WO2008053136A1 - 2-(hetero-)aryl,4-carbonyl substituted pyrazole derivatives as inhibitors of p38 mitogen-activated protein kinase - Google Patents

Abstract

Compounds of formula (I) are inhibitors of p38 MAP kinase activity, and are useful in the treatment of, inter alia, inflammatory and autoimmune disease wherein Ring A is aryl, heteroaryl or heterocyclyl of 5-13 atoms; Ring B is optionally substituted aryl or heteroaryl of 5-13 atoms; Z is (a) a radical of formula -(CH2)z-X1-L1-Y- NHCHR1R2 or (b) a radical of formula -(CH2)z-Y1-L1-R wherein R1R2CHNH- and R are respectively N- and C-linked amino acid or amino acid ester groups as defined in the description, and -Y-L1-X1-(CH2)z- and -L1-Y1-(CH2)z- are linker radicals as defined in the description; R7 is hydrogen or -C(=O)R' where R' is hydrogen, (C1-C6)alkyl, (C3-C6)cycloalkyl or (C1-C6)haloalkyl; R8 is hydrogen or (C1C6)alkyl; R9 is hydrogen, halogen, hydroxyl, (C1-C6)alkoxy, (C1-C6)alkyl; R18 is hydrogen, halogen, hydroxyl, (C1C6)alkoxy (C1-C6)alkyl, - NRaRb where Ra and Rb are hydrogen or (C1-C6)alkyl, or optionally substituted aryl, heteroaryl or heterocyclyl or Ra and Rb when taken together with the nitrogen to which they are attached form a cyclic amino group of up to 6 ring atoms; R19 is hydrogen, halogen, (C1C6)alkoxy, or (C1C6)alkyl.

Description

2-(HETERO-)ARYL,4-CARBONYL SUBSTITUTED PYRAZOLE DERIVATIVES AS INHIBITORS OF P38 MITOGEM-ACTIVATED PROTEIN KINASE

This invention relates to a series of amino acid esters, to compositions containing them, to processes for their preparations and to their uses in medicine as p38 mitogen-activated protein (MAP) kinase inhibitors for the treatment of autoimmune and inflammatory diseases including chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis, diabetes, atopic dermatitis, graft versus host disease, systemic lupus erythematosus.

Background to invention

Inappropriate activation of leukocytes including monocytes, macrophages and neutrophils leading to the production of elevated levels of cytokines such as TNF-α, IL1-β and IL-8, is a feature of the pathogenesis of several inflammatory diseases including rheumatoid arthritis, ulcerative colitis, Crohn's disease, chronic obstructive pulmonary disease (COPD), asthma and psoriasis. The production of cytokines by inflammatory cells is a result of response to a variety of external stimuli, leading to the activation of a number of intracellular signalling mechanisms. Prominent amongst these is the mitogen-activated protein kinase (MAPK) superfamily consisting of highly conserved signalling kinases that regulate cell growth, differentiation and stress responses. Mammalian cells contain at least three families of MAPKs: the p42/44 extracellular signal-regulated kinase (ERK) MAPKs, c-Jun NH2-terminal kinases (JNKs) and p38 MAPK (also termed p38a/Mpk2/RK/SAPK2a/CSBP1/2). p38 MAPK was first cloned following its identification as a kinase that is tyrosine phosphorylated after stimulation of monocytes by lipopolysaccharide (LPS) [Han et al, Science 1994,265,808]. Additional homologues of mammalian p38 have been described and include p38β [Jiang et al, J.Biol.Chem, 1996, 271 , 17920], p38γ [Li et al, Biochem. Biophys. Res. Commun., 1996, 228, 334] and p38δ [Jiang et al, J.Biol.Chem. 1997, 272, 30122]. While p38α and p38β are ubiquitously expressed, p38γ is restricted primarily to skeletal muscle and p38δ is predominantly expressed in lung and kidney.

The release of cytokines by host defence cells and the response of leukocytes to cytokines and other pro-inflammatory stresses are to varying extent regulated by p38 MAPK [Cuenda et al FEBS Lett, 1995, 364, 229-233]. In other cell types, p38 MAPK controls stress responses such as the production of IL-8 by bronchial epithelial cells stimulated by TNF-α, and the up-regulation of the cell adhesion molecule ICAM-1 in LPS-stimulated endothelial cells. Upon activation, via dual phosphorylation of a TGY motif by the dual specificity kinases MKK3 and MKK6, p38 MAPK exerts its effects through phosphorylation of transcription factors and other kinases. MAP kinase-activated protein kinase-2 (MAPKAPK-2) has been identified as a target for p38 phosphorylation. It has been demonstrated that mice [Kotlyarov et al Nat. Cell Biol. 1999, 1 , 94-97] lacking MAPKAPK-2 release reduced levels of TNF-α, IL- 1 β, IL-6, IL-10 and IFN-γ in response to LPS/galactosamine mediated endotoxic shock. The regulation of the levels of these cytokines as well as COX-2 is at the mRNA level. TNF-α levels are regulated through translational control via AU-rich elements of the 3'-UTR of TNF- α mRNA, with MAPKAPK-2 signalling increasing TNF-α mRNA translation. MAPKAPK-2 signalling leads to increased mRNA stability for COX-2, IL-6 and macrophage inflammatory protein. MAPKAPK-2 determines the cellular location of p38 MAPK as well as transducing p38 MAPK signalling, possessing a nuclear localisation signal at its carboxyl terminus and a nuclear export signal as part of its autoinhibitory domain [Engel et al, EMBO J. 1998, 17, 3363-3371]. In stressed cells, MAPKAPK-2 and p38 MAPK migrate to the cytoplasm from the nucleus, this migration only occurring when p38 MAPK is catalytically active. It is believed that this event is driven by the exposure of the MAPKAPK-2 nuclear export signal, as a result of phosphorylation by p38 MAPK [Meng et al, J.Biol.Chem. 2002,277, 37401-37405]. Additionally p38 MAPK either directly or indirectly lead to the phosphorylation of several transcription factors believed to mediate inflammation, including ATF1/2 (activating transcription factors 1/2), CHOP-10/GADD-153 (growth arrest and DNA damage inducible gene 153), SAP-1 (serum response factor accessory protein-1 ) and MEF2C (myocyte enhancer factor-2) [Foster et al, Drug News Perspect. 2000, 13, 488-497].

It has been demonstrated in several instances that the inhibition of p38 MAPK activity by small molecules, is useful for the treatment of several disease states mediated by inappropriate cytokine production including rheumatoid arthritis, COPD, asthma and cerebral ischemia. This modality has been the subject of several reviews [Salituro et al, Current Medicinal Chemistry, 1999, 6, 807-823 and Kumar et al, Nature Reviews Drug Discovery 2003, 2, 717-726]

Inhibitors of p38 MAPK have been shown to be efficacious in animal models of rheumatoid arthritis, such as collagen-induced arthritis in rat [Revesz et al, Biorg. Med. Chem. Lett., 2000, 10, 1261-1364] and adjuvant-induced arthritis in rat [Wadsworth et al, J. Pharmacol. Exp.Ther., 1999, 291 , 1685-1691]. In murine models of pancreatitis-induced lung injury, pre-treatment with a p38 MAPK inhibitor reduced TNF-α release in the airways and pulmonary oedema [Denham et al, Crit. Care Med., 2000, 29, 628 and Yang et al, Surgery, 1999, 126, 216]. Inhibition of p38 MAPK before ovalbumin (OVA) challenge in OVA- sensitized mice decreased cytokine and inflammatory cell accumulation in the airways in an allergic airway model of inflammation, [Underwood et al, J. Pharmacol. Exp.Ther., 2000,293, 281]. Increased activity of p38MAP kinase has been observed in patients suffering from inflammatory bowel disease [Waetzig et al, J. Immunol, 2002,168, 5432-5351]. p38 MAPK inhibitors have been shown to be efficacious in a rat models of cardiac hypertrophy [Behr et al, Circulation, 2001 , 104, 1292-1298] and cerebral focal ischemia [Barone et al, J.Pharmacol.Exp.Ther., 2001 ,296,312-321] .

Brief description of the invention

This invention makes available a group of pyrazole compounds which are potent and selective inhibitors of p38 MAPK (p38oc,Pjand δ) and the isoforms and splice variants thereof especially p38α, p38β and p38β2. The compounds are thus of use in medicine, for example in the treatment and prophylaxis of immune and inflammatory 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 p38 MAP kinase activity of the compound is prolonged and enhanced within the cell. The compounds of the invention are related to the p38 MAP kinase inhibitors encompassed by the disclosures in International Patent Application WO0121591 but differ therefrom in that the present compounds have the amino acid ester motif referred to above.

Detailed description of the invention

The present invention provides a compound of formula (I) or a solvate, Λ/-oxide, hydrate or a pharmaceutically acceptable salt thereof:

(D

wherein:

Ring A is an optionally substituted aryl, heteroaryl or heterocyclyl ring of 5-13 atoms;

Ring B is an optionally substituted aryl or heteroaryl ring of 5-13 atoms;

Z is (a) a radical of formula -(CH₂)_Z-X¹-L¹-Y- NHCHR₁R₂ or (b) a radical of formula -(CH₂)_z-Y¹-L¹-R, wherein:

R is a radical of formula (X) or (Y)

R₁ is a carboxylic acid group (-COOH), or an ester group which is hydrolysable by one or more intracellular esterase enzymes to a carboxylic acid group;

R₆ is hydrogen; or optionally substituted Ci-C₆ alkyl, C₃-C₇ cycloalkyl, aryl or heteroaryl or -(C=O)R₃, -(C=O)OR₃, or -(C=O)NR₃ wherein R₃ is hydrogen or optionally substituted (CrC₆)alkyl.

R₂ is the side chain of a natural or non-natural alpha amino acid;

Y is a bond, -C(=0)-, -S(=0)₂-, -C(=0)0-, -C(=0)NR₃-, -C(=S)-NR₃ , -C(=NH)-NR₃ or -S(=O)₂NR₃- wherein R₃ is hydrogen or optionally substituted C₁-C₆ alkyl;

Y¹ is a bond, -C(=0)-, -S(=0)₂-, -C(=0)0-, -0C(=0)-, -C(=0)NR₃-,

-NR₃(C=O)-, -S(=O)₂NR₃-, -NR₃S(=O)₂-, or -NR₃(C=O)NR₄-, wherein R₃ and R₄ are 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 0, a divalent radical of formula -Q¹-X²- wherein X² is -O-, -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, C₁-C₆ 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 C₁-C₃ alkyl;

X¹ is a bond, -C(=O)-; or -S(=O)₂-; -NR₄C(=O)-, -C(=O)NR₄-,

-NR₄C(=O)-NR₅- , -NR₄S(=O)₂-, or -S(=O)₂NR₄- wherein R₄ and R₅ are independently hydrogen or optionally substituted C₁-C₆ alkyl; and

z is 0 or 1.

R₇ is hydrogen or -C(=O)R' where R' is hydrogen, (Ci-C₆)alkyl, (C₃-C₆)cycloalkyl or (C₁- C₆)haloalkyl;

R₈ is hydrogen or (d-C^alkyl;

R₉ is hydrogen, halogen, hydroxyl, (C_rC₆)alkoxy, (C₁-C₆)alkyl;

R₁₈ is hydrogen, halogen, hydroxyl, (C_rC₆)alkoxy (C₁-C₆)alkyl, -NR^aR^b where R^a and R^b are hydrogen or (d-C^alkyl, or optionally substituted aryl, heteroaryl or heterocyclyl or R^aand R^b when taken together with the nitrogen to which they are attached form a cyclic amino group of up to 6 ring atoms;

R₁₉ is hydrogen, halogen, (C₁-C₆JaIkOXy, or (C_rC₆)alkyl.

In another broad aspect the invention provides the use of a compound of formula (I) as defined above, or an Λ/-oxide, salt, hydrate or solvate thereof in the preparation of a composition for inhibiting the activity p38 MAP kinase enzyme. The compounds with which the invention is concerned may be used for the inhibition of p38 MAP kinase enzyme activity in vitro or in vivo.

Pharmaceutical compositions comprising a compound of the invention together with one or more pharmaceutically acceptable carriers and excipients also form part of the invention.

In one aspect of the invention, the compounds of the invention may be used in the preparation of a composition for the treatment of autoimmune or inflammatory disease, particularly those mentioned above in which p38 MAP kinase activity plays a role.

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.

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 "(C_a-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. A "divalent phenylene, pyridinylene, pyrimidinylene, or pyrazinylene radical" is a benzene, pyridine, pyrimidine or pyrazine ring, with two unsatisfied valencies, and includes 1 ,3- phenylene, 1 ,4-phenylene, and the following:

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, (Ci-C₆)alkyl, (C_rC₆)alkoxy, hydroxy, hydroxy(Cr C₆)alkyl, mercapto, mercapto(d-C₆)alkyl, (CrC₆)alkylthio, phenyl, halo (including fluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, -COOH, -COOR^A, -C0R^A, -SO₂R^A, -CONH₂, -SO₂NH₂, -C0NHR^A, -SO₂NHR^A, -CONR^AR^B, -SO₂NR^AR^B, -NH₂, -NHR^A, -NR^AR^B, -OCONH₂, -0C0NHR^A , -OCONR^AR^B, -NHC0R^A, -NHC00R^A, -NR^BCOOR^A, -NHSO₂OR^A, -NR⁶SO₂OH, -NR^BSO₂OR^A,-NHCONH₂, -NR^ONH₂₁ -NHCONHR⁶ -NR^AC0NHR^B, -NHC0NR^AR^B or -NR^AC0NR^AR^B wherein R^A and R^B are independently a (CrC₆)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 tetrahydropyrrolyl). 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. Λ/-methyl-D-glucamine, choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, Λ/-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, benzenesulfonic, glutamic, lactic, and mandelic acids and the like.

It is expected that compounds of the invention may be recovered in hydrate or solvate form. The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.

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 R₁ 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 A

Ring A is an optionally substituted aryl or heteroaryl or heterocyclic ring or ring system of 5-

13 atoms, for example a divalent phenyl, pyridinyl, pyrimidinyl, or pyrazinyl ring radical. Currently preferred is a phenyl ring.

The ring B

Ring A is also an optionally substituted aryl, heteroaryl or heterocyclic ring or ring system of

5-13 atoms, for example a divalent phenyl, pyridinyl, pyrimidinyl, or pyrazinyl ring radical. Again a phenyl ring is currently preferred.

The substituent R₁

R₇ is hydrogen or -C(=O)R' where R' is hydrogen, (d-C₆)alkyl, such as methyl, ethyl (C₃- C₆)cycloalkyl such as cyclopropyl, cyclobutyl, or haloalkyl such as trifluoromethyl. Currently preferred is when R₇ is hydrogen. The substituent f?«

R₈ is hydrogen or optionally substituted (C_rC₆)alkyl, such as methyl, ethyl or n- or iso-propyl. Currently preferred is when R₈ is hydrogen.

The substituents RQ, R₁R and R₁Q.

R₉ is hydrogen, halogen such as chloro or fluoro, hydroxyl, (C_rC₆)alkoxy such as methoxy or ethoxy, or (CrC₆)alkyl such as methyl, ethyl or n- or iso-propyl.

Ri₈ is hydrogen, halogen such as chloro or fluoro, hydroxyl, (Ci-C₆)alkoxy such as methoxy, (CrC₆)alkyl such as methyl, ethyl or n- or iso-propyl., -NR^aR^b where R^aand R^b are independently hydrogen or (Ci-C₆)alkyl such as methyl, ethyl or n- or iso-propyl, or optionally substituted aryl such as phenyl, or heterocyclic such as pyridyl pyrimidinylor imidazolyl, or R^a and R^b together with the nitrogen to which they are attached form a cyclic amino group such as piperidinyl, morpholinyl or piperazinyl Optional substituents in R₁₈ when optionally substituted (CrC₆)alkyl, aryl, heteroaryl or heterocyclyl include, for example halogen, such as chloro, bromo and fluoro.

Rig is hydrogen, halogen such as chloro or fluoro, hydroxyl, (CrC_δJalkoxy such as methoxy, (d-CeJalkyl such as methyl, ethyl or n- or iso-propyl., -NR^aR^b where R^a and R^b are independently hydrogen or (C_rC₆)alkyl such as methyl, ethyl or n- or iso-propyl, or optionally substituted aryl such as phenyl, or heterocyclic such as pyridyl pyrimidinylor imidazolyl, or R^a and R^b together with the nitrogen to which they are attached form a cyclic amino group such as piperidinyl, morpholinyl or piperazinyl Optional substituents in R₁₈ when optionally substituted (CrCβJalkyl, aryl, heteroaryl or heterocyclyl include, for example halogen, such as chloro, bromo and fluoro.

Specific classes of compounds of the invention include those of formula (IA) or (IB):

wherein R₁₉ is hydrogen, fluoro or chloro, and wherein the other variables are as defined above and further discussed below.

The group Z

(a) Z is a radical of formula -(CHJ₇-X¹ -L¹ -Y- NHCHR₁R₇

In this class of compound of the invention, the amino acid or amino acid ester group -NHCHR₁R₂ is linked to the rest of the molecule via its amino nitrogen. In other words, the amino acid or amino acid ester group is N-linked.

The group R₁ in Z case (a)

R₁ is a carboxylic acid group or an ester group which 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 ester. 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 p38 inhibitor. 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 modulator 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 R₁ include those of formula -(C=O)OR₁₀ wherein R₁₀ is R₁₁Ri₂R₁₃C- wherein (i) R₁₁ is hydrogen or optionally substituted (C₁-C₃)alkyl-(Z¹)_a-[(C₁-C₃)alkyl]_b-₎ (C₂- C₃)alkenyl-(Z¹)_a-[(CrC₃)alkyl]_b- or phenyl-(Z¹)_a-[(Ci-C₃)alkyl]_b-, wherein a and b are independently 0 or 1 and Z¹ is -O-, -S-, or -NR₁₄- wherein R₁₄ is hydrogen or (C₁- C₃)alkyl; and R₁₂ and R₁₃ are independently hydrogen or (C_rC₃)alkyl-;

(ii) R₁₁ is hydrogen or optionally substituted R₁₅R₁₆N-(CrC₃)alkyl- wherein R₁₅ is hydrogen, (d-C₃)alkyl or phenyl, and R₁₆ is hydrogen or (Ci-C₃)alkyl; or R₁₅ and R₁₆ 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 R₁₂ and R₁₃ are independently hydrogen or (CrC₃)alkyl-;or

(iii) R₁₁ and R₁₂ 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 R₁₃ is hydrogen.

Within these classes, R₁₀ may be, for example, methyl, ethyl, n- or iso-propyl, n-, sec- or tert- butyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- or 4-pyridylmethyl, N-methylpiperidin-4-yl, tetrahydrofuran-3-yl, methoxyethyl, indanyl, norbonyl, dimethylaminoethyl, or morpholinoethyl. Currently preferred is where R₁₀ is cyclopentyl.

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 Carraro, Curr Drug Targets lnflamm Allergy, 2005, 3-8 ). Hence agents that selectively target macrophage cell proliferation and function 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 cells that express hCE-1 , in particular, macrophages and other cells derived from the myelo-monocytic lineage such as monocytes, osteoclasts and dendritic cells. This is based on the observation that the way in which the esterase motif is linked to the inhibitor determines whether it is hydrolysed by all three human carboxylesterases or just by hCE-1 , and hence whether or not it accumulates in different cell types. Specifically it has been found that macrophages and other cells derived from the myelo-monocytic lineage, both normal and cancerous, contain the human carboxylesterase hCE-1 whereas other cell types do not. In the general formula (I) when the nitrogen of the esterase motif R₁CH(R₂)NH- is not directly linked to a carbonyl (-C(=O)-), i.e. when Y 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 selectively accumulate in macrophage-related cells.

The amino acid side chain R? in Z case (a)

Subject to the requirement that the ester group Ri be hydrolysable by intracellular carboxylesterase enzymes, the identity of the side chain group R₂ is not critical for non-macrophage selective compounds. For macrophage selective compounds, side chains such as valine, cyclohexylglycine, t-butylserine, t-butylcysteine, proline, phenylalanine, leucine and phenylglycine are preferred.

Examples of amino acid side chains include

(C₁-CeJaIkVl₁ phenyl, 2,- 3-, or 4-hydroxyphenyl, 2,- 3-, or 4-methoxyphenyl, 2,- 3-, or 4-pyridylmethyl, benzyl, phenylethyl, 2-, 3-, or 4-hydroxybenzyl, 2,- 3-, or 4-benzyloxybenzyl, 2,- 3-, or 4- (d-CeJalkoxybenzyl, and benzyloxy(Ci-C₆alkyl)- groups;

the characterising group of a natural α amino acid, in which any functional group may be protected;

groups -[AIk]_nR₁₆ where AIk is a (C_rC₆)alkyl or (C₂-C₆)alkenyl group optionally interrupted by one or more -O-, or -S- atoms or -N(R₁₇)- groups [where R₁₇ is a hydrogen atom or a (C₁- C₆)alkyl group], n is 0 or 1 , and R₁₆ is an optionally substituted cycloalkyl or cycloalkenyl group; a benzyl group substituted in the phenyl ring by a group of formula -OCH₂COR₁₈ where R₁₈ is hydroxyl, amino, (Ci-C₆)alkoxy, phenyKCrCβJalkoxy, (CrC₆)alkylamino, di((C_r C₆)alkyl)amino, pheny^CrC^alkylamino, the residue of an amino acid or acid halide, ester or amide derivative thereof, said residue being linked via an amide bond, said amino acid being selected from glycine, α or β alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histidine, arginine, glutamic acid, and aspartic acid;

a heterocyclic(C₁-C₆)alkyl group, either being unsubstituted or mono- or di-substituted in the heterocyclic ring with halo, nitro, carboxy, (CrC₆)alkoxy, cyano, (CrC₆)alkanoyl, trifluoromethyl (C_rC₆)alkyl, hydroxy, formyl, amino, (C_rC₆)alkylamino, di^d-CeJalkylamino, mercapto, (CrC₆)alkylthio, hydroxy(Ci-C₆)alkyl, mercapto(C₁-C₆)alkyl or (C₁- C₆)alkylphenylmethyl; and

a group -CR₃RbR₀ in which:

each of R₃, R_b and R_c is independently hydrogen, (d-C₆)alkyl, (C₂-C₆)alkenyl, (C₂- C₆)alkynyl, phenyl(C_rC₆)alkyl, (C₃-C₈)cycloalkyl; or

R₀ is hydrogen and R_a and R_b are independently phenyl or heteroaryl such as pyridyl; or

R_c is hydrogen, (C_rC₆)alkyl, (C₂-C₆ )alkenyl, (C₂-C₆)alkynyl, phenyl(C_rC₆)alkyl, or (C₃- C₈)cycloalkyl, and R₃ and R_b together with the carbon atom to which they are attached form a 3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic ring; or

R₃, R_b and R₀ together with the carbon atom to which they are attached form a tricyclic ring (for example adamantyl); or

R₃ and R_b are each independently (CrC₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(CrC₆)alkyl, or a group as defined for R₀ below other than hydrogen, or R₃ and R_b together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic ring, and R_c is hydrogen, -OH₁ -SH, halogen, -CN, -CO₂H, (C₁- C₄)perfluoroalkyl, -CH₂OH, -Cθ₂(C_rC₆)alkyl, -O(C₁-C₆)alkyl, -O(C₂-C₆)alkenyl, -S(C₁- C₆)alkyl, -SO^-QOalkyl, -SO₂(C₁-C₆) alkyl, -S(C₂-C₆)alkenyl, -SO(C₂-C₆)alkenyl, - SO₂(C₂-C₆)alkenyl or a group -Q-W wherein Q represents a bond or -0-, -S-, -SO- or -SO₂- and W represents a phenyl, phenylalkyl, (C₃-C₈)cycloalkyl, (C₃- C₈)cycloalkylalkyl, (C₄-C₈)cycloalkenyl, (C₄-C₈)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, which group W may optionally be substituted by one or more substituents independently selected from, hydroxyl, halogen, -CN, -CO₂H, -CO₂(C₁- C₆)alkyl, -CONH₂, -CONH(C_rC₆)alkyl, -CONH(C₁-C₆alkyl)₂, -CHO, -CH₂OH, (C₁- C₄)perfluoroalkyl, -O(C_rC₆)alkyl, -S(C₁-C₆)alkyl, -SO(C₁-C₆)alkyl,

- NO₂, -NH₂, -NH(C_rC₆)alkyl, -N((C_rC₆)alkyl)₂, -NHCO(C_rC₆)alkyl, (C_rC₆)alkyl, (C₂- C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl, (C₄-C₈)cycloalkenyl, phenyl or benzyl.

Examples of particular R₂ groups include benzyl, phenyl, cyclohexylmethyl, cyclohexyl, pyridin-3-ylmethyl, tert-butoxymethyl, iso-butyl, sec-butyl, tert-butyl, 1-benzylthio-1- methylethyl, 1-methylthio-i-methylethyl, 1-mercapto-i-methylethyl, and phenylethyl. Presently preferred R₂ groups include phenyl, benzyl, iso-butyl, cyclohexyl and t- butoxymethyl.

For compounds of the invention which are to be administered systemically, esters with a slow rate of carboxylesterase cleavage are preferred, since they are less susceptible to pre- systemic metabolism. Their ability to reach their target tissue intact is therefore increased, and the ester can be converted inside the cells of the target tissue into the acid product. However, for local administration, where the ester is either directly applied to the target tissue or directed there by, for example, inhalation, it will often be desirable that the ester has a rapid rate of esterase cleavage, to minimise systemic exposure and consequent unwanted side effects. In the compounds of this invention, if the carbon adjacent to the alpha carbon of the alpha amino acid ester is monosubstituted, ie R₂ is -CH₂R^Z (R^z being the mono- substituent) then the esters tend to be cleaved more rapidly than if that carbon is di- or tri- substituted, as in the case where R₂ is, for example, phenyl or cyclohexyl.

The radical -(CH,),-X¹-L¹-Y- in Z case (a)

This radical (or bond) arises from the particular chemistry strategy chosen to link the amino acid ester motif R₁CH(R₂)NH- to the rest of the molecule. Clearly the chemistry strategy for that coupling may vary widely, and thus many combinations of the variables z, L¹, X¹ and Y are possible. The precise combination of variables 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.

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

With the foregoing general observations in mind, taking the variables making up the radical - (CH₂)_Z-X¹-L¹-Y- in turn: z may be 0 or 1 ;

In the radical L¹, examples of AIk¹ and AIk² radicals, when present, include

-CH₂-, — CH₂CH2-, — CH2CH2CH2-, — CH₂CH₂CH2CH2-, -CH=CH-,

-CH=CHCH₂-, -CH₂CH=CH-, CH₂CH=CHCH₂- C=C-, -C=CCH₂-, CH₂C=C-, and

CH₂C≡CCH₂. Additional examples of 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.

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 0, and at least one of m and p is 1 , the radical is a hydrocarbon chain (optionally substituted and perhaps having an ether, thioether or amino linkage). Presently it is preferred that there be no optional substituents in L¹. When both m and p are 0, L¹ is a divalent mono- or bicyclic carbocyclic or heterocyclic radical with 5 - 13 ring atoms (optionally substituted, but presently preferred to be unsubstituted, and perhaps linked to an adjacent atom through an ether, thioether or amino link (Note: this is when Q is -Q²-X²-, if applicable in this application). 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 (optionally substituted and perhaps having an ether, thioether or amino linkage) and a mono- or bicyclic carbocyclic or heterocyclic radical with 5 - 13 ring atoms (optionally substituted, but presently preferred to be unsubstituted, and perhaps linked to an adjacent atom through an ether, thioether or amino link.

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.

In some embodiments of the invention, L¹, m and p may be 0 with n being 1. In other embodiments, n and p may be 0 with m being 1. In further embodiments, m, n and p may be all 0. In still further embodiments m may be 0, n may be 1 with Q being a monocyclic heterocyclic radical, and p may be 0 or 1. Specifically, AIk¹ and AIk², when present, may be selected from -CH₂-, -CH₂CH₂-, and -CH₂CH₂CH₂- and Q when present may be selected from:

wherein E and G are independently CH or N.

The linkage X¹ represents a bond, -(C=O)-, -S(O₂)-, -NR₄C(=O)-, -C(=O)NR₄-, - NR₄C(=O)-NR₅- , -NR₄S(=O)₂-, or -S(=O)₂NR₄- wherein R₄ and R₅ are independently hydrogen or optionally substituted C₁-C₆ alkyl such as methyl or ethyl.

The linkage Y is a bond, -C(=0)-, -S(=0)₂-, -C(=0)0-, -Cf=O)NR₃-, -C(=S)-NR₃ , -C(=NH)-NR₃ or -S(=O)₂NR₃- wherein R₃ is hydrogen or optionally substituted Ci-C₆ alkyl such as methyl or ethyl.;

Often z will be O and X¹ and Y will each simply be a bond, so that the amino acid esterase motif R₁R₂CHNH- is linked to the ring containing X by the radical L¹ as defined and discussed above.

In particular examples compounds of the invention wherein Z case (a) applies, R₁R₂CHNH-Y-L¹X¹-(CH₂)_z- is selected from R₁R₂CHNH-CH₂-, R₁R₂CHNH-CH₂CH₂-, R₁R₂CHNH-CH₂CH₂CH₂-,. R₁R₂CHNH-CH₂-O-, R₁R₂CHNH-CH₂CH₂-O-, and R₁R₂CHNH-CH₂CH₂CH₂-O-.

In other compounds of the invention R₁R₂CHNH-Y-L¹X^(CH₂)_Z-, is selected from: R₁R₂CHNHSO₂-, R₁R₂CHNHCO-, R₁R₂CHNHCH₂-, R₁R₂CHNH(CH₂)₃O-and the following:

R₁ R₂CHNH- (CH₂)_0i1 -Y V— (CH₂)_0i1 — ( Ψ )_Oi1 -+-

R₁R₂CHNH- (CH₂)O ₁ - ( V-I)_0,,

wherein Y is C or N₁ V is C or N, and V¹ is O, S or NH. For example, RiR₂CHNH-Y- L¹X¹-(CH₂)_Z- may be:

Particular examples of the linker radical -(CH₂)_Z-X¹-L¹-Y- in Z case (a) include (i) -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, and -CH₂CH₂CH₂CH₂- in any of which a carbon is optionally substituted by hydroxy; or (ii) -OCH₂-, -OCH₂CH₂-, -OCH₂CH₂CH₂-, and -OCH₂CH₂CH₂CH₂- wherein the oxygen is linked to the ring A; or (iii) -NHCH₂-, -NHCH₂CH₂-, -NHCH₂CH₂CH₂-, and -NHCH₂CH₂CH₂CH₂- wherein the nitrogen is linked to the ring A; or (iv) a divalent cyclohexyl radical or cyclohexyloxy radical wherein the oxygen is linked to the ring A.

(b) Z /s a radical of formula -(CH₇K-V -L¹ -R

In this class of compound of the invention, the amino acid or amino acid ester group - NHCHR₁R₂ is linked to the rest of the molecule via its side chain. In these compounds the amino acid or amino acid ester group may be regarded as C-linked.

The radical R in Z case (b)

R is a radical of formula (X) or (Y)

In formula (X) and (Y), R₁ is a carboxylic acid group or an ester group which is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group, as defined and discussed above with reference to Z case (a).

The ring D in Z case (b)

When R is a group of formula (Y), examples of R include:

In formula (X) and (Y), R₁ is a carboxylic acid group or an ester group which is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group, as defined and discussed above with reference to Z case (a). Specifically Ri 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, indanyl, norbomyl, dimethylaminoethyl, or morpholinoethyl ester group. In this case (b) where Z is a radical of formula -(CH₂)_z-Y¹-L¹-R, cyclopentyl or tert butyl esters are currently preferred.

The group Re in Z case (b)

The group R₆ is present in the compounds of the invention in this case when R is a radical of formula (X)

R₆ may be optionally substituted C₁-C₆ alkyl, C₃-C₇ cycloalkyl, aryl or heteroaryl, for example methyl, ethyl, n-or isopropyl, cyclopropyl, cyclopentyl, cyclohexyl, phenyl, or pyridyl. In cases where macrophage specificity is not required, R₆ may be hydrogen or -(C=O)R⁰, wherein R^D is optionally substituted (CrC₆)alkyl such as methyl, ethyl, n-or isopropyl, or n-, iso- or sec- butyl, (C₃-C₇)cycloalkyl such as cyclopropyl, cyclopentyl, cyclohexyl, phenyl, pyridyl, thienyl, phenyl(C_rC₆ alkyl)-, thienyl(C_rC₆ alkyl)- or pyridyl(C_rC₆ alkyl)- such as benzyl, 4- methoxyphenylmethylcarbonyl, thienylmethyl or pyridylmethyl.

R₆ may also be, for example -(C=O)OR⁰, or -(C=O)NHR⁰ wherein R° is hydrogen or optionally substituted (CrC₆)alkyl such as methyl, ethyl, or n-or isopropyl.

For compounds of the invention which are to be administered systemically, esters with a slow rate of esterase cleavage are preferred, since they are less susceptible to pre-systemic metabolism. Their ability to reach their target tissue intact is therefore increased, and the ester can be converted inside the cells of the target tissue into the acid product. However, for local administration, where the ester is either directly applied to the target tissue or directed there by, for example, inhalation, it will often be desirable that the ester has a rapid rate of esterase cleavage, to minimise systemic exposure and consequent unwanted side effects. If a carbon atom to which the group R is attached is unsubstituted, ie R is attached to a methylene (-CH₂)- radical, then the esters tend to be cleaved more rapidly than if that carbon is substituted, or is part of a ring system such as a phenyl or cyclohexyl ring.

The radical -L¹-Ϋ-{CH,l- in Z case (b)

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

With the foregoing general observations in mind, taking the variables making up the radical - L¹-Y¹-[CH₂]_Z- in turn:

z may be 0 or 1 , so that a methylene radical linked to the rest of the molecule is optional;

Y¹ may be, for example, -NR₃-, -S-, -O-, -C(=O)NR₃-, - NR₃C(=O)-, or -C(=O)O-, wherein R₃ is hydrogen or optionally substituted C₁-C₆ alkyl such as - CH₂CH₂OH;

In the radical L¹, examples of AIk¹ and AIk² radicals, when present, include

— CH₂-, — CH₂CH₂-, — CH₂CH₂CH₂-, — CH₂CH₂CH₂CH₂-, — CH⁼CH-,

-CH=CHCH₂-, -CH₂CH=CH-, CH₂CH=CHCH₂-, -C≡C-, -C=CCH₂-, CH₂C=C-, and

CH₂C≡CCH₂. Additional examples of 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.

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 0, the radical is a hydrocarbon chain (optionally substituted and perhaps having an ether, thioether or amino linkage). Presently it is preferred that there be no optional substituents in L¹. When both m and p are 0, L¹ is a divalent mono- or bicyclic carbocyclic or heterocyclic radical with 5 - 13 ring atoms (optionally substituted). When n is 1 and at least one of m and p is 1 , L¹ is a divalent radical including a hydrocarbon chain or chains and a mono- or bicyclic carbocyclic or heterocyclic radical with 5 - 13 ring atoms (optionally substituted). When present, Q may be, for example, a divalent phenyl, naphthyl, cyclopropyl, cyclopentyl, or cyclohexyl radical, or a mono-, 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 0 with n being 1. In other embodiments, n and p may be 0 with m being 1. In further embodiments, m, n and p may be all 0. In still further embodiments m may be 0, n may be 1 with Q being a monocyclic heterocyclic radical, and p may be 0 or 1. 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)_L3NH-, - (CH₂)_1-3c(=O)NH-, CH₂C(O)O-, -(CH₂)_L3C(O)O-, -(CH₂J₄NH-,

-(CH₂)_L3S-, -(CH₂)_L3O-, and

As mentioned above, the compounds with which the invention is concerned are inhibitors of p38 MAK kinase activity, and are therefore of use in the treatment of diseases such as psoriasis, inflammatory bowel disease, Crohns disease, ulcerative colitis, chronic obstructive pulmonary disease (COPD), asthma, multiple sclerosis, diabetes, atopic dermatitis, graft versus host disease, or systemic lupus erythematosus and rheumatoid arthritis, in which p38 MAP kinase activity plays a part. For treatment of COPD, compounds of the invention wherein Z case (b) applies are preferred.

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.

The compounds of the invention may be administered in inhaled form. Aerosol generation can be carried out using, for example, pressure-driven jet atomizers or ultrasonic atomizers, preferably using propellant-driven metered aerosols or propellant-free administration of micronized active compounds from, for example, inhalation capsules or other "dry powder" delivery systems.

The active compounds may be dosed as described depending on the inhaler system used. In addition to the active compounds, the administration forms may additionally contain excipients, such as, for example, propellants (e.g. Frigen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, fillers (e.g. lactose in the case of powder inhalers) or, if appropriate, further active compounds.

For the purposes of inhalation, a large number of systems 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 EP-A-0505321 ).

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.

The compounds of the invention may be synthesised by a number of processes generally described below and more specifically in the Examples hereinafter. Thus compounds of general formula (I) may be prepared by, but not restricted to methods set out in Schemes 1- 12.

Abbreviations:

Boc = te/t-butoxycarbonyl

CCI₄ = carbon tetrachloride

DCE = 1 ,2-dichloroethane

DCM = dichloromethane

DIPEA = diisopropylethylamine

DMAP = dimethylamino pyridine

DMF = dimethylformamide

DMSO = dimethyl sulfoxide

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

Et₂O = diethyl ether

EtOAc = ethyl acetate

EtOH = ethanol g = gram(s)

HCI = hydrochloric acid

K₂CO₃ = potassium carbonate

LCMS = high performance liquid chromatography/mass spectrometry

LDA = lithium di-/sopropylamine

MeOH = methanol mg = milligram(s) mL = millilitre(s) mmol = millimole(s) mol = mole(s)

Na₂CO₃ = sodium carbonate NaHCO₃ = sodium hydrogen carbonate NaOH = sodium hydroxide NBS = Λ/-bromosuccinimide NMR = nuclear magnetic resonance Sat = saturated

STAB = sodium triacetoxyborohydride TBAF = tetrabutylammonium fluoride TFA = trifluoroacetic acid THF = tetrahydrofuran μl_ = microlitre(s) μmol = micromole(s)

Commercially available reagents and solvents (HPLC grade) were used without further purification. Solvents were removed using a Buchi rotary evaporator. Microwave irradiation was carried out using a Biotage Initiator™ Eight microwave synthetiser. Purification of compounds by flash chromatography column was performed using silica gel, particle size 40-63 μm (230-400 mesh) obtained from Fluorochem. Purification of compounds by preparative HPLC was performed on Gilson systems using reverse phase Axia™ prep Luna C18 columns (1Ou, 100 x 21.2 mm), gradient 0-100% B (A = water /0.05 % TFA, B = acetonitrile / 0.05% TFA) over 10 min, flow = 25 ml/min, UV detection at 254 nm.

¹H NMR spectra were recorded on a Bruker 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 was performed on an Agilent HP1100 LC system using reverse phase Luna C18 columns (3 μm, 50 x 4.6 mm), gradient 5-95% B ( A = water / 0.1 % Formic acid, B = acetonitrile/ 0.1% Formic acid) over 2.25 min, flow = 2.25 ml/min. UV spectra were recorded at 220 and 254 nm using a G1315B DAD detector. Mass spectra were obtained over the range m/z 150 to 800 on a LC/MSD SL G1956B detector. Data were integrated and reported using ChemStation and ChemStation Data Browser software. Intermediates

All the examples disclosed were synthesised using Intermediate 1, which was prepared as described below in Scheme 1.

Intermediate 1

Scheme 1

Intermediate 1a r5-Amino-1-(4-fluorophenyl)-1AY-pyrazol-4-yll(3- hydroxyphenvDmethanone

Stage 1 - 3-(3-Methoxyphenyl)-3-oxopropanenitrile

LDA (44.2 ml. of a 2M solution in THF, 88.5 mmol) was added dropwise over 20 minutes to a cold (-78 ⁰C) solution of acetonitrile (2.8 ml_, 53.6 mmol) in THF (63 mL) under an atmosphere of nitrogen. The reaction mixture was stirred for 30 minutes and a solution of methyl 3-methoxybenzoate (7.00 g, 42.1 mmol) in THF (63 mL) was added dropwise over 30 minutes. The reaction mixture was stirred for 2 hours, quenched with a saturated aqueous solution of ammonium chloride (100 mL), allowed to warm to room temperature and diluted with Et₂O (300 mL). The aqueous layer was separated and extracted with Et₂O (100 mL). The combined organic layers were washed with 1 M HCI (200 mL), brine, dried (MgSO₄) and concentrated under reduced pressure. The residue was purified by column chromatography (25-30 % EtOAc in heptane) to afford the title compound as a pale orange solid (6.16 g, 83 % yield). 1H NMR (300 MHz, CDCI₃) 7.50-7.42 (3H, m), 7.24-7.20 (1 H, m), 4.10 (2H, s), 3.89 (3H, s).

Stage 2 - (2E^Z)-3-Anilino-2-(3-methoxybenzoyl)acrylonitrile

A mixture of 3-(3-methoxyphenyl)-3-oxopropanenitrile (1.34 g, 7.65 mmol) and Λ/,Λ/-diphenyl formamidine (1.50 g, 7.65 mmol) in p-xylene (10 mL) was heated to reflux for 3 hours, allowed to cool to room temperature and diluted with heptane. A precipitate was collected by filtration, washed with heptane and dried to afford the title compound as a solid (1.76 g, 83 % yield).

¹H NMR (300 MHz, CDCI₃) 12.85-12.70 (1 H, m), 8.11-8.04 (1 H, m), 7.60-7.55 (1 H, m), 7.50-

7.33 (4H, m), 7.30-7.21 (2H, m), 7.14-7.10 (1 H, m), 3.90 (3H, s).

Stage 3 - [5-Amino-1-(4-fluorophenyl)-1 H-pyrazol-4-yl](3-methoxyphenyl)methanone

Triethylamine (970 μl_, 6.96 mmol) was added to a slurry of 4-fluorophenyl hydrazine hydrochloride (1.03 g, 6.32 mmol) in EtOH (50 mL) and the solution was stirred for 10 minutes. (2£/Z)-3-Anilino-2-(3-methoxybenzoyl)acrylonitrile (1.76 g, 6.32 mmol) was added and the reaction was refluxed for 18 hours. The reaction was allowed to cool to room temperature and concentrated under reduced pressure. Purification by column chromatography (20-30 % EtOAc in heptane) afforded the title compound as a pale yellow solid (1.45 g, 74 % yield).

¹H NMR (300 MHz₁ CDCI₃) 7.83 (1 H, s), 7.60-7.56 (2H, m), 7.47-7.40 (2H, m), 7.30-7.22 (2H, m), 7.14-7.10 (1 H, m), 6.05 (2H, br s), 3.90 (3H, s).

Stage 4 - [5-Amino-1-(4-fluorophenyl)-1 H-pyrazol-4-yl](3-hydroxyphenyl)methanone

BBr₃ (4.49 mL of 1 M solution in DCM, 4.49 mmol) was added to a cold (0 ⁰C) solution of [5- amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl](3-methoxyphenyl) methanone (279 mg, 0.90 mmol) in DCM (5 mL). The reaction mixture was allowed to warm to room temperature, stirred for 3 hours, poured into brine and extracted with EtOAc. The organic layer was dried (MgSO₄) filtered and concentrated under reduced pressure. The residue was triturated with heptane, filtered and dried to afford the title compound as a yellow solid (265 mg, 100 % yield).

¹H NMR (300 MHz, DMSO-(J₆) 9.73 (1 H, br s), 7.77 (1 H, s), 7.64-7.58 (2H, m), 7.44-7.30 (3H, m), 7.21-7.10 (4H, m), 6.99-6.95 (1 H, m). Intermediate 1b f5-Amino-1-(2,4-difluorophenvπ-1H-pyrazol-4-yll(3- hydroxyphenvOmethanone

Intermediate 1b was synthesised as described above using 2,4-difluorophenyl-hydrazine hydrochloride instead of 4-fluorophenylhydrazine hydrochloride in Stage 3. 1H NMR (300 MHz, CDCI₃) 7.93 (1 H, s), 7.63-7.51 (1 H₁ m), 7.40-7.39 (2H, m), 7.32 (1 H, br s), 7.13-7.03 (3H, m).

Intermediate 1c r5-Amino-1-(2-chloro-4-fluorophenyl)-1H-Pyrazol-4-yll(3- hydroxyphenvDmethanone

Intermediate 1c was synthesised as described above using 2-chloro-4-fluorophenyl- hydrazine hydrochloride instead of 4-fluorophenylhydrazine hydrochloride in Stage 3. m/z 332/334 [M+H]\

Intermediate 1d r5-Amino-1-(3,4-difluorophenyl)-1H-pyrazol-4-vπ(3- hydroxyphenvDmethanone

Intermediate 1d was synthesised as described above using 3,4-difluorophenyl-hydrazine hydrochloride instead of 4-fluorophenylhydrazine hydrochloride in Stage 3. m/z 316 [M+H]⁺.

Intermediate 2 Cyclopentyl 5-bromo-Λ/-(fe/t-butoxycarbonyl)-L-norvalinate

Intermediate 2 was synthesised as shown below in Scheme 2.

Additional literature references relating to this route can be found within J. Org. Chem. 1984,

49, 3527-3534.

\ ) ; R BODAΔc

i) Ethyl chloroformate, NMM, THF Stage 3 ii) NaBH₄, THF, water

Intermediate 2

Scheme 2

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

Cyclopentanol (4.8 ml_, 53.3 mmol), EDC (9.40 g, 48.9 mmol) and DMAP (543 mg, 4.4 mmol) were added to a cold (0 ⁰C) solution of L-glutamic acid, Λ/-[(1 ,1-dimethyl- ethoxy)carbonyl]-5-(phenylmethyl) ester (15 g, 44.5 mmol) in DCM (220 ml_). The reaction mixture was allowed to warm to room temperature, stirred for 12 hours, diluted with DCM (200 ml_), washed with 1 M HCI, 1 M Na₂CO₃ and brine, dried (MgSO₄), filtered and concentrated under reduced pressure. Purification by column chromatography (20 % EtOAc in heptane) afforded the title compound as a white solid (12.4 g, 69 % yield). 1H NMR (300 MHz, CDCI₃) 7.38 (5H₁ m), 5.70 (1 H, m), 5.10 (2H, s), 5.05 (1 H, m), 4.25 (1 H, m), 2.47 (2H, m), 2.15 (1H, m), 1.95-1.55 (9H, m), 1.47 (9H, s).

Stage 2 - (4S)-4-[(te/?-butoxycarbonyl)amino]-5-(cyclopentyloxy)-5-oxopentanoic acid

5-Benzyl 1-cyclopentyl Λ/-(teAf-butoxycarbonyl)-L-glutamate (12.4 g, 30.5 mmol) was dissolved in EtOAc (200 ml_) and the reaction mixture was purged with nitrogen. 20% Pd(OH)₂ on carbon catalyst (1.3 g) was added and the reaction mixture was stirred at room temperature under an atmosphere of hydrogen for 5 hours. The reaction mixture was filtered through Celite and the filter cake washed with EtOAc (50 ml_). The combined filtrates were concentrated under reduced pressure to afford the title compound as a clear oil (7.73 g, 85 % yield).

¹H NMR (300 MHz, CDCI₃) 10.0 (1 H, br s), 5.70 (2H₁ m), 4.28 (1 H₁ m), 2.47 (2H, m), 2.15 (1 H, m), 1.95-1.55 (9H, m), 1.47 (9H, s).

Stage 3 - Cyclopentyl Λ/-(terf-butoxycarbonyl)-5-hydroxy-L-norvalinate

Ethyl chloroformate (2.45 ml_, 25.6 mmol) was added to a cold (-20 ⁰C) solution of (4S)-4- [(terf-butoxycarbonyl)amino]-5-(cyclopentyloxy)-5-oxopentanoic acid (6.73 g, 21.4 mmol) and Λ/-methyl morpholine (3.05 ml, 27.8 mmol) in THF (50 ml_). A white solid formed. THF (100 mL) was added and the reaction mixture was stirred at -20 ⁰C for 2 hours. The white solid was filtered off and the filtrate was added over a period of 20 minutes to a cold (0 ⁰C) solution of sodium borohydride (2.43 g, 64.1 mmol) in THF (20 mL) and water (5 mL). The reaction mixture was allowed to warm to room temperature and stirred for 4 hours. The reaction mixture was acidified to pH 5 with 1 M HCI and extracted with EtOAc (3x100 mL). The combined organic extracts were washed with brine, dried (MgSO₄), filtered and concentrated under reduced pressure. Purification by column chromatography (0-5% MeOH in DCM) afforded the title compound as a clear oil (5.0 g, 78 % yield).

¹H NMR (300 MHz, CDCI₃) 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-Λ/-(te/t-butoxycarbonyl)-L-norvalinate

A solution of triphenylphosphine (4.87 g, 18.8 mmol) in DCM (15 mL) was added to a suspension of Λ/-bromosuccinimide (3.54 g, 19.9 mmol) in DCM (30 mL). The solution was stirred for 5 minutes. Pyridine (644 μl, 7.96 mmol) and a solution of cyclopentyl N-(tert- butoxycarbonyl)-5-hydroxy-L-norvalinate (2.0 g, 6.64 mmol) in DCM (20 mL) were added. The reaction mixture was stirred for 18 hours, concentrated under reduced pressure and the residual solvent azeotroped with toluene (3x30 mL). The residue was triturated with diethyl ether (30 mL) and ethyl acetate/heptane (1 :9, 2x30 mL). The combined ether and ethyl acetate/heptane solutions were concentrated under reduced pressure. Purification by column chromatography (10-20 % EtOAc in heptane) afforded the title compound as a clear oil (1.34 g, 55 % yield).

¹H NMR (300 MHz, CDCh) 5.25 (1 H, m), 5.05 (1 H, br d), 3.45 (2H, m), 2.00-1.55 (12H, m), 1.45 (9H, s).

Intermediate 3a Cyclopentyl L-leucinate hydrochloride

Intermediate 3a was synthesised using the generic route shown in Scheme 3 below.

Stage 1 Stage 2

^H ₂ ^N V^H BoC₂O, NaHCO₃ Cyclopentanol BocHN THF, water

EDC, DMAP, DMF O t>

Intermed°iartes> 3a/3b

Scheme 3

Stage 1 - Λ/-(terf-Butoxycarbonyl)-L-leucine

Di-terf-butyl dicarbonate (36.4 g, 167 mmol) and NaHCO₃ (65.0 g, 700 mmol) were added to a solution of (L)-Leucine (20.0 g, 152 mmol) in THF (200 ml.) and water (200 ml_). The reaction mixture was stirred at room temperature for 72 hours, concentrated under reduced pressure and the residue was partitioned between water (300 ml_) and EtOAc (300 ml_). The aqueous layer was acidified to pH 4 using 1 M HCI and extracted with EtOAc (2x250 mL). The combined organic extracts were dried (MgSO₄), filtered and concentrated under reduced pressure to afford the title compound as a colourless oil (22.7 g, 64% yield).

Stage 2 - Cyclopentyl Λ/-(te/t-butoxycarbonyl)-L-leucinate

Λ/-(terf-Butoxycarbonyl)-L-leucine (22.7 g, 98.2 mmol) was dissolved in anhydrous DMF (200 mL) under an atmosphere of nitrogen and cyclopentanol (17.82 ml_, 196.4 mmol), EDC (20.7 g, 108.0 mmol) and DMAP (1.20 g, 9.82 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours, concentrated under reduced pressure and partitioned between water (200 mL) and EtOAc (200 mL). The aqueous layer was separated and extracted with EtOAc (200 mL). The combined organic extracts were dried (MgSO₄), filtered and concentrated under reduced pressure. Purification by column chromatography (0-30 % EtOAc in heptane) afforded the title compound as a colourless oil (18.01 g, 61 % yield).

Stage 3 - Cyclopentyl L-leucinate hydrochloride

Cyclopentyl Λ/-(tert-butoxycarbonyl)-L-leucinate (18.01 g, 60.2 mmol) was dissolved in DCM (200 mL) and 4M HCI in dioxane (30.1 mL, 120.4 mmol) was added. The reaction was incomplete after stirring at room temperature for 72 hours, and further 4M HCI in dioxane (15 mL, 60.2 mmol) was added. The reaction mixture was stirred for 6 hours, concentrated under reduced pressure to afford the title compound as a white solid (13.0 g, 92 % yield).

Intermediate 3b Cvclopentyl O-tert-butyl-L-serinate hydrochloride

Intermediates 3a and 3b were synthesised as described above in Scheme 3 and used as their free bases in Examples 2-17.

Intermediate 4 S-Pyridin-2-yl 5-amino-1-(4-fluorophenvh-1H-pyrazole-4-carbothioate

Intermediate 4 was synthesized as shown below in Scheme 4.

Scheme 4

Stage 1 - Ethyl 5-amino-1-(4-fluorophenyl)-1 /-/-pyrazole-4-carboxylate

Ethyl(ethoxymethylene)cyanoacetate (20.80 g, 123 mmol) and triethylamine (17.1 ml_, 123 mmol) were added to a solution of 4-fluorophenylhydrazine hydrochloride (20.00 g, 123 mmol). The reaction mixture was refluxed for 2.5 hours and allowed to cool to room temperature. A solid was collected by filtration, washed with small amounts of ethanol and heptane and allowed to dry under reduced pressure to afford the title compound as a beige solid (22.18 g, 72 % yield). m/z 250 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.80 (1 H, s), 7.58-7.51 (2H, m), 7.27 (2H, m), 5.27 (2H, br s), 4.33 (2H₁ q, J=7.2 Hz), 1.39 (2H, t, J=7.2 Hz).

Stage 2 - 5-Amino-1-(4-fluorophenyl)-1 H-pyrazole-4-carboxylic acid

Ethyl 5-amino-1-(4-fluorophenyl)-1H-pyrazole-4-carboxylate (22.18 g, 89 mmol) was suspended in 1 N aqueous solution of LiOH (178 ml_, 178 mmol) and methanol (200 ml_). The reaction mixture was refluxed for 17 hours, allowed to cool to room temperature and filtered. The filtrate was acidified to pH=7 with 2N HCI. A solid was collected by filtration and allowed to dry under reduced pressure to afford the title compound as a pale yellow solid (18.64 g, 95 % yield), which was used without further purification.

Stage 3 - S-Pyridin-2-yl 5-amino-1-(4-fluorophenyl)-1/-/-pyrazole-4-carbothioate

Triphenylphosphine (11.05 g, 42 mmol) and 2,2'-dipyridyldisulfide (9.28 g, 42 mmol) were added to a solution of 5-amino-1-(4-fluorophenyl)-1H-pyrazole-4-carboxylic acid (9.32 g, 42 mmol) in acetonitrile (1.25 L). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours. A solid was collected by filtration and washed with a small amount of acetonitrile. The filtrate was concentrated under reduced pressure to leave a yellow solid. Trituration with acetonitrile (300 mL) afforded an off-white solid. The solids from filtration and trituration were combined, allowed to dry under reduced pressure to afford the title compound as an off-white solid (8.47 g, 64 % yield). m/z 315 [M+H]⁺.

Intermediate 5 (3-(r5-Amino-1-(ΦfluorophenylHW-Pyrazol-4-yllcarbonyl}phenyl)- acetaldehvde

Intermediate 5 was synthesized as shown below in Scheme 5.

i) Mg, THF. reflux

Stage 3 ii) Intermediate 4

Scheme 5

Stage 1 - 2-(3-Bromophenyl)ethanol

BH₃-Me₂S (7 ml_ of a 2M solution in THF, 14.0 mmol) was added to a cold (0 ⁰C) solution of 3-bromophenylacetic acid (2.00 g, 9.3 mmol). The reaction mixture was allowed to warm to room temperature, stirred for 20 hours and re-cooled to 0 ⁰C. Water (10 ml.) was added dropwise. The organic layer was separated, washed with brine (20 mL), dried (MgSO₄), filtered and concentrated under reduced pressure to leave a colourless oil. Purification by column chromatography (50 % EtOAc in heptane) afforded the title compound as a colourless oil (1.47 g, 79 % yield). m/z 224/226 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.41-7.35 (2H, m), 7.23-7.16 (2H, m), 3.87 (2H, t, J=6.5 Hz), 2.86 (2H, t, J=6.5 Hz).

Stage 2 - [2-(3-Bromophenyl)ethoxy](te/?-butyl)dimethylsilane

Triethylamine (1.3 mL, 9.5 mmol), DMAP (220 mg, 1.8 mmol) and terf-butyl(chloro)- dimethylsilane (1.10 g, 8.8 mmol) were added to a cold (0 °C) solution of 2-(3- bromophenyl)ethanol (1.47 g, 7.3 mmol) in DCM (40 mL). The reaction mixture was stirred at 0 ⁰C for 30 minutes, allowed to warm to room temperature, stirred for an additional 1.5 hour, washed with saturated NH₄CI (40 mL), brine (40 mL), dried (MgSO₄), filtered and concentrated under reduced pressure to leave a colourless oil. Purification by column chromatography (10 % EtOAc in heptane) afforded the title compound as a colourless oil (2.07 g, 90 % yield).

¹H NMR (300 MHz, CDCI₃) 7.40-7.34 (2H, m), 7.18-7.15 (2H, m), 3.82 (2H, t, J=6.8 Hz) , 2.81 (2H, t, J=6.8 Hz), 0.90 (9H, s), 0.00 (6H, s).

Stage 3 - [5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl][3-(2-{[te:t-butyl(dimethyl)silyl]- oxy}ethyl)phenyl]methanone

Magnesium turnings (77 mg, 3.2 mmol) were added to an oven dried flask charged with [2-

(3-bromophenyl)ethoxy](te/t-butyl)dimethylsilane (1.00 g, 3.2 mmol) in anhydrous THF (10 mL) and a crystal of iodine. The reaction mixture was refluxed for 3 hours and allowed to cool to room temperature. S-Pyridin-2-yl 5-amino-1-(4-fluorophenyl)-1 H-pyrazole-4-carbothioate (Intermediate 4) (498 mg, 1.6 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure. The residue was taken up in EtOAc (50 mL), washed with a saturated aqueous solution of NH₄CI (25 mL), brine (25 mL), dried (MgSO₄), filtered and concentrated under reduced pressure to leave a yellow solid. Purification by column chromatography (20 % EtOAc in heptane) afforded the title compound as a white solid (226 mg, 32 % yield). m/z 440 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.79 (1 H, s), 7.68-7.65 (2H, m), 7.59-7.54 (2H, m), 7.43-7.41 (2H, m), 7.58-7.22 (2H, m), 6.03 (2H, br s), 3.87 (2H, t, J=6.9 Hz), 2.92 (2H, t, J=6.9 Hz), 0.88 (9H, s), 0.00 (6H, s).

Stage 4 - [5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl][3-(2-hydroxyethyl)phenyl]-methanone

TBAF (0.62 mL of 1 M solution in THF₁ 0.62 mmol) was added to a solution of [5-amino-1-(4- fluorophenyl)-1/-/-pyrazol-4-yl][3-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-phenyl]methanone (226 mg, 0.51 mmol). The reaction mixture was stirred at room temperature for 2.5 hours, diluted with EtOAc (40 mL), washed with brine (20 mL), dried (MgSO₄) filtered and concentrated under reduced pressure to leave a yellow oil. Purification by column chromatography (80 % EtOAc in heptane) afforded the title compound as a white solid (143 mg, 85 % yield). m/z 326 [M+H]⁺, 348 [M+Na]⁺.

Stage 5 - (3-{[5-Amino-1-(4-fluorophenyl)-1 H-pyrazol-4-yl]carbonyl}phenyl)-acetaldehyde

Dess-Martin periodinane (652 mg, 1.5 mmol) was added to a solution of [5-amino-1-(4- fluorophenyl)-1 H-pyrazol-4-yl][3-(2-hydroxyethyl)phenyl]methanone (417 mg, 1.3 mmol) in DCM (40 ml_). The reaction mixture was stirred at room temperature for 2 hours and quenched with a saturated aqueous solution of sodium thiosulfate (20 ml.) and a saturated aqueous solution of NaHCO₃ (20 ml_). The mixture was vigorously stirred for 30 minutes. The aqueous layer was separated and extracted with DCM (2x30 ml_). The combined organic extracts were dried (MgSO₄), filtered and concentrated under reduced pressure to leave a yellow oil (323 mg), which was used without further purification.

Intermediate 6 1,4-Dioxaspiror4.5ldec-8-yl 4-methylbenzenesulfonate

Intermediate 6 was synthesised using the route shown in Scheme 6.

p-Toluenesulfonyl chloride

NaBH₄, MeOH Pyridine Stage 1 Stage 2

Intermediate 6

Scheme 6

Stage 1 - 1 ,4-Dioxaspiro[4.5]decan-8-ol

NaBH₄ (2.42 g, 64 mmol) was added portionwise to a cold (0 ⁰C) solution of 1 ,4- dioxaspiro[4.5]decan-8-one (5.00 g, 32 mmol) in MeOH (30 ml_). The solution was stirred at 0 ⁰C for 3 hours, concentrated under reduced pressure and 5% aqueous NaOH was added. The solution was extracted with 2-propanol/CHCI₃ (1 :4, 2x100 ml_). The combined organic extracts were dried (MgSO₄), filtered and concentrated under reduced pressure to afford the title compound as an oil (4.74 g, 94 % yield).

¹H NMR (300 MHz, CDCI₃) 4.02-3.92 (4H, m), 3.85-3.77 (1 H, m), 1.94-1.80 (4H, m), 1.73- 1.56 (4H, m).

Stage 2 - 1 ,4-Dioxaspiro[4.5]dec-8-yl 4-methylbenzenesulfonate

p-Toluenesulfonyl chloride (1.46 g, 7.6 mmol) was added in one portion to a solution of 1 ,4- dioxaspiro[4.5]decan-8-ol (1.00 g, 6.35 mmol) in pyridine (10 ml_). The reaction mixture was stirred at room temperature for 18 hours, quenched with brine and extracted with EtOAc. The combined organic extracts were washed with 1 M HCI, a saturated aqueous solution of NaHCO₃, brine, dried (MgSO₄), filtered and concentrated under reduced pressure The residue was purified by column chromatography (30 % EtOAc in heptane) to afford the title compound as a solid (1.50 g, 76 % yield).

¹H NMR (300 MHz, CDCI₃) 7.82 (2H, d, J=8.4 Hz), 7.34 (2H, d, J=8.4 Hz), 4.69-4.63 (1 H, m), 3.98-3.88 (4H, m), 2.46 (3H, s), 1.94-1.76 (6H, m), 1.60-1.50 (2H, m). Examples

Example 1

Cyclopentyl 5-(3-{[5-amino-1 -(4-fluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}phenoxy)-L- norvalinate

Example 1 was synthesised as shown below in Scheme 7.

Intermediate 2

Sch erne 7 Stage 1 - Cyclopentyl 5-(3-{[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]carbonyl}phenoxy)- Λ/-(te/t-butoxycarbonyl)-L-norvalinate

Cyclopentyl 5-bromo-Λ/-(ter£-butoxycarbonyl)-L-norvalinate (Intermediate 2) (152 mg, 0.42 mmol) and K₂CO₃ (185 mg, 1.34 mmol) were added to a solution of [5-amino-1-(4- fluorophenyl)-1 /-/-pyrazol-4-yl](3-hydroxyphenyl) methanone (Intermediate 1a) (100 mg, 0.34 mmol) in DMF (3 ml_). The reaction mixture was stirred at 40 ⁰C for 18 hours, then 80 ⁰C for 5 hours, allowed to cool to room temperature and partitioned between water and EtOAc. The organic layer was separated, washed with brine, dried (MgSO₄), filtered and concentrated under reduced pressure. Purification by column chromatography (30-40% EtOAc in heptane) afforded the title compound as a viscous oil (128 mg, 66 % yield).

¹H NMR (300 MHz, CDCI₃) 7.82 (1 H, s), 7.60-7.54 (2H, m), 7.44-7.40 (2H, m), 7.32-7.24 (3H, m), 7.12-7.07 (1 H, m), 6.05 (2H, br s), 5.27-5.20 (1 H, m), 5.18-5.13 (1 H, m), 4.40-4.38 (1 H, m), 4.06 (2H, t, J=5.7 Hz), 1.95-1.65 (12H, m), 1.47 (9H, s).

Stage 2 - Cyclopentyl 5-(3-{[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]carbonyl}phenoxy)- L-norvalinate dihydrochlohde

4M HCI in dioxane (5 mL) was added to cyclopentyl 5-(3-{[5-amino-1-(4-fluorophenyl)-1H- pyrazol-4-yl]carbonyl}phenoxy)-Λ/-(terf-butoxycarbonyl)-L-norvalinate (128 mg, 0.22 mmol) and the mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure. The residue was triturated with Et₂O to afford the title compound as a yellow solid (75 mg, 61 % yield). m/z 480 [M+H]⁺. ¹H NMR (300 MHz, DMSO-Cf₆) 8.51 (3H, br s), 7.80 (1 H, s), 7.64-7.57 (2H, m), 7.49-7.32 (4H, m), 7.23 (1 H, br s), 7.17 (3H, br s), 5.25-5.15 (1 H, m), 4.15-4.00 (3H, m), 2.05-1.50 (12H, m).

Examples 2-11 were prepared as described below in Scheme 8.

Intermediates 1a-d

Scheme 8

Example 2

Cyclopentyl Λ/-[3-(3-{[5-amino-1 -(4-fluorophenyl)-1 H-pyrazol-4-yl]carbonyl}-phenoxy)propyl]- L-leucinate

Stage 1 - [5-Amino-1-(4-fluorophenyl)-1/-/-pyrazol-4-yl][3-(3-chloropropoxy)phenyl] methanoπe

1-Chloro-3-bromo-propane (200 μl, 2.02 mmol) and K₂CO₃ (278 mg, 2.02 mmol) were added to a solution of [5-amino-1-(4-fluorophenyl)-1 /-/-pyrazol-4-yl](3-hydroxyphenyl) methanone (Intermediate 1a)(200 mg, 0.67 mmol) in DMF (10 ml_). The reaction was stirred at 40 ⁰C for 48 hours. 1-Chloro-3-bromo-propane (67 μl, 0.68 mmol) and K₂CO₃ (93 mg, 0.68 mmol) were added. The reaction mixture was stirred at 40 ⁰C for an additional 5 hours, allowed to cool to room temperature, diluted with EtOAc washed with water, brine, dried (MgSO₄), filtered and concentrated under reduced pressure. Purification by column chromatography (20-30% EtOAc in Heptane) afforded the title compound as an off-white solid (150 mg, 60 % yield). ¹H NMR (300 MHz, CDCI₃) 7.83 (1 H, s), 7.60-7.55 (2H, m), 7.43 (2H, d, J=5.1 Hz), 7.37 (1 H, s), 7.29-7.21 (2H, m), 7.14-7.10 (1 H, m), 6.04 (2H, br s), 4.24-4.18 (2H, m), 3.81-3.62 (1 H, m), 2.40-2.25 (2H, m).

Stage 2 - Cyclopentyl Λ/-[3-(3-{[5-amino-1 -(4-fluorophenyl)-1 H-pyrazol-4- yl]carbonyl}phenoxy)propyl]-L-leucinate

Cyclopentyl L-leucinate (Intermediate 3a) (240 mg, 1.21 mmol), sodium iodide (121 mg, 0.80 mmol), and DIPEA (210 μl, 1.21 mmol) were added to a solution of [5-amino-1-(4- fluorophenyl)-1/-/-pyrazol-4-yl][3-(3-chloropropoxy)phenyl] methanone (150 mg, 0.40 mmol) in DMF (8 ml_). The reaction was stirred at 90 ⁰C for 18 hours, allowed to cool to room temperature, diluted with EtOAc, washed with water, brine, dried (MgSO₄), filtered and concentrated under reduced pressure. Purification by column chromatography (30-40% EtOAc in heptane) followed by preparative HPLC afforded the title compound as a viscous oil (63 mg, 29 % yield). m/z 537 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.79 (1 H, s), 7.59-7.50 (2H, m), 7.40-7.36 (2H, m), 7.31 (1 H, s), 7.27-7.17 (2H₁ m), 7.12-7.04 (1 H₁ m), 6.09 (2H₁ br s), 5.25-5.18 (1 H, m), 4.10 (2H₁ 1, J=6.1 Hz), 2.24 (1 H₁ 1, J=7.4Hz), 2.86-2.76 (1 H₁ m), 2.69-2.60 (1 H₁ m), 2.00-1.56 (13H₁ m), 1.49-1.40 (2H₁ m), 0.95-0.85 (6H₁ m).

Example 3

Cyclopentyl Λ/-[3-(3-{[5-amino-1 -(4-fluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}phenoxy) propyl]- O-tert-butyl-L-serinate

Example 3 was synthesised as described above for Example 2 using cyclopentyl O-tert- butyl-L-serinate (Intermediate 3b) instead of cyclopentyl L-leucinate (Intermediate 3a) in Stage 2 of Scheme 8. m/z 567 [M+H]⁺. ¹H NMR (300 MHz₁ CDCI₃) 7.82 (1 H₁ s), 7.60- 7.54 (2H₁ m), 7.42-7.40 (2H₁ d, J=5.1 Hz), 7.34 (1 H₁ s),7.29-7.23 (2H₁ m), 7.13-7.09 (1 H, m), 6.04 (2H₁ br s), 5.27-5.23 (1 H₁ m), 4.14 (2H₁ 1, J=6.1 Hz), 3.64-3.60 (2H₁ m), 3.40 (1 H, t, J=4.8Hz), 2.95-2.85 (1 H₁ m), 2.80-2.70 (1 H₁ m), 2.10-2.00 (2H₁ m), 1.95-1.55 (10H₁ m), 1.16 (9H₁ s).

Example 4

Cyclopentyl Λ/-[3-(3-{[5-amino-1 -(2,4-difluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}- phenoxy)propyl]-L-leucinate

Example 4 was synthesised as described above for Example 2 using Intermediate 1b in Stagel of Scheme 8. m/z 555 [M+H]⁺.¹H NMR (300 MHz, CDCI₃) 7.87 (1 H, br s), 7.57 (1 H, dd, J=7.1 , 14.9 Hz), 7.44-7.34 (3H, m), 7.13-7.07 (3H, m), 6.00 (2H, br s), 5.24 (1 H, br s), 4.13 (2H, t, J=6.2 Hz), 3.26 (1 H, t, J=7.4 Hz), 2.89-2.81 (1 H, m), 2.73-2.64 (1 H, m), 2.07-1.45 (12H₁ m), 1.28 (1 H, br s), 0.96-0.91 (6H₁ m).

Example 5

Cyclopentyl Λ/-[4-(3-{[5-amino-1 -(2,4-difluorophenyl)-1 H-pyrazol-4-yl]carbonyl}- phenoxy)butyl]-L-leucinate

Example 5 was synthesised as described above for Example 4 using 1-bromo-4- chlorobutane instead of 1-bromo-3-chloropropane in Stage 1 of Scheme 8. m/z 569 [M+H]⁺. ¹H NMR (300 MHz₁ CDCI₃) 7.86 (1 H₁ s), 7.60-7.52 (1 H, m), 7.44-7.38 (2H₁ m), 7.33-7.29 (1 H₁ m), 7.12-7.05 (3H₁ m), 6.01 (2H, br s), 5.25-5.21 (1 H, m), 4.05 (2H, t, J=6.3 Hz)₁ 3.24 (1 H, t, J=7.4 Hz), 2.73-2.64 (1 H₁ m), 2.58-2.50 (1 H₁ m), 1.91-1.60 (13H, m), 1.52-1.41 (2H₁ m), 0.98-0.93 (6H, m). Example 6

Cyclopentyl Λ/-[5-(3-{[5-amino-1 -(2,4-difluorophenyl)-1 H-pyrazol-4-yl]carbonyl}- phenoxy)pentyl]-L-leucinate

Example 6 was synthesised as described above for Example 4 using 1-bromo-5- chloropentane instead of 1-bromo-3-chloropropane in Stage 1 of Scheme 8. m/z 583 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.87 (1 H, br s), 7.60-7.53 (1 H, m), 7.44-7.30 (3H, m), 7.12-7.07 (3H, m), 5.99 (2H, br s), 5.24 (1 H, br s), 4.04 (2H, t, J=6.3 Hz), 3.25 (1 H, t, J=7.4 Hz), 2.71-2.49 (2H, m), 1.98-1.46 (14H₁ m), 1.29 (1 H, br s), 0.98-0.91 (6H, m).

Example 7

Cyclopentyl Λ/-[6-(3-{[5-amino-1 -(2,4-difluorophenyl)-1 H-pyrazol-4-yl]carbonyl}- phenoxy)hexyl]-L-leucinate

Example 7 was synthesised as described above for Example 4 using 1-bromo-6- chlorohexane instead of 1-bromo-3-chloropropane in Stage 1 of Scheme 8. m/z 597 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.87 (1 H, s), 7.87 (1 H, s), 7.60-7.53 (1 H, m), 7.44-7.38 (2H, m), 7.35-7.33 (1 H, m), 7.13-7.06 (3H, m), 6.00 (2H, br s), 5.25-5.21 (1 H, m), 4.03 (2H, t, J=6.5 Hz), 3.22 (1 H, t, J=7.4 Hz), 2.64-2.55 (1 H, m), 2.52-2.43 (1 H, m), 1.90-1.38 (17H, m), 0.99-0.88 (8H, m).

Example 8

Cyclopentyl Λ/-[3-(3-{[5-amino-1 -(2-chloro-4-fluorophenyl)-1 H-pyrazol-4- yl]carbonyl}phenoxy)propyl]-L-leucinate

Example 8 was synthesised as described above for Example 2 using Intermediate 1c in Stagel of Scheme 8. m/z 571/573 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.87 (1 H, s), 7.55-7.50 (1 H, m), 7.43-7.35 (3H, m), 7.23-7.17 (2H, m), 7.13-7.09 (1 H, m), 5.88 (2H, br s), 5.24 (1 H, t, J=6.0 Hz), 4.13 (2H, t, J=6.0 Hz), 3.27 (1 H, t, J=7.2 Hz), 2.90-2.82 (1 H, m), 2.75-2.66 (1 H, m), 2.07-1.48 (12H, m), 1.28 (1 H, t, J=7.2 Hz), 0.98-0.91 (6H, m).

Example 9

Cyclopentyl Λ/-[5-(3-{[5-amino-1 -(2-chloro-4-fluorophenyl)-1 H-pyrazol-4- yl]carbonyl}phenoxy)pentyl]-L-leucinate

Example 9 was synthesised as described above for Example 8 using 1-bromo-5- chloropentane instead of 1-bromo-3-chloropropane in Stage 1 of Scheme 8. m/z 599/601 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.87 (1 H, s), 7.53 (1 H, dd, J=5.4, 8.7 Hz), 7.43-7.34 (3H, m), 7.24-7.17 (2H₁ m), 7.12-7.08 (1 H, m), 5.88 (2H, br s), 5.25 (1 H, br s), 4.04 (2H, t, J=6.3 Hz)₁ 3.31 (1H₁ 1, J=7.2 Hz), 2.67-2.51 (2H₁ m), 1.89-1.47 (14H₁ m), 1.28 (1 H₁ br s), 0.99-0.90 (6H, m).

Example 10

Cyclopentyl Λ/-[3-(3-{[5-amino-1 -(3,-4-difluorophenyl)-1 /-/-pyrazol-4- yl]carbonyl}phenoxy)propyl]-L-leucinate

Example 10 was synthesised as described above for Example 2 using Intermediate 1d in Stage 1 of Scheme 8. m/z 555 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.81 (1 H, s), 7.51-7.28 (6H₁ m), 7.11-7.08 (1 H, m), 6.14 (2H₁ br s), 5.25-5.21 (1 H₁ m), 4.11 (2H, t, J=6.0 Hz)₁ 3.23 (1 H₁ 1, J=7.2 Hz)₁ 2.87- 2.78 (1 H, m), 2.70-2.61 (1 H, m), 2.01-1.38 (13H, m), 0.98-0.89 (6H₁ m).

Example 11

Cyclopentyl Λ/-[5-(3-{[5-amino-1 -(3,4-difluorophenyl)-1 H-pyrazol-4- yl]carbonyl}phenoxy)pentyl]-L-leucinate

Example 11 was synthesised as described above for Example 10 using 1-bromo-5- chloropentane instead of 1-bromo-3-chloropropane in Stage 1 of Scheme 8. m/z 583 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.82 (1 H, s), 7.52-7.41 (1 H, m), 7.39-7.31 (5H, m), 7.11-7.07 (1 H, m), 6.31 (2H, br s), 5.25-5.21 (1 H, m), 4.03 (2H, t, J=6.5 Hz), 3.22 (1 H, t, J=7.4 Hz), 2.64-2.58 (1 H, m), 2.51-2.46 (1 H, m), 1.91-1.38 (15H, m), 1.32-1.25 (2H, m), 0.98-0.89 (6H, m).

Examples 12 and 13 were prepared as described below in Scheme 9.

Scheme 9

Example 12

Cyclopentyl Λ/-[(2R)-3-(3-{[5-amino-1 -(2,4-difluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}phenoxy)- 2-hydroxypropyl]-L-leucinate

Stage 1 - [5-Amino-1-(2,4-difluorophenyl)-1H-pyrazol-4-yl]{3-[(2f?)-oxiran-2- ylmethoxy]phenyl}methanone

Potassium carbonate (164 mg, 1.2 mmol) and (S)-epichlorohydrin (187 μl_, 2.4 mmol) were added to a solution of Intermediate 1b (250 mg, 0.8 mmol) in acetone (2.5 ml_). The reaction mixture was refluxed for 24 hours and (S)-epichlorohydrin (187 μl_, 2.4 mmol) was added. The reaction mixture was refluxed for an additional 24 hours, allowed to cool to room temperature, filtered and concentrated under reduced pressure to leave a brown oil. Purification by column chromatography (50 % EtOAc in heptane) afforded the title compound as a pale yellow solid (220 mg, 75 % yield). m/z 372 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.78 (1 H, s), 7.51-7.44 (1 H, m), 7.38-7.34 (2H, m), 7.28-7.26 (1 H, m), 7.08-6.97 (3H, m), 5.93 (2H, br s), 4.26 (1 H, dd, J=3.0, 5.1 Hz), 3.94 (1 H, dd, J=5.8, 11.1 Hz), 3.35-3.30 (1 H, m), 2.86 (1 H, t, J=4.4 Hz), 2.72 (1 H, dd, J=2.9, 4.9 Hz).

Stage 2 - Cyclopentyl Λ/-[(2f?)-3-(3-{[5-amino-1-(2,4-difluorophenyl)-1 /-/-pyrazol-4- yl]carbonyl}phenoxy)-2-hydroxypropyl]-L-leucinate

Cyclopentyl-L-leucinate (Intermediate 3a) (130 mg, 0.65 mmol) was added to a solution of [5-amino-1-(2,4-difluorophenyl)-1/-/-pyrazol-4-yl]{3-[(2R)-oxiran-2- ylmethoxy]phenyl}methanone (220 mg, 0.59 mmol) in ethanol (2 ml_). The reaction mixture was refluxed for 18 hours, allowed to cool to room temperature and concentrated under reduced pressure to leave a yellow oil. Purification by preparative HPLC afforded the title compound as a pale yellow oil (132 mg, 39 % yield). m/z 571 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.86 (1 H, s), 7.60-7.52 (1 H, m), 7.44-7.41 (2H, m), 7.36-7.34 (1 H, m), 7.16-7.05 (3H, m), 6.01 (2H, s), 5.26-5.20 (1 H, m), 4.08-3.96 (3H, m), 3.24 (1 H, t, J=7.3 Hz), 3.00 (1 H, dd, J=3.6, 12.1 Hz), 2.58 (1 H, dd, J=8.3,12.1 Hz), 1.93-1.57 (9H, m), 1.50-1.45 (2H, m), 0.94 (6H, t, J=6.6 Hz).

Example 13

Cyclopentyl Λ/-[(2S)-3-(3-{[5-amino-1 -(2,4-difluorophenyl)-1 H-pyrazol-4-yl]carbonyl}phenoxy)- 2-hydroxypropyl]-L-leucinate

Example 13 was synthesised as described for Example 12 using (f?)-epichlorohydrin instead of (S)-epichlorohydrin in Stage 1 of Scheme 9 to afford the title compound as a pale yellow oil (139 mg, 31 % yield over 2 steps). m/z 571 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.86 (1 H, s), 7.60-7.51 (1 H, m), 7.43-7.41 (2H, m), 7.36-7.34 (1 H, m), 7.16-7.05 (3H, m), 6.01 (2H, s), 5.28-5.22 (1H, m), 4.08-34.02 (3H, m), 3.26 (1 H, t, J=7.2 Hz), 2.90-2.84 (1 H, m), 2.71 (1 H, dd, J=3.2,12.1 Hz), 1.93-1.59 (9H₁ m), 1.50-1.45 (2H, m), 0.94 (6H, t, J=6.5 Hz).

Examples 14 and 15 were prepared as described below in Scheme 10.

Scheme 10

Example 14

Cyclopentyl Λ/-[2-(3-{[5-amino-1 -(4-fluorophenyl)-1 H-pyrazol-4-yl]carbonyl}-phenyl)ethyl]-L- leucinate

Stage 1 - Cyclopentyl Λ/-[2-(3-{[5-amino-1-(4-fluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}- phenyl)ethyl]-L-leucinate

STAB (635 mg, 3.0 mmol) was added to a solution of crude (3-{[5-amino-1-(4-fluorophenyl)- 1H-pyrazol-4-yl]carbonyl}phenyl)-acetaldehyde (Intermediate 5) (323 mg, 1.0 mmol) and cyclopentyl L-leucinate (Intermediate 3a) (299 mg, 1.5 mmol) in THF (20 ml_). The reaction mixture was stirred at room temperature for 18 hours and quenched with water (25 ml_) and extracted with EtOAc (3x25 ml_). The combined organic extracts were washed with brine (25 ml_), dried (MgSO₄), filtered and concentrated under reduced pressure to leave a yellow oil. Purification by column chromatography (60 % EtOAc in heptane) followed by preparative HPLC afforded the title compound as a pale yellow oil (229 mg, 45 % yield over 2 steps). m/z 507 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.70 (1 H, s), 7.61-7.57 (2H, m), 7.51-7.44 (2H, m), 7.39-7.31 (2H, m), 7.20-7.12 (2H, m), 5.98 (2H, br s), 5.15-5.10 (1 H, m), 3.17 (1 H, t, J=7.4 Hz), 2.90-2.64 (4H, m), 1.79-1.49 (8H₁ m), 1.43-1.30 (2H, m), 0.86-0.79 (6H, m).

Example 15

Cyclopentyl Λ/-[3-(3-{[5-amino-1 -(4-fluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}-phenyl)propyl]-L- leucinate

Example 15 was prepared as described above for Example 14 using 3-(3- bromophenyl)propan-1-ol instead of 2-(3-Bromophenyl)ethanol in Stage 2 of Scheme 5. m/z 521 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.79 (1 H, s), 7.66-7.64 (2H, m), 7.60-7.54 (2H, m), 7.45-7.38 (2H, m), 7.30-7.22 (2H, m), 6.05 (2H, br s), 5.24-5.20 (1 H, m), 3.24 (1 H, t, J=7.4 Hz), 2.81-2.72 (2H, m), 2.70-2.64 (1 H, m), 2.57-2.49 (1 H, m), 1.94-1.45 (13H, m), 0.96-0.84 (6H, m). Examples 16 and 17 were synthesised as shown below in Scheme 11.

ntermedi⁰at

Scheme 11

Example 16

Cyclopentyl Λ/-[4-(3-{[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]carbonyl}- phenoxy)cyclohexyl]-L-leucinate

Stage 1 - [5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl][3-(1 ,4-dioxaspiro[4.5]dec-8- yloxy)phenyl]methanone

1 ,4-Dioxaspiro[4.5]dec-8-yl 4-methylbenzenesulfonate (Intermediate 6) (315 mg, 1.01 mmol) and K₂CO₃ (186 mg, 1.35 mmol) were added to a solution of [5-amino-1-(4-fluorophenyl)-1/-/- pyrazol-4-yl](3-hydroxyphenyl) methanone (Intermediate 1a) (200 mg, 0.67 mmol) in DMF (10 ml_). The reaction mixture was heated at 80 ⁰C for 48 hours and 1 ,4-dioxaspiro[4.5]dec- 8-yl 4-methylbenzenesulfonate (201 mg) and K₂CO₃ (62 mg) were added. The reaction mixture was stirred at 80 ⁰C for an additional 18 hours, allowed to cool to room temperature, diluted with water (25 mL) and extracted with EtOAc (3 x 25 ml_). The combined organic extracts were washed with brine (25 mL), dried (MgSO₄) and concentrated under reduced pressure. Purification by column chromatography (30-35 % EtOAc in heptane) afforded a mixture of the title compound and [5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl](3- hydroxyphenyl) methanone (220 mg), which was used without further purification in Stage 2.

Stage 2 - 4-(3-{[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]carbonyl}phenoxy)- cyclohexanone

A 4M solution of HCI in dioxane (4 mL) was added to a solution of [5-amino-1-(4- fluorophenyl)-1 H-pyrazol-4-yl][3-(1 ,4-dioxaspiro[4.5]dec-8-yloxy)phenyl]methanone and [5- amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl](3-hydroxyphenyl) methanone (220 mg) in THF (4 mL). The reaction mixture was stirred at room temperature for 2.5 hours, concentrated under reduced pressure and partitioned between EtOAc and sat. NaHCO₃. The organic layer was separated, washed with brine, dried (MgSO₄), filtered and concentrated under reduced pressure. The residue was purified by column chromatography (30-40 % EtOAc in Heptane) to afford the title compound as a waxy solid (108 mg, 41 % yield over 2 steps). 1H NMR (300 MHz, CDCI₃) 7.82 (1 H₁ s), 7.60-7.54 (2H, m), 7.47-7.45 (2H, m), 7.41 (1 H, s), 7.30-7.24 (2H, m), 7.20-7.16 (1 H, m), 6.06 (2H, br s), 4.85-4.70 (1 H, m), 2.78-2.67 (2H, m), 2.42-2.31 (4H, m), 2.18-2.07 (2H, m), 1.61 (6H, m).

Stage 3 - Cyclopentyl Λ/-[4-(3-{[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]carbonyl}- phenoxy)cyclohexyl]-L-leucinate

Cyclopentyl L-leucinate (Intermediate 3a) (28 mg, 0.40 mmol) and acetic acid (7 μl, 0.13 mmol) were added to a solution of 4-(3-{[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4- yl]carbonyl}phenoxy) cyclohexanone (50 mg, 0.13 mmol) in DCE (2.5 mL) and the reaction mixture was stirred for 10 minutes. STAB (30 mg, 0.14 mmol) was then added. The reaction mixture was stirred for 18 hours at room temperature, diluted with DCM, washed with a saturated aqueous solution of NaHCO₃, brine, dried (MgSO₄), filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC to afford the title compound as an oil (46 mg, 63 % yield). m/z 577 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.71 (1 H, s), 7.51-7.45 (2H, m), 7.44-7.11 (5H, m), 7.03-6.96 (1 H, m), 5.98 (2H, br s), 5.17-5.12 (1 H, m), 4.45-4.10 (1 H, m), 3.26 (1 H₁ q, J=7.1 Hz), 2.43-2.35 (1 H, m), 2.12-1.90 (2H, m), 1.85-1.45 (15H, m), 1.40-1.28 (2H, m), 0.87- 0.81 (6H, m).

Example 17

Cyclopentyl Λ/-[4-(3-{[5-amino-1 -(4-fluorophenyl)-1 H-pyrazol-4-yl]carbonyl}phenoxy)- cyclohexyl]-O-te/?-butyl-L-serinate

Example 17 was prepared as described for Example 16, using cyclopentyl O-tert-butyl-L- serinate (Intermediate 3b) in Stage 3 of Scheme 11. m/z 607 [M+H]⁺. ¹H NMR (300 MHz, CDCI₃) 7.71 (1 H, s), 7.50-7.45 (2H, m), 7.31-7.27 (2H, m), 7.23 (1 H, s), 7.20-7.12 (2H, m), 7.03-6.98 (1H, m), 5.97 (2H, br s), 5.18-5.12 (1 H, m), 4.50- 4.10 (1H, m), 3.53-3.49 (1H, m), 3.43-3.38 (2H, m), 2.55-2.40 (1 H, m), 2.00-1.75 (6H, m), 1.72-1.50 (10H₁ m), 1.08 (9H, s).

Examples 18-34 were prepared from the corresponding cyclopentyl esters as described below in Scheme 12.

Scheme 12

Example 18

5-(3-{[5-Amino-1-(4-fluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}phenoxy)-L-norvaline

Lithium hydroxide (9 mg, 0.38 mmol) was added to a solution of cyclopentyl 5-(3-{[5-amino-1- (4-fluorophenyl)-1 /7-pyrazol-4-yl]carbonyl}phenoxy)-L-norvalinate dihydrochloride (Example 1) (30 mg, 0.05 mmol) in THF/H₂O (1 :1 , 2 ml_). The reaction mixture was stirred at room temperature for 18 hours and concentrated under reduced pressure. Purification by preparative HPLC afforded the title compound as an off-white solid (15 mg, 67 % yield). A77/Z 413 [M+H]⁺. ¹H NMR (300 MHz, DMSO-d₆) 7.81 (1 H, s), 7.65-7.58 (2H, m), 7.47-7.30 (4H₁ m), 7.23 (1 H, s), 7.16 (2H, s), 4.06 (2H, s), 1.95-1.65 (4H, m).

Example 19

Λ/-[3-(3-{[5-Amino-1-(4-fluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}phenoxy)propyl]-L-leucine

From the compound of Example 2. m/z 496 [M+H]⁺. ¹H NMR (300 MHz, DMSO-Cf₆) 7.83-7.80 (1 H, m), 7.63-7.58 (2H, m), 7.47-

7.33 (4H, m), 7.24 (1 H, s), 7.16 (3H, s), 4.20-4.10 (2H, m), 3.25-3.10 (1 H, m), 3.05-2.85 (2H, m), 2.10-1.98 (2H, m), 1.90-1.70 (1 H, m), 1.55-1.40 (2H, m), 0.91-0.85 (6H, m).

Example 20 Λ/-[3-(3-{[5-Amino-1-(4-fluorophenyl)-1 /-/-pyrazol-4-yl]ca^bonyl}phenoxy)propyl]-0-te/t-butyl-L- serine

Potassium trimethylsilanolate (226 mg, 1.76 mmol) was added to a solution of cyclopentyl N- [S-^-flδ-amino-i-^-fluorophenyO-I H-pyrazol^-yllcarbonylJphenoxyJpropyO-L-leucinate (Example 3) (50 mg, 0.09 mmol) in THF (4 ml_). The reaction was stirred at 50 ⁰C for 1.5 hours, cooled and concentrated under reduced pressure. The residue was dissolved in water (10 ml.) and the pH adjusted to 7 using 1 M HCI. A precipitate was filtered, washed with a small amount of water, diethyl ether and dried to provide the title compound as a pale yellow solid (43 mg, 98 % yield).

A77/Z 499 [M+H]⁺. ¹H NMR (300 MHz, DMSO-d₆) 7.80 (1 H, s), 7.64-7.59 (2H, m), 7.48-7.33 (4H, m), 7.25 (1 H, s), 7.16 (3H, s), 4.15 (2H, t, J=5.9 Hz), 3.77-3.71 (1 H, m), 3.76-3.71 (1 H, dd, J=3.3, 3.6 Hz), 3.61-3.55 (1 H, m), 3.32-3.28 (1 H, dd, J=3.3, 3.6Hz), 3.14-3.05 (2H, m), 2.15-2.05 (2H, m), 1.14 (9H, s).

Example 21

Λ/-[3-(3-{[5-Amino-1-(2,4-difluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}phenoxy)propyl]-L-leucine

From the compound of Example 4. m/z 487 [M+H]⁺. ¹H NMR (300 MHz, DMSO-Cf₆) 7.81 (1 H, s), 7.69-7.54 (2H, m), 7.47-7.42

(1 H, m), 7.36-7.14 (6H, m), 4.13 (2H, t, J=5.9 Hz), 3.14 (1 H, t, J=3.6 Hz), 3.01-2.84 (2H, m), 2.03 (2H, t, J=6.8 Hz), 1.85-1.76 (1 H, m), 1.58-1.49 (1 H, m), 1.45-1.36 (1 H, m), 0.90-086 (6H, m). Example 22

Λ/-[4-(3-{[5-Amino-1-(2,4-difluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}phenoxy)butyl]-L-leucine

From the compound of Example 5. m/z 501 [M+H]⁺. ¹H NMR (300 MHz, DMSO-Of₆, 100 ⁰C) 7.76 (1 H, s), 7.65-7.55 (1 H, m), 7.52-

7.13 (5H, m), 6.94 (1 H, s), 4.16 (2H₁ br s), 3.11 (1 H₁ br s), 2.77-2.59 (2H, m), 1.82 (2H, br s), 1.69-1.63 (1 H, m), 1.56-1.34 (2H, m), 0.91 (6H, br s).

Example 23

Λ/-[5-(3-{[5-Amino-1-(2,4-difluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}phenoxy)pentyl]-L-leucine

From the compound of Example 6. m/z 515 [M+H]\ ¹H NMR (300 MHz, DMSOd₆) 7.71 (1 H, br s), 7.66-7.53 (1 H, m), 7.49-7.06

(6H, m), 6.97 (2H, br s), 4.08 (2H, t, J=6.2 Hz), 3.08 (1 H, t, J=6.4 Hz), 2.77-2.56 (2H, m), 1.85-1.66 (3H, m), 1.62-1.36 (6H, m), 0.92-0.88 (6H, m).

Example 24

Λ/-[6-(3-{[5-Amino-1-(2,4-difluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}phenoxy)hexyl]-L-leucine

From the compound of Example 7. m/z 529 [M+H]⁺. ¹H NMR (300 MHz₁ DMSO-(Z₆, 100 ⁰C) 7.76 (1 H₁ s), 7.66-7.58 (1 H, m), 7.48-

7.12 (5H₁ m), 6.94 (1 H₁ s), 4.08 (2H₁ 1, J=6.1 Hz), 3.09 (1 H, t, J=6.7 Hz), 2.74-2.56 (2H₁ m), 1.85-1.73 (3H, m), 1.54-1.36 (8H₁ m), 0.90 (6H₁ 1, J=5.9 Hz).

Example 25

Λ/-[3-(3-{[5-Amino-1-(2-chloro-4-fluorophenyl)-1H-pyrazol-4-yl]carbonyl}phenoxy)-propyl]-L- leucine

From the compound of Example 8. m/z 503/505 [M+H]⁺. ¹H NMR (300 MHz₁ DMSOd₆) 7.80-7.64 (3H₁ m), 7.47-7.13 (7H₁ m),

4.14 (2H₁ 1, J=6.3 Hz)₁ 3.12 (1 H₁ 1, J=6.9 Hz)₁ 2.96-2.84 (2H, m), 2.05-1.90 (2H, m), 1.84-1.73 (1 H₁ m), 1.55-1.33 (2H₁ m), 0.91-0.86 (6H₁ m).

Example 26

Λ/-[5-(3-{[5-Amino-1-(2-chloro-4-fluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}phenoxy)-pentyl]-L- leucine

From the compound of Example 9. m/z 531/533 [M+H]⁺. ¹H NMR (300 MHz, DMSO-d₆) 7.75 (1 H, s), 7.70-7.60 (2H, m), 7.46-

7.33 (3H, m), 7.25 (1 H, s), 7.14 (1 H, d, J=7.1 Hz), 6.90 (2H, br s), 4.08 (2H, t, J=5.9 Hz), 3.10 (1 H, t, J=6.6 Hz)₁ 2.76-2.59 (2H₁ m), 1.86-1.69 (3H, m), 1.61-1.36 (6H, m), 0.92-0.89 (6H, m).

Example 27

A/-[3-(3-{[5-Amino-1-(3,4-difluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}phenoxy)propyl]-L-leucine

From the compound of Example 10. m/z 487 [M+H]⁺. ¹H NMR (300 MHz, DMSOd₆, 100 ⁰C) 7.77 (1 H, s), 7.68-7.11 (6H, m), 7.05

(1 H₁ s), 4.15 (2H, br s), 3.14 (1 H₁ br s), 2.88-2.65 (2H₁ m), 2.03-1.88 (2H₁ m), 1.85-1.72 (1 H₁ m), 1.55-1.37 (2H₁ m), 0.91 (6H₁ br s).

Example 28

A/-[5-(3-{[5-Amino-1-(3,4-difluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}phenoxy)pentyl]-L-leucine

From the compound of Example 11. m/z 515 [M+H]⁺. ¹H NMR (300 MHz, DMSOd₆, 100 ⁰C) 7.77 (1 H, s), 7.68-7.13 (6H, m), 7.05

(1 H, s), 4.08 (2H₁ 1, J=8.7 Hz), 3.10 (1 H, t, J=8.7 Hz), 2.72-2.57 (2H, m), 1.78 (3H, br s ), 1.63-1.37 (6H, m), 0.91 (6H, t, J=5.1 Hz).

Example 29

Λ/-[(2f?)-3-(3-{[5-Amino-1-(2,4-difluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}phenoxy)-2- hydroxypropyl]-L-leucine

From the compound of Example 12. m/z 503 [M+H]⁺. ¹H NMR (300 MHz, CD₃OD) 7.83 (1 H₁ s), 7.66-7.58 (1 H, m), 7.50-7.20 (6H, m), 4.32-4.24 (1 H, m), 4.41-4.12 (2H, m), 3.59 (1 H, t, J=6.9 Hz), 3.20-3.13 (2H, m), 1.93-1.77 (2H₁ m), 1.69-1.60 (1 H, m), 1.03 (3H, d, J=6.2 Hz), 1.01 (3H, d, J=6.0 Hz). Example 30

/V-[(2S)-3-(3-{[5-Amino-1-(2,4-difluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}phenoxy)-2- hydroxypropyl]-L-leucine

From the compound of Example 13. m/z 503 [M+H]⁺. ¹H NMR (300 MHz, CD₃OD) 7.83 (1 H, s), 7.66-7.58 (1 H, m), 7.51-7.20 (6H, m), 4.32-4.26 (1 H, m), 4.16-4.08 (2H, m), 3.63-3.579 (1 H, m), 3.19-3.08 (2H, m), 1.93-1.79 (2H₁ m), 1.71-1.59 (1 H, m), 1.03 (3H, d, J=6.4 Hz), 1.01 (3H, d, J=6.4 Hz).

Example 31

Λ/-[2-(3-{[5-Amino-1-(4-fluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}phenyl)ethyl]-L-leucine

From the compound of Example 14. m/z 439 [M+H]⁺. ¹H NMR (300 MHz, DMSOd₆) 7.76 (1 H₁ s), 7.70-7.54 (3H, m), 7.45-7.34

(3H, m), 6.90 (2H, br s), 3.17 (1 H, t, J=6.2 Hz), 2.96-2.85 (4H, m), 1.79-1.73 (1 H, m), 1.54- 1.41 (2H, m), 0.89 (6H, t, J=5.6 Hz). Example 32

Λ/-[3-(3-{[5-amino-1-(4-fluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}phenyl)propyl]-L-leucine

From the compound of Example 15. m/z 453 [M+H]⁺. ¹H NMR (300 MHz, DMSO-Cf₆) 7.80 (1 H, s), 7.68-7.56 (3H, m), 7.48-7.37

(3H₁ m), 7.16 (2H, br s), 3.10 (1 H, t, J=6.9 Hz), 2.80-2.73 (4H, m), 1.99-1.77 (3H, m), 1.56- 1.50 (1 H, m), 1.43-1.36 (1 H, m), 0.87 (6H, t, J=7.1 Hz).

Example 33

Λ/-[4-(3-{[5-Amino-1-(4-fluorophenyl)-1/-/-pyrazol-4-yl]carbonyl}phenoxy)cyclohexyl]-L-leucine

From the compound of Example 16. m/z 509 [M+H]⁺. ¹H NMR (300 MHz, CD₃OD) 7.70 (1 H, s), 7.62- 7.57 (2H, m), 7.50-7.09 (6H, m), 4.73-4.03 (1 H, m), 3.96-3.80 (1 H, m), 3.30-3.15 (1 H, m), 2.35-2.15 (2H, m), 2.10-1.50 (9H, m), 1.04 (6H, t, J=6.9 Hz). Example 34

/V-[4-(3-{[5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]carbonyl}phenoxy)cyclohexyl]-O-te/t- butyl-L-serine

From the compound of Example 17. m/z 539 [M+H] ⁺.

Measurement of biological activities

p38 MAP Kinase activity

The ability of compounds to inhibit p38 MAPα Kinase activity was measured in an assay performed by Upstate (Dundee UK). In a final reaction volume of 25 μl_, p38 MAP Kinase α (5-1 OmU) is incubated with 25mM Tris pH 7.5, 0.02mM EGTA₁ 0.33 mg/mL myelin basic protein, 10 mM MgAcetate and [γ-³³P-ATP] (specific activity approx. 500cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 μL of a 3% phosphoric acid solution. 10 μL of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.

Duplicate data points are generated from a 1/3 log dilution series of a stock solution in DMSO. Nine dilutions steps are made from a top concentration of 10 μM, and a 'no compound' blank is included. The standard radiometric filter-binding assay is performed at an ATP concentration at, or close to, the Km. Data from scintillation counts are collected and subjected to free-fit analysis by Prism software. From the curve generated, the concentration giving 50 % inhibition is determined and reported. LPS-stimulation of THP-1 cells

THP- 1 cells were plated in 100 μL at a density of 4 x 10⁴ cells/well in V-bottomed 96 well tissue culture treated plates and incubated at 37 ⁰C in 5% CO₂ for 16 hours. 2 hours after the addition of the inhibitor in 100μl of tissue culture media, the cells were stimulated with LPS (E coli strain 005:B5, Sigma) at a final concentration of 1 μg/mL and incubated at 37 ⁰C in 5 % CO₂ for 6 hours. TNF-α levels were measured from cell-free supernatants by sandwich ELISA (R&D Systems #QTA00B)

LPS-stimulation of human whole blood

Whole blood was taken by venous puncture using heparinised vacutainers (Becton Dickinson) and diluted in an equal volume of RPM11640 tissue culture media (Sigma). 100μl was plated in V-bottomed 96 well tissue culture treated plates. 2hrs after the addition of the inhibitor in 100μl of RPM 11640 media, the blood was stimulated with LPS (E coli strain 005:B5, Sigma) at a final concentration of 100ng/ml and incubated at 37⁰C in 5% CO₂ for 6hrs. TNF-α levels were measured from cell-free supernatants by sandwich ELISA (R&D Systems #QTA00B)

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

Range A: IC50 < 10O nM

Range B: 100 nM < IC50 <1000 nM

Range C: IC50 >1000 nM

NT = not tested

Broken Cell Carboxylesterase Assay

Any given compound of the present invention wherein Ri 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 ⁰C. This was repeated twice and the final cell pellet was re-suspended in 35 ml_ of cold homogenising buffer (Trizma 10 mM, NaC1 130 mM, CaCI₂ 0.5 mM pH 7.0 at 25⁰C). Homogenates were prepared by nitrogen cavitation (700 psi for 50 min at 4 ⁰C). 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 centrifugation at 525 g for 10 min, the resulting supernatant was used as a source of esterase activity and was stored at -80 ⁰C 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 25 °C. At zero time the ester (substrate) was then added at a final concentration of 2.5 μM and the samples were incubated at 37⁰C 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 AcCN (75x2.1 mm) column and a mobile phase of 5-95 % acetonitrile in water /0.1 % formic acid. Rates of hydrolysis are expressed in pg/mUmin.

Table 1 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

Table 1

Claims

Claims:

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

wherein:

Ring A is an aryl, heteroaryl or heterocyclyl of 5-13 atoms;

Ring B is optionally substituted aryl or heteroaryl of 5-13 atoms;

Z is (a) a radical of formula -(CH₂)_Z-X¹-L¹-Y- NHCHR₁R₂ or (b) a radical of formula -(CH₂)_Z-Y¹-L¹-R, wherein:

R is a radical of formula (X) or (Y)

Ri is a carboxylic acid group (-COOH)₁ or an ester group which is hydrolysable by one or more intracellular esterase enzymes to a carboxylic acid group;

R₆ is hydrogen; or optionally substituted C₁-C₆ alkyl, C₃-C₇ cycloalkyl, aryl or heteroaryl or -(C=O)R₃, -(C=O)OR₃, or -(C=O)NR₃ wherein R₃ is hydrogen or optionally substituted (C_rC₆)alkyl. R₂ is the side chain of a natural or non-natural alpha amino acid;

Y is a bond, -C(=O)-, -Sf=O)₂-, -C(=O)O-, -Cf=O)NR₃-, -Cf=S)-NR₃ , -Cf=NH)-NR₃ or -Sf=O)₂NR₃- wherein R₃ is hydrogen or optionally substituted C₁-C₆ alkyl;

Y¹ is a bond, -Cf=O)-, -Sf=O)₂-, -Cf=O)O-, -OCf=O)-, -Cf=O)NR₃-,

-NR₃(C=O)-, -Sf=O)₂NR₃-, -NR₃Sf=O)₂-, or -NR₃(C=O)NR₄-, wherein R₃ and R₄ are independently hydrogen or optionally substituted (Ci-C₆)alkyl,

L¹ is a divalent radical of formula -(Alk¹)_m(Q)_n(Alk²)_p- wherein m, n and p are independently 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, C₁-C₆ alkylene, C₂-C₆ alkenylene, or C₂-C₆ 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 C₁-C₃ alkyl;

X¹ is a bond, -C(=0)-; or -Sf=O)₂-; -NR₄Cf=O)-, -Cf=O)NR₄-,

-NR₄Cf=O)-NR₅- , -NR₄Sf=O)₂-, or -Sf=O)₂NR₄- wherein R₄ and R₅ are independently hydrogen or optionally substituted C₁-C₆ alkyl; and

z is O or 1.

R₇ is hydrogen or -Cf=O)R' where R' is hydrogen, (CrC₆)alkyl, (C₃-C₆)cycloalkyl or (C₁- C₆)haloalkyl;

R₈ is hydrogen or (d-C₆)alkyl;

R₉ is hydrogen, halogen, hydroxyl, (C₁-C₆JaIkOXy, (Ci-CβJalkyl; Ri8 is hydrogen, halogen, hydroxyl, (C₁-C₆JaIkOXy (C_rC₆)alkyl, -NR^aR^b where R^a and R^b are hydrogen or (C₁-C₆JaIKyI, or optionally substituted aryl, heteroaryl or heterocyclyl or R^a and R^b when taken together with the nitrogen to which they are attached form a cyclic amino group of up to 6 ring atoms;

Rig is hydrogen, halogen, (C_rC₆)alkoxy, or (Ci-C₆)alkyl.

2. A compound as claimed in claim 1 wherein ring A and ring B are each phenyl rings.

3. A compound as claimed in claim 1 or claim 2 wherein R₇ is hydrogen.

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

5. A compound as claimed in any of the preceding claims wherein R₉ is hydrogen.

6 A compound as claimed in any of the preceding claims wherein R₁₈ is hydrogen.

7. A compound as claimed in claim 1 which has formula (IA) or (IB):

wherein R_ig is hydrogen, fluoro or chloro, and the other variables are as defined in claim 1.

8. A compound as claimed in any of the preceding claims wherein Z is a radical of formula -(CH₂)_z-X¹-L¹-Y-NHCHR₁ R₂.

9. A compound as claimed in claim 8 wherein R₁ is an ester group of formula -(C=O)OR₁₀ wherein R₁₀ is R₁₁R₁₂R_1SC- wherein (i) R₁₁ is hydrogen or optionally substituted (Ci-C₃)alkyl-(Z¹)_a-[(Ci-C3)alkyl]_b-, (C₂- C₃)alkenyl-(Z¹)_a-[(CrC3)alkyl]_b- or phenyl-(Z¹)_a-[(Ci-C3)^alky']b-. wherein a and b are independently 0 or 1 and Z¹ is -O-, -S-, Or -NR₁₄- wherein R₁₄ is hydrogen or (C₁- C₃)alkyl; and R₁₂ and R₁₃ are independently hydrogen or (CrC^alkyl-;

(ii) R₁₁ is hydrogen or optionally substituted R^R^N-^-QOalkyl- wherein R₁₅ is hydrogen, (CrC₃)alkyl or phenyl, and Ri₆ is hydrogen or (C_rC₃)alkyl; or R₁₅ and R₁₆ 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 R₁₂ and R₁₃ are independently hydrogen or (d-C₃)alkyl-;or

(iii) R₁₁ and R₁₂ 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 R₁₃ is hydrogen.

10. A compound as claimed in claim 8 wherein R₁ is a methyl, ethyl, n- or iso-propyl, n-, sec- or tert-butyl, cyclohexyl, allyl, phenyl, benzyl, phenylethyl, 4-fluorophenylethyl, 2-, 3- or 4-pyridylmethyl, N-methylpiperidin-4-yl, tetrahydrofuran-3-yl, methoxyethyl, indanyl, norbomyl, dimethylaminoethyl, or morpholinoethyl ester group.

1 1. A compound as claimed in claim 8 wherein R₁ is a cyclopentyl ester group.

12. A compound as claimed in any of claims 8 to 11 wherein R₂ is iso-propyl, cyclohexyl, t-butoxymethyl, t-butylthiomethyl, pyrrolidin-2-yl, benzyl, isobutyl or phenyl.

13. A compound as claimed in any of claims 8 to 12 wherein Z is selected from: R₁R₂CHNH-CH₂-, R₁R₂CHNH-CH₂CH₂-, R₁R₂CHNH-CH₂CH₂CH₂-,. R₁R₂CHNH-CH₂-O-, R₁R₂CHNH-CH₂CH₂-O-, R₁R₂CHNH-CH₂CH₂CH₂-O-^₁R₂CHNHSO₂-, R₁R₂CHNHCO-, R₁R₂CHNHCH₂-, R₁R₂CHNH(CH₂)_SO- and the following: R₁R₂CHNH -(CH₂)_{O 1} -Y V— (CH₂)_{0 1} — ( V¹)_{0 1} -)-

R₁R₂CHNH -(CH₂)_{O 1} -/ ^ (CH₂)_{0 1} ( V¹)_Ol1 -f-

wherein Y is C or N₁ V is C or N, and V¹ is O, S or NH.

14. A compound as claimed in any of claims 8 to 12 wherein Z is:

15. A compound as claimed in any of claims 8 to 12 wherein -(CH₂)_Z-X¹-L¹-Y- in Z is selected from (i) -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, and -CH₂CH₂CH₂CH₂- in any of which a carbon is optionally substituted by hydroxy; or (ii) -OCH₂-, -OCH₂CH₂-, -OCH₂CH₂CH₂-, and -OCH₂CH₂CH₂CH₂- wherein the oxygen is linked to the ring A; or (iii) -NHCH₂-, NHCH₂CH₂-, -NHCH₂CH₂CH₂-, and -NHCH₂CH₂CH₂CH₂- wherein the nitrogen is linked to the ring A; or (iv) a divalent cyclohexyl radical.

16. A compound as claimed in any of claims 1 to 7 wherein Z is a radical of formula R-L¹-Y¹-(CH₂)_Z-.

17. A compound as claimed in claim 16 wherein R₁ is an ester group as defined in claim 9.

18. A compound as claimed in claim 16 wherein R₁ 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, norbomyl, dimethylaminoethyl, or morpholinoethyl ester group.

19. A compound as claimed in claim 16 wherein R₁ is a cyclopentyl or tert-butyl ester group.

20. A compound as claimed in any of claims 16 to 19 wherein -(CH₂)_Z-Y¹-L¹- in Z is selected from (i) -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, and -CH₂CH₂CH₂CH₂- in any of which a carbon is optionally substituted by hydroxy; or (ii) -OCH₂-, -OCH₂CH₂-, -OCH₂CH₂CH₂-, and -OCH₂CH₂CH₂CH₂- wherein the oxygen is linked to the ring A; or (iii) -NHCH₂-, NHCH₂CH₂-, -NHCH₂CH₂CH₂-, and -NHCH₂CH₂CH₂CH₂- wherein the nitrogen is linked to the ring A.

21. A compound as claimed in claim 1 selected from the group consisting of:

Cyclopentyl 5-(3-{[5-amino-1 -(4-fluorophenyl)-1 /-/-pyrazol-4-yl]carbonyl}phenoxy)-L- norvalinate,

Cyclopentyl Λ/-[5-(3-{[5-amino-1 -(2,4-difluorophenyl)-1 H-pyrazol-4-yl]carbonyl}- phenoxy)pentyl]-L-leucinate,

Cyclopentyl Λ/-[6-(3-{[5-amino-1 -(2,4-difluorophenyl)-1 /7-pyrazol-4-yl]carbonyl}- phenoxy)hexyl]-L-leucinate,

Cyclopentyl Λ/-[5-(3-{[5-amino-1 -(3,4-difluorophenyl)-1 H-pyrazol-4- yl]carbonyl}phenoxy)pentyl]-L-leucinate,

Cyclopentyl Λ/-[(2/?)-3-(3-{[5-amino-1 -(2,4-difluorophenyl)-1 H-pyrazol-4-yl]carbonyl}phenoxy)- 2-hydroxypropyl]-L-leucinate,

Cyclopentyl Λ/-[(2S)-3-(3-{[5-amino-1-(2,4-difluorophenyl)-1 H-pyrazol-4-yl]carbonyl}phenoxy)- 2-hydroxypropyl]-L-leucinate,

and hydrates, solvates and pharmaceutically acceptable salts thereof.

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

23. The use of a compound as claimed in any of claims 1 to 21 in the preparation of a composition for inhibiting the activity of a p38 MAP kinase enzyme in vitro or in vivo.

24. The use of a compound as claimed in any of claims 1 to 21 in the preparation of a composition for the treatment of autoimmune or inflammatory disease

25. A method of inhibiting the activity of a p38 MAP kinase enzyme comprising contacting the enzyme with an amount of a compound as claimed in any of claims 1 to 21 effective for such inhibition.

26. A method for the treatment of autoimmune or inflammatory disease which comprises administering to a subject suffering such disease an effective amount of a compound as claimed in any of claims 1 to 21.

27. The use as claimed in claim 24 or method as claimed in claim 26 wherein the disease is psoriasis, inflammatory bowel disease, Crohns disease, ulcerative colitis, chronic obstructive pulmonary disease, asthma, multiple sclerosis, diabetes, atopic dermatitis, graft versus host disease, or systemic lupus erythematosus.

28. The use as claimed in claim 24 or method as claimed in claim 26 wherein the disease is rheumatoid arthritis.