P38 inhibitors and methods of use thereof

Oescription

BACKGROUND OF The invention

Field of The invention.

[0001] This invention relatesto novel inhibitorsof p38 MAP kinaseand related kinases, pharmaceutical compositions containing the inhibitors, and methods for preparíng these inhibitors. They are useful for the treatment of inflammation, osteoarthritis, rheumatoid arthritis, psoriasis, Crohn's disease, ínflammatory bowel disease, cancer, autoimmune dis-eases, and for the treatment of other cytokine-medíated diseases.

Description of the state of the art.

[0002] A number of chronic and acute inflammatory conditions have been associated with the overproduction of proinflammatory cytokines. Such cytokines include but are not limited to tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1P), interleukin 8 (IL-8) and interleukin 6 (IL-6). Rheumatoid Arthritis (RA) is a chronic disease where TNF-a and IL-1 β are implicated in the onset of thediseases and in the progression of the bone and joint destruction seen with this debi litating condition. Recently approved therapeutic treatments for RA have included soluble TNF-areceptor (etanercept) and IL-1 receptor antagonist (anakinra). These treatments work by blocking the ability of their respective cytokines to bind to their natural receptors. Alternative methods to treat cytokine-mediated diseases are currently under investigation. One such method involves inhibition of the signaling pathway that regulates the synthesis and production of proinflammatory cytokines like p38.

[0003] P38 (also CSBP or RK) is a serine/threonine mitogen-activated protein kínase (MAPK) that has been shown to regulate pro-inflammatory cytokines. P38 was first identified as a kinase which became tyrosine phosphorylated in mouse monocytes following treatment with Iípopolysaccharide (LPS). A link between p38 and the response of cells to cytokines was first established by Saklatvala J., et al., Cell, 78: 1039-1049 (1994), who showed that IL-1 activates a protein kinase cascade that results in the phosphorylatíon of the small heat shock protein, Hsp27, probably by mitogen-activated protein activated protein kinase 2 (MAPKAP kinase-2). Analysis of peptide sequences derived fram the purified kinase indicated that it was related to the p38 MAPK activated by LPS in mouse monocytes, Han, J., et al., Science, 265: 808-811 (1994). At the same time it was shown that p38 MAPK was itself activated by an upstream kinase in response to a variety of cellular stresses, induding exposure to UV radiation and osmotic shock, and the identity of the kinase that directly phosphorylates Hsp27 was confirmed as MAPKAP kinase-2, Rouse, J., et al., Cell, 78:1027-1037 (1994). Subsequently, workers at SmithKline Beecham showed that p38 MAPK was the molecular target of a seríes of pyridinylimidazole compounds that inhibited' the production of TNF from LPS-challenged human monocytes, Lee, J., et alNature, 372: 739-746. This was a key discovery and has led to the development of a number of selective inhibitors of p38 MAPK and the eluddation of its role in cytokine signaling.

[0004] It is now known that multiple forms of p38 MAPk (α, β, γ, δ), each encoded by a separate gene, form part of a kinase cascade involved in the response of cells to a variety of stimuli, induding osmotic stress, UV light and cytokine mediated events. These four ísoforms of p38 are thought to regulate different aspects of intracellular signaling. Its acti vation is part of a cascade of signaling events that lead to the synthesis and production of pro-inflammatory cytokines likeTNF-a. P38 functions by phosphorylating downstream substrates that include other kinases and transcription factors. Agents that inhibit p38 kínase have been shown to block the production of cytokines induding but not limited to TNF-a, IL-6, IL-8 and IL-1 β in vitro and in vivo models Adams, J. L., et al., Progress in Medidnal Chemistry, 38: 1-60 (2001). [0005] Peripheral blood monocytes (PBMCs) have been shown to express and secrete pro-inflammatory cytokines when stimulated with lipopolysaccharide (LPS) in vítro. P38 inhibitors efficiently block this effect when PBMCs are pretreated with such compounds priorto stimulation with LPS. Lee, J.C., et al., Int. J. Immunopharmacol., 10: 835-843 (1988). The efficacy of p38 inhibitors in animal models of inflammatory disease has prompted an investigation of the underlying mechanism(s) which could account for the effect of these inhibitors. The role of p38 in the response of cells to IL-1 and TNF has been investigated in a number of cells systems relevant to the inflammatory response using a pyridinyl imidazole inhibitor: endothelial cells and IL-8, Hashimoto, S., et al., J, Pharmacol. Exp. Ther., 293: 370-375 (2001), fibroblasts and IL-6/GM-CSF/PGE2 Beyaert, R., et al., EMBO J., 15: 1914-1923 (1996), neutrophils and IL-8 Albanyan, E. A., et al., Infect. Immun., 68: 2053-2060 (2000) macrophages and IL-1 Caivano, M. and Cohen, P., J. Immunol., 164: 3018-3025 (2000), and smooth musde cells and RANTES Maruoka, S., et al„ Am. J. Respir. Crit. Care Med., 161: 659-668 (1999). The destructive effects of many disease states are caused by the over production of proinflammatory cytokines. The ability of p38 inhibitors to regulate this overproduction makes them excellent candidates for disease modifying agents.

[0006] Inhibitors of p38 are active in a variety of widely recognized disease models and show positive effects in a number of standard animal models of inflammation induding rat collagen-induced arthritis, Jackson, J.R., et al., J.

Pharmacol. Exp. Ther., 284: 687-692 (1998); rat adjuvant-induced arthritis, Badger, A. M., et al., Arthritís Rheum., 43: 175-183 (2000); Badger, A. M., et al., J. Pharmacol. Exp. Ther., 279:1453-1461 (1996); and carrageenan-induced paw edema ín the mouse, Nishikori, T., et al., Eur. J. Pharm., 451: 327-333 (2002). Moleculesthat block p38’s function have been shown to be effective in inhibiting bone resorption, inflammation, and other immune and inflammation-based pathologies in these animal models. Thus, a safe and effective p38 ínhibitor would provide a means to treat debilitating diseases that can be regulated by modulation of p38 signaling like, but not limited to, RA.

[0007] P38 inhibitors are well known to those skilled in the art. Reviews of early inhibitors have helped establish the structure activity relationshipsimportantforenhanced activity both in vitro and in vívo. See, Salituro, E. G., etal., Current Medicínal Chemistry, 6: 807-823 (1999) and Foster, M. L., et al., Drug News Perspact., 13: 488-497 (2000). More contemporary reviews have focused on the structural diversity of new In h ibitors being explored as p38 inhi bitors Boehm, J. D. and Adams, J. L., Exp. Opin.Ther. Patents, 10:25-37 (2000). This invention describes a novel seriesofsubstituted 2-aza-[4.3.0]-bicyclic hereroaromatic compounds as p38 inhibitors that are useful for the treatment of inflammation, osteoarthritis, rheumatoid arthritis, cancer, autoimmune diseases, and for the treatment of other cytokine mediated diseases.

SUMMARY OF The invention [0008] This Invention provides compounds and pharmaceutical compositions containing them that inhibit p38 alpha and the associated p38 mediated events such as the inhibition of cytokine production. Such compounds, generally referred to as 2-aza-[4.3.0] bicyclic heteroaromatic rings, have utility as therapeutic agents for diseases that can be treated by the inhibition of the p38 signaling pathway.

BRIEF DESCRIPTION OF THE FIGURES [0009] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate embodiments ofthe present invention, and togetherwith the description, serve to explain the principles of the ínvention, In the figures,

Figures 1A-1C show a reaction scheme for the synthesis of compounds having the generic structure 11g;

Figure 2 shows a reaction scheme for the synthesis of compounds having the generic structure 1j;

Figure 2 shows a reaction scheme for the synthesis of compounds having the generic structure 1m;

Figure 4 shows a reaction scheme for the synthesis of compound 17d.

Figure 5 shows a reaction scheme for the synthesis of compound 26d.

DETAILED DESCRIPTION OF The invention [0010] The present invention provides a compound, including solvates and pharmaceutically acceptable salts thereof, which is selected from:

5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid amide;

[5-(4-fluorophenoxy)-1-isobutyl-1H-indazol-6-yl]morpholin-4-yl-methanone;

[5-(4-fluorophenoxy)-1-isobutyl-1H-indazol-6-yl]-(4-methylpiperazin-1-yl)methanone;

5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (1 -benzyl-piperidin-4-yí)amide;

5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-benzylaminoethyl)amide;

5-(4-fluorophenoxy)-1-isobutyl-1H-indazoie-6-carboxylic acid (2-piperidin-yl-ethyl)amide;

5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-pyrrolidin-1 -ylethyl)amide;

5-(4-fluorophenoxy)-1-isobiityl-1H-indazole-6-carboxylic acid (3-morpholin-4-ylpropyl)amide;

5-(4-fluorophenoxy)-1-isobutyl-1 H-indazole-6-carboxylic acid (3-dimethylaminopropyl)amide;

5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-dimethylaminoethyl)amide;

5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid methyl-(1-methylpiperidin-4-yl)amide;

5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid [3-(methylphenylamino)-propyl]amide;

3- {[5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amino}-pyrrolidine-1-carboxylic acid tert-butyl ester;

5-(4-fluorophenoxy)-1 -(2,2,2-trifluoroethyl)-1 H-indazole-6-carboxylic acid (2-dimethylaminoethyl) amide;

5-(4-fluorophenoxy)-1-methyl-1H-indazole-6-carboxylic acid (2-dimethyl-aminoethyl) amide;

5-(4-fluorophenoxy)-1H-indazole-6-carboxylic acid (2-dimethylaminoethyl) amide;

4- amino-2-{[5-(4-fluorophenoxy)-1-isobutyl-1 W-indazole-6-carbonyl]-amino} butyric acid methyl ester;

4-amino-2-{[5-(4-fluorophenoxy)-1-(2,2,2-trifluoroethyl)-1H-indazole-6-carbonyl]-amino} butyric acid methyl ester;

4- amino-2-{[5-(4-fluorophenoxy)-1-methyl-1H-indazole-6-carbonyl]-amino} butyric acid methyl ester;

(S)-N-(4-amino-1-hydroxybutan-2-yl)-5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxamide;

(S)-methyl 2-(5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxamido)-4-(dimethylamino)butanoate;

(S)-5-(2,4-difluorophenoxy)-N-(4-(dimethylamino)-1-hydroxybutan-2-yl)-1-isobutyl-1H-indazole-6-carboxamide;

(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (1-hydroxymethyl-3-isopropylaminopropyl) amide;

(S)-2-{[5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amíno}-4-dimethylaminobutyríc acid;

(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (1 -hydroxymethyl-3-piperidin-1 -ylpropyl) amide;

(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (3-dimethylamino-1-dimethylcarbamoylpro-pyl)amide;

(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (3-dimethylamino-1-methylcarbamoylpropyl) amide;

(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (1-carbamoyl-3-dimethylaminopropyl)amide;

(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid [1-(2-dimethylaminoethyl)-2-hydroxy-2-meth-ylpropyl]amide;

(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylicacid {1-hydroxymethyl-3-[(2-methoxyethyl)methyl-amino]propyl}amide;

(S)-5-(2,4-difluorophenoxy)-1 -isobutyl-1 H-indazole-6-carboxylic acid [3-dimethylamino-1-(2-hydroxyethylcar-bamoyl)propyl]amide;

5- (2,4-difluorophenoxy)-1 -isobutyl-1 H-indazole-6-sulfonic acíd (3-dimethylaminopropyl)amide;

(S)-methyl 2-(5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-sulfonamido)-4-(dimethylamino)butanoate;

5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-sulfonic acíd [2-(1 -metbylpyrrolidin-2-yl)-ethyl]amide; and

5-(2,4-difluorophenoxy )-1 -isobutyl-1 H-indazole-6-sulfonic acid (2-dimethylaminoethyl)amide.

[0011] The compounds may be represented by the general formula I <img file="IS2675B_D0001.tif" /> [0012] The term "solvate" refers to an aggregate of a molecule with one or more solvent molecules.

[0013] A ''pharmaceutically acceptable salt" is a salt that retains the biological effectiveness of the free acids and bases of the specified compound and that is not biologically orotherwíse undesirable. Acompound of The invention may possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable sale. Examples of pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base, such salts including sulfates, pyrosulfates, bisulfates, sulfites, bísulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlo-rides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, iso-butyrates, caproates, heptanoates, proplolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dio-ates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitro-menzoates, hydroxybenzoates, meth-oxybenzoates, phthalates, sulfonates, xylenesulfonates, pheylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycollates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalone-2-sulfonates, and mandelates.

[0014] If the inventive compound is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acíd, such as acetic acid, maleic acid, succinic acíd, mandelic acid.fumaricacid, malonic acid, pyruvic acid, oxalic acid, glycolicacid, salicylic acid, a pyranosidyl acid, such as glucuronicacid orgalacturonicacid, an alphahydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

[0015] If the inventive compound is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acíd with an inorganic or organíc base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include, but are not límited to, organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amínes, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

[0016] The inventive compounds may be prepared using the reaction routes and synthesis schemes as described below, employing the techniques available In the art using starting materials that are readily available.

[0017] Figures 1A-1C showan exampleofthe synthesis of a specific compound having the general Formula I. In one general synthetic process, compounds of Formula I are prepared as follows. 1-Fluoro-3-methyl-benzene undergoes an addition reaction to form 2-fluoro-4-methylbenzoic acid, followed by nitration to provide 2-fluoro-4-methyl-5-nitrobenzoic acid. The acid group is esterified, and then the fluoro group is replaced by ArO-upon treatment with ArOH.and a strong base. Reduction of the nitro group followed by diazotization and cyclization provides the 5-OAr-S-CO₂Me indazole derivative, which is then treated with RBr in the presence of base to provide the 1-N substituted derivative. Hydrolysis of the ester group followed by amidation provides the 6-amide indazole derivative having Formula I.

[0018] Therapeutically effective amounts of the compounds of The invention may be used to treat diseases mediated by modulation or regulation of protein kinases. An "effective amount" is intended to mean thatamount ofcompound that, when administered to a mammal in need of such treatment, is sufficient to effect treatment for a disease mediated by

<img file="IS2675B_D0002.tif" />

the activity of one or more protein kinases, such as that p38 alpha and the associated p38 mediated events such as cytokine production. Thus, forexample, a therapeutically effective amount of a compound of Formula I or a salt, thereof, is a quantity sufficient to modulate, regulate, or inhibit the activity of one or more proteín kinases such that a disease condition which is mediated by that actívity is reduced or alleviated.

[0019] The amount of a given agent that will correspond tosuch an amount will vary depending uponfactors such as the particula r compound, disease condition and its severity, the identity (e:g„ weíght) of the mammal in need of treatment, but can nevertheless be routi nely determined by one skilled in the art. "Treating" is intended to mean at least the m itigation of a disease condition ín a mammal, such as a human, that is affected, at least in part, by the activity of one or more protein kinases, such as p38, and indudes, but is not limited to, preventing the disease condition from occurríng in a mammal, particularly when themammal isfound tobe predisposed to having thedisease condition buthas not yet been diagnosed as having it; modulating and/or inhibiting the disease condition; and/or alleviating the disease condition. [0020] In order to use a compound of the Formula I, or a pharmaceutically acceptable salt thereof, for the therapeutic treatment (including prophylactic treatment) of mammals including humans, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition. According to this aspect of The invention there is provided a pharmaceutical composition that comprises a compound of the Formula I, or a pharmaceutically acceptable salt thereof, as defined hereinbefore in association with a pharmaceutically acceptable diluent or carrier.

[0021] The compositions of The invention may be In a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example was afinely divided powder) orfor parenteral administration (for exampleas a sterile aqueous or oily solutionfor intravenous, subcutaneous, or intramuscular dosing or as a suppository for rectal dosing). For example, compositions intended for oral use may contain, for example, one or more coloring, sweetening, flavoring and/or preservative agents.

[0022] Suitable pharmaceutically-acceptable excipients for a tablet formulation include, for example, inert díluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and d isi ntegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of theactive ingredient within the gastrointestinal tract, orto improve theirstabilityand/orappearance, in either case, using conventional coating agents and procedures well known ín the art.

[0023] Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

[0024] Aqueous suspensions general ly contain the active ing redient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium algínate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (forexample polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, orcondensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), coloring agents, flavoring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame), [0025] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavoring agents may be added to provida a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0026] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavoring and coloring agents, may also be present.

[0027] The pharmaceutical compositions of The invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture ofany ofthese. Suitable emulsifying agents may be, forexample, naturally-occurring gums such asgum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring and preservative agents.

[0028] Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavoring and/or coloring agent.

[0029] The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.

[0030] Suppository formulations may be prepared by mixing the active ingredient with a suitable non-i rritating excipient which is solid at ordinary temperatures but liquid atthe rectal temperature and wilí therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.

[0031] Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedures well known in the art.

[0032] Compositions for administration by insuff lation may be in the form of a finely divided powder containing particles of average diameter of, for example, 30 pm or much less, the powder itself comprising either active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insufflation is then conveniently retained in a capsule containing, for example, 1 to 50 mg of active ingredient for use with a turboinhaler device, such as is used for insufflation of the known agent sodium cromoglycate.

[0033] Compositions for administration by inhalation may be in the form of a conventional pressurized aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.

[0034] For further information on formulations, see Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0035] The amount of a compound of this invention that is combined with one or more excipients to produce a single dosageform willnecessarilyvarydependinguponthe hosttreatedandtheparticularrouteofadministration.Forexample, a formulation intended fororal administratíon to humans will may contain, forexample, from0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on routes of administration and dosage regimes, see Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0036] The size of the dose for therapeutic or prophylactic purposes of a compound of Formula I will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of admin-istration, according to well known principles of medicine.

[0037] In one aspect of this invention, the compounds of this ínvention or pharmaceutical salts thereof may be formulated into pharmaceutícal compositions for administration to animals or humans to treat or prevent a p38-mediated condition. The term "p38-mediated condition." as used herein means any disease or other deleterious condition in whích p38 is known to play a role. This includes conditions which are known to be caused by IL-1, TNF, IL-6 or IL-8 overproduction. Such conditions include, without limitation, inflammatory diseases, autoimmune díseases, destructive bone disorders, proliferative disorders, infectious diseases, viral disease, and neurodegenerative diseases [0038] Inflammatory diseases which may be treated or prevented include, but are not limited to, acute pancreatitis, chronic pancreatitis, asthma, allergies, and adult respiratory distress syndrome.

[0039] Autoimmune diseases which may be treated or prevented include, but are not limited to, glomeralonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves’ dísease, autoimmune gas-tritis, insulin-dependent diabetes mellitus (Type I), autoimmune hemolytic anemia, autoimmune neutropenia, thrombo-cytopenia, atopic dermatitis, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, psoriasis, or graft vs. host disease.

[0040] Destructive bone disorders which may be treated or prevented inciude, but are not limited to, osteoporosis, osteoarthritis and multiple myeloma-related bone disorder.

[0041] Proliferative diseases which may be treated or prevented include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma.

[0042] Infectious diseases which may be treated or prevented include, but are not limited to, sepsis, septic shock, and Shigellosis.

[0043] Viral díseases which may be treated or prevented include, but are not limited to, acute hepatitis infection (including hepatitis A; hepatitis B and hepatitis C), HIV infection and CMV retinitis.

[0044] Degenerative conditions or diseases which may be treated or prevented by the compounds of this invention include, but are not limited to, Alzheimer’s disease, Parkinson's dísease, cerebral ischemia and other neurodegenerative diseases.

[0045] "p38-mediated conditions" also include ischemia/reperfusion in stroke, heart attacks, myocardial ischemia, organ hypoxia, vascular hyperplasia, cardiac hypertrophy and thrombin-induced platelet aggregation.

[0046] In addition, the p38 inhíbitors of this invention are also capable of inhibiting the expression of inducible proinflammatory proteins such as prostaglandin endoperoxide synthase-2 (PGHS-2), also referred to as cydooxygenase-2 (COX-2). Therefore, other "p38-mediated conditions" are edema, analgesia, fever and pain, such as neuramuscular pain, headache, cancer pain, dental pain and arthritis pain.

[0047] The conditions and diseases that may be treated or prevented by the p38 inhibitors of this invention may also be conveniently grouped by the cytokine (e.g., IL-1, TNF, IL-6, IL-8) that is believed to be responsible forthe disease. [0048] Thus, an IL-1-mediated disease or condition includes rheumatoid arthritis, osteoarthritis, stroke, endotoxemia and/or toxic shock syndrome, inflammatory reaction induced by endotoxin, inflammatory bowel disease, tuberculosis, atherosclerosís, muscel degeneration, cachexia, psoriatic arthritis, Reiter’s syndrome, gout, traumatic arthritis, rubella arthritis, acute synovitis, diabetes, pancreatic β-cell disease and Alzheimer’s disease.

[0049] ATNF-mediated disease or condition includes rheumatoid arthritis, rheumatoid spondy litis, osteoa rthritis, gouty arthritis and other arthritic conditions, sepsis, septicshock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosís, pulmonary sarcoisosís, bone resorption diseases, reperfusion injury, graft vs. host reaction, allograft rejections, fever and myalgias due to infection, cachexia secondary to infection, AIDS, ARC or malignancy, keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis or pyresis. TNF-mediated diseases also include viral infections, such as HIV, CMV, influenza and herpes; and veterinary viral infections, such as lentivirus infections, íncluding, but not limited to equine infectious anaemia virus, caprine arthritis virus, visna virus or maedi virus; or retrovirus infections, including feline immunodeficiency virus, bovine immunodeficiency virus, or canine imrriunodeficiency virus.

[0050] IL-8 mediated disease or condition includes diseases characterized by massive neutrophil infiltration, such as psoriasis, inflammatory bowel disease, asthma, cardiac and renal reperfusion injury, adult respiratory distress syndrome, thrombosis and glomeriilonephritis, [0051] ln addition, the compounds of this infection may be used topically to treat or prevent conditions caused or exacerbated by IL-1 or TNF. Such conditíons ínclude ínflamed joints, eczema, psoriasis, inflammatory skin conditions such as sunburn, inflammatory eye conditions such as conjunctivitis, pyresis, pain and other conditions associated with inflammation.

[0052] The compounds of this invention may be used in combination with other drugs and therapies used in the treatment of disease states which would benefit from the inhibition of cytokines, in particular IL-1, TNF, IL-6 or IL-8. [0053] For example, by virtue of their ability to inhibit cytokines, the compounds of Formula I are of value in the treatment of certain inflammatory and non-inflammatory diseases which are currently treated with a cyclooxygenase-inhibitory non-steroidal anti-inflammatory drug (NSAID) such as indomethacin ketorolac, acetylsalicylic acid, ibuprofen, sulindac, tolmetin and píroxicam. Co-administration of a compound of the Formula I with a NSAID can result in a reduction of the quantity of the latter agent needed to produce a therapeutíc effect, and thus the likelihood of adverse side-effects fram the NSAID such as gastrointestinal effects are reduced. Thus according to a furtherfeature of The invention there is provided a pharmaceutical composition which comprises a compound of Formula I, or a pharmaceutically-acceptable saltthereof, In conjunction or admixture with a cyclooxygenase inhibitory non-steroidal anti-inflammatory agent, and a pharmaceutically-acceptable diluent or carrier.

[0054] The compounds of Formula I may also be used In the treatment of conditions such as rheumatoid arthritis In combination with antiarthritic agents such as gold, methotrexate, steroids and penicillinamine, and In conditions such as osteoarthritis in combination with steroids.

[0055] The compounds of the present invention may also be administered in degradative diseases, for example osteoarthritis, with chondroprotective, anti-degradative and/or reparative agents such as Diacerhein, hyaluronic acid formulations such as Hyalan, Rumalon, Arteparon and glucosamine salts such as Antril.

[0056] The compounds of Formula I may also be used In the treatment of asthma in combination with antiaslhmatic agents such as bronchodilators and leukotriene antagonists.

[0057] Although the compounds of Formula I are primarily of value as therapeutic agents for use In warm-blooded animals (including man), they are also useful whenever it Is required to inhibit the effects of cytokines. Thus, they are useful as pharmacological standards for use in the development of new biologícal tests and in the search for new pharmacological agents.

[0058] The actívity of the compounds ofthis invention may be assayed for p38 inhibition in vitro, in vívo, or in a cell line. In vitro assays include assays that determine inhibition of either the kinase activity or ATPase activity of activated p38. Alternate in vitro assays quantitate the ability of the inhibitor to bind to p38 and may be measured either by radi-olabeliing the inhibitor prior to binding, isolating the inhibitor/p38 complex and determining the amount of radiolcibel bound, or by running a competition experiment where new inhibitors are incubated with p38 bound to known radioligands. These and other useful in vitro and cell culture assays are well known to those of skill in the art.

[0059] Cell culture assays of the inhibitory effect of the compounds of this invention may be used to determine the amounts of TNF-oc, IL-1, IL-6 or IL-8 produced in whole blood or cell fractíons thereof in cells treated with inhibitor as compared to cells treated with negative controls. Level of these cytokines may be determined through the use of commercially available ELISAs or as described in the Biological Examples section below.

BIOLOGICAL EXAMPLES [0060] The biological activities of the compounds of The invention were demonstrate by the following in vitro assays.

p38 Biochemical Assay [0061] P38 activity was assayed at room temperature in a 100 μΙ reaction containing 5 nM activated p38cc enzyme and 1 uM ATF-2 (Activating Transcription Factor 2 fusion protein) as the substrate in 25mM HEPES (pH 7.4), 100 μΜ Vanadate, 1 mM DTT, 10 mM MgCI₂and 10 μΜ [C]-³³P]-ATP (-0.1 μθί P³³/reaction). The reaction was terminated after 30-40 minutes by adding 25% TCA, let stand for 5 minutes and then transferred directly to a GF-B membrane filter plate. The filter was washed twice for 30 seconds with 0.5% phosphoric acid using a Tomtec Mach III Automated Harveslor. After washing, the vacuum was continued for 30 seconds to dry the filter. Approximately 30 μΙ of scintillant was added per well to the filter plate and then read in a Liquid Scintillation Counter (Packard TopCount HTS).

PBMC Assay [0062] The ability of compounds of this invention to inhibit TNF-α production was assessed by using human peripheral blood mononuclear cells ("PBMC") whích synthesize and secrete TNF-oe when stimulated with lipopolysaccharide. [0063] Compound test solutions were made by making 5 fold serial dilutions in DMSO, which dilutions were then diluted to 5x stocks by diluting with MEM, 2% heat inactivated fetal bovine serum ("FBS"), 20 mM HEPES, 2mM L-glutamine, and 1% penicillin/streptomycin.

[0064] PBMC’s were isolated from human blood as follows. Whole blood samples were collected from human volunteers into Vacutainer™ CPT from Becton Dickinson. Tubes were mixed and centrifuged at room temperature (18 - 25° C) in a horizontal rotorfora minimum of 15 minutes at 1500 -1800 RCF (relative centrifugal force). For each donor, the buffy coat layers were pooled into a single tube and washed twice with phosphate buffered saline ("PBS"). The cell pellet was resuspended in MEM, 2% heat inactivated fetal bovine serum ("FBS"), 20 mM HEPES, 2mM L-glutamine, and 1% penicillin/streptomycin. Total cell numberwas determined using a hemocytometerand the cell suspension was adjusted to 2 X 106 cells/mL.

[0065] 0.1 mL of cell suspension was added to each well of a 96-well cell culture plate. 30 μι. of a compound test solution was added, and the cells were incubated in a 37°C/5% CO2 incubatorfor 1 hour. 20 μΙ_ of 7.5 ng/mL lipopoly-saccharide (LPS E. Coli K-235) was then added to each well, and the cells were returned to the 37°C/5% CO₂ incubator for 16-20 hours. The cells were centrifuged for 15 minutes at 1100 RCF. Approximately 0.12 mL ofthe supernatant was transferred into a clean 96 well polypropylene plate. The samples were either assayed immediately or were stored at -80°C until ready for assay. TNF-α levels were determined in each sample using a human TNF-α ELISA assay such as that described below.

[0066] TNF-α levels were determined using the following assay. TNF-alpha antíbody coated plates were prepared by addíng 150 μι. of 2 μο/ητί anti-TNF-α purified mouse monoclonal IgG in Carbonate-Bicarbonate buffer (BupH™ Carbonate-Bicarbonate Buffer Pack) to wells of a 96-well Immulon 4 plate (Immulon 4 ELISA Flat Bottom Plate; Dynex, catalog number 011-010-3855) and incubated overnight at 2 - 8 °C. Coating solution was removed and 200 μί of "blocking buffer" (20 mM HEPES pH 7.4,150 mM NaCI, 2% BSA) was added and plates were stored 2 - 8“C until ready to use. A ten-point recombinant human TNF-α standard curve was prepared by a 1:2 serial dilution in "sample diluent" (20 mM HEPES, pH 7.4, 150 mM NaCI, 2 mM MgCI₂,1 % BSA) with a top concentration of 6000 pg/mL.

[0067] Blocking solution was removed from TNF-α ELISA plates by washing five times wíth 300 μι. of "wash buffer" (20 mM HEPES, pH 7.4, 150 mM NaCI, 2 mM MgCI₂, 0.02% Tween-20). 50 μι. of "sample díluent" was added to all wells, and then either 50 μι. of a TNF-α standard curve solution or test compound supernatant was added to all wells. The plate was incubated at room temperature for one hour with shaking (300 rpm). The plate was washed wash five times with 300 μΙ_ "wash buffer". 100 μΙ. of 0.2 μg/mL biotinylated goat anti-human TNF-α in "antibody diluent" (20 mM HEPES, pH 7,4, 150 mM NaCI, 2 mM MgCI₂, 1 % BSA, 0.02% Tween-20) was added per well, and the plate was incubated at room temperature for one hour with shaking (300 rpm). The plate was washed wash five times wíth 300 μι "wash buffer” per well. 100 μι. of 0.02 p.g/mL streptavidín alkaline phosphatase in "antibody diluent" was added per well, and the plate was incubated at room temperature for one hour with shaking (300 rpm). The plate was washed wash five times with 300 μΙ_ wash buffer per well. 200 μΙ_ of 1 mg/mL pNPP (p-n itropheny I phosphate) in diethanolamine buffer with 0.5 mM MgCI₂ was added per well, and the plate was incubated for 30 to 45 minutes at room temperature with shaking (300 rpm). Reaction progress was monitored by determining optical density: when the top standard reached an OD between 2.0 and 3.0,50 μΙ. of 2N NaOH was added per well. The optical dansity of each well was determined within 30 minutes, using a microtiter plate reader set to 405 nm. The data was analyzed in XL fit using 4-parameter curve fitting. [0068] The following reagents were used in the above-described assays. Dulbecco’s Phosphate Buffered Saline without Calcium or Magnesium (Gibco Catalog No. 14190); Minimum essential medium Eagle (MEM; Gibco Catalog No. 11090); pen ici II in-streptomycin (GibcoCatalog No. 15140); L-glutamine, 200 mM (Gibco Catalog No. 25030); HEPES, 1M (Gibco Catalog No. 15630); fetal bovine serum ("FBS"; HyClone Catalog No. SH30070.03); lipopolysaccharides from Escherichia coli K-235 ("LPS"; Sigma Catalog No. L2048); anti-TNF-α, Purifled Mouse Monoclonal IgG (R&amp;D Systems Catalog No. MAB210); BupH™ Carbonate-Bicarbonate Buffer Paok (Pierce Catalog No. 28382); HEPES (FW 238.3; Sigma Catalog No. H3575); NaCI (Sigma Catalog No. S7653); bovine serum albumin ("BSA"; Jackson Immu-noReseach Catalog No. 001-000-162); polyoxyethylene 20 sorbilan monolaurate (Sigma Catalog No. P2287); magnesium chloride, hexahydrate (Sigma Catalog No. M2670); recombinant human TNF-α (R&amp;D Systems Catalog No. 210TA010); biotinylated TNF-α affinity purified goat IgG (R&amp;D Systems Catalog No. BAF210); streptavidin alkaline phosphatase (Jackson ImmunoResearch Catalog No. 016-050-084); diethanolamine Substrate Buffer (Pierce Catalog No. 34064); p-nitrophenyl phosphate (Sigma Catalog No. N2765).

[0069] T able 3 shows the results of p38 inhibition and inhibition of LPS-índuced TNF-α secretion from human peri pheral blood mononuclear cells ("PBMC'). An "active" compound is defined as a compound having an IC₅₀ below 500 nM.

<img file="IS2675B_D0003.tif" />

PREPARATIVE EXAMPLES [0070] In orderto ilíustrate The invention, the following examples are included.

EXAMPLES [0071] In the examples described below, unless otherwise indicated all temperatures are set forth in degrees Celsius. Reagents were purchased from commercial suppliers such as Aldrich Chemical Company, Lancaster, TCI orMaybridge, and were used without further purification unless otherwise indicated. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF), dichloromethane (DCM), toluene, dioxane and 1,2-difluoroethane were purchased from Aldrich in Sure seal bottles and used as received.

[0072] The reactions set forth below were done generally under a positive pressure of nitrogen or argon or with a drying tube (unless otherwise stated) in anhydrous solvents, and the reaction flasks were typically fitted with rubber septa for the introduction of substrates and reagents via syringe. Glassware was oven dríed and/or heat dried.

[0073] Column chromatography was done on a Biotage system (Manufacturer. Dyax Corporation) having a silica gel column or on a silica SepPak cartridge (Waters).

[0074] 1H-NMR spectra were recorded on a Bruker instrument operating at 300 MHz or on a Varian instrument operating at 400 MHz. ¹H-NMR spectra were obtained as CDCI₃ solutions (reported in ppm), using chloroform as the reference standard (7.25 ppm). Other NMR solvents were used as needed. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets). Coupling constants, when given, are reported in Hertz (Hz).

[0075] Examples 1-16describethesynthesisofamidecompoundofthisinvention having thegeneric Formula I. Figure 1A to 1C shows the reaction scheme forthe synthesis compound having the generic structure 11 g.

<img file="IS2675B_D0004.tif" />

Example I

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid amide (11g-1) [0076] StepA: 1-Fluoro-3-methyl-benzene (compound 1g; 18.7 g, 170 mmol) wasadded to a three neck500 mLflask and cooled to -78’C. Next, solution of potassium t-butoxide (11.0 g, 170 mmol) in THE was added slowly by syringe. After 10 minutes, t-BuLi (19.0 g, 170 mmol) in pentane was added slowly by cannula under nitragen to the reaction. After 2.5 hours of stirring, the reaction was quenched with large amount of crushed fresh dry ice, taken off the -78°C bath and manually stirred with a metal spatula to turn the dark brown material into a much lighter yellow slurry. After 20 minutes of mixing by hand, about 500 mL of water were added and reaction mixture was stirred. The reaction mixture was then washed with Et₂O and then acidified with 6 N HCI to pH less than 3 and extracted with Et₂O. The organic was washed with brine, dried over MgSO₄ filtered and concentrated to yield 10 gm (45% yield) of compound 2g. ¹H NMR (400 MHz, CDCI₃)5 7.90 (t, 1H), 7.04 (d, 1H), 6.97 (d, 1H), 2.39 (S, 3H).

[0077] Step B: Compound 2g (8.0 g, 52 mmol) was added to a 500 mL flask and cooled to salt water ice bath temp. H₂SO₄ (150 mL) was added and the mixture stirred. Next, a mixtureoffreshly prepared H₂SO₄ (6.11 g, 62.3 mmol) and HNO₃ (5.2 g, 83 mmol) was dripped into the reaction mixture over 10 mínutes. After 3 hours at 0°C, the reaction was complete and was added to 1500 ml of ice/ice water and stirred for 1 hour. The reaction was filtered and rinsed several timeswith coldwateranddried underhigh vacuum, yieldinq 8 q (80% yieldlof compound3q. ¹H NMR(400 MHz, CDCL) 5 8.74 (d, 1H), 7.20 (d, 1H), 2.69 (s, 3H).

[0078] Step C: Compound 3g (8 g, 40.0 mmol) was dissolved in MEON and H₂SO₄ (20.0 g, 201 mmol) was slowly added. The reaction was heated to 65°C for 20 hours. The reaction was concentrated, diluted with ice and water, sonicated, filtered, rinsed several times with cold water and dried on high vacuum for 2 days. The crude was material, compound 4g, was used directly in the next step. ¹H NMR (400 MHz, CDCI₃) δ 8.66 (d, 1H), 7.01 (d, 1H), 3.95 (s, 3H), 2.68 (s, 3H).

[0079] Step D: Compound 4g (5.4 g, 41 mmol) was added to THF and cooled to 0°C. To this was added 4-fluorophenol (5.1g, 45 mmol). Next, NaH (60% in oils) (1.8 g, 45 mmol) was added in portions. After 1 hour, the reaction warmed to room temperature and stirred for 2 more hours. The reaction was concentrated and quenched with a large excess of 0.5 N Na₂CO₃ to pH 7.0. The reaction was sonicated for 30 minutes, filtered, and rinsed with more buffer and H₂O. The reaction was dried on high vacuum for 1 hour, then added to THF and MgSO₄ to dry, was fíltered and evaporated to yield approximately 8 g (75% yield) of compound 5g. ¹H NMR (400 MHz, DMSO-d6) δ 8.66 (d, 1H), 7.01 (d, 1H), 3.95 (s, 3H), 2.68 (s, 3H).

[0080] Step E: Compound 5g (10.0 g, 33.0 mmol) and zinc (11.0 g, 164 mmol) were added to methanol and stirred. Acetic acid (4.0 g, 66 mmol) was slowly added. The reaction was stirred overnight, sonicated and passed through Celite. Solution was concentrated to yield approximately 14 g of compound 6g and zinc by-products. The crude material was taken on to the next step.

[0081] Step F: Compound 6g (9.0 g, 33.0 mmol), ammonium tetrafluoroborate (6.0 g, 65 mmol), and HCI (17,0 g, 163 mmol), were added to 200 mL of AcOH/H₂O (2:1) and sonicated. The material was scraped off the sides of round bottom and NaNO₂ (2.7 g, 3 mmol) was added. The reaction was sonicated for 10 minutes turning dark brown while the appearance of a new precipitate formed (product salt). The reaction was allowed to stír for 4 hours. The reaction was concentrated on a speed vacuum at 65°C, then taken up in toluene and evaporated to dryness. The crude material, compound 7g, was taken directly on to the next step without any workup.

[0082] Step G: Compound 7g (11.0 g, 31 mmol), potassium acetate(5.2g, 53 mmol) and 18-crown-6 (0.1 equivalents) were added to chloroform and sonicated for 10 minutes. The reaction ran overnight at room temperature. A column was packed in a 1000 mL filter flask consisting of approximately 2 inches of silica gel, 2 inches of Celite layered on top or the silica gel, a sheet of filter paper on top of the Celite, and one half inch of sand on top of the filter paper. The column was washed with CHCI₃. The crude material was loaded onto the column directly in CHCI₃, and the column was eluted with CHCI₃ until a large amount of yellow material came off. Next, the product was eluted from the column with ethyl aœtate and the ethyl acetate collections were pooled and concentrated to give around 7 g (95 % yield) of compound 8g. MS (ESI+) m/z287 (M+H) detected.

[0083] Step H: Compound 8g (0.250 g, 0.87 mmol), was added to dry DMF, and to this was added isobutyl bromide (0,15 mL, 1,2 mmol), and K₂CO₃ (0.5 g, 3.6 mmol), This reaction mixture was then placed in a septum covered vial and stirred at 95°C overnight. The material was purified by column chromatography with 1:1 diethyl ether/hexanes to provide 0.1 g (33% yield) of compound 9g-1. MS (ESI+) m/z343 (M+H) detected.

[0084] Step I: Compound 9g-1 (0.100 g, 0.292 mmol) was placed in a 1:1 míxture of 1 N LiOH/THF and stirred at 55°C. After4 hours, the THF was evaporated and 1 N HCI was added. The reaction mixture was sonicated and filtered to isolate around 0.075 g (78 % yield) of compound 10g as a pure material. MS (ESI+) m/z329 (M+H) detected. [0085] Step J: A solution of compound 10g (20 mg, 0.061 mmol) in THF (1 mL) was treated with CDI (1.2 equivalents) at room temperature under nitrogen atmosphere, After stirring for 18 hours, the reaction was treated with 0.5 M NH₄ in dioxane (0.11 mL, 0.67 mmol). Afteran additional 18 hours, thesolventwas allowed to slowly evaporate and the mixture was purified In a Sep Pak cartridge eluting with CH₂CI₂ - 5% MeOH/CH₂CI₂ to provide 2.2 mg of compound 11 g-1 as an oil in 12% yíeld. ¹H NMR (400 MHz, DMSO-d6) δ 8.01 (s, 1H), 7.99 (s, 1H), 7.73 (s, 1H), 7.57 (s, 1H), 7.26 (s, 1 H), 7.20 (m, 2H), 7.05 (m, 2H), 4.27 (d, 2H), 2.24 (m, 1H); 0.86 (d, 6Η):

Example 2

Preparation of r5-(4-fluQrophenoxy)-1-isobu1yl-1H-indazol-6-yll-morpholin-4-yl-methanone (11g-2) [0086] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described in Example 1) In THF was treated with carbonyldiimidazole (1.2 equivalents) at room temperature under nitrogen atmosphere. After stirring for 18 hours, the reaction was treated with morpholine (1 equívalent). After an additional 18 hours, the solvent was allowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to MeOH/ CH₂CI₂ to provide compound 11 g-2 as an oil in 93% yield.

Example 3

Preparalion of [5-(4-fluorophenoxy)-1-isobutyl-1H-indazol-6-yl]-(4-methylpiperazin-1-yl)-methanone (11g-3) [0087] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1 H-indazole-8-carboxylic acid (compound 10g, prepared as described in Example 1) in THF was treated with carbonyldiimidazole (1.2 equivalents) at room temperature under n itrogen atmosphere.Afterstírringfor18hours,thereactionwastreatedwith1-methyl-piperazine(1 equivalent).Afteranadditional 18 hours, the solvent was allowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/ CH₂CI₂ to provide compound 11g-3 as an oil In 95% yield.

Example 4

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (1-benzylpiperidin-4-yl)-amide (11q-4) [0088] A solution of 5-(4-fluorophenoxy)-1-ísobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described in Example 1) in THF was treated with carbonyldiimidazole (1.2 equivalents) at room temperature under nitrogen atmosphere. After stirring for 18 hours, the reaction was treated with 1 -benzyl-piperidin-4-yl-amine (1 equivalent). After an additional 18 hours, the solvent was allowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/ CH₂CI₂ to provide compound 11 g-4 as an oil In 97% yield.

Example 5

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-benzylaminoethyl)-amide (11g-5) [0089] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described In Example 1) in THF was treated with carbonyldiimidazole (1.2 equivalents) at room temperature under nitrogen atmosphere. After stirring at for 18 hours, the reaction was treated with N1-benzyl-ethane-1,2-diamine (1 equivalent). After an additional 18 hours, the solvent was allowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to MeOH/ CH₂CI₂ to provide compound 11 g-5 as an oil in 100% yíeld.

Example 6

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-piperidin-yl-ethyl)-amide (11g-6) [0090] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described in Example 1) in THF was treated with carbonyldiímídazole (1.2 equivalents) at room temperature under nitragen atmosphere. After stirring for 18 hours, the reaction was treated with 2-piperid in-1 -yl-ethylamine (1 equivalent). After an additional 18 hours, the solvent was allowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/ CH₂CI₂ to provide compound 11 g-6 as an oil in 100% yield.

Example 7

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide (11g-7) [0091] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described in Example 1) inTHF wastreated with carbonyldiimidazole (1.2 equivalents)atroomtemperature under nitrogen atmosphere. After stirring for 18 hours, the reaction was treated with 2-pyrrolidin-1-yl-ethylamine (1 equivalent). After an additional 18 hours, thesolvent was allowed to slowly evaporate and the residue was purified in a Sep Pakcartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/ CH₂CI₂ to provide compound 11g-7 as an oil in 63% yield.

Example 8

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (3-morpholin-4-yl-propyl)-amide (11q-8) [0092] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carbyxylic acid (compound 10g, prepared as described in Example 1) in THF wastreated with carbonyldiimidazole (1.2 equivalents)at room temperature under nitrogen atmosphere. After stirring for 18 hours, the reaction was treated with 3-morpholin-4-yl-propylamine (1 equivalent). After an additional 18 hours, the solvent was allowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/CH₂CI₂ to provide compound 11 g-8 as an oil In 70% yield

Example 9

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (3-dimethylaminopropyl)-amide (11g-9) [0093] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described in Example 1) in THF was treated with carbonyldiimidazole (1.2 equivalents)at room temperature under nitrogen atmosphere. After stirring for 18 hours, the reaction was treated with N-1-dimethyl-propane-1,3-diamine (1 equivalent). After 18 add itíonal hours, the solvent was allowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/ CH₂CI₂ to provide compound 11g-9 as an oil in 44% yield.

Example 10

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-dimethylaminoethyl)-aminde (11g-10) [0094] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described in Example 1) in THF was treated with carbonyldiimidazole (1.2 equivalents) at room temperature under nitrogen atmosphere. After stirring for 18 hours, the reaction was treated with N1-dimethyl-ethane-1,2-diamine; 1 equivalent). After 18 additional hours, the solvent was allowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/ CH₂CI₂ to provide compound 11 g-10 as an oil in 58% yield.

Example 11

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid methyl-(1-methylpiperidin-4-yl)-amide (Hg-11) [0095] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described In Example 1)inTHFwastreated with carbonyldiimidazole (1.2equivalents)atroom temperature under nitrogen atmosphere. After stirring for 18 hours, the reaction was treated with methyl-methyl-piperidin-4-yl)-amíne (1 equivalent). After 18 additional hours, the solvent was allowed to slowly evaporate and the resídue was purífied in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/ CH₂CI₂ to provide compound 11g-11 as an oil in 3% yield.

Example 12

Preparation of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid [3-(methylphenylamino)-propyl]-amide (113-12) [0096] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described in Example 1) in.THF was treated with carbonyldiimidazole (1.2 equivalents) at room temperature under nitrogen atmosphere. After stirring for 19 hours, the reaction was treated with N1-Methyl-N1-phenyl-propane-1,3-diamine (1 equivalent). After 18 additional hours, the solvent wasallowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/ CH₂CI₂ to provide compound 11g-12 as an oil in 78% yleid.

Example 13

Preparationof 3-{[5-(5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carbonyl1-amino)-pyrrolidine-1-carboxylicacid tertbutyl ester (11g-13) [0097] A solution of 5-(4-fluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (compound 10g, prepared as described In Example 1) In THF was treated with carbonyldiimidazole (1.2 equivalents) at room temperature under nítrogen atmosphere. After stirring for 18 hours, the reaction was treated with 3-amino-pyrrolidine-1-carboxylic acid tert-butyl ester (1 equivalent). After 18 additional hours, the solvent was allowed to slowly evaporate and the residue was purified in a Sep Pak cartridge eluting with a gradient of 100% CH₂CI₂ to 5% MeOH/ CH₂CI₂ to provide compound 11g-13 as an oil in 94% yield.

Example 14

Preparationof5-(4-fluorophenoxy )-1-(2,2,2-trifluoroethyl)-1/-/-indazole-6carboxylicacid(2-dimethylaminoethyl)amide (Hg-14) [0098] Step A: Compound 8g was prepared as described in Example 1.

[0099] Step B: Compound 8g, 2-bromo-1,1,1-tnfluoro-ethane and K₂CO₃ and DMF were combined and the reaction mixture was stirred overnight at 75°C. Two additional equivalents of 2-bromo-1,1,1-trifluoroethane were added and the reaction stirred at 90 °C. Several additional equivalents of 2-bromo-1,1,1-trifluoroethane were added and the reaction stirred at 50°C for 72 hours. The reaction was concentrated, taken up In toluene, and purified by column chromatography (eluted with hexane/Et₂O), yielding 80 mg (24 % yield) of compound 9g-2. MS (ESI+) mlz369 (M+H) detected.

[0100] Step C: Compound 9g-2 (0.075 g, 0.20 mmol) was placed ín a 1:1 mixture of 1 N LiOH/THF and stirred for 18 hours at room temperature. The THF was evaporated and 1 N HCI was added to the reaction mixture, which was then sonicated and filtered to isolate approximately 0.070 g (97 % yield) of compound 10g-2 as pure material. MS (ESI+) ml z355 (M+H) detected.

[0101] Step D: Compound 10g-2 (0.03 g, 0.847 mmol), benzotriazole-1,3-diol (0.022 g, 0.25 mmol) and (3-dimethyl-aminopropyl)-ethylcarbodlimide (0.011 g, 0.10 mmol) were added to dichloroethane and stirred for 5 minutes. Next, A/¹-dimethyl-ethane-1,2-diamine (0.019 g, 0.10 mmol) was added and the reaction stirred for 3 hours. The reaction mixture was concentrated, taken up in dicloromethane, dried under high vacuum and purified by reverse phase HPLC according to method C (see below), yielding 25 mg (56 % yield) of compound 11g-14 as the TFA salt. ¹H NMR (400 MHz, CDCI₃) δ 8.45 (s, 1H), 8.10 (s, 1H), 7.90 (s, 1H), 7.12 (m, 4H), 5.02 (q, 2H), 3.93 (br, 2H), 3.34 (br, 6H), 2.72 (s, 6H).

Example 15

Preparation of 5-(4-fluorophenoxy)-1-methyl-1H-indazole-6-carboxylic acid (2-dimethylaminoethyl) amide (11g-15) [0102] Step A: Compound 8g was prepared as described in Example 1.

[0103] Step B: Compound 8g, iodomethane and K₂CO₃ were added to DMF and heated to about 75°C. After 48 hours the reaction was filtered to remove the K₂CO₃, concentrated, taken up in toluene and purified by column chromatography (eluting with 1:1 Et₂O/hexane), yielding 70 mg (36.7 % yield) of compound 9g-3. MS (ESI+) mlz 301 (M+H) detected. [0104] Step C: Compound 9g-3 (0.075g, 0.25 mmol) was placed ín a 1:1 mixture of 1 N LiOH / THF and stirred for 18 hours at room temperature. The THF was evaporated and 1 N HCI was added to the reaction mixture, which was then sonicated and filtered to provide approxímately 0.060 g (84 % yield) of compound 10g-3 as pure material. MS (ESI+) mlz287 (M+H) detected.

[0105] Step D: Compound 10g-3 (0.030 g, 0.105 mmol), benzotriazole-1,3-diol (0.028 g, 0.31 mmol) and (3-dimeth-ylamino-propyl)-ethyl-carbodiimide (0,019 g, 0.13 mmol) were added to dichloroethane and stirred for 5 minutes. Next, /V¹-dimethyl-ethane-1,2-diamine (0.024 g, 0.13 mmol) was added and the reaction stirred for 3 hours. The reaction mixture was then concentrated, taken up In dichloroethane, dried under high vacuum and purified by reversed phase HPLC according to Method C of Example 20, yielding 25 mg (52 % yield) of compound 11 g-15 as the TFA salt. ¹H NMR (400 MHz, CDCI₃) δ 8 44 (br, 1H), 8.21 (s, 1H), 7.85 (s, 1H); 7.05 (m, 4H), 4.15 (s, 3H), 3.90 (br, 2H), 3.30 (br, 2H), 2.92 (s, 6H).

Example 16

Preparation of 5-(4-fluorophenoxy)-1H-indazole-6-carboxylic acid (2-dimethylaminoethyl) amide (11g-16) [0106] Step A: Compound 8g was prepared asdescribed In Example 1.

[0107] Step B: Compound 8g was stirred in THF, one volume equivalent of 1 N LiOH was added and the reaction stirred at 60°C for 6 hours. The reaction was concentrated, quenched with 1 N HCI, cooled, sonicated, filtered and dríed to give 0.40 g of compound 10g-4 (84% pure material). MS (ESI+) mlz287 (M+H) detected.

[0108] Step C: Compound 10g-4 (0.030 g, 0.110 mmol), benzotriazole-1,3-diol (0.029 g, 0.33 mmol) and (3-dímeth-ylaminopropyl)-ethylcarbodiimide (0.020 g, 0.13 mmol) were added to dichloroethane and stírred for 5 minutes. Next, N¹-dimethyiethane-1,2-diamine (0.025 g, 0.13 mmol) was added and the reaction stirred for 3 hours. The reaction was evaporated, taken up In dichloroethane and dried under high vacuum and purified by reversed phase HPLC according to Method B of Example 20, to provide 25 mg (51 % yield) of compound 11 g-16 as the TFA salt. ¹H NMR (400 MHz, CDCI3)88.45 (br, 1H), 8.22 (s, 1H), 7.91 (s, 1H), 7.09 (s, 1H), 7.06 (m, 3H), 3.85 (br, 2H), 3.20 (br, 2H),2.90 (s, 6H). [0109] Examples 17-19describethesynthesisofaminoacidcompoundsofthegeneral Formula I. Figure 2 shows a synthetic reaction scheme for the synthesis of compounds having the generic structure Ij.

Example 17

Preparation of4-amino-2-{[5-(4-fluorophenoxy)-1-isobutyl-1 ti-indazole-6-carboyll-amino) butyric acid methyl ester (112} [0110] StepA: Compoiind 10g-1 was prepared as described in Example 1.

[0111] StepB: Asolutionofcompound 10g-1 (60mg,0.15mmol) lnTHF(0.5mL)wastreated with CDI (1.1 equivalents) at room temperature under N₂ atmosphere. After stirring for 18 hours, 2-amino-4-ferf-butoxycarbonylamino butyric acid methyl ester (36 mg, 0.165 mmol), was added, followed by the addition of N.N-diisoproylethylamine (29 mg, 0.225 mmol). After stirring for 18 hours, the reactíon was concentrated, the residue taken up in CH₂CI₂ and washed with 1N on HCI. The organiclayerwas filtered through 1PS paperand purified in a SepPak cartridge eluting with 10:1 CH₂CI₂/Et₂O. The desired fractions were concentrated to yield 72 mg of compound 1 j-1 as a beige foam (99 % yield). ¹H NMR (400 MHz, DMSO-d6) δ 8.65 (br, 1H), 8.10 (s, 1H), 7.9 (s, 1H), 7.28 (1H, s), 4.21 (d, 2H), 4.42 (m, 1H), 3.6 (s, 3H), 2.95 (m, 2H). [0112] Step C: A solution of compound 1j-1 (72 mg, 0.13 mmol) in CH₂CI₂ (0.2 mL) was treated with TFA (0.1 mL) at room temperature. After 18 hours, the solvent was concentrated and co-evaporated from ether, yielding 70 mg (98 % yield) of compound 1j-2 as an amber oil. ¹H NMR (400 MHz, DMSO-d6) 5 8.85 (br, 1H); 8.01 (s, 1H), 7.98 (s, 1H), 7.70 (br, 2H), 4.60 (m, 1H), 4.22 (d, 2H), 3.80 (s, 3H), 2.85 (m, 2H).

Example 18

Preparation of 4-amino-2-{[5-(4-fluorophenoxy)-1-(2,2,2-trifliioroethyl)-1H-indazole-6-carbonyl]-amino} butyric acid methyl ester (11-4) [0113] Step A: Compound 10g-2 was prepared as described in Example 14.

[0114] Step B: Compound 10g-2 (0.026 g, 0.073 mmol), benzotriazale-1,3-diol (0.013 g, 0.088 mmol)and (3-dimeth-ylaminopropyl)-ethylcarbodiimide (0.017 g, 0.088 mmol) were added to díchloroethane and mixed for 10 minutes. Next, a heterogeneous mixture of the HCI salt of 2-amino-4-t-butoxycarbonylannino butyric acid methyl ester (0.039 g, 0.147 mmol) and triethylamine (0.030,0.29 mmol) In dichloroethane were added. The reaction mixture was stirred for 3 hours, concentrated and purified by reversed phase HPLC according to Method A of Example 20 to provide approxímately 30 mg of pure compound 1j-3 (71.9% yield). MS (ESI+) m/z569 (M+H) detected.

[0115] Step C: Compound 1 j-3 (0.0012 g, 0.024 mmol) was added to 1:1 CH₂CI₂/TFA for 1.5 hours, then concentrated to provide 2.3 mg (100% yield) of compound 1j-4.¹H NMR (400 MHz, CDCI₃) δ 9.21 (br, 1H), 8.40 (br, 1H), 8.04 (br, 1H), 7.44 (br, 1H), 7.18 (s, 1H), 7.03 (m, 3H), 5.05 (m, 2H), 4.80 (br, 1H), 3.75 (s, 3H), 3.36 (br, 1H), 2.97 (br, 1H), 2.51 (br, 1H), 1.92 (br, 1H).

Example 19

Preparationof4-amino-2-([5-(4-fluorophenoxy)-1-methyl-1H-indazole-6-carbonyl1-amino} butyric acid methyl ester (1j-θ) [0116] Step A: Compound 10g-3 was prepared as described in Example 15.

[0117] Step B: Compound 10g-3 (0.026 g, 0.090 mmol), benzotriazole-1,3-diol (0.017 g, 0.11 mmol) and (3-dimeth-ylaminopropyl)ethylcarbodiimide (0.021 g, 0.017 mmol) were added to dichloroethane and mixed for 10 minutes. Next, a heterogeneous mixture of the HCI salt of 2-amino-4-tert-butoxycarbonylamino butyric acid methyl ester(0.05 g, 0.20 mmol) and triethylamine (0.037,0.36 mmol) in dichloroethane were added. The reaction mixture was stirred for 3 hours and then purified by reversed phase HPLC according to Method A of Example 20 to provide 30 mg (66 % yield) of compound 1j-5 as pure material. MS (ESI+) mlz 501 (M+H) detected.

[0118] Step C: Compound 1 j-5 (0.0012 g, 0.024 mmol) was added to 1:1 CH₂CI₂/TFA for 1.5 hours, then concentrated to provide 1.2 mg (100 % yield) of compound 1j-6.¹H NMR (400 MHz, CDCI₃) δ 9.10 (br, 1H), 8.32 (br, 1H), 8.05 (br, 1H), 7.90 (s, 1H), 7.05 (s, 1H), 7.05 (m, 3H), 4.75 (br, 1 H), 4.14 (s, 3H) 3.65 (s, 3H), 3.30 (br, 1H), 2.92 (br, 1H), 2.51 (br, 1H), 1.82 (br, 1H).

Example 20

Preparative RP-HPLC Conditions

Method A:

[0119] Column: YMC ODS-AQ, 250 X 20 mm i.d., s-10/20 μΓΠ, 12 nm. Solvent A: H₂O with 0.1% TFA. Solvent B: acetonitrile with 0.05% TFA. Collection triggered by mass spectrometer.

<img file="IS2675B_D0005.tif" />

Method B:

[0120] Column: YMC ODS-AQ, 250 X 20 mm i.d., s-10/20 μπι, 12 nm. Solvent A: H₂O with 0.1% TFA. Solvent B: acetonitrile with 0.05% TFA. Collection triggered by mass spectrometer.

% A % B flow rate

0.03 min 95 5 10ml/min

1.50 min 95 5 20ml/min

22.5 min 5 95 20 ml/min

24.0 min 5 95 15ml/min

30.5 min 95 5 15ml/min

Method C:

[0121] Column: YMC ODS-AQ, 250 X 20 mm i.d., s-10/20 μπι, 12 nm. Solvent A: H₂O with 0.1% TFA. Solvent B: acetonitrile with 0.05% TFA. Collection triggered by mass spectrometer.

% A % B flow rate

0.03 min 95 5 10ml/min

1.50 min 95 5 15ml/min

18.5 min 5 95 15ml/min

20.0 min 5 95 15ml/min

20.85 min 95 5 15 ml/min

<img file="IS2675B_D0006.tif" />

Example 21

Preparation of compound 1m-1 [0122] The synthesis of compound 1m-1 is shown ín Figure 3.

[0123] Step A: Compound 1j-7 (0.07g, 0.13 mmol), prepared in a manner similar to that described for compound 1j-3, was treated with sodium borohydride (10 equivalents, 0.049 g, 1.3 mmol) in 1:1 MeOH/THF and heated to 60°C for 3 hours. The reaction mixture was concentrated and then coevaporated with MeOH to provide compound 1 i-1.

[0124] Step B: Compound 11-1 was places in a 1:1 mixture of MeOH/4 M HCI in dioxane for 1.5 hours, and then the reaction mixture was concentrated. The residue was taken up In chloroform, washed with a 0.8 M Na₂CO₃ solution (pH 7.0) and aqueous saturated NaCI, and dríed over MgSO₄. After filtration, the filtrate was evaporated to provide compound 1 m-1 (99% pure) asthe free base. H-NMR (400 MHz), CDCI₃: δ 8.39 (d, 1H), 8.34 (s, 1H), 7.90 (s, 1H), 7.24 (s, 1H), 6.98 (Μ, 4H), 4.27 (m, 1H), 4.20 (d, 2H), 3.64 (m, 2H), 2.65 (m, 1H), 2.39 (m, 1H), 2.37 (m, 1H), 2.18 (m, 1H), 1.59 (m, 1H), 0.93 (d, 6H). (APCI+) m/z251/253 (M/M+2H, 1:1) wasdetected.

Example 22 (Reference)

Preparation of 5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (17d) [0125] The reaction scheme for the synthesis of compound 17d according to this Example is shown in Figure 4. [0126] Step A: 1,2-Dibromo-4-methyl-5-nitrobenzene: 3,4-dibromotoluene (108.11 mL, 800 mmol) was added drop-wise over 4 hours to nitric acid (90%, 280 ml_, 6000 mmol) that was cooled to 0°C under a nitrogen atmosphere with mechanical stirring. The internal temperature of the mixture was maintained below 10°C during the addition and was stirred for 1 hour at 0°C after completion of addition. Water (840 mL) was added drop-wise to the mixture maintaining the internal temperature below 10°C. The crude product was collected by filtration and was washed with water (5 X 500 mL) to remove the excess nitric acid. The solids were dried under high vacuum and purified by recrystallization from ethanol (800 mL) to produce 180.9 g (77% yield) of the desired product as a solid. ¹H NMR (400 mHz, CDCI₃) δ 8.24 (s, 1H), 7.64 (s, 1H), 2.55 (s, 3H) [0127] Step B: 1-Bromo-2-(2,4-difluorophenoxy)-4-methyl-5-nitrobenzene: A mixture of 1,2-dibromo-4-methyl-5-nitrobenzene (84.3 g, 286 mmol), 2,4-difluorophenol (37.2 g, 286 mmol), and K₂CO₃ (43.5 g, 315 mmol) were heated to 100°c for 45 hours. The reaction míxture was cooled to room temperature and then stored In a 5°C refrigerator overnight. The reaction mixture was poured into 1200 mL of ice water all at once. The resulting damp solid was collected, partially ground up, and stírred in 900 mL H₂O for 45 minutes. The solid was collected by filtration and rinsed with 700 mL of water portion-wise. The resulting solid was dried under high vacuum overnight to yield 93.5 g of a brown solid (95%yield). ¹H NMR (400 mHz, CDCI3) δ8.38 (s, 1H), 7.18 (m, 1H), 7.03 (m, 1H), 6.97 (m, 1H), 6.52 (s, 1H), 2.50 (s, 3H). [0128] Step C: 5-Bromo-4-(2,4-difluorophenoxy)-2-methylphenylamine: 1-Bromo-2-(2,4-dífluoro-phenoxy)-4-methyl-5-nitro-benzene (87.0 g, 253 mmol) was dissolved in THF (300 mL) and diluted with MeOH (900 mL). Zinc dust (82.7 g, 1.26 mol) was added and 1 L of saturated NH4CI was added slowly so that the reaction temperature never exceeded 42°C. The reaction was mechanically stirred vigorously for 16 hours. The reaction was filtered through Celite and the filter cake was washed with ethyl acetate. The filtrate was then concentrated with 1.2 L of saturated NH4OAc. When the THF/MeOH was removed, the solids were collected and washed with water. The solids were then stirred in 1 L water for 30 min, then collected via filtration and rinsed with water (1 L) in three portions. The resulting solid was dried underhigh vacuum for 48 hours to produce 64 g ofthe desired product (81 %yield). MS (ESI +) m/z314,316 (M+1, Br pattern) detected; ¹H NMR (400 mHz, CDCI3) δ 6.92 (m, 1H), 6.91 (s, 1H), 6.75 (m, 2H), 6.70 (s, 1H), 3.57 (br. s, 2H), 2.08 (s, 3H).

Step D: 6-Bromo-5-(2,4-difluorophenoxy)-1H-indazole (14d):

[0129] 5-bromo-4-(2,4-difluorophenoxy)-2-methylbenzenediazonium tetrafluoroborate: 5-Bromo-4-(2,4-difluorophe-noxy)-2-methylphenylamine (30.0 g, 96 mmol) was dissolved in 2:1 AcOH/H₂O (960 mL). NH₄BF₄ (20.0 g, 191 mmol) was added and the mixture was cooled to 3°C (—30 min). Concentrated HCI (40 mL) was then added all at once and the mixture warmed to 6°C. The mixture was cooled to 2°C and then NaNO₂ (7.25 g, 105 mmol) was added. The reaction mixture was stirred in the ice bath for 5 minutes and then allowed to stir for 1 hour at room temperature. The mixture was concentrated under reduced pressure and the residue was azeotroped with toluene (3 X 400 mL). The crude material (5-bromo-4-(2,4-difluorophenoxy)-2-methyl-benzenediazonium tetrafluoro borate) was used in the next reaction without further purification.

[0130] 6-Bromo-5-(2,4-difluorophenoxy)-1H-indazole: The crude 5-bromo-4-(2,4-difluorophenoxy)-2-methyl-benzen-ediazonium tetrafluoroborate was suspended in ethyl acetate (650 mL) and treated with 10 equívalents of KOAc. The mixture was vigorously stirred at room temperature for 1,5hours and then filtered and diluted to a 1 L total volume with ethyl acetate. The mixture was washed with saturated NaHCO₃/ brine (800 mL, 1:1). The aqueous phase was extracted with ethyl acetate (400 mL). The organícs were combined, dried (MgSO₄) and concentrated to a brown solid (31 g, 99% yield). ¹H NMR (400 mHz, CDCI₃) δ 10.55 (br. s, 1H), 7.98 (s, 1 H), 7.84 (s, 1H), 7.20 (s, 1H), 6.99 (m, 1H), 6.94 (m, 1H),6.84(m, 1H).

[0131] Step E: 6-Bromo-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole (15d): 6-Bromo-5-(2,4-difluorophe-noxy)-1H-indazole(60.0 g, 185mmol)wasdissolvedin DMF and treatedwith K₂CO₃ (76.5 g, 554 mmol)andwithisobutyl bromide (126.4 g, 923 mmol). The mixture was stirred and heated to 80 °C for 16 hours. An additional 15 g of K₂CO₃were added and the mixture was vigorously stirred for 24 hours more. The reaction mixture was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and dissolved in ether (1 L). The mixture was washed with 1:5 brine/water (2 X 600 mL). The aqueous phases were extracted with ether (300 mL) and the combined organics were dried (MgSO₄) and concentrated under reduced pressure. The crude product was chromatographed on a Biotage Flash 75 in two batches (about 35 g each) eluting with 5% ethyl acetate ín hexanes. The combined purified products yielded 30.1 g of thedesired product as a solid (43% yield). MS (ESI +) m/z 381,383 (M+1, Brpattern) detected; ¹H NMR(400 mHz, CDCI,)87.86 (s, 1H), 7.72 (s, 1H), 7.16 (s, 1H), 6.98 (m, 1H),6.92 (m, 1H), 6.82 (m, 1H), 4.12 (d, 2H), 2.34 (m, 1H), 0.94 (d, 6H).

[0132] Step F: 5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carbonitrile (16d): 6-Bromo-5-(2,4-difluoroph-enoxy)-1-isobutyl-1H-indazole (31.2 g, 82 mmol)and Cu(l)CN (13.9 g, 156 mmol) weredissolved in DMAanddegassed with nitrogen under vacuum. The reaction mixture was heated to 150°C for 16 hours. The mixture was cooled to room temperature and diluted with ethyl acetate before washing twice with 7M NH₄OH. The organics were washed with brine and degassed with nitrogen before being dried over MgSO₄ and concentrated under reduced pressure. The crude product was chromatographed eluting with 10 % ethyl acetate in hexanes to afford 25.1 g of product (95% yield). [0133] Step G: 5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (17d): 5-(2,4-Difluorophe-noxy)-1-isobutyl-1H-indazole-6-carbonitrile (25.1 g, 77 mmol) was suspended in ethanol (620 ml_) and KOH (2.5 M, 310 mL) and heated to reflux for 24 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to remove the ethanol. The resulting aqueous solution was diluted with water and washed with ether. The aqueous layer was acidified with concentrated HCI to pH 1 and extracted with ethyl acetate several times. The organic layers were combined and concentrated under reduced pressure to afford 25.5 g of the product (96% yield). ¹H NMR (400 mHz, CDCI3) δ 8.37 (s, 1H), 7.91 (s, 1H), 7.20 (m, 1H), 7.07 (s, 1H), 7.04 (m, 1H), 6.95 (m, 1H), 4.24 (d, 2H), 2.36 (m, 1H), 0.94 (d, 6H).

Example 23

Preparation of 5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-sulfonic acid (3-dimethylaminopropyl)-amide (26d) [0134] The reaction scheme for the synthesis of compound 26d according to this example is shown in Figure 5. [0135] Step A: 5-(2,4-Difluoro-phenoxy)-1-isobutyl-1H-indazole-6-sulfonyl chloride (25d): To a cooled (-78°C) solution of 6-bromo-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole (15d; prepared as In Example 22, Steps A-E) (2.0 g, 5.2 mmol) in THF (50 mL) under N₂ atmosphere was added n-butyl lithium (1.5 mL of 2.5 M) dropwise: The resulting solution was stirred at -78°C for 5 minutes and then transferred via cannula to a cooled (-78°C) suspension of SO₂ (0.34 g, 5.25 mmol) ín THF (5 mL). The mixture was stirred at -78°Cfor2 hours and then diluted with ether(20 mL)and stirred at room temperature for 1 hour. The suspension was concentrated under reduced pressure and the residue was stirred in an íce bath with saturated NaHCO₃ (15 mL) and NCS (0.77 g, 5.8 mmol) for45 minutes, The reaction mixture was extracted with ethyl acetate (3X) and the combíned organic layers were washed with brine and dried over Na₂SO₄. The solution was concentrated under reduced pressure to afford a highly viscous liquid that was used in the next reaction without further purification.

[0136] Step B: 5-(2,4-Difluorophenoxy)-1-isobutyt-1H-indazole-6-sulfonic acid (3-dimethylaminopro-pyl)-amide (26d): To a cooled (0°C) solution of 5-(2,4-difluorophenoxy)-1 -isobutyl-1 H-indazola-6-sulfonyl chloride (0.20 g, 0.50 mmol) in dichloromethane under N₂ atmosphere was added 3-(dimethylamino)propylamine (0.05 g, 0.50 mmol) and triethylamine (0.15 g, 1.5 mmol) drop-wise. The reaction mixture was stirred for 4 hours and then diluted with dichloromethane (20 mL), washed with water, saturated NaHCO₂ and brine and then dried over MgSO₄. The mixture was concentrated under reduced pressure and chromatographed on preparatory TLC plates eluting with dichlorometh-ane/ MeOH/Et₃N (95:4:1) to afford the 93 mg of final product (40% yield). MS (APCI -) mlz466 detected; ¹H NMR (400 mHz, CDCI₃)88.15 (s, 1H), 7.91 (s, 1H), 7.20 (m, 1H), 7.09 (s, 1H), 7.00 (m, 1H), 6.90 (m, 1H), 4.22 (d, 2H), 3.12 (t, 2H), 2.35 (m, 3H), 2.13 (s, 6H), 1.70 (m, 2H), 0.94 (d, 6H).

Example 24

Preparation of (S)-methyl 2-(5-(2,4-difluorophenoxy)-1 -isobutyl-1 H-indazole-6-sulfonamido)-4-(dimethylamino)bu-tanoate (27d) [0137] Prepared as in Example 23, Steps A and B, substituting 2-amino-4-dimethylamlnobutyric acid methyl ester dihydrochloride for 3-(dimethylamino)propylamlne, The crude product was chromatographed on preparatory TLC plates eluting with hexanes/ethyl acetate/Et₃N (50:50:5) to afford the final product (37% yield). MeOH/Et₃N (95:4:1) to afford the 93 mg of fínal product (40% yield). MS (APCI -) m/z 523 (M-1) detected; ¹H NMR (400 mHz, CDCI₃) δ 8.08 (s, 1H), 7.90 (s, 1H), 7.23 (m, 1H),7.08(s, 1H), 7.00 (m, 1H), 6.89 (m, 1H),4.32 (t, 1H), 4.21 (d, 2H), 3.45 (s, 3H), 2.37 (m, 3H), 2.15 (s, 6H), 2.05 (m, 1H), 1.90 (m, 1H), 0.92 (dd, 6H).

Example 25

Preparation of 5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-sulfonicacid [2-(1-methylpyrrolidin-2-yl)-ethyl]-amide (28d) [0138] Prepared as in Example 23, Steps A and B, substituting 2-( 1 -methylpyrrolidin-2-yl)-ethylamíne for 3-(dimeth-ylamino)propylamine. The crude product was chromatographed on preparatory TLC plates eluting with hexanes/ ethyl acetate/ Et₃N (1:1:0.1) to afford the final product (24% yield). MS (APCI +) mlz493 (M+1) detected; ¹H NMR (400 mHz, CDCI₃) δ 8.14 (s, 1H), 7.93 (s, 1H), 7.19 (m, 1H), 7.12 (s, 1H), 7.00 (m, 1H), 6.90 (m, 1H), 4.23 (d, 2H), 3.12 (m, 3H), 2.44 (m, 1H), 2.36 (m, 1H), 2.34 (s, 3H), 2.24 (m, 1H), 1.92 (m, 1H), 1.77 (m, 4H), 1.51 (m, 1H), 0.94 (d, 6H).

Example 26

Preparation of 5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-sulfonic acid (2-dimethylaminoethyl)-amide (29d) [0139] Prepared as in Example 23, Steps A and B, substituting 2-dimethylaminoethylamine for 3-(dimemylamino) pmpylamine. The crude product was chromatographed on preparatory TLC plates eluting with hexanes/ ethyl acetate/ Et₃N (1:1:0.1) to afford the final product. MS (APCI +) mlz453 (M+1) detected; ¹H NMR (400 mHz, CDCI₃) δ 8.15 (s, 1H), 7.92 (s, 1H). 7.18 (m, 1H), 7.12 (s, 1H), 7.00 (m, 1H), 6.89 (m, 1H), 4.23 (d, 2H), 3.07 (t, 2H), 2.41 (t, 2H), 2.36 (m, 1H), 2.15 (s, 6H), 0.94 (d, 6H).

Example 27

Preparation of (S)-methyl 2-(5-(2,4-difluorophenoxy)-1-isobiityl-1H-indazole-6-, carboxamido)-4-(dimethylamino)bu-tanoate (30d) [0140] <img file="IS2675B_D0007.tif" /> [0141] Step A: (S)-2-(tert-butoxycarbonyl)-4-hydroxybutanoic acid: To a solution of L-homoserine (49.9 g, 419 mmol) in 1N NaOH (460 mL) and EtOH (400 mL) was added a solution of Boc anhydride (100.6 g, 461 mmol) in THF (400 mL) over 15 minutes. The reaction mixture was stirred at room temperature for 16 hours. The mixture was then washed with ether (3 X 500 mL), acídified with 1N HCI to pH 2 and extracted with ethyl acetate (6 X 250 mL). The

<img file="IS2675B_D0008.tif" />

combined organic extracts were washed with brine (2 X 250 mL), dried over MgSO₄, filtered through Celite, and concentrated under reduced pressure to afford 72.6 g of white solid (79 % yield).

[0142] Step B: (S)-2-(tert-butoxycarbonyl)-4-hydroxybutanoic acid-dicyclohexylamine complex: To a solution of (S)-2-(tert-butoxycarbonyl)-4-hydroxybutanoic acid (72.6 g, 331 mmol) in EtOH (500 mL) was added dropwise dicy-clohexylamine (73 mL, 364 mmol). The mixture wasstirredfor2 hours at room temperature and then concentrated under reduced pressure. The white solid wasdried underhigh vacuum and then triturated with ether (1000 mL). Thefine white powder was collected by filtration, washed with ether and dried under high vacuum (125.6 g, 95% yield).

[0143] Step C: (S)-2-tert-Butoxycarbonylamino-4-hydroxybutyric acid methyl ester: To a suspension of (S)-2-(tert-butoxycarbonyl)-4-hydroxybutanoic acid-dicyclohexylamine complex (110 g, 275 mmol) in DMF (900 mL) was added idodomethane (20.5 mL, 330 mmol). The mixture was stirred at room temperature for 16 hours. The clear solution was concentrated under reduced pressure and azeotroped with toluene (5 X 200 mL). The residue was diluted with water (500 mL) and ethyl acetate (500 mL) and stirred for 2 hours before the layers were separated. The aqueous layer was extracted with ethyl acetate (9 X 250 mL). The combined extracts were washed with brine (250 mL), dried over MgSO₄, filtered through Celite, and concentrated under reduced pressure to provide yellow oil. The crude oil was chromatographed on silica eluting with ether/ hexanes (3:1) to afford 53 g colorless oil (83% yield).

[0144] Step D: (S)-4-Bromo-2-tert-butoxycarbonylaminobutyric acid methyl ester: To a cooled (0°C) solution of (S)-2-tert-butoxycarbonylamino-4-hydroxy-butyricacid methyl ester (28.7 g , 123 mmol) in dichloromethane (500 mL) was added CBr₄ (51.0 g,154 mmol). The mixture was stirred for 5 minutes before the portion-wise addition of triphenyl-phosphine (48.41 g, 185 mmol). Tbe mixture continued to stir at 0“C for 1 hour and was then allowed to warm to room temperature. The solvent was removed under reduced pressure and was then diluted with ether (500 mL) and stirred for 30 minutes. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was chromatographed eluting with ether/ hexanes (1:2) to afford 27.5 g of white solid (76% yield).

[0145] Step E: (S)-2-tert-Butoxycarbonylamino-4-dimethylaminobutyric acid methyl ester: To a solution of (S )-4-bromo-2-tert-butoxycarbonylaminobutyric acid methyl ester (27.5 g, 93 mmol) in THF (100 mL) in a pressure reaction vessel was added triethylamine (26 mL) and dimethylamine (93 mL of 2.0 M in THF). The reaction vessel was sealed and heated to 60°C for 16 hours and then cooled to room temperature. The reaction mixture was concentrated under reduced pressure and then dissolved in dichloromethane (500 mL). The solution was washed with water (3 X 200 mL) and brine (200 mL), dried over MgSO₄, filtered through Celite and concentrated under reduced pressure. The residue was dried under high vacuum to afford 23.4 g of yellow oil (97% yield).

[0146] Step F: (S)-methyl 2-amino-4-(dimethylamino)butanoate dihydrochloride: To a cooled (0°C) solution of (S)-2-tert-butoxycarbonylamino-4-dimethylaminobutyric acid methyl ester (23.4 g, 90 mmol) in dioxane (100 mL) was added drop-wise HCI (225 mL, 4M In dioxane). The mixture was warmed to room temperature and stirred for 3 hours. The solid was filtered, washed with ether (3 X100 mL), and dried under high vacuum to afford 20.2 g of product (96% yield). [0147] StepG:(S)-methyl2-(5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxamido)-4-(dimethylami-no)butanoate (30d): 5-(2,4-Difluorophenoxy)-1 -isobutyl-1 H-indazole-6-carboxylic acid (17d; prepared according to Example 22) (0.396 g, 1.14 mmol) was stirred with HOBt (0.192 g, 1.26 mmol) and EDCI (0.241 g, 1.26 mmol) in dichloroethane (2 mL) for 10 minutes at room temperature. This mixture was then added to a suspension of (S)-methyl 2-amino-4-(dimethylamino)butanoate dihydrochloride (0.280 g, 1.20 mmol) and triethylamine (1 mL, 6.9 mmol) In dichloromethane (6 mL). The reaction mixture was stirred for 3 hours and then was concentrated under reduced pressure. The residue was diluted with chloroform (50 mL) and washed with 1N HCI (2 X 25 mL), saturated K₂CO₃ (2 X 50 mL), water (25 mL), brine (25 mL), and dried over MgSO₄. The filtered solution was concentrated under reduced pressure to provide yellow oil. The oil was chromatographed eluting with 5% MeOH in dichloromethane to afford a viscous colorless oil that solidified upon drying under high vacuum (0.393 g, 71% yield). MS (ESI +) mlz 489 (M+1) detected; ¹H NMR (400 mHz, CDCI₃) δ 8.91 (d, 1H), 8.36 (s, 1H), 7.86 (s, 1H), 7.16 (m, 1H), 7.03 (m, 1H), 7.00 (s, 1H), 6.93 (m, 1H), 4.88 (m, 1H), 4.21 (d, 2H), 3.74 (s, 3H), 2.34 (m, 3H), 2.06 (s, 6H), 2.01 (m, 2H), 0.92 (d, 6H).

Example 28

Preparation of (S)-5-(2,4-difluorophenoxy)-N-(4-(dimethylamino)-1-hydroxybLitan-2-yl)-1-isobLityl-1H-indazole-6-car-boxamide (31 d) [0148] A mixture of (S)-methyl 2-(5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxamido)-4-(dimethylamino) butanoate (30d; Example 27, Steps A-E) (0.370 g, 0.76 mmol) and NaBH₄ (0.114 g, 3.04 mmol) in THF/EtOH(20 mL, 3:2) was heated to 60°C for 7 hours. The reaction mixture was concentrated under reduced pressure and diluted with dichloromethane. The slurry was chromatographed on Biotage eluting with 10% MeOH In dichloromethane with 1% triethylamine. The product was obtained as 0.337g of viscous oil (97% yield). MS (ESI +) mlz461 (M+1) detected; ¹H NMR(400 mHz, CDCI₃)8 8.34 (d, 1H), 8.31 (s, 1H), 7.86 (s, 1H),7.13(m, 1H), 7.02 (s, 1H),7.00(m, 1H),6.90(m, 1H), 4.33 (m, 1H), 4.22 (d, 2H), 3.64 (m, 2H)2.37 (m, 1H), 2:17 (s, 6H), 2.10 (m, 1H), 1.82 (m, 1H), 1.07 (m,2H), 0.93 (d, 6H).

Example 29

Preparationof(S)-5-(2,4-difluophenoxy)-1-isobutyl-1H-indazole-6-carboxylicacid(1-hydroxymethyl-3-isopropylamino-propyl)-amide (32d) [0149] Step A: lsopropyl-(4-methoxybenzyl)-amine: A míxture of 4-methoxybenzylamine (1.37 g. 10 mmo!) and acetone (0.81 mL, 11 mmol) in dry dichloroethane (20 mL) were stirred at room temperature for 30 minutes. To the solution was added sodium triacetoxyborohydride (3.18 g. 15 mmol) and the resulting mixture was slirred at room temperature for 17 hours. The reactíon mixture was quenched with 1 N NaOH (50 mL) and the layers were separated. The aqueous layer was extracted with dichloromethane (2 X 20 mL). The combined extracts were washed with water (20 mL), brine (20 mL), dried over MgSO₄, filtered through Celite, and concentrated under reduced pressure. The residue was chromatographed eluting with 10% MeOH in dichloromethane with 1% triethylamine to provide 1.53 g of oil (85% yíeld).

[0150] Step B: (S)-methyl 4-bromo-2-(tert-butoxycarbonyl)butanoate: To a cooled (0°C) solution of (S)-methyl 2-amino-4-bromobutanoate (1.80 g, 6,5 mmol) in THF (20 mL) was added triethylamine (4.53 g, 32.5 immol) and Boc anhydride (1.49 g, 6.83 mmol, solution in 20 mL THF). The mixture was stirred at 0°C for 30 minutes and then warmed to room temperature and stirred for 18 hours. The reaction mixture was quenched with 1N HCI (50 mL) and the layers were separated. The aqueous layer was extracted with ether (2 X 20 mL) and the combined organic extracts were washed with water (20 mL) and brine (20 mL), dried over MgSO₄, filtered through Celite, and concentrated under reduced pressure to provide pale yellow oil. The oíl was chromatographed eluting with ether; hexanes (1:2) to provide 1.45 g of colorless oil that solidified under high vacuum (75% yield).

[0151] Step C: (S)-methyl 4-((4-methoxybenzyl)(isopropyl)amino)-2-(tert-butoxycarbonyl)biitanoate hydro-chloride: A mixture of isopropyl-(4- methoxybenzyl)-amíne (0.111 g, 0.62 mmol), (S)-methyl 4-bromo-2-(tert-butoxy-carbonyl)butanoate (0.150 g, 0.51 mmol), and triethylamine (0.21 mL, 1.52 mmol) in THF (5 mL) was heated to reflux for 65 hours. The mixture was then cooled to room temperature and concentrated under reduced pressure. The residue was chromatographed eluting with 5% MeOH in dichloromethane to provide 0.026 g of colorless toil (18 % yield). The compound was then treated with HCI (1 mL of 4N in dioxane) at room temperature for 3 hours. The mixture was concentrated under reduced pressure and dried under high vacuum.

[0152] Step D: (S)-methyl 4-((4-methoxybenzyl)(isopropyl)amino)-2-(5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxamido)butanoate: 5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (17d; prepared according to Example 22) (0.022 g, 0.064 mmol) was stirred with HOBt (0.011 g, 0.070 mmol) and EDCI (0.014 g, 0.070 mmol) in dichloroethane (1 mL) for 10 minutes at room temperature. To this mixture was then added a suspension of (S)-methyl 4-((4-methoxybenzyl)(isopropyl)amino)-2-(tert-butoxycarbonyl)butanoate hydrochloride (0.025 g, 0.067 mmol) and triethylam ine (0.054 mL, 0.384 mmol) in dichloromethane (2 mL). The mixture was stirred at room temperature for 16 hours. The solution was filtered through Celite and concentrated under reduced pressure. The crude oil was chromatographed eluting with 2% MeOH in dichloromethane with 1% triethylamine to provide 0.035 g of viscous pale yellow oil (89% yield).

[0153] Step E: (S)-N-(4-((4-methoxybenzyl)(isopropyl)amino)-1 •hydroxybutan-2-yl)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxamide: (S)-methyl 4-((4-methoxybenzyl)(isopropyl)amino)-2-(5-(2,4-difluomphe-noxy)-1-isobutyl-1H-indazole-6-carboxamido)butanoate (0.035 g, 0.057 mmol) and NaBH₄ (0.022 g, 0.57 mmol) were dissolved in THF/MeOH (5 mL, 3:2) and heated to 50°C for 3 hours. The mixture was cooled to room temperature and concentrated under reduced pressure. The residue was chromatographed eluting with 5% MeOH in dichloromethane with 1% triethylamine to provide 0.020 g of gel (59% yield).

[0154] Step F: (S)-5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (1-hydroxymethyl-3-iso-propylaminopropyl)-amide (32d): To a solution of (S)-N-(4-((4-methoxybenzyl)(isopropyl)amino)-1-hydroxybutan-2-yl)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxamide (0.020 g, 0.033 mmol) in MeOH (5 mL) was added wet Pd/C (0.020 g, 10% by weight). The mixture was purged with hydrogen several times and then stirred at room temperature under H₂ atmosphere for 5 hours. The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was chromatographed eluting with 10% MeOH In dichloromethane with 2% tri-ethylamine to provide9.4 mg colorlessgel (60% yield). MS (ESI +) mlz475 (M+1) detected; ¹H NMR (400 mHz, CDCI₃) 5 8.40 (d, 1H), 8.31 (s, 1H), 7.87 (s, 1H), 7.13 (m, 1H), 7.03 (s, 1H), 7.01 (m, 1H), 6.91 (m, 1H), 4.33 (m, 1H), 4.22 (d, 2H), 3.69 (m, 2H), 2.69 (m, 2H), 2.37 (m, 2H), 1.32 (m, 2H), 1.09 (d, 3H), 1.01 (d, 3H), 0.93 (d, 6H).

Example 30

Preparationof(S)-2-([5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amino}-4-dimethylaminobutyricacid (33d) [0155] Step A: 5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid 2,5-dioxopyrrolidin-1-yl es-ter: 5-(2,4-Difluorophenoxy)-1-isobiityl-1H-indazolo-6-carboxylic acid (17d; prepared according to Example 22) (25.0 g, 72.2 mmol), EDCI (18.0 g, 93.8 mmol), and 1-hydroxypyrrolidine-2,5-dione (9.97 g, 86.6 mmol) were suspended in dichlommethane and stirred at room temperature for 2 hours. The mixture was diluted with dichloromethane (500 mL) and washed with saturated NH₄CI (2 X 200 mL), saturated NaHCO₃ (2 X 200 mL) and brine (200 mL). The organics were dried over MgSO₄ and concentrated under reduced pressure to afford the crude product as yellow foam.

[0156] Step B: (S)-2-{[5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amino}-4-dimethylami-nobutyric acid methyl ester: To a solution of 5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid 2,5-dioxopyrrolidin-1-yl ester (32.0 g, 72.2 mmol) and 2-amino-4-dimethylaminobutyric acid methyl ester dihydrochloride (19.35 g, 83.0 mmol) in dichloromethane was added triethylamine (35 mL, 253 mmol) and the mixture stirred at room temperature for4 hours. The reaction mixture was concentrated under reduced pressure and diluted with dichloromethane (400 mL). The solution was washed with saturated NH₄CI (2 X 200 mL), saturated NaHCO₃ (2 X 200 mL) and brine (200 mL). The organics were dried over MgSO₄ and concentrated under reduced pressure to afford the crude product.

[0157] Step C: (S)-2-{[5-(2,4-Difluorophenoxy)-1 -isobutyI-1 H-indazole-6-carbonyl]-amino}-4-dimethylami-nobutyric acid (33d): Potassium trimethylsilanolate (1.77 g, 13.8 mmol) was added to a solution of (S)-2-{[5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amino}-4-dimethylaminobutyric acid methyl ester (3.36 g, 6.88 mmol) in THF (5 mL). The reaction mixture was stirred at room temperature for4 hours. HCI (17 mL of 4M in dioxane) was added to the mixture before it was concentrated under reduced pressure. The residue was suspended in dichloromethane and filtered. The filtrate was concentrated under reduced pressure to afford 2.23 g of product (68% yíeld). MS (ESI +)m!z475 (M+1) detected; ¹H NMR(400mHz, DMSO-D_e) δ 8.81 (d, 1H), 8.02 (s, 1H), 7.99 (s, 1H), 7.48 (m, 1H), 7.25 (m, 1H), 7.22 (s, 1H), 7.11 (m, 1H), 4.50 (m, 1H), 4.28 (d, 2H), 3.17 (m, 1H), 3.04 (m, 1H), 2.70 (s, 6H), 2.25 (m, 2H), 2:13 (m,1H), 0.87 (d, 6H).

Example 31

Preparationof(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylicacid (1-hydroxymethyl-3-piperidin-1-yl-propyl)-amide (34d) [0158] Step A: (S)-2-tert-Butoxycarbonylamino-4-piperidin-4-yl-butyric acid methyl ester: (S)-4-Bromo-2-tert-butoxycarbonylaminobutyric acid methyl ester (0.10 g, 0.34 mmol) (prepared as in Example 27, Steps A-D) and pi peridine (1 mL) were heated to 50°C for 16 hours and then cooled to room temperature and concentrated under reduced pressure. The residue was azeotroped with toluene (3 X 10 mL) and then chromatographed eluting with MeOH/ dichloromethane (1:9) to provide 93 mg of colorless oil (97% yield).

[0159] Step B: (S)-2-Amino-4-piperidin-1-yl-butyric acid methyl ester dihydrochloride: HCI (0.45 mL of 4M ín dioxane) was added to (S)-2-tert-Butoxycarbonylamino-4-piperidin-1 -yl-butyric acid methyl ester and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and dried under high vacuum for 16 hours to provide the product (46% yield).

[0160] Step C: (S)-2-{[5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amino}-4-piperidin-1-yl· butyric acid methyl ester: To a solution of 5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (0.050 g, 0.14 mmol), (S)-2-amino-4-piperidin-1 -yl-butyric acid methyl ester dihydrochloride (0.043 g, 0.16 mmol), EDCI (0.033g, 0.17 mmol) and HOBt (0.023 g, 0.17 mmol) in dichloromethane was added dropwise DIEA (0.093 g, 0.72 mmol). The reaction mixture was stirred at room temperature until HPLC analysis showed consumption of the starting material and then was diluted with dichloromethane and washed with saturated NaHCO₃. The organic layer was dried over MgSO₄and concentrated under reduced pressure. The residue was chromatographed to afford 0.051 g of product (67% yield). [0161] StepD: (S)-5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylicacid (1-hydroxymethyl-3-pip-eridin-1-yl-propyl)-amide (34d): Sodium borohydride (0.012 g, 0.31 mmol) was added portion-wise to a heated (50°C) solution of (S)-2-{[5-(2,4-difluoro-phenoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amino}-4-piperidin-1-yl-butyric acid methyl ester (0.022 g, 0.042 mmol) ín MeOH. The reaction mixture was stirred at 50°C until HPLC analysis showed consumption of the starting material. The mixture was cooled to room temperature and concentrated under reduced pressure. The resídue was diluted with ethyl acetate and 1N HCI. The organics were extracted with 1N HCI until the organic layer contained no product by HPLC analysis. The aqueous solution was basified to pH 14 with NaOH and then extracted with dichloromethane several times. The combined organics were dried over MgSO₄ and concentrated under reduced pressure to afford 9.1 mg of oil (44% yield). MS (APCI +) mlz 501 (M+1) detected.

Example 32

Preparation of (S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (3-dimethylamino-1-diiriethylcar-bamoylpropyl)-amide (35d) [0162] To a solution of (S)-2-([5-(2,4-difluorophonoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amino}-4-dimethylami-nobutyríc acid (33d; Example30) (0.100 g, 0.21 mmol), dimethylamine (0.095 g, 2.11 mmol), EDCI (0.053 g, 0.27 mmol), HOBt (0.037 g, 0.27 mmol)dissolved in dichloromethane was added drop-wise DIEA (0.082 g, 0.63 mmol). The reaction mixture was stirred at room temperature until complete consumption of the starting material was observed by HPLC analysis. The reaction mixture was then diluted with dichlommethane and washed with saturated NaHCO₃, dried over MgSO₄, and concentrated under reduced pressure. The residue was chromatographed on Isolute SPE column flash Sl (5g) eluting with a gradientTEA/ dichloromethane (100 mL, 0.3:99.7), TEA/MeOH/ dichloromethane (100 mL, 0.3: 0.5: 99.2), TEA/ MeOH/ dichloromethane (100 mL, 0.3:2.5: 97.2), TEA/MeOH/dichloromethane (100 mL, 0.3: 5:94.7). The final product was obtained in 86% yield. MS (ESI +) mlz 502 (M+1) detected; ¹H NMR (400 mHz, DMSO-D₆) δ 8.63 (d, 1H), 8.01 (s, 1H), 7.96 (s, 1H), 7.48 (m, 1H), 7.23 (m, 1H), 7.20 (s, 1H), 7.09 (m, 1H), 4.98 (m, 1H), 4.26 (d, 2H), 3.07 (s, 3H); 2.84 (s, 3H), 2.23 (m, 2H), 2.13 (m, 1H), 2.03 (s, 6H), 1.80 (m, 1H), 1.65 (m, 1H), 0.86 (d, 6H).

Example 33

Preparation of (S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (3-dimethylamino-1-methylcar-bamoylpropyl)-amide (36d) [0163] Prepared according to the procedure in Example 32, substituting methylamine for dimethylamine. The product was obtained in 78% yield. MS (ESI +) m/z488 (M+1) detected; ¹H NMR (400 mHz, CDCI₃) δ 9.05 (d, 1H), 8.29 (s, 1H), 7.86 (s, 1H), 7.44 (m, 1H), 7.19 (m, 1H), 7.02 (m, 1H), 7.01 (s, 1H), 6.93 (m, 1H), 4.78 (m, 1H), 4.21 (d, 2H), 2.81 (d, 3H), 2.50 (m, 1H), 2.42 (m, 1H), 2.36 (m, 1H), 2.23 (s, 6H), 2.15 (m, 1H), 1.88 (m, 1H), 0.92 (d, 6H).

Example 34

Preparation of (S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (1-carbamoyl-3-dimethylamino-propyl)-amide (37d) [0164] Prepared according to the procedure in Example 32, substituting ammonia for dimethylamine. The product was obtaíned in 70% yield. MS (APCI +) mlz 474 (M+1) detected; ¹H NMR (400 mHz, DMSO-D₆) δ 8.59 (d, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 7.50 (m, 1H), 7.39 (s, 1H), 7.27 (m, 1H), 7.19 (s, 1H), 7.11 (m, 2H), 4.44 (m, 1H), 4.27 (d, 2H), 2.24 (m, 2H), 2.15 (m, 1H), 2.00 (s, 6H), 1.88 (m, 1H), 1.71 (m, 1H), 0.86 (d, 6H).

Example 35

Preparation of (S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid [1-(2-dimethylaminoethyl)-2-hy-droxy-2-methylpropyl]-amide (38d) [0165] To a cooled (0°C) solution of (S)-2-{[5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amino}-4-dimethylaminobutyric acid methyl ester (see Exampe 30, Steps A-B) (0.112 g, 0.229 mmol) in THF (2 ml) was added dropwise methyl magnesium bromide (2.00 mL of 1.4M solution). The reaction mixture was allowed to warm to room temperature and stir for 16 hours under a N₂ atmosphere. The mixture was partitioned between ethyl acetate and saturated NH₄CI. The layers were separated and the aqueous layer was extracted with ethy I acetate twice. The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was chromatographed on isolute SPE column flash Si (5g) el uting with a gradient TEA/CH₂CI₂ (100 mL, 0.3:99.7), TEA/MeOH/CH₂CI₂(100 mL, 0.3: 0.5: 99.2), TEA/MeOH/CH₂CI₂ (100 mL, 0.3: 2.5: 97.2), TEA/MeOH/ CH₂CI₂ (100 mL, 0.3: 5: 94.7). The final product was obtained In 41 % yield. MS (APCI +) mlz489 (M+1) detected; ¹H NMR (400 mHz, DMSO-D₆) δ 8.00 (s, 1H), 7.97 (d, 1H), 7.89(s, 1H), 7.48 (m, 1H), 7.23 (m, 1 H), 7.18 (s, 1H), 7.10 (m, 1H), 4.58 (m, 1H), 4.27 (d, 2H), 3.88 (m, 1H), 2.23 (m, 2H), 2.07 (s, 6H), 1.88 (m, 1H), 1.43 (m, 1H), 1.13 (ε, 3H), 1.04 (s, 3H), 0.86 (dd, 6H).

Example 36

Preparationof(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylicacid(1-hydroxymethyl-3-[(2-methoxye-thyl)-methylamino]-propyl}-amide (39d) [0166] Step A: (S)-2-tert-Butoxycarbonylamino-4-lodobutyric acid methyl ester: A mixture of (S)-4-bromo-2-tert-butoxycarbonylamínobutyric acid methyl ester (1.19 g, 4.0 mmol) (Example 27, Steps A-D) and Nat (6.0 g, 40.0 mmol) in acetone (25 mL) was heated to 70“C for 2 hours. The mixture was then cooled to room temperature, concentrated under reduced pressure and partitioned between water (10 ml_) and ether (40 ml_). The layers were separated and the organic layer was washed with water (10 mL), dried over MgSO₄, filtered through Celite, and concentrated under reduced pressure to afford 1.26 g of the product (92% yield).

[0167] Step B: (S)-2-tert-Butoxycarbonylamino-4-[(2-methoxyethyl)-methylamlno]-butyrlc acid methyl ester:

A mixture of (S)-24ert-butoxycarbonylamino-4-iodobutyric acid methyl ester (0.200 g, 0.58 mmol), (2-methoxyo-thyl)-methylamine (0.062 g, 0.70 mmol), and triethylamine (0.41 mL, 2.9 mmol) in dioxane (1 mL) was stirred at 70°C for 16 hours. The mixture was cooled to room temperature, concentrated under reduced pressure, and dissolved in dichloromethane (20 mL). The solution was washed with water (3X10 mL), brine (10 mL), dried over MgSO₄, filtered through Celite, and concentrated under reduced pressure. The tan oil was chromatographed eluting with ether to provide 0.87 g of pale yellow oil (49% yield).

[0168] StepC: (S)-2-Amino-4-[(2-methoxyethyl)-methylamino]-butyricacid methylesterdihydrochloride: (S)-2-tert-Butoxycarbonylamino-4-[(2-methoxyethyl)-methylamino]-butyríc acid methyl ester (0.086 g, 0.28 mmol) was treated with HCI (1 mL of 4M in díoxane) and sonicated. The mixture was concentrated and dried under high vacuum to provide the product.

[0169] Step D: (S)-2-{[5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carbonyl]-amino}-4-[(2-methoxye-thyl)-methylamino]-butyric acid methyl ester: To a solution of 5-(2,4-difluorophenoxy)-1-isobutyl-1 H-indazole-6-car-boxylic acid (prepared as In Example 22, Steps A-G) (0.094 g, 0.27 mmol), (S)-2-amino-4-[(2-methoxy-ethyly)-methyl-amino]-butyric acid methyl ester dihydrochloride (0.079 g, 0.28 mmol), EDCI (0.057 g, 0.30 mmol) and HOBt.(0.045 g, 0.30 mmol) in dichloroethane (2 mL) was added dropwise triethylami ne (0.23 mL, 1.62 mmol). The reaction mixture was stirred at room temperature for 2.5 hours. The mixture was concentrated under reduced pressure and chromatographed, eluting with ethyl acetate, to afford 0.100 g of product (69% yield).

[0170] Step E: (S)-5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid {1-hydroxymethyl-3-[(2-methoxyethyl)-methylamino]-propyl}-amide (39d): (S)-2-([5-(2,4-Difluorophenoxy)-1-isobutyl-1H-indazole-6-carbo-nyl]-amino}-4-[(2-methoxyethyl)-methylamino]-butyric acid methyl ester (0.023 g, 0.043 mmol) and NaBH4 (0.016 g, 0.43 mmol) were dissolved In THF/MeOH (7 mL, 5:2) and heated to 70°C in a sealed vial for 5 hours. The mixture was cooled to room temperature, concentrated under reduced pressure and chromatographed eluting with TEA/ethyl acetate (1:4) to provide 0.007 g of the product as viscous oil (31% yield). MS (APCI +) m/z505 (M+1) detected.

Example 37

Preparation of_(S)-5-(2,4-difluorophenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid [3-dimetbylamino-1 -(2-hydrox-yethylcarbamoyl)-propyl]-amide (40d) [0171] PreparedaccordingtotheprocedureinExample32,substituting2-aminoethanolfordimethylamine.Theproduct was obtained in 51% yield: MS (APCI +) m/z518 (M+1) detected; ¹H NMR (400 mHz, CDCI₃) δ 9.00 (d, 1H), 8.29 (s, 1H), 7.86 (s, 1H), 7.81 (t, 1H), 7.18 (m, 1H), 7.02 (m, 1H), 7.00 (s, 1H), 6.92 (m, 1H), 4.82 (m, 1H), 4.21 (d, 2H), 3.42 (m, 2H), 2.86 (m, 1H), 2.49 (m, 2H), 2.35 (m, 1H), 2.23.(s, 6H), 2.17 (m, 2H), 1.94 (m, 1H), 0.92 (d, 6H).