NZ577006A - Hydroxamates as inhibitors of histone deacetylase - Google Patents

Abstract

Disclosed is a hydroxamate compound of formula (I) where the substituents are as disclosed in the description. Examples of compound of formula (I) include: cyclopentyl N-{ 1-[4-hydroxycarbamoyl)phenyl]piperidin-4-yl} -L-leucinate; cyclopentyl (2S)-cyclohexyl{ [(4-{ [({ 1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl} methyl)amino]methyl} cyclohexyl)methyl] amino} acetate and cyclopentyl (2S)-cyclohexyl[(4-{ [({ 1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl} methyl)amino]methyl} benzyl)amino]acetate. Also disclosed is a pharmaceutical composition comprising a compound of formula (I) and the use of a compound of formula (I) in the manufacture of a medicament for treating cell proliferation, Huntington disease, Alzheimer disease or rheumatoid arthritis.

Description

New Zealand Paient Spedficaiion for Paient Number 577006 HYDROXAMATES AS INHIBITORS OF HISTONE DEACETYLASE This invention relates to compounds which inhibit members of the histone deacetylase family of enzymes and to their use in the treatment of cell proliferative diseases, including cancers, polyglutamine diseases, for example Huntingdon disease, neurodegenerative diseases for example Alzheimer disease, autoimmune disease for example rheumatoid arthritis and organ transplant rejection, diabetes, haematological disorders, inflammatory disease, cardiovascular disease, atherosclerosis, and the inflammatory sequelae of infection.

Background to the Invention In eukaryotic cells DNA is packaged with histones, to form chromatin. Approximately 150 base pairs of DNA are wrapped twice around an octamer of histones (two each of histones 2A, 2B, 3 and 4) to form a nucleosome, the basic unit of chromatin. The ordered structure of chromatin needs to be modified in order to allow transcription of the associated genes. Transcriptional regulation is key to differentiation, proliferation and apoptosis, and is, therefore, tightly controlled. Control of the changes in chromatin structure (and hence of transcription) is mediated by covalent modifications to histones, most notably of the N-terminal tails. Covalent modifications (for example methylation, acetylation, phosphorylation and ubiquitination) of the side chains of amino acids are enzymatically mediated (A review of the covalent modifications of histones and their role in transcriptional regulation can be found in Berger SL 2001 Oncogene 20, 3007-3013; See Grunstein M 1997 Nature 389, 349-352; Wolffe AP 1996 Science 272, 371-372; and Wade PA et al 1997 Trends Biochem Sci 22, 128-132 for reviews of histone acetylation and transcription).

Acetylation of histones is associated with areas of chromatin that are transcriptionally active, whereas nucleosomes with low acetylation levels are, typically, transcriptionally silent. The acetylation status of histones is controlled by two enzyme classes of opposing activities; histone acetyltransferases (HATs) and histone deacetylases (HDACs). In transformed cells it is believed that inappropriate expression of HDACs results in silencing of tumour suppressor genes (For a review of the potential roles of HDACs in tumorigenesis see Gray SG and Teh BT 2001 Curr Mol Med 1, 401-429). Inhibitors of HDAC enzymes have been described in the literature and shown to induce transcriptional reactivation of certain genes resulting in the inhibition of cancer cell proliferation, induction RECEIVED AT IPONZ 1/12/11 2 of apoptosis and inhibition of tumour growth in animals (For review see Kelly WK et al 2002 Expert Opin Investig Drugs 11, 1695-1713). Such findings suggest that HDAC inhibitors have therapeutic potential in the treatment of proliferative diseases such as cancer (Kramer OH et al 2001 Trends Endocrinol 12, 294-300, Vigushin DM and Coombes RC 2002 Anticancer Drugs 13, 1-13).

In addition, others have proposed that aberrant HDAC activity or histone acetylation is implicated in the following diseases and disorders; polyglutamine disease, for example Huntington disease (Hughes RE 2002 Curr Biol 12, R141-R143; McCampbell A et al 2001 Proc Soc Natl Acad Sci 98, 15179-15184; Hockly E et al 2003 Proc Soc Natl Acad Sci 100, 2041-2046), other neurodegenerative diseases, for example Alzheimer disease (Hempen B and Brion JP 1996, J Neuropathol Exp Neurol 55, 964-972), autoimmune disease and organ transplant rejection (Skov S et al 2003 Blood 101, 14 30-1438; Mishra N et al 2003 J Clin Invest 111, 539-552), diabetes (Mosley AL and Ozcan S 2003 J Biol Chem 278, 19660 - 19666) and diabetic complications, infection (including protozoal infection (Darkin-Rattray, SJ et al 1996 Proc Soc Natl Acad Sci 93, 13143-13147)) and haematological disorders including thalassemia (Witt O et al 2003 Blood 101, 2001-2007). The observations contained in these manuscripts suggest that HDAC inhibition should have therapeutic benefit in these, and other related, diseases Many types of HDAC inhibitor compounds have been suggested, and several such compounds are currently being evaluated clinically, for the treatment of cancers. For example, the following patent publications disclose such compounds: US 5,369,108 and WO 01/18171 US 4,254,220 3 WO 04/092115 WO 04/0224991 WO 05/014588 WO 05/018578 WO 05/019174 WO 05/007091 WO 05/030704 WO 05/013958 WO 05/028447 WO 05/02690 Many of the HDAC inhibitors known in the art have a structural template, which may be represented as in formula (A): wherein ring A is a carbocyclic or heterocyclic ring system with optional substituents R, and [Linker] is a linker radical of various types. The hydroxamate group functions as a metal binding group, interacting with the metal ion at the active site of the HDAC enzyme, which lies at the base of a pocket in the folded enzyme structure. The ring or ring system A lies within or at the entrance to the pocket containing the metal ion, with the -[Linker]-radical extending deeper into that pocket linking A to the metal binding hydroxamic acid group. In the art, and occasionally herein, the ring or ring system A is sometimes informally referred to as the "head group" of the inhibitor.

International Patent application No. PCT/GB2006/001779 describes and claims a new class of HDAC inhibitors whose structures fit the generalised template (A). That new class consists of compounds of formula (B) and their salts, N-oxides, hydrates and solvates: (A) Q=V HO ^ [Linkerl ]—B—[Linker2] W > (B) O wherein Q, V and W independently represent -N= or -C=; B is a divalent radical selected from (B1), (B2), (B3), (B4), and (B5).

RECEIVED at IPONZ on 11 October 2011 4 -N -N N- (B1) (B2) (B3) N— (B4) (B5) wherein the bond marked * is linked to the ring containing Q, V and W through -[Linkerl ]- and the bond marked ** is linked to A through -[Linker2]-; A is an optionally substituted mono-, bi- ortri-cyclic carbocyclic or heterocyclic ring system; and -[Linkerl]- and -[Linker2]- independently represent a bond, or a divalent linker radical.

Brief Description of the Invention This invention is based on the finding that the introduction of an alpha amino acid ester grouping into the HDAC inhibitor molecular template (B) and certain structurally similar templates, facilitates penetration of the agent through the cell membrane, and thereby allows intracellular carboxylesterase activity to hydrolyse the ester to release the parent acid. Being charged, the acid is not readily transported out of the cell, where it therefore accumulates to increase the intracellular concentration of active HDAC inhibitor. This leads to increases in potency and duration of action. The invention therefore makes available a class of compounds which are alpha amino acid conjugates of structures (B) and certain related strcutures. The alpha amino acid ester moiety is a substrate for intracellular carboxylesterase (also referred to herein as an "esterase motif). Such conjugates, and the corresponding de-esterified parent acids, have pharmaceutical utility in the treatment of diseases such as cancers which benefit from intracellular inhibition of HDAC.

Detailed Description of the Invention Broadly disclosed herein, is a compound of formula (I), or a salt, N-oxide, hydrate or solvate thereof: HO Q=V O ■[B]m—[Linker]—[A]n Z1 (I) wherein m and n are independently 0 or 1, provided that at least one of m and n is 1; Q, V and W independently represent -N= or -C=; B is a divalent radical selected from (B1), (B2), (B3), (B4), (B5) and (B6): N *—N N- (B1) (B2) . (B3) -N N- r -N A r v N— j —N V (B4) (B5) (B6) wherein the bond marked * is linked to the ring containing Q, V and W ; A is an optionally substituted mono-, bi- or tri-cyclic carbocyclic or heterocyclic ring system; -[Linker]- represents a bond, or a divalent linker radical; Z1 is (a) a radical of formula R1R2CHNH-Y-L1-X1-(CH2)Z- or (b) a radical of formula R-L1-Y1-(CH2)Z-, wherein: R is a radical of formula (X) or (Y) 6 r. r. hn d (Y) (X) Ri is a carboxylic acid group (-COOH), or an ester group which is hydrolysable by one or more intracellular esterase enzymes to a carboxylic acid group; R6 is hydrogen; or optionally substituted CrC6 alkyl, C3-C7cycloaIkyl, aryl or heteroaryl or -(C=0)R3, -(C=0)0R3, or-(C=0)NR3 wherein R3 is hydrogen or optionally substituted (CrC6)alkyl.

R2 is the side chain of a natural or non-natural alpha amino acid; Y is a bond, -C(=0)-, -S(=0)2-, -C(=0)0-, -C(=0)NR3-, -C(=S)-NR3, -C(=NH)-NR3 or -S(=0)2NR3- wherein R3 is hydrogen or optionally substituted CrC6 alkyl; Y1 is a bond, -(C=0)-, -S(02)-, -C(=0)0-, -0C(=0)-, -(C=0)NR3-, -NR3(C=0)-, -S(02)NR3-, -NR3S(02)-, or -NR3(C=0)NR4-, wherein R3 and R4 are independently hydrogen or optionally substituted (Ci-C6)alkyl, L1 is a divalent radical of formula -(Alk1)m(Q)n(Alk2)p- wherein m, n and p are independently 0 or 1, Q is (i) an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5-13 ring members, or (ii), in the case where p is 0, a divalent radical of formula -Q1-X2- wherein X2 is -0-, -S- or NRA-wherein RA is hydrogen or optionally substituted CrC3 alkyl, and Q1 is an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5-13 ring members, Alk1 and Alk2 independently represent optionally substituted divalent C3-C7 cycloalkyl radicals, or optionally substituted straight or branched, CrC6 alkylene, C2-C6 alkenylene, or C2-C6 alkynylene radicals which may RECEIVED at IPONZ on 11 October 2011 7 optionally contain or terminate in an ether (-O-), thioether (-S-) or amino (-NRa-) link wherein RA is hydrogen or optionally substituted C1-C3 alkyl; X1 is a bond, -C(=0)-; or-S(=0)2-; -NR4C(=0)-, -C(=0)NR4-, -NR4C(=0)-NR5-, -NR4S(=0)2-, or -S(=0)2NR4- wherein R4 and R5 are independently hydrogen or optionally substituted C^-Ce alkyl; and z is 0 or 1.

Specifically, in a first aspect, the present invention provides a compound of formula (I), or a salt, N-oxide, hydrate or solvate thereof: Q=V (I) O wherein n is 0 or 1; Q, V and W independently represent -N= or-C=; B is a divalent radical selected from (B1), (B2), (B3), (B4), (B5) and (B6): (B1) (B2) (B3) N \ / N / I \ (B4) (B5) (B6) (followed by page 7a) RECEIVED at IPONZ on 11 October 2011 7a wherein the bond marked * is linked to the ring containing Q, V and W ; A is selected from the following ring systems, optionally substituted: M N (followed by page 7b) RECEIVED at IPONZ on 11 October 2011 (followed by page 7c) RECEIVED at IPONZ on 11 October 2011 7c o> a c? $ o*- N —■j-vW .N ° ck N Rio — wherein Rio is hydrogen or CrC6 alkyl, the bond intersected by the wavy line connects to the -[Linker]- radical, and Z1 is attached to an available ring atom; -[Linker]- is selected from: (i) a bond; (ii) -0-, -S-, -C(=0)-, -S(=0)2-, -NRC-, -C(=0)NRc-, -S(=0)2NRc-, -NRcC(=0)-, -NRcS(=0)2-,-NRc(CH2)rn-,-NRcC(=0)(CH2)m-,-NRcS(=0)2(CH2)m, -NRDC(=0)NRc-, or -NRcC(=0)(CH2)mAr-, or-NRcS(=0)2(CH2)mAr-wherein Rc and R° are independently hydrogen, CrC4 alkyl or a nitrogen substituent, m is 1, 2 or 3, and Ar is a divalent phenyl radical or a divalent mono-, or bi-cyclic heteroaryl radical having 5 to 13 ring members; and (iii) an optionally substituted, straight or branched, Ci-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene radical which may optionally contain or terminate in an ether (-0-), thioether (-S-) or amino (-NRA-) link wherein RA is hydrogen, CrC3 alkyl or a nitrogen substituent; Z1 is (a) a radical of formula RiR2CHNH-Y-L1-X1-(CH2)z- or (b) a radical of formula R-L1-Y1-(CH2)Z-, wherein: (followed by page 7d) RECEIVED at IPONZ on 11 October 2011 R is a radical of formula (X) or (Y) R R hn d (Y) (X) Ri is an ester group which is hydrolysable by one or more intracellular esterase enzymes to a carboxylic acid group; R6 is hydrogen; or optionally substituted CrC6 alkyl, C3-C7 cycloalkyl, aryl or heteroaryl or-(C=0)R3, -(C=0)0R3, or-(C=0)NR3 wherein R3 Is hydrogen or optionally substituted (Ci-C6)alkyl; Ring D is a pyrrole or piperidine ring; and (i) C-I-C6 alkyl, phenyl, 2,- 3-, or 4-hydroxyphenyl, 2,- 3-, or 4-methoxyphenyl, 2,-3-, or 4-pyridylmethyl, benzyl, phenylethyl, 2-, 3-, or 4-hydroxybenzyl, 2,- 3-, or 4-benzyloxybenzyl, 2,- 3-, or 4- CrC6 alkoxybenzyl, or benzyloxy(C1-C6alkyl)-groups; or (ii) the characterising group of a natural a amino acid, in which any functional group may be protected; or (iii) a group -[Alk]nR6 where Alk is a (CrC6)alkyl or (C2-C6)alkenyl group optionally interrupted by one or more -0-, or -S- atoms or -N(R7)- groups [where R7 is a hydrogen atom or a (C1-C6)alkyl group], n is 0 or 1, and R6 is an optionally substituted cycloalkyl or cycloalkenyl group; or (iv) a benzyl group substituted in the phenyl ring by a group of formula -OCH2COR8 where R8 is hydroxyl, amino, (CrC6)alkoxy, phenyl(Cr R2 is (followed by page 7e) RECEIVED at IPONZ on 11 October 2011 7e C6)alkoxy, (C-i-CeJalkylamino, di((C1-C6)alkyl)amino, phenyl(Cr C6)alkylamino, the residue of an amino acid or acid halide, ester or amide derivative thereof, said residue being linked via an amide bond, said amino acid being selected from glycine, a or/? alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histidine, arginine, glutamic acid, and aspartic acid; or (v) a heterocyclic(CrC6)alkyl group, either being unsubstituted or mono- or di-substituted in the heterocyclic ring with halo, nitro, carboxy, (Cr C6)alkoxy, cyano, (Ci-C6)alkanoyl, trifluoromethyl (C-i-C6)alkyl, hydroxy, formyl, amino, (CrCeJalkylamino, di-(Ci-C6)alkylamino, mercapto, (Cr C6)alkylthio, hydroxy(C1-C6)alkyl, mercapto(CrC6)alkyl or (Cr C6)alkylphenylmethyl; or (vi) a group -CRaRbRc in which: each of Ra, Rb and Rc is independently hydrogen, (Ci-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, pheny^CrCeJalkyl, (C3-C8)cycloalkyl; or Rc is hydrogen and Ra and Rb are independently phenyl or heteroaryl; or Rc is hydrogen, (CrC6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(Cr C6)alkyl, or (C3-C8)cycloalkyl, and Ra and Rb together with the carbon atom to which they are attached form a 3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic ring; or Ra, Rb and Rc together with the carbon atom to which they are attached form a tricyclic ring; or Ra and Rb are each independently (CrC6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyKC-i-CeJalkyl, or a group as defined for Rc below other than hydrogen, or Ra and Rb together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic ring, and Rc is hydrogen, - (followed by page 7f) RECEIVED at IPONZ on 11 October 2011 7f OH, -SH, halogen, -CN, -C02H, (CrC4)perfluoroalkyl, -CH2OH, -C02(Cr C6)alkyl, -0(CrC6)alkyl, -0(C2-C6)alkenyl, -S(CrC6)alkyl, -SO^-C6)alkyl, -S02(C-|-C6) alkyl, -S(C2-C6)alkenyI, -SO(C2-Ce)alkenyl, -S02(C2-C6)alkenyl or a group -Q-W wherein Q represents a bond or -0-, -S-, -SO-or -S02- and W represents a phenyl, phenylalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkylalkyl, (C4-C8)cycloalkenyl, (C4-C8)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, which group W may optionally be substituted by one or more substituents independently selected from, hydroxyl, halogen, -CN, -C02H, -C02(C1-C6)alkyl, -CONH2, -CONH(Cr C6)alkyl, -CONH(C1-C6alkyl)2, -CHO, -CH2OH, (C1-C4)perfluoroalkyl, -O^-Cejalkyl, -S(CrC6)alkyl, -SO(CrC6)alkyl, -SO^CrCeJalkyl, -N02, -NH2, -NH(C1-C6)alkyl, -N((CrC6)alkyl)2, -NHCO(C1-Ce)alkyl, (CrC6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C3-C8)cycloalkyl, (C4-C8)cycloalkenyl, phenyl or benzyl; Y is a bond, -C(=0)-, -S(=0)2-, -C(=0)0-, -C(=0)NR3-, -C(=S)-NR3, -C(=NH)-NR3 or -S(=0)2NR3- wherein R3 is hydrogen or optionally substituted CrCe alkyl; Y1 is a bond, -(C=0)-, -S(02)-, -C(=0)0-, -0C(=0)-, -(C=0)NR3-, -NR3(C=0)-, -S(02)NR3-, -NR3S(02)-, or -NR3(C=0)NR4-, wherein R3 and R4 are independently hydrogen or optionally substituted (CrC6)alkyl, L1 is a divalent radical of formula -(Alk1)m(Q)n(Alk2)p- wherein m, n and p are independently 0 or 1, Q is (i) an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5-13 ring members, or (ii), in the case where p is 0, a divalent radical of formula -Q1-X2- wherein X2 is -0-, -S- or NRA-wherein RA is hydrogen or optionally substituted Q-Cs alkyl, and Q1 is an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5-13 ring members, Alk1 and Alk2 independently represent optionally substituted divalent C3-C7 cycloalkyl radicals, or optionally substituted straight or branched, CrC6 (followed by page 7g) RECEIVED AT IPONZ 1/12/11 7g alkylene, C2-C6 alkenylene, or C2-C6 alkynylene radicals which may optionally contain or terminate in an ether (-0-), thioether (-S-) or amino (-NRa-) link wherein RA is hydrogen or optionally substituted C1-C3 alkyl; X1 is a bond, -C(=0)-; or-S(=0)2-; -NR4C(=0)-, -C(=0)NR4-, -NR4C(=0)-NR5-, -NR4S(=0)2-, or -S(=0)2NR4- wherein R4 and R5 are independently hydrogen or optionally substituted CrC6 alkyl; and z is 0 or 1; and wherein the term "optionally substituted" means substituted with up to four compatible substituents, independently selected from CrC6alkyl, C-pCealkoxy, hydroxy, hydroxyGr C6alkyl, mercapto, mercaptoC-i-Cealkyl, CrC6alkylthio, phenyl, halo including fluoro, bromo and chloro, trifluoromethyl, trifluoromethoxy, nitro, nitrile -CN, oxo, -COOH, -COORa, -CORa, -S02Ra, -CONH2, -S02NH2i -CONHRa, -S02NHRa, -CONRaRb, -SO2NRaRb, -NH2, -NHRa, -NRaRb, -OCONH2, -OCONHRa , -OCONRaRb, -NHCORa, -NHCOORa, -NRbCOORa, -NHS020Ra, -NRbS020H, -NRbS020Ra, -NHCONH2i -NRaCONH2,-NHCONHRb-NRaCONHRb, -NHCONRaRb or-NRACONRARB wherein Ra and RB are independently a CrC6alkyl, (C3-C6) cycloalkyl, phenyl or monocyclic heteroaryl having 5 or 6 ring atoms, or RA and RB when attached to the same nitrogen atom forms a cyclic amino group.

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound according to the first aspect, together with a pharmaceutically acceptable carrier.

In a third aspect, the present invention provides a use of a compound according to the first aspect in the preparation of a composition for the treatment of cancer cell proliferation, Huntington disease, Alzheimer disease or rheumatoid arthritis.

Although the above definition potentially includes molecules of high molecular weight, it is preferable, in line with general principles of medicinal chemistry practice, that the compounds with which this invention is concerned should have molecular weights of no more than 600. (followed by page 7h) RECEIVED at IPONZ on 11 October 2011 7h Also disclosed herein, is the use of a compound of formula (I), or an N-oxide, salt, hydrate or solvate thereof in the preparation of a composition for inhibiting the activity of histone deacetylase.

The compounds of formula (I) may be used for the inhibition of histone deacetylase activity, ex vivo or in vivo.

Also disclosed herein, are compounds of formula (I) which may be used in the preparation of a composition for the treatment of cell-proliferation disease, for example cancer cell proliferation and autoimmune diseases.

Also disclosed herein, is a method for the treatment of the foregoing disease types, which comprises administering to a subject suffering such disease an effective amount of a compound of formula (I) as defined above.

Terminology The term "comprising" as used in this specification means "consisting at least in part of. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. (followed by page 7i) RECEIVED at IPONZ on 11 October 2011 7i In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

As used herein, the term "(Ca-Cb)alkyl" wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms. Thus when a is 1 and b is 6, for example, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl. (followed by page 8) WO 2008/053131 PCT/GB2006/004034 8 As used herein the term "divalent (Ca-Cb)alkylene radical" wherein a and b are integers refers to a saturated hydrocarbon chain having from a to b carbon atoms and two unsatisfied valences.

As used herein the term "(Ca-Cb)alkenyr wherein a and b are integers refers to a straight or branched chain alkenyl moiety having from a to b carbon atoms having at least one double bond of either E or Z stereochemistry where applicable. The term includes, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.

As used herein the term "divalent (Ca-Cb)alkenylene radical" means a hydrocarbon chain having from a to b carbon atoms, at least one double bond, and two unsatisfied valences.

As used herein the term "Ca-Cb alkynyl" wherein a and b are integers refers to straight chain or branched chain hydrocarbon groups having from a to b carbon atoms and having in addition one triple bond. This term would include for example, ethynyl, 1-propynyl, 1- ; and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.

As used herein the term "divalent (Ca-Cb)alkynylene radical" wherein a and b are integers refers to a divalent hydrocarbon chain having from a to b carbon atoms, and at least one triple bond.

As used herein the term "carbocyclic" refers to a mono-, bi- or tricyclic radical having up to 16 ring atoms, all of which are carbon, and includes aryl and cycloalkyl.

As used herein the term "cycloalkyl" refers to a monocyclic saturated carbocyclic radical having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the unqualified term "aryl" refers to a mono-, bi- ortri-cyclic carbocyclic aromatic radical, and includes radicals having two monocyclic carbocyclic aromatic rings which are directly linked by a covalent bond. Illustrative of such radicals are phenyl, biphenyl and napthyl.

As used herein the unqualified term "heteroaryl" refers to a mono-, bi- or tri-cyclic aromatic radical containing one or more heteroatoms selected from S, N and O, and includes WO 2008/053131 PCT/GB2006/004034 9 radicals having two such monocyclic rings, or one such monocyclic ring and one monocyclic aryl ring, which are directly linked by a covalent bond. Illustrative of such radicals are thienyl, benzthienyl, furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.

As used herein the unqualified term "heterocyclyl" or "heterocyclic" includes "heteroaryl" as defined above, and in its non-aromatic meaning relates to a mono-, bi- or tri-cyclic non-aromatic radical containing one or more heteroatoms selected from S, N and O, and to groups consisting of a monocyclic non-aromatic radical containing one or more such heteroatoms which is covalently linked to another such radical or to a monocyclic carbocyclic radical. Illustrative of such radicals are pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido groups.

Unless otherwise specified in the context in which it occurs, the term "substituted" as applied to any moiety herein means substituted with up to four compatible substituents, each of which independently may be, for example, (CrC6)alkyl, (C1-C8)alkoxy, hydroxy, hydroxy(C-rC6)alkyl, mercapto, mercaptoCC-i-C^alkyl, (C1-C6)alkylthio, phenyl, halo (including fluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, -COOH, -COORa, -CORa, -S02Ra, -CONH2i -S02NH2, -CONHRa, -S02NHRa, -conrarb, -so2nrarb, -nh2, -nhra, -nrarb, -oconh2, -OCONHRa , -OCONRaRb, -NHCORa, -NHCOORa, -NRbCOORa, -NHSO2ORa, -NRbS020H, -NRbS020Ra, -nhconh2, -NRaCONH2,-NHCONHRb-NRaCONHRb, -NHCONRaRb or-NRaCONRaRb wherein RA and RB are independently a (C-i-C^alkyl, (C3-C6) cycloalkyl, phenyl or monocyclic heteroaryl having 5 or 6 ring atoms, or RA and RB when attached to the same nitrogen atom form a cyclic amino group(for example morpholino, piperidinyl, piperazinyl, or tetrahydropyrrolyl). An "optional substituent" may be one of the foregoing substituent groups.

As used herein, the term "nitrogen substituent" means a substituent on a nitrogen atom which is selected from the following: WO 2008/053131 PCT/GB2006/004034 amino C-,.6 alkyl eg aminoethyl, C-,_3 alkylamino C^ alkyl, C1.3 dialkylamino C-|.6 alkyl, hydroxy alkyl eg hydroxyethyl, C1.3 alkoxy C -,.6 alkyl eg methoxyethyl, mercapto C-,.3 alkyl, C-,.3 alkylmercapto C-,.6 alkyl, carboxamido C-,.6 alkyl e.g. -CH2CONH2, aminosulphonyl C-|.6 alkyl e.g. -CH2S02NH2, C-,^ alkylaminosulphonyl C^e alkyl e.g. -CH2S02NHMet C^ dialkylaminosulphonyl Ci.6alkyl e.g. -CH2S02NMe2, C-,.6 alkanoyl, Ci.6 alkylsulphonyl, aminosulphonyl (-S02NH2), Ci.6 alkylaminosulphonyl e.g. - S02NHMe, C1.6 dialkylaminosulphonyl e.g. - S02NMe2, optionally substituted phenylaminosulphonyl, carboxamido (-CONH2), C-,.6 alkylaminocarbonyl, C-,_6 dialkylaminocarbonyl, morpholinyl C-,.6 alkyl, imidazolyl C^e alkyl, triazolyl C-,.6 alkyl, or monocyclic heterocycloalkyl C^e alkyl, optionally substituted in the imidazolyl, triazolyl or heterocyclyl ring, eg piperidinyl C-,.6 alkyl, piperazinyl C-,.6 alkyl or 4-(C-,.6 alkyl)piperazinyl C^e alkyl.

As used herein the term "salt" includes base addition, acid addition and quaternary salts. Compounds of the invention which are acidic can form salts, including pharmaceutic,ally , acceptable salts, with bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium :• hydroxides; with organic bases e.g. N-methyl-D-glucamine, choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethyl piperidine, dibenzylamine and the like. Those compounds (I) which are basic can form salts, including pharmaceutical^ acceptable salts with inorganic acids, e.g. with hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like, and with organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic, p-toluenesulphonic, benzoic, benzenesunfonic, glutamic, lactic, and mandelic acids and the like.

Compounds of the invention which contain one or more actual or potential chiral centres, because of the presence of asymmetric carbon atoms, can exist as a number of enantiomers or diastereoisomers with R or S stereochemistry at each chiral centre. The invention includes all such enantioners or diastereoisomers and mixtures thereof.

In the compounds of the invention, in any compatible combination, and bearing in mind that the compounds preferably have a molecular weight of less than 600: The hvdroxamate group -C(=Q)NHOH WO 2008/053131 PCT/GB2006/004034 11 In the compounds of the invention, the hydroxamate group functions as a metal binding group, interacting with the metal ion at the active site of the HDAC enzyme, which lies at the base of a pocket in the folded enzyme structure.

The ring containing Q. V and W Each of Q, V and W may be -C=, or at least one of Q, V and W may be -N=, or Q may be -C= and V and W may each be -N=; Currently preferred is the case where Q is -C=, V and W are each be -N= and the H0NHC(=0)~ radical is attached to the 5-position of the resultant pyrimidin-2-yl radical.

The ring A Ring A radicals may be, for example, optionally substituted aromatic carbocyclic such as optionally substituted phenyl and naphthyl, or optionally substituted heteroaromatic such as optionally.substituted pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl,isoxazolyl, thiazolyl,, isothiazolyl,'pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, 1,2,5-triazolyl, 1,2,3-oxadiazolyl, ..1-,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazole, < pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, . isobenzothienyl, indolyl, isoindolyl, indanyl, 3H-indolyl, benzimidazolyl, indazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolizinyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnolinyl, naphthyridinyl, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl or benzoquinolyl radicals.

Ring A radicals may also be, for example, optionally substituted non aromatic carbocyclic and heterocyclic, such as optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2-cyclobuten-1-yl, 2-cyclopenten-1-yl, 3-cyclopenten-1-yl, 2,4-cyclopentadien-1-yl, 3,5-cyclohexadien-1-yl, tetrahydrofuranyl, pyrroline, eg 2- or 3-pyrrolinyl, pyrrolidinyl, dioxolanyl, eg 1,3-dioxolanyl, imidazolinyl, eg 2-imidazolinyl, imidazolidinyl, pyrazolinyl, eg 2-pyrazolinyl, pyrazolidinyl, pyranyl, eg 2- or4-pyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, piperazinyl, 1,3,5-trithianyl, oxazinyl, eg 2H-1,3-, 6H-1,3-, H-1,2-, 2H-1,2- or4H1,4- oxazinyl, 1,2,5-oxathiazinyl, isoxazinyl, oxathiazinyl, eg 1,2,5 or 1,2,6-oxathiazinyl, or 1,3,5-oxadiazinyl radicals.

Specific ring A radicals include the following ring systems, optionally substituted: 12 13 WO 2008/053131 PCT/GB2006/004034 14 CO 0* Cf ($ (y &(X&° 9- wherein R10 is hydrogen or CrC6 alkyl, the bond intersected by the wavy line connects to the -[Linker]- radical and the radical R in formula (I) is attached to any available ring atom.

Optional substituents in A may be, for example methyl, ethyl, n-propyl, isopropyl, fluorine, chlorine, bromine or iodine atoms, or a methylamino, ethylamino, hydroxymethyl, hydroxyethyl, methylthiol, ethylthiol, methoxy, ethoxy, n-propoxy, 2-hydroxyethoxy, 3-hydroxypropoxy, 2-aminoethoxy, 3-aminopropoxy, 2-(methylamino)ethoxy, 2-(dimethylamino)ethoxy,3-(dimethylamino)propoxy, cyclopentyl, cyclohexyl, cyclohexylamino, trifluoromethyl, trifluoromethoxy, amino (-NH2), aminomethyl, aminoethyl, dimethylamino, diethylamino, ethyl(methyl)amino, propyl(methyl)amino, 2-hydroxyethylamino, 3-hydroxypropylamino, 2-aminoethylamino, 3-aminopropylamino, 2-(methylamino)ethylamino, 2-(ethylamino)ethylamino, 2-(isopropylamino)ethylamino, 3-(isopropylamino)propylamino, 2-(dimethylamino)ethylamino, 2-(diethylamino)ethylamino, 2-(methylamino)ethyl(methyl)amino, 3-(methylamino)propyl(methyl)amino, nitro, cyano, hydroxyl, formyl, carboxyl (-C02H), -CH2C02H, -0CH2C02H, -C02CH3, -C02CH2CH3, -CH2C02CH2CH3, -CH2C02CH2Ph, t-butoxycarbonylmethoxy, acetyl, phenacyl, thio, thiomethyl, thioethyl, sulphonyl, methylsulphonyl, methylaminosulphonyl, ethylaminosulphonyl, dimethylaminosulphonyl, carboxamido, methylaminocarbonyl, ethylaminocarbonyl, dimethylaminocarbonyl, diethylaminocarbonyl, methylaminocarbonylmethyl, -NHC(S)NH2, sulphonylamino (-NHS02H), methylsulphonylamino, dimethylsulphonylamino, aminosulphonylamino, (-NHS02NH2), methylaminosulphonylamino, dimethylaminosulphonylamino, methylaminocarbonylamino, dimethylaminocarbonylamino, acetylamino, phenylcarbonylamino, aminomethylcarbonylamino, acetylaminomethyl, methoxycarbonylamino, t-butoxycarbonylamino, pyrrolidinyl, piperidynyl, piperazinyl, 4-methylpiperazinyl, WO 2008/053131 PCT/GB2006/004034 homopiperazinyl, morpholinyl, imidazolyl, 1,2,4-triazolyl, 1.2.3-triazolyl, 1,3,4-triazolyl, 1,2,5-triazolyl, C-|.6 straight or branched chain alkyl, amino alkyl eg aminoethyl, Ci_3 alkylamino C^e alkyl, C^ dialkylamino alkyl, hydroxyl C^ alkyl eg hydroxyethyl, C-i.3 alkoxyl C -,.6 alkyl eg methoxyethyl, thiol C^ alkyl C^e, C^ alkylthiol Ci.e alkyl, C-,.6 alkanoyl, C-|.6 alkylsulphonyl, aminosulphonyl (-S02NH2), C-i_6 alkylaminosulphonyl e.g. -S02NHMe, C-,.6 dialkylaminosulphonyl e.g. - S02NMe2, optionally substituted phenylaminosulphonyl, carboxamido (-CONH2), carboxamido C^ alkyl e.g. CH2CONH2, Ci.6 alkylaminocarbonyl, C-|.6 dialkylaminocarbonyl, aminosulphonyl C-|.6 alkyl e.g. CH2S02NH2, C-,.3 alkylaminosulphonyl C^e alkyl e.g. CH2S02NHMe, C-|.3 dialkylaminosulphonyl C^e alkyl e.g. CH2S02NMe2, C-^ morpholinyl C^ealkyl, optionally substituted imidazolyl C^e alkyl, optionally substituted triazolyl Ci_6 alkyl, optionally substituted hetero C3.6 cycloalkyl Ci.6 alkyl eg piperidinyl Ci_6 alkyl, piperazinyl C^e alkyl, and 4-(C1-e alkyl)piperazlnyl alkyl.

Currently preferred rings A include optionally substituted phenyl, cyclohexyl, naphthyl, quinolin-2-yl, and 1,3-dihydro-isoindol-2-yl; Substituents which may be present in such preferred rings A include halogen, particularly fluoro and chloro. Specifically the radical -A- in formula (I) above, when present, may be 1,4-phenylene or 1,4-cyclohexylene.

The I,Linker1 radical -[Linker]- serves to link the divalent B radical to the ring A, when present. Thus, it may be selected from the following examples: (i) a bond. This will normally be the case when ring A is not present; (ii) -0-, -S-, -C(=0)-, -S(=0)2-, -NRC-, -C(=0)NRc-, -S(=0)2NRc-, -NRcC(=0)-, -NRcS(=0)2-,-NRc(CH2)m-,-NRGC(=0)(CH2)m-, -NRcS(=0)2(CH2)m, - NRdC(=0)NRc-, -NRcC(=0)(CH2)mAr-, or -NRcS(=0)2(CH2)mAr- wherein Rc and R° are independently hydrogen, C1-C4 alkyl, or a nitrogen substituent, m is 0, 1, 2, 3, 4 or 5 and Ar is a divalent phenyl radical or a divalent mono-, or bi-cyclic heteroaryl radical having 5 to 13 ring members; and (iii) an optionally substituted, straight or branched, CrC6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene radical which may optionally contain or terminate in an ether (-0-), thioether (-S-) or amino (-NRA-) link wherein RA is hydrogen, CrC3 alkyl, or a nitrogen substituent; When -Ar- is present in-[Linker]- it may be a divalent radical selected from the following: 16 /=H wherein X is O, S or NH. For example, -Ar- when present in-[Linker]- may be a divalent phenylene, such as a 1,4-phenylene, radical.

Examples of-[Linker]- include those present in the compounds of the specific examples herein.

The group Z1 Case (a): Z1 is a radical of formula -(CH7)7-X1-L1-Y- NHCHR1R? The group Ri in Z1 case (a) The ester group Fl| must be one which in the compound of the invention is hydrolysable by one or more intracellular carboxyesterase enzymes to a carboxylic acid group. Intracellular carboxyesterase enzymes capable of hydrolysing the ester group of a compound of the invention to the corresponding acid include the three known human enzyme isotypes hCE-1, hCE-2 and hCE-3. Although these are considered to be the main enzymes, other enzymes such as biphenylhydrolase (BPH) may also have a role in hydrolysing the ester. In general, if the carboxyesterase hydrolyses the free amino acid ester to the parent acid it will, subject to the N-carbonyl dependence of hCE-2 and hCE-3 discussed above, also hydrolyse the ester motif when covalently conjugated to the HDAC inhibitor. Hence, the broken cell assay and/or the isolated carboxyesterase assay described herein provide a 16 \\ / n: * // n- 7 1 //> x -n \\ // n-n // n- 7 n=n :n ^ i -n n=n wherein X is O, S or NH. For example, -Ar- when present in-[Linker]- may be a divalent phenylene, such as a 1,4-phenylene, radical.

Examples of-[Linker]- include those present in the compounds of the specific examples herein.

The group Z1 Case (a): Z1 is a radical of formula -(CH?h-X1-L1-Y- NHCHRiR, The group R± in Z1 case (a) The ester group Rt must be one which in the compound of the invention is hydrolysable by one or more intracellular carboxyesterase enzymes to a carboxylic acid group. Intracellular carboxyesterase enzymes capable of hydrolysing the ester group of a compound of the invention to the corresponding acid include the three known human enzyme isotypes hCE-1, hCE-2 and hCE-3. Although these are considered to be the main enzymes, other enzymes such as biphenylhydrolase (BPH) may also have a role in hydrolysing the ester. In general, if the carboxyesterase hydrolyses the free amino acid ester to the parent acid it will, subject to the N-carbonyl dependence of hCE-2 and hCE-3 discussed above, also hydrolyse the ester motif when covalently conjugated to the HDAC inhibitor. Hence, the broken cell assay and/or the isolated carboxyesterase assay described herein provide a WO 2008/053131 PCT/GB2006/004034 17 straightforward, quick and simple first screen for esters which have the required hydrolysis profile. Ester motifs selected in that way may then be re-assayed in the same carboxyesterase assay when conjugated to the inhibitor via the chosen conjugation chemistry, to confirm that it is still a carboxyesterase substrate in that background.

Subject to the requirement that they be hydrolysable by intracellular carboxyesterase enzymes, examples of particular ester groups Ri include those of formula -(C=0)0Rg wherein R9 is R7R8CH- wherein (i) R7 is hydrogen or optionally substituted (C1-C3)alkyl-(Z1)a-[(C1-C3)alkyl]b- or (C2-C3)alkenyl-(Z1)a-[(CrC3)alkyl]b- wherein a and b are independently 0 or 1 and Z1 is -0-, -S-, or-NR10- wherein R10 is hydrogen or CrC3 alkyl, and R8 is hydrogen or (CrC3)alkyl-; (ii) R7 is hydrogen or optionally substituted R10RnN-(CrC3)alkyl- wherein R10 is hydrogen or CrC3 alkyl and R^ is hydrogen or CrC3 alkyl; or R10 and R-n together with the nitrogen to which they are attached form an optionally substituted monocyclic heterocyclic ring of 5- or 6- ring atoms or bicyclic heterocyclic ring system of 8 to 10 ring atoms, and R8 is hydrogen or (CrC3)alkyl-; or (iii) R7 and R8 taken together with the carbon to which they are attached form an optionally substituted monocyclic carbocyclic ring of from 3 to 7 ring atoms or bicyclic carbocyclic ring system of 8 to 10 ring atoms.

Within these classes, R9 may be, for example, methyl, ethyl, n- or iso-propyl, n-, sec-or tert-butyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- or 4-pyridylmethyl, N-methylpiperidin-4-yl, tetrahydrofuran-3-yl, methoxyethyl, indanyl, norbornyl, dimethylaminoethyl, morpholinoethyl. Currently preferred is where Rgo is cyclopentyl.

Macrophages are known to play a key role in inflammatory disorders through the release of cytokines in particular TNFa and IL-1 (van Roon et al Arthritis and Rheumatism , 2003, 1229-1238). In rheumatoid arthritis they are major contributors to the maintenance of joint inflammation and joint destruction. Macrophages are also involved in tumour growth and development (Naldini and Carraro, Curr Drug Targets Inflamm Allergy ,2005, 3-8 ). Hence agents that selectively target macrophage cell proliferation could be of value in the treatment of cancer and autoimmune disease. Targeting specific cell types would be WO 2008/053131 PCT/GB2006/004034 18 expected to lead to reduced side-effects. The inventors have discovered a method of targeting inhibitors to macrophages which is based on the observation that the way in which the esterase motif is linked to the inhibitor determines whether it is hydrolysed, and hence whether or not it accumulates in different cell types. Specifically it has been found that macrophages contain the human carboxylesterase hCE-1 whereas other cell types do not. In the general formula (I) when the nitrogen of the esterase motif R1CH(R2)NH- is not directly linked to a carbonyl (-C(=0)-), ie when Y is not a -C(=0), -C(=0)0- or -C(=0)NR3-radical, the ester will only be hydrolysed by hCE-1 and hence the inhibitors will only accumulate in macrophages.

The amino acid side chain R? in Z1 case (a) Subject to the requirement that the ester group R-i be hydrolysable by intracellular carboxylesterase enzymes, the identity of the side chain group R2 is not critical.

Examples; of amino acid side chains include: C-i-Ce alkyl, phenyl, 2,- 3-, or 4-hydroxyphenyl, 2,- 3-, or 4-methoxyphenyl, 2,-3-, or4-pyridylmethyl, benzyl, phenylethyl, 2-, 3-, or 4-hydroxybenzyl, 2,- 3-, or 4-benzyloxybenzyl, 2,- 3-, or 4- CrC6 alkoxybenzyl, and benzyloxy(CrC6alkyl)-groups; the characterising group of a natural a amino acid, in which any functional group may be protected; groups -[Alk]nR6 where Alk is a (C1-C6)alkyl or (C2-C6)alkenyl group optionally interrupted by one or more -0-, or -S- atoms or -N(R7)- groups [where R7 is a hydrogen atom or a (Cr C6)alkyl group], n is 0 or 1, and R6 is an optionally substituted cycloalkyl or cycloalkenyl group; a benzyl group substituted in the phenyl ring by a group of formula -OCH2COR8 where R8 is hydroxyl, amino, (CrC6)alkoxy, phenyl(CrC6)alkoxy, (C1-C6)alkylamino, di((Cr C6)alkyl)amino, phenyl(Ci-C6)alkylamino, the residue of an amino acid or acid halide, ester or amide derivative thereof, said residue being linked via an amide bond, said amino acid being selected from glycine, a or p alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histidine, arginine, glutamic acid, and aspartic acid; 19 a heterocyclic(C1-C6)alkyl group, either being unsubstituted or mono- or di-substituted in the heterocyclic ring with halo, nitro, carboxy, (CrC6)alkoxy, cyano, (CrC6)alkanoyl, trifluoromethyl (CrC6)alkyl, hydroxy, formyl, amino, (C1-C6)alkylamino, di-(Cr C6)alkylamino, mercapto, (C1-C6)alkylthio, hydroxy(CrC6)alkyl, mercapto(CrC6)alkyl or (CrCeJalkylphenylmethyl; and a group -CRaRbRc in which: each of Ra, Rb and Rc is independently hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(C1-C6)alkyl, (C3-C8)cycloalkyl; or Rc is hydrogen and Ra and Rb are independently phenyl or heteroaryl such as pyridyl; or Rc is hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(CrC6)alkyl, or (C3-C8)cycloalkyl, and Ra and Rb together with the carbon atom to which they are attached form a 3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic ring; or Ra, Rb and Rc together with the carbon atom to which they are attached form a tricyclic ring (for example adamantyl); or Ra and Rb are each independently (Ci-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyKCrCeJalkyl, or a group as defined for Rc below other than hydrogen, or Ra and Rb together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic ring, and Rc is hydrogen, -OH, -SH, halogen, -CN, -C02H, (Cr C4)perfluoroalkyl, -CH2OH, -C02(C1-C6)alkyl, -0(CrC6)alkyl, -0(C2-C6)alkenyl, -S(C1-C6)alkyl, -SO(CrC6)alkyl, -S02(CrC6) alkyl, -S(C2-C6)alkenyl, -SO(C2-C6)alkenyl, -S02(C2-C6)alkenyl or a group -Q-W wherein Q represents a bond or -0-, -S-, -SO- or -S02- and W represents a phenyl, phenylalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkylalkyI, (C4-C8)cycloalkenyl, (C4-C8)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, which group W may optionally be substituted by one or more substituents independently selected from, hydroxy!, halogen, -CN, -C02H, -C02(CrC6)alkyl, -CONH2, -CONH(CrCe)alkyl, -CONH(CrC6alkyl)2, -CHO, -CH2OH, (C-i-C^perfluoroalkyl, -0(CrC6)alkyl, -S(CrC6)alkyl, -SOtCrCeJalkyI, - WO 2008/053131 PCT/GB2006/004034 S02(CrC6)alkyl, -N02, -NH2, -NH(Ci-C6)alkyl, -N((CrC6)alkyl)2, -NHCO(Cr C6)alkyl, (CrC6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C3-C8)cycloalkyl, (C4- C8)cycloalkenyl, phenyl or benzyl.

Examples of particular R2 groups include hydrogen (the glycine "side chain"), benzyl, phenyl, cyclohexylmethyl, cyclohexyl, pyridin-3-ylmethyl, tert-butoxymethyl, iso-butyl, sec-butyl, tert-butyl, 1-benzylthio-1-methylethyl, 1-methylthio-1-methylethyl, 1-mercapto-1-methylethyl, and phenylethyl. Presently preferred R2 groups include phenyl, benzyl, and iso-butyl, cyclohexyl and t-butoxymethyl.

For compounds of the invention which are to be administered systemically, esters with a slow rate of carboxylesterase cleavage are preferred, since they are less susceptible to pre-systemic metabolism. Their ability to reach their target tissue intact is therefore increased, and the ester can be converted inside the cells of the target tissue into the acid product. However, for local administration, where the ester is either directly applied to the target tissue or directed there by, for example, inhalation, it will often be desirable that the ester has a rapid rate of esterase cleavage, to minimise systemic exposure and consequent unwanted side effects. In the compounds of this invention, if the carbon adjacent to the alpha carbon of the alpha amino acid ester ester is monosubstituted, ie R2 is CH2Rz (Rz being the mono-substituent) then the esters tend to be cleaved more rapidly than if that carbon is di- or tri-substituted, as in the case where R2 is, for example, phenyl or cyclohexyl.

The radical -Y-L1 -X1 -[CH?h- in Z1 case (a) This radical (or bond) arises from the particular chemistry strategy chosen to link the amino acid ester motif R1CH(R2)NH- to the ring A (when present), or ring B (via the -[Linker]- radical). Clearly the chemistry strategy for that coupling may vary widely, and thus many combinations of the variables Y, L1, X1and z are possible. However, when the inhibitor is bound to the HDAC enzyme at its active site, the rings B and/or A are located at the top of, or within, the metal-ion-containing pocket of the enzyme, so by linking the amino acid ester motif to those rings it generally extends in a direction away from that pocket, and thus minimises or avoids interference with the binding mode of the inhibitor. Hence the precise combination of variables making up the linking chemistry between the amino acid ester motif and the rings B and/or A will often be irrelevant to the primary binding mode of the compound as a whole. On the other hand, that linkage chemistry may WO 2008/053131 PCT/GB2006/004034 21 in some cases pick up additional binding interactions with the enzyme at the top of, or adjacent to, the metal ion-containing pocket, thereby enhancing binding.

It should also be noted that the benefits of the amino acid ester motif described above (facile entry into the cell, carboxylesterase hydrolysis within the cell, and accumulation within the cell of active carboxylic acid hydrolysis product) are best achieved when the linkage between the amino acid ester motif and the rings B and/or A is not a substrate for peptidase activity within the cell, which might result in cleavage of the amino acid from the molecule. Of course, stability to intracellular peptidases is easily tested by incubating the compound with disrupted cell contents, and analysing for any such cleavage.

With the foregoing general observations in mind, taking the variables making up the radical -Y-L1-X1-[CH2]Z- in turn: z may be 0 or 1, so that a methylene radical linked to the ring A or the ring B is optional; specific preferred examples of Y when macrophage selectivity is not required include-(C=0)-, -(C=0)NH-, and -(C=0)0-; Where macrophage selectivity is required any of the other options for Y, including the case where Y is a bond, are appropriate.

In the radical L1, examples of Alk1 and Alk2 radicals, when present, include -ch2-, -ch2ch2- -ch2ch2ch2-, -ch2ch2ch2ch2-, -ch=ch-, -CH=CHCH2-, -CH2CH=CH-, CH2CH=CHCH2-CeC-, -C=CCH2-, CH2C=C-, and CH2CsCCH2. Additional examples of Alk1 and Alk2 include -CH2W-, -CH2CH2W- -CH2CH2WCH2-, -CH2CH2WCH(CH3)-, -CH2WCH2CH2-, -CH2WCH2CH2WCH2-, and -WCH2CH2- where W is -0-, -S-, -NH-, -N(CH3)-, or -CH2CH2N(CH2CH2OH)CH2-. Further examples of Alk1 and Alk2 include divalent cyclopropyl, cyclopentyl and cyclohexyl radicals.

In L1, when q is 0, the radical is a hydrocarbon chain (optionally substituted and perhaps having an ether, thioether or amino linkage). Presently it is preferred that there be no optional substituents in L1. When both p and r are 0, L1 is a divalent mono- or bicyclic carbocyclic or heterocyclic radical with 5-13 ring atoms (optionally substituted). When n is 1 and at least one of p and r is 1, L1is a divalent RECEIVED at IPONZ on 11 October 2011 22 radical including a hydrocarbon chain or chains and a mono- or bicyclic carbocyclic or heterocyclic radical with 5-13 ring atoms (optionally substituted). When present, Q1 may be, for example, a divalent phenyl, naphthyl, cyclopropyl, cyclopentyl, or cyclohexyl radical, or a mono-, orbi-cyclic heterocyclic! radical having 5 to13 ring members, such as piperidinyl, piperazinyl, indolyl, pyridyl, thienyl, or pyrrolyl radical, but 1,4-phenylene is presently preferred.

Specifically, in some embodiments, L1, p and rmay be 0 with q being 1. In other embodiments, q and r may be 0 with p being 1. In further embodiments, p, q and r may be all 0. In still further embodiments p may be 0, q may be 1 with Q1 being a monocyclic heterocyclic radical, and r may be 0 or 1. Alk1 and Alk2, when present, may be selected from -CH2-, -CH2CH2-, and -CH2CH2CH2- and Q1 may be 1,4-phenylene.

Specific examples of the radical -Y-L1-X1-[CH2]Z- include -C(=0)- and -C(=0)NH- as well as -(CH2V, -(CH2)vO-, -C(=0)-(CH2)v-, -C(=0)-(CH2)v0-, -C(=0)-NH-(CH2)w-, -C(=0)-NH-(CH2)w0- wherein v is 1, 2, 3 or 4 and w is 1, 2 or 3, such as -CH2-, -CH20-, -C(=0)-CH2-, - C(=0)-CH20-, -C(=0)-NH-CH2-, and -C(=0)-NH-CH20-.

Case (b): Z1 is a radical of formula -(CH?)?-Y1-L1-R The radical R in Z1 case (b) R is a radical of formula (X) or (Y) and (X) (Y) WO 2008/053131 PCT/GB2006/004034 23 In formula (X) and (Y), R-, is is a carboxylic acid group or an ester group which is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group, as defined and discussed above with reference to Z1 case (a).

The ring D in Z1 case (b) When R is a group of formula (Y), examples of R include: [Tx ~ h 1 h wherein Ri is as defined and discussed above.

The group R* in Z1 case (b) The group R6 is present in the compounds of the invention in this case when R is a radical of formula (X) As mentioned above, if the modulator is intended to act only in cell types where hCE-1 is present, such as macrophages, the amino group of the carboxylesterase motif should be directly linked to a group other than carbonyl. In such cases R0 may be optionally substituted C^-Ce alkyl, C3-C7 cycloalkyl, aryl or heteroaryl, for example methyl, ethyl, n-or isopropyl, cyclopropyl, cyclopentyl, cyclohexyl, phenyl, or pyridyl. In cases where macrophage specificity is not required, R6 may be hydrogen or -(C=0)RD, wherein R° is optionally substituted (CrC6)alkyl such as methyl, ethyl, n-or isopropyl, or n-, iso- or sec-butyl, (C3-C7)Cycloalkyl such as cyclopropyl, cyclopentyl, cyclohexyl, phenyl, pyridyl, thienyl, phenyl(CrC6 alkyl)-, thienyKC-i-Ce alkyl)- or pyridyl(Ci-C6 alkyl)- such as benzyl, 4-methoxyphenylmethylcarbonyl, thienylmethyl or pyridylmethyl.

R6 may also be, for example -(C=0)0RD, or -(C=0)NHRD wherein R° is hydrogen or optionally substituted (C-t-C6)alkyl such as methyl, ethyl, or n-or isopropyl.

For compounds of the invention which are to be administered systemically, esters with a slow rate of esterase cleavage are preferred, since they are less susceptible to pre-systemic metabolism. Their ability to reach their target tissue intact is therefore increased, and the ester can be converted inside the cells of the target tissue into the acid product.

WO 2008/053131 PCT/GB2006/004034 24 However, for local administration, where the ester is either directly applied to the target tissue or directed there by, for example, inhalation, it will often be desirable that the ester has a rapid rate of esterase cleavage, to minimise systemic exposure and consequent unwanted side effects. If a carbon atom to which the group R is attached is unsubstituted, ie R is attached to a methylene (-CH2)- radical, then the esters tend to be cleaved more rapidly than if that carbon is substituted, or is part of a ring system such as a phenyl or cyclohexyl ring.

The radical -L1 -Y1 -fCH-,h- in Z1 case (b) As in the Z1 case (a), this radical (or bond) arises from the particular chemistry strategy chosen to link the amino acid ester motif R in substituent Y to the rest of the molecule. Clearly the chemistry strategy for that coupling may vary widely, and thus many combinations of the variables Y1, L1, and z are possible. However, when the inhibitor is bound to the enzyme at its active site, the amino acid ester motif generally extends in a direction away from the enzyme, and thus minimises or avoids interference with the binding mode of the inhibitor. Hence the precise combination of variable making up the linking chemistry between the amino acid ester motif and the rest of the molecule will often be irrelevant to the primary binding mode of the compound as a whole.

With the foregoing general observations in mind, taking the variables making up the radical -L1-Y1-[CH2]Z- in turn: z may be 0 or 1, so that a methylene radical linked to the rest of the molecule is optional; Y1 may be, for example, -NR3-, -S-, -0-, -C(=0)NR3-, - NR3C(=0)-, or -C(=0)0-, wherein R3 is hydrogen or optionally substituted CrC6 alkyl such as -CH2CH2OH; In the radical L1, examples of Alk1 and Alk2 radicals, when present, include -CH2-, -CH2CH2- -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH=CH-, -CH=CHCH2-, -CH2CH=CH-, CH2CH=CHCH2-, -C=C-, -C=CCH2-, CH2C=C-, and CH2C=CCH2. Additional examples of Alk1 and Alk2 include -CH2W-, -CH2CH2W- -CH2CH2WCH2-, -CH2CH2WCH(CH3)-, -CH2WCH2CH2-, -CH2WCH2CH2WCH2-, and -WCH2CH2- where W is -0-, -S-, -NH-, RECEIVED at IPONZ on 11 October 2011 -N(CH3)-, or -CH2CH2N(CH2CH20H)CH2-. Further examples of Alk1 and Alk2 include divalent cyclopropyl, cyclopentyl and cyclohexyl radicals.

Alk1 and Alk2 when present may also be branched chain alkyl such as -CH(CH3)-, -C(CH3)2-, or in either orientation -CH2CH(CH3)-, -CH2C(CH3)2-.

In L1, when n is 0, the radical is a hydrocarbon chain (optionally substituted and perhaps having an ether, thioetheror amino linkage). Presently it is preferred that there be no optional substituents in L1. When both m and p are 0, L1 is a divalent mono- or bicyclic carbocyclic or heterocyclic radical with 5-13 ring atoms (optionally substituted). When n is 1 and at least one of m and p is 1, L1 is a divalent radical including a hydrocarbon chain or chains and a mono- or bicyclic carbocyclic or heterocyclic radical with 5-13 ring atoms (optionally substituted). When present, Q may be, for example, a divalent phenyl, naphthyl, cyclopropyl, cyclopentyl, or cyclohexyl radical, or a mono-, orbi-cyclic heterocyclic radical having 5 to13 ring members, such as piperidinyl, piperazinyl, indolyl, pyridyl, thienyl, or pyrrolyl radical, but 1,4-phenylene is presently preferred.

Specifically, in some embodiments, L1, m and p may be 0 with n being 1. In other embodiments, n and p may be 0 with m being 1. In further embodiments, m, n and p may be all 0. In still further embodiments m may be 0, n may be 1 with Q being a monocyclic heterocyclic radical, and p may be 0 or 1. Alk1 and Alk2, when present, may be selected from -CH2-, -CH2CH2-, and -CH2CH2CH2- and Q may be 1,4-phenylene.

Specific examples of the radical -L1-Y1-[CH2]Z- include -(CH2)3NH-, - CH2C(=0)NH-, -CH2CH2C(=0)NH-,-CH2C(0)0- -CH2S-, -CH2CH2C(0)0-, -(CH2)4NH-, -CH2CH2S-, -CH20, -CH2CH2O-, 26 As mentioned above, the compounds with which the invention is concerned are HDAC inhibitors, and may therefore be of use in the treatment of cell proliferative disease, such as cancer, in humans and other mammals.

It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing treatment. Optimum dose levels and frequency of dosing will be determined by clinical trial.

The compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties. The orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

For topical application by inhalation, the drug may be formulated for aerosol delivery for example, by pressure-driven jet atomizers or ultrasonic atomizers, or preferably by WO 2008/053131 PCT/GB2006/004034 27 propellant-driven metered aerosols or propellant-free administration of micronized powders, for example, inhalation capsules or other "dry powder" delivery systems. Excipients, such as, for example, propellants (e.g. Frigen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, and fillers (e.g. lactose in the case of powder inhalers) may be present in such inhaled formulations. For the purposes of inhalation, a large number of apparata are available with which aerosols of optimum particle size can be generated and administered, using an inhalation technique which is appropriate for the patient. In addition to the use of adaptors (spacers, expanders) and pear-shaped containers (e.g. Nebulator®, Volumatic®), and automatic devices emitting a puffer spray (Autohaler®), for metered aerosols, in particular in the case of powder inhalers, a number of technical solutions are available (e.g. Diskhaler®, Rotadisk®, Turbohaler® or the inhalers for example as described in European Patent Application EP 0 505 321).

For topical application to the skin, the drug may be made up into a cream, lotion or ointment. Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.

For topical application to the eye, the drug may be made up into a solution or suspension in a suitable sterile aqueous or non aqueous vehicle. Additives, for instance buffers such as sodium metabisulphite or disodium edeate; preservatives including bactericidal and fungicidal agents such as phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents such as hypromellose may also be included.

The active ingredient may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.

Synthesis There are multiple synthetic strategies for the synthesis of the compounds (I) with which the present invention is concerned, but all rely on known chemistry, known to the synthetic organic chemist. Thus, compounds according to formula (I) can be synthesised according to procedures described in the standard literature and are well-known to the one skilled in WO 2008/053131 PCT/GB2006/004034 28 the art. Typical literature sources are "Advanced organic chemistry", 4th Edition (Wiley), J March, "Comprehensive Organic Transformation", 2nd Edition (Wiley), R.C. Larock , "Handbook of Heterocyclic Chemistry", 2nd Edition (Pergamon), A.R. Katritzky), review articles such as found in "Synthesis", "Acc. Chem. Res.", "Chem. Rev", or primary literature sources identified by standard literature searches online or from secondary sources such as "Chemical Abstracts" or "Beilstein". The synthetic routes used in the preparation of the compounds of the Examples below may be adapted for the preparation of analogous compounds.

Abbreviations MeOH = methanol EtOH = ethanol EtOAc = ethyl acetate Boc = tert-butoxycarbonyl DCM = dichloromethane DMF = dimethylformamide DMSO = dimethyl sulfoxide TFA = trifluoroacetic acid THF = tetrahydrofuran Na2C03 = sodium carbonate K2C03 = potassium carbonate HCI = hydrochloric acid aq = aqueous solution DIPEA = diisopropylethylamine NaH = sodium hydride NaOH = sodium hydroxide NaHC03 = sodium hydrogen carbonate Pd/C = palladium on carbon TBME = tert-butyl methyl ether N2 = nitrogen PyBop = benzotriazole-1-yI-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate Na2S04 = sodium sulphate Et3N = triethylamine NH3 = ammonia TMSCI = trimethylchlorosilane NH4CI = ammonium chloride WO 2008/053131 PCT/GB2006/004034 29 LiAIH4 = lithium aluminium hydride PyBrOP = bromo-tris-pyrrolidino phosphoniumhexafluorophosphate MgS04 = magnesium sulfate nBuLi = n-butyllithium C02 = carbon dioxide EDCI = /V-(3-dimethylaminopropyl)~/\/'-ethylcarbodiimide hydrochloride Et20 = diethyl ether LiOH = lithium hydroxide HOBt = 1 -hydroxybenzotriazole TLC = thin layer chromatography LCMS = liquid chromatography / mass spectrometry mL = millilitre(s) g = gram(s) mg = milligram(s) mol = moles mmol = millimole(s) HPLC = high performance liquid chromatography NMR = nuclear magnetic resonance RT = room temperature h = hour(s) The following Examples illustrate the preparation of specific compounds of the invention, and the HDAC inhibitory properties thereof: WO 2008/053131 PCT/GB2006/004034 Preparation of aminoacid esters (intermediates A to DI Route I. Used for the preparation of Intermediate A and Intermediate B q V/' Stage 1. cyclopentanol Q Stage 2.

BXa.DMAP.DMF HC, H,NV^) Route II. Used for the preparation of Intermediate C o ,o Stage 1. cyclopentanol ^ V' o" l_l pTSA, cyclohexane H N* Tf'0 o Route III. Used for the preparation of Intermediate D V O Stage 1. cyclopentanol n Stage 2. Pd(OH), O f O ( ^ (-'u A\OH H3CI.DMAP.DMF iT0s{2> H2'H0Ac H2N^Y° Scheme 1 WO 2008/053131 Compounds prepared: 31 HCI H2N' v V\ L/ Intermediate A .0 ^Tt) Intermediate C Figure 1 Synthesis of compounds outlined in Figure 1 Route I (exemplified for Intermediate B) Stage 1 - Ester formation Intermediate D To a solution of (S)-2-ferf-butoxycarbonylamino-3-cyclohexyl-propionic acid (5g, 19.4mmol) in DMF (50mL) at 0°C was added cyclopentanol (8.8mL, 97.15mmol), EDCI (4.09g, 21.37mmol) and finally DMAP (237mg, 1.94mmol). The reaction mixture was warmed to RT and stirred for 18h.

The DMF was removed in vacuo to give a clear oil. This was separated between water and EtOAc. The organic phase was dried (MgS04) and concentrated in vacuo. The crude extract was purified by column chromatography (25% EtOAC in heptane) to yield the 32 desired product as a clear oil (14.87g, 55%). 1H NMR (300MHz, d6-DMSO) 5: 7.09 (1H, d), 5.08 (1H, t), 3.76 (1H, t), 1.50-1.85 (1 OH, br m), 1.39 (9H, s), 1.00-1.25 (9H, br m).

Stage 2 - Boc deprotection to yield cyclopentyl (2S)-amino(cyclohexyl)acetate hydrochloride (Intermediate B) Stage 1 product (14.87g, 45.69mmol) was dissolved in DCM (100mL) and treated with 4M HCI/dioxane (22.8mL, 91.38mmol) and the reaction mixture was stirred at RT for 24h. The crude mixture was concentrated under reduced pressure to give an orange oil. This was triturated with Et20 to give a white precipitate. This was further washed with Et20 to.give the desired product as a white powder (7.78g, 65%). 1H NMR (300MHz, c/6-DMSO) 5: 8.45 (3H, br s), 5.22 (1H, t), 3.28 (1H, d), 1.95-1.50 (1 OH, br m), 1.30-0.90 (9H, br m).

Route II Stage 1 - Ester formation to yield (1S)-2-(cyclopentyloxy)-2-oxo-1-phenylethanaminium 4-methylbenzenesulfonate (Intermediate C) To a slurry of (S)-phenylglycine (5g, 33.1mmol) in cyclohexane (150mL) was added cyclopentanol (29.84mL, 331mmol) and p-toluene sulfonic acid (6.92g, 36.4mmol). The reaction was fitted with a Dean-Stark receiver and heated to 135°C for complete dissolution. After 12h, the reaction was cooled to RT leading to the precipitation of a white solid. The solid was filtered and washed with EtOAc before drying under reduced pressure to give the required product as a white powder (11.01 g, 85%). 1H NMR (300MHz, d6- 33 DM SO) 5Q8.82 (2H, brs), 8.73 (1H, brs), 7.47 (7H, m), 7.11 (2H, d), 5.25 (1H, brs), 5.18 ' (1H, m), 2.29 (3H, s), 1.87-1.36 (8H, m).

Route III Stage 1 - Ester formation To a solution of (S)-2-benzyloxycarbony]amino-3-terf-butoxy-propionic acid (25g, 84.65mmol) in DMF (250mL) at 0°C was added cyclopentanol (15.36mL, 169.3mmol), EDCI (17.85g, 93.11mmol) and finally DMAP (1.03g, 8.46mmol). The reaction mixture Was ■ • • i warmed to RT and stirred for 18h.

The DMF was removed in vacuo to give a yellow oil. This was partitioned between water and EtOAc. The organic phase was dried (MgS04) and concentrated in vacuo. The crude extract was purified by column chromatography (25% EtOAC in heptane) to yield the desired product as a clear oil. This was used directly in the next stage without characterization.

Stage 2 - Cbz deprotection to yield cyclopentyl O-ferf-butyl-L-serinate (Intermediate D) Stage 1 product was dissolved in EtOAc (150mL), treated with Pd(OH)2 (10 mol%) and stirred under an atmosphere of hydrogen for 32h. Upon completion, the catalyst was removed by filtration through celite and the filtrate concentrated in vacuo to yield the desired product as a clear oil (15.96g, 82% over two steps). 1H NMR (300MHz, d6-DMSO) 5: 5.17 (1H, t), 3.45 (1H, m), 3.34 (2H, q), 1.90-1.50 (9H, br m), 1.08 (9H, s). 34 Preparation of O-M-isobutoxvethvDhvdroxvlamine (Intermediate E) N-OH + 1.TFA, EtOAc 2. K2C03, n-propylamine O O O Intermediate E Scheme 2 Intermediate E was prepared following the methodology described in WO 01/60785. 1H NMR (300MHz, cfe-DMSO) 5: 0.85 (6H, d), 1.15 (3H, d), 1.75 (1H, m), 3.18 (1H, dd), 3.42 (1H, dd), 4.53 (1H, q), 5.82 (2H, s).

Preparation of ethyl 2-(methvlsurfonvDpyrimidine-5-carboxvlate (intermediate R SMe SMe Stage 1. Zn, benzene/NH4CI ^Cl C02Et C02Et S02Me Stage 2. mCPBA, THF C02Et Intermediate F Stage 1 - Chloro reduction Scheme 3 yo- ^ \=M EtO Ethyl 4-chloro-2-methylthio-5-pyrimidine carboxylate (12.5g, 53.88mmol) and Zn powder (14.1g, 215.52mmol) were combined and benzene (60mL) and 3M NH4CI (140mL) were added. The suspension was stirred vigorously and heated to 80°C for 30h. The reaction mixture was filtered through celite and washed with EtOAc (200mL). The filtrate was concentrated in vacuo to about 50mL and then partitioned between H20 (400mL) and EtOAc (400mL). The aqueous layer was further extracted with EtOAc (250mL). The combined organic layers were dried (MgS04) and concentrated in vacuo to a dark oil. This was purified by column chromatography (neat heptane followed by 1:1:1 heptane/CH2CI2/Et20 and finally 2:2:0.5 heptane/CH2CI2/Et20). The desired product was obtained as a colourless oil (13g, 61%). m/z = 199 [M+H]+, 1H NMR (300MHz, of6-DMSO) 5: 1.30 (3H, t), 2.60 (3H, s), 4.35 (2H, q), 9.0 (2H, s).

Stage 2 - Sulfide oxidation to yield ethyl 2-(methylsulfonyl)pyrimidine-5-carboxylate (Intermediate F) To a stirred solution of stage 1 product (13g, 47.59mmol) in dry THF (250mL) was slowly added over 30 minutes a solution of mCPBA (47.59g, 275.76mmol) in THF (150mL) at 0°C under N2. The reaction mixture was allowed to warm to RT and stirred for 2h. The reaction mixture was then concentrated in vacuo to about 10OmL and the product / benzoic acid mixture pre-absorbed onto silica gel. Purification was achieved via column chromatography (neat hexane initially, then 1:5:3 CH2CI2/heptane/Et20, followed by 1:1:1 CH2CI2/heptane/Et20). The desired compound was obtained as a white solid (10g, 66%). m/z = 231 [M+H]+, 1H NMR (300MHz, d6-DMSO) 5: 1.40 (3H, t), 3.50 (3H, s), 4.40 (2H, q), 9.50 (2H, s).

Preparation of 3-azabicvclor3.1.01hex-6-vlmethanol (Intermediate G) E Ph Ph Stage 3. LiAIH4, THF OH Ph Intermediate G Scheme 4 36 Stage 1 - Diels Alder reaction 0 o N-Benzylmaleimide (50g, 267.1 mmol) was dissolved in Et20 (600mL), treated with ethyldiazoacetate (31 mL, 293.8mmol) and stirred at RT under nitrogen atmosphere for 36h. A white precipitate had formed so it was isolated by filtration, washed with ice-cold Et20 and dried to give the desired compound as a white solid (72g, 89%). m/z = 302 [M+H]\ 1H NMR (300MHz, d6-DMSO) 5: 1.20 (3H, t), 4.15 (2H, q), 4.55 (2H, s), 4.60 (1H, d), 5.00 (1H, d), 7.15-7.35 (5H, m), '9.60 (1H, s).

Stage 2 - Condensation Stage 1 product (72g, 239.2mmol) was heated to 160°C until it melted to a yellow oil. The oil was heated further to 200°C and bubbling started. The oil was heated at 200°C for 30 minutes until the bubbling had subsided. The now amber-coloured oil was cooled to RT and triturated with ice-cold Et20. The resulting precipitate was filtered and washed with more ice-cold Et20 to give the product as a cream solid (37.2g, 57%). 1H NMR (300MHz, c/6-DMSO) 5: 1.20 (3H, t), 2.80 (1H, t), 3.00 (2H, d), 4.15 (2H, q), 4.35 (2H, s), 7.20-7.40 (5H, m).

O 37 Stage 3 - Reduction OH Stage 2 product (37.2g, 136.3mmoi) was dissolved in dry THF (400mL). This solution was added dropwise at 0°C to a suspension of LiAIH4 (20.7g, 545.3mmol) in dry THF (200mL). The resulting brown suspension was heated to 60°C under nitrogen atmosphere for 36h. The mixture was then cooled to 0°C and quenched carefully with saturated NH4Claq. A grey solid formed and more THF was added to allow adequate stirring. Solid Na2S04 was added to the mixture which was stirred at RT for 30 minutes. The mixture was then filtered through celite to give a pale yellow solution. This was concentrated in vacuo to give the desired product as an orange oil (14.1 g, 51%). m/z = 204 [M+H]+, 1H NMR (300MHz, dg-DMSO) 5: 2.25 (2H, d), 2.85 (2H, d), 3.20 (2H, t), 3.55 (2H, s), 4.35 (1H, t), 7.20-7.40 (5H, m).

Stage 4 - Nitrogen deprotection to yield 3-azabicyclo[3.1.0]hex-6-ylmethanol (Intermediate G) Stage 3 product (7.5g, 37.0mmol) was dissolved in dry MeOH (250mL) at RT. Pd(OH)2 (1.5g) was added to the solution and the reaction fitted with a hydrogen balloon. The reaction was degassed twice and flushed with H2. The reaction was allowed to stir for 6h and flushed with a fresh hydrogen balloon and allowed to stir for a further 64h. The reaction mixture was then filtered through celite. The solvent was removed in vacuo and the residue dried to give the product as a white solid (4.1g, 98%). m/z =114 [M+H]+, 1H NMR (300MHz, CDCI3) 8: 0.94 (1H, septet, J=3.3Hz), 1.37 (2H, m), 2.89 (2H, d, J=11.4Hz), 3.02 (2H, d, J=11.4Hz), 3.54 (2H, d, J=6.9Hz).

WO 2008/053131 PCT/GB2006/004034 38 Example 1 S02Me N^N Stage 1. K2C03, DMF EtO CO,Et 0=^JMH N N^N Stage 2. NaOH/THF HO' ?N O Stage 3. Intermediate A, NaBH(OAc)3, DCE N N HO N N N Stage 4. Intermediate E, EDCI HOBt, EtjN, DMF Stage 5. TFA, DCM Stage 6. KOTMS Stage 7. TFA, DCM HO_ I HO_ N .An °T> Scheme 5 WO 2008/053131 Compounds prepared: 39 HOv 1 N^N N Y R H O HOv I N" >f°NR H 8 R = Cyclopentyl (1) R = H (2) R = Cyclopentyl (3) R = H (4) HO.

N" N H 1 A ^ 'N N i N' V°Vr H 5 HOs H II I N' Y°nR H O R = Cyclopentyl (5) R = H (6) R = Cyclopentyl (7) R = H (8) o HOv. n' >r H VN OH N Y R H O R = Cyclopentyl (9) Figure 2 Synthesis of compounds outlined in Figure 2 exemplified for (1) and (2) Stage 1 - Coupling 40 A suspension of piperidine-4-one HCI salt (1.16g, 8.5mmol) and K2CO3 (11.70g, 85.0mmol) was stirred in DMF (50mL) at RT under a nitrogen atmosphere for 10 minutes. Intermediate F (1.97g, 8.5mmol) was then added and stirring continued for a further 10 minutes. The reaction was then diluted with water (150mL) and extracted with EtOAc (2 x 200mL). The combined organic layers were dried (MgS04) and the solvent removed in vacuo to give the product as a yellow solid which was used in the next step without further purification, m/z = 250 [M+H]+.

Stage 2 - Ester hydrolysis Stage 1 product was stirred in 1M NaOHaq (30mL) and THF (30mL) at RT for 4 days. The reaction was then acidified to pH ~ 3 with 1M HCIaq and this was extracted with DCM (2 x 200mL). The combined organic layers were dried (Na2S04) and the solvent removed in vacuo to give the product as a yellow solid, (665mg, 35% over 2 steps), m/z = 222 [M+H]+.

Stage 3 - Reductive amination Stage 2 product (100mg, 0.45mmol) was stirred in DCE (10mL) with intermediate A (106mg, 0.45mmol) and NaBH(OAc)3 (142mg, 0.67mmol) at RT under a nitrogen atmosphere for 3 days. The reaction was then diluted with water (50mL) and extracted with DCM (2x1 OOrnL). The combined organic layers were dried (Na2S04) and the solvent removed in vacuo to give the product as a yellow solid which was used in the next step without further purification, m/z = 405 [M+H]+. o o 41 Stage 4 - Protected hydroxamate formation Stage 3 product (182mg, 0.45mmol) was stirred in DMF (10mL) with EDCI (103mg, 0.54mmol), HOBt (73mg, 0.54mmol), Et3N (314|jL, 2.25mmol) and intermediate E (310(jL, 2.25mmol) for 16h at RT under a nitrogen atmosphere. The reaction was then diluted with water (50mL) and extracted with DCM (2x1 OOmL). The combined organic layers were then dried (MgS04) and the solvent removed in vacuo. The residue was purified by column chromatography (0 to 15% MeOH in DCM) to give the product as a yellow oil (110mg, 47%). m/z = 520 [M+H]+.

Stage 5 - Hydroxamate deprotection to yield cyclopentyl A/-{1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl}-L~leucinate (1) Stage 4 product (110mg, 0.21 mmol) was stirred in DCM (20mL) with TFA (0.5mL) for 1h at RT. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a purple solid (12mg, 11%). LCMS purity 99%, m/z = 420 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 0.91 (6H, m), 1.46-1.84 (13H, m), 2.05 (2H, m), 2.90 (2H, m), 3.42 (2H, m), 4.01 (1H, m), 4.91 (1H, m), 5.27 (1H, m), 8.58 (2H, m).

O WO 2008/053131 Stage 6 - Ester hydrolysis 42 OH O Stage 4 product (182mg, 0.45mmol) was stirred in THF (10mL) with KOTMS (115mg, 0.9mmol) for 4 days at RT under a nitrogen atmosphere. After this time the solvent was removed in vacuo and the residue used in the next step without further purification, m/z = 452 [M+H]+.

Stage 7 - Hydroxamate deprotection to yield A/-{1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl}-L-leucine (2) Stage 6 product (0.45mmol) was stirred in DCM (10mL) with TFA (1mL) at RT for 30 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a pink solid (7mg, 5% over two steps). LCMS purity 95%, m/z = 352 [M+H]+, insoluble in NMR solvents.

The analogues outlined in Figure 2 were prepared by the procedure described for (1) and (2). Data for each analogue is given.

LCMS purity 97%, m/z = 440 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.30-1.61 (9H, m), 1.78-1.90 (4H, m), 2.23 (2H, m), 2.90 (2H, m), 5.00 (1H, m), 5.33 (1H, m), 7.53 (5H, m), 8.69 (2H, s).

O O (3) WO 2008/053131 PCT/GB2006/004034 43 (4) LCMS purity 98%, m/z = 378 [M+H]+, insoluble in NMR solvents. (5) LCMS purity 97%, m/z = 446 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 0.80-1.82 (24H, m), 2.04 (2H, m), 2.94 (2H, m), 4.86 (1H, m), 5.23 (1H, m), 8.58 (2H, s). (6) LCMS purity 95%, m/z = 372 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.20 (2H, m), 1.50 (2H, m), 2.15 (2H, m), 2.80 (3H, m), 4.88 (1H, m), 7.37-7.46 (5H, m), 8.56 (2H, s). (7) LCMS purity 98%, m/z = 450 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.25 (9H, s), 1.58-1.77 (8H, brm), 1.96 (2H, m), 2.22 (2H, m), 3.02 (2H, m), 3.55 (1H, m), 3.87 (1H, dd, J=10.8, 2.7Hz), 3.99 (1H, dd, J=10.8, 2.7Hz), 4.45 (1H, m), 5.03 (2H, m), 5.35 (1H, m), 8.70 (2H, s). (8) LCMS purity 98%, m/z = 382 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.25 (9H, s), 1.65 (2H, m), 2.25 (2H, m), 3.01 (2H, t, J=13.2Hz), 3.57 (1H, m), 3.91 (1H, dd, J=10.5, 2.7Hz), 4.00 (1H, dd, J=10.5, 2.7Hz), 4.38 (1H, m), 5.05 (2H, m), 8.70 (2H, s). (9) LCMS purity 95%, m/z = 394 [M+HJ+, 1H NMR (300MHz, CD3OD) 5: 1.52-1.98 (11H, m), 2.10 (2H, m), 2.87 (3H, m), 3.44 (1H, m), 3.95 (2H, m), 4.18 (1H, m), 5.25 (1H, m), 8.58 (2H, m).

WO 2008/053131 Example 2 44 r X ^ Stage 1. Intermediate G Et0'"^j^^[jl^ K,CO,, DMF N N 0 C02Et Intermediate F Stage 2. Dess-Martln EtO ^ N DCM N N OH HO'^Y^N Vh Stage 5. Intermediate E EDCI, HOBt, Et3N, DMF N^-O-O 6 Stage 3. NaOH, THF Stage 4. Intermediate B j-jq NaBH(OAc)3, DCE N YVSol N N Stage 7. KOTMS, THF HO. fiJvO 6 Stage 6. TFA, MeOH DCM N "If N H "A "N N aJ-0X> 6 VrrV, N iAN 6 0 Stage 8. TFA, DCM HCsAf^N H Ia ^N N LJ <-> a^A, 6 Scheme 6 WO 2008/053131 Compounds prepared: 45 R = Cyclopentyl (10) R = Cyclopentyl (12) R = H (11) R = H (13) R = Cyclopentyl (14) R = H (15) Figure 3 Synthesis of compounds outlined in Figure 3 exemplified for (14) and (15) Stage 1 - Coupling O Intermediate G (0.97g, 8.62mmol) was stirred in DMF (20mL) and MeCN (20mL) with K2C03 (5.96g, 43.10mmol) at RT under a nitrogen atmosphere for 10 minutes. Intermediate F (2.00g, 8.62mmol) was then added and the reaction allowed to stir for a further 20 minutes. The reaction was then diluted with water (100mL) and extracted with EtOAc (2 x 100mL). The combined organic layers were dried (MgS04) and the solvent WO 2008/053131 PCT/GB2006/004034 46 removed in vacuo to give the product as a light yellow solid which was used in the next step without further purification (1.8g, 78%). m/z = 264 [M+H]+.

Stage 2 - Alcohol oxidation O Stage 1 product (1.80g, 6.84mmol) was stirred in DCM (50mL) at 0°C and Dess-Martin periodinane (3.50g, 8.22mmol) added. The reaction was allowed to warm to RT and stir for 4h. It was then quenched with a 1:1 mixture of sat. NaHC03(aq) and sat. Na2S204(aq) and the resulting mixture stirred for 20 minutes. The mixture was then extracted with DCM (2 * 100mL) and the combined organic extracts dried (MgS04) and the solvent removed in vacuo. The residue was purified by column chromatography (0 to 10% MeOH in DCM) to give the product as a yellow oil (1.35g, 72%). m/z = 262 [M+H]+.

Stage 3 - Ester hydrolysis O Stage 2 product (1.35g, 5.17mmol) was stirred in 1M NaOHaq (20mL) and THF (20mL) at RT for 3h. The reaction was then acidified to pH ~ 3 with 1M HCIaq which caused a white solid to precipitate out. This solid was filtered off and dried and retained. The filtrate was then extracted with DCM (2 x 100mL) and the combined organic layers dried (Na2S04) and the solvent removed in vacuo to give a light yellow solid which was combined with the previously obtained solid (990mg, 82%). m/z = 236 [M+H]+. 47 Stage 4 - Reductive amination O HO I^N-\ 6 Stage 3 product (175mg, 0.75mmol) was stirred in DCE (10mL) with intermediate B (196mg, 0.75mmoi) and NaBH(OAc)3 (222mg, 1.05mmoi) at RT under a nitrogen atmosphere for 16h. The reaction was then diluted with H20 (50mL) and extracted with Et20. The combined organic layers were dried (Na2S04) and the solvent removed in vacuo to give the desired product as a yellow oil which was used in the next step without further purification (171 mg, 52%). m/z = 443 [M+H]+.

Stage 5 - Protected hydroxamate formation Stage 4 product (171mg, 0.39mmol) was stirred in DMF (10mL) with intermediate E (539pL, 3.9mmol), EDCI (90mg, 0.47mmol), HOBt (63mg, 0.47mmol) and Et3N (543pL, 3.9mmol) at RT under a nitrogen atmosphere for 16h. The reaction was then diluted with H20 (50mL) and extracted with DCM (2x1 OOmL). The combined organic layers were dried (MgS04) and the solvent removed in vacuo. The residue was purified by column chromatography (0 to 10% MeOH in DCM) to give the product as a colourless oil (91 mg, 42%). m/z = 558 [M+H]+.

O 48 Stage 6 - Hydroxamate deprotection to yield cyclopentyl (2S)-cyclohexyl[({3-[5-(hydroxycarbamoyl)pyrimidin-2-yl]-3-azabicyclo[3.1.0]hex-6-yl}methyl)amino]acetate (14) Stage 5 product (91 mg, 0.163mmol) was stirred in 1:1 MeOH / DCM (4mL) with TFA (2mL) at RT for 1 h. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the desired product as a white solid (15mg, 20%). LCMS purity 98%, m/z = 558 [M+H]+, 1H NMR (300MHz, CD3OD) 5:0.92-1.38 (6H, m), 1.73-1.95 (16H, m), 3.09 (2H, m), 3.62 (2H, d, J=11.4Hz), 3.91 (1H, d, J=3.6Hz), 4.00 (2H, d, J=11.4Hz), 5.51 (1H, m), 8.67 (2H, s).

Stage 7 - Ester hydrolysis Stage 5 product (161mg, 0.29mmol) was stirred in THF (10mL) with KOTMS (74mg, 0.58mmol) at RT under a nitrogen atmosphere for48h. More KOTMS (112mg, 0.87mmol) was then added and the reaction stirred at 50°C for 48h. The solvent was then removed in vacuo and the residue used in the next step without further purification, m/z = 490 [M+H]+.

O 49 Stage 8 - Hydroxamate deprotection to yield (2S)-cyclohexyl[({3-[5-(hydroxycarbamoyl)pyrimidin-2-yl]-3-azabicyclo[3.1.0]hex-6-yl}methyl)amino]acetic acid (15) Stage 7 product (0.29mmol) was stirred in DCM (10mL) with TFA (1mL) at RT for 30 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a white solid (13mg, 12%). LCMS purity 99%, m/z = 390, [M+Hf, 1H NMR (300MHz, CD3OD) 5: 0.96 (1H, m), 1.10-1.47 (6H, m), 1.72-1.87 (6H, rh), 1.99 (1H, m), 2.99 (1H, m), 3.18 (1H, m), 3.63 (2H, dd, J=11.7, 3.3Hz), 3.85 (1H, d, J=3.3Hz), 3.99 (2H, d, J=11.7Hz), 8.67 (2H, s).

The analogues outlined in Figure 3 were prepared by the procedure described for (14) and (15). Data for each analogue is given. (10) LCMS purity 95%, m/z = 452 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.41-1.64 (6H, m), 1.72-1.93 (5H, m), 2.88 (2H, m), 3.60 (2H, m), 3.97 (2H, d, J=10.5Hz), 5.26 (1H, s), 5.32 (1H, m), 7.51 (5H, m), 8.67 (2H, s). (11) LCMS purity 97%, m/z = 384 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 0.94 (1H, m), 1.83 (2H, m), 3.01 (2H, d, J=7.5Hz), 3.62 (2H, m), 3.96 (2H, d, J=11.7Hz), 5.07 (1H, s), 7.53 (5H, m), 8.67 (2H, s). (12) LCMS purity 95%, m/z = 462 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 0.99 (1H, m), 1.24 (9H, s), 3.11 (2H, dd, J=7.5, 2.4Hz), 3.63 (2H, dd, J=12.0, 3.1Hz), 3.91 (2H, ddd, J=24.6, 10.8, 3.3Hz), 4.01 (2H, d, J=11.7Hz), 4.26 (1H, m), 5.35 (1H, m), 8.68 (2H, s).

O 50 (13) LCMS purity 98%, m/z = 394 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.01 (1H, m), 1.25 (9H, s), 1.88 (2H, m), 3.11 (2H, d, J=7.5Hz), 3.64 (2H, dd, J=11.7, 3.9Hz), 3.90 (2H, m), 4.01 (2H, d, J=11.7Hz), 4.14 (1H, m), 8.67 (2H, s).

WO 2008/053131 PCT/GB2006/004034 51 Example 3 OVn v s j 2 HN^I Stage 1. U..H ' II K,CO,, DMF, MeCN EtoXv^tjl C02Et Intermediate F Stage 2. NaOH, THF HO i OH OH Stage 3. Intermediate E EDCI, HOBt, EtjN, DMF N l/V HO. ouuoo 6 Stage 6. TFA, DCM I N^N S-^c 6 Stage 7. a) NaOH, THF b) HCI„ HO.

N N H || 6 Scheme 7 WO 2008/053131 Compounds prepared: 52 ho,.A^, B » X^ M-n-" kAA,A0- N N l u O R R = Cyclopentyl (16) R = Cyclopentyl (18) R = H (17) R = H (19) o H iAN"^ „ O > H o O UL* v -o-R R = Cyclopentyl (20) R = Cyclopentyl (22) R = H (21) R = H (23) HO.

N" H 11 A N N CUU^q'R OH R = Cyclopentyl (24) R = H (25) HO.

N if N » 1 A N N H I] nXR R = Cyclopentyl (26) Figure 4 53 Synthesis of compounds outlined in Figure 4 exemplified for (18) and (19) Stage 1 - Coupling Piperidin-4yl-methanol (2.48g, 21.55mmol) was stirred in 1:1 DMF/MeCN (20mL) with K2C03 (8.9g, 64.65mmol) for 10 minutes at RT under a nitrogen atmosphere. Intermediate F (5g, 21.55mmol) was then added and the reaction allowed to stir for 20 minutes. It was then diluted with H20 (100mL) and extracted with EtOAc (2 * 100mL). The combined organic layers were dried (MgS04) and the solvent removed in vacuo to give the product as an orange solid which was used in the next step without further purification (5.70g, 99%). m/z = 266 [M+H]+.

Stage 2 - Ester hydrolysis Stage 1 product (5.70g, 21.51mmol) was stirred in 1M NaOHaq (20mL) and THF (20mL) at RT for 48h. The reaction was then acidified to pH ~ 3 with 2M HCIaq causing a solid to precipitate out. This was collected and dried in vacuo to give the product as a white solid (4.47g, 89%). m/z = 238 [M+H]+.

Stage 3 - Protected hydroxamate formation O O 54 Stage 2 product (4.47g, 18.86mmol) was stirred in DMF (50mL) with intermediate E (13.00mL, 94.30mmol), EDCI (4.33g, 22.60mmol), HOBt (3.05g, 22.60mmol) and Et3N (13.10mL, 94.30mmol) at RT under a nitrogen atmosphere for 48h. The reaction was then diluted with H20 (200mL) and extracted with DCM (2 x 200mL). The combined organic layers were dried (MgS04) and the solvent removed in vacuo. The residue was purified by column chromatography (0 to 15% MeOH in DCM) to give the product as a yellow oil (5.12g, 77%). m/z = 353 [M+H]+.

Stage 4 - Alcohol oxidation A solution of (COCI)2 (253pl_, 2.90mmol) in DCM (50mL) was stirred under a nitrogen atmosphere and cooled to an internal temperature of -70°C. DMSO (363[jL, 5.11mmol) was then slowly added, maintaining the temperature at -70°C. When the addition was complete, a solution of stage 3 product (1.00g, 2.84mmol) in DCM (50mL) was slowly added, again maintaining the internal temperature at -70°C. When the addition was complete, Et3N (1.70mL, 12.21mmol) was slowly added, again maintaining the internal temperature at -70°C. The reaction was allowed to warm to RT and then the solvent was removed in vacuo. The residue was then purified by column chromatography (0 to 10% MeOH in DCM) to give the product as a colourless oil (890mg, 89%). m/z = 351 [M+H]+.

Stage 5 - Reductive amination 55 Stage 4 product (100mg, 0.28mmol) was stirred in DCE (10mL) with intermediate B (73mg, 0.28mmol) and NaBH3CN (35mg, 0.56mmoi) at RT under a nitrogen atmosphere for 16h. The reaction was then diluted with H20 (50mL) and extracted with DCM (2 x 100mL). The combined organic layers were dried (MgS04) and the solvent removed in vacuo. The residue was purified by column chromatography (0 to 10% MeOH in DCM) to give the product as a colourless oil (145mg, 92%). m/z = 560 [M+H]+.

Stage 6 - Hydroxamate deprotection to yield cyclopentyl (2S)-cyclohexyl[({1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl}methyl)amino]acetate (18) Stage 5 product (145mg, 0.26mmol) was stirred in DCM (10mL) with TFA (0.5mL) at RT for 10 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a light purple solid (11mg, 9%). LCMS purity >95%, m/z 460 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.02-1.37 (8H, m), 1.73-1.94 (19H, m), 3.02 (3H, m), 3.90 (1H, m), 5.37 (1H, m), 8.67 (2H, s).

Stage 7 - Ester hydrolysis and hydroxamate deprotection to yield (2S)-cyclohexyl[({1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl}methyl)amino]acetic acid (19) O O 56 Stage 5 product (78mg, 0.14mmol) was stirred in 1M NaOHaq (10mL) and THF (10mL) for 4 days at 40°C. After this time the reaction was cooled to RT and acidified to pH ~ 3 with 1M HCIaq. This mixture was stirred for 10 minutes and then evaporated to dryness. The residue was purified by preparative HPLC to give the product as a white solid (1mg, 2%). LCMS purity 98%, m/z = 392 [M+H]\ 1H NMR (300MHz, CD3OD) 5: 1.03-1.25 (9H, m), 1.60-1.87 (8H, m), 2.06 (1H, m), 2.85 (4H, m), 3.44 (1H, m), 8.55 (2H, s).

The analogues outlined in Figure 4 were prepared by the procedure described for (18) and (19). Data for each analogue is given. (16) LCMS purity 98%, m/z = 434 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.03 (6H, m), 1.32 (4H, m), 1.71-1.95 (15H, m), 2.90 (1H, m), 3.01 (2H, m), 4.01 (1H, m), 5.37 (1H, m), 8.68 (2H, m). (17) LCMS purity 97%, m/z = 366 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 0.92 (6H, m), 1.19 (3H, m), 1.58 (2H, m), 1.75 (4H, m), 1.98 (1H, m), 2.89 (4H, m), 3.70 (1H, m), 8.55 (2H, s). (20) LCMS purity 98%, m/z = 454 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.19-1.62 (10H, m), 1.79-1.92 (6H, m), 2.09 (1H, m), 2.84 (1H, m), 2.97 (2H, m), 5.32 (1H, m), 7.54 (5H, m), 8.67 (2H, m) (21) LCMS purity 98%, m/z = 386 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.08-1.28 (4H, m), 1.77 (2H, m), 1.98 (1H, m), 2.68-2.87 (4H, m), 4.90 (1H, m), 7.39 (5H, m), 8.54 (2H, s). (22) LCMS purity 98%, m/z = 464 [M+H]\ 1H NMR (300MHz, CD3OD) 5: 1.13 (9H, s), 1.56-1.71 (12H, m), 1.82 (3H, m), 2.04 (1H, m), 2.91 (3H, m), 3.81 (2H, m), 4.14 (1H, m), 5.39 (1H, m), 8.55 (2H, m).

WO 2008/053131 PCT/GB2006/004034 57 (23) LCMS purity 98%, m/z = 396 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.26 (9H, s), 1.31 (4H, m), 1.94 (2H, m), 2.14 (1H, brs), 3.03 (4H, m), 3.95 (2H, m), 4.16 (1H, m), 8.67 (2H, s). (24) LCMS purity 98%, m/z = 408 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.22-1.33 (3H, m), 1.53-1.73 (11H, m), 2.02 (1H, m), 2.93 (4H, m), 3.91 (2H, m), 4.02 (1H, m), 5.23 (1H, m), 8.55 (2H, s). (25) LCMS purity 95%, m/z = 340 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.28 (2H, m), 1.97 (2H, m), 2.15 (1H, m), 3.07 (4H, m), 4.09 (4H, m), 4.93 (1H, m), 8.67 (2H, m). (26) LCMS purity 87%, m/z = 504 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.02-1.48 (7H, br m), 1.67-1.82 (5H, m), 2.01 (1H, m), 2.97 (2H, m), 3.13 (4H, m), 5.19 (2H, m), 7.49 (3H, m), 7.83 (4H, m), 8.59 (2H, s).

WO 2008/053131 PCT/GB2006/004034 58 Example 4 OH H,Ns.

Stage 1.

Boc20, NaHC03 H20, THF °Y° HN Stage 2.

OH UAIHJ.THF dioxane HN.

Stage 3.

OH Mn02, DCM H,N 5t> Stage 5.

HCt in dioxane DCM 0^.0 HN. 5x> Stage 4. Intermediate A j NaBH(0Ac)3, DCE Scheme 8 Compounds prepared: 59 HO.

R = Cyclopentyl (27) R = H (28) H0V 0 R = Cyclopentyl (29) R = H (30) HO.

IN | IN H IA Xuuoo R = Cyclopentyl R = H (31) (32) R = Cyclopentyl (33) R = H (34) Figure 5 Synthesis of compounds outlined in Figure 5 exemplified for (27) and (28) Stage 1 - Boc protection 4-(Aminomethyl)benzoic acid (10.00g, 65.36mmoi) was stirred with Boc20 (28.00g, 130.72mmoi) in H20 (100mL) and THF (100mL) at RT. Sat NaHC03(aq) was added until pH ~ 6 was reached and the reaction was allowed to stir for 16h. The reaction was then carefully acidified to pH ~ 3 with 1M HCIaq which caused a solid to precipitate out. This 60 was filtered and dried to give the product as a white solid (16.1g, 97%). m/z = 274 [M+Na]+.

Stage 2 - Acid reduction Stage 1 product (16.1g, 64.14mmol) was stirred in THF (300ml_) and dioxane (200ml_) at 0°C under a nitrogen atmosphere. LiAIH4 was then added and the reaction allowed to warm to RT and stir for 16h. It was then cooled to 0°C and quenched with sat. NH4Claq. Na2S04 was added and the mixture stirred for 30 minutes. It was then filtered through celite and the filtrate concentrated in vacuo to give the product as a light yellow solid (13.1 g, 94%). m/z = 260 [M+Na]+.

Stage 3 - Alcohol oxidation Stage 2 product (5.87g, 24.73mmol) was stirred in DCM (200mL) with Mn02 (16.71g, 192.20mmol) for 16h at RT. The reaction was then filtered through celite and the solvent removed in vacuo to give the product as a yellow oil which was used in the next step without further purification (4.63g, 80%). m/z = 258 [M+Na]+.

Stage 4 - Reductive amination 61 Stage 3 product (650mg, 2.70mmol) was stirred in DCE (20ml_), intermediate A (634mg, 2.70mmol) and NaBH(OAc)3 (918mg, 4.33mmoi) at RT under a nitrogen atmosphere for 3h. After this time the reaction was diluted with H20 (50mL) and extracted with Et20 (2 x 10OmL). The combined organic extracts were dried (MgS04) and solvent removed in vacuo to give the product as a brown oil which was used in the next step without further purification (1.1g, 98%). m/z = 419 [M+H]+.

Stage 5 - Boc deprotection Stage 4 product (1.1g, 2.63mmol) was stirred in DCM (5mL) with 4M HCI in dioxane (2mL) at RT under a nitrogen atmosphere for 3h. The solvent was removed in vacuo and the residue dried to give the product as a brown solid as the HCI salt (670mg, 100%). m/z = 319 [M+H]+.

Stage 6 - Reductive amination 2-(4-Oxopiperidin-1-yl)pyrimidine-5-carboxylicacid (prepared as described in Example 1 -100mg, 0.45mmo!) was stirred in DCE (10mL) with stage 5 product (159mg, 0.45mmol) and NaBH(OAc)3 (191mg, 0.9mmol) at RT under a nitrogen atmosphere for 64h. The reaction was then diluted with H2Q (100mL) and extracted with DCM (2 x 100mL). The HCI O 62 combined organic layers were dried (Na2S04) and solvent removed in vacuo. The residue was used in the next step without further purification, m/z = 524 [M+H]+.

Stage 7 - Protected hydroxamate formation Stage 6 product (0.45mmol) was stirred in DMF (10mL) with intermediate E (621 pL, 4.50mmol), EDCI (103mg, 0.54mmol), HOBt (73mg, 0.54 mmol) and Et3N (31 3jjL, 2.25mmol) at RT under a nitrogen atmosphere for 40h. The reaction was then diluted with H20 (50mL) and extracted with DCM (2 * 100mL). the combined organic layers were dried (MgS04) and the solvent removed in vacuo to give the product as a yellow oil which was used in the next step without further purification, m/z = 639 [M+H]+.

Stage 8 - Hydroxamate deprotection to yield cyclopentyl /V-{4-[({1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl}amino)methyl]benzyl}-L-leucinate (27) o HOv.

Stage 7 product (0.45mmol) was stirred in DCM (10mL) with TFA (1mL) at RT for 30 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a pink solid (15mg, 6% over 3 steps). LCMS purity 96%, m/z WO 2008/053131 PCT/GB2006/004034 63 = 639 [M+H]+. 1H NMR (300MHz, CD3OD) 5: 1.00 (6H, m), 1.61-1.95 (12H, m), 2.28 (2H, m), 3.04 (3H, m), 4.01 (1H, m), 4.29 (4H, m), 5.04 (2H, m), 5.34 (1H, m), 7.60 (4H, m), 8.70 (2H, s).

Stage 9 - Ester hydrolysis Stage 7 product (0.45mmol) was stirred in THF (10mL) with KOTMS (115mg, 0.9mmol) for 96h at RT under a nitrogen atmosphere. The solvent was then removed in vacuo and the residue used in the next step without further purification, m/z = 571 [M+H]+.

Stage 10 - Hydroxamate deprotection to yield A/-{4-[({1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl}amino)methyl]benzyl}-L-leucine (28) o Stage 9 product was stirred in DCM (1 OmL) with TFA (1 mL) at RT for 30 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a white solid (5mg, 3%). m/z = 471 [M+H]+, 1H NMR (300MHz, d6-DMSO) 5: 0.9 (6H, d, J=4.8Hz), 1.51 (2H, m), 1.71 (2H, m), 2.19 (2H, m), 3.00 (4H, m), 4.42 (2H, WO 2008/053131 PCT/GB2006/004034 64 br s), 3.75 (1H, m), 4.22 (4H, m), 4.80 (2H, m), 7.52 (4H, m), 8.71 (2H, s), 8.98 (2H, br s), 11.01 (1H, s).

The analogues outlined in Figure 5 were prepared by the procedure described for (27) and (28). Data for each analogue is given. (29) LCMS purity 99%, m/z = 565 [M+H]+, 1H NMR (300MHz, cf6-DMSO) 5: 0.88 (2H, m), 1.05-1.30 (5H, m), 1.50-1.85 (18H, m), 2.18 (2H, m), 3.00 (2H, m), 2.25 (2H, m), 4.80 (2H, m), 5.17 (2H, m), 7.54 (4H, m), 8.71 (2H, s), 8.93 (2H, m), 9.45 (1H, brs), 11.10 (1H, s). (30) LCMS purity 95%, m/z = 497 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 0.80-1.34 (6H, m), 1.49-1.68 (5H, m), 1.85 (1H, m), 2.17 (2H, m), 2.93 (4H, m), 3.58 (1H, m), 4.20 (4H, m), 4.93 (2H, m), 7.51 (4H, m), 8.59 (2H, m). (31) LCMS purity 98%, m/z = 569 [M+Hf, 1H NMR (300MHz, CD3OD) 5: 1.22 (9H, s), 1.51-1.84 (11H, m), 2.18 (2H, m), 2.93 (3H, m), 3.44 (1H, m), 3.79 (2H, qd, J = 7.8, 3.3Hz), 4.24 (4H, m), 4.93 (2H, m), 5.22 (1H, m), 7.52 (4H„ m), 8.59 (2H, s). (32) LCMS purity 96%, m/z = 501 [M+Hf, 1H NMR (300MHz, CD3OD) 5: 1.25 (9H, s), 1.62 (2H, m), 2.29 (2H, m), 3.07 (4H, m), 3.85 (2H, m), 4.35 (4H, m), 5.07 (2H, m), 7.63 (4H, m), 8.71 (2H, m). (33) LCMS purity 98%, m/z = 559 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.31-1.93 (12H, m), 2.29 (2H, m), 3.04 (3H, m), 3.56 (1H, m), 4.22 (4H, m), 5.17 (2H, m), 5.30 (1H, m), 7.47-7.43 (9H, m), 8.69 (2H, s). (34) LCMS purity 95%, m/z = 491 [M+Hf, 1H NMR (300MHz, CD3OD) 5: 1.63 (2H, m), 2.29 (2H, m), 3.06 (2H, m), 3.29 (2H, m), 3.56 (1H, m), 4.20 (4H, m), 5.07 (1H, m), 7.54 (9H, m), 8.70 (2H, s).

WO 2008/053131 PCT/GB2006/004034 65 Example 5 stage 1.

N^N-"\ NaBH3CN DCE O Prepared as described Prepared as described in Example 4 in Example 2 O HO JT tT N .0. tXjuCnrYr- Scheme 9 Compounds prepared: (35) Figure 6 WO 2008/053131 Stage 1 - Reductive amination 66 T Cyclopentyl (2S)-{[4-(aminomethyl)benzyl]amino}(phenyl)acetate (prepared as described in Example 4 - 100mg, 0.29mmol) was stirred with 2-(6-formyl-3-azabicyclo[3.1.0]hex-3-yl)-A/-(1-isobutoxyethoxy)pyrimidine-5-carboxamide (prepared as described in Example 2 - 108mg, 0.29mmol) and NaBH3CN (36mg, 0.58mmol) in DCE (10mL) at RT under a nitrogen atmosphere for 16h. The reaction was then diluted with H20 (50mL) and extracted with DCM (2 * 100mL). The combined organic extracts were dried (MgS04) and the solvent removed in vacuo. The residue was purified by column chromatography (0 to 10% MeOH in DCM) to give the product as a yellow oil (56mg, 29%). m/z = 671 [M+H]+.

Stage 2 - Hydroxamate deprotection to yield cyclopentyl (2S)-[(4-{[({3-[5-(hydroxycarbamoyl)pyrimidin-2-yl]-3-azabicyclo[3.1,0]hex-6-yl}methyl)amino]methyl}benzyl)amino](phenyl)acetate (35) Stage 1 product (56mg, 0.08mmol) was stirred in DCM (10mL) with TFA (1mL) at RT for 15 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a pink solid (12mg, 25%). m/z = 571 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 0.98 (1H, m), 1.31-1.64 (6H, m), 1.76-1.93(4H, m), 3.09 (2H, d, J=7.5Hz), 3.61 (2H, d, J=10.5Hz), 3.98 (2H, d, J=10.5Hz), 4.15 (2H, m), 4.27 (3H, m), 5.31 (1H, m), 7.57 (9H, m), 8.66 (2H, s).

HO.

WO 2008/053131 PCT/GB2006/004034 67 Example 6 o °Y°VSp' stage1- NaBH3CN O DCE Prepared as described Prepared as described in Example 4 in Example 3 Scheme 10 WO 2008/053131 Compounds prepared: 68 H(X N N N" Y°"R H O HO.

N t/V nn" Y0sR h 8 R = Cyclopentyl (36) R = H (37) R = Cyclopentyl (38) R = H (39) HO.

X.

N N Ov HO. 1 N^V O.

R = Cyclopentyl (40) R = H (41) R = Cyclopentyl (42) R = H (43) HO^ N N^N' .OH 8 I R = Cyclopentyl (44) R = H (45) Figure 7 Synthesis of compounds outlined in Figure 7 exemplified for (38) and (39) Stage 1 - Reductive amination O CL ^O.

OuuCr»Yx> 69 Cyclopentyl (2S)-{[4-(aminomethyl)benzyl]amino}(4-methylcyclohexyl)acetate (prepared as described in Example 4 - 100mg, 0.29mmol) was stirred with 2-(4-formylpiperidin-1-yl)-/V-(1-isobutoxyethoxy)pyrimidine-5-carboxamide (preprared as described in Example 3 -110mg, 0.29mmol) in DCE (10mL) with NaCNBH3 (36mg, 0.58mmol) at RT under a nitrogen atmosphere for 16h. The reaction was then diluted with H20 (50mL) and extracted with DCM (2 x 100mL). The combined organic extracts were dried (MgS04) and the solvent removed in vacuo to give the product as a yellow oil which was used in the next step without further purification, m/z = 701 [M+Na]+.

Stage 2 - Hydroxamate deprotection to yield cyclopentyl (2S)-cyclohexyl[(4-{[({1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin~4-yl}methyl)amino]methyl}benzyl)amino]acetate (38) Stage 1 product was stirred in DCM (10mL) with TFA (1mL) at RT for 30 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a purple solid (14mg, 8% over two steps). LCMS purity >95%, m/z = 579 [M+Hf, 1H NMR (300MHz, CD3OD) 5: 1.01-1.42 (10H, m), 1.74-2.12 (18H, m), 3.01 (2H, m), 3.82 (1H, m), 4.28 (4H, m), 5.31 (1H, m), 7.61 (4H, m), 8.66 (2H, m).

Stage 3 - Ester hydrolysis HO.

N H Xs O. /O^ T OH Stage 1 product (100mg, 0.28mmol) was stirred in THF (10mL) with KOTMS (180mg, 1,40mmol) at 50°C under a nitrogen atmosphere. The reaction was allowed to cool to RT WO 2008/053131 PCT/GB2006/004034 70 and then the solvent was removed in vacuo and the residue used in the next step without further purification, m/z = 611 [M+H]+.

Stage 4 - Hydroxamate deprotection to yield (2S)-cyclohexyl[(4-{[({1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl}methyl)amino]methyl}benzyl)amino]acetic acid (39) OH Stage 3 product was stirred in DCM (10mL) with TFA (1mL) at RT for 30 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a pink solid (19mg, 13% over two steps). LCMS purity >95%, m/z = 511 [M+H]\ 1H NMR (300MHz, CD3OD) 5: 1.16-1.41 (9H, m), 1.69-1.91 (8H, m), 2.12 (1H, m), 2.99 (4H, m), 4.29 (4H, m), 4.92 (1H, m), 7.62 (4H, m), 8.66 (2H, s).

The analogues outlined in Figure 7 were prepared by the procedure described for (38) and (39). Data for each analogue is given. (36) LCMS purity 98%, m/z = 553 [M+H]+, 1H NMR (300MHz, d6-DMSO) 5: 0.90 (6H, m), 1.12 (2H, m), 1.28 (2H, d, J=6.6Hz), 1.65 (9H, m), 1.72 (3H, m), 2.03 (1H, m), 2.73 (2H, m), 2.96 (2H, t, J=12Hz), 4.20 (4H, m), 4.72 (2H, d, J=13.5Hz), 5.20 (1H, m), 7.54 (4H, m), 8.66 (2H, s), 8.95 (2H, br s), 9.60 (1H, br s), 11.05 (1H, s). (37) LCMS purity 97%, m/z = 485 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 0.87 (6H, m), 1.29 (4H, m), 1.77 (5H, m), 2.00 (1H, m), 2.88 (4H, m), 3.88 (1H, m), 4.18 (4H, m), 7.52 (4H, s), 8.61 (2H, s).

WO 2008/053131 PCT/GB2006/004034 71 (40) LCMS purity 99%, m/z = 583 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.24 (9H, s), 1.41 (2H, d, J=6.6Hz), 1.69-1.95 (12H, br m), 2.15 (1H, brs), 3.00 (4H, m), 3.93 (2H, m), 4.20 (1H, m), 4.31 (4H, m), 5.34 (1H, m), 7.63 (4H, m), 8.66 (2H, s). (41) LCMS purity 99%, m/z = 515 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.25 (9H, s), 1.29 (4H, m), 1.89 (2H, m), 2.12 (1H, brs), 3.01 (4H, m), 3.94 (2H, qd, J=9.0, 3.9Hz), 4.12 (1H, t, J=3.6Hz), 4.33 (4H, app. d, J=18.6Hz), 7.63 (4H, s), 8.66 (2H, s). (42) LCMS purity 98%, m/z = 573 [M+H]+, 1H NMR (300MHz, d6-DMSO) 5: 1.13-1.83 (12H, br m), 2.04 (1H, m), 2.85 (2H, m), 2.96 (2H, t, J=12Hz), 3.99 (2H, m), 4.15 (2H, m), 4.70 (2H, d, J=13.2Hz), 5.16 (2H, m), 7.51 (9H, m), 8.66 (2H, s), 8.97 (2H, brs), 10.13 (1H, brs), 11.05 (1H, s). (43) LCMS purity 99%, m/z = 505 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 0.89 (1H, m), 1.08 (2H, m), 1.77 (3H, m), 2.01 (1H, m), 2.90 (4H, m), 4.08 (4H, m), 4.94 (1H, s), 7.45 (9H, m), 8.55 (2H, s). (44) LCMS purity 99%, m/z = 528 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.30 (3H, m), 1.67-1.78 (11H, m), 2.12 (1H, m), 3.02 (4H, m), 4.06 (3H, m), 4.33 (4H, app. d, J=18Hz), 5.34 (1H, m), 7.66 (4H, m), 8.66 (2H, s). (45) LCMS purity 96%, m/z = 459 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.25 (4H, m), 1.89 (2H, m), 2.15 (1H, m), 2.99 (4H, m), 4.00 (1H, m), 4.09 (2H, m), 4.34 (4H, app. d, J=21.9Hz), 7.64 (4H, m), 8.66 (2H, s).

WO 2008/053131 Example 7 72 H,N OH Stage 1. uaih4,thf H9N OH .

Stage 2.

Boc20, NaOH, dioxane, H20 0^.0 Stage 3.

HN.

{C0CI)2, DMSO, Et3N, DCM Stage 5.

HCI in dioxane DCM 0^0 HN ^o Stage 4. Intermediate B NaBH(0Ac)3, DCE 0^0 HN.

^O Stage 6.

NaBH3CN DCE vck>° O-N ^=N >—' H Prepared as described in Example 3 T ~a T1 N N ouuonnrt) Stage 9. KOTMS, THF 0.^0.

T ITXI N N ,0H Stage 8. TFA, DCM HO., IT H H JL ouuonnro HO., Stage 10. TFA, DCM X.

N N -OH Scheme 11 WO 2008/053131 Compounds prepared: 73 HO O R R = cyclopentyl R = H (46) (47) Figure 8 Stage 1 - Acid reduction OH 4-(Aminomethyl)cyclohexanecarboxylic acid (4.00g, 25.44mmol) was stirred in THF (100mL) at 0°C under a nitrogen atmosphere. LiAIH4 (2.90g, 76.33mmol) was then added and the reaction allowed to warm to RT and stir for 3h. It was then cooled to 0°C and quenched with H20. Na2S04 was then added and the mixture stirred for 10 minutes. It was then filtered through celite and the filtrate concentrated in vacuo to give the product as a colourless oil which solidified on standing to give the product as a white solid (3.72g, 100%). 1H NMR (300MHz, cfe-DMSO) 5: 0.95 (4H, m), 1.22-1.47 (5H, m), 1.86 (4H, m), 2.55 (2H, d, J=6.6Hz), 4.46 (2H, d, J=6.3Hz).

Stage 2 - Boc protection Stage 1 product (3.72g, 26.01 mmol) was stirred with NaOH (1.00g, 26.01 mmol) and di-tert-butyl-dicarbonate (6.24g, 28.61 mmol) in H20 (50mL) and dioxane (50mL) at RT for 16h. The reaction was then concentrated in vacuo. When approximately 50% had been evaporated, a solid precipitated out of solution and was collected and dried to give the 74 product as a white solid (5.5g, 87%). m/z = 266 [M+Na]+, 1H NMR (300MHz, CDCI3) 5: 0.82 (4H, m), 1.28 (2H, m), 1.37 (9H, s), 1.70 (4H, m), 2.76 (2H, t, J=6.3Hz), 3.19 (2H, d, J=6.3Hz), 4.32 (1H, brs), 6.75 (1H, m).

Stage 3 - Alcohol oxidation A solution of DCM (100mL) and (COCI)2 (1.58mL, 18.14mmol) was stirred under a nitrogen atmosphere and cooled to -78°C. DMSO (2.27mL, 32.02mmol) was then added whilst maintaining the temperature below -65°C. A solution of stage 2 product (4.5g, 17.79mmol) in DCM (50mL) was then prepared and added slowly to the reaction mixture, again maintaining the temperature below -65°C. When addition was complete Et3N (9.99mL, 71.69mmol) was slowly added, again maintaining the temperature below -65°C. When addition was complete the reaction was allowed to warm to RT and then the solvent removed in vacuo. The residue was purified by column chromatography (0 to 10% MeOH in DCM) to give the product as a light yellow oil (5g, >100% - contains some Et3N). m/z = 264 [M+Na]+, 1H NMR (300MHz, CDCI3) 5: 1.02 (2H, m), 1.30 (2H, m), 1.45 (9H, s), 1.90 (2H, m), 2.03 (2H, m), 3.01 (2H, t, J=6.3Hz), 4.57 (1H, br s), 9.63 (1H, s).

Stage 4 - Reductive amination Stage 3 product (1.00g, 4.14mmol) was stirred with intermediate B (1.08g, 4.14mmol) and sodium triacetoxyborohydride (1.33g, 6.21 mmol) in DCE (20mL) at RT for 16h. The reaction was then diluted with H20 (100mL) and extracted with DCM (2 * 100mL). The combined organic extracts were dried (MgS04) and the solvent removed in vacuo to give 75 the product as a grey solid which was used in the next step without further purification (1.74g, 94%). m/z = 451 [M+Hf.

Stage 5 - Boc deprotection Stage 4 product (1.74g, 3.87mmol) was stirred in DCM (10mL) with 4M HCI in dioxane (3mL) at RT for 16h. The solvent was then removed in vacuo and the residue dried under vacuum to give the product as a white solid (1.36g, 98%). m/z = 351 [M+H]+, 1H NMR (300MHz, c/6-DMSO) 5: 0.90-1.20 (9H, m), 1.50-2.00 (21H, m), 2.65 (4H, m), 3.85 (1H, m), 5.25 (1H, m), 7.83 (2H, m).

Stage 6 - Reductive amination Stage 5 product (216mg, 0.56mmol) was stirred with 2-(4-formylpiperidin-1-yl)-/V-(1-isobutoxyethoxy)pyrimidine-5-carboxamide (preprared as described in Example 3 -200mg, 0.57mmol) and NaBH3CN (70mg, 1.12mmol) in DCE (10mL) at RT under a nitrogen atmosphere for 48h. The reaction was then diluted with H20 (100mL) and extracted with DCM (2 * 10OmL). The combined organic extracts were dried (MgS04) and the solvent removed in vacuo to give the product as a yellow oil which was used in the next step without further purification.

H 76 Stage 7 - Hydroxamate deprotection to yield cyclopentyl (2S)-cyclohexyl{[(4-{[({1~[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl}methyl)amino]methyl}cyclohexyl)methyl]amino}acetate (46) Stage 6 product (0.28mmol) was stirred in DCM (10mL) with TFA (0.5mL) at RT for 30 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a pink solid (15mg, 9% over two steps). LCMS purity >95%, m/z = 585 [M+Hf, 1H NMR (300MHz, CD3OD) 5: 0.96-1.39 (13H, m), 1.72-2.16 (23H, m), 2.81-3.04 (7H, m), 3.84 (2H, d, J=3.9Hz), 5.36 (1H, m), 8.66 (2H, s).

Stage 8 - Ester hydrolysis Stage 6 product (0.28mmol) was stirred with KOTMS (180mg, 1.4mmol) and THF (10mL) at 50°C under a nitrogen atmosphere for 16h. The reaction was the cooled to RT and the solvent removed in vacuo and the residue used in the next step without further purification m/z = 617 [M+Hf.

OH WO 2008/053131 PCT/GB2006/004034 77 Stage 9 - Hydroxamate deprotection to yield (2S)-cyclohexyl{[(4-{[({1-[5-(hydroxycarbamoyl)pyrimidin-2-yl]piperidin-4-yl}methyl)amino]methyl}cyclohexyl)methyl]amino}acetic acid (47) Stage 8 product (0.28mmol) was stirred in DCM (10mL) with TFA (1mL) at RT for 30 minutes. The solvent was then removed in vacuo and the residue purified by preparative HPLC to give the product as a white solid (5mg, 3% over two steps), m/z = 517 [M+H]+, 1H NMR (300MHz, CD3OD) 5: 1.13-1.44 (11H, m), 1.75-1.94 (15H, m), 2.82-3.09 (8H, m), 3.39 (1H, m), 3.65 (1H, m), 4.94 (1H, m), 8,67 (2H, m).

WO 2008/053131 PCT/GB2006/004034 78 Example 8 II HN'^i * 0 Stage 1. K2C03 MeCN EtO <3 Stage 2. NaOH EtOH O HO Stage 3. HCIaq HO CX -OEt OEt HO Stage 4. Intermediate A STAB, DCE 'T> Stage 5. Intermediate E EDCI, HOBt, Et3N, DMF yAI °x> Stage 7. i) NaOHaq, THF ii) HCIaq Stage 6. TFA, DCM HO.

HO.

OH T> Scheme 12 WO 2008/053131 Compounds prepared: 79 R = cyclopentyl (48) R = H (49) Figure 9 Stage 1 - Coupling Ethyl 4-fluorobenzoate (6.75g, 40.1mmol) and 1,4-dioxa-8-azaspiro[4.5]decane (2.73g, 19.1mmol) were stirred with K2C03 (8.5g, 61.5mmol) in MeCN (10ml_) at 95°C for 1 week. The mixture was added to EtOAc (50mL) and washed with water (3 x 50mL). The organic was dried (MgS04), filtered and concentrated in vacuo. The product was purified by flash chromatography (2%MeOH in DCM) to yield a yellow oil (5.58g, 48%). m/z = 292 [M+H]+.

Stage 2 - Ester hydrolysis OEt Stage 1 product (5.58g, 19.15mmol) was dissolved in EtOH (25mL) and stirred with 50% NaOHaq (25mL) at 60°C for 3h. The mixture was cooled to RT and 2M HCIaq (50mL) 80 added. The mixture was then stirred at 30°C for 3 days. The product was extracted with DCM (4 x 50mL), dried (MgS04), concentrated in vacuo and recrystallised from EtOH to yield a white solid (1.459g, 26%). m/z = 294 [M+H]+.

Stage 3 - Acetal deprotection Stage 2 product (509mg, 1.735mmol) was stirred in 1M HClaq (15ml) at RT for 30 minutes. The mixture was concentrated in vacuo, added to minimal water, filtered and dried in a desiccator. The product was obtained as a white solid (279mg, 73%). m/z = 220 [M+H]+ Stage 4 - Reductive amination Stage 3 product (279mg, 1.27rrimol) and intermediate A (303mg, 1.28mmol) were stirred in 1,2-dichloroethane (10mL) with sodium triacetoxyborohydride (405mg, 1.91 mmol) at RT overnight. The mixture was added to DCM (100mL) and washed with water (2 x 100mL). The combined aqueous layers were re-extracted with DCM (100mL), then the combined organics were dried (Na2S04), filtered and concentrated in vacuo to yield a brown oil (495mg, 97%). m/z = 403 [M+H]+. o o 81 Stage 5 - Protected hydroxamate coupling Stage 4 product (495mg, 1.20mmol), was dissolved in DMF and stirred with intermediate E (0.84mL, 6.12mmol), triethylamine (0.84mL, 6.03 mmol), HOBt (228mg, 1.49mmol) and EDCI (295mg, 1.54mmol) at RT for 2 days. The mixture was added to DCM (100mL) and washed with water (2 x 100mL). The combined aqueous layers was re-extracted with DCM (1 OOmL), then the combined organics were dried (Na2S04), filtered and concentrated in vacuo to yield a yellow oil (638mg, quant), m/z =518 [M+H]\ Stage 6 - Hydroxamate deprotection to yield cyclopentyl /V-{1-[4-(hydroxycarbamoyl) phenyl]piperidin-4-yl}-L-leucinate (48) Stage 5 product (309mg, 597(jmol) was stirred in DCM (5mL) and TFA (0.5mL) at RT for 1h. The reaction mixture was purified directly by preparative HPLC to give a white solid (75mg, 30%). LCMS purity 95%, m/z = 418 [M+H]+. 1H NMR (300MHz, CD3OD) 5: 7.67 (2H, d, J=8.5Hz), 7.02 (2H, d, J=8.5Hz), 5.38 (1H, m), 4.13 (1H, m), 4.04 (2H, d, J=12.4Hz), 2.92 (2H, m), 1.97 (2H, m), 1.88-1.68 (13H, m), 1.04 (6H, dd, J=6.2, 7.8Hz).

O 82 Stage 7 - Ester hydrolysis and hydroxamate deprotection to yield A/-{1-[4-(hydroxycarbamoyl)phenyl]piperidin-4-yl}-L-leucine (49) o HO.

A 'Av OH O Stage 5 product (50mg, 120[jmol) was stirred in THF (5mL) and 1M NaOHaq (5mL, 5mmol) at RT for 3 days. The mixture was acidified to pH ~ 3 with 2M HCIaq, and stirred for 20 minutes, then it was concentrated in vacuo and purified by preparative HPLC to give a white solid (5mg, 12%). LCMS purity 90%, m/z = 350 [M+H]+. 1H NMR (300MHz, d6-DMSO) 5: 10.90 (1H, s), 7.63 (2H, d, J=8.8Hz), 6.93 (2H, d, J=9.0Hz), 3.84 (2H, d, J=12.9Hz), 3.24 (1H, t, J=6.9Hz), 2.97-2.76 (3H, m), 1.96-1.74 (3H, m), 1.54-1.32 (4H, m), 0.89 (6H, t, J=6.4Hz). 83 Example 9 EtO HN .OH Stage 1. K2C03 DMSO .OH Stage 2. NaOH THF Scheme 13 WO 2008/053131 Compounds prepared: 84 IV X0/R R = cyclopentyl (50) R = H (51) Figure 10 Stage 1 - Coupling EtO Ethyl 4-fluorobenzoate (2.740g, 16.29mmol) and 4-piperidinemethanol (1.876g, 16.29mmol) were stirred in DMSO (100mL) with K2C03 (6.765g, 48.95mmol) at 100°C overnight. The mixture was cooled to RT and added to water (100mL). The product was extracted with DCM (2 xlOOmL), dried (Na2S04) and evaporated in vacuo to give a yellow oil. This was used in the next stage without further purification or characterisation.

Stage 2 - Ester hydrolysis Stage 1 product (16.29mmol) was stirred in 2M NaOHaq (40ml_, 80mmol) and THF (40mL) at RT for 2 days. Since the reaction had shown little progress, 50% NaOHaq (10mL) was 85 added, and the mixture stirred at 55°C for 2 further days. The mixture was then cooled to RT and the resulting precipitate was filtered and retained. The filtrate was acidified to pH ~ 3 with 2M HCIaq and stirred for 1 h at RT. A second crop of precipitate was then filtered. Both crops were dried to yield white solids (combined 3.052g, 80%). m/z = 236 [M+H]+. Stage 3 - Protected hydroxamate formation Stage 2 product (3.031 g, 12.17mmol) was stirred in DMF (100mL) with intermediate E (8.0mL, 58.27mmol), triethylamine (8mL, 57.39mmol), HOBt (2.759g, 18.02mmol) and EDCI (3.390g, 17.72mmol) at RT overnight. The mixture was added to DCM (100mL) and washed with water (2 x 10OmL). The combined aqueous layers were re-extracted with DCM (100mL), then the combined organic was dried (Na2S04), and concentrated in vacuo. The crude product was purified by column chromatography (1 to 10% MeOH in DCM) to yield a yellow oil (3.076g, 75%). m/z = 351 [M+H]+.

Stage 4 - Swern oxidation DCM (200mL) was stirred with oxalyl chloride (0.80mL, 9.17mmol) at -72°C.To this DMSO (1.1 mL, 15.50mmol) was added dropwise, maintaining the temperature at -72°C. Stage 3 product (3.08g, 8.78mmol) was dissolved in DCM (100mL) and added to the mixture dropwise, maintaining the temperature at -72°C. The reaction was then stirred at this temperature for a further 5 minutes before being allowed to warm to RT. The mixture was then concentrated in vacuo and purified by flash chromatography (1 to 10% MeOH in DCM) to yield an off white solid (3.05g, quant), m/z = 349 [M+H]+.

WO 2008/053131 Stage 5 - Reductive amination 86 Y N CUXO 6 Stage 4 product (206mg, 0.591 mmol), was stirred with intermediate B (155mg, 0.592mmol) in 1,2-dichloroethane (20mL). To this was added triethylamine (0.85ml, 6.10mmol) and sodium cyanoborohydride (525mg, 8.35mmol) and the mixture stirred at RT for 2 days. The reaction was quenched with water (50mL) and extracted with DCM (2 x oil (313mg, 95%). m/z = 558 [M+H]+.

Stage 6 - Hydroxamate deprotection to yield cyclopentyl (2S)-cyclohexyl[({1-[4-(hydroxycarbamoyl)phenyl]piperidin-4-yl}methyl)amino]acetate (50) Stage 5 product (180mg, 323|jmol) was stirred in DCM (10mL) and TFA (0.5mL) at RT for 1h. The product was purified directly by preparative HPLC to yield a white solid (7.4mg, 5%). LCMS purity 95%, m/z = 458 [M+H]+. 1H NMR (300MHz, CD3OD) 5: 7.67 (2H, d, J=8.3Hz), 7.02 (2H, d, J=8.3Hz), 5.40 (1H, t, J=5.4Hz), 3.93 (3H, m), 3.07-2.85 (4H, m), 2.05-1.70 (20H, m), 1.47-1.06 (8H, m). 50mL). The organic phase was dried (MgS04), and concentrated in vacuo to yield a yellow o 87 Stage 7 - Ester hydrolysis OH 0 Stage 5 product (184mg, 330[jmol) was stirred in THF (10mL) with potassium trimethylsilanolate (428mg, 3.34mmol) at 50°C for 4 days. The mixture was concentrated in vacuo and the residue used crude for next stage.

Stage 8 - Hydroxamate deprotection to yield (2S)-cyclohexyl[({1 -[4-(hydroxycarbamoyl)phenyl]piperidin-4-y|}methyl)amino]acetic acid (51) Stage 7 product was stirred in DCM (10mL) with TFA (0.5mL) at RT for 1h. The mixture was concentrated in vacuo and the product purified by preparative HPLC to yield a white solid (6.8mg, 5%). LCMS purity 93%, m/z = 390 [M+H]+. 1H NMR (300MHz, CD3OD) 5: 7.65 (2H, s), 6.99 (2H, s), 3.90 (2H, d, J=12.5Hz), 3.84 (1H, s), 2.89 (4H, m), 2.01-1.65 (10H,m), 1.45-1.05 (8H, m).

O Example 10 88 ,CL ^0.

V N I H CXo „,N_aMro Prepared as described in Example 9 Stage 2. TFA, DCM HOv Prepared as described in Example 4 o. , o Y it cuucm-o Stage 1. NaCNBH3 Et3N, DCE cujcrnro Stage 3. KOTMS, THF Scheme 14 WO 2008/053131 Compounds prepared 89 HO.

R R = H R = cyclopentyl (52) (53) Figure 11 Stage 1 - Reductive amination 4-(4-Formylpiperidin-1-yl)-A/-(1-isobutoxyethoxy)benzamide (prepared as described in Example 9 -210mg, 0.603mmol), was stirred with cyclopentyl (2S)-{[4-(aminomethyl)benzyl]amino}(cyclohexyl)acetate (prepared as described in Example 4 -230mg, 0.604mmol) in 1,2-dichloroethane (20mL). To this was added triethylamine (0.85mL, 6.10mmol) and sodium cyanoborohydride (540mg, 8.59mmol) and the mixture stirred at RT for 2 days. The mixture was added to water (50mL) and extracted with DCM (2 x 50mL). The organic phase was dried (MgS04), and concentrated in vacuo to yield a brown oil (408mg, 92%). m/z = 677 [M+H]+.

Stage 2 - Hydroxamate deprotection to yield cyclopentyl (2S)-cyclohexyl[(4-{[({1~[4-(hydroxycarbamoyl)phenyl]piperidin-4-yl}methyl)amino]methyl}benzyl)amino]acetate (52) HO. 90 Stage 1 product (226mg, 334|jmol) was stirred in DCM (10mL) and TFA (0.5mL) at RT for 1h. The product was purified by preparative HPLC to yield an off white solid (33.5mg, 17%). LCMS purity 95%, m/z = 577 [M+H]+. 1H NMR (300MHz, CD3OD) 5: 7.64 (6H, m), 7.02 (2H, d, J=8.2Hz), 5.31 (1H, t, J=5.3Hz), 4.30 (4H, brs), 3.86 (3H, m), 3.01 (1H, s), 2.92 (2H, t, J=11.1Hz), 2.05-1.68 (20H, m), 1.48-0.95 (8H, m).

Stage 3 - Ester hydrolysis Stage 1 product (218mg, 322pmol) was stirred in THF (10mL) with potassium trimethylsilanolate (418mg, 3.26mmol) at 50°C for 4 days. The mixture was concentrated in vacuo and the residue used crude in the next stage.

Stage 4 - Hydroxamate deprotection to yield {(2S)-2-cyclohexyl-2-[(4-{[({1-[4- (hydroxycarbamoyl)phenyl]piperidin-4-yl}methyl)amino]methyl}benzyl)amino]acetyl}oxy (53) Stage 3 product was stirred in DCM (10mL) with TFA (0.5mL) at RT for 1 h. The mixture was concentrated in vacuo and the product purified by preparative HPLC to yield a white solid (28.0mg, 17%). LCMS purity 95%, m/z = 509 [M+H]+. 1H NMR (300MHz, CD3OD) 5 7.66 (6H, m), 7.02 (2H, d, J=8.6Hz), 4.30 (4H, s), 3.89 (2H, d, J=12.2Hz), 3.76 (1H, d, J=3.2Hz), 3.01 (2H, d, J=6.4Hz), 2.90 (2H, t, J=12.1Hz), 2.03-1.68 (10H, m), 1.46-1.05 (8H, m).

.OH 91 Example 11 Prepared as described in Example 9 Prepared as described in Example 7 Stage 1. NaCNBH3 Et3N, DCE Stage 2. TFA, DCM H0-.

Co & X) O. .0 Y ti CuuCT"B fi ^ Stage 3. KOTMS, THF Stage 4. DCM, TFA CL .0.

Tff OH HO.

OH Scheme 15 WO 2008/053131 Compounds prepared: 92 R = cyclopentyl (54) R = H (55) Figure 12 Stage 1 - Reductive amination 4-(4-formylpiperidin-1 -yl)-A/-(1 -isobutoxyethoxy)benzamide (prepared as described in Example 9 -212mg, 0.608mmol), was stirred with cyclopentyl (2S)-({[4-(aminomethyl)cyclohexyl]methyl}amino)(cyclohexyl)acetate (prepared as described in Example 7 - 236mg, 0.609mmol) in 1,2-dichloroethane (20mL). To this was added triethylamine (0.85mL, 6.10mmol) and sodium cyanoborohydride (555mg, 8.83mmol) and the mixture stirred at RT for 2 days. The mixture was then added to water (50mL) and extracted with DCM (2 x 50mL). The organic phase was dried (MgS04), and concentrated in vacuo to yield a brown oil (318mg, 77%). m/z = 683 [M+H]+. 93 Stage 2 - Hydroxamate deprotection to yield cyclopentyl (2S)-cyclohexyl{[(4-{[({1-[4-(hydroxycarbamoyl)phenyl]piperidin-4-yl}methyl)amino]methyl}cyclohexyl)methyl]amino}acetate (54) Stage 1 product (257mg, 376|jmol) was stirred in DCM (10mL) and TFA (0.5mL) at RT for 1h. The product was purified by preparative HPLC to yield an off white solid (18.7mg, 9%). LCMS purity 98%, m/z = 583 [M+H]+. 1H NMR (300MHz, CD3OD) 5: 7.68 (2H, d, J=8.5Hz), 7.04 (2H, d, J=8.6Hz), 5.36 (1H, m), 3.04-2.81 (8H, m), 2.06-1.64 (25H, m), 1.53-0.97 (12H, m).

Stage 3 - Ester hydrolysis Stage 1 product (196mg, 287pmol) was stirred in THF (10mL) with potassium trimethylsilanolate (399mg, 3.11 mmol) at 50°C for 4 days. The mixture was concentrated in vacuo and the residue used crude for the next stage.

OH 94 Stage 4 - Hydroxamate deprotection to yield (2S)-cyclohexyl{[(4-{[({1-[4-(hydroxycarbamoyl)phenyl]piperidin-4-yl}methyl)amino]methyl}cyclohexyl)methyl]amino}acetic acid (55) Stage 3 product was stirred in DCM (10mL) with TFA (0.5mL) at RT for 1h. The mixture was concentrated in vacuo and the product purified by preparative HPLC to yield an off white solid (36.7mg, 25%). LCMS purity 98%, m/z = 515 [M+H]+. 1H NMR (300MHz, CD3OD) 5: 7.68 (2H, d, J=8.7Hz), 7.05 (2H, d, J=8.5Hz), 3.89 (2H, d, J=12.6Hz), 3.79 (1H, d, J=3.6Hz), 3.03-2.82 (8H, m), 2.07-1.65 (16H, m), 1.54-1.04 (12H, m).

Broken Cell Carboxylesterase Assay Any given compound of the present invention wherein R-[ is an ester group, may be tested to determine whether it meets the requirement that it be hydrolysed by intracellular esterases, by testing in the following assay.

Preparation of cell extract U937 or HCT 116 tumour cells (~ 109) were washed in 4 volumes of Dulbeccos PBS (~ 1 litre) and pelleted at 525 g for 10 min at 4°C. This was repeated twice and the final cell pellet was resuspended in 35 ml of cold homogenising buffer (Trizma 10 mM, NaC1130 mM, CaCI2 0.5 mM pH 7.0 at 25°C). Homogenates were prepared by nitrogen cavitation (700 psi for 50 min at 4°C). The homogenate was kept on ice and supplemented with a cocktail of inhibitors at final concentrations of: Leupeptin 1 p,M Aprotinin 0.1 |j,M E64 8 jiM Pepstatin 1.5 juM Bestatin 162|j.M WO 2008/053131 PCT/GB2006/004034 95 Chymostatin 33 |u,M After clarification of the cell homogenate by centrifugation at 525 g for 10 min, the resulting supernatant was used as a source of esterase activity and was stored at -80°C until required.

Measurement of ester cleavage Hydrolysis of esters to the corresponding carboxylic acids can be measured using the cell extract, prepared as above. To this effect cell extract (~30 yg / total assay volume of 0.5 ml) was incubated at 37°C in a Tris- HCI 25 mM, 125 mM NaCI buffer, pH 7.5 at 25°C. At zero time the ester (substrate) was then added at a final concentration of 2.5 DM and the samples were incubated at 37°C for the appropriate time (usually 0 or 80 min). Reactions were stopped by the addition of 3 x volumes of acetonitrile. For zero time samples the acetonitrile was added prior to the ester compound. After centrifugation at 12000 g for 5-min, samples were analysed for the ester and its corresponding carboxylic acid at room temperature by LCMS (Sciex API 3000, HP1100 binary pump, CTC PAL). Chromatography was based on an AceCN (75*2.1mm) column and a mobile phase of 5-95 % acetonitrile in water /0.1 % formic acid.

WO 2008/053131 PCT/GB2006/004034 96 Measurement of biological activities Histone deacetylase activity The ability of compounds to inhibit histone deacetylase activities was measured using the commercially available HDAC fluorescent activity assay from Biomol. In brief, the Fluor de Lys™ substrate, a lysine with an epsilon-amino acetylation, is incubated with the source of histone deacetylase activity (HeLa nuclear extract) in the presence or absence of inhibitor. Deacetylation of the substrate sensitises the substrate to Fluor de Lys™ developer, which generates a fiuorophore. Thus, incubation of the substrate with a source of HDAC activity results in an increase in signal that is diminished in the presence of an HDAC inhibitor.

Data are expressed as a percentage of the control, measured in the absence of inhibitor, with background signal being subtracted from all samples, as follows: % activity = [(S' - B) / (S° - B)] x 100 where S' is the signal in the presence of substrate, enzyme and inhibitor, S° is the signal in the presence of substrate, enzyme and the vehicle in which the inhibitor is dissolved, and B is the background signal measured in the absence of enzyme.

IC50 values were determined by non-linear regression analysis, after fitting the results of eight data points to the equation for sigmoidal dose response with variable slope (% activity against log concentration of Compound), using Graphpad Prism software.

Histone deacetylase activity from crude nuclear extract derived from HeLa cells was used for screening. The preparation, purchased from 4C (Seneffe, Belgium), was prepared from HeLa cells harvested whilst in exponential growth phase. The nuclear extract was prepared according to the methodology described by J. D. Dignam, Nucl. Acid. Res., 1983,11, 1475-1489, snap frozen in liquid nitrogen and stored at -80°C. The final buffer composition was 20 mM Hepes, 100 mM KCI, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF and 20 % (v/v) glycerol.

IC50 results were allocated to one of 3 ranges as follows: Range A: IC50<100nM, Range B: IC50from 101nM to 1000nM; WO 2008/053131 and Range C: IC50 >1001nM. 97 U937 and HUT cell inhibition assay Cancer cell lines (U937 and HUT) growing in log phase were harvested and seeded at 1000 - 2000 cells/well (100|jl final volume) into 96-well tissue culture plates. Following 24h of growth cells were treated with Compound. Plates were then re-incubated for a further 72 - 96h before a WST-1 cell viability assay was conducted according to the suppliers (Roche Applied Science) instructions.

Data were expressed as a percentage inhibition of the control, measured in the absence of inhibitor, as follows: % inhibition = 100-[(SVS°)x100] where S' is the signal in the presence of inhibitor and S° is the signal in the presence of DMSO.

Dose response curves were generated from 8 concentrations (top final concentration lOpM, with 3-fold dilutions), using 6 replicates.

IC50 values were determined by non-linear regression analysis, after fitting the results to the equation for sigmoidal dose response with variable slope (% activity against log concentration of Compound), using Graphpad Prism software.

IC50 results were allocated to one of 3 ranges as follows: Range A: IC50<330nM, Range B: IC50 from 331 nM to 3300nM; Range C: ICS0 >3301 nM; and n/d: not determined.

HeLa cell inhibition Assay HeLa cells growing in log phase were harvested and seeded at 1000 cells/well (200jjI final volume) into 96-well tissue culture plates. Following 24h of cell growth cells were treated with Compounds (final concentration of 20pM). Plates were then re-incubated for a further WO 2008/053131 PCT/GB2006/004034 98 72h before a sulphorhodamine B (SRB) cell viability assay was conducted according to the methodology described by Skehan et al, J. Natl. Cane. Inst., 1990, 82,1107-1112.

Data were expressed as a percentage inhibition of the control, measured in the absence of inhibitor, as follows:- % inhibition = 100-[(SVS°)x100] where S1 is the signal in the presence of inhibitor and S° is the signal in the presence of DMSO.

IC50 values were determined by non-linear regression analysis, after fitting the results of eight data points to the equation for sigmoidal dose response with variable slope (% activity against log concentration of Compound), using Graphpad Prism software.

IC50 results were allocated to one of 3 ranges as follows: Range A: IC50<330nM, Range B: IC50 from 331 nM to 3300nM; Range C: IC50 >3301 nM; and n/d: not determined.

Results Table Example HDAC U937 HUT HeLa No. activity activity activity activity 1 B A B C 2 B n/d n/d n/d 3 B B C C 4 C n/d n/d n/d B B B C 6 C n/d n/d n/d 7 B A B C 8 B n/d n/d n/d 9 B B B C A B B B 11 B n/d n/d n/d 12 A A B B 13 B n/d n/d n/d 14 A B B B B n/d n/d n/d 16 A A A B 17 B n/d n/d n/d

Claims (22)

WO 2008/053131 PCT/GB2006/004034 99 18 A A B B 19 A n/d n/d n/d 20 A A B B 21 A n/d n/d n/d 22 A A A B 23 B n/d n/d n/d 24 A A A B 25 B n/d n/d n/d 26 B B B C 27 A A A B 28 B n/d n/d n/d 29 A A B B 30 A n/d n/d n/d 31 B A B B 32 B n/d n/d n/d 33 A A A B 34 A n/d n/d n/d 35 A B B B 36 A A A A 37 A n/d n/d n/d 38 A A B A 39 A n/d n/d n/d 40 A A A B 41 A n/d n/d n/d 42 A A A A 43 A n/d n/d n/d 44 A A A B 45 A n/d n/d n/d 46 B B B n/d 47 A n/d n/d n/d 48 C B C n/d 49 C n/d n/d n/d 50 C C C n/d 51 C n/d n/d n/d 52 C B B n/d 53 C n/d n/d n/d 54 C B B n/d 55 C n/d n/d n/d RECEIVED at IPONZ on 11 October 2011 100 What we claim is:

1. A compound of formula (I), or a salt, N-oxide, hydrate or solvate thereof: HO' Q=V // W B [Linker] [A]n Z1 (I) O wherein n is 0 or 1; Q, V and W independently represent -N= or-C=; B is a divalent radical selected from (B1), (B2), (B3), (B4), (B5) and (B6): *—N N N- (B1) (B2) (B3) -N N- / -N A r v N- -N / V (B4) (B5) (B6) wherein the bond marked * is linked to the ring containing Q, V and W ; A is selected from the following ring systems, optionally substituted: RECEIVED at IPONZ on 11 October 2011 101 RECEIVED at IPONZ on 11 October 2011 102 RECEIVED at IPONZ on 11 October 2011 103 CO O* Cf c^a &-$ CO" wherein R10 is hydrogen or Ci-C6 alkyl, the bond intersected by the wavy line connects to the -[Linker]- radical, and Z1 is attached to an available ring atom; -[Linker]- is selected from: (i) a bond; (ii) -0-, -S-, -C(=0)-, -S(=0)2-, -NRC-, -C(=0)NRc-, -S(=0)2NRc-, -NRcC(=0)-, -NRcS(=0)2-,-NRc(CH2)m-,-NRcC(=0)(CH2)m-,-NRcS(=0)2(CH2)m, -NRdC(=0)NRc-, or -NRcC(=0)(CH2)mAr-, or-NRcS(=0)2(CH2)mAr-wherein Rc and Rd are independently hydrogen, C1-C4 alkyl or a nitrogen substituent, m is 1, 2 or 3, and Ar is a divalent phenyl radical or a divalent mono-, or bi-cyclic heteroaryl radical having 5 to 13 ring members; and (iii) an optionally substituted, straight or branched, Ci-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene radical which may optionally contain or terminate in an ether (-0-), thioether (-S-) or amino (-NRA-) link wherein RA is hydrogen, CrCs alkyl or a nitrogen substituent; Z1 is (a) a radical of formula R1R2CHNH-Y-L1-X1-(CH2)Z- or (b) a radical of formula R-L1-Y1-(CH2)z-, wherein: R is a radical of formula (X) or (Y) RECEIVED at IPONZ on 11 October 2011 104 H (X) Ri is an ester group which is hydrolysable by one or more intracel acid group; lar esterase enzymes to a carboxylic R6 is hydrogen; or optionally substituted CrC6 alkyl, C3-C7 cycloalkyl, aryl or heteroaryl or-(C=0)R3, -(C=0)0R3, or-(C=0)NR3 wherein R3 is hydrogen or optionally substituted (C1-Ce)alkyl; Ring D is a pyrrole or piperidine ring; and R2 is (i) Ci-C6 alkyl, phenyl, 2,- 3-, or 4-hydroxyphenyl, 2,- 3-, or 4-methoxyphenyl, 2,-3-, or 4-pyridylmethyl, benzyl, phenylethyl, 2-, 3-, or 4-hydroxybenzyl, 2,- 3-, or4-benzyloxybenzyl, 2,- 3-, or 4- C1-C6 alkoxybenzyl, or benzyloxy(Ci-C6alkyl)-groups; or (ii) the characterising group of a natural a amino acid, in which any functional group may be protected; or (iii) a group -[Alk]nR6 where Alk is a (CrC6)alkyl or (C2-C6)alkenyl group optionally interrupted by one or more -0-, or -S- atoms or -N(R7)- groups [where R7 is a hydrogen atom or a (Ci-C6)alkyl group], n is 0 or 1, and R6 is an optionally substituted cycloalkyl or cycloalkenyl group; or (iv) a benzyl group substituted in the phenyl ring by a group of formula -OCH2COR8 where R8 is hydroxyl, amino, (CrC6)alkoxy, phenyKCr Ce)alkoxy, (C1-C6)alkylamino, di((Ci-C6)alkyl)amino, pheny^Cr C6)alkylamino, the residue of an amino acid or acid halide, ester or amide RECEIVED at IPONZ on 11 October 2011 105 derivative thereof, said residue being linked via an amide bond, said amino acid being selected from glycine, a or/? alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histidine, arginine, glutamic acid, and aspartic acid; or (v) a heterocyclic(CrC6)alkyl group, either being unsubstituted or mono- or di-substituted in the heterocyclic ring with halo, nitro, carboxy, (Cr C6)alkoxy, cyano, (C-rC6)alkanoyl, trifluoromethyl (C-i-C6)alkyl, hydroxy, formyl, amino, (Ci-C6)alkylamino, di-(Ci-C6)alkylamino, mercapto, (Cr C6)alkylthio, hydroxy(CrC6)alkyl, mercapto(CrC6)alkyl or(Cr C6)alkylphenylmethyl; or (vi) a group -CRaRbRc in which: each of Ra, Rb and Rc is independently hydrogen, (Ci-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(CrC6)alkyl, (C3-C8)cycloalkyl; or Rc is hydrogen and Ra and Rb are independently phenyl or heteroaryl; or Rc is hydrogen, (Ci-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(Cr C6)alkyl, or (C3-C8)cycloalkyl, and Ra and Rb together with the carbon atom to which they are attached form a 3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic ring; or Ra, Rb and Rc together with the carbon atom to which they are attached form a tricyclic ring; or Ra and Rb are each independently (Ci-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(Ci-C6)alkyl, or a group as defined for Rc below other than hydrogen, or Ra and Rb together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic ring, and Rc is hydrogen, -OH, -SH, halogen, -CN, -C02H, (CrC/Operfluoroalkyl, -CH2OH, -C02(Cr C6)alkyl, -0(CrC6)alkyl, -0(C2-C6)alkenyl, -S(CrC6)alkyl, -SO(Cr RECEIVED at IPONZ on 11 October 2011 106 C6)alkyl, -S02(CrC6) alkyl, -S(C2-C6)alkenyl, -SO(C2-C6)alkenyl, -S02(C2-C6)alkenyl or a group -Q-W wherein Q represents a bond or -0-, -S-, -SO-or -S02- and W represents a phenyl, phenylalkyl, (C3-C8)cycloalkyl, (C3-CaJcycloalkylalkyl, (C4-C8)cycloalkenyl, (C4-C8)cycloalkenylalkyl, heteroaryl or heteroaryialkyl group, which group W may optionally be substituted by one or more substituents independently selected from, hydroxyl, halogen, -CN, -C02H, -C02(CrC6)alkyl, -CONH2, -CONH(Ci-C6)alkyl, -CONH(Ci-C6alkyl)2, -CHO, -CH2OH, (C1-C4)perfluoroalkyl, -CKCrCe^lkyl, -S(CrC6)alkyl, -SO(CrC6)alkyl, -S02(CrC6)alkyl, -N02, -NH2, -NHCCrCeJalkyl, -N((CrC6)alkyl)2, -NHCO(Ci-C6)alkyl, (CrC6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C3-C8)cycloalkyl, (C4-C8)cycloalkenyl, phenyl or benzyl; Y is a bond, -C(=0)-, -S(=0)2-, -C(=0)0-, -C(=0)NR3-, -C(=S)-NR3, -C(=NH)-NR3 or -S(=0)2NR3- wherein R3 is hydrogen or optionally substituted Ci-C6 alkyl; Y1 is a bond, -(C=0)-, -S(02)-, -C(=0)0-, -0C(=0)-, -(C=0)NR3-, -NR3(C=0)-, -S(02)NR3-, -NR3S(02)-, or -NRs^OJNR,-, wherein R3 and R4 are independently hydrogen or optionally substituted (Ci-C6)alkyl, L1 is a divalent radical of formula -(Alk1)m(Q)n(Alk2)p- wherein m, n and p are independently 0 or 1, Q is (i) an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5-13 ring members, or (ii), in the case where p is 0, a divalent radical of formula -Q1-X2- wherein X2 is -0-, -S- or NRA-wherein RA is hydrogen or optionally substituted CrC3 alkyl, and Q1 is an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5-13 ring members, Alk1 and Alk2 independently represent optionally substituted divalent C3-C7 cycloalkyl radicals, or optionally substituted straight or branched, CrC6 alkylene, C2-C6 alkenylene, or C2-C6 alkynylene radicals which may RECEIVED at IPONZ on 11 October 2011 107 optionally contain or terminate in an ether (-0-), thioether (-S-) or amino (-NRa-) link wherein RA is hydrogen or optionally substituted CrC3 alkyl; X1 is a bond, -C(=0)-; or-S(=0)2-; -NR4C(=0)-, -C(=0)NR4-, -NR4C(=0)-NR5-, -NR4S(=0)2-, or -S(=0)2NR4- wherein R4 and R5 are independently hydrogen or optionally substituted CrC6 alkyl; and z is 0 or 1; and wherein the term "optionally substituted" means substituted with up to four compatible substituents, independently selected from CrC6alkyl, C-rC6alkoxy, hydroxy, hydroxyC-i-C6alkyl, mercapto, mercaptoC-i-Cealkyl, CTC6alkylthio, phenyl, halo including fluoro, bromo and chloro, trifluoromethyl, trifluoromethoxy, nitro, nitrile -CN, oxo, -COOH, -COORA, -CORA, -S02RA, -CONH2, -S02NH2, -CONHRA, -S02NHRa, -CONRaRb, -S02NRaRb, -NH2, -NHRa, -NRaRb, -OCONH2, -OCONHRa , -OCONRaRb, -NHCORa, -NHCOORa, -NRbCOORa, -NHS020Ra, -NRbS020H, -NRbS020Ra, -NHCONH2, -NRaCONH2, -NHCONHR6, -NRaCONHRb, -NHCONRaRb, or-NRACONRARB wherein RA and RB are independently a CrCealkyl, (C3-C6) cycloalkyl, phenyl or monocyclic heteroaryl having 5 or 6 ring atoms, or RA and RB when attached to the same nitrogen atom form a cyclic amino group.

2. A compound as claimed in claim 1 wherein Ra, Rb and Rc together with the carbon atom to which they are attached form an adamantly ring.

3. A compound as claimed in claim 1 or claim 2 wherein RA and RB when attached to the same nitrogen atom form a morpholino, piperidinyl, piperazinyl, or tetrahydropyrrolyl group.

4. A compound as claimed in any one of claims 1 to 3 wherein Rc is hydrogen and Ra and Rb are independently pyridyl. RECEIVED at IPONZ on 11 October 2011 108

5. A compound as claimed in any one of claims 1 to 4 wherein Q is -C= and V and W are each -N=.

6. A compound as claimed in any one of claims 1 to 5 wherein ring A is selected from optionally substituted phenyl, cyclohexyl, naphthyl, quinolin-2-yl, and 1,3-dihydro-isoindol-2-yl.

7. A compound as claimed in claim 6 wherein optional substituents in ring A are selected from fluoro and chloro.

8. A compound as claimed in any one of the preceding claims wherein z is 0.

9. A compound as claimed in any one of claims 1 to 8 wherein Y is a bond.

10. A compound as claimed in any one of the preceding claims wherein, in the radical L1, Alk1 and Alk2, when present, are selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, and divalent cyclopropyl, cyclopentyl and cyclohexyl radicals.

11. A compound as claimed in any one of the preceding claims wherein, in the radical L1, Q1 is a divalent phenyl radical or a mono-, or bi-cyclic heteroaryl radical having 5 to13 ring members.

12. A compound as claimed in claim 9 wherein Q1 is 1,4-phenylene.

13. A compound as claimed in any one of the preceding claims wherein Ri is an ester group of formula -(C=0)0Rg wherein Rg is R7R8CH- wherein (i) R7 is hydrogen or optionally substituted (C1-C3)alkyl-(Z1)a-[(Ci-C3)alkyl]b-or (C2-C3)alkenyl-(Z1)a-[(C1-C3)alkyl]b- wherein a and b are independently 0 or 1 and Z1 is -0-, -S-, or-NR10- wherein R10 is hydrogen or CrC3 alkyl, and R8 is hydrogen or (CrC3)alkyl-; (ii) R7 is hydrogen or optionally substituted RioRnN-(CrC3)alkyl- wherein R10 is hydrogen or C1-C3 alkyl and Rn is hydrogen or CrC3 alkyl; or R10 and RECEIVED at IPONZ on 11 October 2011 109 Rn together with the nitrogen to which they are attached form an optionally substituted monocyclic heterocyclic ring of 5- or 6- ring atoms or bicyclic heterocyclic ring system of 8 to 10 ring atoms, and R8 is hydrogen or (CrCsJalkyl-; or (iii) R7 and R8 taken together with the carbon to which they are attached form an optionally substituted monocyclic carbocyclic ring of from 3 to 7 ring atoms or bicyclic carbocyclic ring system of 8 to 10 ring atoms.

14. A compound as claimed in claim 13 wherein Rg is methyl, ethyl, n- or iso-propyl, n-, sec-or tert-butyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- or4-pyridylmethyl, N-methylpiperidin-4-yl, tetrahydrofuran-3-yl, methoxyethyl, indanyl, norbornyl, dimethylaminoethyl, or morpholinoethyl.

15. A compound as claimed in claim 13 wherein Rg is cyclopentyl.

16. A compound as claimed in any of claims 1 to 15 wherein Z1 is a radical of formula R1R2CHNH-Y-L1-X1-(CH2)z- and wherein R2 is phenyl, benzyl, iso-butyl, cyclohexyl ort-butoxymethyl.

17. A compound as claimed in claim 1 selected from those of the following structural formulae 1, 7,16, 22, 27 and 31: O 1 o 7 RECEIVED at IPONZ on 11 October 2011 RECEIVED at IPONZ on 20 DECEMBER 2011 111 HO 31 "o

18. A pharmaceutical composition comprising a compound as claimed in any one of the preceding claims, together with a pharmaceutical^ acceptable carrier.

19. Use of a compound of formula (I) as claimed in any one of claims 1 to 17 in the preparation of a composition for the treatment of cancer cell proliferation, Huntington disease, Alzheimer disease or rheumatoid arthritis.

20. A compound as claimed in any one of claims 1 to 17, substantially as herein described with reference to any example thereof.

21. A pharmaceutical composition as claimed in claim 18, substantially as herein described.

22. The use as claimed in claim 19, substantially as herein described.