Pyridinone compounds for the treatment of autoimmune diseases

Abstract

The present invention relates to a compound of formula (I) TIFF2023536339000135.tif41165 (In the formula, R 1 , R 2 , R 3 , R 4 and A is as described herein. and pharmaceutically 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 / or TLR8 and / or 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 Sjögren's syndrome (pSjS), mixed connective tissue disease (MCTD), dermatomyositis / polymyositis (DM / PM), rheumatoid arthritis (RA), and systemic sclerosis (SSc). With the exception of RA, no truly effective and safe treatments are available to patients. SLE, the prototypic CTD with a prevalence of 20–150 per 100,000 people, 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, long-term use of immunosuppressants, such as corticosteroids, is only partially effective and is associated with undesirable toxicities and side effects. Belimumab, the only lupus drug approved by the FDA in the past 50 years, has only modest and delayed efficacy in a small proportion of SLE patients (Navarra, SV et al., Lancet 2011, 377, 721). Other biologics, such as anti-CD20 mAbs, mAbs against specific cytokines, or soluble receptors, have failed in most clinical studies. Therefore, novel therapies are needed that provide sustained improvement in larger patient populations 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 TLRs 7, 8, and 9 recognize nucleic acids derived from viruses and bacteria. Specifically, TLRs 7 / 8 and 9 recognize single-stranded RNA (ssRNA) and single-stranded CpG-DNA, respectively. However, abnormal nucleic acid sensing by TLRs 7, 8, and 9 has been implicated as 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 for 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 activate TLR9. 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 are upregulated in SLE tissues and have been reported to correlate with the chronicity and activity of lupus nephritis, respectively. In B cells from SLE patients, TLR7 expression correlates with anti-RNP antibody production, whereas TLR9 expression correlates with IL-6 levels and anti-double-stranded DNA antibody levels. Consistently, in a lupus mouse model, 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 nephrosis, induction of Th17 cells, and suppression of Treg cells.In addition to the role of TLR9 in promoting B cell autoantibody production, activation of TLR9 by self-DNA in pDCs also leads to the induction of type I IFN and other proinflammatory cytokines. Given the important role of TLR9 in the pathogenesis of autoimmune diseases in both pDCs and B cells and the widespread presence of self-DNA complexes that can readily activate TLR9 in many patients with autoimmune diseases, blocking the self-DNA-mediated TLR9 pathway in addition to inhibiting the TLR7 and TLR8 pathways may be particularly advantageous. In summary, the TLR7, 8, and 9 pathways represent novel therapeutic targets for the treatment of autoimmune and autoinflammatory diseases for which no effective steroid-free, non-cytotoxic oral drugs exist. Inhibiting all of these pathways from their upstream pathways may provide satisfactory therapeutic effects. Therefore, we have developed 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 of formula (I) [ka] (In the formula, R 1 is C 1~6 is alkyl, R 2 is C 1~6 is alkyl, R 3 is C 1~6 Alkyl or haloC 1~6 is alkyl, R 4 is piperazinyl, piperidinyl or 3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazinyl, and the piperazinyl, piperidinyl or 3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazinyl is 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, Phenyl C1~6 alkyl (phenyl substituted with piperazinyl); piperazinyl, Pyrazinyl C 1~6 alkyl (pyrazinyl substituted with piperazinyl); pyridinyl (pyridinyl substituted with piperazinyl), Pyridinyl C 1~6 alkyl (pyridinyl is substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl or piperazinyl); pyrimidinyl (pyrimidinyl substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl), and Pyrimidinyl C 1~6 Alkyl (pyrimidinyl is amino (C 1~6 substituted with alkyl)azetidinyl, 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl, amino-1,4-oxazepan-4-yl or piperazinyl; A is CH or N or a pharmaceutically acceptable salt thereof.

[0005] Another object of the present invention relates to a novel compound of formula (I). Its preparation, a pharmaceutical preparation based on the compound according to the present invention and its preparation, and the use of the compound of formula (I) as a TLR7, TLR8, and TLR9 antagonist, and its use for the treatment or prevention of systemic lupus erythematosus or lupus nephritis. The compound of formula (I) exhibits excellent antagonistic activity against TLR7, TLR8, and TLR9. In addition, the compound of formula (I) also exhibits good cytotoxicity, phototoxicity, solubility, hPBMC, human microsome stability, AO (human cytosolic aldehyde oxidase), and SDPK profiles, as well as low CYP inhibition. DETAILED DESCRIPTION OF THE INVENTION

[0006] 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.

[0007] The terms "halogen" and "halo" are used interchangeably herein to refer to fluoro, chloro, bromo, or iodo.

[0008] "Haro C 1~6 The term "alkyl" refers to 1~6 C in which at least one of the hydrogen atoms of the alkyl group is replaced by the same or different halogen atom, in particular a fluoro atom. 1~6 Indicates an alkyl group. HaloC 1~6 Examples of alkyl include monofluoromethyl, difluoromethyl, or trifluoromethyl, monofluoroethyl, difluoroethyl, or trifluoroethyl, or monofluoropropyl, difluoropropyl, or trifluoropropyl, such as 3,3,3-trifluoropropyl, 2-fluoroethyl, trifluoroethyl, fluoromethyl, difluoromethyl, difluoroethyl, or trifluoromethyl.

[0009] The term "pharmaceutically acceptable salt" means a salt that is not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts.

[0010] The term "pharmaceutically acceptable acid addition salt" means a pharmaceutically acceptable salt such as formed with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and the like, and an organic acid 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, malonic 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.

[0011] 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 naturally occurring 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, N-ethylpiperidine, and polyamine resins.

[0012] The term "pharmaceutically 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 alter their physical properties and biological effects. These metabolic transformations usually affect the polarity of the compounds of the present invention and change how the drug is distributed in and excreted from the body. However, in some cases, drug metabolism is required for therapeutic effect.

[0013] The term "therapeutically effective amount" means 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. A 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.

[0014] The term "pharmaceutical composition" refers to a mixture or solution containing a therapeutically effective amount of an active ingredient together with pharmaceutically acceptable excipients, to be administered to a mammal (e.g., a human) in need thereof.

[0015] TLR7 and / or TLR8 and / or TLR9 antagonists The present invention provides a compound of formula (I): [ka] (In the formula, R 1 is C 1~6 is alkyl, R 2 is C 1~6 is alkyl, R 3 is C 1~6 Alkyl or haloC 1~6 is alkyl, R4 is piperazinyl, piperidinyl or 3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazinyl, and the piperazinyl, piperidinyl or 3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazinyl is 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, Phenyl C 1~6 alkyl (phenyl substituted with piperazinyl); piperazinyl, Pyrazinyl C 1~6 alkyl (pyrazinyl substituted with piperazinyl); pyridinyl (pyridinyl substituted with piperazinyl), Pyridinyl C 1~6 alkyl (pyridinyl is substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl or piperazinyl); pyrimidinyl (pyrimidinyl substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl), and Pyrimidinyl C 1~6 Alkyl (pyrimidinyl is amino (C 1~6 substituted with alkyl)azetidinyl, 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl, amino-1,4-oxazepan-4-yl or piperazinyl; A is CH or N or a pharmaceutically acceptable salt thereof.

[0016] A further embodiment of the invention is (ii) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein A is CH.

[0017] A further embodiment of the present invention is (iii) a compound of formula (I) according to (i) or (i), or a pharmaceutically acceptable salt thereof, R 4 teeth, [ka] and R 5 teeth, 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, Phenyl C 1~6 alkyl (phenyl substituted with piperazinyl); piperazinyl, Pyrazinyl C 1~6 alkyl (pyrazinyl substituted with piperazinyl); pyridinyl (pyridinyl substituted with piperazinyl), Pyridinyl C 1~6 alkyl (pyridinyl is substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl or piperazinyl); pyrimidinyl (pyrimidinyl substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl), and Pyrimidinyl C 1~6 Alkyl (pyrimidinyl is amino (C 1~6 alkyl) azetidinyl, 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl, amino-1,4-oxazepan-4-yl or piperazinyl) is selected from.

[0018] A further embodiment of the present invention is (iv) a compound of formula (I) according to any one of (i) to (iii), or a pharmaceutically acceptable salt thereof, R 4 teeth, [ka] (In the formula, R 5a is 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, ((piperazinyl)phenyl)C 1~6 Alkyl, ((piperazinyl)pyrazinyl)C 1~6 Alkyl, ((piperazinyl)pyridinyl)C 1~6Alkyl, ((5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyridinyl)C 1~6 Alkyl, ((amino(C 1~6 Alkyl)azetidinyl)pyrimidinyl)C 1~6 Alkyl, ((5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl)C 1~6 Alkyl, ((amino-1,4-oxazepan-4-yl)pyrimidinyl)C 1~6 Alkyl, or ((piperazinyl)pyrimidinyl)C 1~6 alkyl), [ka] (In the formula, R 5b is piperazinyl), or [ka] (In the formula, R 5c is piperazinylpyridinyl or (5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl is.

[0019] A further embodiment of the present invention is (v) a compound of formula (I) according to any one of (i) to (iv), R 4 teeth, [ka] (In the formula, R 5ais 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, (4-piperazin-1-ylphenyl)methyl, (3-piperazin-1-ylphenyl)methyl, (5-piperazin-1-ylpyrazin-2-yl)methyl, (5-piperazin-1-yl-2-pyridinyl)methyl, (6-piperazin-1-yl-3-pyridinyl)methyl, [6-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)-3-pyridinyl ]methyl, [2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl, [2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl, [2-[6-amino-1,4-oxazepan-4-yl]pyrimidin-5-yl]methyl, (2-piperazin-1-ylpyrimidin-5-yl)methyl, or (5-piperazin-1-ylpyrimidin-2-yl)methyl), [ka] (In the formula, R 5b is piperazin-1-yl), or [ka] (In the formula, R 5c is 6-piperazin-1-yl-3-pyridinyl or 2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl is.

[0020] A further embodiment of the present invention is (vi) a compound of formula (I) according to any one of (i) to (v) or a pharmaceutically acceptable salt thereof, wherein R 3 is C 1~6 It is alkyl.

[0021] A further embodiment of the present invention is (vii) a compound of formula (I) according to any one of (i) to (vi) or a pharmaceutically acceptable salt thereof, wherein R 3 is ethyl or isopropyl.

[0022] A further embodiment of the present invention is (viii) a compound of formula (I) according to any one of (i) to (vii) or a pharmaceutically acceptable salt thereof, wherein R 4 teeth [ka] (In the formula, R 5a is ((5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl)C 1~6 Alkyl or ((amino-1,4-oxazepan-4-yl)pyrimidinyl)C 1~6 alkyl), or [ka] (In the formula, R 5c is (5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl is.

[0023] A further embodiment of the present invention is a compound of formula (I) according to any one of (ix)(i) to (viii) or a pharmaceutically acceptable salt thereof, wherein R 4 teeth [ka] (In the formula, R 5a is [2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl or [2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl, or [ka] (In the formula, R 5c is 2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl is.

[0024] A further embodiment of the present invention is a compound of formula (I) according to any one of (x)(i) to (ix) or a pharmaceutically acceptable salt thereof, R 1 is C 1~6 is alkyl, R 2 is C 1~6 is alkyl, R 3 is C 1~6 is alkyl, R 4 teeth, [ka] (In the formula, R 5a is ((5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl)C 1~6 Alkyl or ((amino-1,4-oxazepan-4-yl)pyrimidinyl)C 1~6 alkyl), or [ka] (In the formula, R 5c is (5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl), A is CH A compound of formula (I), or a pharmaceutically acceptable salt thereof:

[0025] A further embodiment of the present invention is (xi) a compound of formula (I) according to any one of (i) to (x) or a pharmaceutically acceptable salt thereof, R 1 is methyl, R 2 is methyl, R 3 is ethyl or isopropyl, R 4 teeth [ka] (In the formula, R5a is [2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl or [2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl), or [ka] (In the formula, R 5c is 2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl), A is CH A compound of formula (I), or a pharmaceutically acceptable salt thereof:

[0026] Another embodiment of the present invention is a method for manufacturing a semiconductor device comprising: 5-[2-ethyl-6-[4-[[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-ethyl-6-[4-[[6-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)-3 pyridyl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[6-[4-[[2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[6-[4-[[2-[(6S)-6-amino-1,4-oxazepan-4-yl]pyrimidin-5-yl]methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-ethyl-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[4-[[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[4-[(5-piperazin-1-yl-2-pyridyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[4-[(5-piperazin-1-ylpyrazin-2-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[4-[(2-piperazin-1-ylpyrimidin-5-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[6-[4-[[2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[4-[(6-piperazin-1-yl-3-pyridyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[4-[(5-piperazin-1-ylpyrimidin-2-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[4-[(3-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[4-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-Isopropyl-6-[8-(6-piperazin-1-yl-3-pyridyl)-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-(difluoromethyl)-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-(4-piperazin-1-yl-1-piperidyl)-3-pyridyl]-1,3-dimethyl-pyridin-2-one; and 5-[2-ethyl-6-[2-[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-8-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; or a pharmaceutically acceptable salt thereof.

[0027] synthesis The compounds of the present invention can be prepared by any conventional means. Suitable processes for synthesizing these compounds and their starting materials are provided in the following schemes and examples. All substituents, particularly R 1 , R 2 , R 3 , R 4 and A are as described above unless otherwise indicated. Furthermore, unless expressly stated otherwise, all reactions, reaction conditions, abbreviations and symbols have meanings well known to those skilled in the art of organic chemistry.

[0028] A general synthetic route for preparing compounds of formula (I) is shown below.

[0029] [ka] In the formula, X 1 , X 2 , X 3is a halogen; A is CH or N; PG is a protecting group such as Boc; L is piperazinyl, piperidinyl, piperazinylpiperidinyl, piperidinylpiperazinyl, or 3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl; G 1 is 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, phenyl, piperazinyl, pyrazinyl, pyridinyl or pyrimidinyl; G 2 piperazinyl, 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl, amino(C 1~6 alkyl)azetidinyl or amino-1,4-oxazepan-4-yl.

[0030] Compound (IV) is treated with bis(pinacolato)diboron in the presence of a suitable base, such as KOAc, and a suitable palladium catalyst, such as PdCl(DPPF)-CHCl adduct, to give compound (V). Suzuki-coupling reaction between compound (V) and compound (VI) using a suitable catalyst, such as PdCl(DPPF)-CHCl adduct, and a suitable base, such as KCO, gives compound (VII). Buchwald-Hartwig amination of compound (VII) with compound (VIII) in the presence of a catalyst, such as RuPhos Pd G, and a suitable base, such as CsCO or t-BuONa, gives compound (IX). Deprotection of compound (IX) under acidic conditions, such as TFA, gives compound (I-1). Substitution reaction between compound (I-1) and compound (X) in the presence of a suitable base such as K2CO3 gives compound (XIII). Compound (XIII) can also be obtained by reductive amination between compound (X) and compound (XII) using a reducing agent such as NaBH(OAc)3. Coupling of compound (XIII) and compound (XIV) under Buchwald-Hartwig amination conditions using a catalyst such as RuPhos Pd G2 and a suitable base such as Cs2CO3 or t-BuONa gives compound (XV). Deprotection of compound (XV) under acidic conditions such as TFA gives compound (I-2).

[0031] [ka] In the formula, G 3 is 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl.

[0032] Coupling of compound (I-1) with compound (XVI) under Buchwald-Hartwig amination conditions using a catalyst such as RuPhos Pd G2 and a suitable base such as Cs2CO3 or t-BuONa provides compound (XVII). Deprotection of compound (XVII) under acidic conditions such as TFA provides compound (I-3).

[0033] [ka]

[0034] Compounds of formula (VII) can also be obtained via Scheme 3.

[0035] Treatment of compound (VI) with bis(pinacolato)diboron in the presence of a suitable base, such as KOAc, and a suitable palladium catalyst, such as PdCl(DPPF)-CHCl adduct, provides compound (XIX). Coupling of compound (XIX) with compound (IV) under Suzuki coupling conditions using a suitable catalyst, such as PdCl(DPPF)-CHCl adduct, and a suitable base, such as KCO, provides compound (VII).

[0036] The compounds of the present 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.

[0037] The present invention also provides a method for producing a pharmaceutical composition comprising the steps of: a) Formula (IX) [ka] The compound of formula (I-1) is deprotected using an acid to give the compound of formula (I-1) [ka] obtaining a compound of formula (I), b) Formula (XV) [ka] The compound of formula (I-2) is deprotected using an acid to give the compound of formula (I-3) [ka] obtaining a compound of formula (I), c) Formula (XVII) [ka] The compound of formula (I-3) is deprotected using an acid to give the compound of formula (I-4) [ka] A step of obtaining a compound of (In the preparation method, In steps a), b) and c), the acid may be, for example, TFA. The present invention also relates to a process for preparing a compound of formula (I), comprising:

[0038] Compounds of formula (I) when prepared by the above process are also an object of the present invention.

[0039] Indications and methods of treatment The present invention provides compounds that can be used as antagonists of TLR7 and / or TLR8 and / or TLR9, inhibiting TLR7 and / or TLR8 and / or TLR9-mediated pathway activation and their downstream biological events, including, but not limited to, innate and adaptive immune responses mediated through the production of all types of cytokines and all forms of autoantibodies. Thus, the compounds of the present invention are useful for blocking TLR7 and / or TLR8 and / or TLR9 in all types of cells that express such receptor(s), including, but not limited to, plasmacytoid dendritic cells, B cells, T cells, macrophages, monocytes, neutrophils, keratinocytes, and epithelial cells. Thus, the compounds can be used as therapeutic or prophylactic agents for systemic lupus erythematosus and lupus nephritis.

[0040] The present invention provides methods for the treatment or prevention of systemic lupus erythematosus and lupus nephritis in a patient in need thereof.

[0041] Another embodiment includes a method of treating or preventing systemic lupus erythematosus and lupus nephritis in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a compound of Formula (I), a stereoisomer, a tautomer, a prodrug, or a pharmaceutically acceptable salt thereof. [Example]

[0042] 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.

[0043] 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.

[0044] The abbreviations used herein are as follows: ACN: acetonitrile Boc2O: di-tert-butyl dicarbonate CbzCl: benzyl chloroformate DAST: (Diethylamino)sulfur trifluoride DEA: Diethylamine DIPEA: N,N-diisopropylethylamine DMF: N,N-dimethylformamide EtOAc or EA: Ethyl acetate FA: Formic acid HLM Human Liver Microsomes I C 50 :50% inhibitory concentration LCMS Liquid Chromatography Mass Spectrometry MS: Mass spectrometry PBS Phosphate-buffered saline [Pd(allyl)Cl]2: Allylpalladium(II) chloride dimer 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: Chloro(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) 2nd generation PdCl2(DPPF)-CH2Cl2 adduct: [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) Dichloromethane dichloride adduct DAST: (Diethylamino)sulfur trifluoride SFC: Supercritical Fluid Chromatography TEA: Trimethylamine TFA: Trifluoroacetic acid v / v: volume ratio

[0045] General experimental conditions Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) a Biotage SP1 system and Quad 12 / 25 cartridge module; ii) an ISCO Combi-Flash chromatography instrument. The silica gel brands and pore sizes were: i) KP-SIL 60Å, particle size: 40-60 μm; ii) CAS Registry 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.

[0046] 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) columns on a 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).

[0047] 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) on 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, and UV detection at 254 or 220 nm.

[0048] 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 or 1.5 minutes): Acidic conditions I: A: 0.1% TFA in H2O; B: 0.1% TFA in acetonitrile; Acidic conditions II: A: 0.0375% TFA in H2O; B: 0.01875% TFA in acetonitrile; Basic condition I: A: 0.1% NH3·H2O in H2; B: acetonitrile; Basic conditions II: A: 0.025% NH3·H2O in H2O; B: acetonitrile; Neutral conditions: A: H2O; B: acetonitrile.

[0049] Mass spectra (MS): Generally, only ions representing the parent mass are reported; unless otherwise stated, the mass ions quoted are positive mass ions (M−H). + is.

[0050] NMR spectra were obtained using a Bruker Avance 400 MHz.

[0051] Microwave-assisted reactions were performed in a Biotage Initiator Sixty microwave synthesizer. All reactions involving air-sensitive reagents were carried out under an argon or nitrogen atmosphere. Reagents were obtained as received and used without further purification unless otherwise noted.

[0052] Preparation Examples The following examples are intended to illustrate the meaning of the present invention but do not in any way represent a limitation within the meaning of the present invention.

[0053] Intermediate A1 3-Bromo-6-chloro-2-ethylpyridine [ka]

[0054] Intermediate A1 Step 1: Preparation of 5-bromo-6-ethylpyridin-2-amine [ka]

[0055] Intermediate A1-a A mixture of 6-ethylpyridin-2-amine (5 g, 40.9 mmol, CAS number 21717-29-3, distributor: Bide Pharmatech, catalog BD3776), 1-bromopyrrolidine-2,5-dione (8.01 g, 45 mmol, CAS number 128-08-5, distributor: ALDRICH, catalog B81255) in MeOH (20 mL) was stirred at 0° C. for 16 hours. After the reaction was complete, the mixture was then concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give 5-bromo-6-ethylpyridin-2-amine (4.1 g, 49.8% yield) as an orange solid. MS: calculated 202 (M+H + ), actual value 202 (M+H + ).

[0056] Step 2: Preparation of 3-bromo-6-chloro-2-ethylpyridine [ka]

[0057] Intermediate A1 A mixture of 5-bromo-6-ethylpyridin-2-amine (4.1 g, 20.4 mmol), CuCl (5.48 g, 40.8 mmol), tert-butyl nitrite (5.26 g, 51 mmol, CAS number 540-80-7, vendor: TCI, catalogue N0357) in DCM (40 mL) was stirred at 50 °C for 2 h. After the reaction was complete, the mixture was then concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of PE / EA (0% to 80%) to give 3-bromo-6-chloro-2-ethylpyridine (2.8 g, 62.3% yield) as a yellow liquid. MS: calculated 220 (M+H + ), actual value 220 (M+H + ).

[0058] Intermediate A2 6-chloro-2-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine [ka]

[0059] Intermediate A2 To a mixture of 3-bromo-6-chloro-2-ethylpyridine (2.8 g, 12.7 mmol), KOAc (3.12 g, 31.7 mmol), and bis(pinacolato)diboron (3.55 g, 14 mmol, CAS number 73183-34-3, supplier: Accela ChemBio Inc., catalog SY001323) in dioxane (20 mL), PdCl(DPPF)-CHCl adduct (929 mg, 1.27 mmol, CAS number 95464-05-4, supplier: Accela ChemBio Inc., catalog SY002614) was added, and the mixture was stirred at 90 °C under a N atmosphere for 2 hours. After the reaction was complete, the mixture was then concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of PE / EA (0% to 10%) to give 6-chloro-2-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (1.95 g, 57.4% yield) as a yellow solid. MS: calculated 268 (M+H + ), actual value 268 (M+H + ).

[0060] Intermediate A3 3-Bromo-6-chloro-2-isopropylpyridine [ka]

[0061] Intermediate A3 Intermediate A3 was prepared similarly to the preparation of Intermediate A1 by using 6-isopropylpyridin-2-amine instead of 6-ethylpyridin-2-amine in step 1. MS calculated 234 (M+H + ), actual value 234 (M+H + ).

[0062] Intermediate A4 6-chloro-2-isopropyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine [ka]

[0063] Intermediate A4 Intermediate A4 was prepared in a manner similar to that of Intermediate A2, using 3-bromo-6-chloro-2-isopropyl-pyridine instead of 3-bromo-6-chloro-2-ethylpyridine. MS calculated 282 (M+H + ), actual value 282 (M+H + ).

[0064] Intermediate A5 3-Bromo-6-chloro-2-(difluoromethyl)pyridine [ka]

[0065] Intermediate A5 To a solution of 3-bromo-6-chloropicolinaldehyde (1.5 g, 6.8 mmol, CAS No. 1060815-64-6, distributor: Bide Pharmatech, catalog BD259869) in DCM (40 mL) cooled to −78° C., DAST (4.39 g, 3.6 mL, 27.2 mmol, CAS No. 38078-09-0, distributor: PharmaBlock Sciences (Nanjing), Inc., catalog PBLY8231) was added. After the addition, the mixture was stirred at −78° C. for an additional 30 minutes, then warmed to room temperature and stirred for 10 hours. After the reaction was complete, the mixture was concentrated in vacuo. The residue was then purified by flash column elution with a gradient of PE / EA (0% to 10%) to give 3-bromo-6-chloro-2-(difluoromethyl)pyridine (1.5 g, 91% yield) as a yellow solid. MS: Calculated 242 (M+H + ), actual value 242 (M+H + ).

[0066] Intermediate B1 1,3-Dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-one [ka]

[0067] Intermediate B1 To a mixture of 5-bromo-1,3-dimethylpyridin-2(1H)-one (0.5 g, 2.47 mmol, CAS number 51417-13-1, vendor: ALDRICH, catalog JRD0890), KOAc (291 mg, 2.97 mmol), bis(pinacolato)diboron (754 mg, 2.97 mmol) in dioxane (10 mL), PdCl(DPPF)-CHCl adduct (90.5 mg, 124 μmol) was added, and the mixture was stirred at 90° C. under a N atmosphere for 2 hours. After the reaction was complete, the mixture was then concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give 1,3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-one (616 mg, 100% yield) as a brown solid. MS: calculated 250 (M+H + ), actual value 250 (M+H + ).

[0068] Example 1 5-[2-ethyl-6-[4-[[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0069] The title compound was prepared according to the following scheme.

[0070] [ka]

[0071] Step 1: Preparation of 5-(6-chloro-2-ethyl-3-pyridyl)-1,3-dimethyl-pyridin-2-one [ka]

[0072] To a mixture of 5-bromo-1,3-dimethylpyridin-2(1H)-one (238 mg, 1.19 μmol, CAS number 51417-13-1, supplier: ALDRICH, catalog JRD0890), 6-chloro-2-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (Intermediate A2, 318 mg, 1.19 mmol) and KCO (205 mg, 1.48 mmol) in a mixed solvent of dioxane (5 mL) and water (1 mL), PdCl(DPPF)-CHCl adduct (72.4 mg, 99 μmol, CAS number 95464-05-4, supplier: Accela ChemBio Inc, catalog SY002614) was added, and the mixture was stirred at 80 °C under a N atmosphere for 2 h. After the reaction was completed, the mixture was concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give 5-(6-chloro-2-ethyl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (297 mg, 94.8% yield) as a light brown oil. MS: calculated 263 (M+H + ), actual value 263 (M+H + ).

[0073] Step 2: Preparation of tert-butyl 4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]piperazine-1-carboxylate [ka]

[0074] To a mixture of 5-(6-chloro-2-ethyl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (297 mg, 1.13 mmol), tert-butyl piperazine-1-carboxylate (274 mg, 1.47 mmol, CAS number 77279-24-4, supplier: Bide Pharmatech, catalog B13517) and CsCO (552 mg, 1.7 mmol) in dioxane (5 mL), RuPhos Pd G (43.9 mg, 56.5 μmol, CAS number 1375325-68-0, supplier: ALDRICH, catalog 753246) was added, and the mixture was stirred at 110 °C under a N atmosphere for 16 hours. After the reaction was complete, the mixture was then concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]piperazine-1-carboxylate (287 mg, 61.5% yield) as an orange oil. MS: calculated 413 (M+H + ), actual value 413 (M+H + ).

[0075] Step 3: Preparation of 5-(2-ethyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one [ka]

[0076] To a solution of tert-butyl 4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]piperazine-1-carboxylate (90 mg, 218 μmol) in DCM (4 mL) was added TFA (1 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated in vacuo. The residue was diluted with 2M KOH solution (5 mL), and the resulting mixture was extracted twice with DCM (30 mL). The combined organic layers were concentrated in vacuo to give crude 5-(2-ethyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (68 mg, 99.8% yield) as a yellow oil, which was used directly in the next step without further purification. MS: calculated 313 (M+H + ), actual value 313 (M+H + ).

[0077] Step 4: Preparation of 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0078] A mixture of 5-(2-ethyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (805 mg, 2.58 mmol), 2-chloro-5(chloromethyl)-pyrimidine (2.1 g, 12.9 mmol, CAS number 148406-13-7, supplier: PharmaBlock (Nanjing) R&D Co. Ltd., catalog PBN20120209) and K2CO3 (1.78 g, 12.9 mmol) in MeCN (10 mL) was stirred for 16 hours at 40° C. After the reaction was complete, the mixture was filtered and the filtrate was concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one (260 mg, 23% yield) as a yellow oil. MS: calculated 439 (M+H + ), actual value 439 (M+H + ).

[0079] Step 5: Preparation of tert-butyl 2-[5-[[4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]piperazin-1-yl]methyl]pyrimidin-2-yl]-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate [ka]

[0080] To a mixture of 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one (65 mg, 148 μmol), tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (67.6 mg, 296 μmol, CAS number 1251011-05-8, vendor: PharmaBlock (Nanjing) R&D Co. Ltd., catalog PBN20111063), and CsCO (96.5 mg, 296 μmol) in dioxane (3 mL), Ruphos Pd G (5.75 mg, 7.4 μmol) was added, and the mixture was stirred at 110 °C under a N atmosphere for 16 hours. After the reaction was complete, the mixture was concentrated in vacuo. The residue was then purified by flash column elution with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 2-[5-[[4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]piperazin-1-yl]methyl]pyrimidin-2-yl]-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (37 mg, 39.6% yield) as a yellow oil. MS: calculated 631 (M+H + ), actual value 631 (M+H + ).

[0081] Step 6: Preparation of 5-[2-ethyl-6-[4-[[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0082] To a solution of tert-butyl 2-[5-[[4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]piperazin-1-yl]methyl]pyrimidin-2-yl]-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (37 mg, 58.7 μmol) in DCM (4 mL) was added TFA (1 mL) and the mixture was then stirred at room temperature for 1 h. After the reaction was completed, the mixture was concentrated in vacuo, and then the residue was purified by preparative HPLC to give 5-[2-ethyl-6-[4-[[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one (17 mg, 44.9% yield) as a white powder. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.50(s,2H),7.54-7.48(m,1H),7.46(d,J=2.2Hz,1H),7.36(dd,J=1.1,2.3Hz,1H),6.85(d,J=8.7Hz,1 H),4.35-4.29(m,2H),4.28-4.23(m,2H),4.16-4.11(m,2H),4.11-3.74(m,6H),3.62(s,3H),3.53(s,2H),3.41(br s,4H),3.29-3.23(m,2H),2.71(q,J=7.5Hz,2H),2.16(s,3H),1.19(t,J=7.5Hz,3H).MS: Calculated value 531(M+H + ), actual value 531 (M+H + ).

[0083] Example 2 5-[2-ethyl-6-[4-[[6-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)-3-pyridyl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0084] The title compound was prepared in the same manner as in Example 1, except that in step 4, 2-chloro-5-(chloromethyl)pyridine (CAS number 70258-18-3, vendor: TCI) was used instead of 2-chloro-5-(chloromethyl)pyrimidine (CAS number 148406-13-7, distributor: PharmaBlock (Nanjing) R&D Co. Ltd, catalog PBN 20120209).

[0085] Example 2 (45 mg, 20.6%) was obtained as a white powder. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.20(d,J=1.8Hz,1H),7.90(dd,J=2.2,8.9Hz,1H),7.50-7.44(m,2H),7.36(dd, J=1.1,2.3Hz,1H),6.87-6.80(m,1H),6.77(d,J=8.9Hz,1H),4.36-4.29(m,4H),4.19(d,J= 9.9Hz,2H),4.05-3.75(m,6H),3.65-3.60(m,3H),3.59-3.54(m,2H),3.44-3.34(m,4H),3. 30-3.25(m,2H),2.70(q,J=7.5Hz,2H),2.16(s,3H),1.19(t,J=7.5Hz,3H).MS: Calculated value 530(M+H + ), actual value 530 (M+H + ).

[0086] Example 3 5-[6-[4-[[2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0087] The title compound was prepared in the same manner as in Example 1, except that in step 5, tert-butyl N-(3-methylazetidin-3-yl)carbamate (CAS number 1018443-01-0, supplier: PharmaBlock (Nanjing) R&D Co. Ltd, catalog PB03046) was used instead of the compound tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (CAS number 1251011-05-8, supplier: PharmaBlock (Nanjing) R&D Co. Ltd, catalog PBN 20111063).

[0088] Example 3 (15 mg, 45.8%) was obtained as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.52(s,2H),7.48-7.43(m,2H),7.36(dd,J=1.1,2.4Hz,1H),6.82(d,J=8.7Hz,1H),4.31(s,2H),4.29-4.15(m,4H) ,4.15-3.53(m,7H),3.48-3.33(m,4H),2.69(q,J=7.5Hz,2H),2.16(s,3H),1.69(s,3H),1.19(t,J=7.5Hz,3H).MS: Calculated value 489(M+H) + ), actual value 489 (M+H + ).

[0089] Example 4 5-[6-[4-[[2-[(6S)-6-amino-1,4-oxazepan-4-yl]pyrimidin-5-yl]methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0090] The title compound was prepared in the same manner as in Example 1, except that in step 5, tert-butyl N-[(6S)-1,4-oxazepan-6-yl]carbamate (CAS number 2306247-11-8, supplier: PharmaBlock (Nanjing) R&D Co. Ltd, catalog PB97931) was used instead of tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (CAS number 1251011-05-8, supplier: PharmaBlock (Nanjing) R&D Co. Ltd, catalog PBN20111063).

[0091] Example 4 (7 mg, 29.3%) was obtained as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.51(s,2H),7.45-7.48(m,1H),7.44(d,J=2.2Hz,1H),7.34-7.37(m,1H),6.79-6. 84(m,1H),4.43(dd,J=15.0,4.6Hz,1H),4.31(s,2H),4.10-4.21(m,1H),4.04(s,3H),3.91(br t,J=2.9Hz,7H),3.61(s,3H),3.41(br s,4H),2.69(d,J=7.6Hz,2H),2.16(s,3H),1.19 ppm(t,J=7.5Hz,3H).MS: Calculated value 519(M+H + ), actual value 519 (M+H + ).

[0092] Example 5 5-[2-ethyl-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0093] The title compound was prepared according to the following scheme. [ka]

[0094] Step 1: Preparation of tert-butyl 4-[4-[[4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]piperazin-1-yl]methyl]phenyl]piperazine-1-carboxylate [ka]

[0095] A mixture of NaBH(OAc)3 (369 mg, 1.74 mmol), 5-(2-ethyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (68 mg, 218 μmol), and tert-butyl 4-(4-formylphenyl)piperazine-1-carboxylate (253 mg, 871 μmol, CAS number 197638-83-8, vendor: Accela ChemBio, catalog SY031491) in DCM (10 mL) was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was then concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 4-[4-[[4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]piperazin-1-yl]methyl]phenyl]piperazine-1-carboxylate (19 mg, 14.9% yield) as a yellow oil. MS: calculated 587 (M+H + ), actual value 587 (M+H + ).

[0096] Step 2: Preparation of 5-[2-ethyl-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0097] To a solution of tert-butyl 4-[4-[[4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]piperazin-1-yl]methyl]phenyl]piperazine-1-carboxylate in DCM (4 mL) was added TFA (1 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated in vacuo. The residue was then purified by preparative HPLC to give 5-[2-ethyl-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one (11 mg, 55.4% yield) as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=7.48-7.42(m,4H),7.35(d,J=1.2Hz,1H),7.13(d,J=8.8Hz,2H),6.81-6.76(m,1H),4.32(s,2H),3. 72-3.32(m,15H),3.30-2.78(m,4H),2.67(q,J=7.5Hz,2H),2.16(s,3H),1.18(t,J=7.5Hz,3H).MS: Calculated value 487(M+H + ), actual value 487 (M+H + ).

[0098] Example 6 5-[2-Isopropyl-6-[4-[[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0099] The title compound was prepared according to the following scheme. [ka]

[0100] Step 1: Preparation of 5-(6-chloro-2-isopropyl-3-pyridyl)-1,3-dimethyl-pyridin-2-one [ka]

[0101] Compound 6a was prepared similarly to compound 1a, except that in step 1, 6-chloro-2-ethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (intermediate pair 4) was used instead of 6-chloro-2-isopropyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine. MS: calculated 277 (M+H + ), actual value 277 (M+H + ).

[0102] Step 2: Preparation of 5-(2-isopropyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one [ka]

[0103] Compound 6b was prepared similarly to compound 1c, except that in step 2, 5-(6-chloro-2-isopropyl-3-pyridyl)-1,3-dimethyl-pyridin-2-one was used instead of 5-(6-chloro-2-ethyl-3-pyridyl)-1,3-dimethyl-pyridin-2-one. MS: calculated 327 (M+H + ), actual value 327 (M+H + ).

[0104] Step 3: Preparation of 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0105] A mixture of 2-chloropyrimidine-5-carbaldehyde (180 mg, 1.26 mmol, CAS number 933702-55-7, supplier: PharmaBlock Sciences (Nanjing), Inc., catalog PB01503), 5-(2-isopropyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (300 mg, 919 μmol), NaBH(OAc) (300 mg, 1.42 mmol) in DCM (10 ml) was stirred at 25° C. for 16 hours. After the reaction was completed, the mixture was then concentrated in vacuo, and the residue was purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one (460 mg, 110%) as a yellow oil. MS: calculated 453 (M+H + ), actual value 453 (M+H + ).

[0106] Step 4: Preparation of 5-[2-isopropyl-6-[4-[[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0107] The title compound was prepared similarly to the preparation of Example 1 by using 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one in step 5 instead of 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one.

[0108] Example 6 (30 mg) was obtained as a yellow solid. 1H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.50(s,2H),7.43-7.38(m,2H),7.34-7.31(m,1H),6.76(d,J=8.6H z,1H),4.35-4.21(m,5H),4.13(d,J=10.1Hz,2H),4.04-3.93(m,2H),3.32(br s,10H),3.30-3.03(m,5H),2.16(s,3H),1.18(d,J=6.7Hz,6H).MS: Calculated value 545(M+H + ), actual value 545 (M+H + ).

[0109] Example 7 5-[2-Isopropyl-6-[4-[(5-piperazin-1-yl-2-pyridyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0110] Step 1: Preparation of 5-[6-[4-[(5-bromo-2-pyridyl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0111] Compound 7a was prepared similarly to compound 6c by using 5-bromopyridine-2-carbaldehyde instead of 2-chloropyrimidine-5-carbaldehyde. MS: calculated 496 (M+H + ), actual value 496 (M+H + ).

[0112] Step 2: Preparation of 5-[2-isopropyl-6-[4-[(5-piperazin-1-yl-2-pyridyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0113] The title compound was prepared in a manner similar to that of Example 1, except that in step 5, 5-[6-[4-[(5-bromo-2-pyridyl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl piperazine-1-carboxylate were used instead of 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl piperazine-1-carboxylate.

[0114] Example 7 (6.0 mg) was obtained as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.48(d,J=2.7Hz,1H),7.55-7.50(m,1H),7.48-7.44(m,1H), 7.43-7.38(m,2H),7.34-7.31(m,1H),6.76(d,J=8.7Hz,1H),4.44(s,2H) ),4.10-3.74(m,4H),3.61(s,3H),3.60-3.53(m,4H),3.49-3.39(m,8H) ,3.15-3.05(m,1H),2.16(s,3H),1.18(d,J=6.7Hz,6H).MS: Calculated value 502(M+H + ), actual value 502 (M+H + ).

[0115] Example 8 5-[2-Isopropyl-6-[4-[(5-piperazin-1-ylpyrazin-2-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0116] Step 1: Preparation of 5-[6-[4-[(5-chloropyrazin-2-yl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0117] Compound 8a was prepared similarly to compound 1d, except that 5-(2-isopropyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one and 2-chloro-5-(chloromethyl)pyrazine (CAS number 105985-21-5, vendor: Bide Pharmatech, catalog BD228124) were used instead of 5-(2-ethyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one and 2-chloro-5-(chloromethyl)pyrimidine. MS: calculated value 453 (M + H + ), actual value 453 (M+H + ).

[0118] Step 2: Preparation of 5-[2-isopropyl-6-[4-[(5-piperazin-1-ylpyrazin-2-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0119] The title compound was prepared in a manner similar to that of Example 1, except that in step 5, 5-[6-[4-[(5-chloropyrazin-2-yl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl piperazine-1-carboxylate were used instead of 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate.

[0120] Example 8 (26.0 mg) was obtained as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.54-8.40(m,1H),8.28(d,J=1.3Hz,1H),7.47-7.37(m,2H),7.33(dd,J=1.0,2.3Hz,1H),6.76(d,J=8.7Hz,1H),4.74-4.15(m,4H) ,4.03-3.93(m,4H),3.85-3.31(m,12H),3.29-3.16(m,1H),3.09(qd,J=6.7,13.4Hz,1H),2.16(s,3H),1.18(d,J=6.6Hz,6H).MS: Calculated value 503(M+H) + ), actual value 503 (M+H + ).

[0121] Example 9 5-[2-Isopropyl-6-[4-[(2-piperazin-1-ylpyrimidin-5-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0122] The title compound was prepared similarly to the preparation of Example 1 by using 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl piperazine-1-carboxylate in step 5 instead of 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate.

[0123] Example 9 (28 mg, 51.6%) was obtained as a yellow solid. 1H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.57-8.52(m,2H),7.43-7.39(m,2H),7.34-7.31(m,1H),6.76(d,J=8.6Hz,1H),4.74-4.38(m,2H),4.37-4.26(m,2H),4.21-4. 11(m,4H),4.13-3.99(m,1H),3.85-3.34(m,8H),3.29-2.97(m,4H),2.16(s,3H),1.22-1.17(m,1H),1.18(d,J=6.6Hz,6H).MS: Calculated value 503(M+H) + ), actual value 503 (M+H + ).

[0124] Example 10 5-[6-[4-[[2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0125] The title compound was prepared in the same manner as in Example 1, except that in step 5, 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate were replaced with 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl N-(3-methylazetidin-3-yl)carbamate (CAS number 1018443-01-0, distributor: PharmaBlock (Nanjing) R&D Co. Ltd, catalog PB03046).

[0126] Example 10 (28 mg, 42%) was obtained as an off-white powder. 1H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.52(s,2H),7.43-7.38(m,2H),7.32(dd,J=1.0,2.3Hz,1H),6.75(d,J=8.6Hz,1H),4.44-4.01(m ,7H),3.89-3.32(m,8H),3.29-3.02(m,2H),2.16(s,3H),1.70(s,3H),1.18(d,J=6.7Hz,6H).MS: Calculated value 503(M+H + ), actual value 503 (M+H + ).

[0127] Example 11 5-[2-Isopropyl-6-[4-[(6-piperazin-1-yl-3-pyridyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0128] Step 1: Preparation of 5-[6-[4-[(6-chloro-3-pyridyl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0129] Compound 11a was prepared similarly to compound 1d, except that 5-(2-isopropyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one and 2-chloro-5-(chloromethyl)pyridine (CAS number 70258-18-3, supplier: TCI) were used instead of 5-(2-ethyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one and 2-chloro-5-(chloromethyl)pyrimidine. MS: calculated 452 (M+H + ), actual value 452 (M+H + ).

[0130] Step 2: Preparation of 5-[2-isopropyl-6-[4-[(6-piperazin-1-yl-3-pyridyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0131] The title compound was prepared in a manner similar to that of Example 1, except that in step 5, 5-[6-[4-[(6-chloro-3-pyridyl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl piperazine-1-carboxylate were used instead of 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate.

[0132] Example 11 (28 mg, 53.7%) was obtained as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.30(d,J=2.3Hz,1H),7.79(dd,J=2.4,8.9Hz,1H),7.43-7.38(m,2H),7.34-7.31(m,1H),7.04(d,J=8.9Hz,1H),6.75(d,J=8.6 Hz,1H),4.83-4.07(m,4H),4.01-3.80(m,5H),3.68-3.32(m,10H),3.28-2.92(m,3H),2.16(s,3H),1.18(d,J=6.6Hz,6H).MS: Calculated value 502(M+H) + ), actual value 502 (M+H + ).

[0133] Example 12 5-[2-Isopropyl-6-[4-[(5-piperazin-1-ylpyrimidin-2-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0134] Step 1: Preparation of 5-[6-[4-[(5-chloropyrimidin-2-yl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0135] Compound 12a was prepared in the same manner as compound 6c, except that 2-chloropyrimidine-5-carbaldehyde (CAS No. 933702-55-7, supplier: PharmaBlock Sciences (Nanjing), Inc., catalog PB01503) was used instead of 2-chloropyrimidine-5-carbaldehyde. MS: calculated value 453 (M + H + ), actual value 453 (M+H + ).

[0136] Step 2: Preparation of 5-[2-isopropyl-6-[4-[(5-piperazin-1-ylpyrimidin-2-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0137] The title compound was prepared similarly to the preparation of Example 1 by using 5-[6-[4-[(5-chloropyrimidin-2-yl)methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl piperazine-1-carboxylate in step 5 instead of 5-[6-[4-[(2-chloropyrimidin-5-yl)methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one and tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate.

[0138] Example 12 (30 mg, 42.5%) was obtained as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.63(s,2H),7.44-7.39(m,2H),7.35-7.32(m,1H),6.78(d,J=8.7Hz,1H),4.78-4.01( m,4H),3.91-3.32(m,17H),3.17-3.02(m,1H),2.16(s,3H),1.18(d,J=6.7Hz,6H).MS: Calculated value 503(M+H + ), actual value 503 (M+H + ).

[0139] Example 13 5-[2-Isopropyl-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0140] The title compound was prepared similarly to the preparation of Example 5, by using 5-(2-isopropyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one in place of 5-(2-ethyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one in step 1.

[0141] Example 13 (28 mg, 43.3%) was obtained as a yellow solid. 1H NMR(400MHz,CD3OD,298 K)δ(ppm)=7.45(d,J=8.8Hz,2H),7.42-7.37(m,2H),7.34-7.30(m,1H),7.13(d,J=8.8Hz,2H),6.74(d,J=8.6Hz,1H),4.70-4.41(m,2H) ,4.32(s,2H),3.61(s,3H),3.56-3.44(m,6H),3.41-3.36(m,4H),3.28-2.98(m,5H),2.16(s,3H),1.17(d,J=6.7Hz,6H).MS: Calculated value 501(M+H) + ), actual value 501 (M+H + ).

[0142] Example 14 5-[2-Isopropyl-6-[4-[(3-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0143] The title compound was prepared similarly to the preparation of Example 5, by using 5-(2-isopropyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one and tert-butyl 4-(3-formylphenyl)piperazine-1-carboxylate (CAS number 1257849-25-4, distributor: Bide Pharmatech, catalog BD168751) in step 1 instead of 5-(2-ethyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one and tert-butyl 4-(4-formylphenyl)piperazine-1-carboxylate.

[0144] Example 14 (39 mg, 66.7%) was obtained as a yellow solid. 1H NMR(400MHz,CD3OD,298 K)δ(ppm)=7.65-7.59(m,1H),7.58-7.51(m,1H),7.45-7.31(m,5H),6.73(d,J=8.6Hz,1H),4.60-4.09(m,4 H),3.61(s,3H),3.53-3.32(m,8H),3.28-2.99(m,7H),2.16(s,3H),1.17(d,J=6.7Hz,6H).MS: Calculated value 501(M+H + ), actual value 501 (M+H + ).

[0145] Example 15 5-[2-Isopropyl-6-[4-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0146] The title compound was prepared according to the following scheme. [ka]

[0147] Step 1: Preparation of tert-butyl 2-[4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]piperazin-1-yl]-7,8-dihydro-5H-1,6-naphthyridine-6-carboxylate [ka]

[0148] To a mixture of 5-(2-isopropyl-6-piperazin-1-yl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (100 mg, 306 μmol), tert-butyl 2-chloro-7,8-dihydro-5H-1,6-naphthyridine-6-carboxylate (206 mg, 766 μmol, CAS number 1151665-15-4, distributor: Bide Pharmatech, catalog BD216990), CsCO (299 mg, 919 μmol) in dioxane (5 mL) was added RuPhos Pd G (47.6 mg, 61.3 μmol, CAS number 1375325-68-0, distributor: ALDRICH, catalog 753246) and the mixture was stirred at 110 °C under N atmosphere for 16 h. After the reaction was completed, the mixture was concentrated in vacuo, and the residue was purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 2-[4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]piperazin-1-yl]-7,8-dihydro-5H-1,6-naphthyridine-6-carboxylate (100 mg, 58.4% yield) as a yellow oil. MS: calculated 559 (M+H + ), actual value 559 (M+H + ).

[0149] Step 2: Preparation of 5-[2-isopropyl-6-[4-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0150] To a solution of tert-butyl 2-[4-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]piperazin-1-yl]-7,8-dihydro-5H-1,6-naphthyridine-6-carboxylate in DCM (4 mL) was added TFA (1 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was concentrated in vacuo. The residue was then purified by preparative HPLC to give 5-[2-isopropyl-6-[4-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one (34 mg) as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=7.66(d,J=9.2Hz,1H),7.48-7.42(m,2H),7.35(dd,J=1.0,2.3Hz,1H),7.11(d,J=9.2Hz,1H),6.79(d,J=8.8Hz,1H),4.29(s,2H), 3.89-3.79(m,8H),3.63-3.61(m,3H),3.21-3.15(m,2H),3.15-3.07(m,1H),2.16(s,3H),2.03(s,2H),1.23(d,J=6.7Hz,6H).MS: Calculated value 459(M+H) + ), actual value 459 (M+H + ).

[0151] Example 16 5-[2-Isopropyl-6-[8-(6-piperazin-1-yl-3-pyridyl)-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0152] The title compound was prepared according to the following scheme. [ka]

[0153] Step 1: Preparation of tert-butyl 8-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazine-2-carboxylate [ka]

[0154] To a mixture of 5-(6-chloro-2-isopropyl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (100 mg, 361 μmol), tert-butyl 1,3,4,6,7,8,9,9a-octahydropyrazino[1,2-a]pyrazine-2-carboxylate (87.2 mg, 361 μmol, CAS number 1159825-34-9, supplier: PharmaBlock Sciences (Nanjing), Inc., catalog PB07063) and CsCO (235 mg, 723 μmol) in dioxane (3 mL), RuPhos Pd G (14 mg, 18.1 μmol) was added, and the mixture was stirred at 110 °C under a N atmosphere for 16 hours. After the reaction was complete, the mixture was then concentrated in vacuo. The residue was then purified by flash column elution with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 8-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazine-2-carboxylate (143 mg, 82.2% yield) as a yellow oil. MS: calculated 482 (M+H + ), actual value 482 (M+H + ).

[0155] Step 2: Preparation of 5-[6-(1,3,4,6,7,8,9,9a-octahydropyrazino[1,2-a]pyrazin-2-yl)-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0156] To a solution of tert-butyl 8-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazine-2-carboxylate (143 mg, 297 μmol) in DCM (4 mL) was added TFA (1 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated in vacuo. The residue was diluted with 2M KOH solution (5 mL), and the resulting mixture was extracted twice with DCM (30 mL). The combined organic layers were concentrated in vacuo to give crude 5-[6-(1,3,4,6,7,8,9,9a-octahydropyrazino[1,2-a]pyrazin-2-yl)-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one (87 mg, 76.8% yield) as a light brown oil, which was used directly in the next step without further purification. MS: calculated 382 (M+H + ), actual value 382 (M+H + ).

[0157] Step 3: Preparation of tert-butyl 4-[5-[2-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-8-yl]-2-pyridyl]piperazine-1-carboxylate [ka]

[0158] To a mixture of 5-[6-(1,3,4,6,7,8,9,9a-octahydropyrazino[1,2-a]pyrazin-2-yl)-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one (87 mg, 228 μmol), tert-butyl 4-(5-bromo-2-pyridyl)piperazine-1-carboxylate (156 mg, 456 μmol, CAS number 153747-97-8, vendor: Accela ChemBio Inc., catalog SY101561), and CsCO (149 mg, 456 μmol) in dioxane (3 mL) was added RuPhos Pd G (8.86 mg, 11.4 μmol), and the mixture was stirred at 110 °C under a N atmosphere for 16 hours. After the reaction was complete, the mixture was concentrated in vacuo. The residue was then purified by flash column elution with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 4-[5-[2-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-8-yl]-2-pyridyl]piperazine-1-carboxylate (16 mg, 10.9% yield) as a light brown oil. MS: calculated 643 (M+H + ), actual value 643 (M+H + ).

[0159] Step 4: Preparation of 5-[2-isopropyl-6-[8-(6-piperazin-1-yl-3-pyridyl)-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0160] To a solution of tert-butyl 4-[5-[2-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-8-yl]-2-pyridyl]piperazine-1-carboxylate (16 mg, 0.024 mmol) in DCM (4 mL) was added TFA (1 mL), and the mixture was then stirred at room temperature for 1 h. After the reaction was completed, the mixture was concentrated in vacuo, and then the residue was purified by preparative HPLC to give 5-[2-isopropyl-6-[8-(6-piperazin-1-yl-3-pyridyl)-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one (9 mg, 46.8% yield) as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=7.89(d,J=2.8Hz,1H),7.79(dd,J=9.4,2.9Hz,1H),7.39-7.46(m, 2H),7.31-7.35(m,1H),7.19(d,J=9.5Hz,1H),6.81(d,J=8.7Hz,1H),4.72(br s,2H),3.91(br d,J=12.6Hz,1H),3.58-3.71(m,6H),3.19-3.46(m,12H),2.99-3.19(m,3H),2.16(s,3H),1.16-1.23(m,7H).MS: Calculated value 543(M+H) + ), actual value 543 (M+H + ).

[0161] Example 17 5-[2-(Difluoromethyl)-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0162] The title compound was prepared according to the following scheme. [ka]

[0163] Step 1: Preparation of tert-butyl 4-[5-bromo-6-(difluoromethyl)-2-pyridyl]piperazine-1-carboxylate [ka]

[0164] A mixture of CsCO (302 mg, 928 μmol), 3-bromo-6-chloro-2(difluoromethyl)pyridine (150 mg, 619 μmol), and tert-butyl piperazine-1-carboxylate (173 mg, 928 μmol) in DMF (3 mL) was stirred at 120 °C for 12 h. After the reaction was complete, the mixture was diluted with water (10 mL), and the resulting mixture was extracted twice with DCM (30 mL). The combined organic layers were concentrated in vacuo, and the residue was then purified by flash column elution with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 4-[5-bromo-6-(difluoromethyl)-2-pyridyl]piperazine-1-carboxylate (123 mg, 50.7% yield) as a yellow oil. MS: calculated 393 (M+H + ), actual value 393 (M+H + ).

[0165] Step 2: Preparation of tert-butyl 4-[6-(difluoromethyl)-5-(1,5-dimethyl-6-oxo-3-pyridyl)-2-pyridyl]piperazine-1-carboxylate [ka]

[0166] To a mixture of tert-butyl 4-(5-bromo-6-(difluoromethyl)pyridin-2-yl)piperazine-1-carboxylate (123 mg, 314 μmol), 1,3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one (78.1 mg, 314 μmol), and KCO (65 mg, 470 μmol) in a mixed solvent of dioxane (2 mL) and water (0.5 mL), PdCl(DPPF)-CHCl adduct (11.5 mg, 15.7 μmol) was added, and the mixture was stirred in dioxane / HO (5:1, 2.5 mL) under a N atmosphere at 80 °C for 16 hours. After the reaction was complete, the mixture was concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 4-[6-(difluoromethyl)-5-(1,5-dimethyl-6-oxo-3-pyridyl)-2-pyridyl]piperazine-1-carboxylate (120 mg, 88.2% yield) as an orange solid. MS: calculated 435 (M+H + ), actual value 435 (M+H + ).

[0167] Step 3: Preparation of 5-[2-(difluoromethyl)-6-piperazin-1-yl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0168] To a solution of tert-butyl 4-[6-(difluoromethyl)-5-(1,5-dimethyl-6-oxo-3-pyridyl)-2-pyridyl]piperazine-1-carboxylate (120 mg, 275 μmol) in DCM (4 mL) was added (1 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was then concentrated in vacuo, the residue was diluted with 2M KOH solution (5 mL), and the resulting mixture was extracted twice with DCM (30 mL). The combined organic layers were concentrated in vacuo to give crude 5-[2-(difluoromethyl)-6-piperazin-1-yl-3-pyridyl]-1,3-dimethyl-pyridin-2-one (92.4 mg, 100%) as a yellow oil, which was used directly in the next step without further purification. MS: calculated 335 (M+H + ), actual value 335 (M+H + ).

[0169] Step 4: Preparation of tert-butyl 4-[4-[[4-[6-(difluoromethyl)-5-(1,5-dimethyl-6-oxo-3-pyridyl)-2-pyridyl]piperazin-1-yl]methyl]phenyl]piperazine-1-carboxylate [ka]

[0170] A mixture of 2'-(difluoromethyl)-1,5-dimethyl-6'-(piperazin-1-yl)-[3,3'-bipyridin]-6(1H)-one (92.4 mg, 275 μmol), tert-butyl 4-(4-formylphenyl)piperazine-1-carboxylate (472 mg, 1.62 mmol) and NaBH(OAc) (574 mg, 2.71 mmol) in DCM (5 mL) was stirred at 25 °C for 16 h. After the reaction was completed, the mixture was then concentrated in vacuo, and the residue was purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 4-[4-[[4-[6-(difluoromethyl)-5-(1,5-dimethyl-6-oxo-3-pyridyl)-2-pyridyl]piperazin-1-yl]methyl]phenyl]piperazine-1-carboxylate (124 mg, 75.1% yield) as a yellow oil. MS: calculated 609 (M+H + ), actual value 609 (M+H + ).

[0171] Step 5: Preparation of 5-[2-(difluoromethyl)-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0172] To a solution of tert-butyl 4-[4-[[4-[6-(difluoromethyl)-5-(1,5-dimethyl-6-oxo-3-pyridyl)-2-pyridyl]piperazin-1-yl]methyl]phenyl]piperazine-1-carboxylate (125 mg, 205 μmol) in DCM (4 mL) was added TFA (0.5 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was concentrated in vacuo. The residue was then purified by preparative HPLC to give 5-[2-(difluoromethyl)-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one (15 mg, 11.5% yield) as a yellow solid. 1H NMR(400MHz,CD3OD,298 K)δ(ppm)=7.62(d,J=8.7Hz,1H),7.50-7.43(m,3H),7.38(s,1H),7.16-7.07(m,3H),6.75-6.4 1(m,1H),4.77-4.39(m,2H),4.33(s,2H),3.68-3.33(m,14H),3.31-2.99(m,3H),2.15(s,3H). 19 F NMR(376MHz,CD3OD,298 K)δ(ppm)=-113.52(d,J=54.5Hz,2F).MS: Calculated value 509(M+H + ), actual value 509 (M+H + ).

[0173] Example 18 5-[2-Isopropyl-6-(4-piperazin-1-yl-1-piperidyl)-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0174] The title compound was prepared according to the following scheme. [ka]

[0175] Step 1: Preparation of tert-butyl 4-[1-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]-4-piperidyl]piperazine-1-carboxylate [ka]

[0176] To a mixture of 5-(6-chloro-2-isopropyl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (80 mg, 289 μmol), tert-butyl 4-(4-piperidyl)piperazine-1-carboxylate (100 mg, 371 μmol, CAS number 205059-24-1, vendor: Bide Pharmatech, catalog BD57121), CsCO (150 mg, 425 μmol) in dioxane (5 mL), RuPhos Pd G (15 mg, 19.3 μmol) was added, and the mixture was stirred at 110° C. under a N atmosphere for 16 hours. After the reaction was complete, the mixture was then concentrated in vacuo. The residue was then purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 4-[1-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]-4-piperidyl]piperazine-1-carboxylate (50 mg, 33.9%) as a yellow oil. MS: calculated 510 (M+H + ), actual value 510 (M+H + ).

[0177] Step 2: Preparation of 5-[2-isopropyl-6-(4-piperazin-1-yl-1-piperidyl)-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0178] To a solution of tert-butyl 4-[1-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-isopropyl-2-pyridyl]-4-piperidyl]piperazine-1-carboxylate (50 mg, 0.098 mmol) in DCM (4 mL) was added TFA (1 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated in vacuo. The residue was then purified by preparative HPLC to give 5-[2-isopropyl-6-(4-piperazin-1-yl-1-piperidyl)-3-pyridyl]-1,3-dimethyl-pyridin-2-one (24 mg, 46.7%) as a yellow solid. 1H NMR(400MHz,CD3OD,298 K)δ(ppm)=7.44-7.39(m,2H),7.32(dd,J=1.0,2.3Hz,1H),6.79(d,J=8.7Hz,1H),4.59(br d,J=13.6Hz,2H),3.61(s,3H),3.59-3.50(m,8H),3.49-3.41(m,1H),3.15-3.06(m,1H),2.99(br t,J=11.9Hz,2H),2.24-2.12(m,5H),1.76(br dd,J=4.0,12.2Hz,2H),1.20(d,J=6.7Hz,6H).MS: Calculated value 410(M+H + ), actual value 410 (M+H + ).

[0179] Example 19 5-[2-Ethyl-6-[2-[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-8-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0180] The title compound was prepared according to the following scheme. [ka]

[0181] Step 1: Preparation of tert-butyl 2-(5-bromopyrimidin-2-yl)-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate [ka]

[0182] A mixture of 5-bromo-2-chloro-pyrimidine (300 mg, 1.55 mmol, CAS number 32779-36-5, supplier: Accela ChemBio Inc, catalog 32779-36-5), tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (425 mg, 1.88 mmol) and KCO (322 mg, 2.33 mmol) in DMSO (3 mL) was stirred at 90° C. for 3 hours. After the reaction was complete, the mixture was diluted with water (5 mL) and the resulting mixture was extracted twice with DCM (30 mL). The combined organic layers were concentrated in vacuo, and the residue was purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 2-(5-bromopyrimidin-2-yl)-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (539 mg, 90.2% yield) as a white solid. MS: calculated 385 (M+H + ), actual value 385 (M+H + ).

[0183] Step 2: Preparation of tert-butyl 8-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazine-2-carboxylate [ka]

[0184] To a mixture of 5-(6-chloro-2-ethyl-3-pyridyl)-1,3-dimethyl-pyridin-2-one (555 mg, 2.1 mmol, compound 1a), tert-butyl 1,3,4,6,7,8,9,9a-octahydropyrazino[1,2-a]pyrazine-2-carboxylate (608 mg, 2.5 mmol, CAS number 1159825-34-9, supplier: PharmaBlock Sciences (Nanjing), catalog PB07063) and CsCO (1.0 g, 3.1 mmol) was added RuPhos Pd G (81 mg, 110 μmol), and the mixture was stirred in dioxane (5 mL) under a N atmosphere at 110 °C for 16 hours. After the reaction was completed, the mixture was then concentrated in vacuo, and the residue was purified by flash column eluting with a gradient of MeOH / DCM (0% to 10%) to give tert-butyl 8-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazine-2-carboxylate (517 mg, 52.6% yield) as a yellow oil. MS: calculated 468 (M+H + ), actual value 468 (M+H + ).

[0185] Step 3: Preparation of 5-[6-(1,3,4,6,7,8,9,9a-octahydropyrazino[1,2-a]pyrazin-2-yl)-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0186] To a solution of tert-butyl 8-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazine-2-carboxylate (517 mg, 1.1 mmol) in DCM (4 mL) was added TFA (1 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated in vacuo, and the residue was adjusted to pH 12 by the addition of 2 M KOH solution. The resulting mixture was extracted twice with DCM (30 mL). The combined organic layers were concentrated in vacuo to give crude 5-[6-(1,3,4,6,7,8,9,9a-octahydropyrazino[1,2-a]pyrazin-2-yl)-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one (376 mg, 93% yield) as an orange oil. MS: calculated 368 (M+H + ), actual value 368 (M+H + ).

[0187] Step 4: Preparation of tert-butyl 2-[5-[8-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl]pyrimidin-2-yl]-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate [ka]

[0188] 5-[6-(1,3,4,6,7,8,9,9a-octahydropyrazino[1,2-a]pyrazin-2-yl)-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one (200 mg, 0.544 mmol), tert-butyl 2-(5-bromopyrimidin-2-yl)-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (209.67 mg, 0.544 mmol), Pd2(dba)3 (99.67 mg, 0.109 mmol, CAS number 51364-51-3, distributor: BePharm), 2-(di-t-butylphosphino)biphenyl (64.96 mg, 0.218 mmol, CAS number 224311-51-7, distributor: J&K) in toluene (5 mL). To a mixture of 2,8-dimethyl-3,5-dioxo-3-pyridyl-6-ethyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl]pyrimidin-2-yl]-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (90 mg, 24.6% yield) was added sodium tert-butoxide (209.2 mg, 2.18 mmol). The mixture was stirred at 110 °C under a N atmosphere for 16 h. After the reaction was complete, the mixture was concentrated in vacuo, and the residue was purified by flash column eluting with a gradient of MeOH / DCM (0% to 15%) to give 2-[5-[8-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl]pyrimidin-2-yl]-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (90 mg, 24.6% yield) as a yellow oil. MS: calculated 672 (M+H + ), actual value 672 (M+H + ).

[0189] Step 5: Preparation of 5-[2-ethyl-6-[2-[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-8-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one [ka]

[0190] To a solution of tert-butyl 2-[5-[8-[5-(1,5-dimethyl-6-oxo-3-pyridyl)-6-ethyl-2-pyridyl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl]pyrimidin-2-yl]-5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (90 mg) in DCM (4 mL) was added TFA (1 mL), and the mixture was then stirred at room temperature for 1 h. After the reaction was completed, the mixture was concentrated in vacuo, and then the residue was purified by preparative HPLC to give 6'-(4-((2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)-2'-ethyl-1,5-dimethyl-[3,3'-bipyridin]-6(1H)-one 2,2,2-trifluoroacetate (42 mg) as a yellow solid. 1 H NMR(400MHz,CD3OD,298 K)δ(ppm)=8.32(s,2H),7.56(d,J=8.7Hz,1H),7.48(d,J=2.2Hz,1H),7.37(dd,J=1.0,2.4Hz,1H),6.93(d,J=8.7Hz,1H),4.63(br s,2H),4.20(d,J=10.0Hz,2H),4.08(d,J=9.8Hz,2H),3.97(dd,J=4.1,5.8Hz,2H),3.82-3.74(m,1H),3.73-3.63(m,4H),3.63-3.60(m,3H),3. 52(s,2H),3.47-3.33(m,3H),3.30-3.17(m,4H),3.14-3.03(m,1H),2.74(q,J=7.5Hz,2H),2.16(s,3H),1.21(t,J=7.5Hz,3H).MS: Calculated value 572(M+H) + ), actual value 572 (M+H + ).

[0191] Example 20

[0192] The following test was carried out to determine the activity of compounds of formula (I) and formula (Ia) in the HEK293-Blue-hTLR-7 / 8 / 9 cell assay.

[0193] HEK293-Blue-hTLR-7 cell assay: The 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 human TLR7 stimulation by monitoring NF-κB activation. The 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. Accordingly, reporter expression was reduced by TLR7 antagonists under ligand stimulation, 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.

[0194] HEK293-Blue-hTLR7 cells were incubated at a density of 250,000–450,000 cells / mL in a 170 μL volume in 96-well plates 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 serially diluted test compound in the presence of 1% final DMSO and 10 μL of 20 μM R848 in the DMEM. The incubation was carried out at 37°C in a CO2 incubator for 20 hours. Then, 20 μL of supernatant from each well was incubated with 180 μL of Quanti-blue substrate solution at 37°C for 2 hours, 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.

[0195] HEK293-Blue-hTLR-8 cell assay: The 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 human TLR8 stimulation by monitoring NF-κB activation. The 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 ligand stimulation, such as R848, for 20 hours of incubation. The activity of the SEAP reporter in the cell culture supernatant was measured at a wavelength of 640 nm using the QUANTI-Blue™ kit (catalog number: rep-qb1, Invivogen, San Diego, California, USA), and the detection medium turned purple or blue in the presence of alkaline phosphatase.

[0196] HEK293-Blue-hTLR8 cells were incubated in a 96-well plate at a density of 250,000–450,000 cells / mL in a volume of 170 μL 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 serially diluted test compound in the presence of 1% final DMSO and 10 μL of 60 μM R848 in the DMEM. The incubation was performed at 37°C in a CO2 incubator for 20 hours. 20 μL of supernatant from each well was then incubated with 180 μL of Quanti-Blue substrate solution for 2 hours at 37°C. 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 modified a similar reporter assay to evaluate TLR8 antagonists.

[0197] HEK293-Blue-hTLR-9 cell assay: The 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 human TLR9 stimulation by monitoring NF-κB activation. The 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. Accordingly, reporter expression was reduced by TLR9 antagonists for 20 hours of incubation under ligand stimulation, such as ODN2006 (catalog number: tlrl-2006-1, Invivogen, San Diego, CA, USA). The activity of the SEAP reporter in the cell culture supernatant was measured at a wavelength of 640 nm using the QUANTI-Blue™ kit (catalog number: rep-qb1, Invivogen, San Diego, CA, USA), and the detection medium turned purple or blue in the presence of alkaline phosphatase.

[0198] HEK293-Blue-hTLR9 cells were incubated in a 96-well plate at a density of 250,000–450,000 cells / mL in a volume of 170 μL 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 serially diluted test compound in the presence of 1% final DMSO and 10 μL of 20 μM ODN2006 in the DMEM. The incubation was continued for 20 hours at 37°C in a CO2 incubator. 20 μL of supernatant from each well was then incubated with 180 μL of Quanti-Blue substrate solution for 2 hours at 37°C. The absorbance was measured at 620–655 nm using a spectrophotometer. Because the signaling pathway in which TLR9 activation leads to downstream activation of NF-κB is widely accepted, a similar reporter assay was modified for the evaluation of TLR9 antagonists.

[0199] The compounds of formula (I) have a human TLR7 and / or TLR8 inhibitory activity (IC 50 The activity data of the compounds of the present invention are shown in Table 1. [Table 1]

[0200] Example 21 hERG channel inhibition assay: The hERG channel inhibition assay is a highly sensitive measure to identify compounds that exhibit hERG inhibition associated with in vivo cardiotoxicity. + The channel has been cloned in human and stably expressed in a CHO (Chinese Hamster Ovary) cell line. hERG Cells were used for patch clamp (voltage clamp, whole cell) experiments. Cells were stimulated with voltage patterns to activate hERG channels and induce I KhERGThe cells were allowed to stabilize for several minutes, after which I KhERG The amplitude and kinetics of the I pulses were 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 minutes, at which point the next higher concentration was applied. KhERG The amplitude and kinetics of the responses were recorded at each drug concentration and compared to the control value (set as 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.Devel.5:116-26).

[0201] The hERG results are shown in Table 2. The safety ratio (hERG IC20 / EC 50 ) suggests a sufficient window to discern pharmacology by inhibiting the TLR7 / 8 / 9 pathway from potential hERG-related cardiotoxicity. 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]

[0202] Example 22 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, primarily composed of lymphocytes, monocytes, and dendritic cells. These cells express TLR7, TLR8, or TLR9 and are therefore natural responders to their respective ligand stimulation. Upon activation of these TLRs, PBMCs secrete similar cytokines and chemokines in vitro and in vivo, so the in vitro efficacy of TLR7 / 8 / 9 antagonists in human PBMCs can be readily translated into pharmacodynamic responses in vivo.

[0203] 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 of PBS (Ca) in a 50 mL conical tube with a porous barrier (Leucosep tube, Greiner bio-one). 2+ , Mg2+ The tubes were centrifuged at 800 × g (1946 rpm) for 20 minutes with the brake in the off position to collect PBMCs from the buffy coat. Next, the cells were washed twice with PBS, and red blood cells were lysed in 2 mL of suspension (Red Blood Cell Lysis Buffer, Alfa Aesar) at room temperature for 5–10 minutes. After the final wash with PBS, the cells were diluted to a final concentration of 2 × 10 in RPMI-1640 medium containing GlutaMAX™ (Gibco) supplemented with 10% fetal bovine serum (Sigma). 6 PBMCs were resuspended at 3 × 10 cells / mL and placed in a tissue culture-treated round-bottom 96-well plate (Corning Incorporated) at 150 μL / well (3 × 10 5 Cells were plated at 1000 x g (cells / well).

[0204] Antagonist compounds (compounds of the present invention) solubilized and serially diluted in 100% DMSO were added to cells in duplicate to a final concentration of 1% DMSO (v / v). PBMCs were incubated with the antagonist compounds for 30 minutes at 37°C and 5% CO2, after which 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 protocol (eBioscience, ThermoFisher Scientific). Cell viability was also confirmed by Cell Viability Assay (CellTiter Glo® Luminescent Cell Viability Assay, Promega). [Table 3]

[0205] Example 23 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.

[0206] Human liver microsomes (Corning, USA, Catalog No. 452117; Xenotech, USA, Catalog No. H2610) were preincubated with test compounds in 100 mM potassium phosphate buffer (pH 7.4) at 37°C for 10 minutes. The reaction was initiated by the addition of an NADPH regenerating system. The final incubation mixture contained 1 μM test compound, 0.5 mg / mL liver microsomal protein, 1 mM MgCl, 1 mM NADP, 1 unit / mL isocitrate dehydrogenase, and 6 mM isocitrate in 100 mM potassium phosphate buffer (pH 7.4). After incubation times of 0, 3, 6, 9, 15, and 30 minutes at 37°C, the reaction was terminated by adding 300 μL of cold acetonitrile (containing the internal standard) to 100 μL of the incubation mixture. After precipitation and centrifugation, the amount of compound remaining in the sample was measured by LC-MS / MS. Controls without the NADPH regenerating system were also prepared and analyzed at 0 and 30 minutes. The compounds of the present invention showed good human liver microsome stability as measured in the above assay. The results are shown in Table 4 below. [Table 4]

[0207] Example 24 3T3 in vitro phototoxicity assay Phototoxicity is defined as a toxic response elicited after initial cutaneous exposure to a specific chemical followed by subsequent exposure to light, or similarly elicited by systemic administration of the chemical followed by cutaneous irradiation. The assay used in this study was designed to detect the phototoxic potential of chemicals using a simple in vitro cytotoxicity assay with Balb / c 3T3 mouse fibroblasts. The principle of this test is to compare the cytotoxicity of tested chemicals with and without exposure to a nontoxic dose of UVA light. Cytotoxicity is expressed as a dose-dependent decrease in cell proliferation rate, as determined by uptake of the vital dye neutral red 1 day after treatment.

[0208] 1. Method Preparation of test item stock solutions and dosages Immediately prior to the start of cell exposure, small amounts of material were 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.

[0209] reference material Chlorpromazine (HCL) (Sigma, batch / lot number: 120M1328V), test concentration: 300 μg / mL, solvent: PBS / 3% DMSO

[0210] Measurement of UV absorption spectra Absorption spectra, either by themselves or after UV-A or UV-B pre-irradiation, were recorded between 240 nm and 400 nm using a Lambda-2 spectrophotometer (Perkin Elmer). [Table 5]

[0211] 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 examine the potential phototoxic potential of test articles. This assay is based on the active uptake of neutral red dye into lysosomes of cultured mouse fibroblasts. Because 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.

[0212] Preparation of cell culture Mouse fibroblast clone A31 (ATCC no. CCL163 - passage no. 108) was cultured in a 175 cm culture medium containing sDMEM (Dulbecco's minimal 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 tissue culture grade flasks at 37°C in a humidified atmosphere of 6% CO. Before approaching confluence, the cells were removed from the flasks by trypsinization. Before use in the assay, the cells were diluted to 1 x 10 in 100 μl of sDMEM. 4 The cells were transferred to 96-well microtiter plates at a concentration of 100 cells / well and allowed to attach for 24 hours.

[0213] Exposure to test items For incubation with mouse fibroblasts, test items were diluted in PBS / 3% DMSO (see Results for detailed concentrations).

[0214] The culture medium (Dulbecco's Modified Eagle's 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 the mouse fibroblasts were washed with PBS. Then, 100 μL of PBS / 3% DMSO containing the test item was added, and the target cells were incubated for 1 hour at 37°C in 6% CO2.

[0215] UV exposure For each test item, microtiter plates were prepared according to Table 6. "UVA plates" were prepared at approximately 5 J / cm 2 Plates were exposed to 1000 uV of UVA light, and "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).

[0216] 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]

[0217] A 96-well microtiter plate was prepared as follows: 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 item, for calculation of the standard cell viability curve. Wells labeled U01–U08 contained different concentrations of the test item.

[0218] 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.

[0219] 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.

[0220] 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 vigorously shaking the plate on a microtiter plate shaker for at least 10 minutes until the neutral red was extracted from the cells and formed a homogenous solution, the absorbance of the resulting colored solution was measured at 540 nm using a SPECTRAmax PLUS microtiter plate reader (Molecular Devices).

[0221] 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)

[0222] This allowed the calculation of cell viability after incubation with increasing concentrations of the test chemicals. Chlorpromazine (HCl) served as a positive control for the experiment.

[0223] 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).

[0224] Calculation of the discrimination coefficient for phototoxicity IC determined with and without UV exposure to assess potential phototoxicity 50 The values ​​were compared. Coefficient = IC 50 (-UV) / IC 50 (+UV)

[0225] 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).

[0226] Test compounds that are not cytotoxic to mouse fibroblasts, even at the highest concentrations tested, but that 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 De O, 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).

[0227] The test results are shown below, and the compounds of the present invention showed a very good phototoxicity profile. [Table 7]

[0228] Example 25 Parallel artificial membrane permeability assay (PAMPA) PAMPA (Parallel Artificial Membrane Permeability Assay) is a first-order selective permeability screen for drug candidates. This assay uses artificial phospholipid membranes to mimic transcellular absorption conditions and generates permeability values ​​that can be used for compound ranking and optimization, as well as input parameters for in silico models to predict intestinal absorption.

[0229] Permeation experiments are performed in hydrophobic PVDF 96-well microtiter filter plates (MultiScreen filter plates, Millipore, #MAIPN 4550). Each well is coated with a PVDF membrane, which is prepared with 5 μL dodecane (Sigma, D221104) containing 1% lecithin (Sigma, P3556-1G).

[0230] A typical PAMPA experimental protocol is as follows: 150 μL of 100 mM PBS buffer (2.6 g KH2PO4 and 18.5 g K2HPO4.3H2O) is dissolved in approximately 1000 mL of ultrapure water and mixed thoroughly. The pH is adjusted to 7.40 ± 0.05 using either 1 M sodium hydroxide or 1 M hydrochloric acid containing 5% DMSO. The bottom filter of each acceptor well is filled with 300 μL of 100 mM PBS buffer (2.6 g KH2PO4 and 18.5 g K2HPO4.3H2O are dissolved in approximately 1000 mL of ultrapure water and mixed thoroughly. The pH is adjusted to 7.40 ± 0.05 using either 1 M sodium hydroxide or 1 M hydrochloric acid). The resulting sandwich is incubated at room temperature for 4 hours under constant shaking (300 rpm). The sandwich is then disassembled. Prior to incubation, 20 μL of the dosing solution was spiked and mixed with 250 μL of PBS and 130 μL of quenching solution (acetonitrile) as the TO sample. After incubation, 270 μL of solution was withdrawn from the acceptor chamber, followed by the addition of 130 μL of acetonitrile. 20 μL of solution was withdrawn from the donor chamber, followed by the addition of 250 μL of PBS and 130 μL of acetonitrile. The concentrations of the compounds in all samples were determined by LC-MS / MS, and the permeability (Pe, 10 -6 The formula for calculating cm / s is as follows:

number

[0231] V D is the volume of the donor well, and V Ris the volume of the acceptor well, area is the active surface area of ​​the membrane, time is the incubation time (14,400 seconds in this assay), and C R and C D are the concentrations of the compound in the acceptor and donor solutions, respectively, at the completion of the assay, and C0 is the concentration of the compound in the donor solution before incubation.

[0232] The primary readout of the PAMPA assay is -6 The permeability value Pe is expressed in cm / s. Secondary readouts to be determined are the amount of compound in the donor and acceptor compartments and the compound retention in the membrane. Depending on the permeation rate and membrane retention, compounds are classified as "low" (Pe<0.2 and membrane retention<20%) or "medium and high" (Pe≥0.2; or Pe<0.2 and membrane retention≥20%). Each sample is measured in triplicate, and the standard deviation is determined for the permeability constant Pe. No results are displayed if the sample in the acceptor and donor solutions reaches equilibrium (no kinetic information), if the reference precipitates (turbidity measurement), or if there are analytical limitations. [Table 8]

[0233] Example 26 Single-dose pharmacokinetic (PK) study in male Wistar-Han rats The pharmacokinetic properties of selected compounds were evaluated by a single-dose PK study in male Wistar-Han rats (vendor: Beijing Vital River Laboratory Animal Technology Co., Ltd.). Briefly, two groups of animals received 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 5 minutes (IV only), 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 7 hours, and 24 hours after administration. The blood samples were placed in tubes containing EDTA-K2 anticoagulant and centrifuged at 3000 rpm for 15 minutes at 4°C to separate plasma from the samples. After centrifugation, the resulting plasma was transferred to clean tubes for bioanalysis by LC / MS / MS. Pharmacokinetic parameters were calculated using non-compartmental analysis. The 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 the

[0234] 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.

[0235] The results of the PK parameters after IV and PO administration are shown in Table 9. [Table 9]

[0236] Example 27 Human cytosolic aldehyde oxidase (AO) substrate assay The human cytosol AO substrate assay is designed to assess the metabolic stability of test compounds in human liver cytosol with or without selected aldehyde oxidase (AO) inhibitors.

[0237] Cytosolic incubations were performed in deep-well 96-well plates. Test compound conversion and oxidative metabolite formation were monitored over a 60-minute period. The incubation volume was 0.4 mL / well, with time points at 0.5, 3.5, 6.5, 10, 20, 30, 45, and 60 minutes. Human liver cytosol (1 mg protein / mL, BD UltraPool™ Human Cytosol) and test compound (1 μM in duplicate) or control compound (i.e., a known AO substrate; 1 μM in duplicate) were incubated at 37°C in a water bath. At each corresponding time point, the reaction was stopped by adding 120 μL of quench solution (hydralazine in acetonitrile, 50 μM, total organic concentration ≤1% at the final incubation), and a 40 μL sample was removed. All sample plates were mixed thoroughly and centrifuged at 3220 × g for 10–20 minutes. The supernatant was diluted with water or a buffer appropriate for LC / MS / MS analysis.

[0238] To determine the in vitro excretion rates of test and control compounds, the analyte / internal standard peak area ratio was converted to percent retention using the following formula:

number

[0239] Also, the half-life (T 1 / 2 Estimates of in vitro liver intrinsic clearance (CLint) values ​​were calculated from the rate of substrate disappearance in liver cytosol incubations as follows: CLint(cytosol)=0.693 / half-life / mg cytosol protein / mL. [Table 10]

Claims

1. Formula (I): 【Chemistry 1】 (In the formula, R １ is C １～６ is alkyl, R ２ is C １～６ is alkyl, R ３ is C １～６ Alkyl or haloC １～６ is alkyl, R ４ is piperazinyl, piperidinyl or 3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazinyl, said piperazinyl, piperidinyl or 3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazinyl being 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, Phenyl C １～６ alkyl (phenyl substituted with piperazinyl); piperazinyl, Pyrazinyl C １～６ alkyl (pyrazinyl substituted with piperazinyl); pyridinyl (pyridinyl substituted with piperazinyl), Pyridinyl C １～６ alkyl (pyridinyl is substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl or piperazinyl); pyrimidinyl (pyrimidinyl substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl), and Pyrimidinyl C １～６ Alkyl (pyrimidinyl is amino (C １～６ alkyl) azetidinyl, 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl, amino-1,4-oxazepan-4-yl or piperazinyl) and is substituted with a substituent selected from A is CH or N. or a pharmaceutically acceptable salt thereof.

2. 2. The compound of claim 1, wherein A is CH.

3. R ４ but, 【Chemistry 2】 and R ５ teeth, 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, Phenyl C １～６ alkyl (phenyl substituted with piperazinyl); piperazinyl, Pyrazinyl C １～６ alkyl (pyrazinyl substituted with piperazinyl); pyridinyl (pyridinyl substituted with piperazinyl), Pyridinyl C １～６ alkyl (pyridinyl is substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl or piperazinyl); pyrimidinyl (pyrimidinyl substituted with 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl), and Pyrimidinyl C １～６ Alkyl (pyrimidinyl is amino (C １～６ alkyl) azetidinyl, 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl, amino-1,4-oxazepan-4-yl or piperazinyl) 3. The compound according to claim 1 or 2, selected from:

4. R ４ but, 【Transformation 3】 (In the formula, R ５ａ is 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, ((piperazinyl)phenyl)C １～６ Alkyl, ((piperazinyl)pyrazinyl)C １～６ Alkyl, ((piperazinyl)pyridinyl)C １～６ alkyl, ((5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyridinyl)C １～６ Alkyl, ((amino (C １～６ Alkyl)azetidinyl)pyrimidinyl)C １～６ alkyl, ((5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl)C １～６ Alkyl, ((amino-1,4-oxazepan-4-yl)pyrimidinyl)C １～６ Alkyl, or ((piperazinyl)pyrimidinyl)C １～６ alkyl), 【Chemistry 4】 (In the formula, R ５ｂ is piperazinyl), or 【Transformation 5】 (In the formula, R ５ｃ is piperazinylpyridinyl or (5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl 4. The compound of claim 3, wherein:

5. R ４ but, 【Transformation 6】 (In the formula, R ５ａ is 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, (4-piperazin-1-ylphenyl)methyl, (3-piperazin-1-ylphenyl)methyl, (5-piperazin-1-ylpyrazin-2-yl)methyl, (5-piperazin-1-yl-2-pyridinyl)methyl, (6-piperazin-1-yl-3-pyridinyl)methyl, [6-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)-3-pyridinyl ]methyl, [2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl, [2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl, [2-[6-amino-1,4-oxazepan-4-yl]pyrimidin-5-yl]methyl, (2-piperazin-1-ylpyrimidin-5-yl)methyl, or (5-piperazin-1-ylpyrimidin-2-yl)methyl), 【Transformation 7】 (In the formula, R ５ｂ is piperazin-1-yl), or 【Transformation 8】 (In the formula, R ５ｃ is 6-piperazin-1-yl-3-pyridinyl or 2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl 5. The compound of claim 4, wherein:

6. R ３ is C １～６ 6. The compound of claim 4 or 5, wherein the compound is alkyl.

7. R ３ The compound of claim 6, wherein is ethyl or isopropyl.

8. R ４ but, 【Chemistry 9】 (In the formula, R ５ａ is ((5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl)C １～６ Alkyl or ((amino-1,4-oxazepan-4-yl)pyrimidinyl)C １～６ alkyl), or 【Chemistry 10】 (In the formula, R ５ｃ The compound of claim 6, wherein is (5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl.

9. R ４ but, 【Chemistry 11】 (In the formula, R ５ａ is [2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl or [2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl, or 【Chemistry 12】 (In the formula, R ５ｃ The compound of claim 8, wherein is 2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl.

10. R １ is C １～６ is alkyl, R ２ is C １～６ is alkyl, R ３ is C １～６ is alkyl, R ４ but, 【Chemistry 13】 (In the formula, R ５ａ is ((5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl)C １～６ Alkyl or ((amino-1,4-oxazepan-4-yl)pyrimidinyl)C １～６ alkyl), or 【Chemistry 14】 (In the formula, R ５ｃ is (5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidinyl), 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein A is CH.

11. R １ is methyl, R ２ is methyl, R ３ is ethyl or isopropyl, R ４ but, 【Chemistry 15】 (In the formula, R ５ａ is [2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl or [2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl, or 【Chemistry 16】 (In the formula, R ５ｃ is 2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl; 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, wherein A is CH.

12. 5-[2-ethyl-6-[4-[[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-ethyl-6-[4-[[6-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)-3 pyridyl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[6-[4-[[2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[6-[4-[[2-[(6S)-6-amino-1,4-oxazepan-4-yl]pyrimidin-5-yl]methyl]piperazin-1-yl]-2-ethyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-ethyl-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[4-[[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[4-[(5-piperazin-1-yl-2-pyridyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[4-[(5-piperazin-1-ylpyrazin-2-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[4-[(2-piperazin-1-ylpyrimidin-5-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[6-[4-[[2-(3-amino-3-methyl-azetidin-1-yl)pyrimidin-5-yl]methyl]piperazin-1-yl]-2-isopropyl-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[4-[(6-piperazin-1-yl-3-pyridyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[4-[(5-piperazin-1-ylpyrimidin-2-yl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[4-[(3-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[4-(5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-[8-(6-piperazin-1-yl-3-pyridyl)-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-(difluoromethyl)-6-[4-[(4-piperazin-1-ylphenyl)methyl]piperazin-1-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one; 5-[2-isopropyl-6-(4-piperazin-1-yl-1-piperidyl)-3-pyridyl]-1,3-dimethyl-pyridin-2-one; and 5-[2-ethyl-6-[2-[2-(5-oxa-2,8-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl]-3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-8-yl]-3-pyridyl]-1,3-dimethyl-pyridin-2-one or a pharmaceutically acceptable salt thereof.

13. a) Formula (IX) 【Chemistry 17】 The compound of formula (I-1) is deprotected using an acid to give the compound of formula (I-1) [Chemistry 18] obtaining a compound of formula (I), b) Formula (XV) 【Chemistry 19】 The compound of formula (I-2) is deprotected using an acid to give the compound of formula (I-3) 【Chemistry 20】 obtaining a compound of formula (I), c) Formula (XVII) 【Chemistry 21】 The compound of formula (I-3) is deprotected using an acid to give the compound of formula (I-4) 【Chemistry 22】 A step of obtaining a compound of including any of the following: wherein PG is Boc; L is piperazinyl, piperidinyl, piperazinylpiperidinyl, piperidinylpiperazinyl, or 3,4,6,7,9,9a-hexahydro-1H-pyrazino[1,2-a]pyrazin-2-yl; G １ is 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl, phenyl, piperazinyl, pyrazinyl, pyridinyl or pyrimidinyl; G ２ piperazinyl, 5-oxa-2,8-diazaspiro[3.5]nonan-2-yl, amino (C １～６ alkyl)azetidinyl or amino-1,4-oxazepan-4-yl; G ３ is 5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl; R １ , R ２ , R ３ A process for preparing a compound according to any one of claims 1 to 12, wherein A and B are defined as in any one of claims 1 to 11.

14. 13. A compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, for use as a therapeutically active substance.

15. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 and a therapeutically inert carrier.

16. 16. The pharmaceutical composition according to claim 15 for the treatment or prevention of systemic lupus erythematosus or lupus nephritis.

17. Use of a compound according to any one of claims 1 to 12 for the preparation of a medicament for the treatment or prevention of systemic lupus erythematosus or lupus nephritis.

18. Use of a compound according to any one of claims 1 to 12 for the preparation of a medicament for antagonists of TLR7 and TLR8 and TLR9.

19. 13. A compound or a pharmaceutically acceptable salt according to any one of claims 1 to 12 for the treatment or prevention of systemic lupus erythematosus or lupus nephritis.

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