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

Abstract

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

Description

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

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

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

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

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

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

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

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

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

[0010] US8293923 and US20130274241 disclose IRAK4 inhibitors having a 3-substituted indazole structure, but there is no record of 2-substituted indazole.

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

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

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

[0014]

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

[0016]

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

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

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

[0020] In addition to the above-mentioned substitution forms at positions 1 and 2 on indazole, WO2015104662 also describes indazoles having a substituent at position 6, where R1 is defined as follows: alkyl, cyano, -NR a R b or an optional substituent selected from a cycloalkyl, aryl or heterocyclic group, wherein the substituent is independently alkyl, alkoxy, halogen, hydroxyl, 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, to which priority is claimed, do not disclose any indazole compounds wherein R1 is an alkyl group.

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

[0022]

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

[0024] Examples of the 6-position substituent R1 described in WO2015104662 are cyclopropyl, cyclohexyl, cyano, 3-fluorophenyl and saturated heterocyclic substituents. Indazoles having a hydroxy-substituted alkyl group at the 6-position are not explicitly described in WO2015104662.

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

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

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

[0028] The IRAK4 inhibitors of the present invention are particularly suitable for treating and preventing animal inflammatory diseases characterized by an overreactive immune system. The following diseases should be mentioned in particular: canine atopic dermatitis and flea allergy dermatitis in dogs and cats; inflammatory bowel disease in dogs and cats; osteoarthritis pain and inflammatory pain in dogs, cats, horses and cattle; non-infectious recurrent airway disease in horses (also known as chronic obstructive pulmonary disease, emphysema); insect allergies in horses (also known as sweet itch, summer eczema); cat asthma; bovine respiratory disease; bovine mastitis and endometritis; 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, with recurring episodes throughout their lives. In a 1999 study by Lund et al., which surveyed 31,484 dogs in 52 private practices in the United States, the prevalence of CAD was 8.7%. CAD is the second most common cause of pruritus in dogs after flea allergy dermatitis (FAD).

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

[0032] In all cases, IgE was not a prerequisite for the development of clinical symptoms, and a separate clinical entity called atopic dermatitis was defined as "an inflammatory and pruritic skin disease with clinical features identical to those observed in canine atopic dermatitis in which IgE responses to environmental or other allergens have not been 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, clawing, and excessive chewing of areas such as the groin and armpits. Over time, scratched skin can develop hot spots—raw, inflamed areas—that can become infected.

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

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

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

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

[0039] As the disease progresses, hair loss, broken hair, oozing or crusty sores, pimply bumps, generalized redness, and skin inflammation may occur. The sores can be very painful. In severe cases, the skin can become thickened and darkened, primarily on the dog's back and tail base area. The dog may inflict self-inflicted injuries due to the severe itching.

[0040] In general, prevention and treatment of flea infestations are the treatment of choice. Use the most common neonicotinoids such as imidacloprid, or gamma-aminobutyric acid (GABA)-gated chloride channel blockers such as fipronil. If skin allergic dermatitis symptoms do not resolve, use current treatments mentioned under CAD, such as topical and oral corticosteroids, oral cyclosporine, and oral oclacitinib.

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

[0042]

[0043] In formula (I):

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

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

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

[0047] or a group selected from the following:

[0048]

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

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

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

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

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

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

[0055] R 8 is C1-C6-alkyl, where the C1-C6-alkyl radical is unsubstituted or monosubstituted or identically or differently polysubstituted by halogen.

[0056] In the case of the described uses of the synthetic intermediates and working examples of the invention described below, any compound specified in the form of a salt of the corresponding base or acid is generally a salt of unknown exact stoichiometric composition obtained by a corresponding preparation and / or purification process. Therefore, for such salts, other names and structural formulae such as "hydrochloride", "trifluoroacetate", "sodium salt" or "xHCl", "xCF3COOH", "xNa + " is not to be understood in a stoichiometric sense but is merely an illustrative notation for the salt-forming components present therein.

[0057] This also applies correspondingly in the case of synthesis intermediates or working examples or their salts which are obtained by the preparation and / or purification processes described and whose stoichiometric composition is unknown (if it is of a defined type) in the form of solvates, such as hydrates.

[0058] The compounds of the present invention are compounds of formula (I) and salts, solvates and solvates of salts thereof, compounds covered by formula (I) and of the following structural formulas and salts, solvates and solvates of salts thereof, and compounds covered by formula (I) and mentioned below as embodiments and salts, solvates and solvates of salts thereof, even if the compounds covered by formula (I) and mentioned below are not salts, solvates and solvates of salts thereof.

[0059] In the context of the present invention, preferred salts are physiologically acceptable salts of the compounds according to the invention. However, the present disclosure also encompasses salts which are not suitable for pharmaceutical applications per se but which can be used, for example, for the isolation or purification of the compounds according to the invention.

[0060] Physiologically acceptable salts of the compounds of the invention include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, for example, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

[0061] Physiologically acceptable salts of the compounds according to the invention also include salts with customary bases, such as and preferably alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts), and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, such as and preferably ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.

[0062] In the context of the present invention, solvates are described as those forms of the compounds according to the invention which form solid or liquid complexes by coordination with solvent molecules. Hydrates are a special form of solvates which coordinate with water.

[0063] The compounds according to the invention may, depending on their structure, exist in different stereoisomeric forms, i.e. in the form of configurational isomers or, if appropriate, conformational isomers (enantiomers and / or diastereomers, including atropisomers). The invention therefore includes the use of enantiomers and diastereomers and their respective mixtures. The stereoisomerically homogeneous constituents can be isolated from these mixtures of enantiomers and / or diastereomers in a known manner; for this purpose, preferably chromatography is used, in particular HPLC chromatography on an achiral or chiral phase.

[0064] If the compounds according to the invention can exist in tautomeric forms, the present invention includes the use of all tautomeric forms.

[0065] The present invention also includes the purposes of all suitable isotopic variants of the compounds of the present invention. In this article, the isotopic variants of the compounds of the present invention are understood to mean such compounds: wherein at least one atom in the compounds of the present invention has been replaced with the same atomic number, but the atomic mass is different from another atom of the atomic mass usually or mainly present in nature. Examples of isotopes that can be incorporated into the compounds of the present invention are those isotopes 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. Special isotopic variants of the compounds of the present invention, such as those variants in which one or more radioactive isotopes have been incorporated, can be beneficial for investigating the mechanism of action or active ingredient distribution in vivo; due to relatively easy preparability and detectability, compounds labeled with 3H or 14C isotopes are particularly suitable for this purpose. In addition, the incorporation of isotopes (e.g., deuterium) may produce special therapeutic benefits due to the higher metabolic stability of the compound, such as an extension of the half-life in vivo or a reduction in the required active dose; therefore, in some cases, such modifications to the compounds of the present invention may also constitute a preferred embodiment of the use of the present invention. Isotopic variants of the compounds of the present invention can be prepared by methods known to those skilled in the art, such as by the methods further described below and the methods described in the working examples, by using the corresponding isotopic modifications of the respective reagents and / or starting compounds.

[0066] The present invention further provides for the use of all possible crystalline forms and polymorphs of the compounds according to the invention, wherein the polymorphs may be present as single polymorphs or as mixtures of a plurality of polymorphs within the entire concentration range.

[0067] Furthermore, the present invention also encompasses the use of prodrugs of the compounds according to the invention. In this context, the term "prodrug" refers to a compound which itself may be biologically active or inactive, but is converted (eg metabolically or hydrolytically) into a compound according to the invention during its residence time in the body.

[0068] In the context of the present invention, unless otherwise stated, substituents have the following meanings:

[0069] alkyl In the context of the present invention, it means a straight or branched alkyl group with a specified specific number of carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl, 2-methylpropyl, tert-butyl, n-pentyl, 1-ethylpropyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl and 2-ethylbutyl. Methyl, ethyl, n-propyl, n-butyl, 2-methylbutyl, 3-methylbutyl and 2,2-dimethylpropyl are preferred.

[0070] Cycloalkyl In the context of the present invention are monocyclic saturated alkyl radicals having in each case the indicated number of carbon atoms. Preferred examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0071] Alkoxy In the context of the present invention, a straight or branched alkoxy group having a specified specific number of carbon atoms is represented. Preferably 1 to 6 carbon atoms. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, 1-methylpropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy, 1-ethylpropoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy and n-hexyloxy. Particularly preferably, a straight or branched alkoxy group having 1 to 4 carbon atoms is represented. Examples mentioned as preferred are methoxy, ethoxy, n-propoxy, 1-methylpropoxy, n-butoxy and isobutoxy.

[0072] halogen In the context of the present invention these are fluorine, chlorine and bromine. Preference is given to fluorine.

[0073] Hydroxyl In the context of the present invention this is OH.

[0074] Monocyclic saturated heterocyclic ringIt is a monocyclic saturated heterocycle having 4 to 6 ring atoms and containing a heteroatom or hetero group selected from O, S, SO and SO2. It is preferably a heterocycle having a heteroatom or hetero group 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-dioxotetrahydrothiophene-3-yl, 1,1-dioxotetrahydrothiophene-2-yl, 1,1-dioxothiani-2-yl or 1,1-dioxothiani-3-yl. Oxetane and tetrahydrofuran are particularly preferred in this article. Oxetane-3-yl is very particularly preferred.

[0075] The symbol * on a bond indicates the bonding site in the molecule.

[0076] Unless otherwise indicated, when a group in the compounds of the invention is substituted, the group may be monosubstituted or polysubstituted. In the context of the present invention, all groups occurring more than once are defined independently of one another. Preferably, they are substituted by one, two or three identical or different substituents.

[0077] R 1 A preferred embodiment is a C2-C6-alkyl group substituted by 1, 2 or 3 fluorine atoms. Particular preference is given to 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl and 4,4,4-trifluorobutyl. Very particular preference is given to the 4,4,4-trifluorobutyl group.

[0078] R 1 Another preferred embodiment is a C2-C6-alkyl group substituted by one or two hydroxyl groups or a C1-C3-alkoxy or trifluoro-substituted C1-C3-alkoxy. Particularly preferred is a C2-C5-alkyl group substituted by a hydroxyl or C1-C3-alkoxy or trifluoromethoxy or 2,2,2-trifluoroethoxy. Very particularly preferred is 3-hydroxy-3-methylbutyl, 3-methoxypropyl, 3-hydroxypropyl, 3-trifluoromethoxypropyl, 2-methoxyethyl or 2-hydroxyethyl. Especially preferred is a 3-hydroxy-3-methylbutyl group.

[0079] Also preferably, R 1 is a C2-C6-alkyl group substituted by a C1-C6-alkyl-SO2 group. Particularly preferred is a C2-C4-alkyl group substituted by a methyl-SO2-group. 1 It is 2-(methylsulfonyl)ethyl or 3-(methylsulfonyl)propyl. In the latter group, 2-(methylsulfonyl)ethyl is particularly preferred.

[0080] In addition, preferably, R 1is a C1-C3-alkyl group substituted by an oxetane group, a tetrahydrofuranyl group, a tetrahydro-2H-pyran-4-yl group, a 1,1-dioxotetrahydro-2H-thiopyran-3-yl group, a 1,1-dioxotetrahydro-2H-thiopyran-2-yl group, a 1,1-dioxotetrahydro-2H-thiopyran-4-yl group, a 1,1-dioxotetrahydrothiophen-3-yl group, a 1,1-dioxotetrahydrothiophen-2-yl group, a 1,1-dioxothiane-2-yl group or a 1,1-dioxothiane-3-yl group. A C1-C3-alkyl group substituted by an oxetane group is particularly preferred. R is particularly preferred. 1 It is an oxetan-3-ylmethyl group.

[0081] For R 2 and R 3 , always have the same definition, preferably hydrogen or methyl. Particular preference is given to methyl.

[0082] For R 4 , preferably an unsubstituted or mono- or poly-halogen-substituted C1-C3-alkyl group, or a C1-C3-alkyl group substituted by one hydroxyl group, or a C1-C3-alkyl group substituted by one hydroxyl group and three fluorine atoms.

[0083] For R 4 , particularly preferably the following groups: methyl, ethyl, trifluoro-C1-C3-alkyl, difluoro-C1-C3-alkyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxypropan-2-yl and 2,2,2-trifluoro-1-hydroxyethyl. 4 , particularly preferably methyl, trifluoromethyl and difluoromethyl groups. The trifluoromethyl group is particularly preferred herein.

[0084] R 5 A preferred embodiment of is hydrogen, fluorine, chlorine or C1-C3-alkyl. More preferably, R 5 is hydrogen, fluorine or methyl. Most preferably, R 5 is hydrogen or fluorine.

[0085] R is also particularly preferred 4 is methyl or trifluoromethyl and R 5 Very particularly preferably R 4 is methyl and R 5 A compound of fluorine, wherein R 5 In R 4 The adjacent position.

[0086] For R 6, preferred embodiments include oxetane, 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-dioxotetrahydrothiophene-3-yl, 1,1-dioxotetrahydrothiophene-2-yl, 1,1-dioxothiani-2-yl or 1,1-dioxothiani-3-yl. Oxetane is particularly preferred herein. Oxetane-3-yl is very particularly preferred.

[0087] R 7 Only connected to the functional groups -SO2- and -SO-, that is, R 7 -substituted -SO2- or SO group. 7 Preferably, C1-C4-alkyl, wherein the C1-C4-alkyl group is unsubstituted or monosubstituted by hydroxyl or cyclopropyl or by three fluorine atoms. 7 is a cyclopropyl group. 7 is methyl, ethyl or hydroxyethyl. Very particularly preferably R 7 It is methyl.

[0088] This means that, when R 7 SO2- or R 7 In the case of SO-substituted C1-C6-alkyl groups, R 1 For R, C1-C6-alkyl substituted by C1-C6-alkyl-SO2 or C1-C6-alkyl-SO is preferred. 1 In this context, methylsulfonylethyl and methylsulfonylpropyl are particularly preferred. In this context, methylsulfonylethyl is very particularly preferred.

[0089] 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.

[0090] As mentioned above, the intracellular enzyme interleukin-1 receptor-associated kinase 4 (IRAK4) plays an integral role in the signaling pathways of receptors activated by cytokines and TLR ligands involved in inflammatory processes. In addition to inflammation, IRAK4 is also involved in the signaling of allergic processes. Such allergic processes play an important role in the pathogenesis of allergic skin diseases such as atopic dermatitis.

[0091] For example, IL-33, which was recently added to the IL-1 cytokine family that also includes IL-18 and IL-1, binds to and activates the IL-33 receptor (IL-33R), which in turn is associated with MyD88, IRAK4, and TRF6 (Schmitz et al., Immunity, 2005). IRAK4 is an essential component of this signaling pathway. IL-33R is strongly expressed in T helper cell type 2 (Th2) cells, mast cells, and eosinophils. IL-33 activates these cells and promotes the immune response of Th2 (Schmitz et al., Immunity, 2005). These cell types are all involved in the pathogenesis of atopic dermatitis. The IL-33 content in serum is associated with the severity of human atopic dermatitis and is reduced when treated with topical steroids & calcineurin inhibitors (Tamagawa-Mineoka et al., J American Academy Dermatology, 2014). In a model of acute canine atopic dermatitis, the IL-33 gene has been shown to be significantly upregulated in skin lesions (Schamber et al., G3 (Bethesda), 2014; Olivry et al., Journal of Investigative Dermatology, 2016).

[0092] In addition, the second member of the IL-1 cytokine family, IL-18 is associated with atopic dermatitis. Serum IL-18 levels increase with the severity of atopic dermatitis in children (Sohn et al., Allergy and Asthma Proceedings, 2004). In the model of acute canine atopic dermatitis, it has been shown that the IL-18 gene is significantly upregulated in skin lesions (Schamber et al., G3 (Bethesda), 2014; Olivry et al., Journal of Investigative Dermatology, 2016). In addition, atopic dermatitis inflammation & pruritus is caused by excessive release of IL-18 and accelerated by mouse IL-1 (Konishi et al., Proceedings of the National Academy of Sciences, 2002). In addition, it has been shown that IRAK-4 is an essential component of the IL-18 signal cascade (Suzuki et al., J Immunology, 2003). Similarly, IRAK4 is essential for signaling of IL-1 and TLR ligands (Suzuki et al., Nature, 2002). TLR antagonists are known to cause pruritus (Liu et al., Neuroscience bulletin, 2012) - an important symptom of atopic dermatitis, and anti-IL-1 therapy is used off-label to treat atopic dermatitis. In addition, polymorphs of IRAK4 are associated with elevated total IgE in allergic diseases such as asthma and chronic sinusitis (Tewfik et al., Allergy, 2009). In atopic dermatitis, IgE levels are also elevated.

[0093] Therefore, since IRAK4 is a key part of the signaling pathway activated by many cytokines, TLR ligands, and IRAK4 has polymorphic forms associated with increased IgE levels, inhibition of IRAK4 is an important therapeutic strategy for treating allergic skin diseases such as atopic dermatitis. In addition, in pets (especially dogs and cats), atopic dermatitis and flea allergy dermatitis are appropriate indications because both diseases include type I allergies involving IgE antibodies, Th2 cells, mast cells, and eosinophils. In addition, FAD may include type IV allergies in which IL-1 and IL-18 are involved.

[0094] The compounds of the present invention act as IRAK4 kinase inhibitors and therefore have an unexpectedly useful spectrum of pharmacological activity in the treatment and / or prevention of allergic and / or inflammatory diseases in animals.

[0095] Preferred are compounds of formula (I), and diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts thereof, for use in treating and / or preventing allergic and / or inflammatory diseases in animals, wherein

[0096] R 1 is C1-C6-alkyl, wherein the C1-C6-alkyl group is unsubstituted or substituted by fluorine, hydroxyl, or R 6 , R 7 SO2、R 7 SO or R 8 The O groups are monosubstituted or polysubstituted identically or differently;

[0097] R 2 and R 3 always have the same definition and are hydrogen or C1-C3-alkyl;

[0098] R 4 is halogen, cyano or C1-C3-alkyl, where the C1-C3-alkyl radical is unsubstituted or monosubstituted or identically or differently polysubstituted by halogen or hydroxy;

[0099] R 5 is hydrogen, fluorine, chlorine or C1-C3-alkyl;

[0100] R 6 is oxetane or tetrahydrofuranyl;

[0101] R 7 is C1-C4-alkyl, wherein the C1-C4-alkyl group is unsubstituted, or monosubstituted by hydroxy or cyclopropyl, or substituted by three fluorine atoms;

[0102] R 8 is unsubstituted C1-C4-alkyl or trifluoro-substituted C1-C4-alkyl.

[0103] Also preferred are compounds of formula (I), and diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts thereof, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals, wherein

[0104] R 1 is C2-C6-alkyl, wherein the C2-C6-alkyl is unsubstituted, or

[0105] C2-C6-alkyl is mono-, di- or tri-fluoro-substituted, or

[0106] C2-C6-alkyl is hydroxylated, R 6 , R 7 SO2 or R 8 O single substitution,

[0107] or where R 1 is an oxetane-substituted C1-C3-alkyl group;

[0108] R 2 and R 3 always have the same definition and are either hydrogen or methyl;

[0109] R 4 is an unsubstituted or mono- or poly-halogen-substituted C1-C3-alkyl group, or a C1-C3-alkyl group substituted by one hydroxyl group, or a C1-C3-alkyl group substituted by one hydroxyl group and three fluorine atoms;

[0110] R 5 is hydrogen, fluorine or C1-C3-alkyl;

[0111] R 7 is C1-C3-alkyl;

[0112] R 8 is C1-C4-alkyl, wherein the C1-C4-alkyl group is unsubstituted or mono-, di- or tri-fluoro-substituted.

[0113] Particularly preferred are compounds of the general formula (I), and their diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals.

[0114] R 1 is a C2-C5-alkyl radical substituted by hydroxy, or C1-C3-alkoxy, or trifluoromethoxy, or 2,2,2-trifluoroethoxy, or trifluoromethyl, or

[0115] is a methyl-SO2-substituted C2-C4-alkyl group, or

[0116] is a C1-C2-alkyl group substituted with an oxetane-3-yl group;

[0117] R 2 and R 3 always have the same definition and are either hydrogen or methyl;

[0118] 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

[0119] R 5 is hydrogen, fluorine or methyl.

[0120] Very particular preference is given to the following compounds, and their diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals:

[0121] 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;

[0122] R 2 and R 3 are all methyl or hydrogen, and

[0123] R 4 is difluoromethyl, trifluoromethyl or methyl, and

[0124] R 5 is hydrogen or fluorine.

[0125] Also very particularly preferred are the following compounds, and their diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals:

[0126] R 1 is 3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-(methylsulfonyl)propyl or 2-(methylsulfonyl)

[0127] Ethyl;

[0128] R 2 and R 3 All are methyl;

[0129] R 4 is difluoromethyl or trifluoromethyl; and

[0130] R 5 For hydrogen.

[0131] Particular preference is also given to the following compounds, and their diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts, for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals:

[0132] R 1 is 3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-(methylsulfonyl)propyl or 2-(methylsulfonyl)

[0133] Ethyl;

[0134] R 2 and R 3 All are methyl;

[0135] R 4 is methyl, and

[0136] R 5 is fluorine, where R 5 In R 4 The adjacent position.

[0137] The present invention particularly provides the following compounds for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals:

[0138] 1) N-[6-(2-hydroxypropan-2-yl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

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

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

[0141] 4) N-[6-(hydroxymethyl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)

[0142] Pyridine-2-carboxamide

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

[0144] 6) N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0145] 7) N-[2-(2-hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0146] 8) N-[6-(2-hydroxypropan-2-yl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0147] 9) N-[6-(Hydroxymethyl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0148] 10) N-{6-(2-hydroxypropan-2-yl)-2-[3-(methylsulfonyl)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0149] 11) N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0150] 12) N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0151] 13) 6-(Difluoromethyl)-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0152] 14) 6-(Difluoromethyl)-N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}pyridine-2-carboxamide

[0153] 15) 6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0154] 16) N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0155] 17) N-{6-(2-hydroxypropan-2-yl)-2-[3-(trifluoromethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0156] 18) N-{6-(2-hydroxypropan-2-yl)-2-[3-(2,2,2-trifluoroethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0157] 19) 5-Fluoro-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0158] 20) N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0159] 21) 6-(2-hydroxypropan-2-yl)-N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0160] 22) N-{2-[2-(1-Hydroxycyclopropyl)ethyl]-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide.

[0161] The present invention also provides compounds of general formula (III) and diastereomers, enantiomers, metabolites, salts, solvates or solvates of salts thereof for use in treating and / or preventing allergic and / or inflammatory diseases in animals

[0162]

[0163] In the general formula (III)

[0164] 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;

[0165] R 4 is difluoromethyl, trifluoromethyl or methyl; and

[0166] R 5 is hydrogen or fluorine.

[0167] Particularly preferred are compounds of the following general formula (III) for use in the treatment and / or prevention of allergic and / or inflammatory diseases in animals:

[0168] 5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester and

[0169] Methyl 2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate.

[0170] The compound of general formula (III) is suitable for preparing a part of the compound of general formula (I).

[0171] Furthermore, the compounds of the general formula (III) are inhibitors of interleukin-1 receptor associated kinase-4 (IRAK4).

[0172] The compound of general formula (III) can be prepared from the compound of general formula (II) by reacting (II) with an appropriately substituted alkyl halide or alkyl 4-methylbenzenesulfonate in the presence of potassium carbonate.

[0173]

[0174] in

[0175] R 1 4,4,4-trifluorobutyl, 3-hydroxy-3-methylbutyl, 3-methoxypropyl, 3-hydroxypropyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-trifluoromethoxypropyl, 2-methoxyethyl, 2-hydroxyethyl, 2-(methylsulfonyl)

[0176] Ethyl, 3-(methylsulfonyl)propyl or 2-(1-hydroxycyclopropyl)ethyl;

[0177] R 4 is difluoromethyl, trifluoromethyl or methyl; and

[0178] R 5 is hydrogen or fluorine.

[0179] In addition, the compound of general formula (I) can be prepared from the compound of formula (III) by Grignard reaction with methylmagnesium bromide.

[0180]

[0181] in

[0182] R 1 4,4,4-trifluorobutyl, 3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-methoxypropyl, 3-hydroxypropyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl, 3-trifluoromethoxypropyl, 2-methoxyethyl, 2-hydroxyethyl, 3-(methylsulfonyl)propyl, 2-(1-hydroxycyclopropyl)ethyl;

[0183] R 2 and R 3 is methyl;

[0184] R 4 is difluoromethyl, trifluoromethyl or methyl; and

[0185] R 5 is hydrogen or fluorine.

[0186] The compounds of the present invention act as IRAK4 kinase inhibitors and possess an unexpectedly useful spectrum of pharmacological activity.

[0187] It is further preferred that the compounds of the formula (I), or the compounds specifically mentioned above, are used for the treatment and / or prevention of allergic and / or inflammatory diseases in domestic animals, in particular cats and dogs, and more particularly dogs.

[0188] In the present context, the term "livestock" includes, for example, mammals, such as hamsters, guinea pigs, rats, mice, chinchillas, ferrets or in particular dogs, cats; caged birds; reptiles; amphibians or aquarium fish.

[0189] It is also preferred that the compounds of formula (I), or the compounds specifically mentioned above, are used for the treatment and / or prevention of allergic dermatitis in livestock, in particular canine and feline allergic dermatitis, and more particularly canine allergic dermatitis. It is also preferred that the compounds of formula (I), or the compounds specifically mentioned above, are used for the treatment and / or prevention of allergic and / or inflammatory diseases in farm animals, in particular sheep, goats, horses, cattle and pigs, and more particularly cattle and pigs.

[0190] In the present context, the term "farm animals" includes, for example, mammals such as horses, sheep, goats, buffalo, reindeer, fallow deer or more particularly cattle or pigs.

[0191] It is further preferred that the compounds of formula (I), or the compounds specifically mentioned above, are used in a method for the treatment and / or prevention of the following diseases in animals: atopic dermatitis, flea allergy dermatitis, inflammatory bowel disease, osteoarthritis pain and inflammatory pain, non-infectious recurrent airway diseases, insect allergies, asthma, respiratory diseases, mastitis and endometritis, in particular atopic dermatitis and flea allergy dermatitis.

[0192] Particularly preferably, the compounds of formula (I), or the compounds specifically mentioned above, are used in a method for the treatment and / or prevention of canine atopic dermatitis, flea allergy dermatitis in dogs or cats; inflammatory bowel disease in dogs or cats; osteoarthritis pain and inflammatory pain in dogs, cats, horses or cattle; non-infectious recurrent airway disease in horses; insect allergy in horses; feline asthma; bovine respiratory disease; bovine mastitis; bovine endometritis and porcine respiratory disease.

[0193] Very particular preference is given to using the compounds of the formula (I) or the compounds specifically mentioned above in a method for the treatment and / or prophylaxis of canine atopic dermatitis and flea allergy dermatitis in dogs or cats, more particularly in dogs.

[0194] Very particular preference is further given to using the compounds of the formula (I) or the compounds mentioned specifically above in a method for the treatment and / or prevention of osteoarthritic and inflammatory pain in cattle, respiratory diseases in cattle, bovine mastitis, bovine endometritis and respiratory diseases in pigs.

[0195] With regard to the compounds of formula (III), it is further preferred that the compounds of formula (III) are used for the treatment and / or prevention of allergic and / or inflammatory diseases in domestic animals, in particular cats and dogs, and more particularly dogs.

[0196] The compounds of the formula (III) are further preferably used for the treatment and / or prevention of allergic dermatitis in domestic animals, in particular canine and feline allergic dermatitis, and more particularly canine allergic dermatitis.

[0197] The compounds of the formula (III) are further preferably used for the treatment and / or prevention of allergic and / or inflammatory diseases in farm animals, in particular sheep, goats, horses, cattle and pigs, and more particularly cattle and pigs.

[0198] The compounds of the formula (III) are further preferably used in a method for the treatment and / or prevention of the following diseases in animals: atopic dermatitis, flea allergy dermatitis, inflammatory bowel disease, osteoarthritic pain and inflammatory pain, non-infectious recurrent airway diseases, insect allergies, asthma, respiratory diseases, mastitis and endometritis, in particular atopic dermatitis and flea allergy dermatitis.

[0199] The compounds of formula (III) are particularly preferably used in a method for treating and / or preventing canine atopic dermatitis, flea allergy dermatitis in dogs or cats; inflammatory bowel disease in dogs or cats; osteoarthritis pain and inflammatory pain in dogs, cats, horses or cattle; non-infectious recurrent airway disease in horses; insect allergy in horses; feline asthma; bovine respiratory disease; bovine mastitis; bovine endometritis and porcine respiratory disease.

[0200] Very particular preference is given to using the compounds of the formula (III) in a method for the treatment and / or prophylaxis of canine atopic dermatitis and flea allergy dermatitis in dogs or cats, more particularly in dogs.

[0201] Very particular preference is further given to using the compounds of the formula (III) in a method for the treatment and / or prevention of osteoarthritic pain and inflammatory pain in cattle, respiratory tract disease in cattle, bovine mastitis, bovine endometritis and porcine respiratory tract disease.

[0202] For example, compound examples 11, 12, 13, 19 (as shown below) have been evaluated using recombinant canine IRAK4 enzymes in an in vitro IRAK4 TR-FRET assay described in detail below. The IC50 values ​​of each compound for inhibiting canine IRAK4 have been calculated. It has been confirmed that the example compounds (11, 12, 13, 19) can be used to treat allergic skin diseases such as atopic dermatitis and flea allergy dermatitis in animals, especially dogs and cats. Example compounds 11, 12, 13, 19 are effective inhibitors of canine IRAK4 with IC50 values ​​of 1.7, 9.2, 2.2, 7.6 nM, respectively. For inhibiting human IRAK4, the IC50 values ​​of each of these example compounds are similar to the calculated IC50 values.

[0203] As another example, Example Compound 12 was also evaluated in vitro to determine the efficacy of the compound on lipopolysaccharide (LPS)-induced cytokine production by canine peripheral blood mononuclear cells (PBMCs). Example Compound 12 inhibited the production of proinflammatory cytokine tumor necrosis factor α (TNFα) in a concentration-dependent manner by LPS-induced canine PBMCs. PBMCs 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 TNFα is elevated in patients with atopic dermatitis (Sumimoto et al., Archives of Disease in Childhood, 1992). This example is also evaluated in vitro to determine the efficacy of the compound on lipopolysaccharide (LPS)-induced cytokine production by canine peripheral blood mononuclear cells (PBMCs). Example Compound 12 inhibited the production of proinflammatory cytokine tumor necrosis factor α (TNFα) by LPS-induced canine PBMCs in a concentration-dependent manner. PBMCs 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 TNFα is elevated in patients with atopic dermatitis (Sumimoto et al., Archives of Disease in Childhood, 1992). Figure 7 illustrate.

[0204] Thus, compounds of the invention demonstrated inhibition of recombinant canine IRAK4 enzyme and cytokine production by canine PBMCs, indicating potential therapeutic benefit of these compound examples in canine atopic dermatitis and flea allergy dermatitis.

[0205] In addition, in a house dust mite model, Example Compound 12 was evaluated in vivo in further studies to determine the efficacy of the compound in treating clinical symptoms associated with canine allergic dermatitis, particularly canine atopic dermatitis (CAD). Example Compound 12 significantly reduced the clinical symptoms of CAD-like skin edema and erythema. This example also Fig.11 and Fig.12 illustrate.

[0206] Thus, the compounds of the present invention exhibit a reduction in the characteristic clinical symptoms of canine allergic dermatitis, thereby demonstrating the therapeutic benefit of such compound examples in canine allergic dermatitis, particularly canine atopic dermatitis (CAD). In addition, Example Compound 12 has been evaluated in an in vivo model of canine flea allergy dermatitis (CAD) to determine the anti-itching efficacy of the compound. Subsequent treatment with Example Compound 12 significantly reduced the itch associated with allergic diseases such as flea allergy dermatitis. This example is also Fig.13 illustrate.

[0207] Thus, the compounds of the present invention show a reduction in characteristic clinical symptoms associated with allergic dermatitis, such as dermatitis and pruritus, thereby demonstrating the therapeutic benefit of such compound embodiments in canine allergic dermatitis, particularly flea allergy dermatitis (FAD) and canine atopic dermatitis (CAD).

[0208] In the present context, the term "canine allergic dermatitis" specifically includes canine atopic dermatitis (CAD) and flea allergy dermatitis (FAD).

[0209] As another example, Example Compound 12 has also been evaluated in an ex vivo assay to determine the efficacy of the compound on lipopolysaccharide (LPS)-induced cytokine production by bovine peripheral blood mononuclear cells (PBMCs). Example Compound 12 inhibits the production of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) in a concentration-dependent manner by LPS-induced canine PBMCs. PBMCs include cell types such as dendritic cells, T lymphocytes and B lymphocytes, and monocytes, each of which is associated with inflammatory and infectious diseases with excessive proinflammatory immune responses, such as respiratory diseases (Sterner-Kock, Haider et al., Tropical Animal Health and Production, 2016), intestinal diseases (Pan, Rostagnio et al., Veterinary Immunology and Immunopathology, 2015) and mastitis (Zheng, Xu et al., Free Radical Biology and Medicine, 2016), and TNFα is elevated in patients with these diseases. This example is also Figure 8 and 9 illustrate.

[0210] Thus, compounds of the invention exhibited inhibition of cytokine production by bovine PBMCs, suggesting possible therapeutic benefit of such compound embodiments in inflammatory and / or infectious diseases such as respiratory diseases, intestinal diseases and mastitis.

[0211] As another example, Example Compound 12 has also been evaluated in vitro to determine the efficacy of the compound on lipopolysaccharide (LPS)-induced cytokine production by porcine peripheral blood mononuclear cells (PBMCs). Example Compound 12 inhibits the production of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) by LPS-induced porcine PBMCs. PBMCs include cell types such as dendritic cells, T lymphocytes and B lymphocytes, and monocytes, each of which is associated with inflammatory diseases and infectious diseases such as respiratory and intestinal diseases with excessive proinflammatory immune responses, and TNFα is elevated in patients with these diseases. This example is also evaluated in vitro to determine the efficacy of the compound on lipopolysaccharide (LPS)-induced cytokine production by porcine peripheral blood mononuclear cells (PBMCs). Example Compound 12 inhibits the production of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) by LPS-induced porcine PBMCs. PBMCs include cell types such as dendritic cells, T lymphocytes and B lymphocytes, and monocytes, each of which is associated with inflammatory diseases and infectious diseases such as respiratory and intestinal diseases with excessive proinflammatory immune responses, and TNFα is elevated in patients with these diseases. Fig.10 illustrate.

[0212] Thus, the present compounds showed inhibition of cytokine production by porcine PBMCs, indicating the potential therapeutic benefit of such compound embodiments in inflammatory and / or infectious diseases such as respiratory and intestinal diseases.

[0213] The compounds of the present invention also provide for the prevention and / or treatment of pruritus and pain in animals, particularly acute, chronic, inflammatory and neuropathic pain in animals.

[0214] In addition, the compounds of the present invention are suitable for the treatment and / or prevention of pain diseases in animals, especially acute, chronic, inflammatory and neuropathic pain. The diseases preferably include hyperalgesia, allodynia, arthritis (e.g. osteoarthritis, rheumatoid arthritis and spondyloarthritis) pain, premenstrual pain, endometriosis-related pain, postoperative pain, interstitial cystitis pain, CRPS (complex regional pain syndrome), trigeminal neuralgia, prostatitis pain, pain caused by spinal cord injury, pain caused by inflammation, low back pain, cancer pain, chemotherapy-related pain, HIV-treatment-induced neuropathy, pain caused by burns and chronic pain.

[0215] The present invention also provides a method for treating and / or preventing animal diseases, in particular the above-mentioned diseases, using an effective amount of at least one compound according to the present invention.

[0216] Preferably, there is provided a method for treating and / or preventing allergic and / or inflammatory diseases in animals by administering an effective amount of at least one compound of formula (I) according to the invention as defined above to an animal in need thereof.

[0217] In the context of the present invention, the term "treatment" or "treating" includes inhibiting, delaying, checking, alleviating, mitigating, limiting, reducing, suppressing, resisting or curing a disease, condition, illness, injury or health problem, or the development, progression or evolution of said state and / or the symptoms of said state. The term "therapy" is understood herein to be synonymous with the term "treatment".

[0218] In the context of the present invention, the terms "prevention", "prophylaxis" and "preclusion" are used synonymously and refer to avoiding or reducing the risk of contracting, experiencing, suffering or having a disease, condition, disorder, injury or health problem or the development or progression of such conditions and / or symptoms of such conditions.

[0219] A disease, condition, disorder, injury or health problem may be treated or prevented, in part or in whole.

[0220] The compounds of the present invention can be used alone or, if necessary, in combination with other active ingredients. The present invention also provides a medicament containing 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 the composition include:

[0221] In general, there may be mentioned active ingredients such as antibacterial substances (e.g. penicillin, vancomycin, ciprofloxacin), antiviral substances (e.g. aciclovir, oseltamivir) and antimycotic substances (e.g. naftifin, nystatin) and gamma-globulins; immunomodulatory and immunosuppressive compounds such as cyclosporin, TNF antagonists (eg Etanercept, infliximab), IL-1 inhibitors (e.g., anakinra, canakinumab, rilonacept), phosphodiesterase inhibitors (e.g., apremilast), Jak / STAT inhibitors (e.g., tofacitinib, baricitinib, GLPG0634), leflunomid, cyclophosphamide, rituximab, belimumab, tacrolimus, rapamycin, mycophenolate mofetil mofetil), interferon, corticosteroids (e.g., prednisone, prednisolone, methylprednisolone, hydrocortisone, betamethasone), cyclophosphamide, azathioprine, and sulfasalazine; paracetamol, non-steroidal anti-inflammatory substances (NSAIDS) (aspirin, ibuprofen, naproxen, etodolac, celecoxib, colchicine).

[0222] In addition to those mentioned above, the IRAK4 inhibitor of the present invention may also be combined with the following active ingredients:

[0223] Substances for the treatment of lung diseases, such as beta-2-sympathomimetics (e.g. salbutamol), anticholinergics (e.g. glycopyrronium), methylxanthines (e.g. theophylline), leukotriene receptor antagonists (e.g. montelukast), PDE-4 (phosphodiesterase type 4) inhibitors (e.g. roflumilast), methotrexate, IgE antibodies, azathioprine and cyclophosphamide, preparations containing cortisol; Substances for the treatment of osteoarthritis, such as nonsteroidal anti-inflammatory substances (NSAIDs). In addition to the two therapies mentioned, methotrexate and biologics (eg rituximab, abatacept) for B-cell and T-cell therapy should be mentioned for rheumatoid diseases such as rheumatoid arthritis, spondyloarthritis and juvenile idiopathic arthritis. Neurotrophic substances such as acetylcholinesterase inhibitors (e.g. donepezil), MAO (monoamine oxidase) inhibitors (e.g. selegiline), interferons and anticonvulsives (e.g. gabapentin); active ingredients for the treatment of cardiovascular diseases, such as beta blockers (e.g. metoprolol), ACE inhibitors (e.g. benazepril), angiotensin receptor blockers (e.g. losartan, valsartan), diuretics (e.g. hydrochlorothiazide), calcium channel blockers (e.g. nifedipine), statins (e.g. simvastatin, fluvastatin);Antidiabetic agents, such as metformin, glinides (e.g. nateglinide), DPP-4 (dipeptidyl peptidase-4) inhibitors (e.g. linagliptin, saxagliptin, sitagliptin, vildagliptin), SGLT2 (sodium / glucose cotransporter 2) inhibitors / gliflozins (e.g. dapagliflozin, empagliflozin), incretin mimetics (hormone glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1 (glucagon-like peptide 2) inhibitors, 1) (GLP-1) analogs / agonists) (e.g. exenatide, liraglutide, lixisenatide), α-glucosidase inhibitors (e.g. acarbose, miglitol, voglibiose) and sulfonylureas (e.g. glibenclamide, tolbutamide), insulin sensitizers (e.g. pioglitazone) and insulin therapy (e.g. NPH insulin, insulin lispro). Active ingredients for the treatment of chronic inflammatory bowel disease, such as mesalazine, sulfasalazine, azathioprine, 6-mercaptopurine or methotrexate, probiotics (Mutaflor,; Lactobacillus GG, Lactobacillus plantarum, L. acidophilus, L. casei, Bifidobacterium infantis 35624, Enterococcus fecium SF68, Bifidobacterium longum, Escherichia coli Nissle 1917); antibiotics such as ciprofloxacin and metronidazole; antidiarrheals such as loperamide; or laxatives (bisacodyl). Immunosuppressants used to treat lupus erythematosus such as glucocorticoids and nonsteroidal 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.

[0224] Mention should also be made of medicaments for the treatment and / or prevention of the abovementioned diseases, which comprise at least one compound according to the invention and one or more further active ingredients for the use according to the invention, in particular EP4 inhibitors (prostaglandin E2 receptor 4 inhibitors), P2X3 inhibitors (P2X purinergic receptor 3), PTGES inhibitors (prostaglandin E synthase inhibitors) or AKR1C3 inhibitors (aldodetoreductase family 1 member C3 inhibitors).

[0225] 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 route, via the ear or as an implant or stent.

[0226] The compounds according to the invention can be administered in administration forms suitable for these administration routes.

[0227] Suitable administration forms for oral administration are those which act according to the prior art and release the compound according to the invention quickly and / or in a sustained manner and which contain the compound according to the invention in crystalline and / or amorphous and / or dissolved form, for example tablets (uncoated or coated tablets, the coated tablets having, for example, a gastric-resistant coating or a dissolving-delaying coating or an insoluble coating which controls the release of the compound according to the invention), tablets which disintegrate rapidly in the mouth or flakes / flat tablets, flakes / lyophilisates, capsules (for example hard or soft gelatin capsules), sugar-coated tablets, chewables (for example soft chewables), granules, pills, powders, emulsions, suspensions, aerosols or solutions.

[0228] Parenteral administration can be accomplished by bypassing the reabsorption step (e.g., by intravenous, intraarterial, intracardiac, intraspinal or intralumbar routes) or by including absorption (e.g., intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal routes). Administration forms suitable for parenteral administration include injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilized agents or sterile powders.

[0229] For other routes of administration, suitable examples are inhalable pharmaceutical forms (including powder inhalers, sprays), nasal drops, nasal solutions or nasal sprays; tablets, wafers / flats or capsules for translingual, sublingual or buccal administration; suppositories; otic or ophthalmic preparations, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, pour-ons, transdermal therapeutic systems (e.g. patches), milks, pastes, foams, sprinkling powders, implants or stents.

[0230] Oral administration or parenteral administration is preferred, especially oral administration.

[0231] The compounds according to the invention can be converted into the administration forms mentioned. This can be done in a manner known per se by mixing with inert, nontoxic, pharmaceutically suitable excipients. These excipients include carriers (e.g. microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycol), emulsifiers and dispersants or wetting agents (e.g. sodium lauryl sulfate, polyoxysorbitan oleate), binders (e.g. polyvinylpyrrolidone), synthetic and natural polymers (e.g. albumin), stabilizers (e.g. antioxidants, e.g. ascorbic acid), colorants (e.g. inorganic pigments, e.g. iron oxide) and flavor correctants and / or odor correctants.

[0232] The invention also provides medicaments comprising at least one compound of the invention, typically together with one or more inert, non-toxic, pharmaceutically acceptable excipients, for use in a method for the treatment and / or prevention of allergic and / or inflammatory diseases in animals.

[0233] Generally, it has been found that in the case of parenteral administration, it is advantageous to administer an amount of about 0.001 to 1 mg / kg body weight, preferably about 0.01 to 0.5 mg / kg body weight, to achieve effective results. In the case of oral administration, the dosage is about 0.01 to 100 mg / kg body weight, preferably about 0.01 to 20 mg / kg body weight and most preferably 0.1 to 10 mg / kg body weight.

[0234] However, in some cases, it may be necessary to deviate from the amounts specified, in particular as a function of body weight, route of administration, individual response to the active ingredient, nature of the formulation and the time or interval at which administration occurs. Thus, in some cases, it may be sufficient to proceed with an amount less than the above minimum amount, while in other cases the upper limit mentioned must be exceeded. In the case of larger amounts to be administered, it is advisable to divide it into several individual doses over the day.

[0235] The following working examples illustrate the invention. The invention is not limited to the examples described.

[0236] Unless otherwise stated, the percentages in the following tests and examples are percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentration data for liquid / liquid solutions are in each case based on volume.

[0237] Preparation of compounds

[0238] The preparation of compounds of the present invention is illustrated by the following synthetic schemes.

[0239] The starting materials for the synthesis of the compounds of the invention are carboxylic acids (intermediate V3), which are commercially available or can be prepared by routes known from the literature or analogously to routes known from the literature (see, for example, European Journal of Organic Chemistry 2003, 8, 1559-1568; Chemical and Pharmaceutical Bulletin, 1990, 38, 9, 2446-2458; Synthetic Communications 2012, 42, 658-666; Tetrahedron, 2004, 60, 51, 11869-11874) (see, for example, Synthesis Scheme 1). Some carboxylic acids V3 can be prepared starting from carboxylic acid esters (intermediate V2) by hydrolysis (see, for example, the reaction of ethyl 6-(hydroxymethyl)pyridine-2-carboxylate with aqueous sodium hydroxide in methanol, WO2004113281), or, in the case of tert-butyl esters, by reaction with acids such as hydrochloric acid or trifluoroacetic acid (see, for example, Dalton Transactions, 2014, 43, 19, 7176-7190). Carboxylic acids V3 can also be used in the form of their alkali metal salts. Intermediate V2 can also be optionally prepared from intermediate V1 containing chlorine, bromine or iodine as substituent X1, by reacting in a carbon monoxide atmosphere, optionally under high pressure, in the presence of a phosphine ligand (e.g. 1,3-bis(diphenylphosphino)propane), a palladium compound (e.g. palladium(II) acetate) and a base (e.g. triethylamine), with ethanol or methanol added to a solvent (e.g. dimethyl sulfoxide) (for preparation methods, see, e.g. WO2012112743, WO 2005082866, Chemical Communications (Cambridge, England), 2003, 15, 1948-1949, WO200661715). Intermediate V1 is commercially available or can be prepared by a route 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.

[0240]

[0241] Synthesis Scheme 1

[0242] X 1 Chlorine, bromine or iodine.

[0243] R d It is methyl, ethyl, benzyl or tert-butyl.

[0244] R 4 , R 5 Each is as defined in the general formula (I).

[0245] Analogously to WO 2008 / 001883, 5-amino-1H-indazole-6-carboxylic acid methyl ester (intermediate 2) can be prepared according to synthetic scheme 2 starting from 1H-indazole-6-carboxylic acid methyl ester (intermediate 0) by nitration and reduction of the nitro group of intermediate 1 with hydrogen in the presence of palladium on carbon. 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, Vol. 3 - Building Blocks, Catalysis and Coupling Chemistry, Andrew B. Hughes, Wiley, Chapter 12 - Peptide-Coupling Reagents, 407-442; Chem. Soc. Rev., 2009, 38, 606). For example, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride can be used in combination with the following substances as coupling reagents: 1-hydroxy-1H-benzotriazole hydrate (HOBt, WO2012107475; Bioorg. Med. Chem. Lett., 2008, 18, 2093), (1H-benzotriazol-1-yloxy)(dimethylamino)-N,N-dimethylmethaniminium tetrafluoroborate (TBTU, CAS 12 5700-67-6), (dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)carboximidate hexafluorophosphate (HATU, CAS 148893-10-1), propanephosphonic anhydride (in the form of ethyl acetate solution or DMF solution, CAS 68957-94-8) or di-1H-imidazol-1-ylmethanone (CDI), in each case a base such as triethylamine or N-ethyl-N-isopropylpropan-2-amine is added to the reaction mixture. Preference is given to using TBTU and N-ethyl-N-isopropylpropan-2-amine in THF.

[0246]

[0247] Synthesis Scheme 2

[0248] Substituent R 4 , R 5 Each is as defined in the general formula (I).

[0249] Starting from intermediate 3, 2-substituted indazole derivatives (intermediate 4) (see synthesis scheme 3) can be prepared. For this purpose, useful reactions include those reactions with optionally substituted alkyl chlorides, alkyl bromides, alkyl iodides or 4-methylbenzenesulfonic acid alkyl esters. The alkyl halides or 4-methylbenzenesulfonic acid alkyl esters used are commercially available or can be prepared similarly to the approach known from the literature (for the preparation of 4-methylbenzenesulfonic acid alkyl esters, one embodiment is to react an appropriate amount of alcohol with 4-methylbenzenesulfonyl chloride in the presence of triethylamine or pyridine; See, for example, Bioorganic and Medicinal Chemistry, 2006, 14, 12 4277-4294). Optionally, in the case of using alkyl chlorides or alkyl bromides, alkali metal iodides such as potassium iodide or sodium iodide can also be added. The base used can be, for example, potassium carbonate, cesium carbonate or sodium hydride. In the case of reactive alkyl halides, N-cyclohexyl-N-methylcyclohexylamine can also be used in some cases. Useful solvents include, for example, 1-methylpyrrolidone-2-one, DMF, DMSO or THF. Optionally, the alkyl halide or 4-methylbenzenesulfonic acid alkyl ester used may have a functional group optionally protected in advance with a blocking group (see also PGM Wuts, TW Greene, Greene's Protective Groups in Organic Synthesis, the fourth edition, ISBN: 9780471697541). For example, if an alkyl halide or 4-methylbenzenesulfonic acid alkyl ester with one or more hydroxyl groups is used, these hydroxyl groups may be optionally protected by tert-butyl (dimethyl) silyl or similar silicon-containing blocking groups familiar to those skilled in the art. Alternatively, hydroxyl groups may also be protected with tetrahydro-2H-pyrans (THP) groups or acetyl or benzoyl. Then, after the synthesis of intermediate 4 or after the synthesis of (I), the used blocking group may be removed. For example, if the tert-butyl (dimethylsilyl) group is used as a blocking group, the tetrabutylammonium fluoride used in a solvent such as THF may be removed. For example, the THP protecting group may be removed using 4-methylbenzenesulfonic acid (optionally in the form of the monohydrate). An acetyl or benzoyl group may be removed by treatment with aqueous sodium hydroxide.

[0250] Optionally, the alkyl halide or 4-methylbenzenesulfonic acid alkyl ester used may contain a functional group that can be converted by oxidation or reduction reactions known to those skilled in the art (see, for example, Science of Synthesis, GeorgThieme Verlag). For example, if the functional group is a sulfide group (sulphide group), it can be oxidized to a sulfoxide or sulfone group by methods known in the literature. In the case of a sulfoxide group, it can be oxidized to a sulfone group. For these oxidation steps, for example, 3-chloroperbenzoic acid (CAS 937-14-4) can be used (for this, see, for example, US201094000, wherein 2- (methylsulfanyl) ethyl -1H- pyrazole derivatives are oxidized to 2- (methylsulfinyl) ethyl -1H- pyrazole derivatives, and other 2- (methylsulfanyl) ethyl -1H- pyrazole derivatives are oxidized to 2- (methylsulfonyl) ethyl -1H- pyrazole derivatives). If the alkyl halide or p-toluenesulfonate used contains a keto group, it can be reduced to an alcohol group by a reduction method known to those skilled in the art (see, for example, Chemische Berichte, 1980, 113, 1907-1920 for the use of sodium borohydride). These oxidation or reduction steps can be performed after the synthesis of intermediate 4 or after the synthesis of the compound of general formula (I) of the present invention. Alternatively, intermediate 4 can be prepared by the Mitsunobu reaction of intermediate 3 with an optionally substituted alkyl alcohol (see, for example, KCK Swamy et al., Chem. Rev. 2009, 109, 2551-2651). Various phosphine compounds such as triphenylphosphine, tributylphosphine or 1,2-diphenylphosphinoethane can be used in combination with diisopropyl azodicarboxylate (CAS 2446-83-5) or other diazene derivatives mentioned in the literature (KCK Swamy et al., Chem. Rev. 2009, 109, 2551-2651). Preference is given to using triphenylphosphine and diisopropyl azodicarboxylate. If the alkyl alcohol carries a functional group, then - as in the case of the above reaction with an alkyl halide - known protective group strategies can be carried out (further instructions can be found in PGM Wuts, TW Greene, Greene's Protective Groups in Organic Synthesis, 4th edition, ISBN: 9780471697541) and - as in the case of the above reaction with an alkyl halide - the synthesis of intermediate 4, or the corresponding oxidation and reduction steps after the synthesis of the compound of the general formula (I) according to the invention. The compound of the general formula (I) according to the invention, in which R 2 and R 3 is defined as C1-C6-alkyl (where R 2 and R 3have the same definition), can be obtained from intermediate 4 by Grignard reaction (see, for example, the reaction of 1H-indazole-6-carboxylic acid methyl ester derivatives with methylmagnesium bromide in EP 2489663). For the Grignard reaction, alkylmagnesium halides can be used. Particularly preferred are methylmagnesium chloride or methylmagnesium bromide in THF or diethyl ether or in a mixture of THF and diethyl ether. Alternatively, the compound of the general formula (I) of the present invention, wherein R 2 and R 3 is defined as C1-C6-alkyl (where R 2 and R 3 have the same definition), can be obtained from intermediate 4 by reaction with an alkyl lithium reagent (see, for example, the reaction of 2-amino-4-chloro-1-methyl-1H-benzimidazole-7-carboxylic acid methyl ester derivative with isopropyl lithium or tert-butyl lithium in WO2006116412). Starting from intermediate 4, by reduction with lithium aluminum hydride in THF, lithium borohydride in THF or sodium borohydride in THF (optionally with methanol added), or a mixture of lithium borohydride and sodium borohydride, wherein R 2 and R 3 The compound of the general formula (I) of the present invention is defined as H.

[0251]

[0252] Synthesis Scheme 3

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

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

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

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

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

[0258]

[0259] Synthesis Scheme 4

[0260] Substituent R 1 , R 4 , R 5 are as defined in the general formula (I). 2 and R 3 Always have the same definition and are all C1-C6-alkyl.

[0261] Intermediate 4 can be prepared from Intermediate 1 in an alternative manner (see Synthesis Scheme 5). First, Intermediate 1 is converted to Intermediate 6 by the method as in Synthesis Scheme 3 (Preparation of Intermediate 4 from Intermediate 3).

[0262] Then, intermediate 6 can be converted into intermediate 7 by the reduction of nitro.For example, nitro can be reduced with palladium on carbon under hydrogen atmosphere (see, for example, WO2013174744 about 6-isopropoxy-5-nitro-1H-indazole being reduced to 6-isopropoxy-1H-indazole-5-amine), or reduced with iron and ammonium chloride in water and ethanol (see, for example, Journal of the Chemical Society, 1955, 2412-2419), or reduced with tin chloride (II) (CAS 7772-99-8). Preferably, iron and ammonium chloride in water and ethanol are used.Preparation of intermediate 4 by intermediate 7 can be carried out similarly to synthesis scheme 2 (preparation of intermediate 3 by intermediate 2).

[0263] As described in Scheme 3, in the case of Scheme 5, a protecting group strategy can also be optionally used. Starting from intermediate 6 or intermediate 7, oxidation or reduction reactions known to those skilled in the art (see, for example, Science of Synthesis, Georg Thieme Verlag) can optionally also be carried out as described in Scheme 3.

[0264]

[0265] Synthesis Scheme 5

[0266] Substituent R 1 , R 4 , R 5 Each is as defined in the general formula (I).

[0267] Synthesis of Example Compounds

[0268] Abbreviations and descriptions

[0269] DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide THF Tetrahydrofuran RT Room temperature HPLC High performance liquid chromatography h Hour HCOOH Formic acid MeCN Acetonitrile min minute UPLC Ultra-high performance liquid chromatography DAD Diode Array Detector ELSD Evaporative Light Scattering Detector ESI Electrospray ionization SQ Single quadrupole detector CPG Core-pulled precision glass <![CDATA[NH3]]> ammonia

[0270] The term sodium chloride solution always refers to a saturated aqueous sodium chloride solution.

[0271] The chemical names of the intermediates and examples were generated using ACD / LABS (Batch version 12.01) software.

[0272] method

[0273] In some cases, compounds of the invention and their precursors and / or intermediates were analyzed by LC-MS.

[0274] Method A1: UPLC (MeCN-HCOOH):

[0275] Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity UPLC BEH C18 1.7 50×2.1 mm; eluent A: water + 0.1 volume % formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow rate 0.8 mL / min; temperature: 60°C; injection volume: 2 μL; DAD scan: 210-400 nm; MS ESI+, ESI-, scan range 160-1000 m / z; ELSD.

[0276] Method A2: UPLC (MeCN-NH3):

[0277] Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity UPLC BEH C18 1.7 50×2.1 mm; eluent A: water + 0.2 volume % ammonia (32%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow rate 0.8 mL / min; temperature: 60°C; injection volume: 2 μL; DAD scan: 210-400 nm; MS ESI+, ESI-, scan range 160-1000 m / z; ELSD.

[0278] Method A3: (LC-MS)

[0279] Instrument: Agilent 1290 Infinity LC; column: Acquity UPLC BEH C18 1.750×2.1 mm; eluent A: water + 0.05 volume % formic acid, eluent B: acetonitrile + 0.05 volume % formic acid; gradient: 0-1.7 min 2-90% B, 1.7-2.0 min 90% B; flow rate 1.2 mL / min; temperature: 60°C; injection volume: 2 μL; DAD scan: 190-390 nm; MS: Agilent TOF 6230.

[0280] Method A4: (LC-MS)

[0281] Instrument: Waters Acquity; Column: Kinetex (Phenomenex), 50×2 mm; Eluent A: water + 0.05 volume % formic acid, eluent B: acetonitrile + 0.05 volume % 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.

[0282] In some cases, compounds of the invention and their precursors and / or intermediates were purified by the following illustrative preparative HPLC methods:

[0283] Method P1: System: Waters automated purification system: pump 2545, sample manager 2767, CFO, DAD 2996, ELSD 2424, SQD; column: XBridge C18 5μm 100×30mm; eluent A: water + 0.1 volume % formic acid, eluent B: acetonitrile; gradient: 0-8min 10-100% B, 8-10min 100% B; flow rate: 50mL / min; temperature: room temperature; solution: maximum 250mg / maximum 2.5mL DMSO or DMF; injection volume: 1×2.5mL; detection: DAD scan range 210-400nm; MS ESI+, ESI-, scan range 160-1000m / z.

[0284] Method P2: System: Waters automated purification system: pump 254, sample manager 2767, CFO, DAD 2996, ELSD2424, SQD 3100; column: XBridge C18 5μm 10×30mm; eluent A: water + 0.2 volume % ammonia (32%), eluent B: methanol; gradient: 0-8min 30-70% B; flow rate: 50mL / min; temperature: room temperature; detection: DAD scanning range 210-400nm; MS ESI+, ESI-, scanning range 160-1000m / z; ELSD.

[0285] Method P3: System: Labomatic, Pump: HD-5000, Fraction collector: LABOCOL Vario-4000, UV detector: Knauer UVD 2.1S; Column: XBridge C18 5μm100×30mm; Eluent A: water+0.2vol% ammonia (25%), Eluent B: acetonitrile; Gradient: 0-1min 15% B, 1-6.3min 15-55% B, 6.3-6.4min 55-100% B, 6.4-7.4min 100% B; Flow rate: 60ml / min; Temperature: room temperature; Solution: maximum 250mg / 2mL DMSO; Injection volume: 2×2mL; Detection: UV 218nm; Software: SCPA PrepCon5.

[0286] Method P4: System: Labomatic, Pump: HD-5000, Fraction collector: LABOCOL Vario-4000, UV detector: Knauer UVD 2.1S; Column: Chromatorex RP C18 10 μm 125×30 mm; Eluent A: water + 0.1% volume of formic acid, Eluent B: acetonitrile; Gradient: 0-15 min 65-100% B; Flow rate: 60 mL / min; Temperature: room temperature; Solution: maximum 250 mg / 2 mL DMSO; Injection: 2×2 mL; Detection: UV 254 nm; Software: SCPA PrepCon5.

[0287] 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: 80mL / min; Temperature: 40°C; Solution: maximum 250mg / 2mL DMSO; Injection volume: 5×0.4mL; Detection: UV 254nm.

[0288] 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: 25mL / min; Temperature: 25℃; Solution: 187mg / 8mL ethanol / methanol; Injection volume: 8×1.0mL; Detection: UV280nm.

[0289] Method P7: System: Labomatic, Pump: HD-5000, Fraction collector: LABOCOL Vario-4000, UV detector: Knauer UVD 2.1S; Column: XBridge C18 5μm100×30mm; Eluent A: water + 0.1% volume of formic acid, Eluent B: acetonitrile; Gradient: 0-3min: 65% B isocratic, 3-13min: 65-100% B; Flow rate: 60mL / min; Temperature: room temperature; Solution: maximum 250mg / 2mL DMSO; Injection volume: 2×2mL; Detection: UV 254nm.

[0290] 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.

[0291] In some cases, mixtures of material were purified by silica gel column chromatography.

[0292] 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.

[0293] Starting Materials

[0294] Intermediate V2-1

[0295] 6-(2-Hydroxypropan-2-yl)pyridine-2-carboxylic acid methyl ester

[0296]

[0297] 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.

[0298] UPLC-MS (Method A1): R t=0.76 min (UV detector: TIC), molecular weight measurement value 195.00.

[0299] Intermediate V3-1

[0300] Potassium 6-(2-hydroxypropan-2-yl)pyridine-2-carboxylate

[0301]

[0302] First, 1.60 g of the crude product of Intermediate 0-1 was added to 15 mL of methanol, 0.74 g of potassium hydroxide was added, and the mixture was stirred at 50° C. for 16.5 h. After concentration, 2.1 g of a residue was obtained, which was used without further purification.

[0303] UPLC-MS (Method A1): R t =0.47 min (UV detector: TIC), molecular weight measurement value 181.00.

[0304] Intermediate 1-1

[0305] 5-Nitro-1H-indazole-6-carboxylic acid methyl ester

[0306]

[0307] In a three-necked flask with a CPG stirrer, a dropping funnel and an internal thermometer, 4.60 g (26.1 mmol) of 1H-indazole-6-carboxylic acid methyl ester (CAS No.: 170487-40-8) was dissolved in 120 mL of sulfuric acid (96%) and cooled to -15 ° C. In 15 min, a pre-prepared and cooled nitrating acid (10 mL of 96% sulfuric acid in 5 mL of 65% nitric acid) was added dropwise to the solution. After the addition was complete, the mixture was stirred for another 1 h (internal temperature was -13 ° C). The reaction mixture was added to ice, the precipitate was filtered off with suction, washed with water and dried under reduced pressure in a drying oven at 50 ° C. 5.49 g of the title compound was obtained.

[0308] UPLC-MS (Method A2): R t =0.75min

[0309] MS (ESIpos): m / z = 222 (M + H) +

[0310] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.87 (s, 3H), 7.96 (s, 1H), 8.44 (s, 1H), 8.70 (s, 1H), 13.98 (br.s., 1H).

[0311] Intermediate 2-1

[0312] 5-Amino-1H-indazole-6-carboxylic acid methyl ester

[0313]

[0314] 4.40 g (19.8 mmol) of 5-nitro-1H-indazole-6-carboxylic acid methyl ester (intermediate 1-1) was dissolved in 236 mL of methanol and hydrogenated with 1.06 g (0.99 mmol) of activated carbon-supported palladium at 25 ° C for 3 h under standard hydrogen pressure. The reaction mixture was filtered through diatomaceous earth, the filtrate was washed with methanol, and the filtrate was concentrated. 3.53 g of the title compound was obtained.

[0315] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 3.85 (s, 3H) 6.01 (s, 2H) 6.98 (s, 1H) 7.79-7.91 (m, 1H) 7.99 (s, 1H) 12.84 (br.s., 1H).

[0316] Intermediate 3-1

[0317] 5-({[6-(Trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester

[0318]

[0319] First, 4.95g (25.9mmol) of 6-(trifluoromethyl)pyridine-2-carboxylic acid is added to 45mL THF. Add 9.07g (28.2mmol) of tetrafluoroboric acid O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylurea and 4.92mL (28.2mmol) of N-ethyl-N-isopropylpropan-2-amine, and stir the mixture for 30min at 25 ℃. Subsequently, add 4.50g (23.5mmol) of 5-amino-1H-indazole-6-carboxylic acid methyl ester (intermediate 2-1), and stir the mixture for 24h at 25 ℃. The reaction mixture is filtered by membrane filter, and the solid is washed with THF and water, and dried overnight in a drying oven. Obtain 7.60g of title compound.

[0320] UPLC-MS (Method A2): R t =1.16min

[0321] MS (ESIpos): m / z=365 (M+H) +

[0322] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.97 (s, 3H), 8.13-8.27 (m, 2H), 8.30 (s, 1H), 8 .33-8.45(m, 1H), 8.45-8.51(m, 1H), 9.15(s, 1H), 12.57(s, 1H), 13.44(s, 1H).

[0323] Intermediate 3-2

[0324] 5-({[6-(Difluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester

[0325]

[0326] First, 2.85g (23.5mmol) of 6-(difluoromethyl)pyridine-2-carboxylic acid was added to 30mL of THF. 6.05g (18.8mmol) of tetrafluoroboric acid O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylurea and 3.3mL of N-ethyl-N-isopropylpropane-2-amine were added, and the mixture was stirred at room temperature for 10min. Subsequently, 3.00g (15.7mmol) of 5-amino-1H-indazole-6-carboxylic acid methyl ester was added, and the mixture was stirred at room temperature overnight. The reaction mixture was mixed with water, the precipitate was filtered off by suction, and it was repeatedly washed with water and dichloromethane. 1.53g (27% of theoretical value) of the title compound was obtained. The phases of the separated filtrate were concentrated, the organic phase was mixed with a small amount of dichloromethane and suspended in an ultrasonic bath, and the precipitate was filtered off by suction. This gave 1.03g of the title compound.

[0327] 1 H-NMR (first product fraction, 300 MHz, DMSO-d6): δ [ppm] = 3.99 (s, 3H), 7.09 (t, 1H), 8.00 (d, 1H), 8.21-8.40 (m, 4H), 9.14 (s, 1H), 12.53 (s, 1H), 13.44 (s, 1H).

[0328] Intermediate 3-3

[0329] 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester

[0330]

[0331] First, 2.10g of 6-(2-hydroxypropane-2-yl) pyridine-2-carboxylic acid potassium (intermediate V3-1) was added to 15mL of THF. 3.69g (11.5mmol) of tetrafluoroboric acid O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylurea and 2.00mL of N-ethyl-N-isopropylpropane-2-amine were added, and the mixture was stirred at room temperature for 15min. Subsequently, 1.83g (9.58mmol) of 5-amino-1H-indazole-6-carboxylic acid methyl ester (intermediate 2-1) was added, and the mixture was stirred at room temperature for 19h. The mixture was mixed with water and ethyl acetate, undissolved solids were filtered out, and each phase of the filtrate was separated, and the aqueous phase was extracted twice with ethyl acetate, washed with sodium chloride solution, filtered through a hydrophobic filter, concentrated, and purified by silica gel column chromatography (hexane / ethyl acetate). After removing the solvent, 1.56g of the title compound in yellow foam was obtained.

[0332] UPLC-MS (Method A1): R t =1.00 min (UV detector: TIC Smooth), molecular weight measurement value 354.00.

[0333] 1 H-NMR (500MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 3.97 (s, 3H), 5.37 (s, 1H), 7.90-7.95 (m, 1H) , 8.03-8.07(m, 2H), 8.23(s, 1H), 8.29(s, 1H), 9.19(s, 1H), 12.79(s, 1H), 13.41(br.s., 1H).

[0334] Intermediate 4-1

[0335] 2-(Oxetane-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0336]

[0337] 1.00g (2.66mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was dissolved in 10mL of DMF, and after adding 1.10g (7.99mmol) of potassium carbonate and 221mg (1.33mmol) of potassium iodide, the mixture was stirred at 25°C for 30min. 603mg (3.99mmol) of 3-bromomethyloxetane was added, and the mixture was stirred at 25°C for 24h. The reaction mixture was layered between water and ethyl acetate. The mixture was extracted twice with ethyl acetate, and the combined organic phases were filtered through a hydrophobic filter and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 260mg of the title compound was obtained.

[0338] UPLC-MS (Method A2): R t =1.24min

[0339] MS (ESIpos): m / z=435 (M+H) +

[0340] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.49-3.64 (m, 1H), 3.95 (s, 3H), 4.49 (t, 2H), 4.68 (dd, 2H), 4.81 (d, 2 H), 8.20 (dd, 1H), 8.35-8.41 (m, 1H), 8.43-8.49 (m, 2H), 8.55-8.58 (m, 1H), 9.06 (s, 1H), 12.53 (s, 1H).

[0341] Intermediate 4-2

[0342] 2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0343]

[0344] 1.00g (2.75mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was dissolved in 5mL of DMF, and 387μL (4.12mmol) of 2-bromoethyl methyl ether, 1.14g (8.23mmol) of potassium carbonate and 228mg (1.37mmol) of potassium iodide were added while stirring. The reaction mixture was stirred at 25°C for 24h, diluted with water, and extracted twice with ethyl acetate. The combined organic phases were filtered through a hydrophobic filter and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 12mg of the title compound was obtained.

[0345] UPLC-MS (Method A1): R t =1.24min

[0346] MS (ESIpos): m / z=423 (M+H) +

[0347] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.24 (s, 3H), 3.86 (t, 2H), 3.96 (s, 3H), 4.65 (t, 2H), 8.2 1(dd, 1H), 8.35-8.42(m, 1H), 8.43-8.51(m, 2H), 8.52(d, 1H), 9.06(s, 1H), 12.53(s, 1H).

[0348] Intermediate 4-3

[0349] 2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0350]

[0351] 1.00g (2.75mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was dissolved in 5mL of DMF, and 460μL (4.12mmol) of 1-bromo-3-methoxypropane, 1.14g (8.23mmol) of potassium carbonate and 228mg (1.37mmol) of potassium iodide were added while stirring. The reaction mixture was stirred at 25°C for 72h, diluted with water, and extracted twice with ethyl acetate. The combined organic phases were filtered through a hydrophobic filter and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 28mg of the title compound was obtained.

[0352] UPLC-MS (Method A1): R t =1.29min

[0353] MS (ESIpos): m / z=437 (M+H) +

[0354] 1 H NMR (400 MHz, DMSO-d6): δ [ppm] = 2.17 (quintet, 2H), 3.24 (s, 3H), 3.33-3.36 (m, 2H), 3.96 (s, 3H), 4.53 (t, 2H), 8.21 (dd, 1H), 8.35-8.42 (m, 1H), 8.45-8.49 (m, 2H), 8.54 (d, 1H), 9.06 (s, 1H), 12.54 (s, 1H).

[0355] Intermediate 4-4

[0356] 2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0357] Preparation method 1

[0358]

[0359] First, 930mg (2.55mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1), 1.06g of potassium carbonate and 212mg of potassium iodide were dissolved in 9mL of DMF, and the mixture was stirred for 15min. Then, 0.62mL of 4-bromo-2-methylbutan-2-ol was added, and the mixture was stirred at 60°C for 16h. The mixture was mixed with water and extracted twice with ethyl acetate, and the extract was washed three times with saturated sodium chloride solution, filtered and concentrated. Silica gel column chromatography (hexane / ethyl acetate) purification obtained 424mg of the title compound.

[0360] UPLC-MS (Method A2): R t =1.21 min (UV detector: TIC), molecular weight measurement value 450.00.

[0361] 1H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.16 (s, 6H) 2.02-2.11 (m, 2H) 3.96 (s, 3H) 4.51-4.60 (m, 3H) 8.2 0 (dd, J=7.83, 1.01Hz, 1H) 8.39 (s, 1H) 8.45 (s, 2H) 8.55 (d, J=0.76Hz, 1H) 9.05 (s, 1H) 12.52 (s, 1H).

[0362] Preparation method 2

[0363] First, 1.95g (7.03mmol) of 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester (intermediate 7-1) was added to 30mL of THF. 1.48g (7.37mmol) of 6-(trifluoromethyl)pyridine-2-carboxylic acid, 2.71g (8.44mmol) of tetrafluoroboric acid O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylurea and 1.47mL (8.44mmol) of N-ethyl-N-isopropylpropane-2-amine were added, and the mixture was stirred at 25°C for 20.5h. Water was added, and the mixture was extracted three times with ethyl acetate, and the extract was washed with sodium chloride solution, filtered through a hydrophobic filter and concentrated. The residue was separated by silica gel column chromatography (hexane / ethyl acetate gradient). 2.79g of the title compound was obtained.

[0364] UPLC-MS (Method A1): R t =1.23 min (UV detector: TIC), molecular weight measurement value 450.00.

[0365] Intermediate 4-5

[0366] 2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0367]

[0368] First, 1.00g (2.66mmol, 97%) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was added to 50mL of DMF, 1.10g (7.99mmol) of potassium carbonate and 221mg (1.33mmol) of potassium iodide were added while stirring, and the mixture was stirred at 25°C for 30min. Subsequently, 857μL (3.99mmol) of (2-bromoethoxy) (tert-butyl) dimethylsilane were added, and the mixture was stirred at 25°C for 24h. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic phases were filtered and concentrated through a hydrophobic filter. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 400mg of the title compound was obtained.

[0369] UPLC-MS (Method A1): R t =1.58min

[0370] MS (ESIpos): m / z=523 (M+H) +

[0371] 1 H NMR (300MHz, DMSO-d6): δ[ppm]=-0.18--0.13(m, 6H), 0.74(s, 9H), 3.96(s, 3H), 4.08(t, 2H), 4. 57(t, 2H), 8.15-8.25(m, 1H), 8.32-8.43(m, 1H), 8.43-8.52(m, 3H), 9.07(s, 1H), 12.53(s, 1H).

[0372] Intermediate 4-6

[0373] 2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0374]

[0375] Similarly to intermediate 4-5, 1.00g (2.75mmol) of 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-1) was dissolved in 10mL of DMF, 1.14g (8.24mmol) of potassium carbonate and 228mg (1.37mmol) of potassium iodide were added while stirring, and the mixture was stirred at 25°C for 30min. Subsequently, 1.04g (4.12mmol) of (3-bromopropoxy) (tert-butyl) dimethylsilane was added, and the mixture was stirred at 25°C for 24h. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate. The reaction mixture was layered between water and ethyl acetate, and the aqueous phase was extracted twice with ethyl acetate. The combined organic phase was filtered through a hydrophobic filter and concentrated. The residue was purified by preparative HPLC to obtain 428mg of the title compound.

[0376] UPLC-MS (Method A1): R t =1.63min

[0377] MS (ESIpos): m / z=537 (M+H) +

[0378] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = -0.02-0.06 (m, 6H), 0.87 (s, 9H), 2.14 (quin, 2H), 3.62 (t, 2H), 3.96 (s , 3H), 4.54 (t, 2H), 8.20 (d, 1H), 8.35-8.42 (m, 1H), 8.43-8.48 (m, 3H), 8.49-8.53 (m, 1H), 9.06 (s, 1H).

[0379] Intermediates 4-7

[0380] 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-2-(4,4,4-trifluorobutyl)-2H-indazole-6-carboxylic acid methyl ester

[0381]

[0382] First, 300 mg (0.80 mmol) of 5-({[6-(2-hydroxypropane-2-yl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylic acid methyl ester (intermediate 3-3) was added to 4.5 mL of DMF. 287 mg (1.21 mmol) of 1,1,1-trifluoro-4-iodobutane and 333 mg of potassium carbonate were added, and the mixture was stirred at 100 ° C for 23 h. Water was added and the mixture was extracted three times with ethyl acetate. The mixture was concentrated and the product was purified by preparative HPLC. 72 mg of the title compound was obtained.

[0383] UPLC-MS (Method A1): R t =1.26min (UV detector: TIC), molecular weight measurement value 464.17.

[0384] Intermediate 4-8

[0385] 5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester

[0386]

[0387] Analogously to Intermediate 4-4 (Preparation Method 2), 195 mg (0.46 mmol) of 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester (Intermediate 7-1) was reacted with 78 mg (0.50 mmol) of 5-fluoro-6-methylpyridine-2-carboxylic acid for 19.5 h. After similar aqueous work-up, 228 mg of crude product was obtained.

[0388] UPLC-MS (Method A1): R t =1.20 min (UV detector: TIC), molecular weight measurement value 414.00.

[0389] Intermediate 4-9

[0390] 2-(3-Hydroxy-3-methylbutyl)-5-{[(6-methylpyridin-2-yl)carbonyl]amino}-2H-indazole-6-carboxylic acid methyl ester

[0391]

[0392] Analogously to Intermediate 4-4 (Preparation Method 2), 195 mg (0.46 mmol) of 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester (Intermediate 7-1) was reacted with 70 mg (0.50 mmol) of 6-methylpyridine-2-carboxylic acid for 19.5 h. After similar water treatment, 278 mg of the title compound was obtained as a crude product.

[0393] UPLC-MS (Method A1): R t =1.14 min (UV detector: TIC), mass measurement value 396.00.

[0394] Intermediate 4-10

[0395] 2-[3-(2,2,2-trifluoroethoxy)propyl]-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester

[0396]

[0397] 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.

[0398] UPLC-MS (Method A1): R t =1.39 min (UV detector: TIC), molecular weight measurement value 504.12.

[0399] Intermediate 4-11

[0400] 5-({[6-(Difluoromethyl)pyridin-2-yl]carbonyl}amino)-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester

[0401]

[0402] First, 2.00g of 5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester (intermediate 7-1) is added to 40mL of THF.1.50g of 6-(difluoromethyl)pyridine-2-carboxylic acid, 2.78g of tetrafluoroboric acid O-(benzotriazole-1-yl)-N,N,N',N'-tetramethylurea (TBTU, CAS 125700-67-6) and 1.5mL of N-ethyl-N-isopropylpropane-2-amine are added, and the mixture is stirred at room temperature for 24h.Water is added, the mixture is extracted three times with ethyl acetate, and the organic phase merged is washed with sodium chloride solution and filtered by a hydrophobic filter.Concentrated mixture, and residue is purified by silica gel column chromatography (hexane / ethyl acetate).Obtain the title compound of 3.05g yellow solid.

[0403] UPLC-MS (Method A1): Rt = 1.15 min (UV detector TIC), molecular weight determination value 432.00.

[0404] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.17 (s, 6H), 2.04-2.11 (m, 2H), 3.99 (s, 3H), 4.52-4.60 (m, 3H), 7.1 0 (t, 1H), 8.00 (dd, 1H), 8.28-8.38 (m, 2H), 8.44-8.47 (m, 1H), 8.56 (d, 1H), 9.05 (s, 1H), 12.49 (s, 1H).

[0405] Intermediate 5-1

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

[0407]

[0408] To a solution of 1.50 g (4.12 mmol) of methyl 5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate in 20 mL of THF cooled in an ice-water cooling bath was carefully added 6.9 mL (5 equivalents) of a 3M solution of methylmagnesium bromide in diethyl ether. The mixture was stirred for 1 h while cooling with an ice bath and at room temperature for 19.5 h. 2 equivalents of methylmagnesium bromide solution were added, and the mixture was stirred for another 24 h at room temperature. Saturated aqueous ammonium chloride solution was added, the mixture was stirred, and extracted three times with ethyl acetate. The combined organic phases were washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate). 763 mg of the title compound was obtained.

[0409] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 5.99 (s, 1H), 7.49 (s, 1H), 8.06 (s, 1H), 8.14-8.19 (m, 1H), 8.37 (t, 1H), 8.46 (d, 1H), 8.78 (s, 1H), 12.32 (s, 1H), 12.97 (s, 1H).

[0410] Intermediate 5-2

[0411] 6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]pyridine-2-carboxamide

[0412]

[0413] Analogously to the preparation of intermediate 5-1, 2.40 g (6.93 mmol) of methyl 5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxylate (intermediate 3-2) in 10 mL of THF was reacted with three portions of 3M methylmagnesium bromide in diethyl ether (6.9 mL, then stirred at room temperature for 45 min; 11.6 mL, then stirred at room temperature for 2 h; 6.9 mL, then stirred at room temperature for 2 h). After work-up as for intermediate 5-1, 2.39 g of crude product was obtained, which was used without further purification.

[0414] Intermediate 6-1

[0415] 2-(3-Hydroxy-3-methylbutyl)-5-nitro-2H-indazole-6-carboxylic acid methyl ester

[0416]

[0417] First, 5.00g (22.6mmol) of 5-nitro-1H-indazole-6-carboxylic acid methyl ester (intermediate 1-1) was added to 40mL of DMF. 5.65g (33.9mmol) of 4-bromo-2-methylbutan-2-ol, 9.37g (67.8mmol) of potassium carbonate and 5.63g (33.9mmol) of potassium iodide were added, and the mixture was stirred at 100°C for 20h. Water was added, and the mixture was extracted with ethyl acetate three times, and 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 solid obtained was stirred with diethyl ether, filtered off with suction, washed with diethyl ether and dried. 2.49g of the title compound was obtained.

[0418] UPLC-MS (Method A1): R t =0.93 min (UV detector: TIC), molecular weight measurement value 307.00.

[0419] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.15 (s, 6H), 2.02-2.11 (m, 2H), 3.84 (s, 3H), 4.54(s, 1H), 4.58-4.65(m, 2H), 8.05(s, 1H), 8.69(s, 1H), 8.86(s, 1H).

[0420] Intermediate 7-1

[0421] 5-Amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester

[0422]

[0423] 4.53 g of iron and 217 mg of ammonium chloride are added to a solution of 2.49 g (8.10 mmol) of 2-(3-hydroxy-3-methylbutyl)-5-nitro-2H-indazole-6-carboxylic acid methyl ester (intermediate 6-1) in 30 mL of ethanol and 10 mL of water, and the mixture is stirred at 90 ° C for 21.5 h. The mixture is filtered through diatomaceous earth and washed three times with ethanol, the filtrate is concentrated, and the residue is mixed with water. The extraction is carried out three times with ethyl acetate (sodium chloride solution is added to improve phase separation). The combined organic phases are washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. 1.95 g (85% of theoretical value) of the title compound are obtained.

[0424] UPLC-MS (Method A1): R t =0.67 min (UV detector: TIC), molecular weight measurement value 277.00.

[0425] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.14 (s, 6H), 1.96-2.08 (m, 2H), 3.85 (s, 3H), 4.39-4.51(m, 3H), 5.81(s, 2H), 6.80(s, 1H), 8.05(s, 1H), 8.18(s, 1H).

[0426] Working Example

[0427] Example 1

[0428] N-[6-(2-Hydroxypropan-2-yl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0429]

[0430] 75mg (0.18mmol) of 2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-2) was dissolved in 500μL of THF and mixed with 887μL (0.89mmol) of 1M THF solution of methylmagnesium bromide. The reaction mixture was stirred at 25°C for 60min. Subsequently, 1mL of saturated aqueous ammonium chloride solution was carefully added, and the mixture was filtered. The aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, filtered through a hydrophobic filter, and concentrated. The residue was dissolved in 3mL of DMSO and purified by preparative HPLC. The portion containing the product was freeze-dried. 20mg of the title compound was obtained.

[0431] UPLC-MS (Method A1): R t =1.08min

[0432] MS (ESIpos): m / z=423 (M+H) +

[0433] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 3.22 (s, 3H), 3.82 (t, 2H), 4.55 (t, 2H), 5.96 (s, 1H), 7.57 (s, 1H), 8.16 (d1 H), 8.29-8.42(m, 2H), 8.42-8.50(m, 1H), 8.71(s, 1H), 12.36(s, 1H).

[0434] Example 2

[0435] N-[6-(Hydroxymethyl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0436]

[0437] 13mg (0.36mmol) of lithium aluminum hydride was suspended in 1mL of THF, and the mixture was cooled to 0°C. A solution of 75mg (0.17mmol) of 2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-2) dissolved in 500μL THF was added dropwise, and the mixture was stirred at 25°C for 60min. The mixture was diluted with water and extracted twice with ethyl acetate, and the combined organic phases were washed with sodium chloride solution, filtered through a hydrophobic filter, concentrated, and dried under reduced pressure. 13mg of the title compound was obtained.

[0438] UPLC-MS (Method A2): R t =0.99min

[0439] MS (ESIpos): m / z=394 (M+H) +

[0440] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 3.23 (s, 3H), 3.83 (t, 2H), 4.56 (t, 2H), 4.69 (d, 2H), 5.77 (t, 1 H), 7.57 (s, 1H), 8.19 (d, 1H), 8.33-8.41 (m, 2H), 8.43-8.47 (m, 1H), 8.51 (s, 1H), 11.20 (s, 1H).

[0441] Example 3

[0442] N-[6-(2-Hydroxypropan-2-yl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0443]

[0444] 75mg (0.17mmol) of 2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-3) was dissolved in 500μL of THF and mixed with 859μL (0.86mmol) of 1M THF solution of methylmagnesium bromide. The reaction mixture was stirred at 25°C for 60min. Subsequently, 1mL of saturated ammonium chloride solution was carefully added and the mixture was filtered. The aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, filtered through a hydrophobic filter, and concentrated. The residue was dissolved in 3mL of DMSO and purified by preparative HPLC. The portion containing the product was freeze-dried. 25mg of the title compound was obtained.

[0445] UPLC-MS (Method A1): R t =1.13min

[0446] MS (ESIpos): m / z=437 (M+H) +

[0447] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 2.14 (quin, 2H), 3.23 (s, 3H), 3.26-3.32 (m, 2H), 4.44 (t, 2H) , 5.95 (s, 1H), 7.58 (s, 1H), 8.16 (d, 1H), 8.31-8.40 (m, 2H), 8.43-8.48 (m, 1H), 8.72 (s, 1H), 12.36 (s, 1H).

[0448] Example 4

[0449] N-[6-(Hydroxymethyl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0450]

[0451] 13 mg of lithium aluminum hydride was suspended in THF, and the mixture was cooled to 0 ° C. A solution of 75 mg (0.17 mmol) of 2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-3) in THF was added dropwise, and the mixture was allowed to reach room temperature within 30 min. The mixture was diluted with water and filtered, the residue was washed with ethyl acetate, and the filtrate was extracted with ethyl acetate. The combined ethyl acetate phase was washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC.

[0452] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 2.14 (quin, 2H), 3.23 (s, 3H), 3.29 (t, 2H), 4.45 (t, 2H), 4.68 (d, 2H), 5.77 (t, 1H), 7.58 (s, 1H), 8.18 (d, 1H), 8.32-8.48 (m, 3H), 8.51 (s, 1H), 11.21 (s, 1H).

[0453] Example 5

[0454] N-[2-(2-Hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0455] Step A:

[0456] 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

[0457]

[0458] 100 mg (0.19 mmol) of methyl 2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-5) was dissolved in 1 mL of THF and mixed with 669 μL (0.67 mmol) of a 1 M solution of methylmagnesium bromide in THF. The reaction mixture was stirred at 25° C. for 60 min. 287 μL (0.29 mmol) of a 1 M solution of methylmagnesium bromide in THF was added and the mixture was stirred at 25° C. for 3 h. Subsequently, 20 mL of a saturated ammonium chloride solution was carefully added and the mixture was filtered. The aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, dried over magnesium sulfate, filtered, concentrated, and dried under reduced pressure. 50 mg of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide are obtained.

[0459] UPLC-MS (Method A2): R t =1.51min

[0460] MS (ESIpos): m / z=523 (M+H) +

[0461] 1 H NMR (300MHz, DMSO-d6): δ[ppm]=-0.17--0.09(m, 6H), 0.78(s, 9H), 1.62(s, 6H), 4.04(t, 2H), 4.47(t, 2H), 5.98 (s, 1H), 7.57 (s, 1H), 8.16 (d, 1H), 8.29 (s, 1H), 8.37 (t, 1H), 8.45 (d, 1H), 8.73 (s, 1H), 12.38 (s, 1H).

[0462] Step B:

[0463]

[0464] 50mg (96μmol) of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide was dissolved in 1.0mL of THF and mixed with 144μL (0.14mmol) of 1M tetrabutylammonium fluoride THF solution. The reaction mixture was stirred at room temperature for 1h. The mixture was diluted with water and extracted twice with ethyl acetate, the combined organic phases were washed with saturated sodium chloride solution, filtered through a hydrophobic filter, and concentrated. 36mg of N-[2-(2-hydroxyethyl)-6-(2-hydroxypropyl-2-yl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Example 5) was obtained.

[0465] 1 H-NMR (400MHz, DMSO-d6): d[ppm]=1.62(s, 6H), 3.86(q, 2H), 4.43(t, 2H), 4.95(t, 1H), 5.94(s, 1 H), 7.57(s, 1H), 8.16(dd, 1H), 8.30(s, 1H), 8.37(t, 1H), 8.45(d, 1H), 8.72(s, 1H), 12.36(s, 1H).

[0466] UPLC-MS (Method A2): R t =0.97 min (UV detector: TIC), molecular weight measurement value 408.00.

[0467] Example 6

[0468] N-[6-(2-Hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0469] Step A:

[0470] 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

[0471]

[0472] 50mg (0.09mmol) of 2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-6) was dissolved in 500μL of THF and mixed with 326μL (0.33mmol) of 1M THF solution of methylmagnesium bromide. The reaction mixture was stirred at 25°C for 60min. Subsequently, 20mL of saturated ammonium chloride solution was carefully added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were filtered through a hydrophobic filter, concentrated, and dried under reduced pressure. The residue was purified by preparative HPLC. 40 mg of N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide are obtained.

[0473] UPLC-MS (Method A1): R t =1.58min

[0474] MS (ESIpos): m / z=537 (M+H) +

[0475] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 0.02-0.05 (m, 6H), 0.84-0.91 (m, 9H), 1.62 (s, 6H), 2.02-2.18 (m, 2H), 3.55-3.62 (m, 2H), 4 .45 (t, 2H), 5.96 (s, 1H), 7.57 (s, 1H), 8.16 (d, 1H), 8.31 (s, 1H), 8.33-8.42 (m, 1H), 8.45 (d, 1H), 8.72 (s, 1H), 12.37 (s, 1H).

[0476] Step B:

[0477]

[0478] 37mg (0.07mmol) of N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide was dissolved in 500μL of THF and mixed with 207μL (0.21mmol) of 1M tetrabutylammonium fluoride in THF. The reaction mixture was stirred at 25°C for 2h. The mixture was diluted with water and extracted twice with ethyl acetate, and the combined organic phases were washed with saturated sodium chloride solution, filtered and concentrated. After purification by preparative HPLC, 10mg of N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Example 6, containing minor components) was obtained.

[0479] UPLC-MS (Method A2): R t =1.00min

[0480] MS (ESIpos): m / z=423 (M+H) +

[0481] 1 H NMR selected signals (400 MHz, DMSO-d6): δ [ppm] = 1.61 (s), 2.00-2.12 (m), 3.38 (t, 2H), 4.44 (t, 2H), 4.62 (br. s., 1H), 5.93 (br. s., 1H), 7.55 (s, 1H), 8.13 (d, 1H), 8.27-8.38 (m, 2H), 8.43 (d, 1H), 8.71 (s, 1H), 12.30 (br. s., 1H).

[0482] Example 7

[0483] N-[2-(2-Hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0484] Step A:

[0485] N-[2-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0486]

[0487] 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 2M lithium borohydride solution. The mixture was stirred at 25° C. for 24 h. 14 mg (0.38 mmol) of sodium borohydride and 500 μL of methanol were added, and the mixture was stirred at 25° C. for 4 h. 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 to obtain 30 mg of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide.

[0488] UPLC-MS (Method A2): R t =1.44min

[0489] MS (ESIpos): m / z=495 (M+H) +

[0490] 1 H NMR (300MHz, DMSO-d6): δ[ppm]=-0.16--0.12(m, 6H), 0.75-0.79(m, 9H), 4.05(t, 2H), 4.48(t, 2H), 4.69(d, 2H), 5.7 5-5.77 (m, 1H), 7.57 (s, 1H), 8.18 (dd, 1H), 8.30-8.33 (m, 1H), 8.38 (t, 1H), 8.45 (d, 1H), 8.51 (s, 1H), 11.20 (s, 1H).

[0491] Step B:

[0492]

[0493] 33mg (0.07mmol) of N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide was dissolved in 1mL of THF and mixed with 100μL (0.10mmol) of 1M tetrabutylammonium fluoride in THF solution. The reaction mixture was stirred at 25°C for 1h. The mixture was diluted with water and extracted twice with ethyl acetate, and the combined organic phases were washed with saturated sodium chloride solution, filtered through a hydrophobic filter, concentrated, and dried under reduced pressure. 25mg of N-[2-(2-hydroxyethyl)-6-(hydroxymethyl)-2H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (Example 7) was obtained.

[0494] UPLC-MS (Method A2): R t =0.87min

[0495] MS (ESIpos): m / z=381 (M+H) +

[0496] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 3.87 (q, 2H), 4.44 (t, 2H), 4.69 (d, 2H), 4.98 (t, 1H), 5.70-5.81 (m, 1 H), 7.57 (s, 1H), 8.11-8.23 (m, 1H), 8.31-8.42 (m, 2H), 8.43-8.49 (m, 1H), 8.51 (s, 1H), 11.20 (s, 1H).

[0497] Example 8

[0498] N-[6-(2-Hydroxypropan-2-yl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0499]

[0500] 50mg (0.12mmol) of 2-(oxetanes-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-1) was dissolved in 500μL of THF and mixed with 576μL (0.58mmol) of 1M THF solution of methylmagnesium bromide. The reaction mixture was stirred at 25°C for 60min. Subsequently, 20mL of saturated aqueous ammonium chloride solution was carefully added, and the mixture was concentrated. The aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in 2.0mL of DMSO and purified by preparative HPLC. The fractions containing the product were freeze-dried. 30mg of the title compound were obtained.

[0501] UPLC-MS (Method A2): R t =1.03min

[0502] MS (ESIpos): m / z=435 (M+H) +

[0503] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 3.45-3.61 (m, 1H), 4.48 (t, 2H), 4.66 (dd, 2H), 4.72 (d, 2H), 5.94 (s, 1H), 7.57 (s, 1H), 8.16 (d, 1H), 8.33-8.42 (m, 2H), 8.42-8.47 (m, 1H), 8.72 (s, 1H), 12.36 (s, 1H).

[0504] Example 9

[0505] N-[6-(Hydroxymethyl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0506]

[0507] 75 mg (0.17 mmol) of 2-(oxetane-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-1) was dissolved in 1 mL of THF / methanol (1:1) mixture, and 8 mg (0.21 mmol) of sodium borohydride was added. The mixture was stirred at 25 ° C for 60 min. The reaction mixture was concentrated and the residue was mixed with water. The suspension was vigorously stirred for 15 min, the solid was filtered off, washed twice with water, washed twice with diethyl ether, and dried under reduced pressure. 48 mg of the title compound was obtained.

[0508] UPLC-MS (Method A2): R t =0.94min

[0509] MS (ESIpos): m / z=407 (M+H) +

[0510] 1 H NMR (300MHz, DMSO-d6): δ [ppm] = 3.55 (s, 1H), 4.48 (t, 2H), 4.61-4.77 (m, 6H ), 7.57 (s, 1H), 8.18 (dd, 1H), 8.33-8.49 (m, 3H), 8.51 (s, 1H), 11.21 (s, 1H).

[0511] Example 10

[0512] N-{6-(2-hydroxypropan-2-yl)-2-[3-(methylsulfonyl)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0513]

[0514] A mixture of 500mg (1.32mmol) of N-[6-(2-hydroxypropyl-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (intermediate 5-1), 569mg of potassium carbonate and 114mg of potassium iodide in 5.0mL of DMF was stirred at room temperature for 15min. 414mg of 1-bromo-3-(methylsulfonyl)propane was added, and the mixture was stirred at room temperature overnight. Water was added, the mixture was extracted twice with ethyl acetate, the extract was washed with sodium chloride solution, and concentrated. The residue was purified by column chromatography (dichloromethane / methanol gradient). The product portion was stirred, filtered and dried with diethyl ether. 59mg of the title compound was obtained.

[0515] UPLC-MS (Method A2): R t=1.02min

[0516] MS (ESIpos): m / z = 485 (M+H) +

[0517] 1 H-NMR (300MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 2.26-2.42 (m, 2H), 2.99 (s, 3H), 3.06-3.16 (m, 2H), 4.55(t, 2H), 5.96(s, 1H), 7.60(s, 1H), 8.16(d, 1H), 8.33-8.48(m, 3H), 8.73(s, 1H), 12.37(s, 1H).

[0518] Embodiment 11

[0519] N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0520]

[0521] Preparation method 1

[0522] First, 705 mg (1.57 mmol) of 2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-4) was added to 10 mL of THF and cooled in an ice-water cooling bath. 2.6 mL (5.0 equivalents) of 3M methylmagnesium bromide solution (in diethyl ether) were added, and the mixture was stirred for 1 h while cooling with an ice bath, and stirred at room temperature for 4.5 h. 1 equivalent of methylmagnesium bromide solution was added, and the mixture was stirred at room temperature for 20.5 h. 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 phase was washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. 790 mg of residue was obtained, which was purified by preparative HPLC. 234 mg of the title compound and 164 mg of a product fraction were obtained which was stirred with diethyl ether. After suction filtration and subsequent drying, a further 146 mg of the title compound were obtained.

[0523] UPLC-MS (Method A1): R t =1.10 min (UV detector: TIC), molecular weight measurement value 450.00.

[0524] 1H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.14 (s, 6H), 1.61 (s, 6H), 1.99-2.08 (m, 2H), 4.42-4.55 (m, 3H), 5.9 3 (s, 1H), 7.56 (s, 1H), 8.15 (dd, 1H), 8.32-8.39 (m, 2H), 8.41-8.47 (m, 1H), 8.70 (s, 1H), 12.34 (s, 1H).

[0525] Preparation method 2

[0526] A mixture of 500 mg (1.37 mmol) of N-[6-(2-hydroxypropan-2-yl)-1H-indazole-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 were added, and the mixture was heated to 100° C. for 2 h. 0.5 equivalents of 4-bromo-2-methylbutan-2-ol were also 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 portion was obtained, which was stirred with diethyl ether. The solid was filtered and dried. 60 mg of the title compound was obtained.

[0527] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.14 (s, 6H), 1.61 (s, 6H), 1.99-2.07 (m, 2H), 4.43-4.52 (m, 3H) 5. 94 (s, 1H) 7.57 (s, 1H) 8.15 (dd, 1H) 8.33-8.40 (m, 2H), 8.42-8.48 (m, 1H), 8.71 (s, 1H), 12.35 (s, 1H).

[0528] Example 12

[0529] N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0530]

[0531] 160mg (0.44mmol) of N-[6-(2-hydroxypropyl-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (intermediate 5-1) was suspended in 1.0mL of DMF with 182mg of sodium carbonate and 36mg of potassium iodide, and the mixture was stirred at room temperature for 15min. Then, 123mg of 2-bromoethyl methyl sulfone (0.66mmol) was added, and the mixture was stirred at room temperature overnight. Water was added, the mixture was extracted twice with ethyl acetate, the extract was washed with saturated sodium chloride aqueous solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC to obtain 20mg of the title compound.

[0532] UPLC (Method A2): R t =1.01min;

[0533] MS (ESIpos): m / z = 471 (M + H) +

[0534] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 2.90 (s, 3H), 3.85 (t, 2H), 4.86 (t, 2H), 5.97 (s, 1 H), 7.59 (s, 1H), 8.13-8.19 (m, 1H), 8.37 (s, 1H), 8.41-8.48 (m, 2H), 8.74 (s, 1H), 12.37 (s, 1H).

[0535] Embodiment 13

[0536] 6-(Difluoromethyl)-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0537]

[0538] Preparation method 1

[0539] A mixture of 250 mg of 6-(difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazole-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, the mixture was stirred at 110° C. for 3 h, 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, the mixture was extracted twice with ethyl acetate, the extract was washed with a half-saturated sodium chloride aqueous solution, filtered through a hydrophobic filter, and concentrated. Purification was performed by silica gel column chromatography (hexane / ethyl acetate). 61 mg of the title compound was obtained.

[0540] UPLC-MS (Method A1): R t =1.00 min (UV detector: TIC), molecular weight measurement value 432.00.

[0541] 1 H-NMR (300MHz, DMSO-d6): δ [ppm] = 1.14 (s, 6H), 1.63 (s, 6H), 1.97-2.08 (m, 2H), 4.41-4.55 (m, 3H), 5. 99 (s, 1H), 7.03 (t, 1H), 7.56 (s, 1H), 7.94-8.00 (m, 1H), 8.24-8.38 (m, 3H), 8.71 (s, 1H), 12.49 (s, 1H).

[0542] Preparation method 2

[0543] Analogously 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 a 3M solution of methylmagnesium bromide (in diethyl ether). The crude product was stirred with diethyl ether, filtered, and purified by preparative HPLC to obtain 1.37 g of the title compound.

[0544] Embodiment 14

[0545] 6-(Difluoromethyl)-N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulfonyl)ethyl]-2H-indazol-5-yl}pyridine-2-carboxamide

[0546]

[0547] A mixture of 250 mg 6-(difluoromethyl)-N-[6-(2-hydroxypropyl-2-yl)-1H-indazole-5-yl]pyridine-2-carboxamide (crude product of intermediate 5-2), 144 mg potassium iodide and 239 mg potassium carbonate in 2.5 mL DMF was stirred at room temperature for 15 min. 162 mg 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 extract was washed with a half-saturated sodium chloride aqueous solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC, and the product portion was purified again by silica gel column chromatography (hexane / ethyl acetate). 40 mg of the title compound was obtained.

[0548] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.65 (s, 6H), 2.90 (s, 3H), 3.85 (t, 2H), 4.85 (t, 2H), 6.03 (s, 1H) , 7.04 (t, 1H), 7.59 (s, 1H), 7.98 (d, 1H), 8.25-8.36 (m, 2H), 8.43 (s, 1H), 8.75 (s, 1H), 12.52 (s, 1H).

[0549] Embodiment 15

[0550] 6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0551] Phase A:

[0552] 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

[0553]

[0554] A mixture of 250 mg 6-(difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazole-5-yl]pyridine-2-carboxamide (intermediate 5-2), 48 mg potassium iodide and 239 mg potassium carbonate in 2.5 mL DMF was stirred at room temperature for 15 min. 219 mg (0.87 mmol, 1.5 equivalents) of (3-bromopropoxy) (tert-butyl) dimethylsilane were added, and the mixture was stirred at 110 ° C for 3 h. 1 equivalent of (3-bromopropoxy) (tert-butyl) dimethylsilane was 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 sodium chloride aqueous 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.

[0555] Phase B:

[0556]

[0557] Analogously to the preparation of Example 6, step B, 92 mg of N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(difluoromethyl)pyridine-2-carboxamide was reacted with 0.53 mL of a 1M solution of tetrabutylammonium fluoride in THF for 1 h. Aqueous workup was performed as in Example 6 and purification was performed by preparative HPLC to obtain 46 mg of the title compound.

[0558] UPLC-MS (Method A1): R t =0.92 min (UV detector: TIC), molecular weight measurement value 404.00.

[0559] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.64 (s, 6H), 2.05 (quin, 2H), 3.35-3.46 (m, 2H), 4.45 (t, 2H), 4.64 (t, 1H) ), 5.99 (s, 1H), 7.04 (t, 1H), 7.57 (s, 1H), 7.95-7.99 (m, 1H), 8.25-8.36 (m, 3H), 8.73 (s, 1H), 12.50 (s, 1H).

[0560] Example 16

[0561] N-[6-(2-Hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

[0562]

[0563] A mixture of 210mg (0.58mmol) of N-[6-(2-hydroxypropane-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (intermediate 5-1) in 3mL DMF was mixed with 0.11mL (0.87mmol) of 1,1,1-trifluoro-4-iodobutane and 239mg of potassium carbonate, and the mixture was stirred at 80°C for 6h. After adding water, the mixture was extracted three times with ethyl acetate, and the combined organic phase was washed with a saturated sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The crude product was purified by preparative HPLC. 19mg of the title compound was obtained.

[0564] UPLC-MS (Method A1): R t =1.27 min (UV detector: TIC), molecular weight measurement value 474.15.

[0565] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.62 (s, 6H), 2.10-2.33 (m), 4.49 (t, 2H), 5.94 (s, 1H), 7.5 9(s, 1H), 8.13-8.18(m, 1H), 8.32-8.41(m, 2H), 8.41-8.47(m, 1H), 8.72(s, 1H), 12.35(s, 1H).

[0566] Embodiment 17

[0567] N-{6-(2-hydroxypropan-2-yl)-2-[3-(trifluoromethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0568]

[0569] First 150mg (0.33mmol) N-[6-(2-hydroxypropyl-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl) pyridine-2-carboxamide (intermediate 5-1) is added in 2mL THF. Add 58mg (0.40mmol) of 3-(trifluoromethoxy) propan-1-ol, 131mg triphenylphosphine and 71 μ L diisopropyl azodicarboxylate (DIAD, CAS2446-83-5), and the mixture is stirred at room temperature for 19h. Add 0.83mL of sodium hydroxide solution (2M), and the mixture is stirred at 40 ℃ for 5h. Dilute the mixture with water, and extract three times with ethyl acetate, the organic phase combined is concentrated, and purified by preparative HPLC. Obtain the title compound in 16mg crude product form.

[0570] UPLC-MS (Method A2): R t =1.26min (UV detector: TIC), molecular weight measurement value 490.14.

[0571] 1 H-NMR (400 MHz, DMSO-d6, selected signal): 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).

[0572] Embodiment 18

[0573] N-{6-(2-hydroxypropan-2-yl)-2-[3-(2,2,2-trifluoroethoxy)propyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0574]

[0575] Analogously 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

[0576] UPLC-MS (Method A1): R t =1.25min (UV detector: TIC), molecular weight measurement value 504.16.

[0577] 1 H-NMR (500MHz, DMSO-d6): δ [ppm] = 1.63 (s, 6H), 2.20 (quin, 2H), 3.58 (t, 2H), 4.05 (q, 2H), 4.47 (t, 2H), 5 .94 (s, 1H), 7.58 (s, 1H), 8.15 (dd, 1H), 8.32 (s, 1H), 8.36 (t, 1H), 8.45 (d, 1H), 8.73 (s, 1H), 12.36 (s, 1H).

[0578] Embodiment 19

[0579] 5-Fluoro-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0580]

[0581] First, 228mg (0.31mmol) of 5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-8) was added to 4.5mL THF and cooled with an ice-cooling bath. 0.63mL of 3M methylmagnesium bromide solution (in diethyl ether) was added, and the mixture was stirred for 2h while cooling with an ice bath, and stirred for 21h at room temperature. The reaction mixture was mixed with saturated aqueous ammonium chloride solution and extracted three times with ethyl acetate. The combined organic phase was concentrated. The residue was purified by preparative HPLC. 82mg of the title compound was obtained.

[0582] UPLC-MS (Method A2): R t =1.03min (UV detector: TIC), molecular weight measurement value 414.21.

[0583] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.13 (s, 6H), 1.63 (s, 6H), 1.99-2.05 (m, 2H), 2.55-2.59 (m, 3H), 4.42-4 .50 (m, 3H), 5.95 (s, 1H), 7.54 (s, 1H), 7.83 (t, 1H), 8.05 (dd, 1H), 8.31 (s, 1H), 8.68 (s, 1H), 12.33 (s, 1H).

[0584] Embodiment 20

[0585] N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-methylpyridine-2-carboxamide

[0586]

[0587] First, 278mg (0.48mmol) of 2-(3-hydroxy-3-methylbutyl)-5-{[(6-methylpyridin-2-yl)carbonyl]amino}-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-9) was added to 5.0mL THF and cooled with an ice-cold bath. 0.97mL of 3M methylmagnesium bromide solution (in diethyl ether) was added, and the mixture was stirred for 2h while cooling with an ice bath, and stirred at room temperature for 20.5h. 0.48mL of 3M methylmagnesium bromide solution was added, and the mixture was stirred at room temperature for 67h. The mixture was mixed with saturated aqueous ammonium chloride solution and extracted three times with ethyl acetate, and the extract was washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC. 111mg of the title compound was obtained.

[0588] UPLC-MS (Method A2): R t =0.97 min (UV detector: TIC), molecular weight measurement value 396.22.

[0589] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.15 (s, 6H), 1.64 (s, 6H), 2.00-2.08 (m, 2H), 2.61 (s, 3H), 4.41-4.59 (m , 3H), 5.92 (s, 1H), 7.50 (dd, 1H), 7.56 (s, 1H), 7.90-7.99 (m, 2H), 8.33 (s, 1H), 8.70 (s, 1H), 12.39 (s, 1H).

[0590] Embodiment 21

[0591] 6-(2-Hydroxypropan-2-yl)-N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]pyridine-2-carboxamide

[0592]

[0593] A solution of 72 mg (0.16 mmol) of 5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-2-(4,4,4-trifluorobutyl)-2H-indazole-6-carboxylic acid methyl ester (intermediate 4-7) in 10 mL of THF was cooled in an ice / water cooling bath. 0.26 mL of a 3 M solution of methylmagnesium bromide in diethyl ether was added, and the mixture was stirred for 2 h, and then at room temperature for 20 h. 1 equivalent of a 3 M solution of methylmagnesium bromide was added, and the mixture was stirred at room temperature for 24 h. Saturated aqueous ammonium chloride solution was added, the mixture was extracted three times with ethyl acetate, the extract was washed with sodium chloride solution, and concentrated. 22 mg (31% of theoretical value) of the title compound were obtained by preparative HPLC.

[0594] UPLC-MS (Method A2): R t =1.15min (UV detector: TIC), molecular weight measurement value 464.20.

[0595] 1 H-NMR (400MHz, DMSO-d6): δ [ppm] = 1.56 (s, 6H), 1.64 (s, 6H), 2.07-2.34 (m, 4H), 4.49 (t, 2H), 5.32 (s, 1 H), 6.05 (s, 1H), 7.60 (s, 1H), 7.87 (dd, 1H), 7.99-8.05 (m, 2H), 8.35 (s, 1H), 8.79 (s, 1H), 12.45 (s, 1H).

[0596] Embodiment 22

[0597] N-{2-[2-(1-Hydroxycyclopropyl)ethyl]-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide

[0598]

[0599] First, 250 mg (0.69 mmol) of N-[6-(2-hydroxypropan-2-yl)-1H-indazole-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide (intermediate 5-1) was added to 5 mL of DMSO. 159 mg (0.96 mmol) of 1-(2-bromoethyl)cyclopropanol, 285 mg of potassium carbonate and 171 mg of potassium iodide were added, and the mixture was stirred at 100 ° C for 5 h. Water was added and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with sodium chloride solution, filtered through a hydrophobic filter, and concentrated. The residue was purified by preparative HPLC (column: Waters XBridge C18 5μ100×30 mm, eluent A: water + 0.1 volume % of formic acid (99%), eluent B: acetonitrile). Freeze drying gave 45 mg of the title compound.

[0600] 1 H-NMR (500MHz, DMSO-d6): δ [ppm] = 0.18-0.22 (m, 2H), 0.48-0.52 (m, 2H), 1.62 (s, 6H), 2.08 (t, 2H), 4.54-4.60 (m, 2H) ), 5.36 (s, 1H), 5.96 (s, 1H), 7.57 (s, 1H), 8.16 (dd, 1H), 8.34-8.39 (m, 2H), 8.45 (d, 1H), 8.72 (s, 1H), 12.36 (s, 1H).

[0601] Physiological efficacy assessment

[0602] IRAK4 kinase assay

[0603] The IRAK4-inhibitory activity of the substances according to the invention was determined in the IRAK4 TR-FRET assay (TR-FRET=time resolved fluorescence resonance energy transfer) described below.

[0604] GST (glutathione-S-transferase) from the N-terminus and human IRAK4 recombinant fusion protein were used as enzymes, the recombinant fusion protein was expressed in baculovirus-infected insect cells (Hi5, BTI-TN-5B1-4, cell line purchased from Invitrogen, catalog number B855-02) and purified by affinity chromatography. The substrate for the kinase reaction was the biotinylated peptide biotin-Ahx-KKARFSRFAGSSPSQASFAEPG (C-terminus in amide form), which can be purchased, for example, from Biosyntan GmbH (Berlin-Buch).

[0605] For the assay, 11 different concentrations ranging from 20 μM to 0.073 nM were prepared from a 2 mM DMSO solution of the test substance. 50 nL of the corresponding solution was pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of IRAK4 in assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w / v) bovine gamma-globulin (BGG), 0.04% (v / v) nonidet-P40 (Sigma)] were added, and the mixture was incubated for 15 min to allow the substances to pre-bind to the enzyme before the kinase reaction. The kinase reaction is then started by adding 3 μL of a solution of adenosine triphosphate (ATP, 1.67 mM = final concentration in the assay volume of 5 μL: 1 mM) and peptide substrate (0.83 μM = final concentration in the assay volume of 5 μL: 0.5 μM) in assay buffer and the resulting mixture is incubated at 22° C. for a reaction time of 45 min. The concentration of IRAK4 is adjusted to the respective activity of the enzyme and is set so that the assay is performed in the linear range. Typically the concentration is on the order of about 0.2 nM. The reaction was terminated by adding 5 μL of a solution of TR-FRET detection reagent [0.1 μM streptavidin-XL665 (Cisbio Bioassays; France, catalog number 610SAXLG)], 1.5 nM anti-phosphoserine antibody [Merck Millipore, “STK antibody”, catalog number 35-002], and 0.6 nM LANCE EU-W1024 labeled anti-mouse IgG antibody (Perkin-Elmer, product number AD0077; alternatively terbium cryptate labeled anti-mouse IgG antibody from Cisbio Bioassays can be used) in EDTA aqueous solution (100 mM EDTA, 0.4% (w / v) bovine serum albumin [BSA] in 25 mM HEPES, pH 7.5).

[0606] The resulting mixture was incubated at 22 ° C for 1 h to form a complex of biotinylated phosphorylated substrate and detection reagent. The amount of phosphorylated substrate was then assessed by measuring the resonance energy transferred from the anti-mouse IgG antibody labeled with europium chelate to streptavidin-XL665. For this reason, after excitation at 350 nm in a TR-FRET measuring instrument such as Rubystar (BMG Lab technologies, Offenburg, Germany) or Viewlux (Perkin-Elmer), the fluorescence emission at 620 nm and 665 nm was measured. The emission ratio at 665 nm and 622 nm was used as a measure of the amount of phosphorylated substrate. Data were normalized (enzyme reaction without test substance = 0% inhibition; all other test components but without enzyme = 100% inhibition). Typically, the test substances were tested in the same microtiter plate at 11 different concentrations ranging from 20 μM to 0.073 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 nM and 0.073 nM). The dilution series were prepared by serial dilution prior to the assay (2 mM to 7.3 nM in 100% DMSO). IC was calculated by 4-parameter fitting 50 value.

[0607] Table 1: IC of Example Compounds in IRAK4 Kinase Assay 50 value

[0608]

[0609] The inhibitory activity of the substance of the general formula (III) of the present invention on IRAK4 was also determined in the above-mentioned IRAK4 TR-FRET assay. The following are mentioned by way of example: compound intermediate 4-2, IC 50 =21.7nM; intermediate 4-3, IC 50 =13.0 nM; and intermediate 4-4, IC 50 =6.2nM.

[0610] TNF-α secretion in THP-1 cells

[0611] This test is suitable for testing the ability of substances to inhibit the secretion of TNF-α (tumor necrosis factor α) in THP-1 cells (a human monocytic acute leukemia cell line). TNF-α is a cytokine involved in inflammatory processes. In this test, TNF-α secretion is triggered by incubation with bacterial lipopolysaccharide (LPS).

[0612] THP-1 cells were maintained in a continuous suspension cell culture medium [RPMI 1460 medium containing L-Glutamax (Gibco, catalog number 61870-044), supplemented with 10% fetal calf serum (FCS) (Invitrogen, catalog number 10082-147), 1% penicillin / streptomycin (Gibco BRL, catalog number 15140-114)] and should not exceed 1×10 6 The assay was performed in a cell culture medium (RPMI 1460 medium containing L-Glutamax, supplemented with 10% FCS).

[0613] In each case, 2-2.5 μL of cell suspension (corresponding to 4000 cells) per well was distributed to a 384-well test plate (Greiner, catalog number 784076), wherein in each well, 40-50 nL of the substance was dissolved in 100% DMSO. For each substance, dissolution was completed by 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 of LPS (Sigma, Escherichia coli 055:B5, catalog number L5418) (final concentration 0.05 μg / mL) dissolved in the cell culture medium was distributed to each well. As a neutral control, cells were treated with 0.05 μg / mL of LPS and 1% of DMSO, and as an inhibitor control, only 1% of DMSO was used.

[0614] The plate was centrifuged at 80 g for 30 s and incubated for 17 h at 37 ° C, 5% CO2 and 95% atmospheric humidity. The amount of TNF-α was determined using a TNF-α HTRF Detection Kit (Cisbio, catalog number 62TNFPEB / C). For this purpose, 2 μl of the detection solution, which consisted of anti-TNF-α-XL665 conjugate and anti-TNF-α-cryptate conjugate, was dissolved in reconstitution buffer according to the manufacturer's instructions, was added in each case for HTRF (homogeneous time-resolved fluorescence) testing. After addition, the mixture was incubated at room temperature for 3 h or overnight at 4 ° C. The signal at 620 / 665 nm was then read using a HTRF-enabled measuring instrument such as BMG PheraStar.

[0615] The activity of the substances was expressed as the ratio between the neutral control and the inhibitor control in percent. IC was calculated using a 4-parameter fit 50 value.

[0616] Table 2:IC of Example Compounds on TNF-α Secretion in THP-1 Cells 50 value

[0617]

[0618] In vitro LPS (lipopolysaccharide)-induced cytokine production in human PBMC (peripheral blood mononuclear cells)

[0619] The efficacy of the compounds of the general formula (I) of the present invention on inducible cytokine production in human PBMCs was investigated. Herein, cytokine production is induced by LPS, a TLR4 ligand, which leads to activation of the IRAK4-mediated signaling pathway.

[0620] Human PBMC is obtained from human anticoagulated whole blood. For this purpose, 15mL of Ficoll-Paque (Biochrom, catalog number L6115) is first pipetted into a Leucosep tube, and 20mL of human blood is added. After the blood is centrifuged at room temperature for 15min at 800g, the plasma containing platelets is removed and discarded. PBMC is transferred to a centrifuge tube and supplemented with PBS (phosphate buffered saline) (Gibco, catalog number 14190). The cell suspension is centrifuged at room temperature for 10min at 250g and the supernatant is discarded. PBMC is resuspended in complete medium (RPMI 1640, without L-glutamine (PAA, catalog number E15-039), 10% FCS; 50U / mL penicillin, 50μg / mL streptomycin (PAA, catalog number P11-010) and 1% L-glutamine (Sigma, catalog number G7513)).

[0621] The experiment was also carried out in complete medium. PBMC were cultured at 2.5×10 5The cell density of individual cells / well is inoculated on 96-well plates. The compounds of the present invention are serially diluted, diluted in isometric 100% DMSO, and applied to the test at 8 different concentrations within the range of 10 μM to 3nM, so that the final DMSO concentration is 0.4% DMSO. Then before actual stimulation, the cells are pre-incubated with it for 30 min. To induce cytokine secretion, cells are stimulated for 24 hours with 0.1 μg / mL of LPS (Sigma, Escherichia coli (Escherichia coli) 0128:B12, catalog number L2887). Cell viability is measured according to the manufacturer's instructions using CellTiter-Glo luminescence assay (Promega, catalog number G7571 (G755 / G756A)). The amount of TNF-α secreted in the cell culture supernatant is measured using 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.

[0622] TLR-4 / TLR-7 induced secretion of interleukin (IL)-23 by human dendritic cells (DC) in vitro

[0623] The efficacy of the compounds of the general formula (I) of the present invention on the induction of the production of the proinflammatory cytokine IL-23 was investigated in human DCs, and the proinflammatory cytokine IL-23 plays an important role in the generation of TH-17 cells. It is reported that TH-17 cells play a key role in the pathogenesis of diseases such as rheumatoid arthritis, psoriatic arthritis, Bechterev's disease (ankylosing spondylitis) or multiple sclerosis (Lubberts, Nat. Rev. Rheumatol., 2015; Marinoni et al., Auto. Immun. Highlights, 2014; Isailovic et al., J. Autoimmun., 2015; Staschke et al., J Immunol., 2009). To detect the effect of the compounds of the present invention on IL-23 production, primary human monocytes (using magnetic separation [Miltenyi Biotech, Monocyte Isolation Kit, catalog number 130-091-153] were isolated from human PBMCs and cultured in complete medium (VLE (very low endotoxin) RPMI1640 [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]) were added with growth factors (recombinant human GM-CSF [PeproTech, catalog number 300-03] and IL-4 [PeproTech, catalog number 200-04])) to differentiate into DCs in the culture medium for 6 days. After DCs were collected, they were resuspended in complete culture medium and cultured at 2×10 5The cell density of each cell / well was inoculated into a 96-well plate (Costar, catalog number 3599). The compound of the present invention was serially diluted in an isovolumetric 100% DMSO and used in the test at 9 different concentrations in the range of 10 μM to 1 nM. It was ensured here that for each of the 9 concentrations used, the DMSO concentration present was always 0.1%. DC was pre-incubated with the compound of the present invention for 30 min. Thereafter, 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 can activate the signaling pathway mediated by IRAK4), DC was stimulated in an incubator (37°C, 95% rH, 5% CO2) to produce IL-23 for 24 hours. After 24 hours of incubation, the supernatant was collected and analyzed using a commercially available hIL-23 ELISA (eBiosciences, catalog number 88-7237-88) according to the manufacturer's instructions. Taking Example Compound 12 as an example, the results of the inhibition of IL-23 in human DCs are shown in Figure 1 middle.

[0624] In vitro TLR-7 / 8- or TLR-9-induced IFNα production by human plasmacytoid dendritic cells (pDCs)

[0625] With the aid of this test, the effect of the compounds of the general formula (I) of the present invention on the production of IFNα (interferon α) (a key cytokine in the pathogenesis of systemic lupus erythematosus) in human pDC can be studied (Mathian et al., Arthritis Rheum, 2009; Crow MK, 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 separation kit (Miltenyi Biotech, Plasmacytoid Dendritic Cell Isolation Kit II, catalog number 130-097-415). The obtained pDCs were resuspended in complete culture medium (RPMI 1640+GlutaMax [Gibco, catalog number 61870-010], supplemented with 10% FBS [Gibco, catalog number 10493-106] and 50 U of penicillin / streptomycin [Gibco, catalog number 15140-114]) and cultured at 5×10 4The cell density of individual cells / well is inoculated in a 96-well microtiter plate (Costar, catalog number 3599). The compounds of the present invention are serially diluted in isochoric 100% DMSO and used in the test at 9 different concentrations in the range of 10 μM to 1 nM. For each of 9 test concentrations, it is ensured that the DMSO concentration present is always 0.1%. pDC is pre-incubated for 30 minutes with the compounds of the present invention. Stimulate pDC with TLR7 / 8 ligands (imiquimod, R837, Invivogen, catalog number tlrl-imq) or with TLR-9 ligands (CPG-A, ODN2216, Invivogen, catalog number tlrl-2216-1), which can cause the activation of the signal pathway mediated by IRAK4. After 24 hours of incubation, the cell culture supernatant was removed and analyzed using a commercially available human IFNα ELISA (IFNalpha Multi-Subtype ELISA Kit, pbl Assay Science, catalog number 41105-1). Taking Example Compound 12 as an example, the results of inhibition of IFNα in human plasmacytoid DCs are shown in FIG2 .

[0626] In vivo model of TLR-mediated inflammation

[0627] The in vivo efficacy of the compound of the general formula (I) of the present invention was investigated in an in vivo TLR-mediated inflammation model. Due to the use of an LPS-mediated inflammation model, this mechanism model particularly shows the potential efficacy of the compound of the present invention for TLR4-mediated diseases. In this model, female Balb / c mice (about 8 weeks old; Charles River Laboratory, Germany) were divided into groups of 5 animals per group. The control group was treated with a vehicle (vehicle) in which the substance was dissolved (substance vehicle) and a vehicle in which LPS was dissolved. The substance treatment group and the positive control group were intraperitoneally (ip) administered with 0.2 mg LPS / kg body weight (Sigma, catalog number L4391) (lipopolysaccharide from E.coli 0111: B4). In addition, the positive control group was treated with the above-mentioned substance vehicle. 16 hours before the inflammation was induced by administering LPS, the substance was orally administered. In order to investigate the efficacy of the compound of the present invention on inflammation, blood samples were taken from the animals after 1.5 hours. The concentration of specific cytokines in plasma was determined using the MouseProInflammatory7-Plex Tissue Culture Kit (MSD, Cat. No. K15012B) according to the manufacturer's instructions.IRAK4 inhibitors are effective in TLR-mediated inflammation models. Figure 3The graph shows that the amount of TNF-α in plasma was decreased in a dose-dependent manner by the administration of Example Compound 11, compared with the LPS-induced concentration.

[0628] In vivo model of IL-1β-mediated inflammation

[0629] To evaluate the potential efficacy of the compounds of the general formula (I) of the present invention in IL-1β-mediated diseases, female Balb / c mice (about 8 weeks old; Charles River Laboratory, Germany) were intraperitoneally administered with IL-1β, and the efficacy of the compounds of the present invention for IL-1β-mediated cytokine secretion was investigated. There were 5 animals in each group. The control group was treated with a vehicle capable of dissolving the substance and IL-1β. 90 μg IL-1β / kg body weight (R&D, catalog number 401-ML / CF) was administered intraperitoneally to the substance treatment group and the positive control group, respectively. 6 hours before the administration of IL-1β, the substance or its vehicle was administered to the positive control group. 2 hours after the administration of IL-1β, the TNF-α in plasma separated from the blood was determined using Mouse Pro Inflammatory 7-Plex Tissue Culture Kit (MSD, catalog number K15012B) according to the manufacturer's instructions. Administration of IL-1β resulted in an increase in TNF-α plasma concentration, which was inhibited by treatment with Example Compounds 11 and 12. This is due to Figure 4 illustrate.

[0630] In vivo adjuvant-induced arthritis model

[0631] In order to determine the anti-inflammatory activity of the compounds of the general formula (I) according to the invention, their in vivo efficacy was investigated in an arthritis model. For this purpose, on day 0, each male Lewis rat (approximately 100-125 g, Charles River Laboratories, Germany) was administered subcutaneously at the base of the tail with 100 μL of a complete Freund's adjuvant (CFA) solution (bound to mycobacterium (M. tuberculosis) H37Ra [Difo Lab, catalog number-231141], dissolved in incomplete Freund's adjuvant [Difco Lab, catalog number-263910]). There were n=8 rats in each group. Both healthy and diseased control groups were included in the study. Each control group was treated orally (po) with only the vehicle of the test substance. In a preventive manner, i.e. starting from day 0, the treatment was carried out 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 (arthritis severity grading based on a point system). Here, erythema including joint swelling of both hind paws together was given a score of 0 to 4 (0 = none; 1 = mild; 2 = moderate; 3 = marked; 4 = severe) according to the degree of joint inflammation. To determine the anti-inflammatory efficacy of the compounds, the disease activity of the animals was scored by means of the disease activity score starting from day 8, when the animals first showed signs of arthritis, and then three times a week until the end (day 20). Statistical analysis was performed using one-way analysis of variance (ANOVA) and comparison with the control group was performed by means of multiple comparison analysis (Dunnett's test).

[0632] Subcutaneous administration of CFA in rats resulted in acute arthritis with marked joint inflammation in rats. This induced arthritis was inhibited by treatment with Example Compound 11. Figure 5 illustrate.

[0633] Collagen antibody-induced arthritis model in mice

[0634] The anti-inflammatory efficacy of the compounds of the general formula (I) of the present invention was investigated in another murine arthritis model. For this purpose, on day 0, each female Balb / c mouse (approximately 9 weeks old, Charles River Laboratories, Kingston, Canada) was intravenously injected with 200 μL of a collagen antibody cocktail (10 mg / mL; ArthritoMab, MD Bioproducts) into the tail vein (except for the healthy control group included in the study). Then on day 6, these mice were each given an intraperitoneal injection of 200 μl of LPS. There were n=10 mice in each group. Both the healthy control group and the diseased control group were included in the study. Each control group was treated orally with only the vehicle of the test substance. In a preventive manner, i.e., starting from day 0, the test substance was treated by oral administration with different doses. During the experiment, the degree of disease in all four paws was scored based on a point judgment system for scoring disease activity. In this point assessment, a healthy paw is assessed as no point, and a certain degree of joint inflammation occurring from the toes through the metatarsal joints to the ankle joints is assessed as 1 point [mild inflammation, such as toe inflammation] to 4 points [severe inflammation extending to the entire paw], as explained below:

[0635] 0 = Normal

[0636] 1 = Erythema and mild swelling limited to the tarsus, ankle, or toes

[0637] 2 = Erythema and mild swelling extending from the ankle to the metatarsal bones (stage 2)

[0638] 3 = Erythema and moderate swelling extending from the ankle to distal to the metatarsal joints

[0639] 4 = Erythema and severe swelling involving the metatarsals, feet, and toes

[0640] For this parameter, the starting condition was determined in advance the day before the start of the experiment (day -1), and the disease activity score was subsequently scored three times a week from day 8 onwards. Statistical analysis was performed using one-way analysis of variance (ANOVA) and comparisons with the control group were performed with the aid of multiple comparison analysis (Dunnett's test).

[0641] Intravenous administration of the collagen antibody cocktail in mice, including subsequent intraperitoneal administration of LPS, resulted in acute arthritis with significant joint inflammation. This induced arthritis was suppressed by treatment with Example Compound 12. Figure 6 illustrate.

[0642] In vivo NASH mouse model

[0643] To experimentally induce NASH, 200 μg of streptozotocin (STZ; Sigma-Aldrich, USA) was injected subcutaneously in 45 male 2-day-old C57BL / 6 mice. Starting from 4 weeks of age, the animals were fed a high-fat diet (HFD; 57 kcal% fat, purchased from CLEA, JAPAN #HFD32) ad libitum. At 6 weeks of age, the animals were randomly divided into 3 groups (15 animals per group). One group did not receive any treatment, but the other 2 groups were orally administered vehicle or test substances every day for 4 weeks. After 4 weeks of treatment, all animals were painlessly killed under anesthesia, and the livers were removed and fixed in Bouin's solution for histological studies (H. Denk, "Fixierung histologischer "[Fixing of Histological Preparations],in:P. (Editor): "Romeis Mikroskopische Technik" [Romei's Microscopy Techniques], Urban & Schwarzenberg, Munich-Vienna-Baltimore 1989, 17th edition, page 97, ISBN 3-541-11227-1). Thereafter, the liver samples were embedded in paraffin and 5 μm thick paraffin sections were prepared. Tissue sections of each liver were stained, a) with hematoxylin-eosin (HC) for determination of NAFLD activity score (NAS), b) with Picro-Sirius red (Waldeck, Germany) for determination of liver fibrosis. NAFLD activity score was determined in hematoxylin-eosin sections based on the criteria recommended by DE Kleiner et al., Hepatology 41 (2005), 1313-1321 (Table 1). For histological quantification of fibrotic areas, five digital photographs were taken of each section at 200-fold microscope magnification (DFC280; Leica, Germany), and the percentage of fibrosis was determined using ImageJ Software (National Institutes of Health, USA).

[0644] In vivo db / db mouse model

[0645] 30 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 given a standard diet (RM1 (E) 801492, SDS) and free access to tap water. The animals were randomly divided into 3 groups (10 in each group) and treated orally with the test substance within 6 weeks. During the study, blood was taken from the animals at different time points (before treatment started, 3 weeks after treatment started, and 2 days before treatment ended) to measure insulin sensitivity parameters (such as HbA1c, glucose content, insulin content). In addition, OGTT (oral glucose tolerance test), a parameter for determining insulin sensitivity, was performed 1 day before treatment started and 2 days after treatment ended. In addition, the HOMA-IR index (fasting insulin level (mU / L)*fasting blood glucose level (mmol / L) / 22.5) was calculated.

[0646] In vivo B-cell lymphoma-related xenograft models

[0647] The antitumor activity of the compounds of the general formula (I) of the present invention was studied in a murine xenograft model. For this purpose, female CB-17 SCID mice were subcutaneously implanted with a human B-cell lymphoma cell line, such as TMD-8. In mice with an average tumor size of 20-30 mm 2 In the case of , oral monotherapy with the compound of the invention is started, or the compound of the invention is combined with standard therapy, each of which is administered orally. However, the animals are randomized in advance. Once the untreated control group has a large tumor, the treatment is terminated immediately. Tumor size and body weight are measured three times a week. Weight loss is a measure of treatment-related toxicity (>10% = critical, stop treatment until recovery, >20% = poisoning, terminate). Tumor area [length (mm) × width (mm)] is detected by electronic calipers. At the end of the study, tumor weight is also determined. Antitumor efficacy is defined as the ratio of tumor weight (T / C) of the treatment group to the control group [tumor weight of the treatment group on day x / tumor weight of the control group on day x] or the ratio of tumor area of ​​the treatment group to the control group [tumor area of ​​the treatment group on day x / tumor area of ​​the control group on day x]. Compounds with T / C greater than 0.5 are 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.

[0648] Canine IRAK4 Kinase Assay

[0649] The IRAK-4 inhibitory activity of the compounds according to the invention towards canine IRAK4 was determined in the Irak4 TR-FRET assay (TR-FRET = Time Resolved Fluorescence Resonance Energy Transfer) described hereinafter.

[0650] A recombinant fusion protein of HIS (polyhistidine) from the N-terminus and canine IRAK4, expressed in baculovirus-infected insect cells (Hi5, BTI-TN-5B1-4, cell line purchased from Invitrogen, catalog number B855-02) and purified by affinity chromatography, was used as enzyme. The substrate for the kinase reaction was the biotinylated peptide biotin-Ahx-KKARFSRFAGSSPSQASFAEPG (C-terminus in amide form), which can be purchased, for example, from Biosyntan GmbH (Berlin-Buch).

[0651] For the assay, 11 different concentrations ranging from 20 μM to 0.073 nM were prepared from 2 mM solutions of the test substances in DMSO. 50 nL of the corresponding solution was pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of IRAK4 in assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 μM activated sodium orthovanadate, 0.1% (w / v) bovine gamma-globulin (BGG), 0.04% (v / v) nonidet-P40 (Sigma)] were added, and the mixture was incubated for 15 min to allow the substances to pre-bind to the enzyme before the kinase reaction. The kinase reaction is then started by adding 3 μL of a solution of adenosine triphosphate (ATP, 1.67 mM = final concentration in the assay volume of 5 μL: 1 mM) and the peptide substrate (0.83 μM = final concentration in the assay volume of 5 μL: 0.5 μM) in assay buffer and the resulting mixture is incubated at 22° C. for a reaction time of 45 min. The concentration of Irak4 is adjusted to the respective activity of the enzyme and is set so that the assay is performed in the linear range. Typically the concentration is of the order of about 0.1 nM. The reaction was terminated by adding 5 μL of a solution of TR-FRET detection reagent [0.1 μM streptavidin-XL665 (CisbioBioassays; France, catalog number 610SAXLG)], 1.5 nM anti-phosphoserine antibody [Merck Millipore, “STK antibody”, catalog number 35-002], and 0.6 nM LANCE EU-W1024-labeled anti-mouse IgG antibody (Perkin-Elmer, product number AD0071; alternatively, terbium cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used) in EDTA aqueous solution (100 mM EDTA, 0.4% (w / v) bovine serum albumin [BSA] in 25 mM HEPES, pH 7.5).

[0652] The resulting mixture was incubated at 22 ° C for 1 h to form a complex of biotinylated phosphorylated substrate and detection reagent. The amount of phosphorylated substrate was then assessed by measuring the resonance energy transferred from the anti-mouse IgG antibody labeled with europium chelate to streptavidin-XL665. For this reason, after excitation at 350 nm in a TR-FRET measuring instrument such as Rubystar (BMG Lab technologies, Offenburg, Germany) or Viewlux (Perkin-Elmer), the fluorescence emission at 620 nm and 665 nm was measured. The emission ratio at 665 nm and 622 nm was used as a measure of the amount of phosphorylated substrate. Data were normalized (enzyme reaction without test substance = 0% inhibition; all other test components but without enzyme = 100% inhibition). Typically, the test substances were tested at 11 different concentrations ranging from 20 μM to 0.073 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 nM and 0.073 nM) on the same microtiter plate. The dilution series were prepared by serial dilutions prior to the assay (2 mM to 7.3 nM in 100% DMSO). IC was calculated by 4-parameter fitting 50 value.

[0653] Table 3: IC of two experiments of the example compounds in the IRAK4 canine kinase assay 50 value

[0654]

[0655] In vitro lipopolysaccharide (LPS)-induced cytokine production by canine peripheral blood mononuclear cells (PBMC)

[0656] The compounds of the general formula (I) of the present invention were investigated for their efficacy on the inducible cytokine production in canine PBMCs. Herein, cytokine production is induced by LPS, a TLR4 ligand, which leads to the activation of the IRAK4-mediated signaling pathway.

[0657] Canine PBMCs were obtained from anticoagulated whole blood of dogs. For this purpose, plasma rich in canine leukocytes was prepared by centrifuging 15 mL of dog blood at 4°C, 400 g for 15 min, followed by collection and then suspending the canine PBMC buffy coat in the plasma. 7 (7) mL of Ficoll-Paque Plus (Fischer Scientific, catalog number 11778538) was pipetted into a centrifuge tube, and then 7 mL of plasma rich in canine leukocytes was layered on top of Ficoll-Paque Plus. After centrifuging the tube at 400 g for 20 min at 4°C, canine PBMCs were collected from the interface of canine plasma and Ficoll-Paque Plus. PBMCs were transferred to a new centrifuge tube and supplemented with Ca-free 2+ / Mg 2+ Hanks' Balanced Salt Solution 1x (HBSS) (Sigma-Aldrich, catalog number H9394). The cell suspension was centrifuged at 400 g for 5 min at 4°C and the supernatant was discarded. The cell pellet was then resuspended in 0.2% hypotonic saline to lyse any remaining red blood cells. After 30 seconds, the cell suspension was made isotonic and centrifuged at 400 g for 5 min at 4°C. The cell pellet was then resuspended in Ca-free 2+ / Mg 2+ The final wash was performed in HBSS and centrifuged at 400 g for 5 min at 4° C. PBMCs were then 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)).

[0658] The experiment was also performed in complete medium. PBMC were cultured at 2.5 × 10 5The cell density of each cell / well was inoculated on a 96-well plate. The compound of the present invention was dissolved in DMSO and a series of dilutions were performed in complete medium. The compound example was used in the test at 8 different concentrations within the range of 3nM to 10μM, so that the final DMSO concentration was 0.0003-0.4%. To induce cytokine secretion, cells were stimulated with 0.1μg / mL of LPS (Sigma-Aldrich, Escherichia coli 0111: B4, catalog number L3024) and kept for 24 hours. Cell viability was determined using 0.2% trypan blue (Sigma-Aldrich, catalog number T8154). According to the manufacturer's instructions, the amount of TNF-α secreted in the cell culture supernatant was determined using canine TNFα DuoSet Elisa (R&D Systems, catalog number DY1507). For example, Example Compound 12 inhibits the generation of TNFα stimulated by LPS in canine PBMC.

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

[0660] The compounds of the general formula (I) of the present invention were investigated for their efficacy on the inducible cytokine production in bovine PBMCs. Cytokine production in this context is induced by LPS, a TLR4 ligand, which results in the activation of the IRAK4-mediated signaling pathway.

[0661] Bovine PBMCs are obtained from bovine anticoagulated whole blood. For this purpose, 500 mL of bovine blood is centrifuged at 1000 g for 20 min at room temperature (RT) to prepare plasma rich in bovine leukocytes, which is then collected and then the bovine PBMC buffy coat is suspended in an equal volume of PBS / 5mM EDTA (room temperature). 30 (30) mL of Ficoll-Paque Plus (Fischer Scientific, catalog number 11778538) is pipetted into a Leucosep tube, and then 30 mL of a bovine PBMC buffy coat / PBS / EDTA mixture is layered on top of Ficoll-Paque Plus. After centrifugation at room temperature for 25 min at 800 g, bovine PBMCs are collected from the interface of bovine plasma and Ficoll-Paque Plus. PBMCs are transferred to a new centrifuge tube and supplemented with cold PBS / 5mM EDTA. The cell suspension is centrifuged at 4 ° C for 10 min at 350 g, and the supernatant is discarded. The cell pellet was then resuspended in 0.2% hypotonic saline to lyse any remaining erythrocytes. After 30 seconds, the cell suspension was made isotonic and centrifuged at 500 g for 5 min at 4°C. The cell pellet was then resuspended in complete medium (DMEM with GlutaMAX (ThermoFisher, catalog number 32430100), 10% horse serum ( 30-2040 TM ), 20 μM β-mercaptoethanol (ThermoFisher Cat. No. 31350010 [stock solution: 50 mM]).

[0662] The experiment was also performed in complete medium. PBMC were cultured at 1×10 6 Cells were seeded onto 24-well plates at a density of 10 cells / well. The compounds of the invention were dissolved in DMSO and serially diluted in complete medium. The compound examples were used in the assay at 8 different concentrations ranging from 0.003 μM to 10 μM, resulting in a final DMSO concentration of 0.5%. To induce cytokine secretion, 1 μg / mL ( Figure 8 ) and 0.1 μg / mL( Fig. 9 The cells were stimulated with LPS (LPS from E. coli K12; Invivogen#tlrl-eklps) for 24 hours. The cell viability was determined using Türk solution (Merck Millipore#1092770100).

[0663] The amount of TNFα secreted in the cell culture supernatant of bovine PBMC exposed to LPS was determined using an ELISA read-out based on rabbit anti-bovine TNFα antibody. The ELISA assay was performed in a 384-well ELISA plate coated with 10 μL / well of 5 μg / mL rabbit anti-bovine TNFα antibody (BioRad, AHP2383) in 50 mM Na2CO3 / NaHCO3 buffer, pH 9.6, and overnight at 4°C. After removing the antibody and rinsing the wells three times with 50 μL of washing buffer (PBS, 0.05% (v / v) Tween 20), the wells were incubated at 37°C for 90 min with 40 μL of blocking buffer (PBS, 0.05% (v / v) Tween 20, 1% (w / v) bovine serum albumin). 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. 1 μg / mL of rabbit anti-bovine TNFα biotin-conjugated antibody (BioRad, AHP2383B) in blocking buffer was added to the plate (20 μl / well) incubated for 60 min at 37°C. After removing the biotinylated antibody and rinsing the wells three times with 50 μL of wash buffer, ExtrAvidin diluted 1:10.000 in blocking buffer was added at 37°C. TM -Alkaline phosphatase (Sigma, E2636), 20 μL / well, keep for 1 hour. TM -alkaline phosphatase and after washing the wells three times with 50 μL of washing buffer, the enzymatic reaction / color development was initiated by adding 50 μL / well of color development buffer (5 mM p-nitrophenyl phosphate (pNPP) in 50 mM Na2CO3 / NaHCO3, pH 9.6, 2 mM MgCl2). The optical density 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, Example Compound 12 inhibited the production of TNFα by bovine PBMCs stimulated by LPS. This was Figure 8 and Fig. 9 illustrate.

[0664] In vitro lipopolysaccharide (LPS)-induced cytokine production by porcine peripheral blood mononuclear cells (PBMC)

[0665] As another example, the efficacy of the compounds of the general formula (I) of the present invention on the inducible cytokine production in porcine PBMCs was investigated. Here, cytokine production is induced by LPS, a TLR4 ligand, which leads to the activation of the IRAK4-mediated signaling pathway.

[0666] Porcine PBMCs were obtained from porcine anticoagulated whole blood. For this purpose, 36 mL of porcine blood was centrifuged at 1000 g for 20 min at room temperature (RT) to obtain plasma rich in porcine leukocytes, which was then collected and then suspended in an equal volume of PBS / 5 mM EDTA (room temperature). 30 (30) mL of Ficoll-Paque Plus (Fischer Scientific, catalog number 11778538) was pipetted into a Leucosep tube, and then 30 mL of a porcine PBMC buffy coat / PBS / EDTA mixture was layered on top of the Ficoll-Paque Plus. After centrifugation at 800 g for 25 min at room temperature, porcine PBMCs were collected from the interface of porcine plasma and Ficoll-Paque Plus. PBMCs were transferred to a new centrifuge tube and supplemented with cold PBS / 5 mM EDTA. The cell suspension was centrifuged at 350 g for 10 min at 4 ° C, and the supernatant was discarded. The cell pellet was then resuspended in 0.2% hypotonic saline to lyse any remaining erythrocytes. After 30 seconds, the cell suspension was made isotonic and centrifuged at 500 g for 5 min at 4°C. The PBMC cell pellet was then resuspended in complete medium (DMEM and GlutaMAX (ThermoFisher, catalog number 32430100), 10% horse serum ( 30-2040 TM ), 20 μM β-mercaptoethanol (ThermoFisher Cat. No. 31350010 [stock solution: 50 mM]).

[0667] The experiment was also performed in complete medium. PBMC were cultured at 1×10 6 Cell density of 1 cell / well was inoculated on 24-well plates. The compounds of the invention were dissolved in DMSO and serially diluted in complete medium. The compound examples were used in the test at 8 different concentrations ranging from 0.003 μM to 10 μM, so that the final DMSO concentration was 0.5%. To induce cytokine secretion, cells were stimulated for 24 hours with LPS (LPS from E. coli K12; Invivogen # tlrl-eklps) in a concentration range of 0.01 ng / mL to 1 ng / mL. Cell viability was determined using Türk solution (Merck Millipore # 1092770100).

[0668] The amount of TNFα secreted in the cell culture supernatant of pig PBMC exposed to LPS was determined using an ELISA reader based on rabbit anti-porcine TNFα antibody. The ELISA assay was performed in a 384-well ELISA plate coated with 3 μg / mL of rabbit anti-porcine TNFα antibody (BioRad, AHP2397) in 50 mM Na2CO3 / NaHCO3 buffer, pH 9.6, at 10 μL / well and at 4°C for 48 h. After removing the antibody and rinsing the wells three times with 50 μL of washing buffer (PBS, 0.05% (v / v) Tween 20), the wells were incubated at 37°C for 60 min with 50 μL of blocking buffer (PBS, 0.05% (v / v) Tween 20, 1% (w / v) bovine serum albumin). Thereafter, the blocking buffer was removed and the culture supernatant samples (20 μL / well) were added. After incubation at 37°C for 90 min, the samples were removed and the wells were rinsed three times with 50 μL of wash buffer. 0.25 μg / mL of rabbit anti-porcine TNFα biotin-conjugated antibody (BioRad, AHP2397B) in blocking buffer was added to the plate (20 μl / well) incubated for 60 min at 37°C. After removing the biotinylated antibody and rinsing the wells three times with 50 μL of wash buffer, ExtrAvidin diluted 1:10.000 in blocking buffer was added at 37°C. TM -Alkaline phosphatase (Sigma, E2636), 20 μL / well, keep for 1 hour. TM -alkaline phosphatase and after washing the wells three times with 50 μL of washing buffer, the enzymatic reaction / color development was initiated by adding 90 μL / well of color development buffer (5 mM p-nitrophenyl phosphate (pNPP) in 50 mM Na2CO3 / NaHCO3, pH 9.6, 2 mM MgCl2). The optical density at a wavelength of 405 nm was recorded. For kinetic measurements, data points were recorded every 5 minutes for 1 hour, and endpoint measurements were performed after 2 hours. For example, 10 μM Example Compound 12 inhibited the production of TNFα by bovine PBMCs stimulated by 0.1 ng / mL LPS. This is due to Fig.10 illustrate.

[0669] An in vivo model of canine allergic dermatitis induced by house dust mites

[0670] To evaluate the potential anti-allergic / anti-inflammatory effects of the compounds of formula (I) of the present invention, a model of house dust mite sensitization in Beagle dogs was used. HMD sensitization included a series of subcutaneous injections of HDM antigen (10 μg, Greer Laboratories, Lenoir, NC, USA) and (0.2mL, InvivoGen, SanDiego, CA 921221, USA) as an adjuvant. The sensitization process was monitored and confirmed by intradermal skin testing. Once the dog was positive for HDM intradermal skin testing, one month after the last sensitization, HDM antigen (135μg) was applied topically and pierced into the skin inside the hind legs of adult beagles (with a 2mm long microneedle) to detect the efficacy of the compounds of the present invention on symptoms of allergic dermatitis such as erythema and edema. There were 2 groups, each with 4 animals: 1 group was a placebo control group and 1 group was treated with Example Compound 12. The control group was treated orally with gelatin capsules containing microcellulose, while the group treated with Example Compound 12 was treated orally with gelatin capsules containing Example Compound 12 and microcellulose. Example Compound 12 or placebo was administered 5 days before the attack with HDM antigen and continued until 2 days after the attack. The frequency of treatment was once a day, and in the case of Example Compound 12, the dose was 10mg / kg body weight. Erythema and edema were assessed using VAS (Visual Analog Scale) in both groups 30 min after the start of the challenge and at different time points within 48 h. Plasma samples were analyzed to determine the compound exposure associated with clinical evaluation. Edema and erythema were significantly reduced after treatment with Example Compound 12. This is shown in Tables 4 and 5, as well as Fig.11 and Fig.12 illustrate.

[0671] Table 4: Erythema after treatment with Example Compound 12 and placebo (in VAS units)

[0672]

[0673] Table 5: Edema after treatment with Example Compound 12 and placebo (in VAS units)

[0674]

[0675] In vivo itch model of flea allergy dermatitis

[0676] In order to evaluate the potential antipruritic efficacy of the compounds of the general formula (I) of the present invention, the flea allergy dermatitis (FAD) model was used. Only adult dogs with a history of FAD were included in the study. The life cycle of the study consists of two phases: an itch induction phase (2 weeks) and a subsequent treatment phase (2 weeks). During the two study phases, the dogs were infected with Ctenocephalides fleas twice a week (100 fleas / dog for the first attack and 30 fleas / dog for all subsequent attacks). There were 2 groups, each with 12 animals: 1 group was a placebo control group and 1 group was treated with Example Compound 12. The control group was treated orally with gelatin capsules containing microcellulose, while the group treated with Example Compound 12 was treated orally with gelatin capsules containing Example Compound 12 and microcellulose. 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 pruritus behavior was measured as the number of seconds spent on scratching, licking, and biting. Plasma samples were analyzed to determine the compound exposure related to clinical evaluation. After 10 days of treatment with Example Compound 12, pruritus was significantly reduced. This is shown in Table 6 and Fig.13 illustrate.

[0677] Table 6: Reduction in pruritus behavior relative to baseline following treatment with Example Compound 12 compared to placebo (shown as percent change from baseline on the listed days following treatment)

[0678] 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％

[0679] Figure 1 : Inhibition of IL-23 in DCs generated by human monocytes by Example Compound 12. Data are shown as mean values ​​with standard deviations.

[0680] Figure 2: Inhibition of INF-α in (A) imiquimod (R837)- or (B) CpG-A-stimulated human plasmacytoid DCs by Example Compound 12. Data are shown as mean values ​​with standard deviations.

[0681] Figure 3 : Treatment of LPS-induced inflammation with Example Compound 11 resulted in a decrease in the amount of secreted TNF-α. Data are shown as means with standard deviations.

[0682] Figure 4 : Treatment of IL-1β-induced inflammation with Example compounds 11 (left) and 12 (right) resulted in a dose-dependent reduction in the amount of secreted TNF-α. Data are shown as mean values ​​with standard deviations.

[0683] 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 as determined by disease activity score. Data correspond to mean + standard deviation. One-way ANOVA analysis of variance followed by multiple comparison analysis with CFA control group by Dunnett's test; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0684] Figure 6 : Anti-inflammatory efficacy of Example Compound 12 in an animal model of rheumatoid arthritis (mouse model induced by collagen antibody). Significant and dose-dependent inhibition of rheumatoid arthritis determined based on disease activity score. Data correspond to mean + standard deviation. Statistical significance between the collagen antibody (AK) control group and the treatment group was calculated by one-way ANOVA analysis of variance followed by multiple comparison analysis (Dunnett's test) (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0685] Figure 7 : Inhibition of LPS-induced TNFα production by canine PBMC by Example Compound 12. Data are shown as means with standard deviations.

[0686] Figure 8 : Dose-dependent inhibition of TNFα production by bovine PBMC induced by 1 μg / mL LPS by Example Compound 12 (kinetic assay). Data shown are mean values ​​with standard deviations of triplicate biomaterials, each measured twice. The IC50 value determined from this curve is 120 nM.

[0687] Fig. 9 : Dose-dependent inhibition of TNFα production by bovine PBMC induced by 1 μg / mL LPS by Example Compound 12 (kinetic assay). Data shown are mean values ​​with standard deviations of triplicate biomaterials, each measured twice. The IC50 value determined from this curve is 70.5 nM.

[0688] Fig.10 : Inhibition of TNFα production by porcine PBMCs induced by 0.1 ng / mL LPS by Example Compound 12 at 10 μM (kinetic assay) The data presented are the mean values ​​with standard deviations of triplicate biomaterials, each measured twice.

[0689] Fig.11 : Treatment of the canine allergic dermatitis model induced by house dust mite with Example Compound 12 resulted in a reduction in erythema (a). Data are shown as mean values ​​with standard deviations.

[0690] Fig.12 : Treatment of the canine allergic dermatitis model induced by house dust mite with Example Compound 12 resulted in a reduction in edema (b). Data are shown as mean values ​​with standard deviations.

[0691] Fig.13 : Antipruritic efficacy of Example Compound 12 in an animal model of flea allergy dermatitis. Data are expressed as percentage change from baseline corresponding to the median value.

Claims

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

3. The use according to claim 2, wherein the allergic dermatitis is atopic dermatitis.

4. The use according to claim 2, wherein the allergic dermatitis is flea allergy dermatitis.

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

6. The use according to claim 5, wherein the compound is in the form of a tablet or capsule containing the compound in an amount to deliver a dose of about 10 to 20 mg / kg body weight.

7. The use according to claim 6, wherein the compound is in the form of a tablet or capsule containing the compound in an amount to deliver a dose of about 10 mg / kg body weight.

8. The use according to claim 6, wherein the compound is in the form of a tablet or capsule containing the compound in an amount to deliver a dose of about 20 mg / kg body weight.

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

10. The use according to claim 1, which is therapy.

11. The use according to claim 2, which is therapy.

12. The use according to claim 6, which is therapy.

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

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

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

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