WO2008053157A1 - Aminoheteroaryl compounds as for the treatment of diseases mediated by c-met kinase activity - Google Patents

Abstract

Compounds of formula (I), are inhibitors of c-Met kinase activity, useful inter alia in the treatment of hyperproliferative diseases: wherein X is -N= or -CH=; ring A is optionally substituted mono- or bi-cyclic aryl or heteroaryl having from 5 to 12 ring atoms; ring B is (i) optionally substituted phenyl, (ii) optionally substituted monocyclic heterocyclic having 5 or 6 ring atoms and having one or two nitrogens as the sole ring heteroatoms, (iii) optionally substituted bicyclic ring system of 9 or 10 ring atoms having one two or three ring nitrogens and optionally a ring oxygen as the sole ring heteroatoms; R2 is selected from hydrogen, halogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C3-C6)cycloalkyl, and mono- or bi-cyclic heterocyclic having from 5 to 12 ring atoms, R3 and R4 are (a) independently selected from hydrogen, halogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C3-C6)cycloalkyl, and mono- or bi-cyclic heterocyclic having from 5 to 12 ring atoms; or (b) R3 and R4 taken together with the carbon to which they are attached form a (C3-C6)cycloalkyl ring; or (c) one of R3 and R4 is as defined in case (a) while the other is a divalent radical selected from -(CH2)3- or -(CH2)4- in which one or two non-adjacent carbons are replaced by oxygen, sulfur or -NR3- wherein Ra is hydrogen or (C1-C3) alkyl; R5 is hydrogen and R6 is a radical of formula (IA), or R6 is hydrogen and R5 is a radical of formula -(Z1)w-(CH2)Z-L1-Y1-CH(R7)-NHR8 wherein R7 is a carboxylic acid group (-COOH), or an ester group which is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group; and R8 is hydrogen; or optionally substituted (C1-C6)alkyl, (C3-C7)cycloalkyl, aryl or heteroaryl Or -(C=O)R9, -(C=O)OR9, Or -(C=O)NR9 wherein R9 is hydrogen or optionally substituted (C1-C6)alkyl; and -(Z1)w-(CH2)Z-L1-Y1- is a linker radical as defined in the claims.

Description

AMINOHETEROARYL COMPOUNDS AS FOR THE TREATMENT OF DISEASES MEDIATED BY C-MET KINASE

ACTIVITY

The invention relates to a series of amino acid esters, to compositions containing them, to processes for their preparation and to their use in medicine as inhibitors of c-Met for the treatment of diseases mediated by c-Met kinase activity. Such diseases include hyperproliferative disorders or diseases associated with an increased invasive potential of cells, such as cancer, including the treatment and prevention of tumour metastases and minimal residual disease as well as diseases involving angiogenesis such as diabetic retinopathy, age-related macular degeneration and rheumatoid arthritis. The present invention encompasses compounds that are 2-aminopyridines and 2-amino pyrazines.

Background to Invention

Receptor tyrosine kinases (RTKs) regulate many key processes in mammalian development, cell function and tissue homeostasis. Amongst these are cell growth and survival, neovascularisation, and tissue repair and regeneration. In many human cancers the deregulation of RTK activity by mutation, gene rearrangement, gene amplification and over-expression of both receptor and ligand has been implicated in the progression of disease.

Hepatocyte growth factor (HGF), also known as scatter factor, has been demonstrated to play a key role in cancer [Nakamura et a/, Nature, 1989, 342, 440-443] and is the ligand for c-Met, its high affinity receptor. c-Met is the prototypic member of a sub-family of RTKs which also includes RON. c-Met and HGF are widely expressed in a variety of tissues, and their expression is normally confined to cells of epithelial or mesenchymal origin, respectively [Di Renzo et al, Oncogene 1991 , 6, 1997-2003]. The transduction of signalling and subsequent biologic effects of HGF by c-Met has been shown to be important in epithelial-mesenchymal interaction and regulation of cell migration, invasion, cell proliferation and survival, angiogenesis, morphogenic differentiation, and organisation of three-dimensional tubular structures (e.g. renal tubular cells, gland formation etc.) during development and tissue repair [Comoglio Semin. Cancer Biol 2001 , 11, 153-165]. HGF has been shown to increase the invasiveness of a number of cancer cells. This is likely to be via a number of mechanisms including the direct increase in the migration of cancer cells [Jiang et al, Critical Reviews in Oncology/ Hematology, 2005, 53, 35-69], increase in adhesion to matrix and endothelium, and increase in expression and secretion of proteolytic enzymes from cancer cells including MMP2, MMP7, MMP9 and uPA [Davies et al, Clin. Can. Res., 2001 , 7, 3289]. It has been shown that HGF invasive growth is dependent upon the effective interaction between c-Met and α6β4 in cells. Loss of this integrin results in non-responsiveness of the cell towards HGF, c-Met induced phosphorylation of the integrin facilitates it's interaction with She and PI3 kinase signals [Trusilino et al, Cell, 2001 , 107, 643-654] which in turn enhances ras and PI3K signals.

HGF is a powerful angiogenic factor, and has been shown to up-regulate VEGF and down-regulate thrombospondin-1 (TSP1 ) in the same cell type [Zhang et al, PNAS, 2003, 100, 12718-12723]. In endothelial cells, inducible nitric oxide synthase expression is thought to be another pathway in HGF-induced migration of endothelial cells [Purdie et al, Circ. Res., 2002, 54, 659-668]. HGF is able to stimulate the expression of angiogenic factors in endothelial cells, including MMP1 , HGF itself and VEGF as well as c-Met. This is via induction of Ets-1 [Tomita _ef _a/, Circulation, 2003, 107, 1411-1417]. HGF has been shown to induce expression of VEGF, via the activation of Sp1 phosphorylation, which in turn increases VEGF promoter activity [Reisinger et al J. Cell Sci. 2003, 116, 225-238]. HGF can also induce HGF-1 expression via PI3-K [Tacchini et al, Carcinogenesis 2001 , 22, 225-238].

Missence mutations of c-Met are present in the germline of hereditary papillary renal cell carcinoma families implicating c-Met in the genesis of the disease [Schmidt et al, Cancer Res 1998, 58, 1719-1722]. In addition, kinase domain mutations have been observed in sporadic papillary renal carcinoma, ovarian cancer and childhood hepatocellular carcinoma while juxtamambrane domain mutations are observed in gastric and lung cancers. c-Met has also been found to be genetically altered in lymph node metastases and secondary neoplasms implicating the HGF pathway in the metastatic progression of selected cancers. Transgenic mice engineered to express HGF in their germline were shown to develop a ^■ diverse array of tumours of both epithelial and mesenchymal origin, including mammary carcinoma, malignant melanoma, squamous cell carcinoma and fibrosarcoma [Takayama et al, PNAS 1997, 94, 701-706].. Transgenic expression of the c-Met receptor or its variants in mice also resulted in the appearance of a variety of preneoplastic and neoplastic lesions. Expression of elevated levels of wild-type c-Met targeted to hepatocytes resulted in the development and maintenance of hepatocellular carcinoma [Wang et al, J. Cell Biol. 2001 , 153, 1023-1034].

Initial attempts to identify inhibitors of c-Met kinase led to the characterisation of the staurosporine analogue and broad spectrum kinase inhibitor K252a as having sub- micromolar c-Met activity. K252a demonstrates modulation of wild type and mutant (M1268T) c-Met dependent function and dissemination of tumour cell in vivo, [Morotti et a/, Oncogene 2002, 21, 4885]. c-Met inhibitors defined by an indolin-2-one structure have been described [Christensen et al, Cancer Res. 2003, 63, 7345-7355]. A quinoline- based small-molecule c-Met inhibitor has been described with EC₅₀ values in the low nanomolar range and is reported to inhibit the growth of MKN45 and U87 tumour xenografts in vivo [WO2003000660A1 , Kirin Beer Kabushiki, Japan 2003].

We have now discovered a group of compounds which are potent and selective inhibitors of c-Met and isoforms and splice variants thereof. The compounds are thus of use in medicine, for example in the treatment and prophylaxis of hyperproliferative and angiogenic disorders described herein. The compounds are characterised by the presence in the molecule of an amino acid motif or an amino acid ester motif which is hydrolysable by an intracellular carboxylesterase. Compounds of the invention having the lipophilic amino acid ester motif cross the cell membrane, and are hydrolysed to the acid by the intracellular carboxylesterases. The polar hydrolysis product accumulates in the cell since it does not readily cross the cell membrane. Hence the c-Met activity of the compound is prolonged and enhanced within the cell.

Brief description of the invention

This invention relates to 2-aminopyridine and 2-aminopyrazine compounds which are inhibitors of c-Met kinase activity. The compounds are thus of use in medicine, for example in the treatment of a variety of proliferative disease states, and for control of angiogenesis. The compounds are characterised by the presence in the molecule of an amino acid motif or an amino acid ester motif which is hydrolysable by an intracellular carboxylesterase. Compounds of the invention having the lipophilic amino acid ester motif cross the cell membrane, and are hydrolysed to the acid by the intracellular carboxylesterases. The polar hydrolysis product accumulates in the cell since it does not readily cross the cell membrane. Hence the c-Met kinase inhibitory activity of the compound is prolonged and enhanced within the cell. The compounds of the invention are related to the c-Met kinase inhibitors encompassed by the disclosures in International Patent Application No. WO2006/021881, WO2006/021884 and WO2006/021886 but differ therefrom in that the present compounds have the amino acid ester motif referred to above.

Detailed Description of the Invention

According to the present invention there is provided a compound of formula (I), or a salt, N-oxide, hydrate or solvate thereof:

wherein

X is -N= or -CH=;

ring A is optionally substituted mono- or bi-cyclic aryl or heteroaryl having from 5 to 12 ring atoms;

ring B is (i) optionally substituted phenyl, (ii) optionally substituted monocyclic heterocyclic having 5 or 6 ring atoms and having one or two nitrogens as the sole ring heteroatoms, (iii) optionally substituted bicyclic ring system of 9 or 10 ring atoms having one, two or three ring nitrogens and optionally a ring oxygen as the sole ring heteroatoms;

R₂ is selected from hydrogen, halogen, (d-C₆)alkyl, (C₂-C₆)alkenyl, (C₃-C₆)cycloalkyl, and mono- or bi-cyclic heterocyclic having from 5 to 12 ring atoms;

R₃ and R₄ are (a) independently selected from hydrogen, halogen, (C_rC₆)alkyl, (C₂- C₆)alkenyl, (C₃-C₆)cycloalkyl, and mono- or bi-cyclic heterocyclic having from 5 to 12 ring atoms; or (b) R₃ and R₄ taken together with the carbon to which they are attached form a (C₃-C₆)cycloalkyl ring; or (c) one of R₃ and R₄ is as defined in case (a) while the other is a divalent radical selected from -(CH₂)₃- or -(CHa)₄- in which one or two non-adjacent carbons are replaced by oxygen, sulfur or -NR₃- wherein R_a is hydrogen or (CrC₃) alkyl;

R₅ is hydrogen and R₆ is a radical of formula (IA), or R₆ is hydrogen and R₅ is a radical of formula (IA);

-(ZV(CH₂)_Z-L¹-Y¹-CH(R₇)-NHR₈ (IA) wherein

Z¹ represents an optionally substituted divalent mono- or bi-cyclic carbocyclic or heterocyclic radical having from 3 to 12 ring atoms;

w and z are independently 0 or 1 ;

Y¹ is a bond, -(C=O)-, -S(O₂)-, -(C=O)NR₉-, -NR₉(C=O)-, -S(O₂)NR₉-, -NR₉S(O₂)-, or - NR₉(C=O)NR₉-, wherein each R₉ is independently hydrogen or optionally substituted (C_rC₆)alkyl,

L¹ is a divalent radical of formula -(Alk¹)_m(Q)_n(Alk²)_p- wherein m, n and p are independently O 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 O, a divalent radical of formula -Q¹-X²- wherein X² is -0-, -S- or NR^A- wherein R^A is hydrogen or optionally substituted C₁-C₃ alkyl, and Q¹ is an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5 - 13 ring members,

AIk¹ and AIk² independently represent optionally substituted divalent C₃-C₇ cycloalkyl radicals, or optionally substituted straight or branched, C_rC6 alkylene, C₂-C₆ alkenylene, or C₂-C₆ alkynylene radicals which may optionally contain or terminate in an ether (-O-), thioether (-S-) or amino (-NR^A-) link wherein R^A is hydrogen or optionally substituted (CrC₃)alkyl;

R₇ is a carboxylic acid group (-COOH), or an ester group which is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group; and

R₈ is hydrogen; or optionally substituted (CrCβ)alkyl, (C₃-C₇)cycloalkyl, aryl or heteroaryl or -(C=O)R₉, -(C=O)ORg, or -(C=O)NR₉ wherein R₉ is hydrogen or optionally substituted (d-C₆)alkyl.

In another broad aspect the invention provides the use of a compound of formula (I) as defined above, or an N-oxide, salt, hydrate or solvate thereof in the preparation of a composition for inhibiting the activity of c-Met kinase.

The compounds with which the invention is concerned may be used for the inhibition of c-Met kinase activity ex vivo or in vivo.

In one aspect of the invention, the compounds of the invention may be used in the preparation of a composition for treatment of hyperproliferative disease, disease associated with an increased invasive potential of cells, minimal residual disease, and for modulation of angiogenesis.

Specific examples of hyperproliferative diseases for treatment with compounds of the invention include cancers and tumour metastases. Specific examples of angiogenisis- realted diseases for treatment with compounds of the invention include diabetic retinopathy, age-related macular degeneration and rheumatoid arthritis. 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 a compound of formula (I) as defined above.

Pharmaceutical compositions comprising a compound of formula (I) above together with one or more pharmaceutically acceptable carriers and/or excipients also form part of the invention.

Terminology

As used herein, the term "(C_a-C_b)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.

As used herein the term "divalent (C_a-C_b)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 "(C_a-C_b)alkenyl" 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 (C_a-C_b)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 "C₃-C_b 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 (C_a-C_b)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- or tri-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 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, (C-ι-C6)alkyl, (Ci-C₆)alkoxy, hydroxy, hydroxy(Ci-C₆)alkyl, mercapto, mercapto(C_rC₆)alkyl, (C-|-C₆)alkylthio, phenyl, halo (including fluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, -COOH, -COOR^A, -COR^A, -SO₂R^A, -CONH₂, -SO₂NH₂, -CONHR^A, -SO₂NHR^A, -CONR^AR^B, -SO₂NR^AR^B, -NH₂, -NHR^A, -NR^AR^B, -OCONH₂, -OCONHR^A , -0C0NR^AR^B, -NHCOR^A, -NHCOOR^A, -NR^BCOOR^A, -NHSO₂OR^A, -NR⁸SO₂OH₁ -NR^BSO₂OR^A, -NHCONH₂, -NR^AC0NH₂, -NHCONHR⁶, -NR^AC0NHR^B, -NHCONR^AR^B or -NR^AC0NR^AR^B wherein R^A and R^B are independently a (C_rC₆)alkyl, (C₃-C₆) cycloalkyl , phenyl or monocyclic heteroaryl having 5 or 6 ring atoms, or R^A and R^B when attached to the same nitrogen atom form a cyclic amino group (for example morpholino, piperidinyl, piperazinyl, or tetrahydropyrrole). An "optional substituent" may be one of the foregoing substituent groups.

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 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)arnino-methane, L-arginine, L-lysine, N-ethyl piperidine, dibenzylamine and the like. Those compounds (I) 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, 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 diastereoisomers with R or S stereochemistry at each chiral centre. The invention includes all such diastereoisomers and mixtures thereof.

The term "ester" or "ester group" or "esterified carboxyl group" in connection with substituent R₇ above means a group R_xO(C=O)- in which R_x is the group characterising the ester, notionally derived from the alcohol R_xOH.

In the compounds of the invention, the variable substituents and groups will now be discussed in more detail:

The ring B

The ring B is (i) optionally substituted phenyl, (ii) optionally substituted monocyclic heterocyclic having 5 or 6 ring atoms and having one or two nitrogens as the sole ring heteroatoms, (iii) optionally substituted bicyclic ring system of 9 or 10 ring atoms having one, two or three ring nitrogens and optionally a ring oxygen as the sole ring heteroatoms;

Examples of rings B of type (ii) include the following:

(HF) (HG) (HH) (IU) (HK)

Examples of ring systems B of type (iii) include:

In each of the above examples of type (ii) rings and type (iii) ring systems, the R₅ group may be attached to a ring carbon or may be substituted on a ring NH. Also in each of the above examples of type (ii) rings and type (iii) ring systems, the unsatisfied valency bond may be from a ring carbon or from a ring nitrogen shown in the formulae as NH.

Presently it is preferred that ring B be of type (ii), particularly those of formula (HA) and (HB). In these preferred rings, R₅ is preferably substituted on a ring NH.

Optional substituents in ring B may be any of those referred to as optional substituents above, and include halogen, especially chloro and fluoro, methoxy, ethoxy, methyl, trifuoromethyl, trifluoromethoxy, nitrile, methylamino, ethylamino, dimethylamino, diethylamino, cyclopropyl, morpholinyl, piperidinyl, piperazinyl, and methyl substitued by morpholinyl, piperidinyl, or piperazinyl.

The ring A

Ring A is optionally substituted mono- or bi-cyclic aryl or heteroaryl having from 5 to 12 ring atoms. Examples of ring A include optionally substituted phenyl, or monocyclic heteroaryl having 5 or 6 ring atoms, such as pyridyl, pyrimidinyl, pyrrolyl, thienyl, oxazolyl, thiazolyl, oxadiazolyl, thiadiazolyl, pyrazolyl and imidazolyl. Presently it is preferred that ring A be optionally substitued phenyl. Substituents in ring A may be any of those referred to as optional substituents above and specific examples of such substituents include halogen, especially chloro and fluoro, methoxy, ethoxy, methyl, trifuoromethyl, trifluoromethoxy, nitrile, methylamino, ethylamino, dimethylamino, diethylamino, cyclopropyl, morpholinyl, piperidinyl, piperazinyl, and methyl substitued by morpholinyl, piperidinyl, and piperazinyl.

The substituents R?, Rz and Rd

R₂ is selected from hydrogen, halogen such as chloro and fluoro, (CrC₆)alkyl such as methyl, ethyl and n-or iso-propyl, (C2-Ci₂)alkenyl such as allyl, (C3-Ci₂)cycloalkyl such as cyclopropyl, cyclopentyl and cyclohexyl, and mono- or bi-cyclic heterocyclic having from 5 to 12 ring atoms such as thienyl, benzthienyl, furyl, benzfuryi, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl, indazolyl, pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, and ethylenedioxyphenyl. However, it is presently preferred that R₂ be hydrogen.

R₃ and R₄ are (a) independently selected from hydrogen, halogen, (CrC6)alkyl, (C₂- C₆)alkenyl, (C₃-C₆)cycloalkyl, and mono- or bi-cyclic heterocyclic having from 5 to 12 ring atoms, as discussed in relation to R₂ or (b) R₃ and R₄ taken together with the carbon to which they are attached form a (C₃-C₆)cycloalkyl ring such as a cyclopropyl cyclopentyl or cyclohexyl ring; or (c) one of R₃ and R₄ is as discussed in case (a) while the other is a divalent radical selected from -(CH₂)₃- or -(CH₂)₄- in which one or two non-adjacent carbons are replaced by oxygen, sulfur or -NR₃- wherein R_a is hydrogen or (C_rC₃) alkyl. In one preferred subclass of compounds of the invention, R₄ is hydrogen. In another preferred subclass of compounds of the invention R₃ is methyl and R₄ is hydrogen. One particular subclass of compounds of the invention consists of those of formula (II) and its salts, N-oxide, hydrate or solvate thereof:

wherein R₅ is a radical of formula (IA) as defined above.

The radical (IA) R^NH-CH(Rr)-Y¹ -L¹ -fCHol_r( Z¹L-:

In the compounds with which the invention is concerned, one of R₅ and R₆ is a radical

R₈NH-CH(Rr)-Y* -L¹-[CH₂]_Z~( Z\- defined above, while the other is hydrogen.

The radical RxNH-CH(R₁)-

This radical is an alpha amino acid or alpha amino acid ester moiety which is linked through a linker radical -Y¹-L¹-(CH₂)₂-( Z¹)_w- to the rest of the molecule. The ester compounds of the invention are converted by intracellular esterases to the carboxylic acid. Both the esters and carboxylic acids may have o-Met inhibitory activity in their own right. The compounds of the invention therefore include not only the ester, but also the corresponding carboxylic acid hydrolysis products.

The ester group R₇ must be one which in the compound of the invention is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group. Intracellular carboxylesterase 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 conjugates. In general, if the carboxylesterase hydrolyses the free amino acid ester to the parent acid it will also hydrolyse the ester motif when covalently conjugated to the modulator. Hence, the broken cell assay described herein provides a 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 carboxylesterase assay when conjugated to the c-Met inhibitor via the chosen conjugation chemistry, to confirm that it is still a carboxylesterase substrate in that background.

Subject to the requirement that they be hydrolysable by intracellular carboxylesterase enzymes, examples of particular ester groups R₇ include those of formula -(C=O)OR₁₂ wherein Ri₂ is Ri₃R₁₄R₁₅C- wherein

(i) R-_I3 is hydrogen or optionally substituted (Ci-C₃)alkyl-(Z¹)_a-[(Ci-C₃)alkyl]_b-, (C₂-C₃)alkenyl-(Z¹)_a-[(Ci-C₃)alkyl]_b- or phenyl-(Z¹)_a-[(C_rC₃)alkyl]_b-, wherein a and b are independently 0 or 1 and Z¹ is -O-, -S-, or -NR₁6- wherein Ri₆ is hydrogen or (Ci-Cs)alkyl; and R₁₄ and Ri₅ are independently hydrogen or (C_rC₃)alkyl-; or

(ii) R₁₃ is hydrogen or optionally substituted Ri₇Ri₈N-(C₁-C₃)alkyl- wherein Ri₇ is hydrogen, (Ci-C₃)alkyl or phenyl, and Ri₈ is hydrogen or (C_rC₃)alkyl; or Ri₇ and Ri₈ 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 Ri₄ and R₁₅ are independently hydrogen or (C_rC₃)alkyl-;or

(iii) Ri₃ and Ru 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, and Ri₅ is hydrogen.

Specifically, the ester group R₇ may be, for example, a 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, i-ndanyl, norbornyl, dimethylaminoethyl, or morpholinoethyl ester group. Currently preferred is where R₇ is a cyclopentyl or tert-butyl ester group.

Macrophages are known to play a key role in inflammatory disorders through the release of cytokines in particular TNFα 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 Carrara, Curr Drug Targets lnflamm 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 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 RiCH(R₂)NH- is not directly linked to a carbonyl (-C(=O)-), ie when Y¹ is not a -C(=O), - C(=O)O-, or -C(=O)NR₃- radical, the ester will only be hydrolysed by hCE-1 and hence the inhibitors will only accumulate in macrophages.

The group RB

R₈ may be, for example, optionally substituted (C_rC₆)alkyl, (C₃-C₆)cycloalkyl, aryl or heteroaryl, for example methyl, ethyl, n-or isoprbpyl, cyclopropyl, cyclopentyl, cyclohexyl, phenyl, or pyridyl. R₈ may also be, for example hydrogen or or -(C=O)Ri₈, wherein Ri₈ is optionally substituted (C_rC₆)alkyl such as methyl, ethyl, n-or isopropyl, or n-, iso- or sec- butyl, (C₃-C₆)cycloalkyl such as cyclopropyl, cyclopentyl, cyclohexyl, phenyl, pyridyl, thienyl, phenyl(CrC₆alkyl)-, thienyl(C_rC₆alkyl)- or pyridyl(CrC₆alkyl)- such as benzyl, 4- methoxyphenylmethylcarbonyl, thienylmethyl or pyridylmethyl.

R₈ may also be, for example -(C=O)ORi₉, or -(C=O)NHRi₉ wherein Rig is hydrogen or optionally substituted (d-C₆)alkyl such as methyl, ethyl, or n-or isopropyl.

Currently it is preferred that R₈ be hydrogen.

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. 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 R₈NH-CH(R₇)- is attached is unsubstituted, ie R₈NH-CH(R₇)- 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 -Y¹ -L¹ -[CH₁L-(Z¹ L-

This radical (or bond) arises from the particular chemistry strategy chosen to link the amino acid ester motif R₈NH-CH(R₇)- 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, Z¹, w and z are possible. 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. On the other hand, that linkage chemistry may in some cases pick up additional binding interactions with the enzyme thereby enhancing binding. With the foregoing general observations in mind, taking the variables making up the radical -Y¹-L¹-[CH₂]_Z-(Z¹)_W- in turn:

Z¹ is optionally substituted divalent mono- or bi-cyclic carbocyclic or heterocyclic radical having from 3 to 12 ring atoms. Examples include a divalent phenyl, naphthyl, cyclopropyl, cyclopentyl, or cyclohexyl radical, or a mono-, or a divalent piperidinyl, piperazinyl, indolyl, pyridyl, thienyl, or pyrrolyl radical. In one preferred class of compounds of the invention, R₅ is a radical of formula (1) or (2):

(CH₂)_Z-U-Y¹-CH(R₇)-NH(R₈) (1 )

( CH₂)_Z-U -Y¹ -CH( R₇)-N H( R₈) (2)

w may be 0 or 1. At present, when Re is hydrogen and R₅ is a radical of formula (IA) above, it is preferred that w be 1. At present, when R₅ is hydrogen and R₆ is a radical of formula (IA) above, it is preferred that w be 0.

z may be 0 or 1.

Y¹ may be, for example, a bond, -NR₉-, -S-, -O-, -C(=O)NR₉-, -NR_gC(=O)-, or -C(=O)O-, wherein R_g is hydrogen or optionally substituted (CrC₆)alkyl such as methyl, ethyl Or -CH₂CH₂OH. Often in the compounds of the invention, Y¹ will be a bond.

In the radical L¹, examples of AIk¹ and AIk² radicals, when present, include -CH₂-, -CH₂CH₂-_J-CH₂CH₂CH₂-, -CH₂CHgCH₂CH₂-, -CH⁼CH-, -CH=CHCH₂-, -CH₂CH=CH-, -CH₂CH=CHCH₂-, -C≡C-, -C=CCH₂-, -CH₂C=C-, and CH₂C=CCH₂. Additional examples of AIk¹ and AIk² include -CH₂W-, -CH₂CH₂W-, -CH₂CH₂WCH₂-, -CH₂CH₂WCH(CH₃)-, -CH₂WCH₂CH₂-, -CH₂WCH₂CH₂WCH₂-, and -WCH₂CH₂- where W is -O-, -S-, -NH-, -N(CH₃)-, or -CH₂CH₂N(CH₂CH₂OH)CH₂-. Further examples of AIk¹ and AIk² include divalent cyclopropyl, cyclopentyl and cyclohexyl radicals. At present it is preferred that AIk¹ and AIk² radicals, when present, are selected from -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, -CH₂O-, -CH₂CH₂O-, -CH₂CH₂CH₂O- and divalent cyclopropyl, cyclopentyl and cyclohexyl radicals.

AIk¹ and AIk² when present may also be branched chain alkyl such as -CH(CH₃)-, -C(CHa)₂-, or in either orientation -CH₂CH(CH₃)-, -CH₂C(CH₃)₂-.

In L¹, when n is O, the radical is a hydrocarbon chain (optionally substituted and perhaps including or terminating in an ether, thioether or amino linkage). Presently it is preferred that there be no optional substituents in L¹. When both m and p are O, L¹ is a divalent mono- or bicyclic carbocyclic or heterocyclic radical (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-, or bi-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 of the invention, L¹, m and p may be O 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. AIk¹ and AIk², 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(=O)NH-, -CH₂CH₂C(=O)NH-,-CH₂C(O)O-, -CH₂S-, -CH₂CH₂C(O)O-, -(CHa) ₄NH-, -CH₂CH₂S-, -CH₂O, -CH₂CH₂O-,

In one preferred class of compounds of the invention R5 is hydrogen and Re is a radical (R₈)NH-CH(R7)-Y¹-L¹-[CH₂]z-(ZV, wherein w and z are each 0, Y¹ is a bond, and L¹ is ^■ -CH₂O-, -CH₂CH₂O- or -CH₂CH₂CH₂O-.

In another preferred class of compounds of the invention R₆ is hydrogen and R₅ is a radical of formula (1) or (2) above.

As mentioned above, the compounds with which the invention is concerned are inhibitors of c-Met kinase activity and are therefore of use for treatment of cell proliferative diseases such as cancer.

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 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 propel lant-free administration of micronized powders, for example, inhalation capsules or other "dry powder" delivery systems. Excipients, such as, for example, propellants (e.g. Frigeri 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 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 those skilled in the art. Typical literature sources are "Advanced organic chemistry", 4^th Edition (Wiley), J March, "Comprehensive Organic Transformation", 2^nd Edition (Wiley), R. C. Larock , "Handbook of Heterocyclic Chemistry", 2^nd Edition (Pergamon), A.R. Katritzky; review articles such as found in "Synthesis", "Ace. 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 compounds of the invention may be prepared by a number of processes generally described below and more specifically in the Examples hereinafter. In the reactions described below, it may be necessary to protect reactive functional groups, for example hydroxyl, amino and carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions [see for example Greene, T.W., "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1999]. Conventional protecting groups may be used in conjunction with standard practice. In some instances deprotection may be the final step in the synthesis of a compound of general formula (I), and the processes according to the invention described herein after are understood to extend to such removal of protecting groups.

Thus compounds of general formula (I) may be prepared by, but not restricted to methods set out in the following schemes.

General method for the alkylation of pyrazoles with electrophilic amino acid cvclopentyl ester derivatives and hydrolysis of pyrazole amino acid esters

Reagents: a) X(CH₂)nNHBocCO₂c-pentyl (X=Br, OMs), BEMP, MeCN b) 4M HCI in dioxane c) LiOH, H₂O, THF

Scheme 1

General method for the alkylation of pyrazoles with electrophilic amino acid t- butyl ester derivatives and hydrolysis of the resulting pyrazole esters

Reagents: a) X(CH₂)nNHCbzCO₂t-butyi (X=Br, OMs), BEMP, MeCN b) 10% Pd-C, H₂, EtOH

Scheme 2

General method for the alkylation of piperidines with electrophilic amino acid cvclopentyl ester derivatives and hydrolysis of piperidine esters

Reagents: a) X(CH₂)_nNHBocC0₂c-pentyl (X=Br, OMs), Et₃N, DMF b) 4M HCI in dioxane c) LiOH, H₂O, THF.

Scheme 3 General method for the alleviation of pjperidines with electrophilic amino acid t- butyl ester derivatives

Reagents: a) X(CH₂)_nNHCbzCO₂t-butyl (X=Br, OMs), Et₃N, DMF b) 10%Pd-C, H₂, EtOH

Scheme 4

General method for the alkylation of phenols with electrophilic amino acid cvclopentyl ester derivatives and hydrolysis of phenolic esters

Reagents: a) 3,4-Dimethoxybenzyl alcohol, NaH, DMF, 5O⁰C followed by TFA, CH₂CI₂ b) X(CHz)_nNHBoCCO₂R₂, Cs₂CO₃, TBAI, DMF, c) 4M HCI in dioxane d) LiOH, H₂O, EtOH.

Scheme 5

General method for the synthesis of pyrimidyl alkoxy linked amino ester derivatives and hydrolysis of pyrimidiyl esters

Reagents: a) 2-Methoxypyrimidine-5-ylboronic acid, PdCl2(dppf), DMF b) 4M HCl in dioxane c) X(CH₂)nNHBocCO₂R2, Cs₂CO₃, TBAI, DMF d) 4M HCI in dioxane e) LiOH, H₂O, THF.

Scheme 6

General method for the synthesis of pyrimidyl aminoalkyl linked amino ester derivatives and hydrolysis of pyrimidiyl esters

Reagents: a) Cyclopentyl /^-(δ-bromopyrimidin^-ylJ-Λ^^tørf-butoxycarbonyO-L-ornithinate, PdCI₂(dppf), Na₂CO₃, DME/EtOH b) 4M HCI in dioxane c) LiOH, H₂O, THF.

Scheme 7 General method for the alkylation of phenols with electrophilic amino acid cvclopentyl ester derivatives and hydrolysis of phenolic esters

Reagents: Cyclopentyl W-(terf-butoxycarbonyl)-5-[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaboro lan-2-yl)phenoxy]-L- norvalinate, PdCfedppf, Na₂CO₃, DME/EtOH b) 4M HCI in dioxane c) LiOH, H₂O, THF.

Scheme 8 Experimental

Abbreviations

AcOH = acetic acid

BEMP = 2-te/?-butylimino-2-diethyiamino-1 ,3-dimethyl-perhydro-1 ,3,2-diazaphosphorine

Boc or boc = fe/Y-butoxycarbonyl

BOC₂O = Di-tert-butyldicarbonate

Cbz = benzyloxycarbonyl

CHCI₃ = chloroform

Cs₂CO₃ = ceasium carbonate

DCE = dichloroethane

DCM = dichloromethane

DIPEA = diisopropylethylamine

DMAP = dimethylamino pyridine

DMF = dimethylformamide

DMSO = dimethyl sulfoxide

EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EtOAc = ethyl acetate EtOH = ethanol Et₂O = diethyl ether Et₃N = triethylamine HCI = hydrochloric acid K₂CO₃ = potassium carbonate LiOH = lithium hydroxide MeOH = methanol MgSO₄ = magnesium sulphate Na₂CO₃ = sodium carbonate NaH = sodium hydride NaHCO₃ = sodium hydrogen carbonate NaI = sodium iodide NaOH = sodium hydroxide NBu₄Br = tetrabutylammonium bromide

Pd(dppf)CI₂ = dichloro-(1 ,2-bis-(diphenylphosphino)ethane)-palladium(ll) Pd/C = palladium on carbon Pd(OH)₂ = Palladium hydroxide STAB = sodium triacetoxyborohydride

TBTU = O-benzotriazol-1-yl-Λ/,Λ/,Λ/',Λ/'-tetramethyluronium tetrafluoroborate TFA = trifluoroacetic acid THF = tetrahydrofuran

aq = aqueous g = gram(s)

LC/MS = high performance liquid chromatography/mass spectrometry mg = milligram(s) min = minutes mL = milliliter(s) μl_ = microlitre(s) mol = mole(s) mmol = miHimole(s)

NMR = nuclear magnetic resonance

RT or rt = room temperature sat = saturated 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 CEM Discovery model set at 300W. Purification of compounds by flash chromatography column was performed using silica gel, particle size 40-63μ μm (230-400 mesh) obtained from Fluorochem. Purification of compounds by preparative HPLC was performed on a Agilent prep system using reverse phase Agilent prep-C18 columns (5μm, 50 x 21.2 mm), gradient 0-100% B (A = water / 0.1% ammonia or 0.1% formic acid and B = acetonitrile / 0.1% ammonia or 0.1 % formic acid) over 10 min, flow = 28 mL/min, UV detection at 254 nm.

¹H NMR spectra were recorded on a Bruker 400 or 300 MHz AV 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 were obtained as follows: Agilent Prep-C18 Scalar column, 5 μm (4.6 x 50 mm, flow rate 2.5 mL/min) eluting with a H₂O-MeCN gradient containing 0.1% v/v formic acid over 7 minutes with UV detection at 254 nm. Gradient information: 0.0 - 0.5 min: 95% H₂O-5% MeCN; 0.5 -5.0 min; Ramp from 95% H₂O-5% MeCN to 5% H₂O- 95% MeCN; 5.0 - 5.5 min: Hold at 5% H₂O-95% MeCN; 5.5 - 5.6 min: Hold at 5% H₂O- 95% MeCN, flow rate increased to 3.5 mL/min; 5.6 - 6.6 min: Hold at 5% H₂O-95% MeCN, flow rate 3.5 mL/min; 6.6 - 6.75 min: Return to 95% H₂O-5% MeCN, flow rate 3.5 mL/min; 6.75 - 6.9 min: Hold at 95% H₂O-5% MeCN, flow rate 3.5 mL/min; 6.9 - 7.0 min: Hold at 95% H₂O-5% MeCN, flow rate reduced to 2.5 mL/min. Mass spectra were obtained using an Agilent multimode source in either the positive (APCI + ESI⁺) or negative (APCI + ESI^") mode.

Intermediate 1 : Cvclopentvl (S)-5-bromo-2-tert-butoxvcarobonvlamino pentanoate

The synthesis of cyclopentyl (S)-5-bromo-2-ferf-butoxycarbonylaminopentanoate is outlined below in Scheme 9. Additional literature references relating to this route can be found within J. Org. Chem. 1984, 49, 3527-3534.

Stage 1

Stage 3

Intermediate 1

Scheme 9

Stage 1- 5-Benzyl 1 -cyclopentyl N-(tert-butoxycarbonyl)-L-glutamate

To a solution of (2S)-5-(benzyloxy)-2-[tert-butoxycarbonyl)amino]-5-oxopentanoic acid (15 g, 44.5 mmol) in DCM (220 mL) in an ice-bath, was added cyclopentanol (4.8 mL, 53.3 mmol, 1.2 eq), EDC (9.4 g, 48.9 mmol, 1.1 eq) and DMAP (543 mg, 4.4 mmol, 0.1 eq). The reaction mixture was allowed to warm to room temperature and stirred for 12 hours for complete reaction. The reaction mixture was diluted with DCM (200 mL) and washed with 1M HCI, 1 M Na₂CO₃ and brine. The organic layer was then dried (MgSO₄) and evaporated under reduced pressure. The product was purified by column chromatography using ethyl acetate/heptane (1 :4) to afford the title compound as a white solid (12.4 g, 69% yield). ¹H NMR (300 MHz, CDCI₃) δ: 7.38 (5H₁ m), 5.70 (1 H, m), 5.10 (2H, s), 5.05 (1H₁ m), 4.25 (1H, m), 2.47 (2H, m), 2.15 (1 H₁ m), 1.95-1.55 (9H, m), 1.47 (9H, s). Stage 2- (4S)-4-[(tert-butoxycarbonyl)amino]-5-(cyc!opentyl)-5-oxopentanoic acid

5-Benzyl 1 -cyclopentyl N-(tert-butoxycarbonyl)-L-glutamate (12.4 g, 30.5 mmol) was dissolved in EtOAc (200 ml_) and purged with nitrogen before addition of 20% Pd(OH)₂ on carbon catalyst (1.3 g). The reaction flask was then purged with hydrogen gas for a period of 5 minutes before leaving under a balloon of hydrogen for 5 hours for complete reaction. The catalyst was removed by filtration, washing with EtOAc (50 ml_) and the combined filtrates were evaporated under reduced pressure. The title compound was isolated as a clear oil (7.73 g, 85% yield) and required no further purification. ¹H NMR (300 MHz, CDCI₃) δ: 10.0 (1H, br s), 5.70 (2H, m), 4.28 (1H, m), 2.47 (2H, m), 2.15 (1H, m), 1.95-1.55 (9H, m), 1.47 (9H, s).

Stage 3- Cyclopentyl N-(tert-butoxycarbonyl)-5-hydroxy-L-norvalinate

Ethyl chloroformate (2.45 ml_, 25.6 mmol, 1.2 eq) was added at -20 ⁰C to a stirred solution of (4S)-4-[(tert-butoxycarbonyl)amino]-5-(cyclopentyl)-5-oxopentanoic acid (6.73 g, 21.4 mmol) and /V-methyl morpholine (3.05 mL, 27.8 mmol, 1.3 eq) in THF (50 ml_). The reaction mixture became very thick with precipitation of a white solid. The reaction was therefore diluted further with THF (100 mL) to aid mixing and left stirring at -20 ⁰C for 2 hours. The precipitated mass was filtered off and the filtrate was added over a period of 20 minutes to a solution of sodium borohydride (2.43 g, 64.1 mmol, 3 eq) in THF (20 mL) and water (5 mL) at 0 ⁰C. The reaction mixture was allowed to stir to room temperature and left for 4 hours for complete reaction. The mixture was acidified to pH 5 with 1M HCI and the THF removed under reduced pressure. The aqueous solution was extracted with EtOAc (3 x 100 mL) and dried (MgSO₄). The product was purified by column chromatography (DCM - 5% MeOH/DCM) and isolated as a clear oil (5.0 g, 78% yield). ¹H NMR (300 MHz, CDCI₃) δ: 5.20 (2H, m), 4.25 (1 H, m), 3.65 (2H, m), 2.00-1.57 (12H, m), 1.47 (9H, s).

Stage 4- Cyclopentyl 5-bromo-N-(tert-butoxycarbonyl)-L-norvalinate (Intermediate

1)

To a slurry of Λ/-bromosuccinimide (3.54 g, 19.9 mmol, 3 eq) in DCM (30 mL) was added a solution of triphenylphosphine (4.87 g, 18.8 mmol, 2.8 eq) in DCM (15 mL). The solution was stirred for a further 5 minutes before addition of pyridine (644 μl, 7.96 mmol, 1.2 eq) and a solution of cyclopentyl N-(tert-butoxycarbonyl)-5-hydroxy-L-norvalinate (2.0 g, 6.64 mmol) in DCM (20 ml_). The solution was stirred for 18 hours, concentrated in vacuo and the residual solvent azeotroped with toluene (3 x 30 ml_). The residue was triturated with diethyl ether (30 ml_) and ethyl acetate:heptane (1 :9, 2 x 30 mL). The combined ether and ethyl acetate/heptane solutions was concentrated onto silica and purified by column chromatography using ethyl acetate/heptane (1:9 to 2:8) to provide the title compound as a clear oil (1.34 g, 55% yield). ¹H NMR (300 MHz, CDCI₃) δ: 5.25 (1H, m), 5.05 (1 H, br d), 3.45 (2H, m), 2.00-1.55 (12H, m), 1.45 (9H, s).

Intermediate2: terf-butyl (2S)-2-U(benzyloxy)carbonvnamino)-4-bromobutanoate

The title intermediate was prepared according to the procedure outlined below in Scheme 10.

Stage 1

Stage 2

Stage 3

Scheme 10

Stage 1- (3S)-3-{[(Benzyloxy)carbonyl]amino}-4-tert-butoxy-4-oxobutanoic acid

To a solution of (3S)-3-amino-4-ferf-butoxy-4-oxobutanoic acid (900 mg, 4.75 mmol) and sodium hydroxide (280 mg, 7.13 mmol) in 25% water/dioxane (50 mL) at 0 ⁰C was added benzyl chloroformate (2 g, 4.13 mmol) in dioxane (10 mL). The mixture was stirred at O⁰C for 1 hour and then at RT overnight. Water (10 mL) was added and the mixture was extracted with EtOAc (2 x 20 mL). The organic phase was back extracted with a saturated aqueous solution of NaHCO₃ (2 x 10 mL). The combined aqueous layers were acidified to pH 1 with 1M HCI, and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried (MgSO₄) and concentrated under reduced pressure. The residue was purified by column chromatography (35% EtOAc/heptane) to give the product as a colourless oil (0.76 g, 50% yield). ESMS: m/z 346 [M+23] ⁺

Stage 2- te/f-Butyl Λ/-[(benzyloxy)carbonyl]-L-homoserinate

To a solution of (3S)-3-{[(benzyloxy)carbonyl]amino}-4-te/f-butoxy-4-oxobutanoic acid (600 mg, 1.87 mmol) in anhydrous THF (20 mL) at -20 ⁰C was slowly added Et₃N (32 μL, 2.24 mmol) and ethyl chloroformate (21 μL, 2.24 mmol). The mixture was stirred at -20 ⁰C for 2 hours. The solid formed was filtered off and washed with THF (2 x 10 mL). The filtrate was added dropwise to a solution of sodium borohydride (0.2 g, 5.61 mmol) at 0 ⁰C over 10 minutes and then allowed to warm to RT. The mixture was stirred for an additional 4 hours. The solvent was removed under reduced pressure and the residue was diluted with water (10 mL), acidified to pH 5 with 1 M HCI and extracted with EtOAc (2 x 20 mL). The organic fractions were combined washed with 10% aqueous sodium hydroxide (10 mL), water (10 mL) and brine (10 mL). The organic layer was dried (MgSO₄) and concentrated under reduced pressure to give the product as a clear oil (0.3 g, 51% yield). ESMS: m/z 332 [M+Na]⁺.

Stage 3- terf-butyl (2S)-2-{[(benzyloxy)carbonyl]amino}-4-bromobutanoate (Intermediate 2)

To a solution of N-bromosuccinimide (520 mg, 2.91 mmol) in DCM (10 mL) was slowly added a solution of triphenylphosphine (0.71 g, 2.72 mmol) in DCM (10 mL). The mixture was stirred at RT for 5 minutes before pyridine (94 μL, 1.16 mmol) and a solution of tert- butyl Λ/-[(benzyloxy)carbonyl]-L-homoserinate (0.30 g, 0.97 mmol) in DCM (20 mL) were added dropwise. The mixture was stirred at RT for another 18 hours. The solvent was removed under reduced pressure, the residue was azeotroped with toluene (2 x 15 mL) and triturated with Et₂O (2 x 25 mL) and 10% EtOAc in heptanes. The filtrate from the triturations were combined and concentrated under reduced pressure. The crude product was purified by column chromatography (15% EtOAc/heptanes) to give the title intermediate as a clear oil (0.16 g, 44% yield). ESMS: m/z 395 [M+Na] ⁺. ¹H NMR (300 MHz, CDCI₃) 6: 7.39-7.30 (5H, m), 5.40 (1H, d, J=6.8Hz), 5.12 (2H, s), 4.38 (1H, q, ^• J=7.7Hz), 3.47-3.38 (2H, m), 5.49-2.33 (1H, m), 2.28-2.13 (1 H, m) and 1.48 (9H, s).

Intermediate 3: ferf-butyl /V-r(benzyloxy)carbonvπ-5-bromo-L-norvalinate

The title intermediate was prepared according to the procedure outlined for Intermediate

2 starting with (4S)-4-amino-5-fe/-f-butoxy-5-oxopentanoic acid. ESMS: m/z 409

[M+Na]⁺.

Intermediate 4: Cvclopentyl 3-bromo-ΛMfert-butoxycarbonvO-L-alaninate

The title intermediate was prepared according to the scheme outlined in Scheme 11.

Scheme 11 Stage 1- Cyclopentyl 0-benzyl-/V-(tert-butoxycarbonyl)-L-seriπate To a solution of Boc-Ser(Bn)-OH (5g, 16.93 mmol) in DMF (20 ml), at O⁰C under N₂, was added cyclopentanol (3.07 ml, 33.9 mmol), EDC (3.89 g, 20.3 mmol) and DMAP (0.2 g, 1.69 mmol). The mixture was warmed to room temperature and stirred for 1 δhrs. The . reaction was concentrated in vacuo and the residue was dissolved in EtOAc, washed with 1 M HCI aq, 1 M Na₂CO₃ aq, brine, dried over magnesium sulphate and concentrated to provide the title compound as a yellow oil which solidified upon standing (4.94 g). NMR ¹H (CDCI₃ 300 MHz) δ: 7.39 - 7.30 (5H, m), 5.40 (1H, br d, J=8.4Hz), 5.28 - 5.22 (1H, m), 4.52 (2H, q, J=12.3 & 12.6Hz), 4.42 - 4.36 (1H, m), 3.90 - 3.84 (1H, m), 3.72 - 3.65 (1 H, m), 1.95 - 1.50 (8H, m), 1.47 (9H, s).

Stage 2- Cyclopentyl /V-(fert-butoxycarbonyl)-L-serinate

The benzyl protected alcohol (0.85 g, 2.34 mmol) was dissolved in EtOH (20 ml_) and the flask was purged with nitrogen. Then Pd(OH)₂ on carbon (100mg) was added and the flask was again purged with nitrogen. Hydrogen was bubbled through the mixture and then the mixture was heated to reflux under an atmosphere of hydrogen for 1 h. The mixture was allowed to cool to room temperature before being filtered through Celite®. The filtrate was evaporated to yield the product as a colourless oil, (0.44 g, 69% yield). ESMS: m/z 274 (M+H)⁺

Stage 3- terf-Butyl /V-[(benzyloxy)carbonyl]-5-bromo-L-norvalinate

A solution of triphenylphosphine (2.69 g, 10.24 mmol) in DCM (10 ml.) was added to a suspension of N-bromosuccinimide (1.95 g, 10.98 mmol) in DCM (10 ml_). The mixture became a red solution and was stirred at RT for 5 min before pyridine (0.355 mL, 4.39 mmol) was added, followed by a solution of cyciopentyl Λ/-(fe/t-butoxycarbonyl)-L- serinate (1 g, 3.66 mmol) in DCM (5 mL). The resulting dark solution was left to stir at room temperature overnight. The reaction mixture was concentrated and then subjected to column chromatography (silica gel, 5% EtOAc / isohexane) to yield the product as a colourless oil (1.02 g, 30% yield). ESMS: m/z 336 (M+H)⁺

Intermediate s: 3-ri-(2,6-dichloro-3-fluorophenyl)ethoxy1-5-(1H-pyrazol-4- yl)pyridin-2-amine

This material was prepared by methods described in WO2006021881.

Intermediate β: 3-f1-(2,6-dichloro-3-fluorophenyl)ethoxy1-5-(1-piperidin-4-yl-1H- pyrazol-4-yl)pyridin-2-amine

This material was prepared by methods described in WO2006021881.

Intermediate 7: 5-Bromo-3-ri-(2,6-dichloro-3-fluorophenyl)ethox1pyridin-2-amine

This material was prepared by methods described in WO2006021881.

Intermediate 8: {6-amino-5-H-(2,6-dichloro-3-fluorophenyl)ethoxylpyridin-3- vDboronic acid

This material was prepared by methods described in WO2006021881.

Intermediate 9: Cyclopentyl 5-r4-(4-(6-amino-5-H-(2,6-dichloro-3-fluoro phenyl) ethoxylpyridin-3-yl>-1A/-pyrazol-1-yl)piperidin-1-vπ-yV-(tert-butoxy carbon yl)-L- norvalinate

Intermediate 1 (24.27 mg, 0.067 mmol) in DMF (0.5 ml_) was added dropwise to a stirred mixture of Intermediate 6 (30 mg, 0.067 mmol) and triethylamine (18.72 μl, 0.133 mmol) in DMF (0.5 mL). The resulting solution was stirred at room temperature for 72 h. The reaction was partitioned between EtOAc (50 mL) and brine (30 mL) and the phases separated. The organic phase was washed with brine (4 x 30 mL), dried (MgSO₄) and evaporated to give a viscous yellow oil. Purification of the residue (silica gel, 5% MeOH/DCM) gave (15.1 mg, 31 % yield) as a white crystalline solid. ESMS: m/z 734 (M+H)⁺.

Intermediate 10: Cvclopentyl 5-(4-{6-amino-5-ri-(2,6-dichloro-3-fluorophenv0 ethoxyipyridin-S-ylVIH-pyrazol-i-vD-N-ffe/'f-butoxycarbonvD-L-norvalinate

Intermediate 1 (86.0 mg, 0.33 mmol) in MeCN (6 mL) was added dropwise to a stirred solution of Intermediate 5 (100.0 mg, 0.27 mmol) and BEMP (117 μl, 0.41 mmol) in MeCN (6 mL). The resulting mixture was stirred at RT for 20 h. The mixture was then partitioned between EtOAc (50 mL) and water (50 mL) and the phases separated. The organic phase was washed with brine (50 mL), dried (MgSO₄) and evaporated. The residue was purified (silica gel, 50% EtOAc/petrol) to give the desired material (75 mg, 43% yield). ESMS: m/z 650 (M+H)⁺

Intermediate 11: Cvclopentyl 5-r(5-(6-amino-5-ri-(2,6-dichloro-3-fluoro phenyl) ethoxv1pvridin-3-vl>pvrimidin-2-vl)oxv1-/V-(ferf-butoxvcarbonvπ-L-norvalinate

The title intermediate was prepared by the method outlined in Scheme 12.

Stage 1

Scheme 12

Stage 1- 3-[1-(2,6-Dichloro-3-fluorophenyl)ethoxy]-5-(2-methoxypyrimidin-5-yl) pyridin-2-amine

A solution of Intermediate 7 (224 mg, 0.591 mmol), (2-methoxypyrimidin-5-yl)boronic acid (100 mg, 0.650 mmol) and PdCI₂(dppf) (48.2 mg, 0.059 mmol) was made up in DME/EtOH (1 ml_). To this was added sodium carbonate (68.9 mg, 0.650 mmol) (0.69 mL of a 1 M solution) and the solution was subjected to microwave irradiation for 10 min at 100 ⁰C. The dark mixture was poured onto water and extracted with EtOAc (5 mL). The combined organic phases were washed with water and brine (2 x 3 mL each), dried (MgSO₄) and evaporated. The residue was subjected to column chromatography (40 g) eluting with 40% EtOAc in hexanes to give the desired material (100 mg) as a colourless solid. ESMS: m/z 409 and 411 (M+H)⁺

Stage 2- 5-{6-Amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl} pyrimidin-2-ol

To a solution of 3-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(2-methoxypyrimidin-5- yl)pyridin-2-amine (100 mg, 0.244 mmol) in HCI in dioxane (611 μl, 2.44 mmol) was added water (1 ml_) and the mixture was heated at 90 ⁰C for 3 h. After this time, the solution was cooled to RT and poured onto sat NaHCO_β (1 ml_) and extracted with

EtOAc (2 x 2 mL). The combined organic phases were washed with water and brine (2 x 2 mL each), dried (MgSO₄) and evaporated. The residue was absorbed onto silica and subjected to column chromatography (silica gel, 5-10% MeOH-DCM) to give the desired material (60 mg, 63% yield). ESMS: m/z 395 (M+H)⁺

Stage 3- Cyclopentyl 5-[(5-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy] pyridin-3-yl}pyrimidin-2-yl)oxy]-/V-(fert-butoxycarbonyl)-L-norvalinate

To a solution of 5-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl} pyrimidin-2-ol (160 mg, 0.405 mmol) and Intermediate 1 (162 mg, 0.445 mmol) in DMF (8 mL) was added CS₂CO₃ (264 mg, 0.810 mmol). The reaction was stirred for 2 hours. Complete conversion of starting material into O and N alkylated products was observed. The reaction was quenched by addition of water, and then extracted into EtOAc. Combined organics were washed with water and brine then dried. The residue was purified by column chromatography (silica gel, 3 % MeOH in DCM) to give Intermediate 9 (60 mg, 22% yield). A second fraction was obtained which yielded Intermediate 12. ESMS: m/z 678 and 680 (M+H)⁺

lntemediate 12: Cvclopentyl 5-r5-(6-amino-5-ri-(2,6-dichloro-3-fluorophenyl) ethoxyi pyridin-3-yl)-2-oxopyrinrιidin-1(2H)-vn-N-(tert-butoxycarbonvπ-L- norvalinate

Intermediate 12 was isolated (154 mg, 56% yield) as a by-product in stage 3 of Intermediate 11. ESMS: m/z 678 and 680 (M+H)⁺

Intermediate 13: Cvclopentyl ΛMfert-butoxycarbonyl)-5-r4-(4,4,5,5-tetra methyl- 1,3,2-dioxaborolan-2-yl)phenoxy1-L-norvalinate

To a mixture of 4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenol (69 mg, 0.314 mmol) and K₂CO₃ (44 mg, 0.318 mmol) in DMF (1 ml_) under nitrogen was added a solution of Intermediate 1 (100 mg, 0.275 mmol) in DMF (1 ml_). The reaction was left to stir at RT under nitrogen for 6 h. The reaction mixture was worked up by partitioning between EtOAc (5 ml_) and saturated Na₂COs solution (2 ml_). The organic layer was further washed with saturated sodium carbonate solution and brine (2 x 2 ml. each), before being dried (MgSO₄) and concentrated in vacuo to afford a pink oil. The oil was purified by column chromatography, (silica gel, 9:1 isohexane: ethyl acetate) to give the desired material as a colourless oil. ESMS: m/z 504 (M+H)⁺ Intermediate 14: Cvclopentyl 5-(4-f6-amino-5-H-(2.6-dichloro-3-fluorophenvπ ethoxy1pyridin-3-yl>phenoxy)-Λ/-(tert-butoxycarbonvπ-L-norvalinate

A microwave tube was charged with Intermediate 7 (31.5 mg, 0.083 mmol), Intermediate 13 (50 mg, 0.099 mmol), sodium carbonate (1M solution) (99 μl, 0.099 mmol) and PdCI₂(dppf) (3.38 mg, 4.14 μmol). A 4:1 mixture of DME/ ethanol (750 μl) was added. The reaction mixture was heated in the microwave at 100⁰C for 40 min. The reaction mixture was concentrated in vacuo and the residue purified by chromatography, (silica gel: 1 :1-isohexane: ethyl acetate) to afford the desired material as an orange oil (45 mg, 80%). ESMS: m/z 676 / 678

Intermediate 15: Cvclopentyl Λ/⁵-(5-bromopyrimidin-2-yl)-Λ/²-(fø/f-butoxy carbonyl)- L-ornithinate

The title intermediate was prepared by the method outlined in Scheme 13.

Stage 1

Stage 2

Scheme 13

Stage 1- Cyclopentyl N⁵-(benzoyloxycarbonyl)-N²-(tert-butoxycarbonyl)-L- ornithinate

A solution of N⁵-(benzoyloxycarbonyl)-N²-(tert-butoxycarbonyl)-L-ornithine (0.75 g, 2.047 mmol) in DCM (6 mL) was cooled to 0 ⁰C, and treated sequentially with cyclopentanol (0.223 mL, 2.456 mmol), EDC (0.432 g, 2.252 mmol) and DMAP (0.025 g, 0.205 mmol). The resulting mixture was allowed to warm to room temperature. The reaction was left to stir for 12h at ambient temperature. The reaction mixture was loaded straight onto a silica gel column and eluted (10 to 50 % EtOAc in isohexane) to afford the desired material (883 mg, 99% yield) as a colourless oil. ESMS: m/z 334 (M+H)⁺

Stage 2- Cyclopentyl /^-(tert-butoxycarbonyO-L-ornithinate

10 wt % Pd/C (50 mg) was added to a solution of cyclopentyl N⁵-(benzoyloxycarbonyl)- N²-(tert-butoxycarbonyl)-L-ornithinate (0.333 g, 0.766 mmol) in 9:1 EtOH:CHCI₃ (6 mL). The reaction was evacuated and purged with hydrogen and then heated to 60 ⁰C for 12 hours. The reaction was filtered through Celite® and washed with 9:1 EtOH:CHCI₃ (5 mL). The solvent was removed under reduced pressure to give the desired material (89 mg, 56%) which was used without purification.

Stage 3- Cyclopentyl Λ/⁵-(5-bromopyrimidin-2-yl)-Λ/²-(terf-butoxycarbonyl)-L- ornithinate

Cyclopentyl Λ/²-(ferf-butoxycarbonyl)-L-ornithinate (142 mg, 0.473 mmol), 5-bromo-2- chloropyrimidine (101 mg, 0.520 mmol) and DIPEA (165 μl, 0.945 mmol) were dissolved in 1-methyl-2-pyrrolidinone (2 mL) and heated in the microwave to 50 ⁰C for 10 minutes. The mixture was partitioned between water (4 mL) and ether (4 mL) and the layers were separated. The organic layer was washed with water (6 x 3mL) and brine (3 mL), then dried (MgSO₄) and concentrated. The residue was purified (silica gel: 20 % EtOAc/ iso- hexane) to give the title intermediate (60 mg, 28% yield). ESMS: m/z 458 (M+H)⁺

Intermediate 16: Cyclopentyl Λ/⁵-(5-{6-amino-5-ri-(2,6-dichloro-3-fluorophenyl) ethoxy1pyridin-3-yl)pyrimidin-2-yl)-/V²-(fert-butoxycarbonyl)-L-ornithinate

A solution of Intermediate 8 (70.5 mg, 0.157 mmol), Intermediate 15 (60 mg, 0.131 mmol) and PdCI₂(dppf) (10.7 mg, 0.013 mmol) was made up in DME/EtOH (1 ml_). To this was added 1 M Na₂CO₃ solution (157 μl, 0.157 mmol) (0.69 mL of a 1 M solution) and the solution was subjected to microwave irradiation for 40 min at 100 ⁰C. The dark mixture was poured onto water (2ml_) and extracted with EtOAc (3 x 5 mL). The combined organic phases were washed with water and brine (2 x 5mL), dried (MgSO₄) and evaporated. The residue was purified (silica gel: 3% MeOH in DCM followed by 75 % EtOAc in iso-hexanes) to give the desired material (76mg, 85% yield). ESMS: m/z 677 and 679 (M+H)⁺

Intermediate 17: ferf-Butyl 4-(4-{6-amino-5-ri-(3-{f(4S)-4-lϊferf-butoxy carbonyl) aminol-5-fcvclopentyloxy)-5-oxopentvnoxy>-2,6-dichloro phenvDethoxyl pyridin-3- yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate

The title intermediate was prepared by the method outlined in Scheme 14. Stage 1 Stage 2

Stage 3

Stage 4

Scheme 14

Stage 1- terf-Butyl 4-(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy] pyridin- 3-yl}-1 /y-pyrazol-1 -yl)piperidine-1 -carboxylate

Cs₂CO₃ (932 mg, 2.86 mmol) was added to a solution of fe/Y-butyl 4-[(methylsulfonyl) oxy]piperidine-1 -carboxylate (399 mg, 1.430 mmol) and 3-[1-(2,6-dichloro-3-fluoro phenyl)ethoxy]-5-(1H-pyrazol-4-yl)pyridin-2-amine (350 mg, 0.953 mmol) in DMF (10 ml_) and the resulting white slurry was heated at 80 ⁰C for 18 h.The mixture was cooled, partitioned between EtOAc (100 mL) and brine (100 ml_) and the phases separated. The organic phase was washed with brine (4 x 100 mL), dried (MgSO₄) and evaporated under reduced pressure. The residue was purified (silica gel, 75-100% EtOAc-hexane) to give the desired material (230 mg, 44% yield) as a foam solid. ESMS: m/z 550 (M+H)⁺.

Stage 2- 3-(1-{t2-amino-5-(1-piperidin-4-yl-1//-pyrazol-4-yl)pyridin-3-yl]oxy} ethyl)- 2,4-dichlorophenol

NaH (60% w/w , 4.36 mg, 0.109 mmol) was added to a stirred solution of 3,4- dimethoxybenzyl alcohol (15.63 μl, 0.109 mmol) in DMF (1 mL) in one portion and the mixture stirred for 10 min. førf-Butyl 4-(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl) ethoxyJpyridin-S-ylJ-IH-pyrazol-i-yOpiperidine-i-carboxylate (20 mg, 0.036 mmol) was added and the resulting mixture heated at 50 ⁰C for 2 h. The reaction mixture was then partitioned between EtOAc (25 ml_) and brine (25 ml_) and the phases separated. The organic phase was washed with brine (3 x 25 mL), dried (MgSO₄) and evaporated under reduced pressure. The residue was dissolved in DCM (0.5 mL) and treated with TFA (0.2 mL). The mixture was stirred at room temperature for 30 min. The solvents were evaporated under reduced pressure and the residue purified on a solid extraction cartridge (300 mg) to give the desired material (13.5 mg, 84% yield) as a pale brown solid. ESMS: m/z 448 (M+H)⁺.

Stage 3- terf-Butyl 4-(4-{6-amino-5-[1-(2,6-dichloro-3-hydroxyphenyl)ethoxy] pyridin-3-yl}-1W-pyrazol-1-yl)piperidine-1-carboxylate

3-(1-{[2-amino-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)pyridin-3-yl]oxy}ethyl)-2,4-dichloro phenol (109 mg, 0.243 mmol) was treated with a solution of di-tert-butyl dicarbonate (56.4 μl, 0.243 mmol) in THF (5 mL) and the mixture stirred at room temperature for 30 min. The reaction mixture was diluted with EtOAc (50 mL) and washed with water (25 mL), brine (25 mL), dried (MgSO₄) and evaporated to give the desired material (151 mg, 98 % yield) as a pale brown solid. ESMS: m/z 548 (M+H)⁺.

Stage 4- ferf-Butyl 4-(4-{6-amino-5-[1-(3-{[(4S)-4-[(fetf-butoxycarbonyl) amino]-5-

(cyclopentyloxyJ-S-oxopentylJoxy^.β-dichlorophenyOethoxylpyridin-S-y^-IH- pyrazol-1-yl)piperidine-1-carboxylate fe/f-Butyl 4-(4-{6-amino-5-[1-(2,6-dichloro-3-hydroxyphenyl)ethoxy]pyridin-3-yl}-1H- pyrazol-1-yl)piperidine-1-carboxylate (135 mg, 0.282 mmol) and Cs₂CO₃ (367 mg, 1.127 mmol) were dissolved in DMF (2.5 mL) and allowed to stir at room temp for 5 min. Intermediate 1 (246 mg, 0.68 mmol) in DMF (2.5 mL) was added and the mixture heated at 50 ⁰C for 1 h. Tetrabutylammonium iodide (26.0 mg, 0.070 mmol) was added and heating continued for 90 h. Further Intermediate 1 (62 mg, 0.164 mmol) was added and heating continued for 6 h. The mixture was partitioned between EtOAc (50 mL) and brine (50 mL) and the phases separated. The organic phase was washed with brine (3 x 50 mL), dried (MgSO₄) and evaporated under reduced pressure to give a brown oil. The residue was purified (silica gel; 5% MeOH/DCM) gave the desired material (112 mg) as a dark brown oil. ESMS: m/z 831 (M+H)⁺. Intermediate 18: Cvclopentyl 543-(1-fr2-amino-5-(1H-Pyrazol-4-vπpyridin-3-yr|o^χy) ethvπ^^-dichlorophenoxyi-N-tfert-butoxycarbonvD-L-norvalinate

The title intermediate was prepared by the method outlined in Scheme 15.

Stage 2

Scheme 15

Stage 1- 3-(1-{t2-amino-5-(1H-pyrazol-4-yl)pyridin-3-yl]oxy}ethyl)-2,4-dichlor ophenol

NaH (60% w/w, 82 mg, 2.042 mmol) was added to a stirred solution of 3,4- dimethoxybenzyl alcohol (293 μl, 2.042 mmol) in DMF (10 ml_) portionwise and the mixture stirred for 10 min. 3-[1-(2,6-Dichloro-3-fluorophenyl)ethoxy]-5-(1H-pyrazol-4- yl)pyridin-2-amine (250 mg, 0.681 mmol) was added and the resulting mixture heated at 50 ⁰C for 5 h. The mixture was partitioned between EtOAc (25 ml_) and brine (25 mL) and the phases separated. The organic phase was washed with brine (4 x 25 mL), dried (MgSO₄) and evaporated to give a brown oil. The residue was diluted with DCM (5 mL) and TFA (1.1 mL) was added and the reaction was stirred for 30 mins. The solution was evaporated and the residue purified (silica gel 5-10% MeOH/DCM) to give the desired material (231 mg, 100% yield). ESMS: m/z 333 (M+H)⁺. Stage 2- Cyclopentyl 5-[3-(1-{[2-amino-5-(1H-pyrazol-4-yl)pyridin-3-yl]oxy}ethyl)- 2,4-dichlorophenoxy]-N-(ferf-butoxycarbonyl)-L-norvalinate

3-(1-{[2-amino-5-(1 H-pyrazol-4-yl)pyridin-3-yl]oxy}ethyl)-2,4-dichlorophenol (25 mg, 0.052 mmol) and potassium carbonate (21.63 mg, 0.156 mmol) were dissolved in DMF (0.5 ml_) and allowed to stir at room temp for 5 min. Intermediate 1 (22.80 mg, 0.063 mmol) in DMF (0.5 mL) was added and the mixture heated at 50 ⁰C for 4 h. Additional potassium carbonate (21.63 mg, 0.156 mmol) was added and the mixture heated at 60 °C for a further 2 h. Tetra- butylammonium iodide (4.82 mg, 0.013 mmol) was added and the mixture heated for a further 40 h. Additional Intermediate 1 (22.80'mg, 0.063 mmol) was added and heating continued for a further 72 h. The mixture was partitioned between EtOAc (25 mL) and brine (25 mL) and the phases separated. The organic phase was washed with brine (3 x 25 mL), dried (MgSO₄) and evaporated to a pale brown oil. The residue was purified (silica gel, 5% MeOH/DCM) to give the desired material (11 mg, 34%) as a tan solid. ESMS: m/z 616(M+H)⁺

Intermediate 19: fert-Butyl 5-(4-(6-amino-5-ri-(2,6-dichloro-3-fluorophenyl) ethoxy1pyridin-3-yl>-1H-pyrazol-1-yl)-/V-f(benzyloxy)carbonvπ-L-norvalinate

A mixture of the Intermediate 5 (100 mg, 0.272 mmol) and BEMP (118 μl, 0.408 mmol) in MeCN (1 mL) was heated to 50 ⁰C for 1 h before a solution of Intermediate 3 (316 mg, 0.817 mmol) in MeCN (0.5 mL) was added. The mixture continued to stir at this temperature for 18 h. More Intermediate 3 (100 mg, 0.272 mmol) and BEMP (120 μl, 0.410 mmol) were added and the reaction stirred for 24 h at 50 ⁰C. Further Intermediate 3 (100 mg, 0.272 mmol) and BEMP (120 μl, 0.410 mmol) were added and the reaction stirred for a further 24 h at 50 ⁰C. The mixture was allowed to cool to RT, and then partitioned between EtOAc (5 ml_) and water (2 ml_). The organic phase was washed with brine (2 x 2 mL), dried (MgSO₄) and evaporated to yield the crude product which was purified by chromatography (silica gel: 40% iso-hexane/EtOAc) to give the title intermediate (68 mg, 37% yield) as a colourless oil. ESMS: m/z 672 (M+H)⁺.

Intermediate 20: ferf-Butyl (2S)-4-(4-(6-amino-5-ri-(2,6-dichloro-3-fluoro phenyl) ethoxy1pyridin-3-yl)-1/y-pyrazol-1-yl)-2-(f(benzyloxy)carbonvnamino)butanoate

Intermediate 5 (70 mg, 0.191 mmol) was dissolved in DCM (2 mL). To this was added Intermediate 2 (85 mg, 0.230 mmol) followed by 2-ferf-Butylimino-2-diethylamino-1 ,3- dimethyl-perhydro-1 ,3,2-diazaphosphorine (83 μl_, 0.285 mmol). The reaction was allowed to stir at 4O⁰C overnight. The mixture was pre-absorbed onto silica and purified by column chromatography (1 :1 EtOAc/heptane) to afford the desired product as a white solid (120 mg, 90% yield).. ESMS: m/z 658 [M+H]⁺. 1 H NMR (300 MHz, CD3OD) δ:7.78 (1 H, s), 7.69 (1H, s), 7.57 (1H, s), 7.46 (1 H, dd J=4.9, 8.9Hz), 7.33-7.29 (5H, m), 7.24 (1 H, t J=8.5), 6.94 (1H, d J=1.4), 6.17 (1H, dd, J=6.6, 13.2Hz), 5.32 (2H, s), 4.29 (2H, t J=6.9Hz), 3.30-3.26 (4H, m), 1.88 (2H, d J=6.7Hz), 1.47 (9H, s).

Intermediate 21 : ferf-Butyl 5-f4-(4-f6-amino-5-ri-(2,6-dichloro-3-fluoro phenyl) ethoxy1pyridin-3-yl>-1H-pyrazol-1-vπpiperidin-1-vn-Λ/-f(benzyloxy)carbonvn-L- norvalinate

Intermediate 6 (80 mg, 0.178 mmol) was dissolved in DMF (2 mL). To this was added intermediate 3 (205 mg, 0.533 mmol) followed by triethylamine (250 μl_, 1.78 mmol). The reaction was allowed to stir at 40°C overnight. The mixture was diluted with water (20 mL) and EtOAc (20 mL), the organic layer washed with brine (20 mL), dried (MgSO₄) and concentrated. The crude product was purified by column chromatography (1 :1 EtOAc/heptane) to afford the desired product as a white solid (70 mg, 52% yield). ESMS: m/z 755 [M+H]+. 1H NMR (300 MHz, CDCI₃) δ: 7.75 (1H, s), 7.56 (1 H, s), 7.44 (1 H, m), 7.32-7.30 (5H, m), 7.06 (1 H, ddd J=3.4, 8.1 , 10.3Hz), 6.88-6.79 (2H, m), 6.08 (1H, dd J=6.7, 13.4Hz), 5.16-5.09 (1H, m), 4.79 (2H, s), 4.29-4.26 (1H, m), 3.50 (3H, s), 3.07-2.96 (2H, m), 2.40 (2H, t J=6.4Hz) 2.16 (4H, m), 1.88 (2H, d J=6.8Hz), 1.66-1.59 (4H₁ m), 1.53 (9H, s).

Examples

Example 1: Cvclopentyl 5-r4-(4-(6-amino-5-f1-(2,6-dichloro-3-fluoro phenyl)ethoxylpyridin-3-yl>-1/y-pyra2ol-1-yl)piperidin-1-vn-L-norvalinate

Cyclopentyl 5-[4-(4-{6-amino-5-[1 -(2,6-dichloro-3-fluorophenyl) ethoxy]pyridin-3-yl}-1 H- pyrazol-1-yl)piperidin-1-yl]-Λ/-(tert-butoxycarbonyl)-L-norvalinate Intermediate 9 (148 mg, 0.202 mmol) was treated with 4M HCI in dioxane (2 ml_, 8.00 mmol). Dioxane (3 mL) and DCM (5 mL) were added to the mixture and it was stirred at room temperature for 1 h with periodic trituration. The mixture was partitioned between EtOAc (25 mL) and NaHCO₃ solution (25 mL) and the phases separated. The aqueous phase was extracted with EtOAc (25 mL) and the combined organic phases washed with brine (50 mL), dried (MgSO₄) and evaporated under reduced pressure. The residue was dissolved in DCM (10 mL) and treated dropwise with 4M HCI in dioxane. The mixture was partitioned between EtOAc (25 mL) and NaHCO₃ solution (25 mL) and the phases separated. The aqueous phase was extracted with EtOAc (25 mL) and the combined organic phases washed with brine (50 mL), dried (MgSO₄) and evaporated. Purification (silica gel, 5% MeOH/DCM, then 1% NH₃/MeOH) afforded the title compound (106 mg, 82%) as a viscous yellow oil. ESMS: m/z 634 (M+H)⁺. ¹H NMR (400 MHz, CDCI₃) δ: 7.76 (1 H, d, J=2.0 Hz), 7.56 (1H, s), 7.31 (1 H, dd, J=8.8, 4.9 Hz), 7.05 (1H, dd, J=8.8, 7.8 Hz), 6.87 (1 H, d, J=1.5 Hz), 6.08 (1 H, q, J=6.7 Hz), 5.30 (1H, s), 5.18 - 5.23 (1 H, m), 4.77 (2H, s), 4.08 - 4.16 (1 H, m), 3.40 - 3.44 (1H, m), 3.02 -3.08 (2H, m), 2.41 (2H, t, J=6.8 Hz), 2.00 - 2.19 (6H, m), 1.57 - 1.89 (18H, m). The Examples described in Table 1 were prepared in a manner analogous to Example 1.

Table 1 Example 10: 5-(4-(6-amino-5-ri-f2₁6-dichloro-3-fluorophenyl)ethoxy1pyridin-3-yl>- 1 //-pyrazol-1 -yl)-L-norvaline

Example 2 (80 mg, 0.145 mmol) was dissolved in THF (1 mL) and was treated with lithium hydroxide (17.40 mg, 0.727 mmol). Water was added dropwise until a clear solution was formed. The mixture was stirred at room temperature for 2 h. The mixture was then neutralised with AcOH. The solvents were evaporated and the resulting residue was purified by preparative HPLC to give the title compound (19 mg, 27%) as a pale yellow gum. ESMS: m/z 482 (M+H)⁺. ¹H NMR (400 MHz, DMSO-c/6) δ: 7.86 (1H, d, J=1.5 Hz), 7.73 (1H, d), 7.55 - 7.61 (1H, m), 7.52 (1H, s), 7.41 - 7.47 (1H, m), 6.88 (1H, t, J=2.0 Hz), 6.08 (1 H, q, J=6.7 Hz), 5.64 (2H, s), 4.06 (2H, t, J=6.8 Hz), 3.12 (1H, t, J=6.1 Hz), 1.78 - 1.92 (4H₁ m), 1.64 - 1.73 (1H, m), 1.48 -1.59 (1H, m).

The examples described in Table 2 were prepared in a manner analogous to the compound of Example 10.

Table 2 Example 19: tert-Butyl 5-(4-f6-amino-5-ri-(2,6-dichloro -3-fluorophenvQ ethoxy1pyridin-3-yl)-1W-pyrazoM-vO-L-nόrvalinate

Intermediate 19 (80 mg, 0.120 mmol) was dissolved in EtOAc and to this was carefully added palladium hydroxide on carbon (40 mg, 50% w/w) under an atmosphere of nitrogen. The reaction mixture was evacuated and placed under an atmosphere of H₂. This was repeated a further two times and the reaction allowed to stir under and atmosphere of H₂ for 16 hours. The reaction mixture was filtered through Celite® and concentrated to give crude product which was purified by column chromatography (0- 10% MeOH/DCM)^" to afford the product as a white solid (20 mg, 31% yield). ESMS: m/z 538 [IVHH]⁺. ¹H NMR (300 MHz, CD₃OD) δ:7.79 (1 H, s), 7.68 (1H,s), 7.55 (1 H, s), 7.46 (1 H, dd J=4.9, 9.1Hz), 7.24 (1H, t J=8.9), 6.94 (1H, d J=3.2), 6.17 (1 H, 6.6 J=13.4Hz), 4.22-4.10 (2H, m), 3.35-3.32 (4H, m), 1.88 (2H, d J=6.8Hz), 1.73-1.68 (2H, m), 1.42 (9H, s)

Example 20: ferf-Butyl (2S)-2-amino-4-(4-(6-amino-5-ri-(2,6-dichloro-3- fluorophenyl)ethoxy1pyridin-3-yl}-1//-pyrazol-1-vπbutanoate

Procedure as in Example 19 starting with intermediate 20

(47 mg, 39% yield). ESMS: m/z 524 [M+H]⁺. 1 H NMR (300 MHz, CD3OD) δ:7.78 (1 H, s), 7.69 (1 H, s), 7.57 (1 H, s), 7.46 (1 H, dd J=4.9, 8.9Hz), 7.24 (1 H, t J=8.5), 6.94 (1 H, d J=1.4), 6.17 (1 H, dd, J=6.6, 13.2Hz), 4.29 (2H, t J=6.9Hz), 3.30-3.26 (4H, m), 1.88 (2H, d J=6.7Hz), 1.47 (9H, s)

Example 21: tert-Butyl 5-r4-(4-(6-amino-5-ri-(2,6-dichloro-3-fluorophenyl) ethoxy1pyridin-3-yl>-1A/-pyrazol-1-yl)piperidin-1-vn-L-norvalinate

Procedure as in Example 19 starting with intermediate 21

(37 mg, 64% yield). ESMS: m/z 621 [M+H]+. 1 H NMR (300 MHz, CD3OD) δ: 7.84 (1H, s), 7.69 (1H, s), 7.58 (1H, s), 7.48 (1H, dd J=4.7, 8.7Hz), 7.25 (1H, t J=8.3), 6.95 (1H, d J=1.5), 6.23-6.17 (1H, m), 4.27 (1H, m), 3.70 (1H, m), 3.32 (3H, s), 3.18-3.16 (2H, m), 2.55 (2H, t J=6.4Hz) 2.16 (4H, m), 1.89 (2H, d J=6.8Hz), 1.53 (9H, s) 1.30 (4H, m). Measurement of Biological Activity

Broken Cell Carboxylesterase Assay

Any given compound of the present invention wherein R7 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, NaC1 130 mM, CaCl₂ 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 μM

Aprotinin 0.1 μM

E64 8 μM

Pepstatin 1.5 μM

Bestatin 162 μM

Chymostatin 33 μM

After clarification of the cell homogenate by ceηtrifugation at 525 g for 10 min, the resulting supernatant was used as a source of esterase activity and was stored at -8O⁰C 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 μg / 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 25⁰C. At zero time the ester (substrate) was then added at a final concentration of 2.5 μM 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.

The table below presents data showing that several amino acid ester motifs, conjugated to various intracellular enzyme inhibitors by several different linker chemistries are all hydrolysed by intracellular carboxyesterases to the corresponding acid.

of

WO2006117548

c-Met Kinase Activity

The ability of compounds to inhibit c-Met kinase activity was measured in an assay performed by Invitrogen (Paisley, UK). The Z'-LYTE™ biochemical assay employed a fluorescence-based, coupled-enzyme format and was based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate was labelled with two fluorophores — one at each end — that make up a FRET pair. In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single serine or threonine residue in a synthetic FRET-peptide. In the secondary reaction, a site-specific protease recognizes and cleaves non-phosphorylated FRET-peptides. Phosphorylation of FRET-peptides suppresses cleavage by the Development Reagent. Cleavage disrupts FRET between the donor (i.e. coumarin) and acceptor (i.e. fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET. A radiometric method, which calculates the ratio (the Emission Ratio) of donor emission to acceptor emission after excitation of the donor fluorophore at 400nm, was used to quantitate the reaction progress.

The final 10μL Kinase Reaction consisted of 1.3-10.Ong MET (cMet), 2μM Tyr 06 Peptide and ATP in 5OmM HEPES pH 7.5, 0.01% BRIJ-35, 1OmM MgCI₂, 1mM EGTA. The assay was performed at an ATP concentration at, or close to, the Km. After the 60 minute Kinase Reaction incubation at room temperature, 5μl_ of a 1 :64 dilution of Development Reagent was added. The assay plate was incubated for a further 60 minutes at room temperature and read on a fluorescence plate reader.

Duplicate data points were generated from a 1/3 log dilution series of a stock solution of test compound in DMSO. Nine dilutions steps were made from a top concentration of 1OmM, and a 'no compound' blank is included. Data was collected and analysed using XLfit software from IDBS. The dose response curve was curve fitted to model number 205 (sigmoidal dose-response model). From the curve generated, the concentration giving 50% inhibition was determined and reported.

Cell inhibition Assay (SNU-5)

Cells were seeded in 96W tissue culture plates (1 well = 32mm²) at a density of 1750 cells per well in 50μl of the appropriate culture medium (see below). 24hrs later 150μl of the compound prepared in the same medium was added as 3 fold dilutions to give final concentrations in the range 4.6nM-10,000nM (n=6 for each concentration). The plates were then incubated at 37⁰C, 5% CO₂ for 72hrs. Cell proliferation was assessed by spiking each well for 3 hours with 0.4μCi 3H-Methyl Thymidine (1mCi/mL stock Amersham TRA120). The results were calculated as a percentage of vehicle response and IC50 values represent the concentration of compound that inhibited the vehicle response by 50%.

SNU-5 Culture Medium - Iscoves (Sigma cat no I-3390) plus 20% heat inactivated fetal calf serum (Hyclone SH30071 Thermo Fischer Scientific) containing 2mM Glutamine (Sigma cat no G-7513) and 100 u/mL penicillin and Streptomycin Sulphate (Sigma Cat no P-0781).

Biological Data

IC50 values were allocated to one of three ranges as follows:

Range A: IC50 < 10OnM

Range B: 10OnM < IC50 <1000nM

Range C: IC50 >1000nM

NT = not tested

The results obtained for compounds of the Examples herein are given in the table below.

Claims

Claims:

1. A compound of formula (I), or a salt, N-oxide, hydrate or solvate thereof:

wherein X is -N= or -CH=;

ring A is optionally substituted mono- or bi-cyclic aryl or heteroaryl having from 5 to 12 ring atoms;

ring B is (i) optionally substituted phenyl, (ii) optionally substituted monocyclic heterocyclic having 5 or 6 ring atoms and having one or two nitrogens as the sole ring heteroatoms, (iii) optionally substituted bicyclic ring system of 9 or 10 ring atoms having one two or three ring nitrogens and optionally a ring oxygen as the sole ring heteroatoms;

R₂ is selected from hydrogen, halogen, (C₁-CeJaIkVl, (C₂-C₆)alkenyl, (C₃-C₆)cycloalkyl, and mono- or bi-cyclic heterocyclic having from 5 to 12'ring atoms,

R₃ and R₄ are (a) independently selected from hydrogen, halogen, (Ci-C₆)alkyl, (C₂- C₆)alkenyl, (C₃-C₆)cycloalkyl, and mono- or bi-cyclic heterocyclic having from 5 to 12 ring atoms; or (b) R₃ and R₄ taken together with the carbon to which they are attached form a (C₃-C₆)cycloalkyl ring; or (c) one of R₃ and R₄ is as defined in case (a) while the other is a divalent radical selected from -(CH₂)₃- or -(CH₂)₄- in which one or two non-adjacent carbons are replaced by oxygen, sulfur or-NR_a- wherein R_a is hydrogen or (C₁-C₃) alkyl;

R₅ is hydrogen and R₆ is a radical of formula (IA), or R₆ is hydrogen and R₅ is a radical of formula (IA); -(Z¹)_w-(CH₂)_rL¹-Y¹-CH(R₇)-NHR₈ (IA) wherein

Z¹ represents an optionally substituted divalent mono- or bi-cyclic carbocyclic or heterocyclic radical having from 3 to 12 ring atoms;

w and z are independently 0 or 1 ;

Y¹ is a bond, -(C=O)-, -S(O₂)-, -(C=O)NR₉-, -NR₉(C=O)-, -S(O₂)NR₉-, -NR₉S(O₂)-, or - NR₉(C=O)NR₉-, wherein each Rg is independently hydrogen or optionally substituted (d-C_β)alkyl,

L¹ is a divalent radical of formula -(Alk¹)_m(Q)_π(Alk²)_p- wherein m, n and p are independently O 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 O, a divalent radical of formula -Q¹-X²- wherein X² is -0-, -S- or NR^A- wherein R^A is hydrogen or optionally substituted C₁-C₃ alkyl, and Q¹ is an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5 - 13 ring members,

AIk¹ and AIk² independently represent optionally substituted divalent C₃-C₇ cycloalkyl radicals, or optionally substituted straight or branched, CrCβ alkylene, C₂-C₆ alkenylene, or C₂-Cβ alkynylene radicals which may optionally contain or terminate in an ether (-0-), thioether (-S-) or amino (-NR^A-) link wherein R^A is hydrogen or optionally substituted (CrC₃)alkyl;

R₇ is a carboxylic acid group (-COOH), or an ester group which is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group; and

R₈ is hydrogen; or optionally substituted (Ci-C₆)alkyl, (C₃-C₇)cycloalkyl, aryl or heteroaryl Or -(C=O)R₉, -(C=O)OR₉, or -(C=O)NR₉ wherein R₉ is hydrogen or optionally substituted (CrCe)alkyl.

2. A compound as claimed in claim 1 wherein ring B is of formula (NA) or (NB):

3. A compound as claimed in claim 2 wherein the R₅ group is substituted on the ring NH.

4. A compound as claimed in any of the preceding claims wherein ring A is optionally substitued phenyl.

5. A compound as claimed in claim 4 wherein optional substituents in the phenyl ring are selected from chloro fluoro, methoxy, ethoxy, methyl, trifuoromethyl, trifluoromethoxy, nitrile, methylamino, ethylamino, dimethylamino, diethylamino, and cyclopropyl.

6. A compound as claimed in any of the preceding claims wherein R₂ is hydrogen.

7. A compound as claimed in any of the preceding claims wherein R₃ is methyl and R₄ is hydrogen.

8. A compound as claimed in any of the preceding claims wherein R₆ is hydrogen and R₅ is a radical of formula (1) or (2):

(CH₂)₂-LJ-Y¹-CH(R₇)-NH(R₈) _{(1 )}

-j-N N— (CH₂)_Z-LJ-Y¹-CH(R₇)-NH(R₈) (2)

9. A compound as claimed in any of claims 1 to 7 wherein R₆ is hydrogen and R₅ is attached to a ring carbon in ring B, and R₅ has the formula (R₈)NH-CH(R₇)-(CH₂)_{2 or3}-NH- or (R₈)NH-CH(R7)-(CH₂)_{2 θ}r3-O-.

10. A compound as claimed in any of claims 1 to 7 wherein, in formula (IA), -Y¹-L¹- (CH₂)_Z-(Z₁)_W- is -CH₂CH₂-,-CH₂CH₂CH₂-, Or -CH₂CH₂CH₂CH₂-.

11. A compound as claimed in any of claims 1 to 7 wherein R₅ is hydrogen and R₆ is a radical of formula (IA) as defined in claim 1 wherein w and z are each 0, Y¹ is a bond, and L¹ is -CH₂O-, -CH₂CH₂O- Or -CH₂CH₂CH₂O-.

12 A compound of formula (II) or a salt, N-oxide, hydrate or solvate thereof:

wherein R₅ is a radical of formula (IA) as defined in claim 1 , claim 8, claim 9 or claim 10.

13. A compound as claimed in any of the preceding claims wherein R₇ is of formula

-(C=O)ORi₂ wherein Ri₂ is R₁₃Ri₄R₁₅C- wherein

(i) R₁₃ is hydrogen or optionally substituted (Ci-C3)alkyl-(Z¹)_a-[(CrC3)alkyl]_b-, (C₂-C₃)alkenyl-(Z¹)_a-[(Ci-C₃)alkyl]_b- or phenyl-(Z¹)_a-[(CrC₃)alkyl]_b-, wherein a and b are independently 0 or 1 and Z¹ is -O-, -S-, or -NRi₆- wherein R₁₆ is hydrogen or (C_rC₃)alkyl; and R₁₄ and Ri₅ are independently hydrogen or (C_rC₃)alkyl-; or

(ii) R₁₃ is hydrogen or optionally substituted R₁₇Ri₈N-(C_rC₃)alkyl- wherein R₁₇ is hydrogen, (CrC₃)alkyl or phenyl, and Ri₈ is hydrogen or (Ci-C₃)alkyl; or Ri7 and Ri₈ 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 Ri₄ and Ri₅ are independently hydrogen or (CrC₃)alkyl-;or

(iii) Ri₃ and Ri₄ 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, and Ri₅ is hydrogen.

14. A compound as claimed in any of claims 1 to 12 wherein R₇ is a 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, or morpholinoethyl ester group..

15. A compound as claimed in any of claims 1 to 12 wherein R₇ is a cyclopentyl or tert-butyl ester group.

16. A compound as claimed in any of the preceding claims wherein R₈ is hydrogen.

17. A compound as claimed in claim 1 selected from the group consisting of: cyclopentyl 5-[4-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro phenyl)ethoxy]pyridin-3-yl}-1 H- pyrazol-1 -yl)piperidin-1 -yl]-L-norvalinate,

cyclopentyl 5-(4-{6-amino-5-[1 -(2,6-dichloro -3-fluorophenyl)ethoxy]pyridin-3-yl}-1 H- pyrazol-1 -yl)-L-norvalinate.

5-(4-{6-amino-5-[1 -(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-1 H-pyrazol-1 -yl)-L- norvaline,

tert-Butyl 5-(4-{6-amino-5-[1 -(2,6-dichloro -3-fluorophenyl)ethoxy]pyridin-3-yl}-1 H- pyrazol-1 -yl)-L-norvalinate,

ferf-Butyl (2S)-2-amino-4-(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3- yl}-1 H-pyrazol-1-yl)butanoate tert-Butyl 5-[4-(4-{6-amino-5-[1 -(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-1 H- pyrazol-1 -yl)piperidin-1 -yl]-L-norvalinate,

and salts, N-oxides, solvates and hydrates thereof.

18. A pharmaceutical composition comprising a compound as claimed in any of the preceding claims together with one or more pharmaceutically acceptable carriers and/or excipients.

19. The use of a compound as claimed in any of claims 1 to 17 in the manufacture of a composition for treatment of hyperproliferative disease, disease associated with an increased invasive potential of cells, minimal residual disease, or for modulation of angiogenesis.

20. A method of treatment of hyperproliferative disease, disease associated with an increased invasive potential of cells, minimal residual disease, or of modulation of angiogenesis comprising administering to a subject an effective amount of a compound as claimed in any of claims 1 to 17

21. A use as claimed in claim 19 or a method as claimed in claim 20 for treatment of cancers or tumour metastases, diabetic retinopathy, age-related macular degeneration or rheumatoid arthritis.