Pyrazolo[3,4-b]pyridine compounds for the treatment of autoimmune diseases

Abstract

The present invention relates to a compound represented by formula (I) TIFF2024534567000107.tif60165 (In the formula, R 1 ~R 3 , A, M, W, and Q are as described herein. and pharma- ceutically acceptable salts thereof, as well as compositions containing and methods of using said compounds.

Description

[Technical field]

[0001] The present invention relates to organic compounds useful for the treatment and / or prevention in mammals, in particular antagonists of TLR7 and TLR8 and TLR9 useful for the treatment of systemic lupus erythematosus or lupus nephritis. [Background technology]

[0002] Autoimmune connective tissue diseases (CTDs) include prototypic autoimmune syndromes such as systemic lupus erythematosus (SLE), primary Sjoegren's syndrome (pSjS), mixed connective tissue disease (MCTD), dermatomyositis / polymyositis (DM / PM), rheumatoid arthritis (RA), and systemic sclerosis (SSc). With the exception of RA, there are no really effective and safe treatments available to patients. SLE is the prototypic CTD with a prevalence of 20-150 per 100,000, and causes widespread inflammation and tissue damage in various organs, ranging from symptoms commonly observed in the skin and joints to renal, pulmonary, or cardiac failure. Traditionally, SLE has been treated with nonspecific anti-inflammatory or immunosuppressive drugs. However, the long-term use of immunosuppressive drugs, such as corticosteroids, is only partially effective and is associated with undesirable toxicities and side effects. Belimumab is the only lupus drug approved by the FDA in the past 50 years, but it only works in a small proportion of SLE patients, and only modestly and slowly (Navarra, SV et al Lancet 2011, 377, 721.). Other biologics, such as anti-CD20 mAbs, mAbs against certain cytokines or soluble receptors, have failed in most clinical studies. Therefore, new therapies are needed that provide sustained improvement in a larger patient population and are safer for chronic use in many autoimmune and autoinflammatory diseases.

[0003] Toll-like receptors (TLRs) are an important family of pattern recognition receptors (PRRs) that can initiate a wide range of immune responses in a wide variety of immune cells. As innate host defense sensors, endosomal TLR7, 8, and 9 recognize nucleic acids derived from viruses, bacteria, and specifically, TLR7 / 8 and TLR9 recognize single-stranded RNA (ssRNA) and single-stranded CpG-DNA, respectively. However, aberrant nucleic acid sensing of TLR7, 8, and 9 has been considered a key node in a wide range of autoimmune and autoinflammatory diseases (Krieg, AM et al. Immunol. Rev. 2007, 220, 251. Jimenez-Dalmaroni, MJ et al Autoimmun Rev. 2016, 15, 1. Chen, JQ, et al. Clinical Reviews in Allergy & Immunology 2016, 50, 1.). Anti-RNA and anti-DNA antibodies are well-established diagnostic markers of SLE and can deliver both self-RNA and self-DNA to endosomes. Self-RNA complexes can be recognized by TLR7 and TLR8, whereas self-DNA complexes can trigger TLR9 activation. Indeed, defects in the clearance of self-RNA and self-DNA from blood and / or tissues are evident in SLE (systemic lupus erythematosus) patients. TLR7 and TLR9 have been reported to be upregulated in SLE tissues and correlated with chronicity and activity of lupus nephritis, respectively. In B cells of SLE patients, TLR7 expression correlates with anti-RNP antibody production, whereas TLR9 expression correlates with IL-6 levels and anti-dsDNA antibody levels. Consistently, in lupus mouse models, TLR7 is required for anti-RNA antibodies and TLR9 is required for anti-nucleosome antibodies. Meanwhile, overexpression of TLR7 or human TLR8 in mice promotes autoimmunity and autoinflammation. Moreover, TLR8 activation specifically contributes to mDC / macrophage inflammatory cytokine secretion, neutrophil nematosis, induction of Th17 cells, and suppression of Treg cells.In addition to the described role of TLR9 in promoting B cell autoantibody production, activation of TLR9 by self-DNA in pDCs also leads to induction of type I IFN and other inflammatory cytokines. Given that TLR9 plays such an important role in the pathogenesis of autoimmune diseases in both pDCs and B cells, and that self-DNA complexes that can easily activate TLR9 are widespread in many patients with autoimmune diseases, there may be special advantages to further block the self-DNA-mediated TLR9 pathway in addition to inhibiting the TLR7 and TLR8 pathways. Taken together, the TLR7, 8, 9 pathways are new therapeutic targets for the treatment of autoimmune and autoinflammatory diseases for which there are no effective steroid-free and non-cytotoxic oral drugs, and inhibiting all of these pathways from the very upstream may provide satisfactory therapeutic effects. Therefore, the present inventors have invented oral compounds that target and inhibit TLR7, TLR8, and TLR9 for the treatment of autoimmune and autoinflammatory diseases. Summary of the Invention

[0004] The present invention relates to a compound represented by formula (I) [ka] (In the formula, R 1 teeth, [ka] (In the formula, R 4 is H or C 1~6 is alkyl, R 5 is C 1~6 alkyl), R 2 is H or C 1~6 is alkyl, R 3 is piperazinyl, (C 1~6 Alkoxy C 1~6alkyl)piperazinyl, 3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazinyl, 4,7-diazaspiro[2.5]octanyl, 5-oxa-2,8-diazaspiro[3.5]nonanyl, amino-1,4-oxazepanyl, amino(C 1~6 alkoxy) piperidinyl, amino(C 1~6 alkoxy)pyrrolidinyl, amino(C 1~6 alkyl)azetidinyl or aminohalopiperidinyl; A is N or CR 6 (where R 6 , H, C 1~6 Alkyl or C 1~6 alkoxy), M is N or CR 7 (where R 7 is H or C 1~6 alkyl), W is N or CH; Q is N or CH; However, two or less of A, M, W, and Q are simultaneously N. or a pharma- ceutically acceptable salt thereof.

[0005] Another object of the present invention relates to novel compounds of formula (I) or (Ia). Their preparation, medicaments based on the compounds according to the present invention and their preparation, and the use of compounds of formula (I) or (Ia) as TLR7 and TLR8 and TLR9 antagonists, and their use for the treatment or prevention of systemic lupus erythematosus or lupus nephritis. Compounds of formula (I) or (Ia) show excellent TLR7 and TLR8 and TLR9 antagonistic activity. In addition, compounds of formula (I) or (Ia) also show good cytotoxicity, solubility, hPBMC, human microsome stability, and SDPK profile, as well as low CYP inhibition.

[0006] Novartis Patent WO2018047081 disclosed compounds with the same pyrazolo[3,4-b]pyridinyl moiety as the compounds of the present invention, but the central bicyclic core of 4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine and the terminal substitution with bicyclo[2,2,2]octane / bicyclo[1,1,1]pentane moiety are essential for TLR7 / 8 / 9 activity based on the information disclosed in the Novartis Patent, which was also considered as the main structural difference compared to the compounds of the present invention. Meanwhile, most of the compounds in WO2018047081 had poor TLR9 activity. Unfortunately, few compounds have relatively improved TLR9 activity, such as N79 (as Example 79), which has the best TLR9 activity, and still have poor human liver microsome stability (see Table 5) and therefore poor PK profile.

[0007] Another Novartis patent, WO 2019220390, discloses a polymorph of compound N8 (as Example 8 of WO 2018047081), which is considered the lead compound in the series, and was found to have much lower TLR9 activity and similarly low human liver microsomal stability (see Table 5). [ka] Compound N8 (hPBMC TLR7 / 8 / 9 antagonist IFNα assay IC 50 (μM): 0.004 / 0.166 / 4.28) [ka] Compound N79 (hPBMC TLR7 / 8 / 9 antagonist IFNα assay IC 50 (μM): 0.004 / 0.136 / 0.064) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] definition "C 1~6The term "alkyl" refers to saturated straight or branched chain alkyl groups containing 1 to 6, especially 1 to 4, carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. 1~6 "Alkyl" groups are methyl, ethyl, and n-propyl.

[0009] "C 1-6 The term "alkoxy" refers to 1-6 It represents alkyl-O-.

[0010] The terms "halogen" and "halo" are used interchangeably herein and refer to fluoro, chloro, bromo or iodo.

[0011] The term "halopiperidinyl" refers to a piperidinyl group in which at least one of the hydrogen atoms of the piperidinyl group has been replaced by the same or different halogen atom, in particular a fluoro atom. Examples of halopiperidinyl include fluoropyrrolidinyl and difluoropyrrolidinyl.

[0012] The terms "cis" and "trans" refer to the relative stereochemistry of molecules or moieties. For example, Example 18, as the cis isomer, is [ka] Similarly, Example 19 as the trans isomer refers to a mixture of [ka] It refers to a mixture of.

[0013] The term "pharmaceutically acceptable salt" means a salt that is not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid addition salts and base addition salts.

[0014] The term "pharmaceutically acceptable acid addition salts" refers to pharmaceutically acceptable salts such as those formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and the like, and organic acids selected from aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid.

[0015] The term "pharmaceutically acceptable base addition salt" refers to a pharmaceutically acceptable salt formed with an organic or inorganic base. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines, including natural substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperidine, piperidine, N-ethylpiperidine, and polyamine resins.

[0016] The term "pharmacologically active metabolite" refers to a pharmacologically active product produced through metabolism in the body of a particular compound or its salt. After entering the body, most drugs become substrates for chemical reactions that can change their physical properties and biological effects. These metabolic transformations usually affect the polarity of the compounds of the present invention and change the way the drug is distributed in and excreted from the body. However, in some cases, metabolism of a drug is required for therapeutic effect.

[0017] The term "therapeutically effective amount" refers to an amount of a compound or molecule of the invention that, when administered to a subject, (i) treats or prevents a particular disease, condition or disorder, (ii) reduces, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition or disorder described herein. The therapeutically effective amount will vary depending on the compound, the condition being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors.

[0018] The term "pharmaceutical composition" refers to a mixture or solution containing a therapeutically effective amount of an active ingredient together with pharma- ceutically acceptable excipients to be administered to a mammal (e.g., a human) in need thereof.

[0019] TLR7, TLR8, and TLR9 antagonists

[0020] The present invention relates to a method for producing a compound represented by the formula (I): [ka] (In the formula, R 1 teeth, [ka] (In the formula, R 4 is H or C 1~6 is alkyl, R 5 is C1~6 alkyl), R 2 is H or C 1~6 is alkyl, R 3 is piperazinyl, (C 1~6 Alkoxy C 1~6 alkyl)piperazinyl, 3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazinyl, 4,7-diazaspiro[2.5]octanyl, 5-oxa-2,8-diazaspiro[3.5]nonanyl, amino-1,4-oxazepanyl, amino(C 1~6 alkoxy) piperidinyl, amino(C 1~6 alkoxy)pyrrolidinyl, amino(C 1~6 alkyl)azetidinyl or aminohalopiperidinyl; A is N or CR 6 (where R 6 , H, C 1~6 Alkyl or C 1~6 alkoxy), M is N or CR 7 (where R 7 is H or C 1~6 alkyl), W is N or CH; Q is N or CH; However, two or less of A, M, W, and Q are simultaneously N. or a pharma- ceutically acceptable salt thereof.

[0021] Another embodiment of the present invention is a compound represented by the formula (Ia): [ka] (In the formula, R 1 teeth, [ka] (In the formula, R 4 is H or C 1~6 is alkyl, R5 is C 1~6 alkyl), R 2 is H or C 1~6 is alkyl, R 3 is piperazinyl, (C 1~6 Alkoxy C 1~6 alkyl)piperazinyl, 3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazinyl, 4,7-diazaspiro[2.5]octanyl, 5-oxa-2,8-diazaspiro[3.5]nonanyl, amino-1,4-oxazepanyl, amino(C 1~6 alkoxy) piperidinyl, amino(C 1~6 alkoxy)pyrrolidinyl, amino(C 1~6 alkyl)azetidinyl or aminohalopiperidinyl; A is N or CR 6 (where R 6 , H, C 1~6 Alkyl or C 1~6 alkoxy), M is N or CR 7 (where R 7 is H or C 1~6 alkylalkyl), W is N or CH; Q is N or CH; However, two or less of A, M, W, and Q are simultaneously N. or a pharma- ceutically acceptable salt thereof.

[0022] A further embodiment of the present invention is a compound of formula (I) or (Ia) according to (iii) (i) or (ii), or a pharma- ceutically acceptable salt thereof, wherein R 1 teeth, [ka] and R 4 is C 1~6 is alkyl, R 5 is C 1~6 It is an alkyl.

[0023] A further embodiment of the present invention is a compound of formula (I) or (Ia) as defined in any one of (i) to (iii) or a pharma- ceutically acceptable salt thereof, wherein R 4 is methyl, R 5 is methyl.

[0024] A further embodiment of the present invention is a compound of formula (I) or (Ia) according to any one of (v)(i) to (iv), wherein A is CR 6 and R 6 is H or C 1~6 It is an alkyl.

[0025] A further embodiment of the present invention is a compound of formula (I) or (Ia) as defined in any one of (i) to (v), or a pharma- ceutically acceptable salt thereof, wherein A is CR 6 and R 6 is H or methyl.

[0026] A further embodiment of the present invention is a compound of formula (I) or (Ia) as defined in any one of (i) to (vi), or a pharma- ceutically acceptable salt thereof, wherein M is CR 7 and R 7 is H.

[0027] A further embodiment of the invention is a compound of formula (I) or (Ia) as defined in any one of (viii) (i) to (vii) or a pharma- ceutically acceptable salt thereof, wherein W is CH.

[0028] A further embodiment of the present invention is a compound of formula (I) or (Ia) as defined in any one of (ix)(i) to (viii) or a pharma- ceutically acceptable salt thereof, wherein Q is N.

[0029] A further embodiment of the present invention is a compound of formula (I) or (Ia) as defined in any one of (x)(i) to (ix), or a pharma- ceutically acceptable salt thereof, wherein R 3is amino-1,4-oxazepanyl, amino(C 1~6 alkoxy)pyrrolidinyl or piperazinyl.

[0030] A further embodiment of the present invention is a compound of formula (I) or (Ia) as defined in any one of (x)(i) to (xi), or a pharma- ceutically acceptable salt thereof, wherein R 3 is 6-amino-1,4-oxazepan-4-yl, 3-amino-4-methoxy-pyrrolidin-1-yl, or piperazin-1-yl.

[0031] A further embodiment of the present invention is a compound of formula (I) or formula (Ia) according to any one of (xii) (i) to (xi), R 1 teeth, [ka] (In the formula, R 4 is C 1~6 is alkyl, R 5 is C 1~6 alkyl), R 2 is H or C 1~6 is alkyl, R 3 is amino-1,4-oxazepanyl, amino(C 1~6 alkoxy)pyrrolidinyl or piperazinyl; A is CR 6 (where R 6 is H or C 1~6 alkyl), M is for CR 7 (where R 7 is H), W is CH; Q is N; or a pharma- ceutically acceptable salt thereof.

[0032] A further embodiment of the present invention is a compound of formula (I) or (Ia) according to any one of (xiii) (i) to (xii), R 1 teeth, [ka] (In the formula, R 4 is methyl, R 5 is methyl) R 2 is H or methyl, R 3 is 6-amino-1,4-oxazepan-4-yl, 3-amino-4-methoxy-pyrrolidin-1-yl or piperazin-1-yl, A is CR 6 (where R 6 is H or methyl; M is for CR 7 (where R 7 is H), W is CH; A compound of formula (I) or (Ia), wherein Q is N, or a pharma- ceutically acceptable salt thereof.

[0033] Another embodiment of the present invention is a compound represented by the formula (xiv) [ka] (In the formula, R 1 teeth, [ka] (In the formula, R 4 is C 1~6 is alkyl, R 5 is C 1~6 alkyl), R 2 is C 1~6 is alkyl, R 3 is piperazinyl, (C 1~6 Alkoxy C 1~6alkyl)piperazinyl, 4,7-diazaspiro[2.5]octanyl, 5-oxa-2,8-diazaspiro[3.5]nonanyl, amino-1,4-oxazepanyl, amino(C 1~6 alkoxy) piperidinyl, amino(C 1~6 alkoxy)pyrrolidinyl, amino(C 1~6 alkyl)azetidinyl or aminohalopiperidinyl; A is CH, M is CH; W is CH; Q is N) or a pharma- ceutically acceptable salt thereof.

[0034] A further embodiment of the present invention is a compound of formula (Ib) according to (xv)(xiv), wherein R 4 is methyl, R 5 is methyl, R 2 is methyl.

[0035] A further embodiment of the present invention is a compound of formula (Ib) according to (xvi), (xiv) or (xv), wherein R 3 is amino-1,4-oxazepanyl.

[0036] A further embodiment of the present invention is a compound of formula (Ib) according to any one of (xvii)(xiv) to (xvi), wherein R 3 is 6-amino-1,4-oxazepan-4-yl.

[0037] A further embodiment of the present invention is a compound of formula (Ib) according to any one of (xviii) (xiv) to (xvii), R 1 teeth, [ka] (In the formula, R 4 is C 1~6 is alkyl, R 5 is C 1~6 alkyl), R 2 is C 1~6 is alkyl, R 3 is amino-1,4-oxazepanyl, A is CH, M is CH; W is CH; A compound of formula (Ib) wherein Q is N, or a pharma- ceutically acceptable salt thereof.

[0038] A further embodiment of the present invention is a compound of formula (Ib) according to any one of (xix), (xiv) to (xviii), R 1 teeth, [ka] (In the formula, R 4 is methyl, R 5 is methyl) R 2 is methyl, R 3 is 6-amino-1,4-oxazepan-4-yl, A is CH, M is CH; W is CH; A compound of formula (Ib) wherein Q is N, or a pharma- ceutically acceptable salt thereof.

[0039] Other embodiments of the present invention include the following: 1,6-Dimethyl-4-[4-(4-piperazin-1-ylphenyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[4-(5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[4-(5-piperazin-1-ylpyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[4-(5-piperazin-1-ylpyrimidin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1-Methyl-4-[4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1-Methyl-4-[4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[4-(4-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[4-(2-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[4-(4-methyl-2-piperazin-1-yl-pyrimidin-5-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 4-[4-(3-ethyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; 4-[4-(3-methoxy-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; (3S,4R)-1-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-4-piperidyl]-5-methyl-3-pyridyl]-3-fluoro-piperidin-4-amine; 4-[4-[5-[(3R)-3-(methoxymethyl)piperazin-1-yl]-3-methyl-2-pyridyl]-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; (3S,4S)-1-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-4-piperidyl]-5-methyl-3-pyridyl]-3-methoxy-piperidin-4-amine; 1-Methyl-4-[cis-3-methyl-4-(6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1-Methyl-4-[trans-3-methyl-4-(6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[cis-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[trans-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[cis-3-methyl-4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[trans-3-methyl-4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[cis-3-methyl-4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[trans-3-methyl-4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; (3R,4R)-1-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-4-methoxy-pyrrolidin-3-amine; 2-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-5-oxa-2,8-diazaspiro[3.5]nonane; (6S)-4-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-1,4-oxazepan-6-amine; 4-[(3S,4R)-4-[5-[(3R)-3-(methoxymethyl)piperazin-1-yl]-3-methyl-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; 4-[(3S,4R)-4-[5-(4,7-diazaspiro[2.5]octan-7-yl)-3-methyl-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; (3R,4R)-1-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-3-fluoro-piperidin-4-amine; 1-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-3-methyl-azetidin-3-amine; (4aR,7aR)-6-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazine; 1,6-Dimethyl-4-[(3R,4S)-3-methyl-4-(5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-Dimethyl-4-[(3S,4R)-3-methyl-4-(5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; (3R,4R)-1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-4-methoxy-pyrrolidin-3-amine; 4-[(3R,4S)-4-[5-[(3R)-3-(methoxymethyl)piperazin-1-yl]-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; 2-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-5-oxa-2,8-diazaspiro[3.5]nonane; (3S,4S)-1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-3-methoxy-piperidin-4-amine; (6S)-4-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-1,4-oxazepan-6-amine; (3R,4R)-1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-3-fluoro-piperidin-4-amine; 4-[(3R,4S)-4-[5-(4,7-diazaspiro[2.5]octan-7-yl)-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; and 1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-3-methyl-azetidin-3-amine or a pharma- ceutically acceptable salt thereof.

[0040] Pharmaceutical Compositions and Administration Another embodiment provides pharmaceutical compositions or medicaments containing the compounds of the invention and therapeutically inert carriers, diluents or excipients, and methods of using the compounds of the invention to prepare such compositions and medicaments. In one example, the compounds of formula (I) can be formulated into galenical dosage forms by mixing at ambient temperature, at an appropriate pH, and to the desired degree of purity with a physiologically acceptable carrier, i.e., a carrier that is non-toxic to the recipient at the dosage and concentration used. The pH of the formulation depends primarily on the particular application and the concentration of the compound, but is preferably in the range of about 3 to about 8. In one example, the compounds of formula (I) are formulated in acetate buffer at pH 5. In another embodiment, the compounds of formula (I) are sterile. The compounds can be stored, for example, as solid or amorphous compositions, as lyophilized formulations, or as aqueous solutions.

[0041] The compositions are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the drug, the method of administration, the administration schedule, and other factors known to medical practitioners. The "effective amount" of the compound to be administered is governed by such considerations and is the minimum amount required to inhibit TLR7, 8, 9 interactions with their respective stimulatory ligands, downstream signaling mediated by MYD88, IRF7, IRF5, NFκB, etc., leading to the production of type I interferons and proinflammatory cytokines (e.g., TNFα, IL-6), and activation of all types of immune cells. For example, such an amount may be below an amount that is toxic to normal cells, or to the mammal as a whole.

[0042] In one example, a pharma- ceutical effective amount of a compound of the invention administered parenterally per dose will be in the range of about 0.001-1000 mg / kg of patient body weight per day, alternatively about 0.001-1000 mg / kg of patient body weight per day, with a typical initial range of the compound used being 0.3-15 mg / kg / day. In another embodiment, oral unit dosage forms such as tablets and capsules preferably contain about 0.1 to about 1000 mg of a compound of the invention.

[0043] The compounds of the present invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural, intranasal, and, where localized treatment is desired, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0044] The compounds of the present invention may be administered in any convenient dosage form, such as tablets, powders, capsules, liquids, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain conventional ingredients in pharmaceutical preparations, such as diluents, carriers, pH adjusters, sweeteners, fillers and additional active agents.

[0045] Typical formulation is prepared by mixing the compound of the present invention and carrier or excipient.Suitable carrier and excipient are well known to those skilled in the art and are described in detail in, for example, Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, fragrances, flavorings, diluents, and other known additives to provide for superior presentation of the drug (i.e., the compound of the present invention or a pharmaceutical composition thereof) or to aid in the manufacture of a pharmaceutical product (i.e., a medicament).

[0046] An example of a suitable oral dosage form is a tablet containing about 0.1-1000 mg of the compound of the present invention, formulated with about 0.1-1000 mg of anhydrous lactose, about 0.1-1000 mg of croscarmellose sodium, about 0.1-1000 mg of polyvinylpyrrolidone (PVP) K30, and about 0.1-1000 mg of magnesium stearate. The powdered ingredients are first mixed together and then mixed with a solution of PVP. The resulting composition can be dried, granulated, mixed with magnesium stearate, and compressed into tablet form using conventional equipment. An example of an aerosol formulation can be prepared, for example, by dissolving 0.1-1000 mg of the compound of the present invention in a suitable buffer solution, for example, phosphate buffer, and adding an isotonicity agent, for example, a salt such as sodium chloride, if desired. The solution can be filtered, for example, using a 0.2 micron filter, to remove impurities and contaminants.

[0047] Thus, one embodiment includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharma- ceutically acceptable salt thereof. A further embodiment includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharma- ceutically acceptable salt thereof, together with a pharma- ceutically acceptable carrier or excipient.

[0048] Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in treating an autoimmune disease.Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in treating an autoimmune disease.

[0049] The following embodiments illustrate typical compositions of the present invention and are intended to serve as merely representative thereof.

[0050] Composition A The compounds of the invention can be used, as active ingredient, in a manner known per se to produce tablets of the following composition:

[0051] Per tablet Active ingredient: 200mg Microcrystalline cellulose 155mg Cornstarch 25mg Talc 25mg Hydroxypropyl methylcellulose 20mg 425mg

[0052] Composition B The compounds of the present invention can be used in a manner known per se to produce capsules of the following composition as an active ingredient:

[0053] Per capsule Active ingredient 100.0mg Cornstarch 20.0mg Lactose 95.0mg Talc 4.5mg Magnesium stearate 0.5mg 220.0mg

[0054] synthesis The compounds of the present invention can be prepared by any conventional means. Suitable methods for synthesizing these compounds and their starting materials are illustrated in the following schemes and examples. All substituents, particularly R 1 ~R 5 , A, M, W, and Q are as defined above unless otherwise indicated. Further, unless expressly stated otherwise, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to one of ordinary skill in the art of organic chemistry.

[0055] A general synthetic route for preparing compounds of formula (I) or (Ia) or (Ib) is given below.

[0056] Scheme 1 [ka] In the formula, X is a halogen.

[0057] Coupling of compounds of formula (II) with (III) can be accomplished by direct displacement at elevated temperatures in the presence of a base such as DIPEA or CsF, or via Buchwald-Hartwig CN bond formation (see Acc. Chem. Res. 1998, 31, 805-818; Chem. Rev. 2016, 116, 12564-12649; Current Chemistry, 2002, 219, 131-209 topics; and references cited therein) with a catalyst such as RuPhos Pd G2, Pd2(dba)3 / XantPhos, and a base such as Cs2CO3 or t-BuONa to provide compounds of formula (IV). 3 Coupling with -H can be achieved by direct coupling with a catalyst such as RuPhos Pd G2, Pd2(dba)3 / XantPhos, and a base such as Cs2CO3 or t-BuONa under Buchwald-Hartwig CN bond forming conditions to give a compound of formula (I). In some embodiments, a compound of formula (IV) and R 3 Coupling with -H is R 3 Products may be provided which contain a protecting group derived from -H, such as Boc or Cbz, which is removed prior to providing the final compound of formula (I).

[0058] Scheme 2 [ka] In the formula, X is a halogen.

[0059] Compounds of formula (VI) can be obtained by hydrogenation reaction from compounds of formula (V) under hydrogen atmosphere in the presence of a catalyst such as Pd(OH)2 / C, Pd / C or Wilkinson's catalyst. The Cbz group can also be deprotected under the same conditions. Coupling of compounds of formula (VI) with (II) can then be achieved by direct displacement at elevated temperature in the presence of a base such as DIPEA or CsF, or by Buchwald-Hartwig CN bond formation under conditions of a catalyst such as RuPhos Pd G2, Pd2(dba)3 / XantPhos and a base such as Cs2CO3 or t-BuONa to provide compounds of formula (I). In some embodiments, coupling of compounds of formula (VI) with (II) may give products containing a protecting group, e.g., Boc, derived from (VI), which is removed before giving the final compound of formula (I).

[0060] The compounds of the invention may be obtained as mixtures of diastereomers or enantiomers, which may be separated by methods well known in the art, for example (chiral) HPLC or SFC.

[0061] The present invention also relates to a method for preparing a compound of formula (I) or formula (Ia), the method comprising the steps of: a) reacting a compound represented by formula (IV) in the presence of a catalyst and a base [ka] Compounds of R 3 The process of the Buchwald-Hartwig C-N bond formation reaction between -H, b) reacting a compound of formula (VI) with a compound of formula (VI) in the presence of a catalyst and a base; [ka] and a compound of formula (II) [ka] The process of the Buchwald-Hartwig C-N bond formation reaction between the compounds c) a step of direct substitution reaction between a compound of formula (VI) and a compound of formula (II) in the presence of a base. Including, During the ceremony, In steps a) and b), the catalyst can be, for example, RuPhos Pd G2, Pd2(dba)3 / Xantphos, and the base can be, for example, Cs2CO3 or t-BuONa; In step c), the base can be DIPEA or CsF.

[0062] The compounds of formula (I) or (Ia) when prepared according to the above process are also an object of the present invention.

[0063] Indications and Treatment Methods The present invention provides compounds that can be used as antagonists of TLR7 and TLR8 and TLR9, inhibiting pathway activation via TLR7 and / or TLR8 and / or TLR9, and their respective downstream biological events, including but not limited to innate and adaptive immune responses mediated through the production of any type of cytokine and any form of autoantibody. Thus, the compounds of the present invention are useful for blocking TLR7 and / or TLR8 and / or TLR9 in any type of cell that expresses such receptor(s), including but not limited to plasmacytoid dendritic cells, B cells, T cells, macrophages, monocytes, neutrophils, keratinocytes, epithelial cells. Thus, the compounds can be used as therapeutic or prophylactic agents for systemic lupus erythematosus and lupus nephritis.

[0064] The present invention provides methods for the treatment or prevention of systemic lupus erythematosus and lupus nephritis in a patient in need of such treatment or prevention.

[0065] Another embodiment includes a method of treating or preventing systemic lupus erythematosus and lupus nephritis in a mammal in need of such treatment or prevention, comprising administering to said mammal a therapeutically effective amount of a compound of formula (I), a stereoisomer, a tautomer, a prodrug, or a pharma- ceutically acceptable salt thereof. EXAMPLES

[0066] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the invention.

[0067] Abbreviation The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the invention.

[0068] The abbreviations used herein are as follows: ACN: Acetonitrile AIBN: Azobisisobutyronitrile Boc2O: Di-tert-butyl dicarbonate CbzCl: benzyl chloroformate CyPF-t-Bu: [(R)-1-[(S)-2-(dicyclohexylphosphino)ferrocenyl]ethyl]di-tert-butylphosphine DCM: dichloromethane DEA: Diethylamine DIPEA: N,N-diisopropylethylamine DMA: Dimethylacetamide DMF: N,N-dimethylformamide DMFDMA: N,N-Dimethylformamide dimethyl acetal dtbbpy: 4,4'-di-tert-butyl-2,2'-dipyridyl EtOAc or EA: Ethyl acetate FA: Formic acid HLM Human Liver Microsomes I C 50 :50% inhibitory concentration Ir[dF(CF3)ppy]2(dtbpy)(PF6): [4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-N1,N1']bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl-N]phenyl-C]iridium(III) hexafluorophosphate JackiePhos: Bis(3,5-bis(trifluoromethyl)phenyl)(2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine LCMS Liquid Chromatography Mass Spectrometry MS: Mass spectrometry [Pd(allyl)Cl]2: Allylpalladium(II) chloride dimer Pd2(dba)3: Tris(dibenzylideneacetone)dipalladium Pd[P(o-tol)3]2: Bis(tri-o-tolylphosphine)palladium PE: Petroleum ether prep-HPLC: Preparative High Performance Liquid Chromatography rt: room temperature RuPhos Pd G2: Second generation chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) SFC: Supercritical Fluid Chromatography TCDI: 1,1'-thiocarbonyldiimidazole TEA: Trimethylamine TFA: Trifluoroacetic acid TfO: Trifluoromethanesulfonic anhydride THF: tetrahydrofuran TTMSS: 1,1,1,3,3,3-hexamethyl-2-trimethylsilyl-trisilane v / v: volume ratio Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

[0069] General experimental conditions Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SP1 system and Quad 12 / 25 cartridge module, ii) ISCO combi-flash chromatography instrument. Brand and pore size of silica gel: i) KP-SIL 60 Å, particle size: 40-60 μm; ii) CAS registration number: silica gel: 63231-67-4, particle size: 47-60 micron silica gel; iii) ZCX from Qingdao Haiyang Chemical Co.,Ltd, pore size: 200-300 or 300-400.

[0070] Intermediates and final compounds were purified by preparative HPLC on reversed-phase columns using XBridge™ Prep-C18 (5 μm, OBD™ 30×100 mm) columns, SunFire™ Prep-C18 (5 μm, OBD™ 30×100 mm) columns, Phenomenex Synergi-C18 (10 μm, 25×150 mm) or Phenomenex Gemini-C18 (10 μm, 25×150 mm). Waters AutoP purification system (sample manager 2767, pump 2525, detectors: Micromass ZQ and UV 2487, solvent system: acetonitrile and 0.1% ammonium hydroxide in water; acetonitrile and 0.1% FA in water or acetonitrile and 0.1% TFA in water). or Gilson-281 purification system (Pump 322, Detector: UV 156, Solvent system: acetonitrile and 0.05% ammonium hydroxide in water; acetonitrile and 0.225% FA in water; acetonitrile and 0.05% HCl in water; acetonitrile and 0.075% TFA in water; or acetonitrile and water).

[0071] For SFC chiral separations, intermediates were separated by chiral columns (Daicel chiralpak IC, 5 μm, 30 × 250 mm), AS (10 μm, 30 × 250 mm) or AD (10 μm, 30 × 250 mm) using a Mettler Toledo Multigram III system SFC, Waters 80Q preparative SFC or Thar80 preparative SFC, solvent systems: CO2 and IPA (0.5% TEA in IPA) or CO2 and MeOH (0.1% NH3·H2O in MeOH), back pressure 100 bar, UV detection at 254 or 220 nm.

[0072] LC / MS spectra of the compounds were obtained using an LC / MS (Waters™ Alliance 2795-Micromass ZQ, Shimadzu Alliance 2020-Micromass ZQ, or Agilent Alliance 6110-Micromass ZQ). The LC / MS conditions were as follows (run time 3 min or 1.5 min): Acidic conditions I: A: 0.1% TFA in HO; B: 0.1% TFA in acetonitrile; Acidic conditions II: A: 0.0375% TFA in HO; B: 0.01875% TFA in acetonitrile; Basic condition I: A: 0.1% NH3·H2O in H2O; B: acetonitrile; Basic conditions II: A: 0.025% NH3·H2O in H2O; B: acetonitrile; Neutral conditions: A: H2O; B: acetonitrile. Mass spectra (MS): Generally, only the ions representing the parent mass are reported; unless otherwise stated, the mass ions quoted are positive mass ions (MH). + It is.

[0073] NMR spectra were obtained using a Bruker Avance 400 MHz.

[0074] Microwave-assisted reactions were performed on a Biotage Initiator Sixty microwave synthesizer. All reactions involving air-sensitive reagents were carried out under an argon or nitrogen atmosphere. Reagents were used as received and without further purification unless otherwise noted.

[0075] Preparation Examples The following examples are intended to illustrate the meaning of the invention but do not in any way represent a limitation within the scope of the meaning of the invention.

[0076] Intermediate A 4-[(3S,4R)-4-(5-chloro-2-pyridyl)-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0077] The title compound was prepared according to the following scheme. [ka]

[0078] Step 1: Preparation of tert-butyl (3S)-3-(8-quinolylcarbamoyl)piperidine-1-carboxylate (Compound A3)

[0079] To a solution of 8-aminoquinoline (compound A2, CAS: 578-66-5, Vendor: Accela, 69.17 g, 479.78 mmol) in DMF (1000 mL) was added HATU (190.60 g, 501.59 mmol) and DIPEA (233 mL, 1309 mmol). The reaction was stirred at 0° C. for 1 h. Then (3S)-1-tert-butoxycarbonylpiperidine-3-carboxylic acid (compound A1, : 88495-54-9, Vendor: Accela, 100.00 g, 436.17 mmol) was added. The reaction was stirred at 0° C. for 15 h. EtOAc (200 mL) and ice water (50 mL) were added and separated. The aqueous phase was extracted twice with EtOAc (200 mL). The combined organic layers were washed four times with brine (300 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash column eluting with a gradient of EA / PE (1:5) to give compound A3 (120 g) as a yellow solid. MS: calculated 356 (MH + ), measured value 356(MH + ).

[0080] Step 2: Preparation of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(8-quinolylcarbamoyl)piperidine-1-carboxylate (compound A5) and tert-butyl (3S,4S)-4-(5-chloro-2-pyridyl)-3-(8-quinolylcarbamoyl)piperidine-1-carboxylate (compound A13)

[0081] A mixture of tert-butyl (3S)-3-(8-quinolylcarbamoyl)piperidine-1-carboxylate (compound A3, 50.00 g, 140.67 mmol), 5-chloro-2-iodo-pyridine (compound A4, CAS: 244221-57-6, vendor: Bide Pharmatech, 77.00 g, 322.18 mmol) and Pd(OAc)2 (3.15 g, 14.07 mmol), AgOAc (46.70 g, 281.35 mmol) was degassed with N2, and then stirred at 110°C under N2 atmosphere for 48 hours. After cooling to room temperature, it was filtered. The filtrate was concentrated under vacuum. The residue was further purified by flash column eluting with a gradient of EA / PE (1 / 10 to 1 / 1) to give compound A5 (20.00 g) and compound A13 (15.00 g) as yellow oils. MS: calculated 467 (MH + ), measured value 467(MH + ).

[0082] Step 3: Preparation of (3S,4R)-1-tert-butoxycarbonyl-4-(5-chloro-2-pyridyl)piperidine-3-carboxylic acid (Compound A6)

[0083] To a solution of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(8-quinolylcarbamoyl)piperidine-1-carboxylate (compound A5, 17.00 g, 36.41 mmol) in ethanol (200 mL), sodium hydroxide (14.57 g, 364.22 mmol) was added and then stirred at 85° C. for 16 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was neutralized to pH 6 with HCl (3M). The mixture was extracted three times with DCM (300 mL). The organic phase was concentrated in vacuo. The residue was purified by flash column eluting with a gradient of PE / EA (5 / 1 to 1 / 1) to give compound A6 (11.50 g) as a yellow oil. MS: calculated 341 (MH + ), measured value 285 (M-C4H8+H + ).

[0084] Step 4: Preparation of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(hydroxymethyl)piperidine-1-carboxylate (Compound A7)

[0085] To a solution of (3S,4R)-1-tert-butoxycarbonyl-4-(5-chloro-2-pyridyl)piperidine-3-carboxylic acid (compound A6, 11.50 g, 33.74 mmol) in THF (50 mL) was added BH3.Me2S (13.5 mL, 134.98 mmol) at 0° C. After the addition, the reaction mixture was further stirred at 25° C. under N2 atmosphere for 16 h. The volatiles were removed to give a white solid residue, and then methanol (50 mL) was added. After stirring for 30 min, the reaction mixture was concentrated and methanol (50 mL) was added again. The residue was then treated dropwise with 10% citric acid. The mixture was extracted three times with DCM (200 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash column eluting with a gradient of PE / EA (3 / 1) to give compound A7 (8.00 g) as a colorless gum. MS: calculated 327 [(M+H) + ], measured value 271 (M-C4H8+H + ).

[0086] Step 5: Preparation of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(imidazole-1-carbothioyloxymethyl)piperidine-1-carboxylate (Compound A8)

[0087] To a solution of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(hydroxymethyl)piperidine-1-carboxylate (compound A7, 4.00 g, 12.24 mmol) and TCDI (4.36 g, 24.48 mmol) in THF (60 mL) was added DMAP (299 mg, 2.45 mmol). The mixture was stirred at 25° C. for 3 h. The reaction mixture was then concentrated and the residue was purified by flash column eluting with a gradient of PE / EA (1 / 1) to give compound A8 (5.00 g) as a yellow oil. MS: calculated 437 (MH +), measured value 437(MH + ).

[0088] Step 6: Preparation of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-methyl-piperidine-1-carboxylate (Compound A9)

[0089] To a mixture of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(imidazole-1-carbothioyloxymethyl)piperidine-1-carboxylate (compound A8, 5.00 g, 11.44 mmol) in toluene (250 mL) was added tris(trimethylsilyl)silane (17 mL, 47.56 mmol) and AIBN (1877 mg, 11.44 mmol). The mixture was degassed with N2 for 10 min and stirred at 85 °C for 5 h. After cooling to room temperature, the mixture was concentrated under vacuum. The residue was purified by flash column and preparative HPLC eluting with a gradient of PE / EA (5 / 1) to give compound A9 (combined two batches, 4.00 g) as a yellow oil. MS: calculated 311 (MH + ), measured value 255 (M-C4H8+H + ).

[0090] Step 7: Preparation of 5-chloro-2-[(3S,4R)-3-methyl-4-piperidyl]pyridine (compound A10)

[0091] To a solution of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-methyl-piperidine-1-carboxylate (compound A9, 4.00 g, 12.87 mmol) in DCM (20 mL) was added TFA (31 mL, 123.08 mmol) at 0° C. The mixture was stirred at 25° C. for 16 h. The reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC to give compound A10 (2.70 g) as a white solid. MS: calculated 211 (MH + ), measured value 211(MH + ).

[0092] Step 8: Preparation of 4-fluoro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (Compound A11)

[0093] A mixture of 4-chloro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound A12, CAS: 19867-78-8, vendor: PharmaBlock, 4.60 g, 25.33 mmol) and cesium fluoride (19.24 mg, 126.64 mmol) in DMSO (60 mL) was stirred at 120° C. for 18 h. After cooling to room temperature, the reaction mixture was added to water (100 mL) and extracted three times with EA (100 mL). The combined organic layers were washed twice with brine (200 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by flash column to give compound A11 (4.00 g, contained some compound A12) as a white solid. MS: calculated 166 (MH + ), measured value 166(MH + ).

[0094] Step 9: Preparation of 4-[(3S,4R)-4-(5-chloro-2-pyridyl)-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine (Intermediate A)

[0095] To a mixture of 4-fluoro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound A11, 2.12 g, 12.81 mmol) and 5-chloro-2-[(3S,4R)-3-methyl-4-piperidyl]pyridine (compound A10, 2.70 g, 12.81 mmol) in DMSO (50 mL) was added CsF (3.72 mg, 64.07 mmol). The reaction was stirred at 120° C. under N2 atmosphere for 12 h. After cooling to room temperature, the reaction mixture was poured into ice water (200 mL) and extracted three times with EA (250 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash column eluting with a gradient of PE / EA (1 / 1) to give the crude product, which was further purified by SFC (gradient: 55% MeOH (0.05% DEA) in CO, column: Daicel Chiralpak IC, 250×30 mm, 10 μm) to give intermediate A (2.52 g, slower eluting) as a white solid. MS: calcd 356 (MH + ), measured value 356(MH + ).

[0096] Intermediate B 4-[(3S,4R)-4-(5-chloro-3-methyl-2-pyridyl)-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0097] The title compound was prepared according to the following scheme. [ka]

[0098] Step 1: Preparation of tert-butyl 4-hydroxy-3-methyl-piperidine-1-carboxylate (Compound B2)

[0099] To a solution of tert-butyl 3-methyl-4-oxopiperidine-1-carboxylate (compound B1, CAS: 181269-69-2, Vendor: PharmaBlock, 50.00 g, 234.44 mmol) in methanol (500 mL) was added NaBH4 (13.30 g, 351.67 mmol) in portions at 0°C. After stirring at 0°C for 1 h and then at 20°C for 2.5 h, the reaction mixture was quenched with saturated aqueous ammonium chloride solution and concentrated under reduced pressure to remove methanol. The mixture was then extracted with dichloromethane and the combined organic layers were dried over sodium sulfate, filtered, and concentrated to give compound B2 (50.40 g) as a pale yellow oil.

[0100] Step 2: Preparation of tert-butyl 4-bromo-3-methyl-piperidine-1-carboxylate (Compound B3)

[0101] To a mixture of tert-butyl 4-hydroxy-3-methyl-piperidine-1-carboxylate (compound B2, 45.00 g, 209.02 mmol) in DCE (500 mL) was added Ph3P (71.27 g, 271.73 mmol) at 20 °C, followed by CBr4 (110.91 g, 334.43 mmol) at 20 °C. The flask was degassed and purged with N2 four times. After stirring at 25 °C under N2 atmosphere for 15 h, the reaction mixture was concentrated and the residue was purified by flash column eluted with a gradient of PE / EA (100 / 0 to 100 / 5) to give compound B3 (35.00 g) as a pale yellow oil.

[0102] Step 3: Preparation of tert-butyl-4-(5-chloro-3-methyl-2-pyridyl)-3-methyl-piperidine-1-carboxylate (Compound B5)

[0103] To a 1000 mL vial equipped with a stir bar was added 2-Bromo-5-chloro-3-methyl-pyridine (compound B4, CAS: 65550-77-8, Vendor: Accela, 22.00 g, 106.55 mmol), tert-butyl 4-bromo-3-methyl-piperidine-1-carboxylate (compound B3, 38.53 g, 138.52 mmol), Ir[dF(CF3)ppy]2(dtbpy)(PF6) (0.85 g, 1.07 mmol), NiCl2·dtbbpy (0.21 g, 0.530 mmol), TTMSS (26.50 g, 106.55 mmol), Na2CO3 (22.59 g, 213.11 mmol), and DME (500 mL). The vial was sealed and purged with nitrogen. The mixture was stirred and irradiated with a 34 W blue LED lamp (7 cm away) with a cooling fan to maintain the reaction temperature at 25° C. for 14 h. The reaction was filtered and washed three times with a mixture of solvents (DCM / MeOH=10 / 1, 5 mL). The filtrate was concentrated and the residue was purified by flash column eluting with a gradient of PE / EA (5 / 1) to give compound B5 (7.00 g) as a pale yellow oil. MS: calculated 325 (MH + ), measured value 269 (M-C4H8+H + ).

[0104] Step 4: Preparation of 5-chloro-3-methyl-2-(3-methyl-4-piperidyl)pyridine (Compound B6)

[0105] To a solution of tert-butyl-4-(5-chloro-3-methyl-2-pyridyl)-3-methyl-piperidine-1-carboxylate (compound B5, 7.00 g, 21.55 mmol) in DCM (30 mL) was added TFA (10 mL) dropwise at 0° C. After stirring at 20° C. for 2 h, the reaction mixture was concentrated, the residue was dissolved in DCM (50 mL) and basified with saturated NaHCO3 solution. The mixture was extracted three times with DCM (40 mL). The combined organic layers were washed with 50 mL of brine, dried over Na2SO4 and concentrated to give compound B6 (4.80 g) as a pale yellow oil. MS: calcd. 225 (MH + ), measured value 225(MH + ).

[0106] Step 5: Preparation of 4-[(3S,4R)-4-(5-chloro-3-methyl-2-pyridyl)-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine (Intermediate B)

[0107] To a mixture of 5-chloro-3-methyl-2-(3-methyl-4-piperidyl)pyridine (compound B6, 4.60 g, 20.47 mmol) and 4-fluoro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound A11, 3.72 g, 22.52 mmol) in DMSO (40 mL) was added DIPEA (2.37 g, 40.94 mmol). The reaction was stirred at 120° C. under N2 atmosphere for 3 h. After cooling to room temperature, the reaction mixture was poured into ice water (400 mL) and extracted three times with EA (80 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by flash column eluting with a gradient of EA / PE (1 / 1) to give the mixture (5.40 g) as a light brown solid. The mixture continued to be purified by SFC (gradient: 40% EtOH (0.1% NH3H2O) in CO2, column: Daicel Chiralpak AD, 250 x 30 mm, 10 μm) to give intermediate B (2.10 g) as a pale yellow solid. MS: calculated 370 (MH + ), measured value 370 (MH + ).

[0108] Intermediate C 4-[4-(6-bromo-3-pyridyl)-3-methyl-1-piperidyl]-1-methyl-pyrazolo[3,4-b]pyridine [ka]

[0109] The title compound was prepared according to the following scheme. [ka]

[0110] Step 1: Preparation of tert-butyl-3-methyl-4-(p-tolylsulfonylhydrazono)piperidine-1-carboxylate (Compound C2)

[0111] To a solution of tert-butyl-3-methyl-4-oxopiperidine-1-carboxylate (compound B1, CAS: 181269-69-2, Vendor: PharmaBlock, 300 mg, 1.41 mmol) in ethanol (10 mL) was added 4-methylbenzenesulfonohydrazide (compound C1, 341 mg, 1.83 mmol). The mixture was stirred at 70° C. for 3 hours. After cooling to room temperature, the mixture was concentrated to give the crude product compound C2 (537 mg), which was used in the next step without further purification. MS: calculated 382 (MH + ), measured value 382(MH + ).

[0112] Step 2: Preparation of tert-butyl 4-(6-bromo-3-pyridyl)-3-methyl-piperidine-1-carboxylate (Compound C4)

[0113] To a solution of tert-butyl-3-methyl-4-(p-tolylsulfonylhydrazono)piperidine-1-carboxylate (compound C2, 537 mg, 1.41 mmol) in 1,4-dioxane (5 mL) was added (6-bromopyridin-3-yl)boronic acid (compound C3, CAS: 223463-14-7, vendor: Accela, 369 mg, 1.83 mmol) and Cs2CO3 (1.38 g, 4.22 mmol). The mixture was stirred under N2 at 120 °C for 16 h. After cooling to room temperature, the mixture was filtered and the solid was washed twice with EA (10 mL). The combined organic layers were concentrated and purified by flash column eluting with a gradient of EA / PE (0%-70%) to give the desired product C4 (50 mg) as a colorless oil. MS: calculated 355 (MH + ), measured value 355 (MH + ).

[0114] Step 3: Preparation of 2-bromo-5-(3-methyl-4-piperidyl)pyridine (compound C5)

[0115] A mixture of tert-butyl 4-(6-bromo-3-pyridyl)-3-methyl-piperidine-1-carboxylate (compound C4, 50 mg, 141 μmol) in HCl (1M in EA, 10 ml, 10 mmol) was stirred at room temperature for 2 hours. The mixture was concentrated to give the desired product C5 (41 mg) as a white solid. MS: calculated 255 (MH + ), measured value 255(MH + ).

[0116] Step 4: Preparation of 4-[4-(6-bromo-3-pyridyl)-3-methyl-1-piperidyl]-1-methyl-pyrazolo[3,4-b]pyridine (Intermediate C)

[0117] To a solution of 4-chloro-1-methyl-pyrazolo[3,4-b]pyridine (compound C6, CAS: 1268520-92-8, vendor: PharmaBlock, 35 mg, 211 μmol) and 2-bromo-5-(3-methyl-4-piperidyl)pyridine (compound C5, 41 mg, 141 μmol) in DMSO (15 mL) was added CsF (107 mg, 703 μmol). The reaction mixture was stirred at 130 °C overnight. After cooling to room temperature, the reaction was diluted with EA, washed four times with water (30 mL), dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by flash column eluting with a gradient of EA (containing 10% MeOH) / PE (0% to 70%) to give intermediate C (42 mg). MS: calculated 386 (MH + ), measured value 386(MH + ).

[0118] Intermediate D 4-[(3R,4S)-4-(5-chloro-2-pyridyl)-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0119] The title compound was prepared in a similar manner to the preparation of intermediate A, by using compound A13 instead of compound A5. Intermediate D was obtained as a white solid. MS: calculated 356 (MH + ), measured value 356(MH + ). (Note: When intermediate A13 is treated with sodium hydroxide in ethanol, the stereocenter at the 3-position of the piperidine ring is epimerized from the S configuration to the R configuration to give (3R,4S)-1-butoxycarbonyl-4-(5-chloro-2-pyridyl)piperidine-3-carboxylic acid.)

[0120] Intermediate E 4-[4-(5-chloro-3-methyl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0121] The title compound was prepared according to the following scheme. [ka]

[0122] Step 1: Preparation of tert-butyl 4-(5-chloro-3-methyl-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (Compound E3)

[0123] To a mixture of 2-bromo-5-chloro-3-methyl-pyridine (compound E1, 15.00 g, 72.65 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound E2, 24.71 g, 79.91 mmol) in 1,4-dioxane (400 mL) was added sodium carbonate (15.40 g, 145.30 mmol) and Pd(dppf)Cl2.CH2Cl2 (5.93 g, 7.26 mmol) in water (40 mL). The mixture was stirred at 95 °C under N2 atmosphere for 16 h. After cooling to room temperature, the mixture was filtered and washed three times with 1,4-dioxane (10 mL). The filtrate was concentrated and the crude product was purified by flash column eluted with a gradient of PE / EA (5 / 1 to 3 / 1) to give compound E3 (9.40 g) as a yellow oil. MS: calculated 309 (MH + ), measured value 309(MH + ).

[0124] Step 2: Preparation of tert-butyl 4-(5-chloro-3-methyl-2-pyridyl)piperidine-1-carboxylate (Compound E4)

[0125] To a solution of tert-butyl 4-(5-chloro-3-methyl-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound E3, 6.00 g, 19.43 mmol) in tetrahydrofuran (120 mL) was added tris(triphenylphosphine)rhodium(I) chloride (4.49 g, 4.86 mmol). The flask was degassed and purged with H2 four times. The mixture was stirred under hydrogen atmosphere (45 psi) at 60 °C for 36 h. The mixture was filtered through Celite and the solid was washed with methanol (5 mL). The solvent was removed under vacuum and the crude product was purified by flash column eluting with a gradient of PE / EA (5 / 1 to 3 / 1) to give compound E4 (4.50 g) as an off-white solid. MS: calcd 311 (MH + ), measured value 311 (MH + ).

[0126] Step 3: Preparation of 5-chloro-3-methyl-2-(4-piperidyl)pyridine (Compound E5)

[0127] To a solution of tert-butyl 4-(5-chloro-3-methyl-2-pyridyl)piperidine-1-carboxylate (compound E4, 4.50 g, 14.48 mmol) in DCM (50 mL) was added HCl in dioxane (10 mL) at 0° C. After stirring at 25° C. for 16 h, the reaction mixture was concentrated under reduced pressure to give compound E5 (3.56 g) as a white solid. MS: calculated 211 (MH + ), measured value 211(MH + ).

[0128] Step 4: Preparation of 4-[4-(5-chloro-3-methyl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine (Intermediate E)

[0129] To a mixture of 4-chloro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (1.15 g, 6.33 mmol) and 5-chloro-3-methyl-2-(4-piperidyl)pyridine (compound E5, 1.72 g, 6.97 mmol) in DMSO (30 mL) was added KF (3.67 g, 63.32 mmol). The reaction was stirred at 120 °C under N2 atmosphere for 15 h. After cooling to room temperature, the reaction mixture was poured into ice water (200 mL) and aqueous NaHCO3 (50 mL) and extracted three times with EA (250 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under vacuum. The crude product was purified by flash column eluting with a gradient of PE / EA (5 / 1 to 0 / 1) to give intermediate E (819 mg) as a pale yellow solid. MS: calcd 356 (MH + ), measured value 356(MH + ).

[0130] Example 1 1,6-Dimethyl-4-[4-(4-piperazin-1-ylphenyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0131] The title compound was prepared according to the following scheme. [ka]

[0132] Step 1: Preparation of tert-butyl 4-[4-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)phenyl]piperazine-1-carboxylate (Compound 1c)

[0133] To a mixture of benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (compound 1a, CAS: 286961-15-7, vendor: Accela, 100 mg, 291 μmol), tert-butyl 4-(4-bromophenyl)piperazine-1-carboxylate (compound 1b, CAS: 352437-09-3, vendor: Accela, 99.4 mg, 291 μmol) in 1,4-dioxane (5 mL) and water (1 mL) was added K2CO3 (121 mg, 874 μmol) and PdCl2(dppf)·CH2Cl2 (24 mg, 29 μmol). The mixture was charged with N2 and stirred at 100 °C overnight. After cooling to room temperature, the mixture was dried over Na2SO4, filtered, and the solid was washed twice with EA (10 mL). The combined organic phase was concentrated and purified by flash column eluting with a gradient of EA / PE (0%-60%) to give compound 1c (83 mg). MS: calculated 478 (MH + ), measured value 478(MH + ).

[0134] Step 2: Preparation of tert-butyl 4-[4-(4-piperidyl)phenyl]piperazine-1-carboxylate (compound 1d)

[0135] To a mixture of tert-butyl 4-[4-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)phenyl]piperazine-1-carboxylate (compound 1c, 83 mg, 174 μmol) in MeOH (10 mL) was added Pd(OH)2 (10 wt% in carbon, 12 mg, 87 μmol). The suspension was purged with H2 three times and then stirred overnight at room temperature under a hydrogen balloon. The mixture was filtered and concentrated to give the crude product compound 1d (60 mg), which was used in the next step without further purification. MS: calcd 346 (MH + ), measured value 346(MH + ).

[0136] Step 3: Preparation of tert-butyl 4-[4-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-4-piperidyl]phenyl]piperazine-1-carboxylate (Compound 1f)

[0137] To a mixture of 4-chloro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound 1e, CAS: 19867-78-8, vendor: PharmaBlock, 24 mg, 130 μmol) and tert-butyl 4-[4-(4-piperidyl)phenyl]piperazine-1-carboxylate (compound 1d, 30 mg, 87 μmol) in 1,4-dioxane (2 mL) was added RuPhos Pd G2 (7 mg, 9 μmol) and Cs2CO3 (85 mg, 261 μmol). The mixture was charged with N2 and stirred at 110 °C overnight. After cooling to room temperature, the mixture was filtered and the solid was washed twice with EA (10 mL). The combined organic phase was concentrated and purified by flash column eluting with a gradient of EA (containing 10% MeOH) / PE (0%-60%) to give the desired product 1f. MS: Calculated value 491 (MH + ), measured value 491(MH + ).

[0138] Step 4: Preparation of 1,6-dimethyl-4-[4-(4-piperazin-1-ylphenyl)-1-piperidyl]pyrazolo[3,4-b]pyridine

[0139] Compound 1f was dissolved in DCM (10 mL) and TFA (2 mL) and stirred at room temperature for 2 h. The mixture was concentrated and purified by reverse flash column eluting with a gradient of ACN / water (containing 0.5% TFA) (0-30%) to give Example 1 (36 mg) as a yellow solid. MS: calculated 391 (MH + ), measured value 391(MH + ). 1 H NMR(400 MHz, methanol-d4)δ=8.36(s,1H),7.23-7.18(m,2H),7.01-6.96(m,2H),6.70(s,1H),4.60(br d,J=13.3 Hz,2H),4.07(s,3H),3.56(br t,J=12.7 Hz,2H),3.39-3.33(m,8H),3.01(tt,J=3.8,11.9 Hz,1H),2.62(s,3H),2.14-2.07(m,2H),1.84(dq,J=3.9,12.7 Hz,2H).

[0140] Example 2 1,6-Dimethyl-4-[4-(5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0141] The title compound was prepared in a similar manner to the preparation of Example 1, by using tert-butyl 4-[6-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)-3-pyridyl]piperazine-1-carboxylate (Compound 2b) instead of Compound 1c. Example 2 (44 mg) was obtained as a white solid. MS: calculated 392 (MH + ), measured value 392(MH + ). 1H NMR(400 MHz,CDCl3)δ=8.22(d,J=2.4 Hz,1H),7.90(s,1H),7.15(dd,J=2.6,8.6 Hz,1H),7.04(d,J=8.6 Hz,1H),6.21(s,1H),4.23(br d,J=13.1 Hz,2H),4.04(s,3H),3.25-3.09(m,6H),3.05-2.89(m,5H),2.53(s,3H),2.09-2.01(m,2H),1.98-1.86(m,3H).

[0142] Compound 2b was prepared according to the following scheme: [ka]

[0143] Step 1: Preparation of benzyl 4-(5-chloro-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 2a)

[0144] To a solution of 2-bromo-5-chloropyridine (CAS: 40473-01-6, vendor: Accela, 1.00 g, 5.2 mmol), benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 1a, 1.80 g, 5.24 mmol) and sodium carbonate (1.20 g, 11.32 mmol) in 1,4-dioxane (10 mL) and water (1 mL) was added PdCl2(dppf)·CHCl2 (380 mg, 0.52 mmol) under N2. The mixture was stirred at 80 °C under N2 for 16 h. After cooling to room temperature, the reaction mixture was filtered, the filtrate was concentrated under reduced pressure to remove the solvent, and the residue was purified by flash column eluted with a gradient of EA / PE (1 / 5 to 1 / 0) to give compound 2a (1.50 g) as a brown oil. MS: calculated 329 (MH + ), measured value 329 (MH + ).

[0145] Step 2: Preparation of tert-butyl 4-[6-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)-3-pyridyl]piperazine-1-carboxylate (compound 2b)

[0146] To a solution of benzyl 4-(5-chloro-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 2a, 500 mg, 1.52 mmol), tert-butyl piperazine-1-carboxylate (566 mg, 3.04 mmol) and sodium tert-butoxide (225 mg, 2.34 mmol) in 1,4-dioxane (5 mL), Xphos-Pd-G3 (129 mg, 0.15 mmol) was added under N2. The mixture was stirred at 100 °C under N2 for 3 h. After cooling to room temperature, the reaction mixture was filtered through Celite, the filtrate was concentrated under reduced pressure to remove the solvent, and the crude product was purified by flash column eluting with a gradient of EA / PE (1 / 5 to 1 / 0) to give compound 2b (223 mg) as a yellow solid. MS: calculated 479 (MH + ), measured value 479(MH + ).

[0147] Example 3 1,6-Dimethyl-4-[4-(5-piperazin-1-ylpyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0148] The title compound was prepared in a similar manner to the preparation of Example 1, by using tert-butyl 4-[5-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)pyrazin-2-yl]piperazine-1-carboxylate (Compound 3b) instead of Compound 1c. Example 3 (23 mg) was obtained as a white solid. MS: calculated 393 (MH + ), measured value 393(MH + ). 1H NMR(400 MHz, CDCl3)δ=8.09-8.06(m,1H),8.01-7.98(m,1H),7.93-7.91(m,1H),6.24-6.22(m,1H),4.29-4.21(m,2H),4.07-4.04( m,3H),3.55-3.50(m,4H),3.27-3.18(m,2H),3.02-2.97(m,4H),2.97-2.89(m,1H),2.57-2.54(m,3H),2.07-1.93(m,4H).

[0149] Compound 3b was prepared according to the following scheme: [ka]

[0150] Step 1: Preparation of tert-butyl 4-(5-chloropyrazin-2-yl)piperazine-1-carboxylate (compound 3a)

[0151] To a solution of 2,5-dichloropyrazine (CAS: 19745-07-4, vendor: Accela, 2.00 g, 13.42 mmol) and tert-butyl piperazine-1-carboxylate (2.75 g, 14.77 mmol) in ACN (20 mL) was added potassium carbonate (2.78 g, 20.14 mmol). The mixture was stirred at 80 °C for 16 h. After cooling to room temperature, the reaction mixture was filtered, the filtrate was concentrated under reduced pressure to remove the solvent, and the residue was purified by flash column eluted with a gradient of PE / EA (10 / 1 to 3 / 1) to give compound 3a (1.50 g, 5.02 mmol) as a pale yellow solid. MS: calculated 299 (MH + ), measured value 299 (MH + ).

[0152] Step 2: Preparation of tert-butyl 4-[5-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)pyrazin-2-yl]piperazine-1-carboxylate (compound 3b)

[0153] To a mixture of tert-butyl 4-(5-chloropyrazin-2-yl)piperazine-1-carboxylate (compound 3a, 1.45 g, 4.85 mmol) and benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro 2H-pyridine-1-carboxylate (compound 1a, 1.83 g, 5.34 mmol) in 1,4-dioxane (10 mL) and water (1 mL) was added K2CO3 (1.34 g, 9.71 mmol) and PdCl2(dppf)·CH2Cl2 (198 mg, 0.24 mmol). The flask was degassed and purged with N2 four times. The mixture was stirred at 90 °C under N2 atmosphere for 16 h. After cooling to room temperature, the mixture was diluted with water (10 mL) and extracted three times with EA (20 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated to give the crude product, which was purified by flash column eluting with a gradient of PE / EA (5 / 1 to 3 / 1) to give compound 3b (1.50 g) as a yellow solid. MS: calculated 480 (MH + ), measured value 480(MH + ).

[0154] Example 4 1,6-Dimethyl-4-[4-(5-piperazin-1-ylpyrimidin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0155] The title compound was prepared in a similar manner to the preparation of Example 2, by using 5-bromo-2-iodopyrimidine (CAS: 183438-24-6, vendor: Accela) instead of 2-bromo-5-chloropyridine. Example 4 (3.5 mg) was obtained as a white solid. MS: calculated 393 (MH + ), measured value 393(MH + ). 1H NMR (400 MHz, methanol-d4) δ=8.40(s,2H),8.08(s,1H),6.40(s,1H),5.02-4.90(m,2H),4.33(br d,J=13.2 Hz,2H),3.99(s,3H),3.26-3.20(m,4H),3.18-3.11(m,1H),3.03-2.95(m,4H),2.52(s,3H),2.13-1.94(m,4H).

[0156] Example 5 1-Methyl-4-[4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0157] The title compound was prepared in a similar manner to the preparation of Example 2, by using 2-bromo-5-chloro-3-methyl-pyridine (CAS: 65550-77-8, vendor: Accela) and 4-chloro-1-methyl-pyrazolo[3,4-b]pyridine (CAS: 1268520-92-8, vendor: PharmaBlock) instead of 2-bromo-5-chloropyridine and compound 1e. Example 5 (29 mg) was obtained as a white solid. MS: calculated 392 (MH + ), measured value 392(MH + ). 1 H NMR (400 MHz, methanol-d4)δ=8.16(s,1H),8.10(d,J=5.9 Hz,1H),7.96(d,J=2.7 Hz,1H),7.21(d,J=2.7 Hz,1H),6.50(d,J=5.9 Hz,1H),4.41(br d,J=13.7 Hz,2H),4.00(s,3H),3.41-3.33(m,2H),3.29-3.22(m,1H),3.18-3.10(m,4H),3.04-2.93(m,4H),2.40(s,3H),2.10-1.95(m,2H),1.88(br dd,J=1.5,12.5 Hz,2H).

[0158] Example 6 1-Methyl-4-[4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0159] The title compound was prepared in a similar manner to the preparation of Example 1, by using 4-chloro-1-methyl-pyrazolo[3,4-b]pyridine (CAS: 1268520-92-8, vendor: PharmaBlock) and tert-butyl 4-(5-bromo-4-methyl-2-pyridyl)piperazine-1-carboxylate (CAS: 944582-92-7, vendor: Bide Pharmatech) instead of Compound 1e and Compound 1b. Example 6 (35 mg) was obtained as a pale yellow solid. MS: calculated 392 (MH + ), measured value 392(MH + ). 1 H NMR (400 MHz, methanol-d4) δ=8.45(s,1H),8.06(d,J=7.3 Hz,1H),7.91(s,1H),7.21(s,1H),6.86(d,J=7.5 Hz,1H),4.70-4.62(m,2H),4.08(s,3H),3.94-3.84(m,4H),3.65(br t,J=12.1 Hz,2H),3.44-3.36(m,4H),3.35-3.24(m,1H),2.56(s,3H),2.14(br d,J=12.3 Hz,2H),1.90(dq,J=3.5,12.6 Hz,2H).

[0160] Example 7 1,6-Dimethyl-4-[4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0161] The title compound was prepared in a similar manner to the preparation of Example 2, by using 2-bromo-5-chloro-3-methoxy-pyridine (CAS: 65550-77-8, vendor: Accela) instead of 2-bromo-5-chloropyridine. Example 7 (61 mg) was obtained as a white solid. MS: calculated 406 (MH + ), measured value 406(MH + ). 1 H NMR(400 MHz, methanol-d4)δ=8.07(s,1H),7.96(d,J=2.9 Hz,1H),7.21(d,J=2.7 Hz,1H),6.40(s,1H),4.37(br d,J=13.4 Hz,2H),3.99(s,3H),3.35(br d,J=2.0 Hz,1H),3.29-3.21(m,2H),3.15(br dd,J=3.9,6.1 Hz,4H),3.02-2.94(m,4H),2.52(s,3H),2.40(s,3H),2.08-1.96(m,2H),1.89-1.80(m,2H).

[0162] Example 8 1,6-Dimethyl-4-[4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0163] The title compound was prepared in a similar manner to the preparation of Example 1, by using 4-bromo-1,6-dimethyl-pyrazolo[3,4-b]pyridine (CAS: 1783407-55-5, vendor: Accela) and tert-butyl 4-(5-bromo-4-methyl-2-pyridyl)piperazine-1-carboxylate (CAS: 944582-92-7, vendor: Bide Pharmatech) instead of compound 1e and compound 1b. Example 8 (32 mg) was obtained as a yellow solid. MS: calculated value 406 (MH + ), measured value 406(MH + ). 1H NMR(400 MHz, methanol-d4)δ=8.27(s,1H),7.81(s,1H),7.07(s,1H),6.62(s,1H),4.54(br d,J=13.4 Hz,2H),3.98(s,3H),3.82-3.73(m,4H),3.51(br t,J=12.6 Hz,2H),3.35-3.25(m,4H),3.19-3.12(m,1H),2.53(s,3H),2.45(s,3H),2.03(br d,J=12.3 Hz,2H),1.77(dq,J=3.5,12.6 Hz,2H).

[0164] Example 9 1,6-Dimethyl-4-[4-(4-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0165] Similar to the preparation of Example 3, 5-bromo-2-chloro-4-methylpyridine (CAS: 778611-64-6, vendor: Accela) was used instead of 2,5-dichloropyrazine, and S N The title compound was prepared by replacing the Ar reaction (K2CO3, CAN) with the Buchwald coupling reaction (XantPhos, Pd2(dba)3, t-BuONa, toluene). Example 9 (61 mg) was obtained as a white solid. MS: calculated 406 (MH + ), measured value 406(MH + ). 1 H NMR (400 MHz, methanol-d4) δ=8.09(d,J=3.4 Hz,2H),7.18(s,1H),6.41(s,1H),4.37(br d,J=13.2 Hz,2H),3.99(s,3H),3.30-3.23(m,2H),3.06-3.01(m,4H),3.02-2.99(m,1H),3. 00-2.93(m,4H),2.52(s,3H),2.33(s,3H),2.05-1.97(m,2H),1.97-1.85(m,2H).

[0166] Example 10 1,6-Dimethyl-4-[4-(2-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0167] In the same manner as in the preparation of Example 3, 3-bromo-6-fluoro-2-methylpyridine (CAS: 375368-83-5, vendor: Accela) was used instead of 2,5-dichloropyrazine, and S N The title compound was prepared by replacing Ar reaction conditions (K2CO3, CAN) with (DIPEA, DMF). Example 10 (7 mg) was obtained as a white solid. MS: calculated 406 (MH + ), measured value 406(MH + ). 1 H NMR (400MHz, methanol-d4) δ=8.10(s,1H),7.42(d,J=8.8 Hz,1H),6.61(d,J=8.6 Hz,1H),6.43(s,1H),4.38(br d,J=13.1 Hz,2H),4.01(s,3H),3.49-3.43(m,4H),3.32-3.26(m,2H),3.11-3.01(m,1H),2. 99-2.92(m,4H),2.54(s,3H),2.50(s,3H),1.97-1.89(m,2H),1.86-1.75(m,2H).

[0168] Example 11 1,6-Dimethyl-4-[4-(4-methyl-2-piperazin-1-yl-pyrimidin-5-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0169] The title compound was prepared in a similar manner to the preparation of Example 3, by using 5-bromo-2-chloro-4-methylpyrimidine (CAS: 633328-95-7, vendor: Accela) instead of 2,5-dichloropyrazine. Example 11 (34 mg) was obtained as a white solid. MS: calculated 407 (MH + ), measured value 407(MH + ). 1 H NMR(400 MHz,CDCl3)δ=8.10(s,1H),7.93(s,1H),6.24(s,1H),4.27(br d,J=12.8 Hz,2H),4.07(s,3H),3.81-3.74(m,4H),3.18(dt,J=2.6,12.5 Hz,2H),2.95-2.81(m,5H),2.57(s,3H),2.43(s,3H),1.97-1.82(m,4H).

[0170] Example 12 1,6-Dimethyl-4-[4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0171] The title compound was prepared in a similar manner to the preparation of Example 3, by using 2,5-dichloro-3-methyl-pyrazine (CAS: 107378-41-6, vendor: Bide) instead of 2,5-dichloropyrazine. Example 12 (16 mg) was obtained as a white solid. MS: calculated 407 (MH + ), measured value 407(MH + ). 1H NMR (400MHz, methanol-d4) δ=8.09(s,1H),7.92(s,1H),6.41(s,1H),4.37(br d,J=13.3 Hz,2H),4.00(s,3H),3.59-3.50(m,4H),3.39-3.34(m,1H),3.32-3.28(m,1H),3.28-3.17( m,1H),3.00-2.88(m,4H),2.54(s,3H),2.51(s,3H),2.07-1.94(m,2H),1.93-1.82(m,2H).

[0172] Example 13 4-[4-(3-ethyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0173] The title compound was prepared in a similar manner to the preparation of Example 1, by using tert-butyl 4-[6-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)-5-vinyl-3-pyridyl]piperazine-1-carboxylate (Compound 13c) instead of Compound 1c. Example 13 (5 mg) was obtained as a white solid. MS: calculated 420 (MH + ), measured value 420 (MH + ). 1 H NMR (400MHz, methanol-d4) δ=8.10(s,1H),7.99(d,J=2.8 Hz,1H),7.22(d,J=2.8 Hz,1H),6.42(s,1H),4.39(br d,J=13.4 Hz,2H),4.01(s,3H),3.39-3.34(m,1H),3.32-3.23(m,2H),3.20-3.12(m,4H),3.04-2.95(m,4H),2.77(q,J=7.5 Hz,2H),2.54(s,3H),2.18-2.02(m,2H),1.85(br d,J=11.1 Hz,2H),1.28(t,J=7.5 Hz,3H).

[0174] Compound 13c was prepared according to the following scheme: [ka]

[0175] Step 1: Preparation of benzyl 4-(3-bromo-5-chloro-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 13a)

[0176] To a mixture of benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 1a, 2.53 g, 7.37 mmol) and 2,3-dibromo-5-chloropyridine (CAS: 137628-17-2, Vendor: Accela, 2.00 g, 7.37 mmol) in 1,4-dioxane (10 mL) and water (1 mL), PdCl2(dppf).CH2Cl2 (301 mg, 0.37 mmol) and Na2CO3 (1.56 g, 14.74 mmol) were added. The mixture was stirred at 80 °C under N2 atmosphere for 0.5 h. After cooling to room temperature, the mixture was filtered and the solid was washed three times with 1,4-dioxane (15 mL) and concentrated. The residue was purified by flash column eluted with a gradient of PE / EA (3 / 1) to give compound 13a (680 mg, 1.67 mmol) as a pale yellow solid. MS: calculated 407 (MH + ), measured value 407(MH + ).

[0177] Step 2: Preparation of benzyl 4-(5-chloro-3-vinyl-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 13b)

[0178] To a mixture of benzyl 4-(3-bromo-5-chloro-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 13a, 100 mg, 0.25 mmol), vinylboronic acid pinacol ester (94 mg, 0.61 mmol) and Na2CO3 (20 mg, 0.490 mmol) in toluene (1 mL), ethanol (0.5 mL) and water (0.5 mL), PdCl2(dppf).CH2Cl2 (20 mg, 0.02 mmol) was added and the mixture was stirred at 100 °C for 1 h under N2 atmosphere. This reaction was repeated four times (for 100 mg of compound 13c each). The combined reaction mixture was filtered and washed with EtOH. The filtrate was concentrated. The residue was purified by flash column eluted with a gradient of PE / EA (5 / 1 to 0 / 1) to give compound 13b (260 mg) as a pale yellow oil. MS: calculated 355 (MH + ), measured value 355 (MH + ).

[0179] Step 3: Preparation of tert-butyl 4-[6-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)-5-vinyl-3-pyridyl]piperazine-1-carboxylate (compound 13c)

[0180] To a mixture of tert-butyl piperazine-1-carboxylate (105 mg, 0.56 mmol) and benzyl 4-(5-chloro-3-vinyl-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 13b, 100 mg, 0.28 mmol) in 1,4-dioxane (2.5 mL) was added Xphos-Pd-G3 (24 mg, 0.03 mmol) and t-BuONa (67 mg, 0.70 mmol). After stirring at 100 °C under N2 for 1.5 h, the reaction mixture was combined with another identical batch and worked up, then the mixture was filtered, washed with EtOH and concentrated. The residue was purified by flash column eluting with a gradient of PE / EA (1 / 0 to 1 / 8) to give compound 13c (90 mg) as a pale yellow solid. MS: calculated 505 (MH + ), measured value 505 (MH + ).

[0181] Example 14 4-[4-(3-Methoxy-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0182] The title compound was prepared in a similar manner to the preparation of Example 9, by using 5-bromo-2-chloro-3-methoxy-pyridine (CAS: 286947-03-3, vendor: Accela) instead of 5-bromo-2-chloro-4-methylpyridine. Example 14 (37 mg) was obtained as a white solid. MS: calculated 422 (MH + ), measured value 422(MH + ). 1 H NMR (400MHz, methanol-d4) δ=8.09(s,1H),7.70(d,J=2.3 Hz,1H),6.99(d,J=2.3 Hz,1H),6.41(s,1H),4.37(br d,J=13.2 Hz,2H),4.00(s,3H),3.89(s,3H),3.44(tt,J=3.9,11.7 Hz,1H),3.32-3.25(m,2H),3.20(dd,J=4.0,6.2 Hz,4H),3.03-2.94(m,4H),2.54(s,3H),2.07-1.95(m,2H),1.94-1.85(m,2H).

[0183] Example 15 (3S,4R)-1-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-4-piperidyl]-5-methyl-3-pyridyl]-3-fluoro-piperidin-4-amine [ka]

[0184] The title compound was prepared according to the following scheme. [ka]

[0185] Step 1: Preparation of tert-butyl N-[(3S,4R)-1-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-4-piperidyl]-5-methyl-3-pyridyl]-3-fluoro-4-piperidyl]carbamate (compound 15b)

[0186] To a mixture of 4-[4-(5-chloro-3-methyl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine (intermediate E, 50 mg, 0.14 mmol) and tert-butyl N-[(3S,4R)-3-fluoro-4-piperidyl]carbamate (compound 15a, CAS: 1434126-99-4, vendor: PharmaBlock, 40 mg, 0.18 mmol) in 1,4-dioxane was added RuPhos Pd G2 (11 mg, 0.014 mmol) and cesium carbonate (92 mg, 0.28 mmol). The mixture was heated to 110° C. overnight. After cooling to room temperature, the solid was filtered off and washed twice with EA (10 mL). The combined organic layers were concentrated and purified by flash column eluted with a gradient of EA (containing 10% MeOH) / PE (0% to 80%) to give the desired product 15b, which was used directly in the next step. MS: calculated 538 (MH + ), measured value 538(MH + ).

[0187] Step 2: Preparation of (3S,4R)-1-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-4-piperidyl]-5-methyl-3-pyridyl]-3-fluoro-piperidin-4-amine (Example 15)

[0188] A mixture of compound 15b in DCM (5 mL) and TFA (2 mL) was stirred at room temperature for 2 h. The mixture was concentrated and purified by reverse flash column eluting with a gradient of ACN / water (containing 0.05% TFA) (0-30%) to give Example 15 (56 mg) as a white solid. MS: calculated 438 (MH + ), measured value 438(MH +). 1 H NMR (400 MHz, methanol-d4) δ=8.37(s,1H),8.15(d,J=2.9 Hz,1H),8.02(d,J=2.7 Hz,1H),6.74(s,1H),5.08(d,J=49.2 Hz,1H),4.76-4.61(m,2H),4.48-4.34(m,1H),4.16-4.03(m,4H),3.76-3.59(m,4H),3.43-3. 25(m,1H),3.24-3.09(m,1H),2.64(s,3H),2.59(s,3H),2.22-2.09(m,4H),2.09-2.00(m,2H).

[0189] Example 16 4-[4-[5-[(3R)-3-(methoxymethyl)piperazin-1-yl]-3-methyl-2-pyridyl]-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0190] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl (2R)-2-(methoxymethyl)piperazine-1-carboxylate (CAS: 1023301-73-6, vendor: PharmaBlock) instead of compound 15a. Example 16 (58 mg) was obtained as a white solid. MS: calculated 450 (MH + ), measured value 450 (MH + ). 1 H NMR (400 MHz, methanol-d4) δ=8.37(s,1H),8.13(d,J=2.8 Hz,1H),7.73(d,J=2.6 Hz,1H),6.73(s,1H),4.70-4.62(m,2H),4.08(s,3H),3.98-3.90(m,2H),3.72-3.57(m,6H),3.52-3.46( m,1H),3.45(s,3H),3.36-3.27(m,1H),3.20-3.06(m,2H),2.63(s,3H),2.53(s,3H),2.14-2.03(m,4H).

[0191] Example 17 (3S,4S)-1-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-4-piperidyl]-5-methyl-3-pyridyl]-3-methoxy-piperidin-4-amine [ka]

[0192] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-[(3S,4S)-3-methoxy-4-piperidyl]carbamate (CAS: 907544-19-8, vendor: PharmaBlock) instead of compound 15a. Example 17 (50 mg) was obtained as a white solid. MS: calculated 450 (MH + ), measured value 450 (MH + ). 1 H NMR (400 MHz, methanol-d4) δ=8.37(s,1H),8.17(d,J=2.9 Hz,1H),8.03(d,J=2.8 Hz,1H),6.75(s,1H),4.74-4.65(m,2H),4.39-4.29(m,1H),4.08(s,3H),4.03-3.91(m,1H), 3.75-3.60(m,3H),3.55(s,3H),3.41-3.32(m,1H),3.26-3.14(m,1H),3.00(dt,J=2.7,12.8 Hz,1H),2.72(dd,J=10.1,12.5 Hz,1H),2.64(s,3H),2.60(s,3H),2.21-2.09(m,5H),1.79(dq,J=4.6,12.5 Hz,1H).

[0193] Example 18 and Example 19 1-Methyl-4-[cis-3-methyl-4-(6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 18) and 1-methyl-4-[trans-3-methyl-4-(6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 19) [ka]

[0194] The title compound was prepared similarly to the preparation of Example 15 by using tert-butyl piperazine-1-carboxylate and intermediate C instead of compound 15a and intermediate E. Example 18 and Example 19 were separated by preparative HPLC.

[0195] Example 18 (6 mg) was obtained as a pale yellow solid. MS: calculated 392 (MH + ), measured value 392(MH + ). 1 H NMR (400 MHz, methanol-d4)δ=8.50(s,1H),8.07-7.99(m,2H),7.69(dd,J=2.3,8.9 Hz,1H),7.05(d,J=8.9 Hz,1H),6.86(d,J=7.6 Hz,1H),4.70(br d,J=13.7 Hz,1H),4.54(br d,J=13.4 Hz,1H),4.07(s,3H),3.85-3.77(m,5H),3.68-3.54(m,1H),3.39-3.33(m,5H),2.46-2.28(m,2H),1.98(br dd,J=2.8,13.8 Hz,1H),0.72(d,J=7.0 Hz,3H).

[0196] Example 19 (8 mg) was obtained as a pale yellow solid. MS: calculated 392 (MH + , measured value 392(MH + ). 1H NMR (400 MHz, methanol-d4)δ=8.41(s,1H),8.08-8.02(m,2H),7.70(dd,J=2.3,9.0 Hz,1H),7.07(d,J=9.0 Hz,1H),6.87(d,J=7.5 Hz,1H),4.65(br d,J=13.8 Hz,1H),4.55(br d,J=12.8 Hz,1H),4.08(s,3H),3.85-3.78(m,4H),3.66-3.53(m,1H),3.38-3.33(m,4H),3.28-3.23(m,1H),2.65(dt,J=4.0,11.4 Hz, 1H), 2.09-1.86 (m, 3H), 0.89 (d, J = 6.5 Hz, 3H). The relative configuration of the two molecules was confirmed by 2D-NMR.

[0197] Example 20 and Example 21 1,6-Dimethyl-4-[cis-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 20) and 1,6-Dimethyl-4-[trans-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 21) [ka]

[0198] The title compound was prepared according to the following scheme. [ka]

[0199] Step 1: Preparation of tert-butyl 4-[4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]piperazine-1-carboxylate (compound 20b)

[0200] To a mixture of tert-butyl 4-(5-bromo-4-methylpyridin-2-yl)piperazine-1-carboxylate (compound 20a, CAS: 944582-92-7, vendor: Bide Pharmatech, 100 mg, 281 μmol) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2 dioxaborolane) (107 mg, 421 μmol) in 1,4-dioxane (10 mL) was added potassium acetate (83 mg, 842 μmol) and PdCl2(dppf)·CHCl2 (23 mg, 28 μmol). The mixture was charged with N2 and stirred at 110 °C overnight. After cooling to room temperature, the mixture was filtered off and the solid was washed twice with EA (10 mL). The combined organic layers were concentrated to give crude compound 20b (113 mg), which was used in the next step without further purification. MS: calculated 404 (MH + ), measured value 404(MH + ).

[0201] Step 2: Preparation of 1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-piperidin-4-one (compound 20e)

[0202] To a mixture of 4-chloro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound 20c, CAS: 19867-78-8, vendor: Pharma Block, 511 mg, 2.82 mmol) and 3-methylpiperidin-4-one hydrochloride (compound 20d, CAS: 4629-78-1, vendor: Pharma Block, 351 mg, 2.35 mmol) in 1,4-dioxane (20 mL) was added RuPhos Pd G2 (182 mg, 235 μmol) and Cs2CO3 (2.29 g, 7.04 mmol). The mixture was charged with N2 and stirred at 110 °C overnight. After cooling to room temperature, the solid was filtered off and washed twice with EA (10 mL). The combined mixture was concentrated and purified by flash column eluting with a gradient of EA (containing 10% MeOH) / PE (0% to 80%) to give the desired product 20e (272 mg) as a yellow oil. MS: calculated 259 (MH + ), measured value 259(MH +).

[0203] Step 3: Preparation of [1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]trifluoromethanesulfonate (compound 20f)

[0204] To a solution of 1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-piperidin-4-one (compound 20e, 200 mg, 774 μmol) in THF (10 mL) was added KHMDS (0.5 M in THF, 2.32 mL, 1.16 mmol) at −78° C. and stirred for 0.5 h. Then, 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide (415 mg, 1.16 mmol) was added and the reaction mixture was stirred at −78° C. for another 2 h. After warming to room temperature, the mixture was directly purified by flash column eluting with a gradient of EA (containing 10% MeOH) / PE (0% to 60%) to give the desired product compound 20f (230 mg). MS: calcd 391 (MH + ), measured value 391(MH + ).

[0205] Step 4: Preparation of tert-butyl 4-[5-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]-4-methyl-2-pyridyl]piperazine-1-carboxylate (compound 20g)

[0206] To a mixture of [1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]trifluoromethanesulfonate (compound 20f, 50 mg, 128 μmol) and tert-butyl 4-[4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]piperazine-1-carboxylate (compound 20b, 113 mg, 280 μmol) in 1,4-dioxane (5 mL) and water (1 mL) was added K2CO3 (53 mg, 384 μmol) and PdCl2(dppf)·CH2Cl2 (21 mg, 26 μmol). The mixture was charged with N2 and stirred at 100 °C for 2 h. After cooling to room temperature, the mixture was dried over Na2SO4, filtered off and the solid was washed twice with EA (10 mL). The combined organic layers were concentrated to give the crude product compound 20g (66 mg), which was used in the next step without further purification. MS: calculated 518 (MH + ), measured value 518(MH + ).

[0207] Step 5: Preparation of tert-butyl 4-[5-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-4-methyl-2-pyridyl]piperazine-1-carboxylate (compound 20h)

[0208] To a flask containing tert-butyl 4-[5-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]-4-methyl-2-pyridyl]piperazine-1-carboxylate (compound 20g, 66 mg, 128 μmol), Pd(OH)2 (10 wt% in carbon, 8.99 mg, 64 μmol) and MeOH (10 mL) were added. The suspension was purged with H2 three times and then stirred overnight at room temperature under a hydrogen balloon. The mixture was filtered and concentrated. The residue was purified by flash column eluting with a gradient of EA (containing 10% MeOH) / PE (0% to 80%) to give the desired product 20h (25 mg). MS: calculated 520 (MH + ), measured value 520 (MH+ ).

[0209] Step 6: Preparation of 1,6-dimethyl-4-[cis-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 20) and 1,6-dimethyl-4-[trans-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 21)

[0210] To a mixture of tert-butyl 4-[5-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-4-methyl-2-pyridyl]piperazine-1-carboxylate (compound 20h, 25 mg, 48 μmol) in DCM (10 mL) was added TFA (2 mL). After stirring at room temperature for 2 h, the mixture was concentrated and purified by reverse flash column eluting with a gradient of ACN / water (containing 0.5% TFA) (0% to 30%) to give the desired product. The structures of Example 20 and Example 21 were confirmed by 2D-NMR.

[0211] Example 20 (9 mg) was obtained as a pale yellow solid. MS: calculated 420 (MH + ), measured value 420 (MH + ). 1 H NMR(400 MHz, methanol-d4)δ=8.45(s,1H),7.81(s,1H),7.20(s,1H),6.76(s,1H),4.71(br d,J=13.2 Hz,1H),4.55(br d,J=13.7 Hz,1H),4.10(s,3H),3.97-3.87(m,4H),3.83(br d,J=11.9 Hz,1H),3.62(br t,J=12.4 Hz,1H),3.53(td,J=3.6,12.9 Hz,1H),3.46-3.39(m,4H),2.65(s,3H),2.56(s,3H),2.54-2.41(m,2H),1.93-1.84(m,1H),0.76(d,J=7.0 Hz,3H).

[0212] Example 21 (2 mg) was obtained as a pale yellow solid. MS: calculated 420 (MH + ), measured value 420 (MH + ). 1 H NMR(400 MHz, methanol-d4)δ=8.24(s,1H),7.84(s,1H),6.90(s,1H),6.65(s,1H),4.56-4.43(m,2H),3.98(s,3H) ),3.73-3.68(m,4H),3.54-3.43(m,1H),3.28-3.23(m,4H),3.20-3.13(m,1H),2.83(dt,J=3.9,11.5 Hz,1H),2.54(s,3H),2.36(s,3H),2.10-1.99(m,1H),1.99-1.92(m,1H),1.76-1.65(m,1H),0.81(d,J=6.6 Hz,3H).

[0213] Example 22 and Example 23 1,6-Dimethyl-4-[cis-3-methyl-4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 22) and 1,6-Dimethyl-4-[trans-3-methyl-4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 23) [ka]

[0214] The title compound was prepared by using tert-butyl 4-[5-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-3,6-dihydro-2H-pyridin-4-yl]-6-methyl-pyrazin-2-yl]piperazine-1-carboxylate (compound 22b) instead of compound 24c in the same manner as in the preparation of example 24 and example 25. The structures of example 22 and example 23 were confirmed by 2D-NMR.

[0215] Example 22 (1 mg) was obtained as a yellow solid. MS: calculated 421 (MH + ), measured value 421(MH+ ). 1 H NMR(400 MHz, methanol-d4)δ=8.30(s,1H),8.07(s,1H),6.71(s,1H),4.60(br d,J=13.8 Hz,1H),4.50(br d,J=12.3 Hz,1H),4.07(s,3H),3.87-3.80(m,5H),3.59(br s,1H),3.35-3.33(m,4H),3.07-3.00(m,1H),2.64-2.60(m,4H),2.52(s,4H),1.96(br s,1H),0.85(d,J=6.5 Hz,3H).

[0216] Example 23 (1 mg) was obtained as a yellow solid. MS: calculated 421 (MH + ), measured value 421(MH + ). 1 H NMR (400 MHz, methanol-d4) δ=8.37(s,1H),8.09(s,1H),6.70(s,1H),4.51(br d,J=12.8 Hz,1H),4.43(br d,J=11.4 Hz,1H),4.09-4.06(m,3H),3.90-3.80(m,5H),3.79-3.68(m,1H),3.60-3.53(m,1 H),3.36-3.35(m,1H),3.34-3.32(m,3H),2.62(s,3H),2.56-2.43(m,5H),1.93(br d,J=9.9 Hz,1H),0.72(d,J=7.0 Hz,3H).

[0217] Compound 22b was prepared according to the following scheme: [ka]

[0218] Step 1: Preparation of tert-butyl 4-(5-bromo-6-methyl-pyrazin-2-yl)piperazine-1-carboxylate (compound 22a)

[0219] To a solution of 2-bromo-5-chloro-3-methyl-pyrazine (200 mg, 0.96 mmol) in DMF (3 mL) was added tert-butyl piperazine-1-carboxylate (180 mg, 0.96 mmol) and cesium carbonate (314 mg, 0.96 mmol). The reaction mixture was stirred at 90° C. for 15 h. After cooling to room temperature, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give compound 22a (77 mg) as a yellow oil. MS: calculated 357 (MH + ), measured value 357(MH + ).

[0220] Step 2: Preparation of tert-butyl 4-[5-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]-6-methyl-pyrazin-2-yl]piperazine-1-carboxylate (compound 22b)

[0221] To a solution of 1,6-dimethyl-4-[5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridin-1-yl]pyrazolo[3,4-b]pyridine (compound 24a, 95 mg, 0.26 mmol) in 1,4-dioxane (5 mL) and water (1 mL) was added tert-butyl 4-(5-bromo-6-methyl-pyrazin-2-yl)piperazine-1-carboxylate (compound 22a, 77 mg, 0.22 mmol), Pd(dppf)Cl2.CH2Cl2 (18 mg, 0.22 mmol) and K2CO3 (45 mg, 0.32 mmol). The reaction mixture was stirred at 90 °C under N2 atmosphere for 2 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure, and the residue was purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give compound 22b (130 mg) as a brown oil. MS: calculated 519 (MH + ), measured value 519(MH + The structures of Examples 22 and 23 were confirmed by 2D-NMR.

[0222] Example 24 and Example 25 1,6-Dimethyl-4-[cis-3-methyl-4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 24) and 1,6-Dimethyl-4-[trans-3-methyl-4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 25) [ka]

[0223] The title compound was prepared by using tert-butyl 4-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]-5-methyl-3-pyridyl]piperazine-1-carboxylate (compound 24c) instead of compound 20g in the same manner as in the preparation of example 20 and example 21. The structures of example 24 and example 25 were confirmed by 2D-NMR.

[0224] Example 24 (16 mg) was obtained as a pale yellow solid. MS: calculated 420 (MH + ), measured value 420 (MH + ). 1 H NMR(500 MHz, methanol-d4)δ=8.42(s,1H),8.20-8.14(m,1H),7.99-7.91(m,1H),6.76(s,1H),4.70(br d,J=13.4 Hz,1H),4.53(br d,J=13.3 Hz,1H),4.09(s,3H),3.86(br d,J=13.6 Hz,2H),3.73-3.60(m,5H),3.49-3.33(m,4H),2.76-2.61(m,4H),2.60-2.52(m,4H),2.03-1.93(m,1H),0.76(d,J=7.0 Hz,3H).

[0225] Example 25 (8 mg) was obtained as a pale yellow solid. MS: calculated 420 (MH + ), measured value 420 (MH+ ). 1 H NMR(500 MHz, methanol-d4)δ=8.33(s,1H),8.17(d,J=2.4 Hz,1H),7.78(br s,1H),6.75(s,1H),4.67(br d,J=13.4 Hz,1H),4.57(br d,J=14.6 Hz,1H),4.08(s,3H),3.69-3.53(m,6H),3.44-3.36(m,4H),3.36-3.24(m,1H), 2.63(s,3H),2.53(s,3H),2.45-2.35(m,1H),2.12-2.00(m,2H),0.88(d,J=6.6 Hz,3H).

[0226] Compound 24c was prepared according to the following scheme: [ka]

[0227] Step 1: Preparation of 1,6-dimethyl-4-[5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridin-1-yl]pyrazolo[3,4-b]pyridine (compound 24a)

[0228] To a mixture of [1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]trifluoromethanesulfonate (compound 20f, 300 mg, 0.768 mmol) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2 dioxaborolane) (292.72 mg, 1.15 mmol) in 1,4-dioxane (10 mL) was added PdCl2(dppf)·CHCl2 (63 mg, 0.077 mmol) and potassium acetate (226 mg, 2.31 mmol). The mixture was charged with N2 and stirred at 90 °C for 2 h. After cooling to room temperature, the mixture was filtered and the solid was washed twice with EA (10 mL). The combined organic layers were concentrated to give the crude product Compound 24a (283 mg), which was used in the next step without further purification. MS: calculated 369 (MH+ ), measured value 369 (MH + ).

[0229] Step 2: Preparation of 4-[4-(5-chloro-3-methyl-2-pyridyl)-5-methyl-3,6-dihydro-2H-pyridin-1-yl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound 24b)

[0230] To a mixture of 1,6-dimethyl-4-[5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridin-1-yl]pyrazolo[3,4-b]pyridine (compound 24a, 283 mg, 0.768 mmol) and 2-bromo-5-chloro-3-methyl-pyridine (CAS: 65550-77-8, vendor: Accela, 175 mg, 0.845 mmol) in 1,4-dioxane (10 mL) and water (2 mL) was added PdCl2(dppf)·CHCl2 (31 mg, 0.038 mmol) and KCO3 (212 mg, 1.54 mmol). The mixture was charged with N2 and stirred at 90 °C overnight. After cooling to room temperature, the mixture was extracted with DCM (20 mL) three times, dried over Na2SO4, filtered, concentrated, and purified by flash column eluted with a gradient of EA / PE (0% to 30%) to give the desired product, Compound 24b (260 mg). MS: calculated 368 (MH + ), measured value 368(MH + ).

[0231] Step 3: Preparation of tert-butyl 4-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]-5-methyl-3-pyridyl]piperazine-1-carboxylate (compound 24c)

[0232] To a mixture of 4-[4-(5-chloro-3-methyl-2-pyridyl)-5-methyl-3,6-dihydro-2H-pyridin-1-yl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound 24b, 130 mg, 0.353 mmol) and 1-Boc-piperazine (99 mg, 0.530 mmol) in 1,4-dioxane (5 mL) was added cesium carbonate (230 mg, 0.707 mmol) and RuPhos Pd G2 (27 mg, 0.035 mmol). The mixture was charged with N2 and stirred at 110 °C overnight. After cooling to room temperature, the solid was filtered off and washed twice with EA (10 mL). The combined organic layers were concentrated and purified by flash column eluting with a gradient of EA (containing 10% MeOH) / PE (0% to 80%) to give the desired product 24c (136 mg). MS: calculated 518 (MH + ), measured value 518(MH + ).

[0233] Example 26 (3R,4R)-1-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-4-methoxy-pyrrolidin-3-amine [ka]

[0234] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-[(3R,4R)-4-methoxypyrrolidin-3-yl]carbamate (CAS: 1932066-52-8, vendor: PharmaBlock) and intermediate B instead of compound 15a and intermediate E. Example 26 (75 mg) was obtained as a white solid. MS: calculated 450 (MH + ), measured value 450 (MH + ). 1H NMR(400 MHz, methanol-d4)δ=8.34(s,1 H),7.85(d,J=2.50 Hz,1 H),7.58(d,J=2.50 Hz,1 H),6.77(s,1 H),4.69(br d,J=14.01 Hz,1 H),4.59(br d,J=13.01 Hz,1 H),4.19(dt,J=5.63,2.94 Hz,1 H),4.09(s,3 H),3.99(dt,J=6.25,3.38 Hz,1 H),3.87(dd,J=11.51,5.50 Hz,1 H),3.80(dd,J=11.26,6.25 Hz,1 H),3.68-3.58(m,1 H),3.55(dd,J=11.26,3.25 Hz,1 H),3.51-3.42(m,4 H),3.40-3.32(m,2 H),2.64(s,3 H),2.57(s,3 H),2.44-2.33(m,1 H),2.14-2.04(m,2 H)0.90(d,J=6.50 Hz,3 H).

[0235] Example 27 2-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-5-oxa-2,8-diazaspiro[3.5]nonane [ka]

[0236] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (CAS: 1251005-61-4, vendor: PharmaBlock) and intermediate B instead of compound 15a and intermediate E. Example 27 (65 mg) was obtained as a white solid. MS: calculated 462 (MH + ), measured value 462(MH + ). 1H NMR (400 MHz, methanol-d4)δ=8.06(s,1H),7.60(d,J=2.5 Hz,1H),6.78(d,J=2.4 Hz,1H),6.41(s,1H),4.38(br d,J=13.8 Hz,1H),4.29(br d,J=11.9 Hz,1H),4.00(s,3H),3.91(d,J=8.0 Hz,2H),3.74-3.63(m,4H),3.02(s,3H),2.99-2.96(m,1H),2.92-2.87(m,1H),2.84-2.80(m,2H),2.53(s,3H),2.37(s,3H),2.31(br dd,J=6.5,11.0 Hz,1H),1.98-1.88(m,1H),1.84-1.75(m,1H),0.77(d,J=6.5 Hz,3H).

[0237] Example 28 (6S)-4-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-1,4-oxazepan-6-amine [ka]

[0238] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-[(6S)-1,4-oxazepan-6-yl]carbamate (CAS: 2306247-11-8, vendor: PharmaBlock) and intermediate B instead of compound 15a and intermediate E. Example 28 (75 mg) was obtained as a pale yellow solid. MS: calculated 450 (MH + ), measured value 450 (MH + ). 1H NMR(500 MHz, methanol-d4)δ=8.39(s,1H),8.11(d,J=3.1 Hz,1H),8.02(d,J=2.6 Hz,1H),6.83(s,1H),4.73(br d,J=10.4 Hz,1H),4.63(br d,J=12.4 Hz,1H),4.20-4.09(m,5H),4.05-3.98(m,1H),3.95-3.80(m,5H),3.75-3.62(m,2H),3.47- 3.34(m,2H),2.68(s,3H),2.63(s,3H),2.54-2.45(m,1H),2.23-2.12(m,2H),0.95(d,J=6.6 Hz,3H).

[0239] Example 29 4-[(3S,4R)-4-[5-[(3R)-3-(methoxymethyl)piperazin-1-yl]-3-methyl-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0240] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl (2R)-2-(methoxymethyl)piperazine-1-carboxylate (CAS: 1023301-73-6, vendor: PharmaBlock) and intermediate B instead of compound 15a and intermediate E. Example 29 (147 mg) was obtained as a yellow solid. MS: calculated 464 (MH + ), measured value 464(MH + ). 1H NMR (500 MHz, methanol-d4) δ=8.37(s,1H),8.21(d,J=2.9 Hz,1H),8.11(d,J=2.6 Hz,1H),6.81(s,1H),4.71(br d,J=12.2 Hz,1H),4.65-4.58(m,1H),4.15-4.04(m,5H),3.77-3.70(m,1H),3.70-3.6 2(m,3H),3.54-3.49(m,1H),3.48-3.41(m,4H),3.38-3.31(m,3H),3.25(br dd,J=10.3,13.7 Hz,1H),2.66(s,3H),2.64-2.59(m,3H),2.53-2.44(m,1H),2.20-2.11(m,2H),0.92(d,J=6.6 Hz,3H).

[0241] Example 30 4-[(3S,4R)-4-[5-(4,7-diazaspiro[2.5]octan-7-yl)-3-methyl-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine, [ka]

[0242] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate (CAS: 674792-08-6, vendor: Accela) and intermediate B instead of compound 15a and intermediate E. Example 30 (42 mg) was obtained as a white solid. MS: calculated 446 (MH + ), measured value 446(MH + ). 1H NMR (400 MHz, methanol-d4) δ = 8.34 (s, 1H), 8.20 (d, J = 2.3 Hz, 1H), 7.97 (br s, 1H), 6.76 (s, 1H), 4.73-4.65 (m, 1H), 4.59 (br d, J = 13.3 Hz,1H),4.09(s,3H),3.74-3.69(m,2H),3.68-3.59(m,1H),3.58-3.49(m,4H),3.45-3.33(m,2H), 2.64(s,3H),2.58(s,3H),2.48-2.35(m,1H),2.20-2.03(m,2H),1.22-1.03(m,4H),0.90(d,J=6.6 Hz,3H).

[0243] Example 31 (3R,4R)-1-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-3-fluoro-piperidin-4-amine [ka]

[0244] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-[(3R,4R)-3-fluoropiperidin-4-yl]carbamate (CAS: 1523530-29-1, vendor: PharmaBlock) and intermediate B instead of compound 15a and intermediate E. Example 31 (44 mg) was obtained as a pale yellow solid. MS: calculated 452 (MH + ), measured value 452 (MH + ). 1H NMR (500 MHz, methanol-d4) δ = 8.33 (s, 1H), 8.19 (d, J = 2.7 Hz, 1H), 7.89 (d, J = 2.6 Hz, 1H), 6.75 (s, 1H), 4.78-4.63 (m, 2H), 4.57 (br d, J = 13.0 Hz,1H),4.35-4.27(m,1H),4.08(s,3H),3.98-3.88(m,1H),3.66-3.57(m,1H),3.57-3.46(m,1H),3.37-3.31(m,2H),3.0 8-2.97(m,2H),2.63(s,3H),2.55(s,3H),2.44-2.34(m,1H),2.28-2.19(m,1H),2.13-2.02(m,2H),1.82(dq,J=4.3,12.6 Hz,1H),0.88(d,J=6.6 Hz,3H).

[0245] Example 32 1-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-3-methyl-azetidin-3-amine [ka]

[0246] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-(3-methylazetidin-3-yl)carbamate hydrochloridecarbamate (CAS: 1408076-37-8, vendor: PharmaBlock) and intermediate B instead of compound 15a and intermediate E. Example 32 (66 mg) was obtained as a pale yellow solid. MS: calculated 420 (MH + ), measured value 420 (MH + ). 1H NMR(500 MHz, methanol-d4)δ=8.33(s,1H),7.77(d,J=2.7 Hz,1H),7.42(d,J=2.6 Hz,1H),6.75(s,1H),4.67(br d,J=13.6 Hz,1H),4.58(br d,J=13.1 Hz,1H),4.17(d,J=8.9 Hz,2H),4.12-4.06(m,5H),3.67-3.58(m,1H),3.37-3.31(m,2H),2.63(s,3H), 2.54(s,3H),2.43-2.34(m,1H),2.12-2.05(m,2H),1.69(s,3H),0.88(d,J=6.6 Hz,3H).

[0247] Example 33 (4aR,7aR)-6-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazine [ka]

[0248] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl (4aR,7aR)-octahydropyrrolo[3,4-b]morpholine-4-carboxylate (CAS: 1932337-68-2, vendor: PharmaBlock) and intermediate B instead of compound 15a and intermediate E. Example 33 (66 mg) was obtained as a pale yellow solid. MS: calculated 462 (MH + ), measured value 462(MH + ). 1H NMR (500 MHz, methanol-d4) δ=8.33(s,1H),7.83(d,J=2.9 Hz,1H),7.56(d,J=2.6 Hz,1H),6.75(s,1H),4.68(br d,J=13.3 Hz,1H),4.64-4.52(m,1H),4.25(dd,J=3.7,13.1 Hz,1H),4.21-4.12(m,1H),4.08(s,3H),4.04-3.97(m,1H),3.89-3.80(m,2H),3.63(dt,J=7.6,10.1 Hz,2H),3.52(dd,J=2.1,13.3 Hz,1H),3.49-3.44(m,1H),3.40(dd,J=4.2,12.9 Hz,1H),3.37-3.32(m,3H),2.63(s,3H),2.56(s,3H),2.43-2.34(m,1H),2.14-2.07(m,2H),0.89(d,J=6.6 Hz,3H).

[0249] Example 35 1,6-Dimethyl-4-[(3R,4S)-3-methyl-4-(5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0250] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl piperazine-1-carboxylate and intermediate D instead of compound 15a and intermediate E. Example 35 (5 mg) was obtained as a white solid. MS: calculated 406 (MH + ), measured value 406(MH + ). 1H NMR (400 MHz, methanol-d4) δ=8.35(s,2H),8.05-7.85(m,1H),7.77-7.62(m,1H),6.77(s,1H),4.67(br d,J=13.4 Hz,1H),4.60-4.51(m,1H),4.09(s,3H),3.66-3.56(m,5H),3.44-3.36(m,4H),3.30-3.19( m,1H),3.09-2.95(m,1H),2.64(s,3H),2.33-2.19(m,1H),2.17-1.99(m,2H),0.90(d,J=6.6 Hz,3H).

[0251] Example 34 1,6-Dimethyl-4-[(3S,4R)-3-methyl-4-(5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine [ka]

[0252] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl piperazine-1-carboxylate and intermediate A instead of compound 15a and intermediate E. Example 34 (20 mg) was obtained as a white solid. MS: calculated 406 (MH + ), measured value 406(MH + ). 1 H NMR (400 MHz, methanol-d4) δ=8.35(s,1H),8.32(d,J=2.9 Hz,1H),7.82(dd,J=2.9,9.0 Hz,1H),7.58(d,J=8.8 Hz,1H),6.76(s,1H),4.69-4.62(m,1H),4.55(br dd,J=2.2,13.9 Hz,1H),4.09(s,3H),3.63-3.53(m,5H),3.47-3.36(m,4H),3.29-3.22(m,1H),2.95(dt,J=4.4,11.2 Hz,1H),2.64(s,3H),2.30-2.17(m,1H),2.15-1.98(m,2H),0.89(d,J=6.6 Hz,3H).

[0253] Example 36 (3R,4R)-1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-4-methoxy-pyrrolidin-3-amine [ka]

[0254] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-[(3R,4R)-4-methoxypyrrolidin-3-yl]carbamate (CAS: 1932066-52-8, vendor: PharmaBlock) and intermediate D instead of compound 15a and intermediate E. Example 36 (28 mg) was obtained as a white solid. MS: calculated 436 (MH + ), measured value 436(MH + ). 1 H NMR (400 MHz, methanol-d4)δ=8.35(s,1H),7.99(d,J=2.75 Hz,1 H),7.73-7.67(m,1H),7.67-7.60(m,1H),6.77(s,1H),4.71-4.63(m,1H) ,4.61-4.53(m,1H),4.24-4.15(m,1H),4.09(s,3H),4.00(td,J=3.2,6.3 Hz,1H),3.89(dd,J=5.6,11.1 Hz,1H),3.81(dd,J=6.3,11.3 Hz,1H),3.62-3.54(m,2H),3.50-3.43(m,4H),3.29-3.22(m,1H),3.01(dt,J=4.3,11.4 Hz,1H),2.64(s,3H),2.28-2.02(m,3H),0.90(d,J=6.5 Hz,3H).

[0255] Example 37 4-[(3R,4S)-4-[5-[(3R)-3-(methoxymethyl)piperazin-1-yl]-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0256] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl (2R)-2-(methoxymethyl)piperazine-1-carboxylate (CAS: 1023301-73-6, vendor: PharmaBlock) and intermediate D instead of compound 15a and intermediate E. Example 37 (5 mg) was obtained as a white solid. MS: calculated 450 (MH + ), measured value 450 (MH + ). 1 H NMR (400 MHz, methanol-d4) δ=8.05(d,J=2.8 Hz,1H),7.96(s,1H),7.33-7.27(m,1H),7.11(d,J=8.7 Hz,1H),6.31(s,1H),4.27(br d,J=13.0 Hz,1H),4.18(br d,J=13.6 Hz,1H),3.89(s,3H),3.55(br t,J=12.7 Hz,2H),3.46-3.34(m,2H),3.31(s,3H),3.17-3.09(m,3H),3.01-2.92(m,1H),2.88-2.72( m,2H),2.62-2.47(m,2H),2.43(s,3H),2.09-1.98(m,1H),1.86-1.75(m,2H),0.69(d,J=6.5 Hz,3H).

[0257] Example 38 2-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-5-oxa-2,8-diazaspiro[3.5]nonane [ka]

[0258] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (CAS: 1251005-61-4, vendor: PharmaBlock) and intermediate D instead of compound 15a and intermediate E. Example 38 (11 mg) was obtained as a white solid. MS: calculated 448 (MH + ), measured value 448(MH + ). 1 H NMR (400 MHz, methanol-d4)δ=8.07(s,1H),7.74(d,J=2.8 Hz,1H),7.16(d,J=8.4 Hz,1H),6.95(dd,J=2.8,8.4 Hz,1H),6.42(s,1H),4.38(br d,J=12.8 Hz,1H),4.29(br d,J=11.0 Hz,1H),4.01(s,3H),3.94(d,J=8.0 Hz,2H),3.70(br d,J=7.8 Hz,4H),3.31-3.22(m,1H),3.02(s,2H),2.99-2.89(m,1H),2.85-2.77(m,2H),2. 63-2.56(m,1H),2.54(s,3H),2.20-2.04(m,1H),1.98-1.87(m,2H),0.81(d,J=6.5 Hz,3H).

[0259] Example 39 (3S,4S)-1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-3-methoxy-piperidin-4-amine [ka]

[0260] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-[(3S,4S)-3-methoxy-4-piperidyl]carbamate (CAS: 907544-19-8, vendor: PharmaBlock) and intermediate D instead of compound 15a and intermediate E. Example 39 (19 mg) was obtained as a white solid. MS: calculated 450 (MH+ ), measured value 450 (MH + ). 1 H NMR (400 MHz, methanol-d4) δ=8.37-8.36(m,1H),8.35(s,1H),8.07(dd,J=8.8,2.9 Hz,1H),7.74(d,J=9.3 Hz,1H),6.76(s,1H),4.67(br d,J=14.3 Hz,1H),4.61-4.52(m,1H),4.35(br dd,J=12.2,3.4 Hz,1H),4.09(s,3H),3.98(br d,J=13.2 Hz,1H),3.64-3.57(m,1H),3.55(s,3H),3.40(td,J=10.0,4.4 Hz,1H),3.29-3.14(m,2H),2.95-3.10(m,2H),2.74(dd,J=12.2,10.3 Hz,1H),2.64(s,3H),2.29-2.04(m,4H),1.81(qd,J=12.6,4.7 Hz,1H),0.91(d,J=6.4 Hz,3H).

[0261] Example 40 (6S)-4-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-1,4-oxazepan-6-amine [ka]

[0262] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-[(6S)-1,4-oxazepan-6-yl]carbamate (CAS: 2306247-11-8, vendor: PharmaBlock) and intermediate D instead of compound 15a and intermediate E. Example 40 (70 mg) was obtained as a white solid. MS: calculated 436 (MH + ), measured value 436(MH + ). 1H NMR (500 MHz, methanol-d4) δ=8.28(s,1H),8.18(d,J=2.7 Hz,1H),8.00(dd,J=2.8,9.2 Hz,1H),7.75(d,J=9.3 Hz,1H),6.71(s,1H),4.60(br d,J=11.7 Hz,1H),4.49(br d,J=12.8 Hz,1H),4.10-3.97(m,5H),3.95-3.88(m,1H),3.84-3.78(m,2H),3.77- 3.68(m,3H),3.64-3.47(m,2H),3.27-3.21(m,1H),3.01(dt,J=3.8,11.5 Hz,1H),2.56(s,3H),2.25-2.14(m,1H),2.14-2.07(m,1H),2.06-1.94(m,1H),0.85(d,J=6.6 Hz,3H).

[0263] Example 41 (3R,4R)-1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-3-fluoro-piperidin-4-amine [ka]

[0264] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-[(3R,4R)-3-fluoropiperidin-4-yl]carbamate (CAS: 1523530-29-1, vendor: PharmaBlock) and intermediate D instead of compound 15a and intermediate E. Example 41 (10 mg) was obtained as a white solid. MS: calculated 438 (MH + ), measured value 438(MH + ). 1H NMR (500 MHz, methanol-d4) δ=8.09-8.03(m,1H),7.95(s,1H),7.30(dd,J=3.0,8.6 Hz,1H),7.08(d,J=8.5 Hz,1H),6.29(s,1H),4.38-4.21(m,2H),4.18-4.13(m,1H),3.94-3.82(m,4H),3.55(br dd,J=1.3,12.7 Hz,1H),3.24-3.19(m,2H),3.18-3.09(m,1H),2.92-2.78(m,2H),2.77-2.66(m,2H),2.49(dt,J=4.7,11.1 Hz,1H),2.09-1.89(m,2H),1.86-1.76(m,2H),1.52(br dd,J=4.1,12.4 Hz,1H),1.24-1.14(m,1H),0.68(d,J=6.6 Hz,3H).

[0265] Example 42 4-[(3R,4S)-4-[5-(4,7-diazaspiro[2.5]octan-7-yl)-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine [ka]

[0266] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate (CAS: 674792-08-6, vendor: PharmaBlock) and intermediate D instead of compound 15a and intermediate E. Example 42 (19 mg) was obtained as a white solid. MS: calculated 432 (MH + ), measured value 432(MH + ). 1H NMR (500 MHz, methanol-d4) δ=7.99(d,J=2.7 Hz,1H),7.95(s,1H),7.24(dd,J=2.9,8.7 Hz,1H),7.07(d,J=8.7 Hz,1H),6.30(s,1H),4.26(br d,J=13.1 Hz,1H),4.20-4.11(m,1H),3.89(s,3H),3.19-3.08(m,3H),3.01-2.96(m,2H),2.95(s,2H),2.86- 2.78(m,1H),2.53-2.45(m,1H),2.42(s,3H),2.07-1.97(m,1H),1.89-1.75(m,2H),0.68(d,J=6.6 Hz,3H),0.59(br d,J=8.4 Hz,4H).

[0267] Example 43 1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-3-methyl-azetidin-3-amine [ka]

[0268] The title compound was prepared in a similar manner to the preparation of Example 15, by using tert-butyl N-(3-methylazetidin-3-yl)carbamate hydrochloridecarbamate (CAS: 1408076-37-8, vendor: PharmaBlock) and intermediate D instead of compound 15a and intermediate E. Example 43 (33 mg) was obtained as a white solid. MS: calculated 406 (MH + ), measured value 406(MH + ). 1H NMR (500 MHz, methanol-d4) δ=8.24(s,1H),7.83(d,J=2.7 Hz,1H),7.58(d,J=8.9 Hz,1H),7.41(dd,J=2.9,8.9 Hz,1H),6.66(s,1H),4.56(br d,J=13.6 Hz,1H),4.46(br d,J=12.7 Hz,1H),4.09(d,J=9.0 Hz,2H),4.03-3.95(m,5H),3.48(br t,J=12.0 Hz,1H),3.19-3.11(m,1H),2.91(dt,J=4.2,11.5 Hz,1H),2.54(s,3H),2.19-1.90(m,3H),1.60(s,3H),0.79(d,J=6.6 Hz,3H).

[0269] Example 44

[0270] The following studies were carried out to determine the activity of compounds of formula (I) and formula (Ia) in the HEK293-Blue-hTLR-7 / 8 / 9 cell assay.

[0271] HEK293-Blue-hTLR-7 Cell Assay: A stable HEK293-Blue-hTLR-7 cell line was purchased from InvivoGen (catalog number: hkb-htlr7, San Diego, CA, USA). These cells were originally designed to study the stimulation of human TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of an IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites. SEAP was induced by activating NF-κB and AP-1 by stimulating HEK-Blue hTLR7 cells with TLR7 ligands. Thus, reporter expression was reduced by TLR7 antagonists under stimulation of ligands such as R848 (Resiquimod) for 20 hours of incubation. SEAP reporter activity in cell culture supernatants was measured at a wavelength of 640 nm using the QUANTI-blue™ kit (catalog number: rep-qb1, Invivogen, San Diego, CA, USA), a detection medium that turns purple or blue in the presence of alkaline phosphatase.

[0272] HEK293-Blue-hTLR7 cells were incubated at a density of 250,000 to 450,000 cells / mL in a volume of 170 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g / L glucose, 50 U / mL penicillin, 50 mg / mL streptomycin, 100 mg / mL normocin, 2 mM L-glutamine, 10% (v / v) heat-inactivated fetal bovine serum, and 20 μL of test compound serially diluted in the presence of 1% final DMSO and 20 uM R848 in 10 μL of the above DMEM, and incubated at 37° C. in a CO2 incubator for 20 hours. Then, 20 μL of the supernatant from each well was incubated with 180 μL of Quanti-blue substrate solution for 2 h at 37 °C, and the absorbance was read at 620–655 nm using a spectrophotometer. Because the signaling pathway in which TLR7 activation leads to downstream NF-κB activation is widely accepted, we modified a similar reporter assay to evaluate TLR7 antagonists.

[0273] HEK293-Blue-hTLR-8 Cell Assay: A stable HEK293-Blue-hTLR-8 cell line was purchased from InvivoGen (catalog number: hkb-htlr8, San Diego, CA, USA). These cells were originally designed to study the stimulation of human TLR8 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of an IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites. SEAP was induced by activating NF-κB and AP-1 by stimulating HEK-Blue hTLR8 cells with TLR8 ligands. Thus, reporter expression was reduced by TLR8 antagonists under stimulation of ligands such as R848 during 20 hours of incubation. SEAP reporter activity in cell culture supernatants was measured at a wavelength of 640 nm using the QUANTI-blue™ kit (catalog number: rep-qb1, Invivogen, San Diego, CA, USA), a detection medium that turns purple or blue in the presence of alkaline phosphatase.

[0274] HEK293-Blue-hTLR8 cells were incubated in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g / L glucose, 50 U / mL penicillin, 50 mg / mL streptomycin, 100 mg / mL normocin, 2 mM L-glutamine, 10% (v / v) heat-inactivated fetal bovine serum, 1% final DMSO, and 20 μL of test compound serially diluted in the presence of 60 uM R848 in 10 μL of the above DMEM at a density of 250,000 to 450,000 cells / mL in a volume of 170 μL, and incubated at 37°C in a CO2 incubator for 20 hours. Then, 20 μL of the supernatant from each well was incubated with 180 μL of Quanti-blue substrate solution for 2 h at 37 °C, and the absorbance was read at 620–655 nm using a spectrophotometer. Because the signaling pathway in which TLR8 activation leads to downstream NF-κB activation is widely accepted, we improved a similar reporter assay to evaluate TLR8 antagonists.

[0275] HEK293-Blue-hTLR-9 Cell Assay: A stable HEK293-Blue-hTLR-9 cell line was purchased from InvivoGen (catalog number: hkb-htlr9, San Diego, CA, USA). These cells were originally designed to study the stimulation of human TLR9 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of an IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites. SEAP was induced by activating NF-κB and AP-1 by stimulating HEK-Blue hTLR9 cells with TLR9 ligands. Thus, reporter expression was reduced by TLR9 antagonists during 20 h of incubation under stimulation of ligands such as ODN2006 (catalog number: tlrl-2006-1, Invivogen, San Diego, CA, USA). SEAP reporter activity in cell culture supernatants was measured at a wavelength of 640 nm using the QUANTI-blue™ kit (catalog number: rep-qb1, Invivogen, San Diego, CA, USA), a detection medium that turns purple or blue in the presence of alkaline phosphatase.

[0276] HEK293-Blue-hTLR9 cells were incubated at a density of 250,000 to 450,000 cells / mL in a volume of 170 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g / L glucose, 50 U / mL penicillin, 50 mg / mL streptomycin, 100 mg / mL normocin, 2 mM L-glutamine, 10% (v / v) heat-inactivated fetal bovine serum, 20 μL of test compound serially diluted in the presence of 1% final DMSO and 20 uM ODN2006 in 10 μL of the above DMEM, and incubated at 37° C. in a CO2 incubator for 20 hours. Then, 20 μL of the supernatant from each well was incubated with 180 μL of Quanti-blue substrate solution for 2 h at 37°C, and the absorbance was read at 620–655 nm using a spectrophotometer. Because the signaling pathway in which TLR9 activation leads to downstream NF-κB activation is widely accepted, a similar reporter assay was modified for the evaluation of TLR9 antagonists.

[0277] The compounds of formula (I) or (Ia) have human TLR7 and TLR8 inhibitory activity (IC 50 In addition, some compounds also have human TLR9 inhibitory activity of less than 0.5 μM. Activity data of the compounds of the present invention are shown in Table 2. [Table 1]

[0278] Example 45 hERG Channel Inhibition Assay: The hERG channel inhibition assay is a highly sensitive measure to identify compounds that exhibit hERG inhibition associated with cardiotoxicity in vivo. + The channel has been cloned in human and stably expressed in a CHO (Chinese Hamster Ovary) cell line. hERGThe cells were used for patch clamp (voltage clamp, whole cell) experiments. The cells were stimulated with a voltage pattern to activate the hERG channel and induce I KhERG The cells were allowed to stabilize for a few minutes, after which the I KhERG The amplitude and kinetics of I was recorded at a stimulation frequency of 0.1 Hz (6 bpm). Test compounds were then added to the preparations in increasing concentrations. For each concentration, an attempt was made to reach a steady-state effect, which was usually achieved within 3-10 min, at which point the next higher concentration was applied. KhERGThe amplitude and kinetics of the responses were recorded at each drug concentration and compared to control values ​​(set at 100%). (Reference: Redfern WS, Carlsson L, Davis AS, Lynch WG, MacKenzie I, Palethorpe S, Siegl PK, Strang I, Sullivan AT, Wallis R, Camm AJ, Hammond TG.2003;Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development.Cardiovasc.Res.58:32-45,Sanguinetti MC,Tristani-Firouzi M.2006;hERG potassium channels and cardiac arrhythmia.Nature 440:463-469,Webster R,Leishman D,Walker D.2002;Towards a drug concentration effect relationship for QT prolongation and torsades de pointes.Curr.Opin.Drug Discov. Dev. 5:116-26).

[0279] The results for hERG are shown in Table 3. The safety ratio (hERG IC 20 / EC 50 ) suggest a sufficient window to discern pharmacology by inhibiting the TLR7 / 8 / 9 pathway from potential hERG-related cardiac toxicity. The following hERG IC, which serves as an early selectivity index to assess hERG liability, 20 / TLR7 / 8 / 9 IC 50 According to the calculations of 、Specifically, the reference compounds ER-887258, ER-888285, ER-888286, R1, and R2 have a very narrow safety window compared to the compounds of the present invention. [Table 2]

[0280] Example 46 Human PBMC cell-based assay Unlike HEK reporter cell lines, human peripheral blood mononuclear cells (PBMCs) represent the primary human immune cells in the blood, consisting mainly of lymphocytes, monocytes, and dendritic cells. These cells express TLR7, TLR8, or TLR9 and are therefore natural responders to the respective ligand stimulation. Upon activation of these TLRs, PBMCs secrete similar cytokines and chemokines in vitro and in vivo, so the in vitro potency of TLR7 / 8 / 9 antagonists in human PBMCs can be easily translated to pharmacodynamic responses in vivo.

[0281] Human peripheral blood mononuclear cells (PBMCs) were isolated from freshly collected lithium heparinized (lithium heparin plus blood collection tubes, BD Vacutainer®) healthy donor whole blood by density gradient (Ficoll-Paque™ PLUS, GE Healthcare life Sciences). Briefly, 50 mL of blood was diluted in 25 mL PBS (CaCl2+, 100 mL PBS ... 2+ , Mg 2+The tubes were centrifuged at 800 × g (1946 rpm) for 20 min with the brake in the off position, and PBMCs were collected from the buffy coat. The cells were then washed twice with PBS, and red blood cells were lysed by 2 mL of suspension (Red Blood Cell Lysis Buffer, Alfa Aesar) for 5–10 min at room temperature. After a final wash with PBS, the cells were diluted to a final concentration of 2 × 10 in RPMI-1640 medium containing GlutaMAXTM (Gibco) supplemented with 10% fetal bovine serum (Sigma). 6 PBMCs were resuspended at 3 × 10 cells / mL and plated at 150 μL / well (3 × 10 cells / mL) in tissue culture-treated round-bottom 96-well plates (Corning Incorporated). 5 The cells were plated at 100 x 100 cells / well.

[0282] Antagonist compounds (compounds of the invention) solubilized and serially diluted in 100% DMSO were added to cells in duplicate to obtain a final concentration of 1% DMSO (v / v). PBMCs were incubated with antagonist compounds for 30 minutes at 37°C, 5% CO2, and then various TLR agonist reagents were added in 48 μL of complete medium per well as follows (final concentrations indicated): 1 μM CpG ODN 2216 (InvivoGen) for TLR9, 1 μg / mL ORN 06 / LyoVec (InvivoGen) for TLR8, and 1 μg / mL R848 (InvivoGen) for TLR7 and TLR8. PBMCs were incubated overnight at 37°C with 5% CO2. Cell culture supernatants were collected and the levels of various human cytokines were assessed by Luminex assay (ProcartaPlex™ Multiplex Immunoassay, Invitrogen) or ELISA procedures according to the manufacturer's recommended protocols (eBioscience, ThermoFisher Scientific). Cell viability was also confirmed by Cell Viability Assay (CellTiter Glo® Luminescent Cell Viability Assay, Promega). [Table 3]

[0283] Example 47 Human microsome stability assay The human microsomal stability assay is used to provide an early assessment of the metabolic stability of test compounds in human liver microsomes.

[0284] Human liver microsomes (Cat. No.: 452117, Corning, USA; Cat. No.: H2610, Xenotech, USA) were preincubated with test compounds in 100 mM potassium phosphate buffer, pH 7.4, for 10 min at 37°C. The reaction was initiated by adding NADPH regenerating system. The final incubation mixture contained 1 μM test compound, 0.5 mg / mL liver microsomal protein, 1 mM MgCl2, 1 mM NADP, 1 unit / mL isocitrate dehydrogenase, and 6 mM isocitrate in 100 mM potassium phosphate buffer, pH 7.4. After 0, 3, 6, 9, 15, and 30 min incubation times at 37°C, the reaction was terminated by adding 300 μL of cold acetonitrile (containing internal standard) to 100 μL of the incubation mixture. After precipitation and centrifugation, the amount of compound remaining in the samples was measured by LC-MS / MS. Controls without NADPH regenerating system were also prepared and analyzed at 0 and 30 minutes. Compounds of the present invention showed good human liver microsomal stability as measured in the above assay. The results are shown in Table 5 below. [Table 4]

[0285] Example 48 3T3 in vitro phototoxicity assay Phototoxicity is defined as a toxic response induced after initial exposure of the skin to a particular chemical followed by subsequent exposure to light, or similarly induced by systemic administration of the chemical followed by dermal irradiation. The assay used in this study is designed to detect the phototoxic potential of chemicals using a simple in vitro cytotoxicity assay with Balb / c 3T3 mouse fibroblast cells. The principle of this test is to compare the cytotoxicity of tested chemicals with and without exposure to a non-toxic dose of UVA light. Cytotoxicity is expressed as a dose-dependent decrease in the proliferation rate of cells, as determined by the uptake of the vital dye neutral red 1 day after treatment.

[0286] 1. Method Preparation of test item stock solutions and dosages Just before starting the exposure of the cells, a small amount of material was weighed and freshly formulated in DMSO. This stock solution or an appropriate dilution in DMSO was added to the cell suspension to obtain the required final concentration. All solutions were typically prepared in Eppendorf caps and discarded after use.

[0287] reference material Chlorpromazine (HCL) (Sigma, batch / lot number: 120M 1328V), test concentration: 300 μg / mL, solvent: PBS / 3% DMSO

[0288] Measurement of UV absorption spectrum Absorption spectra by themselves or with UV-A or UV-B pre-irradiation were recorded between 240 nm and 400 nm using a Lambda-2 spectrophotometer (Perkin Elmer). [Table 5]

[0289] Determination of phototoxicity In this study, the Neutral Red Uptake (NRU) assay of Borenfreund and Puerner (Borenfreund, E, Puerner JA. Toxicity determined in vitro by morphological alterations and Neutral Red absorption. Toxicology Lett. 1985;24:119-124.), modified according to INVITTOX protocol No 78 (ERGATT / FRAME data bank of in vitro techniques in toxicology. INVITTOX PROTOCOL No 78. 3T3 NRU Phototoxicity Assay. March 1994), was adapted to investigate the possible phototoxic potential of test items. This assay is based on the active uptake of neutral red dye into lysosomes of cultured mouse fibroblasts. Since lysosomal membranes are known to be the site of action of many phototoxic compounds, this assay can provide a measure of the potential for phototoxic damage.

[0290] 1. Preparation of Cell Culture Mouse fibroblast clone A31 (ATCC no. CCL163-passage no. 108) was cultured in 175 cm cultures containing sDMEM (Dulbecco's minimum essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units / ml penicillin and 100 μg / ml streptavidin). 2 The cells were cultured in 100 μl tissue culture grade flasks at 37 °C in a humidified atmosphere of 6% CO. Before the cells approached confluence, they were removed from the flasks by trypsinization. Before use in the assay, the cells were diluted to 1 × 10 in sDMEM in a volume of 100 μl. 4 The cells were transferred to 96-well microtiter plates at a concentration of 100 cells / well and allowed to attach for 24 hours.

[0291] Exposure to test items For incubation with mouse fibroblasts, test items were diluted in PBS / 3% DMSO (see Results for detailed concentrations).

[0292] Culture medium (Dulbecco's Modified Eagle Medium (DMEM), GlutaMAX (Gibco Ref 21885-025), 10% fetal bovine serum (FBS) (Gibco Ref 10270-106), 100 IU / ml penicillin and 100 μg / ml streptomycin (Gibco Ref 15140-122)) was removed from the wells and mouse fibroblasts were washed with PBS. Then, 100 μL of PBS / 3% DMSO containing the test items was added and target cells were incubated for 1 h at 37°C with 6% CO2.

[0293] UV exposure For each test item, a microtiter plate was prepared according to Table 6. The "UVA plate" was approximately 5 J / cm 2 The plates were exposed to 1000 dl of UVA light, and the "dark plates" were kept in the dark and served as cytotoxicity controls. Plates containing chlorpromazine hydrochloride served as positive controls. UV flux was measured with a UV meter (Dr. Groebel RM21).

[0294] After UV irradiation, the test items were removed from the wells (one washing step with PBS) and replaced with sDMEM. The target cells were then incubated overnight at 37° C. in 6% CO2. [Table 6]

[0295] A 96-well microtiter plate was prepared as follows.

[0296] Each plate contained wells containing cells that were not incubated with neutral red solution (0% standard - S1) or stained with neutral red (100% standard - S2) and solvent but no test items for calculation of standard cell viability curves. Wells labeled U01–U08 contained different test item concentrations.

[0297] Neutral Red Uptake Ready-to-use Neutral Red (NR) staining solution was freshly prepared as follows: • The 0.4% stock solution was protected from light and filtered to remove NR crystals before use. • A 1:40 dilution of the stock solution was then prepared in sDMEM and added to the cells.

[0298] After incubation, assay wells were filled with 100 μL of sDMEM containing neutral red. Target cells were incubated with NR for 3 hours at 37° C. in 6% CO2.

[0299] Measurement of neutral red uptake Uncontaminated neutral red was removed from the target cells by washing the wells with at least 100 μL of PBS. Then, 150 μL of neutral red desorption solution (1% glacial acetic acid, 50% ethanol in aqua bidest) was added to quantitatively extract the contaminated dye. After shaking the plate vigorously for at least 10 min on a microtiter plate shaker until the neutral red was extracted from the cells to form a homogenous solution, the absorbance of the resulting colored solution was measured at 540 nm using a SPECTRAmax PLUS microtiter plate reader (Molecular Devices).

[0300] Calculation of cell viability Cell viability was calculated using the SOFTmax Pro software package (Molecular Devices). First, a two-point standard curve (0% and 100% viability) was calculated using the linear curve fit option of the program based on the following formula: Y = A + (B × X) (A = y-intercept of the line; B = slope of the line; 0% cell viability = cells with solvent but no test item and neutral red. 100% cell viability = cells containing solvent and neutral red but no test item)

[0301] This allowed the calculation of cell viability following incubation with increasing concentrations of the test chemicals. Chlorpromazine (HCl) served as the positive control for the experiment.

[0302] I C 50 Calculating values All calculations were performed using the SOFTmax Pro analysis software package. (Molecular Devices - see http: / / www.mbl.edu / jbpc / files / 2014 / 05 / SoftMax_Pro_User_Guide.pdf for more information).

[0303] Calculation of the discrimination factor for phototoxicity IC determined with and without UV exposure to assess phototoxicity potential 50 The values ​​were compared. Coefficient = IC 50 (-UV) / IC 50 (+UV)

[0304] A cut-off factor of >5 was applied to distinguish between phototoxic and non-phototoxic test chemicals. (Liebsch M, Spielmann H, Balls M, Brand M, Doering B, Dupuis J, Holzhueter HG, Klecak G, L. Eplattenier H, Lovell W, Maurer T, Moldenhauer F, Moore L, Pape W, Pfannenbecker U, Potthast JM, De Silva O, Steiling W, Willshaw A. First results of the EC / COLIPA Validation Project. In Vitro Phototoxicity Testing. In: In Vitro Skin Toxicology: Irritation, Phototoxicity, Sensitization; Vol. 10. Alternative Methods in Toxicology,-Eds. Rougier A, Maibach HI, Goldberg AM; Mary Ann Liebert Publ.: New York, USA 1994, pp.243-251).

[0305] Test items that are not cytotoxic to mouse fibroblasts even at the highest concentrations tested, but show a strong dose-dependent decrease in cell viability after UV exposure, are also considered phototoxic (Spielmann H, Balls M, Dupuis J, Pape WJW, Pechovitch G, Silva DeO, Holzhuetter, HG, Clothier R, Desolle P, Gerberick F, Liebsch M, Lowell WW, Maurer T, Pfannenbecker U, Potthast JM, Csato M, Sladowski D, Steiling W, Brantom P. The international EU / COLIPA in vitro phototoxicity validation study: Results of phase II (blind trial). Part 1: The 3T3 NRU phototoxicity test. Toxicology in Vitro 1998,12:305-327).

[0306] The test results are shown below, and show that the compounds of the present invention exhibit a very good phototoxicity profile. [Table 7]

[0307] Example 49 Embryonic Stem Cell Testing The in vitro mouse embryonic stem cell test (mEST) assay is a routine assay performed at Roche. The original EST was developed in 1997 by Horst Spielmann and his group as an in vitro model to screen for embryotoxicity, based on the permanent embryonic mouse ESC (mESC) D3 cell line derived from a blastocyst derived from the mouse 129 strain, and was validated by the European Centre for the Validation of Alternatives to Animal Testing (ECVAM).

[0308] The inventors have further optimised and modified the approach to allow application of the assay to pharmaceutical compounds.

[0309] Biological Endpoints and Endpoint Measurements: Cytotoxicity (inhibition of growth) of 3T3 fibroblasts representing differentiated cells after 10 days of substance treatment and cytotoxicity of undifferentiated embryonic stem cells (D3) serve as the two assay endpoints. This is determined by the use of a dehydrogenase enzyme present in intact mitochondria of live cells that converts the yellow soluble substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into a dark blue insoluble formazan product that is trapped intracellularly and quantitatively detected using an absorbance reader (570 nm) after solubilizing the cell membrane.

[0310] The third endpoint is the inhibition of ES cell differentiation into cardiac muscle cells, or cardiomyocytes, after 10 days of treatment, as assessed by microscopy for beating cells.

[0311] Materials and Reagents mESC cells: ES-D3 [D3] (ATCC® CRL-1934®) Mouse fibroblasts: BALB / 3T3 clone A31 (ATCC® CCL-163™) Balb / c 3T3 cell clone A31: American Type Culture Collection (ATCC) Catalog No. CCL-163 ES-D3 (D3): American Type Culture Collection (ATCC) catalog number CRL-1934 m-LIF: Sigma, Catalog number L5158-5UG NEAA (100x): Gibco, Catalog No. 11140-035 Trypan Blue 0.04%: Gibco, Catalog No. T10282 MTT: Tocris Bioscience, Catalog Number 5224 / 500 5-Fluorouracil: Sigma, Catalog No. F-6627-5G Penicillin / Streptomycin: Gibco, Catalog Number 15140-122 PBS (-CaCl2 / -MgCl2): Gibco, Catalog No. 14190-094 FCS:Hyclone, Catalog Number SH30070.03 DMEM with glucose, glutamine, and NaHCO3: Gibco, catalog number 41966-029

[0312] method preparation Media and endpoint assay solutions Culture medium: [Table 8] Assay medium: [Table 9] Media for freezing cells: [Table 10] β-Mercaptoethanol (β-ME) (10 mM) Add 17.5 µL of β-ME to 25 mL of PBS Store at 4℃ for up to 1 week FCS Thaw the FCS once in a water bath (37° C.) and prepare aliquots of 100 mL, 50 mL, and 25 mL. Avoid multiple decompressions Store at -20℃ MTT solution 5mg MTT / ml PBS Use sterile filters from Millipore to make 8 mL and 4 mL aliquots. Store at -20℃ MTT-desorption solution 20% SDS in water / DMF, 1:1, pH adjusted to 4.5 with acetic acid Test Compounds Stock solution: 200mM Solvent: 100% DMSO

[0313] Differentiation assay Day 0 1) Cell passaging with trypsin 0.05% EDTA for D3 cells. 2) Assembly of cell suspension: 2.5 x 10 cells in 18 ml medium (for each test) in a 50 mL Falcon tube. 4 Dilute to 1 / mL. 3) Prepare Petri dishes (PD): Add 5-10 mL of sterile Dulbecco's PBS (Gibco) to each dish bottom and distribute throughout the dish. 4) Dilution series of test compounds in Eppendorf tubes: add 5 μL of compound (1:400 dilution) and 5 μL of control solution (DMSO) to 2 mL of cell suspension and vortex. 5) Prepare hanging drops in Petri dishes: vortex tubes, aspirate suspension using automatic pipette and multi-dispense 20 μl droplets onto Petri dish lids, add 2 ml total within concentric circles of droplets (~100 drops); quickly but smoothly rotate cover to mount PD; incubate at 37 °C / 5% CO2 for 3 days.

[0314] Day 3 1) Compound dilution series in 14mL PP tubes 6 tubes for concentration; fill with 5 mL assay medium Fill one tube for DMSO (solvent control) with 5 mL assay medium. 2) Dilution series of compounds in Eppendorf tubes Add 12.5 μL of compound (1:400 dilution) and 12.5 μL of control solution and vortex 3) Transplantation of Embryoid Bodies in Bacterial Petri Dishes Carefully turn the lid of the PD and check for fungal contamination of the droplets Rinse the droplet several times with 5 mL of the prepared solution. Transfer of bacteria to Petri dishes Incubate at 37℃ / 5% CO2 for 3 days.

[0315] Day 5 1) Dilution of compounds in 50 mL tubes 6 tubes for concentration; fill with 25 mL assay medium Fill one tube for DMSO (solvent control) with 25 mL medium. 2) Dilution of compounds in 1.5 mL tubes Add 62.5 μL of compound (1:400 dilution) and control solution (DMSO) and vortex 3) Preparation of 96-well plates Two plates for each compound, see compound plate layout. Add 220 μL of media / compound / solvent mixture to all 96 wells Start with a low concentration 4) Pipetting of Embryoid Bodies Visual control of embryoid bodies in Petri dishes Using a 25 μL tip, pipette one embryoid body into each well. Visually check the plates to ensure that there is at least one embryoid body in each well. Incubate at 37℃ / 5% CO2 for 3 days.

[0316] Day 10 Visualize each well under a microscope for beating cardiomyocytes Assay medium and DMSO controls should show at least 80% beating cardiomyocytes (see acceptance criteria).

[0317] Cytotoxicity assay A stock solution of 0.2 mol / L is prepared for all substances. Test substances are diluted in DMSO solution.

[0318] Day 0 1) Making cell suspensions for D3 and 3T3 cell lines 2) For 3T3, it is 2.5 x 10 4 cells / mL, 1.5 × 10 for D3 cells 4 pieces / mL 3) Pipetting 200 μL of medium into the outer wells (blanks) of a 96-well multiwell plate 4) Add 50 μL cell suspension to the remaining inner wells of the 96-well multiwell plate (samples). 5) Incubate at 37℃ / 5% CO2 for 2 hours to allow cells to adhere. 6) Pipetting of test substance or DMSO control Make a concentration of 2 mL medium and 6.67 μL test substance in a 5 mL tube. 7) Add 150 μL / well of solution to the sample wells (total 200 μL / well) 8) Incubate at 37℃ / 5% CO2 for 3 days.

[0319] Days 3, 5 and 7 1) Dilute 2 mL of medium (3T3 or D3 cell medium) and add 5 μL of test substance (or DMSO control) (1:400) in a mL tube 2) Remove the medium with a vacuum pump without damaging the bottom cell layer. 3) Add 200 μL of diluted test substance (and DMSO control) to appropriate sample wells incubation Day 3: 2 days at 37℃ / 5% CO2 Day 5: 2 days at 37℃ / 5% CO2 Day 7: 3 days at 37℃ / 5% CO2

[0320] Day 10 Preliminary visual observation under a light microscope for changes in cells, deposition of material, or other effects MTT measurement: 1) Add 4 mL of MTT to 40 mL of DMEM and warm to 37°C to create the final MTT solution. 2) Remove the medium from the 96-well plate by carefully discarding the medium. 3) Using a multi-well pipette, add 200 μL of MTT solution to each well. 4) Incubate the plate at 37°C / 5% CO2 for 3 hours. 5) Heat the MTT-desorption solution to 37°C. 6) Carefully remove the MTT solution. 7) Add 130 µL of MTT-desorption solution to each well and incubate the plate at 37 °C in an incubator for 30 min, then place the plate on a plate shaker for at least 2-3 h. 8) Measure the absorbance at 570 nm using a plate reader.

[0321] Pass / Fail Criteria Differentiation endpoint: At least 80% of beating cardiomyocytes in all assays required for valid assay acceptance. Cytotoxicity Endpoints: The determination of the tolerance range of the DMSO control and POS control as well as the OD values ​​of D3 (approximately 1.8-2.2) and 3T3 (0.8-1.0) should be within their appropriate ranges.

[0322] Data analysis Differentiation endpoints: Determination of total number of beating cardiomyocytes (at least one beating cardiomyocyte per well = one positive count, no beating cardiomyocytes per well = one negative count), normalized to positive DMSO control Cytotoxicity Endpoints:

[0323] Determine the average OD570 of the blank (the value indicates the adhesion of the dye to the plastic material and the residual amount of medium). Subtract this value from the sample value and continue the calculation with the corrected value.

[0324] Determination of the mean OD570 of treated sample wells. Determination of the mean OD570 of solvent control wells is set to 100%. Viability is calculated as % normalized to the DMSO solvent control.

[0325] Predictive Model The optical density (OD570) data files generated by the microplate reader were copied into an EXCEL spreadsheet. The mean OD values, standard deviation and viability were calculated automatically. The following endpoints from the assay could be calculated graphically from the concentration response curves in the spreadsheet: IC50 D3 - The concentration of the test substance at which 50% of the D3 cells died IC50: The concentration of a test substance at which 50% of 3T3 cells are killed. ID50 - The concentration of test substance at which the differentiation of D3-D3 cells into contracting cardiomyocytes is reduced by 50%.

[0326] The IC50 values ​​of D3 and 3T3 cells from the cytotoxicity assay and the ID50 of the D3 differentiation assay were entered into a statistical evaluation developed from a modified predictive model used by Scholz et al. 1999a:

number

[0327] Inconclusive results are also possible if, for example, solubility limits the dose range tested to such an extent that IC50 or ID50 values ​​cannot be determined for one or more dose-response curves (Withlow et al. 2007). [Table 11]

[0328] Example 50 Single-dose pharmacokinetic (PK) study in male Wistar-Han rats The pharmacokinetic properties of selected compounds were evaluated by single-dose PK study in male Wistar-Han rats (Vendor: Beijing Vital River Laboratory Animal Technology Co.,Ltd). Briefly, two groups of animals were administered a single dose of each compound at 2 mg / kg intravenously (IV, bolus) or 10 mg / kg orally (PO, gavage). Blood samples (approximately 150 μL) were collected via the jugular vein at 5 min (IV only), 15 min, 30 min, 1 h, 2 h, 4 h, 7 h, and 24 h after administration. The blood samples were placed in tubes containing EDTA-K2 anticoagulant and centrifuged at 3000 rpm for 15 min at 4°C to separate plasma from the samples. After centrifugation, the resulting plasma was transferred to a clean tube for bioanalysis by LC / MS / MS. Pharmacokinetic parameters were calculated using non-compartmental analysis. Volume of distribution (Vss), half-life (T 1 / 2 ) and clearance (CL) were obtained based on the plasma concentration-time curve after IV administration. max ) was recorded directly from experimental observations after PO administration. Area under the plasma concentration-time curve (AUC 0-last ) was calculated using the linear trapezoidal rule to the last detectable concentration. Bioavailability (F) was calculated as the dose-normalized AUC 0-last was calculated based on.

[0329] A drug's Vss represents the extent to which the drug is distributed to body tissues rather than to plasma. Vss is directly proportional to the amount of drug distributed to tissues. A higher Vss indicates a greater amount of tissue distribution.

[0330] The results of PK parameters after IV and PO administration are shown in Table 9. [Table 12]

Claims

1. Formula (I) 【Chemical 1】 or Formula (Ia) 【Chemistry 2】 (In the formula, R １ teeth, 【Chemistry 3】 (In the formula, R ４ is H or C １～６ alkyl, and R ５ is C １～６ alkyl), R ２ is H or C １～６ is alkyl, R ３ is piperazinyl, (C １～６ Alkoxy C １～６ alkyl)piperazinyl, 3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazinyl, 4,7-diazaspiro[2.5]octanyl, 5-oxa-2,8-diazaspiro[3.5]nonanyl, amino-1,4-oxazepanyl, amino(C １～６ alkoxy)piperidinyl, amino (C １～６ alkoxy)pyrrolidinyl, amino (C １～６ alkyl)azetidinyl or aminohalopiperidinyl; A is N or CR ６ (where R ６ is H, C １～６ Alkyl or C １～６ alkoxy), M is N or CR ７ (where R ７ is H or C １～６ alkyl), W is N or CH; Q is N or CH; However, no more than two of A, M, W, and Q are N at the same time. or a pharmaceutically acceptable salt thereof.

2. R １ but, 【Chemistry 4】 (In the formula, R ４ is C １～６ alkyl, and R ５ is C １～６ 2. The compound of claim 1, wherein R is 1 or 2; or a pharmaceutically acceptable salt thereof.

3. R ４ is methyl, and R ５ 3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein is methyl.

4. A is CR ６ and R ６ is H or C １～６ 3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

5. A is CR ６ and R ６ 5. The compound of claim 4, or a pharmaceutically acceptable salt thereof, wherein is H or methyl.

6. M is CR ７ and R ７ or a pharmaceutically acceptable salt thereof.

7. 7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein W is CH.

8. 8. The compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein Q is N.

9. R ３ amino-1,4-oxazepanyl, amino(C １～６ 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, which is (alkoxy)pyrrolidinyl or piperazinyl.

10. R ３ The compound according to claim 9, wherein is 6-amino-1,4-oxazepan-4-yl, 3-amino-4-methoxy-pyrrolidin-1-yl, or piperazin-1-yl, or a pharmaceutically acceptable salt thereof.

11. R １ but, 【Chemistry 5】 (In the formula, R ４ is C １～６ alkyl, and R ５ is C １～６ alkyl), R ２ is H or C １～６ is alkyl, R ３ amino-1,4-oxazepanyl, amino(C １～６ alkoxy)pyrrolidinyl or piperazinyl, A is CR ６ (where R ６ is H or C １～６ alkyl), M is CR ７ (where R ７ is H), W is CH; Q is N; 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof.

12. R １ but, 【Chemistry 6】 (In the formula, R ４ is methyl, and R ５ is methyl), R ２ is H or methyl, R ３ is 6-amino-1,4-oxazepan-4-yl, 3-amino-4-methoxy-pyrrolidin-1-yl or piperazin-1-yl, A is CR ６ (where R ６ is H or methyl; M is CR ７ (where R ７ is H), W is CH; Q is N; 12. The compound of claim 11 or a pharmaceutically acceptable salt thereof.

13. Formula (Ib) 【Chemistry 7】 (In the formula, R １ teeth, 【Chemistry 8】 (In the formula, R ４ is C １～６ alkyl, and R ５ is C １～６ alkyl), R ２ is C １～６ is alkyl, R ３ is piperazinyl, (C １～６ Alkoxy C １～６ alkyl)piperazinyl, 4,7-diazaspiro[2.5]octanyl, 5-oxa-2,8-diazaspiro[3.5]nonanyl, amino-1,4-oxazepanyl, amino(C １～６ alkoxy)piperidinyl, amino (C １～６ alkoxy)pyrrolidinyl, amino (C １～６ alkyl)azetidinyl or aminohalopiperidinyl; A is CH; M is CH; W is CH; Q is N) or a pharmaceutically acceptable salt thereof.

14. R ４ is methyl, and R ５ is methyl, and R ２ 14. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein is methyl.

15. R ３ The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein is amino-1,4-oxazepanyl.

16. R ３ The compound according to claim 13, or a pharmaceutically acceptable salt thereof, wherein is 6-amino-1,4-oxazepan-4-yl.

17. R １ but, 【Chemistry 9】 (In the formula, R ４ is C １～６ alkyl, and R ５ is C １～６ alkyl), R ２ But C １～６ is alkyl, R ３ is amino-1,4-oxazepanyl, A is CH; M is CH; W is CH; Q is N; 14. The compound of claim 13 or a pharmaceutically acceptable salt thereof.

18. R １ but, 【Chemistry 10】 (In the formula, R ４ is methyl, and R ５ is methyl), R ２ is methyl, R ３ is 6-amino-1,4-oxazepan-4-yl, A is CH; M is CH; W is CH; Q is N; 18. The compound of claim 17 or a pharmaceutically acceptable salt thereof.

19. 1,6-dimethyl-4-[4-(4-piperazin-1-ylphenyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[4-(5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[4-(5-piperazin-1-ylpyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[4-(5-piperazin-1-ylpyrimidin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1-methyl-4-[4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1-methyl-4-[4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[4-(4-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[4-(2-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[4-(4-methyl-2-piperazin-1-yl-pyrimidin-5-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 4-[4-(3-ethyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; 4-[4-(3-methoxy-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; (3S,4R)-1-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-4-piperidyl]-5-methyl-3-pyridyl]-3-fluoro-piperidin-4-amine; 4-[4-[5-[(3R)-3-(methoxymethyl)piperazin-1-yl]-3-methyl-2-pyridyl]-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; (3S,4S)-1-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-4-piperidyl]-5-methyl-3-pyridyl]-3-methoxy-piperidin-4-amine; 1-methyl-4-[cis-3-methyl-4-(6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1-methyl-4-[trans-3-methyl-4-(6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[cis-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[trans-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[cis-3-methyl-4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[trans-3-methyl-4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[cis-3-methyl-4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[trans-3-methyl-4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; (3R,4R)-1-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-4-methoxy-pyrrolidin-3-amine; 2-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-5-oxa-2,8-diazaspiro[3.5]nonane; (6S)-4-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-1,4-oxazepan-6-amine; 4-[(3S,4R)-4-[5-[(3R)-3-(methoxymethyl)piperazin-1-yl]-3-methyl-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; 4-[(3S,4R)-4-[5-(4,7-diazaspiro[2.5]octan-7-yl)-3-methyl-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; (3R,4R)-1-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-3-fluoro-piperidin-4-amine; 1-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-3-methyl-azetidin-3-amine; (4aR,7aR)-6-[6-[(3S,4R)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-5-methyl-3-pyridyl]-3,4,4a,5,7,7a-hexahydro-2H-pyrrolo[3,4-b][1,4]oxazine; 1,6-dimethyl-4-[(3R,4S)-3-methyl-4-(5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; 1,6-dimethyl-4-[(3S,4R)-3-methyl-4-(5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine; (3R,4R)-1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-4-methoxy-pyrrolidin-3-amine; 4-[(3R,4S)-4-[5-[(3R)-3-(methoxymethyl)piperazin-1-yl]-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; 2-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-5-oxa-2,8-diazaspiro[3.5]nonane; (3S,4S)-1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-3-methoxy-piperidin-4-amine; (6S)-4-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-1,4-oxazepan-6-amine; (3R,4R)-1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-3-fluoro-piperidin-4-amine; 4-[(3R,4S)-4-[5-(4,7-diazaspiro[2.5]octan-7-yl)-2-pyridyl]-3-methyl-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine; and 1-[6-[(3R,4S)-1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-3-pyridyl]-3-methyl-azetidin-3-amine; or a pharmaceutically acceptable salt thereof.

20. below: a) reacting a compound of formula (IV) with a compound of formula (IV) in the presence of a catalyst and a base 【Chemistry 11】 and a compound of R ３ a Buchwald-Hartwig C—N bond forming reaction between —H, b) reacting a compound of formula (VI) with a compound of formula (VI) in the presence of a catalyst and a base 【Chemistry 12】 and a compound of formula (II) 【Chemistry 13】 a Buchwald-Hartwig C—N bond forming reaction between a compound of c) a step of direct substitution reaction between a compound of formula (VI) and a compound of formula (II) in the presence of a base. Including, During the ceremony, X is a halogen; In steps a) and b), the catalyst is RuPhos Pd G2 or Pd ２ (dba) ３ / Xantphos, and the base is Cs ２ CO ３ or t-BuONa, In step c), the base is DIPEA or CsF; R １ ~R ５ , A, M, W and Q are as defined in any one of claims 1 to 18; A process for preparing a compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof.

21. A compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof for use as a therapeutically active substance.

22. A pharmaceutical composition comprising the compound of any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof.

23. 23. The pharmaceutical composition of claim 22 for treating or preventing systemic lupus erythematosus or lupus nephritis.

24. The pharmaceutical composition of claim 23 for use as an antagonist of TLR7, TLR8 and TLR9.

25. Use of a compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment or prevention of systemic lupus erythematosus or lupus nephritis.

26. 20. Use of a compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for use as an antagonist of TLR7 and TLR8 and TLR9.

27. 20. A compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof for treating or preventing systemic lupus erythematosus or lupus nephritis.

28. A compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof for use as an antagonist of TLR7, TLR8 and TLR9.

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