WO2012025701A1 - Alpha, alpha - di substituted glycine ester derivatives and their use as hdac inhibitors - Google Patents

Abstract

Compounds selected from the following group and their salts are inhibitors of HDAC activity, useful in the treatment of, inter alia, cell proliferative disease and inflammation: Cyclopentyl 1 -[({5-[(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]pyridin-2- yl}methy)amino]cyclopropanecarboxylate; Cyclopentyl 1 -[({5-[(1E)-3-(hydroxyamino)-3- oxoprop-1-en-1-yl]pyridin-2-yl}methy)amino]cyclobutanecarboxylate; Cyclopentyl 1-[({5- [(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]pyridin-2yl}methyl)amino]- cyclopentanecarboxylate; Cyclopentyl 1-[({5-[(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1- yl]pyridin-2-yl}methyl)amino]cyclohexanecarboxylate; Cyclopentyl 4-[({5-[(1E)-3- (hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2-yl}methyl)amino]tetrahydro-2H-pyran-4- carboxylate; Cyclopentyl 4-[({6-[(1E)-3-{[1 -(2-methylpropoxy)ethoxy]amino}-3-oxoprop-1 - en-1 -yl]pyridin-3-yl}methyl)amino]tetrahydro-2H-pyran-4-carboxylate; Cyclopentyl 1 -({4- [(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]-2-methylbenzyl}amino)- cyclohexanecarboxylate; Cyclopentyl 1-({4-[(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]- 2-methylbenzyl}amino)cyclopentanecarboxylate; Cyclopentyl 1-({4-[(1E)-3- (hydroxyamino)-3-oxoprop-1-en-1-yl]-2-methylbenzyl}amino)cyclobutanecarboxylate; Methylcyclopentyl 4-[({6-[(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]pyridin-3- yl}methyl)amino]tetrahydro-2H-pyran-4-carboxylate; Cyclopentyl 4-{[1 -({6-[(1E)-3- (hydroxyamino)-3-oxoprop-1-en-1-yl]pyridin-3-yl}methyl)piperidin-4-yl]amino}tetrahydro- 2H-pyran-4-carboxylate; Cyclopentyl 4-{[1-({5-[(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1- yl]pyridin-2-yl}methyl)piperidin-4-yl]amino}tetrahydro-2H-pyran-4-carboxylate; Cyclopentyl 4-({4-[(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]benzyl}amino)tetrahydro- 2H-pyran-4-carboxylate; Cyclopentyl 4-({3-[(1E)-3-(hydroxyamino)-3-oxoprop-1 -en-1 - yl]benzyl}amino)tetrahydro-2H-pyran-4-carboxylate; and (3R)-Tetrahydrofuran-3-yl N-{4- [(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]benzyl}-2-methyl-D-leucinate.

Description

ALPHA, ALPHA - Dl SUBSTITUTED GLYCINE ESTER DERIVATIVES AND THEIR

USE AS HDAC INHIBITORS

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, neurogenerative diseases, for example Alzheimer disease, autoimmune disease, for example rheumatoid arthritis, diabetes, haematological disorders, inflammatory disease, cardiovascular disease, atherosclerosis, and the inflammatory sequelia 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 S. L. Berger, Oncogene, 2001 , 20, 3007- 3013. See . Grunstein, Nature, 1997, 389, 349-352; A. P. Wotffe, Science, 1996, 272, 371-372; and P. A. Wade et al, Trends Biochem. Sci., 1997, 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 S. G. Gray and B. T. The, Curr. Mol. Med., 2001 , 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 of apoptosis and inhibition of tumour growth in animals (For review see W. K. Kelly ef al, Expert Opin. Investig. Drugs, 2002, 11 , 1695-1713). Such findings suggest that HDAC inhibitors have therapeutic potential in the treatment of proliferative diseases such as cancer (O. H. Kramer et al, Trends Endocrinol., 2001 , 12, 294-300; D. M. Vigushin and R. C. Coombes, Anticancer Drugs, 2002, 13, 1 -13).

In addition, others have proposed that aberrant HDAC activity or histone acetylation is implicated in the following diseases and disorders; inflammatory disorders (F. Leoni ef a/, Proc. Soc. Natl. Acad. Sci. ,2002, 99, 2995-3000), polyglutamine disease, for example Huntingdon disease (R. E. Hughes, Curr Biol, 2002, 12, R141 -R143; A. McCampbell et al, Proc. Soc. Natl. Acad. Sci., 2001 , 98, 15179-15184; E. Hockly et al, Proc. Soc. Natl. Acad. Sci., 2003, 100, 2041-2046), other neurodegenerative diseases, for example Alzheimer disease (B. Hempen and J. P. Brion, J. Neuropathol. Exp. Neurol., 1996, 55, 964-972), autoimmune disease and organ transplant rejection (S. Skov et al, Blood, 2003, 101, 1430-1438; N. Mishra ef al, J. Clin. Invest, 2003, 111 , 539-552), diabetes (A. L. Mosley and S. Ozcan, J. Biol. Chem., 2003, 278, 19660 - 19666) and diabetic complications, infection (including protozoal infection (S. J. Darkin-Rattray et al, Proc. Soc. Natl. Acad. Sci., 1996, 93, 13143-13147)) and haematological disorders including thalassemia (O. Witt et al, Blood, 2003, 101 , 2001-2007). The observations contained in these manuscripts suggest that HDAC inhibition should have therapeutic benefit in these, and other related diseases.

In our published International Patent Application No: WO 2009/060160 we describe and claim covalent conjugates of an α,α-disubstituted glycine ester and a modulator of the activity of a target intracellular enzyme or receptor, wherein the ester group of the conjugate is hydrolysable by one or more intracellular carboxylesterase enzymes to the corresponding acid and the α,α-disubstituted glycine ester is conjugated to the modulator at a position remote from the binding interface between the inhibitor and the target enzyme or receptor. The presence of the α,α-disubstituted glycine ester motif allows the conjugates to pass into cells and the active acid hydrolysis product accumulates within the cells. Examples 7 -11 of that publication disclose compounds which are embodiments of that invention having HDAC inhibitory activity which incorporate the α,α-disubstituted glycine ester motif. In our unpublished International Patent Application No. PCT/GB2010/000337 we describe and claim a class of HDAC inhibitor compounds which incorporate the α,α- disubstituted glycine ester motif and thus embody the invention disclosed in WO 2009/060160.

This invention relates to certain compounds which are members of the HDAC inhibitor class described and claimed in PCT/GB2010/000337, but which are not specifically disclosed therein.

Detailed Description of the Invention

Accordingly, the present invention provides a compound selected from the group consisting of:

Cyclopentyl 1 -[({5-[(1 £)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2- yl}methyl)amino]cyclopropanecarboxylate;

Cyclopentyl 1 -[({5-[(1 £)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2- yl}methyl)amino]cyclobutanecarboxylate; Cyclopentyl 1 -[({5-[(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1 -yl]pyridin-2- yl}methyl)amino]cyclopentanecarboxylate;

Cyclopentyl 1 -[({5-[(1 £)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2- yl}methyl)amino]cyclohexanecarboxylate;

Cyclopentyl 4-[({5-[(1 £)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2- yl}methyl)amino]tetrahydro-2H-pyran-4-carboxylate;

Cyclopentyl 4-[({6-[(1£)-3-{[1-(2-methylpropoxy)ethoxy]amino}-3-oxoprop-1 -en-1 - yl]pyridin-3-yl}methyl)amino]tetrahydro-2H-pyran-4-carboxylate;

Cyclopentyl 1 -({4-[(1 E)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]-2- methylbenzyl}amino)cyclohexanecarboxylate; Cyclopentyl 1 -({4-[(1 E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]-2- methylbenzyl}amino)cyclopentanecarboxylate;

Cyclopentyl 1-({4-[(1 £)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]-2- methylbenzyl}amino)cyclobutanecarboxylate;

Methylcyclopentyl 4-[({6-[(1£)-3-(hydroxyamino)-3-oxoprop-1 -en-1-yl]pyridin-3- yl}methyl )am i no]tetrahyd ro-2 W-pyran-4-carboxylate; Cyclopentyl 4-{[1-({6-[(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]pyridin-3- yl}methyl)piperidin-4-yl]amino}tetrahydro-2H-pyran-4-carboxylate;

Cyclopentyl 4-{[1-({5-[(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]pyridin-2- yl}methyl)piperidin-4-yl]amino}tetrahydro-2H-pyran-4-carboxylate;

Cyclopentyl 4-({4-[(1 E)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]benzyl}amino)tetrahydro- 2/-/-pyran-4-carboxylate;

Cyclopentyl 4-({3-[(1 E)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]benzyl}amino)tetrahydro- 2H-pyran-4-carboxylate; and

(3R)-Tetrahydrofuran-3-yl /V-{4-[(1 £)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]benzyl}-2- methyl-D-leucinate; and salts thereof.

Compounds of the invention may be prepared in the form of salts, especially pharmaceutically acceptable salts, N-oxides, hydrates, solvates and polymorphic forms thereof. Any claim to a compound herein, or reference herein to "compounds of the invention", "compounds with which the invention is concerned", "compounds of formula (I)" and the like, includes salts, N-oxides, hydrates, solvates and polymorphs of such compounds. The ester compounds of the invention are hydrolysed by the intracellular carboxylesterases hCE-1 , which is present only in cells of the macrophage (including monocyte) lineage, after penetrating the cell wall, and are thus converted to the corresponding carboxylic acids. The latter are active HDAC inhibitors when released in the cell, but they are not generally useful as drugs for administration per se to a subject. It is the ester compounds of the invention which are considered useful for administration.

The ester compounds of the invention are not hydrolysed to any significant extent by the intracellular carboxylesterases hCE-2 and hCE-3. Since the latter are not present in macrophages, the compounds of the invention are hydrolysed selectively in

macrophages, and their HDAC inhibitory activity is selectively exerted in macrophages.

Therefore, in another broad aspect the invention provides the use of an ester compound of the invention in the preparation of a composition for inhibiting the activity of histone deacetylase in macrophage cells

The ester compounds with which the invention is concerned may be used for the inhibition of histone deacetylase activity in macrophage cells, ex vivo or in vivo. In one aspect of the invention, the ester compounds of the invention may be used for the treatment of, or in the preparation of a composition for the treatment of, cell-proliferation disease, for example cancer cell proliferation and autoimmune diseases.

In another aspect, the invention provides a method for the treatment of the foregoing disease types, which comprises administering to a subject suffering such disease an effective amount of an ester compound of the invention.

The rate of hydrolysis of the ester compounds of the invention by plasma and tissue carboxylesterases is slow, and effective amounts of the ester compounds are therefore able to penetrate cells for intracellular hydrolysis.

As used herein the term "salt" includes base addition, acid addition and ammonium salts. As briefly mentioned above compounds of the invention which are acidic can form salts, including pharmaceutically 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 of the invention which are basic can form salts, including pharmaceutically 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, trifluoroacetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic, p- toluenesulphonic, benzoic, benzenesulfonic, glutamic, lactic, and mandelic acids and the like. Those compounds (I) which have a basic nitrogen can also form quaternary ammonium salts with a pharmaceutically acceptable counter-ion such as chloride, bromide, acetate, formate, p-toluenesulfonate, succinate, hemi-succinate, naphthalene- bis sulfonate, methanesulfonate, trifluoroacetate, xinafoate, and the like. For a review on salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

It is expected that compounds of the invention may be prepared in the form of hydrates, and solvates. Any reference herein, including the claims herein, to "compounds with which the invention is concerned" or "compounds of the invention" or "the present compounds", and the like, includes reference to salts, hydrates, and solvates of such compounds. The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.

Individual compounds of the invention may exist in an amorphous form and /or several polymorphic forms and may be obtained in different crystal habits. Any reference herein, including the claims herein, to "compounds with which the invention is concerned" or "compounds of the invention" or "the present compounds", and the like, includes reference to the compounds irrespective of amorphous or polymorphic form.

Some compounds of the invention, having a nitrogen atom in an aromatic ring, may form N-oxides, and the invention includes compounds of the invention in their N-oxide form. As stated above, the esters of the invention are primarily prodrugs of the corresponding carboxylic acids to which they are converted by the intracellular esterase hCE-1.

However, for so long as they remain unhydrolysed, the esters may have HDAC inhibitory activity in their own right. The corresponding carboxylic acid hydrolysis products may be the main source of HDAC inhibitory activity in the cells, but it is the esters which are intended for administration to patients.

Utilities

As mentioned above, the compounds with which the invention is concerned are of use for inhibition of HDAC activity in macrophage cells. Inhibition of HDAC activity in macrophages is a mechanism for treatment of a variety of diseases, including cell proliferative disease such as cancer (including malignancies of the monocytic cell lineage, e.g., juvenile myelomonocytic leukaemia) and psoriasis, polyglutamine disease such as Huntingdon's disease, neurogenerative disease such as Alzheimers disease, autoimmune disease such as rheumatoid arthritis (including systemic juvenile idiopathic arthritis), diabetes, haematological disease, inflammatory disease, cardiovascular disease, atherosclerosis, primary biliary cirrhosis, Wegener's granulomatosis, and the inflammatory sequelia of infection.

Autoimmune disease often has an inflammatory component. Such conditions include acute disseminated alopecia universalise, ANCA positive diseases, Behcet's disease, Chagas' disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, inflammatory bowel disease, Crohn's disease, diabetes mellitus type 1 , Fanconi syndrome, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonlein purpura, Kawasaki's disease, systemic lupus

erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reitei^s syndrome, Sjogren's syndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmune haemolytic anemia, interstitial cystitis, lyme disease, morphea, psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo. Other inflammatory conditions which may be treated with the compounds of the invention include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel, skin allografts, skin homografts, and heart valve xengrafts, sewrum sickness, and graft vs host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurative thyroiditis, hypercalcemia associated with cancer, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, astopic dermatitis, drug hypersensistivity reactions, allergic conjunctivitis, keratitis, herpes zoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonary tuberculosis chemotherapy, idiopathic thrombocytopenic purpura in adults, secondary

thrombocytopenia in adults, acquired (autoimmune) haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia of childhood, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, solid organ transplant rejection, sepsis, primary biliary cirrhosis and primary sclerosing cholangitis.

Preferred treatments using compounds of the invention include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, Type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosis, and inflammation accompanying infectious conditions (e.g., sepsis), psoriasis, Crohns disease, ulcerative colitis, chronic obstructive pulmonary disease, multiple sclerosis, atopic dermatitis, and graft versus host disease.

Another preferred use of the compounds of the invention is in the treatment of cancers.

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. However, it is expected that a typical dose will be in the range from about 0.001 to 50 mg per kg of body weight. 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 polyvinyl-pyrrolidone; 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 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 by inhalation, the drug may be formulated for aerosol delivery for example, by pressure-driven jet atomizers or ultrasonic atomizers, or preferably by 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 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 agent can be dissolved in the vehicle.

Compounds of the invention may be prepared, for example, by the methods described below and in the Examples herein.

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 the art. Typical literature sources are "Advanced organic chemistry, 4^th Edition (Wiley), J March; "Comprehensive Organic Transformation", 2 Edition (Wiley), R.C. Larock; "Handbook of Heterocyclic Chemistry", 2^nd 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 = terf-butoxycarbonyl

DCM = dichloromethane

DMF = dimethylformamide

DCE = 1 ,2-dichloroethane

KOTMS = potassium trimethylsilanolate

DMSO = dimethyl sulfoxide

TFA = trifluoroacetic acid

THF = tetrahydrofuran

Na₂C0₃ = sodium carbonate

K2CO3 = potassium carbonate

HCI = hydrochloric acid

aq = aqueous solution

sat = saturated

DIPEA = diisopropylethylamine

DIEA = N,N-diisopropylethylamine

NaH = sodium hydride

NaOH = sodium hydroxide

STAB = sodium triacetoxyborohydride

NaCNBH₃ = sodium cyanoborohydride

NaHC0₃ = sodium hydrogen carbonate

Pd/C = palladium on carbon

TBME = terf-butyl methyl ether TPAP = tetrapropyl ammonium perruthenate

(COCI)₂ = oxalyl chloride

N₂ = nitrogen

PyBop = benzotriazole-l-yl-oxy-fr/^'s-pyrrolidino-phosphonium hexafluorophosphate Na₂SC>4 = sodium sulphate

Et₃N = triethylamine

NH₃ = ammonia

TMSCI = trimethylchlorosilane

NH₄CI = ammonium chloride

UAIH4 = lithium aluminium hydride

PyBrOP = Bromo-tris-pyrrolidino phosphoniumhexafluorophosphate

gS0₄ = magnesium sulfate

"BuLi = n-butyllithium

C0₂ = carbon dioxide

EDCI = /\/-(3-Dimethylaminopropyl)-/S/'-ethylcarbodiimide hydrochloride

Et₂0 = diethyl ether

LiBr = lithium bromide

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 = mole(s)

mmol = millimole(s)

MOMCI = Methyl chloromethyl ether

HPLC = high performance liquid chromatography

NMR = nuclear magnetic resonance

RT = room temperature

h = hour(s)

Commercially available reagents and solvents (HPLC grade) were used without further purification. Solvents were removed using a Buchi rotary evaporator. Microwave irradiation was carried out using a Biotage Initiator™ Eight microwave synthetiser.

Purification of compounds by flash chromatography column was performed using silica gel, particle size 40-63μ μτη (230-400 mesh) obtained from Fluorochem. Automated silica gel column chromatography was performed on a CombiFlash Companion

(Teledyne Isco, Nebraska, USA) using normal phase Biotage^® SNAP cartridges.

Reverse phase column chromatography was performed using a pre-column on Merck liChroprep RP-18 (40-60μητι) before purification on a CombiFlash Companion (Teledyne Isco, Nebraska, USA) using RediSep Rf C18 columns (Presearch, Basingstoke, UK). Purification of compounds by preparative HPLC was performed on Gilson systems using reverse phase Axia™ prep Luna C18 columns (ΙΟμιτιυ, 100 x 21.2 mm), gradient 0- 100% B (A = water / 0.05 % TFA, B = acetonitrile / 0.05% TFA) over 10 min, flow = 25 ml/min, UV detection at 254 nm.

^ΊΗ NMR spectra were recorded on a Bruker 300 MHz A spectrometer in deuterated solvents. Chemical shifts (δ) are in parts per million. Thin-layer chromatography (TLC) analysis was performed with Kieselgel 60 F₂₅₄ (Merck) plates and visualized using UV light. Analytical HPLC/MS was performed on an Agilent HP1 100 LC system using reverse phase Luna C18 columns (3μ μιη, 50 x 4.6 mm), gradient 5-95% B ( A = water / 0.1 % Formic acid, B = acetonitrile / 0.1 % Formic acid) over 2.25 min, flow = 2.25 ml/min. UV spectra were recorded at 220 and 254 nm using a G1315B DAD detector. Mass spectra were obtained over the range m/z 150 to 800 on a LC/MSD SL G1956B detector. Data were integrated and reported using ChemStation and ChemStation Data Browser softwares. Thus, compounds of general formula (8) and (9) may be, but not exclusively, prepared by the methods outlined in Scheme 1.

Scheme 1

Reagents: a) ethyl glyoxalate, Ac₂0 b) BH3. THF c) LiOH, H₂0, EtOH d) NHOR², HOBT, EDC e) Mn0₂ f) H₂NCR³R⁴C0₂R⁵ g) 4N HCI in dioxane h) NaOH, H₂0, MeOH followed by 4N HCI in dioxane

Thus heteroaromatic carboxylic acids such as 6-methylnicotinic acid (1 ) may be used in a condensation reaction with aldehydic reagents such as ethyl glyoxalate in the presence of acetic anhydride in hydrocarbon solvents such as toluene under reflux conditions to give α,β-unsaturated esters of general formula (2). The carboxylic substituent of (2) may be transformed to a hydroxymethylene group by the use of reducing agents such as borane THF complex to give alcohols of general formula (3). α,β-Unsaturated acids of general formula (4) may be obtained from (3) under basic hydrolysis conditions employing an alkali such as sodium or lithium hydroxide in the presence of a water miscible co-solvent such as methyl or ethyl alcohol. O-Protected hydroxamic acids of general formula (5) may be prepared by the coupling of protected hydroxylamines such as O-(l-isobutoxyethyl) hydroxylamine (WO 01/60785) using reagents such as N-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride or Ν,Ν'-diisopropylcarbodiimide. Oxidation of compounds of general formula (5) to the corresponding aldehydes may be performed by the use of reagents such as manganese dioxide. Reductive amination of aldehydes such as (6) with α,α-disubstituted amino acid esters using reagents such as sodium borohydride, sodium cyanoborohydride or sodium triacetoxyborohydride leads to amino acid ester derivatives of general formula (7). Hydroxamic acids of general formula (8) may be prepared by the treatment of (7) under acidic conditions such as hydrochloric acid in solvents such as 1 ,4-dioxane. Amino acid derivatives of general formula (9) may be prepared by the hydrolysis of (7) under aqueous alkaline conditions using for example aqueous sodium hydroxide in the presence of a water miscible co-solvent such as methyl alcohol or tetrahydrofuran. Alternatively compounds of general formula (8) may be prepared by methods described in Scheme 2.

Scheme 2

(13) (14) (8) Reagents: a) LiAIH₄, THF b) OHCCOOR¹, Ac₂0 c) (COCI)₂, DMSO, DCM d) STAB, HzNCF^R^OzR⁵, THF e) NH₂OH.HCI, KOH, MeOH

Thus compounds such as methyl-6-methylnicotinate (10) may be reduced with hydride donors such as lithium aluminium hydride to give alcohols such as (11 ) which possess an activated alkyl group which can be utilized in condensation reactions with aldehydes such as ethyl glyoxalate to give α,β-unsaturated esters such as (12). Compounds such as (12) may be further oxidized under conditions such as those described by Swern [J.Org.Chem. 1976, 47, 3329] employing, for example, oxalyl chloride and DMSO to give aldehydes of general formula (13). In turn aldehydes such as (13) may be converted to amino acid esters of formula (14) by reductive amination procedures such as those described by Borch [ J Am. Chem. Soc. 1969, 9±, 3006] employing cyanoborohydride or triacetoxyborohydride anions. Hydroxamic acids of formula (8) may be prepared by the reaction of compounds of formula (14) with hydroxylamine hydrochloride in the presence of an alkali such as sodium or potassium hydroxide.

Compounds of general formula (21 ) and (23) may be, but not exclusively, prepared by methods outlined in Scheme 3.

Scheme 3

Reagents: a) Trimethylphosphonoacetate, K₂C0₃, THF b) BH₃:THF complex c) KOH, MeOH, H₂0 d) NHOR², HOBT, EDC e) Mn02 f) STAB, H₂/VCf?³R⁴C0₂/?⁵, THF g) NaOH, H₂0, MeOH h) 4N HCI, dioxane Thus α,β-unsaturated esters such as compounds of general formula (16) may be prepared by a Horner-Emmons reaction between a phosphonate carbanion and an aldehyde such as (15) in the presence of an inorganic base such as potassium carbonate under aqueous conditions. Alternatively other bases such as sodium hydride in DMSO or organic bases such as DBU in acetonitrile could be employed for this transformation. Alcohols of general formula (17) can be obtained by reduction of acids such as (16) with hydride-donor reagents such as borane in inert solvents such as THF. Hydrolysis of esters of general formula (17) to acids of general formula (18) may be performed by a mineral base such as sodium or potassium hydroxide under aqueous conditions in the presence of a co-solvent such as methanol. Aldehydes of general formula (19) may be obtained from ( 8) by a coupling reaction with an O-protected hydroxylamine in the presence of reagents such as N-hydroxybenzotriazole and 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide hydrochloride or N,N'-diisopropylcarbodiimide, followed by oxidation of the alcohol substituent of the resulting hydroxamic intermediate with a reagent such as manganese dioxide. Aldehydes of formula (19) may then be reacted with amino acid esters under conditions of reductive amination with reagents such as sodium triacetoxyborohydride or sodium cyanoborohydride to give compounds of general formula (20). Hydroxamic acids of general formula (21 ) may be prepared by deprotection of compounds of type (20), for example where R² is (1-isobutoxyethyl), with acidic reagents such as 4M HCI in dioxane. Amino acids such as (22) may be prepared by treating compounds of general formula (20) with a mineral base such as lithium hydroxide. Hydroxamic acids of general formula (23) may be prepared by treating compounds of formula (22) under acid conditions, for example with hydrochloric acid. Alternatively compounds such as (23) may be obtained by a process such as described in Scheme 4. Thus reagents such as 4-diethoxybenzaldehyde (24) may be reacted with trialkylphosphonoacetates in the presence of salts such as lithium bromide and organic bases such as triethylamine to give aldehydes such as (25) after acid work up. In turn aldehydes such as (25) may be converted to amino acid esters of formula (26) by reductive amination procedures such as those described by Borch [ J Am. Chem. Soc. 1969, 97, 3006] employing cyanoborohydride or triacetoxyborohydride anions.

Hydroxamates of general formula (23) may then be prepared by the treatment of compounds such as (26) with hydroxylamine hydrochloride in the presence of base such as potassium or lithium hydroxide in a solvent such as methanol or ethanol. Scheme 4

(23)

Reagents: a) Trialkylphoshonoacetate, Et₃N, LiBr, THF followed by HCI in Methanol b) STAB, THF c) KOH, NH20H.HCI, KOH, MeOH

Amino acid derivatives of general formula (28) may also be prepared by methods described in Scheme 5. Scheme 5

(27) (28)

Thus esters of general formula (27) [X=CH or N] may be hydrolysed to acids of type (28) [X=CH,N] with alkaline bases such as potassium or sodium hydroxide. In another procedure acids of general formula (28) [X=CH,N] may be prepared by methods described in Scheme 6. Thus aldehydes of general formula (29) [X=CH,N] and amino acids are reacted under conditions of reductive amination to yield intermediates of general formula (30) [X=CH,N]. The protected hydroxamates of formula (30) [X=CH,N] are treated under acid conditions, such as with hydrochloric acid to give acids of general formula (28) [X=CH,N]. Scheme 6

(29) (30) (28)

Reagents: a) H0₂CR¹R²NH₂, STAB or ce-picoline-borane, MeOH b) 4N HCI, dioxane Amino acid esters of general formula (32) may be prepared by a number of methods including those described in Scheme 7. Thus amino acids of formula (31 ) may be heated with the appropriate alcohol (R³OH) in the presence of H₂S0₄ or reacted with the appropriate alcohol (R³OH) under Dean-Stark conditions in the presence of an acid such as para-toluenesulphonic acid to give esters of formula (32).

Scheme 7

(31 ) (32)

Reagents: R³OH, H₂S0₄ orR³OH, PTSA, cyc!ohexane, Dean-Stark Amino acid esters of general formula (37) may be prepared by procedures such as those described in Scheme 8. Thus ketones of formula (33) where X is O, NBoc or N e for example may be reacted with sodium or potassium cyanide in the presence of ammonium carbonate to give hydantoins of general formula (34). Acids of general formula (35) may then be prepared by heating hydantoins of formula (34) with alkaline reagents such as sodium or potassium hydroxide under aqueous conditions in the presence of a co-solvent such as methanol or ethanol. Subsequent treatment with chloroformate reagents such as benzyl chloroformate gives carbamates of general formula (35). Esters of general formula (36) may be prepared by the treatment of (35) with reagents such as thionyl chloride in the presence of an alcohol such as

cyclopentanol. N-Deprotection of compounds of general formula (36) may be achieved by hydrogenolysis with catalysts such as 10% palladium on charcoal or 10% palladium hydroxide on charcoal to give amino acid esters of general formula (37).

Scheme 8

Reagents and conditions: a) KCN, (NH₄)₂C0₃, EtOH, H₂0, 60°C b) KOH, H₂0, 100°C followed by PhCHiOCOCI, acetone c) SOCI_2l ROH. d) 10% Pd-C, H₂, BOAc

Piperidine derivatives of general formula (39) may be prepared by methods shown in Scheme 9. Thus the reductive amination of N-protected piperidones such as N-Boc-4- piperidone with amino acid esters such as (37) using borohydride reagents such as sodium borohydride or sodium cyanoborohydride yields intermediates of general formula (38) which can subsequently be deprotected under acidic conditions such as TFA or HCI in dioxane to give piperidines of general formula (39).

Scheme 9

(37) (38) (39)

Reagents: a) N-Boc-4-piperidone, NaBH(OAc)₃, DCE b) 4M HCI in dioxane Intermediates

Intermediate 1 Ethyl (2E)-3-(5-formylpyridin-2-yl)acrylate

CH₂CI₂ (20 mL) and DMSO (3.4 mL) were charged to a flask and cooled to <-70 °C. Oxalyl chloride (1.47 mL) was charged drop-wise at below -65 °C then the reaction aged for -0.5 h. Ethyl (2£)-3-[5-(hydroxymethyl)pyridin-2-yl]prop-2-enoate (2 g, 9.6 mmol) was dissolved in CH₂CI₂ (20 mL) and charged to the reaction which was then aged at below - 70 °C for ~1 h. Triethylamine (6.7 mL) was charged and the reaction allowed to warm to ambient temperature. Water (40 mL) was charged to the reaction vessel, the layers separated and the aqueous phase extracted with CH2CI2. The combined organics were washed with water, dried (MgS0₄) then concentrated to dryness to afford Intermediate 1 as a brown solid (1.7g). ¹H N R (300 MHz, COCI₃) 5:10.14 (1 H, s), 9.10 (1 H, d), 8.20 (1 H, dd), 7.72 (1 H, d), 7.60 (1 H, d), 7.08 (1 H, d), 4.31 (2H, q), 1.37 (3H, t). Ethyl (2£)-3-f5-(hvdroxymethyl)pyridin-2-yl1prop-2-enoate

(6-Methylpyridin-3-yl)methanol (75 g, 0.61 mol) and acetic anhydride (380 mL) were heated to 80 °C for 1 h 20 min. 50% Ethyl glyoxal in toluene (165 mL, 0.83 mol) was added and the reaction heated to 100°C for 3 h when additional 50% ethyl glyoxal (55 mL, 0.28 mmol) was added. After 2 h, EtOH (250 mL) was added and the reaction heated at reflux for 1.5 h and then concentrated to dryness. The residue was dissolved in ethanol (400mL), concentrated hydrochloric acid (40mL) charged then heated at reflux for 3h, until no further acetylated product was seen. On cooling the reaction was concentrated in vacuo to afford the crude product. Diethyl ether was charged to the concentrate, slurried, then decanted from the brown oily residue. 2N Potassium carbonate solution (400mL) and ethyl acetate (400mL) were charged to the residue then separated. The aqueous phase extracted twice with ethyl acetate (400mL). The combined organic phases were washed with brine (400mL), dried (Na₂S0₄) and concentrated to dryness. Purification by silica chromatography (1 :1 ethyl acetate/ n- heptanes eluant) afforded the desired product (38.6g). ¹H NMR (CDCI₃): 8.62 (1 H, d, J=1 .8). 7.76 (1 H, dd, J=7.8, 2.1 ), 7.69 (1 H, d, J=15.9), 7.44 (1 H, d, J=7.8), 6.90 (1 H, d, J=15.6), 4.78 (2H, s), 4.26 (2H, q, J=7.2), 1 .35 (3H, t, J=7.2). (6-Methylpyridin-3-yl)methanol

The (6-Methylpyridin-3-yl)methanol used in the above process was prepared as follows:THF (4 L) was added to a flask containing LiAIH₄ (150.4 g, 3.96 mol) and the resulting suspension cooled to < 0 °C. Methyl 6-methylnicotinate (500 g, 3.31 mol) in THF (1 L) was added dropwise to the reaction over 2.5 h, maintaining a temperature < 5 °C. Stirring was continued at 0± 5 °C for 2 h and then the reaction was quenched slowly over 1 h 25 min with satd. NaHC0₃ (1 L) at < 10 °C. The reaction was then stirred overnight at ambient temperature. The reaction was then filtered, and the filter cake washed with EtOAc (2.5 L) and the filtrate concentrated to dryness. The residue was partitioned between NaHC0₃ (500 ml_) and EtOAc (1 .5 L). The layers were separated and the aqueous layer extracted with EtOAc (3 x 1 L). The combined organic layers were concentrated to dryness. The residue was dissolved in EtOAc (1 L), dried (Na₂S0₄) and concentrated to dryness to give the title compound (366 g). ¹H NMR (CDCI₃): 8.31 (1H, d, J=2.1 ), 7.59 (1 H, dd, J=7.8, 2.1 ), 7.10 (1 H, d, J=7.8), 4.69 (2H, s), 2.48 (3H, s).

Intermediate 2 (2E)-3-(5-Formylpyridin-2-yl)-A/-(1-isobutoxyethoxy) acrylamide

Intermediate 2 was prepared by methods described in WO2008/040934. Intermediate 3 (20-3-(4-Formylphenyl)-A/»(1-isobutoxyethoxy)acrylamide

Intermediate 3 was prepared by methods described in WO2008/040934.

Intermediate 4 f2E)-3-(6-Formylpyridin-3-yl)-/V-ri-(2-methylpropoxy)ethoxy1prop-2- enamide

(2£)-3-[6-(Hydroxymethyl)pyridin-3-yl]-W-[1 -(2-methylpropoxy)ethoxy]prop-2-enamide (925 mg, 3.15 mmol) was dissolved in anhydrous CHCI₃ (50 mL) under N₂, and manganese dioxide (2.74 g, 31.5 mmol) added. The reaction was stirred for 1 h and then filtered through Celite. The filtrate was concentrated to dryness and the residue used without purification, m/z 293 [M+H]\

The (2E)-3-[6-(hydroxymethyl)pyridin-3-yl]-A/-[1-(2-methylpropoxy)ethoxy]prop-2- enamide used in the above process was prepared by dissolving (2£)-3-[6- (hydroxymethyl)pyridin-3-yl]prop-2-enoic acid (255.7 mg, 1.42 mmol), 0-[1-(2- methylpropoxy)ethyl]hydroxylamine [WO2001/60785] (0.58 mL, 4.29 mmol), EEDC (330.7 mg, 2.13 mmol) and DMAP (catalytic) in CH₂CI₂ (100 mL) and stirring at RT for 1 h. The reaction was washed with water (100 mL) and the organic layer dried (MgS0₄) and concentrated to dryness. The residue was purified by column chromatography (silica gel, MeOH in CH₂CI₂ 5-10%) to give the desired material (140.9 mg). m/z 295 [M+H]⁺. Re-extraction of the aqueous layer with n-butanol gave additional material.

The (2E)-3-[6-(hydroxymethyl)pyridin-3-yl]prop-2-enoic acid used in the above process was prepared by dissolving methyl (2£)-3-[6-(hydroxymethyl)pyridin-3-yl]prop-2-enoate (1.0g, 5.18 mmol) and KOTMS (2.7 g, 20.73 mmol) in anhydrous THF (50 mL) and the mixture left to stir under N₂ for 1 h. The reaction was poured into stirred water (100 mL) and acidified to pH 4 with 1M HCI. The resulting solution was extracted with EtOAc (5 x 200 mL) and the combined organic layers were dried (MgS04) and concentrated to dryness to give the desired material (255 mg). m/z 178 [M-H]^'. Additional product (312 mg) was obtained by extraction of the aqueous layer with n- butanol.

The methyl (2E)-3-[6-(hydroxymethyl)pyridin-3-yl]prop-2-enoate used in the above process was prepared by methods described in WO2008/040934.

Intermediate 5 Cyclopentyl 1-f({5-r(1E)-3-(ri-(2-methylpropoxy)ethoxy1amino>-3- oxoprop-1-en-1-vnpyridin-2-yl>methyl)amino1cvclopropane carboxylate

Intermediate 4 (100 mg, 0.34 mmol) and Intermediate 22 (133 mg, 0.34 mmol) were dissolved in DCE (10 mL) and stirred at RT for 10 min. To the solution was added sodium triacetoxyborohydride (108 mg, 0.51 mmol) and the reaction was then stirred at RT for 2 h. The reaction was quenched by the addition of water and then diluted with CH₂CI₂ (20 mL) and washed with water (2 x 50 mL). The organic layer was dried (MgSC ) and concentrated to dryness. The residue was purified by automated column chromatography (silica gel 12 g column, eluting with methanol in CH₂CI₂, 0-20%) to give the title compound (47.4 mg). m/z 446 [M+Hf. The following compounds were prepared using similar methodology to Intermediate 5:

Intermediate 6 Cyclopentyl 1-r((5-r(1E)-3-methoxy-3-oxoprop-1-en-1-vnpyridin-2- yl)methyl)amino1cyclobutanecarboxylate

From Intermediate 26 (200 mg, 1.05 mmol) and Intermediate 21 (371.6 mg, 1.05 mmol) to give the title compound (228.7 mg, 61 %), m/z 359 [ +H]⁺. Intermediate 7 Cvclopentyl 1-r((5-r(1E)-3-(ri-f2-methylpropoxy)ethoxy1amino>-3- oxoprop-1-e -1-vnpyridin-2-yl)methyl)aminolcvclopentanecarboxylate

From Intermediate 4 (100 mg, 0.34 mmol), Intermediate 20 (126 mg, 0.34 mmol) and sodium triacetoxyborohydride (108mg, 0.56mmol) to give the title compound (36mg, 22%). m/z 474 [M+H]⁺.

Intermediate 8 Cvclopentyl 1-r((5-r(1£)-3-(hvdroxyamino)-3-oxoprop-1-en-1-vn pyridine-2-yl)methyl)amino1cvclohexanecarboxylate

From Intermediate 26 (200mg, 1.05mmol), Intermediate 19 (220mg, 1.05 mmol) and sodium triacetoxyborohydride (333 mg, 1.57 mmol) to give the title compound (251 mg, 62%) m/z 387 [M+H]⁺.

Intermediate 9 Cvclopentyl 4-r(i5-r(ia-3-ai-(2-methylpropoxy¾ethoxy1amino -3- oxoprop-1-en-1-yllpyridin-2-yl)methvnamino1tetrahvdro-2H-pyran-4-carboxylate

From Intermediate 4 (50 mg, 0,23 mmol), Intermediate 23 (67 mg, 0.23 mmol) and sodium cyanoborohydride (21 mg, 0.34 mmol) to give the title compound (31 mg, 27%). m/z 490 [M+H]⁺ Intermediate 10 Cvclopentyl 4-f^5-f 1g)-3- f1-(2-methylpropoxy)ethoxy1amino}-3- oxoprop-1-en-1-vnpyridin-3-yl methvhamino1tetrahydro-2H-pyran-4-carboxylate

From Intermediate 2 (36 mg, 0.17 mmol), Intermediate 23 (50 mg, 0.17 mmol) and sodium cyanoborohydride (16 mg, 0.26 mmol) to give the title compound (47mg, 57%). m/z 490 [ +H]⁺

Intermediate 11 Cvclopentyl 1-((2-methyl-4-r(1E)-3-(ri-(2-methylpropoxy)ethoxy1 amino}-3-oxoprop-1-en-1-yl1benzyl)amino)cvclohexane carboxylate

From Intermediate 17 (100 mg, 0.33 mmol), Intermediate 19 (69 mg, 0.33 mmol) and sodium triacetoxyborohydride (104mg, 0.49 mmol) to give the title compound (48mg, 30%). m/z 501 [M+H]⁺.

Intermediate 12 Cvclopentyl 1-((2-methyl-4-r(1E)-3-{ri-(2-methylpropoxy)ethoxy1 amino)-3-oxoprop-1-en-1-yl1benzyl}amino)cvclopentane carboxylate

From Intermediate 17 (100 mg, 0.33 mmol), Intermediate 20 (122mg, 0.33 mmol) and sodium triacetoxyborohydride (104 mg, 0.49 mmol) to give the title compound (10.7mg). m/z 487 [M+H]⁺. Intermediate 3 Cyclopentyl 1-(/2-methyl-4-r 1-r)-3-f - 2-methylpropoxy)ethoxyl amino}-3-oxoprop-1 -en-1 -yl1benzyl}amino)cvclobutane carboxylate

From Intermediate 17 (100 mg, 0.33 mmol), Intermediate 21 (1 17 mg, 0.33 mmol) and sodium triacetoxyborohydride (104 mg, 0.49 mmol) to give the title compound (22.1 mg). m/z 473 [M+H]⁺

Intermediate 14 Cyclopentyl 4-{[1-((6-r(1 E)-3-(f1-(2-methylpropoxy)ethoxy1amino -

3- oxoprop-1-en-1-vnpyridin-3-yl methyl)piperidin-4-vnamino>tetrahvdro-2H-pyran-

4- carboxylate

From Intermediate 2 (30 mg, 0.1 mmol), Intermediate 25 (30 mg, 0.1 mmol) and sodium triacetoxyborohydride (32 mg, 0.15 mmol) to give the desired material (43 mg) as a brown oil. m/z 573 [M+H]⁺.

Intermediate 15 Cyclopentyl 4-(ri-( 5-f(1 E)-3-(i1-(2-methylpropoxy)ethoxy1amino>- 3-oxoprop-1-en-1-vnpyridin-2-yl)methyl)piperidin-4-vnamino>tetrahvdro-2H-pyran- 4-carboxylate

From Intermediate 4 (30 mg, 0.1 mmol) and Intermediate 25 (30 mg, 0.1 mmol) and sodium triacetoxyborohydride (32 mg, 0.15 mmol) to give the title compound (50 mg) as a yellow oil. m/z 573 [M+H]⁺. Intermediate 16 Cyclopentyl 4-({4-r(1g)-3-fri-(2-methylpropoxy)ethoxy1amino)-3- oxoprop-1-en-1-yllbenzyl)amino tetrahydro-2H-pyran-4-carboxylate

From Intermediate 3 (50 mg, 0.17 mmol), Intermediate 23 (38 mg, 0.17 mmol) and sodium triacetoxyborohydride (43 mg, 0.204 mmol) to give the title compound (45 mg) as a yellow solid, m/z 489 [M+Hf.

Intermediate 17 (2E)-3-(4-formyl-3-methylphenvn-A/-ri-(2-methylpropoxy)ethoxy1 prop-2-enamide

(2£)-3-[4-(Hydroxymethyl)-3-methylphenyl]-/v-[1 -(2-methylpropoxy)ethoxy]prop-2- enamide (358 mg, 1.17 mmol) was dissolved in CHCI3 (40 ml_), manganese dioxide (1.01 g, 11.7 mmol) was added and the reaction stirred under N₂ at RT for 1 h. Further manganese dioxide (200 mg) was added and stirring continued for 0.5 h. The reaction was filtered through Celite and solvent evaporated under reduced pressure. The product was used without purification, m/z 304 [M-H]^".

The (2£)-3-[4-(hydroxymethyl)-3-methylphenyl]-A/-[1-(2-methylpropoxy)ethoxy]prop-2- enamide used in the above process was prepared by adding 0-[1-(2-methylpropoxy) ethyl]hydroxylamine [WO2001/60785] (1.65 mL, 12.03 mmol), DMAP (catalytic amount), and EDC (934 mg, 6.015 mmol) to a stirred solution of (2E>3-[4-(hydroxymethyl)-3- methylphenyl]prop-2-enoic acid in CH₂CI₂ (50 mL). The resulting solution was stirred over 72 h at RT and then washed with water (2 x 100 mL). The organic layer was dried (Na₂S0₄) and the solvent evaporated under reduced pressure. The residue was purified using automated column chromatography (24 g slica gel column, 0-100% EtOAc in heptanes) to give the desired product (358 mg) as a colourless oil. m/z 330 [M+Na]⁺.

The (2E)-3-[4-(hydroxymethyl)-3-methylphenyl]prop-2-enoic acid used in the above process was prepared by stirring terf-butyl (2£)-3-{4-[(methoxymethoxy)methyl]-3- methylphenyl}prop-2-enoate (1.28 g, 4.39 mmol) in THF (50 mL) with 4 HCI in dioxane (5.49 mL) at RT for 4 h. After this time additional 4M HCI in dioxane (5 mL) was added and stirring was continued for 16 h at RT. Further 4 HCI in dioxane (5 mL) was added and the reaction heated to 60 °C for 6 h. The reaction was diluted with EtOAc (100 mL) and washed with water (2 x 100 mL). The organic phase was dried ( gS04) and the solvent evaporated under reduced pressure to give the desired product (770 mg) as an off-white solid which was used without purification, m/z 193 [ +Hf.

The terf-butyl (2E)-3-{4-[(methoxymethoxy)methyl]-3-methylphenyl}prop-2-enoate used in the above process was prepared by adding Pd(PPh₃)₄ (92.4 mg, 0.08 mmol) to a stirred solution of 4-bromo-1 -[(methoxymethoxy)methyl]-2-methylbenzene (1 g, 4.08 mmol), ferf-butyl acrylate (4.2 mL, 28.37 mmol) in DMA (100 mL). The reaction was heated to 140 °C under N₂ for 2 h. The reaction was filtered through Celite, and the solvent removed in vacuo. The residue was dissolved in EtOAc (100 mL) and washed with 0.3M KHS0₄ (100 mL) followed by brine (100 mL). The organic layer was dried (Na₂S0₄) and solvent evaporated under reduced pressure. The residue was purified using automated column chromatography (24 g silica gel column, 0-100% EtOAc in heptane) to give the desired product (1.28 g) as a colourless oil. ¹H NMR (CDCI₃) δ ppm 7.57 (1 H, d), 7.48-7.32 (3H, m), 6.49 (1 H, d), 4.72 (2H, s), 4.61 (2H, s), 3.44 (3H, s), 2.36 (3H, s), 1.56 (9H, s).

The 4-bromo-1-[(methoxymethoxy)methyl]-2-methylbenzene used in the above process was prepared by dissolving (4-bromo-2-methylphenyl)methanol (4.12 g, 20.5 mmol) in CH₂CI₂ (100 mL) followed by the addition of MOMCI (3.11 mL, 41.0 mmol) and DIEA (8.9 mL, 51.3 mmol). The resulting mixture was heated to reflux for 24 h and the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (-150 mL) and washed with 10% citric acid (aq) (1 x 50 mL), satd. NaHC0₃ (2 x 50 mL) and brine (2 x 50 mL). The organic layer was dried (MgS0₄) and evaporated under reduced pressure. The residue was purified by automated column chromatography (50 g silica gel column, 0-100% EtOc in heptane) to give the desired material (4.13 g). ¹H NMR (CDCI₃) δ ppm 7.23-7.18 (3H, m), 7.11 (1 H, d), 4.58 (2H, s), 4.45 (2H, s), 3.32 (3H, s), 2.21 (3H, s).

The (4-bromo-2-methylphenyl)methanol used in the above process was prepared by dropwise addition of BH₃.THF (27.76 mL, 27.76 mmoL) to a stirred solution of 4-bromo- 2-methylbenzoic acid (2.99 g, 13.88 mmol) in THF (20 mL) under N₂ at 0 °C. After complete addition of BH₃.THF the reaction was stirred at RT, for 4 h and then cold water (10 mL) was added. The reaction was washed with satd. NaHC0₃ (60 mL) and the aqueous phase washed with EtOAc (3 x 100 mL). The combined organic layers were dried (MgSC>₄) and the solvent was removed under reduced pressure to give the desired material (2.83 g). ¹H NMR (d₆-DMSO) δ ppm 7.51-7.32 (3H, m), 5.25 (1 H, t), 4.51 (2H,d), 2.31 (3H, s).

Intermediate 18 2-methylcvclopentyl 4-i(f6-r(10-3-(ri-(2-methylpropoxy) ethoxy1amino>-3-oxoprop-1-en-1-vnpyridin-3-yl>methyl)amino1tetrahvdro-2H- pyran -4-carboxyl ate

Intermediate 2 (85.3 mg, 0.29 mmol) and Intermediate 24 (65.8 mg, 0.29 mmol) were dissolved in DCE (5 mL) and the reaction was stirred at RT for 10 min. Sodium triacetoxyborohydride (92 mg, 0.43 mmol) was added and stirring continued for 1 h. Further Intermediate 2 (100mg) was added as some starting Intermediate was observed (LC/MS) and stirring at RT was continued for 2 h. After this time some Intermediate was still observed (LC/MS) and further Intermediate 2 (100 mg) was added and stirring continued at RT overnight (16 h). The reaction was quenched with water and then diluted with CH₂CI₂ (30 mL). The reaction was washed with water (2 x 30 mL), the organic layer dried (MgS0₄) and concentrated to dryness. The residue was purified by automated column chromatography (13 g, reverse phase column, CH₃CN in water, 0- 100%) to give to give the desired material (138.4 mg). m/z 504 [M+H]⁺, 502 [M-H]^".

Intermediate 19 Cvclopentyl 1-aminocvclohexanecarboxylate tosylate

To 1-aminocyclohexanecarboxylic acid (4.2 g, 29 mmol) in cyclohexane (250 mL) was added cyclopentanol (50 mL) and para-toluenesulphonic acid (5.89 g) and the resulting suspension heated at reflux in a Dean-Stark apparatus for 72 h. On cooling to RT the resulting white solid was collected by filtration and washed with cyclohexane (2 x 100 mL) and dried under reduced pressure to give the title compound (tosylate salt) (4.1 g) as a colourless solid, m/z 212.3 [M+H]\

Intermediate 20 Cvclopentyl 1-aminocvclopentanecarboxylate

To a solution of 1-aminocyclopentanecarboxylic acid (2.58 g, 19.97 mmol) in

cyclopentanol (20 ml), was added concentrated sulfuric acid (2.15g, 21.97mmol) and the mixture stirred over night at 70 °C. The reaction was allowed to cool to RT and the cyclopentanol removed under reduced pressure. The residue was dissolved in EtOAc (30 mL) and washed with sat. NaHCO₃ (30 mL) and water (3 x 20 mL) then dried (MgS0 ), filtered and concentrated in vacuo to leave a dark yellow oil. Purification by column chromatography (15% 1.2M NHj/ eOH in EtOAc) afforded the desired product (1.97 g, 50% yield). ¹H NMR (300 MHz, CDCI₃) δ: 5.21-5.17 (1 H, m), 2.15-1.90 (2H, m), 1.85-1.57 (14H, m).

Intermediate 21 Cvclopentyl 1-aminocvclobutanecarboxylate tosylate

1-Amino-1-cyclobutanecarboxylic acid (1 g, 8.7 mmol), cyclopentanol (2.4 mL) and para- toluenesulphonic acid (1.8 g, 9.6 mmol) were stirred in cyclohexane (5 mL) and heated to reflux for 23 h before additional cyclohexane (3 mL) was added. After an additional 80 minutes the reaction was cooled briefly to 70 °C and cyclohexane (5 mL) and cyclopentanol (2.4 mL) were added and heating at reflux was continued until the reaction was complete. On cooling the reaction to ambient temperature methyl t-butyl ether (50 mL) was added and the reaction stirred for 10 min. The resulting solid was collected by filtration and washed with methyl t-butyl ether (20 mL) to give Intermediate 21 (2.83 g) as the tosylate salt, m/z 184 [M+H]⁺ 1 H NMR (300 Hz,d6-D SO) δ: 7.72 (2H, d), 7.25 (2H, d), 5.35 (1 H, m), 2.58-2.67 (2H, m), 2.42-2.48 (2H, m), 2.41 (3H, ), 2.10-2.39 (2H, m), 1.93-2.08 (2H, m), 1.71-1.86 (6H, m)

Intermediate 22 Cyclopentyl 1-aminocyclopropanecarboxylate

Following the method of Intermediate 21 from 1-aminocyclopropane-1-carboxylic acid (500 mg, 4.95 mmol) to give Intermediate 22 (302.9 mg) m/z 170 [M+H]⁺

Intermediate 23 Cyclopentyl 4-arninotetrahvdro-2H-pyran-4-carboxylate

To cyclopentyl 4-{[(benzyloxy)carbonyl]amino}tetrahydro-2H-pyran-4-carboxylate (767 mg, 2.21 mmol) in EtOAc (50 mL) was added 10% palladium on charcoal (70 mg) and the reaction stirred at RT under H₂ gas for 72 h. The reaction was filtered through Celite and the filtrate concentrated in vacuo to give the desired material (497 mg) as an orange oil. m/z 214 [M+H]⁺.

The cyclopentyl 4-{[(benzyloxy)carbonyl]amino}tetrahydro-2/-/-pyran-4-carboxylate used in the above process was prepared by adding thionyl chloride (392 μΙ_, 5.37 mmol) a stirred solution of cyclopentanol (20 mL) and 4-{[(benzyloxy)carbonyl]amino}tetrahydro- 2/-/-pyran-4-carboxylic acid (1.0g, 3.58 mmol) stirred at RT under N₂. The reaction was heated to 90 °C and stirred for 18 h. The solvent was removed in vacuo and water (50 mL) was added to the residue. The pH was adjusted to 8 with solid NaHC0₃ and the resulting mixture was extracted with EtOAc (2 x 50 mL). The combined organic layers were dried (Na₂S0₄) and solvent was removed in vacuo. The residue was purified by column chromatography (silica gel, 0-100% EtOAc in heptanes) to give the desired material (767 mg) as a yellow oil. m/z 348 [M+H]⁺. The 4-{[(benzyloxy)carbonyl]amino}tetrahydro-2H-pyran-4-carboxylic acid used in the above process was prepared by stirring 8-oxa-1 ,3-diazaspiro[4.5]decane-2,4-dione (15.7 g, 85.33 mmol) and potassium hydroxide (28.73 g, 85.33 mmol) in water (300 mL) at 100 °C for 66 h. The reaction was then cooled to 0 °C and diluted with acetone (200 mL). A solution of benzyl chloroformate (48.7 mL) in acetone (100 mL) was added slowly at 0 °C and stirring was continued at RT for 3 h. Acetone was removed in vacuo and the resulting mixture was extracted with EtOAc (2 x 200 mL). The aqueous layer was acidified to pH -1 using 5M HCI and this was extracted with EtOAc (2 x 200 mL). These extracts were dried (Na₂S0₄) and the solvent removed in vacuo to give the desired material (9.01 g) as an orange solid, m/z 280 [M+H]⁺.

The 8-oxa-1 ,3-diazaspiro[4.5]decane-2,4-dione used in the above process was prepared by heating tetrahydro-4-pyrane (10 g, 100 mmol), potassium cyanide (16.22 g, 200 mmol) and ammonium carbonate (38.43 g, 400 mmol) in ethanol/water (1 :1 v/v, 240 mL) at 60 °C for 18 h. The reaction was cooled to RT and the solvent removed in vacuo. The residue was stirred in cold water, filtered and dried to give the desired product (15.7 g) as a white solid, m/z 171 [M+H]^{+ 1}H N R (300 MHz, d6-DMSO) $ ppm; 8.49 (2H, bs), 3.81 (2H, dt, J = 11.7Hz, 4.2Hz), 3.59 (2H, td, J = 11.7Hz, 2.7Hz), 1.83 (2H, m), 1.46 (2H, m). Intermediate 24 2-methylcvclopentyl 4-aminotetrahvdro-2H-pyran-4-carboxylate

2-Methylcyclopentyl 4-{[(benzyloxy)carbonyl]amino}tetrahydro-2H-pyran-4-carboxylate (279 mg, 0.77 mmol) was dissolved in MeOH (20 mL) and Pd(OH)₂ on carbon (27.9 mg) in MeOH (<5 mL) was added as a slurry. The solvent was degassed and then left to stir under a hydrogen atmosphere. After 2 h the reaction was filtered through Celite, and the Celite washed with MeOH (50 mL). The reaction was then concentrated to dryness to give the desired material (130.9 mg). m/z 228 [M+H]⁺. The 2-methylcyclopentyl 4-{[(benzyloxy)carbonyl]amino}tetrahydro-2W-pyran-4- carboxylate used in the above process was prepared by dissolving 4-{[(benzyloxy) carbonyl]amino}tetrahydro-2H-pyran-4-carboxylic acid (250 mg, 0.9 mmol), 2- methylcyclopentanol (1.36 mL, 2.7 mmol), EDC (209 mg, 1.35 mmol) and DMAP (catalytic amount) in CH₂CI₂ (20 mL) and stirring at RT for 4 h. More DMAP and 2- methylcyclopentanol (1 mL) were added and the reaction was heated to 35 °C for 144 h, more DMAP and trans-2-methylcyclopentanol (2 mL) was added. After a further 24 h, the reaction was cooled to RT and diluted with CH₂CI₂. The reaction was washed with water (2 x 30mL) and the organic layer dried (Na₂S0₄) and then concentrated to dryness. The compound was purified using automated column chromatography (25 g, silica gel column, 0-100% EtOAc in heptane) to give the desired material (279 mg). m/z 362

[M+H]\ 384 [M+Na]⁺.

Intermediate 25 Cvclopentyl 4-(piperidin-4-ylamino)tetrahvdro-2H-pyran-4- carboxylate hydrochloride

ferf-Butyl 4-({4-[(cyclopentyloxy)carbonyl]tetrahydro-2H-pyran-4-yl}amino)piperidine-1- carboxylate (81 mg, 0.2 mmol) was stirred in CH₂CI₂ (10 mL) at RT under N₂ and 4M HCI in dioxane (250 μΙ_, 1 mmol) was added. After stirring for 18 h, the solvent was removed under reduced pressure to give the desired product as a brown solid as the

hydrochloride salt, m/z 297 [M+H]⁺.

The ferf-butyl 4-({4-[(cyclopentyloxy)carbonyl]tetrahydro-2H-pyran-4-yl}amino)piperidine- 1 -carboxylate used in the above process was prepared by adding sodium triacetoxy borohydride (132 mg, 0.81 mmol) to a stirred solution of ferf-butyl 4-oxopiperidine-1- carboxylate (107 mg, 0.54 mmol) in DCE (10 mL) at RT under N₂. The reaction was left to stir for 72 h and then partitioned between water and CH₂CI₂ ( 00 mL, 1 :1 v/v). The organic layer was separated and the aqueous layer extracted with CH₂CI₂ (50 mL). The combined organic layers were dried (Na₂S0₄) and the solvent removed under reduced pressure. The residue was purified by column chromatography (silica gel, 0-100% EtOAc in heptane) to give the desired material (81 mg) as a brown oil. m/z 397 [M+H]⁺.

Intermediate 26 Methyl (2El-3-(6-formylpyridin-3-yl)prop-2-enoate

To a solution of methyl (2£)-3-[6-(hydroxymethyl)pyridin-3-yl]prop-2-enoate

[WO2008/040934] (650 mg, 3.37 mmol) in CH₂CI₂ (50 mL) under N₂ was added manganese dioxide (2.92 g, 33.7 mmol) and the reaction stirred at RT for 1 h. The reaction was filtered through Celite, washing with additional CH₂CI₂. The combined filtrate was concentrated to dryness to give the product (assume 100% conversion) which was used without purification.

methylpropoxy)ethoxy1amino)-3-oxoprop-1-en-1-yllbenzyl>-D-leucinate

Following the method of Intermediate 5, from Intermediate 3 (221 mg, 0.76 mmol), Intermediate 28 (163 mg, 0.76 mmol) and sodium triacetoxyborohydride (242 mg, 1.14 mmol) to give the title compound (26 mg) as a yellow oil. m/z 491 [M+H]\

Intermediate 28 (3/?)-Tetrahvdrofuran-3-yl 2-methyl-D-leucinate Tosylate

Following the method of Intermediate 21 from (R)-oc-methylleucine [Nagase & Co., Ltd. 1-1-17 Shinmachi, Nishi-Ku, Osaka City, Osaka, Japan] (500 mg, 3.42 mmol), (R)-(-)-3- hydroxytetrahydrofuran (549 μΙ_, 6.84 mmol) and 4-toluenesulphonic acid (715 mg, 3.76 mmol) to give the title compound (1.32 g) which was used without purification.

Intermediate 29 (2.=n-3-(3-Formylphenyl)- V-ri-(2-methylpropoxy)ethoxylprop-2- enamide

To a solution of (2E)-3-[3-(hydroxymethyl)phenyl]-/V-[1-(2-methylpropoxy)ethoxy]prop-2- enamide (321.2 mg, 1.10 mmol) in anhydrous CHCI₃ (20 mL) at RT was added manganese dioxide (476 mg, 5.48 mmol). The reaction was stirred for 1 h, at which point additional manganese dioxide (500 mg) was added and stirring at RT was continued for 1 h. The reaction was filtered through Celite and the filtrate concentrated to dryness to give the product as a yellow oil (100% conversion assumed), m/z 314 [ +Na]⁺, 605 [2M+Na]⁺. The (2E)-3-[3-(hydroxymethyl)phenyl]-/S/-[1 -(2-methylpropoxy)ethoxy]prop-2-enamide used in the above process was prepared by dissolving (2£)-3-[3-(hydroxymethyl)phenyl] prop-2-enoic acid (1.41 g, 7.93 mmol), 0-[1-(2-methylpropoxy)ethyl]hydroxylamine

[WO2001/60785] (3.27 mL, 23.78 mmol), EDC (1.85 g, 11.89 mmol) and DMAP

(catalytic amount) in CH₂CI₂ at RT . The reaction was stirred overnight and then washed with water (1 x 100ml_) and the aqueous layer extracted with DCM (2 x 100 mL). The combined organic layers were combined, dried (Na₂S0₄) and evaporated to dryness. The residue was purified by automated column chromatography [25 g silica gel column eluting with EtOAc in heptanes (0-100%) to give the desired material (321.2 mg). m/z 316 [M+Na]⁺, 609 [2M+Na]⁺.

The (2E)-3-[3-(hydroxymethyl)phenyl] prop-2-enoic acid used in the above process was prepared by vigorously stirring a solution of methyl (2E)-3-[3-(hydroxymethyl)phenyl] prop-2-enoate (1 ,91 g, 9.94 mmol) in THF (50 mL) with 1 NaOH (50 mL) at RT. The reaction was stirred for 2 h and then acidified to -pH 3 with 1 M HCI. The reaction was extracted with EtOAc (3 x 100 mL), the combined organic layers dried (Na₂S0₄) and concentrated to dryness to give the desired material (1.41 g). m/z 177 [M-H]^".

The methyl (2E)-3-[3-(hydroxymethyl)phenyl] prop-2-enoate used in the above process was prepared by methods described in WO2008/040934.

Examples

Example 1 Cvclopentyl 1-f((5-r(1E)-3-(hvdroxyamino)-3-oxoprop-1-en-1-vnpyridin- 2-yl)methyl)amino1cvclopropanecarboxylate Trifluoroacetate

Intermediate 5 (47 mg, 0.10 mmol) was dissolved in CH₂CI₂/MeOH (11 mL, 10:1 v/v) and 4M HCI in dioxane was added (0.125 mL, 0.5 mmol). The reaction was stirred at RT for 1 h. The solvent was evaporated under reduced pressure and the residue purified by HPLC to give the title compound (5.26 mg) as an orange solid, m/z 346 [M+H]⁺. ¹H NMR (300 MHz, CD₃OD) δ ppm; 8.81 (1 H, s), 8.12 (1 H, d, J = 7.0 Hz), 7.63 (1 H, d, J = 16.0 Hz), 7.53 (1 H, d, J = 8.3 Hz), 6.64 (1H, d, J = 16.0 Hz), 5.30 (1 H, m), 4.60 (2H, s), 1.90 (2H, m), 1.76-1.60 (8H, m), 1.55 (2H, m).

Example 2 Cyclopentyl 1-r((5-r(1E)-3-(hvdroxyamino)-3-oxoprop-1-en-1-vnpyridin- 2-yl}methyl)amino1cvclobutanecarboxylate Trifluoroacetate

To a stirred solution of Intermediate 6 (228.7 mg, 0,63 mmol) and hydroxylamine hydrochloride (131.4 mg, 1.89 mmol) in MeOH (10 mL) at below 5 °C was added a solution of potassium hydroxide (212 mg, 3.78 mmol) in water (3 mL). After stirring at below 5 °C for 6 h, the reaction was allowed to warm to RT and stirring continued overnight (~ 12 h). The solvent was evaporated under reduced pressure and the residue purified by preparative HPLC to give the title compound (24 mg) as brown solid, m/z 360 [M+H]⁺. ¹H NMR (300 MHz, CD₃OD) δ ppm; 8.81 (0.7H, s), 8.75 (0.3H, s), 8.10 (0.7 H, d, J = 8.1 Hz), 8.00 (0.3H, dd, J = 8.0, 2.0 Hz), 7.63 (0.7H, d, J = 15.6 Hz), 7.52 (0.7H, d, J = 7.2 Hz), 7.48 (0.3H, d, J = 7.2 Hz), 6.67 (0.7H, d, = 15.8 Hz), 5.75 (0.3H, t, J = 8.6 Hz), 5.40 (1 H, m), 4.39 (2H, s), 3.19 (0.3H, dd, J = 16.4, 8.1 Hz), 2.90 (0.3H, dd, J = 16.4 Hz, 9.2 Hz), 2.68 (4H, m), 2.32 (1 H, m), 2.17 (1 H, m), 1.98-1.71 (8H, m).

Example 3 Cyclopentyl 1-r((5-r(1£)-3-(hvdroxyamino)-3-oxoprop-1-en-1-vnpyridin- 2-yl)methyl)amino1cvclopentanecarboxylate Trifluoroacetate

Following the method of Example 1 , from Intermediate 7 (36 mg, 0.076 mmol) to give the title compound (13 mg,) after purification by preparative HPLC as a red-orange solid, m/z 374 [ +Η · ¹H NMR (300 MHz, CD₃OD) δ ppm; 8.82 (1 H, s), 8.10 (1 H, m), 7.65 (1 H, d, J = 13.8 Hz), 7.53 (1 H, m), 6.67 (1 H, m), 5.35 (1 H, m), 4.46 (2H, s), 2.67 (2H. m), 2.37 (2H, m), 1.96 (6H, m), 1.79 (6H, m).

Example 4 Cvclopentyl 1-r({5-r(1-=)-3-(hvdroxYamino)-3-oxoprop-1-en-1-vnpyridin- 2-yl>methyl)aminolcvclohexanecarboxylate trifluoroacetate

Following the method of Example 2 from Intermediate 8 (251 mg, 0.65 mmol) and hydroxylamine hydrochloride (136 mg, 1.96 mmol) to give the title compound (1 14 mg) as an orange solid, m/z 388 [M+H]⁺. Ή NMR (300 MHz, CD₃OD) 8 ppm; 8.78 (0.7H, s), 8.74 (0.3H, s), 8.08 (0.7 H, d, J = 7.2 Hz), 8.00 (0.3H, d, J = 8.1 Hz), 7.56 (1.7H, m), 6.66 (0.7H, d, J = 15.8 Hz), 5.74 (0.3H, t, J = 8.5Hz), 5.33 (1 H, m), 4.40 (2H, s), 3.19 (0.3H, dd, J = 15.3, 7.9 Hz), 2.88 (0.3H, dd, J = 16.5, 9.1 Hz), 2.40 (3H, m), 1.93-1.65 (13H, m), 1.44 (2H, m). Example 5 Cvclopentyl 4-r 5-r(1-=)-3-(hvdroxyamino)-3-oxoprop-1-en-1-vnpyridin- 2-yl)methyQamino1tetrahvdro-2 Y-pyran-4-carboxylate trifluoroacetate

Following the method of Example 1 , from Intermediate 9 (31 mg, 0.06 mmol) to give the title compound (6 mg,) as an orange solid, m/z 390 [M+Hf. ¹H NMR (300 MHz,

CD₃OD) δ ppm; 8.82 (1 H, m), 8.12 (1 H, m), 7.63 (1 H, d, J = 15.9Hz), 7.55 (1 H, d, J = 8.4Hz), 6.64 (1 H, d, J = 15.9Hz), 5.39 (1 H, m), 4.38 (2H, m), 4.01 (2H, m), 3.70 (2H, m), 2.33 (2H, m), 2.02 (4H, m), 1.78 (6H, m).

Example 6 Cyclopentyl 4-r(^6-r(1E)-3-(ri-(2-methylpropoxy)ethoxy1amino -3- oxoprop-1-en-1-vnpyridin-3-yl methyl)aminoltetrahvdro-2H-pyran-4-carboxylate

Following the method of Example 1 , from Intermediate 10 (47 mg, 0.1 mmol) to give the title compound (11 mg) as a yellow solid, m/z 390 [M+Hf . ¹H NMR (300 MHz, CD₃OD) δ ppm; 8.72 (1 H, m), 8.00 (1 H, dd, J = 8.1 Hz, 2.1 Hz), 7.69 (1 H, d, J = 8.1 Hz), 7.61 (1 H, d, J = 15.3Hz), 7.04 (1 H, m), 5.44 (1 H, m), 4.31 (2H, s), 4.02 (2H, m), 3.73 (2H, t, J =

11.4Hz), 2.32 (2H, m), 2.05 (4H, m), 1.83 (6H, m). Example 7 Cyclopentyl 1-f 4-f(1£)-3-(hvdroxyamino)-3-oxoprop-1-en-1-ylT-2- methylbenzyl)amino)cyclohexanecarboxylate Trifluoroacetate

Following the method of Example , from Intermediate 11 (48 mg, 0.096 mmol) to give the title compound (22 mg,) as an orange solid, m/z 401 [M+H]⁺ . ¹H NMR (300 MHz, CD- ₃OD) δ ppm; 7.53 (4H, m), 6.56 (1 H, m), 5.12 (1 H, m), 4.19 (2H, s), 2.48 (3H, s), 2.42 (2H, m), 2.01 (2H, m), 1.89-1.59 (13H, m), 1.39 (1 H, m).

Example 8 Cyclopentyl 1-( 4-f(1£)-3-(hvdroxyamino)-3-oxoprop-1-en-1-vn-2- methylbenzyl>amino)cvclopentanecarboxylate Trifluoroacetate

Following the method of Example 1 , from Intermediate 12 (10.7 mg, 0.022 mmol) to give the title compound (4.4 mg,) as an orange solid, m/z 387 [M+H]⁺ . ¹H NMR (300 MHz, CD₃OD) δ ppm; 7.51 (4H, m), 6.61 (1 H, m), 5.51 (1 H, m), 4.1 1 (2H, s), 2.53 (3H, s), 2.40 (2H, m), 2.17-1.75 (14H, m).

Example 9 Cyclopentyl 1-((4-r(1£n-3-(hvdroxyamino)-3-oxoprop-1-en-1-yl1-2- methylbenzyl)amino)cvclobutanecarboxylate Trifluoroacetate

Following the method of Example 1 , from Intermediate 13 (22.1 mg, 0.047 mmol) to give the title compound (8.76 mg) as an orange solid, m/z 373 [M+H]⁺. ¹H NMR (300 MHz, CO₃OD) δ ppm; 7.52 (4H, m), 6.58 (1 H, m), 5.42 (1 H, m), 4.18 (2H, s), 2.66( 4H, m), 2.53 (3H, s), 2.30 (1 H, m), 2.17 (2H, m), 2.01-1.77 (8H, m).

Example 10 Methylcvclopentyl 4-f( 6-r(1-r)-3-(hydroxyamino)-3-oxoprop-1-en-1- vnpyridin-3-yl methyl)amino1tetrahvdro-2H-pyran-4-carboxylate

Following the method of Example 1 , from Intermediate 18 (138.4 mg, 0.27 mmol) and 4 HCI in dioxane (0.21 mL, 0.81 mmol) to give the title compound (49 mg) as a brown- orange solid, m/z 404 [ +H]⁺ . ¹H NMR (300 MHz, C0₃OD) δ ppm; 8.73 (1 H, s), 8.04 (1 H, dd, J = 8.0, 1.8 Hz), 7.70 (1 H, d, J = 8.1 Hz), 7.60 (1 H, d, J = 15.6 Hz), 6.99 (1 H, d, J = 15.6 Hz), 5.03 (1 H, m), 4.13 (2H, s), 4.03 (2H, dd, J = 12.2, 2.7 Hz), 3.73 (2H, t, J =10.7 Hz), 2.35 (2H, d, J = 13.4 Hz), 2.27-1.94 (5H, m), 1.89-1.76 (3H, m), 1.41 -1.29(1 H, m), 1.11-1.05 (3H, d, J = 7 .OHz).

Example 11 Cvclopentyl 4-(f1-^6-r(1-r)-3-(hydroxyamino)-3-oxoprop-1-en-1- yl1pyridin-3-ylTmethyl)piperidin- -yl1amino>tetrarivdro-2H-pyran-4-carboxylate

Trifluoroacetate

Following the method of Example 1 , from Intermediate 14 (43 mg, 0.075 mmol) and 4M HCI in dioxane (75 mL, 0.3 mmol) to give the title compound (7 mg) as purple solid, m/z 473 [M+H]⁺. ¹H NMR (300 MHz, CD₃OD) δ ppm; 8.72 (1 H, m), 8.01 (1 H, m), 7.68 (2H, m), 7.13 (1 H, m), 5.36 (1 H, m), 4.40 (1 H, m), 3.95 (2H, m), 3.64 (4H, m), 3.45-3.15 (4H, m), 2.13 (4H, m), 2.10-1.60 (12H, m). Example 12 Cvclopentyl 4-(ri-^5-r(1E)-3-(hvdroxyamino)-3-oxoprop-1-en-1- vnpyridin-2-yl>methyl)piperidin-4-vnamino>tetrahydro-2H-Pyran-4-carboxylate Trifluoroacetate

Following the method of Example 1 , from Intermediate 15 (50 mg, 0.087 mmol) and 4M HCI in dioxane (87mL, 0.348 mmol) to give the title compound (5 mg) as a yellow solid. m/z 473 [M+H]⁺. H N R (300 MHz, CD₃OD) δ ppm; 8.84 (1 H, s), 8.11 (1 H, m), 7.63 (1 H, d, J = 15.9Hz), 7.54 (1 H, d, = 8.1 Hz), 6.66 (1 H, d, J = 15.9Hz), 5.35 (1 H, m), 4.50 (2H, m), 3.95 (2H, m), 3.62 (4H, m), 3.25 (5H, m), 2.22 (4H, m), 2.08-1.72 (10H, m).

Example 13 Cyclopentyl 4-((4-r(1E)-3-(hydroxyamino)-3-oxoprop-1-en-1- yllbenzyl)amino)tetrahvdro-2H-pyran-4-carboxylate Trifluoroacetate

Following the method of Example 1 , from Intermediate 16 (45 mg, 0.09 mmol) and 4M HCI in dioxane (90 μΙ_, 0.36 mmol) to give the title compound (5 mg) as a purple solid, m/z 389 [M+H]⁺ . ¹H NMR (300 MHz, CD₃OD) δ ; 7.68 (2H, d, J = 8.1 Hz), 7.59 (1 H, m), 7.54 (2H, d, J = 8.1 Hz), 6.64 (1 H, m), 5.44 (1 H, m), 4.20 (2H, s), 3.96 (2H, m), 3.71 (2H, t, J = 12.0Hz), 2.32 (2H, m), 2.03 (4H, m), 1.83 (6H, m).

Example 14 Cyclopentyl 4-((3-r(1-=)-3-(hydroxyamino)-3-oxoprop-1-en-1- yl1benzyl>arnino)tetrahvdro-2H-pyran-4-carboxylate Trifluoroacetate

Following the method of Intermediate 5, from Intermediate 29 (50 mg, 0.17 mmol), Intermediate 23 (37 mg, 0.17 mmol) and sodium triacetoxyborohydride (39 mg, 0.26 mmol) to give the title compound (10 mg) as a brown solid, m/z 389 [M+H]⁺. ¹H NMR (300 MHz, CD₃OD) δ ; 7.81-7.07 (6H, m), 5.45 (1 H, m), 4.23 (2H, s), 4.00 (2H, m), 3.71 (2H, m), 2.33 (2H, d, J = 13.4 Hz), 2.02 (4H, m), 1.94-1.65 (6H, m). Example 15 (3R)-Tetrahvdrofuran-3-yl /V-{4-r(1 -3-(hvdroxyamino)-3-oxoprop-1- en-1 -yllbenzyll-2-methyl-D-leucinate Trifluoroacetate

Following the method of Example 1 from Intermediate 27 (26 mg, 0.05 mmol) and 4 HCI in dioxane (50 ml_, 0.2 mmol) to give the title compound (10 mg) as a purple solid, m/z 391 [M+H]⁺. ¹H N R (300 MHz, CD₃OD) δ ppm; 7.68 (2H, d, J = 8.1 Hz), 7.57 (3H, m), 6.56 (1 H, d, J = 16.2Hz), 5.51 (1 H, m), 4.30 (1 H, d, J = 12.9Hz), 4.14 (1 H, d, J = 12.9Hz), 3.96 (2H, m), 3.89 (2H, m), 2.36 (1 H, m), 2.14 (1H, m), 2.03 (1 H, m), 1.90 (2H, m), 1.74 (3H, s), 1.01 (3H, d, J = 6.6Hz), 0.98 (3H, d, J = 6.6Hz).

Measurement of biological activities

Histone deacetylase activity

The ability of compounds to inhibit histone deacetvlase 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 fluorophore. 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 = [(S¹ - 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. Histone deacetylase activity from crude nuclear extract derived from HeLa cells was used for screening. The preparation, purchased from Ciibiotech ( ons, 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 et al, Nucl. Acid. Res., 1983, 11 , 1475-1489. The final buffer composition was 20 mM HEPES pH7.9, 100 mM KCI, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF and 20 % (v/v) glycerol.

Dose response curves were generated from 8 compound concentrations (top concentration 10μΜ, with 3-fold dilutions), using duplicate points. IC₅₀ results were allocated to one of 3 ranges as follows: Range A: ICso^ OOnM, Range B: IC₅o from 101nM to 1000nM; Range C: IC₅₀ >1001 nM' NT = Not tested to date.

U937 cell inhibition assay

Cancer cell lines (U937) growing in log phase were harvested and seeded at 1000 - 2000 cells/well (100μΙ 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-[(S7S°)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 10μΜ, with 3-fold dilutions), using 6 replicates. IC₅₀ values were determined by nonlinear 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<100nM,;Range B: IC₅₀ from 101 nM to 1000nM; Range C. IC₅₀ >1000nM; NT= Not tested to date. LPS-stimulation of human whole blood

Whole blood was taken by venous puncture using heparinised vacutainers (Becton Dickinson) and diluted in an equal volume of RPMI1640 tissue culture media (Sigma). 100μΙ was plated in V-bottomed 96 well tissue culture treated plates. 2hrs after the addition of the inhibitor in 10ΟμΙ of RPMI1640 media, the blood was stimulated with LPS (E coli strain 005:B5, Sigma) at a final concentration of 100ng/mL and incubated at 37°C in 5% C0₂ for 6hrs. TNF-a levels were measured from cell-free supernatants by sandwich ELISA (R&D Systems #QTA00B)

IC50 values were allocated to one of three ranges as follows: Range A: IC50 < 100nM; Range B: IC₅₀ from 101 nM to 1000nM; Range C: IC50 >1000nM; NT = Not tested to date

Table 1- Results

Inhibitor activity

Inhibitor Inhibitor activity versus

Example activity versus versus U937 human whole

HDAC proliferation blood

TNFa release

1 B C B

2 C C

^A 20

3 B B A

4 B B A

5 C C A

6 B C B

7 C B ^A 25

8 C B A

9 C B A

10 B C NT

11 A B NT

12 B C NT 30

13 C B A

14 NT NT NT

15 C NT NT Broken Cell Carboxylesterase Assay

Any given compound of the present invention wherein 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 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, NaC1 130 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 μΜ

Aprotinin 0.1 μΜ

Ε64 8 μΜ

Pepstatin 1.5 μΜ

Bestatin 162 μΜ

Chymostatin 33 μΜ

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 pg / 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 μΜ 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 RT by LCMS (Sciex API 3000, HP1100 binary pump, CTC PAL). Chromatography was based on an AcCN (75x2.1mm) column and a mobile phase of 5- 95 % acetonitrile in water /0.1 % formic acid.

Claims

Claims:

1. A compound selected from the group consisting of:

Cyclopentyl 1 -[({5-[(1 £)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2- yl}methyl)amino]cyclopropanecarboxylate;

Cyclopentyl 1 -[({5-[(1 E)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2- yl}methyl)amino]cyclobutanecarboxylate;

Cyclopentyl 1 -[({5-[(1 £)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2- yl}methyl)amino]cyclopentanecarboxylate;

Cyclopentyl 1 -[({5-[(1 E)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2- yl}methyl)amino]cyclohexanecarboxylate; Cyclopentyl 4-[({5-[(1 £)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-2- yl}methyl)amino]tetrahydro-2H-pyran-4-carboxylate;

Cyclopentyl 4-[({6-[(1 E)-3-{[1-(2-methylpropoxy)ethoxy]amino}-3-oxoprop-1-en-1- yl]pyridin-3-yl}methyl)amino]tetrahydro-2H-pyran-4-carboxylate;

Cyclopentyl 1-({4-[(1.r)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]-2- methylbenzyl}amino)cyclohexanecarboxylate;

Cyclopentyl 1 -({4-[(1 E)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]-2- methylbenzyl}amino)cyclopentanecarboxylate;

Cyclopentyl 1 -({4-[(1 E)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]-2- methylbenzyl}amino)cyclobutanecarboxylate; Methylcyclopentyl 4-[({6-[(1£)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]pyridin-3- yl}methyl)amino]tetrahydro-2/- -pyran-4-carboxylate;

Cyclopentyl 4-{[1 -({6-[(1 Zr)-3-(hydroxyamino)-3-oxoprop-1 -en-1 -yl]pyridin-3- yl}methyl)piperidin-4-yl]amino}tetrahydro-2 - -pyran-4-carboxylate; Cyclopentyl 4-{[1-({5-[(1 E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]pyridin-2- yl}methyl)piperidin-4-yl]amino}tetrahydro-2H-pyran-4-carboxylate; Cyclopentyl 4-({4-[(1 E)-3-(hydroxyamino)-3-oxoprop-1-en-1 -yl]benzyl}amino)tetrahydro- 2A7-pyran-4-carboxylate;

Cyclopentyl 4-({3-[(1 £)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]benzyl}amino)tetrahydro- 2tf-pyran-4-carboxylate; and

(3 )-Tetrahydrofuran-3-yl A/-{4-[(1£)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl]benzyl}-2- methyl-D-leucinate; and salts thereof.

2. A pharmaceutical composition comprising a compound as claimed in claim 1 , together with one or more pharmaceutically acceptable carriers or excipients..

3. A compound as claimed in claim 1 for use in the inhibition of HDAC activity, or for use in the preparation of a medicament for inhibition of HDAC activity.

4. A compound as claimed in claim 1 for use in the treatment of, or for use in the preparation of a medicament for treatment of, cell-proliferation disease, polyglutamine disease, neurodegenerative disease, autoimmune disease, inflammatory disease, organ transplant rejection, diabetes, haematological disorders or inflammatory sequelia of infection.

5. A method for the treatment of a disease which responds to inhibition of HDAC activity, which comprises administering to a subject suffering such disease an effective amount of a compound as claimed in claim 1.

6. A method as claimed in claim 5 wherein the disease is cell-proliferation disease, polyglutamine disease, neurodegenerative disease, autoimmune disease, inflammatory disease, organ transplant rejection, diabetes, haematological disorders or inflammatory sequelia of infection.

7. A compound for use, or the use, as claimed in claim 3 or claim 4, or a method as claimed in claim 5 or claim 6 wherein the treatment is of cancer cell proliferation,

8. A compound for use, or the use, as claimed in claim 3 or claim 4, or a method as claimed in claim 5 or claim 6 wherein the treatment is of rheumatoid arthritis.