The present invention relates to depsipeptides which act as inhibitors of histone deacetylase (HDAC) and therefore have therapeutic utility. Background of the Invention

HDACs are zinc metalloenzymes that catalyse the hydrolysis of acetylated lysine residues. In histones, this returns lysines to their protonated state and is a global mechanism of eukaryotic transcriptional control, resulting in tight packaging of DNA in the nucleosome. Additionally, reversible lysine acetylation is an important regulatory process for non-histone proteins. Thus, compounds that are able to modulate HDAC have important therapeutic potential.

The natural products FK228 (Structure I) and Spiruchostatin A (Structure II) are depsipeptides that have been reported to have potential as HDAC inhibitors. The term depsipeptide describes a class of oligopeptides or polypeptides that have both ester and peptide links in the chain.

FK228 is a cyclic depsipeptide containing 4 monomer units together with a cross-ring bridge. This compound, under the trade name of Romidepsin (RTM), has been tested as a therapeutic in human trials and shown that it has valuable effects on a number of diseases.

Spiruchostatin A is a cyclic depsipeptide that is structurally related to FK228: it is a cyclic depsipeptide containing a tri-peptide, a statine unit and a cross-ring bridge.

Structure I Structure Il

However, because both FK228 and Spiruchostatin A are natural products, they are not amenable to optimization for use as a therapeutic agent.

Analogues of Spiruchostatin A are disclosed in WO 2008/062232. They may have improved HDAC inhibitory properties with respect to Spiruchostatin A or FK228 or other drug-like properties that make them more useful as medicines. These compounds have the general structures shown in Structures III&IV wherein R ₁ , R ₅ , R ₇ and R ₉ are the same or different and represent hydrogen or an amino acid side chain moiety (from either a natural or an unnatural amino acid), each R _1O is the same or different and represents hydrogen or CrC ₆ alkyl, C ₂ -C ₆ alkenyl or C ₂ -C ₆ alkynyl, Pr ¹ and Pr ² are the same or different and represent hydrogen or a thiol protecting group and Pr ³ is hydrogen or an alcohol protecting group.

Analogues of Spiruchostatin A of structures III and IV in which either R ₁ and/or R ₅ is replaced by a carbon-linked amide, sulphonamide, aryl or heteroaryl group are disclosed in WO 2009/141657.

Analogues of Spiruchostatin A in which either position 6 on the depsipeptide macrocycle (IUPAC nomenclature) and/or position 12 (IUPAC nomenclature) is bis-substituted, containing two amino acid side chain moieties (neither of which is hydrogen) or a spirocyclic moiety, are disclosed in WO 2009/141658.

Analogues of FK228 are disclosed in WO2006/129105. They may have improved HDAC inhibitory properties with respect to FK228 or other drug-like properties that make them more useful as medicines. These compounds have the general structures shown in Structures V & Vl wherein R ₁ , R5, R ₇ and Rg are the same or different and represent hydrogen or an amino acid side chain moiety (from either a natural or an unnatural amino acid), each R ₁₀ is the same or different and represents hydrogen or C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl or C ₂ -C ₆ alkynyl, and Pr ₁ and Pr ₂ are the same or different and represent hydrogen or a thiol protecting group.

Structure V Structure Vl

Analogues of FK228 of structures V and Vl in which either R ₁ and/or R ₅ is replaced by a carbon-linked amide, sulphonamide, aryl or heteroaryl group are disclosed in WO 2009/141657.

Analogues of FK228 in which either position 6 on the depsipeptide macrocycle (IUPAC nomenclature) and/or position 12 (IUPAC nomenclature) is bis-substituted, containing two amino acid side chain moieties (neither of which is hydrogen) or a spirocyclic moiety, are disclosed in WO 2009/141658.

Analogues of FK228 and Spiruchostatin A with modifications in the disulphide containing bridge are disclosed in WO 2008/062201.

Without being constrained by theory, it is believed that Structures VII and VIII are formed inside the cell from Structures I & Il respectively, by reduction of the disulphide bond, and that the 4-thio-butyl-1-ene so formed is a critical part of the mechanism of action of the compound, forming a metallophile capable of binding Zinc in the active site of HDAC.

Structure VII Structure VIII

This concept is supported by the observation that FR-901375, a cyclic depsipeptide HDAC inhibitor with quite a different ring structure, has the same disulphide-containing bridge across the ring as is seen in FK228 and Spiruchostatin A. Summary of the Invention

The present invention provides derivatives of Structures Il & VIII, and of structures related to them in ways that will be apparent to those skilled in the art, in which the -CH(CH ₃ ) ₂ (at position 12 on the depsipeptide macrocycle - IUPAC nomenclature) and/or the -CH ₃ (at position 6 on the depsipeptide macrocycle - IUPAC nomenclature) is replaced by a carbon-linked sulphone or sulphoxide group.

The present invention also provides derivatives of structures I & VII in which the =CH-CH ₃ (at position 6 on the depsipeptide macrocycle - IUPAC nomenclature) and/or the -CH(CH ₃ ) ₂ (at position 12 on the depsipeptide macrocycle - IUPAC nomenclature) is replaced by a carbon-linked sulphone or sulphoxide group.

These compounds, surprisingly, are found to be effective inhibitors of HDAC enzymes, and have properties which indicate that they have good potential as treatments for human disease.

The compounds of the invention are defined by Structures IX and X:

Structure IX or a pharmaceutically acceptable salt thereof wherein:

X is -C( ^I =O)N(R ₁₀ )- or -CH(OPr ₃ ) -;

R ₇ and Rg are independently selected from hydrogen, an amino acid side chain moiety from a natural amino acid, or -(LQ) _m Y; each L is independently a direct bond, C ₁ -Ci ₀ alkylene, C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, arylene or C ₃ -C _1O cycloalkylene; each Q is independently a direct bond, heteroarylene, -0-, -NR ³ -, -C(O)-, -C(O)NR ₃ -, -SO ₂ -, -SO ₂ -NR ³ -, -NR ³ -C(O)-NR ³ -, -NR ³ -SO ₂ -NR ³ -, halogen, -C(halogen)a(R ³ ₍₂ . _a) )-, -NR ¹¹ R ¹² - or -C(O)NR ¹¹ R ¹² , wherein R ¹¹ and R ¹² together with the nitrogen to which they are attached form a 5 to 7-membered heterocycle linker; m is from 0 to 5;

Y is H, CrC ₁₀ alkyl _, C _2- C ₁₀ alkenyl, C _2- C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, aryl, C ₃ -C ₁₀ cycloalkyl, heteroaryl, -OR ³ , -N(R ³ ) ₂ , -C(O)R ³ , -C(O)OR ₃ , -C(O)N(R ³ ) ₂ , -SO ₂ -R ³ , -SO ₂ -N(R ³ ) ₂ , -N-C(O)-N(R ³ ) ₂ , -N-SO ₂ -N(R ³ ) ₂ , halogen, -C(halogen) _b R ³ ₍₃ _ _b) or -NR ¹¹ R ¹² -C(O)NR ¹¹ R ¹² , wherein R ¹¹ and R ¹² together with the nitrogen to which they are attached form a 5- to 7-membered heterocycle; b is from 1 to 3; a is 1 or 2; each R ³ is independently H, C ₁ -C ₁₀ alkyl, aryl or heteroaryl; each R ₁₀ is independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₂ -Ce alkenyl or C ₂ -C ₆ alkynyl;

Pr ₁ and Pr ₂ are independently selected from hydrogen or a protecting group that forms a thioether, a monothio, dithio or aminothioacetal, a thioester or a carbamic acid thioester to protect a thiol group;

Pr ₃ is hydrogen or protecting group that forms an ether, an acetal or aminoacetal, an ester or a carbamic acid ester to protect a hydroxyl group; and either one of R ₁ and R ₅ is a carbon-linked sulphone or sulphoxide group and the other of R ₁ and R ₅ is hydrogen, an amino acid side chain moiety from a natural amino acid or (LQ) _m Y; or both of R ₁ and R ₅ are a carbon-linked sulphone or sulphoxide group.

The compounds defined above are useful as inhibitors of HDAC. Description of the Invention

Synthesis of compounds of Structures IX and X is typically conducted using amino acids of which -CO-CR-NH- forms part of the macrocycle and R is a side-chain moiety. R ₁ , R ₅ and Rg may be introduced in this way. R ₇ may be an amino acid side chain moiety but may not have been derived directly or indirectly from an amino acid as such. Each C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl or C ₂ -Ce alkynyl may be optionally substituted with, for example, an aryl or heteroaryl group. They may also be optionally substituted with each other, e.g. C ₁ -C ₆ alkylene substituted with C ₂ -C ₆ alkenyl.

CrC ₆ alkylene, C ₂ -C ₆ alkenylene and C ₂ -C ₆ alknylene are the divalent radicals and their scope is the divalent equivalent of the respective monovalent radicals.

A C ₁ -C ₆ alkyl group or moiety can be linear or branched. Typically, it is a C ₁ -C ₄ alkyl group or moiety, for example methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl and t-butyl. Preferred examples include methyl, i-propyl and t-butyl.

A C ₂ -C ₆ alkenyl group or moiety can be linear or branched. Typically, it is a C ₂ -C ₄ alkenyl group or moiety. It is preferred that the alkenyl radicals are mono or diunsaturated, more preferably monounsaturated. Examples include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl and 3-butenyl.

A C ₂ -C ₆ alkynyl group or moiety can be linear or branched. Typically, it is a C ₂ -C ₄ alkynyl group or moiety.

In a preferred embodiment, R ₇ and Rg (and the other of Ri and R ₅ if only one of R ₁ and R ₅ is a carbon linked sulphone or sulphoxide group) are independently selected from -(CH ₂ ) ₂ -C(O)-O-C(CH ₃ ) ₃ (terf-butoxy- carbonylmethylanaline), -(CH ₂ ) ₄ -NH-C(O)-O-C(CH ₃ ) ₃ (N _ε -(terf-butoxycarbonyl)- lysine), -(CH ₂ ) ₃ -NH-C(O)NH ₂ (citrulline), -CH ₂ -CH ₂ OH (homoserine) and -(CH ₂ ) ₂ -CH ₂ NH ₂ (ornithine). R ₇ and R ₉ (and the other of R ₁ and R ₅ if only one of R ₁ and R ₅ is a carbon linked sulphone or sulphoxide group) may also independently be hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, aryl, saturated and unsaturated heterocycles, which can be functionalized and unfunctionalized.

Preferably, R ₇ and R ₉ (and the other of R ₁ and R ₅ if one of R ₁ and R ₅ is a carbon-linked sulphone or sulphoxide group) are (LQ)mY. Preferably, each and the subsequent Q are not both a direct bond. For example, where m is 3, i.e. (LQ)-(LQ)-(LQ)-Y, preferably the first L and the first Q are not both a direct bond, and the second L and the second Q are not both a direct bond etc.

For the avoidance of doubt, when m is more than 1 , each L and Q can be the same or different. As used herein a "carbon-linked sulphone or sulphoxide group" is preferably represented by the formula: -(CRi ₃ Ri ₄ ) _x -S(O) _y Ri ₅ , wherein x is an integer from 1 to 10; y is 1 or 2; Ri ₃ and Ri ₄ are independently hydrogen, C _r C ₆ alkyl, aryl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, heteroaryl; and Ri ₅ is hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, aryl, heteroaryl. Each C _r C ₆ alkyl, C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl may be optionally substituted with an aryl or heteroaryl group.

A "carbon-linked sulphone or sulphoxide group" may also be represented by the group -L-S(O) _y R ₁₅ , where L, y and Ri ₅ are as defined above.

When Ri and/or R ₅ are sulphoxide groups, the bond will have significant dipolar character, with the negative charge residing on the oxygen. A lone pair of electrons therefore resides on the sulphur atom, giving it tetrahedral molecular geometry (as for sp ³ carbon). The sulphur is therefore a chiral centre. The sulphoxide may be in the R or S optical configuration. The compound of the invention may be substantially optically pure, or it may be racemic (i.e. both R and S enantiomers present). In the case where both Ri and R ₅ contain sulphoxide groups, this creates diastereoisomers. All possible diastereoisomers are included within the scope of the invention.

As used herein "aryl" means a monocyclic, bicylic or tricyclic monovalent or divalent (i.e. arylene), aromatic radical, such as phenyl, biphenyl, naphthyl, anthracenyl, which can be optionally substituted with up to five substituents, preferably selected from C _r C ₆ alkyl, hydroxy, d-C ₃ hydroxyalkyl, Ci-C ₃ alkoxy, CrC ₃ haloalkoxy, amino, Ci-C ₃ mono alkylamino, C _r C ₃ bis alkylamino, C ₁ -C ₃ acylamino, CrC ₃ aminoalkyl, mono (Ci-C ₃ alkyl) amino C ₁ -C ₃ alkyl , bis (C ₁ -C ₃ alkyl) amino C ₁ -C ₃ alkyl, CrC ₃ -acylamino, C ₁ -C ₃ alkyl sulphonylamino, halo, nitro, cyano, trifluoromethyl, carboxy, C ₁ -C ₃ alkoxycarbonyl, aminocarbonyl, mono C ₁ -C ₃ alkyl aminocarbonyl, bis C _r C ₃ alkyl aminocarbonyl, -SO ₃ H, C ₁ -C ₃ alkylsolphonyl, aminosulphonyl, mono C ₁ -C ₃ alkyl aminosulphonyl and bis C ₁ -C ₃ - alkyl aminosulphonyl. Preferably, the aryl ring contains from 5 to 7 carbon atoms.

As used herein "heteroaryl" means a monocyclic, bicylic or tricyclic monovalent or divalent (i.e. heteroarylene) aromatic radical containing up to four hetroatoms selected from oxygen, nitrogen and sulphur, such as thiazolyl, tetrazolylene, imidazolyl, oxazolyl, isoxazolyl, thienylene, pyrazolyl, pyridinyl, pyrazinylene, pyrimidinyl, indolyl, quinolyl, isoquinolyl, said radical being optionally substituted with up to three substituents preferably selected from C ₁ - C ₆ alkyl, hydroxy, C _r C ₃ hydroxyalkyl, CrC ₃ alkoxy, C ₁ -C ₃ haloalkoxy, amino, C ₁ - C ₃ mono alkylamino, C _r C ₃ bis alkylamino, C _r C ₃ acylamino, CrC ₃ aminoalkyl, mono (C ₁ -C ₃ alkyl) amino CrC ₃ alkyl , bis (CrC ₃ alkyl) amino CrC ₃ alkyl, CrC ₃ - acylamino, C ₁ -C ₃ alkyl sulphonylamino, halo, nitro, cyano, trifluoromethyl, carboxy, CrC ₃ alkoxycarbonyl, aminocarbonyl, mono CrC ₃ alkyl aminocarbonyl, bis CrC ₃ alkyl aminocarbonyl, -SO ₃ H, C ₁ -C ₃ alkylsolphonyl, aminosulphonyl, mono C ₁ -C ₃ alkyl aminosulphonyl and bis CrC ₃ -alkyl aminosulphonyl. Preferably, the total number of atoms in the ring system is from 5 to 7.

As used herein, "heterocycle" is a monocyclic, bicylic or tricyclic monovalent or divalent saturated or unsaturated radical containing up to four hetroatoms selected from oxygen, nitrogen and sulphur, said radical being optionally substituted with up to three substituents preferably selected from C ₁ - Ce alkyl, hydroxy, C ₁ -C ₃ hydroxyalkyl, C ₁ -C ₃ alkoxy, C ₁ -C ₃ haloalkoxy, amino, C ₁ - C ₃ mono alkylamino, C ₁ -C ₃ bis alkylamino, C ₁ -C ₃ acylamino, C ₁ -C ₃ aminoalkyl, mono (C ₁ -C ₃ alkyl) amino C ₁ -C ₃ alkyl , bis (C ₁ -C ₃ alkyl) amino C ₁ -C ₃ alkyl, C ₁ -C ₃ - acylamino, C ₁ -C ₃ alkyl sulphonylamino, halo, nitro, cyano, trifluoromethyl, carboxy, C ₁ -C ₃ alkoxycarbonyl, aminocarbonyl, mono C ₁ -C ₃ alkyl aminocarbonyl, bis C ₁ -C ₃ alkyl aminocarbonyl, -SO ₃ H, C ₁ -C ₃ alkylsolphonyl, aminosulphonyl, mono C ₁ -C ₃ alkyl aminosulphonyl and bis C _r C ₃ -alkyl aminosulphonyl. Preferably, the total number of atoms in the ring system is from 3 to 7.

In a preferred embodiment, the amino acid side chain moieties are those derived from natural amino acids. Examples of amino acid side chain moieties derived from natural amino acids, with the amino acids from which they are derived shown in brackets, are -H (Glycine), -CH ₃ (Alanine), -CH(CH ₃ ) ₂ (Valine), -CH ₂ CH(CH ₃ ) ₂ (Leucine), -CH(CH ₃ )CH ₂ CH ₃ (Isoleucine), -(CH ₂ ) ₄ NH ₂ (Lysine), -(CH ₂ ) ₃ NHC(=NH)NH ₂ (Arginine), -CH ₂ -(5-1 H-imidazolyl) (Histidine), -CH ₂ CONH ₂ (Asparagine), -CH ₂ CH ₂ CONH ₂ (Glutamine), -CH ₂ COOH (Aspartic acid), -CH ₂ CH ₂ COOH (Glutamic acid), -CH ₂ -phenyl (Phenylalanine), -CH ₂ -(4-OH-phenyl) (Tyrosine), -CH ₂ -(3-1H-indolyl) (Tryptophan), -CH ₂ SH (Cysteine), -CH ₂ CH ₂ SCH ₃ (Methioine), -CH ₂ OH (Serine), and -CH(OH)CH ₃ (Threonine).

In one embodiment, each amino acid side chain is an amino acid side chain moiety present in a natural amino acid or is -(CH ₂ ) ₂ -C(O)-O-C(CH ₃ ) ₃ (te/f-butoxy-carbonylmethylalanine), -(CH ₂ ) ₄ -NH-C(O)-O-C(CH ₃ ) ₃

(N _ε -(terfbutoxycarbonyl)-lysine), -(CH ₂ ) ₃ -NH-C(O)NH ₂ (citrulline), -CH ₂ -CH ₂ OH (homoserine) Or -(CH ₂ J ₂ -CH ₂ NH ₂ (ornithine).

Preferably, each L may be independently optionally substituted with R ³ .

The groups Pr ₁ and Pr ₂ represent hydrogen or a thiol-protecting group. Said thiol-protecting group is typically:

(a) a protecting group that forms a thioether to protect a thiol group, for example a benzyl group which is optionally substituted by CrC ₆ alkoxy (for example methoxy), CrCβ acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl, Ci-Cβ acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl);

(b) a protecting group that forms a monothio, dithio or aminothioacetal to protect a thiol group, for example CrC ₆ alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidomethyl, benzamidomethyl;

(c) a protecting group that forms a thioester to protect a thiol group, such as tertiary butoxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives; or

(d) a protecting group that forms a carbamic acid thioester to protect a thiol group, such as carbamoyl, phenylcarbamoyl, C _r C ₆ alkylcarbamoyl (for example methylcarbamoyl and ethylcarbamoyl).

Pri and Pr ₂ are the same or different and each represent hydrogen or a protecting group that forms a thioether, a monothio, dithio or aminothioacetal, a thioester or a carbamic acid thioester to protect a thiol group. Preferably, Pr ¹ and Pr ² are the same or different and each represent hydrogen or a protecting group selected from a benzyl group which is optionally substituted by CrC ₆ alkoxy (for example methoxy), Ci-Cβ acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl, d-Cβ acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl), CrC ₆ alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives, carbamoyl, phenylcarbamoyl and C ₁ -Ce alkylcarbamoyl (for example methylcarbamoyl and ethylcarbamoyl). Most preferably, Pr ₁ and Pr ₂ are hydrogen.

The group Pr ₃ represents hydrogen or a protecting group that forms an ether, an acetal or aminoacetal, an ester or a carbamic acid ester to protect a hydroxyl group. Preferably, Pr ₃ represents hydrogen or a protecting group selected from a benzyl group which is optionally substituted by C _r C ₆ alkoxy (for example methoxy), C ₁ -Ce acyloxy (for example acetoxy), hydroxy and nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl, C _r C ₆ acyloxymethyl (for example pivaloyloxymethyl, tertiary butoxycarbonyloxymethyl), Ci-C ₆ alkoxymethyl (for example methoxymethyl, isobutoxymethyl), tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives, carbamoyl, phenylcarbamoyl and CrCβalkylcarbamoyl (for example methylcarbamoyl and ethylcarbamoyl). Most preferably, Pr ₃ is hydrogen.

In one embodiment, a compound of the invention has structure IX wherein X = -CH(OPr ₃ ) -, R ₁ = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O) ₂ CH ₃ , R ₇ = -H, R ₉ = -H and Rio = -H, Pr ₃ = -H and is shown as Compound Xl:

Compound Xl

In another embodiment, a compound of the invention has structure IX wherein X = -CH(OPr ₃ ) -, R ₁ = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ S(O) ₂ CH ₃ , R ₇ = -H, R ₉ = -H and R ₁₀ = -H, Pr ₃ = -H and is shown as Compound XII:

Compound XII

In another embodiment, a compound of the invention has structure IX wherein X = -CH(OPr ₃ ) -, Ri = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ S(O) ₂ CH ₂ CH ₃ , R ₇ = -H, R ₉ = -H and R ₁₀ = -H, Pr ₃ = -H and is shown as Compound XIII:

Compound XIII

In another embodiment, a compound of the invention has structure IX wherein X = -CH(OPr ₃ ) -, R ₁ = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O) ₂ CH ₂ CH ₃ , R ₇ = -H, R ₉ = -H and R ₁₀ = -H, Pr ₃ = -H and is shown as Compound XIV:

Compound XIV

In another embodiment, a compound of the invention has structure IX wherein X = -CH(OPr ₃ ) -, R ₁ = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O)CH ₃ wherein the sulphur atom is in the R configuration, R ₇ = -H, R ₉ = -H and R ₁₀ = -H, Pr ₃ = -H and is shown as Compound XV:

In another embodiment, a compound of the invention has structure IX wherein X = -CH(OPr ₃ ) -, R ₁ = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O)CH ₃ wherein the sulphur atom is in the S configuration, R ₇ = -H, R ₉ = -H and R ₁₀ = -H, Pr ₃ = -H and is shown as Compound XVI:

Compound XVI

In another embodiment, a compound of the invention has structure IX wherein X = -CH(OPr ₃ ) -, R ₁ = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O)CH ₃ wherein the sulphur atom is in the RS configuration, R ₇ = -H, R ₉ = -H and R ₁₀ = -H, Pr ₃ = -H and is shown as Compound XVII:

Compound XVII

In one embodiment, a compound of the invention has structure IX wherein X is -C(=O)N(R ₁₀ )- , R ₁ = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O) ₂ CH ₃ , R ₇ = -H, R ₉ = -H and Rio = -H and is shown as Compound XVIII:

In another embodiment, a compound of the invention has structure IX wherein X is -C(=O)N(R ₁₀ )- , Ri = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ S(O) ₂ CH ₃ , R ₇ = -H, R ₉ = -H and Ri ₀ = -H and is shown as Compound XIX:

Compound XIX

In another embodiment, a compound of the invention has structure IX wherein X is -C(=O)N(R ₁₀ )- , Ri = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ S(O) ₂ CH ₂ CH ₃ , R ₇ = -H, R ₉ = -H and Ri ₀ = -H and is shown as Compound XX:

Compound XX

In another embodiment, a compound of the invention has structure IX wherein X is -C(=O)N(R ₁₀ )- , Ri = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O) ₂ CH ₂ CH ₃ , R ₇ = -H, R ₉ = -H and R ₁₀ = -H and is shown as Compound XXI:

In another embodiment, a compound of the invention has structure IX wherein X is -C(=O)N(R ₁₀ )- , Ri = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O)CH ₃ wherein the sulphur atom is in the R configuration, R ₇ = -H ₁ R ₉ = -H and Ri ₀ = -H and is shown as Compound XXII:

Compound XXII

In another embodiment, a compound of the invention has structure IX wherein X is -C(=O)N(R ₁₀ )- , Ri = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O)CH ₃ wherein the sulphur atom is in the S configuration, R ₇ = -H, Rg = -H and Ri ₀ = -H and is shown as Compound XXIII:

Compound XXIII

In another embodiment, a compound of the invention has structure IX wherein X is -C(=O)N(R ₁₀ )- , Ri = -CH(CH ₃ ) ₂ , R ₅ = -CH ₂ CH ₂ S(O)CH ₃ wherein the sulphur atom is in the RS configuration, R ₇ = -H, R ₉ = -H and R ₁₀ = -H and is shown as Compound XXIV:

Compound XXIV

As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines or heterocyclic amines.

As used herein, the term "isostere" refers to a compound resulting from the exchange of an atom or a group of atoms with another, broadly similar, atom or group of atoms. In the compounds of Structures IX or X, the moieties which contain isosteric groups are preferably - NR _1O -CHR ₁ -CO-, -NR ₁₀ -CHR ₉ -CO-O- and -NR _1O -CO-CHR ₅ -NR _1O -CO-CHR ₇ -. Examples of such isosteres are compounds of Structures IX or X wherein the moiety -NH- has been replaced by -CH ₂ -, -O- or -S-, the moiety -CO- has been replaced by -CS- or -C(=NH> and the moiety -O- has been replaced by -S-, -CH ₂ - or -NH-.

For the avoidance of doubt, the present invention also embraces pro-drugs which react in vivo to give a compound of the present invention or an isostere or pharmaceutically acceptable salt thereof.

Said pharmaceutical composition typically contains up to 85 wt% of a compound of the invention. More typically, it contains up to 50 wt% of a compound of the invention. Preferred pharmaceutical compositions are sterile and pyrogen free. Further, the pharmaceutical compositions provided by the invention typically contain a compound of the invention which is a substantially pure optical isomer. Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable salt of a compound of Structure IX or X or an isostere thereof.

The compounds of the invention wherein X is -CH(OPr ₃ ) - can be prepared by conventional routes, for example using the following Scheme 1 wherein the functional groups are as defined above and PG represents a nitrogen protective group :

Scheme 1

In Scheme 1 step(a), an Λ/-protected amino acid bearing the side-chain R ₁ is condensed with an ester enolate bearing the side chain R ₉ and the resulting intermediate 1 ,3-diketoester is then reduced to furnish a statine unit, wherein Pr ₃ is H or a removable alcohol-protecting group. In step (b), the Λ/-protecting group is removed, and the statine is coupled to a protected cysteine derivative to furnish a peptide isostere. In step (c), the /V-protecting group is removed, and the peptide isostere is coupled with an Λ/-protected amino acid bearing the side chain R ₅ . In step (d), the Λ/-protecting group is removed, and the resulting intermediate is coupled with a functionalised β-hydroxy acid derivative wherein R ₁₄ is a temporary blocking group which can be removed to produce a compound wherein R ₁₄ is H, and X is a chiral auxiliary as reported in Yurek- George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan, A. J. Am. Chem. Soc.2004, 126, 1030-1031. In step (e), the ester is hydrolysed, and cyclisation is facilitated in step (f), to provide a compound of the invention of Structure X wherein X is -CH(OPr ₃ ) -. Removal of the thiol-protecting groups and disulfide bond formation occurs in step (g) to provide a compound of the invention of Structure IX wherein X is -CH(OPr ₃ ) -.

Compounds of the invention in which R ₁₀ is other than hydrogen can be obtained either by alkylating a corresponding compound of the invention or intermediate in which R ₁₀ is hydrogen or by using appropriately substituted starting materials

The compounds of the invention wherein X is -C(=O)N(R ₁₀ )- may be prepared by conventional routes, for example using the following Scheme 2 wherein the functional groups are as defined above:

Scheme 2

In Scheme 2 step (a), an amino acid ester bearing the side-chain Rg is coupled with another, Λ/-protected amino acid bearing the side chain R ₁ (where PG represents a conventional protecting group) to furnish the Λ/-protected dipeptide ester. In step (b), the Λ/-protecting group is removed, and the resulting dipeptide ester is coupled to a protected cysteine. In step (c), the Λ/-protecting group is removed, and the resulting tripeptide is coupled with an amino acid bearing the side chain R ₅ to liberate an Λ/-protected tetrapeptide ester. In step (d), the /V-protecting group is removed and the resulting tetrapeptide ester is coupled with a functionalized β-hydroxy acid derivative wherein R ₁₄ is a temporary blocking group which can be removed to produce a compound wherein R ₁₄ is H, and X is a chiral auxiliary as reported in Yurek-George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan, A. J. Am. Chem. Soc. 2004, 126, 1030-1031. In step (e), the ester is hydrolysed, and cyclisation is facilitated in step (f) to provide a compound of Structure X wherein X is -C(=O)N(Ri ₀ )-. Removal of the thiol-protecting groups and disulfide bond formation occurs in step (g) to complete the synthesis of a compound of the Structure IX wherein X is -C(=O)N(R ₁₀ )-.

Compounds of formula (X) may be obtained by reaction of the product of step (g) of the above Schemes 1 and 2, i.e. a compound of Structure IX, to cleave the disulfide bond. The cleavage of the disulfide bond is typically achieved using a thiol compound generally used for a reduction treatment of a protein having a disulfide bond, for example mercaptoethanol, thioglycolic acid, 2-mercaptoethylamine, benzenethiol, parathiocresol and dithiothreitol. Preferably, mercaptoethanol and dithiothreitol are used. An excess thiol compound can be removed by for example dialysis or gel filtration. Alternatively, electrolysis, sodium tetrahydroborate, lithium aluminum hydride or sulfite may, for example, be used to cleave the disulfide bond.

Compounds of formula (X) in which Pri and/or Pr ₂ is other than hydrogen may be prepared by introducing a thiol-protecting group into a corresponding compound in which Pri and/or Pr ₂ is/are hydrogen. In this aspect a suitable agent for introducing thiol-protecting group to be used in this reaction is appropriately determined depending on the protecting group to be introduced. Examples include chlorides of the corresponding protecting group (for example benzyl chloride, methoxybenzyl chloride, acetoxybenzyl chloride, nitrobenzyl chloride, picolyl chloride, picolyl chloride-N-oxide, anthryl methyl chloride, isobutoxymethyl chloride, phenylthiomethyl chloride) and alcohols of the corresponding protecting group (for example diphenylmethyl alcohol, adamanthyl alcohol, acetamidemethyl alcohol, benzamidomethyl alcohol), dinitrophenyl, isobutylene, dimethoxymethane, dihydropyran and t-butyl chloroformate.

As the skilled person will appreciate, when one of R ₁ , R ₅ , R ₇ , Rg, Ri ₀ carries a functional group such as -OH, -SH, -NH ₂ or -COOH, then it may be preferred for that group to be protected for one or more of the reaction steps following its introduction. In this case the group in question could be protected in a separate step after its introduction, or, it could be protected already at the time it is introduced. The skilled person will be aware of suitable protecting groups that can be used in this regard.

The compounds of the invention thus obtained may be salified by treatment with an appropriate acid or base. Racemic mixtures obtained by any of the above processes can be resolved by standard techniques, for example elution on a chiral chromatography column.

The N-protected aminoacids bearing the side-chain R ₅ containing a sulphone or sulphoxide group, which are used in step d) of Schemes 1 and 2, can be prepared with known methods, typically involving oxidation of the sulphur atom of an N-protected aminoacid bearing a side-chain containing a thioeter moiety. For example, the synthesis of all stereoisomers of N-protected methionine and ethionine sulphoxides is described in Holland, H. L.; Andreana, P. R.; Brown, F. M., Tetrahedron: Asimmetry, 10 (1999), 2833-2843. N-protected D-methionine sulphone can be obtained by oxidation of N-protected D- methionine with peroxyacids.

The skilled person will appreciate that various assays are suitable for testing for HDAC inhibition and may be used to measure the activity of a compound obtained from Scheme 1 compared to that of the known HDAC inhibitor SAHA. Thus, the IC ₅₀ of a test compound against HDAC can, for example, be determined in an in vitro assay, and compared with the IC ₅₀ of SAHA under the same assay conditions. If a test compound has an IC ₅₀ value equal to or lower than that of SAHA it should be understood as having an HDAC inhibitory activity which is at least equal to that exhibited by SAHA.

In a preferred embodiment the present invention provides a process for selecting a compound which has an HDAC inhibitory activity which is at least equal to that exhibited by SAHA as defined above, wherein following completion of Scheme 1 or 2, the next step is a an in vitro HDAC assay. Typically, said assay comprises contacting a test compound and SAHA, at various concentrations, with diluted HeLa Nuclear Extract to determine the IC ₅₀ of the test compound and of SAHA against HeLa Nuclear Extract. A test compound which has an IC ₅₀ value measured against HeLa Nuclear Extract which is equal to, or lower than, the IC ₅₀ of SAHA under the same assay conditions should be understood as having an inhibitory activity which is at least equal to that exhibited by SAHA. Typically said assay is performed using a HDAC fluorescent activity assay kit (Biomol, UK) and the test compounds are reduced prior to analysis.

In another embodiment the present invention provides a process for selecting a compound which has a human cancer cell growth inhibitory activity which is at least equal to that exhibited by SAHA, which process comprises preparing a compound of Structure IX or X via Scheme 1 or 2 as defined above followed by screening the thus obtained compound to measure its activity as a human cancer cell growth inhibitor.

The skilled person will appreciate that various assays are suitable for testing for human cancer cell growth inhibition and may be used to measure the activity of a compound obtained via Scheme 1 or 2 compared to that of SAHA. Thus, the IC ₅₀ of a test compound against human cancer cell growth can, for example, be determined in an in vitro assay, and compared with the IC _5O of SAHA under the same assay conditions. If a test compound has an IC _5O value equal to or lower than that of SAHA it should be understood as having an inhibitory activity which is at least equal to that exhibited by SAHA. Typically in this embodiment this step comprises an in vitro assay which comprises contacting a test compound and SAHA, at various concentrations, with an MCF7 breast, HUT78 T-cell leukaemia, A2780 ovarian, PC3 or LNCAP prostate cancer cell line to determine the IC ₅₀ of the test compound and of SAHA against the cell line. A test compound which has an IC ₅₀ value measured against any of these cell lines which is equal to, or lower than, the IC ₅₀ of SAHA under the same assay conditions should be understood as having an inhibitory activity at least equal to that of SAHA. Typically in this embodiment, said assay is performed using the CyQuantTM assay system (Molecular Probes, Inc. USA).

In another preferred embodiment the present invention provides a process for selecting a compound which has an anti-inflammatory activity which is at least equal to that exhibited by SAHA, which process comprises preparing a compound of Structure IX or X via Scheme 1 as defined above followed by screening the thus obtained compound to measure its anti-inflammatory activity.

The skilled person will appreciate that various assays are suitable for assessing the anti-inflammatory activity of a compound. The anti-inflammatory activity of a test compound relative to SAHA may, for example, be determined by measuring the activity of a compound in inhibiting the production of TNFα from peripheral blood mononuclear cells (PBMCs) relative to SAHA. Thus, the ability of a test compound to inhibit the production of TNFα from PBMCs can, for example, be determined in an assay, and compared with the activity of SAHA under the same assay conditions. If a test compound has an in vitro inhibitory activity of TNFα production which is equal to or higher than that of SAHA under the same assay conditions it should be understood as having an antiinflammatory activity which is at least equal to that exhibited by SAHA. Typically this step is performed using the Quantikine® Human-α assay kit (R&D systems, Abingdon UK).

In another aspect of this embodiment, the anti-inflammatory activity of a test compound relative to SAHA may be determined by assessing the activity of a compound in inhibiting inflammation in Balb/c mice relative to SAHA. If a test compound has an in vivo inhibitory activity which is equal to or higher than that of SAHA under the same test conditions it should be understood as having an anti-inflammatory activity which is at least equal to that exhibited by SAHA. Typically, in this embodiment this step is performed by assessing the in vivo activity of a test compound and of SAHA in inhibiting inflammation in Balb/c mice induced by a chemical challenge. Typically, said chemical challenge involves the topical administration to the mice of oxalazone or acetone. In this embodiment, the compounds under investigation may be applied before or after the chemical challenge.

In another preferred embodiment the present invention provides a process for selecting a compound which has an activity in inducing a predominant G2/M phase arrest or cell death in MCF7 cells which is at least equal to that exhibited by SAHA, which process comprises preparing a compound of Structure IX or X via Scheme 1 or 2 as defined above followed by screening the thus obtained compound to measure activity in inducing a predominant G2/M phase arrest or cell death in MCF7 cells relative to SAHA.

The compounds of the present invention are found to be inhibitors of HDAC. The compounds of the present invention are therefore therapeutically useful. The compounds of the invention and compositions comprising them may be administered in a variety of dosage forms. In one embodiment, a pharmaceutical composition comprising a compound of the invention may be formulated in a format suitable for oral, rectal, parenteral, intranasal or transdermal administration or administration by inhalation or by suppository. Typical routes of administration are parenteral, intranasal or transdermal administration or administration by inhalation.

The compounds of the invention can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. Preferred pharmaceutical compositions of the invention are compositions suitable for oral administration, for example tablets and capsules.

The compounds of the invention may also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternally, transdermal^ or by infusion techniques. The compounds may also be administered as suppositories.

The compounds of the invention may also be administered by inhalation. An advantages of inhaled medications are their direct delivery to the area of rich blood supply in comparison to many medications taken by oral route. Thus, the absorption is very rapid as the alveoli have an enormous surface area and rich blood supply and first pass metabolism is bypassed. A further advantage may be to treat diseases of the pulmonary system, such that delivering drugs by inhalation delivers them to the proximity of the cells which are required to be treated.

The present invention also provides an inhalation device containing such a pharmaceutical composition. Typically said device is a metered dose inhaler (MDI), which contains a pharmaceutically acceptable chemical propellant to push the medication out of the inhaler.

The compounds of the invention may also be administered by intranasal administration. The nasal cavity's highly permeable tissue is very receptive to medication and absorbs it quickly and efficiently, more so than drugs in tablet form. Nasal drug delivery is less painful and invasive than injections, generating less anxiety among patients. By this method absorption is very rapid and first pass metabolism is usually bypassed, thus reducing inter-patient variability. Further, the present invention also provides an intranasal device containing such a pharmaceutical composition.

The compounds of the invention may also be administered by transdermal administration. The present invention therefore also provides a transdermal patch containing a compound of the invention, or a pharmaceutically acceptable salt thereof.

The compounds of the invention may also be administered by sublingual administration. The present invention therefore also provides a sub-lingual tablet comprising a compound of the invention or a pharmaceutically acceptable salt thereof.

A compound of the invention is typically formulated for administration with a pharmaceutically acceptable carrier or diluent.

A compound of the invention may also be formulated with an agent which reduces degradation of the substance by processes other than the normal metabolism of the patient, such as anti-bacterial agents, or inhibitors of protease enzymes which might be the present in the patient or in commensural or parasite organisms living on or within the patient, and which are capable of degrading the compound.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions. Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspension or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

The compounds of the present invention are therapeutically useful in the treatment or prevention of conditions mediated by HDAC. Accordingly, the present invention provides the use of a compound of the Structure IX or X, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment or prevention of a condition materially affected by the activity of an HDAC. Also provided is a method of treating a patient suffering from or susceptible to a condition mediated by HDAC, which method comprises administering to said patient an effective amount of a compound of Structure IX or X, an isostere thereof or a pharmaceutically acceptable salt thereof.

In one embodiment the compounds of the present invention may be used in combination with another known inhibitor of HDAC, such as SAHA. In this embodiment, the combination product may be formulated such that it comprises each of the medicaments for simultaneous, separate or sequential use.

The present invention therefore also provides the use of compounds according to Structure IX or X or an isostere or pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for use in co-administration with another known inhibitor of HDAC, such as SAHA.

The compounds of the present invention can be used in both the treatment and prevention of cancer and can be used in a monotherapy or in a combination therapy. When used in a combination therapy, the compounds of the present invention are typically used together with small chemical compounds such as platinum complexes, anti-metabolites, DNA topoisomerase inhibitors, radiation, antibody-based therapies (for example herceptin and rituximab), anti-cancer vaccination, gene therapy, cellular therapies, hormone therapies or cytokine therapy.

In one embodiment of the invention a compound of the invention is used in combination with another chemotherapeutic or antineoplastic agent in the treatment of a cancer. Examples of such other chemotherapeutic or antineoplastic agents include mitoxantrone, vinca alkaloids for example vincristine and vinblastine, anthracycline antibiotics for example daunorubicin and doxorubicin, alkylating agents for example chlorambucil and melphalan, taxanes for example paclitaxel, antifolates for example methotrexate and tomudex, epipodophyllotoxins for example etoposide, camptothecins for example irinotecan and its active metabolite SN 38 and DNA methylation inhibitors for example the DNA methylation inhibitors disclosed in WO 02/085400. According to the invention, therefore, products are provided which contain a compound of the invention and another chemotherapeutic or antineoplastic agent as a combined preparation for simultaneous, separate or sequential use in alleviating a cancer. Also provided according to the invention is the use of a compound of Structure IX or X as defined above or an isostere thereof or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the alleviation of cancer by coadministration with another chemotherapeutic or antineoplastic agent.

The compound of the invention and the said other agent may be administrated in any order. In both these cases the compound of the invention and the other agent may be administered together or, if separately, in any order as determined by a physician.

HDAC is believed to contribute to the pathology and/or symptomology of several different diseases such that reduction of the activity of HDAC in a subject through inhibition of HDAC may be used to therapeutically address these disease states. Examples of various diseases that may be treated using the HDAC inhibitors of the present invention are described herein, and the use of compounds of the present invention described by Structure IX or X are included herein. It is noted that additional diseases beyond those disclosed herein may be later identified as applications of the compounds of the present invention, as the biological roles that HDAC play in various pathways becomes more fully understood.

One set of indications that HDAC inhibitors of the present invention may be used to treat are those involving undesirable or uncontrolled cell proliferation. Such indications include benign tumours, various types of cancers such as primary tumours and tumour metastasis, restenosis (e.g. coronary, carotid, and cerebral lesions), abnormal stimulation of endothelial cells (atherosclerosis), insults to body tissue due to surgery, abnormal wound healing, abnormal angiogenesis, diseases that produce fibrosis of tissue, repetitive motion disorders, disorders of tissues that are not highly vascularized, and proliferative responses associated with organ transplants. More specific indications for HDAC inhibitors include, but are not limited to prostate cancer, lung cancer, acute leukaemia, multiple myeloma, bladder carcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma, neuroblastoma and melanoma.

In one embodiment, a method is provided for treating diseases associated with undesired and uncontrolled cell proliferation. The method comprises administering to a subject suffering from uncontrolled cell proliferation a therapeutically effective amount of a HDAC inhibitor according to the present invention, such that said uncontrolled cell proliferation is reduced. The particular dosage of the inhibitor to be used will depend on the severity of the disease state, the route of administration, and related factors that can be determined by the attending physician. Generally, acceptable and effective daily doses are amounts sufficient to effectively slow or eliminate uncontrolled cell proliferation.

HDAC inhibitors according to the present invention may also be used in conjunction with other agents to inhibit undesirable and uncontrolled cell proliferation. Examples of other anti-cell proliferation agents that may be used in conjunction with the HDAC inhibitors of the present invention include, but are not limited to, retinoid acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN(TM) protein, ENDOSTATIN(TM) protein, suramin, squalamine, tissue inhibitor of metalloproteinase-l, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor-1 , plasminogen activator inhibitor-2, cartilage- derived inhibitor, paclitaxel, platelet factor 4, protamine sulfate (clupeine), sulfated chitin derivatives (prepared from queen crab shells), sulfated polysaccharide peptidoglycan complex (sp-pg), staurosporine, modulators of matrix metabolism, including for example, proline analogs (1-azetidine-2- carboxylic acid (LACA), cishydroxyproline, d,l-3,4-dehydroproline, thiaproline), β-aminopropionitrile fumarate, 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; methotrexate, mitoxantrone, heparin, interferons, 2 macroglobulin-serum, chimp- 3, chymostatin, beta.-cyclodextrin tetradecasulfate, eponemycin; fumagillin, gold sodium thiomalate, d-penicillamine (CDPT), beta.-1-anticollagenase-serum, alpha.2-antiplasmin, bisantrene, lobenzarit disodium, N-(2-carboxyphenyl-4- chloroanthranilic acid disodium or "CCA", thalidomide; angostatic steroid, carboxyaminoimidazole; metalloproteinase inhibitors such as BB94. Other anti- angiogenesis agents that may be used include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms, VEGF-C, HGF/SF and Ang-1/Ang-2 (Ferrara N. and Alitalo, K. "Clinical application of angiogenic growth factors and their inhibitors" (1999) Nature Medicine 5:1359-1364).

Generally, cells in benign tumours retain their differentiated features and do not divide in a completely uncontrolled manner. A benign tumour is usually localized and nonmetastatic. Specific types of benign tumours that can be treated using HDAC inhibitors of the present invention include hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas and pyogenic granulomas.

In the case of malignant tumors, cells become undifferentiated, do not respond to the body's growth control signals, and multiply in an uncontrolled manner. Malignant tumors are invasive and capable of spreading to distant sites (metastasizing). Malignant tumors are generally divided into two categories: primary and secondary. Primary tumors arise directly from the tissue in which they are found. Secondary tumors, or metastases, are tumors that originated elsewhere in the body but have now spread to distant organs. Common routes for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems, and tracking along tissue planes and body spaces (peritoneal fluid, cerebrospinal fluid, etc.).

Specific types of cancers or malignant tumors, either primary or secondary, that can be treated using the HDAC inhibitors of the present invention include, but are not limited to, leukaemia, breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilms' tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, glioblastoma multiforme, leukemias, lymphomas, malignant melanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

The HDAC inhibitors of the present invention may also be used to treat abnormal cell proliferation due to insults to body tissue during surgery. These insults may arise as a result of a variety of surgical procedures such as joint surgery, bowel surgery, and cheloid scarring. Diseases that produce fibrotic tissue include emphysema. Repetitive motion disorders that may be treated using the present invention include carpal tunnel syndrome. An example of a cell proliferative disorder that may be treated using the invention is a bone tumor.

Proliferative responses associated with organ transplantation that may be treated using HDAC inhibitors of the invention include proliferative responses contributing to potential organ rejections or associated complications. Specifically, these proliferative responses may occur during transplantation of the heart, lung, liver, kidney, and other body organs or organ systems.

Abnormal angiogenesis that may be treated using this invention include those abnormal angiogenesis accompanying rheumatoid arthritis, ischemic- reperfusion related brain edema and injury, cortical ischemia, ovarian hyperplasia and hypervascularity, (polycystic ovary syndrome), endometriosis, psoriasis, diabetic retinopathy, and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplastic), macular degeneration, corneal graft rejection, neuroscular glaucoma and Oster Webber syndrome.

Examples of diseases associated with uncontrolled angiogenesis that may be treated according to the present invention include, but are not limited to retinal/choroidal neovascularization and corneal neovascularization. Examples of retinal/choroidal neovascularization include, but are not limited to, Bests diseases, myopia, optic pits, Stargarts diseases, Pagets disease, vein occlusion, artery occlusion, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, carotid obstructive diseases, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosus, retinopathy of prematurity, Eales disease, diabetic retinopathy, macular degeneration, Bechets diseases, infections causing a retinitis or chroiditis, presumed ocular histoplasmosis, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications, diseases associated with rubesis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy. Examples of corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, Mooren ulcer, Terrien's marginal degeneration, marginal keratolysis, polyarteritis, Wegener sarcoidosis, Scleritis, pemphigoid radial keratotomy, neovascular glaucoma and retrolental fibroplasia, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections and Kaposi sarcoma.

Chronic inflammatory diseases associated with uncontrolled angiogenesis may also be treated using HDAC inhibitors of the present invention. Chronic inflammation depends on continuous formation of capillary sprouts to maintain an influx of inflammatory cells. The influx and presence of the inflammatory cells produce granulomas and thus maintains the chronic inflammatory state. Inhibition of angiogenesis using a HDAC inhibitor alone or in conjunction with other anti-inflammatory agents may prevent the formation of the granulosmas and thus alleviate the disease. Examples of chronic inflammatory diseases include, but are not limited to, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosis, and rheumatoid arthritis.

Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis are characterized by chronic inflammation and angiogenesis at various sites in the gastrointestinal tract. For example, Crohn's disease occurs as a chronic transmural inflammatory disease that most commonly affects the distal ileum and colon but may also occur in any part of the gastrointestinal tract from the mouth to the anus and perianal area. Patients with Crohn's disease generally have chronic diarrhea associated with abdominal pain, fever, anorexia, weight loss and abdominal swelling. Ulcerative colitis is also a chronic, nonspecific, inflammatory and ulcerative disease arising in the colonic mucosa and is characterized by the presence of bloody diarrhea. These inflammatory bowel diseases are generally caused by chronic granulomatous inflammation throughout the gastrointestinal tract, involving new capillary sprouts surrounded by a cylinder of inflammatory cells. Inhibition of angiogenesis by these inhibitors should inhibit the formation of the sprouts and prevent the formation of granulomas. Inflammatory bowel diseases also exhibit extra intestinal manifestations, such as skin lesions. Such lesions are characterized by inflammation and angiogenesis and can occur at many sites other the gastrointestinal tract. Inhibition of angiogenesis by HDAC inhibitors according to the present invention can reduce the influx of inflammatory cells and prevent lesion formation.

Sarcoidosis, another chronic inflammatory disease, is characterized as a multisystem granulomatous disorder. The granulomas of this disease can form anywhere in the body. Thus, the symptoms depend on the site of the granulomas and whether the disease is active. The granulomas are created by the angiogenic capillary sprouts providing a constant supply of inflammatory cells. By using HDAC inhibitors according to the present invention to inhibit angiogenesis, such granulomas formation can be inhibited. Psoriasis, also a chronic and recurrent inflammatory disease, is characterized by papules and plaques of various sizes. Treatment using these inhibitors alone or in conjunction with other anti-inflammatory agents should prevent the formation of new blood vessels necessary to maintain the characteristic lesions and provide the patient relief from the symptoms.

Rheumatoid arthritis (RA) is also a chronic inflammatory disease characterized by non-specific inflammation of the peripheral joints. It is believed that the blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. The factors involved in angiogenesis may actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis. Treatment using HDAC inhibitors according to the present invention alone or in conjunction with other anti-RA agents may prevent the formation of new blood vessels necessary to maintain the chronic inflammation.

The compounds of the present invention can further be used in the treatment of cardiac/vasculature diseases such as hypertrophy, hypertension, myocardial infarction, reperfusion, ischemic heart disease, angina, arrhythmias, hypercholesterolemia, atherosclerosis and stroke. The compounds can further be used to treat neurodegenerative disorders/CNS disorders such as acute and chronic neurological diseases, including stroke, Huntington's disease, Amyotrophic Lateral Sclerosis and Alzheimer's disease.

The compounds of the present invention can also be used as antimicrobial agents, for example antibacterial agents. The invention therefore also provides a compound for use in the treatment of a bacterial infection. The compounds of the present invention can be used as anti-infectious compounds against viral, bacterial, fungal and parasitic infections. Examples of infections include protozoal parasitic infections (including Plasmodium, Cryptosporidium parvum, toxoplasma gondii, sarcocystis neurona and Eimeria sp.)

The compounds of the present invention are particularly suitable for the treatment of undesirable or uncontrolled cell proliferation, preferably for the treatment of benign tumours/hyperplasias and malignant tumors, more preferably for the treatment of malignant tumors and most preferably for the treatment of CCL, breast cancer and T-cell lymphoma.

In a preferred embodiment of the invention, the compounds of the invention are used to alleviate cancer, cardiac hypertrophy, chronic heart failure, an inflammatory condition, a cardiovascular disease, a haemoglobinopathy, a thalassemia, a sickle cell disease, a CNS disorder, an autoimmune disease, diabetes, osteoporosis, MDS, benign prostatic hyperplasia, oral leukoplakia, a genentically related metabolic disorder, an infection, Rubens-Taybi, fragile X syndrome, or alpha-1 antitrypsin deficiency, or to accelerate wound healing, to protect hair follicles or as an immunosuppressant.

Typically, said inflammatory condition is a skin inflammatory condition (for example psoriasis, acne and eczema), asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn's disease or colitis.

Typically, said cancer is chronic lymphocytic leukaemia, breast cancer, prostate cancer, ovarian cancer, mesothelioma or T-cell lymphoma.

Typically, said cardiovascular disease is hypertension, myocardial infarction (Ml), ischemic heart disease (IHD) (reperfusion), angina pectoris, arrhythmia, hypercholesterolemia, hyperlipidaemia, atherosclerosis, stroke, myocarditis, congestive heart failure, primary and secondary i.e. dilated (congestive) cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, peripheral vascular disease, tachycardia, high blood pressure or thrombosis.

Typically, said genetically related metabolic disorder is cystic fibrosis (CF), peroxisome biogenesis disorder or adrenoleukodystrophy.

Typically, the compounds of the invention are used as an immunosuppressant following organ transplant.

Typically, said infection is a viral, bacterial, fungal or parasitic infection, in particular an infection by S aureus, P acne, Candida or aspergillus.

Typically, said CNS disorder is Huntingdon's disease, Alzheimer's disease, multiple sclerosis or amyotrophic lateral sclerosis.

In this embodiment, the compounds of the invention may be used to alleviate cancer, cardiac hypertrophy, chronic heart failure, an inflammatory condition, a cardiovascular disease, a haemoglobinopathy, a thalassemia, a sickle cell disease, a CNS disorder, an autoimmune disease, diabetes or osteoporosis, or are used as an immunosuppressant.

The compounds of the invention may also be used to alleviate chronic lymphocytic leukaemia, breast cancer, prostate cancer, ovarian cancer, mesothelioma, T-cell lymphoma, cardiac hypertrophy, chronic heart failure or a skin inflammatory condition, in particular psoriasis, acne or eczema.

The compounds of the present invention can be used in the treatment of animals, preferably in the treatment of mammals and more preferably in the treatment of humans.

The compounds of the invention may, where appropriate, be used prophylactically to reduce the incidence of such conditions. A therapeutically effective amount of a compound of the invention is administered to a patient. A typical dose is from about 0.001 to 50 mg per kg of body weight, according to the activity of the specific compound, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.

The following Examples illustrate the invention. Examples

The following abbreviations are used throughout the examples. mCPBA, = meta-chloroperbenzoic acid; DMAP = 4-dimethylaminopyridine; PyBOP = benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate; EDCI = 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; HOBt = hydroxbenzotriazole; DMAP = 4-dimethylaminopyridine; EtOAc = ethyl acetate; THF = tetrahydrofuran. Example A

(£)-(1S,5S,6R,9S,20R)-5-Hydroxy-6-isopropyl-20-(2-

(methylsulphonyl)ethyl)-2-oxa-1 1 ,12-dithia-7,19,22-triaza-bicyclo[7.7.6]docos-15- ene-3,8,18,21-tetraone (Compound Xl)

The synthesis of Compound Xl is depicted in Scheme 3.

Compound Xl

Scheme 3

1) Preparation of (R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4- (methylsulfonyl)butanoic acid, (1)

To a solution of mCPBA (5.5g, 28mmol) in CH ₂ CI ₂ (75mL), Fmoc-D-Met- OH (5g, 13.5mmol) in CH ₂ CI ₂ (5OmL) was added dropwise at O ⁰ C. After 2 agitation for 2 h the desired product (1) was isolated by filtration as a white solid (5 g, 92%).

¹ H NMR (500 MHz, DMSOd ₆ ): δ _H : 7.87-7.90 (m, 3H), 7.72 (m, 2H), 7.62 (d, 1 H), 7.49 (m, 1 H), 7.40 (m, 2H), 7.33 (m, 2H), 4.22-4.30 (m, 3H), 4.02 (q, 1H), 3.20 (m, 1 H), 3.06 (m, 1H), 2.97 (s, 3H), 2.15 (m, 1H), 2.00 (m, 1H). LC- MS: [M+H] ⁺ : 404.2; [M+Na] ⁺ : 426.1.

2) Preparation of (3) To a stirred solution of (3S,4R)-allyl 4-((S)-2-(((9H-fluoren-9- yl)methoxy)carbonylamino)-3-(tritylthio)propanamido)-3-hydro xy-5- methylhexanoate (A) (1Og, 13mmσl; prepared according to the procedure in Doi, T ₁ ; lijima, Y.; Shin-ya, K.; Ganesan, A.; Takahashi, T.; Tet. Lett. 2006, 47, 1177- 1080) in CH ₂ CI ₂ ZCH ₃ CN (25OmL, 4/1) at room temperature was added Et ₂ NH (3OmL) dropwise. After having been stirred for 3 h at room temperature, the solvents were removed in vacuo. The residue was co-evaporated with hexane (3 x 100 mL) and dried on the high-vacuum pump for 2 h to afford crude amine (3S,4R)-allyl 4-((S)-2-amino-3-(tritylthio)propanamido)-3-hydroxy-5- methylhexanoate (2) as a colourless oil which was used directly in the next step.

To a stirred solution of (1) (5.5g, 13mmol) and PyBOP (6.9g, 13mmol) in CH ₂ CI ₂ (20OmL) was added Λ/,Λ/-diisopropylethylamine (5.5mL) at 0 ⁰ C. After agitation for 10 min a solution of the above crude amine (2) in CH ₂ CI ₂ (200 mL) was added and the resulting solution was warmed to room temperature and stirred overnight. The volatiles were removed and the residue was purified by silica gel column chromatography, eluting with EtOAc/petroleum ether (1/1 to 3/1), to give (3) (6g, 50%) as a white solid.

¹ H-NMR (500 MHz, CDCI ₃ ): δ _H : 7.76 (s, 2H), 7.53 (m, 2H), 7.18-7.39 (m, 25H), 6.75 (s, 1H), 5.95 (d, 1 H), 5.83 (m, 2H), 5.28 (d, 1H), 5.20 (d, 1 H), 4.54 (s, 2H), 4.30-4.36 (m, 3H), 3.79-4.10 (m, 4H), 3.15-3.25 (m, 3H), 2.89 (s, 3H), 2.82 (m, 1H), 2.36-2.55 (m, 3H), 1.93-2.07 (m, 2H), 0.86 (d, 6H).

3) Preparation of (7R,11S,14R,17S,18R,E)-allyl 7,18-dihydroxy-17- isopropyl-11 -(2-(methylsulfonyl)ethyl)-9,12,15-trioxo-1 ,1 ,1 -triphenyl-14- (tritylthiomethyl)-2-thia-10,13,16-triazaicos-5-en-20-oate, (5)

To a stirred solution of (3) (12g, 13.6mmol) in CH ₂ CI ₂ /CH ₃ CN (25OmL, 4/1) at room temperature was added Et ₂ NH (3OmL) dropwise. After agitation for 2 h at room temperature, the solvents were removed in vacuo. The residue was co- evaporated with hexane (3 x 10OmL) and dried on the high-vacuum pump for 2 h to afford crude amine (3R,4S)-allyl 4-((R)-2-((S)-2-amino-4- (methylsulfonyl)butanamido)-3-(tritylthio)propanamido)-3-hyd roxy-5- methylhexanoate (4) as a colourless oil which was used directly for next step.

At 0 ⁰ C, to a stirred solution of (E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4- enoic acid (B) prepared according to WO 2008/062201 (8.5g, 20.4mmol) in CH ₂ CI ₂ (25OmL) was added EDCI (4 g, 20.4 mmol) and HOBt (2.8g, 20.4mmol). After stirring for 5 min, a solution of the crude amine (4) in CH ₂ CI ₂ (25OmL) and 4-dimethylaminopyridine (300 mg, 2.4 mmol) were then added and the reaction mixture was warmed to room temperature and stirred overnight. The reaction was washed with water (10OmL), 10% HCI (10OmL), 5% NaHCO ₃ (10OmL) and saturated NaCI (10OmL) solutions, dried (Na ₂ SO ₄ ), filtered, and concentrated and purified by silica gel column chromatography, eluting with EtOAc/petroleum ether (2/1) and then with EtOAc to give (5) (10 g, 67%) as a white solid.

¹ H-NMR (500 MHz, CDCI ₃ ): δ _H : 7.37-7.48 (m, 14H), 7.10-7.30 (m, 24H), 7.09 (d, 2H), 6.04 (d, 1 H), 5.86 (m, 1 H), 5.45 (m, 1 H), 5.27 (d, 1 H), 5.20 (d, 1 H), 4.54 (d, 2H), 4.46 (q, 1 H), 4.32 (t, 1H), 4.01-4.10 (m, 2H), 3.75 (m, 1H), 3.15- 3.18 (m, 2H), 2.79 (s, 3H), 2.50-2.58 (m, 6H), 2.20-2.42 (m, 8H), 2.02-2.08 (m, 4H), 0.83-0.86 (d, 6H).

4) Preparation of (TS.HR.US.^R.IδS.EK.IS-dihydroxy-i?- isopropyl-11 -(2-(methylsulfonyl)ethyl)-9,12,15-trioxo-1 ,1 ,1 -triphenyl-14- (tritylthiomethyl)-2-thia-10,13,16-triazaicos-5-en-20-oic acid, (6)

To a solution of (5) (18.5 g, 16.7 mmol) in dry CH ₃ OH (40OmL) at room temperature was added morpholine (4ml) followed by Pd(PPh ₃ ) ₄ (2.2g). After stirring for 2 h the solvent was evaporated. The crude product was dissolved in CH ₂ CI ₂ (500 mL), washed with 1 M HCI (10OmL), saturated NaHCO ₃ (10OmL) and saturated NaCI (10OmL) solutions. The organic layer was then dried with MgSO ₄ , filtered and evaporated in vacuo. The residue was rinsed with hexane (3 x 2OmL) and purified by silica gel column chromatography eluting with CH ₃ OHZCH ₂ CI ₂ from 1/20 to 1/10 to give (6) (12 g, 70%) as a yellow solid.

¹ H-NMR (500 MHz, CD ₃ OD) δ _H : 7.81 (s, 1 H), 7.39-7.41 (m, 12H), 7.28- 7.32 (m, 12H), 7.20-7.24 (m, 6H), 5.54 (m, 1 H), 5.47 (m, 1 H), 4.48 (m, 1 H), 4.38 (m, 1H), 4.15 (m, 1H), 3.93 (t, 1H), 3.76-3.78 (m, 6H), 3.67 (m, 1H), 3.16 (m, 2H), 3.02 (m, 6H), 2.91 (s, 3H), 2.55 (m, 2H), 2.20-2.42 (m, 6H), 2.06-2.13 (m, 5H), 0.83-0.87 (m, 6H).

5) Preparation of (2S,6R,9S,12R,13S)-13-hydroxy-12-isopropyl-6-(2- (methylsulfonyl)ethyl)-2-((E)-4-(tritylthio)but-1-enyl)-9-(t ritylthiomethyl)-1- oxa-δ.δ.H-triazacyclopentadecane^J.IO.Iδ-tetraone, (7) To a solution of 2-methyl-6-nitrobenzoic anhydride (MNBA) (3.4g, lOmmol) and DMAP (1.3g, lOmmol) in CH ₂ CI ₂ (100OmL) was added dropwise a solution of acid (6) (4.1g, 3.84mmol) in CH ₂ CI ₂ (300OmL) over 4 h. After a further 12 h, the reaction was quenched by the addition of 1 M HCI (60OmL). The organic phase was separated, washed with saturated NaHCO ₃ (60OmL) and brine (60OmL), dried (MgSO ₄ ), filtered and concentrated in vacuo to give a yellow solid. Purification by silica gel column chromatography eluting with CH ₂ CI ₂ /Me0H (1/100-1/50) gave (7) (2.1 g, 52%) as a yellow solid.

¹ H-NMR (500 MHz, CD ₃ OD) δ _H : 7.81 (s, 1H), 7.27-7.29 (m, 15H), 7.09- 7.21 (m, 23H), 5.52 (m, 1H), 5.42 (m, 1H), 5.30 (m, 1 H), 4.21 (t, 1 H), 4.01 (q, 1 H), 3.85-3.99 (m, 1 H), 3.34-3.37 (m, 2H), 3.13-3.16 (m, 4H), 2.68 (s, 3H), 2.36- 2.48 (m, 4H), 2.16 (m, 3H), 2.04-2.08 (m, 5H), 1.92 (m, 4H), 0.71 (d, 3H), 0.66 (d, 3H).

6) Preparation of (1S,5S,6R,9S,20R,E)-5-hydroxy-6-isopropyl-20-(2- (methylsulfonylJethylJ^-oxa-H.^-dithia-Z.IΘ^Z-triazabicyclo fZJ.eidocos- 15-ene-3,8,18,21-tetraone, (Compound Xl)

To a solution of I ₂ (4.3g, 1 mmol) in CH ₂ CI ₂ /MeOH (9:1 , 300OmL) was added dropwise a solution of (7) (1.8g, 1.7mmol) in CH ₂ CI ₂ /Me0H (9:1 , 150OmL) over 2 h. After a further 30 min the reaction was quenched by the addition of sodium thiosulfate (0.05M, 100OmL) and brine (20OmL). The organic phase was separated and the aqueous phase extracted with CH ₂ CI ₂ (3 x 40OmL). The combined organic phase was dried (MgSO ₄ ), filtered and concentrated in vacuo to give a yellow oil. Purification by silica gel column chromatography eluting with MeOH/CH ₂ CI ₂ (1/100 to 1/30) gave Compound Xl (510 mg, 53%).

¹ H-NMR (500 MHz, CD ₃ OD) δ _H :7.83 (s, 1 H), 7.65 (d, 1 H), 6.96 (d, 1 H), 5.96 (m, 1 H), 5.80 (m, 1 H), 5.52 (s, 1 H), 4.65 (m, 1 H), 4.46 (m, 1 H), 4.10 (t, 1 H), 3.40 (m, 1 H), 3.30 (m, 2H), 3.10 (m, 2H), 2.92 (s, 3H), 2.78 (m, 1 H), 2.66 (m, 2H), 2.49-2.55 (m, 3H), 2.2-2.25 (m, 4H), 0.90 (d, 3H), 0.82 (d, 3H). LC-MS: [M+H] ⁺ : 566.2; [M+Na] ⁺ : 588.2. Example B

1 S,7R, 10S.21 R,E)-7-isopropyl-21 -(2-(methylsulfonyl)ethyl)-2-oxa-12, 13- dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,1 9,22-pentaone (Compound XVIII) The synthesis of Compound XVIII is depicted in Scheme 4.

ysTrt

Scheme 4

1) Preparation of (R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-4- methaπesulfonyl-butyric acid, (1)

Solid NaHCθ ₃ (2.785g, 33.11mmol) was added to a suspension of commercially-available H-D-Met(O ₂ )-OH (3g, 16.55mmol) in water (6OmL) at O ⁰ C. After 15 min of stirring, a solution of Fmoc-CI (6.422g, 24.82mmol) in THF (6OmL) was added. After an additional 1 h stirring at 0°C, the reaction mixture was allowed to warm to room temperature and stirred for a further 20 h. Saturated aqueous NaHCO ₃ (15OmL) and EtOAc (15OmL) were then added to the reaction mixture, the phases were separated and the aqueous phase was extracted with EtOAc (10OmL). The aqueous phase was then acidified to pH 1 with aqueous 2M HCI, and was then extracted with EtOAc (3 x 10OmL). The organic phases were combined, dried over MgSO ₄ , filtered, evaporated under reduced pressure, and then dried under high vacuum to yield the protected amino acid (1) as a white solid (5.39g, 80%). The crude product was used in the next step without further purification.

S _H (400MHZ, MeOH-d ₆ ) 7.80 (2H, d, J=7.4Hz), 7.64-7.70 (2H, m), 7.39 (2H, t, J=7.2Hz), 7.32 (2H, t, J=7.4Hz), 4.40 (2H, dt, J=6.5, 3.2Hz), 4.31 (1 H, dd, J=9.0, 4.8Hz), 4.24 (2H, m), 3.10-3.26 (2H, m), 2.97 (3H, s), 2.32-2.48 (1 H, m), 2.08-2.22 (1 H, m). MS (ES): 426.3 (100%, [M+Na] ⁺ ). 2) Preparation of ((R)-2-{(S)-2-[(R)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-4-methane sulfonyl-butyrylamino]-3- tritylsulfanyl-propionylamino}-3-methyl-butyrylamino)-acetic acid methyl ester, (9)

Et ₂ NH (2ml_) was added to {(R)-2-[(S)-2-(9H-fluoren-9- ylmethoxycarbonylamino)-3-tritylsulfanyl-propionylamino]-3-m ethyl- butyrylamino}-acetic acid methyl ester (8) (548mg, 0.72mmol) - prepared according the procedure described in WO/2006/129105 - in CH ₃ CN (18ml_) at room temperature under argon. After 1 h stirring, the solvent was removed under reduced pressure, and the residue re-dissolved, and evaporated with CH ₃ CN (4 x 2OmL) and hexane (2 x 2OmL). The crude amine product was dried under high vacuum for at least 3 h.

N,N-Diisopropylethylamine (0.31 mL, 1.80mmol) was added to (1) (349 mg, 0.86 mmol) and PyBOP (447mg, 0.86mmol) in CH ₂ CI ₂ (25mL) at 0 ⁰ C under argon. After 10 min stirring, the mixture was transferred to the batch of crude amine obtained above from the deprotection of (8), in CH ₃ CN (25mL) at 0°C under argon. The reaction mixture was subsequently warmed to room temperature and after 17 h the reaction mixture was concentrated under reduced pressure and the residue purified by silica gel chromatography, using hexane/EtOAc (1 :1 then 0:1) as eluant, to yield (9) as a white solid (394mg, 60%).

4 (400MHz, MeOH-de) 7.68-7.77 (2H, m), 7.55 (2H, br s), 7.10-7.42 (2OH, m), 4.26-4.39 (2H, m), 4.10-4.23 (3H, m), 3.96 (1H, t, J=6.7Hz), 3.69-3.82 (2H, m), 3.01-3.11 (2H, m), 2.87 (3H, br s), 2.60-2.69 (1 H, m), 2.47-2.60 (1 H, m), 2.03-2.21 (4H, m), 1.18-1.25 (1 H, m), 0.88 (6H, br s). MS (ES): 941.8 (100%, [M+Na] ⁺ ).

3) Preparation of ((R)-2-{(S)-2-[(R)-2-((E)-(S)-3-hydroxy-7-tritylsulfanyl- hept-4-enoylamino)-4-methanesulfonyl-butyrylamino]-3-trityls ulfanyl- propionylamino}-3-methyl-butyrylamino)-acetic acid methyl ester, (10)

Et ₂ NH (2mL) was added to (9) (390mg, 0.42mmol) in CH ₃ CN (18mL) at room temperature under argon. After 45 min, the solvent was removed under reduced pressure and the residue was re-dissolved and evaporated with CH ₃ CN (4 x 2OmL) and hexane (2 x 2OmL). The crude amine product was dried under high vacuum for at least 3 h.

N,N-Diisopropylethylamine (0.183mL, 1.05mmol) was added to a solution of (E)-(S)-3-hydroxy-7-tritylsulfanyl-hept-4-enoic acid (B) (197mg, 0.47mmol) - prepared according to WO 2008/062201) - and PyBOP (243mg, 0.47mmol) in CH ₂ CI ₂ (1OmL) at 0°C under argon. After 10 min, the reaction mixture was transferred to the above crude amine obtained from the deprotection of (9) dissolved in CH ₃ CN (10 mL) at 0 ⁰ C under argon. The reaction mixture was subsequently allowed to warm to room temperature and, after 90 min, was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography using EtOAc as eluant to furnish (10) as a white solid (393mg, 85%). δa (400MHz, CDCI ₃ /Me0H-d ₆ 9:1 v/v) 7.30-7.42 (14H, m), 7.13-7.26 (16H, m), 5.44-5.53 (1 H, m), 5.35 (1 H, dd, J=15.7, 6.4Hz), 4.44 (1 H, dt, J=7.3, 2.9Hz), 4.31 (1 H, dt, J=9.4, 4.9Hz), 4.04-4.13 (2H, m), 3.81-3.90 (2H, m), 3.67-3.74 (2H, m), 3.65 (3H ₁ s), 3.33-3.38 (1 H, m), 3.08 (1 H, m), 2.82 (3H, s), 2.57 (1 H, d, J=7.2Hz), 2.21-2.32 (2H, m), 2.11-2.21 (2H, m), 2.03-2.11 (2H, m), 1.22 (2H, t, J=7.2Hz), 0.86 (6H, t, J=6.9 Hz). MS (ES): 1119.8 (100%, [M+Na] ⁺ ).

4) Preparation of (6R,9S,12R,16R)-6-isopropyl-12-(2-methanesulfonyl- ethyl)-16-((E)-4-tritylsulfanyl-but-1 -enyl)-9-tritylsulfanylmethyl-1 -oxa- 4,7,10,13-tetraaza-cyclohexadecane-2,5,8,11 ,14-pentaone, (11 )

A solution of LiOH (13mg, 0.53mmol) in water (2mL) was added to (10) (10) (390mg, 0.35mmol) in THF (8mL) at 0 ⁰ C. After 90 min of stirring at 0 ⁰ C, the reaction mixture was neutralized with aqueous 0.5M HCI, then treated with brine (5OmL) and EtOAc (5OmL). The phases were separated, and the aqueous phase was extracted with EtOAc (3 x 15mL). The organic phases were combined, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The crude intermediate was dried under high vacuum and was subsequently dissolved in CH ₂ CI ₂ /THF (12:1 v/v, 30OmL) and added dropwise over a period of 3 h to 2- methyl-6-nitrobenzoic anhydride (145mg, 0.42mmol) and DMAP (103mg, 0.84mmol) in CH ₂ CI ₂ (5OmL) at room temperature under argon. After 16 h, the reaction mixture was concentrated under reduced pressure, and the residue purified by flash column chromatography using CH ₂ CI ₂ ZMeOH to yield (11) as a white solid (96 mg, 25%).

Sn (400MHZ, CDCI ₃ /Me0H-d ₆ 9:1 v/v) 7.32-7.40 (14H, m), 7.14-7.27 (16H, m), 5.56 (1 H, dt, J=14.9, 6.9Hz), 5.34-5.41 (1 H, m), 5.28-5.33 (1H, m), 4.26-4.34 (1H, m), 3.95 (1H, dt, J=12.3, 6.2Hz), 3.32-3.35 (1 H, m), 3.14-3.21 (2H, m), 2.94 (2H, d, J=14.3Hz), 2.65-2.68 (1 H, m), 2.63 (3H, s), 2.59 (1 H, dd, J=12.7, 5.0Hz), 2.33-2.44 (4H, m), 2.09-2.22 (4H, m), 1.21 (1 H, t, J=7.2Hz), 0.92 (2H, t, J=5.6 Hz), 0.81 (3H, d, J=6.8Hz), 0.76 (3H, d, J=6.8Hz). MS (ES): 1087.9 (100%, [M+Na] ⁺ ).

5) Preparation of (E)-(I S,7R,10S,21R)-7-lsopropyl-21 -(2- methanesulfonyl-ethyl)-2-oxa-12,13-dithia-5, 8,20, 23-tetraaza- bicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentaone, (Compound XVIII)

A solution of (11) (95mg, 0.09mmol) in CH ₂ CI ₂ /Me0H (9:1 v/v, 55 mL) was added dropwise over a period of 30 min to I ₂ (230mg, 0.89mmol) in CH ₂ CI ₂ /Me0H (9:1 v/v, 165ml_) at room temperature under argon. After 2 h, 0.1 M aqueous Na ₂ S ₂ O ₃ (50OmL) and brine (15OmL) were added. The phases were separated, and the aqueous phase was extracted with CH ₂ CI ₂ (15OmL) and EtOAc (2 x 5OmL). The organic phases were combined, dried over MgSO ₄ , filtered, and then concentrated under reduced pressure. The residue was purified by flash column chromatography with CH ₂ CI ₂ /Me0H to furnish Compound XVIII as a white solid (18.4mg, 35%).

S _n (400MHZ, CDCI ₃ /Me0H-d ₆ 9:1 v/v) 7.28 (1 H, d, J=4.3Hz), 7.22 (1H, d, J=8.2Hz), 5.68 (1 H, dt, J=16.3, 5.0Hz), 4.59 (1 H, ddd, J=11.5, 8.5, 3.8Hz), 4.11- 4.20 (2H, m), 3.98-4.09 (5H, m), 3.72 (1 H, d, J=17.7Hz), 3.13-3.21 (4H, m), 3.05 (1 H, dd, J=15.4, 11.3Hz), 2.88-2.95 (2H, m), 2.86 (3H, s), 2.60-2.72 (2H, m), 2.45-2.55 (2H, m), 2.10-2.34 (2H, m), 0.80 (6H, d, J=6.7Hz). D _c (100MHz, CDCI ₃ /Me0H-d ₆ 9:1 v/v) 176.67 (C=O), 171.63 (C=O), 170.58 (C=O), 169.61 (C=O), 168.01 (C=O), 130.08 (CH), 129.74 (CH), 70.02 (CH), 63.92 (CH), 55.81 (CH), 54.17 (CH), 50.94 (CH2), 41.35 (CH2), 40.19 (CH3), 38.10 (CH2), 37.86 (CH2), 35.00 (CH2), 30.89 (CH2), 26.84 (CH), 22.33 (CH2), 19.96 (CH3), 19.39 (CH3). MS (ES): 601.4 (100%, [M+Na] ⁺ ). Example C: In Vitro HDAC Inhibition Assay In vitro HDAC assays were performed using a HDAC fluorescent activity assay kit (Biomol, UK) according to the manufacturer's instructions. Compounds were reduced prior to analysis; 500μM compound was reduced with 15mM DTT in DMSO overnight at room temperature, protected from light. Reactions were then set up in a 96-well plate. For each reaction 10μl_ compound (5 x required concentration in assay buffer) was mixed with 15 μL diluted HeLa Nuclear Extract (30-fold in assay buffer). Serial dilutions were set up for each compound. Reactions containing HeLa extract only and assay buffer only were also set up. 25 μL diluted Fluor de Lys™ substrate (100-fold in assay buffer) was added to each reaction, which were then incubated at 37°C for 1 hour. The reaction was stopped by addition of 50 μL Fluor de Lys™ Developer (20-fold dilution in assay buffer, plus TSA diluted 100-fold). The reactions were then incubated at room temperature for 10 minutes before fluorescence was measured using a CytoFluor Il Fluorescence Multiwell Plate Reader and CytoFluor Il software with filters set at 360 nM for excitation and 460 nM for emission. Inhibition of in vitro HDAC activity was determined for mean values of duplicate samples as percentages relative to HeLa extract only reactions. IC ₅₀ values were calculated using GraphPad Prism software. Results are shown In Table 1 (structures shown above). Table 1. Inhibition of HDAC activity

Example D: MCF7 Breast Cancer Cell Proliferation Assay

Cell proliferation assays were performed using the CyQuant (RTM) assay system (Invitrogen) according to the manufacturer's instructions. Human MCF7 breast cancer cells were plated in 96 well plates, at a density of 1000 cells in 100μL of cell culture medium per well. Compounds were added a minimum of 5 hours later, in serial dilutions in cell culture medium, in 100 μL volumes at 2x final concentration. Cell culture medium was removed after 6 days by inversion of the plate onto blotting paper and cells were gently washed once with 200μL PBS. Plates were frozen immediately for a minimum of one hour at -8O ⁰ C and then thawed. 200 μL of 1x CyQuant (RTM) cell lysis buffer supplemented with dye, made according to the manufacturer's instruction, was added immediately to each well and incubated at room temperature for 3-5 minutes. Fluorescence was then measured for each well using a Cytofluor Il Fluoresence Multiwell Plate Reader and CytoFluor Il software with filters at 480 nm for excitation and 520 nm for emission maxima. Cell proliferation was determined for mean values of duplicate samples as percentages relative to untreated cell samples (=100%). IC ₅₀ values were calculated using GraphPad Prism software. The results obtained using the MCF7 cell line are shown in Table 2. Table 2. Cell growth inhibition (IC ₅₀ values (nM))