CH ₂ -indol-3-yl). More preferably, R ² is Ci _-6 alkyl (e.g. tert-butyl) or -(CH ₂ ) _m -aryl (e.g. -(CH ₂ ) ₂ -phenyl).

Preferably, m is 1 or 2. More preferably, m is 2.

Preferably, n is 1.

Preferably, R ³ is hydrogen or Ci _-4 alkyl (e.g. methyl). More preferably, R ³ is hydrogen.

Preferably, R ⁴ is Ci _-4 alkyl (e.g. methyl) or heteroaryl (e.g. pyrid-2-yl). More preferably, R ⁴ is Ci _-4 alkyl (e.g. methyl).

Preferably, R ⁵ is hydrogen, hydroxy or methoxy. More preferably, R ⁵ is hydrogen. In specific embodiments, the compound of formula (I) is:

(^-^-[(SJ-S.S-dimethyl-i-^ethylaminoJ-i-oxobutan^-ylJ-λ^-hy droxy^- isobutyl-λ/ ¹ -methylsuccinamide

(1)

(S)-/V*-hydroxy-2-isobutyl-λ/ ¹ -methyl-λ/ ¹ -[(S)-1-(methylamino)-1-oxo-4- phenylbutan-2-yl]succinamide;

(2) or

(2ff,3S)-λ/ ¹ -[(S)-3,3-climethyl-1-(methylamino)-1-oxobutan-2-yl]-/V*,3-d ihydroxy- 2-isobutyl-λ/ ¹ -methylsuccinamide

(3) or a pharmaceutically acceptable salt or solvate of any one thereof.

The term "pharmaceutically acceptable salt" refers to a salt, for example an acid addition salt or, in certain cases salts of an organic and inorganic base such as carboxylate, sulfonate and phosphate salt. All such salts are within the scope of this invention, and references to compounds of the formula (I) include the salt forms of the compounds. Examples of pharmaceutically acceptable

salts are provided in Berge et a/, 1977, "Pharmaceutically Acceptable Salts," J. Pharm. ScL, Vol. 66, pp. 1-19.

The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.

Examples of acid addition salts include salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+)-camphoric, camphorsulfonic, (+)-(1 S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2- disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalenesulfonic (e.g.naphthalene-2-sulfonic), naphthalene-1 ,5-disulfonic, 1 -hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, toluenesulfonic (e.g. p-toluenesulfonic), undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

The compounds of the invention may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed. If the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO ^" ), then a salt may be formed with a suitable cation. Non-limiting examples of suitable inorganic cations include alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ . Non-limiting examples of suitable organic

cations include ammonium ion (i.e., NH ₄ ⁺ ) and substituted ammonium ions (e.g., NH ₃ R ⁺ , NH ₂ R ₂ ⁺ , NHR ₃ ⁺ , NR ₄ ⁺ ).

Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH ₃ J ₄ ⁺ .

Where the compounds of the formula (I) contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of formula (I).

It will be appreciated that compounds of formula (I) may exist in a number of different geometric isomeric, and tautomeric forms and references to compounds of the formula (I) include all such forms.

It will also be appreciated that where compounds of formula (I) contain one or more chiral centres, they may exist in the form of two or more optical isomers, references to compounds of the formula (I) include all optical isomeric forms thereof (e.g. enantiomers, epimers and diastereoisomers), either as individual optical isomers, or mixtures (e.g. racemic mixtures) or two or more optical isomers.

Examples of stereoisomers of compounds of formula (I) are demonstrated by compounds of formulae (l)a and (l)b:

(l)b

When R ⁵ represents an -OR ⁶ group, the compound of formula (I) may have stereochemistry as demonstrated by compounds of formula (l)c and (l)d:

(l)d

In a preferred embodiment, the compound of formula (I) is a compound of formula (l)a. The optical isomers may be characterised and identified by their optical activity (i.e. as + and - isomers, or c/and / isomers) or they may be characterised in terms of their absolute stereochemistry using the "R and S" nomenclature developed by Cahn, lngold and Prelog, see Advanced Organic Chemistry by Jerry March, 4 ^th Edition, John Wiley & Sons, New York, 1992, pages 109-114, and see also Cahn, lngold & Prelog, Angew. Chem. Int. Ed. Engl., 1966, 5, 385- 415.

It will be appreciated that optical isomers may be separated by a number of techniques which are well known to the person skilled in the art, for example, chiral chromatography (chromatography on a chiral support). Compounds of formula (I) are claimed as solids in either amorphous or crystalline form, including all polymorphic forms. Crystalline forms may be prepared by recrystallisation of the compounds from appropriate solvents. Amorphous forms may be prepared e.g. by spray drying a solution of the compounds. Examples of polymorphic forms include solvates (e.g. hydrates), complexes (e.g. inclusion complexes or clathrates with compounds such as

cyclodextrins, or complexes with metals of the compounds), and pro-drugs of the compounds.

By the term 'an inflammatory condition which is characterised by colonisation with Staphylococcus aureus ¹ is meant a condition such as atopic dermatitis where the skin is colonised by Staphylococcus aureus in the majority of cases and where an increase in colonisation or cutaneous infection results in aggravation of the underlying condition and an increase in the inflammatory response.

A further inflammatory condition is Netherton's syndrome, a severe autosomal recessive skin disorder characterised by ichthyosiform erythroderma, atopy (atopic dermatitis and very high IgE levels) and trichorrhexis invaginata. Most patients experience recurrent or persistent bacterial infections.

By the term 'atopic dermatitis' or 'AD' is meant a chronic relapsing inflammatory skin disease characterised by intense pruritis and cutaneous hyperreactivity associated with elevated serum levels of IgE and eosinophils.

In a preferred embodiment, the method or use of the present invention is for the treatment or prevention of atopic dermatitis.

Certain compounds of formula (I) are aureolysin inhibitors, for example, the compounds may be a metalloprotease inhibitor which is capable of inhibiting the proteolytic activity of aureolysin. Preferably, the compounds of formula (I) inhibit allelic type Il and will more preferably inhibit both allelic forms, type I and type Il aureolysin, type Il being prevalent in skin diseases (Sabat A, Infect.

Immun. 68, 973-6, 2000). This inhibitor may or may not also directly inhibit endogenous metzincin metalloproteases; for example, MMPs (M10) (e.g. MMPs 1 , 2, 8, 9) and/or adamalysins (M12). Aureolysin allelic types I and Il are hereinafter referred to as "aureolysin I" and "aureolysin M" respectively.

The ability of a given substance to inhibit aureolysin can be determined using the "Aureolysin enzyme inhibition assay" given in the Examples below.

By "inhibition of aureolysin" or "aureolysin inhibitor" we mean gives an IC ₅₀ value of less than 50 micromolar (μM) in the aureolysin enzyme assay (e.g. the aureolysin Il enzyme assay), preferably less than 5 micromolar especially less than 0.5 micromolar.

By "inhibition of endogenous metzincin metalloproteases" or "endogenous metzincin metalloprotease inhibitor" we mean gives an IC ₅₀ value

of less than 50 micromolar in the corresponding endogenous metzincin metalloprotease assay, preferably less than 5 micromolar especially less than 0.5 micromolar.

Thus in one embodiment of the invention the aureolysin inhibitor does not significantly inhibit endogenous metzincin metalloproteases e.g. MMP-9. By "does not significantly inhibit" is meant that the strength of inhibition (e.g. as measured by IC ₅₀ ) of the inhibitor against endogenous metzincin metalloproteases (e.g. MMP-9) is at least 5 times weaker preferably at least 10 times e.g. at least 50 times weaker than the strength of inhibition of the inhibitor against aureolysin (e.g. aureloysin II). In another embodiment of the invention the aureolysin inhibitor does significantly inhibit endogenous metzincin metalloproteases (e.g. MMP-9). By "does significantly inhibit" is meant that the strength of inhibition (e.g. as measured by IC ₅₀ ) of the inhibitor against endogenous metzincin metalloproteases (e.g. MMP-9) is at least 0.5 times e.g. at least 1 times the strength of inhibition of the inhibitor against aureolysin (e.g. aureolysin II). The strength of inhibition (e.g. as measured by IC ₅₀ ) of the inhibitor against endogenous metzincin metalloproteases (e.g. MMP-9) may for example be at least 10 times, perhaps 100 times or even 1000 times the strength of inhibition of the inhibitor against aureolysin (e.g. aureloysin II). In another embodiment of the invention the strength of inhibition (e.g. as measured by IC ₅₀ ) of the inhibitor against aureolysin (e.g. aureolysin II) is between 2 and 5 times the strength of inhibition of the inhibitor against endogenous metzincin metalloproteases (e.g. MMP-9).

Known examples of endogenous MMPs are discussed in Hande et al. (2004) Clinical Cancer Research 10, 909-915. These include MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-14 (MT-MMP-1 , a membrane bound enzyme) MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-7 (matrilysin), MMP-11 (stromelysin-3), and MMP-13 (collagenase-3). Another example is MMP-8 (collagenase-2). Examples of adamalysins include ADAM10, ADAM17 and ADAM33. As pointed out above, a number of these enzymes have previously been linked to cancer and inflammation and ADAM33 is genetically linked to asthma.

Certain compounds of formula (I) may also indirectly inhibit other tissue- damaging proteases in the skin. Proteases are frequently expressed as inactive

zymogens that require proteolytic cleavage to become active. Indeed, aureolysin itself is believed to be responsible for initiating the activation of the extracellular proteases secreted by Staphylococcus aureus (Shaw et al, Microbiology, 150, 217-28, 2004). Aureolysin is therefore also likely to activate endogenous host proteases present in the skin and therefore to exacerbate diseases such as AD. It is known, for example, that three other members of the M4 family (Pseudomonas aeruginosa elastase, Vibrio cholera proteinase and thermolysin) can activate human MMPs (Okamoto et al, J. Biol. Chem. 272, 6059-66, 1997). A closely related enzyme to aureolysin, bacillolysin, is known to activate pro-urokinase which converts plasminogen to plasmin (Narasaki et al, J. Biol. Chem. 280, 14278-87, 2005). This reference also discloses that bacillolysin converts plasminogen to a mini-plasminogen-like molecule which is more susceptible to conversion to plasmin. Our data shows that aureolysin activates pro-urokinase and inhibition of aurolysin can prevent this activation. Activation of pro-urokinase leads to the activation of the plasminogen pathway, resulting in the production of pro-inflammatory plasmin. Aureolysin activates pro-MMP-1 , and inhibition of aurolysin can prevent this activation. Activation of MMP-1 leads to increased degradation of collagen in the skin, perturbing the normal skin barrier. Relevant enzyme activity assays are described in WO2007/025999.

A process for preparing compounds of formula (I) comprises: (a) reacting a compound of formula (II)

(II) wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined above for compounds of formula (I), with hydroxylamine; or (b) reacting a compound of formula (III)

wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined above for compounds of formula (I), with a compound of formula P ¹ ONH ₂ , wherein P ¹ represents a suitable protecting group (such as an O- benzyl group), followed by a deprotection step;

(C) preparing a compound of formula (I) wherein R ⁵ represents a hydroxy group which comprises reacting a compound of formula

(IV)

(IV) wherein R ¹ , R ² and R ³ and R ⁴ are as defined above for compounds of formula (I) with hydroxylamine;

(d) deprotecting a protected derivative of a compound of formula (I); or

(e) interconversion of a compound of formula (I).

Step (a) typically comprises the use of an excess of hydroxylamine, typically two molar equivalents, in a suitable solvent (such as methanol or d im ethy lacetam ide) .

Step (b) typically comprises the use of peptide coupling conditions, such as performing the reaction in the presence of HOBT, EDAC, and NMM in the presence of a suitable solvent or mixture of solvents (such as DMF/DCM).

Step (c) typically comprises the use of an excess of hydroxylamine, typically four molar equivalents of hydroxylamine in a suitable solvent (such as methanol). Typically the hydroxylamine free base may be generated in situ from

a salt such as hydroxylamine hydrochloride, by reaction with a base, such as sodium methoxide.

In step (d), examples of protecting groups and the means for their removal can be found in T.W. Greene "Protective Groups in Organic Synthesis" (J Wiley and Sons, 1991). For example, in step (d), when P ¹ represents an O- benzyl group, deprotection may typically comprise a hydrogenolysis reaction involving a suitable catalyst such as 10% palladium/carbon and hydrogen in the presence of a suitable solvent (such as ethanol).

Step (e) may be performed using conventional interconversion procedures such as epimerization, oxidation, reduction, alkylation, nucleophilic or electrophilic aromatic substitution, ester hydrolysis, amide bond formation or transition metal mediated coupling reactions.

Compounds of formula (M) wherein R ⁵ represents hydrogen may be prepared in accordance with the following Scheme 1 :

Scheme 1

wherein R ¹ , R ² , R ³ and R ⁴ are as defined above for compounds of formula (I).

Step (i) typically comprises a standard peptide coupling reaction which may be performed in an analogous manner to that described hereinbefore for process (b). Peptide coupling conditions suitable for the preparation of compounds of formula (ll) ^a in step (i) may typically comprise the use of HOBT, DCC and ethyl acetate.

Compounds of formula (Vl) may be prepared in accordance with the following Scheme 2:

Scheme 2

(VII) (VIII)

R ¹ -L ² Step (ii)

(Vl) (IX) wherein Ar represents either 2- or 4-nitrophenyl and, L ¹ represents a suitable leaving group such as a halogen atom (e.g. chlorine), L ² represents a suitable leaving group such as a hydroxy group or a halogen atom (e.g. bromine) and R ¹ , R ² , R ³ and R ⁴ are as defined for compounds of formula (I).

Step (i) typically comprises reaction of a compound of formula (VII) with a compound of formula Ar-SO ₂ -CI in the presence of a suitable base (such as triethylamine) and in the presence of a suitable solvent (such as DCM).

When L ² represents bromine, step (ii) typically comprises the use of caesium carbonate in the presence of a suitable solvent (such as DMF) at a suitable temperature (such as room temperature).

When L ² represents a hydroxy group, step (ii) typically comprises the use of triphenylphosphine and di-tert-butyl azodicarboxylate in the presence of a suitable solvent (such as DCM) at a suitable temperature (such as room temperature).

Step (iii) typically comprises a nucleophilic desulfonylation reaction using a thiolate reagent, for example, phenyl mercaptan and potassium carbonate in the presence of a suitable solvent (such as DMF) at a suitable temperature (such as room temperature). Alternatively, step (iii) typically comprises the use of 2-mercaptoethanol HOCH ₂ CH ₂ SH and, DBU in a suitable solvent such as acetonitrile at a suitable temperature (such as room temperature).

Compounds of formula (Vl) wherein R represents C _2-4 alkyl, may also be prepared in accordance with the following Scheme 3:

Scheme 3

(VII) (X) (Vl)a

wherein R ^1a represents Ci _-3 alkyl, C _2-3 alkenyl or C _2-3 alkynyl and R ² , R ³ and R ⁴ are as defined above for compounds of formula (I).

Step (i) typically comprises reaction of a compound of formula (VII) with an aldehyde under dehydrating conditions. Step (ii) typically comprises a catalytic hydrogenation reaction in the presence of a suitable catalyst such palladium on carbon. Alternatively, step (ii) may comprise a chemical reduction reaction with a suitable reducing agent, such as sodium cyanoborohydride or sodium triacetoxyborohydride in the presence of a suitable solvent, such as THF and, if necessary, in the presence of a proton source such as AcOH.

Compounds of formula (III) may be prepared by reacting a compound of formula (II) with a suitable acid (such as 10% sulfuric acid).

Compounds of formula (IV) may be prepared in accordance with the following Scheme 4:

(XV) (IV)

wherein R ¹ , R ² , R ³ and R ⁴ are as defined above. Step (i) typically comprises reacting a compound of formula (Xl) with a compound of formula (XII) in the presence of a catalytic amount of an acid such as para-toluenesulfonic acid and a suitable solvent (such as DMF).

Step (ii) typically comprises reacting a compound of formula (XIII) with a compound of formula (XIV) in the presence of a coupling agent such as EDC in a suitable solvent (such as DCM).

Step (iii) typically comprises reacting a compound of formula (XV) with a compound of formula (Vl) in the presence of a suitable solvent (such as DMF).

Compounds of formula (V), (VII), (Xl), (XII), (XIV) and are either commercially available or may be prepared in accordance with known methodology.

It is preferred that a compound of formula (I) is formulated for topical administration and it may be administered to a patient in an amount such that from 0.00001 to 10 g, preferably from 0.0001 to 1 g active ingredient is delivered per m ² of the area being treated. Typically, the total amount of inhibitor is from 0.001 to 12 wt%, e.g. from

0.0018 to 11.6 wt%, suitably from 0.0088 to 1.4 wt%, e.g. 0.01-1.0 wt%, more

suitably from 0.05 to 0.2 wt%, for example about 0.1 wt%, based on the total weight of the formulation.

The topical formulation may, for example, take the form of a gel, ointment, cream or lotion. Other example presentations include impregnated dressings, pastes, dusting powders, sprays, oils, transdermal devices etc.

The topical formulation will preferably maximise surface exposure and minimise systemic exposure to the active ingredient(s).

When the formulation is a gel, it typically comprises a hydrophilic polymer such as cross-linked polyethylene glycol, cross-linked starch or polyvinyl pyrrolidone. An ointment, cream or lotion typically contains an aqueous phase and an oleaginous phase in admixture; such formulations may generally be characterised as oil-in-water emulsions or water-in-oil emulsions. Alternatively, the formulation may be entirely oleaginous in the form of an oil, ointment, gel or spray. The formulation may additionally contain one or more emollients, emulsifiers, thickeners and/or preservatives, particularly when it is a cream or ointment.

Emollients suitable for inclusion in creams or ointments are typically long chain alcohols, for example a C ₈ -22 alcohol such as cetyl alcohol, stearyl alcohol and cetearyl alcohol, hydrocarbons such as petrolatum and light mineral oil, or acetylated lanolin. The total amount of emollient in the formulation is preferably about 5 wt% to about 30 wt%, and more preferably about 5 wt% to about 10 wt% based on the total weight of the formulation.

The emulsifier is typically a nonionic surface active agent, e.g., polysorbate 60 (available from ICI Americas), sorbitan monostearate, polyglyceryl-4 oleate and polyoxyethylene(4)lauryl ether. Generally the total amount of emulsifier is about 2 wt% to about 14 wt%, and more preferably about

2 wt% to about 6 wt% by weight based on the total weight of the formulation.

Pharmaceutically acceptable thickeners, such as Veegum (available from R. T. Vanderbilt Company, Inc.), and long chain alcohols (i.e. Ca-22 alcohols such as cetyl alcohol, stearyl alcohol and cetearyl alcohol) can be used. The total amount of thickener present is preferably about 3 wt% to about 12 wt% based on the total weight of the formulation.

Preservatives such as methylparaben, propylparaben and benzyl alcohol can be present in the formulation. Other example preservatives are phenoxyethanol and chlorocresol. The appropriate amount of such preservative(s) is known to those skilled in the art. Optionally, an additional solubilizing agent such as benzyl alcohol, lactic acid, acetic acid, stearic acid or hydrochloric acid can be included in the formulation. If an additional solubilizing agent is used, the amount present is preferably about 1 wt% to about 12 wt% based on the total weight of the formulation. Optionally, the formulation can contain a humectant such as glycerin and a skin penetration enhancer such as butyl stearate, urea and DMSO.

It is known to those skilled in the art that a single ingredient can perform more than one function in a cream, i.e., cetyl alcohol can serve both as an emollient and as a thickener. Suitably, said formulation or medicament is a cream. The cream typically consists of an oil phase and a water phase mixed together to form an emulsion. Preferably, the cream comprises an oil-in-water emulsion. Preferably, the amount of water present in a cream of the invention is about 45 wt% to about 85 wt% based on the total weight of the cream. Where the formulation or medicament is an ointment, it typically comprises a pharmaceutically acceptable ointment base such as petrolatum, or polyethylene glycol 400 (available from Union Carbide) in combination with polyethylene glycol 3350 (available from Union Carbide). The amount of ointment base present in an ointment of the invention is preferably about 60 wt% to about 95 wt% based on the total weight of the ointment.

One exemplary formulation is a cream which comprises an emulsifying ointment (e.g. around 30 wt%) comprising white soft paraffin, emulsifying wax and liquid paraffin made to 100% with purified water and containing preservative (e.g. phenoxyethanol). This formulation may also be buffered to the required pH (e.g. with citric acid and sodium phosphate). The concentration of active may typically be between 0.01 and 1.0 wt%.

In one embodiment, the formulation is a cream which comprises an oil-in- water cream base comprising isostearic acid, cetyl alcohol, stearyl alcohol, white petrolatum, polysorbate 60, sorbiton monostearate, glycerin, xanthum gum,

purified water, benzyl alcohol, methylparaban and propyl-paraban. Such a cream may be in the form of Aldara imiquimod cream which contains 5% imiquimod.

Compounds of formula (I) may be administered in conjunction with further medicaments, such as conventional therapies for the treatment or prevention of inflammatory skin conditions, for example antibiotics, steroids (such as hydrocortisone, clobetasone butyrate, betamethasone valerate, hydrocortisone butyrate, clobetasol propionate, fluticasone propionate, mometasone furoate and dexamethasone), non-steroidal anti-inflammatory drugs, macrolide immunosuppressants (such as cyclosporine A, tacrolimus and pimecrolimus), leukotriene antagonists and phosphodiesterase inhibitors.

These further treatments may be administered by any convenient route. Topical and oral routes are preferred.

Active agents may, where appropriate, be administered in the form of pharmaceutically acceptable salts, or solvates e.g. hydrates.

Combination treatments may be administered simultaneously, sequentially or separately, by the same or by different routes. For example, the further medicament may be administered orally. In another example, the further medicament may be administered topically, e.g. in a combined preparation with the com pound of form ula ( I ) .

For example, the further medicament may be an antibiotic substance which is bacteriocidal for Staphylococcus aureus and which is administered orally or topically.

The method and use described above may be preceded by a method step involving confirming the presence of Staphylococcus aureus. The presence of Staphylococcus aureus may be determined directly by sampling the skin of patients and determining the presence of Staphylococcus aureus through microbiological or genetic methods. In the simplest form of assay, the affected skin is swabbed and the swab is inoculated onto blood agar plates and colonies of Staphylococcus aureus identified through standard microbiological procedures. A quantitative methodology may also be applied to assess the level of colonisation. Genetic methods such as quantitative PCR may also be used to demonstrate the presence of Staphylococcus aureus. The presence of Staphylococcus aureus may also be determined indirectly by determining the

presence of metalloprotease activity, e.g. in skin washings of patients. The presence of metalloproteases and metalloprotease activity may be detected in skin washings from patients by gelatin zymography or enzyme assay. Such assays are described in WO2007/025999. The following Example illustrates the preparation of a compound of the invention. Example 1

Step 1 : Terf-butyl (5)-1-(methoxycarbonvD-2,2-dimethylpropylmethyl carbamate

Boc

_^ N. .CO ₂ Me Me^ ^^

Boc-L-tert-leucine (3.24 g, 14.03 mmol) was dissolved in anhydrous dimethylformamide (100 ml) and stirred under nitrogen with ice-cooling. Sodium hydride (60% dispersion in mineral oil; 2.74 g, 68.5 mmol) was then added against a flow of nitrogen. Once effervescence had ceased, still at O ⁰ C, iodomethane (8.75 ml, 0.14 mol) was added and stirring was continued, allowing to slowly warm to ambient temperature with continued stirring overnight at 2O ⁰ C. (After 16 hours, an aliquot (0.2 ml) was quenched with acetic acid (1 drop), diluted with ether (4 ml) and water-washed (3 x 4 ml). Solvent was removed under vacuum and the residue was analysed. ¹ H NMR (CDCI ₃ ) was consistent with the desired product). Stirring was again continued at room temperature overnight. The suspension was cooled in an ice-bath and quenched by acidification with acetic acid (10 ml). The mixture was equilibrated between water (200 ml) and diethyl ether (200 ml). The aqueous phase was further extracted with ether (3 x 200 ml). The organics were all combined and washed with saturated aqueous sodium bicarbonate solution (200 ml), then brine (200 ml), then dried (MgSO ₄ ) and then rotary evaporated to afford the title compound as a pale yellow oil (6.56 g). ¹ H NMR (CDCI ₃ ) showed the desired product, together with residual dimethylformamide, acetic acid and mineral oil. The material was taken into the next step without further purification.

Step 2: Terf-butyl (S)-1-carboxy-2,2-dimethylpropylmethylcarbamate

Boc

.N- .CO ₂ H Me^ >T

The starting intermediate ester (6.56 g, contains 14mmol maximum) was dissolved in a mixture of tetrahydrofuran (20 ml), methanol (20 ml) and water (10ml). Lithium hydroxide monohydrate (1.76g, 4 mmol) was then added and the mixture stirred under nitrogen whilst maintaining the temperature at 25°C. After stirring overnight, an aliquot (0.1 ml) was removed and acidified with acetic acid (3 drops), then evaporated to dryness, ¹ H NMR analysis showed over 50% conversion, but still a significant amount of SM. Therefore more Lithium hydroxide monohydrate (0.88 g, 21 mmol) and water (5 ml) were added and reaction was continued at 25°C for a further 24h. The reaction was reduced in vacuo and the resulting residual aqueous solution was diluted with water (150 ml) then washed with diethyl ether (2 x 150 ml). The aqueous phase was then acidified by addition of acetic acid (5 ml) and extracted with ethyl acetate (2 x 150ml). These organic extracts were combined, dried (MgSO ₄ ), rotary- evaporated and pumped to give the title compound as a pale oil, 4.08 g. The ¹ H NMR spectrum was consistent with the desired product containing some residual ethyl acetate and acetic acid (about 20% by weight). Further pumping reduced the weight of product to 3.76 g Step 3: Terf-butyl (5)-1-(methylcarbamoyl)-2,2-dimethylpropylmethyl carbamate

,CONHMe

The starting acid (3.76 g, contains 14.0 mmol maximum) was dissolved in anhydrous tetrahydrofuran (140 ml) and then cooled to O ⁰ C in an ice bath, whilst

stirring under nitrogen. Hydroxybenzotriazole hydrate (4.28 g, 28 mmol) was then added in a single portion and stirring at ice-bath temperature was continued for a further 5 minutes. A solution of methylamine in tetrahydrofuran (2M; 28 ml, 56 mmol) was then added, followed by further stirring at 0 ⁰ C for 10 minutes. 1- Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI. HCI) (5.37 g, 28 mmol) was then added and stirring of the reaction suspension was continued at O ⁰ C for 15 minutes. The cooling bath was removed and stirring under nitrogen was continued at ambient temperature. After stirring for 3 days (weekend), an aliquot (0.2 ml) was reduced in vacuo. ¹ H NMR analysis of the residue indicated that the reaction had gone to completion. The reaction was diluted with diethyl ether (200 ml) and washed sequentially with water (200 ml), 0.1 N aqueous sodium hydroxide solution (200 ml) and again with water (200 ml). The organic phase was dried (MgSO ₄ ), rotary-evaporated and pumped to afford the title compound as a white crystalline solid (3.15 g, 87% over 3 steps).

Step 4: (S)-λ/.3,3-Trimethyl-2-(methylamino)butanamide

H ^N^ .CONHMe

Me ::

The starting Boc-protected compound (3.15g, 12.2 mmol) was suspended in a solution of hydrogen chloride in diethyl ether (1 N, 50 ml, 50 mmol), then stirred at ambient temperature under nitrogen, overnight. A sample of the gummy precipitate was removed and washed with ether (10 ml), air-dried and analysed by ¹ H NMR which revealed an incomplete reaction. The reaction mixture was reduced in vacuo to afford a residue. This residue was dissolved in a solution of hydrogen chloride in propan-2-ol (5N, 15 ml) and stirred under a nitrogen atmosphere overnight. ¹ H NMR analysis of an aliquot of sample, prepared as previously reported, showed complete reaction.

The reaction solution was reduced in vacuo, and the resulting residual oil was triturated with diethyl ether (50 ml), causing the oil to separate. The supernatant ether solution was decanted off, leaving a whitish/yellow oil. This

was then azeotroped with toluene (50 ml) and pumped to give the desired hydrochloride salt as a viscous yellow oil, still containing some residual solvent (by ¹ H NMR analysis).

Preparation of the free base was achieved by dissolving this oil in water (30 ml), basifying with 1N aquous sodium hydroxide solution (20 ml) and then extracting with dichloromethane (5 x 30 ml). The combined organics were dried (MgSO ₄ ) and reduced in vacuo to afford the free base compound as a pale yellow oil which solidified to an off-white solid, 1.73 g (90%). ¹ H NMR was consistent with the pure, desired free base. Step 5: (S)-2-((Methoxycarbonyl)methyl)-4-methylpentanoic acid

Me CO ₂ H

The benzylamine salt of the title acid (3.54 g, 12 mmol) was stirred with saturated aqueous sodium bicarbonate solution (100 ml), then extracted with ethyl acetate (3 x 60 ml). The combined organics were then back-extracted with saturated aqueous sodium bicarbonate solution (90 ml). All of the aqueous sodium bicarbonate solutions were then combined, cooled in an ice-bath and acidified to pH 3 with 6N aqueous hydrochloric acid. The final mixture was extracted with ethyl acetate (3 x 60 ml) and the combined extracts were washed with brine (100 ml), then dried (MgSO ₄ ) and finally reduced in vacuo and pumped to afford the free acid as a pale oil (2.28 g, quantitative yield), confirmed by ¹ H NMR spectroscopy.

Step 6: (SV Methyl 3-(λH(S)-1-(methylcarbamovn-2.2-dimethylpropyl)-M- methylcarbamoyl)-5-methylhexanoate

The starting free acid from step 5 (0.94 g, 5.0 mmol) was dissolved in anhydrous dichloromethane (50 ml) and stirred at room temperature under nitrogen, as oxalyl chloride (2.64 ml, 30 mmol) was added. The resulting solution was stirred at room temperature with a slow evolution of gas for 2.5 h. TLC analysis (drop of reaction solution added to ethanol), eluting with diethyl ether, showed virtually complete reaction. The solution was rotary-evaporated and briefly pumped then further anhydrous dichloromethane (50 ml) was added and again the solution was rotary-evaporated and briefly pumped, to remove any residual oxalyl chloride, giving the intermediate acid chloride as a pale oil.

To this acid chloride, under nitrogen and at room temperature, was then added a solution of the amine free base prepared in step 4 (0.79 g, 5.0 mmol) in anhydrous tetrahydrofuran (30 ml). The resulting solution was stirred for 10 minutes before addition of diisopropylethylamine (1.9 ml, 10.9 mmol). The solution was then stirred at room temperature, overnight, under nitrogen atmosphere. The resulting reaction suspension was diluted with diethyl ether (100 ml) and washed sequentially with 1N aqueous hydrochloric acid (100 ml), 1N aqueous sodium hydroxide solution (100 ml) and saturated aqueous sodium chloride solution (100 ml). The organic phase was dried (MgSO ₄ ), rotary- evaporated and briefly pumped to afford an orange solid. The solid was triturated with 2:1 petrol; diethyl ether (5 ml), filtered and washed with more 2:1 petrol; diethyl ether (5 ml), giving a white solid which was dried in vacuo to afford the desired product, 0.66 g. ¹ H NMR (CDCI ₃ ) was consistent with the structure and showed only very minor impurity peaks.

The petrol/diethyl ether filtrate was reduced in vacuo to give a pale brown solid, 0.64 g. ¹ H NMR (CDCI ₃ ) showed a mixture containing more of the desired product as the major component.

Step 7: (aSϊ-λ/^SVHmethylcarbamoyO^^-dimethylpropyO-λλhydroxy^- isobutyl-/V ¹ -methylsuccinamide

Potassium hydroxide (907 mg, 16.2 mmol) was dissolved in water (4 ml) and this was added to a solution of hydroxylamine hydrochloride (577 mg, 8.3 mmol) in water (2 ml). This solution was allowed to stand at room temperature for 10 minutes then chilled in an ice-bath. This solution was then added to a rapidly stirring solution of the ester from step 6 (0.66 g, 2.01 mmol) in tetrahydrofuran (2 ml). After 1h, TLC (diethyl ether) and LCMS analyses showed complete reaction of the starting material and formation of the desired product. After 1.75h, still at ice-bath temperature, 1N aqueous hydrochloric acid was added to adjust the pH to ~7 and the solution was kept at ~5°C overnight. The resulting white crystalline precipitate was collected by filtration and washed with ice-cold water (4 ml) then dried in vacuo at -5O ⁰ C to constant weight, 504 mg. Analyses were consistent with the pure, desired product:

LCMS: A single peak was observed (at 3.34 minutes) showing 330 (M+1) in the ES+ve spectrum and 328 (M-1) in the ES-ve spectrum.

¹ H NMR (de-DMSO, 85 ⁰ C): δ 0.8-0.9 (m, 6H), 1.02 (s, 9H), 1.2 (m, 1H), 1.4-1.6 (m, 2H), 2.1 (m, 1H), 2.3 (m, 1 H), 2.6 (d, J=4Hz, 3H), (3.06 (s, 3H - partly obscured by solvent water peak), 3.05 (m, 1 H), 4.94 (s, 1 H), 7.3 (br s, 1 H), 8.5 (br s, 1H), 10.3 (br s, 1H).

The activity of the product of Example 1 was tested in assays described in WO2007/025999. Its IC50 value wrt Aureolysin Il (6.4 μM) was similar to that for the desmethyl analogue described there, while its MMP-9 activity was essentially eliminated (IC50 = 4% at 0.1 μM).