Substituted indazoles for treatment and prevention of allergic and / or inflammatory diseases in animals

Abstract

The present invention relates to the use of substituted indazoles for the treatment and / or prevention of allergic and / or inflammatory diseases in animals, in particular for the treatment and / or prevention of atopic dermatitis, flea allergic dermatitis, inflammatory bowel diseases, osteoarthritis pain and inflammatory pain, non-infectious recurrent airway diseases, and the use of substituted indazoles for the treatment and / or prevention of allergic and / or inflammatory diseases in animals. Insect allergy, asthma, respiratory system diseases, mastitis and endometritis.

Description

[0001] This application is a divisional application of PCT international application PCT / EP2017 / 062876 filed on May 29, 2017, which entered the Chinese national phase on December 3, 2018, and is a Chinese patent application with Chinese patent application number 201780034405.9 and invention name “Substituted indazoles for treating and preventing allergic and / or inflammatory diseases in animals”. Technical Field

[0002] The present application relates to the use of novel substituted indazoles for the treatment and / or prevention of allergic and / or inflammatory diseases in animals and their use for the preparation of medicaments for the treatment and / or prevention of allergic and / or inflammatory diseases in animals, in particular atopic dermatitis and / or flea allergy dermatitis, and in particular in domestic animals, in particular dogs.

[0003] The present invention relates to the use of novel substituted indazoles of the general formula (I) for inhibiting interleukin-1 receptor associated kinase 4 (IRAK4). Background Art

[0004] Human IRAK4 (interleukin-1 receptor-associated kinase 4) plays a key role in activating the immune system. Therefore, this kinase is an important therapeutic target molecule for the development of inflammation-inhibiting substances. IRAK4 is expressed by many cells and mediates signal transduction of the following receptors: Toll-like receptors (TLRs) (except TLR3), and interleukin (1L)-1β family receptors composed of 1L-1R (receptor), 1L-18R, 1L-33R and 1L-36R (Janeway and Medzhitov, Annu. Rev. Immunol., 2002; Dinarello, Annu. Rev. Immunol., 2009; Flannery and Bowie, Biochemical Pharmacology, 2010).

[0005] IRAK4-deficient mice and human cells from IRAK4-deficient patients do not respond to stimulation by TLRs (except TLR3) and the IL-1β family (Suzuki, Suzuki et al., Nature, 2002; Davidson, Currie et al., The Journal of Immunology, 2006; Ku, von Bernuth et al., JEM, 2007; Kim, Staschke et al., JEM, 2007).

[0006] Binding of TLR ligands or IL-1β family ligands to their respective receptors leads to the recruitment and binding of MyD88 [myeloid differentiation primary response gene (88)] to the receptor. Consequently, MyD88 interacts with IRAK4, leading to the formation of an active complex that interacts with and activates the kinases IRAK1 or IRAK2 (Kollewe, Mackensen et al., Journal of Biological Chemistry, 2004; Precious et al., J. Biol. Chem., 2009). As a result, the NF (nuclear factor) -κB signaling pathway and the MAPK (mitogen-activated protein kinase) signaling pathway are activated (Wang, Deng et al., Nature, 2001). Activation of both the NF-κB signaling pathway and the MAPK signaling pathway results in processes associated with different immune processes. For example, there is an increase in the expression of various inflammatory signaling molecules and enzymes such as cytokines, chemokines and COX-2 (cyclooxygenase-2), as well as enhanced mRNA stability of inflammation-related genes such as COX-2, IL-6 (interleukin-6), and IL-8 (Holtmann, Enninga et al., Journal of Biological Chemistry, 2001; Datta, Novotny et al., The Journal of Immunology, 2004). In addition, these processes can be accompanied by the proliferation and differentiation of certain cell types such as monocytes, macrophages, dendritic cells, T cells and B cells (Wan, Chi et al., Nat Immunol, 2006; McGettrick and J. O'Neill, British Journal of Haematology, 2007).

[0007] The central role of IRAK4 in the pathology of various inflammatory diseases has been shown by direct comparisons of wild-type (WT) mice with genetically modified animals harboring a kinase-inactivated form of IRAK4 (IRAK4 KDKI). In animal models of multiple sclerosis, atherosclerosis, myocardial infarction, and Alzheimer's disease, IRAK4 KDKI animals have improved clinical manifestations (Rekhter, Staschke et al., Biochemical and Biophysical Research Communication, 2008; Maekawa, Mizue et al., Circulation, 2009; Staschke, Dong et al., The Journal of Immunology, 2009; Kim, Febbraio et al., The Journal of Immunology, 2011; Cameron, Tse et al., The Journal of Neuroscience, 2012). Furthermore, it has been found that the loss of IRAK4 in animal models protects against viral-induced myocarditis by improving antiviral responses and simultaneously reducing systemic inflammation (Valaperti, Nishii et al., Circulation, 2013). It has also been shown that the expression of IRAK4 is associated with the disease activity of Vogt-Koyanagi-Harada syndrome (Sun, Yang et al., PLoS ONE, 2014). In addition, IRAK4 has been shown to be highly correlated with the production of IFNα (interferon-α) mediated by immune complexes of plasmacytoid dendritic cells, a key process in the pathogenesis of systemic lupus erythematosus (SLE) (Chiang et al., The Journal of Immunology, 2010). In addition, the signaling pathway is associated with obesity (Ahmad, R., P. Shihab et al., Diabetology & Metabolic Syndrome, 2015). In addition to the important role of IRAK4 in innate immunity, there is also a suggestion that IRAK4 affects the differentiation of Th17 T cells (members of adaptive immunity). In the absence of IRAK4 kinase activity, fewer IL-17-producing T cells (Th17 T cells) are produced compared to WT mice.Inhibition of IRAK4 enables the prevention and / or treatment of atherosclerosis, type 1 diabetes, rheumatoid arthritis, spondyloarthritis (especially psoriatic spondyloarthritis and Bekhterev's disease), lupus erythematosus, psoriasis, vitiligo, giant cell arteritis, chronic inflammatory bowel disease and viral diseases, such as HIV (human immunodeficiency virus), hepatitis viruses (Staschke et al., The Journal of Immunology, 2009; Marquez et al., Ann Rheum Dis, 2014; Zambrano-Zaragoza et al., International Journal of Inflammation, 2014; Wang et al., Experimental and Therapeutic Medicine, 2015; Ciccia et al., Rheumatology, 2015).

[0008] Due to the central role of IRAK4 in the MyD88-mediated signaling cascade of the TLR (except TLR3) and IL-1 receptor families, inhibition of IRAK4 can be used to prevent and / or treat diseases mediated by these receptors.

[0009] The prior art discloses many IRAK4 inhibitors (see, for example, Annual Reports in Medicinal Chemistry (2014), 49, 117-133).

[0010] US8293923 and US20130274241 disclose IRAK4 inhibitors with 3-substituted indazole structures, but there is no record of 2-substituted indazoles.

[0011] WO2013106254 and WO2011153588 disclose 2,3-disubstituted indazole derivatives.

[0012] WO2007091107 describes 2-substituted indazole derivatives for the treatment of Duchenne muscular dystrophy. The disclosed compounds do not have a 6-hydroxyalkyl substituent.

[0013] WO2015091426 describes indazoles substituted at the 2-position with a carboxamide side chain, such as Example 64.

[0014]

[0015] WO2015104662 discloses 2-substituted indazoles of the following general formula:

[0016]

[0017] Wherein, R2 is an alkyl or cycloalkyl group. WO2015104662 clearly describes 2-substituted indazoles having methyl, 2-methoxyethyl, and cyclopentyl groups at the 2-position (Examples 1, 4, and 76). Indazole derivatives having a hydroxyethyl substituent at the 1-position are also described in Example 117. However, there is no description of indazole derivatives having a 3-hydroxy-3-methylbutyl substituent at the 1- or 2-position.

[0018] In WO2015104662, indazoles having a hydroxy-substituted alkyl group at the 2-position are broadly encompassed by the general formula but are not explicitly disclosed.

[0019] In WO2015104662, the general formula and the definition of the R2 substituent do not include indazoles having an alkyl group in position 2, wherein the alkyl group is additionally substituted by a methylsulfonyl group.

[0020] In addition to the above-mentioned substitution forms at positions 1 and 2 on indazole, WO2015104662 also describes indazoles having a substituent at position 6, where R1 is defined as follows: alkyl, cyano, -NR a R b or an optional substituent selected from a cycloalkyl group, an aryl group, or a heterocyclic group, wherein the substituent is independently an alkyl group, an alkoxy group, a halogen group, a hydroxyl group, a hydroxyalkyl group, an amino group, an aminoalkyl group, a nitro group, a cyano group, a haloalkyl group, a haloalkoxy group, -OCOCH2-O-alkyl, -OP(O)(O-alkyl)2, or -CH2-OP(O)(O-alkyl)2. For indazole compounds in which R1 is an alkyl group, the effective filing date is January 7, 2015 (the international filing date of WO2015104662). Indian applications 146 / CHE / 2014 and 3018 / CHE / 2014, from which priority is claimed, do not disclose any indazole compounds in which R1 is an alkyl group.

[0021] Thus, the indazole compound of the following general formula:

[0022]

[0023] Indazole compounds wherein R1 is an optionally substituted alkyl group were first disclosed on January 7, 2015, and thus after the priority date of the present application.

[0024] Examples of the 6-position substituent R1 described in WO2015104662 are cyclopropyl, cyclohexyl, cyano, 3-fluorophenyl, and saturated heterocyclic substituents. In WO2015104662, there is no explicit description of indazoles having a hydroxy-substituted alkyl group at the 6-position.

[0025] The compounds used in the present invention are also described in co-pending patent application PCT / EP2015 / 077596, published on June 2, 2016 as WO2016083433.

[0026] Current treatment options for allergic and / or inflammatory diseases in animals, such as allergic dermatoses, typically include the use of steroids and cyclosporine, both of which have side effects. Recently, janus-kinase (JAK) inhibitors have been approved for canine atopic dermatitis (CAD), providing symptomatic relief of pruritus; however, dosing regimens can again be limited by side effects. The use of disease-modifying agents to treat CAD without treatment-related side effects remains an unmet medical need.

[0027] The problem addressed by the present invention is to provide better treatment options for inflammatory and / or allergic diseases in animals.

[0028] The IRAK4 inhibitors of the present invention are particularly suitable for treating and preventing inflammatory diseases in animals characterized by an overreactive immune system. The following diseases should be mentioned in particular: canine atopic dermatitis and flea allergy dermatitis in dogs and cats; inflammatory bowel disease in dogs and cats; osteoarthritis pain and inflammatory pain in dogs, cats, horses, and cattle; non-infectious recurrent airway disease (also known as chronic obstructive pulmonary disease and emphysema) in horses; insect allergy (also known as sweet itch and summer eczema) in horses; feline asthma; bovine respiratory disease; bovine mastitis and endometritis; and porcine respiratory disease.

[0029] For example, atopic dermatitis is a common disease in pets, particularly cats and dogs.

[0030] As a specific example, canine atopic dermatitis (CAD) is one of the most common diseases in dogs. CAD can affect patients from an early age, with recurring episodes throughout their lives. In a 1999 study by Lund et al., which surveyed 31,484 dogs from 52 private clinics in the United States, the prevalence of CAD was 8.7%. CAD is the second most common cause of pruritus in dogs, after flea allergy dermatitis (FAD).

[0031] Canine atopic dermatitis can be defined as "an inherited susceptibility to an inflammatory and pruritic allergic skin disease with characteristic clinical features associated with IgE, most commonly to environmental allergens" (Halliwell, Veterinary Immunology and Immunopathology, 2006). These allergens, such as dust mites and pollen, are ubiquitous and are surprisingly difficult for pets to avoid, as they are pervasive in outdoor air. Canine atopic dermatitis is a complex, multifactorial disease involving immune dysregulation, allergic sensitization, skin barrier defects, microbial colonization, and environmental factors.

[0032] In all cases, IgE was not a prerequisite for the development of clinical symptoms, and a separate clinical entity called atopic dermatitis was defined as "an inflammatory and pruritic skin disease with clinical features identical to those observed in canine atopic dermatitis in which IgE responses to environmental or other allergens have not been documented" (Nuttall et al., Vet. Record, 2013).

[0033] The most common symptoms of canine atopic dermatitis include itching, excessive scratching, rubbing against carpets, hair loss, greasy or flaky skin with a foul odor, paws, and excessive chewing of areas such as the groin and armpits. Over time, scratched skin can develop hot spots—raw, inflamed areas—that can become infected.

[0034] Currently, treatment of acute flares of atopic dermatitis (AD) should include seeking and then eliminating the cause of the flare, bathing with a mild body wash, and controlling itching and skin lesions with interventions including topical and / or oral glucocorticoids or oclacitinib. For chronic CAD, the first step in management is to identify and avoid the triggering factors and ensure adequate skin and surface hygiene; this may include more frequent bathing and possibly increasing essential fatty acid intake. Currently, the most effective medications for relieving chronic itching and skin lesions are topical and oral glucocorticoids, oral cyclosporine, oral oclacitinib, and, where available, injectable recombinant interferon. Allergen-specific immunotherapy and active intermittent topical glucocorticoid use are the only interventions that may prevent or delay the recurrence of AD flares (Olivry et al., BMC Veterinary Research, 2015).

[0035] As another specific example, flea allergy dermatitis (FAD), or flea bite allergy, is the most common skin disease in domestic dogs (Scott et al., In: Muller and Kirk's Small Animal Dermatology, 2001), caused by the most common flea on dogs and cats, Ctenocephalides felis (Beresford-Jones, J Small Animal Practice, 1981; Chesney, Veterinary Record, 1995). Cats also develop FAD, which is one of the main causes of feline miliary dermatitis.

[0036] FAD is most prevalent in the summer, however, flea infestations can persist year-round in warm climates. In northern temperate regions, the close association of pets and their fleas with human dwellings creates conditions that make it a year-round problem. Extreme temperatures and low humidity often inhibit flea development.

[0037] When feeding, fleas inject saliva containing a variety of histamine compounds, enzymes, peptides and amino acids with a wide range of sizes (40-60kD), thereby inducing type I, type IV and basophilic hypersensitivity. Flea-puppies exposed intermittently to flea bites produce an immediate (15 min) reaction or a delayed (24-48 hours) reaction, or both, and produce detectable levels of circulating IgE and IgG anti-flea antibodies. Dogs exposed continuously to flea bites have low levels of these circulating antibodies and do not produce skin reactions, or they develop later and to a much lower degree. This may indicate that immune tolerance is naturally developed in dogs continuously exposed to flea bites. Although little is known about the pathophysiology of FAD in cats, a similar mechanism may exist.

[0038] Cat fleas (Ctenocephalides felis) cause severe irritation to animals and humans and can cause flea allergy dermatitis. Typical symptoms include itching, skin inflammation, and skin lesions (erythema, scaling, papules, crusting, and lichenification). These lesions are most common on the back and base of the tail.

[0039] As the disease progresses, there may be hair loss, broken hair, oozing or crusty sores, pimply bumps, generalized redness, and skin inflammation. The sores can be very painful. In severe cases, the skin can thicken and darken, primarily on the dog's back and around the base of the tail. Due to the severe itching, the dog may inflict self-harm.

[0040] Generally, prevention and treatment of flea infestations are the treatment of choice. Most commonly, neonicotinoids such as imidacloprid or gamma-aminobutyric acid (GABA)-gated chloride channel blockers such as fipronil are used. If skin allergic dermatitis symptoms persist, current treatments mentioned under CAD, such as topical and oral corticosteroids, oral cyclosporine, and oral oclacitinib, are used. Summary of the Invention

[0041] The present invention provides compounds of general formula (I), and diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts thereof, for use in treating and / or preventing allergic and / or inflammatory diseases in animals.

[0042]

[0043] In formula (I):

[0044] R 1 is C1-C6-alkyl, wherein the C1-C6-alkyl group is unsubstituted or monosubstituted or identically or differently polysubstituted by the following radicals

[0045] Halogen, hydroxy, unsubstituted or mono- or poly-halogen-substituted C3-C6-

[0046] Cycloalkyl, or R 6 、R 7 SO2、R 7 SO or R 8 O group,

[0047] or a group selected from the following:

[0048]

[0049] where * represents the bonding site of the group to the rest of the molecule;

[0050] R 2 and R 3 always have the same definition and are hydrogen or C1-C6-alkyl;

[0051] R 4 is halogen, cyano, unsubstituted C1-C6-alkyl or monosubstituted or identically or differently polysubstituted C1-C6-alkyl, or unsubstituted C3-C6-cycloalkyl or monosubstituted or identically or differently polysubstituted C3-C6-cycloalkyl, the substituents being selected from halogen and hydroxy;

[0052] R 5 is hydrogen, halogen, or unsubstituted or mono- or poly-halogen-substituted C1-C6-alkyl;

[0053] R 6 is an unsubstituted or mono- or di-methyl-substituted monocyclic saturated heterocycle having 4 to 6 ring atoms, which contains a heteroatom or heterogroup selected from O, S, SO and SO2;

[0054] R 7 is C1-C6-alkyl, where the C1-C6-alkyl radical is unsubstituted or monosubstituted or identically or differently polysubstituted by halogen, hydroxy or C3-C6-cycloalkyl,

[0055] or R 7 is a C3-C6-cycloalkyl group;

[0056] R 8 is C1-C6-alkyl, where the C1-C6-alkyl radical is unsubstituted, monosubstituted or identically or differently polysubstituted by halogen.

[0057] In the case of the described uses of the synthetic intermediates and working examples of the invention described hereinafter, any compound specified in the form of a salt of the corresponding base or acid is generally a salt of unknown exact stoichiometric composition obtained by the corresponding preparation and / or purification process. Therefore, for such salts, other names and structural formulas such as "hydrochloride", "trifluoroacetate", "sodium salt" or "xHCl", "xCF3COOH", "xNa" are used unless otherwise specified. + " is not to be understood in a stoichiometric sense, but is merely an illustrative designation of the salt-forming components present therein.

[0058] This also applies correspondingly in the case of synthetic intermediates or working examples or their salts which are obtained by the preparation and / or purification processes described and whose stoichiometric composition is unknown (if it is of a defined type) in the form of solvates, such as hydrates.

[0059] The compounds of the present invention are compounds of formula (I) and salts, solvates and solvates of salts thereof, compounds encompassed by formula (I) and of the following structural formulas and salts, solvates and solvates of salts thereof, and compounds encompassed by formula (I) and mentioned below as embodiments thereof, salts, solvates and solvates of salts thereof, even if the compounds encompassed by formula (I) and mentioned below are not salts, solvates and solvates of salts thereof.

[0060] In the context of the present invention, preferred salts are physiologically acceptable salts of the compounds according to the invention. However, the present disclosure also encompasses salts which are not suitable for pharmaceutical applications per se but which can be used, for example, for the isolation or purification of the compounds according to the invention.

[0061] Physiologically acceptable salts of the compounds of the invention include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, for example, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

[0062] Physiologically acceptable salts of the compounds according to the invention also include salts with customary bases, such as, for example and preferably, alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, such as, for example and preferably, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.

[0063] In the context of the present invention, solvates are described as those forms of the compounds according to the invention which form solid or liquid complexes by coordination with solvent molecules. Hydrates are a special form of solvates which coordinate with water.

[0064] The compounds according to the invention may exist, depending on their structure, in different stereoisomeric forms, i.e. as configurational isomers or, if appropriate, conformational isomers (enantiomers and / or diastereomers, including atropisomers). The present invention therefore encompasses the use of enantiomers and diastereomers and their respective mixtures. Stereoisomerically homogeneous constituents can be isolated from these mixtures of enantiomers and / or diastereomers in a known manner; for this purpose, preferably chromatography is used, in particular HPLC chromatography on an achiral or chiral phase.

[0065] If the compounds according to the invention can exist in tautomeric forms, the present invention includes the use of all tautomeric forms.

[0066] The present invention also includes the purposes of all suitable isotopic variants of the compounds of this invention.Herein, the isotopic variant of the compounds of this invention is understood to mean such a compound: wherein at least one atom in the compounds of this invention has been replaced with an atomic number identical, but the atomic mass is another atom different from the atomic mass usually or mainly present in nature.The example of the isotope that can be incorporated into the compounds of this invention is those isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine, bromine and iodine, such as 2H (deuterium), 3H (tritium), 13C, 14C, 15N, 17O, 18O, 32P, 33P, 33S, 34S, 35S, 36S, 18F, 36Cl, 82Br, 123I, 124I, 129I and 131I. Special isotopic variants of the compounds of the present invention, such as those in which one or more radioactive isotopes have been incorporated, can be beneficial for investigating the mechanism of action or active ingredient distribution in vivo; compounds labeled with 3H or 14C isotopes are particularly suitable for this purpose due to their relatively easy preparability and detectability. In addition, the incorporation of isotopes (e.g., deuterium) may produce special therapeutic benefits due to the higher metabolic stability of the compound, such as an extension of the half-life in vivo or a reduction in the required active dose; therefore, in some cases, such modifications to the compounds of the present invention may also constitute a preferred embodiment of the use of the present invention. Isotopic variants of the compounds of the present invention can be prepared by methods known to those skilled in the art, for example, by using the corresponding isotopic modifications of the respective reagents and / or starting compounds as described further below and in the working examples.

[0067] The present invention further provides for the use of all possible crystalline forms and polymorphs of the compounds according to the invention, wherein said polymorphs may be present as single polymorphs or as mixtures of a plurality of polymorphs within the entire concentration range. DETAILED DESCRIPTION

[0068] In addition, the present invention also includes the use of prodrugs of the compounds of the present invention. In this context, the term "prodrug" refers to a compound that may itself be biologically active or inactive, but is converted (e.g. metabolically or hydrolytically) into a compound of the present invention during its residence time in the body.

[0069] In the context of the present invention, unless otherwise stated, substituents have the following meanings:

[0070] alkyl In the context of the present invention, alkyl refers to a straight or branched alkyl group having a specified number of carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl, 2-methylpropyl, tert-butyl, n-pentyl, 1-ethylpropyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl, and 2-ethylbutyl. Methyl, ethyl, n-propyl, n-butyl, 2-methylbutyl, 3-methylbutyl, and 2,2-dimethylpropyl are preferred.

[0071] Cycloalkyl In the context of the present invention are monocyclic saturated alkyl radicals having in each case the indicated number of carbon atoms. Preferred examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0072] Alkoxy In the context of the present invention, it refers to a straight-chain or branched alkoxy group with a specified number of carbon atoms. Preferably, it has 1 to 6 carbon atoms. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, 1-methylpropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy, 1-ethylpropoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy and n-hexyloxy. Particularly preferred are straight-chain or branched alkoxy groups with 1 to 4 carbon atoms. Preferred examples include methoxy, ethoxy, n-propoxy, 1-methylpropoxy, n-butoxy and isobutoxy.

[0073] halogen In the context of the present invention these are fluorine, chlorine and bromine. Fluorine is preferred.

[0074] Hydroxyl In the context of the present invention this is OH.

[0075] Monocyclic saturated heterocycleIt is a monocyclic saturated heterocycle having 4 to 6 ring atoms and containing a heteroatom or hetero group selected from O, S, SO and SO2. Heterocycles having heteroatoms or hetero groups selected from O, SO and SO2 are preferred. Examples include: oxetane, tetrahydrofuran, tetrahydro-2H-pyran-4-yl, 1,1-dioxotetrahydro-2H-thiopyran-3-yl, 1,1-dioxotetrahydro-2H-thiopyran-2-yl, 1,1-dioxotetrahydro-2H-thiopyran-4-yl, 1,1-dioxotetrahydrothiophen-3-yl, 1,1-dioxotetrahydrothiophen-2-yl, 1,1-dioxathietan-2-yl or 1,1-dioxathietan-3-yl. Oxetane and tetrahydrofuran are particularly preferred in this context. Oxetane-3-yl is very particularly preferred.

[0076] The symbol * on a bond indicates the bonding site in the molecule.

[0077] Unless otherwise indicated, when a group in the compounds of the present invention is substituted, the group may be monosubstituted or polysubstituted. In the context of the present invention, all groups that occur more than once are defined independently of one another. Preferably, they are substituted by one, two or three identical or different substituents.

[0078] R 1 A preferred embodiment is a C2-C6-alkyl group substituted by 1, 2 or 3 fluorine atoms. Particular preference is given to 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl and 4,4,4-trifluorobutyl. Very particular preference is given to 4,4,4-trifluorobutyl.

[0079] R 1 Another preferred embodiment is a C2-C6-alkyl group substituted by one or two hydroxyl groups or one C1-C3-alkoxy or trifluoro-substituted C1-C3-alkoxy. Particularly preferred are C2-C5-alkyl groups substituted by hydroxyl groups or C1-C3-alkoxy groups or trifluoromethoxy groups or 2,2,2-trifluoroethoxy groups. Very particularly preferred are 3-hydroxy-3-methylbutyl, 3-methoxypropyl, 3-hydroxypropyl, 3-trifluoromethoxypropyl, 2-methoxyethyl or 2-hydroxyethyl groups. 3-hydroxy-3-methylbutyl is particularly preferred.

[0080] In addition, preferably, R 1 is a C2-C6-alkyl group substituted by a C1-C6-alkyl-SO2 group. Particularly preferred is a C2-C4-alkyl group substituted by a methyl-SO2-group. 1 is 2-(methylsulfonyl)ethyl or 3-(methylsulfonyl)propyl. Of the latter group, 2-(methylsulfonyl)ethyl is particularly preferred.

[0081] In addition, preferably, R 1is a C1-C3-alkyl group substituted by an oxetane group, a tetrahydrofuranyl group, a tetrahydro-2H-pyran-4-yl group, a 1,1-dioxotetrahydro-2H-thiopyran-3-yl group, a 1,1-dioxotetrahydro-2H-thiopyran-2-yl group, a 1,1-dioxotetrahydro-2H-thiopyran-4-yl group, a 1,1-dioxotetrahydrothiophen-3-yl group, a 1,1-dioxotetrahydrothiophen-2-yl group, a 1,1-dioxathietan-2-yl group or a 1,1-dioxathietan-3-yl group. A C1-C3-alkyl group substituted by an oxetane group is particularly preferred. R is particularly preferred. 1 It is an oxetan-3-ylmethyl group.

[0082] For R 2 and R 3 , always have the same definition, preferably hydrogen or methyl. Particularly preferred is methyl.

[0083] For R 4 , preferably an unsubstituted or mono- or poly-halogen-substituted C1-C3-alkyl group, or a C1-C3-alkyl group substituted by one hydroxyl group, or a C1-C3-alkyl group substituted by one hydroxyl group and three fluorine atoms.

[0084] For R 4 , particularly preferably the following groups: methyl, ethyl, trifluoro-C1-C3-alkyl, difluoro-C1-C3-alkyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyprop-2-yl and 2,2,2-trifluoro-1-hydroxyethyl. 4 , particularly preferably methyl, trifluoromethyl and difluoromethyl groups. Among them, the trifluoromethyl group is particularly preferred.

[0085] R 5 A preferred embodiment of is hydrogen, fluorine, chlorine or C1-C3-alkyl. More preferably, R 5 is hydrogen, fluorine or methyl. Most preferably, R 5 is hydrogen or fluorine.

[0086] R is also particularly preferred 4 is methyl or trifluoromethyl and R 5 A compound in which R is fluorine. 4 is methyl and R 5 A compound of fluorine, wherein R 5 In R 4 The adjacent position.

[0087] For R 6, preferred embodiments include oxetane, tetrahydrofuran, tetrahydro-2H-pyran-4-yl, 1,1-dioxotetrahydro-2H-thiopyran-3-yl, 1,1-dioxotetrahydro-2H-thiopyran-2-yl, 1,1-dioxotetrahydro-2H-thiopyran-4-yl, 1,1-dioxotetrahydrothiophen-3-yl, 1,1-dioxotetrahydrothiophen-2-yl, 1,1-dioxathietan-2-yl or 1,1-dioxathietan-3-yl. Oxetane is particularly preferred herein. Oxetane-3-yl is very particularly preferred.

[0088] R 7 Only connected to the functional groups -SO2- and -SO-, that is, R 7 -substituted -SO2- or SO group. 7 Preferably C1-C4-alkyl, wherein the C1-C4-alkyl group is unsubstituted or monosubstituted by hydroxy or cyclopropyl or substituted by three fluorine atoms. 7 is a cyclopropyl group. 7 is methyl, ethyl or hydroxyethyl. 7 It is a methyl group.

[0089] This means that, when R 7 SO2- or R 7 In the case of SO-substituted C1-C6-alkyl groups, R 1 For R 1 In this context, methylsulfonylethyl and methylsulfonylpropyl are particularly preferred. In this context, methylsulfonylethyl is very particularly preferred.

[0090] For R 8 , preferably an unsubstituted C1-C4-alkyl group or a trifluorosubstituted C1-C4-alkyl group. Particularly preferred are methyl, ethyl, trifluoromethyl or 2,2,2-trifluoroethyl. Very particularly preferred are methyl, trifluoromethyl or 2,2,2-trifluoroethyl.

[0091] As previously mentioned, the intracellular enzyme interleukin-1 receptor-associated kinase 4 (IRAK4) plays an integral role in the signaling pathways of receptors activated by cytokines and TLR ligands, which are involved in inflammatory processes. In addition to inflammation, IRAK4 is also involved in signaling of allergic processes. Such allergic processes play an important role in the pathogenesis of allergic skin diseases such as atopic dermatitis.

[0092] For example, IL-33, which was recently added to the IL-1 cytokine family that also includes IL-18 and IL-1, binds to and activates the IL-33 receptor (IL-33R), which in turn is associated with MyD88, IRAK4, and TRF6 (Schmitz et al., Immunity, 2005). IRAK4 is an essential component of this signaling pathway. IL-33R is strongly expressed in T helper cell type 2 (Th2) cells, mast cells, and eosinophils. IL-33 activates these cells and promotes the immune response of Th2 (Schmitz et al., Immunity, 2005). These cell types are all involved in the pathogenesis of atopic dermatitis. IL-33 levels in serum are correlated with the severity of human atopic dermatitis and are reduced when treated with topical steroids and calcineurin inhibitors (Tamagawa-Mineoka et al., J American Academy Dermatology, 2014). In a model of acute canine atopic dermatitis, the IL-33 gene has been shown to be significantly upregulated in skin lesions (Schamber et al., G3 (Bethesda), 2014; Olivry et al., Journal of Investigative Dermatology, 2016).

[0093] In addition, the second member of the IL-1 cytokine family, IL-18 is associated with atopic dermatitis. Serum IL-18 levels increase with the severity of atopic dermatitis in children (Sohn et al., Allergy and Asthma Proceedings, 2004). In the model of acute canine atopic dermatitis, it has been shown that the IL-18 gene is significantly upregulated in skin lesions (Schamber et al., G3 (Bethesda), 2014; Olivry et al., Journal of Investigative Dermatology, 2016). In addition, atopic dermatitis-like inflammation & pruritus is caused by the excessive release of IL-18 and is accelerated by mouse IL-1 (Konishi et al., Proceedings of the National Academy of Sciences, 2002). In addition, it has been shown that IRAK-4 is an essential component of the IL-18 signal cascade (Suzuki et al., J Immunology, 2003). Similarly, IRAK4 is crucial for signaling of IL-1 and TLR ligands (Suzuki et al., Nature, 2002). TLR antagonists are known to cause pruritus (Liu et al., Neuroscience Bulletin, 2012), a key symptom of atopic dermatitis, and anti-IL-1 therapy is used off-label to treat atopic dermatitis. In addition, polymorphs of IRAK4 are associated with elevated total IgE in allergic diseases such as asthma and chronic sinusitis (Tewfik et al., Allergy, 2009). In atopic dermatitis, IgE levels are also elevated.

[0094] Therefore, since IRAK4 is a key part of the signaling pathway activated by many cytokines and TLR ligands, and IRAK4 has polymorphic forms that are associated with increased IgE levels, inhibiting IRAK4 is an important therapeutic strategy for treating allergic skin diseases such as atopic dermatitis. In addition, in pets (especially dogs and cats), atopic dermatitis and flea allergy dermatitis are both appropriate indications because both diseases include type I allergies involving IgE antibodies, Th2 cells, mast cells, and eosinophils. In addition, FAD may include type IV allergies in which IL-1 and IL-18 are involved.

[0095] The compounds of the present invention act as IRAK4 kinase inhibitors and therefore have an unexpectedly useful spectrum of pharmacological activity in the treatment and / or prevention of allergic and / or inflammatory diseases in animals.

[0096] Preferred are compounds of formula (I), and diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts thereof, for use in treating and / or preventing allergic and / or inflammatory diseases in animals, wherein

[0097] R 1 is C1-C6-alkyl, wherein the C1-C6-alkyl group is unsubstituted or substituted by fluorine, hydroxy, or R 6 、R 7 SO2、R 7 SO or R 8 The O groups are monosubstituted or polysubstituted identically or differently;

[0098] R 2 and R 3 always have the same definition and are hydrogen or C1-C3-alkyl;

[0099] R 4 is halogen, cyano or C1-C3-alkyl, where the C1-C3-alkyl radical is unsubstituted or monosubstituted or identically or differently polysubstituted by halogen or hydroxy;

[0100] R 5 is hydrogen, fluorine, chlorine or C1-C3-alkyl;

[0101] R 6 is oxetane or tetrahydrofuranyl;

[0102] R 7 is C1-C4-alkyl, wherein the C1-C4-alkyl group is unsubstituted, or monosubstituted by hydroxy or cyclopropyl, or substituted by three fluorine atoms;

[0103] R 8 is unsubstituted C1-C4-alkyl or trifluoro-substituted C1-C4-alkyl.

[0104] Furthermore, preferred are compounds of formula (I), and diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts thereof, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals, wherein

[0105] R 1 is C2-C6-alkyl, wherein the C2-C6-alkyl is unsubstituted, or

[0106] C2-C6-alkyl is mono-, di- or tri-fluoro-substituted, or

[0107] C2-C6-alkyl is replaced by hydroxyl, R 6 、R 7 SO2 or R 8 O single substitution,

[0108] or R 1 is an oxetane-substituted C1-C3-alkyl group;

[0109] R 2 and R 3 always have the same definition and are either hydrogen or methyl;

[0110] R 4 is an unsubstituted or mono- or poly-halogen-substituted C1-C3-alkyl group, or a C1-C3-alkyl group substituted by one hydroxyl group, or a C1-C3-alkyl group substituted by one hydroxyl group and three fluorine atoms;

[0111] R 5 is hydrogen, fluorine or C1-C3-alkyl;

[0112] R 7 is C1-C3-alkyl;

[0113] R 8 is C1-C4-alkyl, where the C1-C4-alkyl radical is unsubstituted or mono-, di- or tri-fluoro-substituted.

[0114] Particularly preferred are compounds of the general formula (I), and their diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals, wherein

[0115] R 1 is a C2-C5-alkyl group substituted by hydroxy, or C1-C3-alkoxy, or trifluoromethoxy, or 2,2,2-trifluoroethoxy, or trifluoromethyl, or

[0116] is a methyl-SO2-substituted C2-C4-alkyl group, or

[0117] is a C1-C2-alkyl group substituted with an oxetane-3-yl group;

[0118] R 2 and R 3 always have the same definition and are either hydrogen or methyl;

[0119] R 4 is methyl, ethyl, trifluoro-C1-C3-alkyl, difluoro-C1-C3-alkyl, hydroxymethyl,

[0120] 1-hydroxyethyl, 2-hydroxypropan-2-yl and 2,2,2-trifluoro-1-hydroxyethyl, and

[0121] R 5 is hydrogen, fluorine or methyl.

[0122] Very particular preference is given to the following compounds, and their diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals, wherein

[0123] R 1 is 4,4,4-trifluorobutyl, 3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-methoxypropyl, 3-hydroxypropyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-trifluoromethoxypropyl, 2-methoxyethyl, 2-hydroxyethyl, 2-(methylsulfonyl)ethyl or 3-(methylsulfonyl)propyl;

[0124] R 2 and R 3 are all methyl or hydrogen, and

[0125] R 4 is difluoromethyl, trifluoromethyl or methyl, and

[0126] R 5 is hydrogen or fluorine.

[0127] Very particular preference is also given to the following compounds, and their diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals, wherein

[0128] R 1 is 3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-(methylsulfonyl)propyl or 2-(methylsulfonyl)

[0129] ethyl;

[0130] R 2 and R 3 All are methyl;

[0131] R 4 is difluoromethyl or trifluoromethyl; and

[0132] R 5 For hydrogen.

[0133] Particular preference is also given to the following compounds, and their diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals:

[0134] R 1 is 3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-(methylsulfonyl)propyl or 2-(methylsulfonyl)

[0135] ethyl;

[0136] R 2 and R 3 All are methyl;

[0137] R 4 is methyl, and

[0138] R 5 is fluorine, where R 5 In R 4 The adjacent position.

[0139] The present invention particularly provides the following compounds for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals:

[0140] 1) N-[6-(2-hydroxypropan-2-yl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0141] 2) N-[6-(hydroxymethyl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)

[0142] Pyridine-2-carboxamide

[0143] 3) N-[6-(2-hydroxypropan-2-yl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0144] 4) N-[6-(hydroxymethyl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)

[0145] Pyridine-2-carboxamide

[0146] 5) N-[2-(2-hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0147] 6) N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0148] 7) N-[2-(2-hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0149] 8) N-[6-(2-hydroxypropan-2-yl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0150] 9) N-[6-(hydroxymethyl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0151] 10) N-{6-(2-hydroxypropan-2-yl)-2-[3-(methylsulfonyl)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0152] 11) N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0153] 12) N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0154] 13) 6-(Difluoromethyl)-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0155] 14) 6-(Difluoromethyl)-N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}pyridine-2-carboxamide

[0156] 15) 6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0157] 16) N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0158] 17) N-{6-(2-hydroxypropan-2-yl)-2-[3-(trifluoromethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0159] 18) N-{6-(2-hydroxypropan-2-yl)-2-[3-(2,2,2-trifluoroethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0160] 19) 5-Fluoro-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0161] 20) N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0162] 21) 6-(2-Hydroxypropan-2-yl)-N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0163] 22) N-{2-[2-(1-Hydroxycyclopropyl)ethyl]-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide.

[0164] The present invention also provides compounds of general formula (III) and diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts thereof for use in treating and / or preventing allergic and / or inflammatory diseases in animals

[0165]

[0166] In the general formula (III)

[0167] R 1 is 4,4,4-trifluorobutyl, 3-hydroxy-3-methylbutyl, 3-methoxypropyl, 3-hydroxypropyl, 3-hydroxybutyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-trifluoromethoxypropyl, 2-methoxyethyl, 2-hydroxyethyl, 2-(methylsulfonyl)ethyl, 3-(methylsulfonyl)propyl or 2-(1-hydroxycyclopropyl)ethyl;

[0168] R 4 is difluoromethyl, trifluoromethyl or methyl; and

[0169] R 5 is hydrogen or fluorine.

[0170] Particularly preferred are compounds of the following general formula (III) for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals:

[0171] 5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester and

[0172] Methyl 2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate.

[0173] The compound of general formula (III) is suitable for preparing a portion of the compound of general formula (I).

[0174] Furthermore, the compounds of formula (III) are inhibitors of interleukin-1 receptor associated kinase-4 (IRAK4).

[0175] Compounds of general formula (III) can be prepared from compounds of general formula (II) by reacting (II) with an appropriately substituted alkyl halide or alkyl 4-methylbenzenesulfonate in the presence of potassium carbonate.

[0176]

[0177] in

[0178] R 1 4,4,4-trifluorobutyl, 3-hydroxy-3-methylbutyl, 3-methoxypropyl, 3-hydroxypropyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-trifluoromethoxypropyl, 2-methoxyethyl, 2-hydroxyethyl, 2-(methylsulfonyl)

[0179] Ethyl, 3-(methylsulfonyl)propyl or 2-(1-hydroxycyclopropyl)ethyl;

[0180] R 4 is difluoromethyl, trifluoromethyl or methyl; and

[0181] R 5 is hydrogen or fluorine.

[0182] In addition, the compound of formula (I) can be prepared from the compound of formula (III) by Grignard reaction with methylmagnesium bromide.

[0183]

[0184] in

[0185] R 1 4,4,4-trifluorobutyl, 3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-methoxypropyl, 3-hydroxypropyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-trifluoromethoxypropyl, 2-methoxyethyl, 2-hydroxyethyl, 3-(methylsulfonyl)propyl, 2-(1-hydroxycyclopropyl)ethyl;

[0186] R 2 and R 3 is methyl;

[0187] R 4 is difluoromethyl, trifluoromethyl or methyl; and

[0188] R 5 is hydrogen or fluorine.

[0189] The compounds of the present invention act as IRAK4 kinase inhibitors and possess an unexpectedly useful spectrum of pharmacological activity.

[0190] It is further preferred to use the compounds of formula (I), or the compounds specifically mentioned above, for the treatment and / or prevention of allergic and / or inflammatory diseases in domestic animals, in particular cats and dogs, and more particularly dogs.

[0191] In the present context, the term "livestock" includes, for example, mammals, such as hamsters, guinea pigs, rats, mice, chinchillas, ferrets or in particular dogs, cats; caged birds; reptiles; amphibians or aquarium fish.

[0192] In addition, it is preferred that the compounds of formula (I), or the compounds specifically mentioned above, are used for the treatment and / or prevention of allergic dermatitis in livestock, in particular canine and feline allergic dermatitis, and more particularly canine allergic dermatitis. In addition, it is preferred that the compounds of formula (I), or the compounds specifically mentioned above, are used for the treatment and / or prevention of allergic and / or inflammatory diseases in farm animals, in particular sheep, goats, horses, cattle and pigs, and more particularly cattle and pigs.

[0193] In the present context, the term "farm animals" includes, for example, mammals such as horses, sheep, goats, buffalo, reindeer, fallow deer or more particularly cattle or pigs.

[0194] The compounds of formula (I), or the compounds mentioned above in particular, are further preferably used in a method for the treatment and / or prophylaxis of atopic dermatitis, flea allergy dermatitis, inflammatory bowel disease, osteoarthritic pain and inflammatory pain, non-infectious recurrent airway diseases, insect allergies, asthma, respiratory diseases, mastitis and endometritis in animals, in particular atopic dermatitis and flea allergy dermatitis.

[0195] Particular preference is given to using the compounds of formula (I), or the compounds specifically mentioned above, in a method for the treatment and / or prevention of canine atopic dermatitis, flea allergy dermatitis in dogs or cats; inflammatory bowel disease in dogs or cats; osteoarthritis pain and inflammatory pain in dogs, cats, horses or cattle; non-infectious recurrent airway disease in horses; insect allergy in horses; feline asthma; bovine respiratory disease; bovine mastitis; bovine endometritis and porcine respiratory disease.

[0196] Very particular preference is given to using the compounds of the formula (I) or the compounds specifically mentioned above in a method for the treatment and / or prophylaxis of canine atopic dermatitis and flea allergy dermatitis in dogs or cats, more particularly dogs.

[0197] Very particular preference is further given to using the compounds of the formula (I) or the compounds mentioned above specifically in a method for the treatment and / or prophylaxis of osteoarthritic and inflammatory pain in cattle, bovine respiratory disease, bovine mastitis, bovine endometritis and porcine respiratory disease.

[0198] With regard to the compounds of formula (III), it is further preferred that the compounds of formula (III) are used for the treatment and / or prevention of allergic and / or inflammatory diseases in domestic animals, in particular cats and dogs, and more particularly dogs.

[0199] The compounds of formula (III) are further preferably used for the treatment and / or prevention of allergic dermatitis in domestic animals, in particular canine and feline allergic dermatitis, and more particularly canine allergic dermatitis.

[0200] The compounds of formula (III) are further preferably used for the treatment and / or prevention of allergic and / or inflammatory diseases in farm animals, in particular in sheep, goats, horses, cattle and pigs, and more particularly in cattle and pigs.

[0201] The compounds of the formula (III) are further preferably used in a method for the treatment and / or prevention of the following diseases in animals: atopic dermatitis, flea allergy dermatitis, inflammatory bowel disease, osteoarthritis pain and inflammatory pain, non-infectious recurrent airway diseases, insect allergies, asthma, respiratory diseases, mastitis and endometritis, in particular atopic dermatitis and flea allergy dermatitis.

[0202] Particularly preferably, the compounds of formula (III) are used in a method for treating and / or preventing canine atopic dermatitis, flea allergy dermatitis in dogs or cats; inflammatory bowel disease in dogs or cats; osteoarthritis pain and inflammatory pain in dogs, cats, horses or cattle; non-infectious recurrent airway disease in horses; insect allergy in horses; feline asthma; bovine respiratory disease; bovine mastitis; bovine endometritis and porcine respiratory disease.

[0203] Very particular preference is given to using the compounds of the formula (III) in a method for the treatment and / or prophylaxis of canine atopic dermatitis and flea allergy dermatitis in dogs or cats, more particularly in dogs.

[0204] Very particular preference is further given to using the compounds of the formula (III) in a method for the treatment and / or prophylaxis of osteoarthritic and inflammatory pain in cattle, respiratory disease in cattle, bovine mastitis, bovine endometritis and respiratory disease in pigs.

[0205] For example, compound examples 11, 12, 13, and 19 (shown below) have been evaluated in an in vitro IRAK4 TR-FRET assay described in detail below using recombinant canine IRAK4 enzymes. The IC50 values ​​for each compound for inhibiting canine IRAK4 have been calculated. The example compounds (11, 12, 13, and 19) have been confirmed to be useful for treating allergic skin diseases such as atopic dermatitis and flea allergy dermatitis in animals, particularly dogs and cats. Example compounds 11, 12, 13, and 19 are potent inhibitors of canine IRAK4 with IC50 values ​​of 1.7, 9.2, 2.2, and 7.6 nM, respectively. For the inhibition of human IRAK4, the IC50 values ​​of each of these example compounds were similar to the calculated IC50 values.

[0206] As another example, Example Compound 12 was also evaluated in vitro to determine the compound's efficacy on lipopolysaccharide (LPS)-induced cytokine production by canine peripheral blood mononuclear cells (PBMC). Example Compound 12 inhibited the production of proinflammatory cytokine tumor necrosis factor α (TNFα) in a concentration-dependent manner by LPS-induced canine PBMC. PBMC includes cell types such as dendritic cells, T lymphocytes and B lymphocytes, as well as monocytes, each of which is associated with atopic dermatitis, and TNFα is elevated in patients with atopic dermatitis (Sumimoto et al., Archives of Disease in Childhood, 1992). This example was also evaluated in vitro to determine the compound's efficacy on lipopolysaccharide (LPS)-induced cytokine production by canine peripheral blood mononuclear cells (PBMC). Example Compound 12 inhibited the production of proinflammatory cytokine tumor necrosis factor α (TNFα) in a concentration-dependent manner by LPS-induced canine PBMC. PBMC includes cell types such as dendritic cells, T lymphocytes and B lymphocytes, as well as monocytes, each of which is associated with atopic dermatitis, and TNFα is elevated in patients with atopic dermatitis (Sumimoto et al., Archives of Disease in Childhood, 1992). Figure 7 illustrate.

[0207] Thus, compounds of the present invention demonstrated inhibition of recombinant canine IRAK4 enzyme and cytokine production by canine PBMCs, indicating potential therapeutic benefit of these compound examples in canine atopic dermatitis and flea allergy dermatitis.

[0208] In addition, in a further study, Example Compound 12 was evaluated in vivo in a house dust mite model to determine the efficacy of the compound in treating clinical symptoms associated with canine allergic dermatitis, particularly canine atopic dermatitis (CAD). Example Compound 12 significantly reduced the clinical symptoms of CAD-like skin edema and erythema. This example also Figure 11 and Figure 12 illustrate.

[0209] Thus, the compounds of the present invention exhibited a reduction in the characteristic clinical symptoms of canine allergic dermatitis, thereby demonstrating the therapeutic benefit of such compound examples in canine allergic dermatitis, particularly canine atopic dermatitis (CAD). In addition, Example Compound 12 has been evaluated in an in vivo model of canine flea allergy dermatitis (CAD) to determine the anti-pruritic efficacy of the compound. Subsequent treatment with Example Compound 12 significantly reduced the pruritus associated with allergic diseases such as flea allergy dermatitis. This example also Figure 13 illustrate.

[0210] Thus, the compounds of the present invention show a reduction in characteristic clinical symptoms associated with allergic dermatitis, such as dermatitis and pruritus, thereby demonstrating the therapeutic benefit of such compound embodiments in canine allergic dermatitis, particularly flea allergy dermatitis (FAD) and canine atopic dermatitis (CAD).

[0211] In the present context, the term "canine allergic dermatitis" specifically includes canine atopic dermatitis (CAD) and flea allergy dermatitis (FAD).

[0212] As another example, Example Compound 12 has also been evaluated in an ex vivo assay to determine the efficacy of the compound on lipopolysaccharide (LPS)-induced cytokine production by bovine peripheral blood mononuclear cells (PBMC). Example Compound 12 inhibits the production of proinflammatory cytokine tumor necrosis factor alpha (TNFα) in a concentration-dependent manner by LPS-induced canine PBMC. PBMC includes cell types such as dendritic cells, T lymphocytes and B lymphocytes, and monocytes, each of which is associated with inflammatory and infectious diseases with excessive proinflammatory immune responses, such as respiratory diseases (Sterner-Kock, Haider et al., Tropical Animal Health and Production, 2016), intestinal diseases (Pan, Rostagnio et al., Veterinary Immunology and Immunopathology, 2015) and mastitis (Zheng, Xu et al., Free Radical Biology and Medicine, 2016), in patients with these diseases, where TNFα is elevated. This example is also described by Figure 8 and 9 illustrate.

[0213] Thus, compounds of the present invention exhibit inhibition of cytokine production by bovine PBMCs, suggesting potential therapeutic benefits of such compound embodiments in inflammatory and / or infectious diseases such as respiratory diseases, intestinal diseases, and mastitis.

[0214] As another example, Example Compound 12 has also been evaluated in vitro to determine the compound's efficacy in lipopolysaccharide (LPS)-induced cytokine production by porcine peripheral blood mononuclear cells (PBMC). Example Compound 12 inhibits the production of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) by LPS-induced porcine PBMC. PBMCs include cell types such as dendritic cells, T lymphocytes and B lymphocytes, as well as monocytes, each of which is associated with inflammatory diseases and infectious diseases such as respiratory and intestinal diseases with excessive proinflammatory immune responses, and TNFα is elevated in patients with these diseases. This example also includes Figure 10 illustrate.

[0215] Thus, the present compounds exhibited inhibition of cytokine production by porcine PBMCs, suggesting potential therapeutic benefits of such compound embodiments in inflammatory and / or infectious diseases such as respiratory and intestinal diseases.

[0216] The compounds of the present invention also provide for the prevention and / or treatment of pruritus and pain in animals, particularly acute, chronic, inflammatory and neuropathic pain in animals.

[0217] In addition, the compounds of the present invention are suitable for treating and / or preventing pain disorders in animals, in particular acute, chronic, inflammatory and neuropathic pain. Such disorders preferably include hyperalgesia, allodynia, pain in arthritis (e.g. osteoarthritis, rheumatoid arthritis and spondyloarthritis), premenstrual pain, endometriosis-related pain, postoperative pain, interstitial cystitis pain, CRPS (complex regional pain syndrome), trigeminal neuralgia, prostatitis pain, pain caused by spinal cord injury, pain caused by inflammation, low back pain, cancer pain, chemotherapy-related pain, HIV-treatment-induced neuropathy, pain caused by burns and chronic pain.

[0218] The present invention also provides a method for treating and / or preventing animal diseases, in particular the above-mentioned diseases, using an effective amount of at least one compound according to the present invention.

[0219] Preferably, there is provided a method for treating and / or preventing allergic and / or inflammatory diseases in animals by administering an effective amount of at least one compound of formula (I) according to the invention as defined above to an animal in need thereof.

[0220] In the context of the present invention, the term "treatment" or "treating" includes inhibiting, delaying, checking, alleviating, relieving, limiting, reducing, suppressing, resisting or curing a disease, condition, disorder, injury or health problem, or the development, progression or evolution of said condition and / or the symptoms of said condition. The term "therapy" is understood herein to be synonymous with the term "treatment".

[0221] In the context of the present invention, the terms "prevention," "prophylaxis," and "preclusion" are used synonymously and refer to avoiding or reducing the risk of contracting, experiencing, suffering, or having a disease, condition, disorder, injury, or health problem, or the development or progression of such a condition, and / or the symptoms of such a condition.

[0222] The disease, condition, disorder, injury or health problem may be treated or prevented, either partially or completely.

[0223] The compounds of the present invention may be used alone or, if desired, in combination with other active ingredients. The present invention also provides a medicament comprising at least one compound of the present invention and one or more other active ingredients for use in treating and / or preventing allergic and / or inflammatory diseases in animals. Preferred examples of active ingredients suitable for use in the composition include:

[0224] In general, there may be mentioned active ingredients such as antibacterial substances (e.g. penicillin, vancomycin, ciprofloxacin), antiviral substances (e.g. aciclovir, oseltamivir) and antimycotic substances (e.g. naftifin, nystatin) as well as gamma-globulins; immunomodulatory and immunosuppressive compounds such as cyclosporin, TNF antagonists (e.g. Etanercept, infliximab), IL-1 inhibitors (e.g., anakinra, canakinumab, rilonacept), phosphodiesterase inhibitors (e.g., apremilast), Jak / STAT inhibitors (e.g., tofacitinib, baricitinib, GLPG0634), leflunomid, cyclophosphamide, rituximab, belimumab, tacrolimus, rapamycin, mycophenolate mofetil mofetil), interferon, corticosteroids (e.g., prednisone, prednisolone, methylprednisolone, hydrocortisone, betamethasone), cyclophosphamide, azathioprine, and sulfasalazine; paracetamol, non-steroidal anti-inflammatory substances (NSAIDS) (aspirin, ibuprofen, naproxen, etodolac, celecoxib, colchicine).

[0225] In addition to those mentioned above, the IRAK4 inhibitors of the present invention may also be combined with the following active ingredients:

[0226] Substances used to treat lung diseases, such as beta-2 sympathomimetics (e.g. salbutamol), anticholinergics (e.g. glycopyrronium), methylxanthines (e.g. theophylline), leukotriene receptor antagonists (e.g. montelukast), PDE-4 (phosphodiesterase type 4) inhibitors (e.g. roflumilast), methotrexate, IgE antibodies, azathioprine and cyclophosphamide, preparations containing cortisol; substances used to treat osteoarthritis, such as nonsteroidal anti-inflammatory substances (NSAIDs). In addition to the two therapies mentioned, methotrexate and biological agents (eg rituximab, abatacept) used for B-cell and T-cell therapy should be mentioned for rheumatoid diseases such as rheumatoid arthritis, spondyloarthritis and juvenile idiopathic arthritis. Neurotrophic substances such as acetylcholinesterase inhibitors (e.g. donepezil), MAO (monoamine oxidase) inhibitors (e.g. selegiline), interferons and anticonvulsives (e.g. gabapentin); active ingredients for the treatment of cardiovascular diseases, such as beta-blockers (e.g. metoprolol), ACE inhibitors (e.g. benazepril), angiotensin receptor blockers (e.g. losartan, valsartan), diuretics (e.g. hydrochlorothiazide), calcium channel blockers (e.g. nifedipine), statins (e.g. simvastatin, fluvastatin);Antidiabetic agents, such as metformin, glinides (e.g., nateglinide), DPP-4 (dipeptidyl peptidase-4) inhibitors (e.g., linagliptin, saxagliptin, sitagliptin, vildagliptin), SGLT2 (sodium / glucose cotransporter 2) inhibitors / gliflozins (e.g., dapagliflozin, empagliflozin), incretin mimetics (hormone glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1), 1) (GLP-1) analogs / agonists) (e.g., exenatide, liraglutide, lixisenatide), α-glucosidase inhibitors (e.g., acarbose, miglitol, voglibiose) and sulfonylureas (e.g., glibenclamide, tolbutamide), insulin sensitizers (e.g., pioglitazone) and insulin therapy (e.g., NPH insulin, insulin lispro). Active ingredients for the treatment of chronic inflammatory bowel disease, such as mesalazine, sulfasalazine, azathioprine, 6-mercaptopurine or methotrexate, probiotics (Mutaflor,; Lactobacillus rhamnosus GG, Lactobacillus plantarum, L. acidophilus, L. casei, Bifidobacterium infantis 35624, Enterococcus fecium SF68, Bifidobacterium longum, Escherichia coli (Nissle 1917); antibiotics such as ciprofloxacin and metronidazole; antidiarrheal drugs such as loperamide; or laxatives (bisacodyl). Immunosuppressants used to treat lupus erythematosus, such as glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDs), cortisone, chloroquine, cyclosporine, azathioprine, belimumab, rituximab, and cyclophosphamide. For skin diseases, vitamin D3 analogs, such as calcipotriol, tacalcitol, or calcitriol, salicylic acid, urea, ciclosporin, methotrexate, and efalizumab, are also used.

[0227] Mention should also be made of medicaments for the treatment and / or prevention of the aforementioned diseases, which comprise at least one compound according to the invention and one or more further active ingredients for the use according to the invention, in particular EP4 inhibitors (prostaglandin E2 receptor 4 inhibitors), P2X3 inhibitors (P2X purinergic receptor 3), PTGES inhibitors (prostaglandin E synthase inhibitors) or AKR1C3 inhibitors (aldoketoreductase family 1 member C3 inhibitors).

[0228] The compounds of the present invention can act systemically and / or locally. For this purpose, they can be administered in a suitable manner, for example by oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, cutaneous, transdermal or conjunctival route, through the ear or as an implant or stent.

[0229] The compounds according to the invention can be administered in administration forms suitable for these administration routes.

[0230] Suitable administration forms for oral administration are those which act according to the prior art and release the compound of the invention quickly and / or in a sustained manner and which contain the compound of the invention in crystalline and / or amorphous and / or dissolved form, such as tablets (uncoated or coated tablets, for example with a gastric-resistant coating or a dissolution-delaying coating or an insoluble coating which controls the release of the compound of the invention), tablets or wafers / flat discs which disintegrate rapidly in the mouth, wafers / lyophilisates, capsules (for example hard or soft gelatin capsules), sugar-coated tablets, chewable tablets (for example soft chewable tablets), granules, pills, powders, emulsions, suspensions, aerosols or solutions.

[0231] Parenteral administration can be accomplished by bypassing the reabsorption step (e.g., by intravenous, intraarterial, intracardiac, intraspinal or intralumbar routes) or by including absorption (e.g., intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal routes). Suitable administration forms for parenteral administration include injections and infusions in the form of solutions, suspensions, emulsions, lyophilized agents or sterile powders.

[0232] For other routes of administration, suitable examples are inhalable pharmaceutical forms (including powder inhalers, sprays), nasal drops, nasal solutions or nasal sprays; tablets, wafers / flats or capsules for translingual, sublingual or buccal administration; suppositories; otic or ophthalmic preparations, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, pour-ons, transdermal therapeutic systems (e.g. patches), milks, pastes, foams, sprinkling powders, implants or stents.

[0233] Oral administration or parenteral administration is preferred, especially oral administration.

[0234] The compounds according to the invention can be converted into the administration forms mentioned. This can be accomplished in a manner known per se by mixing with inert, non-toxic, pharmaceutically suitable excipients. These excipients include carriers (e.g. microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycol), emulsifiers and dispersants or wetting agents (e.g. sodium lauryl sulfate, polyoxysorbitan oleate), binders (e.g. polyvinylpyrrolidone), synthetic and natural polymers (e.g. albumin), stabilizers (e.g. antioxidants, e.g. ascorbic acid), colorants (e.g. inorganic pigments, e.g. iron oxide) and flavoring agents and / or odor correctants.

[0235] The present invention also provides medicaments comprising at least one compound of the invention, typically together with one or more inert, non-toxic, pharmaceutically acceptable excipients, for use in a method of treating and / or preventing allergic and / or inflammatory diseases in animals.

[0236] Generally, it has been found that in the case of parenteral administration, it is advantageous to administer an amount of about 0.001 to 1 mg / kg body weight, preferably about 0.01 to 0.5 mg / kg body weight, to achieve effective results. In the case of oral administration, the dosage is about 0.01 to 100 mg / kg body weight, preferably about 0.01 to 20 mg / kg body weight and most preferably 0.1 to 10 mg / kg body weight.

[0237] However, in some cases, it may be necessary to deviate from the amounts specified, in particular depending on body weight, route of administration, individual response to the active ingredient, nature of the formulation and the time or interval at which administration occurs. Thus, in some cases, it may be sufficient to proceed with an amount less than the above-mentioned minimum amount, while in other cases the upper limit mentioned must be exceeded. In the case of larger amounts to be administered, it is advisable to divide it into several individual doses over the day.

[0238] The following working examples illustrate the invention. The invention is not limited to the examples described.

[0239] Unless otherwise stated, percentages in the following tests and examples are percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentration data for liquid / liquid solutions are in each case based on volume.

[0240] Preparation of compounds

[0241] The preparation of the compounds of the present invention is illustrated by the following synthetic schemes.

[0242] The starting materials for the synthesis of the compounds of the present invention are carboxylic acids (intermediate V3), which are commercially available or can be prepared by routes known from the literature or analogously to routes known from the literature (see, for example, European Journal of Organic Chemistry 2003, 8, 1559-1568; Chemical and Pharmaceutical Bulletin, 1990, 38, 9, 2446-2458; Synthetic Communications 2012, 42, 658-666; Tetrahedron, 2004, 60, 51, 11869-11874) (see, for example, Synthesis Scheme 1). Some carboxylic acids V3 can be prepared starting from carboxylic acid esters (intermediate V2) by hydrolysis (see, for example, the reaction of ethyl 6-(hydroxymethyl)pyridine-2-carboxylate with aqueous sodium hydroxide in methanol, WO2004113281), or, in the case of tert-butyl esters, by reaction with acids such as hydrochloric acid or trifluoroacetic acid (see, for example, Dalton Transactions, 2014, 43, 19, 7176-7190). Carboxylic acids V3 can also be used in the form of their alkali metal salts. Intermediate V2 can also be optionally prepared from intermediate V1 containing chlorine, bromine or iodine as a substituent X1 by reacting a phosphine ligand (e.g., 1,3-bis(diphenylphosphino)propane), a palladium compound (e.g., palladium(II) acetate) and a base (e.g., triethylamine) in a carbon monoxide atmosphere, optionally under high pressure, with ethanol or methanol added to a solvent (e.g., dimethyl sulfoxide) (for preparation methods, see, e.g., WO2012112743, WO2005082866, Chemical Communications (Cambridge, England), 2003, 15, 1948-1949, WO200661715). Intermediate V1 is commercially available or can be prepared by routes known from the literature. Exemplary preparation methods are described in detail in the following documents: WO 2012061926; European Journal of Organic Chemistry, 2002, 2, 327-330; Synthesis, 2004, 10, 1619-1624; Journal of the American Chemical Society, 2013, 135, 32, 12122-12134; Bioorganic and Medicinal Chemistry Letters, 2014, 24, 16, 4039-4043; US2007185058; WO2009117421.

[0243]

[0244] X 1 is chlorine, bromine or iodine.

[0245] R d is methyl, ethyl, benzyl or tert-butyl.

[0246] R 4 、R 5 Each is as defined in general formula (I).

[0247] Analogously to WO 2008 / 001883, 5-amino-1H-indazole-6-carboxylic acid methyl ester (intermediate 2) can be prepared from 1H-indazole-6-carboxylic acid methyl ester (intermediate 0) by nitration and reduction of the nitro group of intermediate 1 with hydrogen in the presence of palladium on carbon according to Synthesis Scheme 2. For the preparation of intermediate 3 starting from intermediate 2, various coupling reagents known from the literature can be used (Amino Acids, Peptides and Proteins in Organic Chemistry, Volume 3 - Building Blocks, Catalysis and Coupling Chemistry, Andrew B. Hughes, Wiley, Chapter 12 - Peptide-Coupling Reagents, 407-442; Chem. Soc. Rev., 2009, 38, 606). For example, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride can be used in combination with the following substances as coupling reagents: 1-hydroxy-1H-benzotriazole hydrate (HOBt, WO2012107475; Bioorg. Med. Chem. Lett., 2008, 18, 2093), (1H-benzotriazol-1-yloxy)(dimethylamino)-N,N-dimethylmethaniminium tetrafluoroborate (TBTU, CAS 12 5700-67-6), (dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)carboximidate hexafluorophosphate (HATU, CAS 148893-10-1), propanephosphonic anhydride (in the form of ethyl acetate solution or DMF solution, CAS 68957-94-8) or di-1H-imidazol-1-ylmethanone (CDI), in each case adding a base such as triethylamine or N-ethyl-N-isopropylpropan-2-amine to the reaction mixture. Preference is given to using TBTU and N-ethyl-N-isopropylpropan-2-amine in THF.

[0248]

[0249] Substituent R 4 、R 5 Each is as defined in general formula (I).

[0250] Starting from intermediate 3, 2-substituted indazole derivatives (intermediate 4) (referring to synthetic scheme 3) can be prepared.For this purpose, useful reactions include those reactions with optionally substituted alkyl chlorides, alkyl bromides, alkyl iodides or 4-methylbenzenesulfonic acid alkyl esters. Alkyl halides used or 4-methylbenzenesulfonic acid alkyl esters are commercially available or can be similar to the preparation of approaches known in the literature (for the preparation of 4-methylbenzenesulfonic acid alkyl esters, one embodiment is to react an appropriate amount of alcohol with 4-methylbenzenesulfonyl chloride in the presence of triethylamine or pyridine; See, for example, Bioorganic and Medicinal Chemistry, 2006, 14, 12 4277-4294). Optionally, when using alkyl chlorides or alkyl bromides, alkali metal iodides such as potassium iodide or sodium iodide can also be added. The base used can be, for example, potassium carbonate, cesium carbonate or sodium hydride. In the case of reactive alkyl halides, N-cyclohexyl-N-methylcyclohexylamine can also be used in some cases. Useful solvents include, for example, 1-methylpyrrolidone-2-one, DMF, DMSO or THF. Optionally, the alkyl halide or 4-methylbenzenesulfonic acid alkyl esters used may have a functional group optionally protected in advance with a blocking group (see also PGM Wuts, TW Greene, Greene's Protective Groups in Organic Synthesis, 4th edition, ISBN:9780471697541). For example, if alkyl halide or 4-methylbenzenesulfonic acid alkyl esters with one or more hydroxyl groups are used, these hydroxyl groups can be optionally protected by tert-butyl (dimethyl) silyl or similar silicon-containing blocking groups familiar to those skilled in the art. Alternatively, hydroxyl groups can be protected with tetrahydrochysene-2H-pyrans (THP) group or acetyl or benzoyl. Then, after synthesizing intermediate 4 or after synthesizing (I), the blocking group used can be removed. For example, if tert-butyl (dimethylsilyl) group is used as blocking group, the tetrabutylammonium fluoride used in solvents such as THF can be removed. For example, the THP protecting group can be removed using 4-methylbenzenesulfonic acid (optionally in the form of its monohydrate). An acetyl or benzoyl group can be removed by treatment with aqueous sodium hydroxide.

[0251] Optionally, the alkyl halide or 4-methylbenzenesulfonic acid alkyl ester used may contain a functional group (see, for example, Science of Synthesis, GeorgThieme Verlag) that can be converted by oxidation or reduction reactions known to those skilled in the art. For example, if the functional group is a sulfide group (sulphide group), it can be oxidized to a sulfoxide or sulfone group by methods known in the literature. In the case of a sulfoxide group, it can be oxidized to a sulfone group. For these oxidation steps, for example, 3-chloroperbenzoic acid (CAS 937-14-4) can be used (for this, also see, for example, US201094000, wherein 2- (methylsulfanyl) ethyl -1H- pyrazole derivatives are oxidized to 2- (methylsulfinyl) ethyl -1H- pyrazole derivatives, and other 2- (methylsulfanyl) ethyl -1H- pyrazole derivatives are oxidized to 2- (methylsulfonyl) ethyl -1H- pyrazole derivatives). If the alkyl halide or p-toluenesulfonate used contains a ketone group, it can be reduced to an alcohol group by reduction methods known to those skilled in the art (see, for example, Chemische Berichte, 1980, 113, 1907-1920 about the use of sodium borohydride). These oxidation or reduction steps can be carried out after the synthesis of intermediate 4 or after the synthesis of the compound of general formula (I) of the present invention. Alternatively, intermediate 4 can be prepared by the Mitsunobu reaction of intermediate 3 with an optionally substituted alkyl alcohol (see, for example, KCK Swamy et al., Chem. Rev. 2009, 109, 2551-2651). Various phosphine compounds such as triphenylphosphine, tributylphosphine or 1,2-diphenylphosphinoethane can be used in combination with diisopropyl azodicarboxylate (CAS 2446-83-5) or other diazene derivatives mentioned in the literature (KCK Swamy et al., Chem. Rev. 2009, 109, 2551-2651). Preference is given to using triphenylphosphine and diisopropyl azodicarboxylate. If the alkyl alcohol carries a functional group, then, as in the case of the above-described reaction with an alkyl halide, known protective group strategies can be employed (further instructions can be found in PGM Wuts, TW Greene, Greene's Protective Groups in Organic Synthesis, 4th edition, ISBN: 9780471697541), and, as in the case of the above-described reaction with an alkyl halide, the synthesis of intermediate 4 or the corresponding oxidation and reduction steps after the synthesis of the compound of the general formula (I) according to the invention can be carried out. The compound of the general formula (I) according to the invention, in which R 2 and R 3 is defined as C1-C6-alkyl (where R 2 and R 3have the same definition), can be obtained from intermediate 4 by Grignard reaction (see, for example, the reaction of 1H-indazole-6-carboxylic acid methyl ester derivatives with methylmagnesium bromide in EP 2489663). For the Grignard reaction, alkylmagnesium halides can be used. Particularly preferred are methylmagnesium chloride or methylmagnesium bromide in THF or diethyl ether or in a mixture of THF and diethyl ether. Alternatively, the compound of the general formula (I) of the present invention, wherein R 2 and R 3 is defined as C1-C6-alkyl (where R 2 and R 3 have the same definition), can be obtained from intermediate 4 by reaction with an alkyllithium reagent (see, for example, the reaction of 2-amino-4-chloro-1-methyl-1H-benzimidazole-7-carboxylic acid methyl ester derivatives with isopropyllithium or tert-butyllithium in WO2006116412). Starting from intermediate 4, by reduction with lithium aluminum hydride in THF, lithium borohydride in THF or sodium borohydride in THF (optionally with methanol), or a mixture of lithium borohydride and sodium borohydride, wherein R 2 and R 3 The compound of the general formula (I) of the present invention is defined as H.

[0252]

[0253] Substituent R 1 、R 2 、R 3 、R 4 、R 5 Each is as defined in general formula (I).

[0254] Intermediate 5, wherein R 2 and R 3 is defined as C1-C6-alkyl (where R 2 and R 3 have the same definition), can be obtained from intermediate 3 by Grignard reaction (see, for example, Synthesis Scheme 4). For this purpose, a suitable alkylmagnesium halide can be used, for example methylmagnesium chloride or methylmagnesium bromide in THF or diethyl ether or in a mixture of THF and diethyl ether.

[0255] Then, a portion (Ia) of the compound (I) of the present invention can be prepared from intermediate 5, wherein R 2 and R 3 is defined as C1-C6-alkyl (where R 2 and R 3(Have the same definition). For this purpose, analogously to Synthesis Scheme 3 (Preparation of Intermediate 3), useful reactions are those of Intermediate 5 with optionally substituted alkyl chlorides, alkyl bromides, alkyl iodides or 4-methylbenzenesulfonic acid alkyl esters. Protective group strategies similar to those described in Synthesis Scheme 3 can be used.

[0256] Alternatively, to prepare a 2 and R 3 is defined as C1-C6-alkyl (where R 2 and R 3 A portion (Ia) of the compound (I) of the present invention having the same definitions as those given above) can be prepared using the Mitsunobu reaction of intermediate 5 with an optionally substituted alkyl alcohol (analogously to Synthesis Scheme 3).

[0257] If R in the compound of formula (Ia) 1 Including appropriate functional groups can then optionally be followed by analogy to Synthetic Scheme 3 using oxidation or reduction reactions to prepare other compounds of the invention.

[0258]

[0259] Substituent R 1 、R 4 、R 5 are as defined in general formula (I). 2 and R 3 always have the same definition and are all C1-C6-alkyl.

[0260] Intermediate 4 can be prepared from Intermediate 1 in an alternative manner (see Synthesis Scheme 5). First, Intermediate 1 is converted to Intermediate 6 by the method as in Synthesis Scheme 3 (Preparation of Intermediate 4 from Intermediate 3).

[0261] Then, intermediate 6 can be converted into intermediate 7 by the reduction of nitro group.For example, nitro group can be reduced with carbon-supported palladium under hydrogen atmosphere (see, for example, WO2013174744 about 6-isopropoxy-5-nitro-1H-indazole being reduced to 6-isopropoxy-1H-indazole-5-amine), or with iron and ammonium chloride reduction in water and ethanol (see, for example, Journal of the Chemical Society, 1955, 2412-2419), or using tin chloride (II) (CAS 7772-99-8) reduction.Preferably, iron and ammonium chloride in water and ethanol are used.Preparation of intermediate 4 by intermediate 7 can be similar to synthesis scheme 2 and carry out (preparation of intermediate 3 by intermediate 2).

[0262] As described in Synthesis Scheme 3, in the case of Synthesis Scheme 5, a protecting group strategy can also be optionally used. Starting from Intermediate 6 or Intermediate 7, oxidation or reduction reactions known to those skilled in the art (see, for example, Science of Synthesis, Georg Thieme Verlag) can optionally also be carried out as described in Synthesis Scheme 3.

[0263]

[0264] Substituent R 1 、R 4 、R 5 Each is as defined in general formula (I).

[0265] Synthesis of Example Compounds

[0266] Abbreviations and descriptions

[0267] DMF N,N-Dimethylformamide DMSO dimethyl sulfoxide THF Tetrahydrofuran RT Room temperature HPLC High-performance liquid chromatography h Hour HCOOH Formic acid MeCN Acetonitrile min minute UPLC Ultra-high performance liquid chromatography DAD Diode array detector ELSD Evaporative Light Scattering Detector ESI Electrospray ionization SQD single quadrupole detector CPG core-pulled precision glass <![CDATA[NH3]]> ammonia

[0268] The term sodium chloride solution always refers to a saturated aqueous sodium chloride solution.

[0269] The chemical names of the intermediates and examples were generated using ACD / LABS (Batch version 12.01) software.

[0270] method

[0271] In some cases, compounds of the invention and their precursors and / or intermediates were analyzed by LC-MS.

[0272] Method A1: UPLC (MeCN-HCOOH):

[0273] Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity UPLC BEH C18 1.7 50×2.1 mm; eluent A: water + 0.1 volume % formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow rate: 0.8 mL / min; temperature: 60°C; injection volume: 2 μL; DAD scan: 210-400 nm; MS ESI+, ESI-, scan range 160-1000 m / z; ELSD.

[0274] Method A2: UPLC (MeCN-NH3):

[0275] Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity UPLC BEH C18 1.7 50×2.1 mm; eluent A: water + 0.2 volume% ammonia (32%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow rate: 0.8 mL / min; temperature: 60°C; injection volume: 2 μL; DAD scan: 210-400 nm; MS ESI+, ESI-, scan range 160-1000 m / z; ELSD.

[0276] Method A3: (LC-MS)

[0277] Instrument: Agilent 1290 Infinity LC; Column: Acquity UPLC BEH C18 1.750×2.1 mm; Eluent A: water + 0.05% by volume formic acid, Eluent B: acetonitrile + 0.05% by volume formic acid; Gradient: 0-1.7 min 2-90% B, 1.7-2.0 min 90% B; Flow rate: 1.2 mL / min; Temperature: 60°C; Injection volume: 2 μL; DAD scan: 190-390 nm; MS: Agilent TOF 6230.

[0278] Method A4: (LC-MS)

[0279] Instrument: Waters Acquity; column: Kinetex (Phenomenex), 50×2 mm; eluent A: water + 0.05% by volume of formic acid, eluent B: acetonitrile + 0.05% by volume of formic acid; gradient: 0-1.9 min 1-99% B, 1.9-2.1 min 99% B; flow rate: 1.5 mL / min; temperature: 60°C; injection volume: 0.5 μL; DAD scan: 200-400 nm.

[0280] In some cases, compounds of the invention and their precursors and / or intermediates were purified by the following illustrative preparative HPLC methods:

[0281] Method P1: System: Waters automated purification system: pump 2545, sample manager 2767, CFO, DAD 2996, ELSD 2424, SQD; column: XBridge C18 5μm 100×30mm; eluent A: water + 0.1% by volume formic acid, eluent B: acetonitrile; gradient: 0-8min 10-100% B, 8-10min 100% B; flow rate: 50mL / min; temperature: room temperature; solution: maximum 250mg / maximum 2.5mL DMSO or DMF; injection volume: 1×2.5mL; detection: DAD scan range 210-400nm; MS ESI+, ESI-, scan range 160-1000m / z.

[0282] Method P2: System: Waters automated purification system: pump 254, sample manager 2767, CFO, DAD 2996, ELSD2424, SQD 3100; column: XBridge C18 5μm 10×30mm; eluent A: water + 0.2% by volume ammonia (32%), eluent B: methanol; gradient: 0-8min 30-70% B; flow rate: 50mL / min; temperature: room temperature; detection: DAD scanning range 210-400nm; MS ESI+, ESI-, scanning range 160-1000m / z; ELSD.

[0283] Method P3: System: Labomatic, Pump: HD-5000, Fraction collector: LABOCOL Vario-4000, UV detector: Knauer UVD 2.1S; Column: XBridge C18 5 μm 100×30 mm; Eluent A: water + 0.2% by volume of ammonia (25%), Eluent B: acetonitrile; Gradient: 0-1 min 15% B, 15-55% B from 1-6.3 min, 55-100% B from 6.3-6.4 min, 100% B from 6.4-7.4 min; Flow rate: 60 ml / min; Temperature: room temperature; Solution: maximum 250 mg / 2 mL DMSO; Injection volume: 2×2 mL; Detection: UV 218 nm; Software: SCPA PrepCon5.

[0284] Method P4: System: Labomatic, Pump: HD-5000, Fraction collector: LABOCOL Vario-4000, UV detector: Knauer UVD 2.1S; Column: Chromatorex RP C18 10 μm 125×30 mm; Eluent A: water + 0.1% volume of formic acid, Eluent B: acetonitrile; Gradient: 0-15 min 65-100% B; Flow rate: 60 mL / min; Temperature: Room temperature; Solution: Maximum 250 mg / 2 mL DMSO; Injection: 2×2 mL; Detection: UV 254 nm; Software: SCPA PrepCon5.

[0285] Method P5: System: Sepiatec:Prep SFC100, Column: ChiralpakIA 5μm 250×20mm; Eluent A: carbon dioxide, Eluent B: ethanol; Gradient: isocratic 20% B; Flow rate: 80mL / min; Temperature: 40°C; Solution: maximum 250mg / 2mL DMSO; Injection volume: 5×0.4mL; Detection: UV 254nm.

[0286] Method P6: System: Agilent: Prep 1200, 2x prep pumps, DLA, MWD, Gilson: Liquid Handler 215; Column: Chiralcel OJ-H 5μm 250×20mm; Eluent A: Hexane, Eluent B: Ethanol; Gradient: Isocratic 30% B; Flow rate: 25mL / min; Temperature: 25℃; Solution: 187mg / 8mL ethanol / methanol; Injection volume: 8×1.0mL; Detection: UV280nm.

[0287] Method P7: System: Labomatic, Pump: HD-5000, Fraction collector: LABOCOL Vario-4000, UV detector: Knauer UVD 2.1S; Column: XBridge C18 5 μm 100×30 mm; Eluent A: water + 0.1% volume of formic acid, Eluent B: acetonitrile; Gradient: 0-3 min: 65% B isocratic, 3-13 min: 65-100% B; Flow rate: 60 mL / min; Temperature: Room temperature; Solution: maximum 250 mg / 2 mL DMSO; Injection volume: 2×2 mL; Detection: UV 254 nm.

[0288] Method P8: System: Agilent: Prep 1200, 2×prep pump, DLA, MWD, Gilson: Liquid Handler 215; Column: Chiralpak IF 5μm 250×20mm; Eluent A: ethanol, Eluent B: methanol; Gradient: isocratic 50% B; Flow rate: 25mL / min; Temperature: 25℃; Solution: 600mg / 7mL N,N-dimethylformamide: Injection volume: 10×0.7mL; Detection: UV 254nm.

[0289] In some cases, mixtures of materials were purified by silica gel column chromatography.

[0290] To prepare certain compounds of the present invention and their precursors and / or intermediates, the Equipment Column chromatography purification is performed on silica gel ("flash chromatography"). This is done using cartridges available from Biotage, such as "SNAP Cartridge, KP_SIL" cartridges of various sizes and "Interchim Puriflash Silica HP 15UM flash column" cartridges of various sizes available from Interchim.

[0291] Starting materials

[0292] Intermediate V2-1

[0293] 6-(2-Hydroxypropan-2-yl)pyridine-2-carboxylic acid methyl ester

[0294]

[0295] 2.00g (9.26mmol) of 2-(6-bromopyridin-2-yl)propan-2-ol (CAS 638218-78-7) was dissolved in 20mL of methanol and 20mL of DMSO. Subsequently, 250mg of 1,3-bis(diphenylphosphino)propane, 130mg of palladium (II) acetate and 3mL of triethylamine were added. The reaction mixture was purged with carbon monoxide three times at room temperature and stirred for 30min in a carbon monoxide atmosphere of 13 bar. The carbon monoxide atmosphere was removed by applying a vacuum, and the mixture was stirred for 24h in a carbon monoxide atmosphere of 14 bar at 100°C. The autoclave was decompressed, water was added to the reaction mixture, and the reaction mixture was extracted three times with ethyl acetate, washed with saturated sodium bicarbonate aqueous solution and sodium chloride aqueous solution, filtered through a hydrophobic filter and concentrated. 1.60g of crude product was obtained.

[0296] UPLC-MS (Method A1): R t=0.76 min (UV detector: TIC), molecular weight measurement value 195.00.

[0297] Intermediate V3-1

[0298] Potassium 6-(2-hydroxypropan-2-yl)pyridine-2-carboxylate

[0299]

[0300] First, 1.60 g of the crude product of Intermediate 0-1 was added to 15 mL of methanol, 0.74 g of potassium hydroxide was added, and the mixture was stirred at 50° C. for 16.5 h. After concentration, 2.1 g of a residue was obtained, which was used without further purification.

[0301] UPLC-MS (Method A1): R t =0.47 min (UV detector: TIC), molecular weight measurement value: 181.00.

[0302] Intermediate 1-1

[0303] 5-Nitro-1H-indazole-6-carboxylic acid methyl ester

[0304]

[0305] In a three-necked flask with a CPG stirrer, a dropping funnel, and an internal thermometer, 4.60 g (26.1 mmol) of 1H-indazole-6-carboxylic acid methyl ester (CAS No.: 170487-40-8) was dissolved in 120 mL of sulfuric acid (96%) and cooled to -15°C. Over 15 minutes, a pre-prepared and cooled nitrating acid (10 mL of 96% sulfuric acid in 5 mL of 65% nitric acid) was added dropwise to the solution. After the addition was complete, the mixture was stirred for another 1 hour (internal temperature was -13°C). The reaction mixture was added to ice, the precipitate was filtered off with suction, washed with water, and dried under reduced pressure in a drying oven at 50°C. 5.49 g of the title compound was obtained.

[0306] UPLC-MS (Method A2): R t =0.75min

[0307] MS (ESIpos): m / z = 222 (M+H) +

[0308] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.87 (s, 3H), 7.96 (s, 1H), 8.44 (s, 1H), 8.70 (s, 1H), 13.98 (br.s., 1H).

[0309] Intermediate 2-1

[0310] 5-Amino-1H-indazole-6-carboxylic acid methyl ester

[0311]

[0312] 4.40 g (19.8 mmol) of 5-nitro-1H-indazole-6-carboxylic acid methyl ester (intermediate 1-1) was dissolved in 236 mL of methanol and hydrogenated with 1.06 g (0.99 mmol) of palladium on activated carbon at 25° C. for 3 h under standard hydrogen pressure. The reaction mixture was filtered through celite, the filtrate was washed with methanol, and the filtrate was concentrated to obtain 3.53 g of the title compound.

[0313] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 3.85 (s, 3H) 6.01 (s, 2H) 6.98 (s, 1H) 7.79-7.91 (m, 1H) 7.99 (s, 1H) 12.84 (br.s., 1H).

[0314] Intermediate 3-1

[0315] 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester

[0316]

[0317] At first 4.95g (25.9mmol) of 6-(trifluoromethyl)pyridine-2-carboxylic acid is joined in 45mL THF.Add 9.07g (28.2mmol) of tetrafluoroboric acid O-(benzotriazole-1-yl)-N,N,N ',N '-tetramethylurea and 4.92mL (28.2mmol) of N-ethyl-N-isopropylpropane-2-amine, and the mixture is stirred at 25 ℃ for 30min.Subsequently, add 4.50g (23.5mmol) of 5-amino-1H-indazole-6-carboxylic acid methyl esters (intermediate 2-1), and the mixture is stirred at 25 ℃ for 24h.Reaction mixture is filtered by membrane filter suction, and solid is washed with THF and water, and dried in drying box overnight.Obtain 7.60g title compound.

[0318] UPLC-MS (Method A2): R t =1.16min

[0319] MS (ESIpos): m / z=365 (M+H) +

[0320] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.97 (s, 3H), 8.13-8.27 (m, 2H), 8.30 (s, 1H), 8 .33-8.45(m, 1H), 8.45-8.51(m, 1H), 9.15(s, 1H), 12.57(s, 1H), 13.44(s, 1H).

[0321] Intermediate 3-2

[0322] 5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester

[0323]

[0324] First, 2.85g (23.5mmol) of 6-(difluoromethyl)pyridine-2-carboxylic acid was added to 30mL of THF. 6.05g (18.8mmol) of tetrafluoroboric acid O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylurea and 3.3mL of N-ethyl-N-isopropylpropan-2-amine were added, and the mixture was stirred at room temperature for 10min. Subsequently, 3.00g (15.7mmol) of 5-amino-1H-indazole-6-carboxylic acid methyl ester was added, and the mixture was stirred at room temperature overnight. The reaction mixture was mixed with water, the precipitate was filtered off with suction, and the precipitate was washed repeatedly with water and dichloromethane. 1.53g (27% of theoretical value) of the title compound was obtained. The phases of the filtrate were separated, the organic phase was concentrated, mixed with a small amount of dichloromethane and suspended in an ultrasonic bath, and the precipitate was filtered off with suction. This gave 1.03g of the title compound.

[0325] 1 H-NMR (first product fraction, 300 MHz, DMSO-d6): δ [ppm] = 3.99 (s, 3H), 7.09 (t, 1H), 8.00 (d, 1H), 8.21-8.40 (m, 4H), 9.14 (s, 1H), 12.53 (s, 1H), 13.44 (s, 1H).

[0326] Intermediate 3-3

[0327] 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester

[0328]

[0329] First, 2.10g of 6-(2-hydroxypropyl-2-yl)pyridine-2-carboxylic acid potassium (intermediate V3-1) was added to 15mL of THF. 3.69g (11.5mmol) of tetrafluoroboric acid O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylurea and 2.00mL of N-ethyl-N-isopropylpropane-2-amine were added, and the mixture was stirred at room temperature for 15min. Subsequently, 1.83g (9.58mmol) of 5-amino-1H-indazole-6-carboxylic acid methyl ester (intermediate 2-1) was added, and the mixture was stirred at room temperature for 19h. The mixture was mixed with water and ethyl acetate, undissolved solids were filtered out, and the phases of the filtrate were separated. The aqueous phase was extracted twice with ethyl acetate, washed with sodium chloride solution, filtered through a hydrophobic filter, concentrated, and purified by silica gel column chromatography (hexane / ethyl acetate). After removing the solvent, 1.56g of the yellow foamy title compound was obtained.

[0330] UPLC-MS (Method A1): R t =1.00 min (UV detector: TIC Smooth), molecular weight measurement value: 354.00.

[0331] 1 H-NMR (500MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 3.97 (s, 3H), 5.37 (s, 1H), 7.90-7.95 (m, 1H) , 8.03-8.07(m, 2H), 8.23(s, 1H), 8.29(s, 1H), 9.19(s, 1H), 12.79(s, 1H), 13.41(br.s., 1H).

[0332] Intermediate 4-1

[0333] 2-(Oxetane-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0334]

[0335] 1.00 g (2.66 mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was dissolved in 10 mL of DMF and after adding 1.10 g (7.99 mmol) of potassium carbonate and 221 mg (1.33 mmol) of potassium iodide, the mixture was stirred at 25 ° C for 30 min. 603 mg (3.99 mmol) of 3-bromomethyloxetane was added and the mixture was stirred at 25 ° C for 24 h. The reaction mixture was layered between water and ethyl acetate. The mixture was extracted twice with ethyl acetate, and the combined organic phases were filtered through a hydrophobic filter and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 260 mg of the title compound was obtained.

[0336] UPLC-MS (Method A2): R t =1.24min

[0337] MS (ESIpos): m / z=435 (M+H) +

[0338] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.49-3.64 (m, 1H), 3.95 (s, 3H), 4.49 (t, 2H), 4.68 (dd, 2H), 4.81 (d, 2 H), 8.20 (dd, 1H), 8.35-8.41 (m, 1H), 8.43-8.49 (m, 2H), 8.55-8.58 (m, 1H), 9.06 (s, 1H), 12.53 (s, 1H).

[0339] Intermediate 4-2

[0340] 2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0341]

[0342] 1.00 g (2.75 mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was dissolved in 5 mL of DMF and 387 μL (4.12 mmol) of 2-bromoethyl methyl ether, 1.14 g (8.23 mmol) of potassium carbonate and 228 mg (1.37 mmol) of potassium iodide were added while stirring. The reaction mixture was stirred at 25 ° C for 24 h, diluted with water, and extracted twice with ethyl acetate. The combined organic phases were filtered through a hydrophobic filter and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 12 mg of the title compound was obtained.

[0343] UPLC-MS (Method A1): R t =1.24min

[0344] MS (ESIpos): m / z=423 (M+H) +

[0345] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.24 (s, 3H), 3.86 (t, 2H), 3.96 (s, 3H), 4.65 (t, 2H), 8.2 1(dd, 1H), 8.35-8.42(m, 1H), 8.43-8.51(m, 2H), 8.52(d, 1H), 9.06(s, 1H), 12.53(s, 1H).

[0346] Intermediate 4-3

[0347] 2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0348]

[0349] 1.00 g (2.75 mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was dissolved in 5 mL of DMF and 460 μL (4.12 mmol) of 1-bromo-3-methoxypropane, 1.14 g (8.23 mmol) of potassium carbonate and 228 mg (1.37 mmol) of potassium iodide were added while stirring. The reaction mixture was stirred at 25 ° C for 72 h, diluted with water, and extracted twice with ethyl acetate. The combined organic phase was filtered through a hydrophobic filter and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 28 mg of the title compound was obtained.

[0350] UPLC-MS (Method A1): R t =1.29min

[0351] MS (ESIpos): m / z=437 (M+H) +

[0352] 1 H NMR (400 MHz, DMSO-d6): δ [ppm] = 2.17 (quintet, 2H), 3.24 (s, 3H), 3.33-3.36 (m, 2H), 3.96 (s, 3H), 4.53 (t, 2H), 8.21 (dd, 1H), 8.35-8.42 (m, 1H), 8.45-8.49 (m, 2H), 8.54 (d, 1H), 9.06 (s, 1H), 12.54 (s, 1H).

[0353] Intermediate 4-4

[0354] 2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0355] Preparation method 1

[0356]

[0357] First, 930 mg (2.55 mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1), 1.06 g of potassium carbonate and 212 mg of potassium iodide were dissolved in 9 mL of DMF, and the mixture was stirred for 15 min. Then, 0.62 mL of 4-bromo-2-methylbutan-2-ol was added, and the mixture was stirred at 60 ° C for 16 h. The mixture was mixed with water and extracted twice with ethyl acetate. The extract was washed three times with saturated sodium chloride solution, filtered and concentrated. Silica gel column chromatography (hexane / ethyl acetate) purification gave 424 mg of the title compound.

[0358] UPLC-MS (Method A2): R t =1.21 min (UV detector: TIC), molecular weight measurement value: 450.00.

[0359] 1H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.16 (s, 6H) 2.02-2.11 (m, 2H) 3.96 (s, 3H) 4.51-4.60 (m, 3H) 8.2 0 (dd, J=7.83, 1.01Hz, 1H) 8.39 (s, 1H) 8.45 (s, 2H) 8.55 (d, J=0.76Hz, 1H) 9.05 (s, 1H) 12.52 (s, 1H).

[0360] Preparation method 2

[0361] First, 1.95g (7.03mmol) of 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester (intermediate 7-1) was added to 30mL of THF. 1.48g (7.37mmol) of 6-(trifluoromethyl)pyridine-2-carboxylic acid, 2.71g (8.44mmol) of tetrafluoroboric acid O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylurea and 1.47mL (8.44mmol) of N-ethyl-N-isopropylpropane-2-amine were added, and the mixture was stirred at 25°C for 20.5h. Water was added, and the mixture was extracted three times with ethyl acetate. The extract was washed with sodium chloride solution, filtered through a hydrophobic filter and concentrated. The residue was separated by silica gel column chromatography (hexane / ethyl acetate gradient). 2.79g of the title compound was obtained.

[0362] UPLC-MS (Method A1): R t =1.23 min (UV detector: TIC), molecular weight measurement value: 450.00.

[0363] Intermediate 4-5

[0364] 2-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0365]

[0366] First, 1.00g (2.66mmol, 97%) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was added to 50mL of DMF, and 1.10g (7.99mmol) of potassium carbonate and 221mg (1.33mmol) of potassium iodide were added while stirring, and the mixture was stirred at 25°C for 30min. Subsequently, 857μL (3.99mmol) of (2-bromoethoxy) (tert-butyl) dimethylsilane were added, and the mixture was stirred at 25°C for 24h. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic phases were filtered through a hydrophobic filter and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 400mg of the title compound was obtained.

[0367] UPLC-MS (Method A1): R t =1.58min

[0368] MS (ESIpos): m / z=523 (M+H) +

[0369] 1 H NMR (300MHz, DMSO-d6): δ[ppm]=-0.18--0.13(m, 6H), 0.74(s, 9H), 3.96(s, 3H), 4.08(t, 2H), 4. 57(t, 2H), 8.15-8.25(m, 1H), 8.32-8.43(m, 1H), 8.43-8.52(m, 3H), 9.07(s, 1H), 12.53(s, 1H).

[0370] Intermediates 4-6

[0371] 2-(3-{[tert-Butyl(dimethyl)silyl]oxy}propyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0372]

[0373] Similarly to intermediate 4-5, 1.00g (2.75mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was dissolved in 10mL of DMF, 1.14g (8.24mmol) of potassium carbonate and 228mg (1.37mmol) of potassium iodide were added while stirring, and the mixture was stirred at 25°C for 30min. Subsequently, 1.04g (4.12mmol) of (3-bromopropoxy) (tert-butyl) dimethylsilane was added, and the mixture was stirred at 25°C for 24h. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate. The reaction mixture was layered between water and ethyl acetate, and the aqueous phase was extracted twice with ethyl acetate. The combined organic phase was filtered through a hydrophobic filter and concentrated. The residue was purified by preparative HPLC to obtain 428mg of the title compound.

[0374] UPLC-MS (Method A1): R t =1.63min

[0375] MS (ESIpos): m / z=537 (M+H) +

[0376] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = -0.02-0.06 (m, 6H), 0.87 (s, 9H), 2.14 (quin, 2H), 3.62 (t, 2H), 3.96 (s , 3H), 4.54 (t, 2H), 8.20 (d, 1H), 8.35-8.42 (m, 1H), 8.43-8.48 (m, 3H), 8.49-8.53 (m, 1H), 9.06 (s, 1H).

[0377] Intermediates 4-7

[0378] 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-2-(4,4,4-trifluorobutyl)-2H-indazole-6-carboxylic acid methyl ester

[0379]

[0380] First, 300 mg (0.80 mmol) of 5-({[6-(2-hydroxypropyl-2-yl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-3) was added to 4.5 mL of DMF. 287 mg (1.21 mmol) of 1,1,1-trifluoro-4-iodobutane and 333 mg of potassium carbonate were added, and the mixture was stirred at 100 ° C for 23 h. Water was added and the mixture was extracted three times with ethyl acetate. The mixture was concentrated and the product was purified by preparative HPLC. 72 mg of the title compound was obtained.

[0381] UPLC-MS (Method A1): R t =1.26 min (UV detector: TIC), molecular weight measurement value: 464.17.

[0382] Intermediates 4-8

[0383] 5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester

[0384]

[0385] Analogously to Intermediate 4-4 (Preparation 2), 195 mg (0.46 mmol) of methyl 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate (Intermediate 7-1) was reacted with 78 mg (0.50 mmol) of 5-fluoro-6-methylpyridine-2-carboxylic acid for 19.5 h. After a similar aqueous workup, 228 mg of crude product was obtained.

[0386] UPLC-MS (Method A1): R t =1.20 min (UV detector: TIC), molecular weight measurement value: 414.00.

[0387] Intermediates 4-9

[0388] 2-(3-hydroxy-3-methylbutyl)-5-{[(6-methylpyridin-2-yl)carbonyl]amino}-2H-indazole-6-carboxylic acid methyl ester

[0389]

[0390] Analogously to Intermediate 4-4 (Preparation 2), 195 mg (0.46 mmol) of 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester (Intermediate 7-1) was reacted with 70 mg (0.50 mmol) of 6-methylpyridine-2-carboxylic acid for 19.5 h. After a similar aqueous workup, 278 mg of the title compound was obtained as a crude product.

[0391] UPLC-MS (Method A1): R t =1.14 min (UV detector: TIC), mass measurement value 396.00.

[0392] Intermediates 4-10

[0393] 2-[3-(2,2,2-trifluoroethoxy)propyl]-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0394]

[0395] A mixture of 250 mg (0.58 mmol) of methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (Intermediate 3-1), 193 mg (0.88 mmol) of 3-bromopropyl-2,2,2-trifluoroethyl ether, 242 mg of potassium carbonate, and 145 mg of potassium iodide in 3 mL of DMF was stirred at 100° C. for 20 h. Water was added, the mixture was extracted with ethyl acetate, and the extract was washed with sodium chloride solution and concentrated. Purification by preparative HPLC gave 52 mg of the title compound.

[0396] UPLC-MS (Method A1): R t =1.39 min (UV detector: TIC), molecular weight measurement value 504.12.

[0397] Intermediates 4-11

[0398] 5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester

[0399]

[0400] First 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl esters (intermediate 7-1) of 2.00g is joined in the THF of 40mL.Add 1.50g 6-(difluoromethyl) pyridine-2-carboxylic acid, 2.78g tetrafluoroboric acid O-(benzotriazole-1-yl)-N, N, N ', N '-tetramethylurea (TBTU, CAS 125700-67-6) and 1.5mL N-ethyl-N-isopropylpropane-2-amine, and mixture is at room temperature stirred to 24h.Add water, mixture is extracted three times with ethyl acetate, the organic phase merged is washed with sodium chloride solution and filtered by hydrophobic filter.Enriched mixture, and resistates is passed through silica gel column chromatography (hexane / ethyl acetate) purifying.Obtain the title compound of 3.05g yellow solid.

[0401] UPLC-MS (Method A1): Rt = 1.15 min (UV detector TIC), molecular weight measurement value 432.00.

[0402] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.17 (s, 6H), 2.04-2.11 (m, 2H), 3.99 (s, 3H), 4.52-4.60 (m, 3H), 7.1 0 (t, 1H), 8.00 (dd, 1H), 8.28-8.38 (m, 2H), 8.44-8.47 (m, 1H), 8.56 (d, 1H), 9.05 (s, 1H), 12.49 (s, 1H).

[0403] Intermediate 5-1

[0404] N-[6-(2-Hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0405]

[0406] To a solution of 1.50 g (4.12 mmol) of methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate in 20 mL of THF cooled in an ice-water cooling bath was carefully added 6.9 mL (5 equivalents) of a 3M solution of methylmagnesium bromide in diethyl ether. While cooling with an ice bath, the mixture was stirred for 1 h and at room temperature for 19.5 h. 2 equivalents of methylmagnesium bromide solution were further added, and the mixture was stirred at room temperature for a further 24 h. Saturated aqueous ammonium chloride solution was added, the mixture was stirred, and extracted three times with ethyl acetate. The combined organic phases were washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 763 mg of the title compound were obtained.

[0407] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 5.99 (s, 1H), 7.49 (s, 1H), 8.06 (s, 1H), 8.14-8.19 (m, 1H), 8.37 (t, 1H), 8.46 (d, 1H), 8.78 (s, 1H), 12.32 (s, 1H), 12.97 (s, 1H).

[0408] Intermediate 5-2

[0409] 6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]pyridine-2-carboxamide

[0410]

[0411] Analogously to the preparation of Intermediate 5-1, 2.40 g (6.93 mmol) of methyl 5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (Intermediate 3-2) in 10 mL of THF was reacted with three portions of 3M methylmagnesium bromide in diethyl ether (6.9 mL, then stirred at room temperature for 45 min; 11.6 mL, then stirred at room temperature for 2 h; 6.9 mL, then stirred at room temperature for 2 h). After workup as for Intermediate 5-1, 2.39 g of crude product was obtained, which was used without further purification.

[0412] Intermediate 6-1

[0413] 2-(3-hydroxy-3-methylbutyl)-5-nitro-2H-indazole-6-carboxylic acid methyl ester

[0414]

[0415] First 5.00g (22.6mmol) of 5-nitro-1H-indazole-6-carboxylic acid methyl esters (intermediate 1-1) are added in 40mL of DMF. Add 5.65g (33.9mmol) of 4-bromo-2-methylbutan-2-ol, 9.37g (67.8mmol) of potassium carbonate and 5.63g (33.9mmol) of potassium iodide, and stir the mixture at 100 ℃ for 20h. Add water, the mixture is extracted three times with ethyl acetate, and the extract is washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. Residue is passed through silica gel column chromatography (hexane / ethyl acetate) purification. The solid obtained is stirred with diethyl ether, filtered off with suction, washed with diethyl ether and dried. Obtain 2.49g of title compound.

[0416] UPLC-MS (Method A1): R t =0.93 min (UV detector: TIC), molecular weight measurement value: 307.00.

[0417] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.15 (s, 6H), 2.02-2.11 (m, 2H), 3.84 (s, 3H), 4.54(s, 1H), 4.58-4.65(m, 2H), 8.05(s, 1H), 8.69(s, 1H), 8.86(s, 1H).

[0418] Intermediate 7-1

[0419] 5-Amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester

[0420]

[0421] 4.53 g of iron and 217 mg of ammonium chloride are added to a solution of 2.49 g (8.10 mmol) of methyl 2-(3-hydroxy-3-methylbutyl)-5-nitro-2H-indazole-6-carboxylate (intermediate 6-1) in 30 mL of ethanol and 10 mL of water, and the mixture is stirred at 90 ° C for 21.5 h. The mixture is filtered through celite and washed three times with ethanol, the filtrate is concentrated, and the residue is mixed with water. Extraction is carried out three times with ethyl acetate (sodium chloride solution is added to improve phase separation). The combined organic phases are washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. 1.95 g (85% of theoretical value) of the title compound are obtained.

[0422] UPLC-MS (Method A1): R t =0.67 min (UV detector: TIC), molecular weight measurement value: 277.00.

[0423] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.14 (s, 6H), 1.96-2.08 (m, 2H), 3.85 (s, 3H), 4.39-4.51(m, 3H), 5.81(s, 2H), 6.80(s, 1H), 8.05(s, 1H), 8.18(s, 1H).

[0424] Working Examples

[0425] Example 1

[0426] N-[6-(2-Hydroxypropan-2-yl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0427]

[0428] 75mg (0.18mmol) of 2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-2) was dissolved in 500μL of THF and mixed with 887μL (0.89mmol) of 1M methylmagnesium bromide in THF. The reaction mixture was stirred at 25°C for 60min. Subsequently, 1mL of saturated aqueous ammonium chloride solution was carefully added and the mixture was filtered. The aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, filtered through a hydrophobic filter, and concentrated. The residue was dissolved in 3mL of DMSO and purified by preparative HPLC. The portion containing the product was freeze-dried. 20mg of the title compound was obtained.

[0429] UPLC-MS (Method A1): R t =1.08min

[0430] MS (ESIpos): m / z=423 (M+H) +

[0431] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 3.22 (s, 3H), 3.82 (t, 2H), 4.55 (t, 2H), 5.96 (s, 1H), 7.57 (s, 1H), 8.16 (d1 H), 8.29-8.42(m, 2H), 8.42-8.50(m, 1H), 8.71(s, 1H), 12.36(s, 1H).

[0432] Example 2

[0433] N-[6-(Hydroxymethyl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0434]

[0435] 13mg (0.36mmol) of lithium aluminum hydride was suspended in 1mL of THF, and the mixture was cooled to 0°C. A solution of 75mg (0.17mmol) of 2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl esters (intermediate 4-2) dissolved in 500 μL of THF was added dropwise, and the mixture was stirred at 25°C for 60min. The mixture was diluted with water and extracted twice with ethyl acetate, and the combined organic phases were washed with sodium chloride solution, filtered through a hydrophobic filter, concentrated, and dried under reduced pressure. 13mg of the title compound was obtained.

[0436] UPLC-MS (Method A2): R t =0.99min

[0437] MS (ESIpos): m / z=394 (M+H) +

[0438] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.23 (s, 3H), 3.83 (t, 2H), 4.56 (t, 2H), 4.69 (d, 2H), 5.77 (t, 1 H), 7.57 (s, 1H), 8.19 (d, 1H), 8.33-8.41 (m, 2H), 8.43-8.47 (m, 1H), 8.51 (s, 1H), 11.20 (s, 1H).

[0439] Example 3

[0440] N-[6-(2-Hydroxypropan-2-yl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0441]

[0442] 75mg (0.17mmol) of 2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-3) was dissolved in 500μL of THF and mixed with 859μL (0.86mmol) of 1M methylmagnesium bromide in THF. The reaction mixture was stirred at 25°C for 60min. Subsequently, 1mL of saturated ammonium chloride solution was carefully added and the mixture was filtered. The aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, filtered through a hydrophobic filter, and concentrated. The residue was dissolved in 3mL of DMSO and purified by preparative HPLC. The portion containing the product was freeze-dried. 25mg of the title compound was obtained.

[0443] UPLC-MS (Method A1): R t =1.13min

[0444] MS (ESIpos): m / z=437 (M+H) +

[0445] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 2.14 (quin, 2H), 3.23 (s, 3H), 3.26-3.32 (m, 2H), 4.44 (t, 2H) , 5.95 (s, 1H), 7.58 (s, 1H), 8.16 (d, 1H), 8.31-8.40 (m, 2H), 8.43-8.48 (m, 1H), 8.72 (s, 1H), 12.36 (s, 1H).

[0446] Example 4

[0447] N-[6-(Hydroxymethyl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0448]

[0449] 13 mg of lithium aluminum hydride was suspended in THF, and the mixture was cooled to 0 ° C. A solution of 75 mg (0.17 mmol) of 2- (3-methoxypropyl) -5- ({ [6- (trifluoromethyl) pyridin-2-yl] carbonyl} amino) -2H- indazole -6- carboxylic acid methyl ester (intermediate 4-3) in THF was added dropwise, and the mixture was allowed to reach room temperature within 30 min. The mixture was diluted with water and filtered, the residue was washed with ethyl acetate, and the filtrate was extracted with ethyl acetate. The combined ethyl acetate phase was washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC.

[0450] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 2.14 (quin, 2H), 3.23 (s, 3H), 3.29 (t, 2H), 4.45 (t, 2H), 4.68 (d, 2H), 5.77 (t, 1H), 7.58 (s, 1H), 8.18 (d, 1H), 8.32-8.48 (m, 3H), 8.51 (s, 1H), 11.21 (s, 1H).

[0451] Example 5

[0452] N-[2-(2-Hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0453] Step A:

[0454] Preparation of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0455]

[0456] 100 mg (0.19 mmol) of methyl 2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-5) was dissolved in 1 mL of THF and mixed with 669 μL (0.67 mmol) of a 1 M solution of methylmagnesium bromide in THF. The reaction mixture was stirred at 25° C. for 60 min. 287 μL (0.29 mmol) of a 1 M solution of methylmagnesium bromide in THF were further added, and the mixture was stirred at 25° C. for 3 h. Subsequently, 20 mL of a saturated ammonium chloride solution was carefully added, and the mixture was filtered. The aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, dried over magnesium sulfate, filtered, concentrated, and dried under reduced pressure. 50 mg of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide are obtained.

[0457] UPLC-MS (Method A2): R t =1.51min

[0458] MS (ESIpos): m / z=523 (M+H) +

[0459] 1 H NMR (300MHz, DMSO-d6): δ[ppm]=-0.17--0.09(m, 6H), 0.78(s, 9H), 1.62(s, 6H), 4.04(t, 2H), 4.47(t, 2H), 5.98 (s, 1H), 7.57 (s, 1H), 8.16 (d, 1H), 8.29 (s, 1H), 8.37 (t, 1H), 8.45 (d, 1H), 8.73 (s, 1H), 12.38 (s, 1H).

[0460] Step B:

[0461]

[0462] 50mg (96μmol) of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide was dissolved in 1.0mL of THF and mixed with 144μL (0.14mmol) of 1M tetrabutylammonium fluoride in THF. The reaction mixture was stirred at room temperature for 1h. The mixture was diluted with water and extracted twice with ethyl acetate, the combined organic phases were washed with saturated sodium chloride solution, filtered through a hydrophobic filter, and concentrated. 36mg of N-[2-(2-hydroxyethyl)-6-(2-hydroxypropyl-2-yl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Example 5) was obtained.

[0463] 1 H-NMR (400MHz, DMSO-d6): d[ppm]=1.62(s, 6H), 3.86(q, 2H), 4.43(t, 2H), 4.95(t, 1H), 5.94(s, 1 H), 7.57(s, 1H), 8.16(dd, 1H), 8.30(s, 1H), 8.37(t, 1H), 8.45(d, 1H), 8.72(s, 1H), 12.36(s, 1H).

[0464] UPLC-MS (Method A2): R t =0.97 min (UV detector: TIC), molecular weight measurement value 408.00.

[0465] Example 6

[0466] N-[6-(2-Hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0467] Step A:

[0468] Preparation of N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0469]

[0470] 50 mg (0.09 mmol) of 2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-6) was dissolved in 500 μL of THF and mixed with 326 μL (0.33 mmol) of a 1M THF solution of methylmagnesium bromide. The reaction mixture was stirred at 25° C. for 60 min. Subsequently, 20 mL of saturated ammonium chloride solution was carefully added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were filtered through a hydrophobic filter, concentrated, and dried under reduced pressure. The residue was purified by preparative HPLC. 40 mg of N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide are obtained.

[0471] UPLC-MS (Method A1): R t =1.58min

[0472] MS (ESIpos): m / z=537 (M+H) +

[0473] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 0.02-0.05 (m, 6H), 0.84-0.91 (m, 9H), 1.62 (s, 6H), 2.02-2.18 (m, 2H), 3.55-3.62 (m, 2H), 4 .45 (t, 2H), 5.96 (s, 1H), 7.57 (s, 1H), 8.16 (d, 1H), 8.31 (s, 1H), 8.33-8.42 (m, 1H), 8.45 (d, 1H), 8.72 (s, 1H), 12.37 (s, 1H).

[0474] Step B:

[0475]

[0476] 37 mg (0.07 mmol) of N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxyprop-2-yl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide was dissolved in 500 μL of THF and mixed with 207 μL (0.21 mmol) of a 1 M solution of tetrabutylammonium fluoride in THF. The reaction mixture was stirred at 25 ° C for 2 h. The mixture was diluted with water and extracted twice with ethyl acetate, and the combined organic phases were washed with a saturated sodium chloride solution, filtered and concentrated. After purification by preparative HPLC, 10 mg of N-[6-(2-hydroxyprop-2-yl)-2-(3-hydroxypropyl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Example 6, containing minor components) was obtained.

[0477] UPLC-MS (Method A2): R t =1.00min

[0478] MS (ESIpos): m / z=423 (M+H) +

[0479] 1 H NMR selected signals (400 MHz, DMSO-d6): δ [ppm] = 1.61 (s), 2.00-2.12 (m), 3.38 (t, 2H), 4.44 (t, 2H), 4.62 (br. s., 1H), 5.93 (br. s., 1H), 7.55 (s, 1H), 8.13 (d, 1H), 8.27-8.38 (m, 2H), 8.43 (d, 1H), 8.71 (s, 1H), 12.30 (br. s., 1H).

[0480] Example 7

[0481] N-[2-(2-Hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0482] Step A:

[0483] N-[2-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0484]

[0485] 100 mg (0.19 mmol) of methyl 2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-5) was dissolved in 1 mL of THF and mixed with 191 μL (0.38 mmol) of a 2M lithium borohydride solution. The mixture was stirred at 25° C. for 24 h. 14 mg (0.38 mmol) of sodium borohydride and 500 μL of methanol were added, and the mixture was stirred at 25° C. for 4 h. An additional 14 mg (0.38 mmol) of sodium borohydride was added, and the mixture was stirred at 25° C. for 24 h. Water was carefully added to the reaction mixture, and the organic phase was removed. The mixture was then extracted twice with ethyl acetate, and the combined organic phases were washed with a saturated sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was dissolved in 2 mL of DMSO and purified by preparative HPLC to obtain 30 mg of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide.

[0486] UPLC-MS (Method A2): R t =1.44min

[0487] MS (ESIpos): m / z=495 (M+H) +

[0488] 1 H NMR (300MHz, DMSO-d6): δ[ppm]=-0.16--0.12(m, 6H), 0.75-0.79(m, 9H), 4.05(t, 2H), 4.48(t, 2H), 4.69(d, 2H), 5.7 5-5.77 (m, 1H), 7.57 (s, 1H), 8.18 (dd, 1H), 8.30-8.33 (m, 1H), 8.38 (t, 1H), 8.45 (d, 1H), 8.51 (s, 1H), 11.20 (s, 1H).

[0489] Step B:

[0490]

[0491] 33 mg (0.07 mmol) of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide was dissolved in 1 mL of THF and mixed with 100 μL (0.10 mmol) of a 1 M solution of tetrabutylammonium fluoride in THF. The reaction mixture was stirred at 25 ° C for 1 h. The mixture was diluted with water and extracted twice with ethyl acetate, and the combined organic phases were washed with a saturated sodium chloride solution, filtered through a hydrophobic filter, concentrated, and dried under reduced pressure. 25 mg of N-[2-(2-hydroxyethyl)-6-(hydroxymethyl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Example 7) was obtained.

[0492] UPLC-MS (Method A2): R t =0.87min

[0493] MS (ESIpos): m / z=381 (M+H) +

[0494] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 3.87 (q, 2H), 4.44 (t, 2H), 4.69 (d, 2H), 4.98 (t, 1H), 5.70-5.81 (m, 1 H), 7.57 (s, 1H), 8.11-8.23 (m, 1H), 8.31-8.42 (m, 2H), 8.43-8.49 (m, 1H), 8.51 (s, 1H), 11.20 (s, 1H).

[0495] Example 8

[0496] N-[6-(2-Hydroxypropan-2-yl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0497]

[0498] 50mg (0.12mmol) of 2-(oxetanes-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-1) was dissolved in 500μL of THF and mixed with 576μL (0.58mmol) of 1M methylmagnesium bromide in THF. The reaction mixture was stirred at 25°C for 60min. Subsequently, 20mL of saturated aqueous ammonium chloride solution was carefully added and the mixture was concentrated. The aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in 2.0mL of DMSO and purified by preparative HPLC. The fractions containing the product were freeze-dried. 30mg of the title compound were obtained.

[0499] UPLC-MS (Method A2): R t =1.03min

[0500] MS (ESIpos): m / z=435 (M+H) +

[0501] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 3.45-3.61 (m, 1H), 4.48 (t, 2H), 4.66 (dd, 2H), 4.72 (d, 2H), 5.94 (s, 1H), 7.57 (s, 1H), 8.16 (d, 1H), 8.33-8.42 (m, 2H), 8.42-8.47 (m, 1H), 8.72 (s, 1H), 12.36 (s, 1H).

[0502] Example 9

[0503] N-[6-(Hydroxymethyl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0504]

[0505] 75 mg (0.17 mmol) of 2-(oxetane-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (Intermediate 4-1) was dissolved in 1 mL of a mixture of THF / methanol (1:1) and 8 mg (0.21 mmol) of sodium borohydride was added. The mixture was stirred at 25 ° C for 60 min. The reaction mixture was concentrated and the residue was mixed with water. The suspension was vigorously stirred for 15 min, the solid was filtered off, washed twice with water, washed twice with diethyl ether, and dried under reduced pressure. 48 mg of the title compound was obtained.

[0506] UPLC-MS (Method A2): R t =0.94min

[0507] MS (ESIpos): m / z=407 (M+H) +

[0508] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 3.55 (s, 1H), 4.48 (t, 2H), 4.61-4.77 (m, 6H ), 7.57 (s, 1H), 8.18 (dd, 1H), 8.33-8.49 (m, 3H), 8.51 (s, 1H), 11.21 (s, 1H).

[0509] Example 10

[0510] N-{6-(2-hydroxypropan-2-yl)-2-[3-(methylsulfonyl)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0511]

[0512] A mixture of 500mg (1.32mmol) of N-[6-(2-hydroxypropyl-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (intermediate 5-1), 569mg of potassium carbonate and 114mg of potassium iodide in 5.0mL of DMF was stirred at room temperature for 15min. 414mg of 1-bromo-3-(methylsulfonyl)propane was added, and the mixture was stirred at room temperature overnight. Water was added, the mixture was extracted twice with ethyl acetate, the extract was washed with sodium chloride solution, and concentrated. Residue was purified by column chromatography (dichloromethane / methanol gradient). The product portion was stirred, filtered and dried together with diethyl ether. 59mg of title compound was obtained.

[0513] UPLC-MS (Method A2): R t=1.02min

[0514] MS (ESIpos): m / z=485 (M+H)+

[0515] 1 H-NMR (300MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 2.26-2.42 (m, 2H), 2.99 (s, 3H), 3.06-3.16 (m, 2H), 4.55(t, 2H), 5.96(s, 1H), 7.60(s, 1H), 8.16(d, 1H), 8.33-8.48(m, 3H), 8.73(s, 1H), 12.37(s, 1H).

[0516] Example 11

[0517] N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0518]

[0519] Preparation method 1

[0520] First, 705 mg (1.57 mmol) of methyl 2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (intermediate 4-4) was added to 10 mL of THF and cooled in an ice-water cooling bath. 2.6 mL (5.0 equivalents) of 3M methylmagnesium bromide solution (in diethyl ether) were added, and the mixture was stirred for 1 hour while cooling with an ice bath and stirred at room temperature for 4.5 hours. 1 equivalent of methylmagnesium bromide solution was added, and the mixture was stirred at room temperature for 20.5 hours. 1 equivalent of methylmagnesium bromide solution was added, and the mixture was stirred at room temperature for 22 hours. The reaction mixture was mixed with saturated aqueous ammonium chloride solution, stirred, and extracted three times with ethyl acetate. The combined organic phases were washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. 790 mg of residue was obtained, which was purified by preparative HPLC. 234 mg of the title compound and 164 mg of a product fraction were obtained, which was stirred with diethyl ether, filtered off with suction and then dried, after which a further 146 mg of the title compound were obtained.

[0521] UPLC-MS (Method A1): R t =1.10 min (UV detector: TIC), molecular weight measurement value: 450.00.

[0522] 1H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.14 (s, 6H), 1.61 (s, 6H), 1.99-2.08 (m, 2H), 4.42-4.55 (m, 3H), 5.9 3 (s, 1H), 7.56 (s, 1H), 8.15 (dd, 1H), 8.32-8.39 (m, 2H), 8.41-8.47 (m, 1H), 8.70 (s, 1H), 12.34 (s, 1H).

[0523] Preparation method 2

[0524] A mixture of 500 mg (1.37 mmol) of N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Intermediate 5-1), 569 mg of potassium carbonate, and 114 mg of potassium iodide in 5 mL of DMF was stirred at room temperature for 15 minutes. 344 mg (1.5 equivalents) of 4-bromo-2-methylbutan-2-ol were added, and the mixture was heated to 100° C. for 2 hours. Another 0.5 equivalents of 4-bromo-2-methylbutan-2-ol was added, and the mixture was stirred at room temperature for 16 hours. The mixture was mixed with water and extracted twice with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 100 mg of the product fraction was obtained, which was stirred with diethyl ether. The solid was filtered and dried. 60 mg of the title compound was obtained.

[0525] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.14 (s, 6H), 1.61 (s, 6H), 1.99-2.07 (m, 2H), 4.43-4.52 (m, 3H) 5. 94 (s, 1H) 7.57 (s, 1H) 8.15 (dd, 1H) 8.33-8.40 (m, 2H), 8.42-8.48 (m, 1H), 8.71 (s, 1H), 12.35 (s, 1H).

[0526] Example 12

[0527] N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0528]

[0529] 160mg (0.44mmol) N-[6-(2-hydroxypropyl-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (intermediate 5-1) was suspended in 1.0mL of DMF together with 182mg of sodium carbonate and 36mg of potassium iodide, and the mixture was stirred at room temperature for 15min. Then, 123mg of 2-bromoethyl methyl sulfone (0.66mmol) was added, and the mixture was stirred at room temperature overnight. Water was added, the mixture was extracted twice with ethyl acetate, the extract was washed with saturated sodium chloride aqueous solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC to obtain 20mg of the title compound.

[0530] UPLC (Method A2): R t =1.01min;

[0531] MS (ESIpos): m / z=471 (M+H)+

[0532] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 2.90 (s, 3H), 3.85 (t, 2H), 4.86 (t, 2H), 5.97 (s, 1 H), 7.59 (s, 1H), 8.13-8.19 (m, 1H), 8.37 (s, 1H), 8.41-8.48 (m, 2H), 8.74 (s, 1H), 12.37 (s, 1H).

[0533] Example 13

[0534] 6-(Difluoromethyl)-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0535]

[0536] Preparation method 1

[0537] A mixture of 250 mg of 6-(difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]pyridine-2-carboxamide (crude product of intermediate 5-2), 144 mg of potassium iodide, and 239 mg of potassium carbonate in 2.5 mL of DMF was stirred at room temperature for 15 minutes. 145 mg (0.87 mmol) of 4-bromo-2-methylbutan-2-ol was added, and the mixture was stirred at 110° C. for 3 hours. An additional 96 mg of 4-bromo-2-methylbutan-2-ol was added, and the mixture was stirred at 110° C. for 4 hours. Water was added, and the mixture was extracted twice with ethyl acetate. The extracts were washed with half-saturated sodium chloride solution, filtered through a hydrophobic filter, and concentrated. Purification was performed by silica gel column chromatography (hexane / ethyl acetate). 61 mg of the title compound was obtained.

[0538] UPLC-MS (Method A1): R t =1.00 min (UV detector: TIC), molecular weight measurement value: 432.00.

[0539] 1 H-NMR (300MHz, DMSO-d6): δ [ppm] = 1.14 (s, 6H), 1.63 (s, 6H), 1.97-2.08 (m, 2H), 4.41-4.55 (m, 3H), 5. 99 (s, 1H), 7.03 (t, 1H), 7.56 (s, 1H), 7.94-8.00 (m, 1H), 8.24-8.38 (m, 3H), 8.71 (s, 1H), 12.49 (s, 1H).

[0540] Preparation method 2

[0541] Analogously to the preparation of Example 11 (Preparation 1), 3.00 g of methyl 5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate (Intermediate 4-11) was reacted with a 3 M solution of methylmagnesium bromide in diethyl ether. The crude product was stirred with diethyl ether, filtered, and purified by preparative HPLC to obtain 1.37 g of the title compound.

[0542] Example 14

[0543] 6-(Difluoromethyl)-N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}pyridine-2-carboxamide

[0544]

[0545] A mixture of 250 mg of 6-(difluoromethyl)-N-[6-(2-hydroxypropyl-2-yl)-1H-indazole-5-yl]pyridine-2-carboxamide (the crude product of intermediate 5-2), 144 mg of potassium iodide and 239 mg of potassium carbonate in 2.5 mL of DMF was stirred at room temperature for 15 min. 162 mg of 2-bromoethylmethylsulfone (0.87 mmol) was added and the mixture was stirred at 110° C. for 3 h. Water was added and the mixture was extracted twice with ethyl acetate, and the extract was washed with a half-saturated sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC, and the product portion was purified again by silica gel column chromatography (hexane / ethyl acetate). 40 mg of the title compound was obtained.

[0546] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.65 (s, 6H), 2.90 (s, 3H), 3.85 (t, 2H), 4.85 (t, 2H), 6.03 (s, 1H) , 7.04 (t, 1H), 7.59 (s, 1H), 7.98 (d, 1H), 8.25-8.36 (m, 2H), 8.43 (s, 1H), 8.75 (s, 1H), 12.52 (s, 1H).

[0547] Example 15

[0548] 6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0549] Phase A:

[0550] Preparation of N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(difluoromethyl)pyridine-2-carboxamide

[0551]

[0552] A mixture of 250 mg of 6-(difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]pyridine-2-carboxamide (Intermediate 5-2), 48 mg of potassium iodide, and 239 mg of potassium carbonate in 2.5 mL of DMF was stirred at room temperature for 15 min. 219 mg (0.87 mmol, 1.5 equivalents) of (3-bromopropoxy)(tert-butyl)dimethylsilane were added, and the mixture was stirred at 110° C. for 3 h. 1 equivalent of (3-bromopropoxy)(tert-butyl)dimethylsilane was also added, and the mixture was stirred at 100° C. for 4 h. Water was added, the mixture was extracted with ethyl acetate, and the extract was washed with aqueous sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by column chromatography (hexane / ethyl acetate). 92 mg of the title compound was obtained.

[0553] Phase B:

[0554]

[0555] Analogously to the preparation of Example 6, Step B, 92 mg of N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(difluoromethyl)pyridine-2-carboxamide was reacted with 0.53 mL of a 1 M solution of tetrabutylammonium fluoride in THF for 1 h. Aqueous workup was performed as in Example 6, and purification by preparative HPLC afforded 46 mg of the title compound.

[0556] UPLC-MS (Method A1): R t =0.92 min (UV detector: TIC), molecular weight measurement value 404.00.

[0557] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.64 (s, 6H), 2.05 (quin, 2H), 3.35-3.46 (m, 2H), 4.45 (t, 2H), 4.64 (t, 1H) ), 5.99 (s, 1H), 7.04 (t, 1H), 7.57 (s, 1H), 7.95-7.99 (m, 1H), 8.25-8.36 (m, 3H), 8.73 (s, 1H), 12.50 (s, 1H).

[0558] Example 16

[0559] N-[6-(2-Hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0560]

[0561] A mixture of 210mg (0.58mmol) of N-[6-(2-hydroxypropan-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (intermediate 5-1) in 3mL DMF was mixed with 0.11mL (0.87mmol) of 1,1,1-trifluoro-4-iodobutane and 239mg of potassium carbonate, and the mixture was stirred at 80°C for 6h. After adding water, the mixture was extracted three times with ethyl acetate, and the combined organic phases were washed with saturated sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The crude product was purified by preparative HPLC. 19mg of the title compound was obtained.

[0562] UPLC-MS (Method A1): R t =1.27 min (UV detector: TIC), molecular weight measurement value 474.15.

[0563] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 2.10-2.33 (m), 4.49 (t, 2H), 5.94 (s, 1H), 7.5 9(s, 1H), 8.13-8.18(m, 1H), 8.32-8.41(m, 2H), 8.41-8.47(m, 1H), 8.72(s, 1H), 12.35(s, 1H).

[0564] Example 17

[0565] N-{6-(2-hydroxypropan-2-yl)-2-[3-(trifluoromethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0566]

[0567] First 150mg (0.33mmol) N-[6-(2-hydroxypropyl-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl) pyridine-2-carboxamide (intermediate 5-1) is added in 2mL THF.Add 58mg (0.40mmol) of 3-(trifluoromethoxy) propan-1-ol, 131mg triphenylphosphine and 71 μ L diisopropyl azodicarboxylate (DIAD, CAS2446-83-5), and the mixture is at room temperature stirred for 19h.Add 0.83mL of sodium hydroxide solution (2M), and the mixture is stirred for 5h at 40 ℃.Dilute the mixture with water, and extract three times with ethyl acetate, the organic phase merged is concentrated, and pass through preparation HPLC purification.Obtain the title compound of 16mg crude product form.

[0568] UPLC-MS (Method A2): R t =1.26 min (UV detector: TIC), molecular weight measurement value 490.14.

[0569] 1 H-NMR (400 MHz, DMSO-d6, selected signals): δ [ppm] = 1.61 (s, 6H), 1.84 (d, 1H), 2.32 (quint., 2H), 4.08 (t, 2H), 4.51 (t, 2H), 7.58 (s, 1H), 8.15 (d, 1H), 8.31-8.39 (m, 2H), 8.44 (d, 1H), 8.72 (s, 1H), 12.35 (s, 1H).

[0570] Example 18

[0571] N-{6-(2-hydroxypropan-2-yl)-2-[3-(2,2,2-trifluoroethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0572]

[0573] Analogously to the preparation of Example 11 (Preparation 1), 52 mg (0.10 mmol) of methyl 2-[3-(2,2,2-trifluoroethoxy)propyl]-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-10) in 3 mL of THF was reacted with 2×171 μL of a 3 M solution of magnesium bromide in diethyl ether. Purification by preparative HPLC gave 12 mg of the title compound.

[0574] UPLC-MS (Method A1): R t =1.25 min (UV detector: TIC), molecular weight measurement value 504.16.

[0575] 1 H-NMR (500MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 2.20 (quin, 2H), 3.58 (t, 2H), 4.05 (q, 2H), 4.47 (t, 2H), 5 .94 (s, 1H), 7.58 (s, 1H), 8.15 (dd, 1H), 8.32 (s, 1H), 8.36 (t, 1H), 8.45 (d, 1H), 8.73 (s, 1H), 12.36 (s, 1H).

[0576] Example 19

[0577] 5-Fluoro-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0578]

[0579] First, 228mg (0.31mmol) of 5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-8) was added to 4.5mL THF and cooled with an ice-cooling bath. 0.63mL of 3M methylmagnesium bromide solution (in diethyl ether) was added, and the mixture was stirred for 2h while cooling with an ice bath, and stirred at room temperature for 21h. The reaction mixture was mixed with saturated aqueous ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phase was concentrated. The residue was purified by preparative HPLC. 82mg of the title compound was obtained.

[0580] UPLC-MS (Method A2): R t =1.03 min (UV detector: TIC), molecular weight measurement value: 414.21.

[0581] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.13 (s, 6H), 1.63 (s, 6H), 1.99-2.05 (m, 2H), 2.55-2.59 (m, 3H), 4.42-4 .50 (m, 3H), 5.95 (s, 1H), 7.54 (s, 1H), 7.83 (t, 1H), 8.05 (dd, 1H), 8.31 (s, 1H), 8.68 (s, 1H), 12.33 (s, 1H).

[0582] Example 20

[0583] N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0584]

[0585] First, 278mg (0.48mmol) of 2-(3-hydroxy-3-methylbutyl)-5-{[(6-methylpyridin-2-yl)carbonyl]amino}-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-9) was added to 5.0mL of THF and cooled with an ice-cold bath. 0.97mL of 3M methylmagnesium bromide solution (in diethyl ether) was added, and the mixture was stirred for 2h while cooling with an ice bath, and stirred at room temperature for 20.5h. 0.48mL of 3M methylmagnesium bromide solution was also added, and the mixture was stirred at room temperature for 67h. The mixture was mixed with a saturated aqueous ammonium chloride solution and extracted three times with ethyl acetate. The extract was washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC. 111mg of the title compound was obtained.

[0586] UPLC-MS (Method A2): R t =0.97 min (UV detector: TIC), molecular weight measurement value 396.22.

[0587] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.15 (s, 6H), 1.64 (s, 6H), 2.00-2.08 (m, 2H), 2.61 (s, 3H), 4.41-4.59 (m , 3H), 5.92 (s, 1H), 7.50 (dd, 1H), 7.56 (s, 1H), 7.90-7.99 (m, 2H), 8.33 (s, 1H), 8.70 (s, 1H), 12.39 (s, 1H).

[0588] Example 21

[0589] 6-(2-Hydroxypropan-2-yl)-N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0590]

[0591] A solution of 72 mg (0.16 mmol) of 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-2-(4,4,4-trifluorobutyl)-2H-indazole-6-carboxylic acid methyl ester (Intermediate 4-7) in 10 mL of THF was cooled in an ice / water cooling bath. 0.26 mL of a 3 M solution of methylmagnesium bromide in diethyl ether was added, and the mixture was stirred for 2 h and then at room temperature for 20 h. 1 equivalent of a 3 M solution of methylmagnesium bromide was further added, and the mixture was stirred at room temperature for 24 h. Saturated aqueous ammonium chloride solution was added, the mixture was extracted three times with ethyl acetate, the extract was washed with sodium chloride solution, and concentrated. 22 mg (31% of theoretical value) of the title compound were obtained by preparative HPLC.

[0592] UPLC-MS (Method A2): R t =1.15 min (UV detector: TIC), molecular weight measurement value: 464.20.

[0593] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.56 (s, 6H), 1.64 (s, 6H), 2.07-2.34 (m, 4H), 4.49 (t, 2H), 5.32 (s, 1 H), 6.05 (s, 1H), 7.60 (s, 1H), 7.87 (dd, 1H), 7.99-8.05 (m, 2H), 8.35 (s, 1H), 8.79 (s, 1H), 12.45 (s, 1H).

[0594] Example 22

[0595] N-{2-[2-(1-Hydroxycyclopropyl)ethyl]-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0596]

[0597] First, 250 mg (0.69 mmol) of N-[6-(2-hydroxypropan-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (intermediate 5-1) was added to 5 mL of DMSO. 159 mg (0.96 mmol) of 1-(2-bromoethyl)cyclopropanol, 285 mg of potassium carbonate and 171 mg of potassium iodide were added and the mixture was stirred at 100° C. for 5 h. Water was added and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with sodium chloride solution, filtered through a hydrophobic filter and concentrated. The residue was purified by preparative HPLC (column: Waters XBridge C18 5μ100×30 mm, eluent A: water + 0.1% by volume of formic acid (99%), eluent B: acetonitrile). Freeze-dried to obtain 45 mg of the title compound.

[0598] 1 H-NMR (500MHz, DMSO-d6): δ [ppm] = 0.18-0.22 (m, 2H), 0.48-0.52 (m, 2H), 1.62 (s, 6H), 2.08 (t, 2H), 4.54-4.60 (m, 2H) ), 5.36 (s, 1H), 5.96 (s, 1H), 7.57 (s, 1H), 8.16 (dd, 1H), 8.34-8.39 (m, 2H), 8.45 (d, 1H), 8.72 (s, 1H), 12.36 (s, 1H).

[0599] Physiological efficacy evaluation

[0600] IRAK4 kinase assay

[0601] The IRAK4-inhibitory activity of the substances according to the invention was determined in the IRAK4 TR-FRET assay (TR-FRET=time-resolved fluorescence resonance energy transfer) described below.

[0602] As the enzyme, a recombinant fusion protein of GST (glutathione-S-transferase) and human IRAK4 from the N-terminus was expressed in baculovirus-infected insect cells (Hi5, BTI-TN-5B1-4, a cell line purchased from Invitrogen, catalog number B855-02) and purified by affinity chromatography. The substrate for the kinase reaction was the biotinylated peptide biotin-Ahx-KKARFSRFAGSSPSQASFAEPG (C-terminus in amide form), which can be purchased, for example, from Biosyntan GmbH (Berlin-Buch).

[0603] For the assay, 11 different concentrations ranging from 20 μM to 0.073 nM were prepared from a 2 mM DMSO solution of the test substance. 50 nL of the corresponding solution was pipetted into a black, low-volume, 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of IRAK4 in assay buffer [50 mM HEPES, pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w / v) bovine gamma-globulin (BGG), 0.04% (v / v) nonidet-P40 (Sigma)] was added, and the mixture was incubated for 15 min to allow the substances to pre-bind to the enzyme before the kinase reaction. The kinase reaction is then initiated by adding 3 μL of a solution of adenosine triphosphate (ATP, 1.67 mM = final concentration in a 5 μL assay volume: 1 mM) and the peptide substrate (0.83 μM = final concentration in a 5 μL assay volume: 0.5 μM) in assay buffer. The resulting mixture is incubated at 22°C for a reaction time of 45 minutes. The IRAK4 concentration is adjusted to the respective enzyme activity and is set so that the assay is performed within the linear range. Typically, the concentration is approximately 0.2 nM. The reaction was terminated by adding 5 μL of a solution of TR-FRET detection reagent [0.1 μM streptavidin-XL665 (Cisbio Bioassays; France, catalog number 610SAXLG)], 1.5 nM anti-phosphoserine antibody [Merck Millipore, “STK antibody”, catalog number 35-002], and 0.6 nM LANCE EU-W1024-labeled anti-mouse IgG antibody (Perkin-Elmer, product number AD0077; alternatively, terbium cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used) in aqueous EDTA (100 mM EDTA, 0.4% (w / v) bovine serum albumin [BSA] in 25 mM HEPES, pH 7.5).

[0604] The resulting mixture was incubated at 22 ° C for 1 h to form a complex of biotinylated phosphorylated substrate and detection reagent. The amount of phosphorylated substrate was then assessed by measuring the resonance energy transferred from the anti-mouse IgG antibody labeled with europium chelate to streptavidin-XL665. For this purpose, after excitation at 350 nm in a TR-FRET measuring instrument such as Rubystar (BMG Labtechnologies, Offenburg, Germany) or Viewlux (Perkin-Elmer), the fluorescence emission at 620 nm and 665 nm was measured. The emission ratio at 665 nm and 622 nm was used as a measure of the amount of phosphorylated substrate. The data were normalized (enzyme reaction without test substance = 0% inhibition; all other test components but without enzyme = 100% inhibition). Typically, the test substances were tested at 11 different concentrations ranging from 20 μM to 0.073 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 nM, and 0.073 nM) on the same microtiter plate. The dilution series was prepared by serial dilution prior to the assay (2 mM to 7.3 nM in 100% DMSO). IC values ​​were calculated by 4-parameter fitting. 50 value.

[0605] Table 1: IC of Example Compounds in IRAK4 Kinase Assay 50 value

[0606]

[0607] The inhibitory activity of the substance of the general formula (III) of the present invention against IRAK4 was also determined in the above-mentioned IRAK4 TR-FRET assay. The following are mentioned as examples: compound intermediate 4-2, IC 50 =21.7nM; intermediate 4-3, IC 50 =13.0 nM; and intermediate 4-4, IC 50 =6.2nM.

[0608] TNF-α secretion in THP-1 cells

[0609] This test is suitable for testing the ability of substances to inhibit the secretion of TNF-α (tumor necrosis factor α) in THP-1 cells (a human monocytic acute leukemia cell line). TNF-α is a cytokine involved in inflammatory processes. In this test, TNF-α secretion is triggered by incubation with bacterial lipopolysaccharide (LPS).

[0610] THP-1 cells were maintained in a continuous suspension cell culture medium [RPMI 1460 medium with L-Glutamax (Gibco, catalog number 61870-044), supplemented with 10% fetal calf serum (FCS) (Invitrogen, catalog number 10082-147), 1% penicillin / streptomycin (Gibco BRL, catalog number 15140-114)] and should not exceed 1×10 6 The assay was performed in a cell culture medium (RPMI 1460 medium containing L-Glutamax, supplemented with 10% FCS).

[0611] In each case, 2-2.5 μ L of cell suspension (corresponding to 4000 cells) per well was distributed to a 384-well test plate (Greiner, catalog number (Cat. No.) 784076), wherein in each well, 40-50 nL of the substance was dissolved in 100% DMSO. For each substance, dissolving was accomplished by using 10 different concentrations ranging from 20 μ M to 0.073 nM. The cells were incubated at room temperature for 15 min. 2-2.5 μ L of LPS (Sigma, Escherichia coli 055:B5, catalog number (Cat. No.) L5418) (final concentration 0.05 μ g / mL) dissolved in the cell culture medium was then distributed to each well. As a neutral control, cells were treated with 0.05 μ g / mL of LPS and 1% of DMSO, and as an inhibitor control, only 1% of DMSO was used.

[0612] The plate was centrifuged at 80 g for 30 seconds and incubated at 37°C, 5% CO₂, and 95% atmospheric humidity for 17 hours. The amount of TNF-α was determined using the TNF-α HTRF Detection Kit (Cisbio, catalog number 62TNFPEB / C). To this end, 2 μl of the detection solution, consisting of anti-TNF-α-XL665 conjugate and anti-TNF-α-cryptate conjugate dissolved in reconstitution buffer according to the manufacturer's instructions, was added in each case for HTRF (homogeneous time-resolved fluorescence) measurement. After addition, the mixture was incubated at room temperature for 3 hours or at 4°C overnight. The signal at 620 / 665 nm was then read using an HTRF-capable measuring instrument, such as a BMG PheraStar.

[0613] The activity of the substance is expressed as the ratio between the neutral control and the inhibitor control in percent. IC was calculated using a 4-parameter fit. 50 value.

[0614] Table 2:IC of Example Compounds on TNF-α Secretion in THP-1 Cells 50 value

[0615]

[0616] In vitro LPS (lipopolysaccharide)-induced cytokine production in human PBMC (peripheral blood mononuclear cells)

[0617] The efficacy of the compounds of formula (I) of the present invention on inducible cytokine production in human PBMCs was investigated. Here, cytokine production was induced by LPS, a TLR4 ligand, which leads to activation of the IRAK4-mediated signaling pathway.

[0618] Human PBMC is obtained from human anticoagulated whole blood. For this purpose, 15mL of Ficoll-Paque (Biochrom, catalog number L6115) is first pipetted into a Leucosep tube and 20mL of human blood is added. After the blood is centrifuged at room temperature for 15min at 800g, the plasma containing platelets is removed and discarded. PBMC is transferred to a centrifuge tube and supplemented with PBS (phosphate buffered saline) (Gibco, catalog number 14190). The cell suspension is centrifuged at room temperature for 10min at 250g and the supernatant is discarded. PBMC is resuspended in complete culture medium (RPMI 1640, without L-glutamine (PAA, catalog number E15-039), 10% FCS; 50U / mL penicillin, 50μg / mL streptomycin (PAA, catalog number P11-010) and 1% L-glutamine (Sigma, catalog number G7513)).

[0619] The experiment was also carried out in complete culture medium. PBMC were cultured at 2.5×10 5The cell density of each cell / well was seeded onto a 96-well plate. The compound of the present invention was serially diluted, diluted in an isometric 100% DMSO, and applied to the test at 8 different concentrations within the range of 10 μM to 3nM, so that the final DMSO concentration was 0.4% DMSO. The cells were then pre-incubated with the cells for 30 min before actual stimulation. To induce cytokine secretion, cells were stimulated for 24 hours with 0.1 μg / mL of LPS (Sigma, Escherichia coli 0128:B12, catalog number (Cat. No.) L2887. Cell viability was measured using the CellTiter-Glo luminescence assay (Promega, catalog number (Cat. No.) G7571 (G755 / G756A)) according to the manufacturer's instructions. The amount of TNF-α secreted in the cell culture supernatant was measured using the Human ProInflammatory 9-Plex Tissue Culture Kit (MSD, catalog number (Cat. No.) K15007B) according to the manufacturer's instructions. For example, the activities of Example Compound 11 and Example Compound 12 were ≤ 1 μM.

[0620] TLR-4 / TLR-7-induced interleukin (IL)-23 secretion by human dendritic cells (DC) in vitro

[0621] The efficacy of the compound of the general formula (I) of the present invention in inducing the production of the proinflammatory cytokine IL-23 was investigated in human DCs. The proinflammatory cytokine IL-23 plays an important role in the generation of TH-17 cells. TH-17 cells are reported to play a key role in the pathogenesis of diseases such as rheumatoid arthritis, psoriatic arthritis, Bechterev's disease (ankylosing spondylitis), or multiple sclerosis (Lubberts, Nat. Rev. Rheumatol., 2015; Marinoni et al., Auto. Immun. Highlights, 2014; Isailovic et al., J. Autoimmun., 2015; Staschke et al., J. Immunol., 2009). To examine the effects of the compounds of the present invention on IL-23 production, primary human monocytes (using magnetic separation [Miltenyi Biotech, Monocyte Isolation Kit, catalog number 130-091-153]) were isolated from human PBMCs and cultured in complete medium (VLE (very low endotoxin) RPMI 1640 [Biochrom DCs were differentiated into DCs over 6 days in a culture medium containing 1% FBS [Gibco, catalog number FG1415], 10% fetal bovine serum (FBS) [Gibco, catalog number 10493-106], 50 μM β-mercaptoethanol [Gibco, catalog number 31350], 50 U / mL penicillin and streptomycin [Gibco, catalog number 15140-114], and supplemented with growth factors (recombinant human GM-CSF [PeproTech, catalog number 300-03] and IL-4 [PeproTech, catalog number 200-04]). After DCs were collected, they were resuspended in complete culture medium and plated at 2 × 10 5The cell density of 10 cells / well was seeded into 96-well plates (Costar, catalog number 3599). The compound of the present invention was serially diluted in an isometric 100% DMSO and used in the test at 9 different concentrations within the range of 10 μM to 1 nM. It was ensured here that for each of the 9 concentrations used, the DMSO concentration present was always 0.1%. DC was pre-incubated with the compound of the present invention for 30 min. Thereafter, 10 ng / mL of LPS (Sigma, Escherichia coli serotype 0127:B8, catalog number L3129) (TLR4 ligand) and 2.5 μg / mL of TLR-7 / 8 ligand R848 (Invivogen, catalog number tlrl-r848-5) (both of which can activate the IRAK4-mediated signaling pathway) were added to DCs in an incubator (37°C, 95% rH, 5% CO ) to stimulate the production of IL-23 for 24 hours. After 24 hours of incubation, the supernatant was collected and analyzed using a commercially available hIL-23 ELISA (eBiosciences, catalog number 88-7237-88) according to the manufacturer's instructions. The results of the inhibition of IL-23 in human DCs using Example Compound 12 are shown in FIG. Figure 1 middle.

[0622] In vitro TLR-7 / 8- or TLR-9-induced IFNα production by human plasmacytoid dendritic cells (pDCs)

[0623] This assay can be used to investigate the effects of compounds of the general formula (I) of the present invention on the production of IFNα (interferon α), a key cytokine in the pathogenesis of systemic lupus erythematosus, in human pDCs (Mathian et al., Arthritis Rheum, 2009; Crow MK, Rheum Dis Clin N Am, 2010). For this purpose, human PBMCs were isolated from whole blood as described above, and plasmacytoid DCs (pDCs) were isolated therefrom using a commercially available cell isolation kit (Miltenyi Biotech, Plasmacytoid Dendritic Cell Isolation Kit II, catalog number 130-097-415). The obtained pDCs were resuspended in complete culture medium (RPMI 1640+GlutaMax [Gibco, catalog number 61870-010], supplemented with 10% FBS [Gibco, catalog number 10493-106] and 50 U of penicillin / streptomycin [Gibco, catalog number 15140-114]) and cultured at 5×10 4The cell density of individual cells / well is seeded in a 96-well microtiter plate (Costar, catalog number 3599). The compounds of this invention are serially diluted in isometric 100% DMSO and used for testing at 9 different concentrations within the range of 10 μM to 1nM. For each of 9 test concentrations, it is ensured that the DMSO concentration present is always 0.1%. pDC are pre-incubated for 30 minutes together with the compounds of this invention. With TLR7 / 8 ligands (imiquimod (imiquimod), R837, Invivogen, catalog number tlrl-imq) or with TLR-9 ligands (CPG-A, ODN2216, Invivogen, catalog number tlrl-2216-1) stimulate pDC, which can cause the activation of the signal pathway mediated by IRAK4. After 24 hours of incubation, the cell culture supernatant was removed and analyzed using a commercially available human IFNα ELISA (IFNalpha Multi-Subtype ELISA Kit, pbl Assay Science, catalog number 41105-1). Taking Example Compound 12 as an example, the results of inhibition of IFNα in human plasmacytoid DCs are shown in FIG2 .

[0624] In vivo model of TLR-mediated inflammation

[0625] The in vivo efficacy of the compounds of the general formula (I) of the present invention was investigated in an in vivo TLR-mediated inflammation model. Due to the use of an LPS-mediated inflammation model, this mechanistic model particularly shows the potential efficacy of the compounds of the present invention for TLR4-mediated diseases. In this model, female Balb / c mice (approximately 8 weeks old; Charles River Laboratories, Germany) were grouped into 5 animals per group. The control group was treated with a vehicle (substance vehicle) in which the substance was dissolved and a vehicle in which LPS was dissolved. The substance-treated group and the positive control group were intraperitoneally (ip) administered with 0.2 mg LPS / kg body weight (Sigma, catalog number L4391) (lipopolysaccharide from E. coli 0111:B4). In addition, the positive control group was treated with the above-mentioned substance vehicle. The substance was orally administered 16 hours before inducing inflammation by administering LPS. To investigate the efficacy of the compounds of the present invention on inflammation, blood samples were taken from the animals after 1.5 hours. The concentrations of specific cytokines in plasma were determined using the MousePro Inflammatory 7-Plex Tissue Culture Kit (MSD, catalog number K15012B) according to the manufacturer's instructions. IRAK4 inhibitors are effective in TLR-mediated inflammation models. Figure 3The graph shows that the amount of TNF-α in plasma was reduced in a dose-dependent manner by the administration of Example Compound 11 compared with the LPS-induced concentration.

[0626] In vivo model of IL-1β-mediated inflammation

[0627] To evaluate the potential efficacy of the compound of general formula (I) of the present invention in IL-1β-mediated diseases, female Balb / c mice (about 8 weeks old; Charles River Laboratory, Germany) were intraperitoneally administered with IL-1β, and the efficacy of the compound of the present invention for IL-1β-mediated cytokine secretion was investigated. There were 5 animals in each group. The control group was treated with a vehicle that can be used to dissolve the substance and IL-1β. The substance-treated group and the positive control group were each given 90 μg IL-1β / kg body weight (R&D, catalog number 401-ML / CF) intraperitoneally. 6 hours before administering IL-1β, the substance or its vehicle was administered to the positive control group. 2 hours after administering IL-1β, TNF-α in plasma separated from blood was determined using Mouse Pro Inflammatory 7-Plex Tissue Culture Kit (MSD, catalog number K15012B) according to the manufacturer's instructions. Administration of IL-1β resulted in an increase in TNF-α plasma concentration, which was inhibited by treatment with Example Compounds 11 and 12. This is due to Figure 4 illustrate.

[0628] In vivo adjuvant-induced arthritis model

[0629] To determine the anti-inflammatory activity of the compounds of the general formula (I) according to the present invention, their in vivo efficacy was investigated in an arthritis model. For this purpose, on day 0, male Lewis rats (approximately 100-125 g, Charles River Laboratories, Germany) were administered subcutaneously at the base of the tail with 100 μL of a complete Freund's adjuvant (CFA) solution (conjugated to Mycobacterium tuberculosis H37Ra [Difo Lab, catalog number 231141], dissolved in incomplete Freund's adjuvant [Difco Lab, catalog number 263910]). There were n=8 rats in each group. Both healthy and diseased control groups were included in the study. Each control group was treated orally (po) with only the vehicle of the test substance. The treatment was carried out prophylactically, i.e., starting on day 0, with different doses of the test substance by oral administration. On day 0, the initial condition of the animals was also determined according to the disease activity score (arthritis severity grading based on a point system). Here, the degree of joint inflammation is assessed on a scale of 0 to 4 (0 = none; 1 = mild; 2 = moderate; 3 = marked; 4 = severe) based on the erythema of both hind paws, including joint swelling. To determine the anti-inflammatory efficacy of the compounds, the disease activity of the animals was scored using the disease activity score starting on day 8, when the animals first developed signs of arthritis, and then three times a week until the end (day 20). Statistical analysis was performed using one-way analysis of variance (ANOVA), and comparisons were made with the control group using multiple comparison analysis (Dunnett's test).

[0630] Subcutaneous administration of CFA in rats resulted in acute arthritis with marked joint inflammation. This induced arthritis was inhibited by treatment with Example Compound 11. Figure 5 illustrate.

[0631] Collagen antibody-induced arthritis model in mice

[0632] The anti-inflammatory efficacy of the compounds of general formula (I) of the present invention was investigated in another murine arthritis model. For this purpose, on day 0, each female Balb / c mouse (approximately 9 weeks old, Charles River Laboratories, Kingston, Canada) was intravenously injected with 200 μL of collagen antibody cocktail (10 mg / mL; ArthritoMab, MD Bioproducts) into the tail vein (except for the healthy control group included in the study). Then, on day 6, these mice were each given an intraperitoneal injection of 200 μl of LPS. There were n=10 mice in each group. Both the healthy control group and the diseased control group were included in the study. Each control group was treated orally with only the vehicle of the test substance. In a preventive manner, i.e., starting from day 0, different doses of the test substance were used for oral administration for treatment. During the experiment, the degree of disease in all four paws was scored based on the point judgment system for disease activity scoring. In this point assessment, a healthy paw is assessed as no point, while a certain degree of joint inflammation occurring from the toes through the metatarsal joints to the ankle joint is assessed as 1 point (mild inflammation, such as inflammation of the toes) to 4 points (severe inflammation extending to the entire paw), as follows:

[0633] 0 = Normal

[0634] 1 = Erythema and mild swelling limited to the tarsus, ankle, or toes

[0635] 2 = Erythema and mild swelling extending from the ankle to the metatarsal bones (stage 2)

[0636] 3 = Erythema and moderate swelling extending from the ankle distal to the metatarsal joints

[0637] 4 = Erythema and severe swelling covering the metatarsals, foot, and toes

[0638] For this parameter, the starting condition was determined in advance on the day before the start of the experiment (day -1), and the disease activity score was subsequently scored three times a week from day 8 onwards. Statistical analysis was performed using one-way analysis of variance (ANOVA) and comparisons with the control group were performed with the aid of multiple comparison analysis (Dunnett's test).

[0639] Intravenous administration of a collagen antibody cocktail, followed by intraperitoneal administration of LPS, induced acute arthritis with pronounced joint inflammation in mice. This induced arthritis was suppressed by treatment with Example Compound 12. Figure 6 illustrate.

[0640] In vivo NASH mouse model

[0641] To experimentally induce NASH, 45 male 2-day-old C57BL / 6 mice were subcutaneously injected with 200 μg of streptozotocin (STZ; Sigma-Aldrich, USA). Starting from 4 weeks of age, the animals were fed a high-fat diet (HFD; 57 kcal% fat, purchased from CLEA, JAPAN as #HFD32) ad libitum. At 6 weeks of age, the animals were randomly divided into 3 groups (15 animals per group). One group did not receive any treatment, but the other 2 groups were orally administered with vehicle or test substances every day for 4 weeks. After 4 weeks of treatment, all animals were painlessly killed under anesthesia, and the livers were removed and fixed in Bouin's solution for histological studies (H. Denk, "Fixierung histologischer "[Fixing of Histological Preparations],in:P. (Editor): "Romeis Mikroskopische Technik" [Romei's Microscopy Techniques], Urban & Schwarzenberg, Munich-Vienna-Baltimore 1989, 17th edition, page 97, ISBN 3-541-11227-1). Thereafter, liver samples were embedded in paraffin, and 5 μm thick paraffin sections were prepared. Tissue sections of each liver were stained: a) with hematoxylin-eosin (HC) for determination of the NAFLD activity score (NAS) and b) with picro-sirius red (Waldeck, Germany) for determination of liver fibrosis. The NAFLD activity score was determined in the HC sections based on the criteria recommended by DE Kleiner et al., Hepatology 41 (2005), 1313-1321 (Table 1). For histological quantification of fibrotic areas, five digital photographs were taken of each section at 200-fold microscope magnification (DFC280; Leica, Germany), and the percentage of fibrosis was determined using ImageJ Software (National Institutes of Health, USA).

[0642] In vivo db / db mouse model

[0643] 30 8-week-old male db / db mice were used. This model is a widely accepted obesity, insulin resistance and type 2 diabetes model (Aileen JF King; The use of animal models in diabetes research; British Journal of Pharmacology 166 (2012), 877-894). During the experiment, animals were given a standard diet (RM1 (E) 801492, SDS) and free access to tap water. Animals were randomly divided into 3 groups (10 per group) and treated orally with test substances within 6 weeks. During the study, blood was taken from animals at different time points (before treatment started, 3 weeks after treatment started, and 2 days before treatment ended) to measure insulin sensitivity parameters (such as HbA1c, glucose content, insulin content). In addition, OGTT (oral glucose tolerance test), a parameter for determining insulin sensitivity, was performed 1 day before treatment started and 2 days after treatment ended. In addition, the HOMA-IR index (fasting insulin level (mU / L)*fasting blood glucose level (mmol / L) / 22.5) was calculated.

[0644] In vivo B-cell lymphoma-related xenograft models

[0645] The antitumor activity of the compounds of formula (I) of the present invention was studied in a murine xenograft model. For this purpose, female CB-17 SCID mice were subcutaneously implanted with a human B-cell lymphoma cell line, such as TMD-8. In mice with an average tumor size of 20-30 mm 2 In the case of symptomatic treatment, oral monotherapy with a compound of the invention was started, or the compound of the invention was combined with standard therapy, each of which was administered orally. However, animals were randomized in advance. Once the untreated control group had a large tumor, treatment was terminated immediately. Tumor size and body weight were measured three times a week. Weight loss is a measure of treatment-related toxicity (>10% = critical, stop treatment until recovery, >20% = poisoning, terminate). Tumor area [length (mm) × width (mm)] was measured by electronic calipers. At the end of the study, tumor weight was also measured. Antitumor efficacy was defined as the ratio of tumor weight (T / C) of the treatment group to the control group [tumor weight of the treatment group on day x / tumor weight of the control group on day x] or the ratio of tumor area of ​​the treatment group to the control group [tumor area of ​​the treatment group on day x / tumor area of ​​the control group on day x]. Compounds with a T / C greater than 0.5 were defined as active (effective). Statistical analysis was performed using one-way ANOVA and pairwise comparative analysis (Dunnett's test) was performed by comparison with the control group.

[0646] Canine IRAK4 kinase assay

[0647] The IRAK-4 inhibitory activity of the compounds of the invention against canine IRAK4 was determined in the Irak4 TR-FRET assay (TR-FRET = Time Resolved Fluorescence Resonance Energy Transfer) described below.

[0648] A recombinant fusion protein of canine IRAK4 and HIS (polyhistidine) at the N-terminus, expressed in baculovirus-infected insect cells (Hi5, BTI-TN-5B1-4, cell line purchased from Invitrogen, catalog number B855-02) and purified by affinity chromatography, was used as the enzyme. The substrate for the kinase reaction was the biotinylated peptide biotin-Ahx-KKARFSRFAGSSPSQASFAEPG (C-terminus in amide form), which can be purchased, for example, from Biosyntan GmbH (Berlin-Buch).

[0649] For the assay, 11 different concentrations ranging from 20 μM to 0.073 nM were prepared from a 2 mM solution of the test substance in DMSO. 50 nL of the corresponding solution was pipetted into a black, low-volume, 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of IRAK4 in assay buffer [50 mM HEPES, pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w / v) bovine gamma-globulin (BGG), 0.04% (v / v) Nonidet-P40 (Sigma)] was added, and the mixture was incubated for 15 min to allow the substances to bind to the enzyme before the kinase reaction. The kinase reaction is then initiated by adding 3 μL of a solution of adenosine triphosphate (ATP, 1.67 mM = final concentration in the 5 μL assay volume: 1 mM) and the peptide substrate (0.83 μM = final concentration in the 5 μL assay volume: 0.5 μM) in assay buffer, and the resulting mixture is incubated at 22°C for a reaction time of 45 min. The concentration of Irak4 is adjusted to the respective enzyme activity and is set so that the assay is performed within the linear range. Typically, the concentration is in the order of 0.1 nM. The reaction was terminated by adding 5 μL of a solution of TR-FRET detection reagent [0.1 μM streptavidin-XL665 (CisbioBioassays; France, catalog number 610SAXLG)], 1.5 nM anti-phosphoserine antibody [Merck Millipore, “STK Antibody”, catalog number 35-002], and 0.6 nM LANCE EU-W1024-labeled anti-mouse IgG antibody (Perkin-Elmer, product number AD0071; alternatively, terbium cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used) in aqueous EDTA (100 mM EDTA, 0.4% (w / v) bovine serum albumin [BSA] in 25 mM HEPES, pH 7.5).

[0650] The resulting mixture was incubated at 22 ° C for 1 h to form a complex of biotinylated phosphorylated substrate and detection reagent. The amount of phosphorylated substrate was then assessed by measuring the resonance energy transferred from the anti-mouse IgG antibody labeled with europium chelate to streptavidin-XL665. For this purpose, after excitation at 350 nm in a TR-FRET measuring instrument such as Rubystar (BMG Labtechnologies, Offenburg, Germany) or Viewlux (Perkin-Elmer), the fluorescence emission at 620 nm and 665 nm was measured. The emission ratio at 665 nm and 622 nm was used as a measure of the amount of phosphorylated substrate. The data were normalized (enzyme reaction without test substance = 0% inhibition; all other test components but without enzyme = 100% inhibition). Typically, the test substances were tested at 11 different concentrations ranging from 20 μM to 0.073 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 nM and 0.073 nM) on the same microtiter plate. The dilution series was prepared by serial dilution prior to the assay (2 mM to 7.3 nM in 100% DMSO). IC values ​​were calculated by 4-parameter fitting. 50 value.

[0651] Table 3: IC of two experiments of the example compounds in the IRAK4 canine kinase assay 50 value

[0652]

[0653] In vitro lipopolysaccharide (LPS)-induced cytokine production by canine peripheral blood mononuclear cells (PBMC)

[0654] The efficacy of the compounds of formula (I) of the present invention on inducible cytokine production in canine PBMCs was investigated. Here, cytokine production is induced by LPS, a TLR4 ligand, which leads to activation of the IRAK4-mediated signaling pathway.

[0655] Canine PBMCs were obtained from dog anticoagulated whole blood. For this purpose, canine leukocyte-rich plasma was prepared by centrifuging 15 mL of dog blood at 4°C, 400 g for 15 min, followed by collection of the canine PBMC buffy coat and then suspending the canine PBMCs in the plasma. 7 (7) mL of Ficoll-Paque Plus (Fischer Scientific, catalog number 11778538) was pipetted into a centrifuge tube, and then 7 mL of canine leukocyte-rich plasma was layered on top of the Ficoll-Paque Plus. After centrifuging the tube at 400 g for 20 min at 4°C, canine PBMCs were collected from the interface of the canine plasma and Ficoll-Paque Plus. The PBMCs were transferred to a new centrifuge tube and supplemented with Ca-free HCl. 2+ / Mg 2+ Hanks' Balanced Salt Solution 1x (HBSS) (Sigma-Aldrich, catalog number H9394) was added. The cell suspension was centrifuged at 400 g for 5 min at 4°C, and the supernatant was discarded. The cell pellet was then resuspended in 0.2% hypotonic saline to lyse any remaining red blood cells. After 30 seconds, the cell suspension was made isotonic and centrifuged at 400 g for 5 min at 4°C. The cell pellet was then resuspended in Ca-free PBS. 2+ / Mg 2+ The cells were washed a final time in HBSS and centrifuged at 400 g for 5 min at 4° C. PBMCs were then resuspended in complete culture medium (RPMI 1640 GlutaMax (Sigma-Aldrich, catalog number R0883), 10% FCS; 50 U of penicillin, 50 μg / mL of streptomycin (Sigma-Aldrich, catalog number P4333)).

[0656] The experiment was also performed in complete medium. PBMC were cultured at 2.5×10 5The cell density of individual cells / well was seeded onto a 96-well plate. The compound of the present invention was dissolved in DMSO and a series of dilutions were performed in complete medium. The compound examples were used in the test at 8 different concentrations within the range of 3nM to 10μM, so that the final DMSO concentration was 0.0003-0.4%. To induce cytokine secretion, cells were stimulated with 0.1μg / mL of LPS (Sigma-Aldrich, Escherichia coli 0111: B4, catalog number L3024) and kept for 24 hours. Cell viability was determined using 0.2% trypan blue (Sigma-Aldrich, catalog number T8154). According to the manufacturer's instructions, the amount of TNF-α secreted in the cell culture supernatant was determined using dog TNFα DuoSet Elisa (R&D Systems, catalog number DY1507). For example, Example Compound 12 inhibited the generation of TNFα stimulated by dog ​​PBMC by LPS.

[0657] In vitro lipopolysaccharide (LPS)-induced cytokine production by bovine peripheral blood mononuclear cells (PBMC)

[0658] The efficacy of the compounds of formula (I) of the present invention on inducible cytokine production in bovine PBMCs was investigated. Here, cytokine production is induced by LPS, a TLR4 ligand, which leads to activation of the IRAK4-mediated signaling pathway.

[0659] Bovine PBMCs were obtained from bovine anticoagulated whole blood. For this purpose, 500 mL of bovine blood was centrifuged at 1000 g for 20 min at room temperature (RT) to prepare bovine leukocyte-rich plasma, which was subsequently collected and then suspended in an equal volume of PBS / 5 mM EDTA (room temperature). 30 (30) mL of Ficoll-Paque Plus (Fischer Scientific, catalog number 11778538) was pipetted into a Leucosep tube, and then 30 mL of a bovine PBMC buffy coat / PBS / EDTA mixture was layered on top of the Ficoll-Paque Plus. After centrifugation at 800 g for 25 min at room temperature, bovine PBMCs were collected from the interface of the bovine plasma and Ficoll-Paque Plus. The PBMCs were transferred to a new centrifuge tube and supplemented with cold PBS / 5 mM EDTA. The cell suspension was centrifuged at 350 g for 10 min at 4 ° C, and the supernatant was discarded. The cell pellet was then resuspended in 0.2% hypotonic saline to lyse any remaining erythrocytes. After 30 seconds, the cell suspension was made isotonic and centrifuged at 500 g for 5 min at 4°C. The cell pellet was then resuspended in complete culture medium (DMEM with GlutaMAX (ThermoFisher, catalog number 32430100), 10% horse serum ( 30-2040 TM ), 20 μM β-mercaptoethanol (ThermoFisher Cat. No. 31350010 [stock solution: 50 mM]).

[0660] The experiment was also carried out in complete medium. PBMC were cultured at 1×10 6 Cells were seeded onto 24-well plates at a density of 10 cells / well. The compounds of the present invention were dissolved in DMSO and serially diluted in complete medium. Compound Examples were used in the assay at 8 different concentrations ranging from 0.003 μM to 10 μM, resulting in a final DMSO concentration of 0.5%. To induce cytokine secretion, 1 μg / mL ( Figure 8 ) and 0.1 μg / mL ( Figure 9 The cells were stimulated with LPS (LPS from E. coli K12; Invivogen#tlrl-eklps) for 24 hours. Cell viability was determined using Turk solution (Merck Millipore#1092770100).

[0661] The amount of TNFα secreted in the cell culture supernatant of bovine PBMC exposed to LPS was determined using an ELISA readout based on rabbit anti-bovine TNFα antibody. The ELISA assay was performed in a 384-well ELISA plate coated with 10 μL / well of 5 μg / mL rabbit anti-bovine TNFα antibody (BioRad, AHP2383) in 50 mM Na2CO3 / NaHCO3 buffer, pH 9.6, and incubated overnight at 4°C. After removing the antibody and rinsing the wells three times with 50 μL of wash buffer (PBS, 0.05% (v / v) Tween 20), the wells were incubated with 40 μL of blocking buffer (PBS, 0.05% (v / v) Tween 20, 1% (w / v) bovine serum albumin) at 37°C for 90 minutes. Afterwards, the blocking buffer was removed and the culture supernatant samples (20 μL / well) were added. After incubation at 37°C for 90 min, the samples were removed and the wells were rinsed three times with 50 μL of wash buffer. 1 μg / mL of rabbit anti-bovine TNFα biotin-conjugated antibody (BioRad, AHP2383B) in blocking buffer was added to the plate (20 μl / well) incubated for 60 min at 37°C. After removing the biotinylated antibody and rinsing the wells three times with 50 μL of wash buffer, ExtrAvidin diluted 1:10.000 in blocking buffer was added at 37°C. TM -Alkaline phosphatase (Sigma, E2636), 20 μL / well, keep for 1 hour. TM After the wells were washed three times with 50 μL of washing buffer, the enzymatic reaction / color development was initiated by adding 50 μL / well of color development buffer (5 mM p-nitrophenyl phosphate (pNPP) in 50 mM Na2CO3 / NaHCO3, pH 9.6, 2 mM MgCl2). The optical density at a wavelength of 405 nm was recorded. For kinetic measurements, data points were recorded every 5 minutes for 1 hour, and the endpoint measurement was performed after 2 hours. For example, Example Compound 12 inhibited the production of TNFα by bovine PBMC stimulated by LPS. This was due to Figure 8 and Figure 9 illustrate.

[0662] In vitro lipopolysaccharide (LPS)-induced cytokine production by porcine peripheral blood mononuclear cells (PBMC)

[0663] As another example, the efficacy of the compounds of formula (I) of the present invention on inducible cytokine production in porcine PBMCs was investigated. Here, cytokine production was induced by LPS, a TLR4 ligand, which leads to activation of the IRAK4-mediated signaling pathway.

[0664] Porcine PBMCs were obtained from porcine anticoagulated whole blood. For this purpose, 36 mL of porcine blood was centrifuged at 1000 g for 20 min at room temperature (RT) to obtain porcine leukocyte-rich plasma, which was subsequently collected and then suspended in an equal volume of PBS / 5 mM EDTA (room temperature). 30 (30) mL of Ficoll-Paque Plus (Fischer Scientific, catalog number 11778538) was pipetted into a Leucosep tube, and 30 mL of the porcine PBMC buffy coat / PBS / EDTA mixture was layered on top of the Ficoll-Paque Plus. After centrifugation at 800 g for 25 min at room temperature, porcine PBMCs were collected from the interface of the porcine plasma and Ficoll-Paque Plus. The PBMCs were transferred to a new centrifuge tube and supplemented with cold PBS / 5 mM EDTA. The cell suspension was centrifuged at 350 g for 10 min at 4 ° C, and the supernatant was discarded. The cell pellet was then resuspended in 0.2% hypotonic saline to lyse any remaining erythrocytes. After 30 seconds, the cell suspension was made isotonic and centrifuged at 500 g for 5 min at 4°C. The PBMC cell pellet was then resuspended in complete culture medium (DMEM and GlutaMAX (ThermoFisher, catalog number 32430100), 10% horse serum ( 30-2040 TM ), 20 μM β-mercaptoethanol (ThermoFisher Cat. No. 31350010 [stock solution: 50 mM]).

[0665] The experiment was also carried out in complete medium. PBMC were cultured at 1×10 6 Cells were seeded onto 24-well plates at a density of 10 cells / well. The compounds of the invention were dissolved in DMSO and serially diluted in complete medium. The compound examples were used in the assay at 8 different concentrations ranging from 0.003 μM to 10 μM, resulting in a final DMSO concentration of 0.5%. To induce cytokine secretion, cells were stimulated for 24 hours with LPS (LPS from E. coli K12; Invivogen #tlrl-eklps) at concentrations ranging from 0.01 ng / mL to 1 ng / mL. Cell viability was determined using Türk solution (Merck Millipore #1092770100).

[0666] The amount of TNFα secreted in the cell culture supernatant of porcine PBMC exposed to LPS was determined using an ELISA reader based on a rabbit anti-porcine TNFα antibody. The ELISA assay was performed in a 384-well ELISA plate coated with 10 μL / well of 3 μg / mL rabbit anti-porcine TNFα antibody (BioRad, AHP2397) in 50 mM Na2CO3 / NaHCO3 buffer, pH 9.6, for 48 h at 4°C. After removing the antibody and rinsing the wells three times with 50 μL of wash buffer (PBS, 0.05% (v / v) Tween 20), the wells were incubated with 50 μL of blocking buffer (PBS, 0.05% (v / v) Tween 20, 1% (w / v) bovine serum albumin) at 37°C for 60 min. Thereafter, the blocking buffer was removed and the culture supernatant samples (20 μL / well) were added. After incubation at 37°C for 90 min, the samples were removed and the wells were rinsed three times with 50 μL of wash buffer. 0.25 μg / mL of rabbit anti-porcine TNFα biotin-conjugated antibody (BioRad, AHP2397B) in blocking buffer was added to the plate (20 μl / well) incubated at 37°C for 60 min. After removing the biotinylated antibody and rinsing the wells three times with 50 μL of wash buffer, ExtrAvidin diluted 1:10.000 in blocking buffer was added at 37°C. TM -Alkaline phosphatase (Sigma, E2636), 20 μL / well, keep for 1 hour. TM After the wells were washed three times with 50 μL of washing buffer, the enzymatic reaction / color development was initiated by adding 90 μL / well of color development buffer (5 mM p-nitrophenyl phosphate (pNPP) in 50 mM Na2CO3 / NaHCO3, pH 9.6, 2 mM MgCl2). The optical density at a wavelength of 405 nm was recorded. For kinetic measurements, data points were recorded every 5 minutes for 1 hour, and the endpoint measurement was performed after 2 hours. For example, 10 μM of Example Compound 12 inhibited the production of TNFα by bovine PBMCs stimulated with 0.1 ng / mL LPS. This was due to Figure 10 illustrate.

[0667] An in vivo model of house dust mite-induced allergic dermatitis in dogs

[0668] To evaluate the potential anti-allergic / anti-inflammatory efficacy of the compounds of formula (I) of the present invention, a house dust mite sensitization model for Beagle dogs was used. HMD sensitization consisted of a series of subcutaneous injections of HDM antigen (10 μg, Greer Laboratories, Lenoir, NC, USA) and (0.2mL, InvivoGen, SanDiego, CA 921221, USA) was used as an adjuvant. The sensitization process was monitored and confirmed by intradermal skin testing. Once the dog tested positive for HDM intradermal skin testing, one month after the last sensitization, HDM antigen (135μg) was applied topically and inserted into the skin inside the hind legs of adult beagles (with a 2mm long microneedle) to detect the efficacy of the compounds of the present invention on symptoms of allergic dermatitis such as erythema and edema. There were 2 groups of 4 animals in each group: 1 group was a placebo control group and 1 group was treated with Example Compound 12. The control group was treated orally with gelatin capsules containing microcellulose, while the group treated with Example Compound 12 was treated orally with gelatin capsules containing Example Compound 12 and microcellulose. Example Compound 12 or placebo was administered starting 5 days before the HDM antigen attack and continued until 2 days after the attack. The frequency of treatment was once a day, and in the case of Example Compound 12, the dose was 10mg / kg body weight. Erythema and edema were assessed in both groups using VAS (Visual Analog Scale) at 30 min after the start of the challenge and at various time points within 48 h. Plasma samples were analyzed to determine the compound exposure, which correlated with the clinical assessment. Edema and erythema were significantly reduced after treatment with Example Compound 12. This is shown in Tables 4 and 5, as well as Figure 11 and Figure 12 illustrate.

[0669] Table 4: Erythema after treatment with Example Compound 12 and placebo (in VAS units)

[0670]

[0671] Table 5: Edema after treatment with Example Compound 12 and placebo (in VAS units)

[0672]

[0673] In vivo pruritus model of flea allergy dermatitis

[0674] In order to evaluate the potential antipruritic efficacy of the compounds of the general formula (I) of the present invention, a flea allergy dermatitis (FAD) model was used. Only adult dogs with a history of FAD were included in the study. The life cycle of the study consisted of two phases: an itch induction phase (2 weeks) and a subsequent treatment phase (2 weeks). During the two study phases, the dogs were infested with Ctenocephalides fleas twice a week (100 fleas / dog for the first attack and 30 fleas / dog for all subsequent attacks). There were 2 groups of 12 animals each: 1 placebo control group and 1 group treated with Example Compound 12. The control group was treated orally with gelatin capsules containing microcellulose, while the group treated with Example Compound 12 was treated orally with gelatin capsules containing Example Compound 12 and microcellulose. The treatment frequency was once a day, and in the case of Example Compound 12, the dose was 20 mg / kg body weight. Starting from the first day after treatment, every third day, the dogs were recorded for 4 hours, and the time spent on pruritic behavior was measured as the number of seconds spent on scratching, licking, and biting. Plasma samples were analyzed to determine the compound exposure, which was correlated with the clinical evaluation. After 10 days of treatment with Example Compound 12, pruritus was significantly reduced. This is shown in Table 6 and Table 7. Figure 13 illustrate.

[0675] Table 6: Reduction in pruritus relative to baseline following treatment with Example Compound 12 compared to placebo (shown as percent change from baseline on the listed days following treatment)

[0676] Day 4 Day 7 Day 10 Day 13 Placebo control group -12.0％ -9.9％ 1.7％ -20.0％ Example Compound 12 -26.7％ 5.7％ -57.7％ -48.0％

[0677] Figure 1 : Inhibition of IL-23 in DCs generated by human monocytes by Example Compound 12. Data are shown as means with standard deviations.

[0678] Figure 2: Inhibition of INF-α in (A) Imiquimod (R837)- or (B) CpG-A-stimulated human plasmacytoid DCs by Example Compound 12. Data are shown as means with standard deviations.

[0679] Figure 3 : Treatment of LPS-induced inflammation with Example Compound 11 resulted in a decrease in the amount of secreted TNF-α. Data are presented as means with standard deviations.

[0680] Figure 4 : Treatment of IL-1β-induced inflammation with Example Compounds 11 (left) and 12 (right) resulted in a dose-dependent reduction in the amount of secreted TNF-α. Data are shown as means with standard deviations.

[0681] Figure 5: Anti-inflammatory efficacy of Example Compound 11 in an adjuvant-induced rat model of rheumatoid arthritis. Significant and dose-dependent inhibition of rheumatoid arthritis as measured by disease activity score. Data represent mean + standard deviation. One-way ANOVA analysis of variance followed by Dunnett's test for multiple comparisons with control group CFA; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0682] Figure 6 : Anti-inflammatory efficacy of Example Compound 12 in a rheumatoid arthritis animal model (collagen antibody-induced mouse model). Significant and dose-dependent inhibition of rheumatoid arthritis as measured by disease activity score. Data correspond to mean + standard deviation. Statistical significance between the collagen antibody (AK) control group and the treatment group was calculated by one-way ANOVA analysis of variance followed by multiple comparison analysis (Dunnett's test) (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0683] Figure 7 : Inhibition of LPS-induced TNFα production by canine PBMCs by Example Compound 12. Data are shown as means with standard deviations.

[0684] Figure 8 : Dose-dependent inhibition of TNFα production by bovine PBMC induced by 1 μg / mL LPS by Example Compound 12 (kinetic assay). Data shown are means with standard deviations of triplicate biomaterials, each measured twice. The IC50 value determined from this curve is 120 nM.

[0685] Figure 9 : Dose-dependent inhibition of TNFα production by bovine PBMC induced by 1 μg / mL LPS by Example Compound 12 (kinetic assay). Data shown are means with standard deviations of triplicate biomaterials, each measured twice. The IC50 value determined from this curve is 70.5 nM.

[0686] Figure 10 : Inhibition of TNFα production by porcine PBMCs induced by 0.1 ng / mL LPS by Example Compound 12 at 10 μM (kinetic assay) The data presented are the mean values ​​with standard deviations of triplicate biological materials, each measured twice.

[0687] Figure 11 Treatment of a canine allergic dermatitis model induced by house dust mites with Example Compound 12 resulted in a reduction in erythema (a). Data are shown as means with standard deviations.

[0688] Figure 12 : Treatment of a canine allergic dermatitis model induced by house dust mites with Example Compound 12 resulted in a reduction in edema (b). Data are shown as means with standard deviations.

[0689] Figure 13 : Antipruritic efficacy of Example Compound 12 in an animal model of flea allergy dermatitis. Data are expressed as percentage change from baseline relative to the median value.

Claims

1. Use of a compound of the following formula or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing arthritis in non-human mammals:

2. The use according to claim 1, wherein the arthritis is rheumatoid arthritis.

3. The use according to claim 1, wherein the non-human mammal is a dog.

4. The use according to claim 1, wherein the compound is formulated for oral administration.

5. The use according to claim 1, wherein the compound is formulated for administration in the form of a tablet or a capsule.

6. The use according to claim 5, wherein the tablet or capsule is formulated to deliver a dose of about 0.01 to 20 mg / kg body weight of the non-human mammal.

7. The use of claim 1, wherein the compound is formulated to deliver about 10 mg / kg body weight of the compound to the non-human mammal.

8. The use of claim 1, wherein the compound is formulated to deliver about 20 mg / kg body weight of the compound to the non-human mammal.

9. The use according to claim 5, wherein the tablet or capsule comprises at least one inert, non-toxic, pharmaceutically suitable excipient.

10. The use according to any one of claims 1 to 9, wherein the use is therapy.

11. The use according to any one of claims 1 to 9, wherein the use is prophylactic.

12. The use according to claim 10 or 11, wherein the compound is a pharmaceutically acceptable salt.

13. The use according to any one of claims 1 to 12, wherein the compound is formulated for once-daily administration.

14. The use of claim 1, wherein the non-human mammal is a dog, wherein the administration is oral, and wherein the compound is formulated for administration in the form of a tablet or capsule formulated to deliver a dose of about 0.01 to 20 mg / kg body weight of the dog.

15. The use according to claim 14, wherein the compound is formulated for once-daily administration.

16. Use of a compound of the formula below or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing arthritis in non-human mammals:

17. The use according to claim 16, wherein the arthritis is rheumatoid arthritis.

18. The use according to claim 16, wherein the non-human mammal is a dog.

19. The use according to claim 16, wherein the compound is formulated for oral administration.

20. The use according to claim 16, wherein the compound is formulated for administration in the form of a tablet or capsule.

21. The use of claim 20, wherein the tablet or capsule is formulated to deliver a dose of about 0.01 to 100 mg / kg body weight of the non-human mammal.

22. The use according to claim 20, wherein the tablet or capsule comprises at least one inert, non-toxic, pharmaceutically suitable excipient.

23. The use according to any one of claims 16 to 22, wherein the use is therapy.

24. The use according to any one of claims 16 to 22, wherein the use is prophylactic.

25. The use according to claim 23 or 24, wherein the compound is a pharmaceutically acceptable salt.

26. The use according to any one of claims 16 to 25, wherein the compound is formulated for once-daily administration.

Images