Substituted indazoles for the 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 the following diseases in animals: atopic dermatitis, flea allergic dermatitis, inflammatory bowel disease, osteoarthritis pain and inflammatory pain, non-infectious recurrent airway disease, insect hypersensitivity, asthma, respiratory diseases, mastitis and endometritis.

Description

[0001] 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 allergic dermatitis, and in particular in domestic animals, especially dogs.

[0002] The present invention relates to the use of novel substituted indazoles of general formula (I) which inhibit interleukin-1 receptor-associated kinase 4 (IRAK4).

[0003] Human IRAK4 (interleukin-1 receptor-associated kinase 4) plays a key role in activating the immune system. Thus, this kinase is an important therapeutic target molecule for the development of substances that inhibit inflammation. IRAK4 is expressed by many cells and mediates signal transduction of the following receptors: Toll-like receptors (TLRs) (except TLR3), and the interleukin (IL)-1β family of receptors consisting of IL-1R (receptor), IL-18R, IL-33R, and IL-36R (Janeway and Medzhitov, Annu. Rev. Immunol., 2002; Dinarello, Annu. Rev. Immunol., 2009; Flannery and Bowie, Biochemical Pharmacology, 2010).

[0004] Neither mice lacking IRAK4 nor human cells from patients lacking IRAK4 respond to stimulation of 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).

[0005] Binding of TLR ligands or IL-1β family ligands to their respective receptors results in the recruitment and binding of MyD88 [myeloid differentiation primary response gene (88)] to the receptor. Thereby, 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). Thereby, 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 gives rise to 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).

[0006] The central role of IRAK4 in the pathology of various inflammatory diseases has been demonstrated by direct comparison of wild-type (WT) mice with genetically modified animals having a kinase-inactive form of IRAK4 (IRAK4KDKI). In animal models of multiple sclerosis, atherosclerosis, myocardial infarction, and Alzheimer's disease, IRAK4KDKI 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). In addition, it has been found that deletion of IRAK4 in animal models prevents virus-induced myocarditis by improving the antiviral response and simultaneously reducing systemic inflammation (Valaperti, Nishii et al., Circulation, 2013). It has also been shown that the expression of IRAK4 is associated with disease activity in Vogt-Koyanagi-Harada syndrome (Sun, Yang et al., PLoS ONE, 2014). In addition, a high correlation has been shown between IRAK4 and IFNα (interferon-α) production mediated by immune complexes through 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 an implication 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 generated compared to WT mice.Inhibiting 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 diseases, and viral diseases such as HIV (human immunodeficiency virus), hepatitis virus (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).

[0007] Due to the central role of IRAK4 in the MyD88-mediated signaling cascades of TLRs (except TLR3) and the IL-1 receptor family, inhibiting IRAK4 can be used for the prevention and / or treatment of diseases mediated by said receptors.

[0008] The prior art discloses many IRAK4 inhibitors (see, e.g., Annual Reports in Medicinal Chemistry (2014), 49, 117–133).

[0009] US8293923 and US20130274241 disclose IRAK4 inhibitors having a 3-substituted indazole structure. There is no record of 2-substituted indazoles.

[0010] WO2013106254 and WO2011153588 disclose 2,3-disubstituted indazole derivatives.

[0011] WO2007091107 describes 2-substituted indazole derivatives for the treatment of Duchenne muscular dystrophy. The disclosed compounds do not have a 6-hydroxyalkyl substituent.

[0012] WO2015091426 describes indazoles substituted at the 2-position with a carboxamide side chain, such as in Example 64.

[0013]

[0014] Example 64

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

[0016]

[0017] wherein, R2 is an alkyl or cycloalkyl group. In WO2015104662, 2-substituted indazoles having a methyl group, 2-methoxyethyl group and cyclopentyl group at the 2-position are specifically recited (Examples 1, 4 and 76). An indazole derivative having a hydroxyethyl substituent at the 1-position is also recited by Example 117. However, indazole derivatives having a 3-hydroxy-3-methylbutyl substituent at the 1-position or 2-position are not recited.

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

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

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

[0021] Therefore, indazole compounds of the following general formula:

[0022]

[0023] wherein R1 is an optionally substituted alkyl group were first disclosed on January 7, 2015 and thus are 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. Indazoles having an alkyl group substituted with a hydroxyl group at the 6-position are not explicitly described in WO2015104662.

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

[0026] Current treatment regimens for allergic and / or inflammatory diseases in animals, such as allergic skin diseases, typically involve 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), which relieves pruritus symptomatically; however, the dosing regimen may again be limited by side effects. Treating CAD with disease-modifying agents without treatment-related side effects remains an unmet medical need.

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

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

[0029] For example, atopic dermatitis is a common disease in pets, especially 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 and recur throughout their lives. In a 1999 study by Lund et al., 31,484 dogs in 52 private clinics in the United States were investigated, and the prevalence of CAD was 8.7%. CAD is the second most common cause of canine pruritus after flea allergic dermatitis (FAD).

[0031] Canine atopic dermatitis can be defined as "a genetically predisposed inflammatory and pruritic allergic skin disease with characteristic clinical features related to IgE and most commonly directed against environmental allergens" (Halliwell, Veterinary Immunology and Immunopathology, 2006), such as dust mites and pollen. Since dust mites are almost everywhere and pollen is ubiquitous in outdoor air, it is extremely difficult for pets to avoid them. Canine atopic dermatitis is a complex multifactorial disease involving immune dysregulation, allergic hypersensitivity, skin barrier defects, microbial colonization, and environmental factors.

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

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

[0034] Currently, the treatment of acute flares of atopic dermatitis (AD) should include finding and then eliminating the cause of the flare, bathing with a mild bath solution, and taking interventions including topical and / or oral glucocorticoids or oclacitinib to control itching and skin lesions. For chronic CAD, the first step in management is to identify and avoid flare factors, as well as ensure adequate skin and surface hygiene care; this may include more frequent bathing and possibly increased intake of essential fatty acids. The most effective current medications for reducing chronic itching and skin damage are topical and oral glucocorticoids, oral cyclosporine, oral oclacitinib, and, where available, injectable recombinant interferon. Allergen-specific immunotherapy and active intermittent topical glucocorticoid application 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 allergic dermatitis (FAD), or flea bite allergy, is the most common skin disease in pet dogs (Scott et al., In: Muller and Kirk’s Small Animal Dermatology, 2001), caused by the most common flea on dogs and cats to date: Ctenocephalides felis (Beresford-Jones, J Small Animal Practice, 1981; Chesney, Veterinary Record, 1995). Cats can also develop FAD, which is one of the main causes of feline miliary dermatitis.

[0036] FAD is most prevalent in summer, however flea infestations can persist throughout the year in warm climates. In the north temperate zone, the close association of pets and their fleas with human dwellings creates conditions for year-round problems. Extreme temperatures and low humidity tend to inhibit flea development.

[0037] During feeding, fleas inject saliva containing a variety of histamine-like compounds, enzymes, polypeptides, and amino acids with a wide range of sizes (40–60 kD), thus inducing type I, type IV, and basophil hypersensitivities. Flea-pups intermittently exposed to flea bites develop immediate (15 min) or delayed (24 - 48 hours) reactions, or both, and produce detectable levels of circulating IgE and IgG anti-flea antibodies. Dogs continuously exposed to flea bites have low levels of these circulating antibodies and also do not develop skin reactions, or develop them later and to a considerably lower extent. This may indicate that immune tolerance has developed naturally in dogs continuously exposed to flea bites. Although little is known about the pathophysiology of FAD in cats, a similar mechanism may exist.

[0038] The cat flea (Ctenocephalides felis) causes severe irritation to animals and humans and results in flea allergic dermatitis. Typical symptoms are: itching, skin inflammation, and skin lesions (erythema, scaling, papules, crusting, and lichenification). These lesions are most commonly seen on the back and the base of the tail.

[0039] As the condition progresses, hair loss, broken hairs, oozing or crusty sores, pimply bumps, general redness, and skin inflammation may occur. The ulcers can be very painful. In severe cases, the skin thickens and darkens, mainly in the area of the dog's back and the base of the tail. Due to severe itching, the dog may cause self-inflicted injuries.

[0040] Generally, the treatment options selected are for the prevention and treatment of flea infestations. The most common neonicotinoids such as imidacloprid, or GABA-gated chloride channel blockers such as fipronil are used. If the symptoms of allergic dermatitis do not resolve, the current treatments mentioned under CAD are used, such as topical and oral corticosteroids, oral cyclosporine, and oral olaratumab.

[0041] The present invention provides compounds of general formula (I), and their diastereoisomers, enantiomers, metabolites, salts, solvates or solvates of salts, which are useful for the treatment and / or prevention of allergic and / or inflammatory diseases in animals

[0042]

[0043] In formula (I):

[0044] R 1 is a C1-C6-alkyl group, where the C1-C6-alkyl group is unsubstituted or mono-substituted or identically or differently multi-substituted with

[0045] halogen, hydroxy, unsubstituted or mono- or poly-halogen-substituted C3-C6-cycloalkyl, or R 6 、R 7 SO2、R 7 SO or R 8 O group,

[0046] or a group selected from:

[0047]

[0048] where * represents the bonding site of the group to the rest of the molecule;

[0049] R 2 and R 3 always have the same definition and are each hydrogen or C1-C6-alkyl;

[0050] R 4 is halogen, cyano, unsubstituted C1-C6-alkyl or mono-substituted or identically or differently multi-substituted C1-C6-alkyl, or unsubstituted C3-C6-cycloalkyl or mono-substituted or identically or differently multi-substituted C3-C6-cycloalkyl, with substituents selected from halogen and hydroxy;

[0051] R 5 is hydrogen, halogen, or unsubstituted or mono- or poly-halogen-substituted C1-C6-alkyl;

[0052] R 6is an unsubstituted or mono- or di-methyl-substituted monocyclic saturated heterocycle having 4 to 6 ring atoms and containing a heteroatom or hetero-group selected from O, S, SO and SO2;

[0053] R 7 is C1-C6-alkyl, where the C1-C6-alkyl group is unsubstituted or mono-substituted by halogen, hydroxy or C3-C6-cycloalkyl or multiply substituted identically or differently, or R 7 is C3-C6-cycloalkyl;

[0054] R 8 is C1-C6-alkyl, where the C1-C6-alkyl group is unsubstituted or mono-substituted by halogen or multiply substituted identically or differently.

[0055] In the context of the uses described hereinafter of the synthetic intermediates and working examples of the present invention, any compound designated in the form of the corresponding salt of a base or acid is generally a salt of unknown exact stoichiometric composition obtained by the corresponding preparation and / or purification methods. Thus, for such salts, unless otherwise specified in more detail, additional names and structural formulas such as "hydrochloride", "trifluoroacetate", "sodium salt" or "x HCI", "x CF3COOH", "x Na + " should not be understood in a stoichiometric sense but are merely illustrative symbols for the salt-forming components present therein.

[0056] This also applies mutatis mutandis to the case of synthetic intermediates or working examples or salts thereof in the form of solvates such as hydrates of unknown stoichiometric composition (if of the defined type) obtained by the said preparation and / or purification methods.

[0057] The compounds of the present invention are the compounds of formula (I) and their salts, solvates and solvates of salts, the compounds covered by formula (I) and being the compounds of the following structural formula and their salts, solvates and solvates of salts, and the compounds covered by formula (I) and mentioned hereinafter as embodiments and their salts, solvates and solvates of salts, even if the compounds covered by formula (I) and mentioned hereinafter are not salts, solvates and solvates of salts.

[0058] In the context of the present invention, preferred salts are the physiologically acceptable salts of the compounds of the present invention. However, the present disclosure also covers salts which are not themselves suitable for pharmaceutical use but which can be used, for example, for the isolation or purification of the compounds of the present invention.

[0059] Physiologically acceptable salts of the compounds of the present invention include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, such as salts of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

[0060] Physiologically acceptable salts of the compounds of the present invention also include salts of conventional bases, such as and preferably alkali metal salts (such as sodium salts and potassium salts), alkaline earth metal salts (such as calcium salts and magnesium salts), and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, such as and preferably ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.

[0061] In the context of the present invention, solvates are described as those forms of the compounds of the present invention that form solid or liquid complexes by coordination with solvent molecules. Hydrates are a special form of solvates that are coordinated with water.

[0062] Depending on their structure, the compounds of the present invention can exist in different stereoisomeric forms, i.e., in the form of configurational isomers or (where appropriate) conformational isomers (enantiomers and / or diastereomers, including atropisomers). Accordingly, the present invention includes the use of enantiomers and diastereomers and their respective mixtures. Stereoisomerically homogeneous components can be separated from these mixtures of enantiomers and / or diastereomers in a known manner; for this purpose, chromatography, especially HPLC chromatography on a non-chiral or chiral phase, is preferably used.

[0063] If the compounds of the present invention can exist in tautomeric forms, the present invention includes the use of all tautomeric forms.

[0064] The present invention also encompasses the use of all suitable isotopic variants of the compounds of the present invention. As used herein, an isotopic variant of a compound of the present invention is understood to mean a compound in which at least one atom in the compound of the present invention has been replaced by another atom having the same atomic number but a different atomic mass from that which is normally or predominantly present in nature. Examples of isotopes that can be incorporated into the compounds of the present invention are those of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, 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. Particular isotopic variants of the compounds of the present invention, for example, those in which one or more radioactive isotopes have been incorporated, can be useful for investigating the mechanism of action or the distribution of the active ingredient in vivo; due to their relatively easy preparability and detectability, compounds labeled with 3H or 14C isotopes in particular are suitable for this purpose. In addition, the incorporation of an isotope (such as deuterium) can provide particular therapeutic benefits due to the higher metabolic stability of the compound, such as an extended half-life in vivo or a reduced required active dose; thus, in certain cases, such modifications to the compounds of the present invention can also constitute preferred embodiments of the uses 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 the methods further described hereinafter and the methods described in the working examples, by using the corresponding isotopic modifications of the respective reagents and / or starting compounds.

[0065] The present invention also provides the use of all possible crystalline forms and polymorphs of the compounds of the present invention, wherein the polymorphs can exist as a single polymorph or a mixture of multiple polymorphs in all concentration ranges.

[0066] Furthermore, the present invention also encompasses the use of prodrugs of the compounds of the present invention. In this context, the term "prodrug" refers to a compound that can itself be bioactive or inactive, but is converted (e.g., metabolically or hydrolytically) into a compound of the present invention during residence in the body.

[0067] In the context of the present invention, unless otherwise specified, substituents have the following meanings:

[0068] AlkylIn the context of the present invention, it represents a straight-chain 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. Preferred are methyl, ethyl, n-propyl, n-butyl, 2-methylbutyl, 3-methylbutyl, and 2,2-dimethylpropyl.

[0069] Cycloalkyl In the context of the present invention, it is a monocyclic saturated alkyl group having a specified number of carbon atoms in each case. Preferred examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0070] Alkoxy In the context of the present invention, it represents a straight-chain or branched alkoxy group having a specified number of carbon atoms. Preferably 1 to 6 carbon atoms. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, 1-methylpropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentyloxy, isopentyloxy, 1-ethylpropoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, and n-hexyloxy. Particularly preferred are straight-chain or branched alkoxy groups having 1 to 4 carbon atoms. Examples mentioned as preferred are methoxy, ethoxy, n-propoxy, 1-methylpropoxy, n-butoxy, and isobutoxy.

[0071] Halogen In the context of the present invention, it is fluorine, chlorine, and bromine. Preferred is fluorine.

[0072] Hydroxyl In the context of the present invention, it is OH.

[0073] Monocyclic saturated heterocycle It is a monocyclic saturated heterocycle having 4 to 6 ring atoms and containing a heteroatom or heterogroup selected from O, S, SO, and SO2. Preferred are heterocycles having a heteroatom or heterogroup selected from O, SO, and SO2. 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-dioxothietan-2-yl, or 1,1-dioxothietan-3-yl. Particularly preferred herein are oxetane and tetrahydrofuran. Very particularly preferred is oxetan-3-yl.

[0074] The symbol * on the bond represents the bonding site in the molecule.

[0075] Unless otherwise specified, when the groups in the compounds of the present invention are substituted, the groups can be mono-substituted or multi-substituted. In the context of the present invention, all groups that appear more than once are defined independently of each other. Preferably, they are substituted by one, two or three identical or different substituents.

[0076] R 1 A preferred embodiment of is a C2-C6-alkyl group substituted by 1, 2 or 3 fluorine atoms. Particularly preferred are 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl and 4,4,4-trifluorobutyl. Very particularly preferred is the 4,4,4-trifluorobutyl group.

[0077] R 1 Another preferred embodiment of is a C2-C6-alkyl group substituted by one or two hydroxyl groups or a C1-C3-alkoxy or trifluoro-substituted C1-C3-alkoxy group. Particularly preferred are C2-C5-alkyl groups substituted by hydroxyl or C1-C3-alkoxy or trifluoromethoxy 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. Particularly preferred is the 3-hydroxy-3-methylbutyl group.

[0078] Additionally preferably, R 1 is a C2-C6-alkyl group substituted by a C1-C6-alkyl-SO2 group. Particularly preferred is a C2-C4-alkyl substituted by methyl-SO2-. Particularly preferred for R 1 is 2-(methylsulfonyl)ethyl or 3-(methylsulfonyl)propyl. In the latter group, particularly preferred is 2-(methylsulfonyl)ethyl.

[0079] Furthermore, preferably, R 1 is a C1-C3-alkyl group substituted by the following groups: oxetanyl, tetrahydrofuranyl, 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-dioxothietan-2-yl or 1,1-dioxothietan-3-yl. Particularly preferred is a C1-C3-alkyl group substituted by an oxetanyl group. Particularly preferred for R 1 is the oxetan-3-ylmethyl group.

[0080] For R 2 and R 3 , they always have the same definition, preferably hydrogen or methyl. Particularly preferred is methyl.

[0081] For R4 , preferably an unsubstituted or mono- or poly-halogen-substituted C1-C3-alkyl group, or a C1-C3-alkyl group substituted by a hydroxyl group, or a C1-C3-alkyl group substituted by a hydroxyl group and three fluorine atoms.

[0082] For R 4 , the following groups are particularly preferred: methyl, ethyl, trifluoro-C1-C3-alkyl, difluoro-C1-C3-alkyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxy-2-propyl and 2,2,2-trifluoro-1-hydroxyethyl. For R 4 , methyl, trifluoromethyl and difluoromethyl groups are particularly preferred. In this text, the trifluoromethyl group is particularly preferred.

[0083] R 5 The preferred embodiments of are hydrogen, fluorine, chlorine or C1-C3-alkyl. More preferably, R 5 is hydrogen, fluorine or methyl. Most preferably, R 5 is hydrogen or fluorine.

[0084] Compounds in which R 4 is methyl or trifluoromethyl and R 5 is fluorine are also particularly preferred. Compounds in which R 4 is methyl and R 5 is fluorine are very particularly preferred, where R 5 is in the ortho position to R 4 .

[0085] For R 6 , the preferred embodiments include oxetanyl, tetrahydrofuranyl, 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-dioxothietan-2-yl or 1,1-dioxothietan-3-yl. In this text, oxetanyl is particularly preferred. Oxetan-3-yl is very particularly preferred.

[0086] R 7 is only connected to the functional groups -SO2- and -SO-, i.e., an R 7 -substituted -SO2- or SO group. For this, R 7 is preferably C1-C4-alkyl, where the C1-C4-alkyl group is unsubstituted or mono-substituted by a hydroxyl group or a cyclopropyl group or substituted by three fluorine atoms. Additionally, R 7 is preferably a cyclopropyl group. R 7 is particularly preferably methyl, ethyl or hydroxyethyl. R 7 is very particularly preferably methyl.

[0087] This means that, in the case of a C1-C6-alkyl group substituted by R 7 SO2- or R 7 SO-, with respect to R 1 preferably a C1-C6-alkyl group substituted by C1-C6-alkyl-SO2 or C1-C6-alkyl-SO. For R 1 , methylsulfonylethyl and methylsulfonylpropyl are particularly preferred herein. Methylsulfonylethyl is very particularly preferred herein.

[0088] For R 8 , preferably an unsubstituted C1-C4-alkyl group or a trifluoro-substituted 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.

[0089] As previously mentioned, the intracellular enzyme interleukin-1 receptor-associated kinase 4 (IRAK4) plays an indispensable role in the signal transduction pathway of receptors activated by cytokines and TLR ligands, which cytokines and TLR ligands are involved in the inflammatory process. In addition to inflammation, IRAK4 is also involved in the signal transduction of allergic processes. Such allergic processes play an important role in the pathogenesis of allergic skin diseases such as atopic dermatitis.

[0090] 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 associates with MyD88, IRAK4 and TRF6 (Schmitz et al., Immunity, 2005). IRAK4 is an essential component of this signal transduction pathway. IL-33R is strongly expressed in type 2 T helper (Th2) cells, mast cells and eosinophils. IL-33 activates these cells and promotes the immune response of Th2 (Schmitz et al., Immunity, 2005). All of these cell types are involved in the pathogenesis of atopic dermatitis. The content of IL-33 in serum is correlated with the severity of human atopic dermatitis and is reduced upon treatment with topical steroids & calcineurin inhibitors (Tamagawa-Mineoka et al., J American Academy Dermatology, 2014). In a model of acute canine atopic dermatitis, it has been shown that the IL-33 gene is significantly upregulated in skin lesions (Schamber et al., G3 (Bethesda), 2014; Olivry et al., Journal of Investigative Dermatology, 2016).

[0091] 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 childhood atopic dermatitis (Sohn et al., Allergy and Asthma Proceedings, 2004). In a model of acute canine atopic dermatitis, a significant upregulation of the IL-18 gene has been shown in skin lesions (Schamber et al., G3 (Bethesda), 2014; Olivry et al., Journal of Investigative Dermatology, 2016). In addition, atopic dermatitis-like inflammation & itching is caused by the over-release of IL-18 and is accelerated by murine IL-1 (Konishi et al., Proceedings of the National Academy of Sciences, 2002). In addition, IRAK-4 has been shown to be an essential component of the IL-18 signaling cascade (Suzuki et al., J Immunology, 2003). Similarly, IRAK4 is crucial for the signaling of IL-1 and TLR ligands (Suzuki et al., Nature, 2002). TLR antagonists are known to cause itching (Liu et al., Neuroscience bulletin, 2012) - an important symptom of atopic dermatitis, and off-label anti-IL-1 therapy has been used to treat atopic dermatitis. In addition, polymorphisms of IRAK4 are associated with increased total IgE in allergic diseases such as asthma and chronic rhinosinusitis (Tewfik et al., Allergy, 2009). In atopic dermatitis, IgE levels are also elevated.

[0092] Therefore, since IRAK4 is a key part of the signaling pathways activated by many cytokines and TLR ligands, and IRAK4 has polymorphisms 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 allergic dermatitis are both appropriate indications, as both diseases involve type I allergies involving IgE antibodies, Th2 cells, mast cells, and eosinophils. In addition, FAD can include type IV allergies involving IL-1 and IL-18.

[0093] The compounds of the present invention act as IRAK4 kinase inhibitors and thus have an unexpectedly useful pharmacological activity profile in the treatment and / or prevention of allergic and / or inflammatory diseases in animals.

[0094] Compounds of formula (I), and their diastereoisomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prophylaxis of allergic and / or inflammatory diseases in animals, in formula (I)

[0095] R 1 is C1-C6-alkyl, where the C1-C6-alkyl group is unsubstituted or mono- or polysubstituted, identically or differently, by fluorine, hydroxy, or R 6 、R 7 SO2, R 7 SO or R 8 O groups;

[0096] R 2 and R 3 always have the same definition and are each hydrogen or C1-C3-alkyl;

[0097] R 4 is halogen, cyano or C1-C3-alkyl, where the C1-C3-alkyl group is unsubstituted or mono-substituted, identically or differently, by halogen or hydroxy, or polysubstituted;

[0098] R 5 is hydrogen, fluorine, chlorine or C1-C3-alkyl;

[0099] R 6 is oxetanyl or tetrahydrofuranyl;

[0100] R 7 is C1-C4-alkyl, where the C1-C4-alkyl group is unsubstituted, or mono-substituted by hydroxy or cyclopropyl, or trisubstituted by three fluorine atoms;

[0101] R 8 is unsubstituted C1-C4-alkyl or trifluoro-substituted C1-C4-alkyl.

[0102] Also preferred are compounds of formula (I), and their diastereoisomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prophylaxis of allergic and / or inflammatory diseases in animals, in formula (I)

[0103] R 1 is C2-C6-alkyl, where the C2-C6-alkyl is unsubstituted, or

[0104] the C2-C6-alkyl is mono-, di- or tri-fluoro-substituted, or

[0105] the C2-C6-alkyl is mono-substituted by hydroxy, R 6 、R 7 SO2 or R 8 O;

[0106] or in which R 1 is an oxetanyl-substituted C1-C3-alkyl;

[0107] R 2 and R 3 always have the same definition and are each hydrogen or methyl;

[0108] R 4 is unsubstituted or mono- or poly-halogen-substituted C1-C3-alkyl, or a C1-C3-alkyl group substituted by one hydroxy group, or a C1-C3-alkyl group substituted by one hydroxy group and three fluorine atoms;

[0109] R 5 is hydrogen, fluorine or C1-C3-alkyl;

[0110] R 7 is C1-C3-alkyl;

[0111] R 8 is C1-C4-alkyl, where the C1-C4-alkyl group is unsubstituted or mono-, di- or tri-fluorosubstituted.

[0112] Particularly preferred are also compounds of the general formula (I), and their diastereoisomers, enantiomers, metabolites, salts, solvates or solvates of salts, which are used for the treatment and / or prophylaxis of allergic and / or inflammatory diseases in animals, in the general formula (I)

[0113] 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

[0114] is a methyl-SO2-substituted C2-C4-alkyl group, or

[0115] is an oxetan-3-yl-substituted C1-C2-alkyl group;

[0116] R 2 and R 3 always have the same definition and are each hydrogen or methyl;

[0117] R 4 is methyl, ethyl, trifluoro-C1-C3-alkyl, difluoro-C1-C3-alkyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxypropan-2-yl and 2,2,2-trifluoro-1-hydroxyethyl, and

[0118] R 5 is hydrogen, fluorine or methyl.

[0119] Very particularly preferred are the following compounds, and their diastereoisomers, enantiomers, metabolites, salts, solvates or solvates of salts, which are used for the treatment and / or prevention of allergic and / or inflammatory diseases in animals, wherein

[0120] 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;

[0121] R 2 and R 3 are both methyl or hydrogen, and

[0122] R 4 is difluoromethyl, trifluoromethyl or methyl, and

[0123] R 5 is hydrogen or fluorine.

[0124] Also very particularly preferred are the following compounds, and their diastereoisomers, enantiomers, metabolites, salts, solvates or solvates of salts, which are used for the treatment and / or prevention of allergic and / or inflammatory diseases in animals, wherein

[0125] R 1 is 3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-(methylsulfonyl)propyl or 2-(methylsulfonyl)ethyl;

[0126] R 2 and R 3 are both methyl;

[0127] R 4 is difluoromethyl or trifluoromethyl; and

[0128] R 5 is hydrogen.

[0129] Also particularly preferred are the following compounds, and their diastereoisomers, enantiomers, metabolites, salts, solvates or solvates of salts, which are used for the treatment and / or prevention of allergic and / or inflammatory diseases in animals, wherein

[0130] R 1 is 3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-(methylsulfonyl)propyl or 2-(methylsulfonyl)ethyl;

[0131] R2 and R 3 are both methyl;

[0132] R 4 is methyl, and

[0133] R 5 is fluorine, where R 5 is in the ortho position to R 4 .

[0134] The present invention particularly provides the following compounds, which are used for treating and / or preventing allergic and / or inflammatory diseases in animals:

[0135] 1) N-[6-(2-Hydroxypropan-2-yl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0136] 2) N-[6-(Hydroxymethyl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0137] 3) N-[6-(2-Hydroxypropan-2-yl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0138] 4) N-[6-(Hydroxymethyl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0139] 5) N-[2-(2-Hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0140] 6) N-[6-(2-Hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0141] 7) N-[2-(2-Hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0142] 8) N-[6-(2-Hydroxypropan-2-yl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0143] 9) N-[6-(Hydroxymethyl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0144] 10) N-{6-(2-Hydroxypropan-2-yl)-2-[3-(methylsulfonyl)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0145] 11) N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0146] 12) N-{6-(2-Hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0147] 13) 6-(Difluoromethyl)-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0148] 14) 6-(Difluoromethyl)-N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}pyridine-2-carboxamide

[0149] 15) 6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0150] 16) N-[6-(2-Hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0151] 17) N-{6-(2-Hydroxypropan-2-yl)-2-[3-(trifluoromethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0152] 18) N-{6-(2-Hydroxypropan-2-yl)-2-[3-(2,2,2-trifluoroethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0153] 19) 5-Fluoro-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0154] 20) N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0155] 21) 6-(2-Hydroxypropan-2-yl)-N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0156] 22) N-{2-[2-(1-Hydroxycyclopropyl)ethyl]-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide.

[0157] The present invention also provides compounds of general formula (III), and their diastereoisomers, enantiomers, metabolites, salts, solvates or solvate salts thereof, which are used for the treatment and / or prevention of allergic and / or inflammatory diseases in animals

[0158]

[0159] In general formula (III)

[0160] 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;

[0161] R 4 is difluoromethyl, trifluoromethyl or methyl; and

[0162] R 5 is hydrogen or fluorine.

[0163] Particularly preferred are the compounds of general formula (III) below, which are used for the treatment and / or prevention of allergic and / or inflammatory diseases in animals:

[0164] Methyl 5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate and

[0165] Methyl 2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate.

[0166] The compounds of general formula (III) are suitable for the preparation of part of the compounds of general formula (I).

[0167] In addition, the compounds of general formula (III) are inhibitors of interleukin-1 receptor-associated kinase-4 (IRAK4).

[0168] The compounds of formula (III) can be prepared from the compounds of formula (II) by reacting (II) with a suitably substituted alkyl halide or alkyl 4-methylbenzenesulfonate in the presence of potassium carbonate,

[0169]

[0170] wherein

[0171] R 1 is 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)ethyl, 3-(methylsulfonyl)propyl or 2-(1-hydroxycyclopropyl)ethyl;

[0172] R 4 is difluoromethyl, trifluoromethyl or methyl; and

[0173] R 5 is hydrogen or fluorine.

[0174] In addition, the compounds of formula (I) can be prepared from the compounds of formula (III) by a Grignard reaction with methylmagnesium bromide

[0175]

[0176] wherein

[0177] 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, 3-(methylsulfonyl)propyl, 2-(1-hydroxycyclopropyl)ethyl;

[0178] R 2 and R 3 is methyl;

[0179] R 4 is difluoromethyl, trifluoromethyl or methyl; and

[0180] R 5 is hydrogen or fluorine.

[0181] The compounds of the invention act as IRAK4 kinase inhibitors and have an unexpectedly useful pharmacological activity profile.

[0182] 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, especially cats and dogs, and more especially dogs.

[0183] In this context, the term "domestic animal" includes, for example, mammals such as hamsters, guinea pigs, rats, mice, chinchillas, ferrets, or especially dogs, cats; caged birds; reptiles; amphibians or ornamental fish.

[0184] 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 dermatitis in domestic animals, especially canine and feline allergic dermatitis, and more especially canine allergic dermatitis. 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 farm animals, especially sheep, goats, horses, cattle and pigs, and more especially cattle and pigs.

[0185] In this context, the term "farm animal" includes, for example, mammals such as horses, sheep, goats, water buffalo, reindeer, fawns or more especially cattle or pigs.

[0186] It is further preferred to use the compounds of formula (I), or the compounds specifically mentioned above, in methods for the treatment and / or prevention of the following diseases in animals: atopic dermatitis, flea allergic dermatitis, inflammatory bowel disease, osteoarthritis pain and inflammatory pain, non-infectious recurrent airway disease, insect allergy, asthma, respiratory diseases, mastitis and endometritis, especially atopic dermatitis and flea allergic dermatitis.

[0187] It is particularly preferred to use the compounds of formula (I), or the compounds specifically mentioned above, in methods for the treatment and / or prevention of the following diseases: canine atopic dermatitis and flea allergic 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.

[0188] It is very particularly preferred to use the compounds of formula (I) or the compounds specifically mentioned above in methods for the treatment and / or prevention of canine atopic dermatitis and flea allergic dermatitis in dogs or cats, more particularly dogs.

[0189] It is also very particularly preferred to use the compounds of formula (I) or the compounds specifically mentioned above in methods for the treatment and / or prevention of osteoarthritis pain and inflammatory pain in cattle, bovine respiratory disease, bovine mastitis, bovine endometritis and porcine respiratory disease.

[0190] Regarding the compounds of formula (III), it is further preferred to use the compounds of formula (III) for the treatment and / or prevention of allergic and / or inflammatory diseases in domestic animals, especially cats and dogs, and more particularly dogs.

[0191] It is further preferred to use the compounds of formula (III) for the treatment and / or prevention of allergic dermatitis in domestic animals, especially canine and feline allergic dermatitis, and more particularly canine allergic dermatitis.

[0192] It is further preferred to use the compounds of formula (III) for the treatment and / or prevention of allergic and / or inflammatory diseases in farm animals, especially sheep, goats, horses, cattle and pigs, and more particularly cattle and pigs.

[0193] It is further preferred to use the compounds of formula (III) in a method for the treatment and / or prevention of the following diseases in animals: atopic dermatitis, flea allergic dermatitis, inflammatory bowel disease, osteoarthritis pain and inflammatory pain, non-infectious recurrent airway disease, insect allergy, asthma, respiratory diseases, mastitis and endometritis, especially atopic dermatitis and flea allergic dermatitis.

[0194] It is particularly preferred to use the compounds of formula (III) in a method for the treatment and / or prevention of the following diseases: canine atopic dermatitis and flea allergic 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 diseases; bovine mastitis; bovine endometritis and porcine respiratory diseases.

[0195] It is very particularly preferred to use the compounds of formula (III) in a method for the treatment and / or prevention of canine atopic dermatitis and flea allergic dermatitis in dogs or cats, more particularly dogs.

[0196] It is also very particularly preferred to use the compounds of formula (III) in a method for the treatment and / or prevention of osteoarthritis pain and inflammatory pain, bovine respiratory diseases, bovine mastitis, bovine endometritis and porcine respiratory diseases in cattle.

[0197] For example, Compound Examples 11, 12, 13, 19 (shown below) have been evaluated using recombinant canine IRAK4 enzyme in the in vitro IRAK4 TR-FRET assay detailed below. The IC50 values for the inhibition of canine IRAK4 have been calculated for each compound. It has been confirmed that the exemplified compounds (11, 12, 13, 19) are useful for treating allergic skin diseases in animals, particularly dogs and cats, such as atopic dermatitis and flea allergic dermatitis. Compound Examples 11, 12, 13, 19 are effective 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 value of each of these exemplified compounds is similar to the calculated IC50 value.

[0198] As another example, an in vitro assay was also performed on Compound Example 12 to determine the efficacy of the compound on lipopolysaccharide (LPS)-induced cytokine production by canine peripheral blood mononuclear cells (PBMC). Compound Example 12 inhibited the production of the pro-inflammatory cytokine tumor necrosis factor α (TNFα) in a concentration-dependent manner by LPS-stimulated canine PBMC. PBMC include cell types such as dendritic cells, T lymphocytes, and B lymphocytes, as well as monocytes, each of which is associated with atopic dermatitis and in which TNFα is elevated in patients with atopic dermatitis (Sumimoto et al., Archives of Disease in Childhood, 1992). This example is also illustrated by Figure 7 description.

[0199] Thus, the compounds of the present invention show inhibition of canine PBMC on recombinant canine IRAK4 enzyme and cytokine production, indicating potential therapeutic benefits of these compound examples in canine atopic dermatitis and flea allergic dermatitis.

[0200] Furthermore, in an indoor dust mite model, in a further study, an in vivo evaluation was performed on Compound Example 12 to determine the efficacy of the compound in treating clinical symptoms associated with canine allergic dermatitis, particularly canine atopic dermatitis (CAD). Compound Example 12 significantly alleviated the clinical symptoms of CAD-like skin edema and erythema. This example is also illustrated by Figure 11 and Figure 12 description.

[0201] Accordingly, the compounds of the present invention show a reduction in the characteristic clinical symptoms of canine allergic dermatitis, thus 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 allergic dermatitis (CAD) to determine the anti-itch efficacy of the compound. Subsequent treatment with Example Compound 12 significantly reduced the itch associated with allergic diseases such as flea allergic dermatitis. This example is also illustrated by Figure 13 description.

[0202] Accordingly, the compounds of the present invention show a reduction in the characteristic clinical symptoms associated with allergic dermatitis such as dermatitis and itch, thus demonstrating the therapeutic benefit of such compound examples in canine allergic dermatitis, particularly flea allergic dermatitis (FAD) and canine atopic dermatitis (CAD).

[0203] In this context, the term "canine allergic dermatitis" particularly includes canine atopic dermatitis (CAD) and flea allergic dermatitis (FAD).

[0204] As another example, Example Compound 12 has also been evaluated in ex vivo assays to determine the efficacy of the compound on lipopolysaccharide (LPS)-induced cytokine production by bovine peripheral blood mononuclear cells (PBMC). Example Compound 12 inhibited the production of the pro-inflammatory cytokine tumor necrosis factor α (TNFα) in a concentration-dependent manner by LPS-stimulated canine PBMC. PBMC include cell types such as dendritic cells, T lymphocytes, and B lymphocytes, as well as monocytes, each of which is associated with inflammatory and infectious diseases with an overactive pro-inflammatory immune response 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 which TNFα is elevated in patients with these diseases. This example is also illustrated by Figure 8 and 9 description.

[0205] Accordingly, the compounds of the present invention show inhibition of cytokine production by bovine PBMC, demonstrating the possible therapeutic benefit of such compound examples in inflammatory and / or infectious diseases such as respiratory diseases, intestinal diseases, and mastitis.

[0206] As another example, in vitro assays have also been performed on Example Compound 12 to determine the efficacy of the compound on lipopolysaccharide (LPS)-induced cytokine production by porcine peripheral blood mononuclear cells (PBMC). Example Compound 12 inhibits the production of the pro-inflammatory cytokine tumor necrosis factor α (TNFα) by LPS-induced porcine PBMC. PBMC include cell types such as dendritic cells, T lymphocytes, and B lymphocytes, as well as monocytes, each of which is associated with inflammatory and infectious diseases with an overactive pro-inflammatory immune response, such as respiratory and intestinal diseases, in which TNFα is elevated in patients with these diseases. This example is also illustrated by Figure 10 description.

[0207] Accordingly, the present compounds show inhibition of cytokine production by porcine PBMC, indicating possible therapeutic benefits of such compound examples in inflammatory and / or infectious diseases such as respiratory and intestinal diseases.

[0208] The compounds of the present invention also provide prophylaxis and / or treatment of pruritus and pain in animals, particularly acute, chronic, inflammatory, and neuropathic pain in animals.

[0209] In addition, the compounds of the present invention are suitable for treating and / or preventing pain diseases in animals, particularly acute, chronic, inflammatory, and neuropathic pain. The diseases preferably include hyperalgesia, allodynia, pain of arthritis (such as osteoarthritis, rheumatoid arthritis, and spondyloarthritis), premenstrual pain, endometriosis-associated 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-associated pain, HIV-treatment-induced neuropathy, pain caused by burns, and chronic pain.

[0210] The present invention also provides a method of treating and / or preventing diseases in animals, particularly the above-mentioned diseases, using an effective amount of at least one compound of the present invention.

[0211] Preferably provided is a method of treating and / or preventing allergic and / or inflammatory diseases in animals by administering an effective amount of at least one compound of formula (I) of the present invention as defined above to an animal in need thereof.

[0212] In the context of the present invention, the term "treatment" or "treating" includes inhibiting, delaying, checking, alleviating, reducing, limiting, decreasing, suppressing, combating or curing a disease, disorder, condition, 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 as being synonymous with the term "treatment".

[0213] 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, undergoing, suffering from or having a disease, disorder, condition, injury or health problem or the development or evolution of said condition and / or the symptoms of said condition.

[0214] A disease, disorder, condition, injury or health problem may be treated or prevented in part or in whole.

[0215] 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 treating and / or preventing allergic and / or inflammatory diseases in animals. Preferred examples of active ingredients suitable for use in the compositions include:

[0216] Active ingredients may generally be mentioned 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 γ-globulin; immunomodulatory and immunosuppressive compounds such as cyclosporin, TNF antagonists (e.g. Etanercept, Infliximab, IL-1 inhibitors (such as Anakinra, Canakinumab, Rilonacept), phosphodiesterase inhibitors (such as Apremilast), Jak / STAT inhibitors (such as Tofacitinib, Baricitinib, GLPG0634), leflunomid, cyclophosphamide, rituximab, belimumab, tacrolimus, rapamycin, mycophenolate mofetil, interferon, corticosteroids (such as prednisone, prednisolone, methylprednisolone, hydrocortisone, betamethasone), cyclophosphamide, azathioprine, and sulfasalazine; paracetamol, non-steroidal anti-inflammatory substances (NSAIDs) (aspirin, ibuprofen, naproxen, etodolac, celecoxib, colchicine).

[0217] In addition to those described above, the IRAK4 inhibitors of the present invention may also be combined with the following active ingredients:

[0218] Substances for treating lung diseases, such as β-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, cortisol-containing preparations; substances for treating osteoarthritis, such as non-steroidal anti-inflammatory substances (NSAIDs). In addition to the two aforementioned therapies, for rheumatic diseases, such as rheumatoid arthritis, spondyloarthritis and juvenile idiopathic arthritis, mention should be made of methotrexate and biological agents (e.g., rituximab, abatacept) for B-cell and T-cell therapies. Neurotrophic substances such as acetylcholinesterase inhibitors (e.g., donepezil), MAO (monoaminooxidase) inhibitors (e.g., selegiline), interferons and anticonvulsives (e.g., gabapentin); active ingredients for treating cardiovascular diseases, such as β-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 (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 diseases, such as mesalazine, sulfasalazine, azathioprine, 6-mercaptopurine or methotrexate, probiotics (Mutaflor,; Lactobacillus rhamnosus (Lactobacillus GG), Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium infantis 35624, Enterococcus faecium SF68, Bifidobacterium longum, Escherichia coli Nissle 1917); antibiotics such as ciprofloxacin and metronidazole; antidiarrheal agents such as loperamide; or laxatives (bisacodyl). Immunosuppressants for the treatment of lupus erythematosus such as glucocorticoids and non-steroidal anti-inflammatory substances (NSAIDs), cortisone, chloroquine, cyclosporine, azathioprine, belimumab, rituximab, cyclophosphamide. For skin diseases, vitamin D3 analogs such as calcipotriol, tacalcitol or calcitriol, salicylic acid, urea, ciclosporine, methotrexate, efalizumab.

[0219] Also to be mentioned are medicaments for the treatment and / or prophylaxis of the above-mentioned diseases, which medicaments comprise at least one compound of the invention and one or more other active ingredients for the uses according to the invention, in particular EP4 inhibitors (prostaglandin E2 receptor 4 inhibitors), P2X3 inhibitors (P2X purinoceptor 3), PTGES inhibitors (prostaglandin E synthase inhibitors) or AKR1C3 inhibitors (aldo-keto reductase family 1 member C3 inhibitors).

[0220] The compounds of the 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, dermal, transdermal or conjunctival routes, via the ear or as implants or stents.

[0221] The compounds of the invention can be administered in dosage forms suitable for these administration routes.

[0222] Suitable dosage forms for oral administration are those which act according to the prior art and release the compounds of the invention rapidly and / or in a modified manner and which contain the compounds of the invention in crystalline and / or amorphous and / or dissolved form, such as, for example, tablets (uncoated or coated tablets, the coated tablets having, for example, a gastric juice-resistant coating or a delayed dissolution coating or an insoluble coating which controls the release of the compounds of the invention), tablets or wafers / dragees which disintegrate rapidly in the mouth, wafers / freezedried preparations, capsules (such as, for example, hard or soft gelatin capsules), dragees, chewables (such as, for example, soft chewables), granules, pills, powders, emulsions, suspensions, aerosols or solutions.

[0223] Parenteral administration can be effected by bypassing the resorption step (for example by the intravenous, intra-arterial, intracardiac, intraspinal or intralumbar route) or by including absorption (for example the intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal route). Dosage forms suitable for parenteral administration include injectable and infusion preparations in the form of solutions, suspensions, emulsions, freeze-dried preparations or sterile powders.

[0224] For other routes of administration, suitable examples are inhalable pharmaceutical forms (including powder inhalers, sprays), nasal drops, nasal solutions or nasal sprays; tablets, wafers / dragees or capsules for administration via the tongue, sublingually or orally; suppositories; ear or eye preparations, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, pour-ons, transdermal therapeutic systems (such as patches), milks, pastes, foams, sprinkling powders, implants or stents.

[0225] Oral administration or parenteral administration is preferred, especially oral administration.

[0226] The compounds of the invention can be converted into the dosage forms mentioned. This can be effected in a manner known per se by mixing with inert, non-toxic, pharmaceutically suitable excipients. These excipients include carriers (such as, for example, microcrystalline cellulose, lactose, mannitol), solvents (such as, for example, liquid polyethylene glycol), emulsifiers and dispersants or wetting agents (such as, for example, sodium lauryl sulphate, polyoxy sorbitan oleate), binders (such as, for example, polyvinylpyrrolidone), synthetic and natural polymers (such as, for example, albumin), stabilizers (such as, for example, antioxidants, such as ascorbic acid), colorants (such as, for example, inorganic pigments, such as iron oxide) and flavor correctants and / or odour correctants.

[0227] The present invention also provides a medicament comprising at least one compound of the present invention, usually further comprising one or more inert, non-toxic, pharmaceutically acceptable excipients, for use in a method for treating and / or preventing allergic and / or inflammatory diseases in animals.

[0228] 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 an effective result. In the case of oral administration, the dose 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.

[0229] However, in certain cases, it may be necessary to deviate from the specified amount, in particular depending on body weight, route of administration, individual response to the active ingredient, nature of the formulation and time or interval at which administration takes place. Thus, in certain cases, it may be sufficient to administer an amount less than the above minimum amount, while in other cases, the upper limit mentioned must be exceeded. In the case of larger amounts of administration, it is recommended to divide them into several separate doses within a day.

[0230] The following working examples illustrate the present invention. The present invention is not limited to the described examples.

[0231] Unless otherwise stated, percentages in the following tests and examples are by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentration data for liquid / liquid solutions are based on volume in each case.

[0232] Preparation of the compound

[0233] The preparation of the compounds of the present invention is illustrated by the following synthetic schemes.

[0234] The starting materials for synthesizing the compounds of the present invention are carboxylic acids (Intermediate V3), which are commercially available or can be prepared by methods known from the literature or similar to those 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, Synthetic 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 an acid 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 optionally be prepared from Intermediate V1 containing chlorine, bromine or iodine as substituent X1 by adding ethanol or methanol in a solvent (such as dimethyl sulfoxide) in the presence of a carbon monoxide atmosphere, optionally under high pressure, in the presence of a phosphine ligand (such as 1,3-bis(diphenylphosphino)propane), a palladium compound (such as palladium(II) acetate) and a base (such as triethylamine) (for the preparation method, see, for example, WO2012112743, WO 2005082866, Chemical Communications (Cambridge, England), 2003, 15, 1948–1949, WO200661715). Intermediate V1 is commercially available or can be prepared by methods known from the literature. Exemplary preparation methods are described in detail in the following documents: WO2012061926; 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.

[0235]

[0236] Synthesis Scheme 1

[0237] X 1 is chlorine, bromine or iodine.

[0238] R d is methyl, ethyl, benzyl or tert-butyl.

[0239] R 4 and R 5 are each as defined in general formula (I).

[0240] Similar to WO 2008 / 001883, methyl 5-amino-1H-indazole-6-carboxylate (Intermediate 2) can be prepared according to Synthesis Scheme 2 starting from methyl 1H-indazole-6-carboxylate (Intermediate 0) by nitration and reduction of the nitro group of Intermediate 1 with hydrogen in the presence of palladium on carbon. 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 combined with the following 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 125700-67-6), (dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaniminium hexafluorophosphate (HATU, CAS 148893-10-1), propanephosphonic anhydride (in the form of an ethyl acetate solution or a DMF solution, CAS 68957-94-8) or di-1H-imidazol-1-ylmethanone (CDI), and in each case a base such as triethylamine or N-ethyl-N-isopropylpropan-2-amine is added to the reaction mixture. Preferably, TBTU and N-ethyl-N-isopropylpropan-2-amine in THF are used.

[0241]

[0242] Synthesis Scheme 2

[0243] Substituent R 4 and R 5 are each as defined in general formula (I).

[0244] Starting from intermediate 3, 2-substituted indazole derivatives (intermediate 4) can be prepared (see Synthesis Scheme 3). For this purpose, useful reactions include those with optionally substituted alkyl chlorides, alkyl bromides, alkyl iodides or alkyl 4-methylbenzenesulfonates. The alkyl halides or alkyl 4-methylbenzenesulfonates used are commercially available or can be prepared analogously to known routes from the literature (for the preparation of alkyl 4-methylbenzenesulfonates, one example 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, in the case of using alkyl chlorides or alkyl bromides, an alkali metal iodide 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-methylpyrrolidin-2-one, DMF, DMSO or THF. Optionally, the alkyl halides or alkyl 4-methylbenzenesulfonates used may have functional groups that are optionally pre-protected with protecting groups (see also P.G.M. Wuts, T.W.G. Greene, Greene’s Protective Groups in Organic Synthesis, Fourth Edition, ISBN: 9780471697541). For example, if an alkyl halide or alkyl 4-methylbenzenesulfonate having one or more hydroxyl groups is used, these hydroxyl groups can be optionally protected with tert-butyl(dimethyl)silyl or similar silicon-containing protecting groups familiar to those skilled in the art. Alternatively, the hydroxyl groups can also be protected with a tetrahydro-2H-pyran (THP) group or an acetyl or benzoyl group. Then, the protecting groups used can be removed after the synthesis of intermediate 4 or after the synthesis of (I). For example, if the tert-butyl(dimethyl)silyl group is used as a protecting group, it can be removed with tetrabutylammonium fluoride in a solvent such as THF. For example, the THP protecting group can be removed with 4-methylbenzenesulfonic acid (optionally in the form of the monohydrate). The acetyl or benzoyl group can be removed by treatment with an aqueous sodium hydroxide solution.

[0245] Optionally, the alkyl halide or alkyl 4-methylbenzenesulfonate used may contain functional groups that can be transformed by oxidation or reduction reactions known to those skilled in the art (see, for example, Science of Synthesis, Georg Thieme Verlag). For example, if the functional group is a sulphide group, it can be oxidized to a sulfinyl or sulfonyl group by methods known in the literature. In the case of a sulfinyl group, it can in turn be oxidized to a sulfonyl group. For these oxidation steps, for example, 3-chloroperbenzoic acid (CAS 937-14-4) can be used (for which, also see, for example, US201094000, where 2-(methylthio)ethyl-1H-pyrazole derivatives are oxidized to 2-(methylsulfinyl)ethyl-1H-pyrazole derivatives, and additionally 2-(methylthio)ethyl-1H-pyrazole derivatives are oxidized to 2-(methylsulfonyl)ethyl-1H-pyrazole derivatives). If the alkyl halide or tosylate used contains a keto group, it can be reduced to an alcohol group by reduction methods known to those skilled in the art (see, for example, the use of sodium borohydride in Chemische Berichte, 1980, 113, 1907–1920). These oxidation or reduction steps can be carried out after the synthesis of intermediate 4 or after the synthesis of the compounds 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, K.C.K. 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 (K.C.K. Swamy et al., Chem. Rev. 2009, 109, 2551–2651). Triphenylphosphine and diisopropyl azodicarboxylate are preferably used. If the alkyl alcohol bears a functional group, a known protecting group strategy can be carried out - as in the case of the reaction with the alkyl halide above - (for other indications, see P.G.M. Wuts, T.W.G. Greene, Greene’s Protective Groups in Organic Synthesis, 4th Edition, ISBN: 9780471697541), and - as in the case of the reaction with the alkyl halide above - the oxidation and reduction steps can be carried out accordingly after the synthesis of intermediate 4 or after the synthesis of the compounds of general formula (I) of the present invention. Compounds of general formula (I) of the present invention, where R 2 and R 3 are defined as C1-C6-alkyl (where R 2 and R 3(having the same definition) can be obtained from Intermediate 4 by a Grignard reaction (see, for example, the reaction of a methyl 1H-indazole-6-carboxylate derivative with methylmagnesium bromide in EP 2489663). For the Grignard reaction, an alkylmagnesium halide can be used. Methylmagnesium chloride or methylmagnesium bromide in THF or diethyl ether or in a mixture of THF and diethyl ether is particularly preferred. Alternatively, a compound of general formula (I) according to the invention, wherein R 2 and R 3 are defined as C1-C6-alkyl (wherein 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 a methyl 2-amino-4-chloro-1-methyl-1H-benzimidazole-7-carboxylate derivative with isopropyllithium or tert-butyllithium in WO2006116412). Starting from Intermediate 4, a compound of general formula (I) according to the invention wherein R 2 and R 3 are defined as H can be prepared by reduction with lithium aluminum hydride in THF, lithium borohydride in THF or sodium borohydride in THF (optionally with addition of methanol), or a mixture of lithium borohydride and sodium borohydride.

[0246]

[0247] Synthesis Scheme 3

[0248] The substituents R 1 、R 2 、R 3 、R 4 、R 5 are each as defined in general formula (I).

[0249] Intermediate 5, wherein R 2 and R 3 are defined as C1-C6-alkyl (wherein R 2 and R 3 have the same definition), can be obtained from Intermediate 3 by a Grignard reaction (see, for example, Synthesis Scheme 4). For this purpose, a suitable alkylmagnesium halide can be used, such as methylmagnesium chloride or methylmagnesium bromide in THF or diethyl ether or in a mixture of THF and diethyl ether.

[0250] Then, a part (I-a) of the compound (I) according to the invention can be prepared from Intermediate 5, wherein R 2 and R 3 are defined as C1-C6-alkyl (wherein R 2 and R 3(having the same definition). For this purpose, similar 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 alkyl 4-methylbenzenesulfonates. Protecting group strategies similar to those described in Synthesis Scheme 3 can be used.

[0251] Alternatively, for the preparation of part (I-a) of the compounds of the invention (I) in which R 2 and R 3 are defined as C1-C6-alkyl (wherein R 2 and R 3 have the same definition), the Mitsunobu reaction of Intermediate 5 with an optionally substituted alkyl alcohol can be used (similar to Synthesis Scheme 3).

[0252] If the R in the compound of formula (I-a) 1 includes suitable functional groups, then other compounds of the invention can subsequently be optionally prepared using oxidation or reduction reactions, similar to Synthesis Scheme 3.

[0253]

[0254] Synthesis Scheme 4

[0255] The substituents R 1 、R 4 、R 5 are as defined in general formula (I) respectively. R 2 and R 3 always have the same definition and are both C1-C6-alkyl.

[0256] Intermediate 4 can be prepared from Intermediate 1 in an alternative way (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).

[0257] Then, Intermediate 6 can be converted to Intermediate 7 by reduction of the nitro group. For example, the nitro group can be reduced with palladium on carbon in a hydrogen atmosphere (see, for example, WO2013174744 regarding the reduction of 6-isopropoxy-5-nitro-1H-indazole to 6-isopropoxy-1H-indazol-5-amine), or with iron and ammonium chloride in water and ethanol (see, for example, Journal of the Chemical Society, 1955, 2412-2419), or using tin(II) chloride (CAS 7772-99-8). Reduction with iron and ammonium chloride in water and ethanol is preferably used. Preparation of Intermediate 4 from Intermediate 7 can be carried out similar to Synthesis Scheme 2 (preparation of Intermediate 3 from Intermediate 2).

[0258] 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, optionally, oxidation or reduction reactions known to those skilled in the art can also be carried out as described in Synthesis Scheme 3 (see, for example, Science of Synthesis, Georg Thieme Verlag).

[0259]

[0260] Synthesis Scheme 5

[0261] Substituent R 1 、R 4 、R 5 Each is as defined in General Formula (I).

[0262] Synthesis of the example compound

[0263] Abbreviations and Explanations

[0264] 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 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

[0265] The term sodium chloride solution always refers to a saturated aqueous solution of sodium chloride.

[0266] The chemical names of the intermediates and examples were generated using ACD / LABS (version 12.01) software.

[0267] Methods

[0268] In some cases, the compounds of the present invention and their precursors and / or intermediates are analyzed by LC-MS.

[0269] Method A1: UPLC (MeCN - HCOOH):

[0270] Instrument: Waters Acquity UPLC-MS SQD 3001; Column: Acquity UPLC BEH C18 1.7 50×2.1mm; Eluent A: water + 0.1% by volume of 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.

[0271] Method A2: UPLC (MeCN - NH3):

[0272] Instrument: Waters Acquity UPLC-MS SQD 3001; Column: Acquity UPLC BEH C18 1.7 50×2.1 mm; Eluent A: water + 0.2% by volume of 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.

[0273] Method A3: (LC-MS)

[0274] Instrument: Agilent 1290Infinity LC; Column: Acquity UPLC BEH C18 1.7 50×2.1 mm; Eluent A: water + 0.05% by volume of formic acid; Eluent B: acetonitrile + 0.05% by volume of 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.

[0275] Method A4: (LC-MS)

[0276] 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.

[0277] In certain cases, the compounds of the present invention and their precursors and / or intermediates are purified by the following illustrative preparative HPLC methods:

[0278] Method P1: System: Waters automatic 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 of formic acid, Eluent B: acetonitrile; Gradient: 0 - 8 min 10 - 100% B, 8 - 10 min 100% B; Flow rate: 50 mL / min; Temperature: room temperature; Solution: maximum 250 mg / maximum 2.5 mL DMSO or DMF; Injection volume: 1×2.5 mL; Detection: DAD scan range 210–400 nm; MS ESI+, ESI-, scan range 160 - 1000 m / z.

[0279] Method P2: System: Waters automatic purification system: pump 254, sample manager 2767, CFO, DAD 2996, ELSD 2424, SQD 3100; Column: XBridge C18 5μm 10×30mm; Eluent A: water + 0.2% by volume of ammonia (32%), Eluent B: methanol; Gradient: 0 - 8 min 30 - 70% B; Flow rate: 50 mL / min; Temperature: room temperature; Detection: DAD scan range 210–400 nm; MS ESI+, ESI-, scan range 160 - 1000 m / z; ELSD.

[0280] Method P3: System: Labomatic, pump: HD - 5000, fraction collector: LABOCOL Vario - 4000, UV detector: Knauer UVD 2.1S; Column: XBridge C18 5μm 100×30mm; Eluent A: water + 0.2% by volume of ammonia (25%), Eluent B: acetonitrile; Gradient: 0 - 1 min 15% B, 1 - 6.3 min 15 - 55% B, 6.3 - 6.4 min 55 - 100% B, 6.4 - 7.4 min 100% B; 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.

[0281] Method P4: System: Labomatic, Pump: HD-5000, Fraction Collector: LABOCOL Vario-4000, UV Detector: Knauer UVD 2.1S; Column: Chromatorex RP C18 10μm 125×30mm; Eluent A: Water + 0.1% v / v 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.

[0282] Method P5: System: Sepiatec: Prep SFC100, Column: Chiralpak IA 5μm 250×20mm; Eluent A: Carbon dioxide, Eluent B: Ethanol; Gradient: Isocratic 20% B; Flow rate: 80 mL / min; Temperature: 40 °C; Solution: Maximum 250 mg / 2 mL DMSO; Injection volume: 5×0.4 mL; Detection: UV 254 nm.

[0283] Method P6: System: Agilent: Prep 1200, 2x prep pump, DLA, MWD, Gilson: Liquid Handler215; Column: Chiralcel OJ-H 5μm 250×20mm; Eluent A: Hexane, Eluent B: Ethanol; Gradient: Isocratic 30% B; Flow rate: 25 mL / min; Temperature: 25 °C; Solution: 187 mg / 8 mL ethanol / methanol; Injection volume: 8×1.0 mL; Detection: UV280 nm.

[0284] Method P7: System: Labomatic, Pump: HD-5000, Fraction Collector: LABOCOL Vario-4000, UV Detector: Knauer UVD 2.1S; Column: XBridge C18 5μm 100×30mm; Eluent A: Water + 0.1% v / v 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.

[0285] Method P8: System: Agilent: Prep 1200, 2×prep pump, DLA, MWD, Gilson: Liquid Handler215; 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.

[0286] In some cases, mixtures of material were purified by silica gel column chromatography.

[0287] To prepare certain compounds of the invention and their precursors and / or intermediates, Equipment Column chromatography purification is performed on silica gel ("flash chromatography"). This is done using cartridges from Biotage, such as "SNAP Cartridge, KP_SIL" cartridges of different sizes and "Interchim Puriflash Silica HP 15UM flash column" cartridges of different sizes from Interchim.

[0288] Starting material

[0289] Intermediate V2-1

[0290] 6-(2-Hydroxypropan-2-yl)pyridine-2-carboxylic acid methyl ester

[0291]

[0292] 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 acetate (II) 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 with ethyl acetate three times, washed with saturated sodium bicarbonate aqueous solution and sodium chloride aqueous solution, filtered and concentrated by a hydrophobic filter. 1.60g of crude product was obtained.

[0293] UPLC-MS (Method A1): R t= 0.76 min (UV detector: TIC), molecular weight measured value 195.00.

[0294] Intermediate V3-1

[0295] Potassium 6-(2-hydroxypropan-2-yl)pyridine-2-carboxylate

[0296]

[0297] 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 residue was obtained, which could be used without further purification.

[0298] UPLC-MS (method A1): R t = 0.47 min (UV detector: TIC), molecular weight measured value 181.00.

[0299] Intermediate 1-1

[0300] Methyl 5-nitro-1H-indazole-6-carboxylate

[0301]

[0302] In a three-necked flask equipped with a CPG stirrer, a dropping funnel, and an internal thermometer, 4.60 g (26.1 mmol) of methyl 1H-indazole-6-carboxylate (CAS No.: 170487-40-8) was dissolved in 120 mL of sulfuric acid (96%), and the solution was cooled to -15 °C. Within 15 min, the pre-prepared and cooled nitrating acid (10 mL of 96% sulfuric acid in 5 mL of 65% nitric acid) was added dropwise to this solution. After the addition was complete, the mixture was stirred for another 1 h (internal temperature -13 °C). The reaction mixture was added to ice, the precipitate was filtered off, washed with water, and dried under reduced pressure in an oven at 50 °C. 5.49 g of the title compound was obtained.

[0303] UPLC-MS (method A2): R t = 0.75 min

[0304] MS (ESIpos): m / z = 222 (M + H) +

[0305] 1 H NMR (400 MHz, 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).

[0306] Intermediate 2-1

[0307] Methyl 5-amino-1H-indazole-6-carboxylate

[0308]

[0309] Dissolve 4.40 g (19.8 mmol) of methyl 5-nitro-1H-indazole-6-carboxylate (Intermediate 1-1) in 236 mL of methanol and hydrogenate at 25 °C for 3 h under a standard hydrogen pressure using 1.06 g (0.99 mmol) of palladium on activated carbon. Filter the reaction mixture through diatomaceous earth, wash the filter cake with methanol, and concentrate the filtrate. 3.53 g of the title compound is obtained.

[0310] 1 H NMR (300 MHz, 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).

[0311] Intermediate 3-1

[0312] Methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate

[0313]

[0314] First, add 4.95 g (25.9 mmol) of 6-(trifluoromethyl)pyridine-2-carboxylic acid to 45 mL of THF. Add 9.07 g (28.2 mmol) of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate and 4.92 mL (28.2 mmol) of N-ethyl-N-isopropylpropan-2-amine, and stir the mixture at 25 °C for 30 min. Subsequently, add 4.50 g (23.5 mmol) of methyl 5-amino-1H-indazole-6-carboxylate (Intermediate 2-1), and stir the mixture at 25 °C for 24 h. Filter the reaction mixture by suction through a membrane filter, wash the solid with THF and water, and dry it overnight in a drying oven. 7.60 g of the title compound is obtained.

[0315] UPLC-MS (Method A2): R t = 1.16 min

[0316] MS (ESIpos): m / z = 365 (M+H) +

[0317] 1 1H NMR (400 MHz, 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).

[0318] Intermediate 3-2

[0319] Methyl 5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate

[0320]

[0321] First, 2.85 g (23.5 mmol) of 6-(difluoromethyl)pyridine-2-carboxylic acid was added to 30 mL of THF. 6.05 g (18.8 mmol) of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate and 3.3 mL of N-ethyl-N-isopropylpropan-2-amine were added, and the mixture was stirred at room temperature for 10 min. Subsequently, 3.00 g (15.7 mmol) of methyl 5-amino-1H-indazole-6-carboxylate was added, and the mixture was stirred at room temperature overnight. The reaction mixture was mixed with water, the precipitate was filtered off, and washed repeatedly with water and dichloromethane. 1.53 g (27% of theory) 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. This gave an additional 1.03 g of the title compound.

[0322] 1 1H-NMR (first product portion, 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).

[0323] Intermediate 3-3

[0324] Methyl 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate

[0325]

[0326] First, 2.10 g of potassium 6-(2-hydroxypropan-2-yl)pyridine-2-carboxylate (Intermediate V3-1) was added to 15 mL of THF. 3.69 g (11.5 mmol) of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate and 2.00 mL of N-ethyl-N-isopropylpropan-2-amine were added, and the mixture was stirred at room temperature for 15 min. Subsequently, 1.83 g (9.58 mmol) of methyl 5-amino-1H-indazole-6-carboxylate (Intermediate 2-1) was added, and the mixture was stirred at room temperature for 19 h. The mixture was mixed with water and ethyl acetate, the undissolved solid was filtered out, 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, 1.56 g of the title compound as a yellow foam was obtained.

[0327] UPLC-MS (Method A1): R t = 1.00 min (UV detector: TIC Smooth), molecular weight measured value 354.00.

[0328] 1 1H-NMR (500 MHz, 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).

[0329] Intermediate 4-1

[0330] Methyl 2-(oxetan-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate

[0331]

[0332] Dissolve 1.00 g (2.66 mmol) of methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (Intermediate 3-1) in 10 mL of DMF. After adding 1.10 g (7.99 mmol) of potassium carbonate and 221 mg (1.33 mmol) of potassium iodide, stir the mixture at 25 °C for 30 min. Add 603 mg (3.99 mmol) of 3-(bromomethyl)oxetane and stir the mixture at 25 °C for 24 h. Layer the reaction mixture between water and ethyl acetate. Extract the mixture twice with ethyl acetate, and filter the combined organic phase through a hydrophobic filter and concentrate. Purify the residue by silica gel column chromatography (hexane / ethyl acetate). Obtain 260 mg of the title compound.

[0333] UPLC-MS (Method A2): R t = 1.24 min

[0334] MS (ESIpos): m / z = 435 (M+H) +

[0335] 1 1H NMR (400 MHz, 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, 2H), 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).

[0336] Intermediate 4-2

[0337] Methyl 2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate

[0338]

[0339] Dissolve 1.00 g (2.75 mmol) of methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (Intermediate 3-1) in 5 mL of DMF, and while stirring, add 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. Stir the reaction mixture at 25 °C for 24 h, dilute with water, and extract twice with ethyl acetate. Filter the combined organic phases through a hydrophobic filter and concentrate. Purify the residue by silica gel column chromatography (hexane / ethyl acetate). Obtain 12 mg of the title compound.

[0340] UPLC-MS (Method A1): R t = 1.24 min

[0341] MS (ESIpos): m / z = 423 (M+H) +

[0342] 1 1H NMR (400 MHz, DMSO-d6): δ [ppm] = 3.24 (s, 3H), 3.86 (t, 2H), 3.96 (s, 3H), 4.65 (t, 2H), 8.21 (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).

[0343] Intermediate 4-3

[0344] Methyl 2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate

[0345]

[0346] Dissolve 1.00 g (2.75 mmol) of methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (Intermediate 3-1) in 5 mL of DMF, and while stirring, add 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. Stir the reaction mixture at 25 °C for 72 h, dilute with water, and extract twice with ethyl acetate. Filter the combined organic phases through a hydrophobic filter and concentrate. Purify the residue by silica gel column chromatography (hexane / ethyl acetate). Obtain 28 mg of the title compound.

[0347] UPLC-MS (Method A1): R t = 1.29 min

[0348] MS (ESI pos): m / z = 437 (M+H) +

[0349] 1 1H 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).

[0350] Intermediate 4-4

[0351] Methyl 2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate

[0352] Preparation method 1

[0353]

[0354] First, 930 mg (2.55 mmol) of methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (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. Purification by silica gel column chromatography (hexane / ethyl acetate) gave 424 mg of the title compound.

[0355] UPLC-MS (Method A2): R t = 1.21 min (UV detector: TIC), molecular weight measured value 450.00.

[0356] 11H-NMR (400 MHz, DMSO-d6): δ [ppm] = 1.16 (s, 6H) 2.02 - 2.11 (m, 2H) 3.96 (s, 3H) 4.51 - 4.60 (m, 3H) 8.20 (dd, J = 7.83, 1.01 Hz, 1H) 8.39 (s, 1H) 8.45 (s, 2H) 8.55 (d, J = 0.76 Hz, 1H) 9.05 (s, 1H) 12.52 (s, 1H).

[0357] Preparation Method 2

[0358] First, 1.95 g (7.03 mmol) of methyl 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate (Intermediate 7-1) was added to 30 mL of THF. 1.48 g (7.37 mmol) of 6-(trifluoromethyl)pyridine-2-carboxylic acid, 2.71 g (8.44 mmol) of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, and 1.47 mL (8.44 mmol) of N-ethyl-N-isopropylpropan-2-amine were added, and the mixture was stirred at 25 °C for 20.5 h. Water was added, and the mixture was extracted three times with ethyl acetate. The extract was washed with a sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was separated by silica gel column chromatography (hexane / ethyl acetate gradient). 2.79 g of the title compound was obtained.

[0359] UPLC-MS (Method A1): R t = 1.23 min (UV detector: TIC), molecular weight found 450.00.

[0360] Intermediate 4-5

[0361] Methyl 2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate

[0362]

[0363] First, 1.00 g (2.66 mmol, 97%) of methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (Intermediate 3-1) was added to 50 mL of DMF. While stirring, 1.10 g (7.99 mmol) of potassium carbonate and 221 mg (1.33 mmol) of potassium iodide were added, and the mixture was stirred at 25 °C for 30 min. Subsequently, 857 μL (3.99 mmol) of (2-bromoethoxy)(tert-butyl)dimethylsilane was added, and the mixture was stirred at 25 °C for 24 h. 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). 400 mg of the title compound was obtained.

[0364] UPLC-MS (Method A1): R t = 1.58 min

[0365] MS (ESIpos): m / z = 523 (M+H) +

[0366] 1 1H NMR (300 MHz, 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).

[0367] Intermediate 4-6

[0368] Methyl 2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate

[0369]

[0370] Similarly to Intermediate 4-5, 1.00 g (2.75 mmol) of methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (Intermediate 3-1) was dissolved in 10 mL of DMF. While stirring, 1.14 g (8.24 mmol) of potassium carbonate and 228 mg (1.37 mmol) of potassium iodide were added, and the mixture was stirred at 25 °C for 30 min. Subsequently, 1.04 g (4.12 mmol) of (3-bromopropoxy)(tert-butyl)dimethylsilane was added, and the mixture was stirred at 25 °C for 24 h. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate. The reaction mixture was separated between water and ethyl acetate, and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were filtered through a hydrophobic filter and concentrated. The residue was purified by preparative HPLC to obtain 428 mg of the title compound.

[0371] UPLC-MS (Method A1): R t = 1.63 min

[0372] MS (ESIpos): m / z = 537 (M+H) +

[0373] 1 1H NMR (400 MHz, 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).

[0374] Intermediate 4-7

[0375] Methyl 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-2-(4,4,4-trifluorobutyl)-2H-indazole-6-carboxylate

[0376]

[0377] First, 300 mg (0.80 mmol) of methyl 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (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.

[0378] UPLC-MS (Method A1): R t = 1.26 min (UV detector: TIC), molecular weight found 464.17.

[0379] Intermediate 4-8

[0380] Methyl 5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate

[0381]

[0382] Similar to Intermediate 4-4 (Preparation Method 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 similar treatment with water, 228 mg of the crude product was obtained.

[0383] UPLC-MS (Method A1): R t = 1.20 min (UV detector: TIC), molecular weight found 414.00.

[0384] Intermediate 4-9

[0385] Methyl 2-(3-hydroxy-3-methylbutyl)-5-{[(6-methylpyridin-2-yl)carbonyl]amino}-2H-indazole-6-carboxylate

[0386]

[0387] Similar to Intermediate 4-4 (Preparation Method 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 70 mg (0.50 mmol) of 6-methylpyridine-2-carboxylic acid for 19.5 h. After similar water treatment, 278 mg of the title compound was obtained as a crude product.

[0388] UPLC-MS (Method A1): R t = 1.14 min (UV detector: TIC), mass measured value 396.00.

[0389] Intermediate 4-10

[0390] Methyl 2-[3-(2,2,2-trifluoroethoxy)propyl]-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate

[0391]

[0392] 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.

[0393] UPLC-MS (Method A1): R t = 1.39 min (UV detector: TIC), molecular weight measured value 504.12.

[0394] Intermediate 4-11

[0395] Methyl 5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate

[0396]

[0397] First, 2.00 g of methyl 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate (Intermediate 7-1) was added to 40 mL of THF. 1.50 g of 6-(difluoromethyl)pyridine-2-carboxylic acid, 2.78 g of O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU, CAS No. 125700-67-6), and 1.5 mL of N-ethyl-N-isopropylpropan-2-amine were added, and the mixture was stirred at room temperature for 24 h. Water was added, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with sodium chloride solution and filtered through a hydrophobic filter. The mixture was concentrated, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate). 3.05 g of the title compound as a yellow solid was obtained.

[0398] UPLC-MS (Method A1): Rt = 1.15 min (UV detector TIC), molecular weight found 432.00.

[0399] 1 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 1.17 (s, 6H), 2.04 - 2.11 (m, 2H), 3.99 (s, 3H), 4.52 - 4.60 (m, 3H), 7.10 (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).

[0400] Intermediate 5-1

[0401] N-[6-(2-Hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0402]

[0403] 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 3 M solution of methylmagnesium bromide in diethyl ether. While cooling with an ice bath, the mixture was stirred for 1 h and then stirred at room temperature for 19.5 h. Another 2 equivalents of the methylmagnesium bromide solution was added, and the mixture was stirred at room temperature for an additional 24 h. Saturated aqueous ammonium chloride was added, the mixture was stirred, and extracted three times with ethyl acetate. The combined organic phases were washed with a 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 was obtained.

[0404] 1 1H-NMR (400 MHz, 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).

[0405] Intermediate 5-2

[0406] 6-(difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]pyridine-2-carboxamide

[0407]

[0408] Similar 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 a 3 M solution of 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 the workup as for Intermediate 5-1, 2.39 g of the crude product was obtained, which was used without further purification.

[0409] Intermediate 6-1

[0410] Methyl 2-(3-hydroxy-3-methylbutyl)-5-nitro-2H-indazole-6-carboxylate

[0411]

[0412] First, 5.00 g (22.6 mmol) of methyl 5-nitro-1H-indazole-6-carboxylate (Intermediate 1-1) was added to 40 mL of DMF. 5.65 g (33.9 mmol) of 4-bromo-2-methylbutan-2-ol, 9.37 g (67.8 mmol) of potassium carbonate and 5.63 g (33.9 mmol) of potassium iodide were added, and the mixture was stirred at 100 °C for 20 h. 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 purified by silica gel column chromatography (hexane / ethyl acetate). The obtained solid was stirred with diethyl ether, filtered off, washed with diethyl ether and dried. 2.49 g of the title compound was obtained.

[0413] UPLC-MS (Method A1): R t = 0.93 min (UV detector: TIC), molecular weight found 307.00.

[0414] 1 1H-NMR (400 MHz, 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).

[0415] Intermediate 7-1

[0416] Methyl 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate

[0417]

[0418] 4.53 g of iron and 217 mg of ammonium chloride were 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 was stirred at 90 °C for 21.5 h. The mixture was filtered through diatomaceous earth and washed three times with ethanol. The filtrate was concentrated, and the residue was mixed with water. Extraction was carried out three times with ethyl acetate (sodium chloride solution was added to improve phase separation). The combined organic phases were washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. 1.95 g (85% of theory) of the title compound was obtained.

[0419] UPLC-MS (Method A1): R t = 0.67 min (UV detector: TIC), molecular weight found 277.00.

[0420] 1 H-NMR (400 MHz, 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).

[0421] Working example

[0422] Example 1

[0423] N-[6-(2-Hydroxypropan-2-yl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0424]

[0425] Dissolve 75 mg (0.18 mmol) of methyl 2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-2) in 500 μL of THF and mix with 887 μL (0.89 mmol) of a 1 M solution of methylmagnesium bromide in THF. Stir the reaction mixture at 25 °C for 60 min. Subsequently, carefully add 1 mL of saturated aqueous ammonium chloride solution and filter the mixture. The aqueous phase is extracted twice with ethyl acetate, the organic phases are combined, filtered through a hydrophobic filter, and concentrated. The residue is dissolved in 3 mL of DMSO and purified by preparative HPLC. The fractions containing the product are lyophilized. 20 mg of the title compound is obtained.

[0426] UPLC-MS (Method A1): R t = 1.08 min

[0427] MS (ESIpos): m / z = 423 (M+H) +

[0428] 1 H NMR (300 MHz, 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 (d 1H), 8.29 - 8.42 (m, 2H), 8.42 - 8.50 (m, 1H), 8.71 (s, 1H), 12.36 (s, 1H).

[0429] Example 2

[0430] N-[6-(Hydroxymethyl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0431]

[0432] 13 mg (0.36 mmol) of lithium aluminum hydride was suspended in 1 mL of THF, and the mixture was cooled to 0 °C. A solution of 75 mg (0.17 mmol) of methyl 2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-2) in 500 μL of THF was added dropwise, and the mixture was stirred at 25 °C for 60 min. The mixture was diluted with water and extracted twice with ethyl acetate. The combined organic phases were washed with a sodium chloride solution, filtered through a hydrophobic filter, concentrated, and dried under reduced pressure. 13 mg of the title compound was obtained.

[0433] UPLC-MS (Method A2): R t = 0.99 min

[0434] MS (ESIpos): m / z = 394 (M+H) +

[0435] 1 1H NMR (400 MHz, DMSO-d6): δ [ppm] = 3.23 (s, 3H), 3.83 (t, 2H), 4.56 (t, 2H), 4.69 (d, 2H), 5.77 (t, 1H), 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).

[0436] Example 3

[0437] N-[6-(2-Hydroxypropan-2-yl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0438]

[0439] Dissolve 75 mg (0.17 mmol) of methyl 2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-3) in 500 μL of THF and mix with 859 μL (0.86 mmol) of a 1 M solution of methylmagnesium bromide in THF. Stir the reaction mixture at 25 °C for 60 min. Subsequently, carefully add 1 mL of saturated ammonium chloride solution and filter the mixture. Extract the aqueous phase twice with ethyl acetate, combine the organic phases, filter through a hydrophobic filter, and concentrate. Dissolve the residue in 3 mL of DMSO and purify by preparative HPLC. Freeze-dry the fractions containing the product. 25 mg of the title compound is obtained.

[0440] UPLC-MS (Method A1): R t = 1.13 min

[0441] MS (ESIpos): m / z = 437 (M+H) +

[0442] 1 H NMR (400 MHz, 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).

[0443] Example 4

[0444] N-[6-(Hydroxymethyl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0445]

[0446] Suspend 13 mg of lithium aluminum hydride in THF and cool the mixture to 0 °C. Dropwise add a solution of 75 mg (0.17 mmol) of methyl 2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-3) in THF and allow the mixture to reach room temperature within 30 min. Dilute the mixture with water and filter, wash the residue with ethyl acetate, and extract the filtrate with ethyl acetate. Wash the combined ethyl acetate phases with sodium chloride solution, filter through a hydrophobic filter, and concentrate. Purify the residue by preparative HPLC.

[0447] 1 1H NMR (300 MHz, 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).

[0448] Example 5

[0449] N-[2-(2-Hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0450] Step A:

[0451] 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

[0452]

[0453] 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 THF solution of methylmagnesium bromide. The reaction mixture was stirred at 25 °C for 60 min. Another 287 μL (0.29 mmol) of a 1 M THF solution of methylmagnesium bromide was added, and the mixture was stirred at 25 °C for 3 h. Subsequently, 20 mL of saturated ammonium chloride solution was carefully added, and the mixture was filtered. The aqueous phase was extracted twice with ethyl acetate, the combined organic phases were 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 was obtained.

[0454] UPLC-MS (Method A2): R t = 1.51 min

[0455] MS (ESIpos): m / z = 523 (M+H) +

[0456] 1 1H NMR (300 MHz, 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).

[0457] Step B:

[0458]

[0459] 50 mg (96 μmol) of N-[2-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide was dissolved in 1.0 mL of THF and mixed with 144 μL (0.14 mmol) of a 1 M solution of tetrabutylammonium fluoride in THF. The reaction mixture was stirred at room temperature for 1 h. 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. 36 mg of N-[2-(2-Hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Example 5) was obtained.

[0460] 1 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 3.86 (q, 2H), 4.43 (t, 2H), 4.95 (t, 1H), 5.94 (s, 1H), 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).

[0461] UPLC-MS (Method A2): R t = 0.97 min (UV detector: TIC), molecular weight found 408.00.

[0462] Example 6

[0463] N-[6-(2-Hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0464] Step A:

[0465] 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

[0466]

[0467] Dissolve 50 mg (0.09 mmol) of methyl 2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-6) in 500 μL of THF and mix with 326 μL (0.33 mmol) of a 1 M solution of methylmagnesium bromide in THF. Stir the reaction mixture at 25 °C for 60 min. Subsequently, carefully add 20 mL of saturated ammonium chloride solution and extract the mixture twice with ethyl acetate. Filter the combined organic phases through a hydrophobic filter, concentrate, and dry under reduced pressure. Purify the residue by preparative HPLC. Obtain 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.

[0468] UPLC-MS (Method A1): R t = 1.58 min

[0469] MS (ESIpos): m / z = 537 (M+H) +

[0470] 1 1H NMR (300 MHz, 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).

[0471] Step B:

[0472]

[0473] 37 mg (0.07 mmol) of N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-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. The combined organic phases were washed with saturated sodium chloride solution, filtered and concentrated. After purification by preparative HPLC, 10 mg of N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Example 6, containing minor components) was obtained.

[0474] UPLC-MS (Method A2): R t = 1.00 min

[0475] MS (ESIpos): m / z = 423 (M+H) +

[0476] 1 Selected signals of 1H NMR (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).

[0477] Example 7

[0478] N-[2-(2-Hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0479] Step A:

[0480] N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0481]

[0482] 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 2 M 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. Another 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 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. 30 mg of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide was obtained.

[0483] UPLC-MS (Method A2): R t = 1.44 min

[0484] MS (ESIpos): m / z = 495 (M+H) +

[0485] 1 1H NMR (300 MHz, 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), 5.75 - 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).

[0486] Step B:

[0487]

[0488] 33 mg (0.07 mmol) of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazol-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. The combined organic phases were washed with 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-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Example 7) was obtained.

[0489] UPLC-MS (Method A2): R t = 0.87 min

[0490] MS (ESIpos): m / z = 381 (M+H) +

[0491] 1 1H NMR (300 MHz, 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, 1H), 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).

[0492] Example 8

[0493] N-[6-(2-hydroxypropan-2-yl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0494]

[0495] Dissolve 50 mg (0.12 mmol) of methyl 2-(oxetan-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-1) in 500 μL of THF and mix with 576 μL (0.58 mmol) of a 1 M solution of methylmagnesium bromide in THF. Stir the reaction mixture at 25 °C for 60 min. Subsequently, carefully add 20 mL of saturated aqueous ammonium chloride solution and concentrate the mixture. Extract the aqueous phase twice with ethyl acetate, combine the organic phases, dry over magnesium sulfate, filter, and concentrate. Dissolve the residue in 2.0 mL of DMSO and purify by preparative HPLC. Freeze-dry the fractions containing the product. 30 mg of the title compound is obtained.

[0496] UPLC-MS (Method A2): R t = 1.03 min

[0497] MS (ESIpos): m / z = 435 (M+H) +

[0498] 1 1H NMR (400 MHz, 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).

[0499] Example 9

[0500] N-[6-(Hydroxymethyl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0501]

[0502] Dissolve 75 mg (0.17 mmol) of methyl 2-(oxetan-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-1) in 1 mL of a THF / methanol (1:1) mixture, and add 8 mg (0.21 mmol) of sodium borohydride. Stir the mixture at 25 °C for 60 min. Concentrate the reaction mixture, and mix the residue with water. Stir the suspension vigorously for 15 min, filter off the solid, wash it twice with water and twice with diethyl ether, and dry it under reduced pressure. 48 mg of the title compound is obtained.

[0503] UPLC-MS (Method A2): R t = 0.94 min

[0504] MS (ESIpos): m / z = 407 (M+H) +

[0505] 1 H NMR (300 MHz, 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).

[0506] Example 10

[0507] N-{6-(2-Hydroxypropan-2-yl)-2-[3-(methylsulfonyl)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0508]

[0509] Stir a mixture of 500 mg (1.32 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 5.0 mL of DMF at room temperature for 15 min. Add 414 mg of 1-bromo-3-(methylsulfonyl)propane, and stir the mixture at room temperature overnight. Add water, extract the mixture twice with ethyl acetate, wash the extract with sodium chloride solution, and concentrate. Purify the residue by column chromatography (dichloromethane / methanol gradient). Stir the product portion with diethyl ether, filter and dry. 59 mg of the title compound is obtained.

[0510] UPLC-MS (Method A2): R t= 1.02 min

[0511] MS (ESIpos): m / z = 485 (M+H)+

[0512] 1 1H-NMR (300 MHz, 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).

[0513] Example 11

[0514] N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0515]

[0516] Preparation method 1

[0517] 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 3 M methylmagnesium bromide solution (in diethyl ether) was added, and the mixture was stirred for 1 h while cooling with an ice bath and then stirred at room temperature for 4.5 h. Another 1 equivalent of methylmagnesium bromide solution was added, and the mixture was stirred at room temperature for 20.5 h. Then another 1 equivalent of methylmagnesium bromide solution was added, and the mixture was stirred at room temperature for 22 h. 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 the product fraction were obtained. The product fraction was stirred with diethyl ether. Suction filtration was carried out, and then it was dried. After that, another 146 mg of the title compound was obtained.

[0518] UPLC-MS (Method A1): R t = 1.10 min (UV detector: TIC), molecular weight measured value 450.00.

[0519] 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 1.14 (s, 6H), 1.61 (s, 6H), 1.99 - 2.08 (m, 2H), 4.42 - 4.55 (m, 3H), 5.93 (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).

[0520] Preparation method 2

[0521] 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 min. 344 mg (1.5 equivalents) of 4-bromo-2-methylbutan-2-ol was added, and the mixture was heated to 100 °C and maintained for 2 h. Another 0.5 equivalent of 4-bromo-2-methylbutan-2-ol was added, and the mixture was stirred at room temperature for 16 h. 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, and the product fraction was stirred with diethyl ether. The solid was filtered and dried. 60 mg of the title compound was obtained.

[0522] 1 H-NMR (400 MHz, 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).

[0523] Example 12

[0524] N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0525]

[0526] Suspend 160 mg (0.44 mmol) of N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Intermediate 5-1) together with 182 mg of sodium carbonate and 36 mg of potassium iodide in 1.0 mL of DMF, and stir the mixture at room temperature for 15 min. Then, add 123 mg of 2-bromoethyl methyl sulfone (0.66 mmol), and stir the mixture at room temperature overnight. Add water, extract the mixture twice with ethyl acetate, wash the extract with saturated aqueous sodium chloride, filter through a hydrophobic filter, and concentrate. Purify the residue by preparative HPLC to obtain 20 mg of the title compound.

[0527] UPLC (Method A2): R t = 1.01 min;

[0528] MS (ESIpos): m / z = 471 (M+H)+

[0529] 1 1H NMR (400 MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 2.90 (s, 3H), 3.85 (t, 2H), 4.86 (t, 2H), 5.97 (s, 1H), 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).

[0530] Example 13

[0531] 6-(Difluoromethyl)-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0532]

[0533] Preparation Method 1

[0534] 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 min. 145 mg (0.87 mmol) of 4-bromo-2-methylbutan-2-ol was added, and the mixture was stirred at 110 °C for 3 h. Another 96 mg of 4-bromo-2-methylbutan-2-ol was added, and the mixture was stirred at 110 °C for 4 h. Water was added, and the mixture was extracted twice with ethyl acetate. The extract was washed with a semi-saturated aqueous sodium chloride solution, filtered through a hydrophobic filter, and concentrated. Purification was carried out by silica gel column chromatography (hexane / ethyl acetate). 61 mg of the title compound was obtained.

[0535] UPLC-MS (Method A1): R t = 1.00 min (UV detector: TIC), molecular weight found 432.00.

[0536] 1 1H-NMR (300 MHz, 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).

[0537] Preparation Method 2

[0538] Similar to the preparation of Example 11 (Preparation Method 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 3 M methylmagnesium bromide solution (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.

[0539] Example 14

[0540] 6-(Difluoromethyl)-N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}pyridine-2-carboxamide

[0541]

[0542] 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 min. 162 mg of 2-bromoethyl methyl sulfone (0.87 mmol) was added and the mixture was stirred at 110 °C for 3 h. Water was added, the mixture was extracted twice with ethyl acetate, and the extracts were washed with semi-saturated aqueous sodium chloride solution, filtered through a hydrophobic filter and concentrated. The residue was purified by preparative HPLC and the product fraction was purified again by silica gel column chromatography (hexane / ethyl acetate). 40 mg of the title compound was obtained.

[0543] 1 1H-NMR (400 MHz, 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).

[0544] Example 15

[0545] 6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0546] Stage A:

[0547] 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

[0548]

[0549] 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 eq) of (3-bromopropoxy)(tert-butyl)dimethylsilane was added and the mixture was stirred at 110 °C for 3 h. Another 1 eq of (3-bromopropoxy)(tert-butyl)dimethylsilane was 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 an 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.

[0550] Stage B:

[0551]

[0552] Similar to the preparation in Step B of Example 6, 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 work-up was carried out as in Example 6 and purification was carried out by preparative HPLC to obtain 46 mg of the title compound.

[0553] UPLC-MS (Method A1): R t = 0.92 min (UV detector: TIC), molecular weight found 404.00.

[0554] 1 1H-NMR (400 MHz, 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).

[0555] Example 16

[0556] N-[6-(2-Hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0557]

[0558] A mixture of 210 mg (0.58 mmol) of N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Intermediate 5-1) in 3 mL of DMF was mixed with 0.11 mL (0.87 mmol) of 1,1,1-trifluoro-4-iodobutane and 239 mg of potassium carbonate, and the mixture was stirred at 80 °C for 6 h. 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. 19 mg of the title compound was obtained.

[0559] UPLC-MS (Method A1): R t = 1.27 min (UV detector: TIC), molecular weight found 474.15.

[0560] 1 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 2.10 - 2.33 (m), 4.49 (t, 2H), 5.94 (s, 1H), 7.59 (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).

[0561] Example 17

[0562] N-{6-(2-hydroxypropan-2-yl)-2-[3-(trifluoromethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0563]

[0564] First, 150 mg (0.33 mmol) of N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Intermediate 5-1) was added to 2 mL of THF. 58 mg (0.40 mmol) of 3-(trifluoromethoxy)propan-1-ol, 131 mg of triphenylphosphine, and 71 μL of diisopropyl azodicarboxylate (DIAD, CAS 2446-83-5) were added, and the mixture was stirred at room temperature for 19 h. 0.83 mL of sodium hydroxide solution (2 M) was added, and the mixture was stirred at 40 °C for 5 h. The mixture was diluted with water, extracted three times with ethyl acetate, the combined organic phases were concentrated, and purified by preparative HPLC. 16 mg of the title compound was obtained in crude product form.

[0565] UPLC-MS (Method A2): R t = 1.26 min (UV detector: TIC), measured molecular weight 490.14.

[0566] 1 1H-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).

[0567] Example 18

[0568] N-{6-(2-Hydroxypropan-2-yl)-2-[3-(2,2,2-trifluoroethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0569]

[0570] Similar to the preparation of Example 11 (Preparation Method 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

[0571] UPLC-MS (Method A1): R t = 1.25 min (UV detector: TIC), measured molecular weight 504.16.

[0572] 1 1H-NMR (500 MHz, 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).

[0573] Example 19

[0574] 5-Fluoro-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0575]

[0576] First, 228 mg (0.31 mmol) of methyl 5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate (Intermediate 4-8) was added to 4.5 mL of THF and cooled with an ice-cooling bath. 0.63 mL of 3 M methylmagnesium bromide solution (in diethyl ether) was added, and the mixture was stirred for 2 h while cooling with an ice bath and then stirred at room temperature for 21 h. The reaction mixture was mixed with saturated aqueous ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phases were concentrated. The residue was purified by preparative HPLC. 82 mg of the title compound was obtained.

[0577] UPLC-MS (Method A2): R t = 1.03 min (UV detector: TIC), molecular weight found 414.21.

[0578] 1 1H-NMR (400 MHz, 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).

[0579] Example 20

[0580] N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0581]

[0582] First, 278 mg (0.48 mmol) of methyl 2-(3-hydroxy-3-methylbutyl)-5-{[(6-methylpyridin-2-yl)carbonyl]amino}-2H-indazole-6-carboxylate (Intermediate 4-9) was added to 5.0 mL of THF and cooled with an ice bath. 0.97 mL of 3 M methylmagnesium bromide solution (in diethyl ether) was added, and the mixture was stirred for 2 h while cooling with an ice bath and then stirred at room temperature for 20.5 h. Another 0.48 mL of 3 M methylmagnesium bromide solution was added, and the mixture was stirred at room temperature for 67 h. The mixture was mixed with 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. 111 mg of the title compound was obtained.

[0583] UPLC-MS (Method A2): R t = 0.97 min (UV detector: TIC), molecular weight found 396.22.

[0584] 1 1H-NMR (400 MHz, 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).

[0585] Example 21

[0586] 6-(2-Hydroxypropan-2-yl)-N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0587]

[0588] A solution of 72 mg (0.16 mmol) of methyl 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-2-(4,4,4-trifluorobutyl)-2H-indazole-6-carboxylate (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. Another 1 equivalent of 3 M methylmagnesium bromide solution was added, and the mixture was stirred at room temperature for 24 h. Saturated aqueous ammonium chloride solution was added, and the mixture was extracted three times with ethyl acetate. The extract was washed with sodium chloride solution and concentrated. 22 mg (31% of theory) of the title compound was obtained by preparative HPLC.

[0589] UPLC-MS (Method A2): R t = 1.15 min (UV detector: TIC), molecular weight found 464.20.

[0590] 1 1H-NMR (400 MHz, 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, 1H), 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).

[0591] Example 22

[0592] N-{2-[2-(1-hydroxycyclopropyl)ethyl]-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0593]

[0594] First, 250 mg (0.69 mmol) of N-[6-(2-hydroxypropan-2-yl)-1H-indazol-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 a sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC (column: Waters XBridge C18 5μ 100×30 mm, eluent A: water + 0.1 vol% formic acid (99%), eluent B: acetonitrile). Lyophilization gave 45 mg of the title compound.

[0595] 1 1H-NMR (500 MHz, 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).

[0596] Physiological efficacy evaluation

[0597] IRAK4 kinase assay

[0598] The activity of the substances of the present invention against IRAK4-inhibition was determined in the IRAK4 TR-FRET assay (TR-FRET = time-resolved fluorescence resonance energy transfer) described below.

[0599] The GST (glutathione-S-transferase) and human IRAK4 recombinant fusion protein from the N-terminus were used as the enzyme, and the recombinant fusion protein was expressed in baculovirus-infected insect cells (Hi5, BTI-TN-5B1-4, cell lines purchased from Invitrogen, catalog number B855-02) and purified by affinity chromatography. The substrate for the kinase reaction was biotinylated peptide biotin-Ahx-KKARFSRFAGSSPSQASFAEPG (C-terminus in amide form), which was available from, for example, Biosyntan GmbH (Berlin-Buch).

[0600] For the experiment, 11 different concentrations in the range 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), and 2 μL of a solution of IRAK4 in the assay buffer [50 mM HEPES at pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w / v) bovine γ-globulin (BGG), 0.04% (v / v) Nonidet-P40 (Sigma)] was added, and the mixture was incubated for 15 min to allow the substance to pre-bind to the enzyme before the kinase reaction. Then the kinase reaction was initiated by adding 3 μL of adenosine triphosphate (ATP, 1.67 mM = final concentration in 5 μL assay volume: 1 mM) and the peptide substrate (0.83 μM = final concentration in 5 μL assay volume: 0.5 μM) in a solution of the assay buffer, and the resulting mixture was incubated for a reaction time of 45 min at 22 °C. The concentration of IRAK4 was adjusted to the respective activity of the enzyme and set such that the experiment was carried out in the linear range. Usually the concentration was on the order of about 0.2 nM. The reaction was terminated by adding 5 μL of the TR-FRET detection reagent [0.1 μM streptavidin-XL665 (Cisbio Bioassays; France, catalog number 610SAXLG)] and 1.5 nM anti-phosphoserine antibody [MerckMillipore, "STK antibody", catalog number 35-002] and 0.6 nM LANCE EU-W1024-labeled anti-mouse IgG antibody (Perkin-Elmer, product number AD0077; or a terbium cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used) in a solution of EDTA aqueous solution (100 mM EDTA, 0.4% (w / v) bovine serum albumin [BSA] in 25 mM HEPES at pH 7.5).

[0601] The resulting mixture was incubated at 22 °C for 1 h to form a complex of biotinylated phosphorylated substrate and the detection reagent. Then, the amount of phosphorylated substrate was evaluated by measuring the resonance energy transferred from the europium chelate-labeled anti-mouse IgG antibody 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 emissions at 620 nm and 665 nm were 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). Usually, the test substance was tested at 11 different concentrations in the range of 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 before the test (2 mM to 7.3 nM, in 100% DMSO). The IC 50 value was calculated by 4-parameter fitting.

[0602] Table 1: The IC 50 value of the example compound in the IRAK4 kinase assay

[0603]

[0604] The inhibitory activity of the substances of general formula (III) of the present invention against IRAK4 was also determined in the IRAK4 TR-FRET assay as described above. By way of example, the following are mentioned: compound intermediate 4-2, IC 50 = 21.7 nM; intermediate 4-3, IC 50 = 13.0 nM; and intermediate 4-4, IC 50 = 6.2 nM.

[0605] TNF-α secretion in THP-1 cells

[0606] This assay is applicable to the ability of a test substance to inhibit the secretion of TNF-α (tumor necrosis factor α) in THP-1 cells (human monocytic acute leukemia cell line). TNF-α is a cytokine involved in inflammatory processes. In this assay, the secretion of TNF-α is induced by incubation with bacterial lipopolysaccharide (LPS).

[0607] THP-1 cells were stored in continuous suspension cell culture medium [RPMI 1460 medium containing L-Glutamax (Gibco, catalog number 61870-044), supplemented with 10% fetal bovine serum (FCS) (Invitrogen, catalog number 10082-147), 1% penicillin / streptomycin (Gibco BRL, catalog number 15140-114)], and the cell concentration should not exceed 1×10 6 cells / mL. The tests were performed in cell culture medium (RPMI 1460 medium containing L-Glutamax, supplemented with 10% FCS).

[0608] In each case, 2-2.5 μL of cell suspension (corresponding to 4000 cells) per well was dispensed into a 384-well test plate (Greiner, catalog number 784076), where in each well, 40-50 nL of the substance had been dissolved in 100% DMSO. For each substance, the dissolution was done using 10 different concentrations in the range of 20 μM to 0.073 nM. The cells were incubated at room temperature for 15 min. Then 2-2.5 μL of 0.1 μg / mL LPS (Sigma, Escherichia coli 055:B5, catalog number L5418) dissolved in cell culture medium (final concentration 0.05 μg / mL) was dispensed into each well. As a neutral control, the cells were treated with 0.05 μg / mL LPS and 1% DMSO, and as an inhibitor control, only 1% DMSO was used.

[0609] The plate was centrifuged at 80 g for 30 s and incubated at 37 °C, 5% CO2 and 95% atmospheric humidity for 17 h. The amount of TNF-α was determined using a TNF-α HTRF Detection Kit (Cisbio, catalog number 62TNFPEB / C). For this purpose, in each case, 2 μl of the detection solution, which consists of an anti-TNF-α-XL665 conjugate and an anti-TNF-α-cryptate conjugate, dissolved in the reconstitution buffer according to the manufacturer's instructions, was added for the HTRF (homogeneous time-resolved fluorescence) test. After addition, the mixture was incubated at room temperature for 3 h or at 4 °C overnight. Then the signal at 620 / 665 nm was read using a measurement instrument supporting HTRF such as the BMGPheraStar.

[0610] The activity of the substance was expressed as the ratio between the neutral control and the inhibitor control in percentage. The IC 50 value was calculated using 4-parameter fitting.

[0611] Table 2:IC of the example compound for TNF-α secretion in THP-1 cells 50 value

[0612]

[0613] In vitro generation of cytokines in LPS (lipopolysaccharide)-induced human PBMCs (peripheral blood mononuclear cells)

[0614] The efficacy of the compounds of general formula (I) of the present invention for the generation of inducible cytokines in human PBMCs was investigated. Herein, cytokine production was induced by LPS - a TLR4 ligand, which leads to the activation of the IRAK4-mediated signaling pathway.

[0615] Human PBMCs were obtained from human anticoagulated whole blood. For this purpose, first 15 mL of Ficoll-Paque (Biochrom, catalog number L6115) was pipetted into a Leucosep tube and 20 mL of human blood was added. After centrifuging the blood at 800 g for 15 min at room temperature, the plasma containing platelets was removed and discarded. The PBMCs were transferred to a centrifuge tube and supplemented with PBS (phosphate buffered saline) (Gibco, catalog number 14190). The cell suspension was centrifuged at 250 g for 10 min at room temperature and the supernatant was discarded. The PBMCs were resuspended in complete medium (RPMI1640, without L-glutamine (PAA, catalog number E15-039), 10% FCS; 50 U / mL penicillin, 50 μg / mL streptomycin (PAA, catalog number P11-010) and 1% L-glutamine (Sigma, catalog number G7513)).

[0616] The assays were also performed in complete medium. The PBMCs were seeded at 2.5×10 5Cells were seeded at a density of cells / well onto 96-well plates. The compounds of the present invention were serially diluted in an equal volume of 100% DMSO and applied to the assay at 8 different concentrations in the range of 10 μM to 3 nM such that the final DMSO concentration was 0.4% DMSO. The cells were then pre-incubated with them for 30 min prior to the actual stimulation. To induce cytokine secretion, the cells were stimulated with 0.1 μg / mL of LPS (Sigma, Escherichia coli 0128:B12, catalog number L2887) for 24 h. Cell viability was determined using the CellTiter-Glo luminescence assay (Promega, catalog number G7571 (G755 / G756A)) according to the manufacturer's instructions. The amount of TNF-α secreted in the cell culture supernatant was determined using the Human ProInflammatory 9-Plex Tissue Culture Kit (MSD, catalog number K15007B) according to the manufacturer's instructions. For example, the activities of Example Compound 11 and Example Compound 12 were ≤1 μM.

[0617] Secretion of interleukin (IL)-23 by TLR-4 / TLR-7-induced human dendritic cells (DCs) in vitro

[0618] The efficacy of the compounds of general formula (I) of the present invention on the induced production of the pro-inflammatory cytokine IL-23 was investigated in human DCs. The pro-inflammatory cytokine IL-23 plays an important role in the generation of TH-17 cells. It has been reported that TH-17 cells play a key role in the pathogenesis of diseases such as rheumatoid arthritis, psoriatic arthritis, Bechterew'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 detect the effect of the compounds of the present invention on IL-23 production, human primary monocytes were isolated from human PBMCs using magnetic separation [Miltenyi Biotech, Monocyte Isolation Kit, catalog number 130-091-153] and differentiated into DCs in medium for 6 days in the presence of growth factors (recombinant human GM-CSF [PeproTech, catalog number 300-03] and IL-4 [PeproTech, catalog number 200-04]) in complete medium (VLE (very low endotoxin) RPMI 1640 [Biochrom AG, 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]). After collecting the DCs, they were resuspended in complete medium and at 2×10 5Cells were seeded at a density of Figure 1 cells / well into 96-well plates (Costar, catalog number 3599). The compounds of the present invention were serially diluted in an equal volume of 100% DMSO and used in the assay at 9 different concentrations in the range of 10 μM to 1 nM. Here, it was ensured that for each of the 9 concentrations used, the DMSO concentration present was always 0.1%. The DCs were pre-incubated with the compounds of the present invention for 30 min. Thereafter, the DCs were stimulated to produce IL-23 by adding 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) in an incubator (37 °C, 95% rH, 5% CO2) for 24 hours. After the 24-hour incubation period, the supernatants were collected and analyzed using a commercially available hIL-23 ELISA (eBiosciences, catalog number 88-7237-88) according to the manufacturer's instructions. Taking the example of Example Compound 12, the results of the inhibition of IL-23 in human DCs are shown in Figure 1 in.

[0619] Generation of IFNα in vitro in TLR-7 / 8- or TLR-9-stimulated human plasmacytoid dendritic cells (pDCs)

[0620] Using this assay, the effect of the compounds of general formula (I) of the present invention on the generation of IFNα (interferon α), a key cytokine in the pathogenesis of systemic lupus erythematosus, in human pDCs can be studied (Mathian et al., Arthritis Rheum, 2009; Crow M.K., Rheum Dis Clin N Am, 2010). For this purpose, as described above, human PBMCs were isolated from whole blood 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 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 seeded at 5×10 4Cells were seeded at a density of cells / well in 96-well microtiter plates (Costar, catalog number 3599). The compounds of the present invention were serially diluted in an equal volume of 100% DMSO and used in the test at 9 different concentrations in the range of 10 μM to 1 nM. For each of the 9 test concentrations, the DMSO concentration present was always 0.1%. pDCs were pre-incubated with the compounds of the present invention for 30 minutes. pDCs were stimulated with TLR7 / 8 ligand (imiquimod, R837, Invivogen, catalog number tlrl-imq) or with TLR-9 ligand (CPG-A, ODN2216, Invivogen, catalog number tlrl-2216-1), which results in the activation of the IRAK4-mediated signaling pathway. After incubation for 24 hours, 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 the example of the compound 12 of the embodiment, the results of the inhibition of IFNα in human plasmacytoid DCs are shown in Figure 2.

[0621] In Vivo Model of TLR-Mediated Inflammation

[0622] The in vivo efficacy of the compounds of general formula (I) of the present invention was investigated in an in vivo model of TLR-mediated inflammation. Due to the use of the LPS-mediated inflammation model, this mechanism model particularly demonstrated 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, with 5 animals in each 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 treatment group and the positive control group were administered 0.2 mg LPS / kg body weight (Sigma, catalog number L4391) (lipopolysaccharide from E. coli 0111:B4) intraperitoneally (i.p.). In addition, the positive control group was treated with the above-mentioned substance vehicle. The substance was administered orally 16 hours before inflammation was induced by administering LPS. To investigate the efficacy of the compounds of the present invention on inflammation, blood samples were taken from the animals 1.5 hours later. According to the manufacturer's instructions, the concentration of specific cytokines in the plasma was measured using the MouseProInflammatory 7-Plex Tissue Culture Kit (MSD, catalog number K15012B). The IRAK4 inhibitor is effective in the TLR-mediated inflammation model. Figure 3It shows the amount of TNF-α in plasma, which is reduced in a dose-dependent manner by administration of Example Compound 11 compared to the LPS-induced concentration.

[0623] In vivo model of IL-1β-mediated inflammation

[0624] To evaluate the potential efficacy of the compounds of general formula (I) of the present invention in IL-1β-mediated diseases, female Balb / c mice (about 8 weeks old; Charles River Laboratories, Germany) were intraperitoneally administered IL-1β, and the efficacy of the compounds of the present invention on IL-1β-mediated cytokine secretion was investigated. There were 5 animals in each group. The control group was treated with a vehicle capable of dissolving the substance and IL-1β. The substance treatment group and the positive control group were intraperitoneally administered 90 μg IL-1β / kg body weight (R&D, catalog number 401-ML / CF), respectively. 6 hours before the administration of IL-1β, the substance or its vehicle was administered in the positive control group. 2 hours after the administration of IL-1β, TNF-α in plasma isolated from blood was measured using the Mouse ProInflammatory 7-Plex Tissue Culture Kit (MSD, catalog number K15012B) according to the manufacturer's instructions. The administration of IL-1β led to an increase in the plasma concentration of TNF-α, which was inhibited by treatment with Example Compounds 11 and 12. This is illustrated by Figure 4 description.

[0625] In vivo adjuvant-induced arthritis model

[0626] To determine the anti-inflammatory activity of the compounds of general formula (I) of the present invention, their in vivo efficacy was investigated in an arthritis model. For this purpose, on day 0, each male Lewis rat (about 100 - 125 g, Charles River Laboratories, Germany) was subcutaneously administered 100 μL of a solution of complete Freund's adjuvant (CFA) (combined with Mycobacterium tuberculosis H37Ra [Difo Lab, catalogue number - 231141], dissolved in incomplete Freund's adjuvant [Difco Lab, catalogue number - 263910]) at the base of the tail. There were n = 8 rats in each group. Both a healthy control group and a diseased control group were included in the study. Each control group was treated orally (p.o.) only with the vehicle of the test substance. In a prophylactic manner, i.e., starting from day 0, the animals were treated by oral administration with different doses of the test substance. On day 0, the initial condition of the animals was additionally determined according to the disease activity score (grading of the severity of arthritis based on a point system). Herein, for erythema including joint swelling for both hind paws together, 0 to 4 points were given according to the degree of joint inflammation (0 = none; 1 = mild; 2 = moderate; 3 = marked; 4 = severe). To determine the anti-inflammatory efficacy of the compounds, starting from day 8, when the animals first showed signs of arthritis, the disease activity of the animals was scored by means of the disease activity score, and then three times a week until the end (day 20). Statistical analysis was performed using one-way analysis of variance (ANOVA), and comparison with the control group was carried out by means of multiple comparison analysis (Dunnett's test).

[0627] Subcutaneous administration of CFA in rats results in acute arthritis with marked joint inflammation in the rats. This induced arthritis was inhibited by treatment with Example Compound 11. This is shown by Figure 5 illustrated.

[0628] Collagen antibody-induced arthritis model in mice

[0629] 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 (about 9 weeks old, Charles River Laboratories, Kingston, Canada) was intravenously injected into the tail vein with 200 μL of a collagen antibody mixture (10 mg / mL; ArthritoMab, MD Bioproducts) (except for the healthy control group included in the study). Then on day 6, these mice were further given an intraperitoneal injection of 200 μL 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 orally treated only with the vehicle of the test substance. In a prophylactic manner, that is, starting from day 0, different doses of the test substance were administered orally for treatment. During the experiment, based on the point determination system for disease activity scoring, the degree of disease in all four paws was scored. In this point determination, for healthy paws, 0 points were determined, while for a specific degree of joint inflammation occurring from the toes through the metatarsophalangeal joint to the ankle joint, 1 point [mild inflammation, such as toe inflammation] to 4 points [severe inflammation extending to the entire paw] were determined respectively, as described below:

[0630] ● 0 = normal

[0631] ● 1 = erythema and mild swelling limited to the tarsus or ankle joint or toes

[0632] ● 2 = erythema and mild swelling extending from the ankle joint to the metatarsus (2 stages)

[0633] ● 3 = erythema and moderate swelling extending from the ankle joint to as far as the metatarsophalangeal joint

[0634] ● 4 = erythema and severe swelling covering the metatarsus, foot and toes

[0635] For this parameter, the starting condition was determined in advance one day before the start of the experiment (day -1), and subsequently, starting from day 8, the disease activity score was scored three times a week. Statistical analysis was performed using one-way analysis of variance (ANOVA) and compared with the control group by means of multiple comparison analysis (Dunnett's test).

[0636] Intravenous administration of the collagen antibody mixture in mice, including subsequent intraperitoneal administration of LPS, resulted in acute arthritis with obvious joint inflammation. Treatment with Example Compound 12 inhibited this induced arthritis. This is illustrated by Figure 6 description.

[0637] In vivo NASH mouse model

[0638] To experimentally induce NASH, 200 μg of streptozotocin (STZ; Sigma-Aldrich, USA) was subcutaneously injected into 45 male 2-day-old C57BL / 6 mice. Starting at 4 weeks of age, these animals were fed ad libitum a high-fat diet (HFD; 57 kcal% fat, #HFD32 purchased from CLEA, JAPAN). At 6 weeks of age, the animals were randomly divided into 3 groups (15 animals per group). One group received no treatment, but the other 2 groups were orally administered a vehicle or a test substance daily for 4 weeks. After 4 weeks of treatment, all animals were painlessly sacrificed under anesthesia, the livers were removed and fixed in Bouin's solution for histological studies (H. Denk, " Fixierung histologischer Präparate" [Fixing of Histological Preparations], in: (Editor): "Romeis Mikroskopische Technik" [Romei's Microscopy Techniques], Urban & Schwarzenberg, Munich-Vienna-Baltimore 1989, 17th edition, p. 97, ISBN 3-541-11227-1). Thereafter, liver samples were embedded in paraffin and 5-μm-thick paraffin sections were prepared. The tissue sections of each liver were stained, a) with hematoxylin-eosin (HC) for determination of the NAFLD activity score (NAS), b) with Picro-Sirius red (Waldeck, Germany) for determination of liver fibrosis. The NAFLD activity score was determined in hematoxylin-eosin sections based on the criteria recommended by D.E. Kleiner et al., Hepatology 41 (2005), 1313-1321 (Table 1). For histological quantification of the fibrotic area, 5 digital photographs (DFC280; Leica, Germany) were taken of each section at a microscopic magnification of 200-fold, and the percentage of fibrosis was determined using ImageJ Software (National Institutes of Health, USA).

[0639] In vivo db / db mouse model

[0640] Thirty 8-week-old male db / db mice were used. This model is a widely accepted model of obesity, insulin resistance, and type 2 diabetes (Aileen JF King; The use of animal models in diabetes research; British Journal of Pharmacology 166 (2012), 877–894). During the experiment, the animals were fed a standard diet (RM1(E)801492, SDS) and had free access to tap water. The animals were randomly divided into 3 groups (10 animals per group) and orally treated with the test substance for 6 weeks. During the study, blood was taken from the animals at different time points (before the start of treatment, 3 weeks after the start of treatment, and 2 days before the end of treatment) to determine insulin sensitivity parameters (such as HbA1c, glucose content, insulin content). In addition, an OGTT (oral glucose tolerance test), which is a parameter for determining insulin sensitivity, was performed 1 day before the start of treatment and 2 days after the end of treatment. In addition, the HOMA-IR index was calculated (fasting insulin level (mU / L) * fasting blood glucose level (mmol / L) / 22.5).

[0641] Xenograft model related to B-cell lymphoma in vivo

[0642] The antitumor activity of the compounds of general formula (I) of the present invention was studied in a murine xenograft model. For this purpose, female C.B-17 SCID mice were subcutaneously implanted with a human B-cell lymphoma cell line, such as TMD-8. When the average tumor size was 20 - 30 mm 2 , oral monotherapy with the compounds of the present invention was started, or the compounds of the present invention were combined with standard therapy, each by oral administration. However, the animals were randomized beforehand. Once the untreated control group had large tumors, the treatment was immediately terminated. The 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% = toxic, terminate). The tumor area [length (mm) × width (mm)] was detected by an electronic caliper. At the end of the study, the tumor weight was also determined. The antitumor efficacy was defined as the ratio of the tumor weight of the treatment group to that of the control group (T / C) [tumor weight of the treatment group on day x / tumor weight of the control group on day x] or the ratio of the tumor area of the treatment group to that of 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 comparison analysis (Dunnett's test) was performed by comparison with the control group.

[0643] Canine IRAK4 kinase assay

[0644] The IRAK-4 inhibitory activity of the compounds of the present invention against canine IRAK4 was determined in the Irak4TR-FRET assay (TR-FRET = time-resolved fluorescence resonance energy transfer) described below.

[0645] A recombinant fusion protein of HIS (polyhistidine) from the N-terminus and canine IRAK4, which 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, was used as the enzyme. The substrate for the kinase reaction was biotinylated peptide biotin-Ahx-KKARFSRFAGSSPSQASFAEPG (C-terminus in amide form), which was available from, for example, Biosyntan GmbH (Berlin-Buch).

[0646] For the experiment, 11 different concentrations in the range of 20 μM to 0.073 nM were prepared from a DMSO solution of the test substance at 2 mM. 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 a solution of IRAK4 in the assay buffer [50 mM HEPES at pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w / v) bovine γ-globulin (BGG), 0.04% (v / v) Nonidet-P40 (Sigma)] was added, and the mixture was incubated for 15 min to allow the substance to pre-bind to the enzyme before the kinase reaction. Then the kinase reaction was initiated by adding 3 μL of adenosine triphosphate (ATP, 1.67 mM = final concentration in 5 μL assay volume: 1 mM) and a peptide substrate (0.83 μM = final concentration in 5 μL assay volume: 0.5 μM) in the assay buffer, and the resulting mixture was incubated for a reaction time of 45 min at 22 °C. The concentration of Irak4 was adjusted to the respective activity of the enzyme and set such that the assay was carried out in the linear range. Typically the concentration was on the order of about 0.1 nM. The reaction was terminated by adding 5 μL of a 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 AD0071; or a terbium cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used) in a solution of aqueous EDTA (100 mM EDTA, 0.4% (w / v) bovine serum albumin [BSA] in 25 mM HEPES at pH 7.5).

[0647] The resulting mixture was incubated at 22 °C for 1 h to form a complex of biotinylated phosphorylated substrate and the detection reagent. The amount of phosphorylated substrate was then evaluated by measuring the resonance energy transferred from the europium chelate-labeled anti-mouse IgG antibody 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 emissions at 620 nm and 665 nm were measured. The emission ratio at 665 nm and 622 nm was taken 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 substance was tested at 11 different concentrations in the range 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 before the assay (2 mM to 7.3 nM in 100% DMSO). The IC 50 values were calculated by 4-parameter fitting.

[0648] Table 3: The IC 50 values of two experiments of the example compounds in the IRAK4 canine kinase assay

[0649]

[0650] In vitro lipopolysaccharide (LPS)-induced cytokine production by canine peripheral blood mononuclear cells (PBMCs)

[0651] The efficacy of the compounds of general formula (I) of the present invention on the production of inducible cytokines in canine PBMCs was investigated. Cytokine production herein was induced by LPS - a TLR4 ligand, which leads to the activation of the IRAK4-mediated signaling pathway.

[0652] Canine PBMCs were obtained from dog anticoagulated whole blood. For this purpose, plasma rich in canine leukocytes was prepared by centrifuging 15 mL of dog blood at 400 g for 15 min at 4 °C, then collected, and then the canine PBMC buffy coat was suspended in the plasma. 7 (seven) mL of Ficoll-Paque Plus (Fischer Scientific, catalog number 11778538) was pipetted into a centrifuge tube, and then 7 mL of plasma rich in canine leukocytes 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 between the canine plasma and Ficoll-Paque Plus. The PBMCs were transferred to a new centrifuge tube and supplemented with Hanks' Balanced Salt Solution 1x (HBSS) without Ca 2+ / Mg 2+ (Sigma-Aldrich, catalog number H9394). The cell suspension was centrifuged at 400 g for 5 min at 4 °C, and then the supernatant was discarded. Then the cell pellet was 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. Then the cell pellet was resuspended in HBSS without Ca 2+ / Mg 2+ for a final wash and centrifuged at 400 g for 5 min at 4 °C. Then, the PBMCs were resuspended in complete 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)).

[0653] The experiments were also conducted in complete medium. The PBMCs were seeded at 2.5×10 5Cells were seeded at a density of

[0654] In vitro lipopolysaccharide (LPS)-induced cytokine production by bovine peripheral blood mononuclear cells (PBMCs)

[0655] The efficacy of the compounds of general formula (I) of the present invention on the production of inducible cytokines in bovine PBMCs was investigated. Cytokine production herein was induced by LPS - a TLR4 ligand, which leads to the activation of the IRAK4-mediated signaling pathway.

[0656] Bovine PBMCs were obtained from bovine anticoagulated whole blood. For this purpose, plasma rich in bovine leukocytes was prepared by centrifuging 500 mL of bovine blood at 1000 g for 20 min at room temperature (RT), followed by collection. Then, the buffy coat of bovine PBMCs was suspended in an equal volume of PBS / 5 mM EDTA (at 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 the bovine PBMC buffy coat / PBS / EDTA mixture was layered on top of the Ficoll-Paque Plus. After centrifuging at 800 g for 25 min at room temperature, bovine PBMCs were collected from the interface between 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. Then the cell pellet was 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 500 g for 5 min at 4 °C. Then the cell pellet was resuspended in complete medium (DMEM with GlutaMAX (ThermoFisher, catalog number 32430100), 10% horse serum ( 30-2040 TM ), 20 μM β-mercaptoethanol (ThermoFisher catalog number 31350010 [stock solution: 50 mM])).

[0657] The experiments were also carried out in complete medium. The PBMCs were seeded at a cell density of 1×10 6 cells / well in a 24-well plate. The compounds of the present invention were dissolved in DMSO and serially diluted in complete medium. The compound examples were used at 8 different concentrations in the range of 0.003 μM to 10 μM in the experiments, such that the final DMSO concentration was 0.5%. To induce cytokine secretion, the cells were stimulated with 1 μg / mL ( Figure 8 ) and 0.1 μg / mL ( Figure 9 ) of LPS (LPS from E. coli K12; Invivogen #tlrl-eklps) for 24 hours. Cell viability was determined using Türk solution (Merck Millipore #1092770100).

[0658] The amount of TNFα secreted in the cell culture supernatant of bovine PBMCs exposed to LPS was determined using an ELISA read-out based on a rabbit anti-bovine TNFα antibody. The ELISA assay was performed in 384-well ELISA plates, which were coated with 5 μg / mL rabbit anti-bovine TNFα antibody (BioRad, AHP2383) in 50 mM Na2CO3 / NaHCO3 buffer at pH 9.6 at 10 μL / well 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) for 90 min at 37 °C. Subsequently, the blocking buffer was removed and the culture supernatant samples (20 μL / well) were added. After incubation for 90 min at 37 °C, 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 plates (20 μl / well) and 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 TM -alkaline phosphatase (Sigma, E2636), 20 μL / well, was added and kept for 1 h. After removing ExtrAvidin TM -alkaline phosphatase and rinsing the wells three times with 50 μL of wash buffer, the enzymatic reaction / color development was initiated by adding 50 μL / well of chromogenic buffer (5 mM p-nitrophenyl phosphate (pNPP) in 50 mM Na2CO3 / NaHCO3 at pH 9.6, 2 mM MgCl2). The optical density was recorded at a wavelength of 405 nm. For kinetic measurements, data points were recorded every 5 minutes for 1 hour and an end-point measurement was performed after 2 hours. For example, Example Compound 12 inhibited the production of TNFα by LPS-stimulated bovine PBMCs. This is demonstrated by Figure 8 and Figure 9 as described.

[0659] In vitro lipopolysaccharide (LPS)-induced cytokine production by porcine peripheral blood mononuclear cells (PBMCs)

[0660] As another example, the efficacy of the compounds of general formula (I) of the present invention on the production of inducible cytokines in porcine PBMC was investigated. In this article, cytokine production was induced by LPS - a TLR4 ligand, which leads to the activation of the IRAK4 - mediated signaling pathway.

[0661] Porcine PBMC were obtained from porcine anticoagulated whole blood. For this purpose, plasma rich in porcine leukocytes was prepared by centrifuging 36 mL of porcine blood at 1000 g for 20 min at room temperature (RT), then collected, and then the porcine PBMC buffy coat was 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 the porcine PBMC buffy coat / PBS / EDTA mixture was layered on top of the Ficoll - Paque Plus. After centrifuging at 800 g for 25 min at room temperature, porcine PBMC were collected from the interface between porcine plasma and Ficoll - Paque Plus. The PBMC 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. Then the cell pellet was 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 500 g for 5 min at 4 °C. Then the PBMC cell pellet was resuspended in complete medium (DMEM and GlutaMAX (ThermoFisher, catalog number 32430100), 10% horse serum ( 30 - 2040 TM ), 20 μM of β - mercaptoethanol (ThermoFisher catalog number 31350010 [stock solution: 50 mM])).

[0662] The assays were also carried out in complete medium. PBMC were seeded onto 24 - well plates at a cell density of 1×10 6 cells / well. The compounds of the present invention were dissolved in DMSO and subjected to a series of dilutions in complete medium. Compound examples were used at 8 different concentrations in the range of 0.003 μM to 10 μM in the assays, such that the final DMSO concentration was 0.5%. To induce cytokine secretion, cells were stimulated with LPS (LPS from E. coli K12; Invivogen #tlrl - eklps) at a concentration in the range of 0.01 ng / mL to 1 ng / mL for 24 hours. Cell viability was determined using Türk solution (Merck Millipore #1092770100).

[0663] The amount of TNFα secreted in the cell culture supernatant of porcine PBMCs exposed to LPS was determined using an ELISA readout device based on a rabbit anti-porcine TNFα antibody. The ELISA assay was performed in a 384-well ELISA plate, which was coated with 3 μg / mL of rabbit anti-porcine TNFα antibody (BioRad, AHP2397) in 50 mM Na2CO3 / NaHCO3 buffer at pH 9.6 at 10 μL / well and incubated at 4 °C for 48 h. 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) and 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 TM -Alkaline phosphatase (Sigma, E2636), 20 μL / well, was added and incubated for 1 h. After removing ExtrAvidin TM -Alkaline phosphatase and rinsing the wells three times with 50 μL of wash buffer, the enzymatic reaction / color development was initiated by adding 90 μL / well of chromogenic buffer (5 mM p-nitrophenyl phosphate (pNPP) in 50 mM Na2CO3 / NaHCO3 at pH 9.6, 2 mM MgCl2). The optical density was recorded at a wavelength of 405 nm. For kinetic measurements, data points were recorded every 5 minutes for 1 hour, and endpoint measurements were performed after 2 hours. For example, 10 μM of Example Compound 12 inhibited the production of TNFα in bovine PBMCs stimulated with 0.1 ng / mL LPS. This is demonstrated by Figure 10 description.

[0664] In vivo model of house dust mite-induced canine allergic dermatitis

[0665] To evaluate the potential anti-allergic / anti-inflammatory efficacy of the compounds of general formula (I) of the present invention, a house dust mite (HDM)-sensitized Beagle dog model was used. Among them, HDM sensitization included a series of subcutaneous injections of HDM antigen (10 μg, Greer Laboratories, Lenoir, NC, USA) and (0.2 mL, InvivoGen, San Diego, CA 921221, USA) as an adjuvant at approximately two-week intervals. The sensitization process was monitored and confirmed by intradermal skin testing. Once the dogs were positive for the HDM intradermal skin test, one month after the last sensitization, HDM antigen (135 μg) was topically applied and injected intradermally (using a 2-mm long micro needle) into the skin inside the hind legs of adult Beagle dogs to detect the efficacy of the compounds of the present invention against allergic dermatitis symptoms such as erythema and edema. There were 2 groups, with 4 animals in each group: 1 group was the placebo control group, and 1 group was treated with Example Compound 12. The control group was orally treated with gelatin capsules containing microcrystalline cellulose, while the group treated with Example Compound 12 was orally treated with gelatin capsules containing Example Compound 12 and microcrystalline cellulose. Example Compound 12 or placebo was administered starting 5 days before the HDM antigen challenge and continued until 2 days after the challenge. The treatment frequency was once a day, and in the case of Example Compound 12, the dose was 10 mg / kg body weight. At 30 min after the start of the challenge and at different time points within 48 h, erythema and edema were evaluated using VAS (Visual Analogue Scale) in both groups. Plasma samples were analyzed to determine the exposure of the compounds related to the clinical assessment. After treatment with Example Compound 12, edema and erythema were significantly reduced. This is shown in Tables 4 and 5, as well as Figure 11 and Figure 12 illustrated.

[0666] Table 4: Erythema (in VAS units) after treatment with Example Compound 12 and placebo

[0667]

[0668] Table 5: Edema (in VAS units) after treatment with Example Compound 12 and placebo

[0669]

[0670] In vivo pruritus model of flea allergic dermatitis

[0671] To evaluate the potential antipruritic efficacy of the compounds of general formula (I) of the present invention, a flea allergic dermatitis (FAD) model was used. Only adult dogs with a history of FAD were included in the study. The lifetime of the study consisted of two phases: an itch induction phase (2 weeks) and a subsequent treatment phase (2 weeks). During both study phases, the dogs were infested with Ctenocephalides fleas twice a week (100 fleas / dog for the first infestation and 30 fleas / dog for all subsequent infestations). There were 2 groups of 12 animals each: 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 microcrystalline cellulose, while the group treated with Example Compound 12 was treated orally with gelatin capsules containing Example Compound 12 and microcrystalline cellulose. 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 scratching, licking, and biting. Plasma samples were analyzed to determine the exposure of the compound relevant to clinical evaluation. After 10 days of treatment with Example Compound 12, itching was significantly reduced. This is shown in Table 6 and Figure 13 description.

[0672] Table 6: Reduction of pruritic behavior relative to baseline after treatment with Example Compound 12 compared to placebo (shown as percentage change from baseline on the days listed after treatment)

[0673] 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％

[0674] Figure 1 : Inhibition of IL-23 in DCs generated from human monocytes by Example Compound 12. Data are shown as mean with standard deviation.

[0675] Figure 2: Inhibition of INF-α in human plasmacytoid DCs stimulated with imiquimod (R837)- or (B) CpG-A by Example Compound 12. Data are shown as mean with standard deviation.

[0676] Figure 3 : Treatment with Example Compound 11 for LPS-induced inflammation resulted in a decrease in the amount of TNF-α secreted. Data are shown as mean with standard deviation.

[0677] Figure 4 : Treatment with Example Compound 11 (left) and 12 (right) for IL-1β-induced inflammation resulted in a dose-dependent decrease in the amount of TNF-α secreted. Data are shown as mean with standard deviation.

[0678] Figure 5: Anti-inflammatory efficacy of Example Compound 11 in an animal model of rheumatoid arthritis (adjuvant-induced rat model). Significant and dose-dependent inhibition of rheumatoid arthritis based on disease activity scores. Data correspond to mean + standard deviation. One-way ANOVA followed by multiple comparison analysis with the CFA control group by Dunnett’s test; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0679] Figure 6 : Anti-inflammatory efficacy of Example Compound 12 in an animal model of rheumatoid arthritis (collagen antibody-induced mouse model). Significant and dose-dependent inhibition of rheumatoid arthritis based on disease activity scores. Data correspond to mean + standard deviation. Statistical significance between the collagen antibody (AK) control group and the treatment groups was calculated by one-way ANOVA followed by multiple comparison analysis (Dunnett’s test) (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0680] Figure 7 : Inhibition of LPS-induced TNFα production by canine PBMCs by Example Compound 12. Data shown are mean values with standard deviation.

[0681] Figure 8 : Dose-dependent inhibition (kinetic assay) of TNFα production by bovine PBMCs induced by 1 μg / mL LPS by Example Compound 12. Data shown are mean values with standard deviation of three biological materials, each measured twice. The IC50 value determined from the curve was 120 nM.

[0682] Figure 9 : Dose-dependent inhibition (kinetic assay) of TNFα production by bovine PBMCs induced by 1 μg / mL LPS by Example Compound 12. Data shown are mean values with standard deviation of three biological materials, each measured twice. The IC50 value determined from the curve was 70.5 nM.

[0683] Figure 10 : Inhibition (kinetic assay) of TNFα production by porcine PBMCs induced by 0.1 ng / mL LPS by 10 μM of Example Compound 12. Data shown are mean values with standard deviation of three biological materials, each measured twice.

[0684] Figure 11 : Treatment of an indoor dust mite-induced canine atopic dermatitis model with Example Compound 12 resulted in a reduction in erythema (a). Data shown are mean values with standard deviation.

[0685] Figure 12 Treatment of a canine atopic dermatitis model induced by Dermatophagoides pteronyssinus with Example Compound 12 resulted in a reduction in edema (b). Data are shown as mean with standard deviation.

[0686] Figure 13 :The antipruritic efficacy of Example Compound 12 in an animal model of flea allergic dermatitis. Data are expressed as percent change from baseline corresponding to the median.

Claims

1. Use of a compound of the following formula or a salt thereof in the preparation of a medicament for treating and / or preventing allergic dermatitis in dogs; 2. The use according to claim 1, wherein the allergic dermatitis is atopic dermatitis or flea allergic dermatitis.

3. The use according to claim 1, wherein the allergic dermatitis is atopic dermatitis.

4. The use according to claim 1, wherein the allergic dermatitis is flea allergic dermatitis.

5. The use according to claim 1, which is for treatment.

6. The use according to claim 2, which is for treatment.

7. The use according to claim 1, wherein the compound is a pharmaceutically acceptable salt.

8. The use according to claim 2, wherein the compound is a pharmaceutically acceptable salt.

9. Use of a compound of the following formula or a salt thereof in the preparation of a medicament for treating and / or preventing inflammatory diseases in dogs; 10. The use according to claim 9, which is for treatment.

11. The use according to claim 9, wherein the compound is a pharmaceutically acceptable salt.

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